Handbook of Vanilla Science and Technology

Glucovanillin is a major glycosyl conjugate of vanillin, although trace amounts of other glycosyl conjugates of vanillin or other phenolic compounds containing mannose, galactose, and rhamnose are found in the developing vanilla pods (Leong et al. 1989a,b; Tokoro etal. 1990; Kanisawa etal. 1994; Pu etal. 1998; Dignum 2001a). According to Arana (1944), glucovanillin was first isolated from the vanilla bean in 1858 by Gobley, followed by a demonstration that the compound undergoes hydrolytic cleavage to vanillin and glucose during the curing process (Goris 1924). It is generally accepted that vanillin is formed by β-glucosidase-catalyzed hydrolytic cleavage of glucovanillin, although green vanilla beans contain other glycosyl hydrolases, including α- and β-glucosidase, α- and β-galactosidase, as well as α- and β-mannosidase (results not shown). Because of the importance of vanillin to vanilla flavor, β-glucosidase-catalyzed formation of vanillin is one of the most studied processes in a vanilla bean. The rate of glucovanillin conversion to vanillin and glucose may be measured by the rate of disappearance of glucovanillin and an accompanying accumulation of vanillin. Another approach is based on estimating the activity of β-glucosidase in bean tissue, assuming that the enzyme activity is an index of glucovanillin hydrolysis. Activity of β-glucosidase is measured traditionally with the use of p-nitrophenyl-β-glucopyranoside or with glucovanillin as substrates.

Temperature regimes during the killing and subsequent sweating stages appear to be critical to the activity of β-glucosidase (Marquez and Waliszewski 2008). Our studies revealed that temperature optima for enzymatic activity were 50°C for β-glucosidase, 55°C for α-galactosidase, and 60°C for β-galactosidase. Activity of β-glucosidase and α- and β-galactosidase in curing vanilla beans held at 50°C is substantial and measurable, whereas activity of other glycosyl hydrolases tends to be low (results not shown). Thermal-stability of glucosidases, arising from molecular features of the enzyme polypeptide chain, is discussed elsewhere (Sanz-Aparicio et al. 1998; Hrmova et al. 1999). Glycation of the polypeptide side chain with oligosaccharide is, apparently, another molecular feature conferring thermal stability on glycosyl hydrolases (Nishi and Itoh 1992; Varki 1993). Thermal tolerance of these enzymes is consistent with the empirical exploitation of elevated temperatures during the curing process, for the hydrolytic release of vanillin and perhaps other flavor components from glycol-conjugate precursors.