11
b. Resistant Starch Analysis Goni et al. 1996
Starch sample was weighed 100 mg into a 50-ml centrifuge tube and mixed with 10 ml KCl- HCl buffer 0.1 M pH 1.5 pH adjustment with HCl 2 M or NaOH 0.5 M. The solution was
homogenized and added 0.2 ml pepsin solution 1 g pepsin10 ml buffer KCl-HCl 445 unitsmg solid, mixed well and incubated in water bath 40
o
C for 60 minutes with constant shaking. The mixture was mixed 9 ml Tris-maleate buffer 0.1 M pH 6.9. pH adjustment with 2 M HCl or 0.5 M
NaOH. Then 1 ml of diluted pancreatic α-amylase Sigma, 40 mg enzymeml Tris-maleate buffer
15.4 unitsmg was added to the mixture, homogenized, and incubated in water bath shaker 55
o
C, 120 rpm, for 16 hours. After the incubation, the sample was centrifuged 3000 rpm, 15 minutes and
supernatant was discarded. Residue was washed at least once with 10 ml distilled water, centrifuged again and supernatant was discarded. Then 3 ml distilled water and 3 ml KOH 4 M were added into
the centrifuge tube, mixed well, and left for 30 minutes at room temperature with constant shaking. Next, 5.5 ml HCl 2 M, 3 ml citrate buffer 0.05 M pH 4.5 and 100
μl of diluted amyloglucosidase 0.1 wv 154000 U 1 were added into the centrifuge tube, homogenized, and incubated 55
o
C for 45 minutes. Mixture then was centrifuged 3000 rpm, 15 minutes, supernatant was collected and
saved in a 100 ml volumetric flask. Residue was washed with 10 ml distillated water, centrifuged, and supernatant was combined with that obtained previously. Supernatant was adjusted to 100 ml.
Resistant starch would be measured using phenol sulphuric acid method AOAC 1995.
c. Glucose Analysis by Phenol Sulphuric Acid Analysis AOAC 1995
Diluted solution from previous method was taken for the total sugar measurement using phenol-sulphuric acid method. 50
μl of solution was pipetted into a test tube and diluted until 1 ml. Then 0.5 ml phenol 5 and 2.5 ml concentrated H
2
SO
4
were added into the test tube. The mixture was homogenized and the absorbance was measured using spectrophotometer at 490 nm. The absorbance
was compared with glucose curve standard 10, 20, 30, 40, 50, 60, 65, 80, and 85 ppm which was made by same method. Resistant starch content was obtained by calculating with formula:
.
3. Fermentation of Type 3 Resistant Starch by Clostridium butyricum
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