15
3.2.2.2 Activation Eubacterium Rectale 17629 Purwani and Suhartono, 2009
Culture of Eubacterium rectale 17629 needed to be activated to begin fermentation step. Activated was done by inoculating 10 ml of Eubacterium rectale 17629 strain into 10 ml sterilized
Peptone Yeast Glucose PYG while flushed with CO
2
and incubated 37 C in anaerobic condition for
24 hours. Stock of bacteria culture was preserved by saving it in cold temperature 5 C. Bacteria
culture was activated every once a month or before it was used as a fermentative culture.
3.2.2.3 In Vitro Fermentation Process Purwani and Suhartono, 2009
Experiment I was design to evaluate the growth of Eubacterium rectale 17629, its fermentation capability SCFA production pattern and optimum time that produce the highest butyrate
concentration. Glucose 5 gl 0.5 and RS3 derived from sweet potato of Sukuh variety starch treated with pullulanase in amount of 20 gl 2 were added into medium. The medium with
resistant starch as a substrate was distributed into 20 ml of medium and inoculated with 1 ml of 24 hours pre-culture. Incubation was performed at 37
C for 6, 12, 24, 36, and 48 hours. Experiment II was conducted to analyze the effect of resistant starch concentration on short
chain fatty acid profile during in vitro fermentation of Eubacterium rectale 17629. Fermentation was carried out using glucose 5 gl 0.5 and RS3 10 gl 1 into medium 20 ml. The medium was
inoculated with 1 ml of 24 hours pre-culture of Eubacterium rectale. Incubation was performed at 37
C for 48 hours and also incubated at the optimum time that produced the highest butyrate in the first experiment 36 hours.
3.2.2.4 Analysis of Product Fermentation
a. Measurement of pH value
The pH of the fermentation culture for each sampling was measured using pH-meter. Lower result of pH in the fermentation medium showed higher production of short chain fatty acid by
Eubacterium rectale 17629.
b. Turbidity Measurement Purwani and Suhartono, 2009
Sampling which was performed every 6 hours and fermentation product in optimum time was measured the cell growth. Cell growth was determined qualitatively by using turbidimetry method.
Absorbance of fermentation product suspense was measured using spectrophotometer at 660 nm. Higher result of absorbance showed higher cell turbidity and higher growth of the bacteria cell in the
fermentation medium.
c. Short Chain Fatty Acid Analysis using Gas Chromatography Purwani and Suhartono,
2009 Short chain fatty acid SCFA production was measured using Gas Chromatography Agilent
technologies 7890A. Culture sample was centrifuged at 10000 rpm for 10 minutes. Supernatant was collected into a 1.5-2 ml effendorf tube for storage at 4
C until use. Before injecting sample, 94 µl of sample was spiked 2 µl of acetic acid SIGMA-ALDRICH 71251, 2 µl of propionic acid SIGMA-
ALDRICH 94425, and 2 µl of butyric acid SIGMA-ALDRICH 19215. Standard curve for each compound was also made to determine SCFA concentration in the sample. SCFA standard mixture for
standard curve was shown in Table 5 .
Mixture sample and SCFA spike was injected into a high resolution gas chromatography Agilent Technologies, 7890 GC System equipped with a flame ionization detector and a HP
Innowax 19091-136 column 60 m x 0.250 m. The carrier gas was helium with a flow rateof 1.8 mlmin, and the split ratio was 40:1. The oven temperature was maintained at 90
C for 0.5 min, and
16 then increased to 110
C at a rate of 10 Cmin, increased to 170
C at a rate of 5 Cmin and finally
increased to 210 C at a rate of 20
C. Injector and detector temperatures were set into 275 C. Acetate,
propionate, and butyrate were used for standard and the result was expressed as mmolL. Table 5. SCFA standard mixture for standard curve
Acetic Acid µl
Acetic acid conc. M
Propionic Acid µl
Propionic acid conc.
M Butyric
Acid µl
Butyric acid conc. M
dH
2
O µl
5 0.1743 5 0.1334 5
0.1084 485 10 0.3487 10 0.2667
10 0.2167 470 15 0.5230 15 0.4001
15 0.3250 455 20 0.6974 20 0.5335
20 0.4334 440 25 0.8717 25 0.6669
25 0.5418 425
CHAPTER IV RESULT AND DISCUSSION
4.1 CHEMICAL CHARACTERISTIC OF SUKUH’S SWEET POTATO
STARCH
The moisture content, fat, protein, ash, carbohydrate, crude fiber, and amylose content of Sukuh sweet potato starch are shown in Table 6. Proximate analysis of Jago, Beauregard, and
Evangeline starch are shown in Table 6 as the comparison for each other. Table 6. Proximate analysis result of Beauregard, Evangeline, Jago, and Sukuh sweet potato starch
dry bases
Analyte Beauregard
Evangeline Jago
Sukuh
Moisture 3.97 ± 0.7
1.83 ± 0.4 13.73 16.45
Ash 0.08 ± 0.1
0.07 ± 0.1 0.23 0.20
Crude fat 0.23 ± 0.3
0.11 ± 0.1 0.44 0.65
Protein 0.17 ± 0.0
0.24 ± 0.0 0.56 0.69
Crude Fiber Carbohydrate
0.06 ± 0.1 95.49
0.00 ± 0.00 97.75
0.00 85.04
0.00 82.01
Amylose 23.60 ± 1.20
27.10 ± 0.30 25.83 ± 0.21
29.35 ± 0.67
Source: Futch, 2004; Devega, 2011
Sukuh variety is national superior variety of sweet potato with compact structure. It has 4-4.5 month of harvest age. Production of this variety reach 25-30 tonha. It has spherical and ellipse form
and has short stalk tuber. Sukuh has yellow skin color, white flesh and delicious taste. Sukuh has crude fiber content 0.85, protein content 1.62, total sugar content 4.56, starch content 31.16
RILET, 2008. Proximate analysis showed that Sukuh extracted starch has moisture content 16.45, ash content 0.20, crude fat content 0.65, protein content 0.69, carbohydrate
content 82.01, and crude fiber content 0. Sukuh is potential to be developed in starch and flour industry, include resistant starch industry, because it has high content of starch and flour yield
RILET, 2008. Decreasing of protein and fiber content showed purity of extracted resulted starch. Starch content of sweet potato depended on the variety as well as age during harvest. Sugar
content fell significantly when harvest was done beyond 90 days, but the amylose content was greatly affected by the time of harvest Antarlina and Kumalaningsih, 1990. Sukuh has harvest age 4-5
month 120 days, but it doesn’t influence the amylose content of the starch. The fibre content in sweet potato varies to a great extent depending on varietals variation and
age of the crop, where the fibre content increases with the maturity. Fibre content in starch derived from tuber extractions may vary to greater extent on the techniques and sieves, used for removal of the
fibrous material. Sweet potato flour containing 2-3 fibre had different compositions compared to the isolated starch having 0.1-0.15 fibre Moorthy, 2002.
Sukuh and Jago variety are the example of superior variety which is released by Research Institute for Legume and Tuber crops, Malang. Starch extraction of the sweet potato is obtained and it
has high purity. It can be shown that the amount of protein and dietary fiber in sweet potato tuber was higher than in extraction starch. High purity of starch is needed to make enzyme hydrolisis effectively.
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