Myosin heavy chain degradation during post mortem storage of Atlantic cod (Gadus morhua L.)

Post mortem proteolytic degradation of fish fillets leads to textural changes like muscle softening and gaping. In this study proteolytic degradation of myosin heavy chain (MHC) was monitored during storage of muscle and of isolated myofibrils at different temperatures and pH-values by the use of MHC-specific antibodies. The ability of cathepsin D to associate to myofibrillar proteins was also studied. Muscle stored at 6 °C and isolated myofibrils stored at 0 °C, 6 °C and 20 °C were degraded at pH 6.3 or lower. Cathepsin D could be found associated with extensively washed myofibrils. Inhibition of cathepsin D during storage affected the observed MHC-degradation at pH 5.5, but not at pH 6.3. This indicates that cathepsin D to a less extend than formerly believed, is responsible post mortem degradation of MHC.     

Bovine cathepsin D activity under high pressure

The stability and catalytic activity of bovine cathepsin D in Bis-Tris buffer (pH 6.0) in different pressure–temperature domains (0.1–650 MPa, 20–75 °C) were investigated and described with mathematical models. Cathepsin D inactivation followed first-order kinetics at all pressure–temperature conditions tested. The protease was largely pressure stable at room temperature and heat stable at ambient pressure up to 300 MPa and 55 °C, respectively, causing less than 10% inactivation after 10 min treatment. Pressure and temperature act synergistically on the enzyme inactivation under most conditions. However, at 100 MPa a significant stabilisation of the enzyme against temperature-induced inactivation was observed. Pressure drastically inhibited the cleavage of a synthetic substrate by cathepsin D in Bis-Tris buffer (pH 6.0) causing a reduction of the catalytic rate of more than 50% at 100–400 MPa. Maximal substrate cleavage by cathepsin D was identified at 60 °C and ambient pressure conditions after 20 min treatment.     

High pressure treatment of brine enhanced pork affects endopeptidase activity,protein solubility,and peptide formation

In order to study the effect of high-pressure (HP) treatment and two different methods of brine addition (important for lysosomal membrane destabilisation) on lysosomal enzymes activity and protein degradation, pork semitendinosus muscle was brine enhanced by injection or tumbling, and HP treated at 600 MPa following storage at 2 °C for up to 8 weeks. In this report a novel protocol for SDS gelatin zymography was established, and an increase of cathepsin B and L activity after HP treatment was shown followed by a decrease during storage. No calpain activity was detected following HP treatment. HP treatment was shown to induce a decrease in protein solubility in both myofibrillar and sarcoplasmic fractions. LC–MS analysis of these fractions showed changes in the peptide pattern during storage. Western blot analysis showed that troponin-T was indeed degraded during storage after HP treatment. The results therefore suggest that HP treatment induced an increase in cathepsin activity, which subsequently affected the myofibrillar protein degradation pattern in pork meat.     

Atlantic salmon (Salmo salar) muscle structure integrity and lysosomal cathepsins B and L influenced by dietary n-6 and n-3 fatty acids

This study had two main objectives: first, to evaluate the impact of different types and levels of dietary n-6 and n-3 fatty acids (FAs) on Atlantic salmon muscle structure integrity; second, to highlight a possible role of lysosomes and lysosomal degrading enzymes, cathepsins, in fish muscle structure integrity, in relation to dietary fatty acids. Four groups of Atlantic salmon (90 g starting weight) in fresh water tanks were fed one of four diets containing 23% crude lipids, with 100% of the added oils as either fish oil (FO), rapeseed oil (RO), eicosapentaenoic acid (EPA) enriched-oil or docosahexaenoic acid (DHA) enriched-oil. The RO diet was characterised by low levels of EPA + DHA (10% of total FAs), whereas the EPA and DHA diets were characterised by very high levels of EPA + DHA (>50% of total FAs). Fatty acid composition of the muscle crude lysosomal fraction (CLF) generally reflected the diets. Salmon fed the RO diet presented a muscle CLF FA composition close to the one of the FO group, showing moderate PUFA levels, and comparable cathepsin B and cathepsin L activities, relative gene expression of cathepsin B and cathepsin L in the muscle and rate of myofibre–myofibre detachments post-mortem. Salmon fed the EPA and DHA-enriched-oil diets presented a fairly similar muscle CLF FA composition, but different from the FO and RO groups. In the EPA and DHA groups, the percentage of PUFAs in the muscle CLF, the rate of myofibre–myofibre detachments and the relative gene expression of cathepsin B were higher than in the FO and RO groups. Cathepsin B and cathepsin L total activities in the muscle were however lower in the EPA and DHA groups 0 h post-mortem. Dietary lipids influenced the level of lysosomal degrading enzyme activity cathepsin B and cathepsin L as well as the relative gene expression of cathepsin B. Feeding Atlantic salmon with rapeseed oil and extreme levels of EPA + DHA highlighted the impact of fatty acid composition of the diet on salmon muscle integrity and the complexity of the process involving muscle lysosomes and cathepsins in relation to these dietary fatty acids.     

Changes in actomyosin dissociation and endogenous enzyme activities during heating and their relationship with duck meat tenderness

The objectives of this study, were to examine the relationship between duck meat tenderness, actomyosin dissociation and endogenous enzyme activities when heating the duck breast muscle, to the internal temperature of 30, 40, 50, 60, 70, 80, 90 °C. The shear force increased in the temperature range of 30–50 °C and 70–90 °C and decreased from 50 to 70 °C, which was negatively related with liberated actin (P < 0.05). The activity of cathepsin B and L was stable while heating the meat to a temperature below 50 °C, then it decreased rapidly with temperature increase. The calpain activity kept decreasing with the temperature increase. There was no significant change in the cathepsin D activity below 70 °C but it declined rapidly thereafter, and its activity was strongly correlated with actomyosin dissociation (P < 0.05). The results suggest that actomyosin dissociation and cathepsin D, could contribute to the tenderness of duck meat during the cooking process.     

Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins

The modori phenomenon during surimi production is caused by endogenous proteinases, especially cathepsin L and myofibril-bound serine proteinase (MBSP). Cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies and revealed a single band with molecular mass of 30 kDa on SDS–PAGE. Peptide mass fingerprinting (PMF) obtained three fragments with 48 amino acid residues, which were highly identical to cathepsin L from other fish species. Its optimal pH and temperature were 5.5 and 55 °C, respectively. Meanwhile, MBSP was purified from the skeletal muscle of blue scad, and the roles of cathepsin L and MBSP in the degradation of myofibrillar proteins were compared. The results indicated that MBSP is more effective than cathepsin L in promoting the degradation of myofibrillar proteins, especially myosin heavy chain (MHC), suggesting that MBSP plays a more significant role.     

Effect of season and fishing ground on the activity of lipases in byproducts from cod (Gadus morhua)

Lipase activity at acid and neutral pH has been measured in cut off, liver and viscera from cod caught in the Icelandic Sea (three different seasons) and the Barents Sea (one season). Lipase activity at pH 5 was highest in liver, while at pH 7 lipase activity was highest in viscera. Activity at pH 7 was higher than at pH 5 in liver and viscera, and at the same level in cut off. Lipase activity at pH 5 and 7 (20 °C) was higher in cut off samples from the Icelandic Sea in May–June, compared to October–December. Lipase activity at pH 7 (20 °C) was higher in cut off from the Barents Sea compared to the Icelandic Sea. No significant effect of season or fishing ground was found for liver lipase. Lipase activity at pH 5 and 7 in viscera from the Icelandic Sea was lower in May–June compared to the other seasons. Lipase activity was generally higher in viscera from the Icelandic Sea compared to the Barents Sea.     

Effect of harvesting stress and storage conditions on protein degradation in fillets of farmed gilthead seabream (Sparus aurata): A differential scanning calorimetry study

A trial was undertaken to evaluate Differential Scanning Calorimetry (DSC) as a fast analytical tool to differentiate gilthead seabream subjected to variable conditions of slaughter stress and post-mortem storage. Fish were subjected to different harvesting stress conditions: profound anaesthesia (PA, low stress) and net crowding (NC, high stress). Fish were slaughtered in an ice-salt water slurry, and subsequently stored on ice (7 days). Additional NC fish were frozen (−20 °C) and subjected to a freeze–thaw cycle. Dorsal muscle was assessed for cathepsins activity, liquid loss and DSC analysis. It is demonstrated that DSC analysis is capable of differentiating fresh, frozen and thawed-re-frozen fish, while liquid loss and cathepsin B activity are good markers to distinguish fresh from frozen fish. Harvesting stress had little effect on myosin and actin enthalpy transitions, as observed by DSC at 49 and 74 °C, respectively, but a lower ΔH actin/myosin ratio was found in PA fish, suggesting that intense exercise prior to slaughter promoted partial denaturation of muscle myosin.     

COMPARISON OF CATHEPSIN B, D, H AND L ACTIVITY IN FOUR SPECIES OF PACIFIC FISH

The activities of cathepsins B, D, H and L were compared in crude muscle extracts from four species of fish: Pacific whiting (PW), (Merluccius productus); arrowtooth flounder (ATF), (Atheresthes stomias); Alaska pollock (AP), (Theragra chalcogramma); and Pacific cod (PC), (Gadus macrocephalus). Both PW and ATF are known to undergo softening during post-mortem handling and cooking while AP and PC do not. Cathepsin B and L activities were both higher in extracts of PW and ATF than in AP and PC. Cathepsins B and L activities were both much higher in PW than in ATF. Cathepsin H activity was highest in AP followed by ATF with PC and PW having the lowest activities. Cathepsin D activity was extremely low in all four species. The heat stability of the various cathepsin activities showed cathepsin B to be the most heat labile and was inactivated by 50C heating for 15 min. Cathepsin H activity was inactivated at 60C, while cathepsin L required 70C for inactivation. Cathepsins B and L are the most likely responsible for softening in PW and ATF during holding and subsequent processing. However, during cooking, cathepsin L likely causes the most damage since it requires 70C for inactivation. The difference in heat stability of cathepsins B and L can be used to differentiate between their activities.     

Mild heat and calcium treatment effects on fresh-cut cantaloupe melon during storage

The effect of mild heat fruit pre-treatment on some properties of fresh-cut cantaloupe melon during storage was determined. Whole fruit, previously held at 4 °C, was immersed in heated water (60 °C) with and without dissolved calcium lactate (1%). Fresh-cut processing was done immediately, either after treatment or after storage at 4 °C for 24 h. Headspace gas accumulation during storage indicated reduced respiration in heat-treated fruit. Reduced lipase activity occurred in heat treated fruit during storage at 10 °C, while the fruit that was cut 24 h after treatment had a reduced peroxidase activity, unlike fruit that was processed immediately after heating. Isoelectric focussing indicated production of heat shock proteins (PI = 5.1 and 6.5) as a result of heat-treatment. Textural measurements showed increased hardness, chewiness and cohesiveness, but springiness decreased in heat-treated fruit. Presence of calcium in the treatment solution did not affect respiration and textural changes caused by heat treatment. Lipase activity was, however, higher in fruit heated in calcium solutions. Results indicated the potential improvement of shelf life of cut cantaloupe melon by mild heat pre-treatment of the fruit, and that the addition of calcium to the treatment water did not further improve product quality.     

Quality of superchilled vacuum packed Atlantic salmon (Salmo salar) fillets stored at −1.4 and −3.6 °C

The most important factor for increasing shelf life is the product temperature, and since fish is more highly perishable than meat, the temperature is even more important. In the present study, portions of fillets of farmed Atlantic salmon (Salmo salar) were superchilled at two temperature levels, −1.4 and −3.6 °C. Texture, drip loss, liquid loss, cathepsin activities and protein extractability were investigated during storage and compared to ice chilled and frozen references. Drip loss was not a major problem in superchilled salmon. Textural hardness was significantly higher in superchilled salmon fillets stored at −3.6 °C compared to those stored at −1.4 °C, ice chilled and frozen references. Cathepsins B and B + L were not deactivated at the selected storage temperatures. The storage time of vacuum packed salmon fillets can be doubled by superchilled storage at −1.4 °C and −3.6 °C compared to ice chilled storage.     

Broad‐spectrum inhibition of proteolytic enzymes by allicin and application in mitigating textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets

The inhibitory effect of allicin on proteolytic enzymes and textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets was investigated. The results of in vitro study showed that allicin inhibited the activity of cathepsin B, L and D, calpain and collagenase in crude extract of grass carp muscle. Among endogenous enzymes, cathepsin B, L and collagenase were the most susceptible to allicin. Proteolysis of myofibrillar proteins by either crude enzyme or cathepsin B and L was almost prevented by allicin when employed at a concentration higher than 100 mm . After storage of 21 days, shear force of fillets treated with allicin at 10–100 mm was 39–51% higher than that of control. Myofibrillar proteins of fillets during storage were well protected against degradation when allicin concentration increased to 100 mm , as evidenced by SDS‐PAGE. Therefore, allicin could be a potential broad‐spectrum inhibitor to retard softening of fish fillets via mitigating myofibrillar proteolysis by endogenous enzymes especially cathepsin B and L during ice‐storage.     

Post-mortem changes in viscera of cuttlefish Sepia officinalis L. during storage at two different temperatures

We observed the post-mortem changes in viscera of cuttlefish Sepia officinalis in order to apply the data to autolysate production. Cuttlefish viscera were stored at 4 or 25 °C and sampled regularly over 4 months. Our results showed that total acid proteases and cathepsins were rapidly released to the extracellular medium due to the breakdown of lysosomes. Total alkaline protease activity increased 2 h after death due to the breakdown of zymogene vesicles. The same patterns were found with trypsin, chymotrypsin and α-amylase activities. After 10 days of incubation, no endogenous enzymatic activity was found. After 50 days of storage, the TCA soluble protein levels decreased rapidly to approximately 30% due to protein degradation and aggregation. After 10 days, the pH of viscera stored at 25 °C was alkaline, whereas in the viscera stored at 4 °C the pH increased more slowly. As significant reduction in the protein molecular weight due to autolysis, was also observed.     

Seasonal effects on the physicochemical characteristics of fish sauce made from capelin (Mallotus villosus)

Fresh capelin (Mallotus villosus) was harvested from the North Atlantic during both summer and winter fishing seasons. Reaction conditions for fish sauce processing were optimized with respect to temperature, salt concentration and reaction time, using a response surface methodology (RSM) experimental design. Whole capelin was minced and samples were ground with increasing salt concentrations. RSM optimizations were conducted, ranging from 5% to 30% salt, and incubating at 5° intervals from 0 to 65 °C. Autolytic activity was estimated by extracting the liquid formed by the mixture with trichloroacetic acid and estimating protein content by the Lowry method. Samples for fish sauce production were then prepared under optimized conditions by mixing ground capelin with 10% salt and incubating at 50 °C for up to 270 days for the summer capelin and up to 360 days for the winter capelin. Samples were collected at regular intervals and analyzed for liquid yield, moisture, protein, soluble solids, specific gravity, pH, colour and amino acid content. Kjeldahl protein content in the fish sauce from summer capelin was 2.03% after 250 days of fermentation and twice as high as that in winter capelin fish sauce. Moisture content and pH were lower in the summer capelin fish sauce, but Brix and density were higher than those in fish sauce from winter capelin. Brown colour formation was very rapid in the summer capelin fish sauce but slow in the winter capelin fish sauce. Summer capelin may be successfully utilized for the production of fish sauce without added enzymes.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

Antioxidant activity of yoghurt peptides: Part 1-in vitro assays and evaluation in ω-3 enriched milk

The aim of the present study was to investigate important factors contributing to the high oxidative stability of fish-oil-enriched yoghurt, with particular emphasis on the possible antioxidative effects of peptides released during yoghurt fermentation. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultrafiltration using membranes with cut off sizes of 30 kDa, 10 kDa and 3 kDa. The fractions were tested for antioxidant activity by investigating the inhibition of oxidation in liposome model system, 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, iron-chelating activity, and reducing power. The lower molecular weight fractions were found to be more effective antioxidants than higher molecular weight fractions. The lower molecular fractions were further tested as antioxidants in fish-oil-enriched milk. On the basis of peroxide value, volatiles, tocopherol and sensory characteristics, the lower molecular weight fractions 3–10 kDa and <3 kDa showed protection against oxidation of fish oil to the same extent as caseinophosphopeptides. The oxygen content of the yoghurt was also found to be lower than that of milk. Thus our findings suggests that the higher oxidative stability of yoghurt might be due to antioxidant peptides released during the fermentation of milk by lactic acid bacteria and/or by the lower oxygen content of yoghurt, which subsequently reduces the oxidative stress of fish oil incorporated in the yoghurt. The results show that antioxidant peptides may be used as an ingredient in foods enriched with fish oils to increase their oxidative stability.     

Partial purification and characterization of trypsin-like proteinases in Indian anchovy (Stolephorus spp.)

Four fractions (P111, P21, P31, and P4) of proteinases were obtained from various purification steps including heat treatment (60 °C, 10 min), 30–60% ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. Optimal temperature and pH of all fractions were 50–60 °C and 8.5, respectively. All partially purified proteinases preferably hydrolyzed substrates containing Arg at the P₁ position. All proteinases were inhibited by soybean trypsin inhibitor, leupeptin, and N-tosyl-l-lysine chloromethyl ketone. Partially purified proteinases were stable at 35 °C up to 12 h. However, their activity decreased about 40% when incubated at the optimal temperature (50–55 °C) for 2 h. Only P111 was stable at its optimal temperature (60 °C) up to 12 h. Molecular weight (MW) of P111, P21, and P31 was estimated to be 27, 33, 37, 43, 48, 55, 60, and 65 kDa, while MW of P4 was 39 kDa based on activity staining. All partially purified proteinases hydrolyzed washed anchovy mince at 4.0 M NaCl, pH 8.5, at 35 °C and at their optimal temperatures (50–60 °C).     

Effects of combining microbial transglutaminase and high pressure processing treatments on the mechanical properties of heat-induced gels prepared from arrowtooth flounder (Atheresthes stomias)

The objective of this study was to evaluate the effect of setting conditions (25 °C for 2 h or 40 °C for 30 min) and combining of microbial transglutaminase (MTGase) and high pressure processing (HPP) on the mechanical properties of heat induced gels obtained from paste from arrowtooth flounder (Atheresthes stomias). Treatments included fish paste control without added MTGase, fish paste incubated with MTGase but not pressurized (MTGase + cooking), fish paste incubated with MTGase and pressurized at 600 MPa for 5 min (MTGase + HPP + cooking) and fish paste pressurized at 600 MPa for 5 min and incubated with MTGase (HPP + MTGase + cooking). The controls and the treated samples were then subjected to one of two thermal treatments: 90 °C for 15 min or 60 °C for 30 min before cooking at 90 °C for 15 min. Samples of fish paste heated at 60 °C before cooking could not be used to prepare gels for texture profile analysis (TPA). TPA showed that pressurization improved the mechanical properties of gels made from paste treated with MTGase and set at 25 °C. The opposite was observed for samples set at 40 °C. Setting at 40 °C appeared to induce proteolytic degradation of myofibrillar proteins.     

Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases

The angiotensin I-converting enzyme (ACE) inhibitory activities of protein hydrolysates prepared from heads and viscera of sardinelle (Sardinella aurita) by treatment with various proteases were investigated. Protein hydrolysates were obtained by treatment with Alcalase^®, chymotrypsin, crude enzyme preparations from Bacillus licheniformis NH1 and Aspergillus clavatus ES1, and crude enzyme extract from sardine (Sardina pilchardus) viscera. All hydrolysates exhibited inhibitory activity towards ACE. The alkaline protease extract from the viscera of sardine produced hydrolysate with the highest ACE inhibitory activity (63.2 ± 1.5% at 2 mg/ml). Further, the degrees of hydrolysis and the inhibitory activities of ACE increased with increasing proteolysis time. The protein hydrolysate generated with alkaline proteases from the viscera of sardine was then fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Biological functions of all fractions were assayed, and P₄ was found to display a high ACE inhibitory activity. The IC₅₀ values for ACE inhibitory activities of sardinelle by-products protein hydrolysates and fraction P₄ were 1.2 ± 0.09 and 0.81 ± 0.013 mg/ml, respectively. Further, P₄ showed resistance to in vitro digestion by gastrointestinal proteases. The amino acid analysis by GC/MS showed that P₄ was rich in phenylalanine, arginine, glycine, leucine, methionine, histidine and tyrosine. The added-value of sardinelle by-products may be improved by enzymatic treatment with visceral serine proteases from sardine.