Tài liệu RESEARCH INSTITUTE FOR AQUACULTURE NO2 pptx

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)

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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
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
Assessment of in vitro digestibility of inherent enzyme extract combined with
external enzyme on some ingredients and Tra feed and determine the ratio of

4.

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
ingredients and striped catfish feed

7.

Evaluation of the bacterial digestive enzyme by striped catfish culture in tank and
field study at intensive scale.

the bacterial digestive enzyme on some

( 7 items)
•

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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 3 times
per day.
‰ Tra catfish previously starved for 12
hours before digestive tract removed
and dissected into stomach, intestine,
liver and pancreas
•

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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).
2 4 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

1. 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) 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.

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2. Electrophoresis to detect amylase
2.1. Characterization of amylase in pancreas and intestine

1

2

1

3

I : zymogram

2

3

1

II: zymoprotein

I : zymogram

1

1

I : zymogram

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

3

3

2

2

3

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

2

3

II: zymoprotein

g;

1

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

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2.2. Electrophoresis to detect protease

1

2

3

1

I : zymogram
y g

2

3

1

II: zymoprotein
y p

2: size
cá kích
cỡ 300 g; 3: cá kích cỡ 50 g;
3:
of 600g

1

2

2

3

3

I : zymogram

1

1

2

2

3

1

I : zymogram

2

3

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;

1

2

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

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.

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3. 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 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

600g
126.97

mg

protein/
ml

150

300g

50g

109.73
87.46

100
50
0

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

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3.3.Trypsin activity

U/mg protein
150

141.03

118.1

112.41 111.03
91.13

100

70 93
70.93

50
0

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
0

21.6

17.53
8.67

15.29

6 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 và Chymotrypsin showed in
pancreas and intestine at pH range from 8-10.

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TT

4. In vitro digestibility of crude enzyme extract on some
ingredients and feed
DH% of protein

1

Crude enzyme extract
(CEE)
CEE of 50gram

2

CEE of 300 gram

3

CEE of 600 gram

ingredients

Feed 1 1
Feed 21
Feed 31
Feed 41
Fish meal
Soybean meal
Feed 1 2

Feed 22
Feed 32
Feed 42
Fish meal
Soybean meal
Feed 1 3
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 7
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
3 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.

5. Starch hydrolysis level
item

Crude Enzyme Extract
(CEE)

ingredient

1

CEE of 50gram

Feed 11

41.26

0.85 a ± 0.02

Feed 21

42.13

0.86 a ± 0.04

2

3

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

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VI. CONCLUSION
1. 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.
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. 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
2 – 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.

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4. 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

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