2692 Advances2692-2696, in Life Sciences Advances in Life Sciences 5(7), Print : ISSN 2278-3849, 2016 5(7), 2016
Estimation of Amino Acids, Minerals and Other Chemical Compositions of Azolla D. C. ROY1*, M.C.PAKHIRA1 AND M.ROY2 1
Dept. of ILFC, WBUAFS, Mohanpur, Nadia, West Bengal, 741252 Dept. of Animal Genetics & Breeding, WBUAFS, Mohanpur, Nadia, West Bengal, 741252 *email:
[email protected] 2
ABSTRACT Azolla ‘the green gold mine’ of the nature is a symbiotic association of Azolla-Anabaena in which the endophytic blue-green alga Anabaena zollae lives within the leaf cavities of the water fern Azolla (Lain). The representative samples of Azolla (Azolla pinnata Lain.) was analyzed for estimation of various minerals and proximate principles i.e. moisture, crude protein, ether extract, crude fibre, total ash etc. Amino acids content of Azolla was also estimated through gas chromatography and capillary electrophoresis by wet chemistry (hydrolysis) method. Azolla contained 8.0% dry matter (DM), 24.61% crude protein (CP), 15% crude fibre (CF), 43.8% neutral detergent fibre (NDF), 31.80% acid detergent fibre (ADF), 3.8% ethyle extract (EE), 11.4% lignin, 4.1% starch and 15.9% total ash on dry matter basis. The amount of calcium, phosphorus, potassium, sodium and magnesium in Azolla were 11.0, 6.1, 17.4, 9.0 and 5.0 g/kg dry matter while the content of trace minerals like manganese, zinc, copper and iron were 0.76, 0.038, 0.016 and 3.90 mg/kg of dry matter respectively, indicating to be a rich source of micro nutrients. Out of 18 nos. of amino acids in Azolla, glutamic acid gave the highest amount of 12.6% followed by aspartic acid (9.3%), leucine (8.4%), alanine (6.4%), arginine (5.9%), glycine (5.6%) and valine (5.5%). However, the amount of cystine was found lowest (1.6%) followed by tryptophan (1.8%). Key words
Azolla, CP, CF, NDF, ADF, minerals, amino acid
Azolla commonly known as mosquito fern, duckweed fern, fairy moss or water fern, is a symbiotic association of Azolla-Anabaena in which the endophytic blue-green alga Anabaena azollae lives within the leaf cavities of the water fern Azolla (Lain). Azolla-Anabaena symbiosis is outstanding due to its high biomass production along with its ability to fix atmospheric nitrogen at higher rate. It is a rich source of protein (20-25%), minerals (Ca, Mg, Fe, P, K), vitamins and carotenoid (20-30%). It contains almost all the essential amino acids.
That’s why Azolla is known as the ‘green gold mine’ of the nature. Previously, Azolla was used only as bio-fertilizer in paddy field. But, now-a-days it is widely used as feed for cattle, ducks, poultry, pig and fishes in many countries (Lumpkin and Plucknett 1980). The main parameter responsible for the value of Azolla as feed or fodder of the livestock and fishes is its amino acids content followed by its other compounds like crude protein, fibre and digestible carbohydrates. While lot of researches were involved in maintaining and improving its quality as green manure or bio-fertilizer in paddy crop, the potentiality of Azolla as a good source of amino acids and protein in the livestock feed fetched very little attention of the researchers. The objective of the present study is to analyze the amino acid content of Azolla so that it can be an excellent dietary component of feeds for livestock and fishes.
Role of amino acids Amino acids are generally classified as nutritionally essential (indispensable) or nonessential (dispensable) for animals and humans based on growth or nitrogen balance namely net synthesis of protein in the whole body (Baker, 2009; Wu, 2009). In case of essential amino acids, carbon skeletons are not synthesized by animal cells and therefore, they must be provided from the diet. On the other hand, nonessential amino acids are generally synthesized by the animal cells in a species-dependent manner (Wu. et. al, 2009). It was tactically assumed, without much evidence, that animals or humans could synthesize sufficient amounts of almost all the nonessential amino acids and did not need them in diets for optimal nutrition or health. However, sufficient number of studies on human cell culture and animal cell shows that some of the classified nonessential amino acids like glutamine, glutamate, arginine etc. play important roles in multiple signaling pathways, thereby regulating gene expression, intracellular protein
ROY et al., Estimation of Amino Acids, Minerals and Other Chemical Compositions of Azolla
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Table 1. Methods of proximate analysis Parameters
Methods
Moisture
Loss on drying (103-104°C for 5 hours)
Dry Matter
By calculation
Crude Protein
Crude Protein in animal and pet food (Kjeldahl method)
Crude Fat
Oil in cereal adjuncts (Petroleum ether method)
Crude Fibre
Crude Fibre in animal and pet food (F.G Crucible)
Ash
Ignition at 600°C for 2 hours
turnover, nutrient metabolism, and oxidative defense (Yao et al. 2008). However, classification of amino acids based on nutrition as essential and nonessential has some limitations in the nutrition of protein. It is a matter of relaxation that some medical Institute and organizations do not suggest dietary requirement of nonessential amino acids for livestock and human beings. It was found that some nonessential amino acids like glutamine and arginine, play vital roles in regulating gene expression at both transcriptional and translational levels (Haynes et al. 2009 and Stipanuk et al. 2006). Many literature also shows that some nonessential amino acids take part in cell signaling via mTOR, extracellular signalrelated kinase, Jun kinase, mitogen-activated protein kinase, and gases (NO, CO, and H2S) (Marc and Wu. 2009 and Kimura 2010). Integrated action of these regulatory activities has profound effects on cell proliferation, differentiation, metabolism, homeostasis, survival, and function. Essential amino acid like leucine and tryptophan attain much importance as they activates mTOR to stimulate protein synthesis and inhibit proteolysis and modulates neurological and immunological functions through multiple metabolites including
serotonin and melatonin (Bazer et al. 2010 and Suryawan el al. 2009). This is due to the fact that dietary amino acids are substantially catabolized by the small intestine and the content of amino acids in the diet differs remarkedly from those in skeletal muscle or the body (Wu, 2009). Therefore it can be remarked that the classic concept of ‘the ideal protein’ based solely on amount of essential amino acids in tissue proteins (Baker 2009), should be reoriented by including all nonessential amino acids. It can be summarized that both essential and non-essential amino acids should be taken into consideration in: a)
Formulation of balanced diets to maximize the growth of livestock, poultry, and fish
b)
Recommendation of amino acids requirements for humans to prevent growth retardation and chronic diseases Optimizations of immune and reproductive functions in all species including human beings.
c)
MATERIALS AND METHOD Proximate Analysis of sundried Azolla: a)
The representative sample (20 nos.) of Azolla
Table 2. Chemical compositions of Azolla Parameters Dry matter
Values in % of dry matter 8.0
Crude protein
24.61
Crude fibre
15.0
NDF
43.8
ADF
31.8
Lignin
11.4
Ether extract
3.8
Ash
15.9
Starch
4.1
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Table 3. Mineral content of Azolla Minerals
Values in g/kg of dry matter
Calcium
11.0
Phosphorus
6.1
Potassium
17.4
Sodium
9.0
Magnesium
5.0
Manganese
0.76*
Zinc
0.038*
Copper
0.016 *
Iron
3.90 *
* mg/kg of dry matter
b)
(Azolla pinnata Lain.) was analyzed for proximate principles i.e. moisture, crude protein, ether extract, crude fibre, total ash etc. as per the methods describes by AOAC (1995) in table 1. Fibre fraction viz. NDF, ADF, Cellulose and Hemicelluloses of feeds and faeces were done as per the method Van Soest et al. (1991)
(1995) including modification described by Wolzak et al. (1985). Quantification of aflatoxin B1 was done by using indirect Enzyme Linked Immuno Sorbent Assay (ELISA) method according to the method applied by Riedel de Haen (1997).
RESULTS AND DISCUSSION
Minerals estimation:
Chemical compositions of Azolla
a)
Calcium and total phosphorus content were determined by modified methods of Talapatra et al. (1940).
b)
Sodium and Potassium Dry Oxidation method in Flame Photometer (Systronics 130) Magnesium, copper, zinc, manganese and iron contents were estimated by dry oxidation method (as described in Perkin Elmer A Analyst 100 Atomic Absorption Spectrometer analytical methods).
Form the experimental data it was found that Azolla contained 8.0% dry matter (DM). Parashuramulu et al. (2013), also got 8.7 % of dry matter in Azolla. The result of proximate analysis is given in Table 2. Azolla contained 24.61% crude protein (CP), 15% crude fibre (CF), 43.8% neutral detergent fibre (NDF), 31.80% acid detergent fibre (ADF), 3.8% ethyle extract (EE), 11.4% lignin, 4.1% starch and 15.9% total ash on dry matter basis. The proximate composition of Azolla obtained in the present study was in similar range to values obtained by Ahirwar and Leela (2012) and Parashuramulu et al. (2013). The content of CP is lower than values reported by Basak et al. (2002) and Nwanna and Falaye (1997) but higher than 17.59% CP reported by Querubin et al. (1986) for a strain of Azolla. VanHove and Lopez (1982) noted that the crude protein content of Azolla might vary from 13.0 to 34.5%.
c)
Amino acids analysis in sun dried Azolla: Amino acids in sun dried Azolla (20 nos. sample) was analysed as per the method described by Rutherfurd and Mougham (2000) with the help of Gas Chromatography and Capillary Electrophoresis by wet chemistry (hydrolysis) method. The contents of the different amino acids recovered were expressed as % of protein and subsequently as % of dry matter of Azolla.
Estimation of Aflatoxin B1 in sun dried Azolla: The determination of aflatoxin B1 in sun dried Azolla was done according to the method AOAC
Minerals content of Azolla The amount of calcium, phosphorus, potassium, sodium and magnesium in Azolla were 11.0, 6.1, 17.4, 9.0 and 5.0 g/kg dry matter while the content of manganese, zinc, copper and iron
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2695
Table 4. Amino acid content of Azolla Amino acids
Values as % protein
Values as % of dry matter
Alanine
6.4
1.52
Arginine
5.9
1.45
Aspartic acid
9.3
2.37
Cystine
1.6
0.19
Glutamic acid
12.6
2.73
Glycine
5.6
1.40
Histidine
2.1
0.47
Isoleucine
4.5
1.20
Leucine
8.4
2.15
Lysine
4.7
1.02
Methionine Phenylalanine
1.4 5.4
0.43 1.41
Proline
4.9
1.19
Serine Threonine
4.5 4.7
1.17 1.14
Tryptophan
1.8
0.40
Tyrosine
3.6
0.95
Valine
5.5
1.45
were 0.76, 0.038, 0.016 and 3.90 mg/kg of dry matter respectively, indicating to be a rich source of micro nutrients. The CP was comparable, while crude fibre (CF) content was lower in Azolla in comparison to Lucerne (16-25% CP and 20-30% CF, ICAR, 1998). Thus it indicates that Azolla could be good source of protein having low fibre content compared to legume forages.
Amino acids content of Azolla From the analysis amino acids it was found that Azolla contains as much as 18 nos. of amino acids. Among the various amino acids, glutamic acid gave the highest amount of 12.6% (% of protein) followed by aspartic acid (9.3%), leucine (8.4%), alanine (6.4%), arginine (5.9%), glycine (5.6%) and valine (5.5%). However, the amount of cystine was found lowest (1.6%) followed by tryptophan (1.8%). Data on the amino acid analysis as given by FAO (2015) indicate that lysine, arginine, isoleucine, leucine, phenylalanine, glycine and valine were predominant and the sulphurcontaining amino acids did not meet the recommended (FAO, 1973) value of 3.5g/100g protein. Therefore replacement of conventional high quality ingredients in diets for non-ruminant animals should give recognition to differences in lysine
content and deficiencies of the sulphur-containing amino acids. Sanginga and Van Hove (1989) have indicated that these limiting amino acids must be added to make Azolla a complete source of amino acids.
Aflatoxin content of Azolla Quantification of aflatoxin B1 was done by using indirect Enzyme Linked Immune Sorbent Assay (ELISA) method according to the method applied by Riedel de Haen (1997). The detection limit was 0.01 microgram/kg.
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Accepted on 26-03-2016
