16 Figure 3. Resistant Starch Yield
Resistant starch analysis was conducted according to Goni et al. 1996. The main features of the analytical procedure are removal of protein, removal of digestible starch, solubilization and
enzymatic hydrolysis of RS, and quantification of RS as glucose released x 0.9 where stomach and intestine physiological conditions pH, transit time are approximately simulated. The removal of
protein was introduced to enhance amylase accessibility avoiding starch-protein associations. Moreover, this step is advisable for a better simulation of physiological conditions proteolytic
digestive enzymes, acidic pH. The removal of digestible starch is done to avoid positive mistake when glucose is released.
Purwani et al 2009 reported that resistant starch content of jago sweet potato was 13.77. The analysis showed that resistant starch content of jago sweet potato treated with pullulanase was
28.15, which was lower than Salosa sweet potato starch treated with pullulanase enzyme that contained 38.22 RS Evalin, 2011. Treatment with pullulanase and retrogradation process were
proved could increase resistant starch content of jago sweet potato starch. Purwani et al 2009 reported that sago resistant starch treated with pullulanase contained 31-38 RS and rice resistant
starch treated with pullulanase contained 21-26 RS, whereas Zhao 2009 reported that maize resistant starch treated with pullulanase contained 24.5-32.4 RS. It showed that Jago sweet
potato starch treated with pullulanase contained higher RS content than rice starch treated with pullulanase enzyme, but lower than sago resistant starch and maize resistant starch which also
treated with similar enzyme. The difference could have been caused by several factors such as starch and enzyme used, amylose content, heating and cooling condition, drying method, etc.
C. FERMENTATION OF RS3 by Clostridium butyricum
In vitro fermentation of RS3 was divided into 2 stages. Stage I was carried out to evaluate production pattern of SCFA to determine optimum fermentation time of Clostridium butyricum to
produce high butyric acid. Stage II was carried out to further increase production of SCFA, especially butyric acid content.
17 Two different fermentation conditions were performed in stage I and II. In stage I,
concentration of RS3 was 20 gl 2 and glucose was 1 gl 0.1. Interval time during fermentation was 6 h, 12 h, 24 h, 36 h, and 48 h. This interval was chosen to simulate
physiological condition in the digestive system. Concentration of RS used in stage I referred to Evalin 2011 who reported that concentration of 2 RS3 gived highest concentration of butyric
acid in Salosa sweet potato RS fermentation. Result of fermentation is presented in Figure 4.
Figure 4. Changes of pH and absorbances medium during 48 h fermentation of 2 RS
A decrease of pH was observed after 6 h of fermentation and it remained until 48 h fermentation, but pH value was not decreased significantly. The lowest pH value 4.74 was
achieved at 48 h fermentation. The pH in medium indicated that short chain fatty acid SCFA were formed during bacterial growth Robertfroid, 2001. It was in agreement with Sayar et al
2007 who stated that carbohydrate fermentation result in pH degradation in the colon, caecum, and faeces. pH degradation plays important role in calsium and magnesium absorption, reduce
solubility of secondary bile acid, and inhibit pathogenic bacterial growth. Increased in cell growth was observed during 48 h fermentation. A significant absorbance
increase was observed after 6 h fermentation, absorbance began to increase constantly after 12 h fermentation. The highest absorbance was achieved at 36 h fermentation, but after that absorbance
was decreased. This implied that during fermentation, Clostridium butyricum grew rapidly. The result showed that the environment condition was suitable for bacteria to grow. Absorbance
between 12 h and 36 h increased slightly because of competition of bacteria to get nutrition for their growth. After 36 h fermentation, absorbance began to decrease due to lack of nutrient and
medium pH was too low and thus inhibited the bacterial growth. Absorbance change implied that there are bacterial growth during fermentation.
Clostridium butyricum is one of the colon bateria which can utilize carbohydrate as carbon source. These bacteria can be isolated from human or animal colon and soil, and can be grown in
Reinforce Clostridial Medium RCM medium. In this research, soluble starch as carbon source was replaced with sweet potato resistant starch.
Resistant starch fermentation by Clostridium butyricum resulted in formation of short chain fatty acid such as acetate acid, propionic acid, and butyric acid. Wang et al 1999 reported that
resistant starch has butyrogenic activity, it means that resistant starch can induce butyric acid
18 formation better than other substrates. In this reserach, SCFA profile was determined with Gas
Chromatography Agilent Technoligies 7890 A GC system. The profile of SCFA produced by Clostridim butyricum in stage I is presented in Figure 5.
Figure 5. Profile of SCFA during 48 h fermentation of 2 RS Result of SCFA analysis showed that compared to other acids, butyric acid was the most
dominant of the short chain fatty acid produced by Clostridium butyricum. The production of acetic acid only occured at 6 to 12 h fermentation, up to 24 h the concentration of acetic acid
decreased dramatically. Concentration of acetic acid, propionic and butyric acid had similar pattern profile while concentration of SCFA was increased at 12 h, decreased at 24 h, and
increased at 36 h. By 48 h fermentation, all of SCFA was not detectable. Apperently the SCFA content in the medium was too low and could not be detected.
From the SCFA profile above, it can be concluded that the optimum time of fermentation of sweet potato resistant starch to produce butyric acid was 12 h, where the molar ratio of acetic acid,
propionic acid, and butyric acid were 50 mM : 37 mM : 68 mM 1.3:1:1,8, but this level is considered as low and can be increased.
Stage II was designed to increase the production of SCFA, especially the butyric acid. The concentration of RS3 was decreased to 10gL 1 and glucose was increased to 5gL 0.5. This
fermentation condition referred to Purwani and Suhartono 2009 who reported that 1 RS and 0.5 glucose increased butyric acid concentration in the fermentation of sago starch. The
fermentation time used at this stage was 12 h referred to Stage I and 48 h referred to Purwani and Suhartono, 2009.
19 Figure 6. Changes of pH and absorbances medium during 48 h fermentation of
1 RS From Figure 6, a decrease of pH was observed after 12 h of fermentation and pH decreased
further at 48 h fermentation. Compared to the pH value in stage I after 12 h fermentation, the pH with 1 RS 5.01 was higher than pH using 2 RS 4.80 even though pH after 48 h
fermentation with 1 RS 4.69 was lower than pH with 2 RS 4.74. Different pH will affect the distribution of acids, cell membrane transport behavior, and cell lysis. Relative high pH value
6.0 is beneficial for cell growth and butyric acid biosynthesis, especially in Clostridium butyricum He et al. 2005. Medium pH also affects the specific growth rate, butyric acid
production rate, and consumption of sugars. The higher pH observed in 1 RS medium after 12 h fermentation maybe due to more
glucose being available as the carbon source in the growth medium such that the bacterial grew and the consequence was higher pH achieved because of decreased of the SCFA production. This
explanation was supported by higher medium absorbance which showed that total bacteria in the medium was increased higher than the previous fermentation. After 48 h fermentation pH in 1
RS medium was lower, the bacteria can produce SCFA and as the consequence, there was higher concentration SCFA which lower the pH. Both fermentations showed similar pattern of
absorbance change, while absorbance was increased at the beginning of fermentation and was decreased in the end of fermentation. Decreasing absorbance indicates that the total bacteria was
declined because of the metabolics produced by the bacteria, pH degradation, and competition between bacteria to get nutrition which inhibit bacterial growth. The profile of SCFA produced by
Clostridim butyricum in stage II is presented in Figure 7.
20 Figure7. Profile of SCFA after 48 fermentation of 1 RS
Profile of SCFA above showed that after 48 h fermentation, the molar ratio of acetic acid, propionic acid, and butyric acid were 453 mM : 248 mM : 225 mM 2:1.1:1. It was higher than
the result at 12 h fermentation, where the molar ratio were 226 mM : 162 mM : 192 mM 1.4:1:1.2. Compared to previous fermentation, 1 RS medium fermentation resulted in higher
level of SCFA. Acetic acid was the most dominant of SCFA compared with propionic acid and butyric acid. This result was different than previous fermentation where butyric acid was the most
dominant. It was in agreement with Purwani and Suhartono 2009 who reported that high butyrate level was produced after 48 h fermentation of sago resistant starch where the molar ratio of acetic
acid, propionic acid, and butyric acid were 83 mM : 47 mM : 46 mM 2:1:1, but it was not in agreement with Evalin 2011 who reported that no more butyrate was produced after 48 h
fermentation of Salosa sweet potato resistant starch where the molar ratio of acetic acid, propionic acid, and butyric acid were 591 mM : 0 mM : 0 mM.
Different level of glucose in medium also influence the profile of SCFA produced by Clostridium butyricum. Higher concentration of glucose 5 gl in the Stage II resulted in higher
medium turbidity than in the Stage I 1 gl. It presumably indicated that more glucose as the carbon source in the medium was more useful for Clostridium butyricum to grow than resistant
starch as the carbon source. Glucose is more useful for Clostridium butyricum to grow because it can be utilized directly as the carbon source whereas the resistant starch have to be hydrolyzed
first to utilize the carbon source. The consequence if the medium contains more glucose is the Clostridium butyricum will grow rapidly and form the SCFA in higher concentration.
21 Table 7. Comparison of SCFA Production from Several RS by Clostridium butyricum
RS Source
Fermentation time h
[RS] [Glucose]
Acetic acid mM
Propionic acid mM
Butyric acid mM
Acetic : propionic :
butyric
Jago Sweet Potato
6 2 0.1 34.31 38,13 52.79
1 :
1.1 :
1.5 12
1 0.5 226.79 162.28 192.25 1
: 1.4
: 1.2
2 0.1 50.13 37.83 68.03 1.3
: 1
: 1.8
24 2
0.1 0.55
16.80 18.81
1 : 30 : 38 36 2 0.1 0 29.73
40.57 - 48
1 0.5 453.65 248.64 225.37 2 :
1.1 :
1 2
0.1 0 0 0 -
Salosa Sweet
Potato Evalin
2011 6
2 0.1 85.25 60.00 73.51 1.4
: 1
: 1.2
12 2
0.1 0 0 0 -
24 2
0.1 13.45
16.70 120.33
1 : 1.2 : 9 36 2 0.1
939.12 0 14.81 - 48 2 0.1
591.84 0 -
Sukuh Sweet
Potato Iswani
2011 6 2
0.5 0 10.29 13.57 -
12 2 0.5 0 0 15.32 -
24 2 0.5 0 0.84 4.01 - 36
1 0.5
500.40 476.28
477.97 1.1 : 1 : 1
2 0.5 0 25.17
50.97 - 48
1 0.5 215.21 281.10 343.12 1
: 1.3
: 1.6
2 0.5 0 0 22.83 -
Rice Purwani
et al. 2009 24
0.5 0.5
86.79 1.09
5.74 80 : 1 : 5
48 1
0.1 71.37
1.42 15.89
50 : 1 : 11
Sago Purwani
et al. 2009 24
0.5 0.5
79.32 0.37
6.99 214 : 1 : 19
48 1
0.1 72.74
3.06 11.12
24 : 1 : 3 Corn Goni
et al. 2000
5 1 - 59.70 27.00 13.30
4.5 :
2 :
1 10 1 - 60.90 25.70 13.50
4.5 :
2 :
1 24 1 - 61.80 27.00 11.20
5.5 :
2.5 :
1 48 1 - 62.30 25.90 11.80
5.5 :
2.5 :
1 : microorganism isolated from caecal Wistar rats
SCFA comparison above showed similar pattern of SCFA production during in vitro fermentation where acetic acid and butyric acid were the most dominant fatty acid. Propionic acid
formation followed similar pattern of butyric acid but the concentration was lower. RS concentration and fermentation time were important to influence profile of SCFA. The difference
of concentration and fermentation time resulted difference profile of SCFA.
22 In this research, 2 RS of jago sweet potato fermentation resulted in lower SCFA level
than 2 RS of salosa sweet potato. Fermentation of 2 RS of salosa sweet potato resistant starch during 36 h and 48 h resulted in very high acetic acid level 939.12 mM and 591.84 mM but the
butyric acid level was very low at 48 h fermentation and it was not detected. Fermentation 2 RS of sukuh sweet potato did not result acetic acid and resulted in lower SCFA level than 2 RS of
jago sweet potato. Lower concentration of jago sweet potato RS 1 resulted in higher SCFA level than rice, sago, and corn resistant starch, especially in the acetic acid and the butyric acid.
Molar ratio resulted from fermentation of 1 jago sweet potato RS during 48 h was 453 mM : 248 mM : 225 mM acetate:propionate:butyrate and it was the highest among the substrates. Rice,
sago, and corn resistant starch showed similar molar ratio of SCFA but the difference was in the propionic level. Propionic acid level in the rice and the sago resistant starch were very low and
lower than the butyric acid level whereas in the corn resistant starch, the propionic acid level was higher than the butyric acid. Fermentation of 0.5 rice and sago resistant starch resulted in higher
molar ratio of SCFA than 1 RS. This result indicated that lower concentration of RS resulted in higher formation of SCFA, especially the acetic acid and the butyric acid.
Longer fermentation time also resulted that increasing level of SCFA. Thus data above confirmed that longer fermentation time until 48 h resulted in higher level of SCFA, especially the
acetic acid and the butyric acid. Fermentation of sweet potato resistant starch during 48 h resulted in the highest molar ratio of SCFA 453 mM : 248 mM : 225 mM acetate:propionate:butyrate.
Similar result was also achieved at 48 h fermentation of rice, sago, and corn resistant starch. Fermentation of salosa sweet potato resistant starch during 36 h resulted in higher molar ratio of
SCFA 939 mM : 0 mM : 14 mM acetate:propionate:butyrate than during 48 h fermentation which resulted in molar ratio of SCFA 591 mM : 0 mM : 0 mM acetate:propionate:butyrate. It
indicated that Clostridium butyricum need longer time to produce higher level of the acetic acid and the butyric acid. Longer fermentation time has relationship with growth cycle of
microorganisms where after 48 h fermentation indicated microorganisms have entered death phase which was pointed by decreasing medium turbidity than in the early fermentation.
Besides RS concentration and fermentation time, glucose content in the medium also influenced the profile of SCFA produced by Clostridium butyricum. Data from Table. 7 showed
that fermentation of all the substrates except corn resistant starch with higher content of glucose 0.5 resulted higher level of SCFA. Higher content of glucose in the medium means that
bacteria will have more carbon sources to grow so it can be utilized by the Clostridium butyricum to duplicate itself at the growth phase. Rapid growth of Clostridium butyricum at the beginning of
the fermentation will reduce amount of the carbon source. The consequence, Clostridium butyricum will begin to hydrolize the resistant starch to glucose and ferment the undigest
carbohydrate to form short chain fatty acid. Higher total bacteria in the medium will give more chances for the Clostridium butyricum to form short chain fatty acid.
The differences in physicochemical properties of substrates such as the crystalline structure of starch and hydrogen bonds may affect the accessibility of microbial enzymes and the colonic
fermentation, modifying the quantity of SCFA produced and the rate of fermentation Goni et al. 2000. Simple crystalline structure of starch enable the microbe’s enzyme to convert glucose to
pyruvate as the intermediate compound in further catabolism. More hydrogen bonds indicate more complex structure which means microorganisms need more energy ATP to convert carbohydrate
to glucose. If energy required at higher level, it will reduce energy source for microorganisms to convert glucose to pyruvate which limit the production of SCFA. The result may imply that jago
sweet potato resistant starch have simpler crystalline structure and less of hydrogen bonds than
23 other substrates such as rice resistant starch, sago resistant starch, and corn resistant, and it can be
better utilized by Clostridium butyricum to produce higher level of acetate and butyrate. High level of butyric acid has relationship with the acetic acid level. Increasing level of
butyric acid was possitively correlated with increasing level of acetic acid. Relationship between acetic acid and butyric acid was observed in all substrates fermentation. In this research, increasing
the acetate and the butyrate level occured after 48 h fermentation. This result has similar pattern with rice resistant starch, sago resistant starch, and corn resistant starch. This result indicated that
after 48 h fermentation, SCFA production tend to shift to the acetate. The lack of nutrient in the medium could be one of the factors which cause why Clostridium butyricum would rather produce
acetic acid because more ATP is required to form the butyric acid. Clostridium is an acidogenic bacterium, produce acetate and butyrate as the main
fermentation products. The result of fermentation also showed that acetate and butyrate were the main fermentation product. Sharp and Macfarlane 2000 reported that saccharolytic Clostridia is
well adapted to grow faster in high substrate concentration and resistant starch granules are advantageous for their growth. Figure 8 shows the metabolic pathways of SCFA production.
Figure 8. The metabolic pathways of SCFA production. 1
a
Butyrate kinase; 1
b
Butyryl CoA-Acetate Transferase Zhang et al. 2009 Glucose as the carbon source is metabolized into pyruvate through glycolisis pathway.
Pyruvate is the main substrate for further enzymatic metabolism. In the acetate pathway, one mole of glucose is metabolized into acetyl-CoA with pyruvate-feredoxin oxidoreductase enzyme which
release CO
2
and 2 ATP, then acetyl-CoA is metabolized into acetate with acetate kinase releasing 2 ATP, so 4 ATP are formed during conversion of the glucose to two acetic acids. In the butyric
pathway, acetyl-CoA is metabolized into butyryl CoA then metabolized into butyrate by acetyl- CoA transferase releasing 1 ATP, so 3 ATP are formed during conversion of glucose to butyric.
More energy are released from conversion of the glucose to acetic acid. In this case bacteria prefer
24 to produce acetic acid than butyric acid in the exponential phase Zhang et al. 2009. Beside
through butyrate kinase, glucose can be metabolized to butyric acid with Co-A transferase enzyme which convert butyryl-CoA and acetate into butyric acid and acetyl-CoA.
1
a
pathway is present generally within Clostridium species, such as Clostridium acetobutylicum, Clostridium tetani, Clostridium perferingens, and Clostridium difficile. In this
pathway, butyrate kinase expressing gene is found in Clostridium sp which is isolated from soil and water while in the 1
b
pathway, it is found from bacteria which is isolated from human gut so this pathway is dominant in colon. Louis et al 2004 reported that CoA transferase enzyme
activity is the most dominant pathway in the human colon. Chen and Blaschek 1999 reported that activity of CoA transferase, acetate kinase, and
butyrate kinase influence acid metabolism during fermentation. Some research reported that enzyme activity in human colon is influenced by substrates, pH, and end product level. In this
research, SCFA profile in Stage I showed that butyric acid was the most dominant. This result showed that Co-A transferase activity to form butyric acid was higher than acetate kinase whereas
in Stage II, acetic acid level was higher than the butyric acid. This result indicated that acetate kinase activity was higher than the Co-A transferase. The difference resistant starch which was
used proved that substrate influenced enzyme activity, where fermentation of 1 RS resulted presumably in higher acetate kinase activity and fermentation of 2 RS resulted presumably in
higher butyrate kinase activity. The pH also influenced the enzyme activity, lower pH 4.69 increased acetate kinase activity and decreased CoA transferase activity.
Aman et al 2001 reported that the butyric acid formation by Clostridium butyricum is affected by high level of butyril CoA : CoA ratio. Increasing the acetate level from 226 mM to 453
mM indicated that butyril CoA : CoA ratio was low such that the metabolism aimed to produce acetic acid. Louis et al 2007 reported that limited carbon source in the medium will stimulate
bacteria to produce acetic acid because they will produce more ATP than butyric acid production. This statement explained why the butyrate level was lower than the acetate level in the end of
fermentation. Many factors are able to influence the metabolics pathway of the microorganism during
fermentation. In the case of butyrate-producing Clostridia, the concentrations of glucose, pH, H
2
partial pressure, acetate, and butyrate impact the growth rate, the final product concentration and the distribution of products Kong et al. 2006. Excess the carbon source often affects osmotic
dehydration of the microorganisms in the fermentation process. The significant increase of the butyrate-acetate ratio was observed in the glucose-limited culture without sparging nitrogen into
fermenter in the butyrate fermentation with Clostridium butyricum as the working microorganism Saint-Amans, 1995.
This research result is expected to give information about short chain fatty acid SCFA profile produced by Clostridium butyricum BCC B2571 during fermentation of jago sweet potato
resistant starch. This resistant starch has potency to develop as functional food because result high butyric acid level during fermentation which can be utilized to prevent colon cancer.
25
V. CONCLUSION AND RECOMMENDATION
