Wine Authentication Using Stable Isotope Ratio Analysis: Significance

Chapter 11

Downloaded by UNIV OF GUELPH LIBRARY on June 5, 2012 | http://pubs.acs.org Publication Date: December 28, 2006 | doi: 10.1021/bk-2007-0952.ch011

Wine Authentication Using Stable Isotope Ratio Analysis: Significance of Geographic Origin, Climate, and Viticultural Parameters 1

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N. Christoph , A. Rossmann , C. Schlicht , and S. Voerkelius 1

Department of Beverage Analysis, Bavarian Health and Food Safety Authority, Luitpold Strasse 1, D97082 Würzburg, Germany Isolab GmbH, D-85301 Schweitenkirchen, Germany Bavarian Health and Food Safety Authority, D-85762 Oberschleissheim, Bavaria Hydroisotop GmbH, D-85301 Schweitenkirchen, Germany 2

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Stable isotope ratio analysis of wine by H-SNIF®-NMR, C-, and O-isotope ratio mass spectrometry are official methods in the European Union for the proof of chaptalization, addition of water, sweetening with sugar, and authentication of geographic origin and year of harvest. By evaluation of stable isotope data of authentic reference wines from the German wine-growing regions Franconia and Lake Constance, Hungary, and Croatia as well as wines which were on the German market, the influence of geographical origin, climate, year and date of vintage, and viticultural aspects (soil water status, water stress, irrigation) on isotopefractionationof C, H , and O in water, sugar, and alcohol are discussed. 18

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© 2007 American Chemical Society In Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Review on authentication of wine by stable isotope ratios Authentication of wine is the analytical process by which a wine is verified as in compliance with its label description. Adulteration of wine was already prosecuted and punished at the age of Hamurabi (1792 - 1750 B.C.), but also nowadays mislabelling of geographic origin, addition of sugar and/or water to pretend a better wine quality or to increase its amount are ascertained again and again. Since these adulterations became more and more sophisticated, it was necessary to develop sophisticated analytical methods for a significant proof of wine authenticity. During the last 30 years many studies on the intermolecular and intra-molecular non-statistical distribution of stable isotopes of the bioelements C , H , 0 , N , and S in natural compounds have been published; it was found that the distribution of stable isotopes in bio-molecules is controlled by logical principles including biotic and abioticfractionationprocesses which result in a pattern characteristic for the plant species and its geographical origin (7). The most important biotic fractionation is that of carbon and hydrogen which takes place during photosynthesis and biosynthesis of sugar and other components in the plant. C / C - and H/ H-isotope ratios of sugar and its related ethanol obtained by fermenting in the same water are primarily determined by two different biosynthetic pathways of biological carbohydrate formation, the Hatch-Slack pathway of Copiants such as corn and sugar cane with higher C - and H-concentrations, and the Calvin pathway of C -plants such as wheat, sugar beet, or grapevine. The knowledge of stable isotope fractionation in plants and their fruits promised to be a powerful tool for authentication of food products produced from these raw materials. Cases of adulteration of fruit juice or control of chaptalization of wines with sucrose which is restricted to some northern situated wine-growing regions of the EU, stimulated further research to develop reliable methods for the proof of such oenological treatments. Stable Isotope Ratio Analysis (SIRA) in official wine control started 1990 in the EU by adopting the SNIF®-NMR-Method' for the detection of chaptalization of grape must and wine by H-Nuclear Magnetic Resonance (NMR) of ethanol (2), followed by 0 - and C-Isotope Ratio Mass Spectrometry (IRMS) (3,4). Table I summarizes the relevant methods, molecules, isotopomers, ratios, symbols, and units of SIRA. The principle of the SNIF®-NMR-method, developed by Prof. Martin (5,6,7) is based on the observation that the deuterium of the sugar- and water-molecule is transferred during fermentation by Site-Specific Natural Isotope Fractionation ('SNIF') into the methyl- and the methylene-position of the ethanol molecule. Approximately 85 % of deuterium in the sugar molecule are transferred during fermentation into the methyl-group of ethanol expressed by the (D/H),-ratio and about 75 % of the 13

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In Authentication of Food and Wine; Ebeler, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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deuterium of grape water into the methylene-group of ethanol expressed by the (D/H) -ratio. n

Table I. Stable isotope ratio analysis used for wine authentication Analytical Method


SNIF®NMR 13

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C/ CIRMS

Component Isotopomer

Symbol, unit

CH DCH OH CH3CHDOH

(D/H), ; ppm (D/H)„;ppm

Ethanol, sugar, organic acids glycerol, C 0

Ô C; [%>] V-PDB

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proof of chaptalization geographic origin geographical origin, climate proof of C -sugar, synthetic glycerol, acids, and C 0 geographical origin, year of vintage, addition of water 4

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0/ 0 IRMS

Authentication parameters

Water, ethanol,

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δ 0;[%ο] V-SMOW 18

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D= Deuterium ( H); V-SMOW= Vienna Standard Mean Ocean Water, δ 0= 0 %o\ V-PDB= Vienna Pee Dee Belemnite, 5 C= 0 %o V-PDB 13

The (D/H) ratio represents the botanical origin of the fermented sugar whereas the (D/H)„-ratio is typical for the deuterium content of the grape-water and reflects the climatical conditions related to the geographical origin and the year of vintage. Figure 1 elucidates that the (D/H),-ratio of ethanol from beet sugar (92.5 +/-1 ppm) is significantly lower compared to that of ethanol from wine (98-104 ppm, extreme cases up to 107 ppm). By chaptalization of grape must the original (D/H) ratio of the related ethanol significantly decreases in correlation with the amount of beet-sugar. In the same way the (D/H) ratio increases by use of C -sugars like cane sugar with (D/H),-ratios between 109 and 112 ppm. Since it is possible to use mixtures of beet and cane sugar, in order to simulate wine-typical (D/H),-values, it is always necessary to determine both the (D/H),- and 8 C-value of grape sugar and wine ethanol respectively; the C amount does not change significantly in case of an addition of beet sugar, but dramatically by use of cane sugar. The significance of the 0/ 0-ratio of water in grapes and wine was already discussed in 1982 by Dunbar (8). Due to evaporation of water in the grape during ripening, an enrichment of 0 takes place, leading to values higher than those of the ground water which is transported via roots into the fruits. This process is influenced by geographic origin and climate. The 5 0-value in grape water therefore is a characteristic marker for the geographical origin of the grapevine and its climate, and can be used to prove addition of ground- or tapwater (8 9,10,12,13,17,18). r

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Since authenticity testing of a wine by SIRA must be related to analytical data of a set of reference samples which are as close as possible to origin, year, date of vintage, and cultivar of the sample to be analysed, it was necessary to establish an official databank which contains authentic and representative samples of all EU wine-growing regions. Corresponding to the actual regulation of the European Commission (77) for this analytical EU Wine Databank (EUWDB), about 1400 samples of grapes (15 kg) are taken each year by official controllers in the wine-growing regions of the EU; after micro-vinification the wines are analysed with SIRA in official institutes and the data enter the EUWDB. SIRA actually is not only used for the authentication of wine (7,9,12, 13), fruit juice (14), and spirits (75) but also for authentication of many other food products (16).

Figure 1. Proof of chaptalization or sweetening of wine with beet-, cane-sugar, and mixtures of beet- and cane-sugar by SNIF-NMR® and C-IRMS of ethanol. 13

Material and Methods 13

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The determination of (D/H)- and C/ C-isotope ratios of ethanol in wine and of 0/ 0-isotope ratio in grape and wine water were performed according to the official analytical methods of the EU. For H-SNIF-NMR®-analysis (2) 18

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authentic grape-musts were fermented with yeasts (Saccharomyces Cerevisia). Wines were distilled with spinning band columns using automatic vapour separation. Water content of distillates (6-9 % mas) was determined by densitometry. Acquisition of H-NMR spectra of ethanol was performed by a BRUKER 400ARX spectrometer with a fluorine lock-channel and a probe-head tuned to the deuterium frequency of 61.42 MHz. 2.0 ml distillate and 2.0 ml TMU (Ν,Ν-Tetramethylurea, Reference Standard STA003, Institute for Reference Materials and Measurements, B-2440 Geel, Belgium) were weighed in a bottle and transferred into a 10 mm NMR-tube, adding 50 μΐ CÔFO as a lock substance. The deuterium spectra were recorded with an acquisition time of 6.7 s, a 25 μϊ pulse (90° flip angle) and 10 experiments per sample with 256 scans each. Processing of the spectra and calculation of (D/H) and (D/H)n-ratios versus the certified (D/H)-ratio of TMU was performed with EUROSPEC® software. The standard deviation of (D/H) measurement is less than 0.4 ppm (2). For C/ C-IRMS of ethanol a Vario EL elemental analyser (Elementar Analysensysteme GmbH, Hanau, Germany), equipped with a solid sample autosampler on top of the combustion furnace was used for fully automated stable isotope analysis. Ethanol samples (3 μΐ) and reference materials were filled into gas-tight tin capsules using a microliter-syringe and the capsules were sealed by a mechanic capsule press. The EA was on-line connected with a GVI2003 (GV Instruments Ltd. Manchester, UK) mass spectrometer, suitable for the measurement of stable isotope ratios of carbon, nitrogen, oxygen and sulphur. Control of the analyses and data evaluation was performed by a GVI software. Usually three samples each containing 3 μΐ of equivalent quantity of a distillate, are combusted and the carbon isotope ratio of the CO2 formed is determined. The calibration of combustion and isotopic determination were performed using the international carbon isotope standard NBS-22 (NIST-22), for which a value of -29.8 %o has been accepted. The standard deviation of measurement was less than 0.1 %o for three measurements of the same sample (4). 8 0-values were determined with a Finnigan-MAT261 or Thermo-Finnigan DeltaXLplus-IRMS using the ions m/z 46 ( C 0 0 ) and m/z 44 ( C 0 ) which are obtained after equilibrium of the isotope exchange of water and carbon dioxide. The exchange reaction C 0 + H 0

Wine Authentication Using Stable Isotope Ratio Analysis: Significance

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