Modelling post-mortem tenderisation-III: Role of calpain I in conditioning

A simple model is developed to show how proteolysis by calpain I can account for the variations in tenderness in electrically stimulated and nonstimulated beef pectoralis profundus muscles stored between 0°C and 30°C. As the pH of the muscle falls to about 6·1, calpain I is activated and causes proteolysis and tenderisation. The rate of tenderisation is then proportional to the concentration of calpain I which is autolysed slowly reducing its concentration and the rate of tenderisation. The activation energy for the inactivation (autolysis) of calpain I is slightly higher than that for its activity in tenderisation (proteolysis) and therefore, at higher temperatures, less tenderisation occurs. Proteolysis and tenderisation continue at a rate governed by the concentration of calpain I and the temperature until calpain I is depleted when tenderisation stops. Parameters for the activity and inactivation of calpain I were derived and were shown to predict 68% of the variation in toughness.     

Modelling post-mortem tenderisation-II: Enzyme changes during storage of electrically stimulated and non-stimulated beef

Levels of calpains I and II, cathepsins B and L and β-glucuronidase were determined in extracts of electrically stimulated and control beef M. Pectoralis profundus stored at temperatures between 0 and 30°C and varied to avoid muscle shortening. The level of lysosomal enzymes remained essentially unchanged throughout storage. The levels of calpain II were largely unaffected by the early treatments and decreased slightly throughout ageing. The level of calpain I, in both stimulated and control meats, was unaffected by temperature prior to the attainment of about pH 6·2 and thereafter the loss was accelerated at higher temperatures. In the extreme case studied, that of stimulated meat held at 15°C, 73% of the activity was lost in the first 24 h. After ageing, the level was about 11% of the initial when stored at 1°C and 25% when stored at 15°C. The exponential decay constants for the decrease in the levels of calpain I were 0·01 h(-1) at 1°C and 0·06 h(-1) at 15°C, and were the same as those for the previously determined rate of tenderisation. This suggested that the rate of proteolysis by calpain I was linked to the rate of tenderisation.     

Effect of delay time before chilling on toughness in pork with high or low initial pH

Paired M. longissimus dorsi muscles from 56 carcasses of Danish Landrace and Yorkshire breeds, slaughtered at approximately 90 kg live weight, were utilized to study the potential of cold induced toughness in pork. Based upon the pH value 45 min post stunning, the carcasses were divided in two groups: a low (5·7 ≤ pH < 6·1) and a high one (6·1 ≤ pH ≤ 6·5). The effects on Warner-Bratzler shear force, sarcomere length and myofibril fragmentation of inserting a delay time of 0, 2 and 4 h before carcasses entered the chilling tunnel (operating at -28°C to -22°C) were investigated on early excised muscles as well as on muscles removed 30 h post stunning. The left LD muscle from each carcass served as a control while all right sides were used for treatments. pH and temperature measurements obtained from LD muscles left on carcasses during chilling showed that LD muscles belonging to the high pH group involve a risk of cold shortening even when a 2 h delay was used before passing in to the chilling tunnel. Comparing pH groups, however, sarcomere lengths did not differ in control sides whereas the Warner-Bratzler shear force values were significantly higher in LD muscles taken from the high pH group. Early excision of the LD muscle resulted in shorter sarcomere lengths and increased WB shear force only for carcasses belonging to the high pH group, which, however, could be avoided by introducing a 4 h delay time before rapid chilling. The effect of delay time on tenderness from muscles excised from the carcass 30 h post stunning was much less but a 4 h delay did significantly (P < 0·05) improve tenderness in carcasses with high initial pH. Coefficient of correlation between Warner-Bratzler shear force and sarcomere length was -0·12 and nonsignificant in the low pH group, whereas it was -0·57 and highly significant in the high pH group.     

Variability in rates of pH fall and of lactate production in the muscles on cooling beef carcasses

The rate of pH fall in four major muscles on cooling beef carcasses varied by about twofold. Thus the time taken to reach pH 6·0 from initial pH values of about 7·1 varied between 8 and 16h, and in one extreme case of an LD muscle was as low as 3·5 h. Some of this variability was due to different rates of cooling between muscles in the same animal, but even after correction of the rates to a constant temperature of 38°C the variability was still about twofold in the higher range of pH (7·0-6·7) and slightly lower in the lower range (6·6-5·8). Similar variability was shown in excised TB muscles, kept at a constant temperature of 38°C. Damage to muscles during excision can lead to much higher variability than this (in rabbit LD muscles, to about sixfold). The rate of pH fall is determined by the rate of ATP-turnover, and so the variability in the former rate is likely to be due to varying intracellular free Ca(2+) levels exerting a stimulating effect on the actomyosin ATP-ase. Some of this extra Ca(2+) may arise from calcium release from mitochondria as they become anaerobic after death.     

Heat-induced gelation of actomyosin

Rheological properties of actomyosin gels were markedly affected by protein concentration, pH and heating temperature. Gel strength increased with increasing protein concentration (30-60 mg ml(-1)) and heating temperature (55-75°C), but decreased with increasing pH (5·5-9·0). Low heating temperatures (50-55°C) favoured the formation of more cohesive actomyosin gels than the higher heating temperatures (60-75°C). Gels formed at low pH (5·5 and 6·0) were less cohesive than those formed at high pH (7·5-9·0). Addition of ATP and pyrophosphate (10 mm) prior to heating decreased gel strength and cohesiveness, whereas EDTA (1-5 mM) reduced gel strength but did not affect gel cohesiveness.     

Effect of post-mortem temperature on beef tenderness

Beef adductor muscles were incubated for 4 h post mortem at 10°C and for 4 h and 6 h post mortem at 30°C, 37°C and 42°C. Half of the muscles were cooked just after incubation and the other half was first stored for two days at 4°C and then cooked. Meat kept for 4 h or 6 h at 42°C and for 6 h at 37°C and cooked at once had a significantly (p<0·05) lower shear force than meat kept for 4 h at 37°C, 4 h at 30°C, 6 h at 30°C or 4 h at 10°C. The respective significant differences were also found when the meat was cooked two days after incubation. Organoleptic evaluation showed that meat incubated for 6 h at 37°C or for 4 h at 42°C was not significantly more tender than other samples. However, meat kept for 6 h at 42°C was more tender (p<0·5) than the other samples. After two days of storage, meat incubated for 6 h at 37°C and for 6 h at 42° was more tender (p<0·05) than meat kept for 6 h at 30°C. It was concluded that high temperature conditioning at 37°C or higher for 6 h (4 h at 42°C) just after slaughter makes meat more tender than conventional cooling systems.     

Manipulation of the pre-rigor glycolytic behaviour of bovine M. longissimus dorsi in order to identify causes of inconsistencies in tenderness

The aim of this study was to monitor the effects of the alteration of the pre-rigor environment of the bovine LD muscle using controlled temperature regimes in order to gain an insight into the early post-mortem pH/temperature/time interactions which are important from the point of view of tenderness and to identify possible reasons for inconsistencies in beef tenderness. LD muscles (n=12) were hot-boned within 90min post-slaughter, cut into three pieces which were randomly placed in polyethylene bags and submerged in water baths pre-set at the following temperatures; 0, 5, 10, 15, 20 and 25°C for 8 h post-mortem then stored at 2°C for up to 14-days post-mortem. The rate of pH decline increased with increasing temperature. Muscles incubated at 0 and 5°C were cold shortened however not all of these muscles were tough as indicated by Warner Bratzler shear force values (WBSF). A pH range of 5.9-6.2 at 3 h post-mortem (pH(3)) produced consistently tender beef where cold-shortening was avoided. Cold shortened muscles showed the greatest variation in tenderness at 14 days post-mortem and underwent the greatest amount of tenderisation (ΔWBSF) and proteolysis between days 2 and 14 post-mortem. Proteolysis of cold shortened muscle may induce variation in tenderness in these muscles.     

The tenderising effect of electrical stimulation of beef carcasses

Electrical stimulation (ES) of beef carcasses soon after death has an accelerated tenderising effect on the musculature, under conditions of slow cooling (8 h at 16°C and then storage in still air at 1°C). The effect is large in the LD muscles, reducing the shear force on day 1 of storage from 11 to 6 kg; on day 14, the difference is still 3·3 kg. These differences would be detected by taste panels. The St muscles show a similar, but less pronounced, effect which would probably not be detected by taste panels. The accelerated tenderisation due to ES can be accounted for by the higher temperatures obtaining in stimulated muscles at the onset of rigor. Rapid cooling soon after death reduces the effect almost to zero. Hence, the extra tenderisation cannot be due to muscle damage during ES. Histological examination shows that stimulated muscles have longer sarcomeres than the controls; they do not exhibit damage. However, with slow cooling, irregular bands of denatured sarcoplasmic protein are deposited within the fibres of stimulated muscles, similar to those found in PSE pig muscles. There is also some shortening of sarcomeres in the region of the bands. The protein is deposited on the myofibrillar surfaces. In spite of the PSE-like appearance, there is no significant increase in drip from the stimulated muscles at 48 h after death.     

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.     

The use of proteases from extreme thermophiles for meat tenderisation

The potential use of the thermophile enzymes E A.1 protease (from Bacillus strain E A.1), 4-1.A protease (from Thermus strain Rt4-1.A) and caldolysin (from Thermus strain T-351) in meat tenderisation has been investigated. Temperature-activity relationships illustrated that E A.1 and 4-1.A proteases were more active on collagen than on meat powder at cooking temperatures (70-90°C), whereas caldoysin was more active on meat powder. The potential of E A.1 and 4-1.A proteases was therefore investigated further using sensory and mechanical evaluation. An Instron Universal Testing Machine was used to quantitatively investigate the effect of cooking temperature and protease concentration on homogenised meat patties. With a cooking time of 30 min, the best protease concentrations (of those used) were found to be 0·75 U/g meat for E A.1 protease and 1·5 U/g meat for 4-1.A protease. The optimum cooking temperature was 80°C in both cases. Sensory analysis confirmed that these concentrations (and also 0·38 U/g meat for E A.1 protease) improved the tenderness significantly. At high concentrations the proteases had a detrimental effect on the mouthfeel of the patties. At lower concentrations this effect was less marked, and good tenderisation was obtained.     

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.     

The effect of temperature and ultimate pH on the increase in meat toughness resulting from restraint during cooking

Samples of stretched muscle cooked at 50, 60, 70 or 80°C, while restrained at either their original pre-cooking length or further tensioned at about 130% of their original pre-cooking length, had significantly (P < 0·001) greater Warner-Bratzler (WB) peak shear force values for all temperatures than similar samples cooked without restraint except for those restrained at their original length and cooked at 50°C. Restraint during cooking at 80°C increased the peak shear force values of stretched sheep muscles with ultimate pH values in the range 5·5-7·0. This increase, which has been related to connective tissue strength, was not significantly related to ultimate pH. Both initial yield and peak force values, for samples cooked either restrained or unrestrained, decreased significantly (P < 0·001) and at similar (not significantly different) rates with increase in ultimate pH.     

The effect of cooking temperature on the functional properties of beef proteins: The role of ionic strength, pH, and pyrophosphate

This study examined the effect of ionic strength (0·12 to 0·52), pH (5·50 and 6·00), pyrophosphate (PP) concentration (0 and 0·31%) and cooking temperature (52 to 87°C) on the cook yield (CY) and tensile strength (TS) of beef homogenates. Increasing the ionic strength, pH and pyrophosphate concentration increased the temperature at which cooking loss first occurred and decreased the temperature required for maximum TS. For most treatments, ionic strengths between 0·32 and 0·42 prevented cooking loss at all temperatures; the lower ionic strengths were required at the higher pH and PP concentration. Maximum TS occurred at 66°C for treatments that had no cooking loss between 60° and 75°C. For treatments that had cooking loss in this temperature range, TS increased linearly with increasing temperature; however, the TS values of these treatments were much lower than those in the former category. CY and TS were optimized by heating to 66°C. PP had a positive effect on both functional properties at ionic strengths >0·25 but a negative effect at ionic strengths <0·25.     

Electrical stimulation of rats: A model for evaluating low voltage stimulation parameters

Anaesthetised rats, used either before or after exsanguination, provided a useful preparation with which to measure muscle tension from the M. triceps surae and follow pH changes from the M. longissimus dorsi due to stimulation. With square wave pulses (5 ms duration) the minimum voltage required to elicit a tension response from the M. triceps surae was 0·1 V when applied via the tibial branch of the sciatic nerve and nearly 3 V when applied directly across the muscles. Maximum responses were achieved by 2 V applied to the nerve or 5-8 V across the muscle. The fusion frequency was higher than the ox, occurring at about 30 pulses per second. There was no response (with voltages less than 8 V) from the same preparation when the rats were curarised. Tension responses of curarised rats, approaching those of normal direct stimulation, were achieved around 20-40 V. Stimulation of the whole rat carcass at 20 V resulted in a pH fall in the M. longissimus dorsi and M. triceps surae. most of which occurred in the first 30 s. The rate of fall of pH following stimulation of M. longissimus dorsi was approximately 0·6 pH units per hour, being considerably greater than the 0·4 pH units per hour for unstimulated rats. As the ultimate pH of rats was high (pH 6·1-6·3), short stimulation times had to be used to avoid asymptotic portions of the curve. Although responses of rat muscles are different from those of bovine or ovine muscles, they show many of the principles which can be used to evaluate stimulation characteristics.     

Effect of temperature and pH on the post-mortem degradation of myofibrillar proteins

Incubation of bovine muscle at 37°C promoted a more drastic proteolytic change in myofibrillar proteins, as determined from sodium dodecyl sulphate polyacrylamide gels of isolated myofibrils, than incubation at 4°C. Degradation of myosin and troponin-T were the most noticeable changes at 37°C. Loss of α-actinin was observed in the 4°C incubated muscle. Ground bovine muscle incubated at pH 5·4 and 7 revealed that alterations in myosin and troponin-T were the most noticeable changes at ph 5·4 while troponin-T and α-actinin were altered at pH7. More troponin-T degradation occurred at pH 5·4 and 37°C than at pH7 and 4°C (similar to the degradation of myosin), indicating that troponin-T degradation in post-mortem muscle may be an indicator of overall myofibrillar proteolysis and not responsible for post-mortem tenderisation per se. Myosin degradation in the ground samples incubated at pH 5·4 and in whole samples incubated at 37°C was compared with the digestion of myofibrillar myosin by papain. Both pyrophosphate and Guba-Straub extracts of the 37°C and pH 5·4 treated samples confirmed that myosin degradation did occur to a much greater extent at this temperature and pH than at 4°C and pH7, and, in addition, at pH 5·4 frequent cleavage occurred near the papain sensitive site of myosin.     

The effects of conditioning on meat collagen: Part 4-The use of pre-rigor lactic acid injection to accelerate conditioning in bovine meat

Injection of fresh bovine muscle with 0·1 m lactic acid (to a level of 10% of original muscle weight) resulted in a pH decline to a minimum pH of 5·33 at 15°C only 3 h after injection. Untreated muscle reached the same pH after 26 h when held at the same temperature. Fresh, unconditioned meat colour was unaffected by pre-rigor 0·1 m lactic acid injection as assessed by visual inspection. The percentage of perimysial collagen extracted as the soluble form was significantly higher (P < 0·05) from three muscles of varying quality when pre-injected with 0·1 m lactic acid and conditioned from 1 to 14 days, than from conditioned untreated muscles. SDS-polyacrylamide gel electrophoresis of CNBr peptides from insoluble perimysium obtained from three muscles of varying quality revealed no obvious differences due to pre-rigor lactic acid injection before conditioning. However, analysis of the high molecular weight perimysial collagen CNBr peptides from lactic acid treated muscles by two-dimensional SDS-polyacrylamide gel electrophoresis revealed an increased incidence of degradation in this region compared with untreated controls. These data strongly suggest that pre-rigor injection of beef muscle with lactic acid may accelerate conditioning. The implications of this finding are discussed.     

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.     

Chilling pig carcasses: Effects on temperature, weight loss and ultimate meat quality

Effects of conventional (4°C, air velocity 0·5 m/s) and forced chilling at -5°C (120 min) or -30°C (30 min) with air velocities of 1, 2 or 4 m/s, followed by conventional chilling till 24 h post mortem on temperatures, meat quality and weight losses, were studied. Experiments were carried out in six batches of six slaughter pigs each (crossbred gilts, weighing 105-110 kg. The subcutaneous temperature decreased very rapidly to values below 0°C when 'ultra' rapid chilling (-30°C) at high air velocities (4 m/s was used. Immediately after rapid chilling, when the carcasses were railed into a conventional chiller, the subcutaneous temperature increased above the air temperature, after which the decline in temperature was continued. Temperature inside the biceps femoris muscle decreased from the start of chilling rather slowly according to an asymptotic curve until ultimate values of 4°C were reached. Theoretically calculated temperatures during slaughter and chilling were comparable with the measured values; indicating that a finite-element calculation method in combination with a cylindrical model for heat transport can be used to predict muscle temperatures for various chilling regimes. Losses in carcass weight, 24 h after conventional and forced chilling at -5°C, were about 2%. After 'ultra' rapid chilling (-30°C) the losses were reduced to 1·3% when air velocity was increased to 4 m/s. Meat quality of the longissimus lumborum muscle was not significantly affected by the various chilling regimes except for the variables related to tenderness. The Warner-Bratzler shear forces were higher (P < 0·05) together with shorter sarcomere lengths (P < 0·10) after 'ultra' rapid chilling at a high (4 m/s) air velocity, indicating an increased risk of cold shortening.     

Effect of temperature of comminution on the stability and eating quality of 'English' sausages

Sausages were prepared after equilibrating the ingredients to temperatures in the range 2° to 37°C. Following comminution for 6·5 min the temperatures of the batters ranged from 15 to 33°C and their pHs from 6·25 to 6·48. During storage at -20°C sausages prepared from the high temperature batters lost more weight than those made from batters prepared at the lower temperatures (0·3% compared to 1·2%). Increasing temperature of comminution led to increased cooking losses, softening in texture and darkening in colour. However, even at the highest temperatures complete emulsion breakdown did not occur as cooling losses were still only about 20%. Subjective assessment indicated that at least up to comminution temperatures of 25°C the sausages were acceptable. At temperatures above 30°C off-flavours developed. It is suggested that comminuted meat products can be manufactured in situations where refrigeration is not available, provided a preservation system can be devised to inhibit microbial and chemical spoilage.     

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

The course of rigor mortis (rigor), ageing and tenderness has been evaluated for three beef muscles; M. biceps femoris (BF), M. semimembranosus (SM) and M. semitendinosus (ST), when entering rigor at constant temperatures of 15 and 37°C respectively, with and without electrical stimulation (ES/NS) (85 V, 14 Hz and 32 s). The course of post-mortem changes has been registered by isometric tension, by shortening of unrestrained muscle strips and by following the pH decline and the changes in metabolites, such as ATP and CP. Ageing at +4°C was recorded by measuring Warner-Bratzler (W-B) shear values 2, 8 and 15 days post mortem. On the last occasion, the sensory properties of the cooked meat were also evaluated. Maximum shortening and isometric tension were higher at 37°C as compared to 15°C, whereas ES did not reduce rigor shortening. A high correlation between maximum shortening and the ATP-level at the onset of the shortening rapid phase was found (r = 0·77(???)), which could explain the greater shortening obtained at 37°C compared to 15°C. Rigor shortening is an important phenomenon governing meat tenderness as tenderness is highly affected by rigor temperature but not by ES. This was the case for muscles SM and ST but not for BF muscle. Even though tenderness was measured after ageing (15 days post mortem), shortening during rigor seems to be more important for toughness when rigor mortis occurs at 37°C than any suggested tenderizing effect due to increased proteolysis in this temperature region.