t 3
4 4
i R
o r
0,00 10,00
20,00 30,00
40,00 50,00
60,00
Cocentr ati
o n mM
Figure 8 Table 8. Prop
37 C
Fermentation time hours
6 12
24 36
48 Comp
17629. Based the highest SC
36 h, where th 0:1:2.03, but
4.5 ANA
4.5.1 Turb
Exper increase SCFA
RS3 from Suku optimum time
result and pH c 6
0,00 10,29
13
8. SCFA profile portion of SCFA
n Acetate mM
0.00 0.00
0.00 0.00
0.00 pared with othe
on SCFA prof CFA, mainly bu
he molar ratio this level was
ALYSIS OF bidimetry a
riment II was A production m
uh sweet potat e that produced
change is prese 12
0,00 9
0,0 3,57
A e produced by E
A production b conc.
M Pro
er, butyrate wa file Figure 8,
utyrate, by in v o of acetate: p
considered be
F FERMENT and pH mea
conducted to mainly butyrat
to 10 gl 1 d the highest
ented in Table 2
0,00 00
15,32
H
Acetate Prop
Eubacterium r by Eubacterium
opionate conc. mM
10.29 0.00
0.84 25.17
0.00 as the highest
it could be con vitro fermentat
propionate: but low and could
TATION PR asurement
analyze the ef te. Fermentatio
Incubation w butyrate in th
9. 24
0,0 0,84
2
4,01
Hours
pionate But
rectale 17629 o m rectale 1762
Butyrate mM
13.5 15.3
4.0 50.9
22.8 SCFA that pro
ncluded that th tion of Sukuh
tyrate were 0.0 d be increased.
RODUCT
ffect of resista on was carried
was performed he first experim
36 00
0,0 25,17
50,97
tyrate over 48 h of fer
9 over 48 h of e conc.
M 57
32 01
97 83
oduced by Eub he optimum tim
type-III resista 0 mM: 25.17
EXPERIM
ant starch conc d out using glu
at 37 C for 48
ment 36 hour 48
00 0,00
22,83
rmentation f fermentation a
Propionate: Butyrate
1.0 : 1.32 -
1.0 : 4.77 1.0 : 2.03
- bacterium recta
me that produc ant starch was
mM: 50.97 m
MENT II
centration and ucose 5 gl a
hours and at t rs. Turbidimet
24 at
ale ced
at mM
to and
the try
25 Table 9. Absorbance and pH value over fermentation of Sukuh type-III resistance starch by E.rectale
17629 in experiment II
Substrate Hours pH Absorbance
RS3 of Sukuh sweet potato 1 + glucose
5 gl 36
4.08 ± 0.03 0.73 ± 0.02
48 3.98 ± 0.04
0.77 ± 0.10
4.5.2 Production of Short Chain Fatty Acid
Pattern of SCFA production by E.rectale 17629 and analysis of resistant starch concentration to increase butyrate production could be investigate in this experiment. Table 12 shows production
and proportion of short chain fatty acid by Eubacterium rectale 17629 Experiment II revealed that changes of fermentation condition of E.rectale 17629 resulted in metabolic shift Table 10. Acetate,
propionate, and butyrate level resulted from the Experiment II is higher than in Experiment I. Table 10. Production and proportion of short chain fatty acid by Eubacterium rectale 17629 in
Experiment II
Fermentation time hours
Acetic acid conc. mM
Propionic acid conc. mM
Butyric acid conc. mM
Acetic : Propionic : Butyric
36 500.40
476.28 477.97
1 : 1 : 1 48
215.21 281.10
343.12 1 : 1.3 : 1.6
Acetic acid was identified in this experiment and it reached the highest concentration 500.40 mM among two others. Butyric acid reached the highest concentration at 36 hours 477.97 mM
while at 48 hours produced 343.12 mmolL. Compared with Experiment I, the molar ratio of acetate:propionate:butyrate at 36 hours were 0.0 mM: 25.17 mM: 50.97mM 0: 1.0: 2.03 was
different than in Experiment II 500.40 Mm: 476.28 mM: 477.97 mM 1.05: 1.0: 1.0. In Experiment II, acetate was the most dominant of SCFA than propionate and butyrate while in Experiment I
butyrate was the most dominant among two others. Sharp and Macfarlane 2000 reported that Sachharolitic clostridia were best adapted to fast
growth and high substrate concentration. It was also reported that resistant starch granules are advantageous for their growth. However, this experiment showed that increasing substrate
concentration did not automatically increase SCFA production. Excess carbon source often affects osmotic dehydration of microorganism in a fermentation process.
Difference fermentation condition between Experiment I and Experiment II also resulted in increasing SCFA production. In Experiment II, fermentation was carried out in much larger vessel
than the first experiment. The size of vessel may affect SCFA production, as the H
2
gas production was limited in Experiment I. The inhibition of the gas caused inhibition of SCFA production.
Eubacterium rectale possesses genes for the production of butyrate that show high similarity to genes from other Clostridia. This is because that finished genome sequences were generated from
Eubacterium rectale and E.eligens, which belong to Clostridium Cluster XIVa, one of the most common gut Firmicute clades Mahowald, 2009. Collins et al 1994 also defined that Eubacterium
rectale belongs to Clostridial cluster XIVa based on phylogenetic analysis of 16S rRNA sequences. Because of the similarity genes from other Clostridia, Eubacterium rectale 17629 is probably has
similarity fermentative metabolic pathway with Clostridia Cluster.
26 Table 11. Comparison of SCFA production from different substrate, bacteria, and fermentation time
RS Carbon
Source Medium
Ferment. time h
[RS] [Carbon]
Glucose Acetate
mM Propionate
mM Butyrate
mM Acetate:
Propionate: Butyrate
Sukuh sweet
potato
a
37 C,
sealed bottle
PYG medium: trypton 0.5,
pepton 0.5, yeast ext 1,
beef ext 0.5, glu 0.5,
K
2
HPO
4
0.2, Tween 80 0.1
6 12
24 36
48 2
2 2
2 1
2 1
0.5 0.5
0.5 0.5
0.5 0.5
0.5 0.0
0.0 0.0
0.0 500.40
0.0 215.21
10.29 0.0
0.84 25.17
476.28 0.0
281.10 13.57
15.32 4.01
50.97 477.97
22.83 343.12
- -
- -
1.1 :1 :1 -
1: 1.3: 1.6 Jago sweet
potato 37
C, sealed
bottle Devega,
2011
b
RCM medium: yeast extract
0.3, beef extract powder
1, peptone 0.5, sodium
chloride 0.5, sodium acetate
0.3, cystein- HCl 0.05
6 12
24 36
48 2
1 2
2 2
1 2
0.1 0.5
0.1 0.1
0.1 0.5
0.1 34.31
226.79 50.13
0.55 0.0
453.65 0.0
38.13 162.28
37.83 16.80
29.73 248.64
0.0 52.79
192.25 68.03
18.81 40.57
225.37 0.0
1: 1.1: 1.5 1: 1.4: 1.2
1.3: 1: 1.8 1 : 30: 38
- 2 : 1.1: 1
-
Salossa sweet
potato 37
C, sealed
bottle Evalin,
2011
b
RCM medium: yeast extract
0.3, beef extract powder
1, peptone 0.5, sodium
chloride 0.5, sodium acetate
0.3, cystein- HCl 0.05
6 12
24 36
48 2
2 2
2 2
0.1 0.1
0.1 0.1
0.1 85.25
13.45 939.12
591.84 60.00
16.70 73.51
120.33 14.81
1.4: 1: 1.2 -
1: 1.2: 9 -
-
Rice 37 C,
sealed bottle
Purwani and
Suhartono, 2009
b
RCM medium: yeast extract
0.3, beef extract powder
1, peptone 0.5, sodium
chloride 0.5, sodium acetate
0.3, cystein- HCl 0.05
24 48
0.5 1
0.5 0.1
86.79 71.37
1.09 1.42
5.74 15.89
80: 1: 5 50: 1: 11
27
a
In vitro fermentation by Eubacterium rectale
b
In vitro fermentation by Clostridium butyricum
As a comparison, other studies related to butyric production by C. butyricum and Eubacterium rectale have been done with other carbohydrate source as substrate, different medium,
and different fermentation time is shown in Table 11. Both of bacteria are known as butyrogenic bacteria species which are isolated a range of butyrate-producing strains from human feces that
produced high concentrations 10 mM of butyrate in batch culture in vitro Barcenilla et al, 2000. Acetate and butyrate were dominantly produced than propionate. SCFA profile showed similar pattern
where acetate and butyrate were the most dominant fatty acid. RS source, RS concentration, medium RS
Carbon Source
Medium Ferment.
time h [RS]
[Carbon] Glucose
Acetate mM
Propionate mM
Butyrate mM
Acetate: Propionate:
Butyrate Sago
37 C,
sealed bottle
Purwani and
Suhartono, 2009
a
PYG medium: trypton 0.5,
pepton 0.5, yeast ext 1,
beef ext 0.5, glucose 0.5,
K
2
HPO
4
0.2, Tween 80 0.1
48 1
0.1 93
52 59
1.8 : 1: 1.1
Pectic Oligo-
saccharides 37
C, pH 7.0
Manderson et al, 2005
a
PYG medium: peptone water
0.2, yeast extract 0.2,
NaHCO
3
0.2, L-cystein HCl
0.05, resazurin 0.4,
Tween 80 0.2, hemin
1 5
10
24 0.2
0.2 0.2
0.2 -
- -
- 4.37 ±
1.03 15.19 ±
3.95 17.43 ±
3.20 32.76 ±
1.45 0.41 ± 0.51
3.91 ± 1.28 6.77 ± 1.41
9.23 ± 1.13 1.06 ±
0.15 2.96 ±
1.60 10.69 ±
1.42 18.09 ±
2.14 10.7 : 1: 2.6
5.1: 1.3: 1 2.6: 1: 1.6
3.5: 1: 2 Mixed
amylo- pectin,
pectin, inulin,
dahlia, xylan,
inulin chicory
37 C, pH
6.5 Hungate
Duncan et al, 2003
a
YCFA medium: Casiton 1,
yeast extract 0.25, NaHCO
3
0.4, cystein 0.1, resazurin
0.1, hemin 1, biotin 1,
cobalamin 1, p-aminobenzoic
acid 3, folic acid 5,
pyridoxamine 15
168 hours
7 days 0.5 0.2
24.0 ± 2.3
21.0 ± 2.4 6.6 ± 0.7
3.5 : 3.2: 1
28 composition, glucose concentration, type bacteria, fermentation condition time, pH, temperature,
method and fermentation time were several factors that influence profile and production of SCFA during in vitro fermentation.
The experiment, both using Eubacterium rectale 17629 in 2 Sukuh RS and Clostridium butyricum in 2 Salossa RS Table 11, showed some differentiation. Fermentation of 2 Salossa RS
showed that butyric acid also was produced along with acetic acid while butyric acid was highly produced at the exponential phase 0-24 hours while acetic acid level highly detected during 36 h and
48 h 939.12mM and 591.84 mM. Moreover, in fermentation of 2 Sukuh sweet potato by Eubacterium rectale 17629, acetic acid was not detected by gas chromatography. This phenomenon
could be caused by metabolic pathway in SCFA production. Thus, change in the products of bacterial metabolism corresponded to the differential expression of metabolic pathway genes. Those genes that
were expressed during growth on resistant starch were specifically required in order for the bacterium to use resistant starch as a source of carbon and energy, while maintaining a sink for reducing
equivalent Scott et al., 2008. Lower concentration of Sukuh resistant starch 1 resulted in higher SCFA level than rice
and sago RS, especially in the acetate and butyrate level. Fermentation of 1 Sukuh during 36 h resulted molar ratio 500.40 mM: 476.28 mM: 477.97 mM acetate:propionate:butyrate and it was the
highest result among rice and sago RS. Complexity of the substrate influenced production of SCFA, mainly butyrate. Physicochemical properties substrate like crystalline structure of starch and hydrogen
bonds may affect the accessibility of microbial enzyme and metabolic colonic fermentation, modifying the quantity of SCFA produced and the rate of fermentation Goni et al, 2000. Simple
crystalline structure of starch made easily to access by microbial enzyme and convert glucose to pyruvate as intermediate compound in further catabolism. More hydrogen bonds indicated more
complex structure and made microorganisms need more energy ATP to convert carbohydrate to glucose. If energy which was required is high, it would reduce energy source for microorganisms to
convert glucose to pyruvate such that limit the production of SCFA. In this case, Sukuh resistant starch probably has more simple crystalline structure which made easily to access by microbial
enzyme and metabolic colonic fermentation compared with Jago and Salossa sweet potato, sago, and rice type-III resistant starch.
In Clostridia metabolic pathway, generally pyruvate prefers to be used for acetate production because it generates more ATP than butyrate production. Glucose as the carbon source was
metabolized into piruvate through glycolisis pathway. Pyruvate was the main substrate for enzymatic metabolism further. Glucose metabolism to pyruvate already produces 2 ATPs and an additional ATP
is formed from butyril-phospate pathway. Therefore three ATPs for each butyrate are produced from glucose. In the acetate pathway, two moles acetic acids are produced from one mole of glucose and 2
ATPs are produced from acetyl-P to acetate, so 4 ATPs are formed during the conversion of glucose to acetic acids. This explains why acetate is mostly produced in exponential phase Zhang et al.,
2009. At the end of the exponential growth, a major metabolic pathway switch took place in the organism. The organisms slowed down acetate production and took up excreted acetate and convert it
into butyrate. Beside through butyrate kinase, glucose could be metabolized to butyric acid with Co-A transferase enzyme which converted butyryl-CoA and acetate into butyric acid and acetyl-CoA.
The main products of microbial fermentation in the large intestine can vary significantly in their relative concentrations and production rates depending on diet and site of production Weaver et
al, 1992. In Experiment I, result showed that butyrate was the most dominant produced than two others propionate and butyrate. This result indicated that Co-A transferase activity to form butyrate
was higher than acetate kinase. Result in experiment II showed differentiation with Experiment I
29 while acetate level was dominantly produced than propionate and butyrate. It indicated that acetate
kinase activity was higher than Co-A transferase.
Figure 9. Alternative anaerob fermentative pathway Louis and Flint, 2007 The result in Experiment I showed that butyric acid was highly produced at the exponential
phase 0-24 hours. Therefore, butyric acid production may occur through 1b pathway Figure 9 which was acetate recycling. The enzyme that was responsible for this pathway is butyril CoA-acetate
transferase that change butyril Co-A and acetate to butyrate and Acetyl Co-A red arrow. Louis et al 2004 also defined that metabolic pathway in human colon with butyril Co-A : Acetate Co-A
transferase enzyme activity is dominantly found to form butyrate than butyrate kinase activity. Beside that, some studies showed that acetic acid may be produced up to certain concentration, whereas it
could harm the cells if it produces more than that concentration. It was probably concluded that acetic acid had crossed the concentration limit in 48 hours range time and it was recycled into butyric acid.
Interestingly, almost all 95 of the strains isolated by Barcenilla et al 2000 that showed net acetate utilization proved to be butyrate producers, although only 50 of the butyrate producers
showed net acetate consumption. This suggests a very strong link between acetate utilization and butyrate production in human colonic bacteria. In strains B.fibrisolvens that possess butyryl-CoA:
acetate-CoA transferase, Diez-Gonzalez et al 1999 showed that increasing acetate concentration favored butyrate production and shifted product ratios. It will be of interest to establish whether or not
this effect occurs also in Eubacterium rectale.
30 Figure 10. Overview of SCFA fermentative pathway of Eubacterium rectale. Pts, phosphotransferase
system; Gpd, glycerol 3-phosphate dehydrogenase; Pck, phosphoenlopyruvate Carboxykinase; Por, pyruvate:ferredoxin oxidoreductase; Hyd, hydrogenase; Rnf, NaDH:
ferredoxin oxidoreductase complex; Fd
red
, reduced ferredoxin; Fd
ox
oxidized ferredoxin; Pta, phosphate acetyltransferase; Bcd, butyryl-CoA dehydrogenase; Etf electron transport
flavoproteins; Cat,butyryl Coa: acetate CoA trasferase; Glt, glutamate synthetase; GlnA, glutamine synthetase Gln, glutamine; Glu, glutamate; Mct1, monocarboxylate transporter 1
Mahowald et al., 2009 Mahowald 2009 also explained that fermentative pathway of Eubacterium rectale involves
condensation of 2 molecules of acetyl-CoA to form butyrate and is accompanied by oxidation of NADH to NAD
+
Figure 10. In vitro studies have shown that in the presence of carbohydrate, E.rectale consumes large amounts of acetate for butyrate production Duncan and Flint, 2008.
Duncan and Flint 2008 also showed that fermentation of sugars by E.rectale in YCFA medium containing 0.5 glucose Table 12 produced butyrate as the highest fermentation product.
Table 12. Changes in fermentation products for five strains of E.rectale growth for 24 h on YCFA medium containing 0.5 glucose
Strain Formate mM
Acetate mM Butyrate
mM Lactate mM
Hydrogen mM
A1-86 7.0 - 6.41 13.7 10.3 3.5
M1041 5.1 - 0.06 9.2
9.0 5.3
L2-21 5.7 - 0.05 9.2 8.8 3.5
T1-815 6.0 - 4.90 11.0 9.1 4.3
VPI 0990 4.1
- 3.68 10.3
9.1 3.6
31 Substrate composition, pH, and end product were several factors which are influenced
enzyme activity. Chen and Blaschek 1999 explained that enzyme activity of Co-A transferase, acetate kinase, phospotransbutirilase, and butyrate kinase give great influence to SCFA fermentative
metabolic pathway. They also explained that the effect of adding acetate in the growth medium in preventing strain degeneration could also be a consequence increasingly active Spo0A in the cells,
since increase SPo0A may ensure the expression of CoA transferase and, maybe, other enzyme associated with solventogenesis. Higher level of acetate which are added into medium made higher
level of butyrate produced by bacteria. Medium containing higher concentrations of added acetate appears to be related to higher CoA transferase activity and maybe due to the higher carbohydrate
utilization efficiently of the culture. Under these condition, the increase in glucose utilization may be related to acetate assimilation. Co-A transferase is an important enzyme responsible for acid
reassimilation in solventogenig clostridia. This enzyme converts one molecule of acetate and butyryl- CoA to one molecule of butyrate and acetyl-Coa. Therefore, acid uptake in added acetate medium,
inhibit acetate kinase enzyme activity and acetate produced by bacteria. In this pathway, acetate is used as a co-substrate to form butyrate by Co-A transferase enzyme.
Research showed that production of short chain fatty acid depend on physicochemical of the fermentation substrate Khan and Edward, 2005. Resistant starch is butyrogenic, means that it
induces the production of butyric acid than other carbohydrate substrate Wang et al., 1999. Acetic acid, propionic acid, and butyric acid have metabolic pathway that related each other Amans et al.,
2001, means production one of fatty acid influence production the other fatty acid. Bacteria, enzyme activity and metabolic pathway also gave great influence to production of short chain fatty acid.
Differentiation in fermentation condition substrate composition, pH, and end product and medium composition would change enzyme activity and bacterial metabolic pathway then influence
distribution of final product fermentation. Biosynthesis of acetate kinase enzyme is regulated by end product acetate. Highest activity
was found in the beginning of fermentation and decrease along with increasing of end product. It could be concluded that acetate kinase activity is inhibited by its product product inhibitor.
However, biosynthesis of butyrate kinase is not regulated by end product and constant along fermentation process Ballongue et al, 1986. Generally, increasing ratio of free butyril Co-A Co-A
could be effect on increasing of Co-A transferase activity and highly butyrate produced by bacteria Amans et al, 2001
Generally, this research explained the potential of type-III resistance starch derived from Sukuh sweet potato as fermentation substrate for Eubacterium rectale 17629. The main fermentation
product mainly butyrate was produced highly while it has a particularly important role as the preferred energy source for the colonic epithelium and proposed role in providing protection against colon
cancer and colitis. It indicated that type-III resistant starch of Sukuh sweet potato is potential to be developed as functional food to increase value added of Sukuh as an original variety of sweet potato
in Indonesia.
CHAPTER V CONCLUSION AND RECCOMENDATION
