## Results and discussion

In every step of fractionation the percentage of ellagitannin purification was calculated: firstly by the quantification of ellagic acid released during acidic hydrolysis and secondly by the quantification of the eight main ellagitannins. Figure 1 shows the evolution of the crude oak wood ellagitannin extract to the purified fraction. After the first TSK HW-40 (F) column, the fractionation on C-18 column permitted to obtain a fraction containing the eight main ellagitannins with a very high purity (i.e., 90%).

<figure><img src="image_247.png" /><figcaption>Fig. 1 - UPLC-UV analysis of ellagitannin fraction.</figcaption></figure>

The purified ellagitannin fraction was dry-heated in a lab oven for 1, 2 and 3 hours at 220°C and analyzed by UPLC-MS (Q-TOF). Figure 2 depicts the UPLC-UV profile after each toasting time. It was clear that toasting creates a lot of new compounds, the intensity of which seems to be more important after 1 h of toasting. Even with a higher toasting time, no further compounds appear to be formed. For this reason the time of 1 h was chosen. Among the formed compounds upon toasting, only one was already known, the dehydrocastalagin (i.e., castalagin oxidation product) [2]. Figure 3 indicates that dehydrocastalagin was increasing during toasting and became the major compound after toasting.

<figure><img src="image_248.png" /><figcaption>Fig. 2 - UPLC-UV analysis of ellagitannin fraction before toasting, and after 1, 2 and 3 hours of toasting.</figcaption></figure>
<figure><img src="image_249.png" /><figcaption>**Fig. 3** - Comparative UPLC-UV analysis of dehydrocastagin before and after toasting.</figcaption></figure>

Other ellagitannin derivatives produced during toasting showing [M-H]⁻ ion peak with m/z 1055.0631, 971.0456, and 1011.0813 were detected for the first time (Figure 4). Another fractionation on C-18 was performed to concentrate these newly formed and unknown compounds, in order to identify them by MS/MS analysis. The further purification of the toasted ellagitannin extract permitted to identify one of the unknown detected compounds. Compounds detected with m/z 1055.0631, with the major fragment ions exhibited m/z 753.0586 (deriving from the loss of ellagic acid) and m/z 1011.0743 (resulting of the loss of CO₂), with a molecular formula C₄₇H₂₈O₂₉, a high identity score (i.e., 94.99) and low difference between the measured mass and the calculated mass (i.e., 0.02 ppm) was assigned to Castacrenin E (Figure 5). Similarly, the identification of fragments and their chemical formulas have also been confirmed by high resolution mass analysis since the mass differences between the measured and the calculated masses were around 2 ppm. Up to now Castacrenin E has been identified in Japanese chestnut tree [4] but never in oak wood. Castacrenin E is the oxidative metabolite of Castacrenin D in which a pyrogallol ring of the C-glycosidic ellagitannin unit is replaced by a cyclopentenone ring. Castacrenin D structure exhibits a vescalagin moiety with a gallic acid moiety link on the C1 carbon. This structure suggests that during toasting Castacrenin D is oxidized in Castacrenin E.

<figure><img src="image_250.png" /><figcaption>**Fig. 4** - Extract ion chromatograms of compounds formed after toasting.</figcaption></figure>
<figure><img src="image_251.png" /><figcaption>Fig. 5 - Castacrenin E fragmentation.</figcaption></figure>

Conclusion

Sequential fractionation on a TSK (HW-40F) column followed by a C-18 column permitted to obtain a highly pure ellagitannin fraction from oak wood crude extract. The concentration, the composition as well as the purity of the ellagitannins of this purified fraction was determined by UPLC-UV before being toasted at 220°C. After toasting, in addition to the dehydrocastalagin, other ellagitannins formed during toasting have been observed for the first time. The compound having given ions [M-H]¹/m/z 1055.0631 was assigned to Castacrenin E.

For the identification of the other compounds, the research is still in progress and constitutes the first comprehensive identification of ellagitannin transformation during toasting by UPLC-MS (Q-TOF), for which levels might be underestimated due to the difficulty associated with their analysis

Acknowledgements

The authors gratefully acknowledge Tonnellerie Nadalié (Ludon-Médoc, France) for providing the oak wood.

References

1. Okuda T, Yoshida T, Hatano T et al (2009) Ellagitannins renewed the concept of tannins, pp. 1-47. In S. Quideau (ed.), *Chemistry and biology of ellagitannins: an underestimated class of bioactive plant polyphenols*, World Scientific Publishing.

2. Glabasnia A, Hofmann T (2007) Identification and sensory evaluation of dehydro- and deoxy-ellagitannins formed upon toasting of oak wood (*Quercus alba* L.). *J Agric Food Chem* **55**: 4109-4118.

3. Chira K, Zeng L, Le Floch A et al (2014) Compositional and sensory characterization of grape proanthocyanidins and oak wood ellagitannin. *Tetrahedron* **71**: 2999-3006.

4. Tanaka T, Ueda N, Shinohara H et al (1997) Four new C-glycosidic ellagitannins, castacrenins D-G, from Japanese chestnut wood (Castanea crenata Sieb. et Zucc.). *Chem Pharm Bull* **45**: 1751-1755.
# Influence of closure type and post-bottling oxygen exposure on the evolution of reductive and varietal volatile sulfur compounds in Carmenere wine

M. UGLIANO<fnref n="1" />, G. CASAUBON<fnref n="2" />, M.C. ZUÑIGA<fnref n="2" />, M.I. ESPINOZA<fnref n="3" />, J.-B. DIEVAL<fnref n="1" />, S. VIDAL<fnref n="1" />*, E. AGOSIN<fnref n="2" />,<fnref n="3" />

Closure types – O₂ ingress properties – sulfur compounds – Carmenere wines

## Introduction

The importance of oxygen (O₂) exposure during wine post-bottling storage to wine chemical and sensory characteristics has been investigated in recent years. Closures can vary with respect to their O₂ ingress properties, making them a major factor influencing wine post-bottling evolution. Because of their implication in oxidation reactions, volatile sulfur compounds (VSCs) represent a major challenge for the wine industry. The varietal VSCs 3SH, 3SHA, and 4SMP are thought to contribute positively to wine aroma. Major losses of these compounds can occur upon excessive O₂ exposure in the bottle, leading to the generalized use of closures allowing low O₂ ingress. However, with too little O₂, certain reductive VSCs such as ethyl mercaptan (EtSH) and dimethyl disulfide (DMDS) can accumulate, with potentially detrimental effects to wine aroma.

## Materials and methods

### Wine and closure types

A Carmenere wine was bottled using three closure types allowing different degrees of post-bottling oxygen exposure, namely one natural cork (higher oxygen ingress) and Nomacorc synthetic Select 500 (moderate oxygen ingress) and Select 300 (lower oxygen ingress).

### Chemical analysis

Analyses of the VSCs 3SH, 3SHA, 4SMP, and benzyl mercaptan (BeSH) were carried out by SPME-GC-MS with prior derivatization using pentafluorobenzyl bromide. DMDS and EtSH were analyzed by SPME-GC-MS. Analyses were carried out in duplicate at different time points over a period of 30 months.

## Results and discussion

Initial concentrations of various VSCs in the Carmenere wine analyzed were in several cases higher than their respective odor threshold, indicating that they can contribute to Carmenere aroma. Concentrations of 3SH, 3SHA, and 4SMP generally decreased during storage. The two synthetic closures generally resulted in higher content of these compounds, consistent with their lower O₂ ingress. An example of the evolution of 3SH is given in Figure 1. A minor influence of O₂ and closures on the reductive compound EtSH was observed (data not shown). Conversely, BeSH showed complex response to varying O₂ exposure (Figure 2). Concentration of BeSH initially increased for all closures, with highest levels observed after 6 months in the lower O₂ ingress Select 300. A decline was then observed, with the higher O₂ ingress Select 500 showing higher concentrations at 12 months.

<footnote n="1">¹Nomacorc France, Domaine de Donadille, Av. Yves Cazeaux, 30230, Rodilhan, France</footnote>

<footnote n="2">²Centro de Aromas y Sabores, DICTUC, Macul, Santiago, Chile</footnote>

<footnote n="3">³School of Engineering, Pontificia Universidad Católica de Chile, Santiago, Chile</footnote>
<figure><img src="image_252.png" /><figcaption>Fig. 1 - Evolution of 3SH during bottle storage with three different closures.</figcaption></figure>

<figure><img src="image_253.png" /><figcaption>Fig. 2 - Evolution of benzyl mercaptan during bottle storage with three different closures.</figcaption></figure>

Conclusion

VSCs can contribute to the aroma characteristics of Carmenere wines. During bottle storage, VSCs generally having a favorable sensory role, such as 3SH, 3SHA, and 4SMP, were found to decrease. Closure with lower oxygen ingress allowed increased preservation of these compounds. The evolution of the powerful VSC benzyl mercaptan, potentially contributing to empyreumatic aromas, was strongly affected by closure type.
# Polysaccharide and lignin composition in oak wood used in cooperage

A. LE FLOCH<fnref n="1" />,<fnref n="2" />,<fnref n="3" />, M. JOURDES<fnref n="1" />,<fnref n="2" />, T. GIORDANENGO<fnref n="3" />, N. MOUREY<fnref n="3" />, P.-L. TEISSEDRE<fnref n="1" />,<fnref n="2" />*

Oak wood – cooperage – polysaccharides – seasoning

## Introduction

It is widely accepted that the quality of alcoholic beverages depends on their ageing in premium quality oak wood. Three polymers constitute the main part of oak wood: cellulose, hemicellulose and lignin. This study focuses on the respective parts of these compounds that may undergo hydrolysis or chemical reactions during cooperage processes, which release extractives such as monosaccharides or aromatic precursors having a genuine sensorial interest on wine aged in barrel or in contact with oak products.

Barrels as well as oak wood products (staves, chips, etc.) deriving from different types of wood are commonly used in cooperage. In this work, the influence of Index of Polyphenols (IP), age and oak wood growth ring (grain selection) were studied respectively on non-mature oak wood and wood during seasoning.

Firstly, total extractives were quantified and secondly, the parts of cellulose, hemicellulose and lignin in oak were evaluated by successive extraction methods. Statistical analysis showed that the respective proportions of these compounds in oak wood were modified during seasoning. Thus, the proportions of compounds that have the potential to be hydrolyzed during cooperage processes such as toasting are modified. Wood raw material composition and seasoning are major factors to take into account in the course of barrel fabrication as they determine the final quality. These results highlight the importance of oak wood polysaccharide composition, which can be a source of aromatic precursor compounds or compounds that have genuine sensorial interest.

## Materials and methods

### *Samples*

All the samples in this study were selected from *Quercus petraea* Liebl. [1784, Fl. Fuld., 403] species. 27 oak wood samples in the form of wooden tray have been carefully selected according to 2 factors including the Index of Polyphenols (IP) and oak wood growth ring. IP is a parameter used to classify samples according to their ellagitannin composition determined by near infrared (NIR) through OAKSCAN® [1] tool. 3 types of IP were identified (low potential ≤ 26, medium potential between 26 and 38, and high potential ≥ 38). Growth ring can be spotted with the ring width of annual increments, also called “grain”. It includes in this plan 3 grain categories: blend, less than 1.5 mm; thin, less than 2 mm; and medium, less than 3 mm. Wooden trays were cut into 3 staves and then stored for natural seasoning. This study was carried out on 4 different times of seasoning: T0: non-matured oak wood, T1: 12 month matured, T2: 18 month matured, T3: 24 month matured. For each time, 27 samples were collected in the form of transects in 3 parts. These parts represent another selection factor since oak wood age depending on the position of the transect, thus, 3 age groups of wood were selected for samples. Age group C is the area close to the bark (0-50 years old) while group A is the old one (100-150 years old), at the heart wood of the oak ridge. All the samples were crushed into a fine powder (particle size < 0.6 mm) to facilitate the extraction of compounds.

### *Preparation of parietal residue*

The totality of oak wood extractable compounds was eliminated as described in El-Malki Redouane's thesis [2]. 3 g of oak wood powder was completely encapsulated in 3 heat sealing filter bags (each bag contained 1 g) and successive hot extractions were applied, with 96% ethanol and water alternately. The mass of extractables was determined after 12 h drying in an oven at 105°C.

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

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

<footnote n="3">³R&D Tonnellerie Radoux - Pronektar, 10, avenue Faidherbe, BP 1113 - 17503 Jonzac cedex, France</footnote>

<footnote>*Corresponding author: p.teissedre@u-bordeaux2.fr</footnote>
### Lignin content

300 mg of wood powder previously extracted sustained acid attack with two successive concentrations of H2SO4 and then autoclaving for heat hydrolysis. Soluble lignin was dosed after filtration thanks to a measurement of optical density at 205 nm, while the insoluble lignin was dried in an oven at 105°C. Lignin content corresponded to the sum of insoluble and soluble lignin.

### Cellulose and hemicelluloses content

Wood powder cellulose content is represented by two sub-forms analyzed: holocellulose (mainly cellulose and hemicellulose) and α-cellulose. Hemicellulose content is the result of the difference between holocellulose and α-cellulose values. 1g of parietal residue underwent hot delignification using a solution of sodium chlorite in an acetate buffer to obtain the residue holocellulose. The α-cellulose fraction is obtained after alkaline digestion with NaOH solution on the holocellulose residue. Holocellulose and α-cellulose proportions were calculated using the mass of each residue obtained after 12 h drying in an oven at 105°C.

### Data analysis

Significant differences among the samples were subjected to an analysis of variance (ANOVA) using the STATISTICA program V. 10.0.1011.7 software (Statsoft Inc. 1984-2011). The average values were compared using the Tukey test at 5% probability (p<0.05).

## Results and discussion

### Oak wood general chemical composition

The analytical results, corresponding to the average concentrations determined for 4 components in 72 wood samples, are listed in Table 1. Polysaccharides (mainly cellulose and hemicelluloses) and lignin represent about 85% of dry wood weight. Their respective proportions naturally bring to wood a balance between strength, gas permeability, and hydrophobicity, which represent the most important required qualities in barrel manufacturing. Beyond this aspect, these biomacromolecules are involved in each stage of the manufacturing process such as seasoning, burning or toasting. As of today, the impact of each manufacturing process on these macromolecules is not well known in comparison to advanced research on phenolic (ellagitannins) or volatile compounds.

<table><caption>Table 1 - Analytical results</caption><thead><tr><th rowspan="2"></th><th colspan="2">Extractables</th><th colspan="2">Lignin</th><th colspan="2">Hemicellulose</th><th colspan="2">Cellulose</th></tr><tr><th>Mean</th><th>Sd</th><th>Mean</th><th>Sd</th><th>Mean</th><th>Sd</th><th>Mean</th><th>Sd</th></tr></thead><tbody><tr><th>Non matured</th><td>15,84</td><td>3,23</td><td>19,83</td><td>3,23</td><td>27,61</td><td>2,49</td><td>36,68</td><td>2,49</td></tr><tr><th>12 months matured</th><td>14,36</td><td>2,63</td><td>21,03</td><td>2,63</td><td>27,77</td><td>2,65</td><td>36,83</td><td>2,65</td></tr><tr><th>18 months matured</th><td>13,98</td><td>2,17</td><td>18,26</td><td>2,17</td><td>28,22</td><td>2,74</td><td>39,45</td><td>2,74</td></tr><tr><th>24 months matured</th><td>13,96</td><td>2,05</td><td>18,81</td><td>2,05</td><td>27,56</td><td>2,05</td><td>39,58</td><td>2,05</td></tr><tr><th>Total average</th><td>14,63</td><td></td><td>19,53</td><td></td><td>27,80</td><td></td><td>38,01</td><td></td></tr></tbody></table>

### Correlations analysis

The Spearman rank correlation presented in Table 2 allowed to measure the degree of association between oak wood compounds. In any seasoning step, although average proportions of each compound evolved, the relationship between the compounds was the same. A strong negative correlation was observed between cellulose and each of the other compounds, explaining that as the proportion of cellulose increases, the average proportion of the others decreases in oak wood. Lignin and hemicellulose rates were positively correlated; their respective proportions evolved in the same way, all factors considered.

### Seasoning impact on oak wood composition

Results highlighted a general evolution of oak wood composition during maturation. Statistical test established significant differences in cellulose, extractable and lignin contents between non-matured and each step of wood maturation. These evolutions are presented in Figure 2. Cellulose and extractable behaviors, as shown in correlations above, were strongly anticorrelated over the seasoning.
<table><caption>**Table 2** - Oak wood compound correlation matrix</caption><thead><tr><th></th><th>Extractables</th><th>Lignin</th><th>Cellulose</th><th>Hemicellulose</th></tr></thead><tbody><tr><th>Extractables</th><td>1,00</td><td>0,12</td><td><strong>-0,57</strong></td><td>0,04</td></tr><tr><th>Lignin</th><td>0,12</td><td>1,00</td><td>-0,55</td><td><strong>0,41</strong></td></tr><tr><th>Cellulose</th><td><strong>-0,57</strong></td><td>-0,55</td><td>1,00</td><td>-0,43</td></tr><tr><th>Hemicellulose</th><td>0,04</td><td><strong>0,41</strong></td><td>-0,43</td><td>1,00</td></tr></tbody></table>

Values in boldface represent significant correlations (Tukey test, p<0.05)

<figure><img src="image_254.png" /><figcaption>**Fig. 1** - Oak wood compound correlation.</figcaption></figure>

*Seasoning impact on oak wood composition*

Results highlighted a general evolution of oak wood composition during maturation. Statistical test established significant differences in cellulose, extractable and lignin contents between non-matured and each step of wood maturation. These evolutions are presented in Figure 2. Cellulose and extractable behaviors, as shown in correlations above, were strongly anticorrelated over the seasoning.

**Conclusion**

This study assessed in the first instance mean levels of general oak wood composition in non-matured and oak wood over seasoning. The influence of maturation on these samples will be further studied by successive sampling during several stages of oak maturation. Thus, changes in chemical composition during wood maturation will be modeled. It will be also establish the relative impact of the main selection factors used in cooperage, grain, IP, and oak wood age. Sensory analysis will also determine the influence of studied selection factors and seasoning time on wines. Thus, the role of these compounds should be taken into account and elucidate with the aim to further improve organoleptic qualities of alcoholic beverages ageing in oak wood. Results obtained showed that changes took place in chemical oak wood composition during seasoning. Statistical analysis allowed to highlight these differences and evaluated the impact of all factors taken into account during sampling.
<figure><img src="image_255.png" /><figcaption>Fig. 2 - General evolution of oak wood composition during seasoning.</figcaption></figure>

**Acknowledgements**

PhD Alexandra le Floch is supported by the Company RADOUX and a scholarship CIFRE ANRT (National Agency for Research and Technology) no. 357/2013.

**References**

1. Giordanengo T, Michel J, Gauthier P et al (2012) Application du procédé oakscan® aux bois pour l'œnologie : sélection et influence de la teneur en polyphénols du chêne sur le profil aromatique et structurant du vin. *Rev Fr Oenol 255*: 16-24.

2. Malki RE (2013) Architecture génétique des caractères cibles pour la culture du peuplier en taillis à courte rotation. *PhD thesis*, Agricultural sciences, University of Orléans (France).
# Ellagitannin content, volatile composition and sensory profile of Cabernet Sauvignon wine from three different countries matured in oak barrels subjected to different toasting methods

M.R. GONZÁLEZ-CENTENO, K. CHIRA, P.-L. TEISSEDRE*

UMR 1219 - Œnologie, Institut des Sciences de la Vigne et du Vin, Faculté d'Œnologie,
210 Chemin de Leysotte, CS 50008, 33882 Villenave d'Ornon Cedex, France

Oak wood - toasting - ellagitannins - volatile composition

## Introduction

Oak barrels have long been used in winemaking, initially for storage and transport. Nowadays, it is known and very appreciated the contribution of oak wood from barrel to the organoleptic quality and complexity of wines stored in it. The hydroalcoholic nature of wine allows the diffusion of certain compounds from wood to wine. In fact, the aroma, structure, astringency, bitterness and colour of wines will be modulated by those extracted compounds [1, 2].

The ellagitannins (hydrolysable tannins) are among these wood compounds, together with volatiles such as *cis* and *trans*-whisky lactone, vanillin, and eugenol, among others. Their extraction depends mainly on the potential extractible compounds initially present in barrel wood, which is specially affected by seasoning and toasting; on the contact time between wood and wine; as well as on wine composition [3].

Therefore, the principal aim of this research was to define the chemical (ellagitannins and woody volatile composition) and sensory characteristics of *Cabernet Sauvignon* wine from three different countries (France, Italy and USA) matured in oak barrels subjected to three different toasting methods (medium MT, medium with watering MTAA, and Noisette).

## Materials and methods

### Oak wood origin and drying conditions

All barrels used were made up of French oak from two species (*Quercus robur* and *Quercus petraea*) from the same forest located in the Center region of France. The raw staves (100 cm x 11 cm x 0.12 cm) were naturally seasoned for 24 months in the Tonnellerie Nadalié (Ludon-Médoc, France) wood yard. Once assembled, barrels (225 L) were submitted to different toasting procedures using the traditional way over an oak wood fire. Specifically, three different toasting levels were provided: Noisette, 62 min at 52±3 °C; MT (medium toast) and MTAA (medium toast with watering), 68 min at 57±3 °C. In the case of MTAA toasting, the watering process took place twice (20 L of water in both cases): first, before starting the toasting procedure, and secondly, after 53 min of toasting.

### Wine samples

*Cabernet Sauvignon* (CS) grapes (*Vitis vinifera* L.) were manually harvested at maturity from three different countries (France, Italy and USA) during 2013 vintage. Once the malolactic fermentation was concluded, wines were transferred and kept in oak barrels for ageing during 12 months at a controlled temperature of 15 - 16 °C. All three toasting methods described above were tested with CS wines from the three countries mentioned above. A wine sample was taken as control before being placed into the barrels. During the year of storage, wine was sampled from oak barrels representing different toasting method and country after being in contact for 10 days, 6 and 12 months.

<footnote>*Corresponding author: pierre-louis.teissedre@u-bordeaux.fr</footnote>
*Chemical, sensory and statistical analyses*

Volatile and ellagitannin compounds extracted from oak barrels after a 12-month maceration were studied and compared to wine control without wood contact during this period. The quantitative determination of volatile compounds proceeding from oak wood was adapted from a previously described gas-chromatography method [4]. The wood volatile compounds identified were the two isomers of β-methyl-γ-octalactone (*cis* and *trans*-whiskey lactones), the phenols guaiacol, eugenol and isoeugenol, and the phenolic aldehyde vanillin.

The ellagitannin composition of red wine was determined from ellagitannin fraction obtained after column fractionation according to Michel *et al.* [5]. Ellagitannin identification and quantification was performed on a Thermo-Finnigan Surveyor HPLC coupled to a Thermo-Finnigan LCQ Advantage spectrometer equipped with an ion trap mass analyzer. The quantification of each target compound (castalagin, vescalagin, grandinin and roburins A, B+C, D, E) was performed by using their molecular ion. Results were calculated relative to the response of the chlorogenic acid (20 mg/L) used as an internal standard.

Sensory analysis concerning vanilla, overall woody, spicy, fruity aroma, mouthfeel astringency, bitterness taste and sweetness was also performed according to Chira *et al.* [1].

Two-way ANOVA was performed by the statistical package R version 3.1.1 to evaluate the existence and degree of significant differences among wines from each country with regard to both the contact time wood - wine and the barrel toasting method.

**Results and discussion**

The evolution of the oak aroma composition (Figure 1) in terms of concentration was different depending on country, barrel toasting and/or contact time. Two-way ANOVA of the raw experimental data revealed that the ageing time and the toasting method, as well as the interaction between both factors, had a significant influence (p < 0.05) on oak wood volatile composition of CS wines from all the three countries in study.

Vanillin was generally the major oak wood volatile constituent of the 12-month aged wines, closely followed by the *trans*-whiskey lactone. The *cis*-whiskey lactone generally displayed moderate values, whereas the volatile phenols were present as minor constituents, with concentrations lower than 38 µg/L wine.

Concentration in wines of *cis* and *trans*-whiskey lactones linearly increased during the oak maturation period, except for American wines aged in MTAA and MT barrels, which showed their maximum levels at 10 days of contact. Apart from MTAA wines, which presented higher *cis* isomer content or a similar proportion of both isomers depending on the country, the *trans* isomer was the predominant one after 12-month ageing. Hence, the watering process during toasting had an important impact on the ratio *cis*/*trans* found in wines. At the end of the oak period (12 months) the highest values of the *cis* isomer were found for French and Italian wines aged in Noisette barrels, and for American wines aged in MTAA barrels (p < 0.05). In the case of the *trans* isomer, MT and Noisette wines presented the most important concentrations, respectively, for France and Italy, and USA.

The phenolic aldehyde vanillin is a direct contributor to the flavour quality of barrel-aged wines. Regardless of the barrel toasting, for French and Italian wines, it increased progressively in concentration over time, but staying below its sensory threshold throughout the ageing period. Nonetheless, for MT and Noisette wines from USA, the maximum extraction of vanillin occurred during the first 10 days of contact. Then, values maintained constant for the former, but significantly decreased for the latter (p < 0.05), probably due to vanillin reduction to the corresponding alcohol [6].

Regardless of country and toasting method, a weak ellagitannin extraction from oak barrel wood was observed during the first 6 months compared to the significantly higher ellagitannin transfer to red wines between 6 and 12-month ageing (p < 0.05) (Figure 2). Furthermore, a particular ellagitannin profile for MT, MTAA and Noisette wines from each country was noted in terms of quantification of the individual compounds. In the case of French wines, significant differences (p < 0.05) were detected among all three toasting methods for all individual ellagitannin concentrations. Specifically, Noisette wine stood out clearly
<figure><img src="image_256.png" /></figure>

<figure><img src="image_257.png" /></figure>

<figure><img src="image_258.png" /><figcaption>**Fig. 1** - Evolution of woody volatile compounds in red wines from France (A), Italy (B) and USA (C) aged in oak barrels during a 12-month period (t0, control wine without wood contact; t1, 10-day ageing; t2, 6-month ageing; t3, 12-month ageing).</figcaption></figure>
from the others for its greatest ellagitannin content, followed by MTAA and MT wines. In the case of Italian wines, only MT wine differed significantly from wines aged in MTAA and Noisette barrels (*p* < 0.05), which did not show significant differences between their ellagitannin composition (*p* > 0.05).

<figure><img src="image_259.png" /><figcaption>**Fig. 2** - Ellagitannin profile of red wines from France (A), Italy (B) and USA (C) aged in oak barrels during a 12-month period (t0, control wine without wood contact; t1, 10-day ageing; t2, 6-month ageing; t3, 12-month ageing).</figcaption></figure>
It is worth noting that this is the first study in the literature presenting the evolution of oak wood individual ellagitannins in wines aged in barrels with different toasting methods. Indeed, references about the monitoring of wine ellagitannin composition during ageing are rather scarce, apart from that of Jourdes *et al.* [7] for both oak barrels and chips, and those of Michel *et al.* [5, 8] for oak barrels and oak staves, respectively. However, there are some studies in the literature reporting kinetics of total ellagitannin extraction in wines aged in barrels or matured with wine woods of different toastings [1, 2].

<figure><img src="image_260.png" /></figure>

<figure><img src="image_261.png" /></figure>

<figure><img src="image_262.png" /></figure>

**Fig. 3** - Spider web diagrams obtained from average values of olfactory and gustative descriptors from sensory analysis of French (A), Italian (B) and American (C) wines after 12-month ageing (*, significance of the descriptor at p < 0.05).
Regarding sensory analysis, all three control wines were characterized with less vanilla flavour and perceived as less woody and spicy than oak-aged wines (Figure 3). This behaviour may be attributed to their lower woody volatile levels. Two-way ANOVA of the raw sensory data revealed that the toast level of barrels does not have a significant impact on differentiation among wines according to their fruity and sweetness attributes (*p* > 0.05). However, MT wines from France and Italy were generally perceived as the woodiest ones (Figure 3A, B), probably due to their higher content of oak wood volatiles. In the case of American wines, the toasting level did not lead to significant differences in overall woody perception (Figure 3C), maybe as a result of the similar oak wood aroma of wines matured in different toasted barrels, in terms of concentration. Significant differences were detected for 12-month aged-wines from Italy with regard to their astringency and bitterness (*p* < 0.05).

## Conclusion

According to the obtained results, the barrel toasting method has an important impact on ellagitannin and volatile composition of wines, as well as on their overall woody perception. To the best of our knowledge, this is the first study in the literature which evaluates and compares wines of the same grape variety from different countries matured in the same oak barrels (Tonnellerie Nadalié, France) during a 12-month period.

## Acknowledgements

The authors gratefully acknowledge Tonnellerie Nadalié (Ludon-Médoc, France) for providing the oak barrels and the financial support for this research. They also thank the judges who participated in the sensory analyses.

## References

1. Chira K, Teissedre P-L (2013) Extraction of oak volatiles and ellagitannins compounds and sensory profile of wine aged with French winewoods subjected to different toasting methods: behaviour during storage. *Food Chem* **140**: 168-177.

2. Chira K, Teissedre P-L (2015) Chemical and sensory evaluation of wine matured in oak barrel: effect of oak species involved and toasting process. *Eur Food Res Technol* **240**: 533-547.

3. García R, Soares B, Dias CB et al (2012) Phenolic and furanic compounds of Portuguese chestnut and French, American and Portuguese oak wood chips. *Eur Food Res Technol* **235**: 457-467.

4. Barbe JC, Bertrand A (1996) Quantitative analysis of volatile compounds stemming from oak wood. Application to the ageing of wines in barrels. *J Sci Technol Tonnellerie* **2**: 83-88.

5. Michel J, Jourdes M, Silva MA et al (2011) Impact of concentration of ellagitannins in oak wood on their levels and organoleptic influence in red wine. *J Agric Food Chem* **59**: 5677-5683.

6. Spillman PJ, Pollnitz AP, Liacopoulos D et al (1998) Formation and degradation of furfuryl alcohol, 5-methylfurfuryl alcohol, vanillyl alcohol, and their ethyl ethers in barrel-aged wines. *J Agric Food Chem* **46**: 657-663.

7. Jourdes M, Michel J, Saucier C et al (2011) Identification, amounts, and kinetics of extraction of C-glucosidic ellagitannins during wine aging in oak barrels or in stainless steel tanks with oak chips. *Anal Bioanal Chem* **401**: 1531-1539.

8. Michel J, Jourdes M, Le Floch A et al (2013) Influence of wood barrels classified by NIRS on the ellagitannin content/composition and on the organoleptic properties of wine. *J Agric Food Chem* **61**: 11109-11118.
# Identification of six new odor active alkylpyrazines in oak wood

M. FLOCH<fnref n="1" />, S. SHINKARUK<fnref n="2" />, B. BENNETEAU<fnref n="3" />, P. DARRIET<fnref n="1" />, A. PONS<fnref n="1" />,<fnref n="4" />*

Volatile compounds – rancid off-flavors – oak wood – alkylpyrazines

## Introduction

Oak (*Quercus* sp.) is currently the only wood used in making barrels for fermenting and aging quality wines. The fundamental aspect of aging wines in oak concerns the aromatic compounds extracted from the wood. When these compounds marry perfectly with a wine's intrinsic aromas, they make a significant contribution to the richness and complexity of the bouquet, as well as improving the flavor. Volatile extractive compounds from oak wood are responsible for important pleasant olfactory notes, such as coconut, wood, vanilla, caramel, and spice. Unfortunately, not all of them are pleasant: for example, sawdust or musty are well known off-flavors. Recently, a new off-flavor reminiscent of rancid was more frequently found in certain oak wood.

Therefore, the present study aims at the identification of the most odor-active compounds in oak wood marked by rancid odors and also considers the influence of some technological parameters on the presence of these molecules in oak wood.

## Materials and methods

### Material

Conventional dried and non toasted oak wood material for wine maturation and aging purposes was used for the investigations. The cardboard was stored under normal storage conditions at 20 °C in the dark until use.

### Isolation of volatile compounds

Oak wood (10 g) was ground to obtain an homogenous powder and moistened with distilled hydroalcoholic solution (100 mL, 12% EtOH) at room temperature for 24 h. After filtration the solution was extracted with dichloromethane (3x5 mL, 3x5 min, 750 rpm). The extract was dried and concentrated at ambient temperature under nitrogen to 0.5 mL.

### General features

Nuclear Magnetic Resonance Spectroscopy (NMR): ¹H and ¹³C NMR spectra were recorded on a Bruker AC-300 FT (¹H: 300 MHz, ¹³C: 75 MHz) and referenced using the lock frequency of deuterated solvent.

### High-Resolution Gas Chromatography-Olfactometry (HRGC-O)

HRGC-O analyses were performed with the GC system described above with the following modifications: the compounds eluting at the end of the capillaries were split with a Y-splitter with a ratio 1:1, v/v and transferred via two deactivated capillaries (SGE, Ringwood, Australia) to the FPD or the MS and an ODO-1 sniffing port (SGE). In this experimental context, the oak wood extract was introduced onto a polar BP20 capillary column (SGE, 50 m, 0.25 mm (i.d.), 0.25 µm film thickness) or a SPB1-type fused silica nonpolar capillary column (Supelco, 50 m, 0.25 mm i.d., 0.25 µm).

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

<footnote n="2">²Bordeaux Sciences Agro, 1 Cours du Général de Gaulle, F-33175 Gradignan, France</footnote>

<footnote n="3">³Université Bordeaux, CNRS, UMR 5255 ISM, 351 Cours de la Libération, F-33405 Talence Cedex, France</footnote>

<footnote n="4">⁴Seguin Moreau France, Z.I. Merpins, B.P. 94, F-16103 Cognac, France</footnote>
### Heart-cut Multidimensional Gas Chromatography-Olfactometry – Mass Spectrometry (MDGC-O-MS)
The MDGC separations were performed on three capillary columns with different stationary phases or film thicknesses on two GC ovens: Oven I was a Hewlett Packard 5890 series II, while Oven II was an Agilent 6890 coupled with a JEOL-Accutof JMS T100 (Jeol, France). The two chromatographs were connected with a temperature-controlled transfer line set at 230 °C. The outlet of the precolumn was connected to a sniff port (ODO I; SGE France) to determine odor retention time in this configuration and to the second column, via a Gerstel MCS 2 multicolumn switching system. The end of the second column was split (1:1) via a crosspiece (Gerstel) between MS detection (JEOL) and the sniffing port (ODP II, Gerstel, Germany). Concerning the operating conditions of the JEOL AccuTOF mass spectrometer, it operated in positive-ion mode (70 eV) or in chemical ionization mode (MeOH). The source temperatures were set at 220 °C and 160 °C for EI and CI experiments, respectively.

## Results and discussion

### Identification of volatile compounds associated with rancid flavors in oak wood

A crude concentrated extract obtained from the maceration of dried oak wood marked or not with rancid odors (10 g/L) was analyzed by gas chromatography coupled with olfactometry (GC-O). The analysis performed on many batches revealed that six odorant zones (OZ) were reminiscent of this odor and associated with the oak wood batches marked by this off-flavor (Table 1). In our particular case, six well resolved peaks were obtained during GC-MS; each of them corresponded to the retention time of the six OZ. However the mass spectra were not found in the NIST or Wiley Database. So the goal of the next part was to find the compounds associated with the mass spectra previously obtained.

Pure mass spectra of the compounds associated with the rancid odors were obtained by multidimensional GC coupled with olfactometry and mass spectrometry MDGC-O-MS (TOF). Mass spectrum of each compounds coincided with those of pyrazine derivatives; this hypothesis was also supported by the determination of exact mass of the M⁺ ion and at the end, the molecular formula. The use of GC-O-NPD confirmed this hypothesis by revealing that the compounds associated with these OZ contained nitrogen.

<table><caption>**Table 1** - Odor-active regions in the oak wood extracts marked (OW-R) or not (OW-C) with rancid odors</caption><thead><tr><th rowspan="2">Odoriferous zones</th><th colspan="2">Distribution of OZ</th><th rowspan="2">Odor descriptors<sup>a</sup></th><th rowspan="2">LRI<sup>b</sup></th><th rowspan="2">Compound identified</th></tr><tr><th>OW-C</th><th>OW-R</th></tr></thead><tbody><tr><td>OZ1</td><td>-</td><td>++</td><td>Buttery</td><td>1495</td><td>2,5-di-isopropylpyrazine</td></tr><tr><td>OZ2</td><td>-</td><td>++</td><td>Cigarette smoke, rancid</td><td>1568</td><td>2-(sec-butyl)-5-isopropylpyrazine</td></tr><tr><td>OZ3</td><td>-</td><td>+</td><td>Cigarette smoke, rancid</td><td>1581</td><td>2-isobutyl-5-isopropylpyrazine</td></tr><tr><td>OZ4</td><td>-</td><td>++</td><td>Rancid, sweat</td><td>1640</td><td>2,4-di-sec-butylpyrazine</td></tr><tr><td>OZ5</td><td>-</td><td>++</td><td>Smoke, rancid</td><td>1656</td><td>2-(sec-butyl)-5-isobutylpyrazine</td></tr><tr><td>OZ6</td><td>-</td><td>++</td><td>Buttery</td><td>1667</td><td>2,5-di-isobutylpyrazine</td></tr></tbody></table>

Pyrazine identification is difficult because the substituents can occur at different positions of the heterocycle. While naturally occurring methylpyrazines can carry up to four methyl groups on various locations, those carrying larger alkyl side chains usually favor a 2,5-dialkyl arrangement. Nevertheless, 2,6-isomers also occur, although in smaller quantities because of their biosynthetic origin. The position of the side chains cannot easily be deduced from the mass spectra, but the isomers have different values of LRI. These were synthesized earlier.

<footnote><sup>a</sup> Odor descriptors generated by the two assessors during GC-O. <sup>b</sup> Retention index (LRI) of odor peak on a BP20 (50 m x 0.25 mm, 0.25 µm) column by GC-O.</footnote>
For this purpose and based on recent work of Rojas et al [1], the six pyrazines were prepared *via* a three-step one pot reaction by reducing a mixture of activated amino acids (Val, Leu, *iso*-Leu) with DIBAL-H at low temperature (Figure 1). Pyrazine derivatives were identified by comparison of their respective mass spectra and gas chromatographic retention indices (I) with those of authentic samples. The identification was also based on previous results obtained by Dickschat et al [2] concerning the identification of novel pyrazines produced by the myxobacteria *Chondromyces crocatus*.

<figure><img src="image_263.png" /><figcaption>**Fig. 1** - Synthetic pathway using the three-step one pot method of Rojas et al [1].</figcaption></figure>

<figure><img src="image_264.png" /><figcaption>**Fig. 2** - Mass spectra and fragmentation pattern (EI) of 2,5-di-isopropylpyrazine (1), 2-(sec-butyl)-5-isopropylpyrazine (2), 2-isobutyl-5-isopropylpyrazine (3), 2,5-di-sec-butylpyrazine (4), 2-(sec-butyl)-5-isobutylpyrazine (5), and 2,5-di-isobutylpyrazine (6).</figcaption></figure>
Figure 2 reports on the mass spectra and molecule structures validated by NMR ($^{1}$H and $^{13}$C) identified in this study. Validation of the identification was also performed by co-injecting the pure compound with an oak wood extract. Validation of the retention time on polar and apolar column as well as the descriptors of the compounds permit us to validate the identification of these new pyrazines in oak wood.

**Conclusion**

This work reports the identification in oak wood of six new alkyl pyrazines. Detection of odorant zone by GC-O, obtaining well resolved chromatographic peak with a clear mass spectrum corresponding to the OZ, was achieved by heart-cut multidimensional gas chromatography-olfactometry – mass spectrometry. Interpretation of unknown mass spectrum followed by development of synthesis strategy *ad hoc* was necessary to identify irrevocably these pyrazines. Finally, we show that these odor-active compounds were associated with rancid butter flavors found in certain oak wood. The origins of this off-flavor are still unclear, and further work needs to be envisioned.

**References**

1. Rojas N, Grillasca Y, Acosta A et al (2013) A new method for the synthesis of symmetrical disubstituted pyrazines. *J Heterocycl Chem* **50**: 982-984.

2. Dickschat JS, Reichenbach H, Wagner-Döbler I et al (2005) Novel pyrazines from the myxobacterium *Chondromyces crocatus* and marine bacteria. *Eur J Org Chem* **2005**: 4141-4153.
# A novel method for evaluation of white wine aging potential

M. NIKOLANTONAKI*, C. COELHO, R.D. GOUGEON

UMR PAM Université de Bourgogne/AgroSupDijon, Institut Universitaire de la Vigne et du Vin, Jules Guyot, Rue Claude Ladrey, BP 27877, 21078 Dijon Cedex, France

Wine oxidation – Chardonnay – EPR analysis – aging potential

## Introduction

The reputation of wines with high added value relies on their aging potential. Indeed these wines have the ability to maintain their varietal aromas while developing specific aroma during aging [1, 2]. In the particular case of dry white wines, oxidation further relates to the actual worldwide problem of premature oxidation, which concerns cellar-worthy white wines within just a few years after the vintage. Today, more than a decade after the problem first emerged, its scope remains wide and the causes are poorly understood. To date, most studies dealing with premature oxidation have focused on the characterization of potent volatile oxidation markers without giving any insights in the mechanisms controlling their formation.

In order to gain control of white wine oxidation mechanisms, this study focuses on the fate of major oxidant compounds (i.e. radical species, phenolic compounds) during the whole vinification process and the reactivity of native chemical regulators of oxidation mechanisms like glutathione. In order to estimate wine's natural resistance to oxidation and target the physicochemical/enological parameters that control it, wine samples were compared according to their responses to the chemically initiated oxidation estimated by Electron Paramagnetic Resonance (EPR) spectroscopy. This alone will be very valuable in understanding aging potential and will provide an avenue to better control the process by knowing how it might be possible to change a wine's oxidation potential.

## Materials and methods

### Initiation of oxidation on Chardonnay white wines

Iron (50 µM) and H2O2 (300 µM) were used in all our samples as a continuous source of hydroxyl radicals via the Fenton reaction, as previously described [3]. Oxidation was performed at room temperature (298 K). POBN concentration was set at 30 mM for each oxidation experiment.

### Electron Paramagnetic Resonance Spectroscopy

EPR measurements were performed using a Bruker ER300 EPR spectrometer operating at X-band with a cavity. The instrument settings used in the spin-trapping experiments were as follows: modulation amplitude, 0.9 G; time constant, 0.16 s; scan time, 120 s; modulation frequency, 100 kHz; microwave power, 10 mW; receiver gain, 104; and microwave frequency, 9.48 GHz. The samples were placed in a quartz EPR tube, and spectra were recorded at room temperature (298 K) during one hour, with four scans every two minutes. The kinetics of POBN-HER spin adduct formation was followed by monitoring the intensity of the central characteristic doublet signal, IPOBN-HER, over time in Chardonnay white wines (Figure 1). Each kinetic curve was triplicated to validate the presented results. Initial constant rate K POBN-HER of spin adduct formation is expressed in M/s.

## Results and discussion

Based on the kinetic study of POBN-1-hydroxyethyl spin adduct formation in wines initiated via the Fenton reaction, a novel tool was developed to quantify the resistance of these wines against oxidation. Indeed, the scatterplot diagram (Figure 2) of the relationship between the maximum intensity and the speed rate of

<footnote>*Corresponding author: maria.nikolantonaki@u-bourgogne.fr</footnote>
POBN-1-hydroxyethyl adduct formation after chemical initiation with H₂O₂ and Iron(II) addition at ambient temperature (20 °C) provided good discrimination of the different wine matrices. Wines presenting low Iₘₐₓ POBN-1-HER and *K* values are considered to consume the produced 1-HER more rapidly and are assumed to have better resistance against oxidation.

<figure><img src="image_265.png" /></figure>

**Fig. 1** - (A) EPR spectra of the POBN-1-hydroxyethyl spin adduct in wine characterized by a triplet of doublets presenting a nitrogen hyperfine coupling constant of AN = 15.6 G and a hydrogen hyperfine coupling constant of AH = 2.6 G, in agreement with the literature [3]; (B) kinetic study of POBN-1-hydroxyethyl spin adduct formation in wine after spiking with H₂O₂ and Iron(II) at 20 °C.

<figure><img src="image_266.png" /></figure>

**Fig. 2** - Classification of wines according to the maximum intensity and the speed rate (K) of POBN-1-hydroxyethyl spin adduct (arbitrary units) formation measured by EPR after chemical oxidation initiated by the Fenton reaction. Measures were taken at the end of alcoholic (FA) and malolactic (FML) fermentation. Different colors represent the three different Chardonnay vineyards considered. Different letters correspond to juice treatments (SM: SO₂ addition (6 g/hL) during pressing; OH: SO₂ addition after settling (6 g/hL); S4: SO₂ addition (4 g/hL) before settling; and S6: SO₂ addition (6 g/hL) before settling).

Most interestingly, the mentioned model could readily discriminate wines according to vineyard origin regardless of the different juice O₂/SO₂ treatments during the very first steps of winemaking (grape pressing and juice settling) and barrel aging. Also, wines from O₂/SO₂ treated juices seem to respond against oxidation in a vineyard dependent manner. This result suggests that this novel method can
discriminate wines according to their aging potential and leave open the possibility of identifying new
natural antioxidants. This alone would also provide an avenue to better control the process by knowing how
it might be possible to change a wine's oxidation potential.

Conclusion

Wine oxidability is a complex property that involves multiple factors. A novel tool based on the initiation of chemical oxidation has been developed to discriminate matrices according to their resistance to oxidation and to predict wine aging potential. Our ultimate goal is the identification of markers characterizing wines having different resistance against oxidation.

Acknowledgements

We thank the regional council of Burgundy for their financial support.

References

1. Escudero A, Cacho J, Ferreira V (2000) Isolation and identification of odorants generated in wine during its oxidation: a gas chromatography-olfactometric study. *Eur Food Res Technol* **211**: 105-110.

2. Lavigne V, Pons A, Darriet P, Dubourdieu D (2008) Changes in the sotolon content of dry white wines during barrel and bottle aging. *J Agric Food Chem* **56**: 2688-2693.

3. Elias RJ, Andersen ML, Skibsted LH, Waterhouse AL (2009) Identification of free radical intermediates in oxidized wine using electron paramagnetic resonance spin trapping. *J Agric Food Chem* **57**: 4359-4365.
# Comparison of precipitates from Bordeaux red and white wines by solution NMR

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

Precipitate – NMR – red wine – white wine

## Introduction

Precipitation occurs in both red and white wines but there are certain differences in the precipitates from the two types of wines. In red wines, the precipitate is observed as a dark red coloring matter while in white wines it is more seen as a haze formation [1]. Unstable proteins, mainly chitinases in combination with polyphenols (condensed tannins), or pH have been suggested to cause haze formation in white wine and finally precipitation [1, 2]. In red wines, a similar hypothesis has been suggested, i.e., interaction of proteins with tannins would lead to aggregate or precipitate formation [3, 4]. However, the polyphenol composition of the red and white wines is quite different. In red wines, the major families of polyphenols include tannins and anthocyanins with tannins present up to 3-5 g/L. In white wines, tannins are present in much smaller quantities, about 50-250 mg/L, and anthocyanins are not present at all [4]. The difference in the amount of tannins between red and white wines is due to the fermentation of grape juice in case of white wines as opposed to the inclusion of grape solids in the case of red wines. The composition of the precipitates from red and white wines could therefore be very different due to the polyphenol composition. However, a comparison in precipitate formation between red and white wines has not been performed previously.

To investigate this in further detail, we have performed a study on precipitation in Bordeaux red and white wines from 2012 at the start of the barrel ageing. The aim is to identify and quantify components in precipitates from red and white wines with solution Nuclear Magnetic Resonance (NMR) spectroscopy to provide a comparison on the precipitation between the two wines. NMR spectroscopy is the method of choice for this study since it is quantitative, non-destructive and reliable and allows determination of components in mixtures, both in solution and solid-state environment.

## Materials and methods

### Precipitate preparation

Red wine samples of 1-4 L each were taken from the upper layers of wine in the oak barrels from Merlot and Sauvignon Blanc (Chateau Reynon, Bordeaux), for year 2012 at time (t) = 1 and 2 months counted from the start of barrel ageing. 1-4 L of Merlot and Sauvignon Blanc were used to obtain the Pre-freezing (Pf) precipitate by placing the wine at -4°C for 6 days. In the case of Merlot 2012, 3.2 L of wine were decanted after the cold exposure. 1 L of Sauvignon Blanc after cold exposure and 0.8 L of Merlot after decantation were centrifuged at 5000-9600 rpm in a JLA 8.100 rotor (Beckman Coulter, USA) to yield a dark colored solid pellet in case of Merlot and white colored crystalline particles in case of Sauvignon Blanc. The supernatant from both preparations was discarded. The precipitates were taken in minimum amount of distilled water and lyophilized overnight yielding a dry red and white powder.

### NMR measurements

Sample preparation: 5 mg of the lyophilized Pf precipitates from Merlot and Sauvignon Blanc 2012, respectively, were placed in 990 µL of 90% H₂O and 10% D₂O. 10 µL of 136 mM 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP) were also added as an internal standard. The solution was stirred and left standing for 30 min before centrifuging. The supernatant was taken for

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

<footnote n="2">²BioLaffort, 126 Quai De La Souys, 33100 Bordeaux, France</footnote>
measurements using a 5-mm NMR glass tube. The remaining precipitate was lyophilized and weighed. It could be estimated from the weight of insoluble fraction that 96% (weight %) of Pf precipitate from Merlot and 98% (weight %) of Pf precipitate of Sauvignon Blanc were solubilized in water.

Liquid state NMR measurements: the solution NMR measurements were performed on a 800 MHz Bruker Avance III NMR spectrometer SB (Wissembourg, France) equipped with a 5-mm TCI cryo probe (¹H-¹³C/¹⁵N/²H) and recorded with an excitation sculpting pulse sequence with gradients to suppress the residual water signal (HDO) [5]. The ¹H chemical shifts of the NMR data were calibrated to the chemical shift of the methyl groups of TMSP at 0 ppm.

## Results

Pre-freezing (Pf) precipitates were obtained as described in the Materials and Method section for Merlot and Sauvignon Blanc 2012. A similar amount of the Pf precipitates, 377 mg/L for Merlot 2012 and 365 mg/L for Sauvignon Blanc, were obtained. ¹³C Cross Polarization Magic Angle Spinning (CPMAS) solid state NMR spectra from both the Pf precipitates from Merlot and Cabernet Sauvignon Blanc 2012 were recorded. The spectra were as expected in the first month of ageing, completely dominated by potassium bitartrate, making it impossible to assign other peaks (data not shown here).

<figure><img src="image_267.png" /><figcaption>Fig. 1 - ¹H-NMR spectrum of water solutions of Pf precipitates for Merlot and Sauvignon Blanc 2012, as obtained at a magnetic field strength of 18.8 T, 298 K with an excitation sculpting pulse sequence to suppress the water signal (see text). A) Pf precipitate from Merlot 2012, t = 1 month and B) Pf precipitate from Sauvignon Blanc 2012, t = 2 months. Tentative assignment wherever possible is made in the spectra. The peaks due to the internal standard are marked with an (*). The polyphenol region (6-8 ppm) is shown with a scaling of the intensities (x8) in the insets.</figcaption></figure>

To increase the resolution and thereby assign the peaks in the precipitates, the precipitates were solubilized in water to perform solution NMR spectroscopy. It could be estimated from the weight of insoluble fraction that 96% (weight %) of Pf precipitate from Merlot and 98% (weight %) of Pf precipitate from Sauvignon Blanc were solubilized in water. ¹H NMR spectra from the Pf precipitate solution from Merlot and Sauvignon Blanc 2012 at t = 1 and 2 months in water are shown in Figure 1A and B, respectively. The intensities of both spectra are comparable and therefore the relative quantities of the components can be compared. There are distinct similarities and differences in the Pf precipitates from Merlot and Sauvignon Blanc. The major components identified and assigned in the soluble fraction of both precipitates include bitartrate, glycerol, succinic acid and lactic acid. However, malic acid is observed only in Pf
precipitate from Sauvignon Blanc. Contributions are observed from bitartrate at 4.5 ppm, succinic acid at 2.6 ppm, lactic acid at 1.37 ppm, malic acid at 2.85 and 2.7 ppm, and the three -CHOH groups of glycerol between 3.4-3.8 ppm. Amino acid contributions from proline are observed for both Merlot and Sauvignon Blanc. However, valine is observed only for Merlot. In addition, the peaks from the polyphenolic components are in the region from 6-8 ppm. The polyphenol region also looks different in the two precipitates. Comparing the area under the peaks of the spectra in Figure 1 and therefore the amounts of the components in the precipitates, the relative quantities could be determined. The bitartrate amount is 12% more for Sauvignon Blanc precipitate as compared to Merlot precipitate. Quantities of all the other components are higher in the precipitate from Merlot as compared to Sauvignon Blanc with the main differences observed for succinic acid which is 6 times higher, glycerol which is 4 times higher and lactic acid which is 3 times higher. Polyphenols are almost 2 times higher in the case of Pf precipitate from Merlot.

## Discussion

The results from the NMR study on the solution of the Pre-freezing (precipitates) from Bordeaux red (Merlot) and white (Sauvignon Blanc) wine provide information on the molecular composition of the two precipitates, making it possible to compare them.

Both Pf precipitates are almost completely (>95%) soluble in water. This observation is slightly different from our previous observation, where we observed only up to 70% of solubility for precipitates from Merlot [6]. Since potassium bitartrate is more soluble in water than in water-alcohol medium of wine, this observation points to the abundance of potassium bitartrate in both precipitates even more than the precipitates solubilized previously. This higher amount of bitartrate in these precipitates has also been confirmed by the ¹³C CPMAS solid state NMR measurements performed on precipitates (data not shown here).

Inherently in red wines, during the fermentation step the grape solids are included resulting in the extraction of a large quantity of tannins (3-5 g/L). On the other hand, the fermentation step for white wines is carried out on grape juice, resulting in 10-20 times less quantity of tannins (50-250 mg/L). It is therefore surprising to find only 2 times less quantity of polyphenols in the two precipitates. Tannins that are present in both white wines and red wines include flavan-3-ols (catechin, epicatechin and procyanidins), cinnamic and benzoic acids and flavonols [7]. Flavan-3-ols, flavonols and anthocyanins all have NMR resonances in the region from 6-8.2 ppm [8-10]. However, the red pigment anthocyanins are not present in white wines. Therefore, the difference in the peak pattern in the polyphenol region (inset in Figure 1A and B for the region 6-8 ppm) could be due to anthocyanins. One can also argue about aromatic amino acid subunits from white wine proteins having peaks in this region. The total protein concentration in white wines is in the range of 15-300 mg/L [2] and unstable proteins cause precipitation in white wines, making it a reasonable assumption. However, no resonances were observed in the amide region (8-10 ppm in proton NMR), characteristic of peptide bonds. It is thus reasonable to assume that no amino acid side chains from white wine proteins are observed. On the other hand, free amino acids (proline in case of Sauvignon Blanc precipitate; proline and valine in case of Merlot precipitate) have been identified.

Organic acids such as succinic acid, lactic acid, malic acid and diol such as glycerol are present in large quantities in the precipitates. The observation of more lactic acid in Merlot and only malic acid in Sauvignon Blanc can be explained. Merlot does not contain malic acid due to the second fermentation step which converts malic acid to lactic acid. The surprising difference is the higher amount of glycerol in the Pf precipitate from Merlot as compared to Sauvignon Blanc. Both red and white wines contain a similar amount of glycerol [7]. The reason for this difference is not apparent. It could be that the higher quantity of glycerol in Pf precipitate indicates a slightly different mode of precipitation in Merlot as compared to Sauvignon Blanc.

## Conclusion

In conclusion, it was possible to analyze and identify components in solutions of Pre-freezing (Pf) precipitates from Merlot and Sauvignon Blanc by NMR. Both precipitates are dominated by potassium bitartrate. The other components identified include organic acids (succinic acid, malic acid and lactic acid), amino acids (proline and valine), glycerol and polyphenols.
Acknowledgements

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

References

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2. Dufrechou M, Poncet-Legrand C, Sauvage FX et al (2012) Stability of white wine proteins: combined effect of pH, ionic strength, and temperature on their aggregation. *J Agric Food Chem* **60**: 1308-1319.

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

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

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6. Prakash S, Iturmendi N, Grélard A et al (2015) Characterization of precipitate formation in Bordeaux red wine with solid-state and solution NMR. *In Proceedings of the 10th International Symposium of Oenology "Oeno 2015"*. Bordeaux.

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10. Furusawa M, Tanaka T, Ito T et al (2005) Flavonol glycosides in leaves of two Diospyros species. *Chem Pharm Bull (Tokyo)* **53**: 591-593.
# A comparative study of the bioconversion kinetic profiles of both *p*-coumaric acid isomers (*trans* and *cis*) by strains of *Brettanomyces bruxellensis*

J. KHEIR<fnref n="1" />*, D. SALAMEH<fnref n="2" />, P. STREHAIANO<fnref n="3" />, C. BRANDAM<fnref n="3" />, R. LTEIF<fnref n="2" />

*Trans*-p-coumaric acid – *cis*-p-coumaric acid – *Brettanomyces* – bioconversion

## Introduction

The increase amount of volatile phenols directly affects the wine quality. The presence of vinylphenols (4-vinylphenol and 4-vinylguaiacol) and ethylphenols (4-ethylphenol and 4-ehtylguaiacol) is directly related to a sequential pathway, due to microbial activity (mainly the yeast *Brettanomyces bruxellensis*) and leading to undesirable odors and flavors in wine. The origin of volatile phenols is most likely due to the conversion of hydroxycinnamic acids or their corresponding tartaric esters.

In literature, hydroxycinnamic acids including *p*-coumaric acid are very often cited without specifying the isomer form. The identification of the *cis*-p-CA isomer has been reported in several studies [1, 2] whose objective was the characterization of polyphenolic compounds in wine. The analysis by LC-PAD-MS showed that both isomers have different maximum absorption wavelengths $\lambda_{max}$: 295.4 nm and 309.6 nm for the *cis* and *trans* forms, respectively [3]. Jaitz et al. [4] showed that the isomeric structures of the *cis*-p-CA and *cis*-resveratrol are the result of the aging forms of *trans*-p-CA and *trans*-resveratrol. Other studies showed that the presence of the *cis* isomer depends on the grape variety. Concentrations of each of the isomers are almost the same in the wines made of the same variety, with an exception for those made from Garnacha. However, Betés-Saura et al. [5] and Ibern-Gómez et al. [6] did not detect the *cis*-p-CA isomer in sparkling wines made from the following white grape varieties: Macabeo, Xarel-lo and Parellada [7].

In wine, the *cis* concentration is always less than that of the *trans* form. Fernández de Simón et al. [8] compared concentrations between wines aged in oak barrels originated from Spain, France and the US. It appeared that the *trans* form was always present in higher quantities regardless of the origin. The values were 2.44 mg.L⁻<fnref n="1" /> for the *trans* against 0.14 for the *cis* and sometimes the latter was not detectable in the majority of the wines tested. Similarly, in French barrels, values were 2.69 mg.L⁻<fnref n="1" /> for the *trans* against 0.18 for the *cis* and 2.27 against 0.47 in US barrels. Hernández et al. [3] reported that the *trans*-p-CA concentration was 0.7 mg.L⁻<fnref n="1" /> and 0.85 for the *cis* before the malolactic fermentation and values increased significantly to 21.34 and 7.19, respectively, during the storage in stainless steel tanks and 17.82 and 8.47 when kept in barrels.

The *cis*-p-CA isomer is not commercially available [4] and is quantified using the calibration curve of the *trans*-p-CA [9, 10]. Phenolic compounds that do not have standards can be confirmed using LC-PAD (Liquid Chromatography - Pulsed Amperometric Detection) and LC-MS (Liquid Chromatography - Mass Spectrometry) [3]. Pozo-Bayón et al. [7] obtained the *cis*-p-CA from the isomerization of the *trans*-p-CA after 2 h of exposure to UV light (340 nm).

The main objective of this study is to compare the amount of 4-EP produced by two strains of *B. bruxellensis* when the initial hydroxycinnamic acid is *trans*-p-CA or *cis*-p-CA.

<footnote n="1">¹Université Saint-Joseph, Ecole Supérieure d'Ingénieurs de Beyrouth (ESIB), Mar Roukoz, Mkallès,
PO. Box 11514, Beirut 1107-2050, Lebanon</footnote>

<footnote n="2">²Université Saint-Joseph, Faculté des Sciences, Mar Roukoz, Mkallès, PO. Box 11514,
Beirut 1107-2050, Lebanon</footnote>

<footnote n="3">³Université de Toulouse, Laboratoire de Génie Chimique, UMR CNRS-INPT 5503, INP-ENSIACET,
4 allée Emile Monso, 31432 cedex 4 Toulouse, France</footnote>
# Materials and methods

## *Yeast strains*

Two different strains of *Brettanomyces* originating from Lebanon (L1) and France (F1) have been selected for this study. These strains have been genetically identified as *B. bruxellensis* species [11].

## *Cis-*p-*coumaric acid and its stability*

All bioconversion studies were carried out using the *trans*-p-CA due to the absence of *cis* standard in the market as was stated in literature. We got this isomer on request from Chemos GmbH - 93128 Regenstauf, Germany. However, after a certain storage time, it appeared that this product was unstable. An isomerization "cis → trans" has probably occurred as after 2 months of storage, the same results as the tests with the *trans* were obtained. This could explain the limited number of strains tested with this reagent.

## *Pre-culture and fermentation in synthetic wine media*

Two liquid pre-cultures prior to fermentations are required to reactivate the strain and to ensure an optimal functioning of its metabolism during fermentation. All fermentations were made in synthetic wine media containing the following composition (in g.L⁻¹): 10 glucose, 10 fructose, 0.5 yeast extract, 0.5 (NH₄)₂SO₄, 0.3 citric acid, 3 malic acid, 2 tartaric acid, 5 KH₂PO₄, 0.4 MgSO₄.7H₂O, 6 glycerol and 10% (v/v) of ethanol only added after autoclaving. The main fermentation is inoculated with a concentration of 3.10⁶ cells.mL⁻¹ when the cells reached their mid-exponential growth phase in the second pre-culture medium. All liquid media were adjusted to pH 3.50 ± 0.01 and then incubated at 30°C with continuous stirring at 250 rpm.

## *Bioconversion study and analytical methods*

The evaluation of the production of 4-EP was studied in the synthetic wine media at the beginning of the stationary phase by adding an amount of *p*-CA.

- Biomass: yeast counting and biomass concentration were tested by both turbidimetry at 620 nm and counting on a Thoma hemacytometer. Population's viability was also tested.

- *p*-Coumaric acid detection: the analysis was performed based on the method published in Salameh et al. [12] using a HPLC system, equipped with a UV detector.

- Volatile phenols detection: concentrations of 4-VP and 4-EP were measured using a HPLC coupled to a DAD, which has been described by Sáez et al. [13, 14]. This method was modified in order to improve the detection of each of the volatile compound. We found optimum wavelengths for the determination of 4-VP and 4-EP. These values are 258 and 224 nm, respectively.

# Results and discussion

## *Comparative profile showing consumption of both acid isomers for each strain*

**Figure 1** shows a comparative profile of two different experiments carried out in parallel, one showing the consumption of *trans*-p-CA and the other the consumption of *cis*-p-CA for each strain.

<figure><img src="image_268.png" /></figure>

<figure><img src="image_269.png" /><figcaption>**Fig. 1** - Amount of *p*-CA consumed by F1 & L1 strains. (◊) *trans*-p-CA, (o) *cis*-p-CA.</figcaption></figure>
The amount of acid initially added (0.067 mmol.L⁻¹) to the fermentation media is never found. For the *cis* isomer, the amount is even lower. The percentage disappeared reaches 60% for F1 and 52% for L1 while the same percentage was 20 and 24% for F1 and L1, respectively, with the *trans* isomer. Then, this quantity undergoes a slight increase probably related to the rapid release of the acid to the extracellular medium or to the desorption phenomena of the quantity previously adsorbed on the yeast cell walls.

### Bioconversion kinetic profiles

The bioconversion comparison (Figure 2) shows that the reaction with the *cis* is slow since the *trans* completely disappeared after 50 h while the *cis* remained after 200 h with an amount of 0.017 mmol.L⁻¹. Similarly, the amount of 4-EP produced by F1 is 0.037 mmol.L⁻¹ after 120 h when the *trans* is the reagent and only 0.01 mmol.L⁻¹ in the case of the *cis*. The same phenomena of a slow bioconversion with the *trans* are observed with L1 strain. Regarding the mass balance (sum of the three components), it becomes constant at 0.047 mmol.L⁻¹ for F1 and 0.052 mmol.L⁻¹ for L1. Table 1 clearly resumes all kinetic parameters compared.

<figure><img src="image_270.png" /><figcaption>Fig. 2 - Bioconversion of *cis*-p-CA by F1 & L1 strains. (◇) *cis*-p-CA, (□) 4-VP, (Δ) 4-EP, (x) mass balance.</figcaption></figure>

<table><caption>Table 1 - Amounts of components and kinetic parameters for bioconversions of both isomers of *p*-CA.</caption><thead><tr><th rowspan="2"></th><th colspan="2">Cis</th><th colspan="2">Trans</th></tr><tr><th>F1</th><th>L1</th><th>F1</th><th>L1</th></tr></thead><tbody><tr><td>Amount of p-CA initially detected (mmol.L<sup>-1</sup>) (10<sup>-3</sup>)</td><td>27</td><td>32</td><td>53</td><td>51</td></tr><tr><td>Final amount of p-CA (mmol.L<sup>-1</sup>) (10<sup>-3</sup>)</td><td>16</td><td>18</td><td>0</td><td>2,5</td></tr><tr><td>Final amount of 4-VP (mmol.L<sup>-1</sup>) (10<sup>-3</sup>)</td><td>2</td><td>0.28</td><td>0.99</td><td>0.64</td></tr><tr><td>Final amount of 4-EP (mmol.L<sup>-1</sup>) (10<sup>-3</sup>)</td><td>10</td><td>32</td><td>37</td><td>49</td></tr><tr><td>Final amount of mass balance (10<sup>-3</sup>)</td><td>46</td><td>53</td><td>47</td><td>58</td></tr><tr><td>Specific consumption rate of p-CA (mol.lev<sup>-1</sup>.h<sup>-1</sup>) (10<sup>-12</sup>)</td><td>1.52</td><td>4.98</td><td>3.22</td><td>5.92</td></tr><tr><td>Specific production rate of 4-EP (mol.lev<sup>-1</sup>.h<sup>-1</sup>) (10<sup>-12</sup>)</td><td>0.29</td><td>3.26</td><td>1.81</td><td>4.51</td></tr><tr><td>Percentage of acid initially disappeared (%)</td><td>60</td><td>52</td><td>20</td><td>24</td></tr><tr><td>Conversion rate of p-CA (%)</td><td>76</td><td>73</td><td>100</td><td>96</td></tr><tr><td>Overall product yield (%)</td><td>20</td><td>65</td><td>56</td><td>76</td></tr><tr><td>Percentage of amounts disappeared (%)</td><td>30</td><td>21</td><td>29</td><td>13</td></tr><tr><td>Product yield (%)</td><td>21</td><td>61</td><td>79</td><td>85</td></tr></tbody></table>

*Cis* is less converted than *trans* for both strains. Its entry into the cell or its decarboxylation by hydroxycinnamate decarboxylase enzyme is less effective. For F1 strain, the step associated with the reductase enzyme activity is also less effective than in the case where the *cis* was introduced (yield 21% against 79%). Similarly, in L1, the product yield is lower in the case of the *cis* (yield of 61% against 85%).
**Conclusion**

As reported in literature, there are no previous bioconversion studies performed with the *cis* isomer. Our results have shown that *Brettanomyces* strains consume less rapidly the *cis*-isomer, and this latter form is less converted than the *trans* for both strains so the amount of 4-EP obtained is minor at the end of the fermentation.

It should be noted that usually only the *trans* form is measured in wine, but it must not be forgotten that the *cis* form is also present and could be converted into 4-EP even if the reaction seems slower.

**Abbreviations**

*p*-CA, *p*-coumaric acid; 4-VP, 4-vinylphenol; 4-EP, 4-ethylphenol.

**References**

1. Peña-Neira A, Hernández T, García-Vallejo C et al (2000) A survey of phenolic compounds in Spanish wines of different geographical origin. *Eur Food Res Technol* **210**: 445-448.

2. Moreno MVG, Barroso CG (2002) Comparison of the evolution of low molecular weight phenolic compounds in typical sherry wines: Fino, Amontillado, and Oloroso. *J Agric Food Chem* **50**: 7556-7563.

3. Hernández T, Estrella I, Carlavilla D et al (2006) Phenolic compounds in red wine subjected to industrial malolactic fermentation and ageing on lees. *Anal Chim Acta* **563**: 116-125.

4. Jaitz L, Siegl K, Eder R et al (2010) LC-MS/MS analysis of phenols for classification of red wine according to geographic origin, grape variety and vintage. *Food Chem* **22**: 366-372.

5. Betés-Saura C, Andres-Lacueva C, Lamuela-Raventos RM (1996) Phenolics in white free run juices and wines from Penedès by High-Performance Liquid Chromatography: changes during vinification. *J Agric Food Chem* **44**: 3040-3046.

6. Ibern-Gomez M, Andres-Lacueva C, Lamuela-Raventos RM et al (2002) Rapid HPLC analysis of phenolic compounds in red wines. *Am J Enol Vitic* **53**: 218-221.

7. Pozo-Barón M, Hernández M, Martín-Álvarez P et al (2003) Study of low molecular weight phenolic compounds during the aging of sparkling wines manufactured with red and white grape varieties. *J Agric Food Chem* **51**: 2089-2095.

8. Fernández de Simón B, Hernández T, Cadahía E et al (2002) Phenolic compounds in a Spanish red wine aged in barrels made of Spanish, French and American oak wood. *Eur Food Res Technol* **216**: 150-156.

9. Alonso AM, Guilleán DA, Barroso CG et al (2002) Determination of antioxidant activity of wine byproducts and its correlation with polyphenolic content. *J Agric Food Chem* **50**: 5832-5836.

10. Del Barrio-Galán R, Ortega-Heras M, Sánchez-Iglesias M et al (2012) Interactions of phenolic and volatile compounds with yeast lees, commercial yeast derivatives and non-toasted chips in model solutions and young red wines. *Eur Food Res Technol* **234**: 231-244.

11. Kheir J, Kallassy M, Salameh D et al (2010) *Brettanomyces* yeasts isolated from Lebanese wines showing difference in their molecular pattern. *Eur Sci J* **10**: 434-448.

12. Salameh D, Brandam C, Lteif R et al (2010) Validation of a reversed phase HPLC method for quantitative *p*-coumaric acid analysis in wine. *J Int Sci Vigne Vin* **44**: 117-225.

13. Sáez JS, Lopes CA, Kirs VC et al (2010) Enhanced volatile phenols in wine fermented with *Saccharomyces cerevisiae* and spoiled with *Pichia guilliermondii* and *Dekkera bruxellensis*. *Lett Appl Microbiol* **51**: 170-176.

14. Sáez JS, Lopes CA, Kirs VE et al (2011) Production of volatile phenols by *Pichia manshurica* and *Pichia membranifaciens* isolated from spoiled wines and cellar environment in Patagonia. *Food Microbiol* **28**: 503-509.
# Non-invasive gas monitoring in sparkling wines

D. GOFFETTE, M. VALADE*

Service Vin, Pôle Technique et Environnement, Comité interprofessionnel du vin de Champagne,
5 rue Henri-Martin, boîte postale 135, 51204 Epernay, France, www.champagne.fr

Non-invasive method – oxygen – carbon dioxide – sparkling wine – Champagne

## Introduction

Carbon dioxide and oxygen have a significant impact on the wine production process, until consumption. This is true for still wines and even more for sparkling wines.

Quantification of dissolved oxygen in wine at one precise moment is actually poorly informative. The key indication is the total amount of oxygen assimilated by wine during winemaking and ageing, which determines its evolution across time [1, 2].

For instance, during ageing on lees, in the traditional process of sparkling winemaking, the amount of oxygen received by a wine depends on the crown cap nature of the seal, and outstandingly influences wine sensory properties (figure 1), especially when ageing on lees is longer than 18-24 months [3].

<figure><img src="image_271.png" /><figcaption>Fig. 1 - Crown cap permeability impacts on final wine sensory properties</figcaption></figure>

In spite of this central role, gases have not been extensively studied in the past years because of various obstacles to the analysis process: sampling is difficult and methods of measurement were long and imprecise.

Non-invasive methods have resolved most of those constraints and are indeed efficient tools we have especially adapted for measurements in bottles in our laboratory.

## Laser measure of carbon dioxide

Measurements made with classical aphrometer cumulate many disadvantages: poorly safe, destructive, no possible long term monitoring on the same bottle.

Aphrometers based on infra-red laser technology (figure 2) have allowed crucial improvements in dioxide carbon monitoring. The system emits an infra-red beam and measures its absorption by carbon dioxide molecules. CO₂ partial pressure and total pressure in the bottle headspace can be deduced.

Before being used in routing device in our laboratory, a test of correlation with the referenced device was made. Laser technology showed a very good correlation with the referenced measurements, with a correlation coefficient of 0.986.

<figure><img src="image_272.png" /><figcaption>Fig. 2 - Laser aphrometer (L-Pro) with control screen and laser beam principle</figcaption></figure>
Very recently, the Arol Company has launched a new device, based on the same technology, allowing measurements on production lines up to 10 000 bottles per hour (figure 3). Unlike the L-Pro designed for a laboratory use, the production-line version does not give the operator a pressure, but an index (figure 4). We validated this new technology last year on various Champagne production sites, and tested 3 different positions within the line:

- At the disgorging stage, between the frozen bath and the properly disgorging act;

- After the disgorging step, providing that carbon dioxide in the liquid and gaseous phases is back to balance;

- Just before labelling, checking each individual bottle ready to be dispatched on the market.

Different uses can be considered, such as detection of any incident in the progress of the second fermentation in the bottle (example in figure 4), individual pressure control on the total production, pressure monitoring of old sparkling wines with very long ageing duration.

<figure><img src="image_273.png" /><figcaption>Fig. 3 - Laser aphrometer settled on a production line</figcaption></figure>

<figure><img src="image_274.png" /><figcaption>Fig. 4 - Champagne pressure controls (index) on a batch with problematic *prise de mousse*</figcaption></figure>

## Measure of oxygen by fluorescence

This non-invasive oxygen measurement technology is based upon the fluorescence quenching by oxygen of a metal organic fluorescent dye immobilized in a gas permeable hydrophobic polymer (dot). A dot is attached inside the bottle and then is illuminated with a pulsed blue light from a LED (figure 5).

<figure><img src="image_275.png" /><figcaption>Fig. 5 - Oxysense 210 T (A) and the blue light pulse directed on the dot for measurement (B)</figcaption></figure>

Because the quenching process is a purely collisional dynamic, the oxygen content of the enclosed space is not changed by the measurement process. Hence, long-time monitoring on the same bottle is possible.

Those non-invasive devices can measure oxygen in headspaces as well as in dissolved liquids. We use it for two main applications:
- Permeability characterization of closures, made in bottle filled with water and pressurized

- Oxygen quantification at bottling and disgorging.

In this short article we present the example of permeability characterization of closures.

Fluorescence technology has allowed more precise results regarding cap permeability [4, 5]. They are indeed directly operated from O₂ measurements and not deduced, as previously, from CO₂ flow during only 24 hours. Results are expressed in oxygen passing through the closure system per time unit. A protocol has been worked out by our R&D team, to characterize crown cap and cork closures.

### Crown cap closure

The kinetics of O₂ diffusion comprises two stages.

- In the first one, O₂ entries are important and nonlinear. This corresponds to the time oxygen needs to be balanced through the bidule and the crown cap. It lasts 30 to 40 days (figure 6).

- The second stage is linear and with lower O₂ entries. The slope is the equivalent of the oxygen transfer rate expressed in µg/L/day and then calculated in µg/L/year per each crown cap (figure 7).

<figure><img src="image_276.png" /><figcaption>**Fig. 6** - Oxygen kinetic diffusion of different crown caps</figcaption></figure>

<figure><img src="image_277.png" /><figcaption>**Fig. 7** - Permeability classification of the crown caps used in Champagne winemaking</figcaption></figure>

For longer monitoring, we can imagine that the entry would no longer be linear, as the oxygen gradient would shrink because of the accumulation in the bottle. This gradient can actually be considered as stable in the real wine condition, because oxygen is immediately consumed by wine during ageing on lees.

### Cork closure

Things are more difficult with cork permeability as evidenced in figure 8. In fact, obstacles result in corks' intrinsic nature: they are non-homogeneous and non-inert materials and interact with aromas and with oxidation reactions in wine. Kinetics of oxygen content measured in cork-closed bottles can lead to 3 comments:

- Corks hold air even if it is compressed before the closing operation, and unlike crown caps, corks have a part inside the bottle. Consequently, air is released in the bottle when the cork takes its shape back. The average amount is 3 mg of O₂ per liter [6].

- After this desorption stage, oxygen rate through the cork appears to be very low. On the contrary, a synthetic cork tested presented a rapid and linear O₂ rate (2.2 mg/L/year). This leads to think that O₂ is trapped or consumed through the cork before reaching the wine.

- After 200 days, we always observe, with all the corks that have been tested, a decrease of O₂ content. It is likely due to phenolic compounds that migrate from the cork to the water, and then are oxidized, resulting in O₂ consumption. Besides, water turns lightly brown.
<figure><img src="image_278.png" /><figcaption>Fig. 8 - Oxygen kinetic diffusion of different corks</figcaption></figure>

Conclusion

Gases' impacts in winemaking are widely recognized. Nevertheless, accurate quantifications with portable
sensors like the oximeter are quite recent. Even though this was a revolution in enology, measures were a
simple photography of gas content at a given instant, and they were destructive when run in bottles.
New non-invasive technologies have brought comfort, security, efficiency and quality in the achievement
of those measurements. The laser technology allows direct pressure measurements in different types of
bottles, whatever their color. The measure is performed by a laser beam passing through the headspace.
In the laboratory, this technique has several positive aspects: accuracy (0.1 bars), rapidity and safety for the
operator. The providing company now has an adaptable version for production lines with which it is
possible to perform measures at a speed up to 10 000 bottles per hour. On the other hand, the O₂ non-
invasive method of analysis is based on the fluorescence principle. Transparent bottles must be pre-
equipped with a sensor (dot). Our laboratory has developed a method on pressurized water, which measures
the accumulation of O₂ in water over time, which is the O₂ transfer rate through various closure systems
placed on the equipped bottle. This technique allows the characterization of the crown caps available on the
market by providing their permeability to O₂. This method is also used to measure the O₂ consumption
capacity of a wine. These non-invasive methods open up very promising perspectives in understanding the
ageing process in wine, once bottled. They also help to develop monitoring tools on bottling production
lines to remove any defective bottle, and more importantly to improve the consistency between bottles of
the same lot by controlling the levels of the gas.

References

1. Valade M, Tribaut-Sohier I, Bunner D et al (2006) Les apports d'oxygène en vinification et leurs impacts sur les vins – 1ère partie. *Le Vigneron Champenois* **8**: 48-71.

2. Valade M, Tribaut-Sohier I, Bunner D et al (2006) Les apports d'oxygène en vinification et leurs impacts sur les vins – 2ème partie. *Le Vigneron Champenois* **9**: 60-95.

3. Valade M, Tribaut-Sohier I (2001) Capsules de tirage à joint synthétique : des fournitures très importantes. *Le Vigneron Champenois* **11**: 50-77.

4. Bunner D, Valade M, Langleron E et al (2010) La mesure de l'oxygène dans les bouteilles par chimiluminescence. *Le Vigneron Champenois* **1**: 85-102.

5. Tribaut-Sohier I, Valade M, Moncomble D (2014) Les capsules de tirage, leur caractérisation par mesure des transferts d'oxygène par chimiluminescence. *Le Vigneron Champenois* **3**: 33-50.

6. Valade M, Bunner D, Tribaut-Sohier I et al (2012) Le gaz carbonique et l'oxygène lors de l'élaboration du Champagne. *Le Vigneron Champenois* **6**: 54-77.
# Role of natural cork stoppers concerning oxygen transmission range (OTR) vs cork parameters

A.C. LOPES CARDOSO¹*, A.C. PEREIRA¹,⁴, C.M. OLIVEIRA¹, A.C. SILVA FERREIRA¹,²,³

¹Cork Supply Portugal, S.A., Rua Nova do Fial, 102 - São Paio de Oleiros, 4535-549
São Paio de Oleiros, Portugal

²Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal

³Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa

⁴Chemical Process Engineering and Forest Products Research Centre, Rua Sílvio Lima, Pólo II, 3030-790 Coimbra, Portugal

Natural corks – oxygen permeability – cork parameters – PreSense

## Introduction

Among other topics, winemakers' concerns regarding cork performance have been related with cork oxygen transmission rate (OTR). Several studies have been comparing different types of closures, naming cork as the more variable. In these studies, the number of samples analyzed is usually low and the variables at stake not controlled for corks used. These factors limit the ability to identify parameters that impact on the mentioned variability.

Being aware of the importance of OTR in wine evolution, the cork industry has been working to develop tools to sort natural corks by OTR ranges, to further let the winemakers decide what is best for their wines. Several studies compared different types of closures [1]. In general, the synthetic stoppers are closures that allow oxygen to enter into the bottle at a relatively high rate, while screw caps let in relatively little oxygen. Intermediate values are obtained when natural and technical cork stoppers are used [2]. Regarding the variability, wider range of oxygen transmission rates is assign to natural corks from about 5.0 x 10⁻⁴ to 1.0 cc/day/closure [3]. This is why wine closed in bottles with natural corks from the very same lot might evolve into products with slightly different flavor nuances. Accordingly, the authors have been doing efforts looking for tools that provide the information of OTR of every single cork as published under WO 2011078714.

Several methods have been referred to measure the oxygen ingress rates through closures: coulometric detection [4-6], colorimetric measurements [7-10], titration of sulfur dioxide [11], chemiluminescence [12, 13], mass spectrometry detection [14, 15], and other techniques.

An interesting aspect of oxygen transfer through cork is its evolution over time post-bottling. Using colorimetric measurements Lopes et al. [8] showed that the rate of transfer through natural cork stoppers depends on time, with measurements varying from 0.0357 to 0.0643 mg/day (0.02685 to 0.04836 cc/day) during the first month of storage, from 0.0001 to 0.0087 mg/day (0.00008 to 0.00654 cc/day) between the 2nd and 12th month, and from 0.0001 to 0.0063 mg/day (0.00008 to 0.00474 cc/day) between the 12th and the 36th month. This suggests a decrease in the oxygen ingress through cork closure over time. It could be inferred that the high permeability observed during the initial period is due to a rapid diffusion of the oxygen initially contained in the cork closure, and not to permeation through the cork.

The aim of the current study was to conduct tests on a great number of samples to evaluate the influence of the cork properties: dimensions (38x24; 45x24; and 49x24) and density on OTR variance. For this purpose, 381 natural corks were evaluated through 750-mL glass wine bottles. Measurements were done during 3 months using chemiluminescence detection using an oxygen sensitive luminophore dot glued inside the wall of the glass bottle [12, 13]. The luminescence decay time is detected by an optical fiber used from the outside wall of the bottle. This luminescence decay time is correlated to the oxygen concentration.
# Materials and methods

## Natural corks

A total of 381 natural corks were analyzed. Table 1 reports the number of samples used for length comparison; an extra set of 256 corks, 45x24mm corks, was used for density studies. All groups of corks were submitted to the same surface treatment, following procedures for commercial purposes.

<table><caption>**Table 1** - OTR values through natural cork stoppers with different dimensions (cc/day)</caption><thead><tr><th>Cork dimensions<br>length x diameter (mm)</th><th>38x24<br>(16 data points)</th><th>45x24<br>(84 data points)</th><th>49x24<br>(25 data points)</th></tr></thead><tbody><tr><th colspan="4" style="text-align: center;">Data @ 1<sup>st</sup> week</th></tr><tr><th>Mean</th><td>0.006238</td><td>0.003004</td><td>0.001706</td></tr><tr><th>StdDev</th><td>0.003445</td><td>0.007625</td><td>0.002858</td></tr><tr><th>Median</th><td>0.006152</td><td>0.001387</td><td>0.000583</td></tr><tr><th colspan="4" style="text-align: center;">Data @ 1<sup>st</sup> month</th></tr><tr><th>Mean</th><td>0.006162</td><td>0.002522</td><td>0.000752</td></tr><tr><th>StdDev</th><td>0.003679</td><td>0.005984</td><td>0.001104</td></tr><tr><th>Median</th><td>0.006075</td><td>0.000494</td><td>0.000326</td></tr><tr><th colspan="4" style="text-align: center;">Data @ 3 months</th></tr><tr><th>Mean</th><td>0.00573</td><td>0.002146</td><td>0.000392</td></tr><tr><th>StdDev</th><td>0.00464</td><td>0.004256</td><td>0.000560</td></tr><tr><th>Median</th><td>0.00589</td><td>0.000375</td><td>0.000152</td></tr></tbody></table>

## Bottles

The transparent non-fluorescent bottles used follow CETIE (International Technical Center for Bottling and related Packaging) specifications for internal bore diameter. The glass thickness was less than 12 mm.

## Oxygen measurement

Oxygen concentrations were determined by a non-invasive and non-destructive method based on a chemiluminescence principle, using the Fibox 3LCD - trace v7 equipment (PreSense). The sensors used were PSt6 dots, which have a detection limit of 0.02 hPa and 1 ppb for measuring headspace and dissolved oxygen, respectively. The resolution of equipment at 0.2 and 2 hPa ranges is about 0.009 hPa and 0.015 hPa, respectively. Automatic temperature compensation was used. Measurements were done in empty bottles during 3 months. Carbon dioxide was used to reduce the levels of oxygen below 10 hPa (approx. 0.5ppm) before bottle corking. The increase of $O_2$ concentration inside the bottle was used to estimate the cork OTR together with cork “outgassing” contribution.

# Results and discussion

Figure 1 represents the relation between density and cork visual grade with OTR values during the first month of storage. We can see that, during this time, all tested corks let oxygen flow in similar amounts, independently of the density properties. These results were similar during the 3 months of storage. Outliers for OTR were found on the higher range of density – this observation is to be explored further on new research programs.

These results drive us to consider that density can only be kept in the equation of oxygen permeability prediction as part of the complex structure of the tested material, but not as a parameter that delivers direct cork performance information.

The oxygen transmission rate (OTR) through natural cork stoppers is presented in Table 1. We can see that OTR values increase with the decrease of the cork dimension and depends on time with average measurements varying from: 0.00624 cc/day (38x24), 0.00267 cc/day (45x24), and 0.00157 cc/day (49x24) during the first week of storage to 0.00728 cc/day (38x24), 0.00074 cc/day (45x24), and 0.00053 cc/day (49x24) during the third month of storage.
Figure 2 plots the difference of OTR measurements showing that the 38mm tends to have the double permeability of the other lengths, whereas the difference between 45mm and 49mm lengths tend to disappear over time in bottle. In other words, the differences between 45mm and 49mm are only observed during the first couple of months in bottle.

<figure><img src="image_279.png" /><figcaption>**Fig. 1** - Relation between density and OTR values (calculations with measurements in the first month of storage).</figcaption></figure>

<figure><img src="image_280.png" /><figcaption>**Fig. 2** - Impact of cork length on OTR values - 38mm, 45mm and 49mm.</figcaption></figure>

Conclusion

During the experiment time, all tested corks let oxygen flow in similar amounts, independently of density properties. Therefore, density was not found as a predictive parameter for oxygen ingress through the tested corks. Despite this, a greater variability was observed on the higher density range.
It was also observed that the rate of transfer through natural cork stoppers depends on time of storage. This observation aligns with reported findings [7, 9]. Differences over time are more relevant for the longer corks with OTR ratio (3 months / 1 month) close to 1 for the 38mm corks and 0.5 for the 49mm corks.

Comparing cork dimensions, oxygen transmission rate (OTR) increases with the decrease of the cork dimension: ratios of 2.4 (1 month) to 2.7 (3 months) were recorded between 38mm and 45mm lengths and of 8.2 (1 month) to 14.6 (3 months) when comparing 38mm and 49mm lengths.

## References

1. Karbowiak T, Gougeon RD, Alinc J-B et al (2010) Wine oxidation and the role of cork. *Crit Rev Food Sci Nutr* **50**: 20-52.

2. Lopes P, Silva MA, Pons A et al (2009) Impact of oxygen dissolved at bottling and transmitted through closures on the composition and sensory properties of a Sauvignon Blanc wine during bottle storage. *J Agric Food Chem* **57**: 10261-10270.

3. Hart A, Kleinig A (2005) *The role of oxygen in the aging of bottled wine*. http://thirtyfifty.com/ACFcombinedmediareport.pdf.

4. Silva A, Lambri M, De Faveri MD (2003) Evaluation of the performances of synthetic and cork stoppers up to 24 months post-bottling. *Eur Food Res Technol* **216**: 529-534.

5. Godden P, Lattey K, Francis L et al (2005) Towards offering wine to the consumer in optimal condition-the wine, the closures and other packaging variables: a review of AWRI research examining the changes that occur in wine after bottling. *Aust NZ Wine Ind J* **20**: 20-30.

6. Valade M, Tribaut-Sohier I, Bunner D et al (2006) Les apports d'oxygène en vinification et leurs impacts sur les vins. Le cas particulier du champagne (2ème partie). *Rev Fr Œnol* **222**: 17-28.

7. Lopes P, Saucier C, Glories Y (2005) Nondestructive colorimetric method to determine the oxygen diffusion rate through closures used in winemaking. *J Agric Food Chem* **53**: 6967-6973.

8. Lopes P, Saucier C, Teissedre P-L et al (2006) Impact of storage position on oxygen ingress through different closures into wine bottles. *J Agric Food Chem* **54**: 6741-6747.

9. Lopes P, Saucier C, Teissedre P-L et al (2007) Main routes of oxygen ingress through different closures into wine bottles. *J Agric Food Chem* **55**: 5167-5170.

10. Brotto L, Battistutta F, Tat L et al (2010) Modified nondestructive colorimetric method to evaluate the variability of oxygen diffusion rate through wine bottle closures. *J Agric Food Chem* **58**: 3567-3572.

11. Keenan CP, Gozukara MY, Christie GBY et al (1999) Oxygen permeability of macrocrystalline paraffin wax and relevance to wax coatings on natural corks used as wine bottle closures. *Aust J Grape Wine Res* **5**: 66-70.

12. Vidal J-C, Guillemat B, Chayvialle C (2010) Oxygen transmission rate of screwcaps by chemoluminescence and air/capsule/headspace/acidified water system. *In 33rd World Congress of Wine, 8th General Assembly of the OIV*, Tbilisi; OIV: Paris, France.

13. Bunner D, Landrieux A, Valade M et al (2010) La mesure de l'oxygène dans les bouteilles par chimiluminescence. *Le Vigneron Champenois* **131**: 84-101.

14. Faria DP, Fonseca AL, Pereira H et al (2011) Permeability of cork to gases. *J Agric Food Chem* **59**: 3590-3597.

15. Brazinha C, Fonseca AP, Pereira H et al (2013) Gas transport through cork: modelling gas permeation based on the morphology of a natural polymer material. *J Membr Sci* **428**: 52-62.
# Etude globale de la pénétration en oxygène durant le remplissage et la conservation des BIB® : Mesure de l'impact sur la qualité des vins

J. DUCRUET¹*, B. BACH¹, M. SARRAZIN¹, M. BLIND¹, E. MARTINE², D. LOUVIER²

¹CHANGINS, Haute école de viticulture et œnologie, Route de Duillier 50, CP 1148 – CH-1260 Nyon, Suisse

²HEIG-VD, Laboratoire Emballage & Conditionnement, Route de Cheseaux 1, CH-1400 Yverdon, Suisse

Vins – conditionnement – oxydation – Bag-in-Box® (BIB)

## Introduction

Après des débuts discrets, le Bag-in-Box® (BIB) connaît depuis plus de 10 ans une progression spectaculaire. Solide, pratique, économique et avec une faible empreinte carbone, il se pose aujourd'hui comme une solution alternative au verre pour le conditionnement du vin. Néanmoins, une des propriétés limitantes de ces emballages est la faible protection contre l'oxydation qu'ils procurent au vin. Il est ainsi communément admis que l'apport d'O₂, pendant le remplissage, puis par perméabilité à travers l'emballage, entraine une évolution défavorable des vins due à l'accélération des phénomènes d'oxydation [1, 2]. La détermination des propriétés de transfert de l'O₂ à travers les emballages de type BIB pour conditionner les vins reste un challenge dans la mesure où il n'existe pas d'instrument simple ou de méthodologies standards pouvant être appliquées à cette détermination. De la même manière il existe très peu d'étude mesurant la pénétration globale de l'oxygène pendant son remplissage et sa conservation et l'impact de cet oxygène sur la qualité du vin.

## Matériel et méthodes

Les mesures de Total Package Oxygen ont été réalisées avec un appareil Nomasens et des robinets transparents selon la méthode proposée par Vialis *et al.* en 2011 [3]. Pour mesurer l'impact de l'oxygène de la bulle, du vin a été conditionné en BIB de 3L au laboratoire (Muscat 2012 et Sauvignon Blanc 2013). L'oxygène dissout a été éliminé à l'aide d'un contacteur membranaire et la bulle chassée par l'ouverture du robinet (TPO<0.5 mg/L). De là, des volumes connus d'azote et d'air (Tableau 1) ont été injectés pour obtenir une bulle de 50 mL contenant différentes quantités d'oxygène. Un BIB sans oxygène dissout et sans bulle est réalisé comme témoin positif.

<table><caption>Tableau 1 - Quantité d'oxygène introduite dans les vins</caption><thead><tr><td>Nom variantes</td><td>Volume injecté dans la bulle air mL - N₂ mL</td><td>O₂ de la bulle (%) - mg/L</td></tr></thead><tbody><tr><td>VSB</td><td>0 – 0</td><td></td></tr><tr><td>V0</td><td>0 – 50</td><td>0 – 0</td></tr><tr><td>V25</td><td>12.5 – 37.5</td><td>5.25 – 1.25</td></tr><tr><td>V50</td><td>25 – 25</td><td>10.5 – 2.52</td></tr><tr><td>V75</td><td>37.5 – 12.5</td><td>15.75 – 3.74</td></tr><tr><td>V100</td><td>50 – 0</td><td>21 – 5.03</td></tr></tbody></table>
Résultats et discussion

La mesure de l'oxygène total emprisonné dans la poche pendant le conditionnement est désormais accessible (TPO : Total Package Oxygen). Nos travaux confirment ceux d'autres confrères (Vialis et al. [3]) à savoir, les quantités d'oxygène emprisonnées lors du conditionnement sont importantes et variables en fonction du volume du BIB et du type de machine (2 à 10 mg/L d'O₂ potentiellement emprisonné). Certains systèmes d'inertage s'avérant inefficaces (résultats non communiqués), un prototype d'inertage permettant de limiter ces entrées d'oxygène de plus de la moitié a été développé (Figure 1).

<figure><img src="image_281.png" /><figcaption>Fig. 1 - Influence du système d'inertage développé sur les TPO de BIB de 3L.</figcaption></figure>

L'impact de différentes concentrations en oxygène dans la bulle sur la qualité des vins a été mesuré. Le transfert de l'oxygène de la bulle vers le vin ne contenant initialement pas d'oxygène est très rapide. La variante V100 constituée de 50 mL d'air passe de 21% à 10% d'O₂ en quelques dizaines de minutes. Ce transfert donne une valeur d'oxygène dissout de l'ordre de 4 mg/L. D'autre part, l'ordre des différentes variantes est respecté et les concentrations en oxygène de la bulle et en oxygène dissout décroissent régulièrement avec la diminution des volumes d'injection d'air (résultats non communiqués). Logiquement, la consommation en SO₂ est plus forte sur les BIB contenant le plus d'air (Figure 2). Enfin, la dégradation plus rapide de cet antioxydant induit une oxydation prématurée du vin qui se traduit en analyse sensorielle, d'une part, par un vin dont l'intensité aromatique est plus élevée au niveau de l'oxydation en bouche comme au nez et d'autre part, par un vin plus amer et dont les notes fruité et florale sont moins intenses (Figure 3).

<figure><img src="image_282.png" /><figcaption>Fig. 2 - Evolution du SO₂ des vins (Sauvignon Blanc 2013, 50 mL, 23°C, 50% HR).</figcaption></figure>

Dans un deuxième temps, nous proposons 2 méthodes originales et standardisées (transfert : Gaz/Gaz ou Gaz/Liquide) permettant d'établir les transferts d'O₂ via une mesure directe par électroluminescence de l'O₂ gazeux ou via le suivi de la dégradation d'une molécule anti-oxydante contenue dans une solution modèle. Ces méthodes se veulent simples, faciles d'utilisation et permettent de tester les BIB dans leur ensemble. Là aussi, nous évaluons l'impact de cette perméabilité sur la qualité des vins. Ces travaux montrent que l'O₂ pénètre par perméabilité à travers le film mais aussi à travers le robinet. Etonnement, ce
dernier peut être responsable de la moitié de la pénétration de l'O₂. Ces résultats confirment les travaux de Doyon et al. en 2005 [4]. A température ambiante, cette entrée d'O₂ s'élève à quelques mg/L par mois. Les méthodes mises en place permettent de faire varier les conditions de température et d'hygrométrie, permettant d'étudier leur impact sur le comportement de l'emballage [5].

<figure><img src="image_283.png" /><figcaption>**Fig. 3** - Influence de l'oxygène de la bulle sur l'analyse sensorielle des vins après conservation en BIB (Muscat 2012, 4 mois à 23°C 50% HR).</figcaption></figure>

## Conclusion

La mesure globale de la pénétration d'oxygène lors du remplissage et de la conservation des BIB est un élément essentiel à l'utilisation de ces emballages. Ils sont un outil précieux pour les praticiens, qui peuvent adapter leurs vinifications, leurs modes d'utilisation et les conditions de stockage des vins en connaissance de cause. Ces résultats sont aussi une base indispensable pour que les industriels proposent des voies d'amélioration du conditionnement et des emballages avec des taux de pénétration d'O₂ plus adaptés aux vins.

## Remerciements

Ce travail fait partie d'un projet qui a été réalisé avec le soutien financier de la CTI (12820.1 PFLS-LS), coordonné par Changins (HES-SO) et en collaboration avec : Biolfuid Systems, Technibag, Schenk SA, Stéphane Gros, le Laboratoire Emballage et Conditionnement (HEIG-VD) et l'Institute for Applied Plastics Research (HEIA-FR).

## Références

1. Ghidossi R, Poupot C, Thibon C et al (2012) The influence of packaging on wine conservation. *Food Control* **23**: 302-311.

2. Ducruet J, Deneulin P, Riedo A (2011) Conservation du vin en Bag-In-Box®. *Rev Suisse Vitic Arboric Hortic* **43**: 290-295.

3. Vialis S, Shea P, Vidal JC et al (2011) La mesure de l'oxygène total au conditionnement en BIB, élément clé de la durée de vie du vin en BIB. *Rev Oenol* **140**: 44-46.

4. Doyon G, Clément A, Ribéreau S et al (2005) Canadian Bag-in-Box wine under distribution channel abuse: material fatigue, flexing simulation and total closure/spout leakage investigation. *Packag Technol Sci* **18**: 97-106.

5. Bach B, Blind M, Dethurens L et al (2014) Développement d'une méthode fiable et accessible de mesure de la perméabilité à l'oxygène des poches souples BIB. *Rev Oenol* **152**: 43-46.
# Effet d'un ajout de glutathion au conditionnement sur la qualité aromatique des vins blancs et rosés

F. CHARRIER<fnref n="1" />*, F. DAVAUX<fnref n="2" />, G. MASSON<fnref n="3" />, L. CAYLA<fnref n="3" />, R. SCHNEIDER<fnref n="4" />

Vin – arôme – glutathion – conditionnement

## Introduction

Le glutathion (GSH), tripeptide soufré, est un composé naturellement présent dans les végétaux, et en particulier dans les baies de raisins [1]. Son implication dans les mécanismes d'oxydation enzymatique des moûts de raisins a été clairement décrite [2-4]. Plus récemment, son rôle pour prévenir le vieillissement prématuré des vins blancs secs a été établi, et tout particulièrement pour préserver l'arôme fruité lié à la présence de constituants aromatiques appartenant à la famille des thiols variétaux [5].

Les teneurs en GSH dans les moûts (quelques à plusieurs dizaines de mg/L) varient selon les cépages et les conditions de culture de la vigne et en particulier son alimentation azotée [6]. La technologie de transformation du raisin, et plus particulièrement l'exposition du moût à l'oxygène pendant la phase critique des étapes pré-fermentaires (extraction des jus), les conditions fermentaires et modalités d'élevage (présence de lies), sont également autant de facteurs susceptibles d'impacter les teneurs résiduelles en GSH dans les vins [7, 8].

De fait, outre l'application de techniques susceptibles de préserver le GSH naturel, se pose la question de son emploi comme auxiliaire de vinification, pour doper les teneurs originelles et ainsi tenter d'amplifier son action antioxydante [9].

## Matériel et méthodes

### Cépages et modalités GSH

L'effet d'un ajout de GSH au conditionnement sur l'évolution de la couleur et des arômes de vins blancs et rosés a été testé. Les cépages blancs retenus sont le Melon B. et le Sauvignon B., le Grenache en rosé. Le dispositif expérimental, répété sur trois millésimes, est appliqué à un vin de chacun des cépages considérés, préparé pour un conditionnement en bouteilles (stabilisé et filtré). Trois niveaux d'apports de GSH sous forme réduite (Sigma Aldrich) sont testés : 0, 20 et 50 mg/L. Ils sont réalisés dans trois situations d'exposition au risque d'oxydation. Pour cela, au moment du conditionnement, les facteurs suivants ont été modulés : la teneur en sulfites du vin (20-25 ou 40-45 mg/L de sulfites libres) et la quantité d'oxygène total après conditionnement (2 ou 6 mg/bouteille). L'obturation des bouteilles est réalisée avec une capsule à vis (Stelvin®, joint Saran film étain). Les bouteilles sont stockées en position couchée, à une température de 20°C.

### Analyse des vins

Les caractéristiques analytiques des vins sont mesurées sur trois bouteilles par modalité après 12 mois de conservation : sulfites (iodométrie), couleur (spectrophotométrie ou colorimétrie), GSH (LC-MS/MS et SIDA), thiols variétaux (3-mercaptohexan-1-ol : 3MH et son acétate : A3MH, GC-MS/MS et SIDA), esters éthyliques d'acides gras (C4, C6, C8 et C10, GC-MS et SIDA) et acétates d'alcools supérieurs (acétate d'isoamyle, d'hexyle et de phényléthyle, GC-MS et SIDA).

<footnote n="1">¹Institut Français de la Vigne et du Vin, Château de la Frémoire, 44 120 Vertou, France</footnote>

<footnote n="2">²Institut Français de la Vigne et du Vin, V'Innopôle Sud-Ouest, 81 310 L'Isle sur Tarn, France</footnote>

<footnote n="3">³Centre d'Expérimentation et de Recherche sur le vin Rosé, 70 avenue Wilson, 83 550 Vidauban, France</footnote>

<footnote n="4">⁴Institut Français de la Vigne et du Vin, UMT Qualinov, Domaine de Pech Rouge, 11 430 Gruissan, France</footnote>

<footnote>*Auteur principal : frederic.charrier@vignevin.com</footnote>
## Résultats et discussion

Après un an de conservation, la majeure partie du GSH ajouté a disparu : les quantités résiduelles de glutathion contenues dans les vins n'excèdent pas 6 mg/L. Quoique modestes, elles sont malgré tout plus élevées lorsqu'un ajout de ce composé est opéré au moment du conditionnement.

Aussi bien pour les vins blancs que pour les vins rosés, il n'est pas mis en évidence d'effet significatif de cet apport de GSH sur la couleur ou les teneurs résiduelles en sulfites.

Les composés aromatiques de type fermentaire, esters éthyliques d'acides gras et acétates d'alcools supérieurs, sont en quantités équivalentes quelle que soit la modalité considérée.

Avant tout pour les vins blancs les plus exposés à l'oxydation (présence d'oxygène dissous et/ou peu de sulfites au moment du conditionnement), l'apport de 20 à 50 mg/L de glutathion se traduit après un an par une plus grande quantité de thiols variétaux dans les vins, principalement de 3MH, éventuellement de A3MH (Figure 1). Le constat est plus significatif dans le cas des vins de Sauvignon B., naturellement plus riches en ce type de composés.

<figure><img src="image_284.png" /><figcaption>**Fig. 1** - Teneurs en 3MH (ng/L) dans les vins après 12 mois de conservation (cas les plus significatifs pour chaque cépage considéré). O2 (-) : < 2 mg O₂/bouteille ; O2 (+) : 5 à 6 mg O₂/bouteille ; sulfites (-) : 20 à 25 mg SO₂ libre /L ; sulfites (+) : 40 à 45 mg SO₂ libre /L ; G0 : + 0 mg GSH/L ; G20 : + 20 mg GSH/L ; G50 : + 50 mg GSH/L.</figcaption></figure>

Par contre, dans nos conditions, aucun effet de ce type n'est observé sur les vins rosés.

Sur le plan sensoriel, sans être systématiques, des défauts olfactifs de type réduction ont été notés sur certains vins ayant subi un tel apport de glutathion.

## Conclusion

Il ressort de ces travaux qu'un apport de glutathion au moment du conditionnement (pratique à ce jour non autorisée par la réglementation vitivinicole) pourrait présenter un intérêt dans le cas des vins initialement riches en thiols variétaux. Ceci est en accord avec les connaissances acquises jusqu'alors sur le rôle de ce composé pour préserver ces arômes de l'oxydation. Pour cela, sans doute conviendrait-il de mettre en oeuvre des doses d'apports significatives, de l'ordre de plusieurs dizaines de mg/L.
Remerciements

Nous remercions France Agrimer pour son soutien financier.

Abréviations

GSH, glutathion ; 3MH, 3-mercaptohexan-1-ol ; A3MH, acétate de 3-mercaptohexyle ; C4, C6, C8 et C10, acides butanoïque, hexanoïque, octanoïque et décanoïque ; SIDA, dosage par dilution isotopique ; GC-MS/MS, chromatographie gazeuse - spectrométrie de masse tandem ; GC-MS, chromatographie gazeuse - spectrométrie de masse ; LC-MS/MS, chromatographie liquide - spectrométrie de masse tandem.

Références

1. Cheynier V, Souquet JM, Moutounet M (1989) Glutathione content and glutathione to hydroxycinnamic acid ratio in *Vitis vinifera* grapes and musts. *Am J Enol Vitic* **40**: 320-324.

2. Singleton VL (1987) Oxygen with phenols and related reactions in musts, wines and model systems: observations and practical implications. *Am J Enol Vitic* **38**: 69-77.

3. Cheynier V, Trousdale EK, Singleton VL et al (1986) Characterization of 2-S-glutathionyl caftaric acid and its hydrolysis in relation to grape wines. *J Agric Food Chem* **34**: 217-221.

4. Cheynier V, Basire M, Rigaud J (1989) Mechanism of trans-caffeoyltartaric acid and catechin oxidation in model solutions containing grape polyphenoloxidase. *J Agric Food Chem* **37**: 1069-1071.

5. Lavigne-Cruège V, Pons A, Choné X et al (2003) Rôle du glutathion sur l'évolution aromatique des vins blancs secs. *Actes du 7ème Symposium d'Oenologie de Bordeaux (Oeno 2003)*, pp. 385-388.

6. Choné X, Lavigne-Cruège V, Tominaga T et al (2006) Effect of vine nitrogen status on grape aromatic potential: flavor precursors (S-cysteine conjugates), glutathione and phenolic content in *Vitis vinifera* L. cv. Sauvignon blanc grape juice. *J Int Sci Vigne Vin* **40**: 1-6.

7. Roland A, Vialaret J, Razungles A et al (2010) Evolution of S-cysteinylated and S-glutathionylated thiol precursors during oxidation of Melon B. and Sauvignon blanc musts. *J Agric Food Chem* **58**: 4406-4413.

8. Lavigne V, Dubourdieu D (2004) Rôle du glutathion dans l'élaboration des vins blancs secs. *Actes du Colloque Mondiaviti Bordeaux 2004*, pp. 174-180.

9. Ugliano M, Kwiatkowski M, Vidal S et al (2010) The role of copper and glutathione addition and oxygen exposure in the evaluation of key aroma compounds of Sauvignon Blanc. *Poster presented at the 14th AWITC*, Adelaïde.
# Repetitive oxidation of Sauvignon blanc wine: comparison of chemical and sensory data

C. COETZEE<fnref n="1" />, A.C. SILVA FERREIRA<fnref n="1" />,<fnref n="2" />, W.J. DU TOIT<fnref n="1" />*

Sauvignon blanc – aroma – oxidation – sensory analysis

## Introduction

The shelf life of especially white wine is of great concern for the wine industry. Oxidation may play an important role in white wine's composition and its sensory characteristics. During oxidation, a decrease in certain aroma compounds occurs followed by a change in wine colour [1, 2]. The change in aroma will typically result in the wine initially losing some of the varietal character after which an increase in unwanted oxidation aroma compounds such as aldehydes will occur [3, 4].

The effects of oxygen and aging on various aspects of dry white wines have been investigated [4, 6-10] and the evolution of wine sensory quality is thought to reach a peak after a period in the bottle, however the time period necessary and the amount of oxygen required to reach this peak remain unknown. Understanding a wine's oxygen capacity enables the winemaker to control wine evolution in the bottle by choosing the optimal closure in accordance with the desired wine style and shelf life.

This study monitored Sauvignon blanc wine evolution in a controlled environment after which both chemical and sensory analysis were performed. Successive mild oxygen additions were applied and oxygen concentrations were specifically measured while the wines were kept at a mild temperature over a long period of time. Frequent sampling also allowed the investigation of the evolution of the aromatic and non-aromatic compounds during the time of the study and the inclusion of sensory data at each of these stages provided a comprehensive assessment of wine oxidative aging in terms of chemical composition and sensory effects.

## Materials and methods

### Wine samples and oxygen treatments

A Sauvignon blanc wine was oxidised at various stages over time to compare chemical content and sensory impact. Wine was collected from a commercial cellar (De Grendel Wine Estate, Tygerberg, South Africa) directly after the 2011 harvest. The wine was then divided using carbon dioxide gas (Afrox South Africa) into 33 x 5 litre glass bottles. All treatments were done in triplicate. After distribution of the wine into the bottles, the first treatment (Control 0) was sampled and frozen at -20 °C for analysis. This treatment would serve as the “beginning” of the trial (no time lapse). All of the oxidised treatments (T1-T5 Ox) then received oxygen by using a micro oxygen sparger connected to a cylinder containing high purity (99.5%) medical oxygen (Afrox South Africa). No oxygen was added to the Control samples (T1-T5 Control). During sparging, the oxygen concentration was constantly monitored by using oxygen sensor spots (Pst3; PreSens, Regensburg, Germany) fitted inside each bottle. As soon as all the oxygen in the Ox samples was consumed, the next phase (T1 Control and T1 Ox) was sampled and frozen at -20 °C. The remaining Ox treatments (T2 Ox to T5 Ox) were oxygenated again to the required level of dissolved oxygen and the process was repeated until all treatments were sampled and frozen for analysis. Overall the study proceeded for 7 months.

<footnote n="1">¹Department of Viticulture and Oenology, University of Stellenbosch, Private Bag X1, Matieland, Stellenbosch, 7602, South Africa</footnote>

<footnote n="2">²Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Dr António Bernardino de Almeida, 4200-072, Porto, Portugal</footnote>
### Sensory analyses

The wines were subjected to descriptive analysis for aroma profiling. Twelve judges participated in the evaluation. Four training sessions of two hours each were conducted to obtain consensus between the judges. The wines were then scored on a 100 mm unstructured line scale according to the attribute intensity.

After training, the wines were subjected to testing (no discussion) where the results were collected on paper ballots. Each replication was tested in duplicate. Black ISO wine tasting glasses were used to avoid any potential bias by the panel due to visual cues. Panellists evaluated the samples orthonasally only and regular breaks were taken between samples to prevent fatigue.

### Chemical analyses

Chemical analyses were performed using various analytical methods as specified by Coetzee, 2014 [11].

## Results and discussion

Figure 1 shows the mean values of various compounds during the oxidative aging period. The intensity rating (out of 100) for some of the attributes generated by the sensory panel is also included in the figure.

Total dissolved oxygen concentrations administered during this study varied from an average of 7 mg/L (T1 Ox) up to 30 mg/L (T5 Ox). Volatile thiol concentration decreased during the 7 month storage period. 3-Mercaptohexan-1-ol (3MH) concentration decreased from 648 ng/L in T0 Control to 295 ng/L in T5 Ox. 3-Mercaptohexyl acetate content decreased from 97 ng/L in T0 Control to 30 ng/L in T5 Ox. These decreases were accompanied by a decrease in the “grapefruit” attribute intensity. An increase in concentration of oxidation-related aldehydes such as acetaldehyde and methional resulted in higher sensory intensity ratings of attributes such as “sherry”. 2-Isobutyl-3-methoxypyrazine concentration remained constant throughout the trial at an average of 13 ng/L (results not shown).

<figure><img src="image_285.png" /><figcaption>**Fig. 1** - Evolution of various compounds during oxidative storage of a Sauvignon blanc wine. Sensory intensities of some attributes are also included as bars. Values are means of triplicate.</figcaption></figure>

Considering the concentration of various fruit-driven aromatic compounds (including the volatile thiols) to be higher than the perception threshold even in the later oxidative stages (T3-T5 Ox) it is evident that some aromatic compounds could have powerful odorous influences causing complete masking or suppressing effects leading to significant decrease in the perception of the fruity attributes such as “grapefruit”. So, not only is the loss in fruity characters likely to be caused by oxidative damage to flavour compounds, but also by sensory interactive effects of especially aldehydes (such as acetaldehyde and methional) being formed.
On the other hand, some of the unpleasant oxidation-related compounds that developed remained below the perception threshold of that compound (such as sotolon). Sensory interaction studies investigating these types of effects have recently been published [5].

Sensory evaluation of the Control samples (T1-T5 Control) did not deliver any drastic differences between the samples (results not shown). The only attribute showing a specific tendency is “cooked green”. “Cooked green” increased in intensity from T0 Control to T5 Control with mostly significantly lower intensities in the Ox samples. The compound responsible for the perception of this attribute is not exactly known, however the development of ‘reductive’ odours which are often described to impart “cooked vegetables”, “onions” and “cabbage” aromas are caused by sulphur-containing compounds such as H₂S, mercaptans, disulphides and dimethyl sulphides which occur due to low oxygen content in a wine [6, 12]. These compounds, however not quantified in the present study, have low perception thresholds and are known to negatively influence young white wines, imparting unpleasant odours [12, 13].

The addition of low amounts of oxygen to the wine could indeed improve the wine sensory quality. An improved expression of wine fruity attributes was also observed when Sauvignon blanc wine was exposed to low (< 3 mg/L) or moderate (about 6 mg/L) oxygen exposure in conjunction with a decrease in ‘reductive’ attributes [7]. In the current study, the occurrence of “cooked green” could be an indication of the development of ‘reductive’ odours in the Control samples.

According to the overall sensory results (not shown) T1 Ox was considered to be more fresh and fruity when compared to T1 Control. However, T1 Ox seems to correlate well with the Control samples comparing the comprehensive chemical analyses performed (results not shown). The question is thus how does this sample score fresher and fruitier than the Control samples as seen in the sensory evaluation? The similar chemical composition would suggest that the wine be sensorially more similar to the other Control samples, however the addition of moderate amounts of oxygen to this wine enhanced the sensory experience. This could be due to oxygen addition inhibiting the formation of ‘reductive’ off-odours. This also raises the question of the ability of these ‘reductive’ odours to mask and suppress fresh and fruity odours considering the similar chemical composition otherwise. Recent publications indicate strong suppressive/masking effects of certain compounds [5]. Follow-up studies should investigate the formation of the ‘reductive’ odours and what specific oxygen concentrations to administer to benefit the wine quality. Interactive effects between the ‘reductive’ sulphur-containing odours and other fresh associated odours such as the volatile thiols and the methoxypyrazines need to be investigated.

## Conclusion

The sensory data reported in the present study is supported by previous publications [7, 13], however this is the first study where a comprehensive overview of both chemical (aromatic and non-aromatic) as well as sensory data has been obtained during repetitive oxidation of the wines. This provides insight into the advantages as well as the disadvantages of various levels of oxygen exposure as well as the capacity of a wine to consume oxygen.

In the present study a significant decrease in the volatile thiol concentration was observed in the oxidised samples while the methoxypyrazines maintained a steady concentration. This happened in concurrence with an increase in chemical compounds associated with oxidation such as certain aldehydes.

Determining the complete chemical content of the wine is a daunting task and will most certainly not include all the compounds at this stage. However, studying the main groups known to significantly influence the sensory perception of a wine might give some insight to the reactions occurring during oxidation. Combining the chemical with the sensory analysis confirmed the difficulty in matching the two data sets precisely, as predictions according to chemical data alone did not always coincide with the sensory profiling of the wine. The presence of compounds not measured in the study could play an important role in this observation.

Moreover, recent publications clearly show the complexity of the interactions that occur between various chemical compounds in a wine medium. Masking, suppressing and enhancing effects complicate the interpretation of a wine’s sensory composition when observing from a chemical viewpoint alone. In a complex medium such as wine, these effects should always be considered.
The analytical measurement of aroma compounds to predict the sensory composition of a wine at this stage should thus be used as an approximate tool due to complex interactions as well as the fact that even a comprehensive survey of chemical compounds in the medium would not necessarily include all compounds present.

## Acknowledgements

The author would like to thank Ms Elizma van Wyngaard and Ms Jeanne Brand for general assistance and assistance during sensory analysis as well as Prof Martin Kidd for statistical analysis. We would also like to thank De Grendel wine estate for donation of the wine as well as Winetech, Thrip and NRF for funding. This work is based on the research supported by the National Research Foundation. Any opinion, finding and conclusion or recommendation expressed in this material is that of the author(s) and the NRF does not accept any liability in this regard.

## Abbreviations

3MH, mercaptohexan-1-ol; Ox, oxidised treatments.

## References

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2. Singleton VL, Trousdale E, Zaya J (1979) Oxidation of wines I. Young white wines periodically exposed to air. *Am J Enol Vitic* **30**: 49-54.

3. 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.

4. Silva Ferreira AC, Hogg T, Guedes de Pinho P (2003) Identification of key odorants related to the typical aroma of oxidation-spoiled white wines. *J Agric Food Chem* **51**: 1377-1381.

5. Coetzee C, Brand J, Emerton G et al (2015) Sensory interaction between 3-mercaptohexan-1-ol, 3-isobutyl-2-methoxypyrazine and oxidation-related compounds. *Aust J Grape Wine Res* **21**: 179-188.

6. Brajkovich M, Tibbits N, Peron G et al (2005) Effect of screwcap and cork closures on SO₂ levels and aromas in a Sauvignon blanc wine. *J Agric Food Chem* **53**: 10006-10011.

7. Lopes P, Silva MA, Pons A et al (2009) Impact of oxygen dissolved at bottling and transmitted through closures on the composition and sensory properties of a Sauvignon blanc wine during bottle storage. *J Agric Food Chem* **57**: 10261-10270.

8. Herbst-Johnstone M, Nicolau L, Kilmartin PA (2011) Stability of varietal thiols in commercial Sauvignon blanc wines. *Am J Enol Vitic* **62**: 495-502.

9. Ugliano M, Kwiatkowski M, Vidal S et al (2011) Evolution of 3-mercaptohexanol, hydrogen sulfide, and methyl mercaptan during bottle storage of Sauvignon blanc wines. Effect of glutathione, copper, oxygen exposure, and closure-derived oxygen. *J Agric Food Chem* **59**: 2564-2572.

10. Fracassetti D, Coetzee C, Vanzo A et al (2013) Oxygen consumption in South African Sauvignon blanc wines: role of glutathione, sulphur dioxide and certain phenolics. *S Afr J Enol Vitic* **34**: 156-169.

11. Coetzee C (2014) Oxidation treatments affecting Sauvignon blanc wine's sensory and chemical composition. *Doctor of Philosophy*, Stellenbosch University.

12. Rauhut D (1993) Yeasts-production of sulfur compounds. In *Wine microbiology and biotechnology*, Fleet GH (Ed.), Hardwood Academic.

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# Nano-oxygenation of three Southern Italy red wines: evolution of sensory active phenolics

A. GAMBUTI<fnref n="1" />*, T. SIANI<fnref n="1" />, L. PICARIELLO<fnref n="1" />, A. RINALDI<fnref n="1" />,<fnref n="2" />, M. UGLIANO<fnref n="3" />, L. MOIO<fnref n="1" />

Nano-oxygenation – pigments – proteins reactive tannins – red wine oxygen tolerance

## Introduction

High quality red wines require, in general, a period of aging in bottle before being consumed. During this time, many of the complex reactions involving polyphenols are influenced by exposure to oxygen. As a consequence, modifications of the characteristics of the wine, such as the reduction of astringency, stabilization of the color and the evolution of olfactory profile, occur [1-4]. The low flow of oxygen ingress through the cap allows to identify this form of exposure as nano-oxygenation (NOx). The oxygen transmission rate (OTR - Oxygen Transfer Rate) of the closure may vary depending on the type of closure and strongly influences the evolution of wines during bottle aging. Although it is now well established that little oxygen uptake plays an important role in wine maturation and quality improvement, one of the enologist main tasks remains the individuation of the maximum amount of oxygen that a wine can tolerate. This information could help determining proper aging practices taking into account the wine expected lifetime.

To investigate oxygen tolerance of a red wine it is necessary to consider the wine oxidation chemistry. During the oxidation process, the hydrogen peroxide is produced from oxygen reduction giving free radical intermediates that oxidize mainly ethanol generating acetaldehyde. The latter reacts with several phenolic compounds determining important changes in wine color [5], but when concentration of acetaldehyde is higher than its sensory threshold, it negatively affects wine aroma [6]. Therefore, hydrogen peroxide could be used as a tool to predict those variations related to wine oxidation [7-8].

In this study, with the aim to individuate a possible correlation between oxygen tolerance of a red wine and the effect of NOx treatment, three red wines with different initial phenolic composition and oxygen tolerance determined by their reactivity towards a strong oxidant solution (RSO) were aged for 15 months with closures at increasing OTR.

## Materials and methods

### RSO test

Six samples were prepared and chromatic changes in CIELab units were registered during 72 hours of incubation at 20°C. The control sample exposed to air consisted of a sample of wine into a 15 mL flask with 5 mL headspace corresponding to 44 mmoles of oxygen. The five following oxidative treatments were obtained by adding hydrogen peroxide (30% Fluka- Sigma Aldrich Chemie GmbH Steinheim-France): HP1 = wine + 43.5 mg/L H₂O₂ (eq. to 20 mg/L O₂); HP2 = wine + 108.5 mg/L H₂O₂ (eq. to 50 mg/L O₂); HP3 = wine + 152 mg/L H₂O₂ (eq. to 75 mg/L O₂); HP4 = wine + 217.5 mg/L H₂O₂ (eq. to 100 mg/L O₂); HP5 = wine + 435 mg/L H₂O₂ (eq. to 200 mg/L O₂). The CIELab coordinates, lightness (L*), chroma (C*), hue angle (Hue), red-greenness (a*) and yellow-blueness (b*) were determined according to CIE76 formula and the data were processed with the PANORAMA® software (LabCognition, Analytical Software GmbH & Co.KG for SHIDMAZU Deutschland GmbH, Duisburg).

<footnote n="1">¹University of Naples Federico II, Department of Agricultural Sciences, Division of Grape and Wine Sciences, Avellino, Italy</footnote>

<footnote n="2">²Laffort, BP 17, 33015 Bordeaux Cedex, France</footnote>

<footnote n="3">³Nomacorc France, 30230 Rodilhan, France</footnote>
### NOx trial

Two levels of NOx were applied to each wine: 3.2 and 4.5 mg O₂/L/year, by means of Nomacorc Select 300 and Select 700 closures, respectively. Because A wines were less tolerant to the action of strong oxidants, a closure at lower oxygen permeability (1.6 mg O₂/L/year) was also tested (Nomacorc Select 100). Wines were analyzed after 0, 7 and 15 months of aging in bottle.

### Analyses performed during NOx treatments

Chromatic characteristics, anthocyanins and low and high molecular weight phenolics were determined by spectrophotometric and HPLC [4] methods. Reactivity of phenolics towards proteins was determined by Harbertson-Adams assay (HAa) [9] and Saliva Precipitation Index (SPI) [10].

## Results and discussion

### RSO evaluation

The reactivity of wines towards a strong oxidant solution (RSO) was evaluated by determining the total chromatic changes in CIELab units after 3 and 72 hours of incubation at 20°C with hydrogen peroxide. Changes due to hydrogen peroxide addition were less prominent for CS and P wines because, regardless of the amount of H₂O₂ added (from 20 to 200 mg/L of O₂ eq.), hue values always fell in the range corresponding to a perceived net red color [11]. On the other hand, for A wines, higher was the addition of H₂O₂, higher was the deviation of hue towards yellow tint (hue values higher than 45 units). As always, hue values of A wines were higher than CS and P, we can also assert that A wine resulted in a more advanced oxidative state. To verify whether differences in chromatic characteristic of wines after addition of strong oxidant solutions were large enough to be distinguished by the human eye, the total color differences (ΔE) between each treated wine and control one were calculated. On the basis of changes detected in the total color differences (ΔE) (e.g., after 72h of incubation ΔE values for the addition of 20 mg/L of O₂ eq were: 2.9 for A, 3.1 for P and 1.6 for CS) and on the trend detected for hue, the order of oxygen tolerance for the three wines is A<< P ≤ CS.

### NOx trial

Color intensity and hue, for all wines, did not change during aging (Table 1). In contrast total and native anthocyanins (Table 2) decreased significantly.

<table><caption>**Table 1** - Chromatic characteristics and total monomeric anthocyanins of red wines closed with synthetic closures at low, medium and high oxygen transfer rate (OTR)</caption><thead><tr><th></th><th>time*</th><th>Ads 420 nm</th><th>Ads 520 nm</th><th>Ads 620 nm</th><th>Color Intensity</th><th>Hue</th><th>Totanth (mg/L)</th></tr></thead><tbody><tr><td>AGL IANICO</td><td>0</td><td>3.11 ± 0.05</td><td>3.97 ± 0.03</td><td>0.93 ± 0.02</td><td>8.01 ± 0.07</td><td>0.78 ± 0.01</td><td>198.89 ± 4.28</td></tr><tr><td>AGL100</td><td>7</td><td>3.00 ± 0.03</td><td>3.86 ± 0.03</td><td>0.92 ± 0.01</td><td>7.78 ± 0.07</td><td>0.78 ± 0.00</td><td>131.58 ± 3.93</td></tr><tr><td>AGL300</td><td>7</td><td>3.02 ± 0.06</td><td>3.91 ± 0.04</td><td>0.93 ± 0.02</td><td>7.86 ± 0.12</td><td>0.75 ± 0.01</td><td>135.83 ± 5.44</td></tr><tr><td>AGL700</td><td>7</td><td>3.06 ± 0.01</td><td>3.92 ± 0.02</td><td>0.96 ± 0.02</td><td>7.93 ± 0.03</td><td>0.78 ± 0.00</td><td>136.23 ± 5.77</td></tr><tr><td>AGL100</td><td>15</td><td>3.00 ± 0.03</td><td>3.86 ± 0.03</td><td>0.92 ± 0.01</td><td>7.78 ± 0.07</td><td>0.78 ± 0.00</td><td>131.58 ± 3.93</td></tr><tr><td>AGL300</td><td>15</td><td>3.02 ± 0.06</td><td>3.91 ± 0.04</td><td>0.93 ± 0.02</td><td>7.86 ± 0.12</td><td>0.75 ± 0.01</td><td>135.83 ± 5.44</td></tr><tr><td>AGL700</td><td>15</td><td>3.06 ± 0.01</td><td>3.92 ± 0.02</td><td>0.96 ± 0.02</td><td>7.93 ± 0.03</td><td>0.78 ± 0.00</td><td>136.23 ± 5.77</td></tr><tr><td>CASAVECCHIA</td><td>0</td><td>3.86 ± 0.05</td><td>5.55 ± 0.05</td><td>1.45 ± 0.02</td><td>10.86 ± 0.11</td><td>0.70 ± 0.00</td><td>674.83 ± 3.37</td></tr><tr><td>CAS300</td><td>7</td><td>3.77 ± 0.06</td><td>5.29 ± 0.05</td><td>1.49 ± 0.02</td><td>10.55 ± 0.07</td><td>0.71 ± 0.02</td><td>496.96 ± 2.03</td></tr><tr><td>CAS700</td><td>7</td><td>3.83 ± 0.01</td><td>5.34 ± 0.01</td><td>1.50 ± 0.01</td><td>10.68 ± 0.01</td><td>0.72 ± 0.00</td><td>506.12 ± 27.20</td></tr><tr><td>CAS300</td><td>15</td><td>3.98 ± 0.02</td><td>5.57 ± 0.02</td><td>1.46 ± 0.01</td><td>11.00 ± 0.04</td><td>0.71 ± 0.00</td><td>433.96 ± 6.74</td></tr><tr><td>CAS700</td><td>15</td><td>3.95 ± 0.02</td><td>5.61 ± 0.12</td><td>1.44 ± 0.01</td><td>11.00 ± 0.14</td><td>0.70 ± 0.01</td><td>443.66 ± 4.40</td></tr><tr><td>PALLAGRELLO</td><td>0</td><td>3.83 ± 0.03</td><td>5.57 ± 0.02</td><td>1.39 ± 0.02</td><td>10.78 ± 0.07</td><td>0.69 ± 0.00</td><td>591.82 ± 4.50</td></tr><tr><td>PALL300</td><td>7</td><td>3.95 ± 0.07</td><td>5.43 ± 0.08</td><td>1.59 ± 0.04</td><td>10.97 ± 0.19</td><td>0.73 ± 0.01</td><td>476.00 ± 7.99</td></tr><tr><td>PALL700</td><td>7</td><td>3.86 ± 0.04</td><td>5.39 ± 0.06</td><td>1.47 ± 0.03</td><td>10.72 ± 0.12</td><td>0.72 ± 0.01</td><td>470.34 ± 8.74</td></tr><tr><td>PALL300</td><td>15</td><td>3.87 ± 0.02</td><td>5.48 ± 0.03</td><td>1.35 ± 0.01</td><td>10.71 ± 0.05</td><td>0.71 ± 0.00</td><td>372.31 ± 8.04</td></tr><tr><td>PALL700</td><td>15</td><td>3.88 ± 0.03</td><td>5.54 ± 0.02</td><td>1.38 ± 0.01</td><td>10.79 ± 0.01</td><td>0.70 ± 0.01</td><td>394.55 ± 4.81</td></tr></tbody></table>

<footnote>*months after bottling</footnote>
<table><caption>**Table 2** - Monomeric anthocyanins of red wines closed with synthetic closures at low, medium and high oxygen transfer rate (OTR)</caption><thead><tr><th></th><th>time*</th><th>Dp3glc</th><th>Pt3glc</th><th>Pn3glc</th><th>Mv3glc</th><th>Mv3acglc</th><th>Mv3cmglc</th></tr></thead><tbody><tr><td>AGLIANICO</td><td>0</td><td>29.503 ± 0.4752</td><td>33.887 ± 5.5384</td><td>14.714 ± 3.2027</td><td>238.36 ± 5.0185</td><td>31.526 ± 0.6723</td><td>14.891 ± 0.8878</td></tr><tr><td>AGL100</td><td>7</td><td>13.038 ± 1.2739</td><td>14.274 ± 0.5198</td><td>0 ± 0</td><td>99.215 ± 1.2671</td><td>12.801 ± 1.2748</td><td>nd</td></tr><tr><td>AGL300</td><td>7</td><td>11.074 ± 0.223</td><td>13.045 ± 1.1953</td><td>0 ± 0</td><td>86.847 ± 1.5324</td><td>10.057 ± 0.9119</td><td>nd</td></tr><tr><td>AGL700</td><td>7</td><td>10.656 ± 0.8504</td><td>11.639 ± 0.3693</td><td>0 ± 0</td><td>81.767 ± 1.1698</td><td>9.9416 ± 0.5601</td><td>nd</td></tr><tr><td>AGL100</td><td>15</td><td>7.711 ± 0.4681</td><td>7.7546 ± 0.2897</td><td>2.5107 ± 0.1132</td><td>58.899 ± 1.6884</td><td>8.1682 ± 0.1845</td><td>4.3536 ± 0.5537</td></tr><tr><td>AGL300</td><td>15</td><td>7.0646 ± 0.848</td><td>7.6297 ± 0.3019</td><td>2.5236 ± 0.1565</td><td>60.439 ± 2.0615</td><td>8.028 ± 0.7614</td><td>5.024 ± 0.1883</td></tr><tr><td>AGL700</td><td>15</td><td>6.7232 ± 0.9942</td><td>6.3188 ± 0.3547</td><td>1.5181 ± 0.8997</td><td>53.2 ± 4.295</td><td>7.5324 ± 0.8716</td><td>2.5387 ± 0.4131</td></tr><tr><td>CASAVECCHIA</td><td>0</td><td>75.807 ± 0.504</td><td>125.08 ± 2.8112</td><td>79.549 ± 0.8572</td><td>1164.4 ± 5.7806</td><td>239.06 ± 30.115</td><td>135.69 ± 0.0327</td></tr><tr><td>CAS300</td><td>7</td><td>64.987 ± 3.5654</td><td>98.216 ± 1.1551</td><td>47.203 ± 2.0439</td><td>895.95 ± 11.929</td><td>188.86 ± 2.2694</td><td>10.865 ± 0.6898</td></tr><tr><td>CAS700</td><td>7</td><td>63.669 ± 2.6352</td><td>97.112 ± 1.6798</td><td>45.971 ± 0.6428</td><td>888.47 ± 8.7482</td><td>189.3 ± 2.1497</td><td>12.043 ± 1.3326</td></tr><tr><td>CAS300</td><td>15</td><td>42.866 ± 0.3486</td><td>74.474 ± 1.2353</td><td>32.71 ± 0.4475</td><td>588.62 ± 6.31</td><td>105.82 ± 4.7533</td><td>nd</td></tr><tr><td>CAS700</td><td>15</td><td>43.452 ± 0.646</td><td>75.852 ± 0.8346</td><td>30.626 ± 4.9985</td><td>597.8 ± 6.7964</td><td>111.47 ± 5.827</td><td>nd</td></tr><tr><td>PALLAGRELLO</td><td>0</td><td>97.26 ± 2.1071</td><td>141.52 ± 3.0877</td><td>50.199 ± 2.0611</td><td>108.25 ± 20.782</td><td>255.53 ± 6.9433</td><td>115.85 ± 3.3882</td></tr><tr><td>PALL300</td><td>7</td><td>74.812 ± 1.5315</td><td>107.44 ± 1.8966</td><td>33.242 ± 0.7059</td><td>806.3 ± 6.9367</td><td>175.19 ± 2.5734</td><td>11.487 ± 0.8366</td></tr><tr><td>PALL700</td><td>7</td><td>78.052 ± 1.3818</td><td>110.27 ± 1.9561</td><td>34.382 ± 0.9382</td><td>825.21 ± 12.265</td><td>180.4 ± 3.4062</td><td>14.284 ± 1.112</td></tr><tr><td>PALL300</td><td>15</td><td>53.533 ± 0.534</td><td>76.522 ± 7.1759</td><td>23.243 ± 0.5057</td><td>546.46 ± 9.2751</td><td>97.801 ± 0.5441</td><td>tr</td></tr><tr><td>PALL700</td><td>15</td><td>54.34 ± 3.4719</td><td>81.685 ± 6.0131</td><td>23.754 ± 2.0343</td><td>559.73 ± 43.201</td><td>95.798 ± 1.7922</td><td>tr</td></tr></tbody></table>

*months after bottling

Table 3 shows that during bottle aging an increase in Short Polymeric Pigments (SPP) for Pallagrello and Casavecchia (more for CS than for P) and in Large Polymeric Pigments (LPP) for Aglianico occurred. The formation of these compounds may balance the loss of native anthocyanins contributing to the stability of color intensity of wines in agreement with previous finding [12]. Because a greater formation of LPP compared to SPP during winemaking and aging has been previously reported [9, 13], it is likely that during aging the formation of LPP follows that of SPP. This can indicate that A wine is in a more advanced oxidative state than P and CS as predicted by the RSO test.

<table><caption>**Table 3** - Evolution of BSA reactive tannins, short polymeric pigments (SPP) and large polymeric pigments (LPP) determined by the Harbertson-Adams assay</caption><thead><tr><th></th><th>time*</th><th>BSA Reactive Tannins</th><th>SPP</th><th>LPP</th></tr></thead><tbody><tr><td>AGLIANICO</td><td></td><td></td><td></td><td></td></tr><tr><td>AGL100</td><td>7</td><td>833 ± 74</td><td>0.689 ± 0.003</td><td>0.351 ± 0.025</td></tr><tr><td>AGL300</td><td>7</td><td>854 ± 18</td><td>0.695 ± 0.009</td><td>0.359 ± 0.027</td></tr><tr><td>AGL700</td><td>7</td><td>839 ± 22</td><td>0.687 ± 0.009</td><td>0.362 ± 0.030</td></tr><tr><td>AGL100</td><td>15</td><td>786 ± 12</td><td>0.676 ± 0.008</td><td>0.446 ± 0.006</td></tr><tr><td>AGL300</td><td>15</td><td>746 ± 19</td><td>0.701 ± 0.014</td><td>0.418 ± 0.012</td></tr><tr><td>AGL700</td><td>15</td><td>773 ± 12</td><td>0.699 ± 0.019</td><td>0.463 ± 0.014</td></tr><tr><td>CASAVECCHIA</td><td></td><td></td><td></td><td></td></tr><tr><td>CAS300</td><td>7</td><td>1023 ± 80</td><td>0.682 ± 0.039</td><td>0.316 ± 0.035</td></tr><tr><td>CAS700</td><td>7</td><td>904 ± 30</td><td>0.711 ± 0.057</td><td>0.315 ± 0.055</td></tr><tr><td>CAS300</td><td>15</td><td>720 ± 5</td><td>0.835 ± 0.014</td><td>0.293 ± 0.003</td></tr><tr><td>CAS700</td><td>15</td><td>694 ± 14</td><td>0.904 ± 0.017</td><td>0.232 ± 0.016</td></tr><tr><td>PALLAGRELLO</td><td></td><td></td><td></td><td></td></tr><tr><td>PALL300</td><td>7</td><td>914 ± 26</td><td>0.687 ± 0.019</td><td>0.275 ± 0.017</td></tr><tr><td>PALL700</td><td>7</td><td>893 ± 45</td><td>0.666 ± 0.008</td><td>0.305 ± 0.034</td></tr><tr><td>PALL300</td><td>15</td><td>777 ± 6</td><td>0.756 ± 0.014</td><td>0.306 ± 0.003</td></tr><tr><td>PALL700</td><td>15</td><td>752 ± 25</td><td>0.742 ± 0.011</td><td>0.313 ± 0.016</td></tr></tbody></table>

*months after bottling
About phenolics, a decrease of total tannins and vanillin reactive flavans was observed for all wines (Table 4), indicating the involvement of these polymeric structures in reactions triggered by oxygen [5]. Parameters linked to the reactivity of tannins towards proteins (HAa and SPI) decreased over time in the following order: CS > P > A. After 15 months of bottle aging, significant lower values of HAa and SPI were detected in CS wines bottled with closures at higher OTR, confirming previous findings on the role of NOx in the decrease of tannin reactivity and wine astringency [4]. This confirms that CS wine benefits more than P and much more than A of oxygen action and is in agreement with RSO evaluation.

<table><caption>**Table 4** - Phenolic compounds (mean value ± standard deviation) and SPI (Saliva Precipitation Index) of red wines closed with synthetic closures at low, medium and high oxygen transfer rate (OTR)</caption><thead><tr><th></th><th>time*</th><th>Tot Phenolics (mg/L)</th><th>Tannins (mg/L)</th><th>VRF (mg/L)</th><th>VRF/T</th><th>SPI</th></tr></thead><tbody><tr><td>AGLIANICO</td><td>0</td><td>3640 ± 44</td><td>3813 ± 61</td><td>1704 ± 5</td><td>0.447 ± 0.007</td><td>8.14 ± 0.22</td></tr><tr><td>AGL100</td><td>7</td><td>4058 ± 246</td><td>4242 ± 188</td><td>1580 ± 19</td><td>0.373 ± 0.013</td><td>5.99 ± 0.19</td></tr><tr><td>AGL300</td><td>7</td><td>3855 ± 60</td><td>4255 ± 131</td><td>1611 ± 32</td><td>0.379 ± 0.017</td><td>6.00 ± 0.58</td></tr><tr><td>AGL700</td><td>7</td><td>3948 ± 209</td><td>4329 ± 62</td><td>1568 ± 33</td><td>0.362 ± 0.013</td><td>6.47 ± 0.47</td></tr><tr><td>AGL100</td><td>15</td><td>3510 ± 96</td><td>3952 ± 119</td><td>1464 ± 34</td><td>0.371 ± 0.008</td><td>5.06 ± 0.05</td></tr><tr><td>AGL300</td><td>15</td><td>3605 ± 53</td><td>4045 ± 98</td><td>1403 ± 30</td><td>0.347 ± 0.012</td><td>5.38 ± 0.05</td></tr><tr><td>AGL700</td><td>15</td><td>3595 ± 84</td><td>3691 ± 150</td><td>1385 ± 65</td><td>0.375 ± 0.019</td><td>5.25 ± 0.02</td></tr><tr><td>CASAVECCHIA</td><td>0</td><td>3870 ± 53</td><td>4025 ± 35</td><td>1433 ± 13</td><td>0.356 ± 0.004</td><td>7.11 ± 0.45</td></tr><tr><td>CAS300</td><td>7</td><td>3937 ± 84</td><td>4369 ± 194</td><td>1684 ± 3</td><td>0.386 ± 0.018</td><td>6.94 ± 0.01</td></tr><tr><td>CAS700</td><td>7</td><td>4127 ± 46</td><td>4216 ± 174</td><td>1606 ± 39</td><td>0.382 ± 0.020</td><td>6.46 ± 0.06</td></tr><tr><td>CAS300</td><td>15</td><td>3973 ± 172</td><td>4262 ± 116</td><td>1321 ± 47</td><td>0.310 ± 0.017</td><td>5.60 ± 0.11</td></tr><tr><td>CAS700</td><td>15</td><td>3845 ± 107</td><td>4392 ± 128</td><td>1323 ± 29</td><td>0.302 ± 0.014</td><td>5.43 ± 0.14</td></tr><tr><td>PALLAGRELLO</td><td>0</td><td>3937 ± 45</td><td>4187 ± 18</td><td>1504 ± 10</td><td>0.359 ± 0.004</td><td>6.83 ± 0.14</td></tr><tr><td>PALL300</td><td>7</td><td>3803 ± 137</td><td>4466 ± 154</td><td>1639 ± 92</td><td>0.368 ± 0.030</td><td>6.42 ± 0.05</td></tr><tr><td>PALL700</td><td>7</td><td>3825 ± 97</td><td>4331 ± 45</td><td>1504 ± 40</td><td>0.347 ± 0.011</td><td>6.19 ± 0.07</td></tr><tr><td>PALL300</td><td>15</td><td>3913 ± 67</td><td>4451 ± 312</td><td>1371 ± 33</td><td>0.309 ± 0.021</td><td>6.15 ± 0.03</td></tr><tr><td>PALL700</td><td>15</td><td>3823 ± 76</td><td>4520 ± 81</td><td>1377 ± 45</td><td>0.305 ± 0.011</td><td>6.27 ± 0.01</td></tr></tbody></table>

*months after bottling

## Conclusion

The trend observed for the evolution of polymeric pigments and for the reactivity of phenolics towards proteins of three analyzed wines is in agreement with results of the test performed using a strong oxidant solution. Results obtained in this experiment not only confirm previous finding on the positive role of NOx in wine phenolic evolution during bottle aging but also indicate that the oxygen tolerance of a red wine is a fundamental parameter to choose the NOx level to be applied taking into account the wine expected lifetime in bottle.

## References

1. Caillé S, Samson A, Wirth J et al (2010) Sensory characteristics changes of red Grenache wines submitted to different oxygen exposures pre and post bottling. *Anal Chim Acta* **660**: 35-42.

2. Wirth J, Morel-Salmi C, Souquet JM et al (2010) The impact of oxygen exposure before and after bottling on the polyphenolic composition of red wines. *Food Chem* **123**: 107-116.

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

4. Gambuti A, Rinaldi A, Ugliano M et al (2013) Evolution of phenolic compounds and astringency during aging of red wine: effect of oxygen exposure before and after bottling. *J Agric Food Chem* **61**: 1618-1627.
5. Fulcrand H, Atanasova V, Salas E et al (2004) The fate of anthocyanins in wine: are there determining factors? *In Red Wine Color: Revealing the Mysteries* **886**: 68-88.

6. Joslyn MA, Comar CL (1941) Role of acetaldehyde in red wines. *Ind Eng Chem* **33**: 919-928.

7. Müller-Späth H (1992) Der POM-test. *Deutscher Weinbau* **23**: 1099-1100.

8. Gambuti A, Lisanti MT, Picariello L et al (2014) Prediction test of maximum oxygen tolerance for red wine. *In 37th World Congress of Vine and Wine*, vol. 1, pp. 1-8.

9. Harbertson JF, Picciotto EA, Adams DO (2003) Measurement of polymeric pigments in grape berry extracts and wines using a protein precipitation assay combined with bisulfite bleaching. *Am J Enol Vitic* **54**: 301-306.

10. Rinaldi A, Gambuti A, Moio L (2012) Application of the SPI (Saliva Precipitation Index) to the evaluation of red wine astringency. *Food Chem* **135**: 2498-2504.

11. Lago-Vanzela ES, Procopio DP, Filomeno Fontes EA et al (2014) Aging of red wines made from hybrid grape cv. BRS Violeta: effects of accelerated aging conditions on phenolic composition, color and antioxidant activity. *Food Res Int* **56**: 182-189.

12. Boulton R (2001) The copigmentation of anthocyanins and its role in the color of red wine: a critical review. *Am J Enol Vitic* **52**: 67-87.

13. Versari A, Parpinello GP, Mattioli AU (2007) Characterisation of colour components and polymeric pigments of commercial red wines by using selected UV-Vis spectrophotometric methods. *S Afr J Enol Vitic* **28**: 1-6.
# Modified carbon paste screen printed electrodes for rapid fingerprinting of white wine oxidizable fraction

A. GONZALEZ ZAVALA<fnref n="1" />,<fnref n="2" />, J. WIRTH<fnref n="1" />, J.-B. DIEVAL<fnref n="1" />, S. VIDAL<fnref n="1" />*, M. UGLIANO<fnref n="1" />,<fnref n="3" />

Voltammetry – oxidizable compounds – wine fingerprint

## Introduction

The ability to evaluate the occurrence of different oxidizable compounds in grapes and wines is crucial to the implementation of successful strategies to reduce the risk of early wine oxidation. To date, the existing analytical methods are poorly adapted to the context of wineries as they are too time consuming and require dedicated equipment and procedures. Voltammetric techniques have been successfully used for the analysis of wine antioxidants [1, 2], but their application to routine wine analysis remains limited due to the complexity of their execution, in particular with regard to electrode calibration and cleaning. In this work we have evaluated the potential of disposable carbon paste screen printed electrodes for the rapid evaluation of wine oxidizable compounds.

## Materials and methods

### Electrochemical measurements

Screen printed NomaSense carbon ink electrodes were obtained from Nomacorc (Belgium). The electrode area was defined by printing an insulating layer. Electrochemical measurements were performed using a commercial NomaSense B200 potentiostat electrochemical analyzer (Nomacorc, Belgium). For each measurement, one drop of sample (50 µL) was deposited over the electrode strip. Linear sweep voltammograms were recorded from 0 V to 1.2 V with a scan rate of 100 mV/s under ambient conditions. For each measurement a fresh electrode was employed. All analyses were carried out in duplicate, with no preliminary sample preparation.

### Chemicals

All chemicals were obtained from Sigma Aldrich.

### Wines

Wines (Chardonnay, Riesling, Viognier, and Muscadet) were purchased at local retail outlets.

## Results and discussion

Examples of the voltammograms of pure compounds obtained on the screen printed electrodes are shown in Figure 1. Large differences in anodic current can be observed in the region between 200 and 400 mV, with catechin and gallic acid showing a broad peak starting at approximately 300 mV. This characteristic is consistent with the fact that these molecules are more powerful antioxidant than coumaric acid, characterized by a peak starting at approximately 500 mV.

Analysis of different white wines revealed that the electrochemical fingerprint obtained at the surface of a carbon ink screen printed electrode contains features that are characteristic of the wine type, reflecting its antioxidant composition, particularly phenolic antioxidants. As it can be seen in Figure 2, these fingerprints can be used to successfully discriminate Muscadet and Riesling samples from each other and from samples of Viognier and Chardonnay.

<footnote n="1">¹Nomacorc France, Domaine de Donadille, Av. Yves Cazeaux, 30230, Rodilhan, France</footnote>

<footnote n="2">²ESA Angers, 55 rue Rabelais, 49007, Angers, France</footnote>

<footnote n="3">³Department of Biotechnology, University of Verona, via della Pieve 70, 37029 San Pietro in Cariano, VR, Italy</footnote>
<figure><img src="image_286.png" /><figcaption>**Fig. 1** - Voltammograms of different wine phenolics in model wine (pH 3.2, 12% ethanol).</figcaption></figure>

<figure><img src="image_287.png" /><figcaption>**Fig. 2** - PLS-DA of 18 commercial white wines.</figcaption></figure>

## Conclusion

The results of this initial evaluation shows that screen printed carbon paste electrodes can be successfully used to analyze different phenolic antioxidants present in white wine. The global fingerprint obtained during analysis of white wine shows a good degree of specificity related to grape variety, suggesting that this analytical approach can provide valuable information in the study and characterization of wine oxidizable fraction

## 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.

2. Martins RC, Oliveira R, Bento F et al (2008) Oxidation management of white wines using cyclic voltammetry and multivariate process monitoring. *J Agric Food Chem* **56**: 12092-12098.
# A voltammetric approach for rapid characterization of oxidation patterns of white wines

A. GONZALEZ ZAVALA<fnref n="1" />,<fnref n="2" />, J.-B. DIEVAL<fnref n="1" />, S. VIDAL<fnref n="1" />*, M. UGLIANO<fnref n="1" />,<fnref n="3" />

Voltammetry – oxidizable compounds – fingerprint – white wines

## Introduction

Oxidative reactions occurring upon accidental or deliberate exposure of wine to oxygen are common during wine production and storage, resulting in either beneficial or detrimental effects depending on oxygen quantities and wine type. White wine typically contains a complex pool of oxidizable compounds, including phenolics, ascorbic acid, SO₂, and different thiols. Initial reaction with oxygen involves oxidation of ortho-diphenols to form quinones, which can in turn react with the other oxidizable compounds. The type and extent of these reactions can depend on relative proportions of individual species, the presence of catalysts, temperature, pH, and probably other hitherto unidentified factors. The ability to characterize and predict wine response to oxygen exposure would greatly assist winemakers in better adapting their winemaking practices to wine requirements to achieve specific product styles. Nevertheless, to date there are no analytical approaches allowing rapid characterization of wine response to oxidation.

Voltammetric techniques have been successfully applied to the analysis of different oxidizable compounds, including phenolics which are the primary target of wine oxidation [1, 2].

Recent progresses in this field resulted in the development of disposable electrodes, allowing to bypass the tedious electrode cleaning procedures associated with voltammetric analyses of phenol-rich matrices.

In this study, a voltammetric approach based on the use of disposable carbon-paste screen printed electrodes was investigated as a suitable strategy for rapid characterization of white wine oxidation patterns.

## Materials and methods

Fourteen commercial wines including Muscadet, Chardonnay, Riesling, Viognier, Roussanne, Sauvignon blanc and Grenache blanc were submitted to controlled oxidation. Wines were supplemented with 6.5 mg/L oxygen and oxidation was considered achieved when 5 mg/L had been consumed. Oxygen was monitored using a NomaSense O2 P300 analyzer (Nomacorc, Belgium). The changes occurring upon oxygen consumption were assessed by different means, including a newly developed voltammetric approach using a NomaSense Polyscan potentiostat and NomaSense carbon-paste screen printed electrodes (Nomacorc, Belgium). For each measurement, one drop of sample (50 µL) was deposited over the electrode strip. Linear sweep voltammograms were recorded from 0 V to 1.2 V with a scan rate of 100 mV/s under ambient conditions. For each measurement a fresh electrode was employed. All analyses were carried out in duplicate, with no preliminary sample preparation.

## Results and discussion

The rate of oxygen consumption was found to vary considerably depending on wine. Fast consuming wines exhibited average oxygen consumption rates in the range of 1 mg/L of oxygen consumed per day. At the other end of the observed spectrum, low oxygen consuming wines were in the range of 0.2 mg/L of consumed oxygen daily. An example of this for a selected set of wines is shown in Figure 1.

<footnote n="1">¹Nomacorc France, Av. Yves Cazeaux, 30230, Rodilhan, France</footnote>

<footnote n="2">²ESA Angers, 55 rue Rabelais, 49007, Angers, France</footnote>

<footnote n="3">³Department of Biotechnology, University of Verona, via della Pieve 70, 37029 San Pietro in Cariano, VR, Italy</footnote>
<figure><img src="image_288.png" /><figcaption>Fig. 1 - Oxygen consumption of five different commercial wines.</figcaption></figure>

Average oxygen consumption rates were not correlated with initial SO₂, copper, iron and total phenolic concentration or with initial content of major white wine phenolics (mostly catechins and hydroxycinnamic acids) as measured by HPLC. Examples of the voltammograms of a wine with or without oxidation are shown in Figure 2 for a Sauvignon blanc sample. Each one of the voltammograms reflected the oxidative reactions taking place at the surface of the electrode under identical analytical conditions. For each wine, the differences observed between the two voltammograms can be attributed to the loss of wine components induced by oxygen consumption. Accordingly, subtraction of the voltammograms of oxidized and control samples of each wine allowed obtaining individual oxidation signatures representing the global changes induced by oxygen consumption. These signature curves contain specific information relevant to the oxidation pattern of individual wines. Examples of such curves are given in Figure 3. Several characteristic features can be observed, which, to different extents, were found to be specific to individual wines. Among these, higher charge values in the area 0-400 mV appear highly discriminating and could be associated with fast oxygen consuming wines.

<figure><img src="image_289.png" /><figcaption>Fig. 2 - Voltammograms of a Sauvignon blanc wine with or without consumption of 5 mg/L of oxygen.</figcaption></figure>
Conclusion

The voltammetric method presented in this study allows to obtain characteristic fingerprints representing
the typical oxidative response of individual wines. The method is easy to implement and the analysis is
very rapid. The fingerprints obtained contain features that appear to be correlated with specific
characteristics of individual wines, including their ability to consume oxygen.

<figure><img src="image_290.png" /><figcaption>**Fig. 3** - Oxidation patterns of three commercial white wines. Data is calculated as difference between the voltammograms of each wine with and without oxygen consumption.</figcaption></figure>

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.

2. Martins RC, Oliveira R, Bento F et al (2008) Oxidation management of white wines using cyclic voltammetry and multivariate process monitoring. *J Agric Food Chem* **56**: 12092-12098.
# Study of quinone reactions with wine nucleophiles in wine-model solutions by cyclic voltammetry

C.M. OLIVEIRA<fnref n="1" />,<fnref n="2" />*, A. BARROS<fnref n="1" />, A.C. SILVA FERREIRA<fnref n="2" />,<fnref n="3" />, A.M.S. SILVA<fnref n="1" />

Quinones – nucleophiles – wine-model solutions – cyclic voltammetry

## Introduction

The oxidative processes of wine begin by the oxidation of polyphenols containing a catechol or a galloyl group such as (+)-catechin/(-)-epicatechin, gallocatechin, gallic acid and its esters, and caffeic acid, which are the most readily oxidized wine constituents. These substrates are sequentially oxidized to semiquinone radicals and quinones while oxygen is reduced to hydrogen peroxide. Quinones are therefore key reactive electrophilic oxidation intermediates in wine and can undertake many reactions with wine nucleophiles like sulfur dioxide, ascorbic acid, thiols, amino acids and others, including both the loss of varietal aroma or the formation of new off-flavors. These reactions are very important in wine aging because they mediate oxygen consumption during both production and bottle aging phases.

The influence of sulfur dioxide, glutathione and ascorbic acid on the cyclic voltammograms of wine polyphenols and wines has already been investigated using a glassy carbon electrode [1]. Results have shown that sulfur dioxide increased the anodic current and decreased the cathodic current, pointing to a rapid interaction of SO₂ with the oxidized polyphenol quinones. However, in that study, ascorbic acid produced no additional effect on the cyclic voltammograms of wine polyphenols and wines [1].

Oxidation of wine polyphenols is thought to play a major role in wine thiol degradation. Volatile thiols such as 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) are aromatic molecules having an important organoleptic impact on white wines with respectively boxwood/broom or grapefruit/passionfruit notes. These molecules decrease dramatically with wine oxidation [2-5]. On the other hand, other thiols with undesirable aromas, like 3-(methylsulfanyl)propan-1-ol (methionol) or 2-sulfanylethanol (2-mercaptoethanol) also decrease with wine oxidation. Port wine studies have concluded that old Port wine (barrel aged) never develops "off-flavors" associated with the presence of methionol (cauliflower) or 2-mercaptoethanol (rubber/burnt). In fact, temperature and oxygen are the major factors in the consumption of these molecules [6].

Sulfur dioxide ($HSO_3^-$) can convert *ortho*-quinones back to *ortho*-dihydroxyphenols and react directly with *ortho*-quinones to form sulfonic acids. Interestingly, the potential of ascorbic acid to recycle *ortho*-quinones back to *ortho*-dihydroxyphenols has also been suggested by various authors [7].

In this work, an attempt was made in order to determine the interaction between *ortho*-quinones and wine nucleophiles, like the amino acids phenylalanine and methionine, the thiols 3SH, 4MSP, and furan-2-ylmethanethiol (2FMT), and the antioxidants sulfur dioxide and ascorbic acid, by cyclic voltammetry. For this purpose, wine-model systems with gallic acid, caffeic acid, or (+)-catechin were used. Cyclic voltammograms were taken in the absence and in the presence of nucleophiles.

## Materials and methods

*Model-wine solutions: amino acids, thiols, and antioxidants*

The use of phenylalanine and methionine is due to their important wine formation of potent "Strecker aldehydes". The use of 3SH and 4MSP was due to their important contribution to wine varietal aroma.

<footnote n="1">¹Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal</footnote>

<footnote n="2">²Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal</footnote>

<footnote n="3">³Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa</footnote>
Conversely, the use of 2FMT was due to its negative wine aroma impact with an odoriferous smelling of cooked meat. Sulfur dioxide and ascorbic acid were used due to their recognized wine antioxidant activity.

Wine-model systems [ethanol 12% (v/v) and 0.033 M tartaric acid, pH = 3.6] with gallic acid, caffeic acid, or (+)-catechin at a concentration of 0.6 mM were used. The nucleophilic compounds were added in a concentration of 2.4 mM.

### Cyclic voltammetry

Experiments were performed using a potentiostat (microAutolab Type III with an Autolab Faraday Cage) and voltammograms were obtained with a scan rate of 100 mV/s with an increment potential of 2.4 mV, between -0.5 V and 1.2 V. The working electrode was a 3 mm glassy carbon disk in combination with a Metrohm tipholder, cleaned by polishing with 3 µm alumina powder during 30 s followed by fixing the potential to 1.5 V in a 0.1 mol/L NaOH solution during 5 s, between acquisitions. A saturated calomel electrode was used as a reference electrode in conjunction with a platinum counter electrode. Each acquisition required 15 mL of sample. Oxygen was removed with a N₂ current flow during 5 min prior to analysis. Cyclic voltammetry experiments were controlled by the GPES 4.9 software provided by *Ecochemie*.

<table><caption>**Table 1** - Evaluation of the anodic and cathodic peak potentials at pH 3.6 of the studied polyphenols [Ep,a - anodic peak potential (V); Ep,c - cathodic peak potential (V)] as well as the anodic and cathodic peak currents at pH 3.6 [Ip,a - anodic peak current (A); Ip,c - cathodic peak current (A)] during the cyclic voltammetry experiment</caption><thead><tr><th rowspan="2">Phenolic compound</th><th colspan="3">pH = 3.6</th></tr><tr><th>Ep<sub>1,a</sub></th><th>Ep<sub>2,a</sub></th><th>Ep<sub>1,c</sub></th></tr></thead><tbody><tr><td>gallic acid</td><td>0.354</td><td>0.735</td><td>0.273</td></tr><tr><td>caffeic acid</td><td>0.374</td><td></td><td>0.327</td></tr><tr><td>catechin</td><td>0.354</td><td>0.747</td><td>0.283</td></tr><tr><td></td><td>Ip<sub>1,a</sub> x10<sup>-6</sup></td><td>Ip<sub>2,a</sub> x10<sup>-6</sup></td><td>Ip<sub>1,cx</sub> x10<sup>-6</sup></td></tr><tr><td>gallic acid</td><td>8.08</td><td>6.14</td><td>-1.00</td></tr><tr><td>gallic+Phe</td><td>8.17</td><td>6.10</td><td>-1.00</td></tr><tr><td>gallic+Met</td><td>8.12</td><td>6.02</td><td>-0.95</td></tr><tr><td>gallic+3SH</td><td>7.01</td><td>6.25</td><td>-0.95</td></tr><tr><td>gallic+4MSP</td><td>7.59</td><td>6.65</td><td>-0.93</td></tr><tr><td>gallic+2FMT</td><td>6.03</td><td>5.85</td><td>-0.86</td></tr><tr><td>gallic+SO<sub>2</sub></td><td>9.70</td><td>8.02</td><td>-0.82</td></tr><tr><td>gallic+AA</td><td>12.95</td><td>9.66</td><td>-0.58</td></tr><tr><td>caffeic acid</td><td>9.20</td><td></td><td>-5.38</td></tr><tr><td>caffeic+Phe</td><td>8.79</td><td></td><td>-5.23</td></tr><tr><td>caffeic+Met</td><td>9.26</td><td></td><td>-5.44</td></tr><tr><td>caffeic+3SH</td><td>8.38</td><td></td><td>-4.40</td></tr><tr><td>caffeic+4MSP</td><td>10.61</td><td></td><td>-4.02</td></tr><tr><td>caffeic+2FMT</td><td>5.24</td><td></td><td>-2.57</td></tr><tr><td>caffeic+SO<sub>2</sub></td><td>7.73</td><td></td><td>-1.15</td></tr><tr><td>caffeic+AA</td><td>13.64</td><td></td><td>-2.82</td></tr><tr><td>catechin</td><td>5.08</td><td>6.39</td><td>-3.36</td></tr><tr><td>catechin+Phe</td><td>4.03</td><td>4.97</td><td>-2.72</td></tr><tr><td>catechin+Met</td><td>3.55</td><td>4.84</td><td>-2.39</td></tr><tr><td>catechin+3SH</td><td>3.80</td><td>5.51</td><td>-2.26</td></tr><tr><td>catechin+4MSP</td><td>4.29</td><td>5.98</td><td>-1.89</td></tr><tr><td>catechin+2FMT</td><td>2.98</td><td>4.82</td><td>-1.74</td></tr><tr><td>catechin+SO<sub>2</sub></td><td>3.97</td><td>6.83</td><td>-1.71</td></tr><tr><td>catechin+AA</td><td>7.64</td><td>7.44</td><td>-1.59</td></tr></tbody></table>

Phe, phenylalanine; Met, methionine; 3SH, 3-sulfanylhexan-1-ol; 4MSP, 4-methyl-4-sulfanylpentan-2-one; 2FMT, furan-2-ylmethanethiol; AA, ascorbic acid; SO₂, sulfur dioxide
# Results and discussion

During the cyclic voltammetry experiment, the anodic and cathodic peak potentials at pH 3.6 of the studied polyphenols as well as the anodic and cathodic peak currents at pH 3.6 were evaluated (Table 1). Cyclic voltammetry is a method to produce polyphenol quinones in a controlled manner (oxidation curves). If a rapid interaction of nucleophiles with polyphenol quinones occurs, there will be less available for its reduction at the carbon electrode on the reverse potential sweep (reduction curves). Conversely, if no interaction of nucleophiles with polyphenol quinones occurs, the cathodic peaks will be retained on the reverse potential sweep.

Figures 1 to 3 represent the cyclic voltammograms of respectively the **amino acids** phenylalanine and methionine, the **thiols** 3SH, 4MSP, and 2FMT, and the **antioxidants** sulfur dioxide and ascorbic acid, in the absence and in the presence of three representative phenolic compounds (gallic acid, caffeic acid, and (+)-catechin).

## Amino acids

Results have shown that phenylalanine and methionine have a small influence (till 5%) on the cathodic peak current intensities of gallic and caffeic acids (Figure 1a, b, and Table 1). On another hand, phenylalanine and methionine decreased the current intensities of the cathodic peak of (+)-catechin by 19 and 29%, respectively (Figure 1c and Table 1). These results point to an interaction of the above amino acids with the (+)-catechin quinone.

<figure><img src="image_291.png" /><figcaption>**Fig. 1** - Cyclic voltammograms taken for amino acid interactions with: a) gallic acid; b) caffeic acid; and c) (+)-catechin.</figcaption></figure>

## Thiols

Results have shown that 3SH and 4MSP have decreased the cathodic current intensities of all the analyzed polyphenols. Furthermore, the decrease rate follows, respectively for 3SH and 4MSP, the order: (+)-catechin (by 33 and 44%) > caffeic acid (by 18 and 25%) > gallic acid (by 5 and 7%) (Figure 2 and Table 1). These results indicate that 4MSP is more reactive with quinones than 3SH. Considering 2FMT, the decrease of the cathodic current intensities of all the analyzed polyphenols were higher than the former thiols (14% for gallic acid, 52% for caffeic acid, and 48% for (+) catechin). These results point to a rapid interaction of 2FMT with the caffeic acid and (+)-catechin quinones.

<figure><img src="image_292.png" /><figcaption>**Fig. 2** - Cyclic voltammograms taken for thiol interactions with: a) gallic acid; b) caffeic acid; and c) (+)-catechin.</figcaption></figure>
Antioxidants

Results have shown that ascorbic acid decreased the current intensities of the cathodic peaks of gallic acid, caffeic acid, and (+)-catechin by 42, 48, and 53%, respectively (Figure 3 and Table 1), pointing to a rapid interaction of ascorbic acid with the oxidized polyphenol quinones. In the same way, results have shown that sulfur dioxide decreased the current intensities of the cathodic peaks of gallic acid, caffeic acid, and (+)-catechin by 18, 79, and 49%, respectively (Figure 3 and Table 1), also pointing to a rapid interaction of ascorbic acid with the oxidized polyphenol quinones.

<figure><img src="image_293.png" /><figcaption>Fig. 3 - Cyclic voltammograms taken for antioxidants interactions with: a) gallic acid; b) caffeic acid; and c) (+)-catechin.</figcaption></figure>

Conclusion

As quinones are electrophilic species which can react with various nucleophiles, a nucleophilicity scale related to the cathodic current intensity decrease of gallic acid, caffeic acid, and (+)-catechin quinones with phenylalanine, methionine, 3SH, 4MSP, 2FMT, ascorbic acid, and sulfur dioxide was attempted by cyclic voltammetry. Results have shown that the lowest reactivity with quinones was attributed to amino acids. Concerning thiols, 4MSP was more reactive with quinones than 3SH, but the highest reactivity was attributed to 2FMT. Sulfur dioxide and ascorbic acid had higher and similar reactions with quinones.

References

1. Makhotkina O, Kilmartin PA (2009) Uncovering the influence of antioxidants on polyphenol oxidation in wines using an electrochemical method: cyclic voltammetry. *J Electroanal Chem* **633**: 165-174.

2. 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.

3. Brajkovich M, Tibbits N, Peron G et al (2005) Effect of screwcap and cork closures on SO2 levels and aromas in a Sauvignon Blanc wine. *J Agric Food Chem* **53**: 10006-10011.

4. Lopes P, Silva MA, Pons A et al (2009) Impact of oxygen dissolved at bottling and transmitted through closures on the composition and sensory properties of a Sauvignon Blanc wine during bottle storage. *J Agric Food Chem* **57**: 10261-10270.

5. Makhotkina O (2011) Stability of New Zealand Sauvignon blanc aroma compounds: oxidation versus hydrolysis. *PhD thesis*, University of Auckland.

6. Silva Ferreira AC, Rodrigues P, Hogg T et al (2003) Influence of some technological parameters on the formation of dimethyl sulfide, 2-mercaptoethanol, methionol, and dimethyl sulfone in Port wines. *J Agric Food Chem* **51**: 727-732.

7. Danilewicz JC (2003) Review of reaction mechanisms of oxygen and proposed intermediate reduction products in wine: central role of iron and copper. *Am J Enol Vitic* **54**: 73-85.
# Network Reconstruction: a tool to unravel wine aging

A.R. MONFORTE<fnref n="1" />*, D. JACOBSON<fnref n="2" />, A.C. SILVA FERREIRA<fnref n="1" />

Aging – oxygen – temperature – kinetic parameters

## Introduction

Port is a naturally sweet wine produced by interrupting alcoholic fermentation by the adding of grape spirit. Chemical reactions occurring during Port wine aging can contribute to the changes in the Ports sensorial profile, contributing to the establishment of different categories: bottle-aged (“vintage” category) or barrel aged (“tawny” category) wines. The changes in the composition of volatile compounds during Port wine aging can be related to parameters such as dissolved O₂ [1], pH and temperature [2]. These parameters will affect the aromatic profile of Ports as the result of several chemical mechanisms. Therefore, wine aging is a complex system, which requires more information to be analyzed to better understand the mechanisms at play. Given this, techniques capable to capture information about a broader range of compounds participating in the aging process are necessary to achieve a better understanding of the process.

Chemiomics has been defined as the study of the relationships between as many chemical compounds as possible in a complex chemical (nonenzymatic) system. Network reconstruction has proven to be useful in visualizing the relationships between all the compounds present in a dataset and the changes in their concentration over time. The aim of this study is to use network reconstruction and kinetic parameters in order to extract more information related with aging mechanisms.

## Materials and methods

### Wines

The wines used in this study were as follows. Group 1: real aged Port wines (34 samples) with ages between 1 and 129 years. Group 2: red Port wine made in the year of the experiment (without any oak contact) with a pH of 3.4, 2.5 mg/L of dissolved oxygen, 17 mg/L of free SO₂, 105 g/L of reducing sugars and 20.5% of alcohol.

### Oxygen treatments

Group 2 has been prepared in order to analyze the effect of parameters that are believed to be important in the aromatic modifications of Port wines, namely, oxygen concentration and temperature. The experiment was performed at four different temperatures (20, 30, 35 and 40°C) and a series of sequential oxygen saturation (0 to 10 saturations). Oxygen treatments included: 0 (F1), 3 (F2), 5 (F3) and 10 (F4) saturations. Samples were taken daily during 10 weeks (63 days). Compounds were extracted by a liquid-liquid extraction (LLE) and the extracts were analyzed by gas chromatography-mass spectrometry (GC-MS).

### Network reconstruction

The ASCII file of chromatographic data obtained from each group 1 samples has been extracted and all the m/z channels were normalized to total intensity to yield a total ion current (TIC) chromatogram for each sample. Network reconstruction has been constructed according to Jacobson et al. [3]. Kinetic parameters were calculated with group 2 wines according to Martins et al [4]. An analysis of variance (ANOVA), using MATLAB, was applied to the experimental data; the results were considered to be significant if the associated p value was <0.05.

<footnote n="1">¹Escola Superior de Biotecnologia – Universidade Católica Portuguesa, Porto, Portugal</footnote>

<footnote n="2">²IWBT – DVO University of Stellenbosch, South Africa</footnote>
**Results and discussion**

Figure 1 shows the network derived from the correlation between all peaks. Each node represents the center of a peak (Kovats index) and each edge represents the best correlation between the peaks. Fold changes between 1- and 129-year old wines were calculated for each peal and the nodes colored accordingly with shades of red representing increasing concentration.

<figure><img src="image_294.png" /><figcaption>Fig. 1 - GC-MS network.</figcaption></figure>

The thickness of each edge has been scaled to represent the level of correlation (thicker lines mean higher correlation values). Using pure standards, it is possible to explore and extract more information from the network. Three main branches were observed. Branch A included compounds such as hexanoic, octanoic and decanoic acids, which cluster together. The ANOVA analysis on group 2 wines shows that acids are not dependent on O₂ or temperature. In branch B, were identified: 5-hydroxymethylfurfural (HMF), furfural, ethyl furoate vanillin, γ-ethoxycarbonyl-γ-butyrolactone and organic acid esters such as diethyl tartrate. All the compounds identified tend to increase during aging and ANOVA shows that significant differences were established across the temperature conditions, meaning that all the compounds identified were dependent on temperature.

In branch C were identified compounds such as: benzaldehyde, *cis*- and *trans*- dioxanes and dioxolanes. All these compounds are formed by reactions dependent on oxygen. Benzaldehyde is formed by the oxidation of benzyl alcohol and dioxanes and dioxolanes by the reaction of acetaldehyde and glycerol [5]. In a particular sub-branch of branch C has been identified 3-hydroxy-4, 5-dimethyl-2 (5H)-furanone (sotolon), whose mechanism is not totally fully understood, but it has been proved that in Port wine its formation is dependent on temperature and oxygen [5]. Kinetic parameters have been calculated in order to validate the network connections between the compounds.

These observations would seem to validate the assumptions made that compounds grouped on the same branch of the network have the same time expression and consequently the same kinetic order. It was also observed that compounds grouped together are sensitive to the same technological parameter.
**Conclusion**

The network reconstruction is very useful in visualizing the relationships between all of the compounds detected via GC-MS and their changes in concentration over time. This view of the data should provide considerably more information in an effort to understand the probable kinetic contexts of the molecules represented by peaks in each chromatogram. As such, the approach described here should indeed be a very powerful tool for the further study of mechanisms and kinetic networks in complex mixtures, such as wines.

**Acknowledgements**

This research was funded by the project "Wine Metrics: Revealing the Volatile Molecular Feature Responsible for the Wine Like Aroma - a Critical Task Toward the Wine Quality Definition" (PTDC/AGR-ALI/121062/2010) and partially supported by ESB/UCP pluriannual funds through the POS-Conhecimento Program that includes FEDER funds through the program COMPETE (Programa Operacional Factores de Competitividade), by national funds through FCT (Fundação para a Ciência e a Tecnologia), the Wine Industry Network of Expertise and Technology (Winetech IWBT BI 13/01) and the Technology and Human Resources Program and the South African National Science Foundation.

**References**

1. Silva Ferreira AC, Barbe J-C, Bertrand A (2003) 3-Hydroxy-4,5-dimethyl-2(5H)-furanone: a key odorant of the typical aroma of oxidative aged Port wine. *J Agric Food Chem* **51**: 4356-4363.

2. Silva Ferreira AC, de Pinho PG, Rodrigues P et al (2002) Kinetics of oxidative degradation of white wines and how they are affected by selected technological parameters. *J Agric Food Chem* **50**: 5919-5924.

3. Jacobson D, Monforte AR, Ferreira ACS (2013) Untangling the chemistry of Port wine aging with the use of GC-FID, multivariate statistics, and network reconstruction. *J Agric Food Chem* **61**: 2513-2521.

4. Martins RC, Monforte AR, Silva Ferreira A (2013) Port wine oxidation management: a multiparametric kinetic approach. *J Agric Food Chem* **61**: 5371-5379.

5. da Silva Ferreira AC, Barbe J-C, Bertrand A (2002) Heterocyclic acetals from glycerol and acetaldehyde in Port wines: evolution with aging. *J Agric Food Chem* **50**: 2560-2564.
# Long term effects of pre-fermentative maceration on the phenolic characteristics and sensory attributes of Pinot Noir

P. WEGMANN-HERR*, D. GOLITKO, P. NICKOLAUS, D. DURNER, U. FISCHER

Competence Center for Wine Research, State Education and Research Center for Viticulture & Horticulture (DLR-Rheinpfalz), Breitenweg 71, 67435 Neustadt, Germany

Pinot Noir – pre-fermentative maceration – phenols – sensory perception

## Introduction

World over, Pinot Noir is considered among the most noble red varieties. Pinot Noir grapes typically contain low anthocyanin concentrations in the skins, while at the same time exhibiting high concentrations of seed tannins, such as catechin and epicatechin [1, 2]. This unfavorable anthocyanin to flavan-3-ol ratio can cause significant challenges for winemakers. Maceration plays a key role in steering the quality of the wine, with the goal of avoiding wines lacking fruitiness and color, or alternately over-extracted, tannic wines. Pre-fermentative maceration, or cold soaking (CS), is one technique which aims to lengthen the portion of maceration which takes place in the absence of alcohol. As the extraction of seed tannins requires the presence of ethanol, while anthocyanins are largely water soluble, this method appears to hold particular promise for improving the quality of Pinot Noir wines. By applying this technique, improvements in color and mouthfeel, as well as all manner of other qualities, ranging from changes in aroma to improved aging potential are reported [3]. However, despite such observations, studies which have been performed leave much still unclear as to the precise effects of CS, and particularly how it impacts the sensory perception of the wines.

## Materials and methods

Five days of “cold soaking” at 5°C was included in fermentations of 10 and 15 days, keeping the total duration of maceration, fermentation temperature, and punch down frequency equal. All fermentations were carried out in repetitions. The description and denotation of the experiments are shown in table 1.

<table><caption>Table 1 - Description and denotation of the experiments</caption><thead><tr><th>Experiment</th><th>5+5 Days</th><th>5+10 Days</th><th>10 Days</th><th>15 Days</th></tr></thead><tbody><tr><td rowspan="2">description</td><td>5 days CS</td><td>5 days CS</td><td>no CS</td><td>no CS</td></tr><tr><td>5 days<br>alcoholic<br>maceration</td><td>10 days<br>alcoholic<br>maceration</td><td>10 days<br>alcoholic<br>maceration</td><td>15 days<br>alcoholic<br>maceration</td></tr></tbody></table>

Grapes of *Vitis vinifera* L. c.v. Pinot Noir, clone Auer 2107, were harvested in 2011 from a vineyard of Weingut Bergdolt – Klostergut St. Lamprecht, Palatinate, Germany. Grape and must composition at harvest is shown in table 2.

<table><caption>Table 2 - Grape and must composition at harvest. All measures were made using Foss Grapescan FTiR.</caption><thead><tr><th>Density</th><th>Titratable acidity [g/L]</th><th>pH</th><th>Malic acid [g/L]</th><th>Tartaric acid [g/L]</th></tr></thead><tbody><tr><td>1.089</td><td>7.6</td><td>3.0</td><td>4.8</td><td>4.0</td></tr></tbody></table>

<footnote>*Corresponding author: pascal.wegmann-herr@dlr.rlp.de</footnote>
After de-stemming and crushing the grapes, 50 mg/L of SO₂ was added in the form of 87 mg/L of potassium bisulfite (KDS), and the must thoroughly mixed. The juice was then separated from the pomace and divided equally into eight 50 L inox steel vats, resulting in 15 L of juice and 35 kg of pomace per vat. All treatments received 30 g/hL of Lallemand GoFerm® yeast nutrient and 0.06 g/hL Thiamin, prior to being inoculated with 30 g/hL of Lallemand RC212 yeast rehydrated according to manufacturer recommendations (Lallemand, Montreal, Canada). 800 g of sucrose was dissolved in each vat to raise the potential alcohol level by 10 g/L. After fermentation, pressing, settling and racking, the wines were inoculated with 2 g/hL VP41 lactic acid bacteria according to manufacturer recommendations (Lallemand, Montreal, Canada). Spectrophotometry (color intensity & tone), HPLC-DAD (total anthocyanins and flavan-3-ols), Glories (phenolic maturity), Folin-C, MCP and Harbertson-Adams assays (characterization of phenolic composition), as well as descriptive sensory analysis were used to analyze the differences between the variations.

## Results and discussion

The results of the standard chemical analysis (ethanol, reducing sugars, pH, volatile acidity and total acidity) of the wines after bottling did not show any significant differences with 0 g/L reducing sugars, 105 g/L ethanol and pH 3.7. Phenolic maturity of the grapes, calculated before and after cold soak by Glories method, is shown in Figure 1.

<figure><img src="image_295.png" /><figcaption>Fig. 1 - Phenolic maturity by Glories method before and after cold soaking.</figcaption></figure>

Both the anthocyanin extractability and the contribution of seed tannins to the total phenol content were reduced through pre-fermentative maceration. The amount of not extractable anthocyanins decreased by 23% and the participation of seed tannins to the total phenol content by nearly 30% compared with the values calculated at harvest. This shows that the CS led to an overall improvement in the extractability of compounds in the grape skin, as compared with the grapes prior to soaking. The change in color intensity during maceration, after MLF and after bottling can be seen in Figure 2. In the (5+5 Days) and (5+15 Days) treatments, an increase can be seen during CS, with both variants showing nearly three times higher absorbance values at fermentation start as compared with the non-cold soak treatments.

Though less pronounced thereafter, these higher CI measurements in the cold soak treatments persist through fermentation and can continue to be seen both after MLF and in the bottle, with final values 0.5 absorbance units (AU) higher than in the control wines. After MLF, a large reduction in CI is seen in all treatments through bleaching of anthocyanins and the active removal through adsorption as a function of the addition of SO₂ and filtration. Interestingly, the observed increase of CI during CS is not due to the extraction of monomeric anthocyanins as shown in Figure 3. At bottling, however, these variants show higher concentrations than the control treatments. Contrary to the behavior of the anthocyanins, the CS treatments show a slight increase in flavanol concentration during pre-fermentative maceration (Figure 4).
<figure><img src="image_296.png" /><figcaption>**Fig. 2** - Color intensity, measured in absorbance units (AU). Time of cold soak is shown as negative days relative to start of AF. Error bars show standard deviation between experimental repetitions (n=2).</figcaption></figure>

<figure><img src="image_297.png" /><figcaption>**Fig. 3** - Concentration of monomeric anthocyanins. Time of cold soak is shown as negative days relative to start of AF. Error bars show standard deviation between experimental repetitions (n=2).</figcaption></figure>

<figure><img src="image_298.png" /><figcaption>**Fig. 4** - Flavan-3-ol monomer concentration. Time of cold soak is shown as negative days relative to start of AF. Error bars show standard deviation between experimental repetitions (n=2).</figcaption></figure>
The flavan-3-ol monomer concentration differs greatly between all treatments at bottling, showing apparent influence of total maceration time. Though the highest total maceration time produced the highest flavan-3-ol concentrations, the CS treatments showed significantly lower concentrations than their control wine counterparts. By calculating the flavan-3-ol to anthocyanins ratio (FAR) in the bottled wines values of 0.2 (5+5 Days); 0.4 (10 Days); 0.5 (5+10 Days) and 0.8 (15 Days) can be obtained. After bottling in 2012 and two years later in 2014 the wines underwent a descriptive sensory analysis with 15 and 11 trained judges respectively in repetition each time. Figure 5 shows the results of the descriptive analysis, comparing the four treatments at the two dates.

<figure><img src="image_299.png" /><figcaption>**Fig. 5** - Descriptive analysis for sensory attributes of all treatments. Sensory analysis 2012 left (n=15 judges x 2 replicates), 2014 right (n=11 judges x 2 replicates). Mean scores and significance are shown for all attributes (* indicates p < 0.10, ** p < 0.01, *** p < 0.001, ns = not significant).</figcaption></figure>

In 2012 judges were clearly able to differentiate between the CS and control treatments based on appearance, with very highly significant differences in both hue and color intensity. Several other attributes were also noted as significantly or highly significantly different between treatments, particularly the in mouth modalities. Here, the control treatments were noted as being more astringent and having a harsher mouthfeel, than the CS treatments. The (5+5 Days) treatment was the only observed to have less dry tannins compared with all other treatments, being significantly different in this regard. This treatment also shows the highest significance regarding aroma descriptors, being noted as having more strawberry and mint aromas. Though not significantly different, the (5+10 Days) treatment was also noted as having more strawberry aroma. The sour cherry attribute was observed to be more prominent in the treatments which spent more time in contact with the pomace. Though described as the fruitiest variant, the (5+5 Days) treatment was also the only which was ascribed significantly more noticeable medicinal/meaty aromas. Contrary to the result in 2012 the descriptive analysis three years after harvest revealed less significant differences between the treatments. The sensory profiles drew near during aging and only the attributes color intensity, strawberry and medicinal/meaty revealed significant differences between treatments. Here the (5+10 Days) treatment showed the highest color intensity with significantly perceived stronger strawberry aroma. Nevertheless it can be stated that both CS treatments showed the highest color intensity and therefore superior potential for long term color stability.

The correlation biplot obtained through PLS regression using the analyses of phenolic content as predictors for the sensory evaluation of in mouth modalities and color in 2012 can be seen in Figure 7. Using this statistical method, the differentiation of the four treatments becomes even more distinct, with all wines occupying a separate quadrant along the x and y axes. Correlations between the sensory descriptors and treatments are again clearly shown, with these relationships only modified slightly through inclusion of the chemical parameters as predictors. Using this statistical method, the differentiation of the four treatments becomes even more distinct. Both CS wines are again shown to correspond with both observed increases in color and hue, observations predicted through anthocyanin monomer concentration, hue and chroma measurements. The (15 Days) and (10 Days) treatments, while remaining strongly correlated with all in mouth descriptors, are more clearly differentiated here, based on stronger correlation with phenolic content.
The perception of dry tannins can be seen as more directly related to the concentration of flavan-3-ol monomers, condensed tannins and total phenolic content than the perception of green tannins, astringency and bitter. Higher amounts of monomeric anthocyanins combined with low FAR values at bottling seem to improve long term color stability. Phenolic analyses of the treatments in 2014 still showed clear differences between the applied techniques. The PCA in Figure 7 shows once again the differentiation of the treatments separating them along the axes PC1 and PC2 which predict about 89% of the total variance. Both control treatments can be categorized based on their correlation with caffeic acid and flavan-3-ol monomers. In particular, the CS variants are not only significantly different from the control treatments, but also from one another, lying well separated on the PC1 axis. Color intensity once in sensory analyses and once in chemical analyses are again as relating closely with both CS treatments, while the sum of SPP and LPP correlate with the (5+10 Days) wine and the color intensity measured by sensory analyses. The (5+5 Days) treatment shows the highest amount of monomeric anthocyanins and caftaric acid.

<figure><img src="image_300.png" /><figcaption>**Fig. 6** - PLS correlation of sensory evaluation with analyses of phenolic composition (2012). The biplot shows the variables of the chemical analyses (x), the sensory evaluation (y), and the treatments as observations.</figcaption></figure>

<figure><img src="image_301.png" /><figcaption>**Fig. 7** - Biplot of principal components 1 and 2 for mean scores of the phenolic data and one sensory data.</figcaption></figure>