THESIS

1.2.6 Microscopic images
Microscopic images of the selected RS III samples were examined
using a SEM with magnifications of 2,500X and 6,000X. The procedures are shown
in Appendix B (Atichokudomchai et al. 2000).
1.2.7 X-ray diffraction
X-ray diffraction patterns of native rice starch and the selected RS
III samples were measured with copper K2 radiation (λ =0.154 nm) using a
diffractometer ( JEOL,JDX-3530, Japan). The diffractometer was operated at 300 mA
and 30 kV, 2θ range from 10 to 70.0 o with a step size 0.05 o and a count time of 2s.
The data was analyzed with program MDI Jade 6.5 (Japan). The crystallinity of RS III
samples were calculated as the proportion of crystalline area to total area at angles
between 10 and 45 o 2θ (Cairns et al. 1997). The procedures are shown in Appendix
B.
1.2.8 Resistant starch, digestible starch and total starch analysis
Resistant starch (RS), digestible starch (DS) and total starch (TS)
content in the RS III samples were determined by using a Megazyme Resistant Starch
kit (AOAC Method 2002.02). Briefly, samples (100 mg) were incubated in a shaking
water bath with pancreatic α-amylase and amyloglucosidase for 16 hr at 37 o C to
reduce digestible starch to glucose. The reaction was terminated with 4 ml ethanol and
the RS pellet was recovered by centrifugation (5000 g, 10 min). The supernatant was
decanted and washed with 50% ethanol for two times (digested starch). The pellet was
solubillized in 2ml of 2 M KOH in an ice bath, neutralized with 8 ml sodium acetate
(1.2 M) and the resistant starch hydrolyzed to glucose with of AMG (0.1 ml, 3300
Uml -1 , 50 o C). The glucose oxidase/peroxidase reaction was used to measure glucose
released from digested starch and resistant starch. Absorbance was read (510 nm)
after a 20 minute incubation period at 50 o C. The procedures of resistant starch,
digested starch and total starch determination were shown in Appendix A.
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