Open Access
Issue
BIO Web Conf.
Volume 15, 2019
42nd World Congress of Vine and Wine
Article Number 02022
Number of page(s) 6
Section Oenology
DOI https://doi.org/10.1051/bioconf/20191502022
Published online 23 October 2019

© The Authors, published by EDP Sciences, 2019

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Any aqueous-organic or aqueous-alcoholic solutions, in particular samples of alcoholic products, can be characterized by composition at different hierarchical levels – isotope, atomic, fragment and component. Each of these provides complementary information to authenticate them. Universal method for determining the composition of stable isotopes of elements is Isotope Ratio Mass Spectrometry/Stable Isotope Ratio Analysis (IRMS/SIRA). Classical methods of elemental analysis of organic systems are also universal, well developed and automated. The fragment composition of organic multicomponent systems is carried out by the methods of nuclear magnetic resonance spectroscopy (NMR) and infrared spectrometry or Raman scattering. The quantitative component composition of such objects is determined by various methods – chromatographic, electrophoretic, mass spectrometry, optical and NMR. The last one is the only direct method for determining the absolute molar content of aqueous-organic/aqueous-alcoholic solution components without the use of authentic standard samples.

2. Rapid screening of the wine component composition by 1H NMR spectroscopy

1H NMR spectroscopy for authentication of wine is currently employed to characterize wine in term of targeted components [12] as well as non targeted fingerprint metabolomic analysis [34]. No targeted method requires a large array of preliminary information that is not always available.

Screening analysis implies a simplicity or lack of sample preparation of analyte, quantitative assessment of the content of components, speed for making an immediate decision [5]. 1H NMR is the ideal method for such screening of wines. The specificity of differences in the component composition of wines is determined by the geographical origin and some features of the region of grape growing, the composition of surface and ground water, soil, grape varieties, the technology of wine production. The latter factors are manifested in the content of minor components of wine, such as glycerol, organic and amino acids, etc. Registration of 1H NMR spectrum provides for a short time (few minutes) obtaining of a spectrum that allows calculating the water content (sum of all exchanging protons of groups OH and NH) and ethanol, glycerol, basic acids and residual carbohydrates. The calculation is based on measuring the signal areas of individual structural fragments of molecules of wine components. Here and below structural fragments are mean hydrogen-containing groups, which include only one atom other than hydrogen. For example, H2O, H2S, NH3, CH4, C2H4, C6H6 (benzene), C6H12 (cyclohexane) molecules contain only one chemically equivalent structural fragment, while the ethanol molecule contains three, and the glycerol molecule contains four. 1H and 2H NMR spectra of any compound contain individual signals of each structural fragment, the integral intensity of which is directly proportional to their content. As a quantitative marker for measuring of minor components, it is convenient to use a strong-field satellite of signal CH3 group of ethanol which has intensity 0.55% of main CH3 signal. Although measuring the content of minor components is more effective in the absence of satellite signals with additional irradiation of the sample at a frequency of 13C nuclei.

1H NMR spectrum gives direct quantitative information about the content of individual components in wine. However, it is not enough for the recognition of authentic wine,. 1H NMR spectrum identical to the spectrum of natural wine can correspond to an artificial mixture of water, ethanol and most minor components, in terms of their availability and known target spectrum. Those, 1H NMR spectrum of adulterated product can be indistinguishable from genuine wine. In principle, a similar method of “synthesis of the image” by a combination of different compounds can provide adulteration of the elemental composition of wine, known from the elemental analysis for the corresponding region. Therefore, a fast screening of content of components of wine by 1H NMR spectroscopy makes it possible to roughly evaluate the conformity of its composition to the declared origin, in particular on the content of ethanol, glycerol, acids and residual sugars. More importantly, registering spectrum in presence of a quantitatively introduced additive of an internal or an external standard (in a coaxial tube) allows to accurate measurement of integral intensities of the signal of 1H nuclei participating in a proton exchange, which is 97% or more due to water protons. Knowledge of this exact value is necessary for accurate determination of 2H isotope content in wine water, as an indicator of the absence of exogenous water addition.

3. About hydrogen isotopes and 2H NMR

Hydrogen is represented in nature by two stable isotopes, corresponding to nuclides 1H (protium) and 2H (deuterium) with an average prevalence of 99.985% and 0.015% (150 ppm), respectively. The natural content of 2H varies in the range from 40 to 200 ppm depending on the geographical location of substance, the source of its production and other reasons. Even in individual natural compound extracted from one plant, the relative content of deuterium in different hydrogen-containing structural fragments can differ by 4–5 times. In case of water, the range of variations in 2H content is 90–180 ppm.

Using 1H NMR spectroscopy only to estimate absolute content of 2H isotope in the structural fragment of molecules by reducing of integral intensities of signals 1H of these structural fragments is impossible, since errors of their measurement are two orders of magnitude higher than the variations in the content of nucleus 2H (± 50 ppm, i.e., 0.005%). The use of 2H NMR spectroscopy directly for these purposes has some limitations since the combination of low natural content of 2H isotope (four orders of magnitude lower than 1H) and the low relative sensitivity of the 2H NMR method (100 times relative to 1H) reduces the actual sensitivity of the method 106 times. However, the use of NMR equipment with high sensitivity and long-term stability removes this problem. Fourier spectrometers with operating frequencies for 2H nuclei 60 MHz and more allows receiving a 2H NMR signal with a signal-to-noise ratio of 100 for 1 g molecules up to 100 Da for one hour.

Precise measurement of 2H isotope content is promising for various areas of science: physics, chemistry, biology, medicine etc. For example, in the diagnosis of various pathologies according to the isotopic composition of hydrogen in biological fluids [6], differentiation of nature of juices and the products of their fermentation [78]. An important aspect of commodity examinations is the detection of adulteration of wines by adding water to them, the presence of exogenous ethanol and other components to imitate “fingerprint” of wine. To solve such problems, classical methods of IRMS/SIRA of hydrogen, carbon, and oxygen are widely used and discussed in a recent review [9]. Significantly less attention was paid to the methods of 2H NMR spectroscopy using external [6] or internal [1011] standards for quantitative measurements.

Table 1.

Physical and chemical characteristics of Chardonnay wines (2016 crop).

4. Detection of wine extension with water

The proof of authenticity and geographical origin of wine can be based on the correspondence of content of minor stable 2H, 13C, 17O, 18O isotopes to values characteristic of genuine wines of the corresponding region, which must be obtained for the individual components of wine – water, ethanol, glycerol, etc. Without separation, IRMS/SIRA method can only give the uninformative integral value of 2H/1H (δ2H), 13C/12C (δ13C) and 18O/16O (δ18O) ratios of an entire set of components.

Due to processes of evapotranspiration in plants and some chemical exchange during fermentation of wine water is always enriched in heavy isotopic form versus water absorbed during ripening from the soil. Detection of significant (30% and more) water addition to wine by official methods are based on analysis of isotopic ratio 18O/16O (δ18O) of wine water and comparison of this value with reference data from published scientific and/or official wine databank [1213].

A known approach to identification of much smaller additives in a wine of exogenous water is based on the measurement of values of isotopic ratios 18O/16O by IRMS/SIRA method in ethanol and wine water [14]. In genuine wines, these values differ by a characteristic value, variations of which are small for a studied set of objects of analysis. As in the case of the official method, this approach requires complete extraction or equilibration of water molecules by reference substance. In a conventional application, it is necessary to carry out complete extraction of ethanol. This leads to main drawbacks of separation techniques – the complexity of sample preparation, possible random errors due to errors in its implementation, as well as systematic, associated with the manifestation of kinetic and thermodynamic isotope effects in any separation processes. These reasons motivate the search for other methods without or with a minimum sample preparation.

NMR spectra of 1H, 2H, 13C and 17O of wine contain separate signals of all structural fragments of its macro and micro components. However, the range of variations in the content of these carbon and oxygen isotopes is much less than that of hydrogen and is comparable with errors of their quantitative measurements. This makes their accurate quantitative measurement even more laborious than isolating individual components of a solution for IRMS/SIRA analysis. Therefore, rapid screening of isotopic composition is possible only for 2H isotope.

The water signal in 2H NMR spectrum has a characteristic position (4.8 ppm) and does not overlap with signals of other structural fragments of wine. Its intensity, as in the case of 1H, even for wine containing 16% vol. of ethanol and 40 g/l of carbohydrates are more than 95% due to 2H nuclei of water. In the case of dry wines, about 98% of the intensity of this signal is determined by 2H nuclei of water, the rest is a contribution from 2H exchanging hydrogen atoms of hydroxyl groups of other wine components, primarily from ethanol and glycerol. Accounting for the contribution of the latter is quite simple from analysis of the 1H NMR spectrum. Integral intensity of the 2H signal of wine water is a characteristic of the region of origin of grapes, climatic features and year of its production, fermentation technology. Its use for strict authentication of wine requires a corresponding database of genuine wines of a particular region and year of production. An alternative approach is to measure the value of 2H content in wine water and compare it with an average content of this isotope in surface and ground waters of corresponding region. The wine water must always have 2H isotope content at 8–15 ppm higher than in external water due to transpiration of light isotopes during the growing season and isotopic redistribution during fermentation.

2H content of wine water is calculated by comparing the measured integral intensities of water signal of wine with the signal of the water sample with a known content of 2H isotope, corrected for the decrease in 1H signal integral intensity of wine water in relation to that of the pure water sample. This ensures that other components are accounted for. To obtain this correction from 1H NMR spectrum, various standards can be used – internal (additive of the substance causing reference signal) or external (separate coaxial tube of smaller diameter inside the tube with analyte). Integral intensities of compared signals in spectra should be of the same order, and internal additives should be inert and low volatile. The main advantages of such cumulative screening for water addition detection are the lack of sample preparation, speed due to optimization of measuring procedure and sufficient precision for making a conclusion based on the magnitude of an excess of 2H content of wine water relative to water of the grape growing region. The proposed limitation of this approach is the same as the official methods of EU Regulation. It means that detection of a small percentage of water is difficult.

The approbation of cumulative screening of 2H isotope content in wine water is implemented for a series of natural wines from grapes from some geographic areas of the Krasnodar region. Information for this and other regions of Russia was previously unknown. As follows from a recent review [9] the systematization of such information for wines from other regions of the world is also absent.

Table 2.

Physical and chemical characteristics of Cabernet Sauvignon wines (2016 crop).

5. Experimental approbation

Two series of measurements of 2H isotope content in wine water of the Krasnodar region of Russia were carried out. The first included 10 samples: 9 samples of red dry Cabernet Sauvignon wine produced from grapes of 2015 in its various parts, 1 sample of the same crop from Spain. Experimental parameters and the detailed results of this study were presented earlier [11].

In 2016, we selected 9 samples of grapes Cabernet Sauvignon and Chardonnay in various areas of the Krasnodar region in 30 kg each. The production of wines carried out according to a classical technology using the same yeast cultures for 10 days. Physical and chemical characteristics of wines are presented in Tables 1 and 2. All dry wines had rather high alcohol content – from 11.1 to 14.9% vol., which is due to high sugar accumulation in berries. High alcohol content in test samples was promoted as a high-quality wine and its further conservation of microbiological stability.

NMR spectra were recorded on JEOL JNM-ECA 600 (Japan) spectrometer, with operating frequencies on 1H 600 MHz, and on 2H 92.1 MHz. Calibrated tubes with a diameter of 10 mm and a length of 178 mm were used. Wine samples were filtered to eliminate possible mechanical impurities. As a standard, a coaxial internal tube (external standard) with an external diameter of 3 mm filled with water containing an additive of 0.2 M dissolved europium (III) trifluoromethylsulfonate was used, that shifts a NMR signal of 2H of water in internal tube by 2 ppm in a weak field relative to water signal of analyte. Registration parameters of NMR spectra are presented in Table 3.

6. Results and discussion

The method of measuring 2H isotope content in wine water without its isolation based on cumulative screening of wine by qNMR 1H and 2H was used first time by us in 2003 for searching of differences between Georgian wines and wines of other states [15]. Authentic wines of Georgia (Kakheti Region) were represented by Georgian side interested in protecting their products [15]. Samples of wines from other countries – Russia (Krasnodar region, as the geographic terrain closest to Georgia) and few of Western Europe countries (Germany, Italy, France and Spain) were purchased in Russian retail. The results obtained showed that 2H isotope content in water of Georgian wines has values in the range of 160–166 ppm, which is close to values for wines from France and Italy, somewhat higher values for wines from Germany and much less than for wine samples from Spain (about 170 ppm). In Russian wines values of 2H content in wine water is significantly lower and vary widely in a range of 146–158 ppm. This was explained as a combination of latitudinal and climatic factors and wine extension with water [15].

In 2017 an analysis of 9 samples of Cabernet Sauvignon wine (2015 crop) of the Krasnodar region from among the nominees of the Russian wine award was conducted. 2H isotope content of water varied in the range of 159–166 ppm in them with one exception (Fig. 1). Similar values have had some wines from Crimea peninsula. The highest value was again found to Spanish wine (168 ppm) [11]. Comparison of the results of measurements of 2H content of water in wines of 2002 and 2015 allowed establishing that the reason for the low content of 2H in the first case was wine extension with water.

To obtain reliable results on 2H isotope content in wines of Krasnodar region, 9 red and 9 white wines from grapes (2016 crop) of different areas of the Krasnodar region were made under standard conditions and 2H isotope content in water was measured. Measurement of ground water isotopic composition in areas where grapes were collected was also conducted. In all cases, the content of isotope 2H in ground water does not exceed 148 ppm, whereas its content in wine water of studied samples ranges from 157 to 164 ppm, i.e. more by 9–16 ppm (Figs. 2 and 3). The sum of experimental measurement errors of signal intensities in 1H and 2H spectra of analytes does not exceed ±1 ppm.

thumbnail Figure 2.

Results of determination of 2H isotope content of water in 9 samples of Cabernet Sauvignon wine 2016 (“0” value corresponds to reference water SMOW 155.76 ppm).

This suggests that for authentic wines from this region in 2016 the isotope 2H content value of wine water should not be less than 156 ppm, i.e. content of 2H in reference water SMOW (155.76 ppm). The analysis of an additional set of 20 commercial wines and wines from the Krasnodar region (2015–2018 crops) confirm such conclusion of the minimum value in the content 2H of authentic wine water from this region.
thumbnail Figure 3.

Results of determination of 2H isotope content of water in 9 samples of Chardonnay wine 2016 (“0” value corresponds to reference water SMOW 155.76 ppm).

The estimated criterion of the authenticity for wines from the Krasnodar region wines is not unique. It is easy to calculate that adding about 30% of water containing 148 ppm 2H isotope to an authentic wine with an ethanol content of 12% vol. and 2H isotope content of 160 ppm in wine water produces an adulterated product with an ethanol content of about 10% vol. and content of 2H isotope about 156 ppm, which is within the authentication range. Consequently, represented procedure which is like official methods according to the EU legislation [16], effective enough to detect wine adulterations. However, its labour intensity is disproportionately lower, since does not require sample preparation. Wine extension with water from other regions of Russia, in which content of 2H isotope in water is usually lower by 4–8 ppm, can be detected with significantly fewer water additives.

There is a known approach for detecting the addition of exogenous water to wine is the method of differential measurement of 18O/16O ratio in separated water and wine ethanol using IRMS/SIRA technique, which does not require any reference samples but uses complicated sample preparation technique [14]. It can be assumed that use of a similar approach for 2H NMR of ethanol of wine with a comparison of values of 2H content in CH3-, CH2- and OH-groups of ethanol molecule ((D/H)I, (D/H)II and (D/H)III) may be more informative [11].

Going beyond the question of wine adulteration with water, it should be noted that presented method of fast cumulative screening of 2H isotope content in water is universal for any aqueous-alcoholic or aqueous-organic solutions, for example, foodstuffs (e.g. mineral water, juices, drinks, wine, brandies), biological fluids, pharmaceuticals, etc. To identify the raw material nature of ethanol in wine, we have applied the ALKOSCAN method [17], which uses the same sample preparation and the same approach as SNIF-NMR developed earlier [18], but which has a few fundamental differences that expand its capabilities. The most important of them is the measurement of signal integral intensity instead of their heights, which makes it possible to implement it on any NMR spectrometers without stabilization by 19F signal and allows measuring the content of 2H isotope in the OH group of ethanol. The use of external or internal standards enriched with 2H isotope for calibration allows minimizing its quantity and halving the time of each measurement on any spectrometer.

Table 3.

Registration parameters of NMR 1H and 2H spectra.

thumbnail Figure 1.

Results of determination of 2H isotope content of water in wines of the 2015 crop (“0” value corresponds to water standard SMOW 155.76 ppm).

7. Conclusion

A new approach for screening of wine authenticity by qNMR 1H & 2H has been proposed, which allows a measurement of 2H isotope content in wine water without sample preparation. This allows to make a conclusion about wine authenticity or to identify significant wine extension with water. Having predictive capabilities comparable to the official EU/OIV methods, a new method is significantly simpler and faster. It is applicable for determinations of 2H isotope in water of any aqueous-alcoholic or aqueous-organic solutions. To identify the use of exogenous carbohydrates in the production of wine, a method like SNIF-NMR is convenient, but it has some positive differences that ensure the expansion of its informativeness, universality and sensitivity. Further detailed studies will be conducted on wines from the Crimea peninsula.

References

  • S. Esslinger, C. Fauhl-Hassek, R. Wittkowski, Advances in Wine Res. 6 (2015) [Google Scholar]
  • E. Lopez-Rituerto, F. Savorani, A. Avenoza, J.H. Busto, J.M. Peregrina, S.B. Engelsen, J. Agric. Food Chem. 60, 3452 (2012) [CrossRef] [PubMed] [Google Scholar]
  • M. Amargianitaki, A. Spyros, Chem. Biol. Technol. Agric. 4, 9 (2017) [CrossRef] [Google Scholar]
  • Y.-Sh. Hong, Magn. Res. Chem. 49, 13 (2011) [CrossRef] [Google Scholar]
  • M. Varcarcel, S. Caerdenas, M. Gallego, Trends Anal. Chem. 18, 11 (1999) [Google Scholar]
  • S.S. Dzhimak, A.A. Basov, L.V. Fedulova, I.M. Bykov, V.A. Ivlev, K.I. Melkonyan, A.A. Timakov, Aviakosmicheskaya i Ekologicheskaya Meditsina 50, 42 (2016) [Google Scholar]
  • G.J. Martin, M.L. Martin, F. Mabon, M.J. Michon, Anal. Chem. 54 (1982) [Google Scholar]
  • EC-Regulation 2676/1990, Off. J. Eur. Commun. 64, L272 (1990) [Google Scholar]
  • N. Christoph, A. Hermann, H. Wachter, Bio Web Conf. 5 (2015) [CrossRef] [Google Scholar]
  • X. Ma, P. Deng, X. Wang, C. Zhang, X. Hou, Talanta 97, 45 (2012) [Google Scholar]
  • G.A. Kalabin, V.A. Ivlev, N.A. Komarov, A.Yu. Kolesnov, Analytika 39, 94 (2018) [Google Scholar]
  • G. Calderone, C. Guillou, Food Chem. 106, 1399 (2008) [Google Scholar]
  • F. Tomas, E. Jamin, D. Hammond, J. AOAC Int. 96, 615 (2013) [Google Scholar]
  • M. Perini, F. Camin, J. Food Sci. 78, 839 (2013) [Google Scholar]
  • G.A. Kalabin, Yu.P. Kozlov, R.S. Rykov, N.V. Kulagina, D.F. Kushnarev, A.V. Rokhin, RUDN Univ. Ecol. Life Sci. 9, 77 (2003) [Google Scholar]
  • Council Regulation (EC), No 491/2009 (2009) [Google Scholar]
  • G.A. Kalabin, N.V. Kulagina, R.S. Rykov, Yu.P. Kozlov, D.F. Kushnarev, A.V. Rokhin, RUDN Univ. Ecol. Life Sci. 7, 87 (2003) [Google Scholar]
  • OIV-MA-AS311-05, Method for determination of the deuterium distribution in ethanol derived from fermentation of grape musts, concentrated grape musts, rectified concentrated grape musts and wines by application of nuclear magnetic resonance, Compendium of International Methods of Wine and Must Analysis 1 (2019) [Google Scholar]

All Tables

Table 1.

Physical and chemical characteristics of Chardonnay wines (2016 crop).

Table 2.

Physical and chemical characteristics of Cabernet Sauvignon wines (2016 crop).

Table 3.

Registration parameters of NMR 1H and 2H spectra.

All Figures

thumbnail Figure 1.

Results of determination of 2H isotope content of water in wines of the 2015 crop (“0” value corresponds to water standard SMOW 155.76 ppm).

In the text
thumbnail Figure 2.

Results of determination of 2H isotope content of water in 9 samples of Cabernet Sauvignon wine 2016 (“0” value corresponds to reference water SMOW 155.76 ppm).

In the text
thumbnail Figure 3.

Results of determination of 2H isotope content of water in 9 samples of Chardonnay wine 2016 (“0” value corresponds to reference water SMOW 155.76 ppm).

In the text

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