Handbook of Vanilla Science and Technology

Fig. 6.7 Time-course of change in total protein content in vanilla bean undergoing curing at 50°C. Total proteins were extracted periodically from bean tissue and estimated as previously described (Ranadive etal. 1983). Reproduced with permission from Perfumer & Flavorist magazine, Allured Business Media, Carol Stream, IL.

Fig. 6.8 Time-course of change in proteolytic activity in vanilla bean undergoing curing at 50°C. Fifty ml of crude extract, denoting unit of pod tissue, represents 14.3 mg fresh weight of curing vanilla bean tissue. Reproduced with permission from Perfumer & Flavorist magazine, Allured Business Media, Carol Stream, IL.

Proteolytic activity in the curing vanilla pod may result from the release of cellular proteases, previously compartmentalized in the vacuole (Okamoto 2006; Muentz 2007) or perhaps from other cellular compartments. It is also likely that latent proteolytic activity is triggered by killing-induced protein denaturation, a process leading to surface exposure of the protein hydrophobic core and a mechanism for proteolytic targeting of denatured proteins (Bond and Butler 1987). Denaturation of cellular proteins may occur during killing by heat or freezing and, in addition, by previously compartmentalized cellular constituents that might be deleterious to correct folding and function of cellular proteins. Examples include cytosol acidification by organic acids or denaturation by phenolic compounds that have diffused out of the vacuole. Lipid peroxides formed in cured beans (Figure 6.9), and perhaps other oxidants, may also attack and denature cellular proteins (Bond and Butler 1987).

Fig. 6.9 Change in the content of lipid hydroperoxides in bean tissue during curing at 50°C. Tissue increments of vanilla bean were removed periodically during curing forthe estimation of lipid hydroperoxide, as previously described (Eskin and Frenkel 1976). Reproduced with permission from Havkin-Frenkel, D., French, J.C., Graft, N.M., Pak, F.E., Frenkel, C. and Joel, D.M. (2004) Interrelation of curing and botany in vanilla (Vanilla planifolia) bean. Acta Hort. (ISHS), 629, 93-102.

A marked decrease in protein content after a few hours of curing (Figure 6.7) suggests that the action of proteases diminished the level of enzymes and proteins. However, glycosyl hydrolases that catalyze the hydrolysis of glyco-conjugates, glucovanillin for example, may be temporarily spared from proteolytic degradation, because extracellular glycosyl hydrolases are glycoproteins. The latter, composed of a polypeptide glycated with oligosaccharide side chains (Trincone and Giordano 2006; Lopez-Casado et al. 2008), display resistance to proteolysis (Nishi and Itoh 1992; Varki 1993). This presumption is in keeping with the observation that β-glucosidase activity persists during the harsh curing conditions and is sufficient to carry out hydrolytic cleavage of glucovanillin to near completion (Figure 6.10), a conclusion verified also by Odoux et al. (2006).

Fig. 6.10 Time-course of change in the content of glucovanillin and in vanillin in whole vanilla beans (top) and chopped beans (bottom) undergoing curing at 50°C. Reproduced with permission from Perfumer & Flavorist magazine, Allured Business Media, Carol Stream, IL.

6.5.2 Cell wall hydrolyzing enzymes

Several studies observed that addition of commercial preparations of cell wall degrading enzymes accelerated the hydrolysis of glucovanillin to vanillin in curing vanilla pods (Mane and Zucca 1993; Brunerie 1998; Ruiz-Teran et al. 2001). These results suggest that glucovanillin or other glycosylated phenolic compounds trapped, apparently, in the wall matrix, might be released by the wall degradation and become accessible to their respective hydrolytic enzymes. We observed, accordingly, that addition of pure preparations of pectin-degrading enzymes to chopped green beans accelerated the conversion of glucovanillin to vanillin. However, at the end of the incubation period, the vanillin content was roughly the same in control beans (no enzyme added) as in enzyme-treated beans, the only difference being the rate of vanillin formation in control and treated beans (results not shown). These data suggest that activity of endogenous wall hydrolyzing enzymes is sufficient for the conversion of glucovanillin to vanillin, whereas applied enzymes merely accelerate the rate of the process. Additional studies may reveal whether the wall degradation is essential for accessing and subsequent conversion of glucovanillin to vanillin or whether cell wall dissolution might, alternatively, increase the extractability of released vanillin and other flavor constituents, as we previously found (unpublished data).

6.5.3 Glycosyl hydrolases