Resistant Starch Content Determination

Using the RS assay kit, the resistant starch (RS) content of rice samples was calculated (Megazyme International Ireland Ltd., Bray, Ireland). Briefly, a 100 mg sample was digested in 4 mL of pancreatic amylase (10 mg/mL) with amyloglucosidase (300 U/mL) at 37 °C with continuous agitation at 200 rpm for 16 h, and then 4 mL of 100% ethanol was added with vigorous stirring. Next, centrifugation at 3000 rpm for 10 min followed. The pellet was centrifuged at 3000 rpm for 10 min while suspended in 8 mL of 50% ethanol. The non-resistant starch content of the supernatant was analysed. The pellet was resuspended in 2 M KOH for 20 min, and then 8 mL of 1.2 M sodium acetate buffer and 0.1 mL of amyloglucosidase (3300 U/mL) were added. The tubes were centrifuged at 3000 rpm for 10 min after being incubated at 50 °C for 30 min. A different set of test tubes received aliquots of 0.1 mL of supernatant, to which 3 mL of glucose oxidase/peroxidase (GOPOD) reagent was added. For 20 min, these tubes were incubated at 50 °C. At 510 nm, there was a comparison to a blank for the reagent. Based on the readings, the RS%, NRS%, and TS% were determined [32 (link)].