Glycogen debranching enzyme and some other factors relating to post‐mortem pH decrease in poultry muscles

The objective of this study was to investigate the activity of glycogen debranching enzyme (GDE, EC 3.2.1.68) in chicken pectoralis superficialis (PS) and quadriceps femoris (QF) muscle post‐mortem. In addition, some other factors relating to pH decrease, such as the activity of glycogen phosphorylase (EC 2.4.1.1) and glycolytic potential were studied. Enzyme activities, glycogen content and lactate content were measured in Ross breed broiler chicken muscles obtained 25 min after stunning. An interesting discrepancy in the activities of the two glycogen degrading enzymes, GDE and phosphorylase, between muscles was found. In light PS muscle, phosphorylase activity was higher (P < 0.001), but GDE activity was lower (P < 0.001) than in dark QF muscle. Buffering capacity and glycolytic potential were higher in PS muscle than in QF muscle. The possible need for high buffering capacity and low GDE activity in PS muscle to protect living muscle from rapid pH decrease was discussed. GDE, together with glycolytic potential, accounted for 40% of the variation in ultimate pH in QF muscle and 61% in PS muscle. Considering the low activity of GDE together with the high activity of phosphorylase found in both muscles, the results of the present study support the hypothesis that GDE may restrict the rate of glycogen degradation. Copyright © 2006 Society of Chemical Industry     

Physical and Biochemical Properties of Porcine Muscle as Affected by Exogenous Epinephrine and Prednisolone

SUMMARY— The effects of intramuscular ante-mortem epinephrine of prednisolone plus epinephrine injection upon some physical and biochemical properties of porcine longissimus dorsi muscles were studied. Rate of post-mortem muscle pH decline, ultimate muscle pH, Munsell value and transmission value were not significantly (P > .05) altered by epinephrine or prednisolone plus epinephrine injection. Significantly (P < .05) higher total phosphorylase activity and slightly greater phosphorylase a and % phosphorylase u (% of total phosphorylase activity) were observed in the muscles of epinephrine injected pigs.
Neither total phosphorylase nor phosphorylase a was related to rate of pH decline or incidence of pale, soft, exudative muscle development. Muscle concentrations of AMP and IMP were not significantly (P > .05) different between treatments. IMP concentration was highly related to rate of pH fall. Correlation coefficients between longissimus dorsi muscle pH and IMP levels were −.93 and −.94 at 1.5 and 45 min post-mortem, respectively. Prednisolone treatment resulted in higher initial and ultimate muscle glycogen levels than those from the control or epinephrine injected pigs.     

Temperature shows greater impact on bovine Longissimus dorsi muscle glycogen debranching enzyme activity than does salt concentration

The degradation of glycogen progresses by the co-operation of two enzymes: glycogen phosphorylase (phosphorylase) and glycogen debranching enzyme (GDE). We studied the effect of temperature (4–42 °C) and salt concentration (0–3% NaCl) on bovine M. longissimus dorsi GDE activity. GDE activity (n = 4) decreased significantly with decreasing temperature from about 40–4 °C. GDE exhibited 52% activity at 25 °C and 11% at 4 °C compared to its optimum activity measured at 39 °C. In rapidly chilled meat, the reduction in GDE activity may substantially delay the rate of glycolysis. However, residual GDE activity at 4 °C seems sufficient to enable the attainment of normal ultimate pH if the available time is long enough. An increase in salt concentration from 0% to 2% and to 3% induced a significant (P < 0.001) increase in the ultimate pH of ground bovine meat (n = 6), but showed no effect on GDE activity.     

Activity of porcine muscle glycogen debranching enzyme in relation to pH and temperature

The activity of glycogen debranching enzyme (GDE) was studied in relation to pH value and temperature in porcine masseter and longissimus dorsi muscles. A glycogen limit dextrin was used as the substrate for GDE, and the enzyme was derived from raw meat extracts. In both muscles, the pH only weakly affected the activity of GDE at the pH values found in carcasses post-slaughter. However, the activity of GDE decreased strongly (P<0.001) when the temperature decreased from values of 39 and 42 °C, found just after slaughter to values of 4 and 15 °C. In both muscles the activity of GDE began to fall at temperatures below 39 °C and was almost zero when the temperature decreased to below 15 °C. Thus, the activity of GDE may control the rate of glycogenolysis and glycolysis, but does not block rapid glycolysis and pH decrease when the temperature is high. This may be important in PSE meat, where the pH decreases rapidly at high temperatures, but rapid cooling could limit the activity of GDE and thus glycogenolysis. As expected, GDE was more active in the light longissimus dorsi muscle than in the dark masseter muscle.     

Temperature shows greater impact on bovine Longissimus dorsi muscle glycogen debranching enzyme activity than does salt concentration

The degradation of glycogen progresses by the co-operation of two enzymes: glycogen phosphorylase (phosphorylase) and glycogen debranching enzyme (GDE). We studied the effect of temperature (4–42 °C) and salt concentration (0–3% NaCl) on bovine M. longissimus dorsi GDE activity. GDE activity (n = 4) decreased significantly with decreasing temperature from about 40–4 °C. GDE exhibited 52% activity at 25 °C and 11% at 4 °C compared to its optimum activity measured at 39 °C. In rapidly chilled meat, the reduction in GDE activity may substantially delay the rate of glycolysis. However, residual GDE activity at 4 °C seems sufficient to enable the attainment of normal ultimate pH if the available time is long enough. An increase in salt concentration from 0% to 2% and to 3% induced a significant (P < 0.001) increase in the ultimate pH of ground bovine meat (n = 6), but showed no effect on GDE activity.     

Changes in solubility and enzymic activity of muscle glycogen phosphorylase in PSE-muscles

Comparison of the crude extracts of soluble proteins of PSE (pale, soft and exudative) and normal porcine muscles shows that the solubility and activity of muscle glycogen phosphorylase are diminished in PSE muscles. In the insoluble fractions of the muscle extract, however, an increased amount of a protein with the molecular weight of the phosphorylase subunit (92·500-95·000) can be found by means of SDS-electrophoresis. The PSE conditions with high temperatures of 36-40°C in the first hour post mortem and a low pH of 5·3-5·8 at the same time post mortem are simulated with normal porcine muscles at twenty-four hours post mortem which causes the same change in solubility and activity of muscle glycogen phosphorylase as in PSE muscles. This proves, for the first time, that low pH and high temperature in whole muscles cause a denaturation of phosphorylase, as indicated by both reduced activity and decreased solubility. Previous studies have looked only at one of these aspects. This supports the hypothesis of Bendall & Wismer-Pedersen (1962) that in PSE muscles a partial denaturation of sarcoplasmic protein occurs.     

Activities of metabolic and contractile enzymes in 18 bovine muscles

Contractile and metabolic characteristics of 18 bovine muscles were studied using 10 animals of various ages (2-10 years), sexes (females, males and castrated males) and types (dairy, meat or crossbred). Contractile type was assessed by measuring myofibrillar Ca-Mg-activated ATPase activity. Metabolic type was appraised by determining phosphorylase, glycogen synthetase, hexokinase, haem iron as well as glycolytic and mitochondrial oxidative activities. 'Glycolytic potential' and buffer capacity (as change in lactate concentration as a function of change in pH) were measured. The results showed that myofibrillar ATPase activity was positively related to glycolytic activities and negatively related to oxidative activities. Ultimate pH was negatively correlated with myofibrillar ATPase activity and had little if any relationship to the glycolytic potential. Metabolic and contractile activities were essentially unaffected by sex, age or breed type.     

Myofibrils, sarcoplasmic reticulum and phosphorylase kinase of Pietrain and Large White-landrace pigs

Comparison of fast and slow twitch myofibril preparations by SDS-gel electrophoresis showed no significant differences between Large White and Pietrain pigs. No variants of parvalbumins or calsequestrin (calcium binding proteins) were found. Pietrain phosphorylase kinase has a greater stimulating activity on porcine phosphorylase than does Large White phosphorylase kinase.     

Post-mortem glycolysis in rabbit Longissimus dorsi muscles following electrical stimulation

The effects of both high voltage and low voltage electrical stimulation were studied in rabbit Longissimus dorsi muscles. The rate of fall of pH as well as the activities of phosphorylase a, phosphorylase kinase, and phosphorylase phosphatase were measured. The effects on the yield, ATPase activities, and calcium permeability of the sarcoplasmic reticulum (SR) were also measured. Although only high voltage stimulation increased the post-stimulation rate of pH fall, both types of electrical stimulation increased the phosphorylase a activity, apparently by increasing the activity of phosphorylase kinase and destroying a large part of the phosphorylase phosphatase activity. Electrical stimulation reduced the yield of SR and increased its basal ATPase activity. It also increased the ability of the SR to retain accumulated calcium. We conclude that the different rates of pH fall observed following the two types of stimulation are due to the differential effects of these treatments on one of the ATPase activities, probably the myofibrillar ATPase.     

Effect of post-mortem storage temperatures on isometric tension, pH, ATP, glycogen and glucose-6-phosphate for selected bovine muscles

Relationships between isometric tension development and pH, ATP, glucose-6-phosphate and glycogen levels as a function of post-mortem storage temperature were examined for three bovine muscles. Tension responses in the range of 5-37°C were similar for B. femoris, Vastus lateralis and outer M. semitendinosis. At 0°C, the three muscles developed considerably higher tension than at 5°C. Cold shortening developed only in the outer M. semitendinosis strips at 0°C. At low temperatures the drop in pH lagged compared to the decline in ATP and glycogen and maximum tension was attained several hours after ultimate pH and minimum levels of ATP and glycogen were reached. Glucose-6-phosphate was found to accumulate rapidly at 0°C in both outer M. semitendinosis and B. femoris, being more pronounced in the latter muscle. The responses of glucose-6-phosphate levels suggest that the relative activities of glycolytic enzymes in muscle stored at 0°C are altered compared to those in muscle stored at higher temperatures.     

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 high pressure on the regulation of phosphorylase activity in pre-rigor rabbit muscle

The effects of high pressure (150 MPa) on the regulation of phosphorylase activity in pre-rigor rabbit muscles have been studied at 35° and 0°C. At 35°C muscle contracts, phosphorylase is activated and the muscle pH falls to 5·8 in 2 min. Coinciding with these changes, phosphorylase phosphatase activity falls rapidly, while phosphorylase kinase, although active for longer, loses its activity as the pH falls. Both of these enzymes are completely inactivated after 5 min under pressure, while phosphorylase still retains 80% of its activity under these conditions. The effects of pressure on the activities of these enzymes in white and red muscles of rabbits were compared, with a greater effect being observed in white muscles. At 0°C, muscles subjected to high pressure did not contract, but at this temperature the three enzyme activities (phosphorylase, phosphorylase kinase and phosphorylase phosphatase) were all lost at a greater rate than at 35°C, although the pH of the muscle did not fall below 6·5. The effects of high pressure treatment on isolated phosphorylase a and b and phosphorylase kinase were also studied at both 0° and 35°C and the results obtained closely paralleled those observed in whole muscle.     

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.     

Glycolytic Intermediates and Co-Factors in "Fast-" and "Slow-Glycolyzing" Muscles of the Pig

SUMMARY— The Longissimus dorsi muscles from Chester White, Hampshire and Poland China animals were used to establish certain differences in metabolic intermediate patterns between muscles with "fast" and "slow" rates of post-mortem glycolysis. Metabolic intermediate patterns were consistent with the concept that phosphorylase is the primary control site of postmortem glycolysis. Adenine nucleotide levels appeared to be the primary regulatory factors for phosphorylase. The phosphofructokinase and pyruvate kinase enzymes were also involved in post-mortem glycolytic control. Levels of high-energy intermediates (adenosine triphosphate, phosphocreatine and pyridine nucleotides) were much higher in the "O" hr samples of "slow-glycolyzing" muscles than in similar samples from muscles having "fast" rates of post-mortem glycolysis. No significant differences in levels of lactate or glucose were observed among these three groups in blood samples taken either at or 24 hr prior to the time of exsanguination.     

Biochemical studies on fast glycolysing bovine muscle

Of the psoas major muscles from 1395 carcasses of young bulls 64% showed pH(1) values (30 min post mortem) below 6·0. In 100 muscle samples (selected so that a normal distribution of the pH(1) values was obtained), water-holding capacity (WHC), brightness of colour and the concentration of all glycolytic metabolites were determined. There were correlations between pH(1) and the other characteristics of meat quality, lower pH(1) values were associated with lower WHC, paler colour, lower glycogen levels and higher levels of lactate (30 min post mortem). All these correlations were highly significant. The accelerated breakdown of glycogen-and a simultaneous accumulation of hexose monophosphates-indicate an activation of the phosphorylase system in fast glycolysing bovine muscle. In fast glycolysing muscle, creatine phosphate was almost absent by 1 h post mortem and the ATP level was very low, indicating a high rate of breakdown of high-energy phosphates. The quality deviation of fast glycolysing beef is much less severe than that of PSE pork and therefore might not present a serious economic problem.     

Effects of dietary α-lipoic acid on glycolysis of postmortem muscle

The effects of dietary α-lipoic acid (α-LA) on the pH value, AMP-activated protein kinase (AMPK) activation, and the activities of glycogen phosphorylase and pyruvate kinase in postmortem muscle were investigated. Eighteen C57BL/6J mice were fed diets containing 0%, 0.5%, and 1.0% α-LA. At the end of 2-week feeding trial, the mice were killed and longissimus muscles were sampled at 0-, 1-, and 24-h postmortem for pH determination and enzyme assay. The results showed that dietary α-LA treatment significantly slowed down the decrease of pH values in postmortem muscle. The ultimate pH values in postmortem muscle of mice receiving 0.5% and 1.0% α-LA treatments were 6.40 and 6.63, respectively, significantly higher than that (6.21) of no α-LA treatment (p<0.05). AMPK was activated at the early postmortem stage. Dietary α-LA can suppress the activation of AMPK in postmortem muscle. At 1- and 24-h postmortem, activities of AMPK were much lower in postmortem muscle of mice receiving 0.5% and 1.0% α-LA treatments than that with no α-LA treatment. Between these two dietary α-LA treatments, however, no difference in AMPK activity was observed, indicating that 0.5% dietary α-LA is enough to suppress AMPK in postmortem muscle. Similar to AMPK, glycogen phosphorylase activity was higher in the treatment without dietary α-LA than those with 0.5% and 1.0% dietary α-LA supplements. No difference in the activity of glycogen phosphorylase was observed between the 0.5% and 1.0% dietary α-LA treatments. Dietary α-LA had no significant influence on the activity of pyruvate kinase in postmortem muscle. All these results indicate that AMPK plays a role in glycolysis in postmortem muscle. Dietary α-LA supplementation can suppress the activation of AMPK in postmortem muscle, down-regulate the activity of glycogen phosphorylase, resulting in a higher ultimate pH values in postmortem muscle. Therefore, dietary α-LA supplementation is a potential way to reduce the incidence of pale, soft, exudative (PSE) meat.     

Influence of early pH decline on calpain activity in porcine muscle

This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation. From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3 h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline. The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24 h. Calpain activity and autolysis from 1 to 72 h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation.     

Glycogen content, buffering capacity and resting pH in live muscles of pigs of different halothane genotypes (a pilot project)

The development of PSE (pale, soft and exudative) meat is characterized by a rapid decrease in pH post-mortem and/or a low ultimate pH. We investigated some physiological properties of the live muscle (the glycogen content, the non-bicarbonate buffering capacity and 'resting pH'), which could influence both the decrease in pH and the ultimate pH. Measurements were performed on three halothane genotypes, hal(N)hal(N), hal(N)hal(n) and hal(n)hal(n), with their known predispositions for PSE meat. It was demonstrated that the glycogen content in both the groups of double recessive and heterozygous individuals was higher than the levels in the group of homozygous dominant pigs. No difference was found in non-bicarbonate buffering capacity between the groups. The groups with the highest glycogen levels also had the lowest 'resting pH' values. The results indicate that measurement of glycogen content in vivo may be superior to the halothane test in detecting PSE-prone individuals. The lower pH values of carriers of the hal(n) gene further indicate that the characteristic rapid decrease after slaughter may not be as fast as generally accepted, as even very low pH values can be observed in the muscles of live pigs.     

Influence of breed and muscle metabolic type on muscle glycolytic potential and meat pH in pigs

Glycolytic potential and activities of myofibrillar ATPase, lactate dehydrogenase, citrate synthase, glycogen synthetases and glycogen phosphorylases were measured in five muscles from five Large White, eight Pietrain, seven Belgian Landrace and eight Penshire pigs (Penshire is a composite line including 50% Hampshire, 35% Duroc and 15% Large White blood). Additionally, ultimate pH was measured on the loin and four ham muscles. Muscles could be classified as follows on the basis of enzyme activities: Longissimus dorsi and Semimembranosus as 'fast white', Rectus abdominis as 'intermediate'. Trapezius and Masseter as 'slow red'. Small breed differences were observed in enzyme activities except for citrate synthase activity and degree of activation of glycogen phosphorylase in the 'fast white' muscles. Large Whites, Pietrains and Belgian Landraces did not differ in glycolytic potential whatever the muscle involved. Compared with these three breeds, Penshires had much higher glycolytic potential values in the 'fast white' muscles (+51 to 75%) and in the 'intermediate' muscle (+29 to 56%). However, in the 'slow red' muscles, there was almost no difference in this trait between the four breeds. The Penshire pigs showed lower ultimate pH than the pigs from the other breeds in three of the four ham muscles studied. It is concluded that muscle metabolic type must be considered when assessing breed variation in glycolytic potential, and that the high muscle glycolytic potential characterizing the Hampshire could be inherited as a dominant trait in Hampshire crosses.