But why bother? The average student can see no practical advantage to all the hard work involved - although the clever and ambitious ones might. Compare our meat industry to other industries in the manufacturing sector. How far would electronics, mechanical engineering and metallurgy have progressed if nobody had bothered to master the underlying physics and chemistry of their industry? Not very far, they would still be in the dark ages. And is our meat industry any different? Of course it is not, and those who can understand the science of the material they work with are clearly in a stronger position to develop new methods and products, to optimize the quality of their product, and to achieve a competitive advantage. The objective here is to explain why it is that muscles produce lactic acid or lactate after slaughter so that acidification occurs - in other words, why the pH goes down. This will help us understand important commercial topics such as:
Glycogen is the primary storage carbohydrate in muscle fibres and appears in electron micrographs as single granules or clumps of granules located in the sarcoplasm (cytoplasm) between myofibrils (contractile fibrils) and under the cell membrane. New research using scanning tunnelling microscopy, one of the most powerful types of microscope yet invented, reveals that glycogen granules are ellipsoidal with a laminar structure which suggests they grow from one edge rather than a central point. In longitudinal sections of skeletal muscle, glycogen and its associated enzymes are concentrated at the I bands (one of the transverse bands across the muscle fibre when examined microscopically).
Glycogen is a polysaccharide formed by the linking together of large numbers of glucose units. However, the glycogen from some animal tissues is a proteoglucan (glycoprotein) that may contain other monosaccharides and phosphate ester groups. Straight chains of glycogen are formed by linkages between carbon atoms given numbers 1 and 4, while branch points are formed by 1-6 linkages.
The concentration of glycogen in fetal muscle increases steadily towards the end of gestation and may peak at the time of birth. Pigs have very high muscle glycogen levels at birth but they decline rapidly after birth to reach future adult levels within a week. Glycogenin is a protein that acts as a starting block for the formation of new glycogen.
There is currently a lot of interest in the possibility of post mortem glycogen break-down (glycogenolysis) by pathways other than the normal pathway mentioned above. If appreciable amounts of glycogen may be removed without lactic acid formation, this might account for some of the considerable variability found in post mortem rates of meat acidification or pH decline. About 5% of muscle glycogen may be contained in lysosomes (suicide bags that contain enzymes to destroy damaged parts of the cell) and post mortem glycogenolysis may be a combination of both hydrolysis and phosphorolysis.
Cardiac activity is important in the early stages of exsanguination.
A transverse section of pork, just as most of its glycogen (stained red) has been depleted.
Glycogenolysis is the enzymatic degradation of glycogen, and is the first step in the release of energy by the oxidation of glucose units (glycolysis). Phosphorylase erodes straight chains (from their non-reducing ends at carbon 4) and attaches a phosphate group to the carbon atom at position 1 as it removes a glucose unit. Phosphorylase erodes straight chains until it comes to the fourth glucose unit preceding a branch point. The three glucose units before the fourth one that carries the branch are removed together, and are added to an adjacent free straight chain so that the 1-6 linkage thus exposed at the branch point may be severed. A second enzyme, debranching enzyme, performs this task. Instead of being released as glucose-1-phosphate, the glucose unit released from a branch point remains as free glucose. Thus, total glycogenolysis liberates glucose-1-phosphate and glucose in a ratio indicating the ratio between the mean length of straight chains and the number of branch points.
While the structure of glycogen and the mechanism of glycogenolysis are important in understanding the conversion of muscles to meat, the remainder of the pathway by which glycogen is anaerobically oxidized to lactate need only be covered in general principle. The most important question to be answered is why lactate is produced under anaerobic conditions, yet hardly at all under aerobic conditions. After a series of steps in the glycolytic pathway, molecules with six carbon atoms derived from the glucose units of glycogen are split to produce two molecules of pyruvate, each with three carbon atoms. All the glycolytic enzymes (except for hexokinase) are concentrated in the I band. If aerobic conditions prevail, pyruvate formed in the cytosol of the muscle fibre now enters a mitochondrion (the metabolic furnace of the cell). After entering a mitochondrion, pyruvate is converted to acetyl-CoA which becomes fused to oxaloacetate to form citrate. The citrate then is oxidized in the well known Krebs' cycle, which is completed by the regeneration of oxaloacetate. Continuous activity of the Krebs' cycle is fuelled by a range of carbohydrates, fatty acids and amino acids, and is the primary system for the aerobic generation of energy. Large numbers of molecules of ATP are produced from ADP by a series of reactions, oxidative phosphorylation, that occur in the mitochondrial membrane.
Under aerobic conditions, the production of two pyruvate molecules from a glucose-1-phosphate molecule results in the reduction of 2NAD+ . Thus, somewhere else in the muscle fibre, NADH must be re-oxidized for glycolysis to continue. Aerobically, this occurs as a consequence of mitochondrial Krebs' cycle activity, although NADH and NAD+ do not actually cross the mitochondrial membrane. In anaerobic living muscles and in meat, the Krebs' cycle is halted, and NADH is re-oxidized in the cytosol by lactate dehydrogenase (LDH) during the conversion of pyruvate to lactate. Pyruvate is of no immediate use anaerobically since mitochondrial oxidation has ceased, but its conversion to lactate ensures a continued supply of NAD+ for the continuation of glycogenolysis and anaerobic glycolysis in the cytosol. However, since these events form only the initial stages of complete carbohydrate oxidation, they do not regenerate much ATP. The net gain of ATP is reduced to only two molecules of ATP per molecule of glucose-1-phosphate. Molecules of glucose released from glycogen branch points generate a total net gain of 3ATP.
LDH adds hydrogen to pyruvate to produce lactic acid and exists in a number of isoenzymes separable electrophoretically by their net electrical charge. If LDH-1 is prevalent, it facilitates aerobic metabolism, where possible, since it is inhibited by pyruvate and lactate. LDH-5 is not inhibited by high levels of lactate and pyruvate, and it facilitates anaerobic metabolism. LDH-1 is typical of cardiac muscle while LDH-5 is typical of skeletal muscles, particularly those adapted for anaerobic conditions during contraction. In skeletal muscles, the ratio of LDH-1 to LDH-5 corresponds to the dominant activity pattern of a muscle. For example, muscles capable of sustained activity and which only use aerobic metabolism have high LDH-1. LDH-5 is the dominant isoenzyme in skeletal muscles from slaughter-weight pigs.
The regulation of glycolysis in a muscle fibre of a live animal is integrated with the metabolic state of the fibre and its immediate energy needs. The metabolic state of the fibre is profoundly affected by hormones, particularly adrenaline, and by the extent of recent contractile activity of the fibre. Phosphorylase is particularly important in the conversion of muscle to meat since it may be a primary control site for post mortem glycolysis. Phosphorylase in muscle is most active when it is, itself, phosphorylated (a). When dephosphorylated, it is less active (b). In general terms, therefore, phosphorylase is switched on and off by the addition or removal of its phosphate, with on and off states being relative rather than absolute. The activity of phosphorylase b is dependent on the presence of AMP but the activity of phosphorylase a is not. There are two conflicting requirements that make the mechanism for the activation of phosphorylase rather complex. Firstly, since phosphorylase initiates the release of considerable amounts of chemical energy, there must be safeguards to prevent its uncontrolled activity. In stress-susceptible pigs, for example, the uncontrolled activity of anaerobic glycolysis may lead to excessive heat production and to a level of acidity that may soon prove fatal. The conflicting requirement is that the vast amounts of phosphorylase spread through the muscle mass must be rapidly activated by relatively small amounts of adrenaline. The adrenaline activation of severely frightened animals often is called the "fight or flight" response: neither of these responses is likely to be of much survival value if the anaerobic energy supply to body muscles is delayed.
The conflicting demands for fail-safe but rapid activation are satisfied by two particular features of the activation system. Firstly, the conversion of phosphorylase b to phosphorylase a is inhibited locally in each muscle fibre by high concentrations of ATP and glucose-6-phosphate (an intermediate in the conversion of glycogen to lactate). Thus, if the energy released by phosphorylase is not rapidly consumed, the energy release system shuts down. If the energy is used, however, AMP and phosphate (from ATP ADP + P and from ADP AMP + P) further enhance the activation of phosphorylase. Secondly, to enable the rapid activation of phosphorylase throughout the musculature, the relatively small amounts of adrenaline that arrive at the muscle initiate a series of biochemical changes functioning as an amplifier. A small input leads to a large output. Adrenaline causes adenyl cyclase to increase its formation, from ATP, of cyclic AMP. Then cyclic AMP activates protein kinase. With ATP and magnesium ions present, protein kinase then phosphorylates another enzyme, phosphorylase b kinase b. The active form, phosphorylase b kinase a, in the presence of magnesium ions, finally activates phosphorylase b to phosphorylase a. As a final safety factor, if the supply of inorganic phosphate is inadequate, even phosphorylase a will be relatively inactive but the system will be primed for rapid energy production once muscle contraction is initiated.
When animals require energy anaerobically during normal activity, phosphorylase b kinase is activated by calcium ions released from the sarcoplasmic reticulum - normally the trigger for muscle contraction in living muscle. Glycogen granules are closely related to the sarcoplasmic reticulum and to glycogenolytic enzymes as part of a structural complex. The activation system linking muscle contraction to glycogenolysis is short lived to avoid the continuous use and depletion of glycogen reserves. Glycogen is a rapidly available energy source for both brief muscle activity and the early stages of sustained activity. Phosphorylase activity is curtailed by phosphatase, which dephosphorylates phosphorylase a and phosphorylase b kinase a, when muscle activity ceases or an animal recovers from fright. Many features of the system for activating phosphorylase are shared with the activation system for glycogen synthesis, but the shared features are opposite in effect. Thus, the muscle fibre does not attempt to synthesize new glycogen at the same time that it is breaking it down, and vice versa.
Newborn pigs often have difficulty maintaining their blood glucose levels, and may die of hypoglycaemia after even short periods of starvation. Given that they have high glycogen levels at birth, this is not easy to explain, although stored glycogen may simply be inadequate in amount if milk is not available. Despite an adult predisposition to the excessive accumulation of adipose tissue, newborn pigs have very little adipose tissue so that liberation of free fatty acids is severely limited. Most phosphorylase is in the relatively inactive b form just after birth. In adult pigs, hyperglycaemia readily occurs in response to exercise and adrenaline secretion.
In living animals, the lactate produced by contracting muscles is removed by the circulatory system, but first it must travel from the muscle fibre into the interstitial space. Lactate release may be retarded in situations such as exhaustive exercise. A fraction of the lactate leaving a fibre may be in the form of undissociated lactic acid, and this fraction may increase with a rise in external pH. In living animals, blood flow is increased in active muscles (hyperaemia). When hyperaemia occurs as a secondary response to hypoxia, it may be mediated by the release of adenosine (from AMP A + P) into the interstitial fluid between muscle fibres.
On arriving in the liver, lactate may be converted to glucose-6-phosphate, and then stored as glycogen or released as glucose back into the blood. Circulating glucose is available to muscles to be stored as glycogen or used directly as energy for contraction (Cori cycle). After exhaustive exercise, an animal may continue to consume oxygen at an increased rate for some time (oxygen debt). During this recovery period, some lactate may be completely oxidized to release enough energy for the remaining lactate to be converted to glucose (gluconeogenesis) in the liver. When an animal is exsanguinated, the blood can no longer perform its transport function in the Cori cycle, and lactate accumulates in the musculature. There are, however, a number of other possible fates for lactate in living animals.
Heart muscle receives its own blood supply from the coronary artery, straight from the aorta, and cardiac muscle may use circulating lactate as an energy source. The high oxygen concentration in the aorta usually allows complete oxidation of lactate by cardiac muscle. Any lactate remaining in the aorta may not all get pumped to the liver or kidneys to participate in the Cori cycle, because the aorta supplies other major arteries in addition to the hepatic and renal arteries. Much of the circulating lactate may pass through non-contracting muscles that oxidize lactate as an energy substrate, particularly if circulating fatty acids are unavailable. Resting muscles also may take up circulating lactate and reconvert it directly to glycogen, probably via a pathway which is independent of the mitochondrial Krebs' cycle. Finally, also it is possible for all the lactate consumed during an oxygen debt period to be oxidized to CO2.
Contraction causes an initial drop in muscle fibre pH because of the hydrolysis of ATP, but this may be followed by an increase in pH from the breakdown of creatine phosphate or CP. CP acts as a short term store of energy since it has a phosphate that may be transferred to ADP (CP + ADP ATP + C) by the creatine phosphokinase (CPK). CP is the dominant carrier of energy from mitochondria to myofibrils. CPK and creatine are normally contained within muscle fibres, but may leak into the blood from damaged or diseased fibres. In healthy muscle, creatine is slowly but continuously converted to creatinine. Creatinine is lost in the urine in approximate proportion to the muscle mass of the body. The production of lactate following muscle contraction may cause another drop in pH. The interstitial pH between muscle fibres and the pH at the muscle surface are profoundly affected by overall pH levels in the blood.
Now we can see why the pH of meat generally declines after slaughter - the lactate accumulates as a byproduct of the process that releases energy in an attempt to keep the cell alive. And now we can explain how this affects the quality of the meat, firstly, its water-holding capacity.
If dried meat protein is rehydrated by exposure to increasingly damp air, three water compartments may be detected by the way in which water is taken up: 4% of the water becomes firmly bound as a monolayer round muscle proteins, another 4% is taken up as looser second layer, and 10% of the water accumulates loosely between protein molecules.
Water binding capacity is the ability of meat to bind its own water or, under the influence of external forces such as pressure and heat, to bind added water. Water absorption or gelling capacity is the ability of meat to absorb water spontaneously from an aqueous environment. Water binding capacity and water absorption are closely related. Water absorption may be found from the increase in weight and volume of meat samples placed in an aqueous solution, and water binding capacity may be determined by centrifugation or by pressing the fluid from the meat and measuring it.
Water binding capacity is modified by pH and drops from a high around pH 10 to a low at the isoelectric point of meat proteins between pH 5.0 and 5.1. At its isoelectric point a protein bears no net charge and its solubility is minimal. Below pH 5, a value only attained if the pH of a processed meat product is deliberately lowered , water binding capacity starts to increase again. Water absorption follows water binding capacity in this regard. Thus, as the pH of pork declines post mortem, its water binding capacity decreases, and much of the water associated with muscle proteins is free to leave the muscle fibre.
Techniques such as electron microscopy and x-ray diffraction have now been used to study the structural basis of water holding capacity in meat. X-ray diffraction enables the lateral separation of the myofilaments responsible for muscle contraction to be measured in small samples of meat. Towards their isoelectric point, thick and thin filaments in myofibrils move closer together and reduce the water space between them. Thus, as the pH declines post mortem, filaments move closer together, myofibrils shrink and the volume of sarcoplasm increases. Eventually, muscle fibres depleted all their ATP, their membranes no longer confine the cell water, and fluid is lost from the muscle fibre and may contribute to exudate lost from the meat.
The contribution of ante mortem extracellular fluid to meat exudate is unknown, but the extracellular space in muscle is greatly increased after short periods of muscle activity. Thus, the efflux of fluid from the vasculature into the carcass muscles is of potential interest. A high volume of free water within muscles may accelerate the rate of glycolysis.
Water escapes from the spaces between muscle fibre bundles when they are cut, and the drip loss from PSE (pale, soft, exudative) pork is increased to about 1.70% from a normal value of about 0.77% of trimmed carcass weight. In sliced pork, drip losses increase with storage, from 9% after 1 day to 12.3% after 6 days. This creates a serious weight loss from the carcass. In the USA, the estimated incidence of PSE carcasses (18%) and their typical extra shrink loss (5 to 6%) causes a total national loss of about 95 million dollars. Obviously this is only a rough guess, but it serves to emphasize the commercial importance of pH in pork. The excess weight loss from PSE pork during transport, storage and processing could exceed one million kilograms per annum in the USA. The exudate from PSE pork is wasted in most countries, but in Japan it has been used successfully in manufacturing sausages.
Exudate-filled spaces between muscle fibre bundles contribute to the soft texture and easily separated fibre bundles in PSE pork because the amount of water bound within muscle fibres may have an effect on meat tenderness. X-ray diffraction results indicate that the detachment of myosin molecule heads may contribute to the softness of PSE pork.
Effect of pH on meat quality - meat paleness
The paleness of meat increases is inversely proportional to pH. This is caused by the increased scattering of light within the meat. Myofibrils are strongly birefringent (they can rotate polarized light), particularly the A band, and birefringence is inversely proportional to pH, peaking around the isoelectric point. Thus, as pH declines post mortem, the back scattering of light is increased, so that the meat appears more pale, and there is a decrease in forward transmittance of light. Similar changes in light scattering related to pH may be observed in living muscle recovering from contraction fatigue.
The muscle fibres of severely PSE pork have dark-stained deposits caused by the precipitation of sarcoplasmic proteins such as phosphorylase. Myofibrillar proteins also are affected by low pH: (1) ATPase activity may be reduced, (2) interactions between actin and myosin may be changed, and (3) myofibrils are less easily fragmented because Z line disintegration is reduced. Differential scanning calorimetry (a technique that detects molecular changes as proteins are heated) shows that about 50% of the myosin may be denatured in PSE pork. Thus, protein denaturation in severe PSE may increase light scattering and increase the paleness caused by birefringence.
Myoglobin is denatured in PSE pork, probably as a result of a rapid post mortem decline in pH, and may be yet another cause of meat paleness. The pH range at which myoglobin deterioration occurs is increased from 4.3 (normal pork) up to 5.1, although this point is seldom reached even in PSE pork. The conversion of pink myoglobin to pale brown metmyoglobin is accelerated in PSE pork, and this may cause increase paleness.