Whole Grain Digestibility Protocol

Whole grain samples were digested following established procedures.^{64 (link)} Briefly, 5 g of whole grain were ground into fine powder using Geno/Grinder 2025 (SPEX SamplePrep) at 1600 rpm for 10 min. Then, 2.5 g of sample was mixed with 30 mL of water in a 50 mL Falcon tube for 20 min until fully dispersed. Tubes containing the slurries were immersed in boiling water for 20 min with constant shaking. The slurries were then placed on an orbital shaker (200 rpm) and incubated at 37°C for 40 min. The pH was adjusted to 2.5 with 1 M HCl followed by the addition of 1 mL of 10% (wt/vol) pepsin (P7000; Sigma, St. Louis, MO) in 50 mM HCl. The slurry was then incubated on an orbital shaker (200 rpm) at 37°C for 60 min. The pH was adjusted to 6.9 with 0.5 M NaHCO₃ and 5 mL of 12.5% (wt/vol) pancreatin (P7545; Sigma, St. Louis, MO) in 0.1 M sodium maleate buffer, and 0.2 mL of amyloglucosidase (E-AMGDF, 3,260 U/mL, Megazyme) was added. The slurry was incubated for 6 h at 37°C with orbital shaking at 200 rpm. Following digestion, the material was transferred to dialysis tubing (molecular weight cutoff of 1000 Dalton) and dialyzed against distilled water for 3 days at 4°C with a water change every 12 h. The retentate from the dialysis was collected, freeze-dried and resuspended in 30 mL of sterile distilled water. The resistant starch concentration was determined with a commercial kit using the protocol variation entitled ‘Determination of total starch content of samples containing resistant starch’ (K-TSTA, Total Starch Assay Kit (AA/AMG), Megazyme).