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

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

Assoc Prof DO THI THANH HUONG NGUYEN KHANH LINH

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

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

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

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

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

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

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

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

- Evaluated the total bacteria in the intestine of striped catfish fed with different doses of FOS

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

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

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)

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

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

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,

digestive enzymes such as protease, trypsin and amylase in the small intestine Besides, another study on male broilers had similar results also

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

concluded that dietary FOS had beneficial effects on growth, FCR, and

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

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

the control Besides, amylase activity in the intestine was significantly

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

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)

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

disk, test-tube, cuvet, 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

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)

Crude Protein Crude Lipid Crude Fiber Moisture Ash Phosphorus Energy (Kcal/kg)

Water quality: pH, temperature, oxygen were measured 2times/day TAN

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 20mL of PCA medium Then mix the sample and medium together by

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=

N: total colonies counted in disks

V: volume of sample implant for one disk

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

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)

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