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.     

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.     

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.     

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.     

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.     

THERMAL BEHAVIOR OF PORCINE COLLAGEN AS RELATED TO POST-MORTEM TIME

SUMMARY: DTA (differential thermal analysis) thermograms of epimysial connective tissue from normal and low quality porcine muscle were compared at 0 and 24 hr post-mortem. In addition, the melting characteristics of intramuscular collagen were determined at 0 hr post-mortem. In all tissues studied, collagen from low quality muscle consistently gave slightly lower peak melting points than that from normal muscle. Epimysial collagen had a significantly ( P < .05) lower peak melting point at 24 hr than at 0 hr post-mortem (64.24 vs. 65.77°C). Using epimysial collagen, a significantly ( P <.01) greater proportion of the total melting range occurred at lower temperatures at 24 hr post-mortem as compared to 0 hr (4 1.1 vs. 30.9%). The thermal behavior of intramuscular collagen at 0 hr post-mortem was similar to that of epimysial collagen at 0 hr, but peak melting temperatures were slightly higher for intramuscular connective tissue.     

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.     

EFFECT OF POSTMORTEM CONDITIONS ON CERTAIN CHEMICAL, MORPHOLOGICAL AND ORGANOLEPTIC PROPERTIES OF BOVINE MUSCLE

Paired sides from U.S. Choice grade beef were aged immediately after slaughter at 2 and 16°C. Samples were removed from longissimus and semitendinosus at slaughter and at 1, 3 and 7 days postmortem for ATPase assay, phase microscopy, shear and organoleptic evaluation. Rib steaks from sides aged at 16°C for 1-day postmortem were as tender as steaks from sides aged at 2°C for 7 days postmortem. Flavor development of rib steaks also was more rapid at 16°C than at 2°C. Tenderness of semitendinosus steaks was improved by aging sides at 16°C; the difference in improvement of tenderness of semitendinosus, however, was not as great between 2°nd 16° as it was for rib steaks. Ca⁺⁺, Mg⁺⁺ and EGTA-modified ATPase activity of myofibrils from both muscles increased with postmortem time, with myofibrils from muscles held at 16°C having slightly higher ATPase activity than myofibrils from muscles held at 2° Increased EGTA-modified ATPase activity was indicative of loss of calcium sensitivity of the myofibril. Sarcomeres of myofibrils from longissimus were longer at 1-day postmortem than those from at-death longissimus and they remained essentially unchanged during the remainder of postmortem aging; however, tenderness improved at 16°C for 1 day and at 2°C for 3 days. Also greater fragmentation of myofibrils from longissimus postmortem aged at 16°C for 1 day and at 2°C for 3 days was observed, suggesting that the rate of myofibril fragmentation is an important factor in tenderization.     

The effect of post-mortem conditions on the extractability and adenosine triphosphatase activity of myofibrillar proteins of rabbit muscle

Summary. 1. Washed myofibrils from rabbit muscle have been heated at pH values between 4.8 and 5.6 and temperatures between 35°C and 42°C. It has been found that, under these conditions, myofibrils lose their Ca²⁺ activated adenosine triphosphatase, their Mg²⁺ activated adenosine triphosphatase and also become less extractable in M KCl–30 mM sodium glycerophosphate, pH 6.2.
2. The reactions follow first-order kinetics and the rates are dependent on pH and temperature. The first order rate constants, enthalpies and entropies for the three reactions are sufficiently near each other to suggest that all three reactions are occurring simultaneously.
3. When a muscle is allowed to go into rigor at 37°C the extractability in M KCl–30 mM sodium glycerophosphate is reduced after 4 hr at 37°C when the pH of the muscle has reached 5.55. At the same time the Ca²⁺ adenosine triphosphatase activity falls but the Mg²⁺ adenosine triphosphatase does not. The latter is reduced by prolonging the period at 37°C to 6 hr.
4. It is suggested that there is present in muscle, undergoing rigor at 37°C, myosin which does not bind to actin and is readily denatured. When bound to actin, myosin in the myofibril is more resistant and denatures only after long exposure to a temperature of 37°C.     

Molecular Properties of Post-Mortem Muscle. 7. Changes in Nonprotein Nitrogen and Free Amino Acids of Bovine Muscle

SUMMARY– Proteolysis and its relationship to tenderness were studied by measuring nonprotein nitrogen (NPN), free amino groups, and shear resistance during post-mortem aging of bovine muscle. Both NPN and free amino groups increased during post-mortem aging, indicating some degradation of proteins and/or peptides. However, neither the increase in NPN nor free amino groups was related to post-mortem tenderization since these quantities increased only after most of the improvement in tenderness had occurred. Much of the increase in NPN or free amino groups may originate from degradation of sarcoplasmic proteins or peptides. It is suggested that weakening or breaks at crucial points in the sarcomere, such as at the junction of the Z-line with the thin filaments, occur within the first 48-72 hr post-mortem and that this weakening or cleavage is responsible for tenderization. Cathepsin D may be responsible for this weakening but most of the available evidence is against proteolysis as the primary cause of post-mortem tenderization.     

Thermal Gelation of Brown Trout Myofibrils: Effect of Muscle Type, Heating Rate and Protein Concentration

The thermal gelation properties of myofibril solutions (KCl 0.6M; pH 6.0) from reared brown trout white and red muscles were analyzed by thermal scanning rheometry. With a heating rate of 1°C/min, red muscle myofibrils exhibited a lower gelation capacity than white muscle myofibrils at low temperatures. No difference was observed above 60°C where solid gels were formed from the two myofibril types. Increasing protein concentration or reducing heating rate increased the values of the rheological parameters at 80°C for the two muscle type myofibrils. With a low heating rate (0.25°C/min), white muscle myofibrils formed stronger gels whatever the temperature.     

Post-Mortem Changes in Subcellular Fractions from Normal and Pale, Soft, Exudative Porcine Muscle. 1. Calcium Accumulation and Adenosine Triphosphatase Activities

SUMMARY– Myofibrillar, mitochondrial, heavy sarcoplasmic reticulum and light sarcoplasmic reticulum fractions were isolated by differential centrifugation of homogenates from normal and pale, soft, exudative (PSE) porcine muscle at various times post-mortem. Calcium uptake was measured using a solution containing⁴⁵Ca⁺⁺. The oxalate-stimulated calcium accumulating ability of the subcellular fractions declined 5-10 fold between 0 and 24 hr post-mortem. The major portion of this decline occurred in the first hour after death in fractions from PSE muscle but was more gradual in the normal fractions. The ATPase activities of normal and PSE fractions obtained at death did not differ significantly. These activities increased with time post-mortem in most normal fractions but decreased in those from PSE muscle. The subcellular site of ATP hydrolysis post-mortem was discussed. The results obtained point to the potential importance of the relaxing, factor in muscle post-mortem.     

Studies on Bovine Natural Actomyosin 2. Physico-Chemical Properties and Tenderness of Muscle

SUMMARY: Studies were made of physicochemical characteristics of natural actomyosin from bovine longissimus of different post-mortem ages and tenderness classifications. Reduced viscosity, ATP sensitivity, and "actin" content (polyethylene sulfonate treatment) were higher for natural actomyosin prepared from muscle 12-24 hr post-mortem than from pre-rigor muscle, which confirms previous reports for rabbit natural actomyosin. A higher actin to myosin ratio in actomyosin from muscle 12-24 hr was therefore postulated. A stronger interaction of actin and myosin in actomyosin from muscle 12-24 hr post-mortem than from pre-rigor or aged muscle was also suggested by reduced viscosity and ultracentrifugation data. Reduced viscosity differences between actomyosins from tough and tender muscle suggested a higher gel character in actomyosin from tough muscle. This possibly indicated a higher content of α-actinin. No consistent differences in ATP sensitivity, myosin and actin content of natural actomyosin of tough and tender muscle were found. Natural actomyosin from muscle aged post-mortem showed the appearance during analytical ultracentrifugation of an additional component which sedimented at about 11S to 12S. This component appeared in the actomyosin prepared from tender muscle after 24 hr but did not appear until 10 days in the actomyosin from tough muscle.     

COLD SHORTENING IN CHICKEN BROILER PECTORALIS MAJOR

At 2°C strips of P. major muscle from chicken broilers developed a peak in isometric tension which quickly dissipated and was followed by no subsequent changes in tension during the next 36 hr. Increasing the temperature at which strips were maintained from 2 ° to 23 °C (22 °-25 °C) after 12 or 24 hr resulted in some further tension development but essentially no tension development was observed in strips which had been held at 2 °C for 36 hr before the temperature was raised to 23 °C. Cold shortening did not significantly lower muscle ATP, creatine phosphate or hexose monophosphate levels.     

Cold shock reactions in iced tropical fish

The development of a rigor mortis-like stiffening and the biochemical changes associated with it were investigated in tilapia ( Oreochromis aureus/niloticus hybrid), a tropical freshwater species, and common carp ( Cyprinus carpio ), a temperate freshwater fish, during storage in ice (0°C) and at ambient temperature (22°C). Onset of stiffening in carp occurred between 16 and 17 hr after death at both temperatures but full stiffness developed much later and was a longer duration at 0°C. In tilapia, onset occurred after 7 hr at 22°C and full stiffness was established after 19 hr. However, at 0°C, tilapia experienced a cold shock reaction such that they stiffened within minutes of being placed in ice and were fully rigid within 8 hr. Resolution of stiffness in this species also occurred later at 0°C. The rate of ATP degradation was similar under both storage conditions in tilapia but more rapid at ambient temperature in carp. Although the rate of lactic acid accumulation was faster at the higher temperature in tilapia, it was not nearly so marked as for carp. Objective measurement of contractions in excised muscle fibres from trout ( Salmo gairdnerii ) and tilapia indicated that reducing the temperature delayed the occurrence of the contraction and reduced its intensity. It was concluded that cold shock stiffening and rigor mortis stiffening are different.     

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 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.     

Survival of Escherichia Coli O157:H7 During Storage of Yogurt at Different Temperatures

ABSTRACT: The behavior of E. coli O157:H7 strains during the storage of plain live yogurt at 4, 8, 17, and 22 °C was investigated. Lactic acid bacteria and pH were also studied. Linear regression analysis was carried out to obtain the specific death rate, the death rate and time of death. During the 1st 24 h, the pathogenic strains decreased slightly at 4 and 8 °C. This decrease was greater at 17 °C, and even greater at 22 °C. E. coli O157:H7 was not detected after 312, 168, 28, and 16 h at 4, 8, 17, and 22 °C, respectively. Counts of Lactobacillus and Streptococcus were about 9 log CFU/g during the study. An increase was detected in the values for time of death from refrigeration to room temperatures. A decrease in the values of both the specific death rate and the death rate related with the increase of temperature was observed. Counts of E. coli O157:H7 were higher than those of non-pathogenic E. coli during the storage period at 4 and 8 °C, showing a better and a higher adaptation capacity to acid pH environments at refrigeration temperatures. This behavior was not observed at room temperatures.