Studies on Bovine Natural Actomyosin 1. Relationship of ATPase and Contractility to Tenderness of Muscle

SUMMARY: The effects of muscle tenderness classification and of aging muscle postmortem on ATPase activity and superprecipitation of natural actomyosin were studied. Actomyosin from muscle 12- and 24-hr postmortem had higher ATPase activity than that from 0-hr, 5-day aged or 10-day aged muscle. However, ATPase activity did not usually return to the 0-hr level. No consistent differences were found in actomyosin ATPase activity after the various periods of aging for actomyosins from tough and tender muscle. Superprecipitation of actomyosin was used as a measure of contractility. Actmyosin from 12- and 24-hr postmortem muscle superprecipitated faster than that from 0-hr muscle. However, actomyosin from 5 and 10-day aged muscle superprecipitated less rapidly than that from 12- and 24-hr postmortem muscle. Superprecipitation was more rapid in actomyosin from tough muscle than tender muscle at low KCI concentrations, but this was not true at high KCI concentrations. This observation suggested that actomyosin from tough muscle had a stronger interaction or higher amounts of some protein factor such as α-actinin than did tender muscle.     

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.     

Pre- and Post-Rigor Sectioned and Formed Beef Steaks Manufactured with Different Salt Levels, Mixing Times and Tempering Times

This study was designed to identify processing parameters necessary for the incorporation of pre-rigor beef in sectioned and formed steaks. Meat logs were prepared from pre- or post-rigor beef muscle with 0.0, 0.5 or 1.0% salt, mixed for 0, 6, 12 or 18 min, frozen and tempered to —3°C. Logs were pressed and cleaved after 0, 24, 48 or 72 hr tempering. Tempering at -3°C resulted in sufficient metabolic activity (as measured by pH and R-value) to deplete the ATP content of pre-rigor muscle. The presence of salt hastened the onset of rigor, maintained a higher ultimate muscle pH and improved cooking yields, bind (adhesion) and tenderness. Longer mixing times enhanced bind. These data suggest that, with appropriate modification in the processing procedure, pre-rigor muscle can be incorporated into sectioned and formed products.     

MYOSIN STABILITY IN INTACT CHICKEN MUSCLE AND A PROTEIN COMPONENT RELEASED AFTER AGING

SUMMARY– My osin from both red (myosin-R) and white (myosin-W) chicken muscle was extracted with Hasselbach-Schneider solution after the muscle had aged for 30 min and 24 hr post-mortem. Myosin-W from aged muscle contained a fast moving boundary in sedimentation velocity studies. When this fraction (component T) was separated on a DEAE-Sephadex column, the ATPase activity and sedimentation pattern of the resulting myosin were similar to those of chromatographed myosin from unaged muscle. The numbers of sulfhydryl groups in both myosin-R and myosin-W were not affected by aging. The extinction coefficient and absorption spectrum of component T were different from those of myosin, and component T had neither ATPase nor 5'-adenylic acid deaminase activity. Lowering the pH of myosin extracted from unaged muscle to the pH values found in muscle aged for 24 hr caused aggregation and loss of ATPase activity. These aggregates of myosin differed from component T in both sedimentation velocity and chromatographic pattern.     

CAF Activity, Calcium Concentration, Andy the 30,000-Dalton Component of Tough and Tender Bovine Longissimus Muscle

Four tender and four tough bovine longissimus samples from A-maturity carcasses and four tender and four tough bovine longissimus samples from E-maturity carcasses postmortem aged 10-14 days were used to study calcium-activated factor activity (CAF), calcium concentration, and the 30,000-dalton component. Significant differences in CAF and free calcium were not found between tender and tough for CAF and free calcium, although the tough samples had more free calcium. Tender bovine longissimus muscle from A-maturity carcasses had a 30,000-dalton component and a high ratio of 30,000-dalton component to actin, whereas tough did not. In E-maturity, however, both tender and tough longissimus had a 30,000-dalton component.     

Manipulation of the pre-rigor glycolytic behaviour of bovine M. longissimus dorsi in order to identify causes of inconsistencies in tenderness

The aim of this study was to monitor the effects of the alteration of the pre-rigor environment of the bovine LD muscle using controlled temperature regimes in order to gain an insight into the early post-mortem pH/temperature/time interactions which are important from the point of view of tenderness and to identify possible reasons for inconsistencies in beef tenderness. LD muscles (n=12) were hot-boned within 90min post-slaughter, cut into three pieces which were randomly placed in polyethylene bags and submerged in water baths pre-set at the following temperatures; 0, 5, 10, 15, 20 and 25°C for 8 h post-mortem then stored at 2°C for up to 14-days post-mortem. The rate of pH decline increased with increasing temperature. Muscles incubated at 0 and 5°C were cold shortened however not all of these muscles were tough as indicated by Warner Bratzler shear force values (WBSF). A pH range of 5.9-6.2 at 3 h post-mortem (pH(3)) produced consistently tender beef where cold-shortening was avoided. Cold shortened muscles showed the greatest variation in tenderness at 14 days post-mortem and underwent the greatest amount of tenderisation (ΔWBSF) and proteolysis between days 2 and 14 post-mortem. Proteolysis of cold shortened muscle may induce variation in tenderness in these muscles.     

Properties of actomyosin isolated from cod (Gadus morhua) after various periods of storage in ice

Natural actomyosin was isolated from cod (Gadus morhua L) stored in ice for up to 28 days. The gelling properties, apparent viscosity, Ca^2--ATPase activity and component protein composition by sodium dodecyl sulphate (SDS) electrophoresis were determined for each preparation of natural actomyosin. The apparent viscosity, protease activity, trimethylamine (TMA) content and pH of the fish muscle were also determined. The results showed that the apparent viscosity and Ca^2--ATPase activity tended to decrease slightly during ageing of the fish in ice, whereas some of the gelling properties showed a maximum between 3 and 6 days of storage. However, there was no change in the apparent viscosity of the muscle as a whole even after the fish were considered to be stale according to the TMA values. The ratio of myosin heavy chain to actin in the actomyosin changed with the time of storage of the fish, being highest at 3 days when gelling properties were maximal and decreasing progressively thereafter.     

Effect of ageing on the properties of myofibrils of rabbit muscle

The effect of ageing of rabbit muscle at 4° and 15–18° on the extractability and adenosine triphosphatase activity of the myofibrils has been examined. The amount of protein extracted by M-KCL-4 mM sodium glycerophosphate (pH 6.2) and by 0.1 M sodium tetrapyrophosphate-4 mM MgCl₂-10 mM-KH₂PO₄ (pH 7.2) increased as the muscle aged. By using the amount of Ca²⁺ adenosine triphosphatase extracted, i.e. the enzyme associated with myosin, as a measure of the amount of myosin in the actomyosin extracted, it was possible to show that the buffered potassium chloride did not extract all the actomyosin from the myofibrils of pre-rigor muscle. As the muscle aged, more actomyosin was extracted, together with some tropomyosin. Pyrophosphate, however, extracted all the myosin from the pre-rigor muscle, and the increase in the protein extracted from aged muscle was due to actin and tropomyosin in addition to myosin. It is suggested that these changes are caused by a disruption of the Z-band structure during ageing, perhaps due to the hydrolysis of tropomyosin by proteolytic enzymes. The specific Ca²⁺ adenosine triphosphatase activity of the myofibrils was unaltered by ageing but the specific Mg²⁺-activated adenosine triphosphatase, i.e. the enzyme associated with actomyosin, was reduced by about one-third. This latter result may be caused by a change in the mode of linkage of actin to myosin.     

EFFECTS OF PRE-RIGOR TENSION ON TENDERNESS OF INTACT BOVINE AND OVINE MUSCLE

Mechanical and weighted methods of applying pre-rigor tension to longissimus muscle of ovine and bovine carcasses conventionally hung by a hind shank were studied. The effect of tensioning of carcasses by adding 4.5 kg weights or mechanically stretching the back 5% in length was significant (P < 0.01) and as effective as greater forces in decreasing shear force values, decreasing fiber diameters and increasing sarcomere lengths. Chops and steaks from tensioned carcasses at 48 hr postmortem were as tender as those from non-tensioned carcasses which had been aged 168–240 hr. Further aging of longissimus muscle of tensioned carcasses continued to decrease shear force values at approximately the same rate at which shear force was decreasing in nontensioned carcasses. The pre-rigor tension was also effective in reducing animal-to-animal tenderness variability.     

Dephosphorylation of myosin regulatory light chain modulates actin–myosin interaction adverse to meat tenderness

The objective of this study was to investigate the relationship between the dephosphorylation of myosin regulatory light chain and actin–myosin interaction after muscle homogenate was treated with alkaline phosphatase and phosphatase inhibitor. The myosin regulatory light chain was significantly dephosphorylated by alkaline phosphatase after incubation. Among different groups, dephosphorylated myosin regulatory light chain to a much greater extent leads to a lower actomyosin dissociation degree, thereby raising the actomyosin ATPase activity, whereas it exhibits a higher actomyosin dissociation degree and a lower actomyosin ATPase activity when the myosin regulatory light chain was in a low dephosphorylated state. The data suggest that the dephosphorylation of myosin regulatory light chain modulates actomyosin dissociation (the number of myosin interact with actin) negatively and has a positive influence on actomyosin ATPase activity (the interacting force between myosin and actin).     

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.     

Isolation of Actomyosin and Myosin from Post-Rigor Turkey Breast and Thigh

Myosin/actomyosin (MAM) was extracted from post-rigor turkey breast and thigh muscles using Hasselbach-Schneider solution. Electrophoretic evaluation of the breast muscle protein extract showed it to be 86.4% myosin and 7% actin. Thigh muscle MAM contained 79.4% myosin and 4.7% actin. Slight variations in minor protein bands were observed between the breast and thigh MAM electrophoretic profiles. Gel filtration of MAM on a 2% agarose column separated actomyosin from myosin. Myosin purity was 97.9% and 99.2% for breast and thigh, respectively. Gel filtration data indicated that myosin, unassociated with actin, was the predominate protein extracted from the post-rigor tissue. Myosin was approximately 67-72% of the original MAM.     

Calcium Chelators Influence Some Physical and Chemical Properties of Rabbit and Pig Muscle

SUMMARY: Intravenous antemortem injections of EDTA, EGTA and CDTA significantly inhibited muscle shortening during development of rigor mortis. Post-mortem micro-injections of CaCl₂ increased shortening, but MGCl₂ had no measurable effect. Even though average shear values for muscle from EDTA-treated rabbits were lower than those of untreated controls, the differences were not statistically significant. However, muscle from EDTA-treated pigs was significantly more tender than that from untreated controls. The interrelationships between ATP levels and pH values at 0 and 24 hr post-mortem were investigated and their effects upon muscle shortening and tenderness are considered and explained. A highly significant negative relationship was shown to exist between cooking losses and both initial pH and initial ATP values for both rabbit and pig muscle.     

Ultrastructural Variation in Beef M. longissimus dorsi as an Explanation of the Variation in Beef Tenderness

ABSTRACT: The objective of this experiment was to quantify variation in bovine M. longissimus dorsi tenderness and determine the extent such variation is explained by variation in the ultrastructure of muscles after different postmortem treatments. Eight muscles were hot-boned and aged for 2 d at 2 °C (T1) to achieve very contracted actomyosin crossover and tough beef. Eight carcass sides were tenderstretched for 10 h at 10 °C and a further 38 h at 2 °C (T2) to achieve lengthened actomyosin crossover and tender beef. Both T1 and T2 were compared with conventionally hung carcasses, which underwent similar chilling regimes, C1 ( n = 8) and C2 ( n = 8), respectively. Measurements of sarcomere length, pH, Warner Bratzler shear force (WBSF), and sensory tenderness were taken, and transmission electron microscopy images analyzed. Variances of attributes were analyzed on Bartlett's test. Variances of the 4 groups were homogenous for all attributes except for pH after 24 h postmortem (with T1 [0.00] having lower variances than C1 = 0.04, T2 = 0.06, and C2 = 0.05) and WBSF after 2 d aging (with T2 [74.33] having lower variances than T1 = 236.76, C1 = 398.82, and C2 = 856.74). The variation in the tenderness of beef was quantified through ultrastructural variation in bovine muscle, with tenderstretched moderately chilled beef having the least variable tenderness as a result of more uniform overlap between actin and myosin filaments. Variation in the eating quality of beef was not reduced by hot-boning with fast chilling or conventional hanging with fast or moderate chilling. The development of the uniformity within filaments of tenderstretched muscle requires further analysis as residual variation remains.     

Paramyosin-Myosin-Actin Interactions in Gel Formation of Invertebrate Muscle

Even when actomyosin dissociated into myosin and actin in the paramyosin-natural actomyosin system, the indices of gel properties increased on increasing the paramyosin content; however, their increases were smaller. Also, in the paramyosin-myosin system gels, the indices of gel properties increased on increasing the paramyosin content; however, the increases were smaller than those for the paramyosin-natural actomyosin system gels. In both systems, a high linearity was observed between each index of gel properties and paramyosin. It was likely that the paramyosin-myosin interaction did not bring about the characteristic texture of invertebrate muscle gel directly, but the binding of myosin and actin might be indispensable if the contribution of paramyosin was to occur.     

The effects of hot water pasteurizing treatments on the appearances of pork and beef

Portions of post- and pre-rigor pork and beef were treated by immersion in water of 75 or 85 °C for 5, 10, 15 or 20 s. The appearances of untreated and treated cut muscle, fat, membrane covered and cut bone surfaces, and the overall appearances of treated and untreated meat pieces, were assessed by a 5-member panel 2 hr and 24 hr after each treatment. At those times, CIE L(?)a(?)b(?) values were obtained for cut-muscle surfaces. The appearances of surfaces covered by membrane, of fat and cut bone surfaces of pre-rigor pork and beef, and of fat and cut bone surfaces of post-rigor pork, were not persistently degraded by any treatment. However, the appearances of fat and cut bone surfaces of post-rigor beef were persistently degraded by the harsher treatments, apparently because of pre-treatment staining of fat and changes in the bone marrow. Panel scores for the overall appearance and for the appearance of cut muscle surfaces were generally the same for each meat piece. All treatments caused patchy bleaching of cut muscle. The degraded appearances of pre- and post-rigor pork and post-rigor beef muscle persisted at 24 hr, but the appearances of beef muscle treated pre-rigor improved between 2 and 24 hr. Most treated muscle gave increased CIE L(?) values and decreased a(?) and b(?) values as compared with untreated portions from the same primal cut. However, CIE b(?) values for pre-rigor beef muscle were higher than for untreated muscle 2hr after treatment, but lower than for untreated muscle 24 hr after treatment. It appears that pasteurizing treatments cannot be applied to meat without some degradation of the appearances of cut muscle surfaces, which will persist in pork treated pre- or post-rigor and in beef treated post-rigor, but which will partially resolve during the storage of beef treated pre-rigor.     

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.     

Pelvic suspension and fast post-mortem chilling: Effects on technological and sensory quality of pork - A combined NMR and sensory study

The effects of pre-rigor excision, which results in a fast post-mortem chilling; pelvic suspension, which results in a muscular stretching; control treatment of m. longissimus dorsi on water distribution measured by (1)H NMR relaxometry and on sensory properties were investigated in two sub-studies including a total of 16 pigs. Determination of sarcomere length revealed significant effects of the treatments on the degree of contraction, as pre-rigor excision and pelvic suspension resulted in shorter and longer sarcomere length, respectively. In addition, an effect of treatment on pH measured at 24h post-mortem was found, as pre-rigor excision was associated with a higher ultimate pH. NMR measurements revealed that water distribution in the meat was affected to a minor degree by the various treatments. However, an interaction between treatment and ageing period was observed, as the data demonstrated an effect of pre-rigor excision on water distribution in the cooked samples when meat was only aged for 2 days, but this effect was eliminated when meat was aged for 5 days. The effect of pre-rigor excision on water distribution in cooked meat after 2 days of ageing is suggested to reflect structural constraints in the meat that are eliminated during ageing. Sensory analyses demonstrated strong interactions between treatment and position on the muscle and between treatment and ageing period. However, in general pre-rigor-excised meat was sensed as significantly juicier compared with the other two treatments. Accordingly, the study demonstrates that under the present conditions the ultimate pH is more important for juiciness than the sarcomere length.     

Oxidation desensitizes actomyosin to magnesium pyrophosphate-induced dissociation

This study aimed to establish the influence of protein oxidation on the ability of magnesium pyrophosphate (PP) to dissociate actomyosin. Actomyosin isolated from pork muscle then suspended in 0.1 M NaCl at pH 6.2 was oxidatively stressed with 10 μM FeCl₃/0.1 mM ascorbate/1 mM H₂O₂ for 6 or 12 h at 4 °C. Protein oxidation was evidenced by the loss of myosin and actin, the concomitant formation of disulphide-cross-linked polymers, and elevated myosin ATPase activity. The intrinsic viscosity of oxidized actomyosin had a weaker response to PP-Mg²⁺ than that of non-oxidized actomyosin, indicating the suppression of actomyosin dissociation. Moreover, oxidized actomyosin solutions were devoid of small particles (<10 nm) and the stressed actomyosin exhibited weaker binding of PP-Mg²⁺ than non-oxidized, which further suggested a reduced myosin–PP interaction and subsequent dissociation of the actomyosin complexes.     

BIOCHEMICAL PROPERTIES OF ACTOMYOSIN AND EXPRESSIBLE MOISTURE OF FROZEN STORED ADDUCTOR MUSCLES OF PATAGONIAN SCALLOP (ZYGOCHLAMYS PATAGONICA)

The biochemical properties of actomyosin and the expressible moisture of frozen stored adductor muscles of scallop (Zygochlamys patagonica) were investigated. After freezing (zero time of storage) both the enzymatic activity and the reduced viscosity of actomyosin remained unchanged with respect to those corresponding to actomyosin from unfrozen muscles. Both parameters significantly decreased (P < 0.01) at 4 months of frozen storage and thereafter gradually decreased. No significant differences (P > 0.01) were observed in the relative percentages of myosin, actin, and in the myosin/actin ratio up to 4 months of storage. Thereafter myosin significantly decreased (P < 0.01) up to the end of storage. The decrease in the biochemical properties of actomyosin was accompanied by a loss in the water holding capacity of the muscles.