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).     

Post-mortem Changes in Breast Muscles of Mule Duck

Post-mortem changes in myofibril fragmentation index and degradation of myofibrillar proteins of duck breast muscles at 5°C was investigated. Muscle samples were collected immediately after killing and from the stored carcasses at 5°C for 1, 3, 7, and 14 days post mortem . The results showed that the MFI of the breast muscles increased with time post mortem . SDS-PAGE of myofibrils indicated that the disappearance of troponin-T accompanied concurrently with the appearance of 32–30 kDa components. After 7 days of storage, α-actinin was degraded, and a 98 kDa component appeared. Titin 1 and nebulin also disappeared after post-mortem storage for 3 days.     

Post-mortem changes in myofibrillar proteins of breast and leg muscles from broilers,spent hens and Taiwanese Country Chickens

Post mortem ageing at 4°C was studied in the breast and leg muscles from broilers, White Leghorn spent hens and Taiwanese Country Chickens (TCC). Purified myofibrils were prepared from muscles after 0, 1, 3, 5, and 7 days of post mortem storage at 4°C. SDS-PAGE was used to examine the changes in myofibrillar proteins of the muscles. Results showed that 30 kDa components appealed earlier in the breast muscles than in the leg muscles and in the order: broiler, TCC, and spent hen. The intensity of 30 kDa band increased with post mortem time. In the breast muscles, a decrease in the intensity of the α-actinin band could be observed at 1 day post mortem in broilers and TCC and at 3 days post mortem in spent hens. This decrease, however, could not be found in the leg samples until 5 or 7 day post mortem. Titin 1 band disappeared within 3 day post mortem in the breast samples but within 5 days in the leg samples. Similar results could be observed in the degradation of nebulin although traces of nebulin remained in the teg muscles of TCC after 7 days post mortem.     

Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef

Changes in proteoglycans (PGs) during post-mortem ageing of bovine m. semitendinosus were studied. Electron microscopic observations made it clear that there were two types of PGs in bovine m. semitendinosus immediately post-mortem: PGs were arranged regularly in the basement membrane and PGs associated with collagen fibrils in the perimysium. After 28 days ageing at 4 °C, no PG was observed in the basement membrane, and the greater part of PGs in the perimysium had disappeared. The total amount of PGs decreased with time post-mortem. SDS-polyacrylamide gel electrophoresis indicated that PGs with a high molecular weight disappeared within 7 days post mortem. These results suggest that PGs are degraded during post-mortem ageing of beef. The degradation of PGs seems likely to be the main factor in the weakening of intramuscular connective tissues, i.e., separation of collagen fibrils and fibres from the endomysium and the perimysium, which results in the partial tenderization of beef during post-mortem ageing.     

Separation and amino acid composition of three troponin components from bovine muscle

Three troponin components were isolated from bovine skeletal muscle, and their molecular weights and amino acid composition were studied. Crude troponin prepared from bovine muscle was purified by DEAE-Toyopearl chromatography. The purified troponin was dissociated in the order of tropopins C, I and T by CM-Toyopearl chromatography in the presence of 6 M urea. The molecular weights of troponins C, I and T were 19,500, 23,300 and 40,400, respectively, as determined with SDS-PAGE. The separation of troponin into three components was also achieved using reverse-phase HPLC; however, the elution order of troponins T and C was contrary to that of the cation-exchange chromatography described above. In this study, the amino acid composition of the three troponin components from bovine skeletal muscle was first determined. The amino acid composition of the three troponin components among bovine, rabbit and chicken skeletal muscles showed stronger similarity than that between bovine skeletal and cardiac muscle with a different muscle type. We considered that this method of troponin preparation from bovine muscle must be a very useful technique for investigating the changes in troponin components, especially troponin T, during ageing of beef.     

Survey of conditioning indicators for pork loins: changes in myofibrils, proteins and peptides during postmortem conditioning of vacuum-packed pork loins for 30 days

This study was performed to examine the changes in myofibrils, proteins and peptides during postmortem conditioning of vacuum-packed pork loins at 4 °C for 30 days. The fragmentation of myofibrils has been observed during postmortem aging for 20 days and its ratio increased until 20 days. The 32 kDa component derived from troponin T increased during storage for 20 days, while a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) among sarcoplasmic proteins was significantly degraded during storage for 15 days. Some oligopeptides increased during such storage, two peptides (peptides P1 and P2) significantly increasing during storage for 20 days. Their sequences were APPPPAEVHVHEEVH (P1) and VPTPNVSVVDLT (P2). Homology analysis showed that peptides P1 and P2 were derived from troponin T and GAPDH, respectively. It was concluded that the fragmentation of myofibrils, the increases in the 32 kDa component and peptides P1 and P2, and the decrease in GAPDH were useful as conditioning indicators of progressive degree during the storage of pork loins stored at 4 °C.     

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.     

The postmortem μ‐calpain activity,protein degradation and tenderness of sheep meat from Duolang and Hu breeds

This study aimed to investigate the differences in meat tenderisation between two Chinese native sheep breeds, Duolang and Hu. The tenderness of longissimus thoracis (LT) muscle, μ‐calpain autolysis and proteolysis of myofibrillar protein was measured at 1‐ and 7‐days postmortem storage at 4 °C. At 7‐days postmortem ageing, meat from Duolang sheep was more tender compared to that from Hu sheep. The Warner–Bratzler shear force of Duolang and Hu sheep was reduced by 55.20% and 41.51% at 7 days compared with 1 day, respectively. In Duolang LT, a higher proportion of 80‐kDa μ‐calpain was autolyzed than in the Hu samples at 1‐day postmortem ageing, but they did not differ significantly at 7 days. More titin, desmin and troponin‐T were degraded in Duolang sheep compared to Hu sheep. Additionally, pH values at the different ageing time were not significantly different, indicating that pH may be not a determinant factor contributing to the tenderness difference between the two breeds. Our data suggest the earlier activation of μ‐calpain and a larger extent of proteolysis of key structural proteins may contribute to the higher rate of tenderisation of Duolang compared to Hu sheep during postmortem ageing.     

Effects of high pressure treatments on intramuscular connective tissue

The changes in the morphological structure and proteoglycan (PGs) extractability of the intramuscular connective tissues from bovine skeletal muscle exposed to high pressure (100～400 MPa) were investigated and compared to those in muscle during ageing. SDS-PAGE profiles of the PGs were also studied. During ageing, structural changes in the endomysium and perimysium occured, and disruption of the honeycomb-like structure of endomysium was observed. In the pressurized muscle, deformation of the honeycomb-like structure of endomysium was accelerated with increase of pressure applied to the muscle, and disruption of endomysium was observed in several places in the muscle pressurized at 400 MPa. Changes in the extractability of PGs were not observed in the pressurized muscle, whereas the extractability of PGs decreased with ageing. Changes in the density of the main component in the PGs with molecular mass of more than 3000 kDa on the SDS-PAGE were not observed in the PGs from the pressurized muscle, whereas the density of this band gradually decreased during the ageing. The decrease in the amounts of PGs extracted from the aged muscle seems to be due to degradation of the PGs into small dialysable molecules. From the results, it seems that high pressure treatment has some different effects on intramuscular connective tissue to ageing. ?     

Effect of ionizing radiation on unesterified tocopherols in fresh chicken breast muscle

The water condition of cured and uncured pork, having different ageing times, was studied in the process of heating to 80°C, using nuclear-magnetic resonance methods. In the transverse magnetization decay functions five components could be distinguished, differing from each other by their relative weights and relaxation times. The greatest relative weight (60-70%) was characteristic of the component having relaxation time 35-45 ms ('d'), which could be related to immobilized water. The change of the component's 'd' relative weight while heating was significantly dependent on the ageing time of meat and the factor of curing or not curing before heating. It was shown that the greatest amount of immobilized water and its strongest bonding were observed in uncured pork during the first 6-7 h and after 48 h post mortem. Cured meat possesses the highest amount of immobilized water at room temperature after 48-96 h post mortem, however, its bonding at heating is higher in the case of meat which is cured during the first hours and after 96 h post mortem.     

Structural weakening of intramuscular connective tissue during post mortem ageing of chicken Semitendinosus muscle

The structural changes in intramuscular connective tissues endomysium and perimysium during post mortem ageing in chicken semitendinosus muscle were investigated using an improved technique of scanning electron microscopy. In post mortem chicken aged for 12 h at 4°C, the endomysium resolved into individual collagen fibrils and the perimysial sheets separated into collagen fibres. These results provide direct evidence for the structural weakening of endomysium and perimysium during post mortem ageing. The structural changes in the intramuscular connective tissue were minimal until 6 h post mortem, but clearly observable after 12 h post mortem. It was concluded that this disintegration of the intramuscular connective tissue is the chief mechanism in tenderisation during extended post mortem ageing of chicken.     

Post mortem changes in porcine longissimus muscle incubated with 0.1 M calcium lactate

The effects of calcium lactate incubation at 5 °C on post mortem changes in porcine longissimus muscle were studied. Myofibrils were purified from control (CON) and calcium lactate‐incubated (CL) muscles. Samples were taken at 0, 1, 3, 7 and 14 days post mortem. SDS‐PAGE results showed that the disappearance of titin, nebulin, tropomyosin and troponin‐T and the appearance of a ～30 kDa component were more pronounced in CL samples than in CON samples. Western blots labelled with a monoclonal antibody to desmin also demonstrated that desmin degraded more quickly in CL samples. Our data suggested that calcium lactate incubation might accelerate the post mortem changes in porcine longissimus muscle via activation of calpains. © 2001 Society of Chemical Industry     

Changes in meat quality characteristics of bovine meat during the first 6 days post mortem

The development of some quality parameters in bovine meat during the first 6 days of post mortem ageing was studied in two commercial groups (heifer and bull). In the first day bull meat is harder and springier. Meat pH falls during the first 24 h post mortem in both groups, reaching values around 5.5, and it did not change during the next 5 days. Water-holding capacity (expressed as percentage of expelled water) increased in heifer meat. Instrumental texture measures (texture profile analysis, TPA) showed a decrease in hardness, springiness and chewiness in bull raw meat. Sensory analysis showed that assessors perceived a decrease in hardness and in springiness in bull meat and a decrease in juiciness and in chewiness (number of chewings before swallowing) in heifer meat. Ageing showed no effect on assessors’ pleasantness either in heifers’ or in bulls’ meat. During the first 3 days, heifer meat was juicier. Heifers’ meat produced a greater pleasantness on the sixth day. Both meats were not very different for most quality parameters studied in this work.     

Effect of lactic or acetic acid on degradation of myofibrillar proteins in post‐mortem goose (Anser anser) breast muscle

The effects of lactic acid (LA) and acetic acid (AA) on changes in myofibrillar proteins of post‐mortem goose breast muscle marinated for 24 h at 5 °C were studied. Purified myofibrils were prepared from 0.1 M LA or AA samples and controls (non‐marinated samples) after 0, 1, 3, 7 or 14 days of storage at 5 °C. The changes in myofibrillar proteins of goose muscle were examined by SDS‐PAGE. Goose breast muscle marinated in LA and AA exhibited degradation of myosin heavy chains. The appearance of ∼95 and ∼27 kDa components and the disappearance of titin and nebulin were also more rapid than for control muscle. These results suggest that acid marination enhanced the post‐mortem proteolysis of goose breast muscle. © 2000 Society of Chemical Industry     

Improving meat quality of organic pork through post mortem handling of carcasses: an innovative approach

This study was conducted to examine the best combination of post mortem chilling, suspension and ageing in order to optimize tenderness of organic pork at slaughter, which may be tougher than conventionally produced pork, because of lower daily gain. Combinations of stepwise chilling with a holding period of 6h at 10°C or traditional blast tunnel chilling, suspension in the pelvic bone or Achilles Tendon and ageing 2 or 4 days post mortem were tested. Stepwise chilling and ageing improved tenderness of the loin, and the effects were additive, whereas pelvic suspension was less effective in texture improvements, and non-additive to stepwise chilling. Stepwise chilling improved tenderness to a similar degree as can be obtained within 2-4 days of extended ageing, however, the minimum temperature during the holding period seems to be crucial in order to obtain a positive effect of stepwise chilling, and it should be above 7.5°C.     

Effect of fiber type on postmortem proteolysis in longissimus muscle of Landrace and Korean native black pigs

The current study was conducted to characterize objective meat quality, fiber type and their relations to postmortem proteolysis in longissimus muscle of Landrace and Korean native black (KNP) pigs. Longissimus muscles from each 10 market-weighted male pigs were removed after conventional slaughtering and chilling procedures, and aged for 1 or 7 days at 4 °C to determine WB-shear force, objective meat color, proportion of myosin heavy chain I (MyHC I), intramuscular fat content and rate of proteolysis by a proteomics approach. KNP had a significantly (p < 0.05) higher content of MyHC I, and that concurred with greatly (p < 0.05) higher intramuscular fat content and Hunter a^* value, and significantly (p < 0.05) lower drip loss than those seen in Landrace. One-dimension SDS-PAGE indicated that GAPDH, troponin I and creatine kinase were prominent proteolytic products during chiller ageing. By applying a gel-based proteome analysis, 26 proteins were identified, which showed different degradation properties during ageing between the breeds. Biopsied sample revealed that myosin regulatory light chain 2, myosin light chain isotype v/sb, fatty acid-binding protein and albumin were expressed at a greatly higher level for KNP, but their relation to fiber type (or genetic background) is unclear. It was particular noticeable that different actin isoforms showed various degradation behavior during ageing time.     

Identification of biomarkers of meat tenderisation and its use for early classification of Asturian beef into fast and late tenderising meat

BACKGROUND: The objective of this work was to study the post‐mortem evolution of potential biomarkers (µ‐calpain activity and proteolytic profile) of meat tenderisation in bovine longissimus dorsi (LD) muscle from several biotypes coming from two beef breeds (‘Asturiana de los Valles’ and ‘Asturiana de la Montaña’) and showing different levels of muscular hypertrophy (mh/mh, mh/+, + /+). RESULTS: LD samples were taken at 2, 12, 24 and 48 h and 3, 7, 14 and 21 days post‐mortem. The presence of muscular hypertrophy produced a faster rate of pH decline, faster exhaustion of µ‐calpain activity and earlier occurrence of proteolytic changes. Changes in the electrophoretic pattern of some peptides from sarcoplasmic (glyceraldehyde‐3‐phosphate dehydrogenase) and myofibrillar (troponin T and troponin I) muscle extracts within the first 24 h significantly correlated with meat toughness and allowed accurate discrimination of meat products into two groups: (1) fast tenderising meat, coming from mh‐biotypes, and (2) late tenderising meat, from normal (+/+) biotypes. CONCLUSION: Early monitoring (within 24 h after slaughter) of selected biomarkers in LD muscle allowed accurate prediction of ultimate meat toughness and could be used in the meat industry as a tool for early classification of beef into fast and late tenderising meat. Copyright © 2012 Society of Chemical Industry     

The significance of pre-slaughter stress and diet on colour and colour stability of pork

The influence of pre-slaughter stress and a diet known to affect post mortem muscle metabolism or a standard diet (control pigs) on colour and colour stability of m. longissimus dorsi, m. biceps femoris and m. semimembranosus from 112 female pigs, free of the Halothane gene, was investigated. Pre-slaughter stress increased the early post mortem temperature in the three muscles, as well as the pH decline in control pigs, but not in pigs fed the experimental diet. Colour was measured on sliced samples after 0, 2 and 5 days retail display (1, 3 and 6 days post mortem, respectively) from the three muscles aged 1 day before cutting as well as on sliced m. longissimus dorsi samples aged 8 days before cutting (8, 10 and 13 days post mortem, respectively). Early post mortem pH was not a main determinant of the colour and colour stability, while the degree of pre-slaughter stress and especially its influence on temperature early post mortem was crucial in relation to colour development and colour stability. The discoloration rate was enhanced in m. longissimus dorsi aged for 8 days prior to retail display compared with samples aged for 1 day. However, the extent of the discoloration after 5 days of retail display was not inferior in muscle samples aged for 8 days due to a higher degree of blooming. Finally, present data indicate that 3-4 days ageing of pork prior to retail display results in the optimal colour stability.     

Electrical stimulation - when more is less

This study was conducted to determine whether electrical stimulation per se can be omitted when other electrical inputs to beef carcasses (stunning and immobilisation) are used. In addition, we investigated which sample preparation method at 1 day post mortem (p.m.), cooked fresh, frozen, or after thawing, had the best predictive value for shear force after ageing of the muscle. Beef carcasses were electrically immobilized (75 V, 15 Hz) before and during exsanguination for 20 or 80 s and meat quality characteristics of the longissimus were determined at 1 and 7 days post mortem. Muscles from carcasses receiving the higher electrical input were similar in tenderness at 1 day p.m., but tougher at 7 days p.m. This result could be explained by the effect of muscle shortening and post mortem proteolysis on tenderness. These results indicate that even low electrical input during immobilization can adequately stimulate carcasses and avoid cold shortening. Freezing samples resulted in a considerable improvement in tenderness and cooking samples from the frozen state had the highest predictive value for tenderness after ageing. In a second experiment it was determined that freezing and thawing did not result in appreciable differences in cooking loss or proteolysis. The tenderising effect of freezing may be explained by tissue damage due to ice formation.     

Effect of electrical stimulation, high temperature conditions and ageing on muscle myofibrillar proteins

Electrical stimulation, when accompanied by high temperature incubation, was found to lower the extractability of myofibrillar proteins and increase their degradation, particularly that of the myosin component. Troponin T and myosin light chain 2 were similarly affected. Troponin T and troponins I and C were degraded earlier during ageing in electrically stimulated muscles incubated at high temperature than in conventionally chilled muscles.