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.     

Inhibition of nitric oxide release pre-slaughter increases post-mortem glycolysis and improves tenderness in ovine muscles

The aim of this experiment was to determine the effect of inhibiting the release of nitric oxide (NO) pre-slaughter in lambs on post-slaughter muscle metabolism and meat quality. Exercise was used as a positive control as NO is known to be released in skeletal muscle during exercise. Forty Border Leicester × Merino lambs were assigned to the treatments L-NAME (NO synthase inhibitor) infusion (0 mg/kg vs. 30 mg/kg, 135 min pre-slaughter) and exercise (none vs. 15 min immediately pre-slaughter). The inhibition of NO release using L-NAME reduced Warner–Bratzler shear force (WBSF) in the longissimus thoracis et lumborum (LTL) after 3 days of ageing, while the Semimembranosous (SM) was unaffected. Inhibition of NO release with L-NAME resulted in altered glucose metabolism as indicated by reduced plasma glucose pre-slaughter particularly in exercised lambs, reduced LTL and SM glycogen of non-exercised lambs post-slaughter and increased SM lactate in exercised lambs post-slaughter. In conclusion, inhibition of NO Synthase with L-NAME pre-slaughter increases post-mortem glycolysis and improves tenderness in the loin muscle.     

Variation in the response to manipulation of post-mortem glycolysis in beef muscles by low-voltage electrical stimulation and conditioning temperature

The aim of this study was to investigate how manipulation of glycolytic rate by post-mortem processing conditions influences quality of aged beef of two bovine muscles of different physiological character, longissimus dorsi (LD) and adductor (AD). Post-mortem glycolysis was manipulated by low-voltage electrical stimulation (LV-ES) of half carcasses and by chilling rate of the muscles. Multivariate statistical analysis was used to visualise the data, while ANOVA was used to identify significant effects and interactions. As expected there was a significant effect of LV-ES on the pH decline in the first hours post-mortem in both muscles. Moreover, significant effects of LV-ES on WB shear force measured 2 and 8 days after slaughter were observed for LD at both chilling temperatures, while for AD no effect on WB shear force was observed. Furthermore, the results revealed a large individual variation in the response of LV-ES on both pH decline and WB shear force, and this variation did not always correlate for the two responses. Some animals showed no response of LV-ES on pH decline, but still had an improved WB shear force, and vice versa. The results from this study indicate that there probably are other mechanisms than accelerated pH decline and prevention of cold-shortening, by which LV-ES can affect meat tenderness.     

Electrical stimulation of post-mortem glycolysis in the Semitendinosus muscle of sheep

Changes in pH and phosphorylase a content in control and electrically stimulated Semitendinosus muscles of sheep were studied. The results provided evidence of acceleration of glycolysis during slaughter and sampling. In muscle electrically stimulated soon after slaughter there was a transient increase in phosphorylase a during stimulation. The magnitude of this increase and of the fall in pH during stimulation both depended on the pH of the muscle at the time of stimulation and reduced to zero when this pH was about 6·3. There was little or no phosphorylase a in samples of muscle taken about 35 min post mortem or, at any time, in muscle electrically stimulated about 35 min post mortem. Possible reasons for the transient nature of the increase in phosphorylase a content during stimulation are discussed.     

Bovine Longissimus dorsi muscle glycogen and color response as affected by dietary regimen and post-mortem electrical stimulation in young bulls

Eighty bulls were assigned to one of two groups and fed a dietary regimen to defer or accelerate growth until slaughter. Bulls fed at an accelerated rate of growth (or high energy regimen) had higher post-mortem pH, and lower muscle glycogen stores, with darker lean color, and improved shear and palatability traits compared to deferred fed animals. Bulls fed at an accelerated rate of growth also had a higher degree of marbling and USDA quality grade. Electrical stimulation did not affect (P > 0·05) ultimate pH, but reduced (P < 0·05) glycogen values at 0 and 2 h post mortem. Electrical stimulation also improved muscle color, lowered cooking losses and improved the palatability of cooked longissimus dorsi steaks.     

The phospholipids of pork muscle, and their relation to the post-mortem rate of glycolysis

The phospholipids of pork longissimus dorsi muscle were investigated. The proportions of the major phospholipid components appeared to be constant in different regions of the same muscle, although the total lipid-P varied widely. Homogenates of the muscle were fractionated by differential centrifuging - ‘myofibrillar’, ‘mitochondrial’, ‘reticular’, and ‘final supernatant’ muscle fractions being obtained. Sarcoplasmic reticulum appeared to be present very largely with the fibrils in the ‘myofibrillar’ fraction, which contained 40–78% of the total lipid-P. The percentage composition of the phospholipids was similar in the first three sub-cellular fractions, while the fourth contained less of the ‘kephalin’ component. During fractionation of the muscle, substantial losses of phospholipids occurred; the possible origin and cause of these losses are discussed. The sum of lipid-P in the first and third subcellular fractions, containing sarcoplasmic reticulum, showed a highly significant negative correlation with pH₁, (muscle pH 45 minutes after slaughter) which represents an approximate index of the rate of post-mortem glycolysis in the muscle. The significance of this correlation is discussed.     

The relation between glycogen, lactate content and muscle fiber type composition, and their influence on postmortem glycolytic rate and pork quality

This study examined the relation between glycogen, lactate content and muscle fiber type composition, and evaluated their influence on postmortem glycolytic rate and meat quality. Muscle samples were classified based on their glycogen and lactate content at 45 min postmortem. Muscles with low glycogen and high lactate levels showed low muscle pH_45 min and high R-values. However, muscles with low glycogen and lactate levels showed normal rates of postmortem glycolysis and normal meat quality. On the other hand, muscles with high glycogen and lactate content showed rapid postmortem glycolysis, paler surface color, higher drip loss, and higher extents of protein denaturation than muscles with high glycogen and low lactate content. These results may be partially explained by muscle fiber type composition. Muscles with low glycogen and lactate content at early postmortem are composed of significantly higher fiber type I and lower fiber type IIB as compared to muscles with high glycogen and lactate content.     

Differential effects of electrical stunning on the early post-mortem glycolysis in sheep

Electrical stunning methods have been examined to determine the effect on post-mortem glycolysis in various muscles of sheep. Although there was no effect on the ultimate pH achieved in any of the muscles, there were marked differences in the pH drop which occurred during stunning.

When curare was used to block neuromuscular transmission much of the effect of stunning on glycolysis was removed. In the absence of neuromuscular blockage, head-only stunning produced the least drop in initial pH and the head-to-back and head-to-foreleg methods the greatest drop, especially in the anterior portions of the LD. Gash cutting without prior stunning produced marked drops in pH in most muscles.

Prolonged (120s) head-to-back stunning gave more substantial drops in pH in the LD, BF, SM and ST muscles than did a shorter stun (1·4s). The prolonged stunning did not produce a change in rate of pH fall.

Electrical stunning cannot be considered as a substitute for effective electrical stimulation as a means of hastening glycolysis to avoid cold shortening.     

Effects of sugars on post-mortem glycolysis in bovine muscle mince

Sternomandibularis muscles from steers were minced, within 75 min of slaughter, with sugars or amylases. The minces were held anoxically at 25°C for at least 7 h. At times samples were removed and chemically analysed. Glucose (1·5%) retarded the post-mortem pH fall, with kinetics similar to those of NaCl (2·0%). Typically, 1·5% glucose maintained the pH at 6·1, whereas control mince attained 5·5. Lesser amounts of glucose had a reduced effect. Other sugars were inactive. The metabolic effects of 1·5% added-glucose were compared with those of an iso-osmotic control, 1·5% added-fructose. For the glucose mince there was no change in glucose concentration over 7 h suggesting that the endogenous hexokinase was inactive. Further, more glycogen remained in the glucose mince that in the control, indicating that glucose inhibited glycolysis. This was confirmed by electron microscopy and was reflected in pH and lactate levels. Hexose monophosphates were strongly affected by glucose. In the control, their concentrations each fell in the first 2 h then increased steadily, as expected. In the presence of glucose, their concentrations were initially lower and decreased steadily with time. Concentrations of adenosine phosphates were little affected by glucose, but downstream products of purine metabolism showed marked differences, probably due to enhanced nucleotidase activity above pH 6. Glucose plus yeast hexokinase produced a mince with a rigor pH close to normal. The data indicated that phosphorylase activity was inhibited by glucose, and suggested that glucose was not phosphorylated because of muscle hexokinase inhibitors such as glucose-1,6-diphosphate.     

Effect of muscle type on the rate of post-mortem proteolysis in pigs

Post-mortem proteolysis was examined in muscle homogenates from porcine m. longissimus dorsi (LD), m. semitendinosus (ST), m. semimembranosus (SM), m. vastus intermedius (VI), and m. soleus (S). During post-mortem storage, desmin and troponin-T degraded faster in LD and SM than in ST, VI and S. ST exhibited the same rate of degradation as VI and S. These differences could not be explained solely by differences in fibre type distribution, indicating that other muscle-specific traits independent of fibre type determine myofibrillar degradation post-mortem. Thus, the rate of post-mortem proteolysis seems to depend more on muscle-to-muscle variations than on fibre type composition.     

The significance of the activity of glycogen debranching enzyme in glycolysis in porcine and bovine muscles

The purpose of the study was to examine the activity of glycogen debranching enzyme, GDE, in porcine and bovine muscles differing in rate of contraction and oxidative capacity. The activity of GDE, the activity of phosphorylase, total glucose content, lactate content and pH were measured from meat samples taken 35min post-mortem and ultimate pH 24 or 48h post-mortem. Both GDE and phosphorylase are needed for the complete degradation of glycogen. In porcine muscles the activities of these glycogen degrading enzymes were higher than in bovine muscles. The activities were increasing with the increasing fast twitch and glycolytic character of a muscle of a given species. However, the increase in the activity of phosphorylase was greater than the increase in the activity of GDE. It was concluded that the GDE may restrict the rate of glycolysis in fast twitch muscles.     

Genetic and environmental effects on the muscle structure response post-mortem

This paper reviewed the mechanisms by which glycolytic rate and pre-rigor stretching of muscle impact on meat quality. If muscle is free to shorten during the rigor process extremes in glycolytic rate can impact negatively on meat quality by inducing either cold or rigor shortening. Factors that contribute to variation in glycolytic rate include the glycogen concentration at slaughter and fibre type of the muscle. Glycolysis is highly sensitive to temperature, which is an important factor in heavy grain fed carcasses. An alternative solution to controlling glycolysis is to stretch the muscle pre-rigor so that it cannot shorten, thus providing an insurance against extremes in processing conditions. Results are presented which show a large reduction in variance (both additive and phenotypic) in tenderness caused by pre-rigor stretching. Whilst this did not impact on the heritability of shear force, it did reduce genotype differences. The implications of these results on the magnitude of genotype effects on tenderness is discussed.     

High post-mortem temperature combined with rapid glycolysis induces phosphorylase denaturation and produces pale and exudative characteristics in broiler Pectoralis major muscles

This study investigated the effect of early post-mortem temperature on broiler protein characteristics and meat quality. Muscles were kept at different temperatures (0, 20 and 40 °C) until 4h post-mortem and then stored at 4 °C. Rapid degradation of ATP and glycogen, thus inducing a high rate of lactate formation and pH drop, were found in the 40 °C group during incubation. When extracting proteins, a lower protein content of the sarcoplasmic fraction and a higher protein content of the myofibrillar fraction were found in the 40 °C group at 24h post-mortem; SDS-PAGE and western-blotting results revealed that phosphorylase was associated with the myofibrillar fraction. Furthermore, the 40 °C group had paler surfaces, higher drip loss and lower processing properties. These data suggest that elevated temperature during early post-mortem period, resulting in rapid glycolysis, induced phosphorylase denaturation and association with myofibrillar proteins thus generating pale and exudative characteristics.     

Effect of electrical stimulation and temperature on biochemical changes in beef muscle

The effect of low voltage electrical stimulation and storage temperatures of 2, 15 and 25°C on biochemical changes in M. sternocephalicus from beef carcasses was studied. It was shown that electrical stimulation accelerated the glycolytic processes resulting in an immediate increase in muscle lactate and about 30% reduction in ATP. Combination of stimulation and 15 or 25°C temperature further increased the changes with ATP, reaching 2 μM/g muscle in about 2 h post mortem. At 2°C, the biochemical changes were slow. By ensuring that the muscle temperatures in low voltage stimulated carcasses do not fall below 15°C for 2 to 3 h, this should provide favorable conditions for depletion of ATP and glycogen and the prevention of cold shortening at chilling.     

Effect of heat treatment on protein oxidation in pig meat

We investigated the oxidative mechanisms and identified the target protein induced by heat treatment. The study was carried out on M. longissimus thoracis from Galia and Redone pigs. Post mortem metabolic parameters and drip loss were determined. Heat treatment was performed at 100 °C for 10 and 30 min. Physicochemical state of the protein, TBA-RS and Schiff bases were assessed. Protein aggregates were evaluated and the protein target of oxidation studied. Muscles from Galia had higher residual glycogen and drip loss. Heat treatment increased surface hydrophobicity, carbonyl, protein aggregate and Schiff bases and TBA-RS whatever the treatment time. Immunoblotting revealed oxidized myosin, oxidized actin and high molecular weight proteins after 30 min cooking. Oxidation products were significantly correlated with drip loss, suggesting a possible reduced ability of oxidized proteins to retain water. Moreover, residual glycogen was positively correlated with oxidized myosin, suggesting a possible role of glycogen as a glucose donor.     

Effects of electrical stimulation on post-mortem changes in the activities of two Ca dependent neutral proteinases and their inhibitor in beef muscle

Post-mortem changes in the activities of two calcium-dependent neutral proteinases and their inhibitor were studied in electrically stimulated and control beef Longissimus dorsi muscles.During meat conditioning in control muscles the activity of the high-calcium-requiring proteinase (mmCa ANP) was not affected while a decrease in the activities of the low-calcium-requiring proteinase (μmCa ANP) was observed.After electrical stimulation, the mmCa ANP activity was only slightly decreased, but the μmCa ANP and the inhibitor activities were drastically affected. An 80% decrease in μmCa ANP activity was observed 4 h post electrical stimulation. The post-mortem changes seem to be closely related to the post-mortem pH fall.If the Ca-requiring neutral proteinases are involved in the meat ageing mechanism, it eems that the μmCa ANP is more likely to cause most of the post-mortem changes in the myofibrillar proteins, taking into account the low level of intracellular calcium. But as its activity is drastically reduced during the rigor mortis process, most of the effect of μmCa ANP could occur at the very beginning of the post-mortem changes.     

Electrical stimulation of rats: Part 2-The effect of electrical parameters on muscle tension and post-Mortem glycolysis

Anaesthetized rats were used as a model to determine the effect of changes in electrical stimulation parameters on the pH fall of the M. longissimus dorsi and M. triceps surae and tension development by the M. triceps surae. Stimulation 5 min before decapitation resulted in a reduced post-stimulation fall in pH. Tension development resulting from stimulation of the sciatic nerve decreased rapidly 5 min after decapitation and ceased 30 min after decapitation, whereas a current pathway through the muscle was still effective. Post-stimulation pH fall was less in the leg not in the stimulation pathway, indicating a lack of crossover effect from one leg to the other; but the difference diminished as stimulation voltage increased. For direct stimulation, 20 V produced a maximal response in the stimulated leg; at least twice that voltage was required for a maximal pH fall in the leg not in the stimulation pathway. The total resistance of the rat with a needle electrode at the nape of the neck and crocodile clips attached to the unskinned legs was 3400 ω, whereas with skinned hindlegs the resistance was 860ω. With this high skin resistance at least 40 V was needed for effective stimulation whereas, for low voltage stimulation of much larger animals, only twice this voltage is used. High contact resistance and unknown current density factors make simple correlations of animal size and voltage effects impossible. The optimal stimulation frequency for rats was 20–30 pulses a second. If the cranial electrode was positive, about 15% more tension was developed than when the cranial electrode was negative. With alternating-polarity pulses, there was a high-low tension response, which was more pronounced at low voltages but evened out as the muscles became exhausted. Alternating and cranial-positive pulses produced similar pH falls but cranial-negative pulses were inferior.     

Effect of electrical stimulation on the rheological properties of rabbit skeletal muscle

The effect of electrical stimulation on the rheological properties of rabbit skeletal muscle after death was investigated. The extensibility of electrically stimulated psoas muscles decreased more rapidly than that of non-stimulated muscles. For raw non-stimulated longissimus thoracis muscles excised from the carcasses immediately after slaughter, the penetration force required was greatest 24 h after slaughter and then decreased slightly after 168 h. The corresponding force for stimulated longissimus thoracis muscles increased to a maximum in 12 h and then decreased to values less than non-stimulated muscles. However, in the case of raw longissimus thoracis muscles whichhad been attached to the skeleton until measurement, there was no significant difference in penetration force between stimulated and non-stimulated muscles. In cooked muscles, electrical stimulation resulted in lower penetration forces at 24 h post mortem, but on further storage the differences decreased.