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.     

Relationships Among Shear Values, Sarcomere Lengths and Cooling Procedures in Turkeys

SUMMARY– Studies were conducted to determine the effect of different chilling procedures after slaughter on the tenderness of the breast and thigh muscles of turkeys as measured by shear press values. Measuring sarcomere lengths determined the effect of the chilling procedures on length of muscle fibrils and their correlation with shear press values. Three chilling treatments were used: (1) 16°C for three hr; (2) 16°C for 45 min, 8°C for 45 min, and 0°C for 90 min; and (3) 0°C for 3 hr. The 0°C treatment for 3 hr resulted in a significant increase in shear press values for thigh muscle in both studies. Shear values also increased for breast muscle in the same 0°C treatment group, but not significantly. Shear values for the left thigh muscle were significantly higher than for the right in Experiment I, while in Experiment II hens had significantly higher thigh shear values than toms. In Experiment I with younger birds, shear values were significantly higher in the breast muscle of toms than in hens. The surface slice of 3 slices of breast muscle had higher shear values in both experiments. Chilling treatments resulted in a progressive shortening of sarcomere lengths in breast and thigh muscles with decreasing temperature, and the sarcomere lengths were shorter for breast muscle than for thigh. No significant correlations were found between shear values of breast and thigh muscles, or between shear values and sarcomere length.     

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.     

The Effect of pH-Temperature Treatments on the Calcium Accumulating Ability of Purified Sarcoplasmic Reticulum

SUMMARY: Pig sarcoplasmic reticulum fragments obtained from the longissimus dorsi muscle at 0- and 24-hours post-mortem were purified by salt extraction and density gradient centrifugation. The calcium uptake activity of 0-hour purified preparations was more than 20-fold higher than that from 24-hr old muscles, but there was no significant difference between fractions for calcium activated ATPase activities. When observed electron microscopically after negative staining, the ultrastructures of the 0. and 24-hour membrane fragments were found to be essentially identical. Incubation of isolated sacroplasmic reticulum fragments at pH 7.2 and 37°C or pH 5.6 and 0°C caused negligible inhibitoin of their calcium accumulating ability. However, treatment at pH 5.6 and 37°C for 1 hr almost completely abolished the sarcoplasmic reticulum calcium uptake. Thus it appears that low muscle pH and high temperature may be responsible for the inactivation of the calcium accumulating ability of the sarcoplasmic reticulum that occurs in situ.     

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.     

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.     

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.     

Formation of Myofibrillar Fragments and Reversible Contraction of Sarcomeres in Chicken Pectoral Muscle

SUMMARY— As morphological changes occurred in the pectoral muscles of chicken carcasses during 48 hr storage at 5°C, two notable phenomena were observed: (1) fragmentation of myofibrils and (2) reversible or irreversible contraction of sarcomeres. When blendorized, myofibrils tend to break into small fragments composed of 1-4 sarcomeres with time post-mortem. It was also found that besides an irreversible post-mortem contraction of sarcomeres generally accepted, a reversible contraction can take place under particular conditions.     

Effects of thawing temperature on the physicochemical properties of pre-rigor frozen chicken breast and leg muscles

The objective of this study was to evaluate effects of thawing temperature on the biochemical and physicochemical properties of pre-rigor frozen chicken breast and leg muscles. Breast and leg muscles from 24 broiler chickens were excised within 10min postmortem. Pre-rigor muscles were frozen at -20°C and thawed at 0 and 18°C, and pH, R-value, sarcomere length, muscle shortening, thaw and cook loss, shear force and myofibrillar fragmentation index (MFI) compared with those in pre-rigor or 2°C chilled muscles. The ultimate pH of 18°C thawed muscle was lower than that of 0°C thawed and 2°C chilled muscles. As expected, the shortening of sarcomere length and muscle length of thaw rigor muscles were more than those of chilled muscle, but there were no significant differences between chilled muscle and 0°C thawed muscle. Also, there were no significant differences in R-value (Abs 250/Abs 260) and cook loss due to thawing temperature. Samples thawed at 0°C had higher MFI and lower shear value than samples thawed at 18°C. Shear force value and MFI were not significantly different between chilled muscle and 0°C thawed muscle. By thawing at 0°C, thaw shortening was prevented, and tender meat comparable to the chilled meat was obtained.     

Effect of subcutaneous fat and high temperature conditioning on bovine meat tenderness

Effects of subcutaneous fat cover and high temperature conditioning on tenderness of meat were studied using 16 steer carcasses. Longissimus subcutaneous fat cover was completely removed from eight carcasses and the right and left sides were stored at either 0°C or 26°C. After 6 h at 26°C, the sides were transferred to the 0°C room; and after 24 h, all sides were transferred to a 1°C room for the duration of the experiment. Cold temperature and removal of fat cover reduced (P < 0·05) the longissimus muscle temperature at 6, 9 and 12 h post-mortem. The pH of the longissimus muscle was lower (P < 0·05) as the result of high temperature conditioning and fat cover 6, 9 and 12 h post-mortem. Consequently, conditions existed which would have been expected to promote cold shortening, yet high temperature conditioning and fat cover had no consistent effects on myofibrillar fragmentation index, sarcomere length or shear values.     

Post-mortem Changes in Breast Muscles of Mule Duck

Post-mortem changes in myofibril fragmentation index and degradation of myofibrillar proteins of duck breast muscles at 5°C was investigated. Muscle samples were collected immediately after killing and from the stored carcasses at 5°C for 1, 3, 7, and 14 days post mortem . The results showed that the MFI of the breast muscles increased with time post mortem . SDS-PAGE of myofibrils indicated that the disappearance of troponin-T accompanied concurrently with the appearance of 32–30 kDa components. After 7 days of storage, α-actinin was degraded, and a 98 kDa component appeared. Titin 1 and nebulin also disappeared after post-mortem storage for 3 days.     

Effect of rigor temperature and frozen storage on functional properties of hot-boned manufacturing beef

Within 45 min post-mortem, 10mm thick strips of semitendinosus muscle from both unstimulated and high voltage stimulated heifer sides were held at 0, 5, 10, 25 and 35 °C for 24 hr, during which they entered rigor. Half the samples were frozen and stored at -20 °C for one month. The pH, sarcomere length, drip, total (TPS), myofibrillar (MPS) and sarcoplasmic (SPS) protein solubilities, and Hunter L (?), a (?) and b (?) values were determined at 24 hr and on thawed samples. Electrical stimulation did not significantly affect any of the parameters measured. The ultimate pH of samples entering rigor at 10 and 25 °C was lower (p < 0.001) than that of samples held at the other temperatures. Rather surprisingly, there was no significant difference in sarcomere length due to rigor temperature. Samples entering rigor at 35 °C had lower TPS, MPS and SPS values than samples held at 0 to 25 °C (p < 0.001). The MPS increased with rigor temperature up to 25 °C (p < 0.001). Drip and total moisture losses, and Hunter L (?), a (?) and b (?) values also increased with rigor temperature (p < 0.001) whereas SPS decreased and NMR meat water spin-lattice relaxation time (T1) shortened with increasing rigor temperature (p < 0.001). Hue angle and cook loss decreased with rigor temperature in 24 hr samples but increased with rigor temperature in frozen samples. After frozen storage, SPS, T1, cook loss and Hunter L (?), a (?), b (?) values decreased, but TPS, MPS, drip losses and hue angle increased. There were significant (p < 0.05) correlations between SPS, hue angle, drip losses and T1.     

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.     

Manipulation of the pre-rigor phase to investigate the significance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi

The objective of this study was to evaluate the importance of proteolysis and sarcomere length in determining the tenderness of bovine M. longissimus dorsi (LD) muscle over a 21-day period. This was done by altering the pre-rigor glycolytic behaviour of hot-boned LD muscles using different early post-mortem temperature regimes. Hot-boned LD muscles (n=8) were cut in two, randomised, placed in polythene bags and submerged in a water bath set at 5°C (rapidly chilled) and 15°C (slowly chilled) for 8h post-mortem. All muscles were then stored at 2°C for up to 21 days post-mortem. The temperature regimes altered the glycolytic behaviour of the muscles in the pre-rigor period. The slowly chilled muscles exhibited a faster (P<0.01) pH fall than rapidly chilled muscles. Cold shortening was induced in rapidly chilled muscles as they had shorter (P<0.01) sarcomere lengths than slowly chilled muscles up to day 21 post-mortem. Warner Bratzler shear force values (WBSF) deemed cold-shortened muscles as tougher than noncold shortened up to day 14 post-mortem. Both cold-shortened and noncold-shortened muscles tenderised over time to an extent where there was no significant difference in WBSF values by day 21 post-mortem. SDS-PAGE protein profiles indicated that the rate of proteolysis was faster in slowly chilled muscles when compared to rapidly chilled muscles. However by day 21 post-mortem it appeared that rapidly and slowly chilled muscles underwent proteolysis to the same extent.     

High post-mortem temperature combined with rapid glycolysis induces phosphorylase denaturation and produces pale and exudative characteristics in broiler Pectoralis major muscles

This study investigated the effect of early post-mortem temperature on broiler protein characteristics and meat quality. Muscles were kept at different temperatures (0, 20 and 40 °C) until 4h post-mortem and then stored at 4 °C. Rapid degradation of ATP and glycogen, thus inducing a high rate of lactate formation and pH drop, were found in the 40 °C group during incubation. When extracting proteins, a lower protein content of the sarcoplasmic fraction and a higher protein content of the myofibrillar fraction were found in the 40 °C group at 24h post-mortem; SDS-PAGE and western-blotting results revealed that phosphorylase was associated with the myofibrillar fraction. Furthermore, the 40 °C group had paler surfaces, higher drip loss and lower processing properties. These data suggest that elevated temperature during early post-mortem period, resulting in rapid glycolysis, induced phosphorylase denaturation and association with myofibrillar proteins thus generating pale and exudative characteristics.     

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.     

POSTMORTEM SHORTENING OF LAMB LONGISSIMUS OXIDATIVE AND GLYCOLYTIC FIBERS

Percentages of lamb M. longissimus thoracis et lumborum fiber types were approximately 66 oxidative and 34 nonoxidative, without significant differences among muscle regions. The effects of skeletal restraint and muscle region on sarcomere shortening during rigor development were found to be highly significant; sarcomeres of caudal-ventral location were stretched by skeletal restraint while the rest were all shortened. In excised muscles, both fiber type and postmortem temperature exerted a highly significant effect on sarcomere shortening. Oxidative fibers showed a more intense shortening, already evident at temperatures well above those causing cold shortening. While shortening of glycolytic fibers was gradually more intense as treatment temperature decreased from 20 to 0°C, a maximum contraction of about 40% was reached at 15°C in oxidative fibers and lower temperatures did not cause any further shortening. Average fiber shortening was found to be highly correlated to meat toughening.     

High early post-mortem temperature induces activation of AMP-activated protein kinase and development of pale,soft and exudative characteristics in turkey muscles

This study investigated the effect of early post-mortem temperature on development of a turkey muscle's pale, soft and exudative (PSE) characteristics. Muscles obtained at 20 min post-mortem were incubated at 0, 20 and 40 °C until 4 h post-mortem and then stored at 4 °C. During incubation, the 40 °C group had greater rate of pH decline, lactate accumulation and R-values increase than other groups. Moreover, AMP-activated protein kinase (AMPK) activation at 1 h post-mortem was higher in the 40 °C group than the other groups. At 24 h post-mortem, the 40 °C group had higher L* values and drip loss; SDS-PAGE and western blotting indicated that lower protein solubility (sarcoplasmic, myofibrillar) in the 40 °C group resulted from phosphorylase denaturation and further adhere to myofibrillar fraction. These results suggest that high temperature early post-mortem could induce AMPK activation, which results in rapid glycolysis, thus affecting protein solubility and generating PSE characteristics.     

Alterations of Bovine Sarcoplasmic Proteins as Influenced by High Temperature Aging

SUMMARY— Effects of high temperature aging upon certain characteristics of bovine I. dorsi muscle were studied. Paired wholesale ribs of carcasses were obtained subsequent to slaughter. The left rib of each pair was held at 30°C for 24 hr, then stored at 3°C. Analogous right ribs were immediately stored at 3°C. A sampling schedule of 0, 1, 2, 3, 4, 7 and 10 days was followed.
There were minor variations in moisture, pH, tyrosine-tryptophan indices of non-protein nitrogenous compounds and expressible moisture ratios between treatments and with time. These differences were not statistically significant.
Up to three days storage, extractability of water soluble protein was greatest from muscles held at the elevated temperature. After the third day, however, extractability was greater for muscles held at 3°C.
Color differences between muscles treated via the two storage temperatures were marked. Absorbance ratios (422.280 mp) of extracts showed that muscles held at the high temperature had higher extractable levels of oxymyoglobin than ribs held at 3°C. This difference remained apparent throughout the aging period.
Results of DEAE-cellulose ion exchange chromatography of the sarcoplasmic proteins showed only minor variations in profiles between the two aging treatments. Alterations did appear with time. Profile alterations did not appear related to anticipated increases in tenderness.     

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.