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Calcium Chelators Influence Some Physical and Chemical Properties of Rabbit and Pig Muscle
Journal of Food Science
Abstract
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.
... The importance of particularly Ca 2+ ions to tenderisation was initially suggested when CaCl 2 , chelating agents such as EDTA, or EGTA were injected into post-mortem muscle. Weiner and Pearson (1969) observed longer sarcomeres in pig muscle injected pre-rigor with 0.1 M EDTA and consequently, significantly lower shear force values than in control muscle at 24-h post-mortem. By contrast, injection of 0.1 M CaCl 2 resulted in significant shortening in rabbit muscle compared with muscle injected with 0.1 M MgCl 2. These effects could be attributed to the role of Ca 2+ ions in muscle contraction. ...
... Although the chilling conditions were not given by Koohmaraie et al. (1990), it could be assumed that either stimulation was not effective in this study or the chilling was less severe than in the previous study (Koohmaraie et al. 1989). Curiously, in neither experiment was sarcomere length measured because, given the results of Weiner and Pearson (1969), shortening may have been expected. Indeed, in the report of Farouk et al. (1992), when a solution containing 0.015 M CaCl 2 was infused into lamb carcasses immediately after death, there was some evidence of shortening (Table 2), although the difference from control samples was not significant. ...
This review seeks to examine the current theories of tenderisation with respect to red meat where tenderisation is defined as the reduction in toughness post-rigor. This examination is in the light of recent research on meat biochemistry, and from this, areas of research that may prove fruitful are highlighted. Based on available data, the major candidate to explain tenderisation post-rigor is the calpain protease system. Evidence that change in the binding of actomyosin (the complex of contractile proteins formed at rigor) or cleavage of myofibrillar proteins due to Ca²⁺ ions contributes to tenderisation is far from compelling. Equally it appears that the cathepsin proteases are unlikely to have a role in early post-mortem cleavage of proteins (proteolysis) and thus tenderisation. The mode of action of the calpains is not yet fully defined and questions remain as to the role of m-calpain given the in vitro requirement for a Ca²⁺ ion concentration exceeding that observed in post-mortem muscle. The existence of the calpains in living muscle and other tissues suggests a mode of action more subtle than currently thought. Additionally, the observation that the degradation of myofibrillar proteins occurs in the presence of effective synthetic and natural calpain inhibitors suggests that other enzymes may also have a role in tenderisation. Inevitably, the accumulated evidence points to a complex system likely to involve interacting proteases and ions, and only through open minded investigation with reliance on developments in the medical and biochemical fields will a more complete model of tenderisation be developed.
... The importance of particularly Ca 2+ ions to tenderisation was initially suggested when CaCl 2 , chelating agents such as EDTA, or EGTA were injected into post-mortem muscle. Weiner and Pearson (1969) observed longer sarcomeres in pig muscle injected pre-rigor with 0.1 M EDTA and consequently, significantly lower shear force values than in control muscle at 24-h post-mortem. By contrast, injection of 0.1 M CaCl 2 resulted in significant shortening in rabbit muscle compared with muscle injected with 0.1 M MgCl 2. These effects could be attributed to the role of Ca 2+ ions in muscle contraction. ...
... Although the chilling conditions were not given by Koohmaraie et al. (1990), it could be assumed that either stimulation was not effective in this study or the chilling was less severe than in the previous study (Koohmaraie et al. 1989). Curiously, in neither experiment was sarcomere length measured because, given the results of Weiner and Pearson (1969), shortening may have been expected. Indeed, in the report of Farouk et al. (1992), when a solution containing 0.015 M CaCl 2 was infused into lamb carcasses immediately after death, there was some evidence of shortening (Table 2), although the difference from control samples was not significant. ...
This review seeks to examine the current theories of tenderisation with respect to red meat where tenderisation is defined as the reduction in toughness post-rigor. This examination is in the light of recent research on meat biochemistry, and from this, areas of research that may prove fruitful are highlighted. Based on available data, the major candidate to explain tenderisation post-rigor is the calpain protease system. Evidence that change in the binding of actomyosin (the complex of contractile proteins formed at rigor) or cleavage of myofibrillar proteins due to Ca 2+ ions contributes to tenderisation is far from compelling. Equally it appears that the cathepsin proteases are unlikely to have a role in early post-mortem cleavage of proteins (proteolysis) and thus tenderisation. The mode of action of the calpains is not yet fully defined and questions remain as to the role of m-calpain given the in vitro requirement for a Ca 2+ ion concentration exceeding that observed in post-mortem muscle. The existence of the calpains in living muscle and other tissues suggests a mode of action more subtle than currently thought. Additionally, the observation that the degradation of myofibrillar proteins occurs in the presence of effective synthetic and natural calpain inhibitors suggests that other enzymes may also have a role in tenderisation. Inevitably, the accumulated evidence points to a complex system likely to involve interacting proteases and ions, and only through open minded investigation with reliance on developments in the medical and biochemical fields will a more complete model of tenderisation be developed.
Organismal death is a process of systemic collapse whose mechanisms are less well understood than those of cell death. We previously reported that death in C. elegans is accompanied by a calcium-propagated wave of intestinal necrosis, marked by a wave of blue autofluorescence (death fluorescence). Here, we describe another feature of organismal death, a wave of body wall muscle contraction, or death contraction (DC). This phenomenon is accompanied by a wave of intramuscular Ca²⁺ release and, subsequently, of intestinal necrosis. Correlation of directions of the DC and intestinal necrosis waves implies coupling of these death processes. Long-lived insulin/IGF-1-signaling mutants show reduced DC and delayed intestinal necrosis, suggesting possible resistance to organismal death. DC resembles mammalian rigor mortis, a postmortem necrosis-related process in which Ca²⁺ influx promotes muscle hyper-contraction. In contrast to mammals, DC is an early rather than a late event in C. elegans organismal death.
Video Abstract
The effects of Ca2+ on the integrity of myofibrils and their proteins were studied by isolating myofibrils from minced at-death bovine longissimus muscle samples treated with 1 mM Ca2+ and 10 mM oxalate and stored at either 2°C or 23°C. Myofibrils were isolated from treated and control samples at various postmortem times and characterized with SDS-polyacrylamide gel electrophoresis. In addition, myofibrils were isolated from treated and control samples at various postmortem times and extracted with Hasselbach-Schneider (H-S) solution and with 1 mM Tris, pH 8.5. These extracts were characterized with SDS-polyacrylamide gel electrophoresis. Myofibrils isolated from postmortem muscle samples treated with Ca2+ showed more rapid degradation of troponin T and the concurrent appearance of a 30,000-dalton component during postmortem storage than did control and oxalate samples. Proteins extracted from myofibrils increased during postmortem storage with both H-S and Tris solutions. The salt soluble proteins of Ca2+ treated samples showed more 30,000-dalton component and α-actinin compared with control and oxalate samples. In essence, the extent and nature of changes in myofibrils and in extracted proteins were dependent upon Ca2+ availability. Storage of muscle samples at room temperature accelerated all these changes. Postmortem modifications in myofibrillar proteins were interpreted as being consonant with the hypothesis that proteolysis of myofibrils is caused by a Ca2+ -activated factor endogenous to the muscle fiber.
Prerigor beef longissimus muscles were removed within 40 min postmortem and injected 10% (within 2 h postmortem) with either a chelator (alginate, 3%; haametaphosphate, 3%; EDTA, 0.13%; citrate, 0.13%), meat slurry (45%), or a myofibrillar meat extract (3.7% protein) to evaluate the effects of each injection on the physical and chemical properties compared to a control and a water only injected treatment. Gross muscle shortening was not inhibited The water only, meat extract and citrate treatments had shorter (P<0.05) sarcomeres than the control. The meat shy, alginate, phosphate, and EDTA treatments produced sarcomere lengths equivalent to the control. The control had the lowest (P<0. 05) pH and percentage moisture. However, there were no differences in percentage cooking losses. Although variations in sarcomere length were apparent, these treatments did not improve tenderness (P>0.05) as measured by Warner-Bratzler shear.
The effects of 15 inorganic salts on the tenderness of spent hen muscle were examined. Addition of salts at in-muscle ionic strengths of μ= 0.125, 0.25, and 0.50 equalized charges, enabling examination of specific ion effects. Criteria measured included water holding capacity, yield, moisture, and shear. Lithium, magnesium, and sodium salts were the most effective for increasing tenderness. Calcium salts decreased tenderness. Overall ranking for tenderizing effects of anions was Cl−1 > Br−1 > I−1 > F−1, and for cations Li+1= Na+1= Mg+2 > K+1 > Ca+2. There were no differences in tenderness among ionic strengths, μ= 0.25 or 0.50. Similar tenderness effects were observed at μ= 0.125. Previously frozen samples were significantly affected by treatments, while unfrozen samples showed no effect due to treatment.
Rigor mortis was induced in frog sartorii from female Rana pipiens by incubating the isolated muscles in 0.38 mm 2,4-dinitrofluorobenzene Ringer's (frog) solution for 90 min at 0°. This compound completely inhibits adenosine triphosphatecreatine phosphotransferase in vivo and eventually induces rigor mortis as characterized by shortening of the muscle to 50% of its rest length and the development of stiffness. As the muscle began to shorten the rate of ⁴⁵Ca efflux increased and the ATP level fell from 3.80 to 0.0 µmoles per g. There was a lack of correspondence between ATP breakdown and appearance of inorganic phosphate, which can be explained by leakage of inorganic phosphate from the muscle and by an increase in the concentration of glucose 6-phosphate, fructose 6-phosphate, and glucose 1-phosphate. The frog sartorius muscle breaks down ATP during a working contraction and in the shortening phase of rigor mortis. There is no ATP requirement for tension maintenance in rigor mortis after the muscle has finished shortening.
1.1. The dependence of nucleosidetriphosphatase (NTPase) activity of actomyosin and L-myosin on the ionic conditions has been investigated.2.2. The following holds with regard to the NTPase activity of calcium-activated L-myosin: 2.1.(a) Ca ions activate much more strongly the splitting of 6-OH-NTP by L-myosin gel and L-myosin sol than the splitting of 6-NH2-NTP.2.2.(b) The order in which the 6-OH-NTP is split at an increasing speed is very markedly dependent on the ionic strength.2.3.(c) The order in which the 6-NH2-NTP is split is independent of the ionic strength. ATP is always split more rapidly than CTP.2.4.(d) All the NTPs are split more rapidly by L-myosin gel than by L-myosin sol.3.3. The following holds with regard to NTPase activity of L-myosin in the presence of magnesium ions: 3.1.(a) In the presence of Mg++ the rates of splitting by L-myosin are low (< 0.1 μmole P × mg protein−1 × min−1).3.2.(b) Mg ions activate the splitting of guanosine triphosphate (GTP) and inosine triphosphate (ITP) by gel and sol. The splitting of uridine triphosphate (UTP) and cytidine triphosphate (CTP) by the gel is only insignificantly altered. The splitting of both NTPs by the sol is inhibited by magnesium. The splitting of adenosine triphosphate (ATP) is always inhibited by Mg ions.3.3.(c) The rate at which L-myosin gel and L-myosin sol split NTP decreases in the order: GTP > ITP > UTP > CTP ≳ ATP3.4.(d) The order of the rate of splitting of NTP is independent of the ionic strength. With increasing ionic strength the rates decrease only slightly.4.4. In the absence of alkaline earth and in the presence of chelating agents L-myosin sol splits 6-NH2-NTP more rapidly than 6-OH-NTP. 4.1.(a) Without addition of alkaline earth the rates for all NTPs are low.4.2.(b) Addition of chelating agents activates the splitting of ATP and CTP very strongly and the splitting of UTP only slightly. The splitting of ITP and GTP is inhibited.5.5. The NTPase activity of actomyosin gel is fundamentally different from that of actomyosin sol and L-myosin preparations.6.6. Both in the absence of alkaline earth and in the presence of Mg and Ca, the gel splits NTP at a decreasing rate in the order ATP ≳ CTP > UTP > ITP > GTP. Splitting of all the NTPs, with the exception of ATP, is more strongly activated by 10−3M Mg++ than by 10−2M Ca++. EDTA inhibits the splitting of all NTP by actomyosin gel.7.7. When the ionic strength increases from 0.1 to 0.4 μ the NTPase activity of actomyosin gel becomes converted to the NTPase activity of actomyosin sol.8.8. At an ionic strength of 0.4 μ, in the presence of Mg++, all the NTPs dissociate actomyosin. The dissociated L-myosin has the same NTPase activity as the L-myosin sol.9.9. When no Mg++ is added the actomyosin sol is dissociated completely only by ATP. CTP causes a stronger partial dissociation than UTP. ITP and GTP are ineffective.10.10. In the absence of alkaline earth and in the presence of Ca, the ATPase and CTPase activities of actomyosin and L-myosin sol are therefore equal and the UTPase activity of both proteins is very similar.11.11. When no alkaline earth is added ITP, GTP and UTP are split by the undissociated actomyosin sol and the L-myosin sol at a similar low rate.12.12. In the presence of Ca++, GTP is split more rapidly than ITP by actomyosin sol-NTPase, while ITP is split more rapidly than GTP by L-myosin-NTPase.13.13. All NTPs are split more slowly by actomyosin sol than by L-myosin sol.14.14. In the presence of EDTA also, ATP dissociates actomyosin completely. UTP, ITP and GTP do not bring about dissociation in EDTA-containing actomyosin sol.15.15. The EDTA-activation of the ATP-splitting by actomyosin sol is less than that by L-myosin sol. UTP-, ITP- and GTP-splitting by undissociated actomyosin sol is inhibited by EDTA.
Tenderness of semitendinosus and psoas major bovine muscles was markedly affected by: (1) allowing the muscle to undergo rigor mortis and the associated contraction, following pre-rigor excision, or by (2) pre-rigor excision followed by restraint in a stretched state while the muscle undergoes rigor mortis. The extent of stretch or contraction induced by pre-rigor treatment was reflected by the sarcomere length. The average sarcomere length of the semitendinosus and psoas major muscles differed widely when samples were removed post-rigor from the carcass. The data indicate that the state of contraction (measured by sarcomere length), when altered in different portions of the same muscle by treatment, or when varying naturally in different muscles, was associated with tenderness.
The tenderness of meat removed from the carcass in a pre‐rigor condition is highly dependent on the extent of the cold shortening which occurs after excision. The relationship between shortening and tenderness is complex. A decrease of up to 20% of the initial excised length does not exert a significant effect, hut toughness increases rapidly with further shortening beyond this point, reaching a peak of several times its original value (in terms of shear force required) at a shortening of about 40%. With yet further shortening, the meat becomes progressively more tender until, at about 55‐60% shortening, it is cleaved about as easily as meat in which less than 20% shortening has occurred.
The presence of intact skeletal attachments does not necessarily overcome the development of shortening‐induced toughness. It is shown that a pre‐rigor muscle which is absolutely fixed in over‐all length is still capable of appreciable shortening in one zone, with compensating lengthening elsewhere, if the application of cold is uneven along its surface.
Interrelationships of fiber diameter, sarcomere length, and tenderness were studied in 12 bovine muscles of horizontally placed and vertically suspended carcass sides. In comparison with the horizontally placed sides, the vertically suspended sides had greater sarcomere lengths in the psoas major, latissimus dorsi, and rectus femoris muscles. Conversely, vertical suspension permitted the longissimus dorsi, gluteus medius, adductor, biceps femoris, and semitendinosus muscles to shorten in sarcomere length. In general the differences in sareomere lengths of muscles (between sides) were associated (r=–.82 P <.01) with differences in fiber diameter. Differences in fiber diameter (between sides) were highly related to differences in shear force (r= .73, P <.01, as were differences in sarcomere length (T = -.80, P <.01). When museles shortened, there were corresponding decreases in sarcomere length, increases in fiber diameter, and decreases in tenderness.
The conception of Huxley concerning the structural basis of muscular contraction is universally accepted. It shows a close correlation between ATP and the fibrillar proteins, also existing during the first phase of the post-mortem changes. Rigor development has been followed in whole fish and isolated beef muscles by measuring the torsion elasticity. Often there are great individual deviations in rigor development within a single species. Generally, at corresponding temperatures, rigor development lasts longer in mirror carp than in the gastrocnemius of beef; rosefish needs a longer time to reach maximum rigor than cod. Evidently, the rapid phase of ATP breakdown and increasing rigidity of muscles is initiated by inactivation of the Marsh-Bendall factor in the post-mortem period. Normally, contraction occurs when ATP is added to fiber fragments of aged meat. This implies that the aetomyosin complex formed during rigor development becomes dissociated, or at least may become dissociated easily, in aged meat, and that tenderness changes in the aging period are correlated to this process. ATP breakdown in fish muscle is highly activated by freezing and thawing (“biochemischer Verletzungseffekt”) and seems to be caused by inactivation of the relaxing factor. Fish (whole fish or fillets) frozen under normal commercial conditions immediately after death show an insignificant degree of thaw contracture. In cod and rosefish no significant difference has been found in the extractability of the actomyosin fraction, if the time passing between death and freezing was considered. In frozen muscle tissue stored below -18°C, ATPase activity and contractability decrease very slowly. This shows that the actin and myosin filaments are not subject to great structural changes by freezing and thawing. In the freezer-burn area of muscle tissue the structure proteins lose the ability to contract on ATP addition more and more with increasing storage time; finally, even the plasticizing effect of ATP on the fibrillar proteins disappears, the fibrils scarcely change their original orientation.
Isolated fresh beef muscles have been found to shorten more at 2° than at 37°. Minimum shortening occurs in the temperature region of 14–19°. At higher temperatures shortening coincides with the onset of rigor mortis but at low temperatures it begins rapidly and usually immediately. This shortening is reversible. Three different beef muscles show this effect but two rabbit muscles do not.
A lethal intravenous injection of EDTA in rabbits inhibited the post-mortem shortening of the semitendinosus muscle and prevented the inextensibility that normally occurs during development of rigor mortis. It was also shown that no appreciable difference in pH and ATP values existed between the EDTA-treated rabbits and the control rabbits at either 1 hour or 20 hours. These results thus indicate that EDTA inhibited the onset of rigor mortis, but did not prevent the breakdown of ATP.
IF an excised beef muscle is exposed to a temperature of 0°-10° C soon after death, a shortening of considerable magnitude occurs during the subsequent 1-5 h1. Both the extent and the rate of shortening are less with higher temperature in this range, and also with longer delay before exposure to the cold environment. By appropriate selection of temperature and delay, therefore, shortening values ranging from nothing to more than 60 per cent of the initial length can be obtained.
1.1. Three types of light-measuring devices have been used to measure the effect of various chemical agents on firefly luminescence. They are the commercially available Farrand photofluorometer, a recording DC IP21 photomultiplier, and a liquid-nitrogen-temperature quantum counter whose ultimate sensitivity is of the order of 10−9 g. ATP/ml.2.2. It has been possible to use both the crude and partially purified water extracts of Photinus pyralis to measure light production in the presence of “potential” or actual ATP by using arsenate buffer.3.3. Differential-determination of ATP and ADP in the presence of each other is possible through the use of myokinase.4.4. Phosphocreatine may be determined by the use of the appropriate transphosphorylase and adenylic acid.5.5. Glucose may be determined by the use of hexokinase and ATP, measuring the depression in luminescence.6.6. Chloride ion has a strong inhibitory effect on luminescence which must be controlled rigorously.7.7. Myokinase, hexokinase, apyrase, and creatine adenylic transphosphorylase may be determined in small concentrations with this system.8.8. Preliminary examination of certain biological processes with this tool suggests its wide applicability for routine surveys of phosphorylative energetic mechanisms.
This chapter briefly reviews the biochemistry and physiology of rigor mortis in regard to the development of pale, soft, exudative (PSE) muscle. It is restricted to PSE musculature and specific closely allied studies. Extremely rapid glycolytic rates, with low pH values being achieved at high or approximately body temperatures, are associated with the development of a pale (white), soft, exudative condition. Regardless of the temperature dependence of the pH values of the buffer systems, it seems pertinent that this elevation in pH may be frequent under normal processing conditions. This depression and elevation in pH may serve, in part, to explain the wide variations noted in the ultimate pH values of PSE muscle. pH changes in any muscle that ultimately has variations in color, gross morphology, and rigor mortis show wide fluctuations and reflect at least six distinct types of pH patterns. The effect of environment on the incidence of PSE muscle has also been studied. In a study of post-mortem changes in muscle, not only are major variations between muscles readily noted but variations within muscles are frequently easily discernible. In a severely PSE carcass the variations among, as well as within, muscles are probably at a minimum. When the condition of the carcass is less severe, variations within muscles are more noticeable. Specific experiments have been conducted to determine the effect of ration on the development of PSE muscle. As with enzymes, it is impossible to cover hormonal considerations of glycolysis and contraction. Consequently, only those hormones that have been studied in relation to PSE muscle have been discussed.
The length‐tension diagram of single vertebrate striated muscle fibres
- Gorden A.M.
Action of relaxing agents on the actomyosin‐ATP system
- Maruyama K.
Sarcoplasmm reticulum. I. The uptake of Ca++ by sarcoplasmic reticulum fragments
- Martonosi A.
Local muscular shortening by intracellularly applied calcium
- Niedergerke R.