Chapter 5
7. PROFILES OF PROTEIN, AMINO ACIDS AND FATTY ACIDS
7.1. Introduction
Proteins are large, complex, organic compounds made up of amino acids.
Amino acids are the building blocks of proteins and serve as body builders. They are
utilized to form various cell structures as key components and serves as source of energy
(Babsky et al., 1989). In addition, the amino acid composition and concentration in the
muscle of prawns may affect the quality of the prawn (Wang et al., 2004).
Amino acids are precursors of proteins and also act as an energy source.
Deficiencies or excess of one or more EAA limit protein synthesis and growth or both
(Litaay et al., 2001). Terrestrial and aquatic animals require dietary amino acids for
metabolic purposes and growth. One of the major purposes of amino acids is as
building blocks for body protein synthesis (e.g. building muscle, organs and functional
proteins such as enzymes, hormones, or immunoglobulins). Some of the amino acids
such as lysine, methionine, threonine, tryptophan, arginine, valine, isoleucine, leucine,
histidine and phenylalanine are considered essential because they cannot be synthesized
by the animal and need therefore to be provided with feed. In addition, some amino acids
are required not only as building block but have other metabolic functions in addition to
building protein. For example, methionine has a central role as methyl group (CH3)
donator (Lemme, 2010). The optimal dietary amino acid profile will depend on the amino
acid requirement of an animal for protein synthesis and the use of individual
amino acids as energy substrates or for other purposes (Ronnestad and Fyhn, 1993).
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Deshimaru and Shigeno (1972) and Ogata et al. (1985) suggested that the amino acid
composition of the food should be very similar to that of the animal’s proteins.
The essential fatty acid (EFA) requirements of freshwater and marine fish species
have been extensively studied over the past 20 years and are known to vary both
qualitatively and quantitatively (Sargent et al., 1989, 1995 and 2002). Lipids are regarded
as the most important energy source in animal tissues, generally stored as triacylglycerols, in
depot organs or adipose tissue. The polyunsaturated fatty acids (PUFA) of the linoleic (n-6)
and linolenic (n-3) families have been recognized as important nutrients for growth and
reproduction in fish (Sargent et al., 1999; Izquierdo et al., 2000), crustaceans (Sheen and
Wu, 1999; Jeffs et al., 2002) and molluscs (Caers et al., 2000; Navarro and Villanueva, 2000;
Nelson et al., 2002, Durazo-Beltran et al., 2003). All terrestrial and aquatic organisms are
able to synthesize unsaturated fatty acids of the n-9 family de novo (Cook, 1996).
However, fatty acids from n-3 and/or n-6 series are synthesized de novo only by
photosynthetic organisms and insects. Some aquatic species can elongate and
desaturate
dietary
18:2n-6
or 18:3n-3 to satisfy or partially contribute to their
nutritional requirements for highly unsaturated fatty acids (HUFAs) like 20:4n-6,
20:5n-3 and 22:6n-3, and this biosynthetic ability varies from species to species
(Sargent et al., 1995; Buzzi et al., 1996). The work in the present chapter was conducted
for analysing the profiles of essential amino acids, fatty acids and protein in 50% of S.
platensis, C. vulgaris and A. pinnata incorporated feeds and these feeds fed
M. rosenbergii PL groups.
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7.2. Materials and Methods
Feeding experiment
Macrobrachium rosenbergii (PL-30) with the length and weight range of 1.56 ±
0.29 cm and 0.22 ± 0.039 g respectively were used for feeding experiment. Thirty PL for
each diet in triplicate were maintained in plastic tanks with 40 L water. One group served
as control. The experimental groups were fed with the respective concentration of 50% of
FM replaced with 50% of S. platensis, C. vulgaris and A. pinnata incorporated diets.
The feeding was adjusted to two times a day (6:00 am and 6:00 pm). The daily ration was
given at the rate of 10% of the body weight of PL with two equal half throughout the
experimental period. The feeding experiment was prolonged for 90 days; mild aeration
was given continuously in order to maintain the optimal oxygen level.
Analysis of the profiles of protein
The tissue samples, first defrosted in homogenization phosphate buffer (137 mM
Nacl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 and pH-7.4) at 4ºC, were
homogenized, and then centrifuged at 1500 rpm at 5 min. The protein content was
determined in supernatant by Lowery et al. (1951).
Total proteins were sepaprated in denaturing polyacyrl amide gel according to
Laemmli, (1970). The gels were stained with Coomassie blue G-250 (9% acetic acid, 45%
methanol, 0.1% Coomassie blue G-250). The molecular weight marker contained six bands
known proteins like β-Galactosidase, E. coli (116 kDa), Bovine serum albumin (66 kDa),
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ovalbumin (45 kDa), carbonic anhydrase (29 kDa), soyabean Trypsin Inhibitor (20 kDa)
and Lysozyme, chicken egg white (14 kDa). The patterns were compared by using
information on apparent molecular masses of bands and their intensity.
Profile of Amino acid
The profiles of amino acids were done following high performance thin layer
chromatographic (HPTLC) method (Hess and Sherma, 2004). The prawns were dried
(80ºC for 3 h), digested with 6 M aqueous hydrochloric acid and dried under vacuum.
The powdered sample was dissolved in distilled water and 5 µl of sample was loaded on
8 mm thick pre-coated Silica gel 60F254 TLC plate (20 cm × 15 cm) and processed in
CAMAG-LINOMAT 5 instrument. The plate was developed in butane-AmmoniaPyridine-Water (3.9:1:3.4:2.6) mobile phase. The plate was sprayed with ninhydrin
reagent prepared in propan-2-ol and dried. The developed plate was documented using
photo-documentation chamber (CAMAG-REPROSTAR 3) at UV 254 nm and UV 366 nm
lights. Finally, the plate was scanned at 500 nm using CAMAG-TLC SCANNER 3.
The peak area of the samples were compared with standard amino acids and quantified.
All the twenty standard amino acids were classified into following four groups based on
their Rf values to avoid merging of individual amino acids while elution. These are
Group-1: asparagin, glutamine, serine, proline and metheonine; Group-2: aspartic acid,
glutamic acid, alanine, valine and phenyl alanine; Group-3: lysine, glycine, threonine,
tyrosine and isoleucine; Group-4: histidine, argentine, cystine, tryphtophan and leucine
(Plate 5.6). Each group consisted of 1 mg of each 5 amino acids dissolved with 5 ml
distilled water.
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Profile of Fatty acid
The profile of fatty acids was done following Gas Chromatography (GC) method
(Nichols et al., 1993). Fatty acids were obtained from lipids by saponification using
NaOH dissolved in methanol H2O mixture (hydrolysis with alkali). They were then
methylated into fatty acid methyl ester using HCl and methanol mixture, which can be
easily identified by GC. The fatty acid methyl ester was separated using mixture of
hexane and anhydrous diethyl ether. For the organic phase aqueous NaOH was used as
base wash and the upper organic layer was separated. Two µl of sample was injected and
analyzed using Chemito 8610 Gas chromatography, with BPX70 capillary column and
flame ionization detector. Nitrogen was used as carrier gas. The chromatogram was used
for calculation. Standard fatty acids were analyzed simultaneously. Based on the
retention time and peak area of the standard fatty acids, each fatty acid in the unknown
sample was identified.
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7.3. Results
Profiles of protein in formulated feed fed M. rosenbergii PL
Extracted protein samples of prawns fed with S. platensis (50%), C. vulgaris
(50%), A. pinnata (50%) included feed and control feed were examined by SDS-PAGE
for separation and analysis of various protein bands as shown in the Plate 5.1.
The present protein separation study showed polypeptides between 59 kDa to 14 kDa.
A total of ten polypeptide bands were observed in the groups fed with C. vulgaris and
S. platensis whereas only a nine polypeptide bands were observed in the groups fed with
A. pinnata and control diet. The intensity of the polypeptide bands of C. vulgaris and
S. platensis was found to be almost similar in experimental groups, the control group
showed low intensity of polypeptide bands.
Amino acid profile in formulated feed
Fourteen amino acids were detected in control and formulated experimental feeds,
among these isoleucine, leucine, lycine, methionine, phenylalanine, thrionine and valine
are essential amino acids; alanine, arginine, glutamic acid, histidine, proline, serine and
tyrosine are non-essential amino acids. In this study, all the essential and non-essential
amino acid categories were found to be significantly higher (P<0.05) in experimental
feeds when compared with control feed.
This was in the order of C. vulgaris >
S. platensis > A. pinnata incorporated feeds when compared with control feed
(Table 7.3.1, Plate 5.2 & 5.3).
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Amino acid profile in M. rosenbergii PL
Fifteen amino acids were detected in control and formulated experimental feed
fed M. rosenbergii PL, among these arginine, isoleucine, lysine, tryptophan,
phenylalanine, threonine and histidine are essential amino acids; asparagine, alanine,
glutamic acid, cystine, tyrosine, aspartic acid, glycine and proline are non essential amino
acids. In this study, all the essential and non essential amino acid categories were found
to be significantly higher (P<0.05) in experimental groups when compared with control
group. This was in the order of C. vulgaris > S. platensis > A. pinnata incorporated feeds
when compared with control feed (Table 7.3.2, Plate 5.4 & 5.5).
Profile of fatty acids in formulated feed
There were 12 fatty acids detected, which include both essential (unsaturated) and
saturated fatty acid (Table 7.3.3, Plate - 5.7 & 5.8). There were six saturated fatty acids
(myristic acid, palmitic acid, stearic acid, behanic acid and lignoceric acid), remaining
seven were unsaturated fatty acids (oleic acid, linoleic acid, linolenic acid, arachidic acid,
EPA and DHA). In the present study, the following fatty acids such as palmitic acid,
stearic acid, oliec acid, linoleic acid, linolenic acid, EPA, lignoceric and DHA were found
to be significantly higher in 50% of C. vulgaris incorporated diet followed by the 50%
S. platensis when compared with control feed. The lauric acid, myristic acid, and
arachidic acid showed significantly higher in S. platensis incorporated feed followed by
the C. vulgaris feed. The behanic acid content was higher in A. pinnata incorporated feed,
other fatty acids in A. pinnata feed showed significantly lower level when compared with
control feed.
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Profile of fatty acids in formulated feed fed PL
In the present study, There were 12 fatty acids were detected in muscle tissue of
formulated feed fed M. rosenbergii PL, which include both essential (unsaturated) and
saturated fatty acid (Table 7.3.4, Plate - 5.9 & 5.10). In this fatty acid profile six saturated
fatty acids (myristic acid, palmitic acid, stearic acid, behanic acid and lignoceric acid),
remaining seven were unsaturated fatty acids (oleic acid, linoleic acid, linolenic acid,
arachidic acid, EPA and DHA). In the present study, 11 fatty acids (except lauric) were
found to be significantly higher in 50% of C. vulgaris incorporated feed fed group
followed by the 50% of S. platensis and A. pinnata
incorporated feed fed groups.
The lauric acid was significantly higher in 50% A. pinnata incorporated feed fed group
followed by the 50% of C. vulgaris and S. platensis feed fed group when compared with
control feed fed group.
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PLATE – 5.1
10% SDS-PAGE of formulated feed fed M. rosenbergii PL muscle tissue
Lane M, Marker
Lane 1, Control feed fed M. rosenbergii PL muscle tissue
Lane 2, 50% of A. pinnata incorporated feed fed M. rosenbergii PL muscle tissue
Lane 3, 50% of S. platensis incorporated feed fed M. rosenbergii PL muscle tissue
Lane 4, 50% of C. vulgaris incorporated feed fed M. rosenbergii PL muscle tissue
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Table 7.3.1. Concentration of amino acids in formulated experimental diet (g/100g
dry weight)
Assigned
substance
Isoleucine*
Leucine*
Lycine*
Methionine *
Phenylalanine*
Thrionine*
Valine*
Alanine**
Arginine**
Control
S. platensis
C. vulgaris
(BI+FM)
(BI+FM50+R50)
0.98 ± 0.15b
b
1.13 ± 0.10
0.96 ± 0.07b
0.86 ± 0.05c
0.36 ± 0.08b
1.19 ± 0.09b
0.89 ± 0.13c
1.91 ± 0.15b
1.25 ± 0.19a
c
Glutamic
acid**
1.13 ± 0.12
Histidine**
1.07 ± 0.05b
Proline**
Serine**
Tyrosine**
0.87 ± 0.10b
2.08 ± 0.11c
0.54 ± 0.10b
( BI+FM50+R50)
A. pinnata
(BI+FM50+R50)
F value
1.12 ± 0.05b
1.49 ± 0.14a
0.99 ± 0.11b
16.853
-2.425 (0.136)
-88.335 (0.000)
-0.433 (0.707)
b
a
1.24 ± 0.09b
1.17 ± 0.18
1.57 ± 0.15
-.0.866 (0.478)
-15.242 (0.004)
-19.053 (0.003)
1.04 ± 0.05b
1.20 ± 0.04a
1.03 ± 0.05b
-6.928 (0.020)
-13.856 (0.005)
-6.062 (0.026)
1.18 ± 0.08b
1.38 ± 0.12a
1.08 ± 0.10b
-18.475 (0.003)
-12.867 (0.006)
-7.621 (0.017)
0.62 ± 0.12a
0.71 ± 0.11a
0.60 ± 0.10a
-11.258 (0.008)
-20.207 (0.002)
-20.785 (0.002)
1.40 ± 0.15b
1.95 ± 0.16a
1.25 ± 0.11b
-6.062 (0.026)
-18.805 (0.003)
-5.196 (0.035)
1.35 ± 0.11b
1.98 ± 0.18a
1.78 ± 0.21a
-39.837 (0.001)
-37.759 (0.001)
-19.269 (0.003)
2.20 ± 0.08a
2.25 ± 0.05a
2.12 ± 0.07a
-7.176 (0.019)
-5.889 (0.028)
-4.547 (0.045)
1.19 ± 0.10a
1.30 ± 0.08a
1.27 ± 0.11a
1.155 (0.368)
-0.787 (0.514)
-0.433 (0.707)
b
a
1.28 ± 0.10c
1.56 ± 0.10
1.85 ± 0.13
-37.239 (0.001)
-124.70 (0.000)
-12.990 (0.006)
1.17 ± 0.11b
1.67 ± 0.13a
1.19 ± 0.06b
-2.887 (0.102)
-12.990 (0.006)
-20.785 (0.002)
0.90 ± 0.05b
1.14 ± 0.10a
1.02 ± 0.06b
-1.039 (0.408)
-46.765 (0.000)
-6.495 (0.023)
2.26 ± 0.03ab
2.38 ± 0.05a
2.15 ± 0.09bc
-3.897 (0.060)
-8.660 (0.013)
-6.062 (0.026)
0.75 ± 0.15ab
0.81 ± 0.05a
0.61 ± 0.11ab
-7.275 (0.018)
-9.353 (0.011)
-12.124 (0.007)
6.254
10.739
8.758
24.650
7.000
26.669
6.590
0.401
7.427
23.649
21.088
12.056
3.930
Each value is a mean ± SD of three replicate analysis, within each row means with different
superscripts letters are statistically significant P<0.05 (one way ANOVA and subsequently post
hoc multiple comparison with DMRT, paired sample ‘t’ test also applied).
*essential amino acid, **non essential amino acid.
BI- Basal ingredients; FM- Fishmeal; R- Replacement.
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PLATE – 5.2
HPTLC analyses of amino acid profile of formulated feeds
Chromatogram of after derivatization
Sample code
A
- Sample coded as control feed
B
- Sample coded as 50% of Spirulina inclusion feed
C
- Sample coded as 50% of Azolla inclusion feed
D
- Sample coded as 50% of Chlorella inclusion feed
G1
- Standard amino-acids Group 1
G2
- Standard amino-acids Group 2
G3
- Standard amino-acids Group 3
G4
- Standard amino-acids Group 4
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PLATE – 5.3
HPTLC Peak densitogram display of amino acid profile of formulated feed
a, Peak densitogram of control feed;
b, Peak densitogram of S. platensis incorporated feed;
c, Peak densitogram of C. vulgaris incorporated feed
d, Peak densitogram of A. pinnata incorporated feed;
e, 3D display of all tracks.
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Table 7.3.2. Concentration of amino acids in formulated feed fed with
M. rosenbergi PL (g/100g dry weight)
Control
(BI+FM)
S. platensis
(BI+FM50+R50)
C. vulgaris
(BI+FM50+R50)
A. pinnata
(BI+FM50+R50)
Arginine*
2.28 ± 0.25c
2.92 ± 0.11b
-7.918 (0.016)
3.44 ± 0.12a
-15.455 (0.004)
2.72 ± 0.10b
-5.081 (0.037)
28.057
Isoleucine*
0.68 ± 0.12c
1.92 ± 0.07b
-42.955 (0.001)
2.52 ± 0.11a
-318.69 (0.000)
0.84 ± 0.06c
-4.619 (0.044)
264.64
Lysine*
1.14 ± 0.05d
1.68 ± 0.02b
-31.177 (0.001)
1.84 ± 0.04a
-121.24 (0.000)
1.52 ± 0.11c
-10.970 (0.008)
65.036
Tryptophan*
1.11 ± 0.17b
1.43 ± 0.14a
-18.475 (0.003)
1.59 ± 0.11a
-13.856 (0.005)
1.08 ± 0.05b
0.433 (0.707)
11.758
Phenylalanine*
1.06 ± 0.12c
1.73 ± 0.19b
-16.578 (0.004)
1.99 ± 0.11a
-161.08 (0.000)
1.18 ± 0.06c
-3.464 (0.074)
35.565
Threonine*
1.12 ± 0.05b
1.54 ± 0.14b
-8.083 (0.015)
1.21 ± 0.11a
-2.598 (0.122)
0.72 ± 0.08c
23.094 (0.002)
33.643
Histidine*
0.99 ± 0.12b
1.71 ± 0.15a
-41.569 (0.001)
1.55 ± 0.11a
-96.995 (0.000)
1.48 ± 0.08a
-21.218 (0.002)
20.857
Asparagine**
1.92 ± 0.08b
2.43 ± 0.11a
-29.445 (0.001)
2.65 ± 0.17a
-14.049 (0.005)
1.74 ± 0.13b
6.235 ()0.025
33.872
Alanine**
1.12 ± 0.11b
1.72 ± 0.13a
-51.962 (0.000)
1.72 ± 0.11a
-103.92 (0.000)
1.64 ± 0.05a
-15.011 (0.004)
23.009
Glutamic acid**
1.12 ± 0.20b
1.72 ± 0.10a
-10.392 (0.009)
1.72 ± 0.14a
-17.321 (0.003)
1.64 ± 0.09a
-8.188 (0.015)
12.911
Cystine**
2.32 ± 0.15c
3.56 ± 0.13ab
-107.38 (0.000)
3.65 ± 0.10 a
-46.073 (0.000)
3.34 ± 0.12b
-58.890 (0.000)
70.525
Tyrosine**
1.76 ± 0.21d
4.08 ± 0.14b
-57.405 (0.000)
5.28 ± 0.15a
-101.61 (0.000)
3.72 ± 0.09c
-28.290 (0.001)
271.66
Aspartic acid **
1.75 ± 0.17c
2.14 ± 0.14b
-22.517 (0.002)
2.45 ± 0.15a
-60.622 (0.000)
2.1 ± 0.11b
-10.104 (0.010)
11.851
Glycine **
0.83 ± 0.15c
1.16 ± 0.11b
-14.289 (0.005)
1.43 ± 0.09a
-17.321 (0.003)
1.12 ± 0.13b
-25.115 (0.002)
12.141
Proline**
2.32 ± 0.14b
3.09 ± 0.15a
-133.36 (0.000)
2.83 ± 0.10a
-22.084 (0.002)
2.11 ± 0.19b
7.275 (0.018)
27.704
Assigned
substance
F value
Each value is a mean ± SD of three replicate analysis, within each row means with different
superscripts letters are statistically significant P<0.05 (one way ANOVA and subsequently post
hoc multiple comparison with DMRT, paired sample ‘t’ test also applied).
*essential amino acid, **non essential amino acid.
BI- Basal ingredients; FM- Fishmeal; R- Replacement.
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PLATE – 5.4
HPTLC Analyses for amino acid profile of formulated feed fed M. rosenbergii PL
tissue
Chromatogram of after derivatization
Sample code
GI
- Standard amino-acids Group 1
GII
- Standard amino-acids Group 2
GIII
- Standard amino-acids Group 3
GIV
- Standard amino-acids Group 4
A
- Sample coded as Control feed fed M. rosenbergii PL.
B
- Sample coded as 50% of Spirulina inclusion feed fed M. rosenbergii PL.
C
- Sample coded as 50% of Chlorella inclusion feed fed M. rosenbergii PL.
D
- Sample coded as 50% of Azolla inclusion feed fed M. rosenbergii PL.
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PLATE – 5.5
HPTLC Peak densitogram display of amino acid profile of control, S. platensis, C.
vulgaris and A. pinnata incorporate feed fed M. rosenbergii PL tissue
a, Peak densitogram of control feed fed PL;
b, Peak densitogram of S. platensis feed fed PL
c, Peak densitogram of C. vulgaris feed fed PL
d, Peak densitogram of A. pinnata feed fed PL
e, 3D display of all tracks.
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PLATE – 5.6
HPTLC Peak densitogram display of standard amino acid
a, Peak densitogram of Group 1 standard amino acid
b, Peak densitogram of Group 2 standard amino acid
c, Peak densitogram of Group 3 standard amino acid
d, Peak densitogram of Group 4 standard amino acid
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Table 7.3.3. Profile of fatty acids in formulated feed (%/µl methylated fatty acid
samples)
Control
(BI+FM)
S. platensis
(BI+FM50+R50)
C. vulgaris
(BI+FM50+R50)
A. pinnata
(BI+FM50+R50)
0.036 ± 0.007d
1.03 ± 0.025a
-95.648 (0.000)
0.268 ± 0.02c
-30.910 (0.001)
0.311 ± 0.029b
-21.651(0.002)
1.158
Myristic acid*
3.12 ± 0.11a
2.92 ± 0.13a
17.321 (0.003)
1.25 ± 0.16b
64.779 (0.000)
1.33 ± 0.19b
38.755 (0.001)
133.014
Palmitic acid*
17.90 ± 1.19b
22.86 ± 0.94a
-34.364 (0.001)
24.18 ± 0.13a
-10.262 (0.009)
23.62 ± 0.11a
-9.173 (0.012)
42.681
Stearic acid*
2.76 ± 0.21b
4.26 ± 0.12a
-28.868 (0.001)
4.44 ± 0.13a
-36.373 (0.001)
4.3 ± 0.21a
*
62.741
Oliec acid**
27.1 ± 1.26c
31.47 ± 1.14b
-63.076 (0.000)
34.09 ± 1.03a
-52.639 (0.000)
29.61 ± 1.25b
-434.74 (0.000)
18.991
Linoleic acid**
15.78 ± 1.06c
19.65 ± 0.96b
-67.030 (0.000)
23.85 ± 1.26a
-69.888 (0.000)
17.91 ± 0.81b
-14.757 (0.005)
32.780
Linolenic
acid**
1.56 ± 0.17b
1.88 ± 0.09a
-6.928 (0.020)
2.08 ± 0.15a
-45.033 (0.000)
1.04 ± 0.035c
6.672 (0.022)
40.682
Arachidic
acid**
0.245 ± 0.012c
0.662 ± 0.022a
-72.227 (0.000)
0.442 ± 0.035b
-14.835 (0.005)
0.255 ± 0.011c
-17.321 (0.003)
233.949
Behanic acid*
0.549 ± 0.12b
0.162 ± 0.11c
67.030 (0.000)
0.507 ± 0.19b
1.039 (0.408)
1.08 ± 0.15a
-30.657 (0.001)
20.253
EPA**
3.48 ± 0.21b
6.78 ± 0.13a
-9.310 (0.011)
4.213 ± 0.19a
-63.480 (0.000)
1.551 ± 0.12c
37.124 (0.001)
154.136
Lignoceric*
0.372 ± 0.02b
0.385 ± 0.01b
-2.252 (0.153)
0.422 ± 0.03a
-8.660 (0.013)
0.253 ± 0.01c
20.611 (0.002)
42.789
DHA**
4.15 ± 0.34c
5.95 ± 0.15b
-16.409 (0.004)
6.72 ± 0.14a
-22.257 (0.002)
3.49 ± 0.09d
4.573 (0.045)
165.005
Fatty acids
Lauric*
F value
Each value is a mean ± SD of three replicate analysis, within each row means with different
superscripts letters are statistically significant P<0.05 (one way ANOVA and subsequently post
hoc multiple comparison with DMRT, paired sample ‘t’ test also applied).
* Saturated Fatty Acids, **Unsaturated Fatty Acids.
BI- Basal ingredients; FM- Fishmeal; R- Replacement.
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PLATE 5.7
GC fatty acid profile chromatogram of control feed.
GC fatty acid profile chromatogram of 50% S. platensis incorporated feed.
PLATE – 5.8
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GC fatty acid profile chromatogram of 50% C. vulgaris incorporated feed.
GC fatty acid profile chromatogram of 50% A. pinnata incorporated feed.
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Table 7.3.4. Profile of essential fatty acids in formulated feed fed M. rosenbergii PL
(%/µl methylated fatty acid samples)
Fatty acids
Lauric*
Control
(BI+FM)
S. platensis
(BI+FM50+R50)
C. vulgaris
(BI+FM50+R50)
A. pinnata
(BI+FM50+R50)
F value
0.108 ±0.034d
0.263 ± 0.024c
0.594 ± 0.045b
1.13 ± 0.09a
206.903
-26.847 (0.001)
-76.525 (0.000)
-31.610 (0.001)
1.828 ± 0.16b
3.88 ± 0.11a
2.19 ± 0.15c
-38.076 (0.001)
-260.58 (0.000)
-23.680 (0.002)
Myristic acid*
0.871 ± 0.09d
Palmitic acid*
d
Stearic acid*
Oliec acid**
Linoleic acid**
19.68 ± 0.16
9.68 ± 0.16c
25.31 ± 0.28d
13.36 ± 0.51c
Linolenic
acid**
0.057 ± 0.011b
Arachidic
acid**
0.36 ± 0.013c
Behanic acid*
2.32 ± 0.15d
EPA**
4.652 ± 0.32c
21.99 ± 0.13
b
22.96 ± 0.24
Lignoceric*
DHA**
0.07 ± 0.01
4.65 ± 0.18c
16.9 ± 0.29c
-133.36 (0.000)
-71.014 (0.000)
37.039 (0.001)
11.8 ± 0.23b
14.48 ± 0.17a
8.32 ± 0.11d
-52.456 (0.000)
-831.38 (0.000)
47.112 (0.000)
30.6 ± 0.23b
32.1 ± 0.15a
28.58 ± 0.16c
-183.25 (0.000)
-90.466 (0.000)
-47.198 (0.000)
15.84 ± 0.26b
26.1 ± 0.16a
13.33 ± 0.36d
-17.182 (0.003)
-63.047 (0.000)
(*)
0.668 ± 0.02a
0.75 ± 0.09a
-117.58 (0.000)
-15.194 (0.004)
0.452 ± 0.03b
0.544 ± 0.022a
0.354 ± 0.012c
-9.373 (0.011)
-35.411 (0.001)
10.392 (0.009)
3.036 ± 0.19b
4.932 ± 0.11a
2.494 ± 0.12c
-31.004 (0.001)
-113.10 (0.000)
-10.046 (0.010)
6.2 ± 0.29b
6.82 ± 0.16a
4.95 ± 0.14c
-23.469 (0.002)
-2.868 (0.103)
-89.37 (0.000)
c
a
0.278 ± 0.03
b
0.432 ± 0.04
a
Trace level
0.262 ± 0.02b
-18.013 (0.003)
-20.90 (0.002)
-33.255 (0.001)
5.07 ± 0.24b
5.79 ± 0.15a
3.22 ± 0.14d
-12.124 (0.007)
-65.818 (0.000)
61.921 (0.000)
276.831
474.460
724.874
576.934
917.580
217.175
56.879
202.090
54.506
88.015
106.485
Each value is a mean ± SD of three replicate analysis, within each row means with different
superscripts letters are statistically significant P<0.05 (one way ANOVA and subsequently post
hoc multiple comparison with DMRT, paired sample ‘t’ test also applied).
* Saturated Fatty Acids, **Unsaturated Fatty Acids.
BI- Basal ingredients; FM- Fishmeal; R- Replacement.
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PLATE – 5.9
GC fatty acid profile chromatogram of control feed fed M. rosenbergii PL muscle
tissue.
GC fatty acid profile chromatogram of 50% S. platensis incorporated feed fed M.
rosenbergii PL muscle tissue.
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PLATE – 5.10
GC fatty acid profile chromatogram of 50% C. vulgaris incorporated feed fed M.
rosenbergii PL muscle tissue.
GC fatty acid profile chromatogram of 50% A. pinnata incorporated feed fed M.
rosenbergii PL muscle tissue.
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7.4. Discussion
Profiles of protein in formulated feed fed M. rosenbergii PL
Crustaceans fed with high concentrations of protein tend to grow and survive better.
Presumably, they take advantage of the protein content in food and acquire more building
blocks of tissue construction and energy reserves for metabolic functions (Koshio et al., 1993;
Moullac and van Wormhoudt, 1994). Sandbank and Hepher, (1978) reported that Spirulina
as a substitute protein source for fishmeal protein replacement for C. carpio. Zeinhom
(2004) found that, Inclusion of algae in fish feed improve the whole fish body dry matter
and crude protein. El-Hadidy et al. (1993) and El-Sayed (1994) mentioned that, the fish
diet containing
algae cause a significant variation in carcass CP in Nile tilapia
(O. niloticus) in T. mossambica (Olevera-Novoa et al., (1998) in H. discus discus
(Stott et al., 2004). Tartiel, (2005), Janczyk et al. (2006), and Vaikosen et al. 2007
reported that C. vulgaris have higher protein content. Tartiel (2008) reported that,
Chlorella Sp. incorporation diets improve the body carcass protein in Nile tilapia
(O. niloticus). Azolla can be utilized as a fish feed for carps (Maity and Patra, 2008) and
it can convert its raw protein into best edible protein (Lejeune et al., 1999). Sudaryono (2006)
reported that A. pinnata is another economical plant protein for P. monodon diet. In the
present study, S. platensis, C. vulgaris and A. pinnata incorporated feed fed PL group
showed higher intensity of polypeptide band and more kDa proteins when compared with
control group.
Amino acids profile
Essential amino acids are precursors of proteins and also act as an energy source
(Litaay et al., 2001). Animals must consume dietary protein to obtain a continual supply
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of amino acids. After ingestion, it is digested or hydrolyzed to release free amino acids
that are absorbed from the intestinal tract, and then distributed to the various organs and
tissues. Amino acids are used by the tissues to synthesize new protein, thus animals do not
necessarily require protein, but do require the amino acid which comprise proteins. Since
high protein diets are needed for good growth of most aquatic animals (NRC, 1993),
estimation of minimum requirement of EAA is indispensable to formulate cost-effective
diets. The quantitative EAA requirements of fish and crustaceans are often determined by
feeding experiments with diets containing graded levels of the particular amino acid to be
examined (Wilson, 1999). Deshimaru and Shigueno (1972) were reported that the amino
acid composition of the dietary protein should match of prawn tissue. The consumed
protein is digested or hydrolyzed to release free amino acids that are absorbed from the
intestinal tract of the animal and distributed by the blood to various organs and tissues;
Amino acid patterns (A/E ratio) have shown increased arginine and decreased
phenylalanine content with growth in the tiger prawn. A significant change in free amino
acid pool occurs during a moult cycle in P. keratharus (Torres, 1979).
The present study revealed that the presence of essential amino acid (EAA) like
valine, lysine, threonine, isoleusine, tyrosine, arginine, histidine and leucine, the non
essential amino acid such as glutamine, serine, proline, glycine and alanine were
identified in formulated feeds. The percentages of this amino acid were varied
remarkably with respect to formulated diet. Hence, it was evidenced that, the formulated
diets are highly enriched aminoacid profile with that of the control diet. In the present
study, presence of these amino acids are observed in laboratory cultured S. platensis,
C. vulgaris and A. pinnata it was already discussed in chapter 1. Uslu et al. (2009) and
Babadzhanov et al. (2004) has been reported that the amino acids are presented in
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S. platensis. Similarly, Janczyk et al. (2005) stated that presence of these amino acids in novel
processing method treated C. vulgaris. Dawah et al. (2002a, b) noted that, five amino acids
(aspartic acid, serine, alanine, leucine and glycine) were collectively responsible for 50%
or more than the total dry matter content of Chlorella species. Sanginga and VanHove
(1989) have indicated that these limiting amino acids can be added to make Azolla as a
complete source of amino acids. Also, Abou et al. (2011) and Huggins (2007), reported
that presences of these amino acids are rich in Azolla meal.
Crustacean muscles contain high concentration of free amino acids, such as
arginine, glycine, proline, glutamine and alanine (Cobb et al., 1975). The free amino
acids have been shown to function in osmoregulation (Fang et al., 1992) and also have a
major contribution to the flavor of sea foods (Thompson et al., 1980). Each aminoacid
has its own biological function and metabolism. Regarding the function of single
aminoacid, leucine is ketone-producing aminoacid. It could be transformed into
acetyl-CoA and acetyl-acetic acid, which are important intermediates in carbohydrate and
lipid metabolisms (Shen and Wang, 1990).
Arginine was proven to be crucial in energy metabolism by maintaining
glycolysis under hypoxic conditions (Gade and Grieshaber, 1986). Arginine plays an
important role in cell division, the healing of wounds, removing ammonia from the body,
immune function, and the release of hormones (Tapiero et al., 2002; Stechmiller et al., 2005;
Witte and Barbul, 2003). Glutamic acid turned into glutamine, which is deaminated to
produce NH3 (Shen and Wang, 1990). NH3 can be excreted along with Cl-. An increase in
the content of NH4Cl after the blastula stage also suggests that NH4+ and Cl- are being
excreted together. Tyrosine can be used to synthesize melanin (Shen and Wang, 1990),
which plays a central role in the accumulation of compound eye pigments. Valine is
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involved in many metabolic pathways and is considered indispensable for protein
synthesis and optimal growth (Wilson, 2002). Valine is a carbohydrate producing amino
acid and may be associated with carbohydrate metabolism through citric cycle. Histidine
is also an indispensable amino acid involved in many metabolic functions including the
production of histamines, which take part in allergic and inflammatory reactions. It plays
a very important role in maintaining the osmoregulatory process and is related to energy
production or is used in other metabolic pathways during certain emergencies/harsh
conditions (Abe and Ohmama, 1987). In consistent with the statements mentioned above,
the present study showed that the presence of EAA like valine, lysine, threonine,
isoleusine, tyrosine, arginine, histidine and leucine. However, the non essential amino
acid such as glutamine, serine, proline, glycine and alanine were identified in formulated
feed fed PL. The percentages of these aminoacid were elevated in S. platensis,
C. vulgaris and A. pinnata incorporated feed fed group. Similary, Bhavan et al. (2010b)
reported that presence of these amino acids in commercial available Spirulina powder
enriched Artemia nauplii fed M. rosenbergii PL. Abou et al. (2011) noted that presence of
these amino acids in Azolla sp. incorporated feed fed O. niloticus.
Profile of fatty acids
Highly unsaturated fatty acids (HUFA), especially eicosapentaenoic acid (20:5n-3)
(EPA) and docosahexaenoic acid (22:6n-3) (DHA) have been identified as important
nutrients for the early growth of fish and crustaceans. In crustaceans, the importance and
essentiality of several poly-unsaturated fatty acids (PUFA) such as linoleic acid (18:2n-6),
linolenic acid (18:3n-3), EPA and DHA to increase growth and survival of larvae and
juveniles, to promote ovarian maturation in broodstock and to promote production of
better quality eggs has been well known. De novo synthesis of these PUFA has not been
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observed in M. rosenbergii (Reigh and Stickney 1989), as well as in other shrimp species
such as P. monodon and P. merguiensis (Kanazawa et al., 1979a,b). The Spirulina components
which are responsible for these therapeutic properties are thought to be compounds with
antioxidant abilities such as polyunsaturated fatty acids (Estrada et al., 2001).
In the present study, palmitic acid, myristic acid, lauric acid, linolenic, linoleic,
lignoceric acid, behanic acid, aracihdic acid, EPA and DHA were identified in the formulated
feed. The presence of fatty acids showed the formulated feeds are enriched source of fatty
acids. Spirulina as a potential source of GLA (Alonso and Maroto, 2000; Quoc et al., 1994)
and the growth conditions needed to increase GLA (Quoc et al., 1994). Colla et al. (2004)
suggested that Spirulina is a rich source of polyunsaturated fatty acids (especially GLA),
it seems that the best way to use Spirulina is by its direct consumption as a nutritional
supplement. Spirulina can be used either as a food supplement or taken in capsule
form, capsules appearing to be the preferred form at present. Tsuzuki et al. (1990) and
Yusof et al. (2011) reported that C. vulgaris have higher concentration of saturated and
unsaturated essential fatty acids. Similarly, Abou et al. (2011) reported that presence of
these fatty acids in Azolla sp.
The fatty acid profile of body tissue is a key factor as it has been proposed for
evaluating quality of seed (Arellano, 1990). The polyunsaturated fatty acids (PUFA) of
the linoleic (n-6) and highly unsaturated fatty acids (HUFA) linolenic, EPA and DHA (n-3)
have been recognized as important nutrients for growth and reproduction of fishes,
crustaceans and mollusks (Izquierdo et al., 2000; Caers et al., 2000; Navarro and
Villanueva, 2000; Jeffs et al., 2002; Nelson et al., 2002). Fatty acid composition of the
animal body tissue, mainly n-3 HUFA, is correlated with their susceptibility to various
diseases i.e. immunity, and ability to tolerate the unfavorable environmental factors.
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If their ability to synthesize those fatty acids is lacking and/or very poor, providing those
fatty acids exogenously (Watanabe et al., 1974; Pillai et al., 2003) will minimize the
problem. Palmitic acid (C16:0) was the major fatty acid among saturated fatty acid group
in the PL fed with all types of feed. Palmitic acid (16:0) is the final product of fatty acid
synthesis in animal tissues, and is the most abundant saturated fatty acid in plankton and fish.
It is a biosynthetic precursor of long-chain saturated fatty acids and the denovo synthesis of
unsaturated n9 fatty acids (Sargent, 1976; Holland, 1978). Querijero et al. (1997a, b)
determined that dietary stearic acid (18:0) and oleic acid (18:1) are used as sources of
energy. Larval M. rosenbergii seemed to be able to convert linolenic (18:3n-3) acid to
eicosapentaenoic (20:5n-3) acid, as was evident by a much higher level of larval
eicosapentaenoic (20:5n-3) acid than the dietary content. The importance of
eicosapentaenoic (20:5n-3) acid as a structural component of juvenile M. rosenbergii has
been reported (Reigh and Stickney, 1989). In the present study, the fatty acids such as
palmitic acid, myristic acid, lauric acid, linolenic, linoleic, lignoceric acid, behanic acid,
arachihdic acid, EPA and DHA were elevated in experimental feeds fed PL groups. It is
evidenced that the formulated feed contains essential fatty acids and they were well
utilized by the prawn PL.
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7.5 Conclusion
The present study, 50% of fishmeal replaced with S. platensis, C. vulgaris and
A. pinnata incorporated feeds showed maximum levels of essential amino acids and fatty
acids. Concurrently, these feeds fed PL groups also gained good growth and production
which could be attributed due to the presence of enhanced levels of protein, essential
amino acids and fatty acids.
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