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Recent Advances in Food and Flavor Chemistry
Food Flavors and Encapsulation, Health Benefits, Analytical Methods, and Molecular Biology of Functional Foods
By C.-T. Ho, C. J. Mussinan, F. Shahidi, E. Tratras Contis The Royal Society of Chemistry
Copyright © 2010 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84755-201-3
CHAPTER 1
HISTORICAL LOOK AT THE USE OF ISOTOPIC ANALYSES FOR FLAVOR AUTHENTICATION
P.G. Hoffman and J.E. Noakes
1 INTRODUCTION
The Flavor and Extract Manufacturers' Association of the United States (FEMA) and the University of Georgia (UofGa) have had almost a quarter of a century professional relationship. During this period FEMA assisted the UofGa in forming and operating a "center of excellence" at the University's Center for Applied Isotopic Studies (CAIS). This cooperative effort is involved with development and application of isotopic methods for the authentication of flavor materials. The following is a historical description of this synergistically beneficial activity.
2 BACKGROUND
Radiocarbon (C) analysis is used in order to date archeological artifacts because of the known and uniform decay of the unstable carbon isotope with a half-life of about 5730 years. It was this method, which was used to disprove the claim that the Shroud of Turin covered the body of Christ as the cloth had a Medieval carbon date. In the 1960's, C14 analysis was applied outside of archeology by Allen, et. al at Coca Cola when they realized synthetic petroleum derived caffeine could be differentiated from modern kola bean derived caffeine with liquid scintillation analysis, the technique used to measure C14 content. Therefore, an adulterated "natural cola" could be identified and eliminated from the market place.
In the 1970's, as a result of efforts to refine radiocarbon dating, the discovery was made that carbon was incorporated into plant materials by several biosynthetic pathways. The C14 as well as C13 and C14 carbon isotope concentrations were altered from ambient levels during these biosyntheses according to the pathway a plant utilized. In addition to the use of this biosynthetic stable isotopic variation for the refinement of radiocarbon dating, these stable isotope ratios (C13/C12) could be used to identify the botanical source of a product. The major biosynthetic pathways are the Calvin and Hatch-Slack cycles as well as a combination of the two, the crassulacean acid metabolism (CAM). A good example of the use of these variable stable isotope ratios in establishing the origin of a product is demonstrated by vanillin. Vanillin from vanilla beans is produced via the intermediate CAM biosynthetic pathway and can be differentiated front vanillin produced synthetically from lignin, a by-product of the paper industry. Lignin is a product of coniferous or pine trees which biosynthesize via the more complex Calvin cycle. Stable isotope ratio analysis (SIRA) is the technique used in order to determine isotopic values. The analysis is performed with an isotope ratio mass spectrometer (IRMS). Synthetic vanillin from lignin which is produced via the more complex biosynthesis has an SIRA of @ -27, indicative of a product depleted in C13. Bean derived vanillin, produced via the intermediate biosynthesis, has an SIRA of @ -20. Products from the more primitive Hatch-Slack pathway have the less negative SIRA of @ -10. Examples of products in this category include corn and com products such as high fructose com syrup (HFCS). These last two values indicate a lesser C13 depletion.
In the mid 1970s researchers with the French government, used C12/C13 SIRA in order to differentiation synthetic and vanilla bean derived vanillin. They also demonstrated that deuterium/hydrogen (D/H) SIRA could also be used in the detection of the adulteration of vanilla products. Vanillin from vanilla beans has a D/H SIRA value @ -100 while that from lignin is @ -180 and from guaiacol is @ -20. The more negative values, as in carbon analyses, indicate a depletion in the heavier hydrogen isotope, deuterium (D). Earlier, Bricout, et al. also used these methods to differentiate cane sugar and beet sugar (C13/C12) and to detect the addition of tap water to orange juice (D/H). In the 1970's applications of SIRA included differentiation among the various origins of citral, detection of the addition of high fructose com syrup (HFCS) to honey and sugar added to maple syrup. From here the applications of isotopic methods for the prevention of adulteration of a variety of products has expanded exponentially including flavor applications beyond vanilla/vanillin. In addition, oxygen (O18/O16) and even sulfur (S34/S32) and nitrogen (N15/N14) stable isotopes have and are being investigated, for example fruit juices with oxygen, allyl isocyanate with sulfur and nitrogen.
Similarly, the applications of C14 radiocarbon analyses expanded to include products such as vinegar/ethanol and cinnamaldehyde/cinnamon. The methodology involved liquid scintillation counting which over an extended period of time individually counts the beta particles which occur when C14 atoms decay to N14. A synthetic product derived from a petroleum sourced material, which because of its age is considered "dead", has only background C14 activity while a modern botanical derived material should have @ 14 dpm – 17 dpm (disintegrations per minute).
The steady state C14 activity was disrupted in the 1950s and 1960s due to the atmospheric nuclear testing by Russia and the US and later China and France. This dramatic increase, by as much as 200%, created a unique biomarker phenomenon. Over the next several decades this increased activity steadily declined due to burning of fossil fuels with the subsequent injection of CO2 with no C14 activity and the substantial oceanic absorption of CO2. These changes in C14 are used to further identify source as well as production of a variety of botanical products.
In addition, the unstable isotope of hydrogen, tritium (H3), has been tested for certain applications, for example benzaldehyde and vanillin. This method was able to differentiate vanilla bean vanillin from that derived from petroleum/guaiacol. While this was successful, it requires an inordinate amount of material 5 -10 gms and an elaborate sample preparation requiring an electrolytic concentration process. The analysis is accomplished in a gas proportional counter (GPC).
A last indication of the complexity of the application of these techniques is the lengths unscrupulous competitors will go to circumvent these techniques. Attempts were quickly made to alter the isotopic signature by isotopically labeling the synthetic material in order to mimic the natural material. This lead to authentication methods which cleaved functional groups, e.g. aldehyde or methoxy, and isotopically analyzed the residues by liquid scintillation or SIRA as appropriate.'
3 FLAVOR AND EXTRACT MANUFACTURERS' ASSOCIATION (FEMA) AND UNIVERSITY OF GEORGIA'S CENTER FOR APPLIED ISOTOPE STUDIES (CAIS)
In 1985, a bitter almond oil crisis overtook the flavor industry. In the US, the Flavor and Extract Manufacturers' Association (FEMA) took the lead in addressing this crisis which involved the sale of synthetic benzaldehyde as natural bitter almond oil. The Flavor Labeling Analytical Subcommittee (FLASC) was formed by FEMA and coordinated with the University of Georgia's Center for Applied Isotope Studies (CAIS) in order to develop the analytical means to differentiate the origins of benzaldehyde as well as other flavors. In order to expand the Center's existing liquid scintillation (LS) capabilities to perform radiocarbon (C14) analysis, FEMA assisted in the purchase and operation of an isotope ratio mass spectrometer (IRMS) in order to carry out stable isotope ratio analyses (SIRA).
Initially, four flavors were targeted. In addition to benzaldehyde, methods for cinnamaldehyde, anethole and methyl silicylate were developed. The Center's early success in developing and implementing the methods to analyze and differentiate the various origins of these four flavor materials were apparent and lead to an expansion of the targeted flavors. Twenty-three flavor compounds were targeted as well as others requested by various FEMA members. (Table 1)
The initial collaborative program between FEMA and CAIS was for five (5) years and was then extended for three (3) additional years. Beyond these eight intensive years, FEMA and CAIS's close cooperation and collaboration continues and has been coordinated by the Isotopic Studies Committee (ISC) which evolved from FLASC. The ISC was expanded from the original five members of FLASC to sixteen (16) member companies.(Table 2) The efforts with the FEMA supported batch IRMS have produced a multitude of data. The FEMA/CAIS isotopic database is focused on three analyses, radiocarbon C14 analyses, and the stable isotopes of C13/C12 and D/H.
While these efforts were productive as indicated by the database of isotopic analyses, it became apparent that a more efficient and faster sample throughput was necessary. Also, since current analyses required the isolation of between 3 – 5 mgs, this precluded, without major efforts, the analyses of these flavor compounds in finished products where they are found at parts per million (ppm) levels. FEMA charged the ISC to assist CAIS in meeting these additional challenges.
In 1992 under the guidance of the ISC, FEMA again teamed with the University of Georgia and provided a gas chromatograph – isotope ratio mass spectrometer (GC-IRMS) to the CAIS. This permits fifty (50) analyses a day including database building samples as well as industry samples. In addition, a flavor could be isolated from a complex matrix by GC separation and directly analyzed by IRMS for carbon and hydrogen SIRA.
Toward the end of the twentieth century, the Center had an opportunity to significantly increase its capability through the acquisition of an Accelerator Mass Spectrometer (AMS). The instrument uses a particle accelerator in tandem with ion sources, large magnets, and detectors to enable the direct counting of the number of atoms in a sample, instead of measuring isotopic decay (liquid scintillation). Therefore, even extremely small (microgram size) samples can be used for quantitative determinations of C14 as well as other elements.
Precision of C-14 by AMS is in the range of 0.5%. The sensitivity of the Center's instrument is comparable to that of much larger units, with theoretical detection limits as low as 4 attomoles (4X10-18 moles) of C-14. The AMS has a broad range of applications in earth, life and environmental sciences, including oceanography, marine science, geology, hydrology, archeology, climatology, soil science, environmental monitoring, biomedicine, pharmacology, and toxicology as well as the applications here for flavor analyses.
The Center began efforts to establish an AMS facility in 1998. CAIS approached FEMA in order to assist in its request and join with the University of Georgia and the Georgia Research Alliance in order to acquire the AMS instrumentation. Because of the obvious use for the authentication of flavors as well as the real potential to be able to investigate flavors in the finished product, FEMA provided enthusiastic and financial support, identifying the flavor industries uses for this type of analyses. This industrial support and identified uses were significant factors in obtaining this instrument for the Center. Thus, this academic-research-industrial group participated in the establishment of the AMS facility at the CAIS.
The National Electrostatics Corporation Model 1.5 SDH-1 Pelletron Accelerator Mass Spectrometer was purchased in 2000 and installed in early 2001. This equipment provides for the precise analyses of carbon isotopes including C-12, C-13 and C-14 at extremely low (parts per quadrillion) concentrations levels.
In support of the flavor industry this allows high precision analysis of C-14 for 3,000 flavor samples per year. Testing and development of 10-100 microgram carbon samples provides a capability to analyze GC-prepped and collected isolates from complex mixtures for unique "biomarker" authentication.
4 CONCLUSION
This cooperative effort between FEMA and CAIS not only demonstrates the flavor industry's dedication to maintaining and enhancing its integrity, but also illustrates the Center's continuing efforts to improve and stretch the limits of applied isotopic science and technology. The flavor industry and the University of Georgia can take great pride in the accomplishment of creating an applied isotopic research center of excellence at the CAIS.
CHAPTER 2
TWO DECADES OF FLAVOUR ANALYSIS: TRENDS REVEALED BY RADIOCARBON (14C) AND STABLE ISOTOPE (δ13C AND δD) ANALYSIS
R. A. Culp and J. E. Noakes
Center for Applied Isotope Studies
University of Georgia
Athens, Georgia USA
1 INTRODUCTION
Isotopic analysis for authenticity testing of food and flavouring products has been in use since the 1970s. Initially used to augment the classical chemical analyses, such as atomic absorption spectroscopy and gas chromatography, isotopic analysis has found its niche in the chemist's repertoire of analytical techniques'. Most notably, radiocarbon (14C) has been found to be the unequivocal determinant of fossil fuel derived carbon. Materials lacking proper apportionment of 14C in their carbon chemistry are composed of some definable proportion of fossil fuel derived or synthetic carbon. To define the natural character of a product, or that portion not synthetically derived, a reference level of C in modern botanically derived products must be accurately determined for comparison.
Changes in the atmospheric concentration of 14CO2 have occurred over time. Perturbations to the natural equilibration level of 14CO2 in the atmosphere have come from fossil fuel combustion since the industrial revolution and nuclear weapons testing in the late 1950s and early 1960s to name a few. Coupled with the global increase in atmospheric CO2 concentration via increased fossil fuel and biomass combustion and reduction of CO2 through biomass photosynthesis and oceanic uptake, an accurate single proxy for current 14C levels is difficult to obtain.
We present here 14C data along with stable carbon (δ13C) and hydrogen (δD) isotope data obtained by our laboratory over the last two decades on seven different flavour compounds of modem botanical origin. Data generated from over 3800 samples including cassia oil, benzaldehyde, acetaldehyde, vanillin, maltol, ethyl butyrate and ethyl acetate reveal an accurate determination of the annual 14C activity level in botanically extracted samples over the last two decades. Stable isotope data reveals trends in product precursors and changes in source material and processing over this time period.
The importance of an accurate assessment of current 14C activity level is two-fold; first, to substantiate truth in advertising and authenticate natural products of recent botanical origin, and second, to accurately determine the quantity of bio-based material in manufactured products. To assist in authenticating natural products and their process of formation, 14C activity has been combined with stable isotope abundance of carbon and hydrogen and ranges of acceptance have been defined. Beginning with research funded by the United States Flavour and Extract Manufacturers Association (FEMA) in 1986, the University of Georgia developed isotopic ranges of acceptance for numerous compounds of interest. By 1998, nearly 1200 samples had been tested for FEMA and associated companies and were represented by over 35 individual flavour compounds. As of 2008, the number of samples and flavours analyzed have grown 10-fold and bolstered our ability to refine isotopic ranges of acceptance. The seven flavour compounds represented in this study are major compounds of interest and the isotopic trends in 14C, δ13C and δD for that fraction reported, or confirmed by testing, to be of natural origin over the last two decades are revealed.
In contrast to distinguishing the source and process of manufacture, bio-based product testing substantiates the bio-base content that can replace those made from fossil fuels. As part of the mandated government initiative to produce materials from bio-based resources, promote the use of renewable resources and lessen our dependence on manufactured goods made from fossil fuels, accurate determination of current 14C activity is critical to proper bio-base content apportionment.
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Excerpted from Recent Advances in Food and Flavor Chemistry by C.-T. Ho, C. J. Mussinan, F. Shahidi, E. Tratras Contis. Copyright © 2010 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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