Correspondence/Rebuttal pubs.acs.org/JAFC
Comment on Authenticity and Traceability of Vanilla Flavors by Analysis of Stable Isotopes of Carbon and Hydrogen Vanilla species are a source of one of the most popular flavors in the world with Vanilla planifolia of exceptional importance to the flavor and food industry. In Europe, extract from Vanilla tahitensis is much valued in the production of ice cream. Due to an ever-increasing demand and limited sources, the price of vanilla has greatly increased over the years, and this has led to adulteration of genuine vanilla, in particular by the fraudulent addition of non-natural vanillin to Vanilla extracts. In a recent paper published in this Journal, Hansen et al.1 reported their findings on the assessment of authenticity and traceability of vanilla flavor using compound-specific isotope ratio mass spectrometry measuring the carbon and hydrogen stable isotope signatures of vanillin. The analytical procedures employed in the study appear sound, and extracts from 79 natural vanilla pods from 12 different regions throughout the world were analyzed. However, the authors have greatly over interpreted their data set and thus the subsequent conclusions drawn now require critical comment and discussion. Characteristic differences in the δ13C values of vanillin in extracts from V. planifolia (n = 54) and V. tahitensis (n = 15) are reported for samples collected from several regions throughout the world. This finding is in accordance with the literature.2 Vanilla species differentiation is important not only for the quality assessment of the vanilla flavor but also for consumer confidence and protection. Hansen et al. attempted to use both the δ13C and δ2H values of the vanillin from the vanilla extracts not only for species verification but also to determine the geographical origin (traceability) of vanilla pods. However, despite what the authors claim, from Figure 4 it is clearly obvious that the values measured for V. planifolia from almost all geographical regions overlap one another and thus no inference about geographical origin can be drawn for this species. All that can be deduced from this data set is that it is possible to differentiate the two species investigated and to distinguish between the two geographical origins of V. tahitensis. At this point it is worth emphasizing that from the viewpoint of European Union food law the authenticity of flavors is of considerably more importance than their traceability. Thus, authentic vanilla flavor solely comprises vanillin originating from pods of the tropical orchid Vanilla species. In terms of European Union food law definitions, natural flavoring substances are flavor active substances formed by biosynthesis.3 It is irrelevant if these substances are produced in plant or animal tissues or by microorganisms supplemented with natural substrates, for instance, by microbial enzymatic oxidation of naturally occurring 4-hydroxy-3-methoxycinnamic acid (ferulic acid). Because ferulic acid is a natural readily accessible substance in rice bran, the cost of production of natural vanillin microbially from this source is considerably lower when compared with that extracted from vanilla pods. This microbially derived flavor compound may be obtained only by authorized preparation processes whereby the biogenic generated structure of the substance remains unaltered during © XXXX American Chemical Society
the preparation process. European legislation (EC 178/2002, article 8)4 requires that vanillin produced by such processes must not be labeled as the popular “Bourbon Vanilla” as this would be fraudulent and also mislead the consumer. To ensure that the legislation is adhered to and properly policed, new methodologies have recently been developed. For vanillin this has led to position-specific isotope measurements employing site-specific natural isotope fractionation−nuclear magnetic resonance (SNIF-NMR) and gas chromatography isotope ratio mass spectrometry (GC-IRMS).5−8 Our group developed a procedure for the selective analysis of δ13C and δ2H of plant methoxyl groups (O−CH3).9,10 The procedure is based on the “Zeisel method”,11 whereby methoxyl groups are transformed to methyl iodide using hydriodic acid and isotope measurements are made on the halomethane. In 2010 we adapted and applied the method for selective analysis of the 3methoxyl group of vanillin.5 In the 2010 study we reported the δ13C and δ2H values of both vanillin bulk and vanillin methoxyl groups and demonstrated that correlations between these values can be used to fully differentiate between natural vanillin extracted from pods of the tropical orchid vanilla and biotechnological vanillin produced from, for example, ferulic acid (from rice bran) by microorganisms or synthetic vanillin of any kind of chemical synthesis.5 Furthermore, the information gained from two-dimensional plots of the δ13C and δ2H values makes it virtually impossible for adulterated vanillin to remain undetected, even when sophisticated adulteration techniques are employed to change the isotopic content of the vanillin. Moreover, we would point out that because the two vanilla species, V. planifolia and V. tahitensis, used in our 2010 study showed much larger isotopic differences in the δ13C and δ2H values of their methoxyl values when compared to those observed for their bulk values, we consider the carbon and hydrogen signatures of methoxyl groups to be better suited for studying the geographical origin of vanilla species. It is most unfortunate that even though in their paper Hansen et al. cited several studies, including ours, on vanillin authenticity (references 16−18), they did not use the information contained therein to fully discuss and evaluate their data set. Had they done so, we are certain that despite the evident interesting trends in the carbon and hydrogen δ values of vanillin, the authors would have realized that these values are not sufficient for verification of the authenticity of vanilla flavors. In conclusion, we consider position-specific analysis of vanillin to be the best approach for the authenticity assessment of vanillin because it clearly distinguishes between vanillin from vanilla, from biotechnology or chemical synthesis. Moreover, we highly recommend that Hansen et al. consider analyzing the Received: December 22, 2014
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DOI: 10.1021/jf506172q J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Correspondence/Rebuttal
relatively large number of vanilla pod samples from different geographical locations using position-specific analysis. From a scientific point of view, these additional data would add significantly to the current database of known stable isotope ratios of natural vanillin and thus be of immense benefit to future authenticity studies and may also assist with the determination of the geographical origin of vanilla pods.
Markus Greule*,†,§ Armin Mosandl§ John T. G. Hamilton# Frank Keppler†,§ †
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Institute of Earth Sciences, Ruprecht Karls University Heidelberg, Im Neuenheimer Feld 234-236, D-69120 Heidelberg, Germany § Max Planck Institute for Chemistry, Hahn-Meitner-Weg 1, D-55128 Mainz, Germany # School of Biological Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast BT9 7BL, United Kingdom
AUTHOR INFORMATION
Corresponding Author
*(M.G.) Phone: +49 6221 545228. E-mail: markus.greule@ geow.uni-heidelberg.de. Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Hansen, A.-M. S.; Fromberg, A.; Frandsen, H. L. Authenticity and traceability of vanilla flavors by analysis of stable isotopes of carbon and hydrogen. J. Agric. Food Chem. 2014, 62, 10326−10331. (2) Scharrer, A.; Mosandl, A. Progress in the Authenticity assessment of vanilla. 2. δ13CV-PDB correlations and methodical optimisations. Dtsch. Leb. Rundschau 2002, 98, 117−121. (3) Council Regulation (EC) 1334/2008 of 16 December 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods and amending Council Regulation (EEC) No. 1601/91, Regulations (EC) No. 2232/96 and (EC) No. 110/2008 and Directive 2000/13/EC [2008] OJ L354/34. (4) Council Regulation (EC) 178/2002 of 28 January 2002 laying down the general principles and requirements of food Law, establishing the European Food Safety Authority and laying down procedures in matters of food safety [2002] OJ L31/1. (5) Greule, M.; Tumino, L. D.; Kronewald, T.; Hener, U.; Schleucher, J.; Mosandl, A.; Keppler, F. Improved rapid authentication of vanillin using δ13C and δ2H values. Eur. Food Res. Technol. 2010, 231, 933−941. (6) John, T. V.; Jamin, E. Chemical investigation and authenticity of Indian vanilla beans. J. Agric. Food Chem. 2004, 52, 7644−7650. (7) Tenailleau, E. J.; Lancelin, P.; Robins, R. J.; Akoka, S. Authentication of the origin of vanillin using quantitative natural abundance 13C NMR. J. Agric. Food Chem. 2004, 52, 7782−7787. (8) Remaud, G. S.; Martin, Y. L.; Martin, G. G.; Martin, G. J. Detection of sophisticated adulterations of natural vanilla flavors and extracts: application of the SNIF-NMR method to vanillin and phydroxybenzaldehyde. J. Agric. Food Chem. 1997, 45, 859−866. (9) Greule, M.; Mosandl, A.; Hamilton, J. T. G.; Keppler, F. A simple rapid method to precisely determine 13C/12C ratios of plant methoxyl groups. Rapid Commun. Mass Spectrom. 2009, 23, 1710−1714. (10) Greule, M.; Mosandl, A.; Hamilton, J. T. G.; Keppler, F. A rapid and precise method for determination of D/H ratios of plant methoxyl groups. Rapid Commun. Mass Spectrom. 2008, 22, 3983−3988. (11) Zeisel, S. Ü ber Ein Verfahren Zum Quantitativen Nachweise von Methoxyl. Monatsh. Chem. 1885, 6, 989−997.
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DOI: 10.1021/jf506172q J. Agric. Food Chem. XXXX, XXX, XXX−XXX
