Post-mortem calpain-system kinetics in lamb: Effects of clenbuterol and preslaughter exercise

The effect of dietary supplementation with clenbuterol for either 8 days or 55 days in lambs was studied. The 55-day treatment was combined with an immediate preslaughter exercise regime. The effect of these treatments on post-mortem calpain system activities, meat ageing and meat quality was studied. Neither short-nor long-term supplementation had an effect on the rate of pH fall post mortem. Short-term supplementation had no effect on the initial nor the final shear force values but these were higher at intermediate times. In contrast, prolonged supplementation increased shear force values at all times post mortem. Preslaughter exercise, while influencing the rate of pH decrease in both control and supplemented groups, did not affect meat tenderness. After short-term clenbuterol-supplementation, the in-vitro μ-calpain activity was significantly lower in the supplemented group at 6 and 24 hr post mortem, while m-calpain and calpastatin activities were largely unaffected. In contrast, 55-day clenbuterol supplementation resulted in significantly higher levels of calpastatin activity at all times post mortem. These data imply that clenbuterol results in toughened meat due to alterations in the calpain/calpastatin system, the mechanisms of which are dependent upon the duration of supplementation.     

Early post-mortem conditions and the calpain/calpastatin system in relation to tenderness of double-muscled beef

Early post mortem temperature, pH, sarcomere length, colour, water holding capacity, calpain/calpastatin activities and myofibrillar protein concentrations (semi-quantative SDS-PAGE), measured at different times post mortem on 153 double-muscled Belgian Blue White bulls, were related to shear force (SF) measurements. The M. longissimus thoracis tenderised up to 8 days post mortem. SF was slightly correlated with temperature at 3 h post mortem (r = 0.20, p = 0.015), but not with pH early post mortem. Higher ultimate pH values measured at 24 h post mortem were related to darker meat, lower cooking losses and shorter sarcomere lengths. Sarcomere length was significantly related to SF, even after 12 days of ageing, suggesting that proteolytic activity was not able to overcome shortening. Up to 12 days post mortem, calpastatin and m-calpain activities were significantly correlated with SF suggesting a considerable role of calpains in meat tenderization of double-muscled Belgian Blue White bulls, although at 24 h post mortem, μ-calpain could no longer be detected. Titin, nebulin, filamin and troponin-T were degraded during the ageing period. Troponin-T was degraded by 80% between day 1 and 8. At 8 days post mortem its concentration was significantly correlated with 30 kDa (r = 0.63, p = 0.00), shear force (r = 0.39, p = 0.00), calpastatin activity at 1 day post mortem (r = 0.29, p = 0.01) and with the m-calpain/ calpastatin activity ratio at 1 day post mortem (r = -0.43, p = 0.00).     

Effect of early post-mortem cooling on temperature, pH fall and meat quality in pigs

The aim of this study was to investigate how early cooling of carcasses after slaughter by showering with cold water affected the rate of the pH fall post mortem, protein denaturation and drip loss. Eighty pigs were selected in pairs at debleeding according to sex and farm of origin. All pigs were halothane genotyped and glycolytic potential in LD analysed. One of each pair was cooled 30 min post mortem by showering with 10-12 °C water for 12 min. The control pig was treated normally except for the same delay before batch chilling commenced. The initial pH fall in LD and BF did not depend on the glycolytic potential in LD but at 5 to 6 and 24 hr post mortem pigs with the lowest glycolytic potential had the highest pH. Weight and lean meat content did not affect the cooling curve, i.e. the temperature fall. The results showed that it was possible to reduce the temperature in BF and LD by cooling at slaughter. The maximum difference in temperature between control and cooled carcasses 2hr post mortem was 2 and 1 °C in LD and BF respectively. The lowering of the muscle temperature early post mortem resulted in a reduced rate of the pH fall and a higher pH from 2 to 6 hr in the cooled carcasses. The rate of the pH fall in LD and BF seemed to be independent of temperature at levels above approximately 37 °C, but decreased linearly as the temperature dropped below approximately 37 °C. The cooling procedure used here did not result in a significant reduction in protein denaturation or drip loss, although there was a tendency towards lower drip loss in LD and BF in cooled carcasses.     

Effects of cryogenic chilling on beef carcass grade, shrinkage and palatability characteristics

Alternate right or left sides of 90 carcasses were randomly selected and chilled at -70°C for 5 h, held at +16°C for 4 h and held at 1°C for 15 h (rapid chill-RC). The remaining sides were chilled at -7°C for 24 h (conventional chill-CC). Physical measurements and USDA quality grade data were obtained at 24 h post mortem and palatability samples were collected at 3-5 days post mortem. RC sides had 0·9% less shrinkage (P < 0·05) at 24 h post mortem when compared to paired CC sides. Beef sides that were given the RC treatment had a darker, softer lean (P < 0·01) and received higher marbling scores (P < 0·01) at 24 h post mortem than did CC sides. Loin steaks from RC sides had longer sarcomeres, less shear resistance and higher sensory panel tenderness ratings. It may be possible to rapidly chill carcasses with less carcass shrinkage and with no detrimental effects on USDA quality grade or on beef palatability.     

Predicting variability of ageing and toughness in beef M. Longissimus lumborum et thoracis

The object of this study was to determine muscle characteristics which might predict meat toughness. Eleven Charolais cattle were slaughtered at approximately 26 months of age and the Longissimus lumborum et thoracis muscle was taken 1 hr post mortem and stored at 12 °C for 24 hr and then at 4 °C.

The average half-life for ageing in these raw muscles was 4.6 days but the toughness varied widely between the animals. Toughness varied 3-fold and the rate of ageing varied 20-fold between animals.

Correlations were done to determine which characteristics might explain this variability. Toughness was correlated positively with increase in oxidative status of muscle and the initial levels of calpastatin. Toughness was correlated negatively with the initial levels of μ- and m-calpains and cysteine and serine proteinase inhibitors, the initial pH values and the rates of their decline. The rates of ageing were highly correlated positively with the initial levels of proteinase inhibitors and the rates of decline of calpastatin and negatively with the ultimate amounts of expressible juice.

There was a wide variability in tenderness in M. Longissimus lumborum et thoracis from similar animals. Variations in metabolism and enzyme activity controlled by inhibitors and calpains appear to be largely responsible for this variability.     

The influence of low temperature, type of muscle and electrical stimulation on the course of rigor mortis, ageing and tenderness of beef muscles

The course of rigor mortis, ageing and tenderness have been evaluated for two beef muscles, M. semimembranosus (SM) and M. longissimus dorsi (LD), when entering rigor at constant temperatures in the cold-shortening region (1, 4, 7 and 10°C). The influence of electrical stimulation (ES) was also examined. Post-mortem changes were registered by shortening and isometric tension and by following the decline of pH, ATP and creatine phosphate. The effect of ageing on tenderness was recorded by measuring shear-force (2, 8 and 15 days post mortem) and the sensory properties were assessed 15 days post mortem. It was found that shortening increased with decreasing temperature, resulting in decreased tenderness. Tenderness for LD, but not for SM, was improved by ES at 1 and 4°C, whereas ES did not give rise to any decrease in the degree of shortening during rigor mortis development. This suggests that ES influences tenderization more than it prevents cold-shortening. The samples with a pre-rigor mortis temperature of 1°C could not be tenderized, when stored up to 15 days, whereas this was the case for the muscles entering rigor mortis at the other higher temperatures. The results show that under the conditions used in this study, the course of rigor mortis is more important for the ultimate tenderness than the course of ageing.     

Effects of stress and high voltage electrical stimulation on tenderness of lamb m. longissimus

This study was conducted to investigate the reported effect of pre-slaughter stress on meat tenderness independent from its effect on ultimate pH, and its interaction with electrical stimulation. From a group of 80 Coopworth lamb, 40 were stressed by subjecting the animals to a swim wash 3 h before slaughter and the use of dogs to assemble the animals to the access ramp of the abbatoir. Half of the carcasses of each group was electrically stimulated within 30 min post mortem. Temperature and pH decline of the longissimus was monitored and shear force of the cooked muscle was determined at 2 days post mortem and after 6 weeks vacuum storage at 1°C. To investigate an effect of stress independent of ultimate pH, 10 muscles with an ultimate pH below 5.8 were selected from each group for detailed analysis. This analysis consisted of determination of calpastatin activity and sarcomere length, and immunoblotting of μ-calpain and calpain substrates. The stress treatment led to an increase in the number of muscles with an ultimate pH above 5.8 (32.5 vs 15%), and muscles with an ultimate pH above 5.8 were significantly tougher than muscles with an ultimate pH below 5.8 at 2 days post mortem. Electrical stimulation improved tenderness at two days post mortem. This effect could be attributed to an effect on muscle contraction, but not on post mortem proteolysis of calpain substrates. A large variation in tenderness at 2 days post mortem was observed and this was not reduced by electrical stimulation. Six weeks of vacuum storage resulted in a 6 kgF drop in mean shear force and a uniformly tender product. Despite the fact that the stress treatment was similar to those in earlier studies, we failed to observe an effect of stress independent of ultimate pH on tenderness. The reason for this is unclear, but differences in the response to stress between breeds may be responsible. The results of the present study underscore the importance of minimizing pre-slaughter stress and adequate post mortem storage for meat quality.     

Tenderization of wether lambs meat through pre-rigor infusion of calcium ions

A study involving 36 wether lamb carcasses was conducted in order to evaluate the effects of 0.3 M CaCl(2) injection on final tenderness in muscle Longissimus thoracis et lumborum. Injection of wether lamb carcasses with CaCl(2) accelerated the post mortem tenderization process. Both control and distilled water injected animals had similar Ca(2+)-dependent proteases (μ-calpain and m-calpain) and their inhibitor (calpastatin) activities, whereas these were all significantly decreased in CaCl(2) injected animals. The effect of CaCl(2) injection on tenderness was recorded by measuring shear force values 2 and 6 days post mortem. Tenderness was significantly improved by CaCl(2) injection at both at 2 and 6 days post mortem. It was concluded that activation of μ-calpain and m-calpain was responsible for the observed post mortem proteolysis and tenderization.     

Tenderisation of pork as affected by degree of cold-induced shortening

Porcine M. longissimus dorsi from 12 carcasses were used to study the effect of ante mortem exercise treatment, time of boning and different ageing periods on tenderness and tenderisation in pork. The pigs were randomly allocated into three groups, of which the pigs (in two of them) were exposed to work on a treadmill: (1) 30 min rest after work, then slaughtered; (2) slaughtered without rest after work; and (3) controls, which were not exposed to work before slaughter. At 1 and 6 h post-stunning, samples from right side of each carcass were excised and immediately chilled in icewater until 24 h post-stunning. The left side loins were used as controls and were chilled on the carcasses at 12-14°C for 90 min and then at 2°C before boning at 24 h post-stunning. The effect of ageing, 0 or 7 days at 2°C, was studied. No parameter in this study was significantly affected by ante mortem treatment. WB shear force decreased significantly with increasing boning time and was correlated to the degree of muscle shortening in unaged excised samples. Muscle shortening for the 1 h excised cuts showed an average value of 27·4%, while an average value of only 6·1% was obtained for 6 h excised cuts. Ageing for 7 days resulted in only minor (7·3%) tenderness improvement for the 1 h excised cuts as compared to the 24·5% decrease in WB shear force obtained from the 6 h excised, less cold-shortened cuts. These differences in ageing rate, however, did not result in detectable changes in the degradation profile of myofibrillar proteins as analysed by SDS-PAGE.     

Electrical stimulation and ultra-rapid chilling of pork

Six pigs were stimulated at 5 min post mortem and six remained unstimulated. All the pigs were split hot and one side from each pig was rapidly chilled in two stages (air at -40°C and 1 m/s for 80 min followed by 0°C and 0·5 m/s for 130 min) and the other side was conventionally chilled (air at 0°C and 1 m/s for 24 h). The weight loss from rapidly chilled sides was approximately 1% less than that from conventionally chilled controls. Cooked samples of Longissimus dorsi were tougher from unstimulated rapidly chilled sides (0·23 J) than from unstimulated conventionally chilled sides (0·18 J), whilst samples from both stimulated treatments were significantly more tender (0·15 J). These differences in toughness are thought to be due to a combination of cold shortening and lack of conditioning. The average pH in the longissimus dorsi of both rapidly and conventionally chilled stimulated sides at 50 min post mortem was 5·57 and samples from these muscles were slightly paler and more watery than the unstimulated controls.     

Post-mortem kinetics of meat tenderness and the components of the calpain system in bull skeletal muscle

Eight strip loins (M. longissimus dorsi) from pasture fed Friesian bulls were aged at 15 °C for a range of times from 1 to 120 h. pH declined from 6.29 (SE 0.119) one hour post slaughter to an ultimate pH of 5.48 (SE 0.013). The activities of the components of the calpain system (μ-calpain, m-calpain and calpastatin) were determined after separation on a DEAE-sephacel column. There was a dramatic decline in μ-calpain activity post slaughter with a complete disappearance within 48 h. The rates of decline in m-calpain and calpastatin activity were slower with 30% and 50% remaining 120 h post slaughter, respectively. The rapid decline in μ-calpain activity relative to the calpastatin activity is likely to reduce the degree of tenderisation and ultimate tenderness of the meat.     

Tenderisation of lamb meat: Effect of rapid postmortem temperature drop on muscle conditioning and aging

Lamb Longissimus dorsi muscles were excised from carcasses of 9-12 kg and 16-20 kg weight and brought to internal temperatures of either 0°C, 4°C, 10°C, 15°C, 20°C or 36°C within 3-4h post mortem. After rigor completion they were allowed to age at 4°C for 7 days. Sensory panel scores obtained on the first day of post-rigor aging showed that meat from heavier animals was tougher than that of lighter ones at any temperature. In all cases aging for 7 days had a marked tenderising effect which was similar at all studied temperatures, and greater in heavier animals. Tenderness was also dependent on the temperature at conditioning, as shortening caused by either high or low temperature resulted in meat toughening. The sole and surprising exception to this fact was, however, the high sensory scores obtained in 0°C experiments, even though shortening occurred as expected. In fact, a more intense proteolysis was evident in this case, which was already apparent on the first day of aging. The effect of a rapid drop of muscle temperature to 0°C on the fast and intense proteolysis, capable of even overcoming toughness due to cold shortening, was explained by the higher pH and the dramatic increase of the sarcoplasmic Ca(++) level induced early post mortem by chilling and which might result in the activation of calpain.     

Comparison of Dehiding versus Scalding and Singeing: Effect on Temperature, pH and Meat Quality in Pigs

The aim of this work was to investigate the temperature and pH changes taking place at the slaughter line and during the chilling process, and the subsequent effect on meat and eating quality in pig carcasses that had either been scalded and singed or dehided. Both processes were followed by fast chilling. 219 halothane-gene-free Duroc Sire and Landrace-Yorkshire pigs were delivered from three farms and slaughtered over 2 weeks at either a dehiding or a scalding singeing slaughterhouse. Temperature and pH were measured at intervals from exsanguination until 6 hours post mortem. Ultimate pH, internal reflection, drip loss and colour (Minolta) were measured the day after slaughter. Colour (JPCS scale) was evaluated after freezing and thawing, and eating quality was estimated on unaged and aged (4 days at 4°C) m. longissimus dorsi (LD), using a trained taste panel. At the slaughter line the dehided carcasses had an almost constant and lower temperature compared to the scalded and singed carcasses. During chilling there is a shift in temperature curves between treatments, where the scalded carcasses had the lowest muscle temperature in LD and m. biceps femoris (BF) from 2hr post mortem and throughout the measuring period. In spite of the shift in temperature, the dehided carcasses had the highest pH in LD and BF from exsanguination and throughout the measuring period. The rate of pH fall in LD and BF was slowest in the dehided carcasses from exsanguination until 3 and 2hr respectively, post mortem. Dehided carcasses showed a 40% lower drip loss, a darker meat colour and a lower internal reflection in LD and BF compared to scalded and singed carcasses, thus indicating less protein denaturation in dehided carcasses. Scalding and singeing, however, lead to increased tenderness compared to dehiding, and this difference persisted even after ageing for 4 days at 4°C. The differences in toughness may be caused by increased proteolysis by released lysosomal cathepsins.     

Rapid tenderisation of lamb M. longissimus with very fast chilling depends on rapidly achieving sub-zero temperatures

A study was undertaken to determine whether variations within the defined temperature-by-time profile for very fast chilling (VFC), might explain variations in tenderness found with VFC. Loins from 32 lambs were subjected to one of five cooling regimes; defined by the average temperature between the meat surface and centre reached at a specific time post mortem. These were: -0.3 °C at 22 h (Control), 2.6 °C at 1.5 h (Fast(supra-zero)), 0.7 °C at 5.5 h (Slow(supra-zero)), -1.6 °C at 1.5 h (Fast(sub-zero)) and -2.3 °C at 5.5 h (Slow(sub-zero)), respectively. Shear force values considered very tender by consumers (less than 50 N, MIRINZ tenderometer) were found 2 days post mortem in Fast(sub-zero) loins only. Both time and temperature at the end of the cooling period contributed to variations in shear force. To achieve low shear force, the loins needed to be cooled to less than 0 °C at 1.5 h post mortem.     

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.     

The influence of elevated temperature conditioning on bison (Bison bison bison) meat quality

Elevated temperature conditioning (ETC: 10°C until 10 h post mortem) was effectively employed as a means of cooling bison carcasses in order to avoid the cold-induced meat quality defects that are a risk with conventional bison carcass chilling (0-2°C for 24 h). The ETC treatment maintained internal M. Longissimus lumborum and M. Semimembranosus temperature above 10°C within the first 10 h post mortem. The time/temperature combination did not result in significant evaporative loss, although loss of weight during carcass cooling can represent a practical economic loss. ETC accelerated post-mortem glycolysis and pH decline, and resulted in samples of lighter, more intense red colour than those conventionally chilled. Significant improvement in both initial tenderness and tenderization during ageing was realized with the use of ETC.     

High pre rigor temperature limits the ageing potential of beef that is not completely overcome by electrical stimulation and muscle restraining

Two simultaneous trials were conducted to determine the effects of electrical input [electrical stunning and stimulation (ES)], wrapping, pre rigor temperature (15 °C and 38 °C) and different post rigor chilling rates on beef quality using M. longissimus lumborum (n=100). The high pre rigor temperature induced a faster pH decline than ES. The loins at 38 °C had significantly greater protein denaturation, more purge and drip loss, higher shear force values and less desmin degradation compared with the loins at 15 °C. No difference in sarcomere length was determined between the pre rigor temperatures regardless of ES and wrapping. Different post rigor chilling rates did not play a substantial role in water-holding capacity, proteolysis, or shear force values during ageing. These results suggest that high pre rigor temperature induces temperature-related toughness of muscle due to protein denaturation with subsequent limitation of proteolysis by μ-calpain, regardless of ES and wrapping treatments.     

Effect of chilling applied to suckling lamb carcasses on hygienic, physicochemical and sensory meat quality

The effect of post mortem temperature treatment on suckling lamb carcass and meat quality was study. Conventional (2°C for 24h), ultra-rapid (-20°C for 3.5h, 2°C until 24h) and slow chillings (12°C for 7h, 2°C until 24h) were compared. Total viable counts (TVC), weight losses, and pH and temperature falls were recorded on carcasses. Meat colour, water holding capacity (WHC), Warner-Bratzler shear force, sarcomere length and sensory analysis were evaluated in M. longissimus. Ultra-rapid treatment reduced TVC and weight losses. The pH decline was faster in slow chilled carcasses than in faster chilled carcasses. No significant differences were found for colour and WHC. Slow treatment carcasses showed significantly lower shear force and higher sarcomere length. In the sensory analysis, tasters also rated the early post mortem slow-treated meat as more tender, less fibrous and chewy. Therefore, delay chilling in suckling lamb carcasses made it possible to obtain meat with better organoleptic characteristics, without affecting weight loss or hygienic quality.     

Breakdown of connectin during cooking of meat

Light-scattering studies on extracts of meat have confirmed the heat-induced breakdown of connectin previously observed by SDS gel electrophoresis. Because of the high subunit MW (～10(6)) of connectin, the weight-average molecular weight of whole muscle undergoes a relatively large decrease when connectin is broken down during heating of meat. In cold-shortened muscle, breakdown of connectin by proteolysis was as rapid as in control samples, suggesting that connectin exists in an exposed environment rather than as a core to thick filaments. The breakdown of connectin during heating at 60 or 80°C for 40 min was more extensive than during ageing for 3 weeks at 2°C. Hence, the partial proteolysis of connectin during storage at 2°C is unlikely to be responsible for tenderisation induced by ageing.     

Functional Properties of Myofibrillar Proteins from Cold-Shortened and Thaw-Rigor Bovine Muscles

Prerigor bovine sternomandibularis muscles were stored at 15, 0 and ?29°C to examine cold-shortening and thaw-rigor effects on myofibrillar protein extractability and gelation properties of myofibrils and salt-soluble protein (SSP). Frozen muscle that underwent severe contraction at thawing showed greater protein extractability (35%) than muscles stored at 0 and 15°C (27% extractability). Of the three tempered muscles, thaw-rigor muscle produced the strongest myofibril gel and cold-shortened muscle formed the most elastic SSP gel as determined by dynamic shear and penetration measurements. However, thermally induced changes in gel viscoelastic moduli for all protein samples followed similar patterns. Results indicated that physicochemical changes accompanying muscle contraction affected protein network formation during gelation.