Handbook of Vanilla Science and Technology

Importantly, browning in a curing vanilla pod appears to be carried out by enzymatic activity, in agreement with the observation that browning was arrested by harsh killing conditions, prolonged or extreme heating, for example (Rabak 1916), apparently due to heat-denaturation of oxidative enzyme(s). A similar study showed arrest of browning in green beans using autoclaving at 120°C and, furthermore, restoration of browning in autoclaved and non-browning beans upon the addition of oxidative enzymes of fungal origin (Jones and Vincente, 1949b). We also observed that while killing by freezing resulted in typical browning in mature green beans, excessive boiling in post-frozen bean resulted in the inhibition of browning (results not shown). Polyphenol oxidase (PPO) and peroxidase are two major enzyme systems that may catalyze browning processes in killed vanilla beans (Broderick 1956b). PPO, represented by a family of enzymes, utilizes molecular oxygen to catalyze the hydroxylation of monophenol to O-diphenol and subsequent removal of hydrogen atoms from O-diphenol to give O-quinone. The latter might spontaneously polymerize, resulting in the formation of dark oxidation products (Toscano et al. 2003). PPO-driven browning in vanilla pods may result mostly from the oxidation of tyrosine, caffeic, and chlorogenic acid, as well as other phenolic compounds (Schwimmer 1981). PPO-induced browning in killed vanilla pods, representing tissues in a state of stress, complies with the emergence of PPO activity in injured or stressed plants (Schwimmer 1981). Peroxidative enzymes, by comparison, utilize H₂O₂ and other hydroperoxides as well as molecular oxygen to catalyze the oxidation of various cellular substrates, including oxidation of aromatic compounds (Schwimmer 1981), oxidative bleaching of carotenoids (Ben-Aziz et al. 1971), discoloration of anthocyanins (Grommeck and Markakis 1964), or degradation of ascorbic acid (Blundstone et al. 1971). Because peroxidases catalyze the degradation of a wide array of cellular substrates, the role of the enzyme may be wider than just a contribution to browning. For example, peroxidase-driven peroxidation of unsaturated fatty acids, catalyzed by the heme group in the enzyme (Lilly and Sharp 1968), may lead to the utilization of lipids, apparently membrane-lipid, for the apoplastic production of H₂O₂ (Lindsay and Fry 2007; Kaerkoenen et al. 2009). Peroxidase-catalyzed production of peroxides might account for a marked increase in lipid hydroperoxides in killed vanilla beans (Figure 6.9), arising perhaps from the oxidation of abundant lipid bodies in placental hairs. Moreover, spontaneous propagation of formed lipid hydroperoxides might further amplify the oxidant effect of the enzyme.

Activities of PPO and peroxidase increase steadily during vanilla pod development (Wild-Altamirano 1969; Ranadive et al. 1983), although the enzyme activity is apparently latent and is not expressed in mature green beans. Activity of these enzymes appears to be unleashed by various killing methods, as occurs naturally in senescence or induced by ethylene. Several studies confirmed that activity of PPO remained high in vanilla beans following killing and during subsequent curing stages (Balls and Arana 1941a, 1942; Jones and Vincente, 1949c) or in tissue extracts of vanilla beans (Dignum 2001a). Though Jiang et al. (2000) observed low PPO levels in cured beans, we found that PPO activity decreased during curing by approximately 50% but remained steady and substantial (Figure 6.12). Peroxidase activity, by comparison, increased with time of curing, suggesting that the enzyme protein resists proteolytic degradation occurring during the killing and subsequent curing stages (Figure 6.12). Persistence of peroxidases well into the conditioning stage (Broderick 1956b) may stem also from the enzyme heat-stability shown by continued peroxidative action, even after dipping green beans in 80°C for 20 minutes (Dignum 2001b) or restoration of peroxidative activity after autoclaving vanilla pod tissue at 120° C (Broderick 1956b), further indicating stability of peroxidative action, even under extreme conditions.

Fig. 6.12 Time-course change in the activity of polyphenoloxidase (top) and peroxidase (bottom) 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.