Cryoprotection Affects Physiochemical Attributes of Rainbow Trout Fillets

ABSTRACT: Stabilization of trout myofibrillar proteins during −20°C, 90-d storage and after soaking fillets in water, 8.0% sucrose/sorbitol, or 1.0% sodium lactate was investigated with or without 0.5% phosphate and with or without 0.05% MgCl₂. Fillets not soaked were the control. Compared with the control, cryoprotectants increased total protein and myofibrillar protein solubility; decreased surface hydrophobicity, total, free, and disulfide sulfhydryl content, and myosin susceptibility to thermal denaturation. There were no differences in total protein solubility and actin susceptibility to thermal denaturation between cryoprotectants and the water treatment. Phosphate minimized frozen storage effects on actin solubility and reduced protein surface hydrophobicity and myosin susceptibility to thermal denaturation, while MgCl₂ increased the negative effects of frozen storage.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE: EFFECT OF SULFHYDRYL REAGENTS AND POST-MORTEM STORAGE ON CHANGES IN MYOSIN B

Studies to determine the relationship of SH groups to certain changes of the myofibrillar proteins of post-mortem muscle were carried out with myosin B from at-death and post-mortem stored rabbit skeletal muscle (2° C and 25° C for 3 days) and with SH reagent modified myosin B from at-death and post-mortem stored muscle. Quantitative SH analysis, ATPase activity, turbidity rate and analytical ultracentrifugation were employed to determine the changes in myosin B associated with changes in SH groups. Post-mortem storage of muscle at 2°C for 3 days had no effect on SH content of myosin B; a decrease in SH groups, however, was observed in myosin B from muscle stored at 25°C for 3 days and for iodoacetamide (IAA) and N-ethylmaleimide (NEM) modified myosin B. ATPase activity was inhibited by reacting myosin B with enough NEM or IAA to block all SH groups. Dialysis of myosin B from at-death and post-mortem muscle against MCE to restore SH groups resulted in partial reversal of Mg++ and EGTA-activated ATPase of myosin B from post-mortem muscle and a less rapid rate of turbidity development. These results suggest that the state and nature of SH groups are partly involved in the actin-myosin interaction of post-mortem muscle; other constituents, however, in addition to SH groups are evidently modified and, in some instances, irreversibly modified, under certain post-mortem storage conditions.     

Effect of frozen storage on the structure and enzymatic activities of myofibrillar proteins of rabbit skeletal muscle

The effect of frozen storage on the biochemical properties of myofibrils, and of their major constituents, actin and myosin, was investigated. Extractability of myofibrillar proteins increased slightly for 3 weeks during frozen storage of muscle, decreasing thereafter. The change in myofibrillar ATPase activity during frozen storage was consistent with that of a reconstituted acto-heavy meromyosin (HMM) complex prepared from frozen stored muscle at the same weight ratio of actin to myosin as in situ. However, myosin ATPase activity showed a different pattern of change when compared with myofibrillar ATPase activity. The maximum velocity of acto-HMM ATPase activity and the apparent dissociation constant of the acto-HMM complex decreased for 1 week during frozen storage, increasing thereafter, indicating that the affinity of actin for myosin was greatest in muscle which had been frozen for 1 week.     

Application of competitive indirect enzyme-linked immunosorbent assay (Ci-ELISA) for monitoring the degree of frozen denaturation of bovine myosin

The denaturation of myosin on freezing and frozen storage was monitored using competitive indirect enzyme-linked immunosorbent assay (Ci-ELISA) formatted with polyclonal antibodies anti-MWM IgG, anti-S-1 IgG and anti-LMM IgG raised against the antigens (Ags) bovine myosin whole molecules (MWMs), heavy meromyosin S-1 (myosin head part, S-1) and light meromyosin (myosin tail part, LMM) respectively. Beef slices and cuts stored at −20 °C or −50 °C lost immune affinity with all antibodies, in particular anti-LMM IgG. Repeated thawing–refreezing treatment caused more myosin denaturation than simple freezing. Myosin from beef stored at −20 °C was denatured more than that stored at −50 °C. The immune affinities between anti-LMM IgG and thawed samples were similar to those from anti-MWM IgG. We were unable to differentiate reliably between fresh and thawed beef using anti-S-1 IgG. Myosin was denatured by freezing, in particular its tail part (LMM).     

Thermal stability of fish myofibrils: a differential scanning calorimetric study

The variation in myofibrillar protein thermostability was compared for various fish species, using differential scanning calorimetry. The tropical fish, catfish ( Clarius gariepinus ), carp ( Cyprinus carpio ), Nile perch ( Lates niloticus ), red snapper ( Lutianus sebae ), red mullet ( Parpeneus barberinus ), sea bream ( Gymnocranius rivalatus ), and cold-water reared trout ( Salmo gairdneri ) and cod ( Gadus morhua ) were analysed. Onset temperature of myofibrillar protein denaturation occurred at up to 11°C higher for tropical species (43.5°C, catfish), than cod (32.6°C) at pH 7 and low ionic strength (I). As pH (6.0-8.0) and I (0.05-1.00) were increased, thermal denaturation temperatures of myosins from tropical, but not cold-water, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and I. Only one thermal transition was detected for myosin at pH 6 and low I, increasing to three as pH and I were increased. Changes in thermal characteristics of myosin subunits over iced and frozen storage suggest more rapid deterioration in cold-water than in tropical fish. The differences in myofibrillar stability of fish from different habitat temperatures have implications for the processing and storage of tropical fish.     

Effect of Ice Storage on the Physicochemical and Dynamic Viscoelastic Properties of Ribbonfish (Trichiurus spp) Meat

ABSTRACT: Changes in physicochemical and dynamic viscoelastic properties of ribbonfish ( Trichiurus spp) meat during different periods of ice storage were investigated. The differential scanning calorimetry profile of fresh ribbonfish meat revealed transitions at 33.17 °C, 48.85 °C, and 60.96 °C, indicating denaturation temperature of different protein fractions. The effect of cornstarch or tapioca starch at 9% level on the viscoelastic properties of ribbonfish meat stored in ice for different periods was also evaluated. Total volatile basic nitrogen (TVBN) increased significantly ( P < 0.05) during ice storage for 24 d. However, the myosin heavy chain concentration was unaltered during the ice storage period, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophore-sis (SDS-PAGE) pattern. A significant ( P < 0.05) decrease in protein solubility (in phosphate buffer 50 m M , pH 7.5, containing 1 M NaCl), calcium-activated adenosine triphosphatase (ATPase) activity, and an increase in reduced viscosity at a protein concentration of 5 mg/mL was observed after 10 d of ice storage indicating protein denaturation and aggregation. The addition of tapioca and cornstarch enhanced storage modulus values of fresh ribbonfish meat. The gelatinization temperature of tapioca starch solution was found to be in the range of 60 °C to 65 °C and for cornstarch 67 °C to 70 °C, as revealed by the differential scanning calorimetry (DSC) profile and dynamic rheological testing. The viscoelastic properties of ribbonfish meat was altered significantly ( P < 0.05), both due to the addition of starch and ice storage period as revealed by frequency sweep of prepared gels.     

Molecular Properties of Post-Mortem Muscle.

SUMMARY— Post-mortem changes in nucleoside triphosphatase activity of bovine myosin B have been studied by using several different modifiers with either 5 mM ATP or 5 mM ITP as substrate at ionic strengths (r/2) of 0.09, 0.19, or 0.52. Enzymic activity was determined by measuring the release of inorganic phosphate. There was very little difference in enzymic activity between myosin B isolated from prerigor, rigor (24 hr post-mortem) or post-rigor (312 hr post-mortem) muscle stored at either 2° or 16°C except that the specific activity of myosin B prepared from muscle stored for 12–24, hr post-mortem was higher than activity of myosin B prepared immediately after death. This increase cannot be explained in terms of rigor shortening, but suggests that a change in myosin conformation or in the nature of the actin-myosin interaction occurs in post-mortem muscle. If an actin-myosin interaction occurs during rigor mortis and if this association remains unchanged during extraction of myosin B, then the very low Mg⁺⁺-modified myosin B enzymic activities obtained at Γ/2 = 0.19 and 0.52 indicate that this interaction is not irreversible. Extraction in the absence of ATP produced a myosin B whose ATPase activity was markedly inhibited by trace amounts of Mg⁺⁺. This may be due to the absence of a-actinin in these myosin B preparations. No consistent differences in activation energies were found either at Γ/2 = 0.19 or 0.52 among the NTPase reactions of myosin B samples prepared from muscle after various times of post-mortem storage.     

Interaction of fish myoglobin and myofibrillar proteins

ABSTRACT:  Interactions between fish myoglobin (Mb) and myofibrillar proteins were investigated in a Mb-natural actomyosin (NAM) model at 4 °C. Increases in metmyoglobin (MetMb) formation and the relative content of bound Mb were observed, as were decreases in whiteness and Ca²⁺-ATPase activity ( P < 0.05). During the first 6 h of incubation, Mb bound preferably to myosin at domains other than the head portion, as evidenced by measurable ATPase activity. The potential binding of Mb to myosin heads occurred after 24-h incubation as evidenced by the marked decrease in Ca²⁺-ATPase activity of the NAM–Mb mixture when compared to that of NAM alone ( P < 0.05). The interaction between fish Mb and myofibrillar proteins was more pronounced with increased storage time; formation of high-molecular-weight aggregates (> 206 kDa) also increased with time. Electrophoretic study revealed that disulfide bonds were not involved in Mb–NAM interactions.     

Morphological changes in tilapia muscle following freezing by airblast and liquid nitrogen methods

The cross-section spacing between the muscle fibre bundles of fresh tilapia chunks was ≈3.06 μm. After freezing by airblast at −20 and −36°C, and by liquid nitrogen at −87 and −128°C at freezing rates of 0.25, 1.53, 9.74 and 19.4 cm h⁻¹, respectively, the spacing increased to 3.21–7.69 μm, which was 5 to 151% greater than that in the fresh samples. The spacing further increased with storage time. Liquid nitrogen freezing resulted in smaller increases in spacing than airblast freezing. On freezing at constant temperatures of −20 to −128°C followed by storage at −20°C for 1 month, the extracellular spacings were 7.38–13.8 μm, and increased to 22.16–29.38 μm after 2 months. After storage at −20°C or −40°C for 6 months, the muscle fibre bundles showed fragmentation in both the airblast and the liquid nitrogen frozen tilapia chunks. The integrity of muscle structure was maintained better with liquid nitrogen freezing than with airblast freezing. All the differences resulting from freezing methods or freezing rates disappeared upon prolonged frozen storage at −20°C or −40°C. The correlations between the freezing temperature and extracellular spacing, and the activation energy (E_a) was calculated. The time required for freezing-temperature-induced differences in crystal growth, or in the extracellular spacing of muscle fibre bundles to disappear when E_a= 0 can be considered as the high-ultrastructural quality shelf-life, which is predicted to be 2.7 months at −20°C for tilapia frozen with liquid nitrogen.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE. 9. Effect of Temperature and pH on Tropomyosin-Troponin and Purified α-Actinin from Rabbit Muscle

SUMMARY– A study was done on the effects of in vitro storage of purified α-actinin, troponin, tropomyosin, and the tropomyosin-troponin complex on the activity of these protein fractions in the ATPase and superprecipitation assays. Storage was done at various combinations of temperatures between 0 and 40°C and pH values between 5.7 and 7.0. Even after 40 hr of storage, activities of purified tropomyosin and the tropomyosin-troponin complex were not affected by any combination of temperature and pH included in this study, but activities of purified α-actinin and troponin were almost completely lost after 16 hr at 40°C and pH 5.7. Storage for 40 hr at low pH (5.7) and low temperatures (0°C) did not affect the activity of either α-actinin or troponin, but 40 hr of storage at high temperatures (40°C) and neutral pH caused some loss in activity for both these proteins. This loss of activity caused by 40°C, pH 7.0 storage was much more noticeable in the case of troponin than in the case of α-actinin. Storage periods of 40 hr or longer were required before any loss of α-actinin activity could be detected at pH 7.0 and 40°C. Since most meat animal carcasses are chilled soon after exsanguination and attain muscle temperatures of 25°C or lower before the pH falls below 6.2, it is probable that α-actinin and tropomyosin-troponin activity remain almost unchanged in meat handled through normal market channels. However, myofibrillar tissue in those porcine animals whose musculature undergoes a very rapid post-mortem decline in pH so that values of 5.7 or less are reached while muscle temperatures are still 37°C or higher may lose much of its α-actinin and tropomyosin-troponin activity during the first 24 hr post-mortem.     

Molecular Properties of Post-Mortem Muscle. 6. Effect of Temperature on Protein Solubility of Rabbit and Bovine Muscle

SUMMARY– Studies were conducted to investigate the effect of temperature on the actin-myosin interaction of rabbit and bovine muscle during rigor and post-rigor shortening. Muscle was stored at four different temperatures (2°, 16°, 25° and 37°), corresponding to three types of post-mortem muscle shortening: cold, minimal and high temperature. These three types of shortening are presumably related to different states of the actin-myosin interaction in post-mortem muscle. Post-mortem tenderization may be the result of either actin-myosin dissociation or F-actin depolymerization.
To detect the occurrence of either of these possible changes, two salt solutions, differing widely in their myofibrillar protein extracting abilities, were used to compare post-mortem myofibrillar protein solubility after different times of post-mortem storage and to provide information about the actin-myosin complex. Myofibrillar protein solubility of both rabbit and beef muscle in 0.5M KCl, 0.1M phosphate, pH 7.4, increased markedly with increasing post-mortem storage at temperatures up to 25deg;. Similar solubility changes were obtained with 1.1M Kl, 0.1M K phosphate, pH 7.4, but these changes were much smaller in magnitude. Solubility in both salt solutions, in general, decreased for muscle stored at 37°.
Although time and temperature of post-mortem storage caused appreciable alterations in protein solubility, these alterations could not be directly related to changes in tenderness or sarcomere length or to species differences in the effects of temperature on post-mortem shortening. Viscosity, analytical ultracentrifugation, and ATPase assays all indicated the absence of "normal" actomyosin in all myofibrillar protein extracts in this study. It was suggested that the 1.1 M KI extracts contained G-actomyosin, but the available evidence indicated the presence of only myosin in 3-hr, 0.5 M KCI extracts.     

CHARACTERISTICS OF MUSCLE FROM TWO SPECIES OF BIGEYE SNAPPER, PRIACANTHUS TAYENUS AND PRIACANTHUS MACRACANTHUS

Composition and some properties of muscle from two species of bigeye snapper, P. tayenus and P. macracanthus, were investigated. Both species had a similar composition with the same myofibrillar protein content. However, muscle proteins from P. tayenus had higher thermal stability than those from P. macracanthus, as indicated by the higher enthalpy for transitions as well as the lower inactivation rate constant (K_D). Upon 15 days of iced storage, natural actomyosin Ca^2*‐ATP ase and Mg²⁺‐Ca²⁺‐ATPase activities decreased, whereas Mg²⁺‐EGTA‐ATPase activity increased, suggesting the denaturation of myosin, actomyosin and troponin/tropomyosin complexes, respectively. Increased surface hydrophobicity and decreased sulfhydryl groups indicated the denaturation possibly occurred via hydrophobic interaction and disulfide formation. Heading and eviscerating offish retarded the denaturation and physicochemical changes of proteins during iced storage. The results indicated that a rapid and proper post harvest handling was of importance to maintain the muscle quality of bigeye snapper.     

Protein Denaturation and Structural Damage During High-Pressure-Shift Freezing of Porcine and Bovine Muscle

ABSTRACT: Pork and beef muscles were subjected to 200 MPa and −20 °C with or without water freezing. Both tissues responded to the treatment with similar behavior. Protein denaturation was greater when freezing occurred. Pressure-induced cold denaturation was complete for actin and very considerable for myosin and other muscle proteins. Connective proteins remained practically unaltered by pressurization and/or freezing. Structural changes in the muscle at sarcomere levels caused by pressurization were more severe when freezing occurred. Color, drip loss, and textural properties on the pressurized samples also revealed an additional deleterious influence of freezing. Pressurization alone and pressure-shift freezing resulted unsuitable for muscle preservation.     

Changes in firmness and thermal behavior of ice-stored muscle of jumbo squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>)

Changes in firmness, muscle total protease activity, and the thermal behavior of jumbo squid (Dosidicus gigas) were measured throughout 15 days of ice-storage. A significant decrease (p<0.05) in the shear force of raw mantle muscle was observed after 7-days ice-storage. The highest total protease activity detected was 1.24 U/g mantle. The thermograms obtained at day zero showed four transition states. The first three transition states were endothermic and correspond to myosin (50 °C), sarcoplasmic proteins (69 °C), and actin (79 °C). The fourth transition state was exothermic at 107 °C, and was probably associated with protein aggregation. The thermal behavior of the muscle showed a decreasing trend in temperature and enthalpy of transition for myosin, sarcoplasmic proteins, and actin with storage time. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a change in the myofibrillar protein pattern, which with the shear force, and differential scanning calorimetry data, suggests a partial denaturation of that protein fraction during ice-storage.     

QUANTITATIVE CHANGES IN WHOLE MYOFIBRILS AND MYOFIBRILLAR PROTEINS DURING FROZEN STORAGE OF TRUE COD

The extractability of whole myofibrils from true cod decreased more rapidly during frozen storage at −40°C than did the extractability of the component myofibrillar proteins. There was a 95% decrease in extractable myofibrils after 6 months in storage, but only a 23% decrease in extractable myofibrillar proteins.     

Cryoprotective additives and cryostabilisation effects on muscle fillets of the freshwater teleost fish Rohu carp (Labeo rohita) during prolonged frozen storage

The effects of various cryoprotective additives separately and in combination were studied on the myofibrillar protein integrity, biochemical enzyme activity levels and muscle ultrastructure in the freshwater teleost fish Rohu carp (Labeo rohita). Fish muscle samples were divided into eight groups and immersed in different mixtures of cryoprotective additives (S1–S8), then frozen at ? 20 or ? 30 °C for 24 months. Electrophoretic studies revealed early (within 6 months) alteration of the myofibrillar proteins myosin light chain, α‐actinin and tropomyosin. Reduction of the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that fish muscle treated with cryoprotective mixture S8 (40 g L^?1 sorbitol/3 g L^?1 sodium tripolyphosphate/4 g L^?1 sodium alginate) showed minimal post mortem changes in myofibrillar proteins. Ultrastructural results also revealed post mortem damage to the muscle, seen earliest (within 6 months) in the sample frozen‐stored without additives (S2), as compared with the normal, unfrozen muscle (S1). The influence of cryoprotectants alone and in combination on fish muscle structural proteins, myosin and actin filaments (A and I bands), during prolonged frozen storage was investigated. After 12 months, samples frozen‐stored with various cryoprotective additives (S2‐S7), except S8, showed signs of myofibrillar disintegration. Beyond that time the degradative processes started showing up in all samples, with minimal muscle ultrastructural damage in sample S8. Again, reducing the storage temperature from ? 20 to ? 30 °C slowed down the degradative processes. Ultrastructural results correlated well with levels of biochemical enzymes (Ca²⁺ myofibrillar ATPase and succinic dehydrogenase) during frozen storage. This is the first report of the cryoprotective effects of these additives on this popular edible fish species. Of the various combinations of additives tested, cryoprotective mixture S8 was found to preserve the muscle structure longest under frozen storage conditions. However, even this mixture was only effective for 18 months at ? 30 °C. Beyond that time the myofibrillar degradative processes were apparent with correlative electrophoretic, biochemical and ultrastructural studies. Copyright © 2006 Society of Chemical Industry     

Changes in myofibrillar proteins during processing of pastirma (Turkish dry meat product) produced with commercial starter cultures

The effects of different commercial starter cultures (Staphylococcus carnosus, S. carnosus + Lactobacillus pentosus and Staphylococcus xylosus + Lactobacillus sakei), on myofibrillar proteins were investigated using differential scanning calorimetry (DSC) during the processing of pastirma. The stage of pastirma production significantly decreased the thermal stabilities of myosin and actin. Actin was less affected than myosin. The myofibrillar fraction of pastirma was hardly denaturated by S. carnosus, but more pronounced denaturation was obtained with S. carnosus + L. pentosus.     

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.     

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.     

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