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.     

Proteolytic activities of ventral muscle and intestinal content of North Sea herring (Clupea harengus) with full and emptied stomachs

The aim of the work was to examine the effect of allowing herring (Clupea harengus) to empty the stomachs prior to being taken on board the fishing vessels, on the proteolytic activities in extracts of ventral muscle and of intestinal content. The proteolytic activities were examined by gelatin zymography and by incubating the extracts with isolated myosin heavy chain (MHC) protein. Extracts from herring with full stomachs showed strong gelatinolytic and MHC degrading activities particularly in extracts from intestinal contents. Extracts from fish that had been allowed to empty their digestive system for 19 h had reduced activities, which was most noticeable in the ventral muscle extracts. The activities from the ventral muscle did not originate endogenously post-mortem, as shown by the fact that ice storage for 24 h of isolated ventral muscle did not display the activities, while ventral muscle extract from ice-stored whole fish for 24 h did.     

Muscle structure responses and lysosomal cathepsins B and L in farmed Atlantic salmon (Salmo salar L.) pre- and post-rigor fillets exposed to short and long-term crowding stress

Atlantic salmon (average body weight 2.9 ± 0.1 kg) were subjected to either minimal pre-slaughter crowding stress (NS group), 20 min (short-term stress, SS group) or about 24 h pre-slaughter crowding stress (long-term stress, LS group). Significant negative effects were mostly seen due to long-term stress (LS) at early stages post-mortem, but also short-term stress (SS) had significant negative impacts on muscle quality of pre- and post-rigor fillets. Pre-slaughter long-term stress (LS) significantly lowered muscle pH, softened the fillets and increased muscle cathepsin L gene expression, immediately post-mortem. A tendency of increased cathepsin B gene expression and cathepsin B total activity was also seen. Stress further accelerated the incidence of myofibre–myofibre detachments, increased the percentage of myofibre–myocommata detachments over the storage period and increased the percentage of myofibre breakages and contracted myofibres 96 h post-mortem. Significant correlations were observed between muscle pH and cathepsin B + L activity, muscle texture and muscle degradation parameters. Cathepsin B activity was correlated to muscle degradation and cathepsin L gene expression to muscle degradation and texture. Pre-slaughter stress, especially long-term stress (LS), thus seems to accelerate cathepsin activity, resulting in faster muscle degradation, directly or indirectly connected to the low initial muscle pH. No significant variation was observed between pre-rigor and post-rigor filleting, except that pre-rigor filleting significantly increased the percentage of myofibre breakages.     

Effect of endogenous acid proteinases on the properties of edible films prepared from Alaska pollack surimi

The existence of endogenous acid proteinases in Alaska pollack surimi and their effect on mechanical properties of surimi films were investigated. The optimum pH of acid proteinases involved in the degradation of myosin heavy chain (MHC) was 3.0, and the optimum temperature was 45 °C. The degradation of MHC was completely inhibited by pepstatin A together with any one of cysteine proteinase inhibitors, suggesting that acid proteinases present in surimi are mainly cathepsin D and cysteine proteinases. The concomitant decrease of surimi film strength with the extent of MHC degradation was observed, but surimi films were formed even when most of MHC was degraded. The main associative forces responsible for the surimi films prepared at pH 3.0 were ionic bonds and hydrophobic interactions.     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.     

Identification of small troponin T peptides generated in dry-cured ham

Troponin T (TnT) is a myofibrillar protein present in striated muscle that develops important structural and regulatory functions. This protein is easily degraded during post-mortem ageing but details of the TnT hydrolysis occurring during much longer processes, e.g. the dry-cured processing, as well as the specific sequences of the generated fragments, still remain undetermined. In the present study, the isolation and identification of 27 peptides, generated from TnT during dry-cured processing, are reported for the first time. This study has been developed using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) and it evidences the extensive degradation of TnT occurring during the processing of dry-cured ham. Some of the cleavage sites, corresponding to these sequenced fragments, confirm the sites reported by other authors both in vitro and during post-mortem ageing of meat.     

Partial characterisation of gelatinolytic activities in herring (Clupea harengus) and sardine (Sardina pilchardus) possibly involved in post-mortem autolysis of ventral muscle

The present work aims at identifying enzymatic activities that may contribute to the post-mortem autolysis of the ventral muscle, known as belly bursting, in herring (Clupea harengus). Gelatinolytic proteases extracted from several herring tissues and also from a sardine (Sardina pilchardus) tissue were partially characterised using gelatin zymography, inhibitor analysis, immunodetection with anti-trypsin antibody and MALDI-TOF/TOF peptide sequencing. The results indicate the presence of gelatinolytic trypsin-like serine proteases and metalloproteases in several samples including the ventral muscle of herring. MS/MS analysis gave partial sequences of peptides from some of the proteases, and these were identified as elastase, trypsin and aspartyl aminopeptidase. It is likely that belly bursting in herring is caused by leakage of enzyme-rich fluids from the intestine and/or pyloric caeca which may also contain exogenous proteases from the digestive systems of the prey.     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

Proteins and proteolytic activity changes during refrigerated storage in sea bass (Dicentrarchus labrax L.) muscle after high-pressure treatment

Contrary to other preservation methods like thermal treatments, high pressure can destroy microorganisms without affecting the nutritional quality, color, or food texture. The firm texture of fish flesh is an important quality parameter. During the refrigerated storage, the tissue becomes softer and the muscle is deteriorated by different proteases. The aim of this study was to study the modification of the fish muscle proteins after high-pressure treatment during the refrigerated storage and to evaluate the effect of high-pressure treatment level on the post-mortem protein changes and enzyme activities. The calpain activity decreased with the high-pressure treatment and evolved differently during the refrigerated storage, depending on the level of pressurisation. Its inhibitor, the calpastatin was not affected by high pressure, but its inhibiting potential decreased during the post-mortem storage. The activities of cathepsins were modified by the high-pressure treatment and the time of storage, but depended according to their class. The electrophoresis profiles showed that sarcoplasmic proteins were modified according both the high-pressure treatment and the period of storage. For the myofibrillar proteins, the only changes were due to the high-pressure treatment.     

Physicochemical properties and tenderness of meat samples using proteolytic extract from Calotropis procera latex

This study was conducted in order to tenderise muscle foods (pork, beef and chicken) by using crude enzyme extract from Calotropis procera latex. Chunks of knuckle muscle from pork and beef as well as of breast muscle from chicken were marinated with distiled water (control) and 0.05%, 0.1%, 0.2%, 0.3% and 0.5% (w/w) of crude enzyme extract powder for 60 min at 4 °C. The marinated samples were then subjected to various physical and chemical property determinations. A decrease in moisture content was observed when the crude enzyme extract was added. Firmness and toughness of the muscle samples significantly decreased with the increased addition of crude enzyme extract (p < 0.05). The water holding capacity and cooking yield of the treated samples showed no significant difference throughout the crude enzyme extract addition (p > 0.05). Crude enzyme extract had no effect on the pH of the pork sample, but it slightly increased the pH in the beef and chicken. An increase in protein solubility and TCA-soluble peptides content was observed in all of the treated samples. The electrophoresis pattern of the muscle treated samples also revealed extensive proteolysis occurring in each muscle type. From the results, it is determined that latex from Calotropis procera can be used as an alternative source of proteolytic enzymes for the effective tenderising of meat.     

Sensory,physical and chemical characteristics of cooked ham manufactured from rapidly chilled and earlier deboned M. semimembranosus

Effects of rapid chilling of carcasses (at − 31 °C in the first 3 h of chilling, and then at 2–4 °C) and earlier deboning (8 h post-mortem), compared to rapid (till 24 h post-mortem) and conventional chilling (at 2–4 °C, till 24 h post-mortem), on quality characteristics of pork M. semimebranosus and cooked ham were investigated. Quality measurements included pH value, colour (CIEL*a*b* values) and total aerobic count of M. semimebranosus, as well as sensory (colour, juiciness, texture, and flavour), physical (pH value, colour – CIEL*a*b* values and texture – Warner–Bratzler shear and penetration forces) and chemical (protein, total fat, and moisture content) characteristics of cooked ham. The cooked ham was manufactured from pieces of M. semimebranosus with ultimate lightness (CIEL* value) lower than 50. Rapid chilling and earlier deboning significantly increased quantity of M. semimebranosus desirable for cooked ham manufacturing. Earlier start of pork fabrication did not affect important quality characteristics of cooked ham.     

Characterization of proteolysis in muscle tissues of sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The proteolysis in muscle tissues of sea cucumber Stichopus japonicus (sjMTs) was characterized. The proteins from sjMTs were primarily myosin heavy chains (MHCs), paramyosin (Pm), and actin (Ac) having a molecular mass of approximately 200, 98, and 42 kDa, respectively. Based on SDS-PAGE analysis and quantification of trichloroacetic acid (TCA)-soluble peptides released, degradation of muscle proteins from sjMTs was favorable at pH 5 and 50°C. Proteolysis of MHCs was mostly inhibited by cysteine protease inhibitors, including trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64) and antipain (AP). E-64 and AP completely inhibited the degradation of Pm and Ac, while iodoacetic acid showed a partially inhibitory effect. These results indicated that the proteolysis of sjMTs was mainly attributed to cysteine proteases. Avoidance of setting the tissues at 40–50°C and slightly acidic condition and inhibition of cysteine proteases are helpful for decreasing sea cucumber autolysis.     

Protein Solubility in Pacific Whiting Affected by Proteolysis During Storage

The effects of post-harvest storage temperatures and times on proteolysis of Pacific whiting (Merluccius productus) and relationship to changes in protein solubility were evaluated. Myosin heavy chain (MHC) degraded rapidly during post-harvest storage at low temperatures (0-5°C). Greater degradation of MHC occurred at elevated temperatures. Actin degradation was similar to that of MHC but to a lesser degree. Both log-percent MHC and actin degradation were highly correlated (R² 5 0.88 and 0.74) to protein solubility. Degraded proteins were not completely removed by washing, resulting in a lower MHC content in washed mince.     

Variation in bull beef quality due to ultimate muscle pH is correlated to endopeptidase and small heat shock protein levels

This study set out to determine if ultimate pH (pH_u) affected the performance of intracellular small heat shock protein and endopeptidase dynamics in muscle during beef ageing. Longissimus dorsi muscles from 39 Angus or Limousin × Angus bulls were examined to see if pH_u achieved at 22 h post mortem (rigor) affected tenderness and water holding capacity of beef. Samples were segregated into three pH_u groups termed high (pH > 6.3), intermediate (5.7 < pH < 6.3) and low (pH < 5.7) pH_u beef. More than 30% of bull beef did not achieve acceptable tenderness at 8 days post mortem with this ageing regime. No significant differences in calpain or cathepsin enzyme levels due to meat pH were observed until after 22 h post mortem, but low pH_u beef had elevated caspase 3/7 activity soon after slaughter. At 22 h post mortem, greater levels of μ-calpain enzyme were found in the high and intermediate pH_u beef and cathepsin B levels were superior in the low pH_u beef after 2 days post mortem. Different rates of desmin and troponin T protein degradation were also observed in aged bull beef. Both proteins were degraded within 6 h post mortem for high pH_u beef, but took >3 days post mortem for intermediate pH_u beef. High levels of alpha β-crystallin (aβC) at 22 h post mortem coincided with delayed muscle protein degradation for low pH_u beef. Our results support the hypothesis that aβC shields myofibrils and buffers against endopeptidase degradation of beef structure during ageing.     

Physicochemical Changes in Pacific Whiting Muscle Proteins during Iced Storage

No changes in actomyosin Ca²⁺-, Mg²⁺-, or Mg²⁺-Ca²⁺-ATPase activities were observed during iced storage of Pacific whiting fillets, but Mg²⁺-EGTA-ATPase increased with a loss of Ca²⁺-sensitivity. Surface hydrophobicity of actomyosin increased substantially within 2 days, but not total sulfhydryl (SH) content. During longer storage, the SH content decreased gradually, but surface hydrophobicity remained constant. Autolytic degradation products increased in fish muscle with storage time. Myosin heavy chain (MHC) was degraded by 45% within 8 days, but no noticeable difference was observed in actin. Results indicated that autolysis may be the main cause of physicochemical changes in Pacific whiting muscle proteins during iced storage.     

Proteins degradation value in cured meat product made from M. Cutaneous-omo brachialis muscle of bovine

We analyzed differences in some physicochemical parameters of proteins in shoulder rose (M. Cutaneous-omo brachialis) muscle as a result of beef processing to produce pastirma. Samples from processed muscles showed significantly increased concentrations of extracted proteins, especially of H₂O P-ex and Guba-Straub–adenosine triphosphate solution (P < 0.01), as a result of the salting–curing process. The salt-curing process was likely to have an important effect on the extractability of muscle proteins such as myosin heavy chain (MHC) and water-soluble proteins, possibly as a result of releasing some proteins from each other and cleaving the structures between certain proteins. The fluorescence intensities of processed samples were higher than those of the control samples at all guanidine hydrochloride concentrations. The hydrophobicity also increased on account of new compounds that were created during the pastirma-making process. Since the process of making pastirma lasts about 4 weeks (drying), the metmyoglobin content was greatly increased in pastirma samples compared with the unprocessed samples (by as much as 57 %). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results conceived that during processing of pastirma products, MHC and other muscle proteins degraded into polypeptides with smaller molecular weight lower than 15-kDa. The results of this study demonstrated that meat processing promoted the enzymatic digestion of some proteins, and the differences in composition between the control and pastirma samples were thus likely to be owing to protein degradation. The traditional pastirma-making process hence has no negative impact on the structure of the muscle and produces a firmer-textured.     

Protein degradation of olive flounder (Paralichthys olivaceus) muscle after postmortem superchilled and refrigerated storage

The aim of this study was to investigate the effect of superchilling at ?2°C in comparison with refrigerated storage at 4°C on the protein degradation of olive flounder (Paralichthys olivaceus) muscle. Flounder muscle softened and shear force value decreased markedly (P < 0.05) with prolonged storage time, while values of electrical conductivity, TCA-soluble peptide, free amino acids, and proteolysis index increased (P < 0.05). The changes were slowed down significantly in samples superchilled at ?2°C (P < 0.05). The fracture of muscle fibre and formation of cracks were accelerated in the samples refrigerated at 4°C, and intercellular spaces were observed after 9 days of storage. Moreover, protein bands of myosin heavy chain (MHC), actin, tropomyosin, 97 kDa, 50 ~ 60 kDa and 35 ~ 36 kDa occurred in varying degrees of degradation with storage time. The results demonstrated that significant postmortem degradation of muscle proteins occurred with extending storage time, while the changes were retarded obviously in samples during superchilled storage.     

Thermal degradation kinetics of xanthophylls from blood orange in model and real food systems

Thermal degradation kinetics of the major blood orange xanthophylls (cis-violaxanthin, lutein, β-cryptoxanthin, zeaxanthin and cis-antheraxanthin) were investigated at 45, 60, 75, and 90 °C in real juice and three model systems formulated to evaluate the impact of xanthophyll form (esterified or free) and pH (acid or neutral). Xanthophylls were monitored by HPLC-DAD and kinetic parameters were identified by non-linear regression. A second order model best fitted the degradation curves of xanthophylls. All degradation rates were the lowest in real juice. Esterified forms were more stable than were the free forms. In all acidic media, β-cryptoxanthin exhibited the lowest degradation rates followed by lutein and zeaxanthin. In comparison, the epoxy carotenoids cis-violaxanthin and cis-antheraxanthin degraded around 3-fold faster in their esterified form. In their free form, cis-antheraxanthin degraded 30-fold faster while cis-violaxanthin instantaneously disappeared because of the isomerisation of its 5,6-epoxy groups into 5,8-epoxy. By contrast, in neutral medium, free epoxy-xanthophylls were about 2-fold more stable than were the free hydroxy xanthophylls lutein, zeaxanthin and β-cryptoxanthin. Kinetic behaviours of xanthophylls were closely dependent on their chemical structures.     

Proteolytic degradation of myofibrillar components by carp cathepsin L

Carp cathepsin L, which is the best candidate to produce textural change in the arai-treated carp fillet, exhibited maximum hydrolytic activity for Z-Phe-Arg-MCA and soluble casein at pH 5·0–5·5. The proteolytic action of the enzyme was evaluated by complete degradation of various carp myofibrils at pH 5·0 over 30 min and by potent degradation of the same proteins at pH 5·5–6·0 over 20 h. All myofibrillar components were partially degraded by the enzyme at pH 6·5–7·0, but varying amounts of them remained undegraded after 20 h. These findings indicate that carp cathepsin L degrades not only carp myofibrillar components but also their resultant products between pH 5·0 and 7·0 and that it markedly acts on myosin heavy chain, α-actinin and troponin-T and -I. Carp cathepsin L likely contributes to postmortem muscle tenderisation of carp fillet over an extensive pH range during storage. © 1998 SCI.     

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS‐PAGE and shear force were measured in non‐injected, 0.5 M and 1.0 M lactic‐acid‐injected samples during a 21 days ageing period. The activities of cathepsin B + L and β‐glucuronidase in the soluble fraction increased with level of lactic acid and with time post‐mortem (P < 0.001). Lactic acid and storage decreased densities of SDS‐PAGE bands migrating at the position of myosin heavy chain (MHC) and α‐actinin and increased densities of a 150 kDa band (P < 0.01). SDS‐PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P < 0.001). The cathepsin B + L activity in the soluble fraction correlated to shear force (r = −0.8), the degradation of MHC and α‐actinin (r = −0.88 and −0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24 h post‐mortem, and was therefore due to a rapid process, eg pH‐induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. © 1999 Society of Chemical Industry