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
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