Resistant starch type III was isola

Resistant starch type III was isolated by modification of
the enzymatic-gravimetric procedure for the determination
of total dietary fibre (Eerlingen et al., 1993a; Shamai et al.,
2003). Enzyme preparations were purchased from Sigma–
Aldrich Chemie GmbH (Steinheim, Germany). Samples
were removed from the incubator and suspended in
50 mL phosphate buffer (pH 6.0) at 37 C before adding
a-amylase (0.4 g/g starch, Sigma A-3176) and incubated
at 37 C for 16 h. The samples were cooled to 25 C before
adjusting the pH to 4.5 using 2 mL/100 mL phosphoric
acid solution. Amyloglucosidase (1 mL, Sigma A-7095)
was added and the samples incubated at 60 C for
30 min. After enzymatic digestion, the samples were transferred
into pre-weighed centrifuge tubes and centrifuged
using a Biofuge Stratos centrifuge (Kendro Laboratory
Products GmbH, Langenselbold, Germany) at 5000 rpm
for 15 min and the residue washed twice with phosphate
buffer (pH 7.5) before re-suspending in 50 mL phosphate
buffer (pH 7.5). Protease (Sigma P-2143) was added
(1 mL of solution containing 16 mg protease/100 mL phosphate
buffer, pH 7.5) and the residue incubated at 42 C for
4 h. The sample was centrifuged at 5000 rpm for 15 min in
two cycles while washing with distilled water then dried to
constant weight at 60 C. The RS III was determined as the
insoluble residue after enzymatic digestion of starch and
removal of amylolytic enzymes with protease. The results
were expressed as percent RS III on dry matter basis