THESIS

8.2.3 Measurement of resistant starch
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Resistant starch content in the RS III samples and control were
determined as following section.
1) Hydrolysis and solubilisation of non-resistant starch
1. Accurately weigh a 100 ± 5 mg sample directly into
each screw cap tube and gently tap the tube to ensure that the sample falls to the
bottom.
2. Add 4.0 ml of pancreatic α-amylase (10 mg/ml)
containing AMG (3 unit/ml to each tube.
3. Tightly cap the tubes, mix them on a vortex mixer and
attach them horizontally in a shaking water bath, aligned in the direction of motion.
4. Incubate tubes at 37°C with continuous shaking (200
strokes/ min) for exactly 16 hr.
5. Remove the tubes rom the water bath and remove
excess surface water with paper towel. Remove the tube caps and treat the contents
with 4.0 ml of ethanol (99%) with vigorous stirring on a vortex mixer.
6. Centrifuge the tubes at 1,500 g (approx. 3,000 rpm) for
10 min (non-capped).
7. Carefully decant supernatants and re-suspend the pellets
in 2 ml of 50% ethanol with vigorous stirring on a vortex mixer. Add a further 6 ml of
50% ethanol, mix the tubes and centrifuge again at 1,500 g for 10 min.
8. Decant the supernatants and repeat this suspension and
centrifugation step once more.
9. Carefully decant the supernatant and invert the tubes on
absorbent paper to drain excess liquid.