inosine monophosphate (IMP), inosine and eventually hypoxanthine.
As more muscles enter rigor mortis the whole carcass becomes stiffer and ‘sets’. This ‘setting’ is to a degree aided by the fat in the carcass becoming firmer as it cools.
10°C increase results in a more than doubling of the final tenderness achieved in a certain time. Chicken meat achieves 80% of its maximum tenderness about 8 h after death of the bird, whereas beef takes 10 days to reach the same level of tenderness (Table 5.2).
These differences in the rate of tenderization lead to different recommended ‘ageing’ times prior to cooking the meat. Keeping meat at refrigerated temperatures is expensive. There is the cost of the storage space, the cost of refrigeration and the inevitable weight loss from the surface of the carcass by evaporation of water, or the loss of exudate from butchered joints. A compromise has therefore to be reached commercially to produce meat with acceptable tenderness in a reasonable period of time (Table 5.3).
The ageing process continues irrespective of the size of the joints or at what stage of the marketing chain the meat has reached. Ageing time can therefore include time spent in distribution and retailing.
However, it does not proceed in the frozen state but will continue on thawing if the normal ageing process was interrupted by the freezing.
The optimization of the tenderization process was discussed by Dransfield (1994a).
The process of conditioning
Conditioning is the term applied to this natural process of tenderization when meat is stored or aged post-rigor. The tenderization could be attributable to two types of process: changes in the connective tissue components of the meat or weakening of the myofibrils. Nishimura et al. (1998) have suggested that conditioning takes place in two phases.
Table 5.2. The rate of tenderization of meat from different species [from Dransfield (1994a) based on figures in Dransfield and MacFie (1980) and Dransfield et al.
(1981)].
Species Days at 1°C to reach 80% of ‘maximum’ tenderness
Beef 10.0
Rabbit 9.5
Sheep 7.7
Pig 4.2
Chicken 0.3
Table 5.3. Recommended conditioning times (days) for pork, lamb and beef.
Pork 4–10
Lamb 7–14
Beef 10–21
There is a rapid first phase caused by changes in the myofibrillar component and a slower second phase caused by structural weakening of the intramuscular connective tissue. However, of these, the changes in the myofibrillar component are generally thought to be the more important and, in fact, only very small changes can be seen in the major connective tissue components such as collagen. Nevertheless, there is some cleavage of collagen cross-links and small structural changes can be seen under the electron microscope after extended ageing times.
In contrast, larger changes in the myofibrillar component can be seen. The attachments of the thin (actin) filaments to the Z discs show some breakdown (Boyer-Berri and Greaser, 1998) and there is an increase in the amount of water-soluble nitrogen compounds. However, the muscle does not become more extensible during conditioning and therefore the conditioning process is not associated with any dissociation of the actomyosin. The thick and thin filaments remain locked together by the myosin cross-bridges. Tenderization is not caused by the filaments regaining the ability to slide over one another.
The mechanism of tenderization Calpains and cathepsins
Tenderization results from the activities of proteolytic enzymes present in the muscles. Their normal role is in the breakdown and recycling of proteins which occurs continuously in all living tissues. There are two main sorts of enzyme involved, cathepsins and calpains, of which, at least in red meat species and poultry, the calpains are thought to be more important. However, cathepsins may be more important in the post-mortem degradation of fish muscle and possibly in the tenderiza- tion that occurs in meat kept at high temperatures. Cathepsins occur in the lysosomes in the sarcoplasm. They are released post mortemand have maximum activity in mildly acid conditions. They are known to degrade troponin-T, some collagen cross-links and mucopoly- saccharides of the connective tissue ground substance. They only appear to degrade actin and myosin below a pH of 5 so this is unlikely to occur under normal conditions in meat. The calpains are activated by calcium ions and have maximum activity in neutral to alkaline conditions. Originally they were referred to as calcium-activated sarco- plasmic factor (CASF). They occur in two forms, m-calpain activated by high (millimolar) concentrations of calcium ions (1–2 mM), and µ-calpain activated by low (micromolar) concentrations (50–100 µM).
The relative importance of the two forms in promoting tenderization is a little unclear. It has generally been thought that the m-calpains were the more important. However, this view has recently been questioned in a paper which also gives an excellent review of our current
knowledge of calpain biochemistry (Boehm et al., 1998). The calpains are located in the region of the Z lines, which they degrade as well as promoting breakdown of other proteins such as tropomyosin and titin (connectin). The calpains are inhibited by calpastatin. High calpastatin activity reduces the extent of proteolysis in muscles.
Evidence for the importance of the calpains
There are various pieces of evidence supporting the importance of the calpains in the tenderization of meat by breaking down parts of the myofibrillar component of the muscle. Substances that inhibit proteo- lysis by calpains also inhibit tenderization. The rates of proteolysis in pork, lamb and beef correlate with overall differences in inherent tender- ness. Thus, pork has the highest rate and in general is most tender. The extent of calpain-induced proteolysis in humped types of cattle (Bos indicus, e.g. Brahman) and European cattle (Bos taurus) correlates with texture. Proteolysis is greater in B. taurus, the meat from which is more tender. Bos indicus cattle have higher activities of the calpastatin inhibitor in their muscles (Shackelford et al., 1991). The meat from animals treated with !-adrenergic agonists undergoes little or no proteo- lysis and is relatively tough (Wheeler and Koohmaraie, 1992). As we have seen previously (Chapter 2), !-adrenergic agonists are growth modifying compounds that enhance muscle development. It is likely that they do this by reducing the normal activity of the proteolytic enzymes involved in the continual processes of accretion and breakdown of the muscle proteins. This reduction in activity probably persists post mortem. The ‘callipyge’ gene in sheep causes muscle hypertrophy.
However, meat from lambs carrying the gene is tougher than normal and undergoes conditioning at a slower rate. This has been attributed to the higher calpastatin activity in the muscles (Koohmaraie et al., 1995).
Stress just before slaughter may increase calpastatin levels and produce tougher meat through reduced conditioning (Sensky et al., 1998).
Possible action of calpains post mortem
After exhaustion of ATP and the development of rigor mortis, the membrane systems of the sarcoplasmic reticulum and mitochondria no longer take up or sequester calcium ions. These are released into the sarcoplasm and bathe the myofibrils (Jeacocke, 1993). The increased calcium concentration activates the µ-calpains allowing proteolysis to proceed. Normally the calpains are inhibited by being bound to calpastatin. Calcium removes this inhibition. Calpastatin is itself eventually broken down by the calpains and the m-calpain may also be converted to µ-calpain by hydrolysis. Calpain activity is promoted by higher calcium levels, higher pH and temperature, and reduced calpastatin activity. From a practical point of view the enzyme activity in meat can be enhanced by infusing the carcasses post mortem with
0.3 M calcium chloride (see Chapter 8). Conversely, infusion of 10 mM EDTA (ethylene diamine tetra-acetic acid), which sequesters calcium, blocks enzyme activity. The effect of temperature is particularly important. From 0° to 40°C the rate of activity more than doubles for every 10°C rise. Keeping meat at high temperatures post-rigor can thus promote rapid tenderization. At 10°C, beef can be adequately aged in 4 days rather than the 10 days at about 1°C. However, these high temperatures are likely to have implications for spoilage. Koohmaraie (1996) gives a very good review of the biochemistry of tenderization.
Other possible mechanisms of tenderization
Many of the processes associated with tenderization, and especially the role and control of the calpains, are still poorly understood. Other mechanisms for tenderization that do not involve the calpains have been postulated. Calcium might, by stimulating muscle contraction pre-rigor, mechanically damage the meat structure and so make it more tender.
Calcium might also cause disruption of the lysosomes and release cathepsins, which then could perhaps cause some protein breakdown.