2.2. Analytical methods
2.2.1. Resistant starch and starch
AOAC Method 2002.02; AOAC Method 32–40 (RSTAR II/02,
Megazyme).
Samples are incubated in the presence of pancreatic alpha-amylase
and amyloglucosidase for 16 h at 37 C. During this time nonresistant
starch is solubilised and hydrolysed to glucose by the synergistic
action of the two enzymes. By the addition of ethanol RS
and subsequent centrifugation RS is obtained as a pellet, while obtained
supernatant is further used for measuring total starch. Pellet
is dissolved in 2 M KOH and solution is neutralised with acetate
buffer. RS is quantitatively hydrolysed to glucose with amyloglucosidase.
Glucose is assayed with glucose oxidase/peroxidase reagent
(GOPOD), and the absorbance is measured at 510 nm. Soluble nonresistant
starch is determined from supernatant, while absorbance
is measured at the same wavelength as for RS. This method offers
the most approximative results, those that can be as far as possible
reflected in vivo and can be considered physiologically significant.