11 stage, a little amount of water is transferred through flask wall and shaken carefully to resform the
crystal. Solution was transferred to the destillation ware, rinsed 5-6 times with 1-2 distilled water, followed by adding distilled water to distillation ware and 8-10 ml of 60 NaOH - 5 Na
2
S
2
O
3
. Erlenmeyer was placed under the condencer with 5 ml H
3
BO
3
and 2-4 drops of red-methylene blue added.end of condenser must be soaked in H
3
BO
3
solution. A titration stage, sample solution was diluted into 50 ml, and then titrated with 0.02 N standardized HCl until grey color appears. The borate
anions formed in previous stage was titrated with standardized acid HCl which is proportional to the amount of nitrogen.
e. Carbohydrate content by difference method AOAC, 1995
Carbohydrate = 100 - protein + water + ash + fat
f. Amylose Content Analysis Apriyantono et al., 1989
Determination of amylose content began with make standard curve of pure amylose. The standard curve was made by dissolving 40 mg pure amylose into 1 ml 95 ethanol and 9 ml NaOH
1N solution. The mixture was boiled for 10 minutes to gelatinize the solution. After it was cooled, it was adjusted with distilled water into 100 ml by volumetric flask, prepared series of dilution in the
100 ml of volumetric flask: 1.0; 2.0; 3.0; 4.0; and 5.0 ml of amylose solution, 0.2; 0.4; 0.6; 0.8; and 1.0 ml of acetic acid 1N, respectively. The series of dilution was added 2 ml iodine solution 0.2 g
iodine and 2 g KI were diluted into 100 ml distilled water for each dilution and was added distilled water to adjust into 100 ml. The mixture was incubated for 20 minutes. Intensity of blue color
complex for each dilution was measured at 620 nm using Spectrophotometer Spectronic 20. The data was plotted to make standard curve.
Amylose content was measured by dissolving 100 mg starch sample into reaction tube, added 1 ml ethanol 95 and 9 ml NaOH 1N solution. Sample was boiled for 10 minutes to gelatinize starch
solution. After it was cooled, starch paste was adjusted with distilled water into 100 ml by volumetric flask. In the amount of 5 ml of the dilution is moved into 100 ml volumetric flask, added 1 ml acetic
acid 1N, 2 ml iodine solution and distilled water to adjust into 100 ml. After incubated for 20 minutes, the absorbance was measured with spectrophotometer at 625 nm. The absorbance of the sample was
compared to the standard curve to determine the amylose content.
3.2.1.2 Production Process of Type-III Resistant Starch
a. Analysis of Pullulanase Enzyme Activity Hartoyo et al., 2009
The standard curve was made by dissolving 0.2 g glucose and adjusted by adding distilled water in 100 ml of volumetric flask, then prepared series of dilution: 0.0; 0.1; 0.2; 0.3; 0.4; and 0.5 ml
glucose solution and added acetate buffer 20 mM pH 5.0: 0.5; 0.4; 0.3; 0.2; 0.1; and 0.0 ml, respectively and mixed well. The mixtures were incubated 25
C for 10 minutes. Then, the mixture was added by 0.5 ml DNS solution 3 g NaOH, 34.6 g NaK-tartarat, 3 g DNS were diluted into 300 ml
distilled water for each dilution, then mixed well. The mixture was boiled for 5 minutes. After it was cooled, it was added 2 ml of distilled water and absorbance of the color complex was measured at 550
nm using spectrophotometer Spectronic 20. The data was plotted to make standard curve. Substrate pullulan solution 2 was made by dissolving 0.02 g pullulan substrate into 1 ml
of distilled water. The mixture was divided into 2 parts:
Sample : Amount of 0.05 ml of the pullulan solution 2 was dissolved by adding 0.35 ml of acetate
buffer then mixed well. The mixture was incubated at 25 C for 10 minutes. Then, it was added by 0.1
ml pullulanase enzyme and was incubated at 25 C for 10 minutes. Then, the mixture mixed by 0.5 ml
12 of DNS solution then mixed well. The mixture was boiled for 5 minutes. After it was cooled, 2 ml of
distilled water added to the mixture and absorbance of the color complex was measured at 550 nm using spectrophotometer Spectronic 20. The data was plotted to make standard curve.
Control : Amount of 0.05 ml of the pullulan solution 2 is dissolved by adding 0.35 ml of acetate
buffer then mixed well. The mixture was incubated at 25 C for 10 minutes. Then, the mixture was
added by 0.5 ml DNS of solution and 0.1 ml pullulanase enzyme and mixed well. After that, the mixture was boiled for 5 minutes and mixed with 2 ml distilled water. After it was cooled, absorbance
of the color complex was measured at 550 nm using spectrophotometer Spectronic 20.
b. Production Process of Type-III Resistant Vatanasuchart et al, 2010
