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Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan 731 235, West Bengal, India
Microbiology Laboratory, Department of Botany, Visva-Bharati University, Santiniketan 731 235, West Bengal, India
Received 29 December 2001; received in revised form 20 February 2002; accepted 12 March 2002
Abstract
Eight isonitrogenous (35% crude protein approximately) and isocaloric (4.2 kcal g1 approximately) diets were formulated including raw and fermented duckweed (Lemna polyrhiza) leaf meal at 10%, 20%, 30% and 40% levels. A particular bacterial strain
(Bacillus sp.) isolated from carp (Cyprinus carpio) intestine and having extracellular amylolytic, cellulolytic, proteolytic and lipolytic
activities was used for leaf meal fermentation for 15 days at 37 C. The bre content of leaf meal reduced from 11.0% to 7.5% and
the antinutritional factors, tannin and phytic acid, were reduced from 1.0% to 0.02% and 1.23% to 0.09%, respectively after fermentation. However, the available reducing sugars, free amino acids and fatty acids increased in the fermented leaf meal. The
response of rohu, Labeo rohita, ngerlings fed the experimental diets for 80 days was compared with sh fed a sh meal based
reference diet. On the basis of growth response, food conversion ratio and protein eciency ratio, 30% fermented Lemna leaf meal
incorporated in the diet resulted in the best performance of rohu ngerlings. In general, growth and feed utilization eciencies of
sh fed fermented leaf meal containing diets were superior to those fed diets containing raw leaf meal. The apparent protein digestibility (APD) decreased with increasing levels of leaf meal irrespective of treatment. The APD for raw leaf meal was lower at all
levels of inclusion in comparison to those for the fermented meals. The highest carcass protein and lipid deposition was recorded in
sh fed the diet containing 30% fermented leaf meal. The results showed that fermented Lemna leaf meal can be incorporated into
carp diets up to 30% level compared to 10% level of raw meal. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Lemna polyrhiza; Intestinal bacteria; Fermentation; Diets; Growth performance; Labeo rohita ngerlings
1. Introduction
Considering the importance of nutritionally balanced
and cost-eective articial diets for sh, there is an increasing research eort to evaluate the nutritive value
of dierent non-conventional feed resources, including terrestrial and aquatic macrophytes (Edwards et al.,
1985; Wee and Wang, 1987; Patra and Ray, 1988; Ray
and Das, 1992, 1993, 1995; Mondal and Ray, 1999).
Aquatic and terrestrial macrophytes have been used as
supplementary feeds in sh farming since the early times
of freshwater sh culture (Bardach et al., 1972) and still
play an important role as sh feed in extensive culture systems (Edwards, 1987). The aquatic weeds have
been shown to contain substantial amounts of protein
Corresponding author.
0960-8524/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 6 7 - 6
18
2. Methods
2.1. Isolation and characterization of sh intestinal
bacteria
The bacterial strain used for fermentation of Lemna
leaf was isolated from the intestine of common carp,
Cyprinus carpio, on sterilized carboxymethylcellulose
(CMC)-agar medium (pH 7) containing (g l1 ), CMC,
10; KH2 PO4 , 4; Na2 HPO4 , 4; MgSO4 7H2 O, 0.2; CaCl2 ,
0.001; FeSO4 7H2 O, 0.004; Agar, 15. CMC-ase producing isolates were identied on CMC plates after
ooding the plates with 5 ml of Congo red dye prepared
in 0.7% agarose (Seakem HGT agarose) essentially
according to the method of Teather and Wood (1982).
The bacterial isolate was further screened for the production of extracellular amylase (Bernfeld, 1955), cellulase (Denison and Koehn, 1977), protease (Walter, 1984)
and lipase (Colowick and Kaplan, 1955). The strain was
identied and characterized by carrying out the tests
described in the Mannual of Microbiological Methods
(Society of American Bacteriologists, 1957).
2.2. Preparation of bacterial seed culture
The selected bacterium was grown in shake bottles in
4% tryptone soya broth (Hi-media, India) for seed culture. After 24 h of growth at 37 C, an average viable
count was about 107 cells/ml of broth. This was used as
bacterial seed for Lemna leaf fermentation.
19
3. Results
The proximate compositions of feed ingredients and
experimental diets are presented in Tables 1 and 2, respectively. Fermentation of Lemna leaf meal resulted in a
signicant decrease in the levels of crude bre and antinutritional factors, tannin and phytic acid, whereas,
there was increase in the levels of free amino acids and
fatty acids. A comparison of the proximate composition
of the reference diet, and Lemna leaf meal incorporated
diets indicated that the crude bre level in the control diet
was 11.80%, whereas, it ranged from 7.87.9% in diets
containing raw Lemna leaf meal, and reduced to 3.8
5.8% in diets containing fermented Lemna leaf meal. In
diets containing fermented Lemna leaf meal the tannin
and phytic acid contents were below detection limit.
The growth performance and feed utilization of L.
rohita ngerlings in terms of percentage weight gain,
SGR, FCR, PER, ANPU and APD are presented in
Table 3. The average weight of the sh increased in all
the dietary treatments, in comparison to the reference
diet excepting in diet D4, which contained 40% raw
Lemna leaf meal. The highest attainment in sh body
weight, average percentage live weight gain and SGR
were recorded in the group of sh reared on diet D7
Table 1
Proximate compositions of feed ingredients (% dry matter basis)
Nutrients
Fish meal
Mustard oilcake
Rice bran
Moisture
Dry matter
Crude protein
Crude lipid
Ash
Crude bre
NFE
Gross energy
(kcal g1 )
Tannin
Phytic acid
Total free amino
acid
Total free fatty acid
2.26
97.04
58.50
8.91
11.50
3.93
14.20
4.19
14.00
86.00
35.95
7.00
8.37
5.53
29.17
4.11
4.45
95.55
13.00
5.14
21.41
25.50
30.50
3.52
32.50
67.50
18.60
1.50
2.50
11.00
83.90
5.04
11.38
1.00
7.50
1.00
1.23
0.32
0.02
0.09
0.95
2.0
6.0
20
Table 2
Ingredient composition (% dry weight) and proximate analyses of experimental diets (on dry matter basis)
RD
Ingredients
Fish meal
Mustard oilcake
Rice bran
Lemna meal
Premixa
Chromic oxide
Proximate composition
Dry matter
Crude protein
Crude lipid
Ash
Crude bre
NFEc
Gross energy
(kcal g1 )
Tannins
Phytic acid
Chromic oxide
Fermented
D1
D2
D3
D4
D5
D6
D7
D8
40.0
23.0
35.0
1.0
1.0
36.0
25.0
27.0
10.0
1.0
1.0
34.0
24.0
20.0
20.0
1.0
1.0
32.0
24.0
12.0
30.0
1.0
1.0
30.0
28.0
40.0
1.0
1.0
36.0
25.0
27.0
10.0
1.0
1.0
34.0
24.0
20.0
20.0
1.0
1.0
32.0
24.0
12.0
30.0
1.0
1.0
30.0
28.0
40.0
1.0
1.0
(%)b
98.0
35.95
8.50
10.0
11.80
31.50
4.6
94.0
35.41
8.50
12.0
7.80
30.89
4.2
98.0
34.93
8.50
14.0
7.90
32.83
4.3
98.0
34.88
8.0
14.0
7.80
32.80
4.4
98.0
35.26
8.50
20.0
7.90
26.20
4.2
96.0
34.68
5.0
12.0
3.80
50.70
4.1
98.0
34.58
5.5
16.0
4.00
51.50
3.9
94.0
33.29
6.50
16.0
4.80
38.72
3.9
96.0
32.97
6.0
20.0
5.80
35.91
3.9
ND
ND
0.97
0.1
0.12
0.92
0.2
0.24
0.95
0.3
0.36
0.97
0.4
0.48
1.0
ND
ND
1.01
ND
ND
1.02
ND
ND
1.01
ND
ND
1.03
ND Not detectable.
a
Vitamin and mineral mixture (Vitaminetes Forte, Roche Products Ltd, 24/28 Pt. M.M. Malaviya Road, Mumbai 400 034, India).
b
Number of samples for each determination 3.
c
Nitrogen-free extract.
4. Discussion
It is evident from the present study that fermented
duckweed meal can be utilized as a feed ingredient in
the diets for the rohu, L. rohita ngerlings eectively up
to 30% level of incorporation without compromising
growth. Growth performance, PER and FCR of rohu
ngerlings were better with 40% fermented Lemna leaf
meal incorporated diet than in the reference diet. Das
and Ray (1989) demonstrated the possibility of incorporation of dried Lemna polyrhiza as a feed ingredient
for L. rohita ngerlings, and recorded higher carbohydrate digestibility in relation to that of the control diet
although the SGR and percent weight gain in sh fed
Lemna meal were slightly lower than with the control
diet. Shireman and Smith (1983) considered duckweed
(Lemna sp.) as a highly nutritious vegetable food for
grass carp because of its tenderness and high protein
content compared to other aquatic weeds. In the present
investigation, the protein content of Lemna polyrhiza
was estimated to be 18.60%. However, the presence of
antinutritional factors limits direct use of the weed as a
Table 3
Growth, feed utilization eciencies and APD in L. rohita ngerling fed experimental diets for 80 days. Data are mean values SE (n 5)
Diets with Lemna leaf meal diet
RD
D1
D2
D3
D4
D5
D6
D7
D8
6.4 0.31
11.02 0.29a
72.18 0.12b
1.65
6.4 0.32
11.22 0.31a
75.31 0.16e
1.43
6.4 0.31
11.20 0.33a
75.00 0.14d
1.48
6.4 0.34
11.18 0.28a
74.78 0.10c
1.52
6.4 0.33
10.80 0.34a
68.75 0.18a
1.52
6.4 0.33
11.72 0.32b
83.12 0.11g
1.54
6.4 0.32
11.89 0.30b
85.78 0.18h
1.47
6.4 0.31
11.93 0.31b
86.50 0.16i
1.48
6.4 0.34
11.54 0.33b
80.31 0.15j
1.47
0.905 0.03a
3.17 0.10c
0.87 0.06a
11.47 0.50c
89.46 4.25b
0.935 0.03b
2.68 0.19ab
1.05 0.05b
4.62 0.21b
86.78 4.24a
0.931 0.02b
2.79 0.06b
1.03 0.06b
2.58 0.12a
90.30 4.22b
0.931 0.04b
2.87 0.15b
1.00 0.04a
37.60 1.08f
86.20 4.22a
0.876 0.02a
3.01 0.13c
0.94 0.04a
31.89 1.50e
82.30 3.21a
1.00 0.03c
2.73 0.09ab
1.05 0.05b
26.34 1.30d
94.43 4.24b
1.031 0.02c
2.56 0.11a
1.13 0.05b
72.87 3.16g
93.90 4.21b
1.038 0.03c
2.55 0.12a
1.18 0.06b
98.71 4.52h
90.20 4.19b
0.981 0.04b
2.66 0.15ab
1.14 0.06b
5.58 0.25b
85.60 4.06a
Means with same superscripts in the same row were not signicantly dierent (P < 0:05).
BW: body weight.
SGR ln final weight ln initial weight=Days on trial 100; PER Wet weight gain of the fish=Protein consumed; FCR Dry weight of the feed given=Increase in wet weight of
fish; ANPU Net increase in carcass protein=Amount of protein consumed 100; APD 100 100 %Cr2 O3 in diet=%Cr2 O3 in faeces %protein in faeces=%protein in diet.
1
Statistical analysis was not possible as determinations were performed on pooled samples.
Table 4
Proximate carcass compositions (% wet weight) of the experimental sh at the start and end of the 80 day feeding experiment. Data are mean values SE (n 5)
Carcass composition
Initial
RD
Diets
D1
D2
D3
D4
D5
D6
D7
D8
Moisture
Crude protein
Crude lipid
Ash
76.31
12.90
2.26
4.96
75.27 0.17c
13.50 0.58a
2.66 0.04c
3.23 0.06a
75.19 0.13c
13.11 0.40a
2.16 0.08b
3.40 0.09ab
74.80 0.15b
13.02 0.48a
2.03 0.07a
3.68 0.11b
76.06 0.15ef
14.69 0.50b
1.83 0.12a
3.49 0.20b
75.60 0.09d
14.38 0.52b
1.83 0.18a
3.19 0.26a
75.86 0.10e
14.22 0.45b
2.23 0.11b
4.20 0.25c
73.23 0.16a
16.42 0.60c
2.30 0.13b
5.03 0.09d
75.93 0.13e
17.52 0.65d
2.70 0.09c
5.00 0.14d
76.18 0.14f
13.15 0.42a
2.16 0.15b
4.24 0.09c
Mean values
Means with same superscripts in the same row were not signicantly dierent (P < 0:05).
21
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5. Conclusion
It was concluded from the present study that fermented Lemna leaf meal can be recommended as a dietary ingredient in diets of L. rohita ngerlings up to
30% incorporation level without any adverse eect on
growth of the sh. The growth and feed utilization efciencies of sh fed diets incorporating fermented
Lemna leaf meal were better than those fed raw Lemna
leaf meal at the same level of inclusion.
Acknowledgements
We are grateful to the Indian Council of Agricultural
Research, New Delhi (project F. no. 4 (11)/98-ASR-I)
and University Grants Commission, New Delhi (DSA
Programme to the Department of Zoology) for nancial
support.
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