CHAPTER III METHODOLOGY
3.1 MATERIALS AND INSTRUMENT
Starch sample used in this research was sweet potato Sukuh strain, which was obtained from Indonesian Center of Agricultural Post Harvest Research and Development, Bogor. Starch degrading
enzyme was obtained from NOVO Nordisk through the PT Halim Sakti Pratama, Jakarta. The enzyme was Promozyme® which has standard activity 177, 222.11 IUml
and density was 1.20 gml, respectively. Strain of colonic butyrate producing bacteria used Eubacterium rectale 17629 was
obtained from culture collection of Balitvet, Bogor, West Java. The material prepared to make basal medium PYG for maintaining E.rectale 17629
composed of tryptone, bacteriological peptone, yeast extract, beef extract, glucose, Tween 80, resazurin, CaCl
2
, MgSO
4
, K
2
HPO
4
, KH
2
PO
4
, NaHCO
3
, NaCl, Vitamin K1,CO
2
. Beside that, chemical reagent used in this research were ethanol, pure amylose, NaOH, acetic acid, iodine, KI, KCl-HCl
buffer, pepsin, Tris-maleat buffer, pancreatic α-amylase, KOH, HCl, citrate buffer, amyloglucosidase,
phenol, H
2
SO
4
, glucose, HCl, butyrate standard Fluka, acetate standard Fluka, propionate standard Fluka.
The instrument used in this research were gas chromatography Agilent technologies 7890A, spray drier, aluminum foil, spectrophotometer, shaking water bath, incubator, micropipette, reaction
tube, syringe 1µl, centrifugation, centrifuge tube, autoclave, volumetric flask, refrigerator, and fermentation tube.
3.2 METHODOLOGY
This research was divided into two steps: 1 production of type-III resistant starch from Sukuh’s sweet potato chemical characteristic of Sukuh’s starch, preparation enzyme, production
process, and analysis of product RS content and 2 in vitro fermentation process by Eubacterium rectale 17629 medium preparation, bacteria activation, fermentation process, and SCFA profile
analysis of fermentation product. Generally, step of this research could be seen in Figure 3.
3.2.1 PRODUCTION OF TYPE-III RESISTANT STARCH 3.2.1.1 Chemical Characteristic of Sukuh Sweet Potato’s Starch
Chemical characteristic of sweet potato’s starch of Sukuh variety were known by proximate and amylose content analysis. Proximate analysis included moisture content oven method AOAC,
1995; ash content dry ash method AOAC, 1995; protein content Kjeldahl method AOAC, 1995; fat content Soxhlet method AOAC, 1995; crude fiber content AOAC, 1995; carbohydrate
analysis by difference method AOAC, 1995.
a. Moisture content by Oven Method AOAC, 1995
Sample was weighed 1-2 g into a aluminum crucible. The sample was dried into oven at 100
C for 1 hour. Then, it was cooled in desiccators and weighed after reaching room temperature. Sample weighing was repeated until reach constantly weigh. The loss of weigh is used to calculate the
moisture content of the sample.
10 Figure 3. Flow chart of the research
b. Ash content by Drying Ashing Method AOAC, 1995
Sample was weighed 5-10 g into a tared porcelain crucible. The porcelain crucible was ignited 12-18 hours or overnight at about 550
C. Then, muffle furnace was turned off and wait to open it until the temperature has dropped to at least 250
C, preferably lower. Using safety tongs, porcelain crucible was transferred quickly into desiccators with a porcelain plate and desicant, allow it
to cool prior to weighing. Sample weighing was repeated until reach constantly weigh. The loss of weigh is used to calculate the ash content of the sample.
c. Fat content by Soxhlet Method AOAC, 1995
Sample was weighed 2 g into pre dried extraction thimble and dried 6-18 hours at 100 C. The
thimble containing dried sample was placed in soxhlet. Amount of 150 ml hexane was added into soxhlet and extracted at a rate of 5 or 6 drops per second condensation for about 4 hours. Then, flask
was removed and the solvent was evaporated by heating. Boiling flask with extracted fat was dried in an air oven 100
C for 1.5-2 hours, then cooled in desiccators and weighed.
d. Protein content by Kjeldahl Method AOAC, 1995
Sample of 100-250 mg was placed into Kjeldahl flask. Then 1.0 ± 0.1 g K
2
SO
4
, 40 ± 10 mg HgO, and 2.0 ± 0.1 ml H
2
SO
4
were added. The solution was boiled for 1-1.5 hours. At distilattion Proximate
analysis Analysis
of amylose content
Sukuh starch
Production of type-III resistant starch
Preparation of fermentation medium with Sukuh’s sweet potato type III resistant starch
as a substrate Activation of Eubacterium rectale 17629
culture Analysis of
resistant starch content
In vitro fermentation of Eubacterium rectale 17629 bacteria in medium an-aerob
condition Analysis of
Pullulanase enzyme activity
Fermentation product pH
measurement Absorbance
measurement Short chain fatty
acid analysis using gas chromatography
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
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