Resistant starch kit

The total amount of starch in the grain was measured by enzymatic methods using the enzymes in Resistant Starch kit (Megazyme, K-RSTAR). A grain was ground in 500 μL of 80%(v/v) ethanol using the Multi-beads Shocker MB2000. The sample was incubated at 85 °C for 5 min. After the centrifugation at 1800 × g, the pellet was resuspended in 1 mL of 80% (v/v) ethanol and centrifuged to obtain a pellet. The pellet was resuspended in 1 mL of 100 mM sodium maleate buffer (pH 6.0) and centrifuged at 1800 × g. After washing three times, the pellet was incubated in 1 mL of 100 mM sodium maleate buffer (pH 6.0) containing 5 mg mL⁻¹ pancreatic α-amylase and 1.5 unit mL⁻¹ amyloglucosidase at 37 °C for 16 h. The supernatant was recovered after centrifugation at 1800 × g for 5 min. The pellet was resuspended with 1 mL of 50% (v/v) ethanol and centrifuged again. The supernatant was recovered and put together with the previous supernatant. This procedure was repeated three times. Finally, the supernatant was adjusted to 10 mL with water. The supernatant (110 μL) was mixed with 900 μL of 100 mM sodium acetate buffer (pH 4.5). The 110 μL of the mix was treated with amyloglucosidase at 50 °C for 20 min. The glucose amount in the sample was measured using GOPOD reagent (Megazyme).