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
Fermentation was carried out in two different conditions. First condition was designed to evaluate production pattern of SCFA where resistant starch 20gL and glucose 1gL were added
into the medium. Second condition was designed in order to increase production of SCFA, especially butyric acid, where resistant starch 10gL and glucose 5gL was added into medium.
a. Fermentation Preparation Purwani et al. 2009
Fermentation preparation was devided into 4 steps. The steps include preparation of Reinforced Clostridial Medium RCM as growing medium for Clostridium butyricum, culture refresh of
Clostridium butyricum, making of growth medium containing resistant starch, and fermentation type 3 resistant starch by Clostridium butyricum.
The first step was preparation of Reinforced Clostridial Medium RCM. All of the ingredients were dissolved in 100 ml distilled water in erlenmeyer. The mixture was adjusted to pH 6.8 with
NH
4
OH or HCl and placed the mixture into butyl rubber bottle and sterilizied at 121
o
C for 15 minutes. The remaining medium was stored in refrigerator for the next uses. Medium composition could be
Resistant Starch = mg glucose x 0.9 x V ml x dilution factor x 100 mg sample
12 seen in Table 3. Second step was culture refresh of Clostridium butyricum. 5 ml Reinforced
Clostridial Medium RCM sterile and 5 ml of pure culture of Clostridium butyricum were added into tube and flushed with CO
2
to keep anaerob condition. The mixture was incubated at 37
o
C for 24 hours. The presence of turbidity and sediment showed the growth of Clostridium butyricum. The third
step was making of growth medium containing type 3 resistant starch. Medium composition used was similar like Reinforced Clostridial Medium RCM, but the difference was that the soluble starch was
replaced with resistant starch at 1 wv. All the ingredients were mixed and adjusted to pH 6.8 with NH
4
OH or HCl and sterilized at 121
o
C for 15 minutes. The fourth step was fermentation of type 3 resistant starch by Clostridium butyricum. A 24-hours culture of Clostridium butyricum was
inoculated into sterilized Reinforced Clostridial Medium RCM while flushed with CO
2
and incubated 37
o
C in anaerobic condition. Table 4. RCM Composition
Source : Dewi, 2009
b. Fermentation Analysis 1 pH Measurement Purwani et al. 2009
pH measurement is conducted to observe the changing of pH during fermentation. pH of cultures is determined using pH meter.
2 Turbidity Measurement Purwani et al. 2009
Measurement was conducted to observe the turbidity of cultures caused by bacterial growth. Turbidity of cultures is determined using spectrophotometer on 660 nm.
4.
Short Chain Fatty Acid SFCA Analysis Purwani et al. 2009
Short chain fatty acid profile SCFA was analyzed using Gas Chromatography Agilent Technoligies 7890 A. Sample result of fermentation were centrifuged at 13000 rpm for 10 min.
Then supernatant was filtered through a 0.2 μm filter into a 1.5 ml eppendorf tube for storage at 4
o
C until use. Before injecting the samples, 94
μl of sample was spiked by adding 2 μl of acetic acid SIGMA-ALDRICH 71251, propionic acid SIGMA-ALDRICH 94425, and butyric acid SIGMA-
ALDRICH 19215. Standard curve for each compound was also made to determine SCFA concentration in the sample Table 5.
Sample of 1 μl were injected into a high resolution gas chromatography Agilent Technologist,
7890A GC System equipped with a flame ionization detector and a HP Innowax 19091N-136 column 60 m x 0.250 mm. The carrier gas was helium with a flow rate of 1.8 mlmin, and the split ratio was
40:1. The oven temperature was maintained at 90
o
C for 0.5 min, and then increased to 110
o
C at a rate Ingredients Concentration
gL Yeast Extract
Beef Extract Powder Peptone
Glucose Soluble Starch
Sodium Chlorida Sodium Acetate
Cystein Hidrocloride 3.0
10.0 5.0
1.0 2.0
5.0 3.0
0.5
13 of 10
o
Cmin, increased to 170
o
C at a rate of 5
o
Cmin and finally increased to 210
o
C at a rate of 20
o
Cmin. Injector and detector temperatures were set into 275
o
C. Acetate, propionate and butyrate were used for standard and the result was expressed as mmolL.
Table 5. SCFA standard mixture for standard curve Concentration
μl ml Acetic acid
μl Propionic acid
μl Butyric Acid
μl H
2
O μl
0 0 0 0 0 10 5 5
5 485 20 10 10 10 470
30 15 15 15 455 40 20 20 20 440
50 25 25 25 425
14
IV. RESULT AND DISCUSSION
A. CHEMICAL ANALYSIS
The amylose and proximate analysis was done to identify amylose content and identify its water, ash, protein, fat, and carbohydrate content of sweet potato starch. Table 6 shows the
amylose and proximate content. Table 6. Major Chemical Composition of Jago Sweet Potato Starch
Chemical Component Sweet Potato Starch
Water Ash
Protein Fat
Carbohydrate Amylose
13.73 0.23
0.44 0.56
85.04 25.83
The water content of sweet potato starch is 13.73, protein content is 0.44, ash content is 0.23, fat content is 0.56, and carbohydrate content is 85.04. Sweet potato is classified as
tubers group so it contains high carbohydrate. The variation of water content of the sweet potato starch depend on drying method, drying time, and storage condition. Sweet potato starch also
contains protein, fat, and ash even though the contents are low. High purity of the starch is very important to keep hydrolisis enzyme runs smoothly.
Starch which is resistant to digestive enzyme in the small intestine is not only affected by processing but also but its chemical structure. Starch which contains more amylose have higher
ability to form amorphous structure because of its hydrogen bond intensiveness. The consequence is starch which can not be swelled or gelatinized better during cooking time will slowly digested
Panlasigui et al. 1991. Therefore, information about amylose content in food is important to produce optimum yield of resistant starch.
Principal of amylose analysis is iodine bound in the spiral shaped starch molecule which yields blue color then measured the absorbance by spectophotometer at 620 nm Winarno, 1997.
The result of amylose analysis showed that amylose content of jago sweet potato starch was 25.83. It was higher than wheat starch 25 and potato starch 20, but lower than corn starch
26 Pomeranz, 1992. Collado and Corke 1997 reported that amylose content in sweet potato starch have variation in range 15-27, depend on variety.
Generally, sweet potato pasta characteristic have tendency to easily retrogradated which showed by pasta viscosity in cooling phase or classified as type C based on classification by
Srichuwong 2005. The other characteristics are the very viscous pasta, stable, and it doe not show a peak in heating phase. Increasing viscosity in cooling phase indicate that retrogradation
process will be undertaken easily. Intensive of retrogradation level is required in RS3 formation. Purwani et al. 2009.
Based on amylose content analysis, it can be concluded that ingredient to produce resistant starch in this research can be classified as high amylose starch and it was expected that jago sweet
potato can be potential ingredient to produce high yield resistant starch.
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