The calculations described here are ment to help to rationalise several key aspects of processing practice and to explain certain previously puzzling phenomena connected with PSE. Seemingly unrelated features of processing to achieve optimal meat quality have been brought together under a reasonably simple and predictive model. This could be used to predict the amount of drip loss associated with various treatments of the carcass, thereby define the critical conditions that have to be fulfilled if the severity of PSE is to be ameliorated.

In order to carry out the calculations, the conditions have been deliberately simplified, for example assuming a linear fall in pH, in order to provide an understanding of broad principles. In order to make more accurate predictions for real carcasses, it would be necessary to feed in detailed analytical data on the time-course of pH and temperature. It is doubtful whether this would be warranted until simplifying assumptions that have been made about the kinetics of myosin denaturation have been critically tested.

Evidence that PSE is caused by protein denaturation

In a classic experiment suggesting that PSE is caused by protein denaturation, muscles were held at 37^oC for several hours post mortem, after which they exhibited the typical characteristics of PSE meat. However, it has been found that the 37^oC treatment do not consistently result in PSE meat and cited examples where a carcass with a slow rate of pH fall became almost as PSE as one with very high rate of glycolysis. It was concluded that a low pH at a high muscle temperature was not a causal factor in the development of PSE. However, by reference to the curve in Fig. 6 for the unchilled muscle, we can see that when muscles are not chilled, it is indeed the more slowly glycolysin muscle that are most vulnerable to myosin denaturation. Thus, far from disproving the hypotesis that protein denaturaion is the cause of PSE, other experiments are broadly consistent with it. It is simply that if muscles are maintained at a high temperature post mortem, a very rapidly glycolysing muscle will experience each small interval of pH between the initial and final pH for a shorter time than a slowly glycolysing muscle, and so tha total amount of myosin denatured is less, not more.

The calculations here of the amount of myosin denaturation in carcasses with different rates and extents of glycolysis and different chilling regimes were based on rate constants of denaturation for rabbit myosin found in vitro. It is important to note that the calculated fractions of myosin denatured under temperature and pH conditions simulating those present in a PSE pig carcass are substantial, although considerably less than one, and are of the same order of magnitude as those found experimentally. There is therefore no reason to think that pig myosin is expecially sensitive to denaturation. A detailed comparison between the predicted and the experimental values for myosin denaturation is not possible since the precise chilling conditions have not usually been defined in the experimental work. There is also the problem that the myosin in the H-zone of a sarcomere will not be protected against denaturation at rigor onset by combination with actin and the absence of ATP will rapidly denature post rigor. Such denaturation in the H-zone would not be expected to affect the water-holding and softness of the meat, but it may cause the calculated fraction of myosin denatured at rigor to be appreciably less than that experimentally determined many hours after rigor. There is clearly a need to compare theoretical and experimental amounts of myosin denaturation under rigorously defined chilling conditions and to distinguish between denaturation of myosin in the H-zones and the denaturation of myosin bound to actin filaments.

The notation that myosin denaturation is the cause of PSE on the basis that in muscle exhibiting PSE the fraction of myosin denaturation was only about 0.2, has been criticised. However, we see no reason to suppose that complete denaturation of myosin is required to produce PSE symptoms. It is possible that the muscles refering to above might have produced even more drip if they had been treated so that more denaturation was produced; it was not show that the muscles produced the maximum amount of drip that the muscles were capable of. Even if they had, it is possible that maximum shrinkage of the myofibrils taken place when only a small fraction of the myosin is denatured. Each half of the thick filament contains about 150 myosin molecules and is surrounded by six thin filaments. Hence the distance between a thick filament and a thin filament is determined by the length of 25 pairs of myosin heads, as well as by the pH. The native myosin head length favours one filament spacing characteristic of normal rigor muscle, the enatured state favours a smaller spacing. It is not obvious how the filament spacing will depend on the fraction of heads denatured. It is possible that the spacing reaches a minimum when only a small fraction of the heads are denatured, and that further denaturation causes no further shrinkage of the filament lattice.

Time-course of temperature

Figure 1 showed a comparison of the time-course of temperature change in normal and PSE pig carcasses. For the higher rates of pH fall, the temperature of the muscle would rise before falling due to the liberation of the metabolic heat associated with glycolysis. With immerdiately exponential chilling of pig carcasses and a half-cooling time of 180 min, such a rise would be expected to occure with rates of pH fall in excess of 0.067 units/min. If the chilling is slower or delayed, the temperature will also rise even with the slower rates of pH fall.

The model was designed to mimic the heat evolution in PSE carcasses. Unfortunately, there is little detailed information available on the time-course of the temperature change at an early stage and the contribution to the temperature rise by the scalding operation normally applied to pig carcasses has not yet bee defined. In as early as in 1954 the Danish scientist Ludvigsen observed temperatures as high as 44-45^oC a few minutes after death in PSE carcasses. In carcasses with a very fast rate of glycolysis there is an early rise of about 2-3^oC. In addition to this, it has been found that 15 min after slaughter, the temperature in the longissimus dorsi muscle of pigs of a stress-resistant breed (Large White), was 36.8^oC but in pigs of a more stress-susceptible breed (Pietrain pigs), the temperature was 38.2^oC. However, if a beta-adrenergic blocking agent is administrated to the Pietrain pigs before slaughter, there is no difference in the temperatures. In fast glycolytic muscles the temperature 15 min post slaughter is about 42.6^oC, compared with 40.3^oC for normal muscles. What is know is that the total metabolic heat liberated in the conversion of glycogen to lactic acid and the hydrolysis of creatine phosphate and ATP is sufficient to raise the carcass temperature to 3^oC. It is clearly highly desirable to have more information on the cooling curves of normal and PSE carcasses.

Effect of rate of glycolysis on myosin denaturation and water holding

It has generally been assumed that the faster the rate of glycolysis, the greater the degree of protein denaturation there would be, and therefore the greater the amount of drip loss. Figures 6, 7 and 8 show that this is not the case. There is a rate of glycolysis, dependent on the chilling conditions, at which the greatest amount of myosin denaturation would be expected; the amount of myosin denaturation would be expected to decline at either slower or faster rates that this, although only moderately at the faster rates. This result follows naturally from the assumption that, once the muscle is in rigor, the myosin is pretected from further denaturation by combination with actin. If the rate of glycolysis is very high, although the muscle experiences particularly severe denaturating conditions so that the rate constant of denaturation reaches high levels, these are experienced for a sufficient short time that the total amount of myosin denatured is lower rather than higher. This raises the question of how we should define the severity of PSE conditions: (1) by the rate of post-mortem glycolysis or (2) by the amount of drip formed? The two measures would not be expected to produce concordant results.

Figure 7 can be compared with experimental data on the effect on myosin denaturation in pig longissimus dorsi muscles of the rate of pH fall, as measured by the pH at 90 min. Little denaturation was observed until the pH₉₀ fell to about 5.9, but below 5.9 the denaturation increased substantiailly. The form of the calculated curve for a half-cooling time of 200 min is similar to the experimental curve, but there is too much scatter in the experimental points for a detailed comparison. For muscles with a pH₉₀ value of 5.6, corresponding to a pH₄₅ value of about 6.3 if the pH fall is linear, the fraction of myoain denatured ranges from about 0.3 to 0.7. Such values are higher than the calculated values (0.16). Part of the reason for this difference may be that myosin in the H-zone of the sarcomeres is not protected against denaturation by combination with actin at rigor.

Comparisons may also be made between the dependence on pH₄₅ of our calculated myosin denaturation and the experimentally observed water-holding capacity and drip loss. It must, however, be appreciated that some drip loss is to be expected even if no myosin is denatured, the myofibrillar lattice shrinks post mortem expelling water, partly due to the fall in pH from rest to rigor and partly due to the attachment of myosin heads at rigor. The shrinkage of the myosin heads due to denaturation causes additional shrinkage of the myofibrils and therefore increases the amount of extraccellular water available for drip formation. The shrinkage in cross-sectional area of the myofibrils at rigor is about twice as high in PSE pig muscles as in normal muscles, and therefore the extracellular water avaiable for drip formation in PSE muscles may be higher by this factor.

Water-holding capacity of pig longissimus dorsi muscles decrease with fall of pH₄₅ down to 6.2 but at pH₄₅ values below this there is no significant effect. This observation was echoed in later studies of drip loss which showed that loss increases greatly as the pH₄₅ value dropped from 7.0 to 6.1, but below 6.1 the drip loss do not increase, or increase only slightly, with further decrease in the pH₄₅ value. A detailed comparison of the experimental observed drip levels is made in Fig. 11. At a pH₄₅ value of 7 very little myosin would be denatured, but the drip loss is appreciable (~5%). It seems reasonable to assume that this amount is the background level that occures even with no denaturation. The maximum drip observed was about three times higher, so the extra drip formed due to myosin denaturation can be at least twice the background level. This is consistant with the measurements of the filament lattice in normal and PSE meat mentioned above. The scale in Fig. 11 have been adjusted to make the curvescoincide at the higher and lower pH₄₅ values. The curves are broadly similar in shape, but since the thermal history of the experimental carcasses is not known, too much should not be made of the correspondence. In particular, too much should not be placed on the precise value of the fraction of myosin denaturation required for a given drip loss (a fraction of 0.1 to give an additional 6% drip loss above background in the experiment cited).

There are several important conclusions that can be drawn from this comparison.

• Firstly, the fact that the drip curves and calculated fractions of myosin denaturation parallell one another suggest that the amount of drip loss may, at least approximately, be linearly related to the amount of myosin denaturated up to a fraction of 0.2. This tentative conclusion needs to be rigorously tested, and it remains to be seen wheather such linearity exists right up to complete myosin denaturation (see above). If correct, it would be extremely useful as it would enable us to use calculations of myosin denaturation to predict drip loss.
• Secondly, although the experimental points presented above could be fitted well by two stright lines intersecting at pH 6.1, the relationship is probably in reality a continous curve.
• Thirdly, there is unlikely to be anything absolute about the pH₄₅ value where a marked change of slope occurs. As shown in Fig. 7 this would be expected to alter markedly with the chilling regime.
• Fourthly, the near-maximum drip loss occuring at a pH₄₅ value of 6.1 does not arise because at this value all myosin is denatured. Figure 11 suggests that only a fraction of the myosin in a muscle is denatured under standard commercial chilling conditions even at the most damaging rate of glycolysis.

Effect of chilling regime

Figure 6, 7, 8 and 9 show clearly the effect of chilling in reducing the amount of myosin denaturation, and by implication the amount of drip loss. Aside from the problem of avoiding cold-shortening and promoting conditioning, faster chilling is always beneficial in this respect, but it should be emphasised that the benefits are much greater with the slower and intermediate rates of pH fall than with very fast rates.

Pig carcasses

For a normal rate of glycolysis, 0.01 units/min, the fraction of myosin denatured at rigor would be 0.82 if the carcass were unchilled, whereas with a half-cooling time of 200 min, this would be reduced to 0.12 (Fig. 6). With a rate of pH fall corresponding to a marginal case of PSE (0.02 units/min), the same difference in chilling regime would reduce the fraction denatured from 0.58 to 0.18. With a rate of pH fall corresponding to a moderate case of PSE (0.03 units/min), it would reduce the fraction denatured from 0.44 to 0.18. This contrast with the situation with a very fast rate of pH fall, 0.1 units/min, where in the unchilled carcass the fraction of myosin denatured would be 0.16, but this would fall only to 0.12 with this chilling. Thus the conditions present in a normal or marginally PSE pig carcass are such as to make them particularly susceptible to poor chilling: the fraction denatured at a half-cooling time (say) 500 min would be expected to cause very exudative meat even with a normal rate of glycolysis. It may thus be seen that the benefits of rapid chilling are readily apparent in normal pig carcasses and in marginal and moderate cases of PSE, but can alleviate the effects of a very high rate of glycolysis only to a small degree. The reason for this is that at the very high rates of pH fall, rigor is attained very quickly before the carcass has cooled appreciably and the speed of chilling has very little influence on the carcass temperature in the critical pre-rigor period. Particular attention clearly needs to be paid to the critical pre-rigor period. Particular attention clearly needs to be paid to the starting temperature. A decrease of 2^oC in the starting temperature would reduce the initial rate of denaturation by 37%, with a corresponding reduction in drip loss. In this regard, more attention should perhaps be given to avoiding the scalding operation or reducing its effect on raising the initial temperature and delaying the chilling. Attention may also need to be paid to reducing the stress on the animals immediately to slaughter to avoid the temperature rise resulting from aerobic metabolism. It is also possible that the air temperature in the slaughterhouse can make an appreciable difference.

Prompt and rapid chilling of pig carcasses after slaughter has been demonstrated to reduce drip loss. In a fast chilling regime, chilling commenced 30 min post mortem and chilling can reach half way after about 280 min in the deep leg. In a slower regime, when chilling is delayed for 6 h and the temperature of the deep leg can reach half way only after 520 min. Faster chilling of PSE pork carcasses (those with a pH₃₀ value <6.1) decreased the average drip loss from 0.80% to 0.38%. The smaller effects of faster chilling on the more rapidly glycolysing carcasses is well explained by the results shown in Fig. 6.

With a slow chilling regime (cooling to 34^oC in 138 min) a moderatly PSE pig longissimus dorsi muscle can give 9.9% drip in 0.3 day whereas faster chilling (cooling to 34^oC in 45 min) can reduce this to 1.1%. A delay before chilling can commence, even as short as 0.5 h, can increase drip loss.

Beef carcasses

Although beef is not usually regarded as being particularly susceptible to the PSE state because of its slow rate of post-mortem glycolysis, the calculations shown in Fig. 5b indicate that significant amounts of myosin denaturation are to be expected when the chilling is slow, such as would occur in the deep musculature. In fact, the amount of myosin denaturation to be expected in a beef leg with a rate of pH fall of 0.001 units/min and a half-cooling time of 700 min is similar to that expected in a pig carcass with a rate of pH fall of 0.01 units/min and a half-cooling time of 180 min. Experimental evidence suggesting that myosin is denatured even in beef carcasses and as a result drip increases, is that faster chilling of beef carcasses reduce drip losses to only a third of the value with the slowest chilling. In an detailed experimental study of the effect of chilling rate on drip loss, solid carbon dioxide was applied at one end of a cylinder of beef semitendinosus muscle to obtain a large range of coolinf rates throughout the length of the muscle. The drip formed from a slice chilled (but not frozen) at the fastest rate was half that in the slice furthest from the coolant. The time for cooling to 7^oC for these slices varied from 11 to 25 h,and over much of this range there was a linear relation between the drip and the cooling time.

In beef carcasses the greater of the denaturation occurs early on in the pre-rigor period whose total duration is ~24 h. In pig carcasses somewhat more denaturation occurs in the half of the pre-rigor period but the more total duration of this period is much shorter, ~2-3 h or less. Thus in both cases there is clearly a need to commence chilling as rapidly as practicable after salughter and to chill as fast as possible, if drip is to be minimised. The problem is that, if chilling is both prompt and rapid, there is a danger, particulary in beef, of causing cild-shortening in the superficial musculature. The obvious solution to this problem is to avoid the large temperature gradients that occur in the carcass by the use of hot boning but only if the meat is boxed in relatively small quantities.

More information is needed on weather or not the exudative properties of beef muscles which have been slowely chilled are accompanied by softness and realtive paleness.

Effect of final pH

Already in 1958 it was found that pig longissimus dorsi muscles exhibiting the PSE state often had an ultimate pH of 5.1 or below, the lowest recorded value being 4.78. It is unclear what factors were responsible for these very low final pHs since in Hampshire pigs, where muscle glycogen levels are twice as high as in other breeds, only about half the glycogen is metabolised post mortem and the final pH is only slightly lower than that in other breeds (5.3 and 5.5, respectively).

Drip loss in pig carcasses increase not only with rate of glycolysis but also with decrease of the final pH. Figure 12 shows a comparison between the amount of drip loss and the calculated fraction of myosin denatured as a function of the final pH assuming that the half-cooling time 1s 180 min and the rate of pH fall 1s 0.01 units/min. It should be noted that the ordinate scale for myosin denaturation and drip loss have been chosen to have the same relationship as those in Fig. 11 (a fraction of 0.1 of myosin denatured corresponding to 6% extra drip). This tests the hypotesis that, regardless of its cause, a given increase in the fraction of myosin denatured produces the same increase in drip loss. The scale have been positioned so that their origins are at the same level. This has been done because at a final pH of about 6, the contribution to drip loss from myosin denaturation would be expected to be very low an therefore it is expected that the curves nearly to coincide at this pH. For both curves there is a rise with fall of final pH, but the curve for drip loss is consistently higher adn rises more steeply than does the curve for myosin denaturation. This is to be expected, since the rise in drip with fall in final pH is caused not only by increased myosin denaturation but also by decrease in the charge on the filaments. The comparison shown in Fig. 12 is therefore broadly consistent with the hypotesis that, regardless of its cause, a given increase in the level of myosin denaturation causes the same increase in the amount of drip formation.

Prediction of PSE

The results of the model calculations show that the amount of myosin denaturation depends on the rate of pH fall, final pH and the chilling regime, but the relationship is complex. For example, at high rates of pH fall, the amount of myosin denaturation is insensitive to changes in the rate of pH fall, but at low rates it is very sensitive. The chilling rate is important at low or moderate rates of pH fall but has little effect at high rates of pH fall. Conversely, the final pH is important at high rates of pH fall but unimportant at low rates. Morover the same degreee of myosin denaturation may be given both by a high and a low rate of pH fall (Fig. 6). It is clear that the effects of these three parameters are not additive and that it is unlikely that a simple prediction of the severity of the PSE state can be made without making calculations along the lines of this paper.

Attempts have been made to predict the severity of the PSE state from a knowledge of the final pH and pH₄₅ alone. While this may be satisfactory when the chilling is standardised, the results of the calculations presented here show that in general this will be inadequate and that knowledge of the complex thermal history of the carcass from the point of slaughter is an essential prerequisite for such a prediction.

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