Vanilla

Jens-Michael Hilmer, Franz-Josef Hammerschmidt, and Gerd Lösing

Introduction

Vanilla belongs to the most valued as well as the most expensive spices of the world. It represents the most important aromatic flavor, whose production constitutes a multimillion dollar a year for the industry (Krueger and Krueger, 1983). Worldwide production of vanilla observed is 2000 metric tons per year. Vanilla extracts are used extensively in chocolate and baked products, but most commonly in ice cream. The main flavor-active ingredient of vanilla extracts is vanillin. Industrial chemical synthesis of vanillin started more than 130 years ago (Tiemann and Haarmann, 1874). It is one of the most widely used flavoring components currently used in the flavor industry. The total consumption of vanillin is estimated to be at 12,000 metric tons per year (Eurofins Newsletter, 2003), about 82% for flavor purposes, 5% for fragrances and cosmetics, and 13% for pharmaceutical intermediates. The demand for it far outweighs its natural supply. Bensaid (Eurofins Newsletter, 2003) states that only 0.33% of the consumed vanillin originate from vanilla beans. Natural vanillin can be 100 times more expensive than vanillin from synthetic origin—in some years even more, depending on the harvest (Eurofins Food Newsletter, 2007). This price difference in combination with the “biotrend” and the demand for natural products has induced the flavor industry to develop alternative sources of natural vanillin, for example, based on the biotransformation of natural compounds.

Therefore, adulteration of vanilla extracts is a major problem in the commercial market for this product category. Consequently, there are plenty of governmental regulations concerning the authenticity of vanilla extracts to detect adulterations and to avoid cases of fraud.

In order to protect food manufacturers and consumers from adulteration of vanilla products, it is necessary to have powerful analytical tools available to prove authenticity of the respective vanilla products. Depending on the nature of the expected fraudulent activities as well as the specific product categories affected (such as vanilla beans, vanilla extracts, vanilla flavors, or vanilla food products), different strategies to detect these adulterations are applied.

There are numerous possibilities known about how the legal and sensory status of a vanilla product can be affected:

• Addition of non-vanilla-derived compounds to impart flavor; this includes:

• nature-identical/artificial vanillin from synthetic origin

• artificial ethyl vanillin

• tonka bean extract

• coumarin

• Adulteration of vanilla beans: addition of iron particles such as nails or even elementary mercury in order to increase the weight of the very precious vanilla beans (this was especially a topic, when vanilla bean prices were very high and reached several hundred dollars a kilogram some years ago).

• Adulteration by incorrect botanical origin: there are more than 100 different vanilla species known, and only three of them have a commercial relevance: Vanilla planifolia Jacks./Andrews, Vanilla tahitiensis J.W. Moore, and Vanilla pompona Schiede. For example, Bourbon Vanilla is related to V. planifolia species.

• Adulteration by incorrect geographical origin: since the geographical origin \of vanilla beans is relevant for regulatory reasons [e.g., to claim “Bourbon Vanilla” is only allowed when using vanilla extracts derived from vanilla grown in Madagascar, Reunion Island (formerly called “Ile Bourbon”), the Comoros, Seychelles, or Mauritius].

• Correct concentration indication of x-fold vanilla extracts: The amount of vanilla beans used for the production of vanilla extracts is relevant, for example, in the US market. This is expressed in the “Standard of Identity” (FDA Code of Federal Regulations). A total of 100 g of vanilla beans used for the production of 1 kg vanilla extract gives a so-called onefold extract.

The analytical methods that could be applied to indicate an authentic vanilla product can be based on the identification of single ingredients as well as on the combination of ingredients typically present in vanilla. As an example, the main flavor-active compound present in vanilla, vanillin, can be monitored specifically with methods such as GC, HPLC, IRMS, or specific NMR methods (see below). In addition, the presence of typical by-products in vanilla, such as vanillic acid, p- hydroxybenzaldehyde, or p-hydroxybenzoic acid, is determined. The ratio of these components is also used to judge the authenticity of vanilla and vanilla products. Also, the absence of compounds, which are usually not present in vanilla, such as (artificial) ethyl vanillin or vanillin derived from chemical synthesis can be used to assess the quality. In order to detect adulterations indicated, various specific analytical methods were developed and presented in the following sections.

Both consumers as well as food manufacturers can be protected from fraudulent activities by using state-of-the-art comprehensive analytical techniques for the evaluation of the authenticity of food raw materials as well as food products.

HPLC Analysis

The HPLC analysis is nowadays the routinely applied instrumental technique to analyze flavor compounds in vanilla products or their extracts. HPLC analysis is used to identify and quantify relevant compounds as well as in the further evaluation of authenticity or adulteration. Other techniques such as GC analyses have been published (Mosandl and Scharrer, 2001) but not been established in quality control and authenticity checks. An authenticity check based on the enantio selective analysis of the volatile fraction of vanilla extracts has been investigated (Mosandl and Hener, 2001). Among others, γ-nonalactone is one of the minor chiral compounds in vanilla and was extracted and separated into the R- and S-enantiomer. The low enantiomeric excess of R-γ-nonalactone of 45–63% was judged to be insignificantly high enough to provide an unequivocal proof for adulteration with racemic γ-nonalactone. HPLC methods established for vanilla are robust, rapid, and well suited for routine analysis. Sample preparation of the HPLC analysis is done by extraction in case of the beans, or simply dilution for extracts. The effects of the sample preparation techniques on the analytical results have been investigated (Ehlers et al., 1999). These results indicate that the ratios of typical vanilla ingredients such as vanillin, vanillic acid, p-hydroxybenzaldehyde, and p-hydroxybenzoic acid can vary depending not only on the raw material but also on the sample preparation method used.

Different HPLC methods have been developed and published and finally being adopted as official methods (Taylor, 1993; ISO, 1999) or even being part of the food law in France (Arrêté du juin 11, 1987).

The stationary phases are dominantly reversed-phase materials, mostly RP-18 materials but also RP-8 and others such as alkyl halogen-modified silica gel (Taylor, 1993).

The methods applying reversed-phase separation typically run with methanol/ water mobile phases in isocratic mode or more often with gradient elution at acidic pH values and finally using UV- or DAD-detection (diode array detector). The quantification can be based on internal standards, and also on external calibration as most of the detected compounds are available as pure chemicals. Run times are around 20 min; however, with special columns with selected stationary phases, the run time can be reduced to less than 5 min (Tracy et al., 2008).

Typical parameters for HPLC analysis of vanilla extracts are given in Table 15.1. The compounds that have been identified are also subject to publications and are described elsewhere in this book (see Chapter 12). Typical compounds that are detected with these analyses are vanillin, vanillic acid, p-hydroxybenzaldehyde, and p-hydroxy benzoic acid. These analytes are seen as impact compounds and represent a typical profile (Figure 15.1). A simple authenticity check can be performed based on the presence of untypical compounds such as ethyl vanillin but also on the absence of one or more of these impact compounds. Other minor compounds that are detected by HPLC, such as anisaldehyde, may serve as indicators to distinguish V. planifolia from V. tahitiensis (Ehlers et al., 1994; Oberdieck, 1998).

FIGURE 15.1 HPLC chromatogram of vanilla extract compounds.

Their quantitative distribution is typical for the kind of vanilla product and the extraction technique. Different studies have investigated the profile resulting from these impact compounds in vanilla extracts and found to be specific and therefore suitable to evaluate the authenticity of a vanilla extract (Fayet et al., 1987; Juergens, 1981). To transform the profile of these compounds into a numerical scale, the ratios of their concentration were calculated and limits were suggested. Five ratios of vanilla compounds have been introduced into French law to evaluate the authenticity (République Française, 1988) of vanilla products. In 2003, the ratios have been amended by the Direction Générale de la Concurrence, de la Consommation et de la Répression des Fraudes (DGCCRF) to reflect findings over a longer period of time and additional information from recent crops at that time (Note d’information, 2003). The limits are shown in Table 15.2. This evaluation is suitable only for alcoholic extracts of cured whole vanilla beans. Changes in the solvent composition or the use of alternative extraction agents such as CO₂ lead to ratios that do not necessarily comply with the limits given in Table 15.2 (Quirin and Gerard, 1998). Also the source of the vanilla beans used to prepare the extracts influence the profile and herewith the ratios. Further studies have partly disproved the conclusion of fraud when a ratio

^a République Française (1988).

^b Note d’information (2003).

is out of the ranges given (John and Jamin, 2004; Littmann-Nienstedt and Ehlers, 2005; Gassenmeier et al., 2008) and the suitability of the so-called ratios has been argued. In an information letter, the International Organization of the Flavor Industry (IOFI) has expressed their reservation toward the applicability of the ratios and overestimation of their validity (IOFI Information Letter, 2000) despite the fact that these ratios are sometimes erroneously cited as “IOFI values.” The questionable validity of the ratios has been applied not only to vanilla beans and extracts but also to vanilla flavors and even flavored food. An interpretation of ratios for flavors and food is far beyond of what has seriously been investigated at the scientific level. Thus, the so-called ratios are a precheck for the authenticity of vanilla beans and extracts thereof, in case the conditions of extraction are well known to the evaluator. Other techniques such as stable isotope ratio mass spectrometry (IRMS) or quantitative site-specific nuclear magnetic resonance spectroscopy provide much clearer indication of adulteration of vanilla products (Kempe and Kohnen, 1999).

IRMS

A very advanced analytical method for the authentification of vanilla extracts and vanilla flavors is stable isotope ratio analysis (SIRA). It was developed in the 1970s and became the most important tool for authenticity testing of nonchiral compounds. This analysis can be performed either by IRMS, mainly combined with a gas chromatographic separation, or by quantitative NMR measurement of the natural abundance at individual atomic sites (Schmidt et al., 2007).

In nature, the main bioelements such as hydrogen, carbon, oxygen, and nitrogen occur as mixtures of isotopes. The natural abundances of the stable isotopes have global average values. Owing to physical processes, (bio)chemical reactions such as photosynthesis, geographic parameters, and climate, their relative ratio can vary. Always the “light” isotopes (¹H, ¹²C, and ¹⁶O) are by far dominant compared with the abundance of the “heavy” ones (²H, ¹³C, and ¹⁸O). The products formed in plants (or animals) and the ingredients of the extracts or the food prepared from them obtain a characteristic isotope ratio, which allows to correlate it to the photosynthetic pathways, and the climatic and geographic conditions.

Compounds with an aromatic ring—for example, vanillin—are generally synthesized in plants through the shikimic acid pathway from erythrose-4-phosphate and phosphoenol pyruvate. The formation of the products of this pathway is accompanied by a corresponding depletion of ¹³C relative to the primary plant products, the carbohydrates.

These carbohydrates are produced during photosynthesis of plants utilizing CO₂ and water. The primary step is enzymatically catalyzed, with ¹³CO₂ reacting somewhat more slowly than ¹²CO₂. This phenomenon is named the “kinetic isotope effect.”

However, the ¹³C deficit is not identical for all plants.

Three major photosynthetic pathways for the CO₂ fixation are known for plants: C3, C4, and CAM. The so-called C3 plants (e.g., wheat, barley, sugar beet, and most trees) use the ribulosebisphosphate-carboxylase reaction, the Calvin pathway, while C4 plants (e.g., sugarcane, maize, sorghum, and millet) use the phosphoenolpyruvate-carboxylase reaction, the Hatch–Slack pathway. The CAM plants such as succulents, orchids (e.g., V. planifolia), and some tropical grasses have the Crassulacean acid metabolism. Each group shows different values of the ¹³C/¹²C, ²H/¹H, and ¹⁸O/¹⁶O ratios for their metabolites as can be shown by IRMS (e.g., carbohydrates, fatty acids, isoprenoids, amino acids, phenylpropanes, etc.). By analyzing these ratios, it is possible to distinguish between compounds produced by plants during the biochemical pathways and those produced by synthesis.

The changes in the isotope ratio caused by these effects are very small. So they are not indicated in the atom% scale. These minimal changes are compared with the values of international standards and expressed in per million (‰) as a variance from standard, the so-called delta value (Schmidt, 2003):

δ¹³C (‰) = (([ ¹³C_sample ] / [ ¹²C_sample ]) / ([ ¹³C_standard ] / [ ¹²C_standard ]) − 1) × 1000

The standard for the ¹³C/¹²C ratio is δ¹³C: V-PDB (Vienna-PeeDee Belemnite). The analysis of δ¹³C values for C3 plants (the so-called “light plants”) give values between −30‰ and −24‰, for C4 plants (the so-called “heavy plants”) between −16‰ and −10‰. The CAM plants have δ¹³C values from −10‰ to −30‰, making a differentiation from the other two types very difficult.

The authenticity proof of flavor substances by determination of their average δ¹³C value implies the combustion at about 1000°C of the purified organic samples in order to convert them into CO₂. An online coupling of gas chromatography and IRMS via a combustion interface reduced the sample amount and made measurements easier. Later on, an online coupling of gas chromatography and IRMS via a pyrolysis interface (about 1300°C) was developed, primarily for ¹⁸O analysis, allowing the simultaneous online measurement of δ¹⁸O and δ¹³C values. This interface also affords the determination of δ²H when applying the pyrolysis at about 1450°C. Hener et al. (1998) compared the δ¹³C values of different vanillin samples measured via CO₂ and CO and showed that the values are, in most cases, in good agreement. Nowadays, mainly the online coupling of gas chromatography with combustion or reductive pyrolysis to isotope ratio mass spectrometry (GC-C-IRMS or GC-P-IRMS) is used for the determination of the δ-values of carbon ¹³C, hydrogen ²H, and oxygen ¹⁸O.

V. planifolia belongs to the CAM plants, and vanillin ex-beans shows δ¹³C values in the range from −16.8‰ (V. tahitiensis) to −22.0‰ (V. planifolia) (see Table 15.3).

^a Scharrer and Mosandl (2002).

^b Gatfield et al. (2007).

^c Bensaid et al. (2002).

^d Schmidt et al. (2007).

^e IOFI (1989).

^f Kempe and Kohnen (1999).

^g Koziet (1997).

^h Hoffman and Salb (1979).

ⁱ Culp and Noakes (1992).

^j Hoffman (1997).

^k Brenninkmeijer and Mook (1982).

^l Werner (1998).

^m Note d’information (2003).

ⁿ Krammer et al. (2000).

Apart from the extraction of vanilla beans, vanillin can also be produced by chemical synthesis or biotechnological pathways from different sources (lignin, guaiacol, eugenol, curcumin, and ferulic acid).

The δ¹³C values of vanillin derived from other sources than vanilla beans or its extracts are in the range from −24.9 to −37.0‰. Therefore, vanillin in vanilla extracts or derived from vanilla beans can be analytically differentiated from other sources by its δ¹³C value. It can be used to indicate the authenticity of vanilla products based on vanilla or its extracts.

According to a recommendation of IOFI, the authenticity of vanilla flavors is estimated by the determination of the δ¹³C value for vanillin. δ¹³C values in the region of –21.0‰ ± 0.5‰ are considered to indicate the plant origin of the analyzed vanillin ex-vanilla beans (IOFI Information Letter, 1989). The variances resulting from the applied isolation techniques are currently under investigation through ring tests performed by a dedicated analytical working group of the German Chemical Society (GDCh).

As long as the vanillin is derived from biotechnological processes such as by fermentation starting from natural ferulic acid, it can be considered as natural and subsequently applied in natural flavors. Owing to the fact, that the δ¹³C values of vanillin derived from natural ferulic acid by fermentation are very low, this quality can usually be differentiated from synthetic vanillin qualities that do not meet the naturalness requirements.

In order to detect an adulteration of vanilla bean extract with vanillin from other sources, it is possible to apply multielement GC-IRMS; this means, to also determine the ratios ²H/¹H and ¹⁸O/¹⁶O and to evaluate this tridimensional dataset. The standard for these two ratios is Vienna-Standard Mean Ocean Water (V-SMOW). In a recent paper, Bensaid et al. (2002) show that the sp₂ oxygen atom in the aldehydic position can chemically exchange with water in industrial or laboratory procedures. Therefore, the authors used the δ¹⁸O value of guaiacol, which is formed by the degradation of the vanillin molecule. However, they state that in practice, oxygen isotope ratios do not play an important role in authentication procedures.

The degradation of the vanillin molecule was already used by Krueger and Krueger (1983), when the instrumentation for the multielement IRMS was not yet well developed. Fraudulent adulterators had learned to imitate the δ¹³C value of natural vanillin by blending it with “nature-identical” vanillin, with products artificially enriched in ¹³C concentration (Krueger and Krueger, 1983, 1985).

IRMS measures the average δ¹³C value of the whole vanillin molecule. Since it is relatively simple to enrich its methyl or carbonyl group in ¹³C, synthetic vanillin blended with a ¹³C-enriched fraction may be mistakenly taken as natural.

To check for [methyl-¹³C]-vanillin, the methyl carbon is removed as CH₃I in refluxing HI. Then, IRMS is performed on the CH₃I (Krueger and Krueger, 1983).

For analyzing [carbonyl-¹³C]-vanillin, the molecule has to be oxidized to vanillic acid. After decarboxylation, the CO₂ formed is analyzed by IRMS (Krueger and Krueger, 1985).

NMR

Presently, the checking for enriched portions is more easily analyzed by quantitative NMR measurements of the abundance of ²H or ¹³C in the different positions of the molecule. This method was named site-specific natural isotope fractionation nuclear magnetic resonance spectroscopy (SNIF-NMR^®) and it is based on the fact that the distribution of ²H or ¹³C at the different sites of the molecule is not statistical and depends on the origin of the particular compound.

SNIF-²H-NMR spectroscopy is a powerful tool and allows a deeper insight into the biochemical mechanisms by the determination of isotope contents at specific molecule sites. In this respect IRMS, which usually needs to burn the sample before analysis, is not suited to the measurement of an “intramolecular” isotope distribution. Basic research has shown that deuterium is far from being randomly distributed in organic molecules (Martin and Martin, 1981). Therefore, the D/H-ratios of the different positions of the molecule have to be determined.

The relative sensitivity of NMR for ¹³C is theoretically about 100 times higher than that for deuterium. But due to the fact that the kinetic isotope effects for carbon are much smaller than those for deuterium and due to shorter relaxation time for deuterium, quantitative ²H-NMR became the best tool for the authenticity control in the flavor field.

The first application of this method to the vanillin molecule appeared in 1983 (Toulemonde et al., 1983). Now SNIF-²H-NMR^® is accepted as the official method by AOAC (No. 2006.05) for the authentication of vanillin. SNIF-²H-NMR combined with ¹³C-IRMS also allows the characterization of biotechnological vanillin derived from ferulic acid, eugenol/isoeugenol, or curcumin.

The vanillin molecule has six monodeuterated isotopomers. Five signals can be seen in the ²H-NMR-spectra of the three different vanillin qualities in Figure 15.2, the two deuteriums in the ortho-position fall into one signal. Table 15.4 shows that vanil-lin synthesized from guaiacol (2-methoxyphenol), by introducing an aldehyde group, is clearly separated from the other variants by a high degree of deuterium enrich ment on the carbonyl function. Vanillin from lignin exhibits deuterium depletion at all positions compared with natural vanillin derived from the vanilla bean. Experimental values for the hydroxyl group are not included in Table 15.4, since they can easily be falsified by H/D exchange processes.

FIGURE 15.2 ²H-NMR spectra of vanillin ex-vanilla beans, vanillin from synthetic origin (ex-lignin, ex-guaiacol), and vanillin from biotechnological origin ex-ferulic acid.

^a Remaud et al. (1997).

^b John and Jamin (2004).

Also, the development of quantitative ¹³C-NMR is ongoing. Therefore, first characteristic ¹³C-distributions in vanillin qualities of different origins have already been obtained (Caer et al., 1991; Tenailleau et al., 2004a, 2004b).

Conclusion

The analytical determination of typical vanilla (extract) ingredients by HPLC and the calculation of their ratios can be considered as a precheck for the authenticity assessment for vanilla. IRMS is a powerful tool to prove the authenticity of vanilla products. Especially since the online technique provides a coupling of GC to IRMS, these measurements have become available without cumbersome isolation steps. The multielement assay with datasets of δ¹³C-, δ²H-, and δ¹⁸O-values should allow to detect trials of fraud.

When a sufficient amount of vanillin can be isolated, quantitative ²H- and ¹³C-NMR measurements are particularly efficient for distinguishing the different origins of vanillin.

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