Effects of Postmortem Storage and Temperature on Muscle Protein Degradation: Analysis by SDS Gel Electrophoresis

The proteolytic breakdown of the major contractile proteins of bovine longissimus muscle was examined during postmortem storage by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Samples of muscle stored at 4°C for 14 days exhibited little proteolysis of the major contractile proteins; however, samples stored at 37°C for 1 day showed significant degradation of myosin heavy chains and almost complete proteolysis of this protein by day 14. Major degradation products of the myosin heavy chains included a series of polypeptides having molecular weights between 145,000 and 125,000. These experiments demonstrate that substantial degradation of the myosin heavy chain and other muscle proteins can occur during the storage of meat, and this phenomenon was highly temperature dependent.     

An immunological method to assess protein degradation in post-mortem muscle

A method for determining proteolysis of any specific protein in muscle is demonstrated. The protocol involves the preparation of a specific antibody which is used in immunoblotting total protein extracts from post-mortem muscle. In the present study an antibody to bovine myosin heavy chain was prepared that reacts with intact myosin heavy chain as well as proteolytic fragments that are generated by proteases. We show that little, if any, myosin heavy chain fragments are generated during prolonged aging of muscle at 4°C. In contrast, storage of muscle at 37°C results in the rapid breakdwon of myosin heavy chain to immunologically detectable peptides. Using a monoclonal antibody to titin, we demonstrate that this protein is degraded at 4°C during the aging period, and that, between 2 and 3 weeks following slaughter, no undergraded titin is detectable. This method is suitable for the analysis of any protein that can be separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE).     

Denaturation of Tilapia Myosin Fragments by High Hydrostatic Pressure

ABSTRACT: Hydrostatic pressures (50 to 300 MPa) were applied at 0 °C for 50 min to determine the aggregation and viscoelastic properties of tilapia ( Orechromis niloticus ) myosin fragments: subfragment-1 (S-1) and rod. With pressure lower than 150-MPa treatment, S-1 underwent intermolecular interaction to increase its elastic properties. After a 200-MPa treatment, S-1 unfolded, aggregated, and decreased its solubility. A turbid solution was obtained after pressurization over 200 MPa. Further, S-1 transformed to a more elastic gel with pressure increasing. At 200 MPa, S-1 denatured entirely with no change of enthalpy detected by differential scanning calorimetry (DSC). However, all properties of rod did not change during pressurization. This study reveals that S-1 contributes the initiation of gel formation in myosin, and the role of rod is not clear. Keywords: hydrostatic pressure, S-1, rod, denaturation     

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.     

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 Transitions of Myosins/Subfragments from Black Marlin (Makaira mazara) Ordinary and Dark Muscles

Thermal transitions were studied by means of differential scanning calorimetry (DSC) and a spectrophotometric method. Three endothermic peaks (40, 43, 50°C: ordinary muscle; 46, 54, 62°C: dark muscle) were observed in DSC thermograms of both myosins. Thermograms of S-l fragments showed one peak (41°C: ordinary muscle, 43°C: dark muscle). But ordinary and dark muscle rod fragments gave two peaks (41, 62°C) and one peak (58°C), respectively. The spectrophotometric results also showed two thermal transitions for both myosins and one transition for their S-1 fragments. However, the rod from ordinary muscle myosin had two transitions, whereas that from dark muscle myosin had one transition.     

The surface active properties of myosin and its proteolytic fragments

The surface active properties of myosin and its proteolytic fragments, light meromyosin (LMM), heavy meromyosin (HMM), subfragment-1 (S-1) and myosin rod, at initial bulk phase concentrations in the range of 10(-4)% to 10(-2)% w/v were determined by the drop volume method. Overall, S-1 was the most effective surface tension depressor, whereas the tail portions of myosin, i.e. LMM and myosin rod were less surface active than the parent myosin molecule. The surface pressures attained after 40 min, at an initial bulk phase concentration of 10(-2)% (w/v), were 22·00, 21·77, 21·02, 16·77 and 16·77 mNm(-1) for S-1, HMM, myosin, LMM and myosin rod, respectively. Furthermore, S-1 effected the most rapid change in surface pressure during the initial 5 min period.     

Influence of Endogenous Proteases and Transglutaminase on Thermal Gelation of Salted Squid Muscle Paste

ABSTRACT: Thermal gelation of salted squid mantle muscle paste was studied in relation to endogenous proteases and transglutaminase. Myosin in the paste was preferentially degraded into 130-kDa and 90-kDa fragments at an optimum temperature of 30 °C. Degradation was inhibited with EDTA or 1,10 phenanthroline, suggesting the presence of metalloproteases. Myosin degradation was markedly reduced above 40 °C. Although 10 mM Ca²⁺ increased cross-linking of myosin heavy chains by activating the endogenous transglutaminase, setting effect on thermal gelation of the paste was offset by degradation induced by simultaneously activated calpains. Ca2+ and the alpain inhibitor, E64, significantly improved the breaking strength and strain of thermal gels preincubated at 40 °C.     

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.     

宰后成熟期间结构蛋白对秦川牛肉嫩度的影响

为探究宰后成熟期间结构蛋白对秦川牛背最长肌嫩度的影响,测定不同贮藏期（0、2、4、6、8 d）内秦川牛背最长肌剪切力、肌原纤维小片化指数（myofibrillar fragmentation index,MFI）和蛋白含量等,并利用4D-非标记定量（4D-label free quantification,4D-LFQ）蛋白质组学技术分析蛋白质组学变化。结果表明：在贮藏期0～8 d内,剪切力总体呈先升后降的趋势（P＜0.01）,上升的幅度小于下降的幅度;秦川牛背最长肌MFI呈极显著上升趋势（P＜0.01）,总体增长了250.81%;总可溶性蛋白含量呈显著下降趋势（P＜0.05）,总体下降了34.60%;通过宰后肌肉组织代谢变化,肌肉组织结构蛋白发生降解,可能会影响到嫩度的形成,肌原纤维蛋白含量呈显著下降趋势（P＜0.05）,前期下降速度较快,后期下降速度减缓,总体下降了50.56%。在贮藏期0～4 d内,通过骨骼肌组织发育过程调控钙离子结合和细胞骨架蛋白结合途径,4 种蛋白（α-肌动蛋白-1、牛肌球蛋白重链9、牛肌球蛋白轻链2、肌球蛋白调节轻链12B）丰富度发生变化;在贮藏期0～8 d内,通过肌肉器官发育和横纹肌组织发育过程调控钙离子结合途径,8 种蛋白（肌球蛋白调节轻链2、肌球蛋白重链6、α-肌动蛋白-1、心肌肌动蛋白α1、牛肌球蛋白轻链2、肌钙蛋白I 1型、肌钙蛋白I 2型、肌球蛋白重链15）丰富度发生变化,通过肌球蛋白结合、钙离子结合、细胞骨架蛋白结合的肌原纤维组装、骨骼肌组织发育、肌肉器官发育、横纹肌组织发育过程等途径调控细胞的生理状态,结构蛋白降解造成肌原纤维小片化升高,进而促使嫩度提升。     

Identification of Cross-linking Site(s) of Myosin Heavy Chains in Oxidatively Stressed Chicken Myofibrils

ABSTRACT: The cross-linking site(s) of myosin heavy chains (MHC) in chicken myofibrils exposed to non-enzymatic, hydroxyl radical-generation oxidizing systems (HRGS) was investigated by means of chymotryptic digestion and subsequent electrophoresis. Oxidation of the chymotryptic digests resulted in cross-linking of the rod or light meromyosin (LMM) segment of MHC mostly via disulfide bonds, while subfragment-1 (S-1) or heavy meromyosin (HMM) was not affected. A mixture of cross-linked rod or LMM and uncross-linked S-1 or HMM was also produced when myofibrils were 1st oxidized and then digested with chymotrypsin, confirming that cross-linking of myosin in HRGS-oxidized myofibrils occurred initially in the LMM portion of the myosin rod.     

Gelation Characteristics of Paddlefish (Polyodon spathula) Surimi Under Different Heating Conditions

Gelation properties of paddlefish surimi were investigated with different heating procedures. Without pre-incubation, gel strength of paddlefish surimi increased as temperature increased from 40 to 60 °C. Pre-incubation at 40 °C caused myosin degradation and reduced gel strength by 55% compared to the control. Pre-incubation at 70 °C followed by cooking at 90 °C produced gels with maximum strength. Isothermal heating between 40 and 50 °C produced rheological transitions between 0 and 15 min. Beef plasma powder reduced myosin degradation and enhanced gelation of surimi incubated around 40 °C. These results indicated that the gel-weakening phenomenon in paddlefish surimi was due to the degradation of myosin by some endogenous protease(s).     

Effect of frozen storage on protein denaturation in bovine muscle 1. Myofibrillar ATPase activity and differential scanning calorimetric studies

The effect of frozen storage on myofibrillar ATPase activity and thermal transitions in bovine muscle was investigated. Myofibrillar ATPase activity and total enthalpy of denaturation (ΔH) decreased with time of storage. The rate of decrease was lower at −20°C than at −5°C or −10°C. Differences in behaviour during storage of muscle after fast or slow freezing could be attributed to differences in ice recrystallization. The observed decreases in area of the first peak seen in the thermograms and Ca²⁺-myo-fibrillar ATPase activity show that the myosin head denaturated progressively during storage. The myosin tail also denaturated during storage but the thin filament remained unaltered. Kinetic analysis suggested that the denaturation of the myofibrillar proteins took place through two consecutive first order reactions; an initial rapid reaction followed by a slower one.     

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.     

Effect of Endogenous Transglutaminase on Threadfin Bream Surimi Gelation

ABSTRACT: Transglutaminase(TGase) activity of threadfin bream mince was 99.6 units/g of dry weight. After washing and screw-pressed dewatering, 44% residual activity was retained. Covalent cross-linking of myosin heavy chain (MHC) was observed at both 25 and 40°C and supported by increased gel strength. When pre-incubation at 40°C was prolonged to 4 h, breaking force and MHC decreased due to endogenous proteinase(s). TGase activity towards MHC and synthetic substrates was effectively inhibited by iodoacetic acid (IAA). Autolytic activity and degradation of MHC was inhibited by phenylmethanesulfonyl fluoride (PMSF). Addition of 0.2% Ca²⁺ significantly improved breaking force and increased MHC cross-linking of surimi gels pre-incubated at 40°C for 2 h. Keywords: transglutaminase, myosin heavy chain, cross-linking, threadfin bream     

An Immunological Assessment of Myosin Degradation in Pressurized Chicken Muscle

ABSTRACT: The factors affecting myosin degradation that occurred during aging following high-pressure treatment over a pressure range from 200 to 600 MPa were investigated by using SDS-PAGE and immunoblotting analysis. The immunoblot pattern of myosin in muscle stored at 37°C for 48 h after pressure treatment at 0. 1 MPa (atmospheric pressure) or 200 MPa for 5 min was similar to that of native myosin incubated with cathepsin D, whereas at 400 or 600 MPa the pattern was close to that of native myosin incubated with cathepsin B. This phenomenon was reflected in the pressure-susceptibilities of cathepsins B and D as reported in the literature (Homma and others 1994). However, these catheptic enzymes released by pressure treatment are unlikely to play a role in pressure-induced tenderization of meat.     

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.     

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 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.     

Changes in myofibrillar proteins during processing of salted cod (Gadus morhua) as determined by electrophoresis and differential scanning calorimetry

The effects of salt-curing, drying and rehydration on muscle proteins in cod (Gadus morhua) were studied during the processing of heavily salted cod or “bacalhau”. The aim was to observe conformational stability and possible degradation or denaturation, with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC). The salting process significantly decreased the heat stabilities of both myosin and actin. The decrease in water content during dry-salting did shift the transition temperatures slightly back to higher temperatures. The results, from the SDS-PAGE, showed that the myosin heavy chain (MHC) was cleaved into smaller sub fragments in the salting process with the two heavy meromyosin fractions (HMM S1 and S2) and the light meromyosin (LLM) fraction being the most abundant. Actin was less affected than myosin.