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
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