3.6. In Vitro Starch Digestibility

The flour of two to four foxtail millet accessions from each AC level was analyzed by using in vitro starch hydrolysis based on Englyst et al. (1999) with some modifications. In rice, IR8, TN11, and Hung-No, representing high AC, intermediate AC, and waxy type were included for comparison. Approximately 0.5 g flour was dispersed in 10.0 mL of freshly prepared pepsin solution (50 mg of pepsin and 50 of guar gum in 10 mL of 0.05 M HCl) and incubated at 37 °C for 30 min at 200 rpm. Subsequently, 10.0 mL of 0.1 M acetate buffer (pH 5.5, 37 °C) was added to form a buffer at pH 5.2. The enzyme mixture was prepared by dispersing 3.0 g of pancreatin in 20.0 mL of H₂O in the tube, shaking for 10 min, and then centrifuging at 1500 g for 10 min; 15.0 mL of pancreatin supernatant was saved, subsequently adding 0.75 mL of amyloglucosidase (1200 U mL⁻¹) and 1 mL of invertase (3000 U mL⁻¹). Starch digestion was initiated by adding 5 mL of the enzyme mixture and then incubating at 37 °C for 3 h under horizontal shaking at 200 rpm. During the incubation, 0.5 mL of each sample was added into 10 mL of absolute ethanol and mixed well every 20 min. These digested samples were collected at 7 time points, specifically 20 (G₂₀), 40, 60, 80, 100, 120 (G₁₂₀), and 180 min (TG), and the supernatant was saved after centrifugation at 1500× g for 5 min. The glucose portion in the supernatant was measured according to the glucose oxidase-peroxidase method by using a GOD-POD diagnostic kit (Applygen Technologies, Beijing, China). The content of three starch fractions, RDS, SDS, and RS was calculated on a dry basis with the following equations and expressed as percentage of total starch [50]:

The estimated glycemic index (eGI) was calculated based on glucose measurements at 7 time points mentioned above during starch hydrolysis. The kinetics of in vitro starch digestion was calculated according to the first order equation [51]:

where C and t correspond to the percentage of starch hydrolyzed at t time. C∞ is the equilibrium concentration and k is the kinetic constant in t time. Area under curve (AUC) was obtained each sample by using the equation:

where tf and t0 are the final (180 min) and initiate (0 min) hydrolysis time, respectively.

The hydrolysis index (HI) was calculated by dividing the area under curve (AUC) value of each sample with the AUC of the reference food, japonica rice TN11. The expected (eGI) was converted by following equation:

which H₁₀₀ is the glucose content measured at 100 min.