THESIS

7.2 Method determination
205
Reducing sugar content of starch hydrolysis samples was determined by
the Park and Johnson method, according to Hizukuri (1995). Reducing sugar for the
native starches, debranched products and RS III sample were purified and
destructured by dissolving in DMSO prior dilution. In sample tubes, the sample tested
was completely solution and contained 0 – 5 µg/ml by dilution. Duplicate 1.0 ml of
starch solution/ standard glucose/ blank were pipette into test tube. An aliquot of 1.0
ml samples/ blank/ standard with 0.5 ml of ferriccyanide reagent and 0.5 ml of NaCO3
–NaHCO3-KCN reagent were heated in a boiling water for 15 min. This solution was
quickly cooled for 10 min, and 2.50 ml of ferric ammonium sulphate reagent were
then added and mixed well. After 20 min incubation at room temperature,
the
absorbance of the colored solution was measured at 715 nm. The absorbance of the
samples
/ standard/ blank were measured using a spectrophotometer (Model UV-160
A, Shimadzu, Japan) with a path length of 10 nm quartz cuvette cell. The reducing
sugar content
was calculated by comparison to a glucose standard curve (Appendix
Figure A3) by using equation:
Reducing sugar (µg/ml) = Absorbance of sample × Dilution
O.D. (715 nm )
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.104x + 0.003
R 2 = 0.999
Slope
0 1 2 3 4 5 6
Gulcose Concentration (ug/ml)
Appendix Figure A 3 Standard curve for reducing sugar determination