effect of fructooligosaccharides (fos) on digestive enzymes of striped catfish (pangasianodon hypophthalmus)

CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES






EFFECT OF Fructo-Oligosaccharides (FOS) ON
DIGESTIVE ENZYMES OF STRIPED CATFISH
(Pangasianodon hypophthalmus)




By
NGUYEN KHANH LINH









A thesis submitted in partial fulfilment of the requirements for
the degree of Bachelor of Aquaculture

Can Tho, December 2013


CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES





EFFECT OF Fructo-Oligosaccharides (FOS) ON
DIGESTIVE ENZYMES OF STRIPED CATFISH
(Pangasianodon hypophthalmus)




By
NGUYEN KHANH LINH





A thesis submitted in partial fulfilment of the requirements for

the degree of Bachelor of Aquaculture




Supervisor
Assoc. Prof. Dr. DO THI THANH HUONG





Can Tho, December 2013


APPROVEMENT
The thesis “Effect of fructooligosaccharides (FOS) on digestive enzymes of
striped catfish (Pangasianodon hypophthalmus)” defended by Nguyen
Khanh Linh,which was edited and passed by the committee on 12-27-2013.

Sign of Supervisor Sign of Student


Assoc. Prof. DO THI THANH HUONG NGUYEN KHANH LINH
i

ACKNOWLEDGEMENT
Firstly, I want to express special thanks to my supervisor, Assoc. Prof. Dr.
Do Thi Thanh Huong for her invaluable guidance, advice, and
encouragement. I would also like to dedicate my great appreciation to

Nguyen Thi Kim Ha for her kind help in finishing the research.
Secondly, many thanks are also given to all other doctors of the college of
aquaculture and fisheries, and especially to those of the department of
Aquatic Nutrition and Products Processing for providing me with great
working and learning conditions.
Thirdly, I would love to express my sincere appreciation to many friends,
especially Le Thi Mai Anh, Nguyen Minh Thuat, Dang Minh Quan, Tran
Thi Be Gam, Nguyen Chi, Vo Van Dao and Tran Thi Bich Thuan for their
unconditionally kind help throughout the experimental period.
Finally, I want to thank my academic adviser, Dr. Duong Thuy Yen, who
was guiding and encouraging me over the last four years, and my family for
their great lifetime support which makes everything possible for me.
Thank you very much,
Nguyen Khanh Linh









ii

ABSTRACT
The aim of the experiment was conducted to assess the impact of the
supplement of fructooligosaccharide on on digestive enzyme activities and
total bacteria in the intestine of stripped catfish fingerling. The experiments
were consists of 5 concentrations (0%; 0.5; 1%; 1.5% and 2%/kg food).

Each treatment was three replicated; the test period was 90 days. The
digestive enzymes activities as pepsine, trypsine, chymotrypsine and
amylase in the stomach and intestine of the fish were measured at 0, 1, 3, 7,
10, 30, 60 and 90 days and the total bacteria in these organs also counted.
The results show that the fish eating with supplement FOS were improved
on digestive enzyme activities and total bacteria in the intestine at the
treatment of 0.5% and 1 % of FOS.

















iii

TABLE OF CONTENTS
Acknowledgement i
Abstract ii
Table of contents iii

List of figures v
List of tables vi
List of abbreviations vii
CHAPTER 1: Introduction 1
1.1 General introduction 1
1.2 Research Objective 1
1.3 Research Content 1
CHAPTER 2: Literature reveiw 2
2.1 Tra Catfish (Pangasianodon hypophthalmus) 2
2.2 Prebiotic 2
2.3 FOS (Frustooligosaccharides) 3
2.4 Application of FOS in terrestrial animals 4
2.5 Application of FOS in aquatic animals 4
2.6 Digestive enzymes in fish 5
CHAPTER 3: Research methodology 6
3.1 Time and place 6
3.2 Materials 6
3.3 Methods 6
3.3.1 Experimental design 6
3.3.2 Evaluate the total microorganisms in intestine 7
3.3.3 Evaluate enzyme in stomach and intestine 8
3.4 Data collection, calculation, and analysis 9
CHAPTER 4: Results and disscussions 10
4.1 Environment parameters 10
4.2 Total bacterial count 11
4.3 Enzymes activity 12
CHAPTER 5: Conclusions and recommendations 17
iv

REFFERNCES 18

APPENDICES 21
Appendix 1: Enzyme analysis 21
Appendix 2: Chemicals preparation 24
Appendix 3: Total bacterial count 26
Appendix 4: Enzyme activity 26




















v

LIST OF TABLES
Table 3.1: Composition of diets 7
Table 4.1: Temperature, DO, pH measured in the morning and afternoon 10

Table 4.2: TAN and NO2- 10
Table 7.1: Total bacterial counted in fish’s intestine 26
Table 7.2: α–amylase in fish’s stomach 26
Table 7.3: α–Amylase in fish’s intestine 27
Table 7.4: Pepsine in fish’s stomach 27
Table 7.5: Trypsine in fish’s intestine 28
Table 7.6: Chymotrypsine in fish’s intestine 28

vi

LIST OF FIGURES
Figure 2.1: Chemical structure for FOS 3
Figure 3.1: Total bacterial count in the intestine 8
Figure 4.1: Total bacterial counted in intestine of the fish 12
Figure 4.2: α – Amylase in stomach of the fish 13
Figure 4.3: α – Amylase in intestine of the fish 13
Figure 4.4: Pepsine in stomach of the fish 14
Figure 4.5: Trypsine in intestine of the fish 15
Figure 4.6: Chymotrypsine in intestine of the fish 15

vii

LIST OF ABBREVIATIONS
FOS Fructooligosaccharide
XOS Xylooligosaccharide
GOS Galactooligosaccharides
MOS Manooligosaccharide
TOS Trans-galacto-oligosaccharides
ScFOS Short chain Fructooligosaccharides
DO Dissolved oxygen
FCR Feed conversion ratio
1

CHAPTER 1
INTRODUCTION
1.1 General introduction
In the recent years, many countries in the word including Vietnam have
been developed aquaculture to contribute to the food consumption.
Compared to many species that cultured in the Mekong Delta, striped
catfish is one of important export products. Until the end of 2012, seed
production of striped catfish was nearly 4.6 billion fingerlings (increase two
times compare with 2011), culture area was about 5,910 ha, grow-out

production was about 1,255,500 tons, and export turnover was about 1.744
billion USD (decrease 3.4% compare to 2011) (Directorate of Fisheries,
2012).
Since Vietnam becomes number one striped catfish export country, striped
catfish become the important species. Many research about nutrition,
genetic, physiology, and diseases were investigated. Striped catfish is
intensively cultured in pond of about 40-60 fish/m
2
(FAO, 2011). Striped
catfish can growth fast and reach harvest size from 1 to 1.5kg in 6 months
or less (FAO, 2011). The feed conversion ratio of P. hypophthalmus fed
commercial pellets is typically 1.7-1.9 (FAO, 2011). Because of high FCR,
antibiotic was used as one kind of animal growth-promoter for its broad-
spectrum antimicrobial effect, but it causes some negative effect for
animals and environment. Therefore, some probiotics and prebiotics (such
as FOS, XOS, GOS, inulin, etc.) are the good choices for improving feed
formula to optimize fish digestibility, reduce the FCR, increase growth and
fish health also.
1.2 Research objectives
The aim of the study is to find out the activity levels of enzymes in stomach
and intestine of striped catfish fed with various doses of FOS in order to
enhance the growth and feed utilization.
1.3 Research contents
- Evaluated the enzymes activities in the stomach (pepsine and amylase)
and in the intestine (trypsine, chymotrypsine and amylase) of striped catfish
fed with different doses of FOS.
- Evaluated the total bacteria in the intestine of striped catfish fed with
different doses of FOS.
2

CHAPTER 2
LITERATURE REVIEW
2.1 Striped Catfish (Pangasianodon hypophthalmus)
Striped catfish (Pangasianodon hypophthalmus) have compressed body
with a short dorsal fin (one or two spines), a developed adipose fin, long
anus, strong pectoral spines and two pairs of barbels. There are six
branched dorsal fin rays and the pelvic fins have eight to nine soft rays. The
gill rakers are described as being normally developed. There is also having
the swimbladder with a single chamber extending posteriorly above anal fin
(Thuong, 2008).
Striped catfish is one of thirty species of Pangasiidae that distributed
naturally in the wild at Cambodia, Laos, Vietnam, and Thailand. Striped
catfish is cultured in pond of Southeast Asia. It is omnivorous species that
can eat plant, fruit, and some mollusk. Striped catfish can also eat artificial
feed so we can add chemicals such as drugs, enzyme for the fish. Striped
catfish is high tolerance with low DO, bad water quality (high nitrate and
ammonia), and the suitable temperature is 22-26
o
C, and optimal pH is 6.5-
7.5 (FAO, 2011).
2.2 Prebiotic:
At the present, many countries in the world recognized the important of
sustainable aquaculture, so many researches in certain year are mention in
the chemicals that friendly with the environment and human health.
Prebiotics are non-digestible but fermentable oligosaccharides that are
specifically changed the composition and activity of the intestinal
microbiota with the prospect to promote the health of the host. Dietary fiber
and non-digestible oligosaccharides are the main growth substrates of gut
microorganisms. Their fermentation results in the acidification of the
colonic con-tents and the formation of short chain fatty acids which serve

as fuels in different tissues and may play a role in the regulation of cellular
processes. Prebiotics specifically stimulate the growth of endogenous
microbial population groups such as bifidobacteria and lactobacilli which
are perceived as being beneficial to human health because according to
Rivero-Urgell, Santamaria-Orleans (2001) complex oligosaccharides have
the capacity of inhibiting the binding of pathogens to cell surface because
they act as competitive receptors. In spite of the interesting nutritional
properties of prebiotics it is questionable whether a wholesome diet rich in
3

fruit and vegetables needs to be supplemented with prebiotics for optimal
health effects (Blaut, 2002).
2.3 Fructooligosaccharides (FOS)
FOS is the prebiotic that has the origin of the plant. FOS is found naturally
in Jerusalem artichoke, burdock, chicory, leeks, onions, and asparagus.
FOS is fiber widely distributed in fruits, vegetables and plants, which is
classified as a food ingredient (not as an additive) and considered to be safe
to eat (Farnworth, 1997).
The term “oligosaccharide” refers to a short chain of sugar molecules.
Fructo-oligosaccharides (FOS) consist of short chains of fructose
molecules. Oligosaccharides are consumed; the undigested portion serves
as food for bacteria, such as Bifidobacteria and Lactobacillus species
(Blaut, 2002).
Besides, FOS has the health benefits, including lowering of blood
cholesterol, lowering of high blood cholesterol, lowering of high blood
pressure, protective effects again infections, controlling arthritis and
enhancing antitumor properties. In addition, prebiotic can cause negative
effects are laxative; increase the gas-producing bacteria in digestive system
(Science and Technology Information Net, 2010)

Figure 2.1: Chemical structure for FOS (Yun and Song, 1999)
4

In over the word, many researchers had the experiments about effect of
FOS on human heal and there are many positive effected such as the
research of Wang and Gibson (1993) showed that oligofructose and inulin
can stimulatory effect on numbers of the health-promoting
genus Bifidobacterium, and maintaining populations of potential pathogens
(Escherichia coli, Clostridium) at relatively low levels.
2.4 Application of FOS in terrestrial animals
There are many researches about applied FOS into terrestrial diet. Example,
the experiment of Xu et al., 2003 about the effect of different
concentrations of FOS (0, 2, 4, 6 gKg
-1
) of growing pig, they founded that
supplementation with 4 and 6g/kg FOS significantly improved average
daily gain and feed conversion ratio. As compared to control,
supplementation with FOS increased the available of Bifidobacterium and
Lactobacillus (health-promoting genus) and reduced Clostridium and
Escherichia coli (unprofitable) in the small intestine. Moreover,
supplementation with 4 and 6gKg
-1
FOS improved the activities of
digestive enzymes such as protease, trypsin and amylase in the small
intestine. Besides, another study on male broilers had similar results also
when they applied 0, 2, 4, 8gKg
-1
diet however the supplementation of
8gKg
-1

reduced the growth of this species.
2.5 Application of FOS in aquatic animals
The experiment of Mahious et al (2006) was done to compare the effect of
cellulose powder, inulin, oligofuctose, they had control treatment included
2% cellulose powder, treatment 1 included 2% inulin, treatment 2 involves
2% oligofructose or 2% lactosucrose, and treatment 3 comprises 2%
lactosucrose. The result showed that, the growth rate in diet contained
oligofructose was highest and the growth of Bacillus sp. than the others.
In 2007, the research of Hui Hui-yuan et al about Effect of dietary short
chain fructo-oligosaccharides (scFOS) on hybrid tilapia was done to find
out the suitable concentrations (control, 0.8, 1.2gKg
-1
) of scFOS. And they
concluded that dietary FOS had beneficial effects on growth, FCR, and
intestinal microflora in tilapia at the 1.2gKg
-1
of scFOS in fish diet. In
addition, the experiment of higher concentration of FOS (control, 1.5 and
3% FOS) showed the similar results that supplementation 1.5 and 3% of
FOS significant increased growth rate, blood RBC count and haemoglobin
(Hb) level of oxyeleotris lineolatus compared to control group. Besides,
digestive enzyme in the stomach, small intestine such as protase, lipase, and
amylase also increased (Renjie et al., 2010). Besides, another research used
600 caspian roach fries to compare the different concentration of FOS (the
control, 1%, 2%, and 3% FOS). After 7 week, immune parameters and
growth rate were significantly higher in 2% and 3% FOS fed fish. All
5

dietary FOS levels were significant increased resistance to a salinity stress.
In addition, digestive enzyme activities were significantly increased

(Soleimani et al., 2012).
Another research of Xu et al (2009) was done in 45 days, they concluded
that there was significant differences increase in the daily weight gain
(DWG) of diets contained 50, 100, 200mgKg
-1
of XOS were compared with
the control. Besides, amylase activity in the intestine was significantly
higher for 100mgKg
-1
compared to diet 50mgKg
-1
and the control.
2.6 Digestive enzymes in fish
According to Huong and Tu (2010), pepsine is an enzyme whose
proenzyme (pepsinogen) is released by the stomach and that degrades
food proteins into peptides. Pepsin is most active in acidic environments
(pH 1.45-3) and temperature between 30°C and 50°C. Although water
temperature is not suitable for pepsin activity, but changing in food supply
for a long time will change in enzyme activity. Trypsin and
chymotrypsin are endopeptidase found in the pancreas, where it breaks
down protein into smaller peptides. The peptide products are then further
hydrolyzed into amino acids. Trypsin and chymotrypsin has an optimal
operating pH about 7-11. Trypsin is produced as
the proenzyme trypsinogen. Amylase is release from the pancreas that
breaks down long chain of starch into oligosaccharides, and maltose will
degrade oligosaccharides into monosaccharides. Amylase present mostly in
the intestine of omnivorous fish. Because of the important function of these
enzymes, there are many researches were done to provide the information
on digestive enzymes activity in fish that fed with different kind of
prebiotics. Huong and Tu also mention in some factors that affect the fish’s

digestion such as quantity of food intake, quality of food intake, and fish
age.
Protease refers to a group of enzymes whose catalytic function is to
hydrolyze (breakdown) peptide bonds of proteins. They are also called
proteolytic enzymes or proteinases. Examples of proteases include: fungal
protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin.
Proteolytic enzymes are very important in digestion as they breakdown the
protein foods to liberate the amino acids needed by the body. (Enzyme
essentials, 2013)

6

CHAPTER 3
RESEARCH METHODOLOGY
3.1 Time and place
The experiment was carried out for 90 days from May to August, 2013 at
the Department of Aquatic Nutrition and Products Processing, College of
Aquaculture and Fisheries, Can Tho University.
3.2 Materials
- Striped catfish fingerlings (size: 10-15g) were bought from a hatchery in
Cantho city and acclimated about 7 or 10 days in laboratory conditions.
- The fish were be fed with commercial feed from Nafa company contained
30% protein
- FOS was purchased from Meiji CO., LD that contained 95% of FOS and
5% of glucose, fructose, and sucrose.
- 15 composite tanks (0.5m
3
/tank), spectrometer, incubator, auto clave, petri
disk, test-tube, cuvet, etc.
- Chemicals: NaCl, KH

2
PO
4
, NaH
2
PO
4
, PCR medium, NaCl, etc.
3.3 Methods
3.3.1 Experimental design
There were 5 treatments in this study:
+ Treatment 1: without FOS
+ Treatment 2: add 0.5% of FOS in the diet
+ Treatment 3: add 1% of FOS in the diet
+ Treatment 4: add 1.5% of FOS in the diet
+ Treatment 5: add 2% of FOS in the diet
The experiment was designed randomly and each treatment was triplicated.
The water exchanged about 50% each tank weekly.
Fish samples were taken at the 0, 1, 3, 7, 10, 30, 60, 90 days in experiment
time.
Experimental animal: The fish had initial body weight 10-15g. After
acclimated about 7 or 10 days in the laboratory condition, fish were chosen
as healthy, same size, normal shape and stocked in the experimental tanks
with the density of 70fish/0.5m
3
.
7

Experimental diets: FOS in powder form was mixed with water depending
on the dose of each treatment (400mL water/5kg of commercial feed).

Then, this mixture was pray with the commercial feed. Finally, squid oil
was covered (ratio: 2%) to make the FOS inside the feed, after that hand-
fed to saturation twice per day.
Table 3.1: Composition of diets (Nafa label with 30% crude protein)
Ingredients
%
Crude Protein
Crude Lipid
Crude Fiber
Moisture
Ash
Phosphorus
Energy (Kcal/kg)
30
5
7
11
12
1
2.400
Water quality: pH, temperature, oxygen were measured 2times/day. TAN
was measured by Indophenol Blueand method and NO
2
-
is determined by
Griess Ilosvay method weekly.
Fish sampling:
+ Intestine and stomach samples were used to determine the
activities of digestive enzyme such as pepsine, chymotrypsine, and trypsine
and α - amylase (3fish/tank).

+ Intestine samples were used to determine the total bacteria in the
intestine (3fish/tank).
3.3.2 Determined the total microorganisms in intestine
The total plate count method was used to count total bacteria in the
intestine.
The fish was operated to take the full intestine. Intestines were weighted
then place into saline peptone water (8.5g NaCl/L of distilled water). The
supernatant diluted with difference concentrates depending on the density
of bacteria in the sample; each concentration was separated 10 times and
implanted the sample in PCA medium (26.5g anhydrous PCA media/L
distilled water)
Place 0.1 mL of diluted concentration into the petri disk containing 15-
20mL of PCA medium. Then mix the sample and medium together by
clockwise or anticlockwise rotation and incubate at 37
o
C in 48 or 72 hours.
8

After 48 or 72 hours, the colonies were counted (acceptable rate is from 25
to 250 colonies /disk). Bacteria density is calculated by using the formula:
A=



Note: A: total colonies (CFU) in 1g of fish sample
N: total colonies counted in disks
n
i
: number of disks at concentration i
V: volume of sample implant for one disk

f
i
: diluted solution at concentration i

Figure 3.1: Total bacterial count in the intestine
The total microorganism in intestine among treatments was evaluated and
compared the results to measure the effect of FOS on microorganisms in
intestine of catfish that fed with various doses of FOS.
3.3.3 Measure the enzyme activities in stomach and intestine
Sampling method: three fish of each tank were operated. After that the
hint gut, and stomach were taken away, stored in eppendorf and frozen at -
80
o
C until future work. Before analysis, melted the sample, and crushed it
in the pH buffer 6.9. Then centrifuged the samples at 4200 rpm at 4
0
C for
30 min, and eliminated upper solution and placed it in the new eppendorf.
Enzyme analytical methods:
Protein (Bradford, 1976)
α – Amylase (Bernfeld, 1951)
Pepsin (Worthington, T.M, 1982)
Trypsin (Tseng, Grendell et Rothman, 1982)
Chymotrypsin (Worthington, 1982)
9

Note: α – Amylase and pepsine were measured by spectrometer
model Helios epsilon
Trypsine and chymotrypsine were measured by spectrometer model
UV1102M195

3.4 Data collection, calculation, and analysis
Data were manually collected throughout the experiment period. Standard
error, standard deviation, and mean were calculated by Excel 2010. Spss
16.0 was used for statistical analysis (one way ANOVA, DUCAN). All
statements of significance were based on probability of P<0.05.

















10

CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 Water quality
In the experimental period, temperature, DO, and pH were stable; the
temperature in the morning and afternoon were swigged about 2
o

C among
the treatments (27.5±0.95 in the morning and 29.3±1.50
o
C in the
afternoon). Because in this experiment aeration system was used
continuously, therefore DO was little differential among the treatments
(5.80±0.97 in the morning and 6.90±3.91 mg/L in the afternoon). Similarly,
pH was not much fluctuation among treatments (7.00±0.52 in the morning
and 7.66±0.64 in the afternoon). According to FAO (2011) the suitable
temperature range for striped catfish is from 22-26
o
C and pH is from 6.5-
7.5, hence the temperature and pH were ideal for fish growth.
Table 4.1: Temperature, DO, pH measured in the morning and afternoon




Note: Values are presented as mean± STD
Striped catfish is high tolerance with low DO, poor water quality (high
nitrate and ammonia) (FAO, 2011). TAN was the highest at 1% and 2% of
FOS-diet (0.43±0.26 and 0.43±0.27 mg/L) compared to the control, 0.5 and
1.5% of FOS-diet. Nitrite in this research quite low, it fluctuated between
0.36±0.23 and 0.42±0.22 mg/L; TAN and nitrite concentrations were
fluctuated, but still in suitable range for fish growth (Boyd, 1998).
Table 4.2: TAN and NO
2
-

Treatment

TAN (mg/L)
NO
2
-
(mg/L)
Control
0.40±0.22

0.38±0.21

0.5%
0.39±0.23
0.42±0.22
1.0%
0.43±0.26
0.38±0.24
1.5%
0.39±0.25
0.36±0.23
2.0%
0.43±0.27
0.37±0.18
Note: Values are presented as mean± STD

Temperature (
o
C)
DO (mg/L)
pH
Morning

Afternoon
Morning
Afternoon
Morning
Afternoon
Control
27.6±1.23

29.1±1.12

5.80±0.97

6.47±1.24

7.00±0.52

7.55±0.66

0.5%
27.6±1.84
29.3±1.50
5.97±0.93
6.52±1.11
7.11±0.51
7.57±0.60
1.0%
27.5±1.19
29.2±1.47
5.88±0.95
6.54±1.27

7.01±0.44
7.55±0.60
1.5%
27.6±1.23
29.2±1.44
5.81±0.97
6.59±1.34
7.08±0.55
7.60±0.54
2.0%
27.5±0.95
28.5±1.09
5.82±1.10
6.90±3.91
7.07±0.55
7.66±0.64
11

4.2 Total bacterial count
On the whole, the total bacterial count in the finger 4.1 showed the
increasing trend with fish’s age over 90 days FOS was administrated. The
intestinal microflora from day 0 to day 10 was not significantly different
among treatments. But in day 30, the total bacterial count at 0.5% FOS was
significantly higher than the other treatments. At the same time, the result
showed that the control group had lowest total bacterial count with
6.10±0.58 log (CFU/g). Moreover, after 60 days administrated FOS at 0.5
and 1% group were significantly higher than control, 1.5 and 2% group
(6.98±0.07 and 7.02±0.07 log (CFU/g)), in addition, 1.5% FOS-diet had the
lowest level compared to other treatments (6.30±0.03 log (CFU/g)). And
after 60 days, the bacterial count peaked at 0.5 and 1% of FOS diet (7.20

and 7.30 log (CFU/g)). Besides, there was no significantly difference
among other treatments. the gastrointestinal tract was very heavily
populated with bacteria, mainly of strictly anaerobic bacteria (Renjie,
2010). This microbial community was extremely complex, both in terms of
the number of organisms, approximately 10
13
in total. This result was
similar with the research of Hui-yuan et al (2007); they concluded that
ScFOS could improve the growth of beneficial bacteria in a certain
concentration but selectively. And the researches of Xu et al (2003);
Mahious et al (2006); Hui-yuan et al (2007) also concluded that the
bacterial population in small intestine can be affected by the
supplementation of FOS. Besides, bacterial growth in the research of Wang
and Gibson (1993) showed that oligofructose and inulin exerted a
preferential stimulatory effect on numbers of the health-promoting
genus Bifidobacterium, and maintaining populations of potential pathogens
(Escherichia coli, Clostridium) at relatively low levels. Although in this
research the health-promoting bacteria and potential pathogen bacteria were
not identified but Gibson and Roberfroid (1995) prebiotics is a non-
digestible food ingredient that beneficially affects the host by selectively
stimulating the growth and activity of one or a limited number of bacteria
in the colon (Gibson and Roberfroid, 1995). In the other hand, complex
oligosaccharides have the capacity of inhibiting the binding of pathogens to
cell surface because they act as competitive receptors (Rivero-Urgell,
Santamaria-Orleans, 2001). In addition, Science and Technology
Information Net (2010) found that if we added too much prebiotic into diet,
it could cause laxative; increase the gas-producing bacteria in digestive
12

system. Therefore, the result is indicated that the fish health could be

improved when supplemented at 0.5 and 1%.

Figure 4.1: Total bacterial count
4.3 Enzyme activity
4.3.1 α–Amylase activity in stomach and intestine of the fish
The amylase in stomach of fish activity in the beginning was 3.18±0.22
mU/min/mg protein and the concentration decreased in first day of
experiment 3.15±0.31 mU/min/mg protein at 2% of FOS-diet, but
generally, the amylase activity had the ascending trend with fish age. In the
beginning, the amylase activity was not significant different among
treatments (P>0.05). But the activity of α - amylase in fish’s stomach was
significantly increased 30 days after the FOS was administrated and peaked
at 0.5 and 1% of FOS diet after 90 days of experiment (6.37 and 6.74
mU/min/mg protein) (P<0.05) (Fig. 4.2). In the other hand, at 1.5 and 2%
of administrated FOS, the amylase activity was no significant different
compared to the control group. The results in accordance with that of the
research of Renjie et al (2010); they also had the similar results that
amylase activity in fish’s stomach applied FOS- diet was significantly
increased compared to control group.
0
2
4
6
8
0 1 3 7 10 30 60 90
log(CFU/g)
Days
control
0,5%
1,0%

1,5%
2,0%
13


Figure 4.2: α – Amylase activity in stomach of the fish
According Huong and Tu, 2010, amylase present mostly in the intestine of
omnivorous fish. Therefore, Amylase concentration in fish’s intestine of
this research is higher than in fish’s stomach. And similarly amylase in
fish’s stomach, the amylase in fish’s intestine was increased with fish age.
From day 0 to day 7, there were no significantly different among
treatments. And after 10 days fed with FOS administration, 0.5 and 1%
FOS treatment were significantly higher than control, 1.5 and 2% (4.13 and
4.35 mU/min/mg protein). In the other hand, after 90 days administrated,
amylase level is reached highest at 0.5 and 1% of FOS diet (14.2 and 14.3
mU/min/mg protein). Besides, applied 1.5 and 2% of FOS-diet, the amylase
activity were no significant different compared to the control group. The
results in accordance with that of Xu et al (2002, 2009) and Renjie et al
(2010)

Figure 4.3: α – Amylase activity in intestine of the fish
0
1
2
3
4
5
6
7
8

0 1 3 7 10 30 60 90
mU/min/mg protein
Days
control
0.50%
1%
1.50%
2%
0
2
4
6
8
10
12
14
16
0 1 3 7 10 30 60 90
mU/min/mg protein
Days
control
0.50%
1%
1.50%
2%
14

4.3.2 Pepsine activity in stomach of the fish
Pepsine actives in acid environment only, suitable pH for pepsine activity is
1.45 -3 (Huong and Tu, 2010). Striped catfish is omnivorous fish and they

have a real stomach also. Therefore, they have pepsine enzyme. Pepsine
activity in stomach of the fish was presented in Fig. 4.4. Generally, the
pepsine levels were augmented with fish age and after 3 days applied 0.5
and 1% FOS-diet, pepsine activities were significantly higher than those of
control, 1.5, 2% treatment (0.131±0.02 and 0.132±0.02 mU/mL/mg
protein), at that time, the lowest level of pepsine was 0.092±0.004
mU/mL/mg protein at 1.5% FOS-diet. 10 days post supplied 1% FOS-diet;
the pepsine levels reached higher level 0.191±0.008 mU/mL/mg protein
compared to other treatment. At the end of experiment, the highest levels of
pepsine activity were 0.337±0.0340 mU/mL/mg protein and 0.356±0.015
mU/mL/mg protein for 0.5 and 1% treatments, respectively.







Figure 4.4: Pepsine activity in stomach of the fish
4.3.3 Trypsine activity in intestine of the fish
No significant in trypsine activities was found within 10 days feeding of
FOS. However, after 30 days fish fed 0.5 and 1% FOS showed trypsine
significantly increased. The research of Xu et al (2003) also concluded that
application with 4 and 6kg FOS/kg significantly improved the trysine
activity in pig.
0
0.05
0.1
0.15
0.2

0.25
0.3
0.35
0.4
0 1 3 7 10 30 60 90
mU/mL/mg protein
Days
control
0.50%
1%
1.50%
2%
15


Figure 4.5: Trypsine activity in intestine of the fish
4.3.4 Chymotrypsine activity in intestine of the fish
Basically, chymotrypsine level showed the increasing trend with the
increase of fish age. However, after one day, the chemontrypsine level was
varying among treatments. But after that, the chymotrypsine concentration
was stable. And after 10 days of experiment 0.5 and 1% of FOS
administrated, chymotrypsine activity were significantly went up (325±21.1
and 330±3.30 mU/mL/min). In addition, at the end of experiment,
chymotrypsine activity got highest at 0.5 and 1% FOS diet (606±3.43 and
609±8.12 mU/mL/min).

Figure 4.6: Chymotrypsine activity in intestine of the fish
0
2
4

6
8
10
12
14
16
0 1 3 7 10 30 60 90
U/mg protein
Days
control
0.50%
1%
1.50%
2%
0
100
200
300
400
500
600
700
0 1 3 7 10 30 60 90
mU/mL/min
Days
control
0.50%
1%
1.50%
2%