THESIS

1.3 Effect of pullulanase enzyme concentration on physicochemical
properties of RS III formation
A pullulanase hydrolysis condition for RS III formation was chosen by
means of the previous experiment. A concentration of 0, 8, 10, 12, 14 and 16 units /
game starch and a hydrolysis period of 16 hr at 55 o C were employed for investigation
of pullulanase enzyme concentration effects on RS III propertes. The RS III was
produced as described in section 1.2.1. Three replicates of all experiments will be
performed. Degree of pullulanase hydrolysis and degree of syneresis were examined
as described in section 1.2 .2 and 1.2.3.
1.3.1 β-amylolysis (%)
The β-amylolysis (%) of native high amylose rice starch and RS
III prepared from the treatments of debranched high amylose rice starch at different
degree of hydrolysis was evaluated in order to investigate degree of debranching. The
analysis procedure was slightly modified from that of Hood and Mercier (1978).
Native high amylose rice starch and debranching treatments with different enzyme
concentration (4 mg) dissolved in 1 ml of dimethylsulfoxide by heating in boiling
bath for 15 min and then cooling and diluted to 3 ml with distilled water. The solution
(0.45 ml) was taken and mixed with an acetate buffer solution pH 6.0 (50 µl; 0.1 M).
β-amylase enzyme (1 µl; 20 units /ml) was added and mixed well. The solution was
incubated at 30 o C for 12-hr and boiled for 5 min for deactivated enzyme. The
reducing sugar (Rds) and total sugar (Ts) of the β-amylolysis product were measured
according to the Park- Johnson method (Hizukuri, 1995) and the phenol-sulfuric acid
reagent method (Dubois et al. 1956), respectively. β-amylolysis (%) was calculated:
Rds of sample after hydrolysis by β-amylase – Rds of β-amylase blank × 100
Ts of sample after hydrolysis by β-amylase – Ts of β-amylase blank
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