## 3 Materials and Methods
### Determination of the Enzymatic Activities  
Extraction and assay of \(\beta\)-glucosidase were performed according to the method described by [PERSON] et al. [26]. Two grams of vanilla beans at different curing stages were chopped into 1 cm pieces.BisTris-propane[1,3-bis[tris(hydroxymethyl)methylamino]-propane; BTP] buffer (150 mM) at 20 mL was used at pH 8.0. Ethylene Diamine Tetraacetic Acid (EDTA) and dithiothreitol (DTT) at 2 and 3 mM, respectively, were added to the buffer. Grinding was performed after adding 100 mg of PVPP. Extracts were centrifuged and then stored at -80 \({}^{\circ}\)C in the presence of 15% glycerol before enzyme analysis. The activity of \(\beta\)-glucosidase was determined at 30 \({}^{\circ}\)C according to [PERSON] et al. The reaction mixture contained 250 \(\upmu\)L enzyme extract and 475 \(\upmu\)L 2.1 mM _p_-nitrophenyl-\(\beta\)-n-glucopyranoside in 0.1 M phosphate buffer (pH 6.3). To inactivate the enzyme, 800 \(\upmu\)L 1 M sodium carbonate was added after 30 min. The absorption was measured at 400 nm. The activity was calculated by using the formula \(\varepsilon\)400 = 18500/M cm, which was expressed by assuming that the mean of enzyme activity in fresh vanilla beans was 100%.  
The extraction of peroxidase was the same as that of \(\beta\)-glucosidase. The activity was determined following the procedure explained by [PERSON] et al. [26]. A cuvette containing 0.3 mL guaiacol (40 mM) and 0.6 mL hydrogen peroxide (1.5 mM) in McIlvaine buffer (pH 3.8) were prepared. With addition of the 200 uL enzyme extracts, the mixture was reacted for exactly 3 min, where the absorbance was measured at 470 nm (\(\varepsilon\)470 = 26600/M cm). The activity was expressed by assuming that the mean enzyme activity in fresh vanilla beans was 100%.  
Polyphenol oxidase activity was assayed using the method described by [PERSON] and [PERSON] [53]. The extraction involved chopping of beans into 1 cm pieces, of which 2 g was extracted with 20 mL of 0.1 M phosphate buffer (pH 6.5) and ground for homogenization. The extract was centrifuged at 75,000 g for 20 min. For the assay:1.5 mL catechol (0.01 M) prepared with 0.1 M phosphate buffer (pH 6.5) was added to 1 mL enzyme extract. After blending, the absorbance was measured at 495 nm, and the data was recorded in each case for 3 min at 30 s intervals. The activity was expressed by assuming that the mean enzyme activity in fresh vanilla beans was 100%.  
Cellulose was performed according to the procedure of [PERSON] et al [4]. For extraction, 2 g of vanilla beans at different stages was chopped into 1 cm pieces and extracted at 4 \({}^{\circ}\)C with 20 mL sodium citrate buffer (pH 5). The extract was centrifuged twice at 5000 g for 15 min and the supernatant was used as the enzyme source. Cellulose activity was determined by incubating 0.5 mL of 0.1% (_w/v_) carboxymethylcellulose sodium (CMC) and 0.25 mL of sodium citrate buffer (pH 5) with 2.5 mL of enzyme extract for 1 h at 37 \({}^{\circ}\)C. The reaction was stopped by the addition of 1 mL NaOH (2M). After the addition of 1 mL DNS, the mixture reacted for 5 min in boiling water, and the absorbance was read at 490 nm. One unit of cellulase activity was the amount of enzyme that catalyzed the formation of 1 reducing group/min. The activity is expressed by assuming that the mean enzyme activity in fresh vanilla beans was 100%.