2.9. In vitro digestibility

The in vitro digestibility of starch was determined as described by Englyst et al. [22] with slight modifications. A starch sample (500 mg, dry basis weight) was added to a capped tube containing 20 mL distilled water, cooked in a boiling water bath for 30 min, and cooled to 37 °C. A mixture of 4 mL α-amylase solution (3600 U/mL) and 1 mL of amyloglucosidase solution (2500 U/mL) was added to each tube, then incubated in 37 °C water bath with 200 r/min agitation. Every 20 min, 1 mL digestive juice was taken, mixed with 1 mL 95 % ethanol, and centrifuged for 10 min at 5000 r/min. The hydrolyzed glucose content was measured using the glucose oxidase-peroxidase reagent (Megazyme). The digestion characteristics of starch were characterized by rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). The calculation formulas were as follows:

where G_t represent the reducing sugar content (mg) in the reaction solution at sampling time point; G₂₀ and G₁₂₀ represent the reducing sugar content (mg) produced by hydrolysis at 20 min and 120 min, respectively; TS represents the total starch content (mg), and FG represents the enzyme content of free reducing sugars in the sample before hydrolysis (mg).