**Conclusion**

While color intensity increased during CS, only slight change in anthocyanin and flavan-3-ol monomers was noted. Despite that the total phenols rise (data not shown), this might suggest that the increase in CI could be related to the extraction of co-pigments, such as caffeic acid or quercetin. Combining the observed changes in phenolic maturity (Glories method), the behavior of anthocyanins during CS and the analysis of the wines after bottling, the extractability of anthocyanins as primary factor improved through CS must be pointed out. Either the cold temperatures during CS or the activity of natural pectolytic enzymes during the lengthened period before AF are likely to have increased the permeability of the cell membranes in the grape skins. The wines which underwent cold soaking were, at the time of bottling, more intensely colored, smoother and somewhat fruitier than the comparative control wines. Even if the differences between the wines in regard of sensory perception were much smaller two years after bottling the difference in color intensity remained, with both CS treatments showing the highest values. When viewed in conjunction with the changes brought through a lengthening of maceration time, it seems clear that CS can be used as an effective tool in managing the phenolic composition and style of Pinot Noir. This technique seems especially well suited to fruit that is less than optimally ripe, offering a means of improving the extractability once alcoholic fermentation is initiated.

**Acknowledgements**

We would like to thank Weingut Bergdolt – Klostergut St. Lamprecht, Palatinate, Germany – for the grapes, as well as Anette Schormann, Sandra Klink and both sensory panels for the help during the sensory evaluation.

**Abbreviations**

CS, cold soaking; AF, alcoholic fermentation; MLF, malolactic fermentation; SPP, small polymeric pigments; LPP, large polymeric pigments; CI, color intensity; FAR, flavan-3-ol to anthocyanins ratio.

**References**

1. Fischer U, Strasser M, Gutzler K (2000) Impact of fermentation technology on the phenolic and volatile composition of German red wines. *Int J Food Sci Technol* **35**: 81-94.

2. Sacchi KL, Bisson LF, Adams DO (2005) A review of the effect of winemaking techniques on phenolic extraction in red wines. *Am J Enol Vitic* **56**: 197-206.

3. Patterson T (2009) Cold soak purists reveal reasoning. *In Wines & Vines*. Wine Communications Group, Inc., San Rafael.
# Pre-fermentation heat treatment of grapes at 50°C: a promising alternative to produce red wines with fruity varietal character

O. GEFFROY<fnref n="1" />*, R. LOPEZ<fnref n="2" />, F. VIOLEAU<fnref n="3" />, D. KLEIBER<fnref n="3" />, J.-L. FAVAREL<fnref n="4" />, V. FERREIRA<fnref n="2" />

Pre-fermentation heat treatment – thermovinification – aroma compounds – varietal character

## Introduction

There is an increasing interest in the world wine market in red wines with fruity characteristics. Thermovinification or Pre-fermentation heat treatment of grapes is becoming more widespread in France to produce wines that can fulfill these expectations. It is known to produce wines with a standardized sensory profile often described as “banana yogurt” by winemakers. Research works on volatile composition showed that maceration heat treatment allowed the elimination of a high amount of 3-isobutyl-2-methoxypyrazine [1] and that fermentation conditions of thermovinified wines particularly enhanced ester formation [2]. More recently, research works [3] suggest thermal degradation of several grape-derived aroma compounds or their precursors (i.e. some varietal thiols, monoterpenols and norisoprenoids) when grapes are heated during 3 hours at 70°C. Following these findings an experiment was launched in 2014 on Spanish Carignan and French Fer healthy grapes in order to study the impact of a pre-fermentation heat treatment of grapes at 50°C. The aim of the study was to modulate the sensory profile of thermovinified wines while maintaining the softness and the sweetness in the palate that characterized this kind of wines.

## Materials and methods

### Grapes and thermovinification treatments

Carignan and Fer grapes used for this study were obtained from two commercial vineyards located in Font-rubí (DO Penedès, Spain) and Lisle-sur-Tarn (AOP Gaillac, France) respectively. Grapes were destemmed manually and for each cultivar 8 homogenous lots of 800 g were constituted. Two levels of temperature (50°C and 75°C) and length of heating (30 minutes and 3 hours) were investigated in duplicate for each variety. Analyses were performed in musts and wines prepared under micro-vinification techniques (1L Erlenmeyer). After the grapes were crushed and received a sulfur addition (400 mg/L), the pre-fermentation heat treatment was undertaken using a water bath system. The rise in temperature of the grapes from room temperature up to the desired temperature was fast, taking exactly 40 minutes. Grapes were then pressed under controlled conditions (200 kPa for 2 min) using a laboratory press and musts were centrifuged (14 000 × g for 6 min).

### Enological and microbiological analyses

100 mL of musts were sampled to perform classical enological (°Brix, TA, pH, tartaric and malic acids, NH4+, amino acids, K+, TPI, anthocyanins, color hue, color intensity) and microbiological analyses mainly according to the OIV methods [4]. After 10 days of alcoholic fermentation, whose kinetics was followed daily by manual weighting of the flasks, wines were centrifuged, received a sulfite addition of 80 mg/L before being bottled and stored at 4°C until analysis. Major, minor, polyfunctional mercaptans and alkylmethoxypyrazines (in Fer only) were determined in wines using the methods proposed and validated in our laboratory [5-8]. Statistical analyses were conducted with XLstat software (Addinsoft, Paris, France). Principal component analysis (PCA) was employed using means of 2 replicates for each of the aroma

<footnote n="1">¹IFV Sud-Ouest, F-81310 Lisle Sur Tarn, France</footnote>

<footnote n="2">²LAAE, Universidad Zaragoza, E-50009 Zaragoza, Spain</footnote>

<footnote n="3">³LAP, Ecole d'Ingénieurs de Purpan, F-31076 Toulouse, France</footnote>

<footnote n="4">⁴Pera, F-34510 Florensac, France</footnote>
compounds measured in wines (except 3-isobutyl-2-methoxypyrazine - IBMP). Analytical data with the exception of IBMP were subjected to a three-way analysis of variance (ANOVA) treatment with first-order interaction. IBMP data were treated by a two-way ANOVA with interaction and then because the interaction was not significant, subjected to a two-way ANOVA.

## Results and discussion

The PCA treatment (Figure 1) shows that even if the *variety* remains the principal factor that allows discriminating the experimental wines, both *temperature* and *length* of heating have a large influence on the aroma compound composition of wines. While Carignan wines are grouped by temperature, Fer wines are organized on the plot by length of heating which suggests that temperature is a key factor for making thermovinified wines from Carignan, while for Fer length of heating has a larger impact. Differences between grape varieties in kinetics of extraction of skin compounds (i.e. polyphenolic compounds) from grapes heated at different levels of temperature for distinct periods have been previously described [9]. As grape-derived aroma compounds found in wines are either directly extracted from the skin or derived from precursors located in the pericarp, such results could be expected.

<figure><img src="image_302.png" /><figcaption>Fig. 1 - Principal component analysis of aroma compounds measured in experimental wines.</figcaption></figure>

The impact of *variety*, *temperature* and *length* of heating on a selection of compounds measured in experimental wines is shown in Table 1. This selection which was based on results of previous research works [4] included some grape-derived aroma compounds, one fatty acid and one acetate that are known to have a significant contribution in the “banana yogurt” typical sensory profile of thermovinified wines. As expected, in accordance with previous research works [4], higher temperature and extended length of heating enhanced the extraction from the skin of amino acids and polyphenolic compounds as reflected by TPI values. Surprisingly, such increase in nitrogen content of the must did not impact isoamyl acetate and hexanoic acid concentrations in wines. In Fer wines, no differences in IBMP concentrations were identified between the experimental wines which show that a heating at 50°C – which corresponds to the boiling point of this compound [1] – is enough when making thermovinified wines to get rid of most of the “removable” pyrazine. The same kind of conclusion can be drawn for *Saccharomyces* yeasts as neither *temperature* nor *length* of heating impacted the final population in musts that remains low.

β-citronellol whose concentration in wines is the resultant of free and bound forms, decreases with increase in temperature. It can be explained by the destruction of the associated β-glycosidase activity and/or thermal degradation of precursors/free compounds at 75°C. For other parameters shown in Table 1, such degradation can also be identified and final concentration in wines seems to be the resultant of both extraction from the skin and degradation. The negative impact of temperature of heating is particularly obvious for anthocyanins, β-damascenone, and 3-mercaptohexanol in Carignan wines. Carignan wines
<table><caption>**Table 1** - Impact of cultivar, temperature and length of heating on a selection of parameters measured in musts and wines</caption><thead><tr><th rowspan="3">Parameter</th><th colspan="4">Fer N</th><th colspan="4">Carignan N</th><th colspan="6">P-values</th></tr><tr><th colspan="2">50°C</th><th colspan="2">75°C</th><th colspan="2">50°C</th><th colspan="2">75°C</th><th rowspan="2">Variety (V)</th><th rowspan="2">T°C (T)</th><th rowspan="2">Length (L)</th><th rowspan="2">VxT</th><th rowspan="2">VxL</th><th rowspan="2">TxL</th></tr><tr><th>30'</th><th>3h</th><th>30'</th><th>3h</th><th>30'</th><th>3h</th><th>30'</th><th>3h</th></tr></thead><tbody><tr><td>Amino acids<sup>m</sup> (mg/L)</td><td>134<sup>a</sup></td><td>186</td><td>196</td><td>245</td><td>91</td><td>120</td><td>123</td><td>146</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>ns</td></tr><tr><td>TPI<sup>m</sup></td><td>23,4</td><td>45,1</td><td>57,2</td><td>82,4</td><td>51,7</td><td>68,0</td><td>70,4</td><td>80,8</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>0,01</td><td>ns</td></tr><tr><td>Anthocyanins<sup>m</sup> (mg/L)</td><td>358</td><td>1141</td><td>1416</td><td>1527</td><td>952</td><td>1448</td><td>1483</td><td>1329</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td></tr><tr><td>Saccharomyces<sup>m</sup> (CFU/mL)</td><td>13,5</td><td>17,5</td><td>2,0</td><td>15,0</td><td>609</td><td>12</td><td>10</td><td>8,5</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Isoamyl acetate<sup>w</sup> (mg/L)</td><td>1,27</td><td>1,86</td><td>2,26</td><td>1,40</td><td>0,34</td><td>0,22</td><td>0,37</td><td>0,54</td><td>&lt;0,001</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>Hexanoic acid<sup>w</sup> (mg/L)</td><td>1,07</td><td>1,41</td><td>1,71</td><td>0,77</td><td>1,04</td><td>0,99</td><td>0,89</td><td>1,13</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>β-damascenone<sup>w</sup> (µg/L)</td><td>3,49</td><td>0,17</td><td>1,49</td><td>0,48</td><td>5,42</td><td>3,21</td><td>2,43</td><td>1,11</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>&lt;0,001</td><td>ns</td><td>&lt;0,001</td></tr><tr><td>β-citronellol<sup>w</sup> (µg/L)</td><td>10,11</td><td>9,84</td><td>10,20</td><td>8,15</td><td>8,65</td><td>8,67</td><td>6,94</td><td>7,79</td><td>&lt;0,01</td><td>&lt;0,05</td><td>ns</td><td>ns</td><td>ns</td><td>ns</td></tr><tr><td>3-mercapto-hexanol<sup>w</sup> (ng/L)</td><td>1360</td><td>338</td><td>1133</td><td>417</td><td>2395</td><td>2188</td><td>1131</td><td>613</td><td>&lt;0,01</td><td>&lt;0,01</td><td>&lt;0,05</td><td>&lt;0,05</td><td>ns</td><td>ns</td></tr><tr><td>IBMP<sup>b,w</sup> (ng/L)</td><td>18,2</td><td>16,1</td><td>16,5</td><td>18,4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>ns</td><td>ns</td><td>-</td><td>-</td><td>-</td></tr></tbody></table>

<footnote><sup>a</sup>Mean of 2 replicates; <sup>b</sup>3-isobutyl-2-methoxypyrazine, <sup>m</sup>analyzed in musts; <sup>w</sup>analyzed in wines; ns non-significant</footnote>
made from grapes heated at 50°C for 3 hours presented significantly higher concentrations in anthocyanins (+119 mg/L), 3-mercapto-hexanol (+257%), and β-damascenone (+189%) in comparison with wines made after a 3 hour heat treatment of grapes at 75°C, a couple T°C/length of heating frequently implemented in industrial conditions. Consequently a large impact on the sensory profile of Carignan wines is expected. However the same conclusions could not be drawn for Fer which lead us to think that mechanisms involved are complex and deserve further study. Even if the experimental musts were deeply centrifuged prior to fermentation, presented a very low and comparable solid contents, conditions of fermentation may also play a role in the release of aroma compounds from their precursors.

## Conclusion

Our results show that a pre-fermentation heat treatment of grapes at 50°C for 3 hours could be implemented on Carignan healthy grapes to produce colored red wines with high concentrations in varietal aroma compounds (i.e. β-damascenone, 3-mercaptohexanol). The overall impact expected on the sensory profile of wines is large and will be evaluated soon by the tasting of wines elaborated under micro-vinification conditions (75 kg). The same results could not be observed in Fer wines which suggests that i) mechanisms involved are complex and aroma compound concentrations in wines are the resultant of both extraction from the skin and thermal degradation, and ii) temperature and length of heating must be adapted to each grape variety. The impact of these two factors on polyphenolic composition was also investigated in 2014 and results will be available soon. Alternatively, as this range of temperature could never be implemented on botrytised grapes, a partial Flash-détente process is currently under study in collaboration with Pera. It consists in heating the grapes briefly at 75°C to inhibit laccase activity and to limit thermal degradation, and then after vacuum and release at atmospheric pressure, the maceration is maintained with the skin for several hours at 50°C.

## Acknowledgements

This study was carried out with financial support from the Communauté de Travail des Pyrénées (CTP), the Région Midi-Pyrénées and the Gobierno de Aragón.

## References

1. Roujou de Boubée D (2000) Recherche sur la 2-méthoxy-3-isobutylpyrazine dans les raisins et les vins. Approches analytique, biologique et agronomique. *PhD Thesis*, University of Bordeaux 2.

2. Girard B, Kopt TG, Reynolds AG (1997) Influence of vinification treatments on aroma constituents and sensory descriptors of Pinot noir wines. *Am J Enol Vitic* **48**: 198-206.

3. Geffroy O, Lopez R, Serrano E et al (2015) Changes in analytical and volatile compositions of red wines induced by pre-fermentation heat treatment of grapes. *Food Chem* **187**: 243-253.

4. OIV (2009) *Recueil des méthodes internationales d'analyse des vins et des moûts*, Organisation Internationale de la Vigne et du Vin.

5. Ortega C, Lopez R, Cacho J et al (2001) Fast analysis of important wine volatile compounds: development and validation of a new method based on gas chromatographic-flame ionisation detection analysis of dichloromethane microextracts. *J Chromatogr A* **923**: 205-214.

6. Lopez R, Aznar M, Cacho J et al (2002) Determination of minor and trace volatile compounds in wine by solid-phase extraction and gas chromatography with mass spectrometric detection. *J Chromatogr A* **966**: 167-177.

7. Mateo-Vivaracho L, Cacho J, Ferreira V (2008) Improved solid-phase extraction procedure for the isolation and in-sorbent pentafluorobenzyl alkylation of polyfunctional mercaptans: optimized procedure and analytical applications. *J Chromatogr A* **1185**: 9-18.

8. Lopez R, Gracia-Moreno E, Cacho J et al (2010) Development of a mixed-mode solid phase extraction method and further gas chromatography mass spectrometry for the analysis of 3-alkyl-2-methoxypyrazines in wine. *J Chromatogr A* **1218**: 842-848.

9. Feilhes C, Favarel JL, Durand S et al (2013) Etude de la macération préfermentaire à chaud : Application à 3 cépages du Sud-Ouest. Méthodologie de suivi et optimisation des conditions opératoires. *Rev Oenol* **148**: 17-20.
# Réduction du SO₂ dans les vins rouges

J. COULON<fnref n="1" />*, B. NAZARIS<fnref n="2" />, C. MANSOUR<fnref n="1" />, V. MOINE<fnref n="1" />

## SO₂ – diminution des intrants – molécules combinant le SO₂

### Introduction

La diminution de l'utilisation du SO₂ est une thématique de plus en plus recherchée par les vinificateurs. Les raisons qui sous-tendent ce choix sont nombreuses, et concernent entre autres le souhait de diminution d'un intrant considéré comme de moins en moins actif vis-à-vis des altérations microbiologiques ; des critères organoleptiques ou des optimisations des caractéristiques chromatiques ; ou bien en réponse à un marché souhaitant la diminution des allergènes dans les vins.

Dans le cas de cette étude, il s'agit, non pas d'annuler totalement l'utilisation de SO₂, mais de limiter son emploi, en ayant pour objectif de potentialiser son action. L'activité du SO₂ dépend d'une part de critères chimiques intrinsèques au vin (pH et TAV notamment), mais aussi de la présence de molécules combinant le SO₂. Les travaux présentés ici visent donc non seulement à limiter l'emploi de SO₂, mais aussi la synthèse de molécules le combinant lors des fermentations et de l'élevage du vin.

Divers essais assortis d'analyses microbiologiques et chimiques permettent d'observer les conséquences de la diminution du SO₂ et de proposer des alternatives lors de plusieurs essais à l'échelle pilote. Des marqueurs microbiologiques (populations levuriennes et bactériennes) mais aussi chimiques comme la mesure du niveau de molécules combinant le SO₂ ont été appréhendés.

### Matériel et méthodes

#### Mise en place des essais

Différents essais ont été réalisés en chai, sur des volumes allant de 2 hL à 4 hL. Les cépages utilisés sont le merlot, le cabernet sauvignon et le petit verdot, sur vendange ramassée et triée manuellement ou mécaniquement (précisions dans le texte ou légendes). Pour les différentes modalités composant les essais, la vendange a été répartie de façon homogène dans les différentes cuves. Lorsqu'ils sont ensemencés, les moûts le sont le plus rapidement possible selon les préconisations du fabriquant, dès l'encuvage, et les cuves préalablement inertées. Les cinétiques fermentaires sont appréciées par densimétrie.

#### Analyses microbiologiques

Les populations de levures totales et les levures non-*Saccharomyces* sont dénombrées sur des milieux gélosés spécifiques (SARCO). Les populations de *B. bruxellensis* sont déterminées par Q PCR (Bio-Rad).

#### Mesure du SO₂ total et libre, calcul du TL35

Les teneurs en SO₂ total et en SO₂ libre sont appréciées par dosages colorimétriques en flux continu. Les incertitudes sont respectivement de 7% et 8%.

Le TL35 (incertitude 15%) est un indice mesuré après ajouts dosés de SO₂ dans l'échantillon, sur une gamme allant de 0 à 200 mg/L : Après dosage du SO₂ total et du SO₂ libre, une droite SO₂ total = f (SO₂ libre) est obtenue et permet de calculer la dose théorique de SO₂ total qu'il faut dans le vin pour obtenir un SO₂ libre à 35 mg/L. Cette mesure est donc un indicateur du niveau de composés combinant le SO₂ dans les vins. Plus il est élevé, plus leur concentration est importante.

<footnote n="1">¹BioLaffort, 126 Quai de la Souys, 33100 Bordeaux Cedex, France</footnote>

<footnote n="2">²Laffort, 126 Quai de la Souys, 33100 Bordeaux Cedex, France</footnote>
# Résultats

## Maitrise microbiologique du moût par le levurage

Lors de vinifications de cépages rouges, un des rôles essentiels du sulfitage de la vendange est la maîtrise microbiologique du moût, afin d'assurer l'implantation de la seule espèce véritablement capable de mener à bien la fermentation alcoolique, S. cerevisiae. La figure 1 montre l'importance du levurage sur les dynamiques de levures non-Saccharomyces et indique que ce dernier participe davantage que le sulfitage à la maîtrise de la population indigène, qui à lui seul, et à des niveaux de 2 à 4 g/hL, ne suffit pas à pondérer son développement. De plus, le développement de levures non-Saccharomyces indigènes participe directement à la synthèse de molécules combinant le SO₂ (tableau 1).

<figure><img src="image_303.png" /><figcaption>**Fig. 1** - Dynamiques de levures non-Saccharomyces. Deux niveaux de sulfitage de la vendange ont été pratiqués, accompagnés ou non d'un levurage précoce (FX10, Laffort, appliquée à 20 g/hL) dès l'encuvage. Les essais ont été réalisés lors du millésime 2012, sur des volumes de 4 hL, à partir d'une vendange de merlot saine et ramassée manuellement.</figcaption></figure>

<table><caption>**Tableau 1** - Populations maximales en cours de FA de levures non-Saccharomyces (NS) et lien avec le niveau de molécules combinant le SO₂. Ces résultats proviennent du même essai que celui décrit à la figure 1.</caption><thead><tr><th></th><th>Vendange sulfitée 4 g/hL non ensemencée</th><th>Vendange sulfitée 4 g/hL ensemencée</th></tr></thead><tbody><tr><td>Population maximale levures NS (UFC/mL) en cours de FA</td><td>2,1.10<sup>6</sup></td><td>5,6.10<sup>4</sup></td></tr><tr><td>TL35 fin FA (mg/L)</td><td>84</td><td>71</td></tr></tbody></table>

## Dégradation de molécules combinant le SO₂ lors de la fermentation malolactique

Les bactéries lactiques sont capables de dégrader lors de la fermentation malolactique diverses molécules combinant le SO₂ dont l'éthanol, l'acide pyruvique et l'acide 2-oxoglutarique essentiellement formés lors de la fermentation alcoolique lors de la phase de vinification. Néanmoins, la vitesse de la FML a un rôle déterminant dans ce processus (figure 2). Selon le délai de son achèvement, le TL35 peut diminuer, indiquant une dégradation nette de molécules combinant le SO₂, ou bien au contraire augmenter. Cette augmentation est soit liée à un métabolisme atténué des composés combinant le SO₂ par les bactéries lactiques, soit à une oxydation chimique de l'éthanol en éthanal plus importante que sa dégradation par les bactéries.
Les outils de stabilisation des vins post-FML

Les données précédentes indiquent l'importance du levurage et la maîtrise de la FML (par le biais d'un ensemencement en bactéries lactiques) dans la maîtrise des composés combinant le SO2. Afin de poursuivre cet effort, et optimiser le premier sulfitage d'élevage, il peut être envisagé de diminuer dans un premier temps la flore post-fermentaire à l'aide d'agents de stabilisation microbiologique autres que le SO2. Le chitosane est une molécule utilisée en œnologie afin de diminuer les populations de B. bruxellensis mais son champ d'action est en réalité plus large et cible aussi bien les levures que les bactéries lactiques. La figure 3 indique que l'application à 8 g/hL d'un produit à base de chitosane (OenoBrett®, Laffort), permet en effet de limiter la présence de molécules combinant le SO2 alors qu'un sulfitage immédiat après FML (ici appliqué à une dose de 2 g/hL), au contraire, augmente leur synthèse.

<figure><img src="image_304.png" /><figcaption>**Fig. 2** - Relation entre le délai d'achèvement de la fermentation malolactique et le niveau de combinaison des vins vis-à-vis du SO2. Ces résultats proviennent du même essai que celui décrit à la figure 1. En fin de FA, le TL35 des vins se situe entre 70 et 85 mg/L. Les points pour lesquels le TL35 sont les plus faibles correspondent à des essais ensemencés à 1 g/hL avec des bactéries lactiques sélectionnées (450 PreAc, Laffort).</figcaption></figure>

<figure><img src="image_305.png" /><figcaption>**Fig. 3** - Effet de la stabilisation microbiologique post FML à l'aide d'OenoBrett® (Laffort) sur l'évolution de la teneur des vins en composés combinant le SO2. Les essais à itinéraire réduit en SO2 sont comparés à des témoins sulfités à la vendange ainsi qu'après FML. Les essais ont été réalisés lors du millésime 2014, sur cabernet-sauvignon-petit verdot ramassé et trié mécaniquement.</figcaption></figure>
## Discussion et conclusion

La limitation de l'utilisation du SO₂ concerne la recherche de moyens œnologiques afin de limiter la présence de molécules le combinant et le rendre ainsi le plus actif possible. Il s'agit donc d'un cercle vertueux dont le point de départ est la maitrise de la flore fermentaire. En effet, ces travaux montrent que le développement des levures non-Saccharomyces, pondéré par le levurage, s'accompagne d'une augmentation des molécules combinant le SO₂. Ces levures indigènes, en général plus sensibles au SO₂ que l'espèce S. cerevisiae, répondent au sulfitage de la vendange par une synthèse accrue de ces composés. Des études d'écologie microbienne sur moûts et vins en cours de FA associées à la détermination fine de la sensibilité de différentes espèces et souches de levures non-Saccharomyces vis-à-vis du SO₂ permettraient de préciser la participation de la microflore indigène vis-à-vis de leur synthèse.

D'autre part, ces molécules peuvent ensuite être dégradées par les bactéries lactiques lors de la fermentation malolactique, mais aussi dans les jours qui la suivent [1]. Cette dégradation pourrait donc être optimisée en repoussant de quelques jours la stabilisation du vin après la fin de la FML. De plus, si la vitesse de la FML est clairement un élément important dans cette dégradation, il reste encore à appréhender l'impact de la souche de bactérie sur la qualité de cette dégradation.

Enfin, pour limiter leur synthèse par les microorganismes en début d'élevage, il peut être envisagé dans un premier temps, de stabiliser les vins par d'autres moyens que le sulfitage, par exemple à l'aide de produits à base de chitosane, ce qui ne rendra que plus actif le SO₂ rajouté par la suite.

## Abréviations

FA, fermentation alcoolique ; FML, fermentation malolactique.

## Références

1. Jackowetz JN, Mira de Orduña R (2012) Metabolism of SO₂ binding compounds by *Oenococcus oeni* during and after malolactic fermentation in white wine. *Int J Food Microbiol* 155: 153-157.
# A combination of biotechnology tools and cooper elements for an alternative to post-MLF SO₂ addition

V. RENOUF*, M. MIRABEL, A.-C. MONTEAU

Chêne & Cie Research department of Taransaud, Canton, Kadar cooperages and Xtrachêne

SO₂ – microbiology (*Brettanomyces*, lactic acid bacteria) – wood antioxidant capacity

## Introduction

The post-MLF (malolactic fermentation) SO₂ addition is an essential event of the transition between fermentations and aging. At this stage, the SO₂ plays two fundamental actions: the antimicrobial action and the antioxidant effect. Nowadays, the high pH found in most wines reduces the benefit of the molecular SO₂ from a microbial point of view. Recent works on *Brettanomyces* strains have shown that some of them are resistant up to 1.2 mg/L of molecular SO₂ [1]. These highly SO₂ resistant strains take benefits from the elimination of other sensitive microorganisms when the molecular SO₂ is only 0.4/0.5 mg/L. Perhaps that, contrary to the desired effect, the SO₂ (associated with excessively low doses and / or too high pH) is partly responsible for contamination by *Brettanomyces* during wines aging.

Biotechnological products are now available for effective antimicrobial actions at the end of MLF. Lysozyme inhibits correctly the lactic acid bacteria (LAB). Chitosan also acts on the LAB but also efficiently on *Brettanomyces*.

Regarding the antioxidant effect we thought that wood barrels could potentially play a protective role. The development of an electrochemical sensor was used to estimate the influence of different cooperage factors on the antioxidant capacity (CaOx) suitable to be conferred by the wood to the wine. Optimized CaOx barrels were designed [2].

Tests were carried out to compare classical post-MLF SO₂ additions (5 g/hL) in classic new barrels (control modalities) to a treatment with a mixture of lysozyme and chitosan in CaOx optimized barrels (test modalities) during different types of aging.

## Materials and methods

### Trials

The trials presented in this work were conducted during the vintage 2014 in two different estates of the Bordeaux area, one located in the Saint-Emilion appellation, the other in the Pauillac appellation. In Saint-Emilion, the test was conducted on a Merlot wine after a MLF performed in barrels. In Pauillac, the test was conducted on a Cabernet-Sauvignon wine after a MLF performed in a stainless tank. In the control modalities, 5 g/hL of SO₂ were added at the end of the MLF. In the test modalities, Lysozyme (Laffort) was added at 25 g/hL and chitosan in the form of the commercial product Oenobrett (Laffort) was added to the equivalent dose of 4 g/hL. The barrels used for control modalities were classic new barrels and the barrels used for test modalities were new barrels produced with optimized CaOx [2]. Each modality was duplicated.

### Microbiological monitoring

Microbiological monitoring was done by numeration of LAB and *Brettanomyces* populations on nutritive selective media [3]. Serial dilutions of each wine sample were used to inoculate in triplicate plates of two different nutritive media. The yeast populations were cultivated on YPG medium (glucose 20 g/L, bactotryptone 10 g/L, yeast extract 10 g/L and agar 25 g/L, pH adjusted to 5.0 using orthophosphoric acid). To count *Brettanomyces* population, after sterilization, the medium was supplemented with biphenyl (Fluka) (0.015% w/v), chloramphenicol (Sigma Aldrich) (0.01% w/v) and cycloheximide (Sigma Aldrich)
(0.5% w/v). The plates were incubated at 25°C for 7 days. After culturing on plates, yeasts belonging to B. bruxellensis species were confirmed by PCR [4]. LAB were isolated on MRS modified medium (Lactobacilli MRS broth (Difco) 55 g/L, D-L malic acid (Prolabo) 10 g/L, agar 20 g/L, pH 4.8 with NaOH 10N, supplemented with 20% of pasteurized commercial apple juice) on Petri dishes; yeast growth was inhibited by adding 50 mg/L of pimaricine (Delvocid, DSM Food Specialties) and acetic acid bacteria growth was inhibited by incubation under anaerobic conditions using an anaerobic system with a palladium catalyst (BBL). LAB plates were incubated at 25°C for 7 days.

### Chemical and dissolved O₂ analyses

Conventional chemical assays, free and total SO₂, volatile acidity and color parameters were done by following the official methods recommended by the “International Organisation of Vine and Wine” (OIV) [5]. The level of volatile phenols was determined by SBSE/GC/MS. Levels of diacetyl (SBME/GC/MS) were also compared. The stability of the color was estimated by a cold test (measurement of the turbidity before and after the cold treatment, internal method of the laboratory SARCO). All the barrels were also fitted with an innovative device allowing to measure the rate of dissolved oxygen without opening the bung including a specific equipment inserted into the wood specially and exclusively developed by us, allowing the use of the oxo-luminescence technology manufactured by NomaSense® (Nomacorc). In this way, the oxygen dissolved in the wine present in barrels was monitored without opening the bung of the barrels.

## Results

Figure 1 illustrates the evolution of the microbial populations in the trial realized at the Pauillac estate after the end of the MLF in the two modalities. This result is confirmed by the numeration done for the Saint-Emilion trial, which is compiled in table 1. The results show that, in the two cases, the test wines without SO₂ are microbiologically more stable than the control wines (with SO₂).

<figure><img src="image_306.png" /><figcaption>**Fig. 1** - Microbial population in the two modalities of the Pauillac trial. Day 0 was the day of SO₂ addition in the control modalities and the day of chitosan and lysozyme addition in the test modalities (average of two duplicates).</figcaption></figure>
<table><caption>**Table 1** - Microbial numerations for the Saint-Emilion trial each month after the end of the MFL (* population below 1 CFU for 10 mL)</caption><thead><tr><th rowspan="2"></th><th colspan="2">Lactic acid bacteria<br>population (CFU/mL)</th><th colspan="2">Non-Saccharomyces ("Brettanomyces")<br>population (CFU/mL)</th></tr><tr><th>Control</th><th>Test</th><th>Control</th><th>Test</th></tr></thead><tbody><tr><td>1 month after the end<br>of the MLF</td><td>1,75.10<sup>5</sup></td><td>3,20.10<sup>1</sup></td><td>&lt;1*</td><td>&lt;1*</td></tr><tr><td>2 months after the end<br>of the MLF</td><td>1,10.10<sup>5</sup></td><td>1,26.10<sup>2</sup></td><td>1</td><td>2</td></tr><tr><td>3 months after the end<br>of the MLF</td><td>2,60.10<sup>5</sup></td><td>7,65.10<sup>1</sup></td><td>10</td><td>1</td></tr><tr><td>4 months after the end<br>of the MLF</td><td>1,80.10<sup>5</sup></td><td>4,55.10<sup>2</sup></td><td>3,0.10<sup>1</sup></td><td>1</td></tr></tbody></table>

The analysis of the metabolites suitable to be produced by the residual microbial population (volatile phenols for the *Brettanomyces* and diacetyl for the LAB) for the Pauillac trial (Table 2) shows that the reduction of the microbial populations in the test modalities has an effect on the level of these metabolites.

<table><caption>**Table 2** - Chemical analyses performed 3 months after the end of the MLF during the Pauillac trial</caption><thead><tr><th></th><th>Control</th><th>Test</th></tr></thead><tbody><tr><td>Diacetyl (mg/L)</td><td>8,8 +/- 1,2</td><td>1,8 +/- 0,2</td></tr><tr><td>4-ethyphenol + 4-ethylguaiacol (µg/L)</td><td>88 +/- 16</td><td>Not detected</td></tr><tr><td>Volatile acidity (g/L of H<sub>2</sub>SO<sub>4</sub>)</td><td>0,38 +/- 0,04</td><td>0,24 +/- 0,01</td></tr><tr><td>Turbidity</td><td>168 +/- 22</td><td>88 +/- 12</td></tr></tbody></table>

Figure 2 shows the result of the color stability test done 3 months after the end of the MLF in the Saint-Emilion trial. By this test, a significant delta of turbidity demonstrates a high instability of the coloring matter. In our case, the color is more unstable in control barrels than in test barrels.

<figure><img src="image_307.png" /><figcaption>**Fig. 2** - Analysis of the color stability 3 months after the end of the MLF in the Saint-Emilion trial.</figcaption></figure>

Figure 3 deals with the measurement of the O₂ dissolved in the wines during the Saint-Emilion trial. Mainly at the beginning of the monitoring, the level of dissolved O₂ was significantly lower in wines from the CaOx optimized barrels than in those from the control barrels.
<figure><img src="image_308.png" /><figcaption>Fig. 3 - Measurement of dissolved oxygen in wines during the Saint-Emilion trial.</figcaption></figure>

## Conclusion

The results obtained in these two trials are very promising. They suggest that the use of biotechnology agents such as lysozyme and chitosan is more effective from a microbial standpoint than the use of SO₂ at the end of the malolactic fermentation. The wines of the test modalities were kept several months without SO₂ and without microbial deviations. That does not mean that we should prohibit the use of SO₂, but rather that SO₂ should be added at more appropriate times. No addition of SO₂ in wines at the end of MLF seems also to promote a better stabilization of the color. From a dissolved oxygen standpoint, it also appears that the selection of wooden barrels with high antioxidant capacity limits the dissolved oxygen in the wine more effectively than the presence of free SO₂.

Therefore the synergistic effects of chitosan, lysozyme and CaOx optimized barrels seem very interesting. New tests will be conducted to confirm these first observations and to better understand certain variables. The two trials differed by the management of MLF (in tank or in barrels, with a malolactic starter or with indigenous flora) and aging (racking frequency). It is also possible that these phenomena influence the results obtained with this protocol.

## References

1. Duckitt E (2012) Investigating the impact of sulphur dioxide on *Brettanomyces bruxellensis* at a molecular and cellular level. *PhD Thesis*, University of Stellenbosch, http://scholar.sun.ac.za.

2. Mirabel M, Renouf V (2015) La protection naturelle des vins blancs contre l'oxydation prématurée lors de l'élevage en fûts. Utilisation d'un outil innovant de mesure de capacité antioxydante (CAOX). *Rev Oenol* **154**: 29-32.

3. Lonvaud-Funel A, Renouf V, Strehaiano P (2010) *Microbiologie du vin, bases fondamentales et applications*, Editions Tec&Doc, Lavoisier, Paris, France.

4. Ibeas JI, Lozano I, Perdigones F et al (1996) Detection of *Dekkera-Brettanomyces* strains in sherry by a nested PCR method. *Appl Environ Microbiol* **62**: 998-1003.

5. OIV (2009) *Compendium of international methods of wine and must analysis*, International Organisation of Vine and Wine, Paris.
# Procédé innovant de régénération de barriques par Ultrasons Haute Puissance : impact sur la qualité du vin

P. RENAULT<sup>1,2,3*</sup>, A. TRILLAUD<sup>3</sup>, F. MEUNIER<sup>4</sup>, A. MASSOT<sup>4</sup>, M. MIETTON-PEUCHOT<sup>1,2,4</sup>, R. GHIDOSSY<sup>1,2</sup>

Barriques – ultrasons – hygiène – *Brettanomyces*

## Introduction

La barrique est un élément essentiel lors de l'élaboration du vin notamment grâce à la complexité et aux caractéristiques gustatives qu'elle apporte. En effet, au cours de l'élevage en fût, l'oxygénation ménagée procure une meilleure stabilité physico-chimique et les échanges vin/bois procurent finesse, équilibre et complexité aromatique très appréciés du consommateur. La barrique "d'occasion" est donc, pour de nombreuses structures viticoles, une réponse bien adaptée pour le viticulteur à la recherche de l'équilibre vin/bois et de l'ajustement financier de sa production.

Cependant, il est primordial de bien entretenir les barriques car l'accumulation des dépôts de tartre au cours des élevages successifs peut perturber les échanges bois/vin et oxygène/vin, en créant une véritable barrière mécanique. De plus, l'utilisation des barriques sur plusieurs élevages représente un facteur de risque important sur le plan microbiologique, puisque les reliquats de vins retenus dans les pores du bois (environ 5L pour une barrique de 225L), représentent un substrat nutritif idéal pour le développement et la survie de microorganismes indésirables pouvant être transmis au vin au cours des élevages ultérieurs. Le principal problème des vinificateurs lors de l'élevage en barriques usagées est le développement de levures d'altération du genre *Brettanomyces* responsables de l'apparition du caractère phénolé due à des teneurs excessives en éthyl-4-phénol et éthyl-4-gaïcol. En effet, lorsque la contamination est avérée, il est très difficile de nettoyer et désinfecter correctement le bois en profondeur ce qui est pourtant essentiel puisque les levures d'altération *Brettanomyces* pénètrent dans le bois jusqu'à 8 mm de profondeur [1]. Il existe de nombreuses méthodes de nettoyage ou désinfection de barriques : eau chaude, vapeur, traitement chimique, méchage, ozone, eau sulfitée et grattage-rebrûlage mais les nombreuses études à ce sujet ont toutes démontrées qu'elles ne permettaient pas une décontamination efficace dans la profondeur du bois [1-3].

Dans ce contexte, la société Dyogéna a développé un procédé de régénération de barriques basé sur l'utilisation d'Ultrasons Haute Puissance. En effet, les HPU (High Power Ultrasound) sont utilisés depuis peu dans de nombreux procédés industriels [4, 5] et sont caractérisés par des intensités supérieures à 1 W/cm<fnref n="2" /> et des fréquences comprises entre 20 et 100 kHz [6, 7]. Quand ils sont émis dans un liquide cela se traduit par la formation de micro bulles de haute énergie (phénomène de cavitation acoustique) [8, 9] qui génèrent localement des températures très élevées et des pressions supérieures à 50 MPa [6]. Le phénomène de cavitation généré par les HPU permet la mort des bactéries et levures par désintégration cellulaire [10, 11]. Enfin, les traitements HPU et thermiques (45-60°C) ont des effets synergiques sur l'inactivation des microorganismes [12].

Le but de cette étude est d'étudier l'impact du procédé de régénération par ultrasons haute puissance sur la qualité des vins élevés en barriques traitées. Ainsi, l'effet du procédé sur les microorganismes d'altération (en surface et en profondeur), sur le détartrage des barriques et sur son impact aromatique au cours de l'élevage du vin a été évalué et comparé aux résultats obtenus par nettoyage des barriques à la vapeur.

<footnote n="1"><sup>1</sup>Univ. Bordeaux, ISVV, EA 4577, Unité de recherche Œnologie, 33140 Villenave d'Ornon, France</footnote>

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

<footnote n="3"><sup>3</sup>Dyogéna, 33290 Blanquefort, France</footnote>

<footnote n="4"><sup>4</sup>Amarante Process, 33140 Villenave d'Ornon, France</footnote>

<footnote>*Auteur principal : philippe.renault33@gmail.com</footnote>
# Matériel et méthodes

Le principe du procédé consiste à remplir la barrique d'eau chaude (chauffée à 60°C par un système autonome) et à y appliquer des ultrasons haute puissance (20 KHz, 4 KW) par l'intermédiaire d'une sonotrode qui s'insère dans le trou de bonde du fût. La propagation des ultrasons va entraîner une légère dépression dans l'eau et la formation de bulles de vapeur d'eau qui, en même temps que la pression descend, vont imploser en libérant une énergie très élevée. Ces micro-bulles de cavitation (diamètre de l'ordre d'un micromètre) agissent en tout point du fluide de façon homogène (loi Pascal) et pénètrent en profondeur dans tous les trous et pores du bois. Cette vibration puissante va permettre de dissoudre le tartre et de briser les parois cellulaires des microorganismes en surface et en profondeur. L'eau utilisée pour le traitement de la barrique est filtrée et renvoyée dans le circuit afin d'être réutilisée (limitation des consommations). Ce système s'inscrit dans le cadre de la réduction des intrants oenologiques (utilisation d'eau chaude sans additifs ou produits chimiques).

# Résultats et discussion

## Détartrage / oxygénation ménagée

Le traitement aux ultrasons permet de détartrer complètement les barriques (Figure 1). Le nettoyage en profondeur des barriques permet ainsi d'augmenter sensiblement l'oxygénation ménagée en comparaison au traitement vapeur (mesure de la vitesse de consommation d'oxygène à l'aide d'une méthode non intrusive : pastille Presens-pst3) (données non montrées).

<figure><img src="image_309.png" /><figcaption>**Fig. 1** - Photographie de l'intérieur d'une barrique ayant contenu du vin blanc durant un (A et B) et deux élevages (C et D) avant (A et C) et après (B et D) traitement de 6 minutes aux ultrasons haute puissance.</figcaption></figure>

## Impact aromatique

Afin d'évaluer l'impact aromatique du procédé, des barriques (chêne français) de différents âges (1, 2 et 3 ans) et différentes marques ont été utilisées. Pour chaque comparaison, un vin issu d'un même lot a été entonné dans un même lot de barriques, seul le traitement initial avant entonnage (HPU 6 minutes ou
vapeur 6 minutes) était différent. Ces expériences ont été réalisées en triplicata (3 barriques par modalités) dans différents châteaux de la région bordelaise. L'impact aromatique du procédé a été évalué en réalisant des analyses sensorielles et des analyses chimiques des composés volatils du bois (GC-MS) à plusieurs reprises sur les 12 mois d'élevage.

Les analyses sensorielles ont été réalisées selon la méthode décrite par Martin et de Revel [13]. Le panel était composé de 30 dégustateurs professionnels (14 femmes et 16 hommes) âgés entre 23 et 58 ans.

Après 12 mois d'élevage, quelque soit l'âge de la barrique, les vins élevés dans les barriques traitées par ultrasons sont perçus d'un point de vue olfactif comme étant significativement plus intenses avec notamment des notes grillées/fumées plus importantes, mais aussi beaucoup plus nets et complexes (Figure 2). En bouche, les vins élevés dans les barriques traitées par ultrasons présentent un volume (caractère enrobant) plus important, sont mieux équilibrés et surtout moins asséchants que les vins élevés dans les barriques nettoyées à la vapeur. Il est intéressant de noter que ces différences organoleptiques étaient déjà perceptibles après 4 et 8 mois d'élevage mais elles sont beaucoup plus nettes après 12 mois.

<figure><img src="image_310.png" /><figcaption>**Fig. 2** - Analyse sensorielle descriptive pour un même vin rouge élevé pendant 12 mois dans des barriques de 3 élevages traitées par ultrasons haute puissance ou nettoyées à la vapeur.
Echelle des descripteurs graduée de 0 (faible intensité) à 7 (forte intensité).
\* signifie une différence significative (Test Student, p<0,05).</figcaption></figure>

<figure><img src="image_311.png" /></figure>

Ces résultats sensoriels sont confirmés d'un point de vue analytique où les vins élevés dans les barriques traitées par ultrasons ont des concentrations en composés volatils du bois supérieures de 20% à 60% aux vins élevés dans les barriques nettoyées à la vapeur (somme des composés suivants : furfural, 5-méthylfurfural, gaïacol, trans et cis-whiskylactone, eugénol, isoeugénol, syringol, vanilline, acétovanilline, syringaldéhyde, et acétosyringone) (Figure 3). L'augmentation de l'apport boisé est variable d'un type de barrique à une autre (influence du type de chauffe, de l'âge de la barrique, de la vinification). De même, l'augmentation de l'extraction des différents composés volatils est variable, même si 4 composés semblent toujours être extraits de manière significativement plus importante : le furfural, le 5-méthylfurfural, la vanilline et la trans-whiskylactone (Figure 4).

<figure><img src="image_312.png" /><figcaption>**Fig. 3** - Concentrations totales en composés volatils du bois dans le vin après 12 mois d'élevage dans des barriques de 1, 2 ou 3 ans nettoyées par ultrasons (HPU) ou vapeur.</figcaption></figure>
<figure><img src="image_313.png" /><figcaption>**Fig. 4** - Evolution de la concentration en *trans*-whiskylactone dans des vins après 12 mois d'élevage dans des barriques de 1, 2 ou 3 ans nettoyées par ultrasons(HPU) ou vapeur. Valeurs moyennes de 3 répétitions, écart type maximal ≤ 10%.</figcaption></figure>

Cette augmentation de l'extraction des composés volatils du bois semble due à 2 phénomènes. Tout d'abord le procédé de régénération de barriques par HPU permet un nettoyage dans la profondeur du bois permettant d'extirper une grande quantité de vin retenu à l'intérieur des douelles et ainsi d'augmenter la surface d'échange entre la chauffe de la barrique (qui reste intacte) et le vin. Deuxièmement, le nettoyage vapeur induit un "lessivage" des barriques entrainant une perte d'aromes importante contrairement au nettoyage par HPU (données non montrées).

Pour finir, le traitement des barriques par HPU n'a eu aucun impact au cours des 12 mois d'élevage sur les concentrations en polyphénols totaux (IPT), les valeurs de l'intensité colorante (IC) ainsi que les concentrations en SO₂ libre, acidité volatile et éthanol.

*Elimination de *Brettanomyces*.*

Afin d'étudier l'impact du procédé de régénération de barriques par HPU, nous avons choisi de travailler sur des douelles contaminées artificiellement en laboratoire. En effet, les barriques présentent toujours une hétérogénéité au niveau des contaminations microbiennes (d'une barrique à l'autre voire d'une douelle à l'autre sur une même barrique) et engendre des difficultés pour prélever de manière stérile et répétable.

Des douelles provenant de barrique d'un et deux élevages ont ainsi été stérilisées par autoclavage puis introduit dans un bouillon de culture contenant des levures *Brettanomyces bruxellensis*. Après 8 jours dans ce "bain" de contamination, la concentration cellulaire en levure *Brettanomyces* a été évaluée par étalement sur milieu gélosé spécifique pour 3 profondeurs de bois différentes : 0 à 2 mm, 2 à 5 mm et 5 à 9 mm (Tableau 1). Les douelles ont ensuite été insérées dans des barriques support et ont subit 2 types de traitement : un traitement de 5 minutes par HPU (température de l'eau = 60°C) ou un traitement vapeur pendant 10 minute. Après traitement, la concentration cellulaire en levure *Brettanomyces* a de nouveaux été évaluée par étalement sur milieu gélosé spécifique pour les 3 profondeurs de bois différentes (Tableau 1).

Le traitement par ultrasons des douelles contaminées a permis d'éliminer en profondeur toutes les levures d'altération *Brettanomyces* (jusqu'à 9 mm). Cette efficacité est due d'une part au fait que le procédé permet d'agir dans la profondeur du bois, d'autre part à l'effet biocide des HPU déjà mis en évidence pour d'autres applications par différents auteurs [10, 11] mais aussi à l'effet synergique entre le traitement aux ultrasons et l'effet thermique de l'eau à 60°C mise en contact des *Brettanomyces* nichées dans la profondeur du bois [12, 14].

Au contraire, le traitement à la vapeur permet d'éliminer les microorganismes présents dans les 2 premiers millimètres du bois mais laisse des microorganismes viables dans la profondeur des douelles ce qui représente un risque évident de recontamination du vin. Ces résultats sont dus à l'inertie thermique du bois. En effet, lors d'un traitement à la vapeur, les premiers millimètres du bois atteignent une température proche de 100°C permettant une élimination des levures *Brettanomyces*, alors qu'à 5 mm la température atteinte est inférieure à 50°C ne permettant pas une élimination totale de ces levures d'altération [12, 15].
<table><caption>**Tableau 1** - Concentrations cellulaires en levures *Brettanomyces* (cellules/gramme de bois) dans des douelles avant et après traitement HPU et vapeur</caption><thead><tr><th rowspan="2">Douelles</th><th rowspan="2">Traitements</th><th rowspan="2">Profondeurs prélèvements</th><th colspan="2">Concentration (cell/g de bois)</th></tr><tr><th>Avant traitement</th><th>Après traitement</th></tr></thead><tbody><tr><td rowspan="6">1 élevage</td><td rowspan="3">HPU 5 mn</td><td>0-2 mm</td><td>5,4.10<sup>7</sup></td><td>0</td></tr><tr><td>2-5 mm</td><td>7,8.10<sup>5</sup></td><td>0</td></tr><tr><td>5-9 mm</td><td>1,7.10<sup>4</sup></td><td>0</td></tr><tr><td rowspan="3">Vapeur 10 mn</td><td>0-2 mm</td><td>6,2.10<sup>7</sup></td><td>0</td></tr><tr><td>2-5 mm</td><td>5,1.10<sup>5</sup></td><td>8,3.10<sup>4</sup></td></tr><tr><td>5-9 mm</td><td>4,3.10<sup>4</sup></td><td>3,9.10<sup>4</sup></td></tr><tr><td rowspan="6">2 élevages</td><td rowspan="3">HPU 5mn</td><td>0-2 mm</td><td>1,3.10<sup>8</sup></td><td>0</td></tr><tr><td>2-5 mm</td><td>1,2.10<sup>6</sup></td><td>0</td></tr><tr><td>5-9 mm</td><td>4,1.10<sup>5</sup></td><td>0</td></tr><tr><td rowspan="3">Vapeur 10 mn</td><td>0-2 mm</td><td>8,2.10<sup>7</sup></td><td>0</td></tr><tr><td>2-5 mm</td><td>5,6.10<sup>6</sup></td><td>9,1.10<sup>4</sup></td></tr><tr><td>5-9 mm</td><td>5,1.10<sup>5</sup></td><td>4,3.10<sup>5</sup></td></tr></tbody></table>

## Conclusion

L'utilisation de barriques durant plusieurs élevages successifs présente un risque de déviation organoleptique principalement lié au développement de levures d'altération du genre *Brettanomyces*. De plus, l'accumulation des dépôts de tartre peut perturber les échanges bois/vin et oxygène/vin, en créant une véritable barrière mécanique. Il est donc primordial de bien nettoyer et désinfecter en profondeur les barriques.

Le procédé de régénération de barriques par Ultrasons Haute Puissance (HPU) présente plusieurs avantages qualitatifs. En effet, il permet de détartrer complètement la barrique et ainsi restaurer l'oxygénation ménagée. Ce procédé a également un impact aromatique positif puisque les vins élevés dans les barriques traitées par ultrasons sont perçus d'un point de vue olfactif comme étant significativement plus intenses avec des notes grillées/fumées plus importantes, mais aussi beaucoup plus nets et complexes que des vins identiques mais élevés dans des barriques nettoyées à la vapeur. Ces résultats sensoriels sont confirmés d'un point de vue analytique où les vins élevés dans les barriques traitées par ultrasons ont des concentrations en composés volatils du bois supérieures de 20 à 60% aux vins élevés dans les barriques nettoyées à la vapeur. En bouche, les vins élevés dans les barriques traitées par ultrasons présentent des volumes plus importants, sont mieux équilibrés et surtout moins asséchants.

Enfin, ce procédé, grâce aux effets synergiques entre traitement thermique (eau à 60°C) et effet biocide des HPU, se révèle très efficace pour l'élimination des levures d'altération du genre *Brettanomyces* en surface mais surtout dans la profondeur du bois (jusqu'à 9 mm).

## Références

1. Malfeito-Ferreira M, Laureano P, Barata A et al (2004) Effect of different barrique sanitation procedures on yeasts isolated from the inner layers of wood. *In ASEV 55th Annual Meeting 55*: 304A.

2. Arvik T, Henick-Kling T (2002) Overview: *Brettanomyces bruxellensis* occurrence, growth, and effect on wine flavor. *Pract Winery Vineyard May/June*: 117-123.
3. Yap A, Jiranek V, Grbin P et al (2007) Studies on the application of high-power ultrasonics for barrel and plank cleaning and disinfection. *Aust NZ Wine Ind J* **22**: 95-104.

4. Patist A, Bates D (2008) Ultrasonic innovations in the food industry: from the laboratory to commercial production. *Innov Food Sci Technol* **9**: 147-154.

5. McClements DJ (1995) Advances in the application of ultrasound in food analysis and processing. *Trends Food Sci* **6**: 293-299.

6. Leighton TG (1998) The principles of cavitation. In Povey MJW, Mason TJ (eds) *Ultrasound in food processing*. Blackie Academic and Professional, London, pp 151-178.

7. Villamiel M, de Jong P (2000) Influence of high-intensity ultrasound and heat treatment in continuous flow on fat, proteins and native enzymes of milk. *J Agric Food Chem* **48**: 472-478.

8. Maisonhaute E, Prado C, White PC et al (2002) Surface acoustic cavitation understood via nanosecond electrochemistry. Part III: shear stress in ultrasonic cleaning. *Ultrason Sonochem* **9**: 297-303.

9. Krefting D, Mettin R, Lauterborn W (2004) High-speed observation of acoustic cavitation erosion in multibubble systems. *Ultrason Sonochem* **11**: 119-123.

10. Piyasena P, Mohareb E, McKellar RC (2003) Inactivation of microbes using ultrasound: a review. *Int J Food Microbiol* **87**: 207-216.

11. Jiranek V, Grbin P, Yap A et al (2008) High power ultrasonics as a novel tool offering new opportunities for managing wine microbiology. *Biotechnol Lett* **30**: 1-6.

12. Lopez-Malo A, Guerrero S, Santiesteban A et al (2005) Inactivation kinetics of *Saccharomyces cerevisiae* and *Listeria monocytogenes* in apple juice processed by novel technologies. *In Proceedings of the 2nd Mercosur Congress on Chemical Engineering*.

13. Martin N, de Revel G (1999) Sensory evaluation: scientific bases and oenological applications. *J Int Sci Vigne Vin Special Issue, Wine-tasting*: 81-93.

14. Couto JA, Neves F, Campos F et al (2005) Thermal inactivation of the wine spoilage yeasts *Dekkera / Brettanomyces*. *Int J Food Microbiol* **104**: 337-344.

15. Chatonnet P (2010) Nettoyage et désinfection appliqués aux contenants vinaires en bois destinés à la vinification et à l'élevage des vins. *Rev Oenol* **137**: 28-34.
# Effect of potassium polyaspartate on the tartaric stability of white and red wines as a function of their physicochemical composition

A. ASPROUDI, M. GUAITA, L. PANERO, S. MOTTA, M. PETROZZIELLO, A. BOSSO*

*Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di Ricerca per l'Enologia, via Pietro Micca 35, 14100 Asti, Italy*

Tartaric stability – polyaspartate – cold test – mini-contact test

## Introduction

The precipitation of potassium bitartrate (KHT) represents the main cause for the formation of deposits in bottled wines. The tartaric stabilization of wines is normally achieved by subtractive techniques (cold treatment, electrodialysis and cation-exchange resins) [1]. In terms of sustainability of the winemaking process, the use of additives to prevent the formation and precipitation of KHT could be an interesting solution to reduce the costs for energy and amortization still maintaining wine quality.

To date the authorized additives are metatartaric acid (MTA), mannoproteins (MP), and cellulose gums (CMC), but all of them are limited in their applicability: low stability over time (MTA), destabilizing effect for red wine color (CMC), and relative effectiveness as a function of wine composition (MP) [2-5]. New products based on polyaspartates have recently been studied in order to verify their effect on tartaric stability. Recent researches [6] have verified that the polyaspartates have stabilizing properties similar to those of the MTA, but are much more stable over time. The present work studied the influence of a potassium polyaspartate on KHT precipitation in 3 wines, whose physicochemical composition was adjusted at 2 levels according to a complete factorial design: pH, alcohol content and dose effect for the Montepulciano red wine and the Cortese white wine; pH and polyphenol content for the Pecorino white wine.

## Materials and methods

### Experimental design

The work studied the effect of 3 different factors (pH, alcohol and polyphenol content) on the tartaric stability of the wines and on the stabilizing effectiveness of a potassium polyaspartate (K-PASP) at 2 different doses. Three different experiments were carried out: in the first two, the influence of pH, alcohol content and dose of K-PASP were studied in a Montepulciano red wine and in a Cortese white wine. A complete factorial design was planned by varying upon 2 levels the factors pH (3.00 and 3.70), alcoholic content (11.5% and 15%) and dose of K-PASP (100 and 200 mg/L).

In the third experiment, the effect of pH and polyphenol content was evaluated before and after the addition of 100 mg/L of K-PASP to a Pecorino white wine. A complete factorial design was planned by varying upon 2 levels the factors pH (3.00 and 3.70) and polyphenol content (zero and 2600 mg/L of added polyphenols). The polyphenolic extract added to the Pecorino white wine was obtained as follows from an Albarossa red wine: passage through a C18-column, washing with water, elution with methanol of the phenolic fraction, concentration to dryness and re-suspension with water:ethanol 1:1 v/v.

### Tartaric stability tests

The wines used were limpid and with a variable degree of tartaric instability; the white wines were stable against protein precipitations. The evaluation of tartaric stability was performed with the cold test and the mini-contact test before and after the addition of K-PASP to the wines. The cold test consists in measuring the drop of tartaric acid concentration in wines by HPLC [7] before and after storage at -4°C for 6 days [4].

<footnote>*Corresponding author: antonella.bosso@entecra.it</footnote>
The mini-contact test consists in measuring the drop of electric conductivity (µS/cm) of 100 mL wine, maintained under stirring at 0°C, 4 min after the addition of finely micronized KHT (10 g/L) as a precipitating agent (Check Stab α2014 easy, Delta waters, Italy).

## Results and discussion

The modifications of the physicochemical composition of the wines influenced their degree of tartaric instability, measured with the cold test and the mini-contact test (Figures 1 and 2).

<figure><img src="image_314.png" /><figcaption>**Fig. 1** - Influence of pH and alcohol content on the tartaric stability of the wines: cold test results. Different letters indicate trials that are significantly different from one another (p<0.05, Tukey's test).</figcaption></figure>

<figure><img src="image_315.png" /><figcaption>**Fig. 2** - Influence of pH and alcohol content on the tartaric stability of the wines: mini-contact test results. Different letters indicate trials that are significantly different from one another (p<0.05, Tukey's test).</figcaption></figure>

The pH affects the dissociation equilibrium of tartaric acid: at equal tartaric acid content, a reduction of wine pH caused a decrease in bitartrate ion content, which is responsible for the formation of insoluble salts with potassium, thus decreasing the degree of wine tartaric instability. The pH effect was statistically significant for all the 3 wines, with both analytical tests employed to measure the tartaric instability (Figures 1 and 2). The pH effect is therefore appreciable both when the primary crystallization of KHT is caused by cold (cold test: effect on the solubility product of KHT) and when the secondary crystallization is caused by the addition of KHT salts in a micronized form (mini-contact test: effect on crystal growth).

It is widely reported in the literature that the alcohol content affects the solubility of KHT in wines, which decreases by increasing the alcohol content: at equal composition, the higher the alcohol content in wines, the higher is their instability. During our experiment we found that the alcohol effect was statistically
significant when the tartaric instability was measured with the cold test (effect on KHT solubility, and therefore on the primary nucleation of KHT crystals) (Figure 1). On the contrary, the effect of the alcohol content on the tartaric instability was scarcely or not appreciable when the mini-contact test was used (Figure 2).

The polyphenolic compounds can act as protective colloids [8] by slowing the KHT crystal growth in wines: their addition leads to a decrease in tartaric instability. In the course of the experiment two limit situations were compared for the same wine composition: a white wine as such and with the addition of a polyphenolic extract (from an Albarossa red wine), in order to increase the total polyphenol content up to about 2600 mg/L. In these conditions the intake of polyphenols played a statistically significant role in the tartaric stability, which was appreciable both with the cold test (before addition: average $\Delta H_2T$ = 0.1 g/L; after addition: average $\Delta H_2T$ = 0.02 g/L) and with the mini-contact test (before addition: $\Delta \chi$ = 113.3 $\mu S/cm$; after addition: $\Delta \chi$ = 36.2 $\mu S/cm$).

<figure><img src="image_316.png" /><figcaption>**Fig. 3** - Influence of pH, alcohol content and dose of added K-PASP on the tartaric stability of the Cortese white wine (mean values of $\Delta H_2T$ during the cold test).</figcaption></figure>

<table><caption>**Table 1** - Influence of pH, alcohol content and dose of added K-PASP on the tartaric stability of the Montepulciano and Pecorino wines after the addition of K-PASP (mean values of the cold test and the mini-contact test). Different letters on the same row (separately for each factor and wine) indicate trials that are significantly different from one another (p<0.05, Tukey's test).</caption><thead><tr><th rowspan="2">Factors Level</th><th colspan="4">Montepulciano red wine</th><th colspan="4">Pecorino white wine</th></tr><tr><th colspan="2">pH</th><th colspan="2">Alcohol (%)</th><th colspan="2">Dose (mg/L)</th><th colspan="2">Polyphenol addition</th></tr><tr><th>Δχ (μS/cm)</th><th>3.00</th><th>3.70</th><th>11.5</th><th>15.0</th><th>100</th><th>200</th><th>3.00</th><th>3.70</th><th>no</th><th>Yes</th></tr></thead><tbody><tr><th>ΔH<sub>2</sub>T (g/L)</th><td>31.3 a</td><td>57.3 b</td><td>44.20</td><td>44.5</td><td>47.04 b</td><td>41.63 a</td><td>26.05</td><td>33.98</td><td>33.4</td><td>26.63</td></tr><tr><th></th><td>0.018</td><td>0.011</td><td>0.098</td><td>0.02</td><td>0.011</td><td>0.018</td><td>0.025</td><td>0.033</td><td>0.035</td><td>0.023</td></tr></tbody></table>

The effectiveness of the addition of K-PASP on the tartaric stability of the different trials was also tested: the addition of 100 mg/L K-PASP stabilized all wines (cold test). Figure 3 reports the average results of the cold test for the various trials of Cortese white wine (the most unstable among the wines), and Table 1 reports in more concise form the results of the cold test and of the mini-contact test for the other 2 wines after the addition of K-PASP.

The dose effect of K-PASP was also studied with 2 wines (Cortese and Montepulciano), by comparing the doses of 100 mg/L and 200 mg/L. Since the dose of 100 mg/L was enough to stabilize the wines (cold test) (Figure 3 and Table 1), the only statistically significant differences found were related to the results of the mini-contact test (Table 1 and Figure 4), which revealed the higher effectiveness of the 200 mg/L dose in slowing the growth rate of the KHT crystals added to the wines.
<figure><img src="image_317.png" /><figcaption>Fig. 4 - Influence of pH, alcohol content and dose of added K-PASP on the tartaric stability of the Cortese white wine (mean Δχ values of the mini-contact test).</figcaption></figure>

## Conclusion

These results proved the excellent effectiveness of polyaspartates for the limitation of tartaric precipitation in wines with different physicochemical composition, confirming what was reported in a previous work comparing different types of polyaspartates [6].

Moreover, the reduction of wine pH caused a decrease of wine tartaric instability and this effect was evidenced with both analytical tests (cold test and mini-contact test). A higher alcohol content contributed to a higher tartaric instability of the wines when measured with the cold test, while no alcohol effect was noticed when the tartaric instability was measured with the mini-contact test.

## Acknowledgements

The work was funded by the European Project STABIWINE "Use of biopolymers for sustainable stabilization of quality wine" - Grant Agreement no. 314903 (Seventh framework Programme. Theme: SME-2012-2. Research for SME associations).

## References

1. Ribéreau-Gayon P, Glories Y, Maujean A et al (2006) *Handbook of Enology*, Chichester, UK, John Wiley & Sons, Ltd.

2. Moutounet M, Battle JL, Saint-Pierre B et al (2000) Stabilisation tartrique. Détermination du degré d'instabilité des vins. *In 6ème Symposium d'Oenologie*, Paris Lavoisier, pp 531-534.

3. Crachereau JC, Gabas N, Blouin J et al (2001) Tartaric stabilisation of wines by carboxymethylcellulose. *Bull OIV 841/842*: 151-159.

4. Motta S, Bollito A, Guaita M et al (2009) Impiego della carbossimetilcellulosa e uso di altri additivi nella stabilizzazione tartarica dei vini rossi. *Bull OIV 941/943*: 367-377.

5. Claus H, Tenzer S, Sobe M et al (2014) Effect of carboxymethyl cellulose on tartrate salt, protein and colour stability of red wine. *Aust J Grape Wine Res* 20: 186-193.

6. Bosso A, Panero L, Petrozziello M et al (2015) Use of polyaspartate as inhibitor of tartaric precipitations in wines. *Food Chem* **185**: 1-6.

7. Cane P (1990) Il controllo di qualità dei vini mediante HPLC: determinazione degli acidi organici. *Enotecnico* **26**: 69-72.

8. Usseglio-Tomasset L, Ubigli M, Barbero L (1992) L'état de sursaturations des vins en tartrate acide de potassium. *Bull OIV 65*: 703-719.
# Recent developments of membrane technologies in the wine industry

Y. EL RAYESS¹,²*, M. MIETTON-PEUCHOT³,⁴

¹Faculty of Agricultural and Food Sciences, Holy Spirit University of Kaslik, Jounieh, Lebanon

²Centre de Viticulture et d'Œnologie de Midi-Pyrénées, Avenue de l'Agrobiopôle,
31320 Castanet-Tolosan, France

³Université de Bordeaux, ISVV, EA 4577, Unité de recherche Œnologie,
33882 Villenave d'Ornon, France

⁴INRA, ISVV, USC1219, Œnologie, 33882 Villenave d'Ornon, France

Membrane technologies – crossflow microfiltration – membrane contactor – electrodialysis

## Introduction

Over the last 30 years, membrane technologies have become an important tool in the wine industry. Membrane-based processes are playing a critical role in the field of separation/purification, clarification, stabilization, concentration and de-alcoholization of wine products. They are starting to be an integral part of the winemaking process.

Nowadays, membrane operations are replacing several conventional processes. For example, the crossflow microfiltration has replaced the traditional filtration techniques like the diatomaceous-earth filtration previously used for wine's clarification. For sugar enrichment, reverse osmosis is more frequently used than chaptalization or addition of rectified concentrated must. The electrodialysis is now used for tartrate stabilization instead of chilling or cold stabilization. These techniques constitute real alternatives to some traditional enological practices even if some techniques like the membrane contactor are still in the experimental stage.

The objective of this paper is to provide an overview of the recent developments in the field of membrane processes for wine treatment.

## Crossflow microfiltration

Crossflow microfiltration is a very well implemented process in wine cellars. It is replacing the traditional filtration techniques using the diatomaceous earth. The advantage of the crossflow concept is that the shear exerted by the feed solution flowing parallel to the membrane surface can sweep the deposited particles towards the concentrate side so that the cake layer remains relatively thin (Figure 1).

Despite the crossflow concept advantage, the membrane fouling remains a major drawback. As wine is a complex medium, its constituents, especially polysaccharides, polyphenols and microorganisms, are the major contributors to membrane fouling.

Various hydraulic and mechanical methods were developed in order to reduce the resistance caused by membrane fouling. These techniques include back-flushing, cross-flushing and back-pulsing. The back-flushing is the common practice used to minimize fouling in wine filtration. The effectiveness of these methods is limited to the adhering deposit at the top surface of the membrane. They are ineffective in the removal of adhesive foulants.

The dynamic filtration technology is a new technology applied to wine filtration and may be a promising approach to resolve fouling problems. The dynamic or the shear-enhanced filtration consists in creating a shear rate at the membrane surface by a moving part such as an impeller. The latter is a disk rotating near a fixed membrane or vibrating membranes (Figure 1). It permits to generate very high shear rates without large feed flow rates and pressure drops.

<footnote>*Corresponding author: youssefrayess@usek.edu.lb</footnote>
<figure><img src="image_318.png" /><figcaption>Fig. 1 - The different types of filtration in the wine industry.</figcaption></figure>

El Rayess [1] studied the impact of rotating and vibrating filtration in wine clarification and showed that the effectiveness of this technology depended on the membrane material and on the frequency of rotation to eliminate fouling. PES hydrophilic membranes showed an increase in membrane permeability when applying the agitation due to the impeller rotation. This technology seemed to be ineffective with the hydrophobic PTFE membranes because the interactions between wine molecules and the membrane appeared to be stronger than the de-fouling action of the impeller.

TMCI Padovan introduced the first rotating dynamic crossflow filter (Dynamos®, Figure 2) into the wine industry. This filter is formed by ceramic disk membranes. It is equipped with a back-pulse system and is covered by an intercontinental patent (WO2011/033537). In this system, two stacks of rotating membranes are used. Preferably the two shafts rotate counter-current to create the maximum shear forces at the membrane surface. This membrane overlapping (Figure 2) can increase the permeate flux considerably (the magnitude depends very strongly on the type of feed and the crossflow conditions.

<figure><img src="image_319.png" /><figcaption>Fig. 2 - The Dynamos® rotating crossflow filter and the membrane overlapping zone.</figcaption></figure>
## Membrane contactor

The membrane contactors are defined as devices that achieve gas/liquid or liquid/liquid mass transfer without dispersion of one phase within the other (Figure 3). Membrane contactors represent a technology in which porous membranes are used as tools for inter-phase mass transfer. The membrane does not act as a selective barrier and the separation is based on the principles of phase equilibrium (Figure 3). Membrane contactors are typically fabricated with hydrophobic hollow-fiber microporous membranes. Therefore, water will not easily pass through the pores.

De-alcoholization and gas transfer are the main applications of membrane contactor in the wine industry. The wine de-alcoholization process using membrane contactor is called osmotic distillation and it is also known as osmotic evaporation, membrane evaporation and isothermal membrane distillation. In osmotic distillation, the membrane acts as a vapor gap between the two liquid phases. The membrane surface is in contact with a concentrated solution (feed) on one side and a dilute aqueous solution (stripper agent) on the other (Figure 3). Across the membrane, any volatile component is free to migrate by either convection or diffusion. The driving potential for such transport is the difference in vapor pressure of each component over each of the contacting liquid phases.

<figure><img src="image_320.png" /><figcaption>Fig. 3 - Membrane contactor principle.</figcaption></figure>

Osmotic distillation of a high-alcohol-content wine at a temperature of 10–20°C using plain water as the stripping liquid can rapidly reduce its alcohol content to levels down to 6% (v/v). The main advantage of osmotic distillation lies in its ability to work at low temperatures and pressures, thus avoiding mechanical damage and thermal degradation of the components and aroma of wines.

Membrane contactors can be used lonely for wine de-alcoholization or coupled to another membrane process. For example, the Australian process marketed by Memstar consists of alcohol reduction by a two-stage process of reverse osmosis followed by a membrane module known as a membrane contactor (Liqui-Cel®). Wine to be treated is first separated by reverse osmosis into concentrate and permeate streams. The wine concentrate contains all the precious wine characters. The alcohol rich permeate is passed through the membrane contactor. On the other side of the contactor, there is a counter-flow of treated strip water. Alcohol passes through the membrane from the permeate into the water. The de-alcoholized permeate is then cooled and recombined with the wine, lowering the alcohol of the blend [2].

At the experimental stage, the membrane contactors are studied in order to monitor the transfer of gases into wines. They can adjust the levels of CO₂ and O₂ and they can efficiently deoxygenate during one passage. They are also tested for the monitoring the wine microoxygenation.
Electrodialysis and bipolar electrodialysis

The electrodialysis process is used as an alternative to the traditional tartrate stabilization process.
The principle of this process consists into the application of an electric field by two electrodes placed on
both sides of the cell. This will lead to ion migration. Electrodialysis differs from a normal ion exchange
process by utilizing ion-selective semipermeable membranes. Three types of ion-exchange membranes
are commonly used in electrodialysis: anion-exchange membranes, cation-exchange membranes and
bipolar membranes.

During wine treatment by electrodialysis and when the electric field is applied, the anions (TH⁻ and T²⁻) are attracted to the positive charge electrode but they are stopped by the cation-exchange membrane and trapped in the brine stream. Similarly, cations (K⁺ and Ca⁺⁺) move in the opposite direction and are stopped by the anion-exchange membrane. Wine circulates along one side of the membrane and on the other an electrolyte that removes the ions. The treated wine will constitute the dilute compartment and the eliminated ions will constitute the concentrate or brine compartment.

The bipolar membranes (association of cation and anion-exchange membranes) are not used for tartrate stabilization but for the regulation of wine pH. In order to lower the pH, a stack of bipolar membranes and cation-exchange membranes are used. Only the cations are eliminated from wines and the salty form of tartaric acid is lowered. On the contrary, an association of bipolar membranes and anion-exchange membranes are used to increase the pH of the wines.

References

1. El Rayess Y (2011) Microfiltration tangentielle appliquée à l’œnologie : maîtrise et compréhension des phénomènes de colmatage. *PhD thesis*, Institut National Polytechnique de Toulouse.

2. El Rayess Y, Mietton-Peachot M (2015) Membrane technologies in wine industry: an overview of applications and recent developments. *Crit Rev Food Sci Nutr* DOI:10.1080/10408398.2013.809566.
# Subcritical water extraction of polyphenols from red grape pomace coupled with membrane filtration

S. YAMMINE<fnref n="1" />,<fnref n="2" />*, R. RABAGLIATO<fnref n="1" />,<fnref n="2" />, C. DELSART<fnref n="1" />,<fnref n="2" />, X. VITRAC<fnref n="3" />, R. GHIDOSSI<fnref n="1" />,<fnref n="2" />, M. MIETTON PEUCHOT<fnref n="1" />,<fnref n="2" />

Subcritical water extraction – membrane process purification – grape pomace – polyphenols

## Introduction

Grape by-products (including skins, seeds, stems and vine shoots) are rich in health promoting polyphenols [1-4]. Their extraction from winery waste and their following purification are of special interest for the production of extracts with high added value compounds [5].

Meanwhile, the growing concern over environmental problems associated with economic constraints requires the development of environmentally sustainable extraction technologies. The extraction using semi-continuous subcritical water (SWE) as a natural solvent at high temperature and high pressure is a promising “green” technology (environmentally friendly, energy efficient) that improves the extraction process in plant tissues [6]. SWE is established at temperatures between 100°C and 374°C (the critical temperature) under high pressure (usually from 1 to 6 MPa) where water stays in the liquid state. At the boundaries higher extraction efficiency are obtained due to superior mass transfer properties of subcritical water. The variability of the dielectric constant with temperature is an important variable to consider [7]. At room temperature the dielectric constant of water is close to 80. To obtain values of about 27, ethanol at ambient temperature, water must be heated to 250°C [8]. In our study subcritical water extraction of polyphenols from red and white grape pomace from Dunkelfelder, Cabernet Franc, Merlot, and Chardonnay was performed. The semi-continuous extraction of subcritical water usually leads to crude extracts rich in different families of polyphenols.

A purification step prior to industrial usage is essential. Coupling subcritical water extraction with membrane processes, due to the large range of flexibility, offers a solution for the purification and fractioning of the crude extracts.

## Materials and methods

### Subcritical water extraction system

Grape pomace provided by the engineering school of Changins, Switzerland was stored at 4°C until further use. The schematic diagram of the apparatus used for the extraction of polyphenolic compounds using subcritical water is shown below (Figure. 1). In the extraction system, an HPLC pump was used for water delivery, pressurization and controlling the pressure of the system. A pressure transducer and thermocouple were installed in the custom-made high-pressure vessel to monitor both the pressure and temperature of the system.

### Extraction parameters

In each run, pomace was loaded into the high-pressure vessel which was then placed in an oven at a predetermined temperature. The outlet valve of the extraction vessel was then closed and the system was pressurized to a desired pressure at a constant flow rate. The combined effects of extraction temperature (60 to 200°C), pressure (25 to 100 bar), flow rate (1 to 10 mL/min), and sample mass (5, 70, 1000 g) were investigated and compared to traditional solvent extraction (1/1 ethanol/water).

<footnote n="1">¹Université de Bordeaux, Institut des Sciences de la Vigne et du Vin, EA 4577, Unité de recherche œnologie, 210 Chemin de Leysotte, CS 50008, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="2">²INRA, ISVV, USC 1366 Œnologie, 210 Chemin de Leysotte, CS 50008, F-33882 Villenave d'Ornon, France</footnote>

<footnote n="3">³Laboratoire PHENOBIO SAS, Centre Montesquieu, 1 Allée Jean Rostand, 33650 Martillac, France</footnote>

<footnote>*Corresponding author: sami.yammine@u-bordeaux.fr</footnote>
<figure><img src="image_321.png" /><figcaption>Fig. 1 - Schematic diagram of the apparatus used for subcritical water extraction.</figcaption></figure>

### Purification parameters

Subsequently, ten nanofiltration flat-sheet membranes and eight tight ultrafiltration membranes with nominal MWCOs ranging from 0.2 to 150 kDa were tested to fraction and concentrate anthocyanin extract from grape pomace extract. A pilot crossflow filtration system SEPA CF II was used, which featured a membrane cell with an effective area of 0.014 m². Permeate fluxes were recorded for transmembrane pressures between 0.5 and 3.0 MPa, while keeping all other operating conditions constant (volumetric reduction ratio 1, 35°C).

### Chemical analysis

All of the produced samples were analysed for their Colouring Intensity (CI) and Total Polyphenol index (TPI) and concentrations of total phenolic compounds (TP), which were measured using Folin–Ciocalteu assay [9]. The major anthocyanins and tannins were quantified by HPLC [10]. Some samples were analysed for their volatile compound content by GC-MS. The antioxidant activity was measured by the Oxygen Radical Absorbance Capacity (ORAC) assay [11].

## Results and discussion

The subcritical water extraction process was more efficient than using an organic solvent and aqueous based system at atmospheric pressure for recovery of antioxidants, leading to double the extraction yield. By increasing the extraction temperature, the quantities of total polyphenols increased regardless of the pressure applied (Figure. 2), reaching 2.7 mg/100 mg of polyphenols from dry extract. Further analysis of main families of polyphenols showed SWE extracts had much higher levels of total anthocyanin as compared to the traditional method of organic solvent at low temperatures (100-110°C) with crude extracts containing 130 mg/100 g DW (+61% compared to traditional methods of extraction). By increasing the temperatures, the anthocyanins were degraded. On the contrary, procyanidin extraction was optimum at a much higher temperature (175°C) with crude extract leading to (+23%) 2077 mg/100 g DW. On the other hand, comparing GC-MS results between SWEX and traditional method of extraction showed that during extraction furfural compounds are produced and volatile esters are degraded. Compiling the information needed showed that the optimal extraction conditions were found to be 150°C regardless of the pressure used.

During filtration of crude extract, two types of products are formed: the permeate which passes through the membrane and the concentrate which is held up by the molecular weight cutoff of the membrane. This has allowed membrane processes to become a powerful alternative for the concentration of natural grape extracts (concentrate), due to their flexibility and mild operating conditions. Retention values of total soluble solids, acidity and anthocyanins increased with transmembrane pressure. With similar permeate fluxes at average transmembrane pressure, retention of anthocyanins is significantly higher for nanofiltration (up to 3.2 folds the initial concentration of polyphenols) membranes than for ultrafiltration membranes.
<figure><img src="image_322.png" /><figcaption>Fig. 2 - Polyphenol concentration as a function of SWE extracts at a specific temperature and pressure of extraction.</figcaption></figure>

<table><caption>**Table 1** - Rejection rate percentages of polyphenol concentration in permeate after membrane filtration</caption><thead><tr><th>Molecules</th><th>Average molecular weight</th><th>1 kDa PEF</th><th>BQ-01 (500-1000Da)</th><th>MX-07 (300-600Da)</th><th>NF-Laval (200-400Da)</th><th>NF-HL (150-300Da)</th><th>MS-19 (125-200Da)</th></tr></thead><tbody><tr><td>Gallic acid</td><td>198.17</td><td>25</td><td>36</td><td>38</td><td>57</td><td>64</td><td>86</td></tr><tr><td>Dimer B2</td><td>578.25</td><td>59</td><td>70</td><td>95</td><td>100</td><td>100</td><td>100</td></tr><tr><td>Catechin</td><td>290 - 442</td><td>22</td><td>54</td><td>42</td><td>95</td><td>98</td><td>100</td></tr><tr><td>Quercetin</td><td>302 - 478</td><td>62</td><td>89</td><td>91</td><td>100</td><td>100</td><td>100</td></tr><tr><td>Taxifolin</td><td>450.39</td><td>36</td><td>61</td><td>76</td><td>100</td><td>100</td><td>100</td></tr><tr><td>Anthocyanins</td><td>465 - 639</td><td>62</td><td>52</td><td>86</td><td>100</td><td>100</td><td>96</td></tr></tbody></table>

The tested membranes also had differential selectivity for polysaccharides, proteins, and different families of phenolic compounds (pentamers, trimers, anthocyanin, and phenolic acids) that pass through the membranes. As shown in Table 1, gallic acid for example was separated by nanofiltration membrane having molecular weight cutoff between 200 and 400 Da leading to high extract purity (95%), thus confirming the applicability of membrane separation for the fractionation and purification of pomace extracts. More research is needed to validate the industrial up scaling and the intended application of the produced extracts.

## Conclusion

Subcritical water extraction process showed a higher efficiency than organic solvent. 150°C appears to be the optimum extraction parameter, regardless of the pressure used. An increase in temperature increases the tannin and decreases the anthocyanin extraction. Crossflow ultrafiltration and nanofiltration of all studied extracts was governed by the multiple operating parameters. Concentration was achieved in the retentate and selective retention of polyphenols in the permeate was achieved. SWE combined with membrane filtration demonstrated the effectiveness for the green extraction and purification of polyphenols from grape pomace.
References

1. Craft BD, Kerrihard AL, Amarowicz R et al (2012) Phenol-based antioxidants and the in vitro methods used for their assessment. *Compr Rev Food Sci Food Saf* **11**: 148-173.

2. Kähkönen MP, Heinonen M (2003) Antioxidant activity of anthocyanins and their aglycons. *J Agric Food Chem* **51**: 628-633.

3. Quideau S, Deffieux D, Douat-Casassus C et al (2011) Plant polyphenols: chemical properties, biological activities, and synthesis. *Angew Chem Int Ed Engl* **50**: 586-621.

4. Stintzing FC, Stintzing AS, Carle R et al (2002) Color and antioxidant properties of cyanidin-based anthocyanin pigments. *J Agric Food Chem* **50**: 6172-6181.

5. Yammine S, Ghidossi R, Mietton-Peachot M (2014) Extraction and purification of phenolic compounds from by-products of the winemaking process. In *Wine: Phenolic Composition, Classification and Health Benefits*, Y. El Rayess (Ed.), NOVA science publishers, pp 313-330.

6. Aliakbarian B, Fathi A, Perego P et al (2012) Extraction of antioxidants from winery wastes using subcritical water. *J Supercrit Fluids* **65**: 18-24.

7. Carr AG, Mammucari R, Foster NR (2011) A review of subcritical water as a solvent and its utilisation for the processing of hydrophobic organic compounds. *Chem Eng J* **172**: 1-17.

8. Ju ZY, Howard LR (2003) Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin. *J Agric Food Chem* **51**: 5207-5213.

9. Swain T, Hillis WE (1959) The phenolic constituents of *Prunus domestica*. I.—The quantitative analysis of phenolic constituents. *J Sci Food Agric* **10**: 63-68.

10. Dudonné S, Vitrac X, Coutière P et al (2009) Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. *J Agric Food Chem* **57**: 1768-1774.

11. Dudonné S (2009) Développement d'extraits végétaux antioxydants à usage nutraceutique. *PhD thesis*, Bordeaux University.
# Alcoholic fermentation in new oak tanks: effects on the colour, chemical composition and sensory profile of Shiraz wine, before and after barrel ageing

A.C. MONTEAU<fnref n="1" />, V. RENOUF<fnref n="1" />*, N. SCRIMGEOUR<fnref n="2" />, N. TIQUET-LAVANDIER<fnref n="1" />

New oak tanks – stainless steel tanks – Shiraz wine – sensory and chemical analysis

## Introduction

A large proportion of high quality wines are fermented in stainless steel tanks before being aged in barrels. Several intern studies showed that fermenting Cabernet Sauvignon in oak tanks provides an alternative method that could be used to achieve a better final integration of oak tannins and aromas.

The goal of this study was to observe the impact of an early contact between oak and Shiraz must on the final wine chemical profile, colour and sensory profile. The trial in Australia was set up in the Margaret River region to follow the impact of fermenting Shiraz must in new oak tanks and stainless steel tanks of the same capacity.

## Materials and methods

### Experimental design

The trial in Australia was set up in the Margaret River region to follow the impact of fermenting 20 tons of Syrah in 3x30hL new oak tanks and 3 stainless steel (s/s) tanks of the same capacity. These tanks were filled alternatively to ensure the homogeneity between the different replicates. Cellar operations were exactly the same within the six batches. The homogeneity of the replicates was checked before filling in six French oak barrels per modality.

### Chemical analyses

Each modality was sampled after alcoholic fermentation (AF) and after approximately 3, 6, 9 and 12 months of barrel ageing. Chemical analyses were performed with GC-MS chromatography by the AWRI laboratory in Australia, on composite samples per modality. The colour intensity and stability of the wine and the oak and wine polyphenols were analysed using turbidity measurements and optical density measurements (UV-Vis).

The analysis results were tested with Variance analyses (ANOVA) to ensure that the differences measured were significant.

### Sensory analyses

Triangular tests were performed after 6 and 10 months of ageing for the Australian Shiraz following the Australian Standard: AS 2542.2.2. Samples were presented to panellists in 30 mL aliquots in 3-digit-coded, covered ISO standard wine glasses at 22 – 24°C, in isolated booths under colour masking sodium lighting, with randomised presentation order across judges.

After 10 months of ageing, a blind descriptive sensory analysis was performed by a professional panel on a blend of each modality. The data were analysed with Student t-tests to ensure that the differences observed between the two modalities were significant. The panellists were asked to select the sample that seemed to present the highest global quality. A Wilcoxon Rank-Sum Test was used to analyse the data.

<footnote n="1">¹R&D department Chêne & Cie</footnote>

<footnote n="2">²AWRI, Adelaide, SA, Australia</footnote>
# Results and discussion

## Chemical analyses

After the alcoholic fermentation, both cis-oak lactone and furfural showed significant differences between the two treatment sets at the 90% confidence interval, with the oak vats fermented wines exhibiting higher concentrations of cis-oak lactone and lower concentrations of furfural (Figure 1).

The data shows that, on average, there were few differences between the two sets of wines at each time-point. However, colour density, pigmented tannin, total ellagitannins and cis-oak lactone were consistently observed to be significant or were approaching significance at the 90% confidence interval throughout maturation.

The wines fermented in oak vats exhibited slightly higher, but not significantly different concentrations of tannins and phenolics, with pigment, free anthocyanin and pigmented tannin levels similar across the two sets of replicate wines.

The chemical analysis data collected after 3, 6 and 10 months of ageing in barrel shows consistent significant differences (α=0,10) for colour density, pigmented tannin, total ellagitannins and cis-oak lactone. The values were found higher for the wines fermented in oak vats.

<figure><img src="image_323.png" /><figcaption>**Fig. 1** - Significant difference observed on several parameters. A) After AF, B) After 10 months of ageing in neutral barrels.</figcaption></figure>

## Sensory analyses

Two triangular tests were set up after 6 and 10 months of ageing (Table 1). The results were not significant (P>0,05).

<table><caption>**Table 1** - Triangular test results after 6 and 10 months of ageing</caption><thead><tr><td>Sample</td><td>6 months</td><td>10 months</td></tr></thead><tbody><tr><td>No. responses (panellists)</td><td>31</td><td>34</td></tr><tr><td>Correct answers</td><td>9</td><td>8</td></tr><tr><td>Significance</td><td>ns</td><td>ns</td></tr></tbody></table>

After 10 months of ageing, a blind descriptive analysis was performed (Table 2). Two composite samples were presented and each descriptor was marked, depending on the intensity of the sensation described.

It appears that the wine fermented in oak vats presents a better integration to the bouquet of the oak aromas. The length of the mouthfeel seems to be improved when AF is performed in oak vats.

However, fruity aromas are less perceived, and the mouthfeel seems less balanced than the one of stainless steel fermented wine.

The wine fermented in oak tank and then aged in neutral barrels seems to present a higher quality potential than the other modality (Table 3).
<table><caption>**Table 2** - Sensory analysis results after 10 months of ageing</caption><thead><tr><th></th><th>Descriptor</th><th>AF in s/s tanks</th><th>AF in oak tanks</th><th>Significance</th></tr></thead><tbody><tr><td rowspan="4">Nose</td><td>Fruity aromas (/5)</td><td>4,00</td><td>3,27</td><td>α=0,05</td></tr><tr><td>Oak integration (/5)</td><td>3,91</td><td>3,27</td><td>α=0,08</td></tr><tr><td>Freshness (/5)</td><td>3,55</td><td>3,00</td><td>ns</td></tr><tr><td>Complexity (/5)</td><td>3,18</td><td>2,82</td><td>ns</td></tr><tr><td rowspan="6">Mouthfeel</td><td>Depth (/5)</td><td>3,09</td><td>3,27</td><td>ns</td></tr><tr><td>Astringency (/5)</td><td>2,27</td><td>3,00</td><td>ns</td></tr><tr><td>Bitterness (/5)</td><td>1,55</td><td>1,73</td><td>ns</td></tr><tr><td>Roundness (/5)</td><td>2,73</td><td>2,64</td><td>ns</td></tr><tr><td>Length (/5)</td><td>2,55</td><td>3,27</td><td>α=0,05</td></tr><tr><td>Balance (/5)</td><td>3,36</td><td>2,73</td><td>α=0,05</td></tr></tbody></table>

<table><caption>**Table 3** - Wilcoxon Rank-Sum Test results after 10 months of ageing</caption><thead><tr><th>Modality</th><th>AF in s/s tanks</th><th>AF in oak tanks</th></tr></thead><tbody><tr><td>Rank Sum</td><td>13</td><td>20</td></tr><tr><td>P-value</td><td colspan="2">P&lt;0,05</td></tr></tbody></table>

## Conclusion

After AF and 3, 6 and 10 months of ageing in neutral barrels, the Shiraz wines fermented in oak vats showed some differences from those fermented in stainless steel, as colour density, pigmented tannin, total ellagitannins and cis-oak lactone.

A blind descriptive analysis after 10 months of ageing showed that the wine fermented in oak tank presented significantly better oak aroma integration to the bouquet and a better length. It may imply that performing the alcoholic fermentation in oak vats allows preparing the wine for ageing in barrels, by enhancing the integration of oak aromas and a better colour density. Similar results were found on a Cabernet Sauvignon from Bordeaux. A last screening of the samples will be done at the end of ageing (12 months).

## Abbreviations

AF, alcoholic fermentation; s/s, stainless steel.
# Prevention of wine oxidation during barrel aging: an innovative method to measure antioxidant compounds released by oak

M. MIRABEL*, V. RENOUF

Chêne & Cie, Research Department of Taransaud, Canton and Kadar cooperages and Xtrachêne,
5 rue Magellan, 75008 Paris, France

Wine oxidation – oak antioxidant compounds – electrochemical sensor – optimized barrel

## Introduction

Wine oxidation is a problem that affects the freshness, the aromatic profile, the color and also the mouthfeel of the wine. It mainly concerns white wines. Oxygen interactions with wine compounds lead to the phenomena cited above that are responsible for the depreciation of these wines. Barrel aging is a crucial step in the wine process because it allows many modifications such as wine enrichment, color stabilization, clarification and also a slow oxygenation of the wine. Effects of the oak barrel have to be known to prevent oxidation of the wine. We have been interested in the main antioxidant compounds released by oak barrels to the wine and we have developed an innovative method to reach directly these antioxidant compounds at the oak stave surface.

## Materials and methods

### Electrochemical method and sensor: principle

The method is based on an innovative sensor that can be applied directly on the oak and measure several parts of the stave at the same time. The apparatus needed for this sensor management is a classical electrochemical system, namely a potentiostat instrument, reference and counter electrodes. The working electrode used is this innovative sensor made from twelve gold ultramicroelectrodes. Direct application of the sensor on the oak stave leads to the recording of a current. This is due to the release of electrons from oak compounds. Those are oxidized due to the electrochemical method and they will provide protection to the wine by being oxidized instead of wine compounds. The current recorded while scanning potential leads to a voltammogram (Figure 1). It is an oxidation wave shape as currently observed in electrochemistry and is due to oxidation of phenolic compounds [1].

The amount of electrons released can be reached thanks to the integration of the voltammogram between potentials 0,1V and 1,2V. This is what we called antioxidant capacity (CAOX). It represents the ability of the oak barrel to protect wine from oxidation. The higher it is, the better the wine is protected. Another index of interest is the inflexion potential. It is the potential value for which electrons are released and oxidation occurs. The lower it is, the faster the oxidation process occurs.

This electrochemical method has been performed with the specific and innovative sensor developed. It has been applied directly onto the oak surface and small currents have been registered. Moreover this analysis does not need sample preparation and is not destructive. The oak analyzed can go back in the cooperage process.

### Development of an optimized barrel

The CAOX is depending on the amount of oak compounds able to release electrons and therefore oxidize. It is easily understood that many parameters encountered in cooperage can influence the CAOX: oak species, origins, type of grain, length of seasoning, toasting intensity and so on. In order to check their incidence many murrains and staves were analyzed directly with the innovative sensor. Some results are presented and they have led to the development of a new barrel where oak antioxidant release is optimized.
<figure><img src="image_324.png" /><figcaption>Fig. 1 - Voltammogram obtained with the innovative sensor and its interpretation.</figcaption></figure>

## Results and discussion

Oak seasoning and toasting are the main important steps in cooperage process. They are different from one cooper to another. Knowing the influence of these parameters on the CAOX can help making barrels with enological purposes. The CAOX measured on murrains put in the yard is decreasing along the seasoning duration (Figure 2).

<figure><img src="image_325.png" /><figcaption>Fig. 2 - CAOX measured on murrains according to their seasoning duration.</figcaption></figure>

Taking into account these results and also others performed on toasted staves, we have made a barrel that optimizes all parameters in order to provide the best protection against oxidation without contributing a strong woody character.

We tested the first prototypes on white wines of several wineries from Burgundy. The electrochemical method and sensor developed for oak can also be applied to wines. Therefore, CAOX analyses were performed on the same wine (Chardonnay) put into prototype and reference barrels in 3 wineries (named X, Y and Z, Figure 3). The CAOX of the wines aged in a prototype barrel was always higher than that of the same wine aged in a reference barrel after a few months of barrel aging. Moreover, those wines were more appreciated because of their freshness, respect of the fruit, aromatic purity and tautness.
<figure><img src="image_326.png" /><figcaption>Fig. 3 - CAOX measured on wines aged in reference and prototype barrels for 3 Burgundy wineries (X, Y and Z).</figcaption></figure>

## Conclusion

The electrochemical method and sensor developed is non destructive and efficient for the measure of oak compounds that can oxidize. We manage to make a barrel that can release these compounds and prevent wine oxidation. It has been a success for white wines and has led to the sale of a new barrel named Pure T. A "neighbor" prototype is now being tested for red wines in order to minimize the sulfites added along barrel aging.

## Acknowledgements

The development of the innovative sensor would not have been possible without the work of William Giraud and Maurice Comtat.

## References

1. Kilmartin PA, Zou H, Waterhouse AL (2001) A cyclic voltammetry method suitable for characterizing antioxidant properties of wine and wine phenolics. *J Agric Food Chem* **49**: 1957-1965.
# A protein extracted from potato as non-allergenic fining agent for juices and wines

N. ITURMENDI<fnref n="1" />, A. RINALDI<fnref n="2" />, A. GAMBUTTI<fnref n="2" />, V. MOINE<fnref n="1" />*, L. MOIO<fnref n="2" />

Patatin-rich protein – fining – white juice – red wine

## Introduction

Wine and must fining is currently used as a treatment for reducing the turbidity, colloidal stabilization and for modifying the aromatic profile. In red wine, fining treatment is carried out during wine ageing but in white wine, it is mainly carried out in juice after enzyme treatment. This difference is due to the role of polyphenols in white/rosé wine, thus they could be oxidized by enzymatic or chemical reactions. These oxidized compounds, named quinones, are highly unstable and they combine to other compounds such as varietal aroma compounds. Thiols are documented as reactive molecules under wine oxidation conditions during ageing [1] or in botrytized wines [2]. Among the thiol compounds, the 3-sulfanylhexan-1-ol (3SH) is highly affected by this phenomenon [1]. Regarding phenolic compounds in wine, the oxidized flavan-3-ols appear to be the most reactive compounds with volatile thiols [3, 4]. Another oxidation consequence is the brown coloured products, whose formation is not yet well established; it appears that it is a combination of hydroxycinnamate quinones with flavan-3-ols [5]. Therefore, the polyphenol elimination in juice by fining could prevent these mechanisms concerning the loss of varietal aroma and colour modifications.

Since November 2013, proteins extracted from potato can be used as fining agents in juices and wines [6, 7]. This extract is rich in patatin, which is one of the glycoprotein families obtained from *Solanum tuberosum* tubers. Potato products are largely used for food consumption and are not listed as food allergens. During the last years, a potato derivate has been tested as a processing aid for juice and wine fining. In red wine, patatin-rich protein has a higher reactivity with tannin-binding salivary proteins than other common protein fining agents and does not modify the chromatic characteristics of the wines [8]. In white juice, the first results indicate that this new protein origin has a high clarification ability and a high reactivity with oxidized phenolic compounds [9, 10].

The aim of this work is to show the main effects of the use of this extract in white juice settling during alcoholic fermentation on the removal of phenolic compounds and oxidation of colour components.

## Materials and methods

### Trials

For trial 1, the pressing juice from Sauvignon Blanc was treated with enzymes (pectin lysate), racked and then settled with Vegecoll® at 10 g/hL. After fining, it was racked and inoculated with yeast (not required) in barrel.

For trial 2, the pressing juice from Sauvignon Blanc was treated with enzymes (pectin lysate à 3 g/hL), racked, and then turbidity adjustment was made at 150 NTUs by lee addition. The juice was inoculated with Yeast A (Zymaflore® X5) and Yeast B (Zymaflore® Delta) at 20 g/hL. Vegecoll® at 3 g/hL was added at density -20.

For trial 3, the wine juice from Rolle (2014) was treated with enzymes (pectin lysate), racked and then inoculated with Yeast B (Zymaflore® Delta) at 20 g/hL. During the alcoholic fermentation, the juice was fined with Vegecoll® at 20 g/hL. Another deposit was fined with liquid pea protein at 50 g/hL.

<footnote n="1">¹Biolaffort, 126 Quay de la Souys, 33100 Bordeaux Cedex, France</footnote>

<footnote n="2">²Università degli Studi di Napoli Federico II, Dipartimento di Agraria, Sezione di Scienze della Vigna e del Vino, Viale Italia, angolo via Perrottelli, 83100, Avellino, Italy</footnote>
## Chemical analysis

After alcoholic fermentation, the alcoholic degree (%), reduced sugars (g/L), total acidity (g H₂SO₄/L), volatile acidity (g H₂SO₄/L), free and total SO₂, Colour Intensity, Total Polyphenol Index and phenol acid estimation were measured at the SARCO Laboratory (Floirac, Gironde, France) according to COFRAC accreditation. The thiols 3-sulfanylhexan-1-ol (3SH), its acetate (3SHA) and the 4-methyl-4-sulfylpentanol (4MSP) were measured by gas-chromatography according to Tominaga *et al.* [11] at the SARCO Laboratory.

## Results

The first trial compares the wine settled with the patatin-rich protein before alcoholic fermentation and the same juice without fining. Analyses were made after alcoholic fermentation. No differences were observed in total and volatile acidity (data not shown). Fining reduced the wine colour, however, no modifications were observed in total polyphenol and phenol acid content (Table 1). The optical density at 420 nm could well express the browning formation due to the oxidation effect. Fining with patatin-rich protein reduced this value by 45%, which indicates the high reactivity of this protein with the oxidized compounds. This reduction of brown compounds has an impact on the varietal aroma of Sauvignon Blanc. The main three thiols which participate in browning are the 3-sulfanylhexan-1-ol (3SH), its acetate (3SHA) and the 4-methyl-4-sulfylpentanol (4MSP). Fining with Vegecoll® mainly preserved the compounds derived from 3SH (Figure 1), around 30% of 3SH and 23% of 3SHA.

<table><caption>**Table 1** - Wine analysis of trial 1 after alcoholic fermentation</caption><thead><tr><th>Analysis of trial 1</th><th>Non-fined wine</th><th>Wine fined with Vegecoll® at 10 g/hL</th></tr></thead><tbody><tr><td>OD<sub>420</sub></td><td>0.170</td><td>0.103</td></tr><tr><td>OD<sub>520</sub></td><td>0.080</td><td>0.038</td></tr><tr><td>OD<sub>620</sub></td><td>0.049</td><td>0.021</td></tr><tr><td>Colour Intensity</td><td>0.299</td><td>0.162</td></tr><tr><td>Total Polyphenol Index (OD<sub>280</sub>)</td><td>8</td><td>8</td></tr><tr><td>Phenol acids (OD<sub>320</sub>)</td><td>6.3</td><td>6.3</td></tr></tbody></table>

<figure><img src="image_327.png" /><figcaption>**Fig. 1** - Volatile thiols (4MSP, 3SH and 3SHA) of a Sauvignon Blanc wine fined with Vegecoll® at 10 g/hL before alcoholic fermentation.</figcaption></figure>
In the second trial, the aim was to increase the volatile aroma by yeast and modulate it by fining with Vegecoll®. The non-fined wines were compared to fined wines after alcoholic fermentation with yeast A and B. Fermentation conditions (turbidity and temperature) were equivalent for all trials.

There were no differences in alcoholic degree, total acidity and volatile acidity between samples after alcoholic fermentation (data not shown). However, the samples had important differences in volatile aroma (Figure 2). The results showed different aroma profile according to the yeast. Wines fermented with Yeast A were more balanced in varietal aroma than those fermented with Yeast B. Wines fermented with Yeast B were richer in fruity compounds (3SH and 3SHA). Independently of this yeast influence, the fining agent could increase this aromatic profile. Fining had no effect on 4MSP. However, the fruity aromas were more influenced by this treatment. Fining with Vegecoll® preserved 18% of thiols in wine fermented with Yeast A and 42% in those fermented with Yeast B. This increase in thiols is due totally to 3SH and its acetate. Differences with control wine were much more important in wine fermented with Yeast B. Specifically, in wine with Yeast B the 3SH and 3SHA increments were 40% and 52% respectively and with Yeast A 19% and 12%.

<figure><img src="image_328.png" /><figcaption>**Fig. 2** - Volatile thiols (4MSP, 3SH and 3SHA) of a Sauvignon Blanc wine fermented with Yeast A and B. Comparison between non-fined and fined with Vegecoll® at 3 g/hL during alcoholic fermentation.</figcaption></figure>

## Discussion

This patatin-rich protein was used in white pressing juices from Sauvignon Blanc and Rolle during the harvest of 2014. The white pressing juice contains fruity grape aromas and their precursors obtained from grape skin, but it also contains several undesirable compounds such as phenolic molecules and vegetal odours associated to solid part of the grapes [12]. Extraction techniques and specific treatments must be well established to avoid undesirable results. Fining of white juice is one of the specific treatments that could prevent chemical oxidation mechanisms that happen during winemaking.

In previous studies, laboratory tests with the patatin-rich protein have shown its high reactivity with tannins in red and white wines [8-10]. In the 2014 harvest, this product became commercially available and different trials at large scale could confirm previous tests in laboratory scale.

In this study, the patatin-rich protein has been tested in two varieties of white wine, Sauvignon Blanc and Rolle. Thiols are the most representative aromas of Sauvignon Blanc but they could be present over their threshold in other non-Sauvignon varieties such as Rolle and others (Ugni Blanc, Colombard, etc). Due to their nucleophilic nature, these volatile compounds can combine by a Michael addition reaction with quinones, a derivate of phenolic compounds under oxidation conditions [13].
<figure><img src="image_329.png" /><figcaption>**Fig. 3** - Colour intensity (a) and volatile thiols (b-d; 4MSP, 3SH and 3SHA) of a Sauvignon Blanc wine fined with Pea protein at 50 g/hL and Vegecoll® at 20 g/hL during alcoholic fermentation.</figcaption></figure>

In the third trial, the patatin-rich protein is compared to the pea protein used during alcoholic fermentation in juice from Rolle 2014. The patatin-rich protein was used at 20 g/hL in comparison to pea protein at 50 g/hL. After alcoholic fermentation, the wine fined with Vegecoll® was slightly lighter than that fined with pea protein (Figure 3a). This colour difference was not expressed in thiol composition (Figure 3b-d). Fining with Vegecoll® had a high impact on 3SH and 3SH acetate thiols. It preserved 22% 3SH and 92% 3SHA more than the same wine fined with pea protein.

The results of this study show that fining with patatin-rich protein could prevent this oxidative process by reducing phenolic compounds. The main effect is on 3SH and its acetate according to works carried out in wine-model solution by Nikolantonaki *et al.* [3]. That work has established that the 2FMT and 3SH react more rapidly with quinones than 4MSP. Our results reaffirm these wine-model observations since great variations in 3SH were observed after fining. The preservation depends on grape variety, juice quality and yeast transformation yield, but the fining action plays an interesting role in this process. Moreover, fining is more effective during alcoholic fermentation than settling before alcoholic fermentation, trials 2 and 3 compared to trial 1. This assumption is not related to wine conditions but to a best protein-wine contact which improves the fining action.

Concerning the differences observed in phenolic composition and colour intensity, no conclusions can be drawn. On the one hand, the differences in colour and phenolic compounds cannot explain directly the thiol preservation. Although several studies have observed the main role of flavan-3-ol monomers in oxidation process and consequently in aroma trap [3, 4], in large scale this interaction is not always observed. On the other hand, differences are observed between the two vegetal proteins (pea protein and patatin-rich protein). In the literature, the interaction polyphenol-protein is modulated by hydrophobic interactions and hydrogen bindings [14]. This interaction also depends on the molecular mass and molecule flexibility [15]. Therefore, previous works with vegetal proteins used as fining agents in red wine have observed that the vegetal proteins with low molecular mass are more selective with tannins [16]. The molecular mass of Patatin protein is 40 kDa, while pea protein has a large molecular mass distribution from 10 to 100 kDa. This characteristic could explain the observed differences of colour due to interaction with phenolic compounds.
Conclusion

Fining with patatin-rich proteins contributes to the preservation of volatile aromas, precisely the 3-sulfanylhexan-1-ol (3SH) and its acetate (3SHA). This preservation comes from the protein interaction with phenolic compounds during fining. This protein appears to be highly reactive due to its high protein concentration and its specific molecular mass. However, the phenolic analyses do not explain completely the thiol preservation. Including in some cases, non-modifications of phenolic analyses are observed. Also this protein appears to be highly reactive with oxidized phenolic compounds. A further study of these compounds could be interesting to understand their effect on volatile compounds.

Acknowledgements

The authors would especially like to thank Château La Louvière (AOC Pessac-Léognan), Château Villa Bel-Air (AOC Graves) and Château de Vauclaire (Côteaux d'Aix-en-Provence) for their participation in these trials.

References

1. Blanchard L, Darriet P, Dubourdieu D (2004) Reactivity of 3-mercaptohexanol in red wine: impact of oxygen, phenolic fractions and sulfur dioxide. *Am J Enol Vitic* **55**: 115-120.

2. Sarrazin E, Shinkaruk S, Pons A et al (2010) Elucidation of the 1,3-sulfanylalcohol oxidation mechanism: an unusual identification of the disulfide of 3-sulfanylhexanol in Sauternes botrytized wines. *J Agric Food Chem* **58**: 10606-10613.

3. Nikolantonaki M, Chichuc I, Teissedre PL et al (2010) Reactivity of volatile thiols with polyphenols in a wine-model medium: impact of oxygen, iron and sulfur dioxide. *Anal Chim Acta* **660**: 102-109.

4. 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.

5. Cheynier V, Fulcrand H, Guyot S et al (1995) Reactions of enzymically generated quinones in relation to browning in grape musts and wines. In *Enzymatic browning and its prevention*, Lee C.Y. & Whitaker J.R. (eds.), ACS symposium series, **600**, pp 130-143.

6. Resolution OIV-Oeno 495/2013 (2013) Monograph on protein plant origin/ International code of oenological practices". In *11<sup>th</sup> General Assembly of the International Organisation of Vine and Wine (OIV)*, Bucharest (Romania).

7. Commission implementing Regulation (EU) No. 1251/2013 amending Regulation No. 606/2009 as regards certain oenological practices and Regulation (EC) No. 436/2009 as regards the registering of these practices in the wine sector registers, *Official Journal of the European Union*, L 323, 28-30.

8. Gambuti A, Rinaldi A, Moio L (2012) Use of patatin, a protein extracted from potato, as alternative to animal proteins in fining of red wine. *Eur Food Res Technol* **235**: 753-765.

9. Renouf V, Louazil P, Iturmendi N et al (2013) Nouvelles alternatives pour le collage des moûts de raisins blancs. *Rev Œnol* **147**: 30-33.

10. Iturmendi N, Moine V, Renouf V et al (2013) Agent de collage des moûts et des vins : une nouvelle source de protéine végétale, la pomme de terre. *Rev Œnol* **149**: 25-28.

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

12. Ribéreau-Gayon P, Dubourdieu D, Donèche B et al (1998) *Traité d'ænologie. Tome 1 : Microbiologie du vin. Vinifications*, Dunod, Paris.

13. Cheynier N, Moine V, Renouf V et al (2013) Agent de collage des moûts et des vins : une nouvelle source de protéine végétale, la pomme de terre. *Rev Œnol* **149**: 25-28.

14. Oh HI, Hoff JE, Armstrong GS et al (1980) Hydrophobic interaction in tannin-protein complexes. *J Agric Food Chem* **28**: 394-398.

15. McManus JP, Davis KG, Beart SH et al (1985) Polyphenol interactions. Part 1. Introduction: some observations on the reversible complexation of polyphenols with proteins and polysaccharides. *J Chem Soc Perkin Trans 2* **1985**: 1429-1438.

16. Maury C, Sarni-Manchado P, Lefebvre S et al (2003) Influence of fining with plant proteins on proanthocyanidin composition of red wines. *Am J Enol Vitic* **54**: 105-111.
# Anthocyanin-derived pigments and tannins of red wines from Rioja and Bordeaux

N. PRIETO-PEREA<fnref n="1" />*, C. QUAGLIERI<fnref n="2" />*, L.A. BERRUETA<fnref n="1" />, B. GALLO<fnref n="1" />, M. JOURDES<fnref n="2" />, P.L. TEISSEDRE<fnref n="2" />

Wine quality – organoleptic characters – phenolic compounds – Rioja and Bordeaux wines

## Introduction

Phenolic compounds are one of the most important wine quality parameters, and their composition in grapes and wines are influenced by the grape variety, climatic conditions, cultural practices and technology. Moreover, other factors such as temperature, light, bottle position and oxygen content can affect the phenolic composition during storage [1].

Anthocyanins and flavanols play an important role in the quality of wine by contributing to sensory aspects such as colour, astringency or bitterness, and they can help in the differentiation between grape varieties and growing conditions of the grapes.

Colour evolution during vinification and ageing has been attributed to the progressive changes of phenolic compounds extracted from grapes. Anthocyanins can react with other compounds found in must and wine, such as flavanol monomers (+)catechin, (-)epicatechin, (+)gallocatechin, (-)epigallocatechin) and flavanol oligomers, to form new anthocyanin-derived pigments that change the initial colour of must and young wine [2]. These reactions can occur either directly or indirectly through an acetaldehyde molecule which generates an ethylidene bridge between both parts. Anthocyanin-tannin condensation products and different types of pyranoanthocyanin classes are the most important anthocyanin-derived pigments; the first ones show less stability during ageing than pyranoanthocyanins because the pyranic ring provides a protection against nucleophilic attack, increasing their stability [3].

Other major organoleptic characters of wine include astringency and bitterness, tannins being the molecules to which these characters are mainly attributed. Astringency is a dryness or roughness feeling that results from increased friction between the tongue and the surfaces inside the mouth. As opposed to astringency, bitterness is a taste induced by large molecules [4]. The chemistry of tannins in wine is also complex. Wine contains not only polymers of different polymerization index, but also species with different constituents' monomers (catechins to form the so-called procyanidins, gallocatechins to form prodelphinidins, or even mixed polymers with both types of monomers) and different bond types (B, A and both types).

## Materials and methods

7 anthocyanins by high performance liquid chromatography with diode array detection (HPLC-DAD), 15 anthocyanin-derived pigments by HPLC with tandem mass spectrometric detection (HPLC-MS/MS) and 29 tannins by HPLC-MS/MS were measured in 192 red wine samples: 96 from Rioja (32 R. Alavesa, 32 R. Alta and 32 R. Baja; 24 young, 24 crianza, 24 reserva and 24 gran reserva) and 96 from Bordeaux (36 Médoc, 27 Libournais, Blayais and Bourgeais, 2 Entre-deux-Mers, 13 Graves and 13 Bordeaux and Bordeaux Supérieur).

Anthocyanins and their derivatives were quantified by external calibration with Mv-3-O-glc and tannin derivatives with an external calibration of catechin.

<footnote n="1">¹Department of Analytical Chemistry, Faculty of Science and Technology, Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain</footnote>

<footnote n="2">²Unité de recherche Oenologie, Institut des Sciences de la Vigne et du Vin, Université de Bordeaux, 210 chemin de Leysotte, CS 50008, 33882 Villenave d'Ornon cedex, France</footnote>

<footnote>(*) both authors contributed equally</footnote>
**Results and discussion**

In general, Rioja wines have higher levels of free anthocyanins than those from the different regions of Bordeaux (Figure 1). This observation could be seen in generic Bordeaux wines (Bordeaux and Bordeaux Supérieur), which have less ageing time in barrel than the ones with a specific Bordeaux denomination.

<figure><img src="image_330.png" /><figcaption>Fig. 1 - Levels of anthocyanins for the different wines of Bordeaux and Rioja.</figcaption></figure>

Rioja wines have higher levels of non-acylated and coumaroylated anthocyanins, and lower levels of acetylated ones than Bordeaux wines (Figure 2). These results are in accordance with other authors, as a consequence of the different grape cultivars used in each region. On the one hand, in Cabernet Sauvignon and Merlot (the main cultivars in Bordeaux), acetylated anthocyanins represent 20-30% of total anthocyanin content, whereas in Tempranillo they represent only 2-6%. On the other hand, the coumaroylated anthocyanins represent 9% of total anthocyanin content in Cabernet Sauvignon and Merlot, while in Tempranillo and Graciano they represent 38 and 17%, respectively [5, 6].

<figure><img src="image_331.png" /><figcaption>Fig. 2 - Levels of non-acylated, coumaroylated and acetylated anthocyanins for the different wines of Bordeaux and Rioja.</figcaption></figure>

In general, Bordeaux wines present higher levels of tannin-anthocyanin condensation products and pyranoanthocyanins formed by reaction of anthocyanins with pyruvic acid (vitisins A), but lower levels of those formed by reaction with acetaldehyde (vitisins B) (Figures 3 and 4).
<figure><img src="image_332.png" /><figcaption>Fig. 3 - Levels of pyranoanthocyanins for the different wines of Bordeaux and Rioja.</figcaption></figure>

<figure><img src="image_333.png" /><figcaption>Fig. 4 - An example of the levels of anthocyanin-tannin derivatives (Epi-Mv-3-glc) for the different wines of Bordeaux and Rioja.</figcaption></figure>

Bordeaux wines have, in general, higher levels of tannins than Rioja wines. Catechin is the monomer with the highest concentration in both regions, followed by epicatechin.

The dimer procyanidin B1 is present at higher levels than the dimer procyanidin B2 in wines of both regions (Figure 5). B1 dimer is present in higher levels in the grape skin, while B2 is the major dimer in the seed. Consequently, procyanidin B1 levels in wine are higher as the skin's extraction during maceration is easier than the seed's extraction.

Mixed dimers with B bond have higher levels than prodelphinidin dimers with B bond in wines of both regions (Figure 6). Also mixed dimers with A bond have higher levels than procyanidin dimers with A bond in both regions. However, the procyanidin dimer A levels are higher in Rioja than in Bordeaux wines (Figure 7). Indirect condensation dimers with a furfuryl bridge present significant levels in wines of both regions.
<figure><img src="image_334.png" /><figcaption>Fig. 5 - Levels of procyanidin B1 and B2 for the different wines of Bordeaux and Rioja.</figcaption></figure>

<figure><img src="image_335.png" /><figcaption>Fig. 6 - Levels of mixed dimers B and prodelphinidin dimers B for the different wines of Bordeaux and Rioja.</figcaption></figure>

<figure><img src="image_336.png" /><figcaption>Fig. 7 - Levels of mixed dimers A and procyanidin dimers A for the different wines of Bordeaux and Rioja.</figcaption></figure>
**Conclusion**

Bordeaux wines have higher levels of acetylated anthocyanins, tannin-anthocyanin condensation products, vitisins A and tannins, while Rioja wines present higher levels of non-acylated and coumaroylated anthocyanins and vitisins B. These differences may be due to the grape variety used in each region and to the climatic conditions.

**Acknowledgements**

The authors thank to Eurorregión Aquitania-Euskadi for financial support.

**References**

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

2. Sánchez-Ilárduya MB, Sánchez-Fernández C, Garmón-Lobato S et al (2014) Detection of non-coloured anthocyanin–flavanol derivatives in Rioja aged red wines by liquid chromatography–mass spectrometry. *Talanta* **121**: 81-88.

3. Alcalde-Eon C, Escribano-Bailón MT, Santos-Buelga C et al (2006) Changes in the detailed pigment composition of red wine during maturity and ageing: a comprehensive study. *Anal Chim Acta* **563**: 238-254.

4. Chira K, Pacella N, Jourdes M et al (2011) Chemical and sensory evaluation of Bordeaux wines (Cabernet-Sauvignon and Merlot) and correlation with wine age. *Food Chem* **126**: 1971-1977.

5. Ryan JM, Revilla E (2003) Anthocyanin composition of Cabernet Sauvignon and Tempranillo grapes at different stages of ripening. *J Agric Food Chem* **51**: 3372-3378.

6. Nuñez V, Monagas M, Gomez-Cordovés MC et al (2004) *Vitis vinifera* L. cv. Graciano grapes characterized by its anthocyanin profile. *Postharvest Biol Technol* **31**: 69-79.
# Bee pollen application as fermentative activator in red winemaking

A. AMORES-ARROCHA*, A.M. ROLDÁN, R. GHIDOSSI, I. CARO, M. MIETTON-PEUCHOT, V.M. PALACIOS

Department of Chemical Engineering and Food Technology, Faculty of Sciences, University of Cadiz,
Polygono Río San Pedro, s/n, 11510 Cádiz, Spain

Bee pollen – fermentative activator – yeast nutrient – red winemaking

## Introduction

The composition of red grape berries (sugar levels, acidity, pH, specific aroma compounds, metabolite levels and proteins) changes during berry ripening. Over-ripening of red grapes may involve an excessive sugar content or lack of nutrients [1] leading to a higher potential alcoholic content [2] or musts deficient in growth factors for yeast (caused by over maturation or absence of maturity) such as YAN (yeast assimilable nitrogen) or thiamin (B1) [3]. Both phenomena, among others, may be the cause of fermentation stops and problems during red vinification.

For this reason, it is necessary to have an equilibrated grape must composition and a good fermentation development to obtain high quality in the physicochemical characteristics and sensory profiles of red wines. To avoid fermentative problems, commercial yeast (ADWY, Active Dry Wine Yeast) and commercial activators are used in the wine industry. So, to enhance nutrient deficiency, nitrogen supplementation with organic nitrogen preparations or inorganic nitrogen salts are used [4]. Yeast metabolism may be affected by the absence of nitrogen, and its addition at different doses in musts influences the fermentation development, ethanol production, volatile acidity, aroma compounds [5] and higher alcohol composition [6] of wines.

Previous studies in meads have shown that addition of bee pollen as fermentation activator benefits not only fermentation development but also the sensorial quality of the products obtained [7]. Bee pollen is a natural product rich in carbohydrates (35%), protein (25%), lipids (6%), minerals and vitamins (~ 3%) [8]. It also has high levels of essential amino acids such as proline, aspartic acid, phenylalanine and glutamic acid [9]; essential fatty acids such as linoleic and linolenic acids; sterols, phospholipids and carotenoids [10]. This research suggests that the use of bee pollen could be of interest as an alternative to commercial activators. Furthermore, this opens up the possibility that pollen could be used as activator in organic or biodynamic winemaking.

## Materials and methods

### Microvinifications

Microvinifications (7 kg grapes) control and using bee pollen (20 g L⁻¹) and a commercial product as fermentative activator (20 g L⁻¹) were carried out with two red varieties: *Cabernet sauvignon* (fermentable sugar: 225.1±1.7 g L⁻¹; pH: 3.59±0.01; total acidity: 5.9±0.2 g L⁻¹ tartaric acid) and *Merlot* (fermentable sugar: 237.6±5.5 g L⁻¹; pH: 3.82±0.04; total acidity: 4.2±0.3 g L⁻¹ tartaric acid). Stainless steel fermentation tanks (5 L) in controlled temperature chamber (21°C) and dry commercial yeasts *Saccharomyces cerevisiae* (20 g L⁻¹) were used to carry out the fermentation. Daily monitoring of viable yeast cells and kinetic parameter controls (density and temperature) were carried out. To determine cell viability, yeast cells were cultured on Petri dishes with chloramphenicol and biphenyl (99%) as bacterial antibiotics. Density was determined using a DMA 35N density meter (Anton Paar).

### Physicochemical and sensory analyses

Once malolactic fermentation was completed, a physicochemical and sensory analysis of the wines obtained was performed. Ethanol (%, v/v), reducing sugar (glu+fru, g L⁻¹), total acidity (g L⁻¹), pH, volatile
acidity (g L⁻¹) and malic acid (g L⁻¹) were determined using Fourier transform infrared spectroscopy (FTIR, OenoFoss™). Total phenolic content was measured according to the Folin-Ciocalteu (F.C.) method and antioxidant capacity through DPPH and FRAP assays [11] and expressed as mg of Trolox equivalents per L⁻¹ of wine, using a microplate spectrophotometer. All wines were sensory evaluated according to triangular test and descriptive characterization. Control-bee pollen, control-commercial and bee pollen-commercial combinations attending to UNI EN ISO 4120 [12] triangular test were carried out using a panel of tasters (n = 20) with extensive experience and familiarity with the aroma and mouthfeel attributes associated with *Cabernet sauvignon* and *Merlot* wines.

## Results and discussion

The main physicochemical parameters of *Cabernet sauvignon* and *Merlot* wines are shown in Table 1 and Table 2, respectively. As can be seen, all of the obtained wines showed similar physicochemical values. Compared to control wine, both varieties showed similar values of ethanol, residual sugars (gluc + fru), total acidity, pH, malic acid and total phenolic content. However, a decrease of volatile acidity content in both types of wine relative to its control was observed. This decrease was more pronounced in *Cabernet sauvignon* wines but with values similar to those obtained using the commercial activator. Addition of nutrients such as amino acids, saturated and unsaturated fatty acids to the yeasts may have an impact leading to lower values of volatile acidity in wines [13]. The use of commercial activators and bee pollen involves a nutrient supply which leads to wines of physicochemical characteristics similar to their control wines.

<table><caption>**Table 1** - Physicochemical profile of control (C.Cs.), bee pollen (B.P.Cs.) and commercial activator (C.A.Cs.) *Cabernet sauvignon* wines</caption><thead><tr><th></th><th>C.Cs.</th><th>B.P.Cs.</th><th>C.A.Cs.</th></tr></thead><tbody><tr><td>Pollen doses (g/hL)</td><td>0</td><td>20</td><td>0</td></tr><tr><td>Commercial activator (g/hL)</td><td>0</td><td>0</td><td>20</td></tr><tr><td>Ethanol (%, v/v)<sup>a</sup></td><td>12.42 ± 0.39</td><td>13.31 ± 0.13</td><td>12.86 ± 0.39</td></tr><tr><td>Reducing sugar (glu+fru, g/L)<sup>a</sup></td><td>1.4 ± 0.3</td><td>1.0 ± 0.1</td><td>1.2 ± 0.2</td></tr><tr><td>Total acidity (g/L)<sup>a</sup></td><td>5.3 ± 0.2</td><td>5.3 ± 0.2</td><td>5.1 ± 0.2</td></tr><tr><td>pH<sup>a</sup></td><td>3.98 ± 0.04</td><td>3.96 ± 0.02</td><td>3.96 ± 0.04</td></tr><tr><td>Volatile acidity (g/L)<sup>a</sup></td><td>0.30 ± 0.02</td><td>0.16 ± 0.02</td><td>0.16 ± 0.02</td></tr><tr><td>Malic acid (g/L)<sup>a</sup></td><td>0.5 ± 0.08</td><td>0.5 ± 0.05</td><td>0.4 ± 0.05</td></tr><tr><td>Total phenolic content (mg/L)<sup>a</sup></td><td>2253.79 ± 258.23</td><td>2452.13 ± 186.26</td><td>2668.23 ± 336.24</td></tr></tbody></table>

<table><caption>**Table 2** - Physicochemical profile of control (C.M.), bee pollen (B.P.M.) and commercial activator (C.A.M.) *Merlot* wines</caption><thead><tr><th></th><th>C.M.</th><th>B.P.M.</th><th>C.A.M.</th></tr></thead><tbody><tr><td>Pollen doses (g/hL)</td><td>0</td><td>20</td><td>0</td></tr><tr><td>Commercial activator (g/hL)</td><td>0</td><td>0</td><td>20</td></tr><tr><td>Ethanol (%, v/v)<sup>a</sup></td><td>14.18 ± 0.12</td><td>14.09 ± 0.24</td><td>14.35 ± 0.19</td></tr><tr><td>Reducing sugar (glu+fru, g/L)<sup>a</sup></td><td>1.9 ± 0.02</td><td>1.7 ± 0.04</td><td>1.3 ± 0.20</td></tr><tr><td>Total acidity (g/L)<sup>a</sup></td><td>5.9 ± 0.2</td><td>5.8 ± 0.2</td><td>5.9 ± 0.4</td></tr><tr><td>pH<sup>a</sup></td><td>3.67 ± 0.05</td><td>3.71 ± 0.04</td><td>3.71 ± 0.06</td></tr><tr><td>Volatile acidity (g/L)<sup>a</sup></td><td>0.13 ± 0.01</td><td>0.10 ± 0.02</td><td>0.14 ± 0.02</td></tr><tr><td>Malic acid (g/L)<sup>a</sup></td><td>1.2 ± 0.2</td><td>1.2 ± 0.1</td><td>1.6 ± 0.2</td></tr><tr><td>Total phenolic content (mg/L)<sup>a</sup></td><td>2164.02 ± 88.29</td><td>2201.78 ± 171.08</td><td>2237.87 ± 8.77</td></tr></tbody></table>

On the other hand, the same results were observed for the antioxidant capacity (Table 3). *Cabernet sauvignon* wines showed higher antioxidant capacity (DPPH and FRAP) than *Merlot* wines related to their higher total phenolic content (Tables 1 and 2). Same doses of pollen and commercial activator slightly increased the total phenolic content of wines and, in turn, their antioxidant capacity.

<footnote>a Each value is mean ± SD (n = 3).</footnote>

<footnote>a Each value is mean ± SD (n = 3).</footnote>
<table><caption>**Table 3** - Antioxidant capacity using DPPH and FRAP assays (mg Trolox/L) of control (C.), bee pollen (B.P.) and commercial activator (C.A.) in *Merlot* (M) and *Cabernet sauvignon* (Cs) wines</caption><thead><tr><th></th><th colspan="2">DPPH</th><th colspan="2">FRAP</th></tr></thead><tbody><tr><td>C.M.<sup>a</sup></td><td>2556.55</td><td>±</td><td>2611.78</td><td>±</td><td>77.23</td></tr><tr><td>B.P.M.<sup>a</sup></td><td>2683.58</td><td>±</td><td>2644.34</td><td>±</td><td>173.83</td></tr><tr><td>C.A.M.<sup>a</sup></td><td>2642.75</td><td>±</td><td>2630.72</td><td>±</td><td>101.35</td></tr><tr><td>C.Cs.<sup>a</sup></td><td>3646.20</td><td>±</td><td>3187.08</td><td>±</td><td>133.21</td></tr><tr><td>B.P.Cs.<sup>a</sup></td><td>3897.06</td><td>±</td><td>3581.96</td><td>±</td><td>210.65</td></tr><tr><td>C.A.Cs.<sup>a</sup></td><td>3956.09</td><td>±</td><td>3652.12</td><td>±</td><td>127.36</td></tr></tbody></table>

Considering the fermentation kinetics, Figures 1 and 2 show viable yeast monitoring during alcoholic fermentation. As can be seen, yeast growth was greatly influenced by the use of pollen and commercial activator. Both fermentations (*Cabernet sauvignon* and *Merlot*) using pollen and commercial activator improved the exponential phase of yeast growth and increased cell population. Therefore, pollen appears to have an important role in yeast growth and survival similar to commercial activator.

<figure><img src="image_337.png" /><figcaption>**Fig. 1** - Yeast population monitoring during alcoholic fermentation of *Cabernet sauvignon* control, bee pollen and commercial activator wines.</figcaption></figure>

<figure><img src="image_338.png" /><figcaption>**Fig. 2** - Yeast population monitoring during alcoholic fermentation of *Merlot* control, bee pollen and commercial activator wines.</figcaption></figure>

<footnote>a Each value is mean ± SD (n = 4).</footnote>
The triangular test results (Table 4) showed statistical significance comparing bee pollen and commercial activator wines with control (a, b), but no statistical significances were found between the two activator wines (c). The sensory attributes analyzed by the testing panel were divided in two groups: aroma (fruity and floral) and mouthfeel attributes (acidity, sweetness, bitterness and astringency).

<table><caption>**Table 4** - Triangle test (α risk) on control, bee pollen and commercial activator in *Merlot* and *Cabernet sauvignon* wines</caption><thead><tr><th></th><th>(a)<br>Bee pollen - Control</th><th>(b)<br>Commercial - Control</th><th>(c)<br>Bee pollen - Commercial</th></tr></thead><tbody><tr><td><strong>Merlot</strong></td><td>0.001</td><td>0.05</td><td>—</td></tr><tr><td><strong>Cabernet sauvignon</strong></td><td>0.2</td><td>0.01</td><td>—</td></tr></tbody></table>

As can be seen in Figures 3 and 4, the use of pollen had greater influence on sensory characteristics of wines than on their physicochemical profile and antioxidant capacity. So, *Cabernet sauvignon* wines with pollen addition (Figure 3) showed higher values in floral aroma (*gardenia* and *roses*), fruity character (*exotic fruit* and *red berries*) and astringency than commercial activator and control wines. Moreover, *Merlot* wines with pollen significantly enhanced fruity and floral character (Figure 4) showing a similar profile to commercial activator. These improvements confer to wines added with bee pollen better aroma and mouthfeel attributes compared with control and commercial activator wines.

<figure><img src="image_339.png" /><figcaption>**Fig. 3** - Spider plots of sensory attributes determined by a panel (n = 20) in *Cabernet sauvignon* control, bee pollen and commercial activator wines.</figcaption></figure>
<figure><img src="image_340.png" /><figcaption>Fig. 4 - Spider plots of sensory attributes determined by a panel (n = 20) in *Merlot* control, bee pollen and commercial activator wines.</figcaption></figure>

## Conclusion

The use of bee pollen as fermentation activator involves a nutrient supply which leads to wines of similar physicochemical characteristics and antioxidant capacity to their control and commercial activator wines. Furthermore, addition of pollen provides nutrients that enhance the development and survival of the yeast, resulting in a good development of the fermentation and production of less volatile acid. In this regard, the pollen is a good substitute for commercial fermentation activators. The tasting session results showed that wines fermented with pollen were highlighted by floral and fruity notes, especially *Cabernet sauvignon*. Similar behavior between pollen and commercial fermentative activator suggests an interesting potential use of bee pollen as an alternative activator in traditional red winemaking. Besides, the use of pollen as fermentation activator would open a new field to organic or biodynamic winemaking.

## Acknowledgements

A.A.A. gratefully thanks the University of Cadiz for a Ph.D. studentship under the program 2013-023/PU/EPIF-FPI-CT/CP. The authors thank the Institut des Sciences de la Vigne et du Vin (ISVV) and the Laboratoire de Génie des Procédés et Environment, Université Victor Segalen Bordeaux 2, for technical support. The authors also thank the members of the test panel for their collaboration.

## Abbreviations

ADWY, active dry wine yeast; B.P.Cs., bee pollen *Cabernet sauvignon* wine; B.P.M., bee pollen *Merlot* wine; C.A.Cs., commercial activator *Cabernet sauvignon* wine; C.A.M., commercial activator *Merlot* wine; C.Cs., control *Cabernet sauvignon* wine; C.M., control *Merlot* wine; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; F.C., Folin–Ciocalteu method; FRAP, ferric reducing ability of plasma, also ferric ion reducing antioxidant power; FTIR, Fourier transform infrared spectroscopy; ISVV, Institut des Sciences de la Vigne et du Vin, Bordeaux University.
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5. Barbosa C, Falco V, Mendes-Faia A et al (2009) Nitrogen addition influences formation of aroma compounds, volatile acidity and ethanol in nitrogen deficient media fermented by *Saccharomyces cerevisiae* wine strains. *J Biosci Bioeng* **108**: 99-104.

6. Lonvaud-Funel A, Renouf V, Strehaiano P (2010) *Microbiologie du vin : Bases fondamentales et applications*. Editions TEC&DOC, Lavoisier, pp 91-93.

7. Roldán A, van Muiswinkel GCJ, Lasanta C et al (2011) Influence of pollen addition on mead elaboration: physicochemical and sensory characteristics. *Food Chem* **126**: 574-582.

8. Almeida-Muradian LB, Pamplona LC, Coimbra S et al (2005) Chemical composition and botanical evaluation of dried bee pollen pellets. *J Food Compos Anal* **18**: 105-111.

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13. Bely M, Rinaldi A, Dubourdieu D (2003) Influence of assimilable nitrogen on volatile acidity production by *Saccharomyces cerevisiae* during high sugar fermentation. *J Biosci Bioeng* **96**: 507-512.
# Characterization and prognosis of the oxidative behavior of wine using chemiluminescence, fluorescence and the colorimetric reducing power method

P. SCHONENBERGER<fnref n="1" />*, B. BACH<fnref n="1" />, J.-P. BOURGEOIS<fnref n="2" />, J.-M. SEGURA<fnref n="3" />, J. HERITIER<fnref n="3" />, J. DUCRUET<fnref n="1" />

Wine oxidation – chemiluminescence – ORAC-FL assay – reducing power method

## Introduction

Oxidation is a common process that touches organisms and biological products alike. In regard to wine, oxygen can be very beneficial or detrimental to its quality. Micro-quantities of oxygen may enhance the wine's quality, whereas prolonged exposure easily leads to its spoilage. Historically, those micro-quantities were transferred into wine simply by using oak barrels for bulk aging. Pore sizes of these wooden barrels were shown to be ideally suited for the so called micro-oxygenation. Similar maturation effects were observed during bottle aging by using natural corks for its cap material. Oak and cork withstood the test over time, however, sometimes large variations between barrels or between corks led to the development of new technologies which offered new solutions for oxygen addition and concentration. The addition of defined micro-quantities of oxygen to a wine is very well mastered today and comprehensively summarized in recent publications [1, 2], but the total amount of oxygen needed over a longer period of time is still subject of debates (depending on grape variety, wine type, and wine style) and generally decided in accordance with the results of a quick sensory evaluation session. The method is heavily dependent on the taster's judgment and opinion. The relationship between sensory evaluation data and analytical control parameters is not very well understood and single parameters to objectively predict the capacity of a wine to receive small amounts of oxygen are not known. Sensory evaluation remains, despite its high degree of subjectivity, the principal method to determine the "healthy" amount of oxygen in a wine.

This project aimed at confronting three different analysis methods for a simple and objective test of a wine's capacity to consume small amounts of oxygen. All three methods were well established, but were originally developed to characterize the resistance to oxygen of solids and/or liquids other than wine. The reported tests were carried out with identical wine samples and compared to the sensory analysis data obtained at the same time by experienced judges in terms of the wine's oxidation levels and aging potentials.

## Materials and methods

### Wine selection

Four wineries were each asked to propose two red wines from their collection: one wine with a weak resistance against oxygen and one wine with a strong resistance against oxygen. Two additional wines from other wineries were added at random. With this procedure, 10 red wines were preselected for a relatively wide range of oxygen resistance levels and used for the experimental analysis and the tasting session. Classic wine analysis and color measurements were performed by InterRhône (Orange, France), as well as comprehensive polyphenolic compound analysis of all 10 red wines.

### Sensory evaluation

The tasting session included 17 participating judges. The 10 wines were served to the judges in coded glasses. Two of the 10 wines were served twice for a total of 12 samples. The judges were asked to rank and score the wine samples on an ordinal scale from 1 through 10 according to their resistance against oxygen.

<footnote n="1">¹CHANGINS, Viticulture and Enology, University of Applied Sciences and Arts, Western Switzerland</footnote>

<footnote n="2">²College of Engineering and Architecture of Fribourg, Switzerland</footnote>

<footnote n="3">³University of Applied Sciences of Valais, Switzerland</footnote>
## Analytical methods

Early applications of Chemiluminescence (CL) in beverages include the detection of light emission during beer oxidation [3]. Navas and Jimenez [4] summarized the applications of CL methods in the context of alcoholic beverage analysis in general. The Oxygen Radical Absorbance Capacity Assay (ORAC-FL assay) introduced by Prior et al. [5, 6] has been used as a particularly pertinent test for wines. The Reducing Power method (RP) method was developed by M. L. Chapon for the brewing industry and expresses the quantity of reducing components in beer. Cavin et al [7] carried out RP tests with wine, in order to find an alternative method for the redox potential analysis of wines.

# Results and discussion

## Sensory evaluation

Each of the 12 samples got 17 scores from the 17 judges and overall scores were calculated. These overall scores and the corresponding samples were then ranked in ascending order. Samples underlined by the same bar were not different (Fig. 1). It can be seen that six distinct oxygen resistant levels were obtained: the least resistant wines being the 2010 Gamay VD and four of the five Pinot noirs. On the other hand, the most resistant wines were Bordeaux varieties and a dark colored Regent from the Zurich region. The two doubled served wines had been ranked close together, rank number 3 and number 5 for the Pinot noir 2012, as well as number 9 and number 11 for the Merlot 2011, respectively.

<figure><img src="image_341.png" /><figcaption>**Fig. 1** - Average oxygen resistance scores of corresponding samples, ranked in ascending order. Significance is indicated with red lines. Samples underlined by the same line were not significantly different.</figcaption></figure>

## Fluorescence (ORAC-FL) method

While a slight trend of increasing ORAC-FL values with higher ranking of the tasting assessment seemed visually apparent, it was not statistically significant (p = 0.56, Fig. 2). However, the ORAC-FL assay significantly correlated (p < 0.001) with the total polyphenolic index, which are the main anti-oxidant properties of the wines.

## Reducing power method (RP)

Correlations of the RP values with the ranking for the 10 wines obtained by the tasting assessment did not show a significant relationship (p = 0.12, Fig. 3). However, as seen for the ORAC-FL method, the total polyphenolic index correlated significantly with the RP value (p < 0.05). These findings indicated that the ORAC-FL assay and the RP assay might give similar, but not necessarily identical, information.

## Chemiluminescence (CL) method

The CL method was designed for the measurement of solid samples. All experiments that were carried out on red wine showed no clear CL signal. Modifications of the instrument were partly successful, i.e. they worked well for liquid samples like olive oil, but not for red wines.
<figure><img src="image_342.png" /><figcaption>**Fig. 2** - ORAC values of the selected wines were measured and compared to the assessment of the tasting panel. While a general trend of increasing ORAC values for increasing assessed resistance is visible, this relationship is not statistically significant (p > 0.56).</figcaption></figure>

<figure><img src="image_343.png" /><figcaption>**Fig. 3** - The values measured using the RP method were compared to the assessment of the tasting panel: the results of the tasting panel and RP values correlated well (R2 = 0.26; n = 10).</figcaption></figure>

## Conclusion

Each of the tested methods included several critical steps that had to be followed in order to maintain accuracy and precision. The reported work allowed the development of standard protocols for the analysis of wine oxidation reactions with the ORAC-FL method and the RP method. Further exploration is needed for the CL method. At this point, the correlations of the tasting results and the results of the ORAC-FL and RP methods did not show strong and significant relationships, although positive tendencies can be seen. Due to the limited number of wine samples, the presented results of these global approaches are preliminary, additional experiments will be carried out.

## References

1. Du Toit WJ, Marais J, Pretorius IS et al (2006) Oxygen in must and wine: a review. *S Afr J Enol Vitic* **27**: 76-94.

2. Biondi Bartolini A, Cavini F, de Basquiat M (2008) *Oxygène et vin : du rôle de l'oxygène à la technique de micro-oxygénation*, Parsec édition.

3. Kaneda H, Kano Y, Kamimura M et al (1990) Detection of chemiluminescence produced during beer oxidation. *J Food Sci* **55**: 881-882.

4. Navas MJ, Jimenez AM (1999) Chemiluminescent methods in alcoholic beverage analysis. *J Agric Food Chem* **47**: 183-189.

5. Ou B, Hampsch-Woodill M, Prior RL (2001) Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. *J Agric Food Chem* **49**: 4619-4626.

6. Cao G, Verdon CP, Wu AH et al (1995) Automated assay of oxygen radical absorbance capacity with the COBAS FARA II. *Clin Chem* **41**: 1738-1744.

7. Cavin S, Romanelli A, Fabre S (2004) Application de la mesure du pouvoir réducteur du vin par colorimétrie. *Rev Oenol* **113**: 17-20.
# Colour evolution of wines aged in different containers by CIELAB: discrimination between type of container and contact time

M. BASALEKOU<fnref n="1" />,<fnref n="2" />*, E. KONTAXAKIS<fnref n="2" />, A. STRATARIDAKI<fnref n="2" />, D. LYDAKIS<fnref n="2" />, S. KALLITHRAKA<fnref n="1" />

CIELAB – barrel – wine – colour

## Introduction

Colour is considered one of the principal parameters of wine quality as it provides a great deal of information on its current status or even aging potential [1]. For this reason, colour analysis is routinely performed in wineries using spectrophotometric measurements, even though there is no simple relationship between these measurements and human colour perception. For red wines, the colour determination is based on the calculation of absorbance at three different wavelengths (420, 520 and 620 nm), while the colour of white wines is evaluated based only on the measurement of the absorbance at 420 nm [2, 3]. The Commission Internationale de l'Eclairage (CIE) established a new system which uses the whole visible spectrum and can provide information on a wine's tonality, luminosity and chromatism [4]. This system has been used in several studies with regard to colour stability, monitoring the fermentation procedure, and the colour evolution during bottle aging [5-7]. During barrel aging, colour is also used to provide information, but given the complex transformations of phenolic compounds and the extraction of various substances from the barrel, the analyses performed concentrate mostly on studying the phenolic content of the wines [8] and the type of phenols extracted, which is greatly influenced by the type of barrel used (size, type of wood) and the time of contact [9]. The aim of this study was to evaluate the use of CIELAB on the differentiation of wines aging in different containers for different periods of time.

## Materials and methods

### *Cultivars and fermentation conditions*

The wines were 2013 vintage from the region of Peza in Crete, Greece. The indigenous grape varieties of Vilana, Dafni (white varieties), Kotsifali and Mandilari (red varieties) were used to produce four wines (oenological parameters of the wines are shown in Table 1). After completing fermentation in stainless steel tanks, all wines were transferred to the aging containers. The containers used were Stainless Steel Tank with oak chips, and barrels made of French Oak, American Oak, Acacia and - only for the case of red wines - Chestnut (all barrels were 225 l with a medium toast, oak chips were Oenostick® V18). Control wines were transferred to stainless steel tanks, and samples from all containers were taken after 3, 6 and 12 months of maturation.

<table><caption>**Table 1 - Oenological parameters of wine samples**</caption><thead><tr><td>Wine</td><td>Ethanol %(v/v)</td><td>Total acidity (g/L in tartaric acid)</td><td>pH</td><td>Volatile acidity (g/L in acetic acid)</td></tr></thead><tbody><tr><td>Vilana</td><td>14,3</td><td>6,07</td><td>3,41</td><td>0,43</td></tr><tr><td>Dafni</td><td>12,7</td><td>5,25</td><td>3,35</td><td>0,40</td></tr><tr><td>Kotsifali</td><td>13,6</td><td>4,95</td><td>3,45</td><td>0,47</td></tr><tr><td>Mandilari</td><td>12,9</td><td>5,17</td><td>3,41</td><td>0,31</td></tr></tbody></table>

<footnote n="1">¹Department of Food Science & Human Nutrition, Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece</footnote>

<footnote n="2">²School of Agricultural Technology, Technological and Educational Institute of Crete, P.O. Box 1939, 71004 Heraklion, Crete, Greece</footnote>
### Colour analysis

Absorbance measurements were done on a Hitachii 2000 spectrophotometer. Total Phenolic Index (TPI) (A280) was determined by measuring the absorption at 280 nm [10], A420 by measuring the absorption at 420 nm [2] and Total Tannins by acid hydrolysis [11]. Wine colour was assessed with a tristimulus colorimeter (Minolta CR-400) using the L*a*b* and L*Ch systems from the CIELAB colour space. The following parameters were evaluated: L*, which is a measure of lightness; a* and b* coordinates, which represent the degree of red (when a*>0), green (when a*<0), yellow (when b*>0), and blue (when b*<0); C* (Chroma), which is a measure of saturation; and h, which represents the hue. The parameters were determined with a D65 light source.

### Data analysis

Data processing was performed with the JMP statistical analysis software (Statistical Discovery software, version 11).

## Results and discussion

Discriminant analysis performed on the CIELAB values showed a significant discrimination (80%) regarding contact time (Figure 1, Table 2).

<figure><img src="image_344.png" /><figcaption>Fig. 1 - Discrimination results regarding contact time, based on CIELAB values (white wines).</figcaption></figure>

<table><caption>**Table 2** - Contact time classification data based on CIELAB values (white wines)</caption><tr><td>Number of Misclassified</td><td>6</td></tr><tr><td>Percent of Misclassified</td><td>20</td></tr><tr><td>-2LogLikelihood</td><td>28,73</td></tr></table>

In the case of the red wines, CIELAB values resulted in a significant classification of 69% of the samples (Table 3).

<table><caption>**Table 3** - Contact time classification data based on CIELAB values (red wines)</caption><thead><tr><td></td><td><strong>Training</strong></td></tr></thead><tbody><tr><td>Number of Misclassified</td><td>14</td></tr><tr><td>Percent of Misclassified</td><td>38,89</td></tr><tr><td>-2LogLikelihood</td><td>53,13</td></tr></tbody></table>

Regarding the type of container used in white wine aging, CIELAB allowed a complete discrimination (92%) of the wines aging in tanks from wines aging in wooden barrels or in tanks with oak chips (Figure 2, Table 4).
<figure><img src="image_345.png" /><figcaption>**Fig. 2** - Discrimination results regarding type of container, based on CIELAB values (white wines).</figcaption></figure>

<table><caption>**Table 4** - Container type classification data based on CIELAB values (white wines)</caption><thead><tr><td></td><td>Training</td></tr></thead><tbody><tr><td>Number of Misclassified</td><td>3</td></tr><tr><td>Percent of Misclassified</td><td>7,5</td></tr><tr><td>-2LogLikelihood</td><td>18,07</td></tr></tbody></table>

Spectrophotometric measurements resulted in a significant discrimination of the Acacia barrel from all other types of containers, with 87% of the samples correctly classified (Figure 3, Table 5).

<figure><img src="image_346.png" /><figcaption>**Fig. 3** - Discrimination results regarding type of container, based on spectrophotometric measurements (white wines).</figcaption></figure>

<table><caption>**Table 5** - Contact time classification data based on spectrophotometric measurements (white wines)</caption><thead><tr><td></td><td>Training</td></tr></thead><tbody><tr><td>Number of Misclassified</td><td>5</td></tr><tr><td>Percent of Misclassified</td><td>12,5</td></tr><tr><td>-2LogLikelihood</td><td>30,11</td></tr></tbody></table>

Moreover, no significantly strong correlation could be established between spectrophotometric data and CIELAB values for the white wines, except for the absorbance at 280 nm and the Chroma value (C*) (Table 6).
<table><caption>Table 6 - CIELAB values and spectrophotometric data correlation</caption><thead><tr><th></th><th>L*</th><th>C*</th><th>h*</th><th>L*(Lab)</th><th>a*</th><th>b*</th><th>A420</th><th>A280</th><th>TT</th></tr></thead><tbody><tr><th>L*</th><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>C*</th><td>0,3898</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>h*</th><td>-0,0124</td><td>-0,0724</td><td>1</td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>L*(Lab)</th><td>0,6517</td><td>0,3606</td><td>0,0667</td><td>1</td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>a*</th><td>-0,0868</td><td>-0,1217</td><td>-0,9562</td><td>-0,1456</td><td>1</td><td></td><td></td><td></td><td></td></tr><tr><th>b*</th><td>0,4253</td><td>0,9316</td><td>-0,0049</td><td>0,5599</td><td>-0,1461</td><td>1</td><td></td><td></td><td></td></tr><tr><th>A420</th><td>-0,097</td><td>0,4509</td><td>0,1721</td><td>-0,2841</td><td>-0,2158</td><td>0,3792</td><td>1</td><td></td><td></td></tr><tr><th>A280</th><td>-0,0297</td><td>0,7418*</td><td>-0,224</td><td>-0,147</td><td>0,0837</td><td>0,5974*</td><td>0,4666</td><td>1</td><td></td></tr><tr><th>TT</th><td>0,1095</td><td>0,6644*</td><td>-0,051</td><td>-0,0376</td><td>-0,0895</td><td>0,5465</td><td>0,4001</td><td>0,8326</td><td>1</td></tr></tbody></table>

## Conclusion

CIELAB colour values allowed the discrimination of wines aged for different time periods but also the discrimination of white wines aged with or without wood contact (barrel or oak chips), whereas the spectrophotometric measurements that evaluate the phenolic content and the A420 measurement allowed the significant discrimination of the Acacia barrel from all other containers. This could be due to the fact that wood contact is perceived by the colorimeter even when the phenols extracted are not providing a significant change in the total phenol content or the browning index (A420) of a wine. These data, along with more research, could prove CIELAB colour space analysis to be a fast and easily applicable method that could be used to provide reliable data on aging conditions.

## Acknowledgements

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: **THALES**. Investing in knowledge society through the European Social Fund.

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1. Bakker J, Timberlake CF (1986) The mechanism of color changes in aging port wine. *Am J Enol Vitic* **37**: 288-292.

2. Glories Y (1984) *La couleur des vins rouges, 2ème partie. Mesure, origine et interpretation*. Conn Vigne Vin **18**: 195-217.

3. Peng Z, Duncan B, Pocock KF et al (1998) The effect of ascorbic acid on oxidative browning of white wines and model wines. *Aust J Grape Wine Res* **4**: 127-135.

4. O.I.V. (2006) *Resolution OENO 1/2006: determination of the chromatic characteristics of wine according to CIELab*. Paris, France.

5. Skouroumounis GK, Kwiatkowski MJ, Francis IL et al (2005) The influence of ascorbic acid on the composition, colour and flavour properties of a Riesling and a wooded Chardonnay wine during five years' storage. *Aust J Grape Wine Res* **11**: 355-368.

6. Gil-Muñoz R, Gómez-Plaza E, Martínez A et al (1997) Evolution of the CIELAB and other spectrophotometric parameters during wine fermentation. Influence of some pre and postfermentative factors. *Food Res Int* **30**: 699-705.

7. Monagas M, Martín-Álvarez PJ, Gómez-Cordovés C et al (2006) Time course of the colour of young red wines from *Vitis vinifera* L. during aging in bottle. *Int J Food Sci Technol* **41**: 892-899.

8. Fulcrand H, Duenas M, Salas E et al (2006) Phenolic reactions during winemaking and aging. *Am J Enol Vitic* **57**: 289-297.

9. Sanza MA, Escudero JAF, Torio RC (2004) Changes in phenolic compounds and color parameters of red wine aged with oak chips and in oak barrels. *Food Sci Technol Int* **10**: 233-241.

10. Ribéreau-Gayon P (1970) Les dosages des composés phénoliques totaux dans le vin rouge. *Chim Anal* **52**: 627-631.

11. Ribéreau-Gayon P, Glories Y, Maujean A et al (2006) *Handbook of enology - volume 2. The chemistry of wine: stabilization and treatments*, John Wiley & Sons Ltd, England.
# Determination of pesticide residues from vine leaves and grapes to the wine

C. FRANC<sup>1,2*</sup>, L. RIQUIER<sup>1,2</sup>, G. JABS<sup>1,2</sup>, V. PELTIER<sup>1,2</sup>, G. DE REVEL<sup>1,2</sup>

<sup>1</sup>Univ. de Bordeaux, ISVV, EA 4577, Unité de recherche OENOLOGIE, F-33882 Villenave d'Ornon, France

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

Pesticides – extraction – quantification – method comparison

## Introduction

Nowadays, food safety is at the heart of consumers, producers and regulators' concerns. Regarding viticulture, a large number of active ingredients are authorised for vine phytosanitary treatments and new substances appear on the market regularly. Some phytosanitary products, principally fungicides, applied close to the harvest date may leave residues on the grapes and may further be transferred to the wines. Although until now no Maximum Residue Limits (MRL) have been set for the wine, powerful analysis methods have to be developed in order to detect a great number of ingredients potentially used in vineyard and to quantify low levels of residues and prohibited molecules in particular. Due to the complexity of the matrices, an efficient extraction-concentration step is required before chromatographic separation. Moreover, concerning solid samples, the grinding method has to be defined.

We previously studied the scope and limitations of the stir bar sorptive extraction (SBSE) of pesticide residues from wine followed by gas chromatography separation and mass spectrometry detection (GC-MS) [1]. Due to the specific features of SBSE and GC techniques, only sufficiently apolar, volatile and thermostable molecules could be analysed. Finally, a quantitative method was optimised for 37 compounds widely used for vineyard protection whereas 80 compounds were tested.

To overcome these specificities and to extend the range of pesticide residues quantified, we turned to liquid chromatography (LC). Moreover, coupling with tandem mass spectrometry (MS/MS) ensures a greater confidence in the detection. QuEChERS (Quick, Easy, Efficient, Rugged and Safe) [2] resolution OIV-OENO 436-2012, Solid-Phase Extraction (SPE) and direct injection (DI) with or without dilution were compared for LC-MS/MS analysis for nearly 200 compounds.

## Materials and methods

### Pesticide residue analysis

A 1260 liquid chromatography system coupled with a 6430 Triple Quadrupole mass spectrometer from Agilent Technologies (Massy, France) was used.

Solid-Phase Extractions were performed by a MultiPurpose System (MPS) from Gerstel (Müllheim an der Ruhr, Germany).

### Vine leaf samples

Vine leaves treated with phytosanitary products during the summer 2013 were used to compare the effect of the sample preparation. Each preparation was performed in triplicate.

### Fermentation conditions

A must was obtained by pressing grape berries with a small pneumatic press. Supplemented musts were prepared by addition of a solution of pesticide standards. The alcoholic fermentations were conducted on 3 samples of each supplemented must. A control, for which fermentation was blocked, was maintained in the same temperature conditions.
Results and discussion

When comparing chromatograms of a supplemented red wine sample obtained after direct injection, QuEChERS extraction and SPE all of them followed by LC-MS/MS analysis (Figure 1), a strong enhancement is noticed for SPE (except for the most polar analytes), whereas direct injection and QuEChERS extraction give similar results. However, QuEChERS extraction is often preferred due to the existence of signal suppression depending on the wine matrix for direct injection.

<figure><img src="image_347.png" /><figcaption>Fig. 1 - Chromatograms of a supplemented wine for three extraction types.</figcaption></figure>

The accuracy of the methods was checked through inter-laboratory experiments (13 laboratories). Quarterly, 45 pesticide residues were sought in a white and a red wine by QuEChERS-LC-MS/MS, SPE-LC-MS/MS and SBSE-GC-MS. For most of the compounds and analyses, the results are in good agreement with the mean values calculated from the inter-laboratory results, with less than 50 % deviation. Vinclozolin could not be analysed by LC-MS/MS. For chlorpyriphos, chlorpyriphos-Me, fenitrothion and procymidone the sensitivity obtained after QuEChERS extraction is very poor. Concerning spiroxamine, unreliable results were generally obtained after SPE-LC-MS/MS.

When considering solid samples, additional parameters have to be taken into account. First of all, the sampling must be well representative of the parcel. Secondly, the size of the solid particles greatly influences the extraction step. To estimate this effect for the extraction of incurred pesticide residues from vine leaves, three grinding techniques were tested: grinding with a kitchen blender at room temperature or with liquid nitrogen and grinding with a mixer mill (ball mill) and liquid nitrogen. Depending on the molecule, the best results were obtained when the leaves were grinded frozen with liquid nitrogen either with a mixer mill or a kitchen blender (Figure 2). However, the use of a mixer mill is laborious and the kitchen blender was preferred.

Regarding the extraction step, three QuEChERS procedures were selected: 5 g leaves and 10 mL water, 5 g leaves and 5 mL water, 2 g leaves and 10 mL water (Figure 3). Among them, 2 g leaves and 10 mL water gave the smallest responses as expected. From a general point of view there is no difference between addition of 5 or 10 mL of water when starting from 5 g of leaves.

Concerning the d-SPE cleaning, four procedures were tested: PSA, PSA followed by acidification, PSA and GCB, PSA and GCB followed by acidification. No effect was observed for the whole set of pesticides except for 2-aminobenzimidazole for which the response decreases with the use of formic acid and even more with GCB or worse with the combination of both. However, the use of GCB is recommended to protect the ion source. Moreover, the addition of formic acid to readjust the pH after d-SPE generally slightly increases the response for all other compounds.
<figure><img src="image_348.png" /><figcaption>Fig. 2 - Grinding conditions for vine leaves.</figcaption></figure>

<figure><img src="image_349.png" /><figcaption>Fig. 3 - QuEChERS extraction conditions for vine leaves.</figcaption></figure>

The evolution of some pesticide residues during the vinification process was monitored with SPE-LC-MS/MS. Each sample was extracted and analysed in triplicate. From Figure 4, it can be seen that for carbendazim a degradation occurs during fermentation whereas for fluazinam the degradation happens over the time. For the other compounds there is no effect of the alcoholic fermentation under the conditions used.

<figure><img src="image_350.png" /><figcaption>Fig. 4 - Monitoring of pesticide residues with SPE-LC-MS/MS from must to wine.</figcaption></figure>
The SPE results were compared to those of the QuEChERS extraction with and without centrifugation of the samples. For most compounds and samples there is a significant loss of residues after centrifugation (Figure 5). In general, the concentrations measured after SPE or QuEChERS with centrifugation of the sample are in good accordance.

<figure><img src="image_351.png" /><figcaption>Fig. 5 - Comparison of SPE and QuEChERS with or without centrifugation from must to wine.</figcaption></figure>

## Conclusion

A LC-MS/MS method was optimised and implemented for the detection of 178 pesticide molecules. This analytical method was used to compare direct injection, SPE and QuEChERS extraction of pesticide residues in wines, showing the much higher sensitivity of the second one. The accuracy of the QuEChERS-LC-MS/MS and SPE-LC-MS/MS results are regularly checked and compared to those of the SBSE-GC-MS for compounds in common.

Considering vine leaves, some parameters were optimised and the method is now operational.

Regarding the evolution of pesticide residues from grapes to the wines, analyses are in progress, but preliminary results confirmed that some residues are lost with the separation of solid parts and that the stability of each residue varies.

## Acknowledgements

The Regional Council of Aquitaine, CIVB (Conseil Interprofessionnel du Vin de Bordeaux) and FranceAgriMer are warmly thanked for their financial support.

## References

1. Franc C, de Revel G (2010) Quantification of pesticides residues in wine: development of a SBSE-GC-MS-SIM method. *In 8th European Pesticide Residue Workshop*, Strasbourg, France.

2. Anastassiades M, Lehotay SJ, Stajnbaher D et al (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. *J AOAC Int* **86**: 412-431.
# Effect of ageing and variety on dimethyl sulfide levels

M. KYRALEOU¹*, S. KALLITHRAKA¹, P. TARANTILIS¹, T. GARDE-CERDAN², Y. KOTSERIDIS¹

¹Department of Food Science & Human Nutrition, Laboratory of Enology,
Agricultural University of Athens, 75 Iera Odos, 11855 Athens, Greece

²Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja-CSIC-Universidad de La Rioja),
Ctra. Mendavia-Logroño NA 134, Km. 90, 26071 Logroño, La Rioja, Spain

Dimethyl sulfide (DMS) – GC-MS – volatile compound – grapes – wine

## Introduction

Aroma is a factor that influences the quality of the wine and depends on a very large number of compounds which are responsible for the character of the wine. During bottle ageing, a lot of chemical changes occur which affect the development of the sensory characteristics of the wine. The composition of wine aroma depends also on the grape variety, the alcoholic fermentation and the production process. In some varieties it was determined a characteristic aromatic compound, such as 4-methyl-4-sulfanylpentan-2-one in Sauvignon Blanc, while other varieties have a characteristic aromatic profile, such as Cabernet Sauvignon and Chardonnay [1].

In this study, the role of dimethyl sulfide (DMS) was investigated. DMS has been reported as a highly volatile sulfur compound found in a wide range of foods and beverages such as corn [2], beer [3], malt [4] and wine [5]. The formation of DMS during the alcoholic fermentation is linked to various amino acids (cysteine, cystine, glutathione, methionine) and DMSO [6], but in a recent study S-methylmethionine (SMM) was identified as the major DMS precursor in grape [7]. Although the aroma of DMS was described as corn, cabbage, asparagus and molasses, it has been demonstrated as an enhancer of the berry fruit note [5, 8]. The positive or negative effect of DMS on the bouquet depends on its concentration in the wines and relates to the grape variety [5, 9].

## Materials and methods

### Wine samples

The samples were commercial wines from the Greek variety cv Xinomavro from the region of Naoussa, Northern Greece and from different vintages (1992, 1996, 1997, 1999, 2005, 2006 and 2008), while the grape samples were from the same vintage (2010) and were collected at maturity.

### DMS analysis

Free and potential DMS were analyzed using a method based on that described by Segurel et al. [5] with some modifications. Free Dimethyl Sulfide (fDMS) and Potential Dimethyl Sulfide (pDMS) were analyzed by GC-MS using the Head-Space Solid Phase Micro Extraction (HS-SPME) methodology.

For the quantification of fDMS, a volume of 25 mL of wine spiked with the internal standards, 2.5 µg [²H]₆-DMS were placed into a 40-mL vial, then supplemented with 3 g NaCl, a magnetic stir bar and sealed with a screw-top cap with a silicon septa. The vial was placed on a heating stir plate and the solution was equilibrated by magnetic stirring at 750 rpm for 5 min at 30°C. The SPME fiber (CAR/PDMS 75 µm) was exposed to the headspace of the sample for 30 min at 30°C and then inserted into the injector of GC for thermal desorption for 10 min.

For the quantification of pDMS, 25 mL of the wine samples or 5 g of homogenated grapes with 10 mL of H₂O were placed into a 40-mL vial at 20°C with a magnetic stir bar. fDMS that already exists in the samples was removed by bubbling nitrogen at 100 mL/min flow rate for 15 min with magnetic stirring at 750 rpm. pDMS was released by heat-alkaline treatment. After the solution got cold, 2.5 µg [²H]₆-DMS were added through the septa. Then the SPME needle was inserted manually through the vial septum and the aforementioned procedure was followed.
Analysis was performed using a Hewlett-Packard 5890 II GC equipped with a Hewlett-Packard 5972 MS detector. The column used was an HP-5 capillary column (30 m × 0.25 mm i.d., 0.25-µm film thickness) and the gas carrier was helium with a flow rate of 1 mL min⁻¹. The injector and MS-transfer line were maintained at 250°C and 260°C, respectively. Oven temperature was held at 30°C for 3 min and raised to 40°C at 1°C min⁻¹ and then to 250°C at 20°C min⁻¹. The selective ion monitoring (SIM) mode was applied and as quantifiers ions were used *m/z* 62 (999) and *m/z* 68 (999) for DMS and [²H]₆-DMS, respectively. As qualifiers ions were used *m/z* 47 (875), 61 (308) and *m/z* 50 (972), 66 (275) for DMS and [²H]₆-DMS, respectively.

### Data analysis

Data were subjected to one-way analysis of variance (ANOVA) using Statistica V.7 Software (Statsoft Inc., Tulsa, OK). Comparison of mean values was performed using Tukey's HSD test when samples were significantly different after ANOVA (p < 0.05).

## Results and discussion

DMS is an important compound for the aromatic character of the wine due to its low perception threshold, which was determined at 27 µg L⁻¹ for red wines [10]. It was also observed that in low concentrations, below the odor threshold, DMS enhanced fruity aroma [5, 8]. High levels of fDMS were already recorded previously in Xinomavro wines [10]. In this study, the levels of DMS in wines exceeded the odor threshold and the values determined ranged from 37 µg L⁻¹ in 2008 up to very high levels in 1992 (234 µg L⁻¹). Results showed that fDMS was characterized by a tendency to increase with ageing (Figure 1A), which is in accordance with previous studies [10, 11]. Fedrizzi et al. [11] compared Merlot with Cabernet Sauvignon and observed higher concentrations in Merlot after 8 years of ageing, but the levels were lower compared to Xinomavro wines and determined at maximum of 80 µg L⁻¹. In contrast, De Mora et al. [12] observed fluctuation in DMS concentrations in aged wines from the same variety and reported no correlation with the years of ageing as a consequence of different viticultural treatments or vinification techniques. It was also observed a decrease of pDMS during the years (Figure 1B), which could be correlated with the increase of fDMS.

<table><caption>**Table 1** - pDMS concentration in grapes of different varieties. Values with different letter are significantly different (Tukey's test, p < 0.05).</caption><thead><tr><td></td><td>Variety</td><td>pDMS µg g<sup>-1</sup> berry</td></tr></thead><tbody><tr><td>1.</td><td>Xinomavro</td><td>0.95 ± 0.03 a</td></tr><tr><td>2.</td><td>Syrah</td><td>0.44 ± 0.06 b</td></tr><tr><td>3.</td><td>Merlot</td><td>0.22 ± 0.01 c</td></tr><tr><td>4.</td><td>Cabernet Sauvignon</td><td>0.11 ± 0.01 d</td></tr><tr><td>5.</td><td>Mavrotragano</td><td>0.05 ± 0.002 e</td></tr><tr><td>6.</td><td>Agiorgitiko</td><td>0.02 ± 0.004 f</td></tr></tbody></table>

Concerning the pDMS concentrations in grapes, differences among the varieties were observed and a potential impact of the variety on DMS levels was observed, which is in accordance with previous studies [5, 8, 11]. As aforementioned, higher concentrations of DMS have been reported in Merlot wines compared to Cabernet Sauvignon [11], which is related to the pDMS levels found in the grape samples of this study. pDMS levels in Merlot grapes were two times higher compared to Cabernet Sauvignon pDMS levels (Table 1). Furthermore, in Syrah grapes the concentration was determined at 0.44 µg g⁻¹ berry. As illustrated in Table 1, Xinomavro had the highest concentration of pDMS, estimated at 0.95 µg g⁻¹ berry, while the lowest concentration was determined in Agiorgitiko (0.02 µg g⁻¹ berry).
A.

<figure><img src="image_352.png" /></figure>

B.

<figure><img src="image_353.png" /></figure>

**Fig. 1** - Concentrations of fDMS (A) and pDMS (B) during ageing. Bars indicate ± Std.Er. of the mean value.

Conclusion

Results have shown that fDMS and pDMS were characterized by a tendency to increase and decrease respectively along with ageing. Variety can influence the concentration of DMS in wines, since differences were observed for potential DMS levels among the studied varieties. DMS can also have a main contribution to Xinomavro wines due to high levels in grapes and wines after ageing.

References

1. Jackson RS (2002) *Wine tasting: a professional handbook*. Elsevier Academic Press, San Diego, California.

2. Breeden DC, Juvik JA (1992) An extraction method for the determination of dimethyl sulfide in cooked corn. *J Food Compos Anal* **5**: 134-138.

3. Otter G E, Marsh AS (1982) An alternative method for the determination of dimethyl sulfide in beer. *J Inst Brew* **88**: 76-79.

4. Drozd J, Havlova P, Novak V (1990) Quantitative evaluation of headspace determination of dimethyl sulfide in malt. *J Inst Brew* **96**: 69-73.

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

6. Ugliano M, Fedrizzi B, Siebert T et al (2009) Effect of nitrogen supplementation and *Saccharomyces* species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine. *J Agric Food Chem* **57**: 4948-4955.

7. Loscos N, Ségurel M, Dagan L et al (2008) Identification of S-methylmethionine in Petit Manseng grapes as dimethyl sulphide precursor in wine. *Anal Chim Acta* **621**: 24-29.

8. Escudero A, Campo E, Farina L et al (2007) Analytical characterization of the aroma of five premium red wines. Insights into the role of odor families and the concept of fruitiness of wines. *J Agric Food Chem* **55**: 4501-4510.

9. Swan HB (2000) Determination of existing and potential dimethyl sulphide in red wines by gas chromatography atomic emission spectroscopy. *J Food Compos Anal* **13**: 207-217.

10. Anocibar Beloqui A, Kotseridis Y, Bertrand A (1996) Détermination de la teneur en sulfure de diméthyle dans quelques vins rouges. *J Int Sci Vigne Vin* **30**: 167-170.

11. Fedrizzi B, Magno F, Badocco D et al (2007) Aging effects and grape variety dependence on the content of sulfur volatiles in wine. *J Agric Food Chem* **55**: 10880-10887.

12. De Mora SJ, Knowles SJ, Eschenbruch R et al (1987) Dimethyl sulphide in some Australian red wines. *Vitis* **26**: 79-84.
# Grape marcs: could their composition be affected by the grape cultivar or the enological technique?

R. APOLINAR-VALIENTE*, I. ROMERO-CASCALES, E. GÓMEZ-PLAZA, J.M. LÓPEZ-ROCA, J.M. ROS-GARCÍA

Department of Food Science & Technology and Human Nutrition, University of Murcia, 30100 Murcia, Spain

Grape marcs – cell wall – cultivar – enological techniques

## Introduction

Great efforts have recently been directed at exploiting by-products from food industries. In the case of enology, the objective is to obtain high value-added products from the press wine processing residues. The grape marc cell wall is a complex network of neutral polysaccharides, acidic pectin substances, insoluble proanthocyanidins, lignin and structural proteins and phenolic compounds, all of which could be used as functional ingredients in several processing industries. However, the differences in marc composition according to the grape variety and the influence of the enological techniques on the grape marc chemical composition have not yet been extensively studied. The present paper provides information on skin cell walls from Cabernet Sauvignon, Syrah and Monastrell grapes. The effect of enzymatic preparation and β-galactosidase separately added and the use of dry ice on the changes that these structures experience during the winemaking process was studied.

## Materials and methods

### Grape samples

Cabernet Sauvignon, Syrah and Monastrell grapes grown in Murcia (southern Spain) were carefully harvested at commercial maturity during the 2007 vintage. Three 90-kg replicate lots of grapes from the three different cultivars were destemmed, crushed, and distributed into nine tanks to yield triplicate control lots. At the same time, potassium metabisulfite was added. This was the basic procedure applicable to all the wines produced.

### Winemaking conditions and treatments

The following three treatments were applied to three wines. Commercial enzyme preparation was added into the nine tanks (3 g/100 kg), giving rise to the three replicate lots. Besides, a mixed enzyme consisting of α and β-galactosidase activity was added into the nine tanks (1 g/100 kg), giving the triplicate lots. Finally, dry ice was added into tanks (-78°C) at a rate of 100 kg per tank directly into the nine tanks and mixed with the crushed grapes. The dry ice kept the must frozen for 3 days at a temperature lower than -3°C. Commercial dry *Saccharomyces cerevisiae* yeast was hydrated and inoculated to start all fermentations. The fermentative pomace contact period lasted ten days in all vinifications. During this time the cap was punched down twice a day.

### Grape marcs samples

When alcoholic fermentation was finished, the wine was strained off and the grape marcs were pressed. Grape marc samples were frozen and peeled with the help of a scalpel, completely separating the skin from the pulp remains. The skins were stored at -80°C until the CWM was isolated.

### Marc chemical composition analysis

CWM was isolated following the procedure of de Vries et al [1]. The neutral sugar composition of the CWM was determined by GLC after pre-treatment, hydrolysis and conversion of the products into alditol acetates [2]. Uronic acid contents were determined by the colorimetric 3,5-dimethylphenol assay [3]. The protein and total phenolic compound contents were determined by colorimetry [4, 5]. The methanol and acetic acid content of the CWM was determined by HPLC [6]. All extractions were done in triplicate.

<footnote>*Corresponding author: rafael.apolinar.valiente@gmail.com</footnote>
Average values and statistical significance according to LSD and Tukey test (*p* < 0.05) were calculated using the package Statgraphics Plus 5.1. (Statpoint Technologies, Inc., Warrenton, UA, USA).

## Results and discussion

The amount of CWM isolated was significantly higher in Cabernet Sauvignon marcs (164 mg g⁻¹ of grape marc) compared with Syrah (137 mg g⁻¹ of grape marc) and Monastrell (140 mg g⁻¹ of grape marc) marcs. It could be explained by the lowest protein content in Cabernet Sauvignon grape skins, which would represent a weaker structural rigidity [7].

Cabernet Sauvignon marcs presented significantly lower protein content (113 mg of BSA g⁻¹ of cell wall). Concerning phenolic compounds, Cabernet Sauvignon and Syrah marcs showed the highest values (165 and 160 mg of gallic acid g⁻¹ of cell wall, respectively) (Figure 1).

<figure><img src="image_354.png" /><figcaption>**Fig. 1** - Two-way ANOVA data showing mean separation of isolation efficiency (| ; mg of cell wall g⁻¹ of grape fresh skin), protein (| ; mg of BSA g⁻¹ of cell wall) and phenolic compound (| ; mg of gallic acid g⁻¹ of cell wall) contents of the CWM isolated from the grape marc from different cultivars (A) and different winemaking techniques (B). Different letters indicate significant differences according to a Tukey test (p<0.05).</figcaption></figure>

The CWM from Monastrell marcs presented the highest total sugar content (325 mg of sugars g⁻¹ of skin cell wall). Cabernet Sauvignon marcs showed a higher DM (57%), in comparison with Monastrell marcs (47%) (Figure 2). On the other hand, Syrah marcs showed the highest DA (22%). Regarding the applied treatments, the lowest total sugar content was found when enzymatic preparation had been used (263 mg of sugars g⁻¹ of skin cell wall). This result was not surprising. The action of the enzymes on the pectin matrix, together with the activities of the enzymatic preparation applied could explain the decrease in total sugar content [8]. Both enzymatic treatments led to significantly higher DM (56% and 54%, respectively) compared with control samples (42%) independently of the cultivar, which is coherent with the results obtained by Romero-Cascales et al. [8].

In order to check whether the measured variables could distinguish among the pre-established groups (grape cultivar and vinification technique), a multivariate discriminant analysis was used (Figure 3). Regarding grape cultivar, two discriminant functions were established (100% correct classification).
The separation was very good, indicating that the differences among cultivars are maintained after the vinification process. Differences were also observed when considering the vinification technique. Three discriminant functions were established, which allowed 88% correct classification of the samples. Samples were misclassified mainly when trying to assign them to the enzymatic preparation treatment or the galactosidase treatment group, indicating that these two treatments affected in a similar way the composition of the marcs for the three cultivars.

<figure><img src="image_355.png" /><figcaption>**Fig. 2** - Two-way ANOVA data showing mean separation of degree of methylation (| ; %) and degree of acetylation (‖ ; %) of the skin CWM isolated from the grape marc from different cultivars (A) and different winemaking techniques (B). Different letters indicate significant differences according to a Tukey test (p<0.05).</figcaption></figure>

<figure><img src="image_356.png" /><figcaption>**Fig. 3** - Distribution of the grape marc samples in the coordinate system defined by the discriminant functions used to differentiate among cultivars (A) and vinification techniques (B). CA: (□), SY (×), MO (△), control (+), enzymatic preparation (–), galactosidase enzyme (◇) and dry ice addition (*).</figcaption></figure>
**Conclusion**

A significant cultivar effect was observed as regards the composition of the CWM isolated from the skin separated from the marc after alcoholic fermentation. The enological technique also seems to influence the composition of the marc CWM. The grape marcs could represent a good source of valuable compounds for several applications.

**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; CO, control; EP, enzymatic preparation; GE, galactosidase enzyme; DIA, dry ice addition; CWM, cell wall material; DM, degree of methylation; DA, degree of acetylation.

**References**

1. de Vries JA, Rombouts FM, Voragen AGJ et al (1981) Extraction and purification of pectins from alcohol insoluble solids from ripe and unripe apples. *Carbohydr Polym* **1**: 117-127.

2. Blakeney AB, Harris PJ, Henry RJ et al (1983) A simple and rapid preparation of alditol acetates for monosaccharide analysis. *Carbohydr Res* **113**: 291-299.

3. Scott RW (1979) Colorimetric determination of hexuronic acids in plant materials. *Anal Chem* **51**: 936-941.

4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. *Anal Biochem* **72**: 248-254.

5. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics and phosphomolybdic-phosphotungstic acid reagents. *Am J Enol Vitic* **16**: 144-158.

6. Huang XM, Huang HB, Wang HC (2005) Cell walls of loosening skin in post-veraison grape berries lose structural polysaccharides and calcium while accumulate structural proteins. *Sci Hort* **104**: 249-263.

7. Arnous A, Meyer AS (2009) Quantitative prediction of cell wall polysaccharide composition in grape (*Vitis vinifera* L.) and apple (*Malus domestica*) skins from acid hydrolysis monosaccharide profiles. *J Agric Food Chem* **57**: 3611-3619.

8. Romero-Cascales I, Ros-García JM, López-Roca JM et al (2012) The effect of a commercial pectolytic enzyme on grape skin cell wall degradation and colour evolution during the maceration process. *Food Chem* **130**: 626-631.
# Use of innovative tools to select and toast oak wood and the impact on wine quality

M.-L. BADET-MURAT<fnref n="1" />*, J.-C. VICARD<fnref n="2" />, B. LEAUTE<fnref n="3" />, A.A. WATRELOT<fnref n="4" />, J.A. KENNEDY<fnref n="4" />

Ellagitannins – NIRS – oak aromatic compounds – condensed tannins

## Introduction

The variability in chemical composition of oak wood both at an inter-individual level [1-5] and within the same tree [4-8] is well documented and accepted by the scientific community. While the selection criteria traditionally used by cooperage such as the width of the growth rings (grain) and the type of silviculture (often associated with geographical origin) allow some control on the quality, these parameters do not accurately reflect the heterogeneity of oak wood chemical composition. To achieve this, it is necessary to chemically analyze the composition of each barrel stave. Recent research based on the classification of oak cooperage according to the content of ellagitannin confirmed that a large variation exists, both inter-individual and according to the position of the piece of wood in the tree [5]. In addition, oak wood undergoes significant changes in wood chemical composition during toasting: aside from the formation of aromatic compounds, ellagitannins are partially degraded. For this reason, and because of the temperature sensitivity of these reactions, it is clear that without the precise management of the toasting process, upstream wood classification makes little sense.

The Vicard Group has developed a particularly innovative toasting tool whose originality lies in the implementation of a fully automated radiant heat process. In 2010, a research and development program was set up in order to strengthen the selection criteria for oak barrels. This research, based upon the determination of ellagitannin content of each barrel stave using near infrared spectroscopy (NIRS), culminated in 2013 with the introduction of a specific product range: *Vicard Generation 7*. To date, three classes of tannin potential (TP) corresponding to distinct ellagitannin content in untoasted wood are now available: Low TP, or LTP < 4000, Medium TP, or MTP from 4000 to 6000 and High TP, or HTP > 6000 µg of ellagic acid equivalent / g of dry wood. In this article, after detailing the methodology of sorting wood according to its TP and explaining the toasting process, we present the first results obtained using these innovative tools and illustrate their impact on wine quality.

## Materials and methods

### Wood samples

The wood used in this study came exclusively from Vicard Group's stocks (French forests and neighboring countries, 30 months of natural maturation).

### Wines

The wines and experimental set-up are presented in table 1.

### Wood classification

Wood classification according to its ellagitannin content was performed using near-infrared spectroscopy (NIRS) using an innovative detection technique based on the use of an acousto-optic tunable filter (AOTF).

### Toasting methodology

Toasting made after steam bending was controlled by computer and performed using radiant heat. That system ensures heating accuracy to +/- 3 °C and has led to the development of innovative and

<footnote n="1">¹Œnologie by MLM, 25 rue Aurel Chazeau, 33160 Saint-Médard-en-Jalles, France</footnote>

<footnote n="2">²Vicard generation 7, 287 route de Jarnouzeaux, 16100 Cognac, France</footnote>

<footnote n="3">³Laboratory LEC, 130 Rue Jules Brisson, 16100 Cognac, France</footnote>

<footnote n="4">⁴Department of Viticulture and Enology, California State University, 2360 East Barstow Avenue, MS VR 89, Fresno, CA 93740-8003, United States</footnote>

<footnote>*Corresponding author: marielaurebadet@gmail.com</footnote>