Handbook of Vanilla Science and Technology

A German research group (Schwarz and Hoffman 2009) investigated non-volatile taste contributing constituents in vanilla. Specifically, they were attempting to detect components beyond vanillin and divanillin that contribute to the “mouth-feel” of this product. A Madagascar vanilla extract was extensively extracted by partitioning with pentane and then ethyl acetate. Further fractionation of the solvent free ethyl acetate fraction by gel permeation chromatography (sephadex LH-20) with varying concentrations of aqueous methanol produced nine sub-fractions. Several targeted fractions were further purified by HPLC and nine “active” constituents were isolated, including vanillin and divanillin. All were fully characterized and their sensory impact determined with taste dilution analysis (TD) with the half-tongue test. The constituent with the most mouth-feel was 4’, 6’ -dihydroxy - 3’, 5’ - dimethoxy - (1, 1’ - biphenyl) - 3 - carboxaldehyde. These researchers proposed that quantitative monitoring of these compounds could be used in order to tailor the curing process to enhance these orosensory active molecules.

10.3 BIOCHEMISTRY AND GENETIC RESEARCH ON VANILLA

During the 1990s, research into the biochemistry of vanilla flavor was undertaken. In 1989, a French group (Leong et al. 1989) synthesized the glycosides of the four major vanilla flavor phenolics. The analytical characteristics (m.p., RF, UV, HPLC, anδ¹³C-NMR) of each of these glucosides were determined. These synthetic glucosides were then used to confirm the presence and quantity of these glycosides in green vanilla beans from Comoros, Madagascar,

Reunion, and Indonesia. They also showed the absence of free aldehydes and acids in green uncured vanilla beans.

Ranadive (1992) demonstrated that the major vanilla flavor constituents were present as glycosides prior to curing. Using reverse phase HPLC, he determined the amount of vanillin, p-hydroxybenzaldehyde, vanillic acid, and p-hydroxybenzoic acid and also identified vanillyl alcohol and p-hydroxybenzyl alcohol. He investigated vanilla beans from seven growing regions and showed that added glucosidase increased the amount of these flavor phenolics when added to heat treated enzyme-deactivated green Jamaican beans.

Brodelius (1994) also used reverse-phase HPLC to study the formation of phenolic flavor compounds in developing vanilla fruits following hand pollination. He found that vanillin formation began at 16 weeks and maximized at 26 weeks after pollination. The other three major vanilla phenolics were also detected.

In the late 1990s, three studies were undertaken by Chinese researchers. Pu et al. (1998a) demonstrated that the treatment of green beans with the enzyme b-glucosidase under optimized conditions improved the hydrolysis of the glycoside and increased vanillin content, as well as the content of the other phenolic flavor compounds also tied up as glycosides. The second study (Pu et al. 1998b) investigated b-glucosidase activity at different stages of green bean maturity. This information could be used for improving the quality of vanilla beans as well as the curing process. The third study (Jiang et al. 1999) investigated vanilla enzymes. b-glucosidase had greater activity in the seeds than the pod. Peroxidase had high activity in the pod and little in the seeds. Jiang et al. (2005) extended this research by investigating changes in b-glucosidase and peroxidase activity during different processing conditions, which included different deactivation of enzyme and roasting processes.

Early in the twenty-first century, a Dutch group (Dignum et al. 2002) used a laboratory model of curing to investigate the effect of the curing processes on the activities of the variety of enzymes found in vanilla beans. b-Glucosidase, peroxidase, protease, and phenylalanine ammonia-lyase were studied. They later investigated the source of the key minor flavor constituents in vanilla (Dignum et al. 2004). They were curious from where p-cresol, creosol, guaiacol, and 2-phenylethanol were derived. Were they derived from glucosides or were they formed during the curing process? Glucosides of p-cresol and creosol were detected as minor compounds in green beans, but glucosides of guaiacol and 2-phenylethanol were not detected. In addition, they found glucosides of vanillin, vanillyl alcohol, vanillic acid, p-hydroxybenzaldehyde, and two tartrates. With all these glucosides, these researchers investigated the b-glycosidase enzyme action on these compounds. This was an intensive investigation of enzyme activity on a series of natural and synthetic glycosides.