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

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

The relationship between tenderness, proteolysis, muscle contraction and dissociation of actomyosin

The right side m. longmissius thoracis et lumborum (LTL) of 18 10-month-old male lambs was injected with the protease inhibitor E-64, and the left side was injected with isotonic saline within 30 min of stunning. Carcasses were hung during chilling by one of three methods; Achilles tendon; pubic symphysis or pubic symphysis with 2-kg weights attached to each hindleg. The LTL section was divided into three portions and aged for either 1, 3 or 7 days. The tenderness, myofibrillar fragmentation and dissociation of extracted actomyosin by pyrophosphate were determined for each portion of LTL. The protease inhibitor increased shear force values by 57%, with a decrease in shear force in the cranial-caudal direction along the LTL. Muscle from Achilles hung carcasses was the toughest. For sarcomere length hanging method had the greatest effect, with muscle from tenderstretched/weighted carcasses having the longest sarcomeres. Injection of an inhibitor caused a significant reduction in myofibrillar fragmentation (P< 0.001) and hanging method and ageing period also significantly affected this characteristic (P< 0.05). Osmolality of samples aged for 1 day was unaffected by any main effect. The amount of actomyosin (mg/g of muscle sample) extracted from muscles injected with the inhibitor was significantly less (P< 0.001) and there was also a significant effect (P< 0.05) of portion on this variable. The relationship between pyrophosphate and the percentage of myosin dissociated from the actomyosin complex was modelled using an exponential function; Y=A - B exp(-kx). Analysis of A, B and k for the 69 samples fitted with an exponential function showed that there was no significant effect (P>0.05) on A and k of any main effect or their interactions. There was, however, a significant effect (P<0.05) of portion on B and also a significant interaction between injection and hanging method (P<0.05). It was found that B did not explain any additional variance in shear force or myofibrillar fragmentation over the main effects and interactions previously found significant. The inhibitor E-64 was effective at preventing tenderisation indicating the role of cysteine proteases in proteolytic degradation. There was no apparent effect of dissociation on tenderness as measured in this experiment and therefore a causal relationship cannot be identified.     

The relationship between post-mortem calcium concentration or pH and indicators of proteolysis in ovine muscle

The relationship between post-mortem calcium concentration in muscle and indicators of proteolysis was examined and compared with pH as a predictor of these changes. Muscle samples (m. longissimus et thoracis lumborum; LTL) were obtained from the left side of 24 lamb carcasses at pH 6.2 and 6.0 and then at 1 and 2 days post-mortem (n=96). Alternate carcasses were electrically stimulated (low voltage) within 15 min of death. The myofibrillar fragmentation index (MFI) was determined on samples taken at pH 6.2 and 1 and 2 days post-mortem (n=72). Protein solubility, pH and free calcium concentration were measured on all samples (n=96). The post-mortem degradation of myofibrillar proteins was followed using SDS electrophoresis on all samples (n=96) from which the relative amounts of the 30 kDa fragment and a protein designated M1 were quantified. Transformation of data values for each variable (MFI, protein solubility, 30 kDa fragment and protein M1) improved the normality of the residuals and increased the variance explained by either calcium concentration or pH. pH was a better predictor of MFI and the 30-kDa fragment than calcium concentration and this was reversed when protein solubility and the protein designated M1 was predicted. Of the variables protein solubility could be predicted with the greatest accuracy using calcium concentration (R²=0.64; R.S.D.=0.81) or pH (R²=0.60; R.S.D.=0.85). However overall calcium concentration was not superior to pH as a predictor of the changes in the different indicators of proteolysis examined in this study.     

The impact of new generation pre-dressing medium-voltage electrical stimulation on tenderness and colour stability in lamb meat

Previous work identified that a large number of domestic lamb carcases processed in Australia do not meet the recommended pH/temperature window during chilling. New medium voltage electrical stimulation technology has been developed to alleviate this problem. This study used an optimised setting (800mA with a pulse width 0.5ms) to evaluate the effects on meat quality with a focus on tenderness and meat colour. In total 40 (electrically stimulated) and 40 (non electrically stimulated) lambs from 11 lots killed over 2 days were evaluated. There was a significant difference (P<0.05) between stimulation treatments for initial pH, rate of pH decline and the predicted temperature at pH 6.0. There was also a large difference in the number of carcases that met the recommended window (pH of 6.0, between 18 and 25°C), with an average 67.5% of stimulated carcases and 25% of unstimulated carcases meeting or falling just above the recommended window. The stimulation treatment had no significant (P>0.05) effect on sarcomere length or myofibrillar fragmentation index (MFI) of the m. longissimus (LL). After 1day of ageing LL samples from stimulated carcases had a significantly lower (P<0.05) shear force than non stimulated samples, but there was no difference between stimulation treatments after 5 days ageing. However, ageing period did have a significant effect (P<0.001) on both MFI and shear force, such that 5days aged product had higher MFI and lower shear force values. There were minimal effects of stimulation on colour stability.     

Calpain-calpastatin and toughness in M. longissimus from electrically stimulated lamb and beef carcasses

Shear strength, pH, temperature, μ-calpain, m-calpain and calpastatin levels were measured over a two-week post-slaughter period in Longissimus lumborum et thoracis (LD) from six lamb and six beef carcasses. All carcasses were subjected to high voltage electrical stimulation. The toughness of the beef LD determined by a MIRINZ tenderometer at 24 h post-slaughter showed a strong correlation (r=0.91) with pH of the LD at 3 h. Beef LD toughness at 14 days was correlated (r=0.84) with initial m-calpain levels. In both lamb and beef, LD toughness at 4 and 14 days respectively was also correlated with initial levels of calpastatin (r=0.85, 0.83, respectively). The strong correlation between calpastatin and the rate of tenderisation indicates that the calpain system is closely linked to the proteolytic breakdown of myofibrillar proteins. There is also evidence of an interaction between pH and μ-calpain activity. The μ-calpain, m-calpain, calpastatin, pH and temperature kinetic changes which occurred during the post-mortem ageing of beef and lamb LD were applied to a computer program which predicted rate of meat tenderisation by calculating in situ calpain activity. The closeness of fit between the predicted rate of meat tenderisation and the observed tenderness values of beef and lamb LD indicates that the post-mortem activity of μ-calpain is the major determinant of variations in tenderness. However, application of the meat tenderisation predictive program to LD from individual animals revealed that the program was not sufficiently robust for this use.     

苏尼特羊宰后成熟过程中肌原纤维蛋白特性与肉品质的变化分析

以6月龄苏尼特羊股二头肌为材料,测定其宰后成熟过程中(0～3 d)的肉品质、肌原纤维蛋白功能特性和蛋白降解相关指标,旨在探究肌原纤维蛋白对宰后肉品质的影响,并确定苏尼特羊合适的宰后成熟时间.结果表明,4项肉品质指标(pH值、色泽、剪切力和蒸煮损失率)中,pH值、蒸煮损失率和剪切力在成熟2 d时达到最优值,并与成熟3 d...     

Up- and down-regulation of longissimus tenderness parallels changes in the myofibril-bound calpain 3 protein

The objective of this study was to utilize Ca(2+) and Zn(2+) treatments of meat to critically explore the possible role of calpain 3 in meat tenderisation. Calpains 1 and 2 were also examined for comparative purpose. Control animals plus animals infused with CaCl(2), ZnCl(2) or H(2)O were used (six lambs per treatment) to determine the temporal changes in muscle calpain 3 protein in the Longissimus thoracis et lumborum (LTL) during post-mortem storage. Concurrently, the temporal changes of; (1) shear force, (2) sarcomere length, (3) proteolysis of titin and nebulin and (4) calpains 1 and 2 proteins were also determined. Infusing LTL with Ca(2+) or Zn(2+) caused significant up- and down-regulation of LTL tenderisation, respectively, compared to water infusion and the control animals. Furthermore, the rate of breakdown of calpain 3, the rate of proteolysis of titin and nebulin and the rate of meat tenderisation during post-mortem storage of LTL in the various treatments were highly correlated. These studies suggest that calpain 3, like calpain 1, may be involved in the tenderisation of meat through limited proteolysis of specific muscle structural proteins such as titin and nebulin.     

Meat quality in suckling lambs: effect of pre-slaughter handling

Thirty-three suckling lambs of the Manchega breed were used to compare the effects of pre-slaughter handling (PSH) on initial meat quality and at 7 days post-mortem. Lambs were distributed into three groups: electrically stunned (ESL; n=15), stunned using CO(2) (GSL; n=10) and slaughtered without previous stunning (USL; n=8). Meat quality was evaluated by examining pH, colour (L*, a*, b* values), water holding capacity (WHC), cooking loss (CL), shear force (SF) and drip loss (DL). At 24 h post-mortem, the highest pH corresponded to the USL group (P<0.05) whereas the lowest pH decline (pH(0)-pH(24)) corresponded to the GSL group (P<0.05). Differences in pH among groups disappeared after 7 days post-mortem. In general, PSH did not affect values of WHC, CL and colour parameters. After 7 days post-mortem, there were significant differences between groups in DL, this being lowest (P<0.05) in stunned animals. GSL resulted in more tender meat (lower SF value) than ESL and USL (P<0.05). Moreover, there was no blood splash in any of the carcasses of the GSL group.     

The relationship between early post-mortem pH and the tenderisation of beef muscles

The rate of early post-mortem pH fall in bovine muscle was studied to determine its influence on the rate and extent of the tenderisation process. The pH of M. longissimus dorsi (LD) and M. semimembranosus (SM) muscles of Hereford cross Friesian heifers (n = 127) was taken up to 24 h post mortem. Twenty-four LD and 24 SM muscles were selected according to their rate of pH fall; slow (n = 8), intermediate (n = 8) and fast (n = 8) and were sampled at 2, 7 and 14 days post mortem for sensory, mechanical, physicochemical and biochemical analysis. Fast glycolysing LD muscles were rated more tender in sensory analysis and texture assessment whereas slow glycolysing muscles were considered significantly tougher (p < 0.001). Fast glycolysing LD had a significantly lower shear force (p < 0.001) at each stage of ageing. Slow glycolysing LD and SM had the highest shear force values. No significant difference was found in intramuscular fat or moisture content between the groups. Slow glycolysing LD muscles had shorter sarcomere lengths at 2 days post mortem. SDS-PAGE electrophoresis patterns showed increased proteolysis, such as the earlier appearance of the 30 kDa fragment, which is believed to be a good indicator of tenderness, in fast glycolysing muscle. These results suggest that the rate of post-mortem pH fall plays an important role in proteolysis and tenderisation.     

Influence of ultimate pH on bovine meat tenderness during ageing

The aim of this work was to evaluate the influence of ultimate pH and ageing at 2±2°C on the tenderness of beef. The m.longissimus thoracis et lumborum from 23 young bulls excised at 28 h post mortem were grouped into: Normal (pH 5.5 to 5.8) moderate DFD (mod DFD) (5.8p<0.05) lower in the DFD group than in the Normal one and a significant (p<0.001) linear relationship was found between ultimate pH and tenderness as evaluated by both methods. The myofibrillar protein solubility (MPS) at pH 7.0 was significantly higher in the DFD group at all post mortem times whereas for myofibrillar fragmentation index (MFI) higher values (p<0.05) were only found in the DFD group at day 1. In all three groups toughness decreased (p<0.05) from 1 to 6 days, with no further decrease to 13 days. This decrease in toughness was associated with an increase of MFI in all pH groups. Collagen solubility in all three groups was not affected by ageing. No significant (p>0.05) differences in soluble collagen and myofibrillar protein solubility (MPS) at pH 5.5 were found between the pH groups at any time. The tenderness evaluated by both methods was significantly (p<0.05) related to MFI, MPS at pH 7.0, cooking loss and juiciness. Total and soluble collagen, sarcomere length, intramuscular fat and MPS at pH 5.5 were not significantly (p>0.05) related with tenderness.     

Pre-rigor infusion with kiwifruit juice improves lamb tenderness

The ability of pre-rigor infusion of kiwifruit juice to improve the tenderness of lamb was investigated. Lamb carcasses were infused (10% body weight) with fresh kiwifruit juice (Ac), water (W) and a non-infusion control (C) treatment. Infusion treatment had no effect on lamb hot carcass weight, cold carcass weight and chilling evaporative losses. The infused treatment carcasses of Ac and W had lower (P < 0.05) pH values than C carcasses during the initial 12 h post-mortem. The LD muscles from Ac carcasses were more tender with significantly lower shear force (P < 0.001) compared with C and W carcasses during the six days following infusion with the kiwifruit juice. The enhanced proteolytic activity (P = 0.002) resulting from the infused kiwifruit juice in Ac carcasses was associated with significant degradation of the myofibrillar proteins, appearance of new peptides and activation of m-calpain during post-mortem ageing. Thus, kiwifruit juice is powerful and easily prepared meat tenderizer, which could contribute efficiently and effectively to the meat tenderization process.     

Rapid chilling has no detrimental effect on the tenderness of low-voltage electrically stimulated M. longissimus in Chinese bulls

This study investigated the effect of pre-rigor rapid chilling (RC) on the rate of pH and temperature decline, shear force values and ultrastructure of M. longissimus from beef carcasses exposed to electrical stimulation (ES). Chinese bull carcasses were electrically stimulated, and the alternate sides of the carcasses either were chilled conventionally (CC, 0-4°C, air speed 0.5 m/s for 24 h) or they underwent RC (-14±1°C, air speed 3 m/s for 2 h and then cooled under CC conditions until 24 h post-mortem). The results showed that RC increased the rate of temperature decline (P<0.05) and decreased the rate of pH decline (P<0.05). There were no significant differences in the shear force value and sarcomere length of M. longissimus between the two treatments (P>0.05). The results of this study indicate that RC has no detrimental impact on the tenderness of beef carcasses exposed to ES.     

Fresh beef tumbling at different post-mortem times to improve tenderness and proteolytic features of M. longissimus lumborum

The aim of this study was to determine the effects of tumbling at different post-mortem times on the proteolytic features and quality attributes of beef loins (M. longissimus lumborum). Loins (n = 12) were cut into 4 sections and assigned to tumbling at 1, 6 or 11 days post-mortem or non-tumbled control. Upon tumbling, additional ageing was applied to a common post-mortem time of 16 days. In general, tumbling had no considerable impacts on water-holding ability of samples. Tumbling resulted in an immediate decrease in shear force values (WBSF) of beef samples. Tumbling at 1 day post-mortem with no ageing had similar WBSF compared to the non-tumbled controls at 16 days (P > 0.05). With ageing, tumbling increased amounts of protein degradation, myofibrillar fragmentation and calpain-1 autolysis of samples. These results suggest that early post-mortem tumbling coupled with ageing can synergistically impact the tenderness development of beef loins and shorten the necessary ageing period.     

The effects of the ultimate pH of meat on tenderness changes during ageing

A range of ultimate pH (pH(u)) values, from 5.4 to 7.2, was produced in the m. longissimus thoracicum et lumborum (LD) of 55 sheep by subcutaneous injections of adrenaline, or by injection and subsequent exercise. The rate constant of ageing for each of 47 animals was calculated from shear force measurements, taken at intervals for up to 5 days from the LD held at 12 dgC. The relationship between the ageing rate and pH(u) could be adequately described by the equation rate constant (k) = 4.109 - 1.349(pH) + 0.1111(pH)(2); r(2) = 0.707, with a minimum rate constant at pH 6.07. The loin muscles from eight carcasses were selected to determine the effects of ultimate pH on the initial, unaged toughness value. Calpain activity was inhibited by intramuscular injection of ZnCl(2) dissolved in saline, and the muscles were aged at 10-12 °C on the carcass for up to 6 days. At all values of pH(u) (5.4-6.7), tenderness as measured by shear force were equivalent, from which it was concluded that the initial toughness of unaged lamb was not affected by pH(u). In the contralateral, untreated muscle, ageing was measured using both shear force measurement and changes in the myofibrillar fragmentation index (MFI). After 1 day, the intermediate pH range (5.8-6.2) has the highest shear force and the lowest fragmentation index value. However, in contrast to the shear force values, the ultimate MFI value in aged muscle was lowest in the high pH(u) range (6.4-6.7; minimum value at 6.4). The highest fragmentation values were found at the low pH(u) range. These observations suggest that pH(u)-related tenderness differences in meat result from different rates of ageing in the post rigor period, and therefore that these differences disappear following a sufficient ageing period. The slowest rate of tenderization measured on cooked meat was observed at pH(u) values around 6.07, but the least fragmentation of raw meat was observed at pH(u) values around 6.4, a difference of almost 0.33 pH unit.     

Singular and combined effects of electrical stimulation, post-mortem ageing and blade tenderisation on the palatability attributes of beef from young bulls

Thirty Santa Gertrudis bulls (approximately 15-18 months old) were slaughtered, dressed and split into siides. The right side of each carcass was electrically stimulated (ES) with seventeen impulses (1·8s impulse duration; 1·8s interval between impulses) of 550 V (AC) and 5 A while the left side served as a non-stimulated control (not-ES). At 24h post morten, USDA quality and yield grade data were obtained from each side. On the second day post mortem, all sides were fabricated and strip loins, top sirloin butts and ribeyes were obtained from each side for post-mortem ageing and blade tenderisation studies. Steaks were removed after a post-mortem ageing period of 4 or 18 days and before (not-BT) or after blade tenderisation (BT) for sensory panel evaluations or shear force determinations. ES sides had more youthful lean maturity (P < 0·0001), higher marbling (P < 0.·002), higher USDA quality grades (P < 0·0.0001) and finer-textured lean (P < 0·002) than did not-stimulated (not-ES) sides. ES significantly improved (P < 0·05) palatability traits in two of twenty-four comparisons; BT significantly improved palatability traits in twelve of twenty-four comparisons and 18-day post-mortem ageing significantly improved palatability traits in seven of twelve comparisons. No significant reductions (P < 0·05) in shear force values were observed for steaks from ES versus not-ES sides while significant reductions (P < 0·05) were observed for steaks from BT versus non-BT cuts (four of six comparisons) and for steaks from cuts aged for 18 versus 4 days (ten of twelve comparisons). BT and 18-day post-mortem ageing were more effective for increasing palatability or for decreasing shear force requirements than was ES; however, ES greatly improved lean colour of meat from bulls.     

Pre-slaughter stress arising from on-farm handling and its interactions with electrical stimulation on tenderness of lambs

The effect of electrical stimulation of lamb carcasses (n=269) or its absence (n=257) on shear force of m. longissimus thoracis et lumborum (LT) was monitored during ageing in pasture-fed merino lambs (n=526). The lambs were slaughtered on four different days allowing durations of between one to 10 days of recovery from pre-slaughter handling (yarding, weighing and crutching) that affected ultimate pH (pH(u)). The right LT was removed 20-40min post-slaughter, tightly-wrapped in cling film (prevents the muscle cross-section increasing and thus minimising shortening) and rapidly cooled to 15°C to enter rigor mortis and age. At 0, 4, 24 and 72h post-slaughter, pH measurements and samples for shear force measurement were taken. Pre-slaughter handling had a significant negative effect on pH(u) and several days recovery were required for pH(u) to reach values associated with optimal meat quality as reflected by pH(u). Lambs with one and three days recovery (no significant difference between them) had a pH(u)>5.7 in 50% of the muscles and 19.4%>pH(u) 5.8. Whereas, in lambs with 8-10 days recovery (no significant difference between them), only 8% had a pH(u)>5.7 and 3.1%>pH(u) 5.8. Within each slaughter day electrically stimulated lambs were always more tender than non-stimulated lambs. For non-stimulated muscles at 72h, shear force values >40N occurred for 11.2% of the muscles: for electrically stimulated muscles at 72h, shear force values >40N occurred for 1.9% of the muscles. The rates of tenderisation were slower for intermediate pH(u) values resulting in higher shear force values at all ageing durations. With ageing at 72h for intermediate pH(u), non-stimulated muscles (n=38) 17.64% were >40N and for stimulated muscles (n=34), 7.9% were >40N.     

Should electrical stimulation be applied when cold shortening is not a risk?

This experiment was designed to show whether delayed high voltage stimulation (ES) is more beneficial than no stimulation (NS) to secure tenderness under circumstances where rigor conditions are difficult to control due to variations in carcass size, fatness and/or chilling capacity. Ten Charolais carcasses were split during slaughter, the left sides were stimulated at 45 min post-mortem for 45 s, and the right sides were left unstimulated. The carcass sides were then chilled at a medium chilling rate. Sarcomere length measurements confirm that there was neither cold nor heat shortening in the M. longissimus (LD). LD from ES sides aged for 2 days was more tender than non-stimulated LD (NES), although prolonged ageing eroded the advantage of ES to a non-significant advantage after 14 days. Initial tenderness differences coincided with lower 24 h calpain activity, suggesting an early onset of proteolysis and ageing (tenderisation). In contrast to conventional early ES, delayed ES, appears to be beneficial for the early development of tenderness without too much interference with enzyme. Myofibril fragment length (MFL) was a good indicator of the development of tenderness during prolonged ageing but not for the early post-mortem variation in tenderness. No colour (L*, a*, b*) differences, occurred due to stimulation treatment, while drip loss was slightly higher at 24 h post-mortem for ES meat.     

Effect of muscle restraint on sheep meat tenderness with rigor mortis at 18°C

The effect on shear force of skeletal restraint and removing muscles from lamb m. longissimus thoracis et lumborum (LT) immediately after slaughter and electrical stimulation was undertaken at a rigor temperature of 18°C (n=15). The temperature of 18°C was achieved through chilling of electrically stimulated sheep carcasses in air at 12°C, air flow 1-1.5 ms(-2). In other groups, the muscle was removed at 2.5 h post-mortem and either wrapped or left non-wrapped before being placed back on the carcass to follow carcass cooling regimes. Following rigor mortis, the meat was aged for 0, 16, 40 and 65 h at 15°C and frozen. For the non-stimulated samples, the meat was aged for 0, 12, 36 and 60 h before being frozen. The frozen meat was cooked to 75°C in an 85°C water bath and shear force values obtained from a 1 × 1 cm cross-section. Commencement of ageing was considered to take place at rigor mortis and this was taken as zero aged meat. There were no significant differences in the rate of tenderisation and initial shear force for all treatments. The 23% cook loss was similar for all wrapped and non-wrapped situations and the values decreased slightly with longer ageing durations. Wrapping was shown to mimic meat left intact on the carcass, as it prevented significant prerigor shortening. Such techniques allows muscles to be removed and placed in a controlled temperature environment to enable precise studies of ageing processes.     

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.     

Effect of stress-induced high post-mortem pH on protease activity and tenderness of beef

Forty-four Swiss Brown young bulls were stressed by regrouping unfamiliar animals before slaughter. M. longissimus thoracis (6-9th ribs) of carcasses were analysed for post-mortem pH, protease activities (m- and α-calpain, calpastatin and cathepsin B + L), Warner-Bratzler shear force and sensory tenderness and juiciness. Muscles were classified into three groups, according to ultimate pH values: > 6.3, 6.3-5.8 and < 5.8. The most significant difference related to high pH was a higher activity of m-calpain at 7th day post mortem. It was also found that meat showing the highest pH was significantly more tender and juicy. Sensory tenderness was highly correlated with activity of m-calpain at 7th day post mortem (r = 0.776) and with ultimate pH (r = 0.708). It is concluded that high ultimate pH induced by stress significantly increases m-calpain activity, and this results in a greatly enhanced tenderisation of beef meat.