Changes in structure of psoas major and minor and semitendinosus muscles of calves, heifers and cows during post-mortem ageing

Changes occurring during post-mortem ageing in structure of psoas major and minor (PM) and semitendinosus (ST) muscles of calves, heifers and cows were observed. Samples from muscles taken 3 h after slaughter and after 6 and 12 days of storage at the temperature of 4?°C were analysed using light and transmission electron microscopy. Three hours after slaughter muscle fibres were close to each other and sarcomeres showed normal structure of A- and I-bands, M-lines and Z-disks. The space between myofibrils and sarcolemma and also between single myofibrils in muscle fibres increased during storage. Structure of sarcomeres underwent continuous degradation, length of sarcomeres increased due to enlargement of I-bands accompanied by Z-disk degradation. During ageing structural changes in myofibrils took place faster, were more intensive in muscles of younger animals and were more extensive and faster in PM than in ST muscles.     

Treatment and Post-Mortem Aging Effects on the Z-line of Myofibrils from Chicken Pectoral Muscle

SUMMARY: Changes in the morphology of myofibrils prepared from chicken pectoral muscle during post-mortem storage at 5°C were examined by light and electron microscopy. When the 24-hr stored samples were blendorized, electron micrographs showed two types of destruction in the Z-lines of sarcomeres and myofibrillar fragments: (1) The degradation and/or disappearance of Z-lines. (2) The breakdown of the junction of Z-line and I-filaments. A change in the state of the Z-line and the junction of the Z-line and I-filaments appeared to be indispensable for the fragmentation of the myofibrils. It was also shown through phase contrast microscopic observations that sarcoplasmic proteins, participating in the glycolytic cycle, may play a role in the fragmentation of the myofibrils. Evidence has not been obtained, to date, on the participation of proteolytic enzymes in the fragmentation phenomenon.     

Molecular Properties of Post-Mortem Muscle. 3. Electron Microscopy of Myofibrils

SUMMARY— A study was made of the fine structure of myofibril suspensions prepared from seven heifers immediately after death and after various times post-mortem. Studies on myofibrils sampled immediately after death showed that sucrose isolation gave the best structural preservation as indicated by maintenance of Z-line structure. Although the appearance of resting muscle was maintained in both sucrose and KCI preparations, several myofibrils from the KCI-treated preparations showed stretched sarcomeres. Glycerol-treated myofibrils usually had shorter sarcomere lengths than myofibrils prepared with the other two solvents. Although fibrillar preservation seemed adequate when glycerol was used, Z-line structure was seldom well-preserved with glycerol.
Myofibrils from muscle sampled 24 hr post-mortem at 2°C were supercontracted with thick filaments pushed against or through the Z-line, and no trace of l-bands remained. Myofibrils from muscle sampled 24 hr post-mortem at 16°C were contracted, but to a much lesser extent than 2°C-24 hr myofibrils. Storage at 2°C for 312 hr after death resulted in myofibrils that were contracted and that were structurally in a much poorer state of preservation than their 16°C counterparts. The 16°C-312 hr myofibrils were slightly contracted as indicated by the absence of H-zones and the presence of prominent, although narrowed, I-bands. All observations showed that shortening accompanying rigor mortis caused changes in banding patterns similar, and probably identical, to those predicted by Huxley's sliding filament model for contracting muscle.     

Effect of Washing on the Texture and Microstructure of Frozen Fish Mince

Textural hardening of fish mince during storage was investigated by examining texture and structure of unwashed and washed fish mince. Washing reduced TMAO and increased water-binding ability, but did not prevent development of hard, rubbery texture in fish mince during frozen storage. Transmission electron microscopy showed more shrinkage of myofibrillar units (sarcomeres) occurred in washed than unwashed mince. Textural hardening of washed mince was thus related to increased freeze-induced contraction of myofibrils. Removal of water-soluble sarcoplasmic proteins facilitated freeze-induced contraction of myofibrils, leading to textural hardening. This suggested that water soluble proteins retard sarcomere shrinkage resulting from freeze-induced contraction/protein cross-linking.     

Effects of ultrarapid chilling and ageing on length of sarcomeres, and tenderness of pork

In this study, the m. longissimus dorsi and the m. semimembranosus of pigs with normal and accelerated glycolysis (pale, soft and exudative, PSE) were used to evaluate the effect of ultrarapid chilling methods (with short-term freezing of the muscle surface) and ageing up to 72 h post-mortem on length of sarcomeres, Warner Bratzler shear force and firmness. The short-term freezing after ultrarapid chilling of musculature with normal glycolysis led to cold shortening with contraction of the sarcomeres by 33·5% (m. longissimus dorsi) and 38·3% (m. semimembranosus). At the same time, a massive increase in shear force and firmness was observed. After ageing up to 48h post-mortem the sarcomeres increased in length, whereby, in comparison with the measurements before the beginning of the chilling, 88 and 84% of the original length was attained, respectively. In addition, ageing led to a significant improvement of tenderness. After ageing for more than 72h the shear force and the firmness of meat specimens chilled under normal and ultrarapid conditions became more similar. The lenghts of the sarcomeres and the shear force of musculature with accelerated glycolysis were glycolysis were not affected by the chilling procedure.     

Structural studies of rigor bovine myofibrils using fluorescence microscopy. II. Influence of sarcomere length on the binding of myosin subfragment-1, alpha-actinin and G-actin to rigor myofibrils

Sarcomere length influences the textural quality of meat, yet the molecular basis for the mechanism of post-mortem shortening and the toughness associated with shortened muscle remain obscure. Bovine and rabbit myofibrillar structure was investigated over a range of sarcomere lengths (SL), using the high affinity of the myosin head for actin and of alpha-actinin for the Z-line. Myofibrils were incubated with fluorescent conjugates of myosin subfragment-1 (S1), alpha-actinin and actin. When S1 and alpha-actinin were added together, S1 bound in the I-band and A-band but not at the Z-line, while alpha-actinin bound at the Z-line. The pattern of S1 binding was highly influenced by SL, and could be explained using a model with the ratio of myofibrillar actin to myosin heads in the overlap region of 2:1 and thin filament penetration into opposing half sarcomeres. Double staining with S1 and actin demonstrated that, once the tip of the thin filament reached the bare zone, few intrinsic myosin heads were available for fluorescent actin. The patterns observed for both S1 and actin staining suggest that myofibrillar rigor bonds form even at very short SL. These observations lead to the hypothesis that the toughness associated with short sarcomeres is due to thick-filament interactions and not to the density of rigor bonds in the myofibril. Regulation of S1 binding was investigated by incubating myofibrils with low levels of fluorescent S1 in the presence and absence of calcium; S1 binding was in the overlap region when calcium was absent, but in the I-band when it was present. These results suggest that the thin filament can be activated for muscle shortening by the binding of myosin heads, a mechanism that may contribute to post-mortem muscle shortening.     

Relationship between the translocation of paratropomyosin and the restoration of rigor-shortened sarcomeres during post-mortem ageing of meat-A molecular mechanism of meat tenderization

The weakening effect of paratropomyosin on rigor linkages formed between actin and myosin was determined by measuring the restoration of rigor-shortened sarcomeres of chicken, pork and beef. We observed the rate of the post-mortem translocation of paratropomyosin from the A-I junction region of sarcomeres onto the thin filaments in the A-band, where rigor linkages had been formed; this agreed well with the rate of increase in length of rigor-shortened sarcomeres. The sarcomere lengths were found to be maximum at 1, 7 and 10 days post-mortem in chicken, pork and beef, respectively. Thus, translocated paratropomyosin weakens rigor linkages and brings about the recovery in the length of rigor-shortened sarcomeres. Paratropomyosin stimulates the resolution of rigor mortis, and is a key factor in meat tenderization during post-rigor ageing. These results powerfully support the ‘Calcium theory of meat tenderization’ which we have proposed.     

Contribution of major structural changes in myofibrils to rabbit meat tenderisation during ageing

The contribution of major structural (myofibrillar fragmentation upon mechanical treatment) and ultra-structural (Z-line degradation, loss of electron density of M-line, transversal disruption of sarcomeres at N(2)-line level, longitudinal fissure of myofibrils, and loss of transversal alignments of Z- and M-lines) changes in myofibrils to rabbit (Oryctolagus cuniculus L.) meat tenderisation, during the ageing period (9 days at +4?°C), was studied for different types of muscle (type I, semimembranosus proprius; type IIB, semimembranosus accessorius; and type IID, psoas major). The results strongly suggest that myofibrillar structure weakening at N(2)-line level (evaluated by myofibrillar fragmentation upon mechanical homogenisation and observed by transversal disruption of sarcomeres), which is very likely mediated by cysteine endopeptidases, might be the major structural change responsible for rabbit meat tenderisation during ageing. Both myofibrillar fragmentation and transversal disruption of sarcomeres are good ageing indices for rabbit meat. The other major ultra-structural changes in myofibrils appear to have no major role in rabbit meat tenderisation at refrigeration temperatures. Finally, it is proposed that meat tenderisation during ageing depends mainly on the specific cleavage of titin molecules/filaments and nebulin molecules, at their susceptible sites located at or very close to the N(2)-line region (extensible segment and near C-terminus, respectively), mediated by cysteine endopeptidases (possibly calpains).     

Sarcomere shortening of prerigor muscles and its influence on drip loss

Detailed studies of muscle shortening post mortem at incubation temperatures between −2°C and +38°C revealed that the sarcomeres in unrestrained, excised red bovine muscle (M. sternomandibularis) shortened less than 10 % in the prerigor state between 6°C and 18°C. Below 6°C, sarcomeres contracted up to 70%. Between 20°C and 38°C sarcomere shortening of 40% was observed. In the red porcine M. cleidooccipitalis the minimum of shortening was measured at about 10°C, a higher degree of shortening—up to 50%—being obtained above and below this temperature. The drip loss of both muscle types increased linearly with increasing prerigor shortening.

This latter relationship is discussed with regard to changes within the muscle post mortem. The influence of three events on water movement from the interfilamental space into the interfibrillar fluid and from there into the extracellular space is critically evaluated. These events are: (1) the prerigor contraction of sarcomeres depending on the temperature of storage, (2) the changes due to the falling pH post mortem and (3) the onset of rigor mortis, with its irreversible association of actin and myosin.     

Protein extractability and thermal gel formability of myofibrils isolated from skeletal and cardiac muscles at different post-mortem periods

The effects of the post-mortem ageing period on the extractability of myofibrillar proteins from pork cardiac and rabbit skeletal muscles under various conditions of pH and ionic strength were studied with particular reference to the changes in the solubility of individual myofibrillar proteins and their denaturation characteristics. The ultimate influence of these changes on the heat-induced gel forming ability of myofibrils isolated from cardiac and skeletal muscles at different post-mortem stages was also investigated. Results showed that pork cardiac myofibrils always exhibited lower solubility than those from rabbit skeletal muscles under identical conditions of pH, ionic strength and temperature. SDS-PAGE profiles indicated several quantitative differences in the relative proportion of individual protein species present in cardiac and skeletal myofibrils. The solubility of various proteins present in myofibrils was also affected differently on heating in 0-1 and 0-6 _M NaCl solution at various pH values. Thermal denaturation of cardiac myofibrils occurred at about 10°C higher than that of skeletal myofibrils as revealed by differential scanning calorimetry. Cardiac myofibrils formed much weaker heat-induced gels than those produced by skeletal myofibrils under identical conditions of temperature, pH, ionic strength and protein content.     

Post-Mortem Changes in Subcelllular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 2. Electron Microscopy.

SUMMARY— Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum, and light sarcoplasmic reticulum fractions were isolated from homogenates of normal and pale, soft, exudative (PSE) porcine muscle at 0 and 24 hr post-mortem and examined by electron microscopy. No differences were observed between normal and PSE myofibrils obtained at death. PSE myofibrils prepared at 24 hr post-mortem had more granular appearing filaments and wider Z lines than normal myofibrils at 24 hr. The PSE heavy sarcoplasmic reticulum fraction obtained at death had a higher proportion of granular material than the same fraction from normal muscle. Several structural differences between the other PSE and normal fractions were also observed, especially at 24 hr postmortem. This study indicated that the composition of the subcellular fractions changed with time post-mortem and that this change should be considered when analyzing biochemical data from these fractions. However, the differences observed could not explain the large changes in calcium accumulating ability that have been shown to occur post-mortem.     

LOCALIZATION OF PARATROPOMYOSIN IN CHICKEN SKELETAL MUSCLE MYOFIBRILS AND ITS BEHAVIOR DURING POSTMORTEM STORAGE OF MUSCLES REVEALED BY IMMUNOELECTRON MICROSCOPY

Paratropomyosin is a minor myofibrillar protein which in freshly prepared myofibrils is exclusively localized at the A-I junction region of sarcomeres. We investigated the ultrastructural localization of paratropomyosin in intact and postrigor myofibrils by immunoelectron microscopy. Paratropomyosin was localized as two distinct stripes at the A-I junction in intact myofibrils. It also was localized at the position corresponding to the original A-I junction in thick filament-free myofibrils (I-Z-I brushes). However, following postmortem storage, paratropomyosin was found broadly distributed in thin filaments of myofibrils.     

Effects of electrical stimulation, chilling temperature and hot-boning on the tenderness of bovine muscles

The objective of this study was to determine the effects of hot-boning, low voltage electrical stimulation (ES) and chilling temperature on the tenderness of bovine M. longissimus dorsi (LD) and M. semimembranosus (SM) muscles. LD (n=32) and SM (n=32) muscles were subjected to different post-mortem treatments; hot-boning (before 90min post-mortem), cold-boning (at 48h post-mortem), low voltage ES and rapid or slow chilling. Hot-boned muscles which were not electrically stimulated (NES) had higher Warner Bratzler shear force (WBSF) values (P<0.001) and shorter sarcomeres (P<0.001) than cold-boned muscles. Hot-boned muscles subjected to ES had lower pH values (P<0.001) post-stimulation and up to 8h post-mortem than NES muscles. At both chilling temperatures WBSF values were lower in ES hot-boned LD and SM muscles at days 2, 7 and 14 post-mortem than NES muscles. Hot-boned muscles subjected to slow chilling had longer sarcomeres (P<0.001) than those subjected to fast chilling. In hot-boned SM muscles, ES resulted in longer sarcomere lengths (P<0.001). However, ES did not have a significant effect on the sarcomere length of LD muscles. As indicated by WBSF values all muscles tenderised during ageing, including muscles which were 'cold shortened'. Proteolysis was not the main reason for differences in WBSF values between ES and NES muscles as judged by qualitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A combination of slow chilling and ES had a positive effect on hot-boned muscles with respect to WBSF values.     

A "Cold Shortening" Effect in Avian Muscle

SUMMARY— Experiments were conducted to determine the effect of post-mortem temperatures between 0 and 20°C on the degree of shortening in isolated pectoralis major muscles of chickens and turkeys. A "cold shortening" effect in these muscles is described and compared to post-mortem pH, average sarcomere length of isolated myofibrils, and relative solubility of myofibrillar and sarcoplasmic proteins.
The degree of muscle shortening at each temperature after various periods post-mortem indicated that shortening was essentially complete after 3 hr in chickens and 5 hr in turkeys. Shortening in muscles stored at 0°C was significantly greater (P < .01) than in the 12–18°C temperature range. Shortening was greatest in muscles stored at 20°C. The degree of gross shortening observed was directly related to the average sarcomere length of isolated myofibrils. Post-mortem decline in pH was not significantly correlated (P > .05) with shortening. Extractability of myofibrillar and sarcoplasmic proteins after 5 hr at either 0 or 16°C was determined and found to be unrelated to the degree of post-mortem shortening.     

Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels

The structure of muscle injected with specific cysteine protease inhibitors was examined to determine whether inhibitors cause denaturation and the degradation post-mortem of myofibrillar proteins was followed using SDS electrophoresis. Given the central role of calcium in theories of tenderisation the level of free calcium was measured during the early post-mortem period. The protease enzyme inhibitor E-64 was injected into the m. longissimus et thoracis lumborum (LTL) on the right side of 12 lamb carcasses within 15 min of death and in another 12 carcasses with the protease inhibitor Z-Phe-Ala-CHN(2). The left LTL (control) was injected with saline (0.25 M NaCl). Muscle samples were obtained at death, pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=215). Muscle samples were selected from eight portions of the LTL (1-day post-mortem, from six different carcasses) for examination by transmission electron microscopy. Matching light images of myofibrils were obtained after determination of myofibrillar fragmentation. Free calcium concentration was determined for all samples (n=191) using an ion selective electrode excluding those 'at death'. Light images of myofibrils from treated samples showed normal striations and no evidence of denaturation or aggregation compared to control samples. This also applied to the samples processed for examination by electron microscopy. Appearance of the 30-kDa subunit increased with time (P<0.001) post-mortem. The interaction between ageing and stimulation had an effect (P<0.001) on the amount of a protein designated M1. The amount of M1 measured pre-rigor was greater for stimulated muscle, but the rate of decline was also greater through to day 2 post-mortem. Proteolysis was very rapid in the first 24 h post-mortem in ovine muscle. Ageing had an effect (P<0.001) on the free calcium concentration, which increased as muscle aged. As a covariate pH also had an effect (P< 0.05). Based on a non-linear model when the concentration of free calcium reached a plateau (～110 μM) the predicted pH was 5.5 (ultimate). From the qualitative observation of images and the levels of free calcium in injected muscle there is no support for the view that the inhibitors bind to sarcomere proteins, occupying sites to which calcium might bind. The levels of free calcium do not provide support for the view that m-calpain has a role in post-mortem tenderisation, but do suggest along with results of protein degradation that activation of μ-calpain is likely to occur before the pH drops to 6.2-6.1.     

The effects of Ca ions, EGTA and storage time on myofibrillar protein degradation, levels of Ca(2+)-dependent proteases and cathepsins B, H, L and D of ostrich skeletal muscle

The effect of Ca ions and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) on myofibrillar protein degradation showed that when ostrich iliotibialis lateralis muscle was incubated with 10 mM EGTA at 2–4 °C for 24 hr, the activity of extracted cathepsin H was unchanged compared with a buffer-incubated sample. Ca⁺⁺ had no effect on extracted cathepsin H activity, while that of Ca²⁺-dependent protease (CDP) decreased significantly (p < 0.05). Ca²⁺-treatment enhanced post-mortem changes observed in myofibrillar protein patterns (production of fragments around 30 K) that were not observed in EGTA-incubated myofibrils. The effect of storage time on shear force, CDP activity, cathepsin B, D, H and L activities and the SDS-PAGE pattern of myofibrils showed a time-dependent reduction in CDP activity. Of the cathepsins studied only cathepsin H showed a reduction (40%) in activity. The most prominent component appearing on storage at 2–4 °C had a M_r of 27 K. The incubation of myofibrils with CDP mimicked the post-mortem changes. CDP may be responsible for some of the post-mortem changes observed, although shear force measurements suggest these changes do not lead to significant tenderisation.     

Ultrastructural changes in pre- and post-rigor beef muscle caused by conventional and microwave cookery

Examination of cooked pre-rigor muscle by TEM (transmission electron microscopy) and SEM (scanning electron microscopy) revealed that all cookery methods resulted in the development of supercontraction bands alternating with areas showing tissue fragmentation and tearing. Microwave cookery produced smaller and less dense supercontraction nodes in pre-rigor muscle with less tearing and fragmentation but more fibre separation. Although cold-shortened pre-rigor muscle cooked by all methods also exhibited supercontraction bands with some tearing and fragmentation in adjacent sarcomeres, the samples cooked by microwaves showed a more uniform repeating pattern of small stretched areas alternating with dense contracted areas. Cooking of muscle in full rigor resulted in myofibrillar protein coagulation and shrinkage but supercontraction nodes were absent. Cold-shortened bicarbonate treated muscle was relatively intact after cooking, exhibiting fusion of the myofibrils and an absence of intermyofibrillar spaces. Results are discussed in relation to possible effects upon tenderness.     

Comparative effects of post-mortem storage and low-calcium-requiring neutral proteinase on bovine and rabbit myofibrillar proteins

The effects of post-mortem storage and of a low-calcium-requiring neutral proteinase on the myofibrils of beef Longissimus dorsi and Rectus abdominis muscles and rabbit Longissimus dorsi muscle were studied by measuring the Mg-Ca-enhanced myofibrillar ATPase activity and the changes in banding patterns on electrophoresis in sodium dodecyl sulphate. During ageing, the changes in the ATPase activity and myofibrillar proteins were qualitatively different between rabbit and bovine muscles, whilst differences in intensity were only observed between the two bovine muscles. The most prevalent component appearing upon conditioning had a mol. wt of 27 000 in rabbit muscle and of 30 000 in bovine muscles. In both species, the incubation of myofibrils with the low-calcium-requiring neutral proteinase mimicked the post-mortem changes. This would suggest that this enzyme has a qualitatively different effect on rabbit and bovine myofibrils.     

Muscle tissue structural changes and texture development in sea bream, Sparus aurata L., during post-mortem storage

Sea bream, Sparus aurata L., specimens were studied in pre-rigor (3 h) and during the following post-mortem days: 1, 5, 10, 15 and 22. Muscle and textural parameters were evaluated on 6 specimens/stage. Structural results showed scarce fibre-to-fibre detachment on pre-rigor, which increased during the post-mortem degradation. Ultrastructural changes revealed rapid muscle degradation. In pre-rigor myofibrils were detached to both sarcolemma and endomysium. Intermyofibrillar spaces increased and some mitochondriae and sarcoplasmic reticulum were swollen. After 1 day, the sarcolemma appeared occasionally disrupted and the interfibrillar spaces increased. From 5 to 10 days, the I-band and Z line presented some alterations, although these were more severe at 15-22 days. Thus, in these two last stages, loss of I-band, Z line and actin filaments was observed, that coincides with the alteration of the hexagonal arrangement in these advanced stages. Also, the fragmentation of myofibrils increased from 5 to 10 days on. Sarcolemma and endomysium were gradually disrupted throughout the post-mortem stages with total loss at 22 days. Consequently, the interfibrillar spaces increased at last stages. Autophagic mechanisms increased from 5 days on, with an intense destruction of all the intracytoplasmic organelles. Textural parameters decreased from pre-rigor until 5-10 days, mainly associated to detachment of myofibers to sarcolemma-endomysium.     

Effect of post-mortem storage on Ca(++)-dependent proteases, their inhibitor and myofibril fragmentation

Post-mortem changes in two calcium-dependent proteases, their inhibitor, myofibril fragmentation index (MFI) and collagen (amount and solubility) were studied. Whereas the activity of high Ca(++)-requiring calcium-dependent protease (CDP-II) remained nearly constant throughout post-mortem storage, there was a progressive decrease in the activities of low Ca(++)-requiring calcium-dependent protease (CDP-I) and their specific inhibitor, with the inhibitor being the most susceptible to post-mortem storage. Results indicated that the greatest changes in MFI occur within the first 24 h of post-mortem storage. There were no detectable changes in either total or soluble collagen content with post-mortem storage. Hence, it was concluded that improvement in tenderness resulting from post-mortem storage must be derived from changes in the myofibrils and since CDP-I activities paralleled the myofibrillar changes, it seems reasonable to suggest that CDP-I, not CDP-II, plays an important role in the fragmentation of myofibrils and consequently in improvement of meat tenderness resulting from post-mortem storage.