Tài liệu RESEARCH INSTITUTE FOR AQUACULTURE NO2 pptx

STUDY ON PRODUCING BACTERIAL Research institute for Aquaculture No2 116 Nguyen Dinh Chieu, Dist 1, HCM city STUDY ON PRODUCING BACTERIAL DIGESTIVE ENZYME FOR USE IN STRIPED CATFISH FEE

STUDY ON PRODUCING BACTERIAL

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 Bach Quynh Mai Phạm Duy Hai, Giap Văn Thang

OUTLINE

II CONTENT

III MATERIAL & METHOD

IV RESULT & DISCUSSION

V CONCLUSION

(5 sections)

I INTRODUCTION

™ Currently, Striped catfish is one of the most

important aquaculture species.

™ Feed cost is a large component in striped

™ 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

1 To establish the scheme to collect the crude enzyme extract from internal organ

of striped catfish.

2 Determine the digestive enzyme activities in each organ of the tract of fish

which range from (50gr, 300gr and 600gr).

3 Assessment of in vitro digestibility of inherent enzyme extract some ingredients

and striped catfish feed

4 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

5 Produce the bacterial digestive enzymes including protease, alpha amylase, phytase

and beta glucanase to add in striped catfish feed.

6. Research on in vitro digestibility of the bacterial digestive enzyme on some

ingredients and striped catfish feed

7 Evaluation of the bacterial digestive enzyme by striped catfish culture in tank and

field study at intensive scale.

( 7 items)

•

Approach Diagram

Striped catfish

ingredi ents

Strippe

d catfish feed

in vitro

di ib in vivol

Field farming

Crude enzyme

Commercial

Protease

Mixtu

re A

Bi

commercial

Alpha amilase

commercial

Phytase

ivity

digestib

farming

y extract

Mixture of bac.digest E + crude E Extract

preparation

Bac.Digest.E preparation

Mixtu

re B

mixtu

re C

Biomass containg protease

Commercial

Beta

glucanase

Biomass containg alpha amilase

Biomass containing phytase

Biomass containing beta glucana

ferment ation ferment ation

ferment ation

ferment ation

Bacillus subtilis Bacillus subtilis Aspergillu s niger Trichoderm a viride

mixtu

re D

III

III MATERIAL AND METHOD MATERIAL AND METHOD

1.Material:

‰ Sampling : Tra catfish ranging from 50,

300 and 600g were collected.

‰ Compounded pellet feed were used for

feeding only and routinely fed on 3 times

per day.

‰ Tra catfish previously starved for 12

hours before digestive tract removed

and dissected into stomach, intestine,

liver and pancreas

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).

2.4 Trypsin Activity: method of USP.

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

striped catfish

Killed by Submersion in cold water

1 The scheme to collect the

crude digestive enzyme

extract

Stomach Gut

Pancreas

Dissection

membrane separated)

Homogenized by Foss machine, 10.000

rounds/minutes

Homogenized with glass powder

Liver

Centrifugation, 4 0 C;

10.000 rounds/min , in 20 minutes

Supernatant filled in eppendoff 1.5 ml Stored at  -30 0 C

According to Alarcon et al.,1998; Hidolgo et al.,1999, Sampling 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) và 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) và Uys and Hecht, (1987) conducted to homogenize and filter for collecting the supernatant.

2

2 Electrophoresis to detect amylase Electrophoresis to detect amylase

1 2 3

2.1 Characterization of amylase in pancreas and intestine

1 2 3

1 2 3 1 2 3 1 2 3

Tụy cá tra;1: cá kích cỡ 50 g;

2: cá kích cỡ 300 g; 3: cá kích cỡ 600 g;

Ruột cá tra;1: cá kích cỡ 50 g;

2: cá kích cỡ 300 g; 3: cá kích cỡ 600 g;

I : zymogram II: zymoprotein

I : zymogram II: zymoprotein

Pancreas 1: size 50g, 2: size 300g and

3: size of 600g

Intestine, 1: size 50g, 2: size 300g and 3: size of 600g

2 3 1 2 3 1

I : zymogram II: zymoprotein

Dạ dày cá tra;1: cá kích cỡ 300 g;

2: cá kích cỡ 600 g; 3: cá kích cỡ 50 g;

I : zymogram II: zymoprotein Gan cá tra kích cỡ 600 g;

stomach 1: size 50g, 2: size 300g and

3: size of 600g

Liver : size of 600g

¾Amylase 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

1 2 3 1 2 3 1 2 3 1 2 3

I : zymogram II: zymoprotein y g y p I : zymogram II: zymoprotein

Dạ dày cá tra;1: cá kích cỡ 600 g;

2: cá kích cỡ 300 g; 3: cá kích cỡ 50 g;

I : zymogram II: zymoprotein

Ruột cá tra;1: cá kích cỡ 600 g;

2: cá kích cỡ 300 g; 3: cá kích cỡ 50 g;

stomach 1: size 50g, 2: size 300g and

3: size of 600g

Intestine, 1: size 50g, 2: size 300g and 3: size of 600g

1 2 3 1 2 3

1 2 3 1 2 3

Tụy cá tra;1: cá kích cỡ 600 g;

2: cá kích cỡ 300 g; 3: cá kích cỡ 50 g;

I : zymogram II: zymoprotein I : zymogram II: zymoprotein

Gan cá tra kích cỡ 600 g;

Pancreas 1: size 50g, 2: size 300g and

3: size of 600g

Liver : size of 600g

¾ Protease activity is different in each organ of digestive tract and

highest activity in stomach

3

3 ENZYME activities ENZYME activities

3.1 α-amylase

128.43

83.57

80.00

100.00

120.00

140.00 UI/ml 50g 300g 600g

31.87 26.65

10.78 12.49

48.97

33.85

11.0711.64 12.7911.83

0.00

20.00

40.00

60.00

Liver Pancreas Stomach Intestine

¾No 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 và 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

0 50 100 150

126.97

109.73

87.46

600g 300g 50g

mg protein/ ml

Stomach

¾Pepsin activity was detected in stomach only and in pH of 2 – 3.5

3.3.Trypsin activity

141.03

91.13

112.41

70 93

111.03 U/mg protein

0

50

100

70.93

600g 300g 50g

¾Tripsin acitivity was detected in pancreas and

intestine Moreover, it’s increase correspondent

with increasing in fish weight.

3.4 Chymotrypsin activity

20

30 17.53 21.6

0 10 20

6.76

600g 300g 50g

¾Chymotrypsin activity detected in pancreas and

intestine also In addition it’s increase correspondent

with fish weight increase

Pepsin detected in stomach at optimal pH range from 2.5 – 3.5 and Trypsin và Chymotrypsin showed in

pancreas and intestine at pH range from 8-10

4 In vitro digestibility of crude enzyme extract on some

ingredients and feed

DH% of protein

TT Crude enzyme extract

(CEE)

ingredients protein content

(%)

DH% (statistical analysis)

1 CEE of 50gram Feed 1 1 33.5 59.29 a ± 3.65

Feed 21 31.4 57.52 a ± 4.08 Feed 31 30.6 58.16 a ± 2.12 Feed 41 29.8 57.32 a ± 3.48 Fish meal 54.6 79.21 b ± 2.88 Fish meal 54.6 79.21 ± 2.88 Soybean meal 38.4 62.14 c ± 3.71

2 CEE of 300 gram Feed 1 2 26.7 55.87 ad ± 2.48

Feed 22 26 55.31 ad ± 3.31 Feed 32 26.4 55.24 ad ± 4.91 Feed 42 26 55.12 ad ± 2.99 Fish meal 54.6 77.42 b ± 5.11 Soybean meal 38.4 64.14 c ± 4.08

3 CEE of 600 gram Feed 1 3 22.3 54.35 d ± 3.82

Feed 23 21.5 54.46 d ± 2.50 Feed 33 20.9 54.22 d ± 4.37

F d 43 21 7 53 81 d 3 82 Feed 43 21.7 53.81 d ± 3.82 Fish meal 54.6 77.87 b ± 5.12 Soybean meal 38.4 66.42 c ± 4.82

kinds of feed in a size of fish

¾ Highest DH% in Fish meal detected.

5 Starch hydrolysis level

item Crude Enzyme Extract

(CEE)

ingredient Cabonhydrate (%) Size of starch hydrolyzed circle

(cm)

1 CEE of 50gram Feed 11 41.26 0.85 a ± 0.02

Feed 21 42.13 0.86 a ± 0.04 Feed 31 42.87 0.82 a ± 0.03 Feed 41 42.07 0.83 a ± 0.03 Cassava 92.6 0.92 b ± 0.05 Rice bran 72.87 0.68 c ± 0.04

2 CEE of 300 gram Feed 12 45.10 0.91 b ± 0.02

Feed 22 46.80 0.90 b ± 0.03 Feed 32 45.11 0.90 b ± 0.03 Feed 42 45.18 0.90 b ± 0.02 Cassava 92.6 1.12 d ± 0.02 Rice bran 72.87 0.75 e ± 0.03

3 CEE of 600 gram 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 1.20 g ± 0.03 Rice bran 72.87 0.79 e ± 0.05

VI CONCLUSION

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.

2 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

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

2 – 3.5, range from 87- 126mg protein/ml.

3.5 Tripsin and Chymotrypsin activity was detected in p y yp y

pancreas and intestine at suitable pH range from 8-10

Moreover, the activity of these enzymes increase

correspondent with increasing in fish weight.

4 In vitro digestibility of crude enzyme

extract on some ingredients and feed

(54- 57%) between kinds of feed in

a size of fish

4.3 No significant differences of

starch hydrolysis (0 8-0 9cm)

between kinds of feed in a size of

fish.

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