Animal Feed Science and Technology 105 (2003) 199204

Short communication

Amino acid composition of processed sh silage using different raw materials

Rose Meire Vidotti a, , Elisabete Maria Macedo Viegas a , Dalton Jos Carneiro b
b

Departamento de Zootecnia da Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de So Paulo, P.O. Box 23, ZIP 13.635-900 Pirassununga, SP, Brazil Centro de Aqicultura, Universidade Estadual Paulista, Campus de Jaboticabal, Via de Acesso Prof. Paulo Donato Castellane, s/n., Km 5. CEP, 14884-900 Jaboticabal, SP, Brazil

Received 7 January 2002; received in revised form 4 February 2003; accepted 4 February 2003

Abstract The objective was to evaluate amino acid composition of silages produced from three raw materials. Commercial marine sh waste, commercial freshwater sh waste, and tilapia lleting residue were used to produce sh silage by acid digestion (20 ml/kg formic acid and 20 ml/kg sulfuric acid) and anaerobic fermentation (50 g/kg Lactobacillus plantarum, 150 g/kg sugar cane molasses). Protein content and amino acid composition were determined for raw materials and silage. Marine sh waste had higher crude protein content (776.7 g/kg) compared to freshwater sh waste (496.2 g/kg) and tilapia lleting residue (429.9 g/kg). All silages lacked up to three amino acids for each product according to FAO standards for essential amino acids. However, considering as the limiting factor only the amino acids below the 30% minimum requirement for sh in general, all products were satisfactory with respect to essential amino acids. Therefore, the results suggest that all products investigated are appropriate for use in balanced sh diets. 2003 Elsevier Science B.V. All rights reserved.
Keywords: Essential amino acids; Fish silages; Fermented silage; Acid silage

1. Introduction Large quantities of sh are wasted in tropical countries during capture, commercialization, and industrialization processes. Methodologies to use these materials as a protein source for human consumption are necessary and urgent. Several studies are being
Corresponding author. Tel.: +55-1935-654245; fax: +55-1935-654114. E-mail addresses: rosevi@usp.br (R.M. Vidotti), emviegas@usp.br (E.M.M. Viegas), daltonjc@caunesp.unesp.br (D.J. Carneiro).

0377-8401/03/$ see front matter 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0377-8401(03)00056-7

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conducted with this objective; in the meantime these materials are commonly used in animal product food. Fish waste is used to produce shmeal, but sh silage may be a feasible alternative for use of these residues (Disney et al., 1977). By-products from the shing industry have great potential to be used as protein supplements in aquaculture feeds. The nutritional value of a balanced diet is determined mainly by essential amino acid composition and ratio. Vegetable protein sources are often decient in some essential amino acids. However, the composition of these ingredients may be improved by adding protein rich products such as shmeal, silages, or hydrolysate. Fish silage is an excellent protein product of high biological value for animal feeding, which can be produced from dead sh, sub-utilized species, by-products from marine shing, commercial sh waste and industrial residues. These are considered low quality raw materials, that if not used may cause environmental, health, and economical problems. During silage processing, enzymes found in muscles hydrolyze proteins and nitrogen becomes more soluble. Proteins are hydrolyzed to free amino acids, thus making silage the most available amino acid source for protein biosynthesis (Espe et al., 1989). The objective was to evaluate amino acid composition of silage produced from three raw materials (commercial marine sh waste, commercial freshwater sh waste, and tilapia lleting residue), as ingredients for use in sh diets.

2. Materials and methods 2.1. Silage production Initially, an experiment to produce acid and fermented silage was carried out using three types of raw materials: commercial waste (sh unt for human consumption, but not yet decomposing) of saltwater (SW) and freshwater sh (FW), as well as residue from the Nile tilapia lleting industry (TR). These residues were ground using an industrial meat-grinding machine, C.A.F. Picadores de Carne, model 22S, motor 1 1/4 CV, cylindrical shape with approximately 30 mm diameter and 30 mm length. To obtain fermented silage (F), the following mixture was added to the raw material in a w/w basis: 15% sugar cane molasses, 5% Lactobacillus plantarum culture material (108 109 CFU/g) and 0.25% sorbic acid (w/w), as a fungicide. To obtain acid silage (A), the following mixture was added to the raw material: 2% formic acid (w/v) and 2% sulfuric acid (w/v). Silages were then stored at room temperature (2832 C) in 3 l plastic buckets with closed lids. For the rst 10 days, pH was measured every other day, and thereafter on the 17th, 24th and 31st days. On the 9th day after processing, acid silage pH stabilized at 2.50, 2.78 and 2.82 (freshwater, saltwater sh and tilapia residue, respectively) and fermented silage pH at 4.06, 4.26 and 4.48 (lleting residue, freshwater and saltwater sh, respectively).

3. Raw material and silage characterization Samples of raw material and silages were lyophilized for determination of crude protein and amino acid composition. Crude protein content of the raw materials and

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silages were determined by the micro-Kjedahl method, according to AOACcode 981.10 (1990). Amino acid composition of the ensiled material and silages was determined by liquid chromatography, using a cationic exchange resin column and ninhidrine post-column derivation in auto-analyzer. These analyses were carried out, in duplicate, at Laboratorio de Quimica de Proteinas, in Faculdade de Medicina de Ribeirao Preto, USP. Samples were hydrolyzed with HCl 6N, for 22 h at 110 C to quantify amino acids as described by Moore and Stein (1963). Since acid hydrolysis destroys tryptophan, samples were also hydrolyzed with lithium hydroxide 4N to determine tryptophan, as described by Lucas and Sotel (1980). 4. Results and discussion Silage protein content was lower than the protein content of the raw materials (Table 1). Similar results were also observed by Morales-Ulloa and Oetterer (1997) for biological
Table 1 Amino acid composition (g/100 g CP) and protein levels of commercial saltwater, freshwater sh waste, tilapia lleting residue and sh silages Amino acids Tryptophan Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine 1/2 Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine CPj (g/kg)
a b

SWa 0.79 10.12 5.24 3.03 9.05 2.85 2.71 13.57 3.19 6.49 8.60 0.81 6.42 6.88 5.31 9.16 1.78 3.99 776.7

FSWb 0.65 9.16 5.85 2.19 10.79 4.97 3.23 14.45 3.66 5.87 7.41 0.69 5.77 6.03 5.05 8.00 1.90 4.32 596.1

ASWc 0.66 7.90 5.70 6.11 7.83 4.58 4.49 14.04 5.74 8.17 7.39 1.54 4.16 3.75 3.10 7.33 3.45 4.08 699.1

FWd 0.97 7.48 2.65 3.62 10.17 3.18 3.39 16.18 4.37 6.20 9.27 0.97 5.95 3.19 5.38 9.61 2.40 5.02 496.2

FFWe 0.87 9.92 3.08 1.80 9.62 5.12 3.52 13.83 5.57 6.32 8.12 1.03 5.83 4.97 5.00 9.31 2.02 4.07 420.9

AFWf 1.34 9.09 2.75 7.72 6.20 5.28 5.53 9.26 7.78 11.55 6.00 0.63 3.92 5.31 3.10 7.57 2.73 4.26 443.8

TRg 0.52 9.75 2.02 2.46 10.16 2.76 2.04 13.88 7.75 7.50 8.81 1.40 6.62 2.80 6.24 10.32 1.22 3.76 429.9

FTRh 0.61 5.94 2.52 2.49 11.79 4.68 3.72 14.76 7.22 9.22 8.92 0.86 5.06 5.54 4.63 6.72 1.70 3.63 358.4

ATRi 0.43 6.77 2.20 7.27 8.98 4.72 5.11 13.10 5.94 12.32 7.63 1.34 4.31 5.37 2.51 6.23 2.43 3.35 395.9

SW, commercial saltwater sh waste. FSW, fermented saltwater sh silage. c ASW, acid saltwater sh silage. d FW, commercial freshwater sh waste. e FFW, fermented freshwater sh silage. f AFW, acid freshwater sh silage. g TR, tilapia lleting residue. h FTR, fermented tilapia residue silage. i ATR, acid tilapia residue silage. j CP, crude protein (dry matter).

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and enzymatic chemical silages. Acid silages produced from whole sh had higher protein content 69.91% (saltwater), 44.38% (freshwater) and 39.59% (tilapia residue) compared to fermented silages, 59.61% (SW), 42.09% (FW) and 35.84% (TR), respectively. The lower crude protein values obtained for fermented silages may be attributed to the addition of 15% sugar cane molasses and 5% L. plantarum; therefore, proportionally diminishing crude protein levels in the nal product. Although less markedly, crude protein levels also diminished in the acid silages since less acid was added to the raw materials (4%). Amino acid composition did not display a homogeneous trend, levels uctuated compared to raw materials. A similar result was observed by Morales-Ulloa and Oetterer (1997) and Arason (1994). The comparison of amino acid composition of silages and raw materials (Table 1) showed an increase in histidine, threonine and serine levels, for both processes and all three raw materials used, where as levels of valine, isoleucine, and leucine decreased in all products. This reduction might have occurred during ensiling due to chemical reactions between amino and aldehyde groups present in amino acids (Johnson et al., 1985). Table 2 compares silage chemical scores with FAO/WHO (1985) standards and Nile tilapia requirements (Oreochromis niloticus), listed by NRC (1993). Results show that
Table 2 Chemical score of the main limiting amino acids according to FAO/WHO standards (1985)a and the requirements for Nile tilapia according to NRC (1993)b Silages TRP ARG 0.44 1.22 0.32 1.54 0.50 1.45 0.52 1.45 0.43 1.84 0.59 1.73 VAL 1.15 0.83 1.17 0.78 1.01 0.86 2.06 1.48 2.08 1.40 1.81 1.54 MET 1.72 1.07 1.42 1.52 1.58 1.53 2.25 1.40 1.85 1.98 2.07 2.00 ILE 1.26 0.78 1.25 0.77 1.16 0.63 1.62 1.00 1.61 1.00 1.49 0.81 LYS 1.66 1.44 1.80 1.65 1.08 1.28 1.78 1.54 1.93 1.77 1.16 1.32 LEU 1.14 1.05 1.33 1.08 0.96 0.89 2.36 2.16 2.75 2.23 1.98 1.84

FAO/WHO standards (1985) 0.65d FSWc ASWe 0.66 0.87 FFWf AFWg 1.34 0.61 FTRh ATRi 0.43 NRC standard (1993) FSW 0.65 ASW 0.66 FFW 0.87 AFW 1.34 FTR 0.64 ATR 0.43

a Essential amino acids of the standard protein according to FAO/WHO (1985): tryptophan 1.00, lysine 5.50, histidine 2.00, arginine 5.00, threonine 4.00, valine 5.00, methionine 3.50, isoleucine 4.00, leucine 7.00 and phenylalanine + tyrosine 4.29. b Essential amino acids requirements of Nile tilapia according to NRC (1993): tryptophan 1.00, lysine 5.12, histidine 1.72, arginine 4.20, threonine 3.75, valine 2.80, methionine 2.68, isoleucine 3.11, leucine 3.39 and phenylalanine + tyrosine 3.75. c FSW, fermented saltwater sh silage. d Chemical score = (g amino acid/100 g test protein)/(g amino acid/100 g standard protein). e ASW, acid saltwater sh silage. f FFW, fermented freshwater sh silage. g AFW, acid freshwater sh silage. h FTR, fermented tilapia residue silage. i ATR, acid tilapia residue silage.

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lysine, histidine, methionine and phenylalanine + tyrosine are present in higher concentrations, where as valine and isoleucine are decient in acid silages and arginine is decient in fermented silages. Tryptophan level was above FAO and NRC standards only in acid silage from whole freshwater sh. The nutritive value of protein of any ingredient depends mainly on the proteins capacity to fulll the needs of organisms with respect to essential amino acids. A parameter called chemical score has been used to evaluate the nutritive value of a protein. This parameter compares levels of essential amino acids between the test and standard proteins (Sgarbieri, 1987). In the current study, the chemical scores of each product were calculated using FAO/WHO standard (1985) and Nile tilapia requirements listed by NRC (1993) as references. From the results it can be seen that humid fermented silages presented arginine as the limiting amino acid (the lowest chemical score). On the other hand with the exception of freshwater sh acid silage (Table 1), tryptophan was the limiting amino acid in acid silages. The chemical scores of silages showed that tryptophan is the limiting amino acid. There are indications that tryptophan is unstable in acid medium, therefore it becomes the rst limiting amino acid in sh silage (Arason, 1994). According to Strom and Eggum (1981) lysine, cystine, and methionine are the most important amino acids for sh from a nutritional viewpoint. From the results presented, all the silages satisfy amino acid requirements according to FAO and Nile tilapia standards (NRC, 1993). Furthermore, when considering amino acid limiting level, the 30% minimum requirement in sh diets rather than FAO standards, this study shows that all silages fullled this requirement (Tacon, 1994). Finally, results suggest that humid silages, in spite of minor deciencies in certain essential amino acids, do not lose their nutritional value. This fact is even more important if silages are to be considered as an ingredient in balanced diets.

Acknowledgements The authors wish to thank FAPESP, Fundacao de Amparo a Pesquisa e Desenvolvimento do Estado de Sao Paulo, for the nancial support that made the execution of this work possible. Thanks are also due to CAUNESP, Centro de Aquicultura da Universidade Estadual Paulista, where the experiments were conducted. References
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