Effect of lactic or acetic acid on degradation of myofibrillar proteins in post‐mortem goose (Anser anser) breast muscle

The effects of lactic acid (LA) and acetic acid (AA) on changes in myofibrillar proteins of post‐mortem goose breast muscle marinated for 24 h at 5 °C were studied. Purified myofibrils were prepared from 0.1 M LA or AA samples and controls (non‐marinated samples) after 0, 1, 3, 7 or 14 days of storage at 5 °C. The changes in myofibrillar proteins of goose muscle were examined by SDS‐PAGE. Goose breast muscle marinated in LA and AA exhibited degradation of myosin heavy chains. The appearance of ∼95 and ∼27 kDa components and the disappearance of titin and nebulin were also more rapid than for control muscle. These results suggest that acid marination enhanced the post‐mortem proteolysis of goose breast muscle. © 2000 Society of Chemical Industry     

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.     

Post mortemRelease of Fish White Muscle α-Actinin as a Marker of Disorganisation

α-Actinin release and its degradation from myofibrils Z-line were studied in post mortem white dorsal muscle from bass and sea trout stored at 4°C and 10°C. Using α-actinin specific antibodies, we show that this protein is rapidly released within the first 24 h for the two species, and reaches a plateau within 4 days. Proteolysis take place very rapidly in bass muscle yielding 80 and 40 kDa fragments from α-actinin as major bands of proteolysis. Sea trout muscle is more resistant, and muscle stored at 4°C is not significantly α-actinin degraded even 10 days after death. In the case of sea trout muscle stored at 10°C, an increasing quantity of 80 and 40 kDa fragment can be observed after the third day. These results show that release and proteolysis of α-actinin are time- and temperature-dependent processes that take place at the early stages of fish storage. Furthermore, we observed that proteolysis of α-actinin seems to be dependent on fish species. In both species studied, the early release of α-actinin comes before the degradation of released molecules, and appears as a biphasic process throughout the disorganisation of post mortem muscle in fish cold-stored above 0°C.     

Myofibrillar protein degradation of carp (Labeo rohita (Hamilton)) muscle after post‐mortem unfrozen and frozen storage

The degradation of myofibrillar proteins of rohu carp (Labeo rohita (Hamilton)) muscle was analysed after post‐mortem storage. Muscle fillets were kept either unfrozen at 2 °C for up to 15 days or frozen at ?8 °C or ?20 °C for up to 6 months. A co‐ordinated histochemical, biochemical and electrophoretic study showed a differential response of the carp muscle, revealing clear degenerative/degradative changes specific to the post‐mortem storage temperatures. The myofibrillar protein fractions, namely myosin light chains and α‐actinin, showed degradative changes during the above storage conditions, whereas other protein fractions in the high‐molecular‐weight range fragmented to give lower‐molecular‐weight proteins. The importance of the post‐mortem storage temperature for controlling the degradation of the myofibrillar proteins was emphasised. This is the first report on this popular fish species, known for its culinary importance, showing that specific protein fractions of the myofibrils degrade during post‐mortem storage. © 2001 Society of Chemical Industry     

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.     

Assessment of protein changes in farmed giant catfish (Pangasianodon gigas) muscles during refrigerated storage

Farmed giant catfish (Pangasianodon gigas) muscles (dorsal and ventral sites) were stored in a refrigerator (at 4 °C) for 14 days to determine the effect of refrigerated storage on biochemical and physical changes. The analyses were carried out at 0, 2, 4, 7, 10 and 14 days of storage. At day 14, Ca²⁺ ‐ATPase activity markedly decreased when compared to its value at day 1 (>90%), while a small decrease was observed for surface hydrophobicity and reactive sulfhydryls content. Total volatile basic nitrogen and trichloroacetic‐soluble peptide content gradually increased when the storage period was extended. The myosin heavy chain decreased slightly on SDS‐PAGE for both meat cuts with increased storage time. Expressible drip and cooking loss were highest during the first day of storage and slightly decreased with storage time. Instrumental hardness was significantly higher in the ventral compared to the dorsal muscle, while the toughness was the highest at the second day of storage. The muscle bundles with scanning electron microscopy were less attached, resulting in the observed big gaps over increasing storage time. Results indicated that changes of proteins have detrimental effects on the quality attributes of farmed giant catfish muscles during refrigerated storage, particularly physical and biochemical properties.     

Effects of Postmortem pH and Temperature Muscle Structure and Meat Tenderness

Bovine longissimus muscles with postmortem pH in the range 5.5 - 7.0 were subjected to different postmortem temperatures of 1°, 4°, 25° and 37°C. Intact beef sides with different postmortem pH were also subjected to two different environmental temperatures of 1° and 25°C. High pH muscles exhibited an extensive degradation of Z-lines, whereas low pH muscles showed a preferential degradation of M-lines and myosin heavy chains. Intermediate pH muscles did not show much degradation of muscle proteins, resulting in tougher meat than either low or high pH muscles. High postmortem temperatures enhanced the degradation of muscle proteins in excised and incubated muscle strips, but the delayed chilling of intact beef sides at 25°C for 8-hr did not affect either the structural changes or meat tenderness.     

Monitoring Myosin Degradation During Conditioning in Chicken Meat Using an Immunological Method

ABSTRACT: Changes of chicken breast myosin during storage at 2°C and 37°C were monitored immunochemically. Anti-myosin subfragment-1 (S-1) monoclonal antibody, which recognized epitopes within the 27 kDa fragment of S-1, and the anti-myosin rod polyclonal antiserum, were prepared. Myosin degradation products were not detected in muscle extracts stored for 3 weeks at 2°C. In contrast, storage at 37°C brought about the degradation of myosin heavy chain to immunologically detectable small fragments. While, myosin rod produced during the conditioning period was not decomposed into any small filaments. Namely, storage of muscle at 37°C resulted in minor amounts of myosin heavy chain degradation, with initial conversion to rod and S-1 fragments, and subsequent breakdown occurred in the S-1 region only. Immunoblot assay also suggested that the pattern of changes in myosin heavy chain in muscle incubated at 37°C was similar to that produced by in vitro digestion with cathepsin D.     

Studies on fish muscle protein. Nutritional consequences of the changes occurring during frozen storage

Samples of cod, whiting, herring, lemon sole and skate muscle were frozen and stored at ? 8°C for up to 20 months. Samples of cod muscle were also stored at ? 30°C. During storage at ?8°C the decrease in the solubility of the muscle proteins in 5% NaCl (denaturation) was most rapid in whiting followed in order by skate, cod, herring and lemon sole. Little change was detected in the NaCl solubility of cod muscle protein stored at ? 30°C. An increase in the pH was found to occur in all the species examined. The relationship between these changes and the toughening that occurs in fish muscle during frozen storage is considered. No nutritionally significant decrease in the lysine availability or the in vitro digestibility of the muscle proteins was found to occur in any of the species examined. In whiting, lemon sole and skate very small, statistically significant, increases in the available lysine content were detected after 7 months of storage at ?8°C and this may indicate that unfolding of the protein chains had occurred.     

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.     

Extractability and thermal stability of frozen hake (Merluccius merluccius) fillets stored at −10 and −30 °C

The relationship between thermal stability changes and functionality loss was monitored in hake muscle fillets stored for 40 weeks at ?10 and ?30 °C. The evolution of changes in apparent viscosity, dimethylamine formation and extractability of muscle proteins in NaCl, sodium dodecyl sulphate (SDS) or SDS plus mercaptoethanol showed drastic differences as a function of temperature. At the higher storage temperature, both myosin heavy chain and collagen were the most severely unextracted in salt and SDS solutions, with actin becoming unextractable at the end of storage. Differential scanning calorimetry showed differences with storage time and temperature in both onset temperature and thermal denaturation enthalpy, mostly affecting the myosin transitions. Some protein denaturation occurred with little or no functionality loss. A considerably high fraction of hake muscle proteins remained in the native‐like condition even at the higher frozen storage temperature. In these conditions both apparent viscosity and myosin and actin extractability in NaCl were very low. © 2002 Society of Chemical Industry     

Storage and Bacterial Contamination Effects on Myofibrillar Proteins and Shear Force of Beef

Thirty-two steaks from the longissimus muscle, fifth rib to third lumbar vertebra, were obtained from youthful carcass beef. Half were sterilized by ultraviolet light and all vacuum packaged and stored for 1, 14, 28 or 57 days at 2°C. After storage, steaks were examined for microbial populations, myofibril fragmentation index (MFI), cooking characteristics and shear force (SF). Aerobic and anaerobic counts decreased during storage. Psychrotrophic counts were low throughout. Sterilization had no effect on SF or MFI. Cooking loss tended (P < 0.09) to increase with time of storage. SF values decreased and MFI values increased through day 14, but remained relatively constant after that. Results of SDS-PAGE, SF and MFI indicate major changes in proteolysis of myofibrils and tenderness were completed by day 14.     

Properties of myofibrillar protein from Japanese stingfish (Sebastes inermis) dorsal muscle

The biochemical and physicochemical properties of myofibrillar proteins from Japanese stingfish dorsal muscle were investigated. On SDS-PAGE, the molecular weights of each protein were as follows: myosin, 200 kDa; actin, 42 kDa; heavy meromyosin, 125 kDa, and light meromyosin, 66 and 77 kDa, respectively. Ca-ATPase activity of heavy meromyosin was higher than that of myosin, while EDTA-ATPase and Mg-ATPase activities were similar to those of myosin. The KCl-dependence of enzymatic activity was similar for both myosin and heavy meromyosin. Heavy meromyosin was more stable than myosin during storage at 4°C. The transition temperatures of each protein as determined by differential scanning calorimetry were as follows: myosin, 40.9°C, actin, 61.1°C, heavy meromyosin, 40.9 and 59.3°C, and light meromyosin, 62.2°C, respectively. When myosin was digested with trypsin, the portion corresponding to subfragment-1, the head portion of myosin, had a similar transition temperature to myosin. The portion corresponding to subfragment-2, the central region of myosin, corresponded to the high temperature-peak. The head portion has ATPase activity. On the other hand, LMM and the central region does not show this activity. These results indicated that the sensitivity of myosin constituents increased by trypsin digestion and suggested that the portion having ATPase activity had a high heat-sensitivity.     

Elucidation of protein aggregation in frozen cod and haddock by transmission electron microscopy/immunocytochemistry,light microscopy and atomic force microscopy

Polyclonal antibodies raised to both native cod myosin and actin as well as to aggregated proteins obtained from frozen cod stored for 11 months at ?10 °C were used to investigate disposition of muscle proteins in frozen cod and haddock fillets by transmission electron microscopy. Specimens from cod and haddock fillets, stored at ?10 °C, treated with anti‐aggregate antibody as the primary antibody, showed significantly more gold particles, especially around the protein aggregates and muscle fibres compared with fish stored at ?30 °C. Samples that were treated with anti‐myosin or anti‐actin antibody showed opposite results. Similar binding properties were observed in ELISA experiments involving the reaction of actin and myosin to both native and aggregate antibodies; thus immunological tests can be used for monitoring aggregate and texture changes in frozen stored fish. In addition, atomic force microscopy images obtained from cod muscle also indicated structural changes in frozen cod muscle proteins. The mica surface was covered with a continuous layer of muscle proteins comprising mainly small globular particles and a few large particles for the control cod sample stored at ?30 °C for 11 months. In contrast, cod fillets stored at ?10 °C showed a thin layer of proteins with small holes and an increased number of large particles denoting aggregates. Formation of ice crystals between the muscle fibres of frozen cod and haddock muscle was monitored without thawing by light microscopy at ?20 °C. The micrographs showed a greater proportion of large ice crystals and extensive protein fibre changes in fillets stored at ?10 °C compared with the control at ?30 °C. Copyright © 2004 Society of Chemical Industry     

Vacuolar (Storage) Proteins of Cocoa Seeds and their Degradation during Germination and Fermentation

Investigations of proteolysis during anaerobic cocoa seed incubation have been extended by disc and SDS-gel electrophoretic protein analysis. Two protein bands (2.6 × 10⁴ and 4.4 × 10⁴ Dalton) were found to be vacuolar storage proteins, which accumulated during seed ripening (90 to 160 days after pollination) and which were specifically utilised during germination. Although the storage proteins are poorly soluble at pH 3.5–4.5, proteolysis during incubation of acetone dry powders is highest in this pH range. All proteins are digested at 50°C, pH 4.5. During seed incubation at 50°C, pH 4.5, however, the storage proteins are degraded preferentially although the cells are dead at 50°C. This degradation is increased by preincubation at 40°C instead of 50°C. The results are discussed in the light of structural peculiarities in the seed tissue and the possible role of specific endopeptidases and peptides in the formation of flavour precursors during fermentation.     

Time and Temperature Effect on Stability of Moroccan Processed Orange Juice during Storage

The loss of quality of processed pasteurized orange juice stored at 4°C, 22.5°C, 35°C, and 45°C for up to 14 wks was evaluated. The results showed that parameters such as pH, total solids, titratable acidity, formal index, and total sugars did not significantly change during storage at all temperatures. However, a major change was observed for ascorbic acid content, reducing sugars and furfural production, except for storage temperature at 4°C. Ascorbic acid degradation, sucrose hydrolysis, and furfural build-up followed pseudo-zero order reaction kinetics. The minimal change of formol number and total sugars suggested that nonenzymatic browning was mainly due to ascorbic acid degradation. Fufurfal formation during storage was found to be much higher than that reported in the literature.     

Effect of Cathepsin D on Bovine Myofibrils under Different Conditions of pH and Temperature

Purified cathepsin D was incubated with bovine skeletal muscle myofibrils under in virro conditions resembling those found in postmortem muscle. SDS-PAGE analysis of myofibrils treated at pH 5.5 and 37°C and the sedimented, showed degradation of myosin heavy chains and titin. A small amount of actin, tropomyosin, troponins T and I, and myosin light chains also were degraded. The cathepsin D treated myofibrils were not fragmented to any greater extend than untreated myofibrils. Raising the pH and/or lowering the temperature greatly reduced the effectiveness of cathepsin D suggesting that the enzyme does not play a principal role in the tenderization process occurring in muscle postmortem.     

Collagenase activity and protein hydrolysis as related to spoilage of iced cod (Gadus morhua)

Collagenase activity and changes in muscular protein of iced Atlantic cod stored for 9 days were studied. The crude fish muscle extract showed maximum collagenase-like activity against bovine insoluble tendon collagen at 48 h of incubation at 37 °C. Collagenase activity against synthetic substrate increased (P<0.05), especially for fish in initial and advanced stages of decomposition. These results suggest that endogenous collagenases and other proteases may be responsible for the destruction of fine collagenous fibrils in the skeletal muscle of cod. The content of titin 1 decreased when decomposition was advanced. Moreover, a progressive degradation of sarcoplasmic proteins with a molecular weight of 100, 94, 85 and 80 kDa was observed. Results suggest that softening of cod muscle during iced storage is caused more by collagenase activity than by proteolysis of myofibrils.     

Biochemical and Functional Properties of Myofibrils from Preand Post-Spawned Hake (Merluccius hubbsi Marini) Stored on Ice

Enzymatic activities assayed at beginning of storage in myofibrils from post-spawned hake were 3 X those in myofibrils from pre-spawned hake. Ca²⁺ sensitivity of myofibrils from pre-spawned hake was 40% less than that of myofibrils from post-spawned hake. The profiles of SDS-PAGE gels of pre-spawned myofibrils at beginning of storage showed a partially denatured myosin heavy chain, and polypeptide bands under myosin heavy chain. They probably represent proteolytic fragments produced by degradation of MHC in vivo. No proteolysis was detected in myofibrils during storage. These results help predict functional properties of fish proteins and changes during storage.     

Studies on the Subunits Involved in the Interaction of Soybean 11S Protein and Myosin

Protein-protein interaction between soybean 11S protein and myosin in a buffer system was studied using gel filtration chromatography and electrophoresis after incubating the single or combined proteins at temperatures between 4°C and 100°C. The elution profiles of 11S protein and myosin indicated that interaction between these two proteins occurred only at temperatures between 85° C and 100°C. The degree of interaction increased as temperature increased from 85 to 100°C. The interaction was not between native soy 11S and myosin, but between partially dissociated soy 11S (intermediary subunits, IS) or fully dissociated soy 11S (basic subunits) and myosin heavy chains. The rate of interaction was proposed as being more rapid between myosin and the basic subunits than between myosin and IS.