*3.8. Rapidly and Slowly Digested Starch Contents*

The rapidly digested starch (RDS) and slowly digested starch (SDS) levels were determined by measuring reducing sugars released during in vitro digestion of biscuits according to the procedure described by Soong, Tan, Leong, and Henry [54]. Briefly, sugars released during in vitro digestion were evaluated as monosaccharides using a dinitrosalicylic acid (DNS) colorimetric method with slight modifications. To complete the depolymerization into monosaccharides, 50 µL of the supernatant was mixed with 0.25 mL of acetate buffer containing 0.4% invertase and 1% amyloglucosidase. Incubation was carried out at room temperature for 30 min. Next, 0.75 mL of DNS mixture containing 0.5 mg/mL glucose, 4 M NaOH, and DNS reagent at a 1:1:5 ratio was added and heated for 15 min at 95–100 ◦C, cooled, and then diluted with 4 mL distilled water. The absorbance of the samples and standards was read at 530 nM against the blank.

The amount of sugars released was calculated in mg of glucose/g of each sample using the absorbance values. The rapidly digested starch (RDS) was evaluated as the amount of reducing sugars determined in the sample aliquot after 20 min from the start of pancreatic digestion, whereas slowly digested starch (SDS) was calculated as the difference between the amount of reducing sugars measured at 120 min and RDS.
