14
3.2.2 IN VITRO FERMENTATION
3.2.2.1 Peptone Yeast Glucose Preparation Purwani and Suhartono, 2009
Peptone Yeast Glucose medium composition and mineral mix could be seen in Table 3 and Table 4. All the ingredients, except cystein hydrochloride, haemin solution, and vitamin K solution
were mixed. The mixture was boiled to dissolve the ingredient and it was cooled by spraying it with CO
2
gas. After that, the medium solution was added by Vitamin K, haemin solution, and Cystein-HCl x H
2
O. The medium was adjusted to pH 7.2 with NaOH 8 N. The medium was distributed into 20 ml amount into serum bottle flushed with CO
2
. The bottle was sealed with a butyl rubber septum and autoclaved at 121
C for 15 minutes to sterilize. The remaining medium could be store in refrigerator before next uses.
Table 3. Medium composition of PYG for 1000 ml
Component Amount Unit
Tripticase peptone trypton 5.00
g Peptone 5.00
g Yeast extract
10.00 g
Beef extract 5.00
g Glucose 5.00
g K
2
HPO
4
2.00 g
Tween 80 1.00
ml Resazurin 1.00
mg Salt solution mineral mix solution
40 ml
Distilled water 950
ml Haemin solution
10 ml
Vitamin K solution 0.20 ml
Cystein-HCl x H
2
O 0.50 g
Vitamin K, haemin solution, Cystein-HCl x H
2
O was added after it was boiled and cooled by spraying it with CO
2
gas. Medium was adjusted to pH 7.2 with NaOH 8 N. The medium was flushed again with CO
2
and autoclaved at 121 C for 15 minutes to sterilize.
Table 4. Mineral mix solution composition
Mineral Amount Unit
CaCl
2
x 2H
2
O 0,25 g
MgSO
4
x 7H
2
O 0.50 g
K
2
HPO
4
1 g
KH
2
PO
4
1 g
NaHCO
3
10 g
NaCl 2 g
Aquades 1000 ml
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
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