2.2. Starch composition
Starch composition was determined enzymatically using the
Megazyme RS assay procedure (KRSTAR test kit, Megazyme International,
County Wicklow, Ireland). Briefly, 100 mg of milled sample
were incubated in a shaking water bath with thermo-stable
pancreatic a-amylase and AMG for 16 h at 37 C. During this incubation
the non-resistant starch (non-RS) is solubilised and
hydrolysed to glucose by the two enzymes. The reaction was
terminated by the addition of equal volume of ethanol and the RS
was recovered as a pellet on centrifugation (3000 g) for 10 min. The
supernatants of this centrifugation and those of two consecutive
washings were removed by decantation and stored. RS pellets were
dissolved in 2 mol/L KOH and stirred for 20 min in an ice/water bath
over a magnetic stirrer. Sodium acetate buffer (1.2 mol/L, pH 3.8)
was added, the starch was quantitatively hydrolysed to glucose with AMG. The absorbance of the released glucose was spectrophotometrically
determined at 510 nm using glucose oxidaseeperoxidase
reagent (GOPOD) method (GOPOD Format Assay Kit,
Megazyme International, County Wicklow, Ireland). Glucose
release of non-RS was equally determined by previously pooling
the supernatant and the twowashings and adjusting the volume to
100 mL. Total starch was calculated as the sum of RS and non-RS.