12/24/2008 Research institute for Aquaculture No2 116 Nguyen Dinh Chieu, Dist 1, HCM city STUDY ON PRODUCING BACTERIAL DIGESTIVE ENZYME FOR USE IN STRIPED CATFISH FEED TO IMPROVE FEED UTILIZATION Nguyen Van Nguyen, Nguyen Bach Quynh Mai Phạm Duy Hai, Giap Văn Thang OUTLINE I INTRODUCTION II CONTENT III MATERIAL & METHOD IV RESULT & DISCUSSION V CONCLUSION (5 sections) 12/24/2008 I INTRODUCTION ™Currently, Striped catfish is one of the most important aquaculture species ™ Feed cost is a large component in striped catfish production cost ™Improving feed utilization is concerning to efficiency and sustainable aquaculture • Consequently, our general objective that improving feed utilization in striped catfish by addition of bacterial digestive enzymes II CONTENT To establish the scheme to collect the crude enzyme extract from internal organ of striped catfish Determine the digestive enzyme activities in each organ of the tract of fish which range from (50gr, 300gr and 600gr) Assessment of in vitro digestibility of inherent enzyme extract some ingredients and striped catfish feed Assessment of in vitro digestibility of inherent enzyme extract combined with external enzyme on some ingredients and Tra feed and determine the ratio of external digestive enzyme to add in striped catfish feed Produce the bacterial digestive enzymes including protease, alpha amylase, phytase and beta glucanase to add in striped catfish feed Research on in vitro digestibility of ingredients and striped catfish feed Evaluation of the bacterial digestive enzyme by striped catfish culture in tank and field study at intensive scale the bacterial digestive enzyme on some ( items) • 12/24/2008 Approach Diagram ingredi ents Striped catfish Commercial Protease commercial Alpha amilase commercial Phytase Commercial Beta glucanase Mixtu re A Crude enzyme y extract in vitro di digestib ib ivity Mixtu re B mixtu re C mixtu re D ferment ation Bacillus subtilis in vivo culture l Field farming Bac.Digest.E preparation Mixture of bac.digest E + crude E Extract Biomass Bi containg protease Strippe d catfish feed Bi Biomass containg alpha amilase ferment ation Bacillus subtilis Biomass Bi containing phytase Biomass Bi containing beta glucana ferment ation ferment ation Aspergillu s niger Trichoderm a viride III MATERIAL AND METHOD 1.Material: ‰Sampling:Tra catfish ranging from 50, 300 and d 600g 600 were collected ll t d ‰Compounded pellet feed were used for feeding only and routinely fed on times per day ‰ Tra catfish previously starved for 12 hours before digestive tract removed and dissected into stomach, intestine, liver and pancreas • 12/24/2008 2.METHOD 2.1 Determine the enzyme activities in the striped catfish digestive organs 2.1.1 PAGE Electrophoresis was used to characterize alpha amylase and presence of protease in the crude enzyme extract 2.1.2 Biochemical analysis to determine digestive enzyme activities: 2.1.Pepsin activity: EC.3.4.23.1 method 2.2 Alpha amylase activity: method of I.S.E.S.r.l Italian Company 2.3 Chymotrypsin activity: method of USP (United States Phamacopeia) Trypsin 2.4 i Activity: A i i method h d off USP S 2.2 in vitro Digestibility of Crude enzyme extract on some ingredients and feed 2.2.1 pH stat titration method (Dimes and Haard, 1994) 2.2.2 hydrolysis level of starch was determined by size of starch hydrolyzed circle VI.RESULT AND DISCUSSION The scheme to collect the crude digestive enzyme extract striped catfish Stomach Gut Pancreas Killed by Submersion in cold water Dissection Liver Intestine and stomach (mucous membrane separated) Pancreas, liver Homogenized by Foss machine, 10.000 rounds/minutes Centrifugation, 40C; 10.000 rounds/min , in 20 minutes Supernatant filled in eppendoff 1.5 ml Stored at  -300C Homogenized with glass powder According to Alarcon et al.,1998; Hidolgo et al.,1999, Sampling and process stage for collecting crude enzyme extract are two main factors effect to enzyme activity Following Glass et al (1989), Das and Tripathi (1991); Dimes et al (1994), the starved time of striped catfish relates with enzyme activity Some researches of Torrissen (1984); Das and Tripathi (1991); Dimes et al (1994) Chakrabarti et al (1995), they also mixed organs with glass powder and centrifuge with different speeds to collect Crude enzyme extract Contrary to them, Hofer and Schiemer (1981) Uys and Hecht, (1987) conducted to homogenize and filter for collecting the supernatant 12/24/2008 Electrophoresis to detect amylase 2.1 Characterization of amylase in pancreas and intestine I : zymogram II: zymoprotein I : zymogram 1 I : zymogram 2:3:cásize kích cỡ 300 g; 3: cá kích cỡ 600 g; of 600g 3 2 II: zymoprotein Ruột cá50g, tra;1: cá300g kíchand cỡ 50 g; Intestine, 1: size 2: size 2: cá kích cỡ 300 g; 3: cá kích cỡ 600 3: size of 600g Pancreas size 50g, sizecỡ 300g Tụy cá1:tra;1: cá 2: kích 50 and g; 2 II: zymoprotein g; I : zymogram Dạ dày cá50g, tra;1: cá kích stomach 1: size 2: size 300g cỡ and300 g; II: zymoprotein Livercá : tra size of 600g Gan kích cỡ 600 g; 2: cá kích cỡ 600 g; 3: cá kích cỡ 50 g; 3: size of 600g ¾ Amylase was detected in all organs of digestive tract ¾Striped catfish can digest with high levels of starch ¾There are many kind of amylase in the digestive tract 12/24/2008 2.2 Electrophoresis to detect protease I : zymogram y g II: zymoprotein y p 2: size cá kích cỡ 300 g; 3: cá kích cỡ 50 g; 3: of 600g 2 3 I : zymogram 1 2 I : zymogram II: zymoprotein Intestine, 1: size 50g, 2: size 300g and stomach size 50g, 2: 300g Dạ dày1:cá tra;1: cásize kích cỡ and 600 g; Ruột cá tra;1: cá kích cỡ 600 g; 3: size of 600g 2: cá kích cỡ 300 g; 3: cá kích cỡ 50 g; 3 II: zymoprotein Pancreas 50g,cá 2: kích size 300g andg; Tụy 1: cásize tra;1: cỡ 600 I : zymogram II: zymoprotein Gan cá tra cỡ600g 600 g; Liver : kích size of cá of kích cỡ 300 g; 3: cá kích cỡ 50 g; 3:2:size 600g ¾ Protease activity is different in each organ of digestive tract and highest activity in stomach 12/24/2008 ENZYME activities 3.1 α-amylase 140.00 UI/ml 50g 128.43 300g 600g 120.00 100.00 83.57 80.00 60.00 48.97 31.87 40.00 26.65 33.85 20.00 10.78 11.07 11.64 12.49 12.79 11.83 Stomach Intestine 0.00 Liver Pancreas ¾ No significant differences between alpha amylase in stomach and intestine at different sizes of fish ¾ alpha amylase activity in liver and pancreas increase correspondent with fish weight increase - Kuz’mina V.V , Golovanova I.L (2004) found α-amylase in pancreas and intestine of Samon it’s highest in pancreas - Uys Hecht (1987) studied on (clariid catfish),indicated that α-amylase was found in pancreas and intestine but highest activity in pancreas also 3.2 Pepsin activity 600g 126.97 mg protein/ ml 150 300g 50g 109.73 87.46 100 50 Stomach ¾ Pepsin activity was detected in stomach only and in pH of – 3.5 12/24/2008 3.3.Trypsin activity U/mg protein 150 141.03 118.1 112.41 111.03 91.13 100 70 93 70.93 50 Intestine Pancreas 600g 300g 50g ¾Tripsin acitivity was detected in pancreas and intestine Moreover, it’s increase correspondent with increasing in fish weight UI/mg protein 3.4 Chymotrypsin activity 30 20 10 21.6 17.53 8.67 15.29 76 6.76 Intestine 600g 12.17 Pancreas 300g 50g ¾ Chymotrypsin activity detected in pancreas and intestine also also In addition addition, it s increase correspondent it’s with fish weight increase Pepsin detected in stomach at optimal pH range from 2.5 – 3.5 and Trypsin Chymotrypsin showed in pancreas and intestine at pH range from 8-10 12/24/2008 TT In vitro digestibility of crude enzyme extract on some ingredients and feed DH% of protein Crude enzyme extract (CEE) CEE of 50gram CEE of 300 gram CEE of 600 gram ingredients Feed 1 Feed 21 Feed 31 Feed 41 Fish meal Soybean meal Feed Feed 22 Feed 32 Feed 42 Fish meal Soybean meal Feed Feed 23 Feed 33 F d 43 Feed Fish meal Soybean meal protein content (%) 33.5 31.4 30.6 29.8 54.6 38.4 26.7 26 26.4 26 54.6 38.4 22.3 21.5 20.9 21 21.7 54.6 38.4 DH% (statistical analysis) 59.29a ± 3.65 57.52a ± 4.08 58.16a ± 2.12 57.32a ± 3.48 79.21b ± 2.88 62.14c ± 3.71 55.87ad ± 2.48 55.31ad ± 3.31 55.24ad ± 4.91 55.12ad ± 2.99 77.42b ± 5.11 64.14c ± 4.08 54.35d ± 3.82 54.46d ± 2.50 54.22d ± 4.37 53 81d ± 3.82 53.81 82 77.87b ± 5.12 c 66.42 ± 4.82 No significant differences of DH% between kinds of feed in a size of fish ¾ ¾ Highest DH% in Fish meal detected Starch hydrolysis level item Crude Enzyme Extract (CEE) ingredient CEE of 50gram Feed 11 41.26 0.85 a ± 0.02 Feed 21 42.13 0.86 a ± 0.04 CEE of 300 gram CEE of 600 gram Cabonhydrate (%) Size of starch hydrolyzed circle (cm) Feed 31 42.87 0.82 a ± 0.03 Feed 41 42.07 0.83 a ± 0.03 Cassava 92.6 0.92b ± 0.05 0.68c ± 0.04 Rice bran 72.87 Feed 12 45.10 0.91b ± 0.02 Feed 22 46.80 0.90 b ± 0.03 Feed 32 45.11 0.90b ± 0.03 Feed 42 45.18 0.90 b ± 0.02 Cassava 92.6 1.12d ± 0.02 Rice bran 72.87 0.75e ± 0.03 Feed 13 51.17 0.97 f ± 0.02 Feed 23 54.72 0.96 f ± 0.01 Feed 33 53.42 0.96 f ± 0.02 Feed 43 52.39 0.97 f ± 0.01 Cassava 92.6 1.20g ± 0.03 Rice bran 72.87 0.79e ± 0.05 ¾No significant differences of starch hydrolysis between kinds of feed in a size of fish ¾Highest hydrolysis level of starch in cassava powder detected 12/24/2008 VI CONCLUSION Scheme to collect crude enzyme extract: The starved time of fish, sampling, glass powder used for sample homogenization and speed of centrifuge effect to the digestive enzyme activity of the organs Enzyme detection 2.1.Amylase was detected in all organs of digestive tract and Striped catfish can digest with hight starch level 2.2.Protease activity is different in each organ of digestive tract and highest activity in stomach Biochemical analysis: 3.1.No significant differences between alpha amylase in stomach and intestine at a sizes of fish and alpha amylase is highest activity in the liver (128.43UI/ml) 3.3.alpha amylase activity in liver and pancreas range from 26.65 – 128.43UI/ml and increase correspondent with fish weight increase (50g-600g) 3.4 Pepsin activity was detected in stomach only and in pH of – 3.5, range from 87- 126mg protein/ml p and Chymotrypsin y yp activity y was detected in 3.5 Tripsin pancreas and intestine at suitable pH range from 8-10 Moreover, the activity of these enzymes increase correspondent with increasing in fish weight 10 12/24/2008 In vitro digestibility of crude enzyme extract on some ingredients and feed 4.1 No significant differences of DH% (54- 57%) b (54 between t ki kinds d off ffeed d iin a size of fish 4.3 No significant differences of starch hydrolysis (0.8-0.9cm) (0 8-0 9cm) between kinds of feed in a size of fish THANK YOU 11 ... collected ll t d ‰Compounded pellet feed were used for feeding only and routinely fed on times per day ‰ Tra catfish previously starved for 12 hours before digestive tract removed and dissected into... most important aquaculture species ™ Feed cost is a large component in striped catfish production cost ™Improving feed utilization is concerning to efficiency and sustainable aquaculture • Consequently,... enzymes including protease, alpha amylase, phytase and beta glucanase to add in striped catfish feed Research on in vitro digestibility of ingredients and striped catfish feed Evaluation of the bacterial