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 Km of 1.1 mM with pNPG and 20 mM with glucovanillin, and a Vm of around 5 μkatal mg−1 protein with the two substrates.
Hanum (1997) obtained a Km value of 0.38 mM with pNPG and Dignum et al. (2004) obtained a Km 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).