Open Access
Issue
BIO Web Conf.
Volume 9, 2017
40th World Congress of Vine and Wine
Article Number 02020
Number of page(s) 8
Section Oenology
DOI https://doi.org/10.1051/bioconf/20170902020
Published online 04 July 2017

© The Authors, published by EDP Sciences 2017

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).

1. General climatic conditions in the areas of the study

1.1. Black sea

The grapes samples from two main regions of the Black Sea have been used in the study – Crimean peninsula and South West Coast of the Greater Caucasus (Fig. 1). The sampling of the authentic fresh grapes was carried out in 5 viticulture areas of the Crimean Peninsula – Western Maritime-Steppe area (villages Romashkino and Plodovoye), Western Foothills-Maritime area (village Uglovoye), Eastern Mountain-Valleey Maritime zone of the South Coast area (village Morskoye) and East foothill zone of the South Coast area (village Koktebel). The total area of sampling vineyards made 512.0 ha. The main areas on the South West Coast of the Greater Caucasus are the Temryuk, Krymsk and Anapa districts, and also areas close Novorossiysk. The total area of vineyards on South West Coast, in which in a season of 2016 grapes samples have been taken, has made 215.02 ha. Scientific data on the Black Sea areas, including climate, soils and grapes varieties have been reported in the previous publication about results of the study of 2015 [1].

thumbnail Figure 1.

Black Sea areas.

1.2. Don basin

The grapes were studied in two areas – Tsimlyansky (Tsimlyansky Reservoir coast) and Martynovsky districts (Fig. 2). The total area of the surveyed vineyards has made 80.60 ha.

thumbnail Figure 2.

Don Basin areas.

thumbnail Figure 3.

Western Caspian.

1.3. Western caspian

The main area of viticulture is the The Republic of Dagestan. The list of areas of this region, in which grapes samples have been taken, had included the Derbent, Sergokalinsky, Suleyman-Stalsky and Magaramkentsky districts. The total area of the surveyed vineyards has made 375.04 ha.

2. Grape samples

2.1. Varieties

The samples of the 9 white and red grape varieties were used for the study – Aligote, Rkatsiteli, Cabernet Sauvi- gnon, Sauvignon Green (Crimean Peninsula); Cabernet Sauvignon, Chardonnay (South West Coast – Krasnodar region); Bianka, Saperavi Severniy, Tsimlyansky Cherniy (Don Basin); Rkatsiteli, Riesling, Cabernet Sauvignon (Western Caspian – The Republic of Dagestan). The sampling of the authentic fresh grapes was carried out in 15 viticulture areas in shown four regions. The total area of sampling vineyards made 1182.66 ha. The sampling was conducted according to the requirements described in [2].

2.2. Main physical-chemical characteristics

The research program included 43 samples of grapes of the above stated varieties. The fresh musts were obtained under the laboratory conditions. Gentle conditions for the extraction of musts exclude any changes in the natural composition of carbon and oxygen isotopes in the target components of grapes – carbohydrates (sugars) and intracellular water. In order to ensure the microbiological stability the musts samples have been pasteurized and filled in glass containers under aseptic conditions. Before the isotopic measurements the must samples were stored at a temperature from 2 to 4 ◦C in sealed sterile glass containers without access of air.

36 samples of dry white and red wines (residual sugar - from 0.5 to 3.6 g/l) have been made from the fresh musts under laboratory conditions. The fermentation of fresh musts was carried out using a pure 47 K yeast culture, which was added in an amount of 2% of the must volume. The SO2 in an amount of 75–100 mg/l was added to musts before fermentation. Prior to the isotopic measurements the dry wines were stored at a temperature from 2 to 4 ◦C in sealed sterile glass containers without access of air.

The mass concentration of reducing sugars in the studied grape samples has been from 17.5 to 25.0 g/100 ml, titrated acids concentration (based on tartaric acid) - from 6.0 to 9.1 g/l, the buffer capacity 34.1–63.2 mg- Eq/l. Red and white wine made from respective grapes contained from 0.5 to 3.6 g/l of residual sugar; from 11.1 to 14.5% ethanol by volume; buffer capacity was 35.2–52.6 mg-Eq/l. The additional scientific data about buffer capacity measurements in musts and wines are published in [3, 4].

3. Stable isotopes compositions of studied grape samples

Scientific study of compositions of stable isotopes of carbon (13C/12C) and oxygen (18O/16O) was carried out using two types of IRMS/SIRA1 technique - redox transformation (Flash Combustion - FC-IRMS/SIRA) and isotopic exchange/ equilibration (Equilibration - EQ-IRMS/SIRA). Methodological principles of the FC- IRMS/SIRA and EQ-IRMS-SIRA methods have been described in previous publication [1] and in OIV methods [5, 6].

3.1. Materials and methods

3.1.1. Musts and wine samples

The musts and dry wines were used for measurements of the composition of the carbon isotopes 13C/12C in carbohydrates (sugars) and ethanol by the FC-IRMS/SIRA method. The composition of oxygen isotopes 18O/16O was studied by the EQ-IRMS/ SIRA method directly in musts and dry wines without prior isolation of water from the samples.

3.1.2. Water samples

To study the composition of stable isotopes of oxygen (18O/16O) 19 water samples were taken in all studied areas - two samples from the Crimean Peninsula (water from atmospheric precipitations & underground source), two samples from the Don Basin (water from atmospheric precipitations & underground source), twelve samples form the South West Coast (water from atmospheric precipitations, tap water used for irrigation of vineyards, water from the Anapka river, city tap water and water from underground sources) and three samples from the Western Caspian (water from atmospheric precipitations, city tap water and water from underground sources).

The water samples were stored before the isotopic measurements at a temperature from 2 to 4 ◦C in sealed sterile glass containers without access of air.

3.1.3. FC-IRMS/SIRA method for δ13CVPDB analysis

The study of fractionation effect of the stable isotopes of carbon (13C/12C) in grape and wine components (carbohy- drates (sugars), ethanol) using the FC-IRMS/SIRA method was carried out by using of the following laboratory facilities for stable isotopes measurements:

  1. an elemental analyzer FlashEA1112® with oxida- tion & reduction reactors and autosampler (Thermo Fisher Scientific, Germany);

  2. an isotopic interface Conflo III® (Thermo Fisher Scientific, Germany);

  3. an IRMS/SIRA isotopic mass spectrometer Delta V Advantage® (Thermo Fisher Scientific, Germany);

  4. a gas system Sigm-Plus® for the supply of the analytical devices with highly purified gases (Sigm- Plus Ltd., Russian Federation);

  5. a PC workstation Optiplex 745® (Dell, USA) for data registration and processing by the high level software Isodat NT 2.5® (Thermo Fisher Scientific, Germany).

The reference substance IAEA-600 (Caffeine, δ13C = −27.771%) was used for the calibration of the working reference gas (WRG) – carbon dioxide 99.9999% (Linde Gas RUS, Russian Federation). The high purity helium 99.9999% (Linde Gas RUS, Russian Federation) was used in the study as carrier gas.

Sample preparation – extraction and purification of carbohydrates (sugars) from musts obtained from fresh grapes – was performed according to the method described in [7]. The isolated and purified preparations of carbohydrates (sugars) were freeze-dried at −50oC and 0.035 mbar pressure in the laboratory lyophilic facility FreeZone12 (Labconco Corporation, USA). The preparations were stored at a temperature no higher than -20 ◦C in a sealed glass container. Ethanol was extracted from dry wines by using of the laboratory glass distillation unit according to the conditions for the ethanol extraction as described in [5, 8]. Extracted ethanol samples were stored at a temperature no higher than −20 ◦C in a sealed glass container.

The above mentioned laboratory facilities have been used for isotope measurements. All measurements were carried out taking into account guidelines published in [1, 911]. Joint hinges of preparations of carbohydrates (sugars) containing from 40 to 100 ng of carbon were placed in tin capsules before carrying out the measurements. The capsules were thoroughly covered with the help of microtweezers, providing its full integrity and eliminating the contact of organic matter with the atmospheric air. The encapsulated hinges (in five replicates for each sample) were placed into the autosampler mounted on the oxidation reactor of the elemental analyzer (A). Along with the hinges of carbohydrates (sugars) the encapsulated reference substance was loaded into the autosampler for control of reliability of measurement’s results. Ethanol sapmles were injected directly into oxidation reactor of the elemental analyzer (A) by using of a microsyringe. The injection volume of each ethanol sample was from 0.08 to 0.1 μl.

The conditions of carring out measurements of isotope composition of carbon in the preparations of carbohydrates (sugars) and ethanol by using the stated above laboratory facilities corresponded to the optimum modes which are developed during the preliminary runs. Quantitative combustion of carbohydrates (sugars) and ethanol was performed in a helium flow (flow rate 100 ml/min) in the oxidation and redox reactors of the elemental analyzer (A) at the temperature of 900 and 650 ◦C in the presence of molecular high purity oxygen 99.999% (Linde Gas RUS, Russian Federation). The separation of elementary gases resulting from the combustion of carbohydrates (sugars) or ethanol in the redox reactor was carried out in automatic mode by the chromatographic purification in the elemental analyzer (A). Water contained in the gas flow was removed by the trap column filled with the magnesium perchlorate.

For the quantitative measurement of carbon isotopes 13C/12C, the isolated carbon dioxide is directed through isotopic interface (B) to the IRMS/SIRA mass spectrom- eter Delta V Advantage® (C). Accelerating voltage of the mass spectrometer was 3.07 kV, pressure in the ion source −1.8 × 106 kPa, ionization method - electron impact (electron energy 124 eV).

The conducting of a simultaneous correction of measured values of signal strength is an important aspect for control of accuracy of results to exclude the possible influence of isobars 12C17O17O and 13C17O16O which share is determined at the hardware level by the measurement of a signal of carbon dioxide of mass 45 taking into account the extent of the distribution of the isotopes 13C and 17O in the nature (respectively 1.11 and 0.0375%).

3.1.4. EQ-IRMS/SIRA method for δ18OVSMOW analysis

The study of fractionation effect of the stable isotopes of oxygen (18O/16O) in the intracellular water in grapes (musts) and water samples using the EQ-IRMS/SIRA method was carried out by using of the laboratory facilities for stable isotopes measurements:

  1. a module used for sample preparation for the analysis (Thermo Fisher Scientific, Germany);

  2. a control unit for chromatographic purification and samples injection Gasbench II® (Thermo Fisher Scientific, Germany);

  3. an IRMS/SIRA isotopic mass spectrometer Delta V Advantage® (Thermo Fisher Scientific, Germany);

  4. a gas system for the supply of the analytical devices with highly purified gases Sigm-Plus® (Sigm-Plus Ltd., Russian Federation);

  5. a PC workstation Optiplex 960® (Dell, USA) for data registration and processing by the high level software Isodat 3.0® (Thermo Fisher Scientific, Germany).

The reference substance IAEA-600 (Caffeine, δ13C = −27.771%) was used for the calibration of the working reference gas (WRG) – carbon dioxide 99.9999% (Linde Gas RUS, Russian Federation). The high purity helium 99.9999% (Linde Gas RUS, Russian Federation) was used in the study as carrier gas. The additional reference substances (all from the IAEA, Austria) with known composition of oxygen isotopes 18O/16O were used for adjustment of measurement’s results: VSMOW2 (Vienna-Standard Mean Ocean Water, δ18O = 0.00%); GISP (Greenland Ice Sheet Precipitation, δ18OVSMOW = −24.76%); SLAP2 (Standard Light Antarctic Precipita- tion, δ18OVSMOW = −55.50%).

High purity gases were used in the study – flushing gas – helium 99.9999%, containing 0.4–0.5% of high purity carbon dioxide (99.9999%), carrier gas - helium 99.9999% (all from Linde Gas RUS, Russian Federation).

Sample preparation for the direct measurement of oxygen isotopes in intracellular water was carried out by isotopic equilibration technique (EQ-IRMS/SIRA method). For isotopic equilibration reaction, 0.500 ml of grape must or 0.200 ml of water was added into a 12 ml round-bottom borosilicate glass vial. The tubes were hermetically sealed by disposable screw cap with Teflon- silicone membrane. 0.200 ml of reference substances VSMOW2, GISP and SLAP2 were added separately in similar tubes.

To carry out measurement, we formed series (groups) of tubes. Each group included the required number of sample tubes and test tubes with reference substances. A batch of tubes was placed in the cells of the sample preparation module (A). Results of preliminary experiments on the duration of the isotopic equilibration reactions show that the equilibrium state occurs after at least 18 hours. The optimum temperature for the reaction was 24.0 ± 0.1◦C.

At the end of the isotope exchange reaction the gas mixture from each tube was transferred to the control unit Gasbench II® (B). Gas mixture was dried over by molecular sieve membrane Nafion® and transferred to the gas chromatograph installed in the control unit Gasbench II® (B). Chromatographic separation is carried out in helium flow (2 ml/min), column used – PLOT Fused Silica, stationary phase CP-PoraPLOT Q, length 25 m, diameter 0.32 mm (Varian, USA), column temperature 70 ◦C. Temperature of samples in the sample module (1) was maintained at a constant level of 24.0 ± 0.1 ◦C until the end of the measurements.

Quantitative measurements of oxygen isotopes 18O/16O were performed according to conditions described above and in [12]. During the measurement cycle the WRG calibrated against a reference substance is introduced into the mass spectrometer – five portions WRG at the beginning of the cycle and three portions WRG at the end. Measurement of each sample was performed in triplicate in series of 10 impulses. For subsequent calculation of the δ18OVSMOW value (%), the intensities of 7, 8 and 9th impulses of the carbon dioxide with masses 46 and 44 should be used. Registration and processing of measurements results, as well as control for all devices of the analytical system is done via a PC workstation (E).

The results of measurements calculated on the basis of the recorded signals for the masses 44 and 46 are subjected to further adjustment. This adjustment takes into account the linear relationship between the values δ18OVSMOW of used reference substances VSMOW2, GISP, SLAP2 and the values measured for these references in each batch of samples. The correlation coefficient of the linear dependence must be not lower than 0.99999. Otherwise, the measurement for a particular series of samples, including measurements of reference substance included in this batch, have to be repeated until the above conditions are met.

4. Results and discussion

4.1. Compositions of stable isotopes in grapes, dry wines and waters

The results of measurements of isotope compositions of stable carbon & oxygen isotopes presented as δ13CVPDB and δ18OVSMOW values, are shown below in Tables 19. The tables contain the average values obtained in a batch of five parallel determinations of carbon isotopes measurements for carbohydrates (sugars) and ethanol of each studied grape variety and dry wines samples, as well as in a batch of three parallel determinations of oxygen isotopes measurements in the intracellular water of each grape variety and water samples from surface, atmospheric and underground sources.

Table 1.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Crimean Peninsula (crop of 2016).

Table 2.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the South West Coast of the Greater Caucasus (crop of 2016).

Table 3.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Don Basin (crop of 2016).

Table 4.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Western Caspian (crop of 2016).

Table 5.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Crimean Peninsula (crop of 2016).

Table 6.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the South West Coast of the Greater Caucasus (crop of 2016).

Table 7.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Don Basin (crop of 2016).

Table 8.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Western Caspian (crop of 2016).

Table 9.

Composition of stable oxygen isotopes (18O/ 16O) in water samples from selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (study of 2016).

The expanded measurement uncertainty (EMU) of each batch of determinations did not exceed 0.400 (for carbon isotopes) and 0.420 (for oxygen isotopes). For the calculation of the EMU the coverage factor of 2 was used that corresponds to a confidence level of 95%.

5. Conclusions

Summarizing the data clusters obtained within the research program on the white and red grape’s samples taken in the Crimean Peninsula, South West Coast of the Greater Caucasus (Krasnodar region), Don Basin and Western Caspian allows to set the ranges of the natural fractionation of stable carbon and oxygen isotopes in components of this plant within the season of 2016 (Tables 1011).

Table 10.

Ranges for δ13CVPDB and δ18OVSMOW in carbohydrates (sugars) and intracellular water of white and red grape varieties of the selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (crop of 2016).

Table 11.

Range for δ13CVPDB in ethanol of dry white and red wines made from grape varieties of the selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (crop of 2016).

The results of our study characterizing the fractionation of carbon isotopes in native carbohydrates (sugars) and also oxygen isotopes in intracellular water as well as in wines correlate very well with the results of our study of 2015 in the Crimean Peninsula [1] and the published data of other researches, which were conducted in other geographical regions with similar agroclimatic conditions, frequent water deficiency and an irregular irrigation (or total absence of that) during the vegetative period. It was shown that the carbohydrates (sugars) of grapes which are grown up in the conditions of water deficiency and high level of active temperatures are enriched by “heavy” 13C carbon isotopes [1323]. For example, C. van Leeuwen et al. suggested in [15], according to the results of their studies, the following dependence of the range for δ13C of sugars on the water status during the grape cultivation:

  • “no water deficit” … <−26.0%,

  • “weak water deficit” … from −24.5 to −26.0%,

  • “moderate to weak water deficit” … from −23.0 to −24.5%,

  • “moderate to severe water deficit” … from −21.5 to −23.0%,

  • “severe water deficit” …>−21.5%.

In our study the width of the range of δ13CVPDB values for natural carbohydrates (sugars) in 9 white and red grape varieties amounts to 6.09%.

Carbohydrates (sugars) of grape varieties from four geographical regions regarding the carbon isotope composition are almost equivalent in accordance with the experimental data from this study, taking into account the quantitative levels of measurement uncertainty. In this part the results of the study correlate well with published data from other scientific researches [1820].

The research results for water samples show the δ18OVSMOW values, which are characteristic for the natural fractionation of oxygen isotopes in the atmospheric and geological underground waters (Table 9).

The results of the study can be used in practice for quality assessment, including identification of commercial products offered on national and international foodstuffs markets (e.g. wines from the regions with a Geographical Indication (GI) or Designation of Origin (DO)).

However, in applying the results of this study, including above shown intervals of δ13CVPDB and δ18OVSMOW for grapes from the Crimean Peninsula, South West Coast of Greater Caucasus, Don Basin and Western Caspian, for example, when planning a scientific or an applied work, it is necessary to take into account interrelations of the obtained experimental data with agroclimatic conditions of cultivation and harvest of 2016, and also to consider the size of a data matrix which is defined by a quantity of samples, climate peculiarities sampling areas and grape varieties.

We thank Non-profit Self-regulating Organization “Winegrowers & Winemakers” (Krasnodar Region), Union of Winegrowers & Winemakers of Russia (SVVR), winegrowers and wineries from the Crimean Peninsula, Don Basin and Western Caspian regions for supporting of the scientific study, especially for assistance with sampling of authentic grapes.


1

IRMS/SIRA = Isotope Ratio Mass Spectrometry/Stable Isotope Ratio Analysis.

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All Tables

Table 1.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Crimean Peninsula (crop of 2016).

Table 2.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the South West Coast of the Greater Caucasus (crop of 2016).

Table 3.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Don Basin (crop of 2016).

Table 4.

Composition of stable carbon isotopes (13C/12C) in carbohydrates (sugars) of grape varieties and ethanol of dry wines of the selected areas of the Western Caspian (crop of 2016).

Table 5.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Crimean Peninsula (crop of 2016).

Table 6.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the South West Coast of the Greater Caucasus (crop of 2016).

Table 7.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Don Basin (crop of 2016).

Table 8.

Composition of stable oxygen isotopes (18O/ 16O) in intracellular water of grape varieties from selected areas of the Western Caspian (crop of 2016).

Table 9.

Composition of stable oxygen isotopes (18O/ 16O) in water samples from selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (study of 2016).

Table 10.

Ranges for δ13CVPDB and δ18OVSMOW in carbohydrates (sugars) and intracellular water of white and red grape varieties of the selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (crop of 2016).

Table 11.

Range for δ13CVPDB in ethanol of dry white and red wines made from grape varieties of the selected areas of the Crimean Peninsula, South West Coast of the Greater Caucasus, Don Basin and Western Caspian (crop of 2016).

All Figures

thumbnail Figure 1.

Black Sea areas.

In the text
thumbnail Figure 2.

Don Basin areas.

In the text
thumbnail Figure 3.

Western Caspian.

In the text

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