Processing of seed coat material. Vanilla seed and pod were received as a mixture from a natural vanilla processing plant (Bakto Flavors LLC). The mixture was sifted, and the lower-density remaining pod powder was blown away using a heat gun (set on cold). Preparation of vanilla seed coat NMR samples was via methods described previously (22). Briefly, isolated vanilla seed coats (4 × 300 mg) were ball-milled (30 × 10 min, 5-min cooling cycle) using a Retsch PM100 ball mill vibrating at 600 rpm with ZrO2 vessels containing ZrO2 ball bearings. Preground seed coat was extracted using a modified Bligh and Dyer extraction (41) to remove oils and extractives.

Modified Bligh and Dyer extraction. Vanilla seed material (100 g in total) was shaker-milled (MM400, Retsch) at 3600 rpm for 5 min using a 50-ml stainless steel jar and a single 20-mm ball bearing. The milled sample was transferred to a 1-liter volumetric flask, and a magnetic stir bar was added. Deionized (DI) water (80 ml), chloroform (100 ml), and methanol (200 ml) were added, and the mixture was stirred at 50°C for 30 min. To the mixture was then added 100 ml more of chloroform, and then, after another 30 min, 100 ml of DI water was added. The stirring was continued at 50°C for 24 hours, and the insoluble material was removed by centrifugation (3800 rpm for 15 min), retaining the solids by decanting off the solvent and keeping the filtrate as well. The residue was extracted again by the same method. The filtrates were combined, and the solvents were removed by rotary evaporation to produce the extractives fraction for analysis.

EL from vanilla seed coat. The ball-milled extract-free vanilla seed coat material (1 g) was placed in centrifuge tubes and digested at 35°C with crude cellulases [CELLULYSIN cellulases, Trichoderma viride; sample (50 mg/g) in acetate buffer (pH 5.0); two times over 3 days; fresh buffer and enzyme were added each time; catalog no. D00074989, Calbiochem], leaving all of the phenolic polymers and residual polysaccharides totaling 859 mg (85.9%) (table S1).

Acidic LiBr pretreatment of C-lignin from vanilla seed coat. C-LBL was prepared using the acidic LiBr trihydrate method described previously (23). Briefly, ball-milled extract-free vanilla seed coat material (1 g) was added into a 40-ml glass vial with a polytetrafluoroethylene (PTFE) lined cap, together with 4.50 ml of acidic 60 weight % (wt %) LiBr solution containing 0.04 M HCl. The vial was immersed into an oil bath preheated at 110°C under magnetic stirring. The mixture was filtered under vacuum and washed with water. The residues were dried at 40°C under reduced pressure (yield, 72.4%; table S1).

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