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Chapter 12. Developing the Aromatic Quality of Cured Vanilla Beans (Vanilla planifolia G. Jackson)

Eric Odoux

COMPOSITION OF VOLATILE COMPOUNDS IN CURED VANILLA BEANS

The analysis of the volatile fraction of cured vanilla, which, paradoxically, has been the subject of little research, has made it possible to detect and identify over 150 molecules belonging to numerous chemical classes (Klimes and Lamparsky, 1976; Galetto and Hoffman, 1978; Schulte-Elte et al., 1978; Nakasawa et al., 1981; Vidal et al., 1989; Adedeji et al., 1993; Kiridena et al., 1994; Ramaroson-Raonizafinimanana et al., 1997; Werkhoff and Günter, 1997; Sostaric et al., 2000; Perez-Silva et al., 2006).

From a chemical viewpoint, these different compounds belong to the following classes: hydrocarbons, alcohols, aldehydes, ketones, esters, lactones, acids, terpenoids, heterocyclics, and phenols.

Among the hydrocarbons, a large number of alkanes have been identified (such as n-pentacosane), methylated and ethylated derivatives of these alkanes, alkenes (such as 1-hentriacontene), terpenoids (such as α-pinene or limonene), and aromatic rings (such as benzene and some of its derivatives) (Figure 12.1).

Aliphatic acids (such as acetic acid and linoleic acid) are also represented by a number of molecules, as well as aromatic acids (such as benzoic acid or cinnamic acid) (Figure 12.1).

FIGURE 12.1 Chemical structures of some volatile compounds from different chemical classes identified in cured vanilla beans.

Aliphatic esters and aromatic esters (such as linoleic acid ethyl ester or cinnamic acid methyl ester) are also found, and even terpene esters (such as acetic acid α-terpinyl ester) (Figure 12.1).

Aliphatic alcohols (such as 2,3-butanediol), aromatic alcohols (such as benzyl alcohol), and even terpenoids (such as linalol) have been identified (Figure 12.1).

Aliphatic aldehydes (such as 2-heptenal), terpene aldehydes (such as β-cycloci-tral), aliphatic ketones (such as 3-hydroxy-2-butanone), and aromatic ketones (such as acetophenone) are also present (Figure 12.1).

Several lactones have also been reported as being a component of the volatile fraction of vanilla (such as γ-butyrolactone), along with heterocyclics (such as furfural or cis-vitispirane) (Figure 12.1).

Finally, phenols, which are the major representatives (both qualitatively and quantitatively) of the volatile fraction of cured vanilla, and which may also bear the aldehyde functions (such as 3-methoxy-4-hydroxy-benzaldehyde or vanillin), acid functions (such as 4-hydroxybenzoic acid), alcohol functions (such as 4-hydroxybenzyl alcohol), and even ketone functions (such as acetovanillone) (Figure 12.1); numerous esters exist (such as salicylic acid methyl ester) as well as ethers (such as 4-hydroxy benzyl methyl ether) (Figure 12.1).

A fairly exhaustive list of the compounds identified in cured vanilla can be consulted in two recent review articles by Dignum et al. (2001) and Ranadive (2006).

This composition may vary according to the geographical origin (the concept of “terroir” in the broad sense) and botanical origin of the samples (Adedeji et al., 1993).

Concerning the botanical aspect, only the species V. planifolia, Vanilla tahitensis (see Chapter 13), and to a lesser extent V. pompona (Ehlers and Pfister, 1997), have been the subject of research on their aromatic composition. But even in the case of V. planifolia, much research remains to be done using well-defined genetic material and a standardized curing process.

Finally, it should be remembered that extraction and analysis techniques may also result in variability in the aromatic composition (and also in artifacts).

Quantitatively, the major component in cured vanilla is vanillin (3-methoxy-4-hydroxybenzaldehyde, Figure 12.2), which may reach concentrations of several tens of grams per kilogram of dry weight (in other words, several tens of thousands of ppm) (ISO 5565-1). Three other compounds are also commonly quantified, as they are considered as indicators of quality and authenticity (see Chapter 15); these are p-hydroxybenzaldehyde and vanillic acid (Figure 12.2), whose concentrations are generally measured at around 1 g/kg of dry weight (or around 1000 ppm), in other words, around 10 times lower than that of vanillin, and finally p-hydroxybenzoic acid (Figure 12.2), whose average concentration is around 100 mg/kg of dry weight (or around 100 ppm), in other words, 100 times lower than that of vanillin. These figures may of course vary considerably, depending on the quality of the product (Gassenmeier et al., 2008; Saltron et al., 2002).

Other compounds have also been measured at concentrations of more than 100 ppm, such as acetic acid or 4-hydroxy benzyl methyl ether (Klimes and Lamparsky, 1976) and also hexadecanoic acid and linoleic acid (Perez-Silva et al., 2006) (Figure 12.2). Adedeji et al. (1993) even report a large number of compounds with concentrations of more than 1000 ppm, such as 2-furfural (which is in fact the second most abundant compound after vanillin in a sample of Mexican vanilla) or 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one (Figure 12.2), which exceeds 3000 ppm in most of the samples analyzed, in other words, concentrations that are higher than those of p-hydroxybenzaldehyde and vanillic acid.

FIGURE 12.2 Chemical structures of the major compounds (quantitatively) found in the volatile fraction of cured vanilla beans.

It should, nevertheless, be noted that techniques for analyzing and measuring these compounds vary greatly from one author to another, and are also very different from the standardized techniques (ISO 5565-2), which explains the differences observed.

In fact, it is generally acknowledged that 95% of the volatile compounds in cured vanilla are found at concentrations of less than 10 ppm (Hoffman et al., 2005).

Although vanillin is the major component quantitatively, this molecule alone does not explain the quality of the global aroma of vanilla; it is even difficult to show a correlation between the vanillin content and the sensory profile of vanilla extract (Gassenmeier et al., 2008). In fact, rather surprisingly, very few studies have been published on the contribution of different aroma compounds to the quality of the aroma and flavor of cured vanilla.

Using GC-olfactometry (but without providing details of the methodology employed), Dignum et al. (2004) found that p-cresol, 2-phenylethanol, guaiacol, and 4-creosol (Figure 12.3) had a considerable impact on vanilla aroma. Also using GC-olfactometry, Perez-Silva et al. (2006) detected 26 aroma-active compounds in a Mexican vanilla extract; of these compounds, 13 are derivatives of the phenylpropanoid pathway (see Chapter 10), of which some, such as guaiacol, 4-creosol, acetovanillone, and salicylic acid methyl ester (Figure 12.3), are similar in intensity to vanillin, while their concentrations are 1000 times lower (sometimes even less) in the extract. It should be noted that p-cresol, cinnamic acid methyl ester, and anisyl alcohol (Figure 12.3) have intermediate intensities, even though they have concentrations of just a few ppm. In general, these compounds are responsible for sweet, woody, balsamic, spicy, vanilla-like, and toasted notes. Certain aliphatic aldehydes, alcohols, and acids also have intermediate intensities despite concentrations of less than 10 ppm.

In a study aimed at correlating sensory analysis and chemical analysis in order to classify the quality of vanilla extracts, Hoffman et al. (2005) identified 14 compounds that were detected in almost all the extracts studied (55 samples in total) and contributed positively or negatively to the aroma and flavor.

Acetic acid ethyl ester, hexanoic ethyl ester, octanoic ethyl ester, nonanoic ethyl ester, and hexanal ethyl acetal (Figure 12.3) are highly correlated with the “age-related compounds” criterion; 4-hydroxy benzyl ethyl ether, vanillyl ethyl ether, and acetaldehyde ethyl acetal (Figure 12.3) are highly correlated with the “rummy/resinous” criterion; and p-hydroxybenzaldehyde and vanillin (Figure 12.3) are highly correlated with the “vanillin” criterion. All of these compounds are therefore associated with positive criteria, whereas p-hydroxybenzoic acid, vanillic acid, guaiacol, and nonanoic acid (Figure 12.3) are highly correlated with the “smoky/phenolic” criterion, in other words, a negative descriptor.

FIGURE 12.3 Chemical structures of some aroma-active compounds detected in cured vanilla beans.

Finally, less volatile molecules should be mentioned, which are consequently involved in the quality of the flavor rather than the aroma, and are the subject of recent publications (Gatfield et al., 2006; Schwarz and Hofmann, 2009). Seven molecules (Figure 12.4), including divanillin, were identified in cured vanilla and in extracts, and are involved in the velvety mouth-coating sensation. Divanillin, for example, was found at a concentration of around 170 ppm in Madagascan vanilla (Gatfield et al., 2006). These molecules are mostly the products of condensation between two phenolic compounds.

FIGURE 12.4 Chemical structures of compounds involved in the velvety mouth-coating sensation identified in cured vanilla beans.

COMPOSITION OF GLUCOSYLATED COMPOUNDS IN GREEN VANILLA BEANS

As mentioned in the previous section, the most important compounds in the volatile fraction, both quantitatively and qualitatively, have an aromatic ring and are derived from the phenylpropanoid pathway. Many of them are also found in the green fruit in the form of glycosides—in other words, bound by a glycosidic bond (such as O-glycoside) to one or several sugar groups—and have no olfactory properties.

Historically, the presence of glucosylated aroma precursors in the green vanilla bean was long suspected. After much debate (Lecomte, 1901, 1913; Goris, 1924), it was established that “vanilloside” (glucovanillin) was the direct precursor of vanillin, and that three other precursors also existed, but in smaller quantities. Different publications describe the attempts made to isolate and identify them (Goris, 1947; Janot, 1954). These are glucosides of vanillyl alcohol (“vanilloloside”), of protocatechuic aldehyde (3,4-dihydroxy benzaldehyde), and of an unidentified ester (Figure 12.5).

More recently, glucosides of vanillin, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and vanillic acid (Figure 12.5) were identified in the green bean (V. planifolia origin Comoros, Réunion, Madagascar, Indonesia), using modern analytical techniques (Leong et al., 1989a,b; Leong, 1991). Moreover, glucose seems to be the major sugar, since the acid hydrolysis of a prepurified extract of vanilla glycosides followed by an acetylation assay makes it possible to obtain around 20% glucose, 1% mannose, and traces of rhamnose (percentage expressed in relation to initial dry matter).

FIGURE 12.5 Chemical structures of the different aglycones found as glucosides in the green Vanilla beans according to different authors.

Kanisawa (1993) identified the glucosides of 31 molecules (Figure 12.5) in the green vanilla bean (V. planifolia origin Indonesia), including the vanillin, p- hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and vanillyl alcohol already identified by the previous authors. Compounds such as 2-phenyl ethanol, salicylic acid methyl ester, p-cresol, and acetovanillone—aroma active compounds— were also found in glucosylated form.

This author also isolated and identified p-hydroxybenzyl alcohol and two digluco-sides, bis[4-(β-d-glucopyranosyloxy)-benzyl] 2-isopropyl tartrate (glucoside A), and bis[4-(β-d-glucopyranosyloxy)-benzyl] 2-(1-methyl-propyl) tartrate (glucoside B), which are nevertheless only intermediaries in the biosynthetic pathway of the phenolic compounds.

In a later publication, however, Kanisawa et al. (1994) did not confirm the identification of the glucosides of 4 methyl esters: ferulic acid methyl ester, p-hydroxyben-zoic acid methyl ester, vanillic acid methyl ester, and p-hydroxycinnamic acid methyl ester. More surprisingly, nor did they confirm the presence of glucosides of p-hydroxybenzoic acid and benzyl alcohol in this publication.

In a recently published study, Dignum et al. (2004) looked for different glucosides in the green bean (V. planifolia origin Indonesia). The different glucosides identified are those of vanillin, vanillic acid, p-hydroxybenzaldehyde, p-cresol, creosol, vanil-lyl alcohol, and the two diglucosides A and B (Figure 12.5). In a previous study (Dignum et al., 2002), these authors also found guaiacol glucoside, a compound that they were subsequently unable to isolate.

Finally, Perez-Silva (2006) identified 17 glucosides in green vanilla beans from Mexico, including the glucoside of anisyl alcohol (Figure 12.5), which was reported for the first time.

However, it is important to note that the presence of many of the aforementioned molecules must be confirmed, as only the glucosides of vanillin, p-hydroxybenzal-dehyde, p-hydroxybenzoic acid, vanillic acid, and the glucosides A and B have been formally isolated and identified. Most of the other glucosides mentioned were first hydrolyzed by β-glucosidase action, and the free aglycones were identified. This kind of approach is likely to produce artifacts due to the reactivity of aglycones after their release.

FORMATION OF AROMA COMPOUNDS DURING CURING

GLUCOSIDES

As mentioned in Chapter 11, the best-known reaction in the development of the aromatic quality of vanilla during curing is the hydrolysis of the different glucosides that are precursors of the aforementioned aroma compounds. This is the fundamental reaction in the aroma development of vanilla, as it allows an aglycone that may have olfactory properties to be released from a glucoside that has none.

This reaction can be achieved either chemically under acidic conditions or enzy-matically using a β-glucosidase (or more generally, with a glycosidase, depending on the nature of the sugar linked to the aglycone). In the case of glucovanillin, despite some debate (see Chapter 11), it is now acknowledged that this hydrolysis is the result of one or several β-glucosidases that are endogenous to the fruit.

This idea was first put forward by Miller in 1754 (quoted by Janot [1954]). Lecomte (1901) was then able to prove the existence of ferments hydratant et oxydant— probably the origin of the term fermentation, which is still often incorrectly associated with vanilla curing—and studied their role in the development of the vanilla aroma.

After the glucovanillin (and other glucosides) had been isolated and the glucosi-dase activity in the raw extracts of the green bean had been measured, it seemed clear that the main reaction involved in the aromatic development of vanilla was the hydrolysis of the glucosylated compounds by a glucosidase (Arana, 1943): Lecomte’s ferment hydratant (1901). Many authors subsequently measured this enzymatic activity as part of their studies on the transformation process, the physiology of the fruit, and so on, but no research has been published on the purification and characterization of this glucosidase.

Kanisawa et al. (1994) mentioned the existence of two β-glucosidases in green vanilla beans: one is very specific to glucovanillin and p-hydroxybenzaldehyde glucoside, and the other has a much broader spectrum of activity. These results were obtained after precipitation using ammonium sulfate and cation-exchange chromatography. However, the experimental results are not included in the publication.

Odoux et al. (2003) purified and characterized a β-glucosidase of vanilla. An enzyme was isolated, with a native molecular weight of 200 kDa, an optimal pH of 6.5, an optimal temperature of 40°C, a K_m of 1.1 mM with pNPG and 20 mM with glucovanillin, and a V_m of around 5 μkatal mg⁻¹ protein with the two substrates.

Hanum (1997) obtained a K_m value of 0.38 mM with pNPG and Dignum et al. (2004) obtained a K_m value of 3.3 mM from a raw enzyme extract of green beans.

The latter also studied the kinetic parameters of the raw enzyme extract with different glucosides.

The findings show that the enzyme has greater affinity for the glucosides of vanillin, ferulic acid, and p-hydroxybenzaldehyde than for the glucosides of vanillic acid, guaiacol and creosol, and no activity for the glucosides of p-cresol and 2-phenyl ethanol.

A previous study (Dignum et al., 2002), based on monitoring aroma compounds during curing under laboratory conditions for a batch of vanilla from Indonesia, nevertheless, appeared to show a reduction in the glucoside of guaiacol (at least of the glucoside identified as such), which seemed less evident for the glucoside of van-illic acid. The results obtained for other compounds (aglycone form only) led these authors to conclude that the formation of the minor compounds, such as p-cresol and 2-phenyl ethanol, appears to occur via a chemical process rather than by an enzymatic one, where the lack of enzyme activity on the glucosides of these two compounds seems to confirm.

Research monitoring aroma compounds, during traditional curing in Mexico, Perez-Silva (2006) observed that of the 17 glucosides identified, 11 are only slightly hydrolyzed, if at all, which seems to confirm the findings of Dignum et al. (2002). These 11 compounds are the glucosides of creosol, 4-vinyl guaiacol, methyl salicy-late, p-hydroxybenzoic acid, p-cresol, anisyl alcohol, 2-phenyl ethanol, phenyl propanol, benzyl alcohol, and cinnamyl alcohol (Figure 12.5).

To sum up, the action of a β-glucosidase on a certain number of aroma precursor glucosides is clearly established in the development of vanilla aroma during curing, but is far less evident for certain others, even though their corresponding aglycones may be present (see below).

It must nevertheless be stressed that given the very low concentrations of these compounds, the natural variability of these concentrations and their reactivity (especially the free aglycones), and so on, monitoring during curing is very difficult to implement and requires special attention regarding the sample size in order to avoid the sometimes erratic biases and fluctuations observed. This implies specific studies that are often lacking.

Approaches that consist in studying the kinetic parameters of the different aroma precursor glucosides on purified β-glucosidase(s) are undoubtedly more demonstrative for establishing once and for all whether these glucosides are substrates of the enzyme, but clearly imply obtaining glucosides and purified enzyme, which represents a good deal of work in itself.

It is also possible to work on raw enzyme extracts rather than on purified enzymes in order to simplify the task and to be sure to have all the β-glucosidase activities present in the green bean (if several exist). It should nevertheless be remembered that the enzyme(s) may be put into contact with products that affect the kinetic parameters (activators/inhibitors), which would otherwise not come in contact in the green bean or during curing.

From experience, we observed during the development of our enzymatic test that extracting the enzyme with low buffer/beans ratios (i.e., 4:1 v/w) led to rapid and considerable losses in β-glucosidase activity (even using a protective agent, such as antiprotease, antioxidants, etc.), while very high buffer/beans ratios (2000:1 v/w) made it possible to achieve high stability for over 4 h of storage at ambient temperature without any protective agent in the extraction buffer (unpublished results). These observations suggest the presence of compounds that inhibit β-glucosidase activity in moderately diluted green vanilla extracts.

OTHER COMPOUNDS

In most cases, the reaction mechanisms that lead to the appearance and/or disappearance of the other aroma compounds during curing remain to be determined.

For example, aldehydes are not found in green vanilla beans (Perez-Silva, 2006), but appear in advanced stages of curing when their content peaks, and then disappear with only traces found in cured vanilla. Some of these aldehydes could be products of the autooxidation of oleic acid and linoleic acid (found in large quantities in green vanilla).

Aliphatic acids on the contrary, of which linoleic acid is the major representative, are found in the green beans and their concentration tends to decrease during curing. Only acetic acid is not found in the green beans, but appears at a concentration of around 700 ppm after the first heat treatment, then decreases and stabilizes at around 200 ppm. The formation of acetic acid could be of microbial origin (Perez-Silva, 2006).

Aliphatic alcohols also appear after heat treatment, whereas the esters and hydrocarbons are already found in the green beans and their concentrations gradually decrease during curing.

As previously mentioned, among the compounds from the phenylpropanoid pathway, a certain number of aglycones are present despite the (apparent) lack of hydrolysis of the corresponding glucoside (as with p-cresol), or are present in far higher proportions than their concentration in glucosylated form suggested (as with vanillic acid), or are even present when the glucosylated form is absent (as with guaiacol). In fact, although the glucosylated forms are known to be relatively chemically unreactive, the aglycone released after hydrolysis may on the contrary become the substrate for chemical and/or enzymatic reactions, and thus be oxidized, reduced, decarboxylated, methylated, and so forth, and interconversions of one phenolic into another are therefore possible and even common.

For example, concentrations of vanillic acid higher than those expected can be explained by the oxidation of vanillin, and vanillic acid may also undergo decarboxy-lation into guaiacol (Perez-Silva, 2006), which could also explain the lack of glucoside of guaiacol. A solution of vanillin kept at 80°C at pH 5 in the presence of oxygen for several days also reveals the presence, in addition to the aforementioned molecules, of 2-methoxyhydroquinone, which can form quinones and semiquinones, which may lead to dimers (Perez-Silva, 2006). Different molecules of this kind, including divan-illin, have been identified in cured vanilla (Figure 12.4) and are considered to contribute positively to the flavor of the product (Gatfield et al., 2006; Schwarz and Hofmann, 2009). Gatfield et al. (2006) suggest that the formation of divanillin is the result of the action of a peroxidase, a form of which has recently been purified (Marquez et al., 2008). This kind of reaction can also lead to the formation of large polymers that are at least partly responsible for the brown color of the vanilla.

These different reactions, which all start from vanillin, partly explain the considerable losses of this compound during curing (Odoux, 2000; Gatfield et al., 2007) that were already mentioned in Chapter 11.

Other reactions with no direct relationship to aroma formation may also explain these losses of vanillin, such as sublimation, coevaporation with water (Frenkel and Havkin-Frenkel, 2006), or the formation of a covalent bond with the lignin of the bean (Gatfield et al., 2007).

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