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Composition, functional properties and in vitro antioxidative activities of protein hydrolysates prepared from muscle of sardinelle (Sardinella aurita) were investigated. Sardinelle protein hydrolysates (SPH) were obtained by treatment with crude enzyme preparations from Bacillus pumilus A1 (SPHA1), Bacillus mojavensis A21 (SPHA21) and crude enzyme extract from sardinelle (Sardinella aurita) viscera (SPHEE). The protein hydrolysates SPHA1, SPHA21 and SPHEE contained high protein content 79.1%, 78.25% and 74.37%, respectively. The protein hydrolysates had an excellent solubility and possessed interfacial properties, which were governed by their concentrations. The antioxidant activities of protein hydrolysates at different concentrations were evaluated using various in vitro antioxidant assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, chelating activity, β-carotene bleaching and DNA nicking assay. All protein hydrolysates showed varying degrees of antioxidant activity. SPHA21 had the highest DPPH radical scavenging activity (89% at 6 mg/ml) and higher ability to prevent bleaching of β-carotene than SPHA1 and SPHEE (p<0.05). However, SPHEE exhibited the highest metal chelating activity (89% at 1 mg/ml) and the strongest protection against hydroxyl radical induced DNA breakage (p<0.05).

BACKGROUND: In Tunisia, sardinelle (Sardinella aurita) catches totalled about 13 300 t in 2002. During processing, solid wastes including heads and viscera are generated, representing about 30% of the original raw material. Viscera, one of the most important by-products of the fishing industry, are recognised as a potential source of digestive enzymes, especially proteases with high activity over a wide range of pH and temperature conditions. This paper describes the purification procedure and some biochemical characterisation of trypsin from S. aurita viscera.

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The antioxidant activities of protein hydrolysates prepared from heads and/or viscera of sardinelle (Sardinella aurita) by treatment with different proteases were evaluated using various in vitro antioxidant assays. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activity. The hydrolysates obtained by treatment with crude enzyme from Mustelus mustelus intestines exhibited the highest radical‐scavenging activity. However, Alcalase hydrolysates displayed the greater reducing power activities. Further, sardinelle heads protein hydrolysates were found to strongly suppress the discoloration of β‐carotene compared with control. Both Alcalase protein hydrolysates obtained from heads or viscera were then fractionated by size exclusion chromatography on a Sephadex G‐25, into two and four major peptide fractions, respectively. All fractions exhibited antioxidant activity, and fraction P4 with molecular mass around 3.5 kDa from sardinelle viscera protein hydrolysates was found to exhibit the highest radical‐scavenging activity. PRACTICAL APPLICATIONS In Tunisia, sardinelle (Sardinella aurita) catches were about 13,300 tons in 2002. During processing, solid wastes including heads and viscera are generated and can be seen as 30% of the original raw material. These by‐products constitute an important source of proteins. An interesting alternative to valorize these by‐products is to transform sardinelle proteins by‐products into biologically active peptides by protease treatments. The results clearly demonstrated that sardinelle by‐products protein hydrolysates have excellent antioxidant activities, and thus, they have great potential as a source for natural antioxidants.

Three trypsin isoforms A, B and C were purified to homogeneity from the viscera of sardinelle (Sardinella aurita). Purification was achieved by ammonium sulfate precipitation (20-70% (w/v)), Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography. The molecular weights of these purified enzymes were estimated to be 28.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Based on the native PAGE and casein-zymography, each purified trypsin appeared as a single band. Trypsins A and C exhibited the maximal activity at 55°C, while trypsin B at 50°C. All isoforms showed the same optimal pH (pH 9.0) using Nα-benzoyl-DL: -arginine-p-nitroanilide (BAPNA) as a substrate. The three trypsins were stable at temperatures below 40°C and over a broad pH range (7.0-11.0). The activities of the three isoforms were strongly inhibited by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, a serine protease inhibitor, and partially inhibited by ethylenediaminetetraacetic acid, a metalloenzyme inhibitor. Kinetic constants of trypsins A, B and C for BAPNA were evaluated at 25°C and pH 9.0. The values of K (m) and k (cat) were 0.125, 0.083 and 0.10 mM, and 2.24, 1.21 and 5.76 s(-1), respectively. The N-terminal sequences of the first 10 amino acids were "I V G G Y E C Q K Y" for trypsin A and "I V G G Y E A Q S Y" for trypsins B and C. These sequences showed highly homology to other fish trypsins.