7. profiles of protein, amino acids and fatty...

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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-Ammonia-

Pyridine-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)

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.

Assigned substance

Control

(BI+FM)

S. platensis

(BI+FM50+R50)

C. vulgaris

( BI+FM50+R50) A. pinnata

(BI+FM50+R50) F value

Isoleucine* 0.98 ± 0.15b 1.12 ± 0.05b

-2.425 (0.136) 1.49 ± 0.14a

-88.335 (0.000) 0.99 ± 0.11b

-0.433 (0.707) 16.853

Leucine* 1.13 ± 0.10b 1.17 ± 0.18b

-.0.866 (0.478) 1.57 ± 0.15a

-15.242 (0.004) 1.24 ± 0.09b

-19.053 (0.003) 6.254

Lycine* 0.96 ± 0.07b

1.04 ± 0.05b

-6.928 (0.020) 1.20 ± 0.04a

-13.856 (0.005) 1.03 ± 0.05b

-6.062 (0.026) 10.739

Methionine * 0.86 ± 0.05c 1.18 ± 0.08b

-18.475 (0.003) 1.38 ± 0.12a

-12.867 (0.006) 1.08 ± 0.10b

-7.621 (0.017) 8.758

Phenylalanine* 0.36 ± 0.08b 0.62 ± 0.12a

-11.258 (0.008) 0.71 ± 0.11a

-20.207 (0.002) 0.60 ± 0.10a

-20.785 (0.002) 24.650

Thrionine* 1.19 ± 0.09b 1.40 ± 0.15b

-6.062 (0.026) 1.95 ± 0.16a

-18.805 (0.003) 1.25 ± 0.11b

-5.196 (0.035) 7.000

Valine* 0.89 ± 0.13c 1.35 ± 0.11b

-39.837 (0.001) 1.98 ± 0.18a

-37.759 (0.001) 1.78 ± 0.21a

-19.269 (0.003) 26.669

Alanine** 1.91 ± 0.15b 2.20 ± 0.08a

-7.176 (0.019) 2.25 ± 0.05a

-5.889 (0.028) 2.12 ± 0.07a

-4.547 (0.045) 6.590

Arginine** 1.25 ± 0.19a 1.19 ± 0.10a

1.155 (0.368) 1.30 ± 0.08a

-0.787 (0.514) 1.27 ± 0.11a

-0.433 (0.707) 0.401

Glutamic acid**

1.13 ± 0.12c 1.56 ± 0.10b

-37.239 (0.001) 1.85 ± 0.13a

-124.70 (0.000) 1.28 ± 0.10c

-12.990 (0.006) 7.427

Histidine** 1.07 ± 0.05b 1.17 ± 0.11b

-2.887 (0.102) 1.67 ± 0.13a

-12.990 (0.006) 1.19 ± 0.06b

-20.785 (0.002) 23.649

Proline** 0.87 ± 0.10b 0.90 ± 0.05b

-1.039 (0.408) 1.14 ± 0.10a

-46.765 (0.000) 1.02 ± 0.06b

-6.495 (0.023) 21.088

Serine** 2.08 ± 0.11c 2.26 ± 0.03ab

-3.897 (0.060) 2.38 ± 0.05a

-8.660 (0.013) 2.15 ± 0.09bc

-6.062 (0.026) 12.056

Tyrosine** 0.54 ± 0.10b 0.75 ± 0.15ab

-7.275 (0.018) 0.81 ± 0.05a

-9.353 (0.011) 0.61 ± 0.11ab

-12.124 (0.007) 3.930


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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





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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)

Assigned substance

Control (BI+FM)

S. platensis (BI+FM50+R50)

C. vulgaris (BI+FM50+R50)

A. pinnata (BI+FM50+R50) F value

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

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)

Fatty acids Control

(BI+FM) S. platensis

(BI+FM50+R50) C. vulgaris

(BI+FM50+R50) A. pinnata

(BI+FM50+R50) F value

Lauric* 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

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.


146

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


147

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 Control (BI+FM)

S. platensis (BI+FM50+R50)

C. vulgaris (BI+FM50+R50)

A. pinnata (BI+FM50+R50) F value

Lauric* 0.108 ±0.034d 0.263 ± 0.024c -26.847 (0.001)

0.594 ± 0.045b -76.525 (0.000)

1.13 ± 0.09a -31.610 (0.001)

206.903

Myristic acid* 0.871 ± 0.09d 1.828 ± 0.16b

-38.076 (0.001) 3.88 ± 0.11a

-260.58 (0.000) 2.19 ± 0.15c

-23.680 (0.002) 276.831

Palmitic acid* 19.68 ± 0.16d 21.99 ± 0.13b -133.36 (0.000)

22.96 ± 0.24a -71.014 (0.000)

16.9 ± 0.29c 37.039 (0.001)

474.460

Stearic acid* 9.68 ± 0.16c 11.8 ± 0.23b -52.456 (0.000)

14.48 ± 0.17a -831.38 (0.000)

8.32 ± 0.11d 47.112 (0.000)

724.874

Oliec acid** 25.31 ± 0.28d

30.6 ± 0.23b -183.25 (0.000)

32.1 ± 0.15a -90.466 (0.000)

28.58 ± 0.16c -47.198 (0.000)

576.934

Linoleic acid** 13.36 ± 0.51c

15.84 ± 0.26b -17.182 (0.003)

26.1 ± 0.16a

-63.047 (0.000) 13.33 ± 0.36d

(*) 917.580

Linolenic acid**

0.057 ± 0.011b

0.668 ± 0.02a -117.58 (0.000)

0.75 ± 0.09a

-15.194 (0.004) Trace level

217.175

Arachidic acid**

0.36 ± 0.013c 0.452 ± 0.03b -9.373 (0.011)

0.544 ± 0.022a -35.411 (0.001)

0.354 ± 0.012c 10.392 (0.009)

56.879

Behanic acid* 2.32 ± 0.15d 3.036 ± 0.19b -31.004 (0.001)

4.932 ± 0.11a -113.10 (0.000)

2.494 ± 0.12c -10.046 (0.010)

202.090

EPA** 4.652 ± 0.32c 6.2 ± 0.29b -89.37 (0.000)

6.82 ± 0.16a -23.469 (0.002)

4.95 ± 0.14c -2.868 (0.103)

54.506

Lignoceric* 0.07 ± 0.01c 0.278 ± 0.03b -18.013 (0.003)

0.432 ± 0.04a -20.90 (0.002)

0.262 ± 0.02b -33.255 (0.001)

88.015

DHA** 4.65 ± 0.18c

5.07 ± 0.24b -12.124 (0.007)

5.79 ± 0.15a -65.818 (0.000)

3.22 ± 0.14d 61.921 (0.000)

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.


150

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


152

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


155

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.


156

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.


7. profiles of protein, amino acids and fatty...

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