Extraction and fractionation of lipolytic enzyme from viscera of Monterey sardine (Sardinops sagax caerulea)

In this study, lipolytic activity of a semi-purified lipolytic enzyme (SLE) from the viscera of sardine ( Sardinops sagax caerulea ) was screened on the lipolysis of olive, Menhaden and sardine oil. A lipolytic enzyme was partially purified from the crude extract of sardine viscera by fractional precipitation followed by ultrafiltration (30 kDa). The main tissues found in sardine viscera were pyloric caeca (19.0% w/w), digestive tract (13.0% w/w), liver (4.8% w/w) and pancreas (1.5% w/w). Results show that pancreas had the highest lipolytic activity. There were no significant differences in lipolytic activity between pyloric caeca, intestine and liver ( P  < 0.05). Specific activity of the SLE increased 47.0-fold after extraction and fractionation, with a yield of 0.34% calculated for the whole viscera weight. Lipolytic activity of SLE from sardine viscera increased threefold when sardine oil was used as substrate. The results of this study confirm the potential importance of lipases from marine sources.     

The effects of the combination of freezing and the use of natural antioxidant technology on the quality of frozen sardine fillets (Sardinella aurita)

The effects of combination of freezing and the use of antioxidant technology on the quality of frozen sardine fillets were investigated in terms of sensory, biochemical [thiobarbituric acid (TBA), total volatile basic nitrogen (TVB‐N), peroxide value (PV) and free fatty acids (FFA)] and microbiological analyses [total viable count (TVC)]. Fish were filleted and divided into three groups. The first group was used as the control (C) without rosemary extract, the second group was treated with 1% rosemary extracts for 2 min (R1) and the third was treated with 2% rosemary extracts for 2 min (R2). All groups were frozen at ?18 °C over the storage period of 6 months. The results obtained from this study showed that the combination of antioxidant and frozen storage resulted in significant reduction of bacterial growth and stabilised the biochemical characteristics, especially for R2. However, the use of antioxidant at the level of 2% (R2) gave a bitter taste according to sensory assessment whereas the panellists mostly preferred R1.     

Effect of ice storage on the functional properties of pink perch and oil sardine meat

Ice storage of dressed pink perch (Nemipterus japonicus) and Indian oil sardine (Sardinella longiceps) for 16 and 20 days, respectively, resulted in a decrease in emulsifying capacity (EC), protein solubility (PS), relative viscosity (RV) of salt‐soluble proteins (SSP) and water‐soluble proteins (WSP), water binding capacity (WBC) in terms of absorbed moisture in water (AM_w), absorbed moisture in brine lpar;AM_b), retained moisture in water (RM_w) and retained moisture in brine (RM_b), WSP and SSP, and increase in cook loss (CL). Decrease in protein solubility influenced the EC, RV, CL and WBC in both the species of fish. Significant (P<0.05) correlations existed among various functional properties analysed, in both the fishes during the ice storage. © 1999 Society of Chemical Industry     

Purification and biochemical characterization of chymotrypsin from the viscera of Monterey sardine (Sardinops sagax caeruleus)

Chymotrypsin was isolated from the viscera of Monterey sardine by ammonium sulphate fractionation, gel filtration, and ionic exchange chromatography. The approximate molecular weight was 26,000 and its isoelectric point was about 5. Identity as chymotrypsin was established by its catalytic specificity for amide or ester bonds on the synthetic substrates succinyl-l-ala-ala-pro-l-pheilalanine-p-nitroanilide and benzoyl-l-tyrosine-ethyl-ester, showing esterase activity 3.2-fold higher than amidase. It was inhibited by phenylmethylsulfonyl-fluoride and soybean trypsin inhibitor, partly inhibited by the specific chymotrypsin inhibitor N-toluenesulfonyl-l-phenylalanine chloromethyl-ketone, but not inhibited by EDTA or Benzamidine. Chymotrypsin showed its maximum activity at pH 8.0 and 50 °C for the hydrolysis of SAAPNA. The Michaelis–Menten constant was 0.074 mM with a catalysis constant of 18.6 seg⁻¹, and catalytic efficiency of 252 seg⁻¹ mM⁻¹. Results indicated that Monterey sardine chymotrypsin is a good catalyst and could be used as a biotechnological tool in food processing and using sardine industry wastes as a material for production of fine reagents.     

Preparation of eicosapentaenoic acid concentrates from sardine oil by Bacillus circulans lipase

An extracellular lipase derived from Bacillus circulans, isolated from marine macroalga, Turbinaraia conoides, was used to prepare n-3 polyunsaturated fatty acid (PUFA) concentrates from sardine oil triglycerides. The enzyme was purified 132-fold with specific activity of 386 LU/mg. The purified lipase was able to enrich sardine oil with 37.7 ± 1.98% 20:5n-3 and 5.11 ± 0.14% 18:3n-3 in the triglyceride fraction after 3 h of hydrolysis. Lower hydrophobic constants of n-3 fatty acids (18:3n-3_logP = 5.65; 20:5n-3_logP = 5.85, respectively) than n-6 (20:4n-6_logP = 6.16) resulted in higher hydrolytic resistance of the former toward lipase, leading to their enrichment in the triglyceride fraction. Lipase-catalysed hydrolysis of sardine oil for 3 h, followed by urea complexation, provided free fatty acids containing 51.3 ± 4.65% 20:5n-3. The purified methyl ester of 20:5n-3 (68.29 ± 2.15%) from the urea concentrate was attained by chromatography on argentated neutral alumina.     

Thermoactivity and effects of organic solvents on digestive lipase from hepatopancreas of the green crab

Unlike classical digestive lipases, the crab digestive lipase (CDL) displayed its maximal activity at a high temperature. The CDL activity’s optimal temperature, when using emulsified or monomolecular film as substrate, was 60 °C. To our knowledge, this is the first report of an animal digestive lipase having such an optimal temperature. The maximum activity of CDL appeared at pH 8. Lipase activity was compatible with the presence of organic solvents, except for butanol. Furthermore, the hydrolysis was found to be specifically dependent on the presence of Ca²⁺ ions, since no significant CDL activity was detected in the presence of ion chelators such as EDTA. Nevertheless, the CDL does not require Ca²⁺ to trigger the hydrolysis of tributyrin emulsion. Interestingly, Zn²⁺ and Cu²⁺ ions acted as strong inhibitors of CDL activity when using tributyrin as substrate. Lipase stability in the presence of organic solvents, as well as at high temperatures, makes it a good candidate for application in non-aqueous catalysis.     

Effectiveness of rutin and its lipophilic ester in improving oxidative stability of sardine oil containing trace water

Poor oxidative stability exhibited by n‐3 polyunsaturated fatty acid rich sardine oil is a major challenge for its utilisation in industry. Considering the fact that water is always present in bulk oil in trace amounts during storage, an effort was made to understand and compare the effectiveness of rutin and its corresponding lipophilic ester in enhancing oxidative stability of refined sardine oil containing trace water (0.16% w/w). Peroxide value, conjugated diene value, p‐anisidine value and thiobarbituric acid reactive substances (TBARS) value were determined during 20 days storage. Rutin fatty ester showed 50% reduction in primary oxidation and 42.46% reduction in secondary oxidation, whereas rutin showed 20.6% and 20.43% reduction in primary and secondary oxidation, respectively, by the end of 20 days storage. Thus, it is clearly established that rutin fatty ester is more effective than hydrophilic rutin in sardine oil containing trace water, which contradicts the polar paradox theory.     

The presence of bioactive peptides in hydrolysates prepared from processing waste of sardine (Sardina pilchardus)

A proteolytic enzyme, Alcalase®, was used to produce partly digested proteins from cooked wastes of sardine (Sardina pilchardus). The presence of biologically active molecules was investigated in these hydrolysates prepared under various conditions of time and enzyme/substrate ratio. By means of radioimmunoassays and mitogenic and radioreceptor assays, the presence of molecules related to secretagogue peptides, growth factors and calcitonin gene‐related peptide (CGRP) respectively was detected in the hydrolysates. Exclusion chromatography permits the conclusion that the biological activity of the different fractions is related to their size. © 2001 Society of Chemical Industry     

Physical,chemical and sensory analysis of sardine (Sardina pilchardus) stored in ice

The shelf-life of iced sardine (Sardina pilchardus) was studied. The main changes which take place in raw fish were investigated by means of organoleptic assessments, chemical analyses (total volatile nitrogen (TVB-N), trimethylamine (TMA-N), trimethylamine oxide (OTMA-N) and hypoxanthine) and physical measurements (GR Torrymeter readings and pH). The influence of both seasonal changes and fat deterioration were also considered. The results obtained indicate that TVB-N and TMA-N parameters are not good freshness indicators for this species, but Torrymeter readings and hypoxanthine values can be used as indicators of freshness. However, they must always be confirmed by a sensory evaluation. From the different combinations tried, the most highly significant degree of correlation was obtained between sensory evaluation and Torrymeter readings.     

The impact of applying natural clinoptilolite (zeolite) on the chemical,sensory and microbiological changes of vacuum packed sardine fillets

Different doses (1% and 5%) of natural zeolite were applied to determine quality changes in vacuum packaged sardine fillets during 19 days at 4 ± 1 °C. Zeolite had an effect to improve sensory quality of sardine especially for removing off‐odour. The acceptable shelf life of vacuum packaged sardine was 8 days for control and 12 days for groups treated with 1% and 5% zeolite. The zeolite application resulted in significant reduction in total volatile basic nitrogen values, except for group treated with 5% zeolite at 15 days. Although the effect of zeolite depended on dose and specific storage days, application of zeolite had no effect on free fatty acid analysis. The use of zeolite significantly reduced ammonia and biogenic amine accumulation, especially for histamine and tyramine. The result of the study showed that the efficacy of zeolite as natural antimicrobials was high and lower dose of zeolite has to be applied to get maximum preservation effect.     

Effects of rosemary and sage tea extract on biogenic amines formation of sardine (Sardina pilchardus) fillets

Natural antimicrobials and antioxidants from rosemary (Rosmarinus officinalis) and sage tea (Salvia officinalis) were produced using solvent extraction method. The effect of two extracts on ammonia (AMN) and biogenic amines (BAs) formation in vacuum packed sardine (Sardina pilchardus) fillets stored at 3 ± 1 °C was investigated for 20 days. Although the effect of extracts was dependent on specific amine and storage time, phenolic compounds from rosemary and sage tea generally resulted in lower AMN and BAs accumulation in sardine muscle. Putrescine (PUT) and cadaverine (CAD) were the most abundant amines, while histamine (HIS) concentration ranged from 2.05 to 28.77 mg 100g^?1. Rosemary and sage tea extracts significantly reduced HIS, PUT, CAD and trimethylamine accumulation in the fish muscle (P < 0.05) while stimulating effect of extracts was observed on serotonin and agmatine formation. At the end of the storage period, PUT and CAD contents of control were 100‐fold higher than those of treated groups.     

Response surface modelling of the production of structured lipids from soybean oil using Rhizomucor miehei lipase

The production of structured lipids (SLs) by the acidolysis of soybean oil (SO) with a free fatty acid (FFA) mixture obtained from Brazilian sardine oil, catalysed by Rhizomucor miehei lipase (Lipozyme RM IM) in a solvent-free medium, was optimised by response surface methodology (RSM) using a three-factor central composite rotatable design. The best reaction conditions to achieve an adequate n-6/n-3 FA ratio were: sardine-FFA:SO mole ratio of 3:1, initial water content of the enzyme of 0.87% w/w, reaction time of 12 h, reaction temperature of 40 °C and 10% by weight of the enzyme (% w/w). Under these conditions, the incorporation of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) into the soybean oil reached 9.2% (% of the total FAs), leading to a significant reduction in the n-6/n-3 FA ratio from 11:1 to 3:1. Analysis of variance (ANOVA) showed that 95% (R² = 0.95) of the observed variation was explained by the model. Lack of fit analysis revealed a non-significant value for the model equation, indicating that the regression equation was adequate for predicting the degree of EPA + DHA incorporation under any combination of values of the variables. Easy ambient sonic-spray ionisation mass spectrometry (EASI-MS) was used for instantaneous characterisation of TAGs. After the enzymatic reaction, a great variety of new TAGs were formed containing EPA, DHA or both in the same molecule.     

Purification and characterization of trypsin from the viscera of sardine (Sardina pilchardus)

Trypsin from the viscera of Sardina pilchardus was purified by fractionation with ammonium sulphate, heat treatment and Sephadex G-100 gel filtration with a ninefold increase in specific activity and 9% recovery. The molecular weight of the enzyme was estimated to be 25,000 Da on SDS–PAGE. This enzyme showed esterase-specific activity on Nα-benzoyl-l-arginine ethyl ester (BAEE). The purified enzyme was inhibited by benzamidine, a synthetic trypsin inhibitor, and phenylmethylsulphonyl fluoride (PMSF) a serine-protease inhibitor, but was not inhibited by the β-mercaptoethanol. The optimum pH and temperature for the enzyme activity were pH 8.0 and 60 °C, respectively. The relative activity at pH 9.0 was 95.5% and the enzyme showed pH stability between 6.0 and 9.0. The N-terminal amino acid sequence of the first 12 amino acids of the purified trypsin was IVGGYECQKYSQ. S. pilchardus trypsin, which showed high homology to other fish trypsins, had a charged Lys residue at position 9, where Pro or Ala are common in fish trypsins. The enzyme was strongly inhibited by Zn²⁺ and Cu²⁺.     

Effects of rosemary and sage tea extracts on the sensory,chemical and microbiological changes of vacuum‐packed and refrigerated sardine (Sardina pilchardus) fillets

The effect of the natural antioxidants and antimicrobials from ethanol extracts of rosemary and sage tea on sensory, chemical [Total volatile basic nitrogen (TVB‐N), thiobarbituric acid reactive substances (TBARs), peroxide value (PV), free fatty acid (FFA)] and microbiological (total viable count‐TVC and total coliform count) changes of vacuum‐packaged sardine (Sardina pilchardus) fillets stored at 3 ± 1 °C was investigated for 20 days. The fish fillets were divided into three groups: untreated group (control, C) and treated groups that were immersed in a 1 L of distilled water containing 10 g rosemary (R group) or sage tea (S group) extracts for 4 min. The shelf life of sardine fillets was found to be 13 days for control (C), 20 days for R and S groups according to sensory assessment results, whose corresponded microbiological assessment showed a shorter shelf life (5 days for C group, 9 for R and S groups). At the end of storage period, TBARs values were 0.98 mg malonaldehyde kg^?1 for C group, 0.66 malonaldehyde kg^?1 for R group and 1.44 mg malonaldehyde kg^?1 for S group. Microbiological results showed that natural compounds from rosemary and sage tea resulted in a lower bacterial growth in fish fillets during the storage period.     

Optimisation of oil extraction from sardine (Sardina pilchardus) by hydraulic pressing

The aim of this study was to evaluate the influence of the processing conditions (pretreatment temperature: 5–55 °C, pressure: 60–120 bar and number of pressing stages: 1–3) on the yield and quality (free fatty acids, peroxide value, p‐anisidine and Rancimat induction period) of the oil extracted from whole sardine by hydraulic pressing. Experimental factors were investigated by a designed experiment and optimised by response surface methodology. A maximum yield of oil, 12.47%, was obtained at 55 °C, 60 bar and two pressing stages. Regarding oil quality, it was found minimum values for acidity (0.25% oleic at 55 °C, 60 bar and one pressing stage) and for peroxide value (0.29 meq kg^?1 oil at 5 °C, 60 bar and one pressing stage). Hence, the opposite effect of pretreatment temperature and number of pressing stages on the yield of oil and on its oxidation parameters suggested applying the weighted‐sum method as multiobjective optimisation technique.     

Thermodynamic activation and structural analysis of trypsin I from Monterey sardine (Sardinops sagax caerulea)

In this work, we report the molecular characterisation of trypsin I (Try I) from Monterey sardine (Sardinops sagax caerulea). Aspects such as thermodynamic activation parameters, molecular model and cDNA-deduced amino acid sequence allow a more in depth understanding of its activity at low temperatures. The analysis of the thermodynamic activation parameters suggests that this molecule is a cold-adapted protease. From the molecular cloning, we deduced the amino acid sequence and predicted a theoretical structural model of sardine Try I with a classical trypsin fold. Cold-adaptation of this enzyme probably comes from amino acid replacement of key residues to improve flexibility at low temperature, thus increasing k_cat. The cold-adaptation of sardine Try I opens a wide range of biotechnological applications for this protease and also it is interesting from the structure function relationship point of view of serine protease proteins.     

Physicochemical properties, gel-forming ability and myoglobin content of sardine (Sardinella gibbosa) and mackerel (Rastrelliger kanagurta) surimi produced by conventional method and alkaline solubilisation process

Characteristics and gel properties of sardine and mackerel surimi produced by conventional washing process and alkaline solubilising process were investigated. The decrease in Ca²⁺-ATPase activity with the changes in the surface hydrophobicity was found in surimi produced by alkaline solubilising process (p<0.05), suggesting the denaturation of protein induced by this process. The alkal-ine solubilising process with prewashing could remove myoglobin most effectively from sardine muscle, whereas the process without prewashing resulted in the greatest myoglobin removal in mackerel muscle (p<0.05). Surimi conventionally prepared by water or NaCl washing showed the gel with greater breaking force and deformation than that from alkaline solubilising process (p<0.05). The hig-her expressible moisture was found in the gels of surimi pre-pared by alkaline process, indicating the poor water holding capacity of the gel matrix. The highest whiteness was found in the gel of sardine surimi produced by alkaline process with prewashing but the highest whiteness was obtained in the gel of mackerel surimi washed with distilled water.     

Purification and properties of polyphenol oxidase from oil bean (Pentaclethra macrophylla Benth) seeds

Polyphenol oxidase extracted from oil bean (Pentaclethra macrophylla) seeds was purified 165-fold over the crude enzyme extract. The apparent molecular weight of the enzyme by gel filtration was 110.8 k ± 9.0 k while SDS-PAGE indicated a single species of molecular weight 28.0 k. A copper content of 1.9 mg g^?1 corresponds to one copper atom for each of the four subunits. The purified enzyme oxidised pyrogallol, catechol, 4-methylcatechol and L-dihydroxyphenylalanine but had low activity towards tyrosine. p-Cresol, caffeic acid and cholorogenic acid were not oxidised. Thio-compounds were strong inhibitors of the enzyme. The phenolic compounds tyrosine, resorcinol and orcinol inhibited catechol oxidation but became oxidised in the process.     

Alcoholysis of salicornia oil using free and covalently bound lipase onto chitosan beads

Porcine pancreatic lipase was immobilized on chitosan by covalent binding and retention of its activity was examined. The activities of free and immobilized lipase were determined using olive oil as substrate. The free and immobilized enzymes showed pH 9 as optimum and retained 50% of activity after five cycles. When the substrate concentration was kept constant and enzyme concentration was varied, the K_m and V_max were observed to be 4.0 × 10⁻⁷ and 0.32, and 3.32 × 10⁻⁷ and 0.32, respectively, for free and covalently bound enzyme. This indicates that there is no possibility of conformational change during immobilization. Immobilized enzyme showed improved thermal and storage stability. Alcoholysis of salicornia oil, mediated by free and immobilized lipase, was carried out at 25 °C using methanol in hexane and acetone. Free and immobilized enzyme in hexane produced, respectively, 45% and 55% of fatty acid methyl ester after 12 h.