Effect of fructo-oligosaccharides (fos) on digestive enzymes of striped catfish (pangasianodon hypophthalmus)

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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 Assoc. Prof. DO THI THANH HUONG Sign of Student 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 i 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. ii 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 iii 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 iv 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 v 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 vi 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 vii 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/m2 (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 broadspectrum 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. 1 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 22-26oC, and optimal pH is 6.57.5 (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 2 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) 3 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 4 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) 5 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 (0.5m3/tank), spectrometer, incubator, auto clave, petri disk, test-tube, cuvet, etc. - Chemicals: NaCl, KH2PO4, NaH2PO4, 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/0.5m3. 6 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 handfed to saturation twice per day. Table 3.1: Composition of diets (Nafa label with 30% crude protein) Ingredients % Crude Protein 30 Crude Lipid 5 Crude Fiber 7 Moisture 11 Ash 12 Phosphorus 1 Energy (Kcal/kg) 2.400 Water quality: pH, temperature, oxygen were measured 2times/day. TAN was measured by Indophenol Blueand method and NO2- 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 1520mL of PCA medium. Then mix the sample and medium together by clockwise or anticlockwise rotation and incubate at 37oC in 48 or 72 hours. 7 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 ni: number of disks at concentration i V: volume of sample implant for one disk fi: 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 80oC 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 40C 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) 8 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. 9 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 2oC among the treatments (27.5±0.95 in the morning and 29.3±1.50oC 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 22-26oC and pH is from 6.57.5, hence the temperature and pH were ideal for fish growth. Table 4.1: Temperature, DO, pH measured in the morning and afternoon Temperature (oC) DO (mg/L) Morning Afternoon Morning Afternoon Control 27.6±1.23 29.1±1.12 5.80±0.97 6.47±1.24 0.5% 27.6±1.84 29.3±1.50 5.97±0.93 6.52±1.11 1.0% 27.5±1.19 29.2±1.47 5.88±0.95 6.54±1.27 1.5% 27.6±1.23 29.2±1.44 5.81±0.97 6.59±1.34 2.0% 27.5±0.95 28.5±1.09 5.82±1.10 6.90±3.91 Note: Values are presented as mean± STD pH Morning Afternoon 7.00±0.52 7.55±0.66 7.11±0.51 7.57±0.60 7.01±0.44 7.55±0.60 7.08±0.55 7.60±0.54 7.07±0.55 7.66±0.64 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 NO2Treatment Control 0.5% 1.0% 1.5% 2.0% TAN (mg/L) NO2-(mg/L) 0.40±0.22 0.39±0.23 0.43±0.26 0.39±0.25 0.43±0.27 0.38±0.21 0.42±0.22 0.38±0.24 0.36±0.23 0.37±0.18 Note: Values are presented as mean± STD 10
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