© The Authors, published by EDP Sciences 2017

1. Introduction^*

Isotope fractionation in plants is well-known, particularly for ¹⁸O and ²H and is a result of the water cycle [1]. In vines, this fractionation is accentuated by transpiration during grape ripening. Carbon isotope fractionation also occurs during plant photosynthesis which selectively uses a light carbon isotope (i.e., ¹²CO₂) for molecule synthesis. Previous studies demonstrated that during water deficit periods the plants close their stomata in order to limit transpiration. As a result, the equilibrium with air CO₂ composition, in terms of isotope concentration (¹²CO₂ & ¹³CO₂) is modified and the plant need to consume more ¹³CO₂ to maintain its photosynthetic activity. As a result, water deficit is characterized by an increase in carbon-13 in photosynthesized compounds [2].

Previous studies demonstrated the impact of water deficit on sugar carbon-13 content at grape level [3]. Another study showed the average δ¹³C discrepancy between sugar and its ethanol, resulting from the fermentation process [4]. Because this shift is constant, ethanol δ¹³C can be used as an indicator of vine water status during grape ripening (Fig. 1).

Figure 1. Water deficit thresholds with respect to δ13C ratio adapted from REF 3.

Many studies have been performed on grapes but, surprisingly, the final product, the wine, has not been addressed in these studies. The lack of tools to estimate the grape ripening conditions directly on wine is regrettable because water deficit during grape ripening usually provides potentially high quality red wines [5]. Moreover, recent studies correlated this parameter with aging bouquet typicality of red Bordeaux wines [6].

The aim of this study, previously published elsewhere [7], was to propose a rapid and reliable method directly applicable to wine, able to provide an estimation of grape ripening conditions with regard to vine water status.

2. Methods

2.1. Samples

Two set of samples have been used for this study: the first one based on 34 authentic samples (equally red and white wines) elaborated from grapes in the laboratory according to a previously described protocol [8]. A second set of 28 authentic wines for which the predawn leaf water potential was followed every 2 weeks (from July to end of September) during grape ripening. All samples have been distilled to recover ethanol, allowing analysis to be carried out on the bulk wine and the ethanol, respectively.

2.2. irm-EA/MS measurements

Ψ Measurements have been performed using an elemental analyser (VarioMicroCube, Elementar) coupled to an isotope ratio monitoring by mass spectrometry (Isoprime, Elementar). Masses measured are m/z 44 and 45 corresponding to the CO₂ isotopologues. The values are expressed in ‰ versus Vienna-Pee Dee Belemnite (V-PDB). The provided data correspond to two measurement average if the deviation between the two measurements is lower than 0.3‰.

2.3. irm-¹³C NMR measurements

A Bruker 400 NMR spectrometer was used to record quantitative ¹³C spectra at 100.6 MHz (5-mm dual+ probe, no rotation, 30 ◦C) Intramolecular ¹³C composition are described in [9]. Positional ¹³C distribution on ethanol skeleton is quantified from the ¹³C mole fraction (f_i= S_i/S_tot,S_i the ¹³C signal and S_tot the sum of the signals). Considering the statistical mode fraction (F_i), the position- specific relative deviation in ¹³C abundance for any C atom in position “i” is d_i = f_i/F_i − 1. Using the isotope composition of the whole ethanol molecule, d_i is then converted to δ¹³C_i (‰).

3. Results

The first step of this work was devoted to determine the relation between ethanol and bulk wine δ¹³C. A set of 31 authentic samples – without any oenological treatment – have been studied. They corresponds to wine samples elaborated, from grapes, in our laboratory [8]. All these wines, coming from various regions of France were distilled; the recovered ethanol and bulk wine were analyzed by irm-EA/MS to quantify the δ¹³C. The results, plotted on Fig. 2, show the full correlation between these two measurements. This result is not surprising as a wine is composed, in average, of 84% of water and nearly 16% of organic and inorganic compounds, ethanol corresponding to 11% of the wine.

Figure 2. Relation between δ13C values of authentic wines and their ethanol for red (squares) and white (circles) wines.

The second step has been the study of 28 wines for which vine water status during grape ripening was known. Vine water deficit can be characterized by the quantifiable value of the minimum pre-dawn leaf water potential (Ψ_PDmin). Ψ_PDmin is the maximum level of water deficit experienced by the vine during grape ripening; the more negative this value, the higher the vine water deficit. Carbon-13 isotope ratio was quantified on bulk wine by irm-EA/MS. The results are plotted in Fig. 3 as a function of the minimum pre-dawn leaf water potential. A correlation coefficient of 0.69 is computed revealing the link between wine ethanol δ¹³C value and Ψ_min, i.e., vine water status during grape ripening.

Figure 3. Relation between ΨPDmin and global ethanol δ13C ratio (black) determined by irm-EA/MS and with position-specific 13C deviation of ethanol (δ13CCH2: squares, δ13CCH3 triangles) determined by irm-13C NMR.

As photosynthetic is a fractionating process, it was interesting to investigate if it has some impact on the carbon-13 repartition of the ethanol carbonated skeleton and then, determine if a better correlation could be established between stereospecific carbon-13 composition and pre-dawn leaf water potential. These measurements have been performed on the ethanol recuperated by distillation of the wine previously described by irm-¹³C NMR. A typical ¹³C NMR spectrum is presented on Fig. 4. The first peak (18 ppm) corresponds to the methyl carbon (C_I) and the second signal (58 ppm), to the methylene carbon (C_II). The quantification for each carbon is possible knowing δ¹³C ratio of the ethanol quantified by irm-EA/MS.

Figure 4. 13C-NMR spectrum of ethanol at natural abundance in DMSO-d6.

The concentration of carbon-13 is significantly different (Δ = 4.8 ± 0.8‰) between the two position CI and CII but it does not seem to be related to vine water status as the difference appears to be quite homogeneous. Moreover, this carbon-13 content heterogeneity on the ethanol carbonated skeleton is in accordance with previous studies [9, 10]. The results plotted on Fig. 3 show the correlation between intramolecular carbon-13 content and minimum pre-dawn leaf water potential. This correlation is slightly better with the C_II position which is in agreement with a previous work showing a significant correlation between δ¹³C_II ratio and the mean of atmospheric temperature during the last three months before harvest [10].

4. Conclusion

The first finding of this work is the similarity between δ¹³C of ethanol and bulk wine, as far as the wine has not been supplemented by oenological products.

The second result highlights the correlation between ethanol δ¹³C and vine water status. This correlation is also observable for intramolecular ¹³C distribution. As a result, two techniques can provide information on vine water status conditions during grape ripening.

The coupling irm-EA/MS is preferred as it is less expensive, faster and because the measurements can be performed directly on the wine.

References

All Figures

	Figure 1. Water deficit thresholds with respect to δ13C ratio adapted from REF 3.
In the text

	Figure 2. Relation between δ13C values of authentic wines and their ethanol for red (squares) and white (circles) wines.
In the text

	Figure 3. Relation between ΨPDmin and global ethanol δ13C ratio (black) determined by irm-EA/MS and with position-specific 13C deviation of ethanol (δ13CCH2: squares, δ13CCH3 triangles) determined by irm-13C NMR.
In the text

	Figure 4. 13C-NMR spectrum of ethanol at natural abundance in DMSO-d6.
In the text