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TENDERIZING MECHANISMS | Enzymatic
Enzymatic E Huff-Lonergan, Iowa State University, Ames, IA, USA r 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by DL Hopkins, E Huff-Lonergan, volume 3, pp 1363–1369, © 2004, Elsevier Ltd.
Glossary Calpains A group of enzymes, including m-calpain and m-calpain, that when activated by calcium degrade the cytoskeletal proteins. Calpastatins Endogenous inhibitor of m- and m-calpains. Cathepsins Cysteine proteases that degrade many different types of proteins. Contractile proteins Muscle proteins, such as actin and myosin, that are directly involved in the contractile process and minimally involved in tenderization.
Cytoskeletal proteins A set of structural proteins (includes titin, nebulin, and desmin) are denatured by calpains during tenderization. Endogenous proteases Proteases involved in muscle remodeling in life that take on the role of degrading cytoskeletal proteins postmortem. Exogenous enzymes Protease enzymes usually of plant material that when applied to meat degrade myofibrillar and connective tissue proteins.
Introduction
Enzymatic Tenderization
The process of meat tenderization is a complex phenomenon. It is accomplished naturally by the presence of endogenous enzymes and it can be augmented by the application of exogenous enzymes, typically purified from plant sources. In postmortem muscle, natural tenderization begins at slaughter and continues to occur while the meat is held at refrigerated temperatures over the next 2–3 weeks. This natural tenderizing process is often referred to as ‘aging’ the product. Tenderization via natural aging is done by the actions of enzymes in the muscle that function to regulate the growth and repair of living muscle, often by serving a role in the initiation of the removal of damaged proteins so that new proteins can be inserted into the appropriate muscle structure. The majority of these endogenous enzymes act on the structures of the myofibril, the main contractile organelle of the muscle cell. The tenderization that occurs via these endogenous enzymes starts at death and is essentially slowed or has ceased by 7 days postmortem in most species. Tenderization via the application of exogenous enzymes is generally less specific than endogenous enzymes. These enzymes target not only myofibrillar proteins but also the connective tissue proteins of the muscle. Most plant enzymes have the capacity to tenderize the product to a greater degree than is possible with endogenous enzymes, leading to the need for the processor to carefully monitor the process to avoid ‘overtenderizing’ the product and creating an overly soft, or even ‘mushy’ texture to the meat. Both types of enzymes are important in the production of tender meat products. An appropriate understanding of the enzyme systems being relied on for tenderization is important to allow maximal tenderness development in a given product.
Role of Endogenous Proteases
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During aging of meat, major structural changes occur in the muscle tissue. Many of these changes are associated with myofibrils, the contractile elements of muscle cells, and their linkages to the cell membrane (sarcolemma) through the cytoskeletal network. As myofibrils make up approximately 80% of the volume of the muscle cell, disruption of myofibrillar structure and, in particular, the cytoskeletal network has the greatest influence on meat tenderness during aging. Changes in the connective tissue are minimal during aging, although the amount of connective tissue that varies between different cuts influences the basic tenderness. Degradation of some proteins linking myofibrils to the sarcolemma and to each other has been observed during the early postmortem period. Other changes that are correlated with increased tenderness include breakages within the myofibrils themselves, particularly within the I-band. These breakages lead to increased fragility and fragmentation of the myofibrils. The histochemical and biochemical evidence indicates that much of the tenderization associated with postmortem aging is due to the action of the enzymes, which are known to be endogenous to the muscle. Some of the major myofibrillar and cytoskeletal proteins that are known to be degraded early during postmortem aging include (but are not limited to) titin, nebulin, desmin, and troponin-T. Interestingly, the most abundant proteins of the myofibril, actin and myosin, are not significantly degraded during postmortem aging.
Cathepsins Early research on the mechanism responsible for the development of meat tenderness during aging focused on the cathepsins. The cathepsins are endogenous proteases found in
Encyclopedia of Meat Sciences, Volume 3
doi:10.1016/B978-0-12-384731-7.00248-8
Tenderizing Mechanisms | Enzymatic lysosomes in living muscles. The most frequently studied cathepsins with respect to meat tenderness include cathepsins B, D, L, and H. The majority of the cathepsins are active at acidic pH values (usually between pH 5 and 6). These proteases were originally of interest because in living tissue lysosomes are one of the major sites of protein degradation. Additionally, these enzymes are active at acidic pH values, near the pH values found in postrigor meat.
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be stressed that other enzyme systems like the serine proteases and the proteasomes may have a role in postmortem proteolysis. Recent studies on the proteasome endopeptidase complex (EC 3.4.25.1) in bovine skeletal muscle have suggested that it could play a role in protein degradation late in the aging process. At this stage, however, there is insufficient evidence to substantiate involvement of these other enzyme systems.
Characteristics of the cathepsins Cathepsin B (EC 3.4.22.1) is a glycoprotein that has a molecular weight of approximately 25 000. It is a cysteine protease and has been shown to have activity over the pH range of 4–6.5. Cathepsin B degrades many proteins in the muscle, including myosin and actin. Cathepsin D (EC 3.4.23.5) is an aspartyl protease with an approximate molecular weight of 42 000. This glycoprotein has been shown to have activity over the pH range 2.5–5.0. Like cathepsin B, cathepsins D and L (EC 3.4.22.15) will degrade myosin and actin. Cathepsin L has also been shown to have activity against α-actinin, troponin-T, and troponin-I and is active over the pH range 3.0–6.5. Cathepsin H (EC 3.4.22.16) is a cysteine protease with a molecular weight of approximately 25 000 and is active from pH 5.5 to 6.5. Like all of the cathepsins mentioned in this article, cathepsin H has a high specific activity against myosin. In addition to degrading many myofibrillar proteins, several of the cathepsins have the ability to hydrolyze connective tissues, especially cathepsins B and L. Cathepsin B has been shown to have activity against collagen and proteoglycans. Cathepsin L has been shown to have activity against collagen, proteoglycans, and elastin. Some studies have indicated that limited proteolytic alteration of collagen occurs especially after long periods of aging.
Cystatin Muscle also contains a family of potent cysteine-type protease inhibitors collectively known as cystatins. These cystatins are found distributed throughout the muscle cell and in the living muscle are thought to modulate the activity of cysteine proteases. In postmortem muscle, the pH should favor the activity of many of the cathepsins and because of this it might be expected that myosin and possibly actin would be among the proteins degraded. However, there is little evidence of either myosin or actin degradation in postmortem muscle. The presence of cystatins may help explain why there is little evidence of cathepsin activity in postmortem muscle. Although there is some evidence that proteins like myosin and actin can be degraded when meat is stored at relatively high temperatures or for extremely long periods of time, myosin and actin are not degraded in the first week after slaughter under normal storage conditions. Therefore, the current evidence implicating the cathepsins in the tenderization of meat during the early stages of normal postmortem aging (when most tenderization occurs) is somewhat limited. Owing to this, in recent years, a much larger effort has been focused on the calpain enzyme system, a system that seems to degrade the same proteins that are degraded in postmortem muscle under normal meat storage conditions. Unlike the cathepsins, calpains degrade the majority of proteins into relatively few fragments, which is similar to what is seen in meat. It should
Calpain System The endogenous calpain system has been implicated as playing a major role in the proteolysis of muscle proteins under postmortem conditions. Some of the proteins that have been shown to be substrates of calpains include titin, nebulin, troponin-T, desmin, synemin, talin, and vinculin. Most of these proteins have structural roles within the muscle cell. Degradation of these proteins has been associated with a weakening of the muscle cell and the myofibrillar structure and with tenderness.
Calpain enzymes The calpain system is composed of several isoforms of tissuespecific and ubiquitous calcium-dependent cysteine proteases (calpains, EC 3.4.22.17), and their specific competitive inhibitor, calpastatin. The two best-characterized isoforms of calpains are the so-called ubiquitous forms m-calpain and m-calpain. They are referred to as ubiquitous because they are found in most tissues. These proteases are named m-calpain and m-calpain in reference to the amount of calcium they require for activation in vitro. m-Calpain requires between 3 and 50-mM calcium for half-maximal activity, whereas m-calpain requires between 0.4 and 0.8-mM calcium for half-maximal activity. Both m- and m-calpain are heterodimers composed of an 80- and a 28-kDa subunit. The 28-kDa subunit is identical in both m-calpain and m-calpain. The C-terminus of this subunit has four sets of amino acid sequences that predict calcium-binding EF hand structures; however, the exact function of this subunit is not known. The 80-kDa subunits of m- and m-calpains are similar, but are encoded for by different genes. The 80-kDa subunit is composed of four domains. Domain I, the N-terminal domain, has no sequence homology to any known polypeptide. Domain II is the catalytic domain and contains a cysteine residue as well as a histidine and asparagine residue that form a catalytic triad similar to that seen in other cysteine proteinases (including papain). Recent determination of the crystal structure of m-calpain has shown that in the absence of calcium, critical regions of the catalytic domain, domain II may be misaligned. The region of domain II (referred to as domain IIa) that contains the cysteine residue and the region of domain II that contains the critical histidine and aspargine residues (domain IIb) appear to be held slightly apart and rotated, potentially rendering the protease inactive. Release of specific structural constraints, possibly triggered by calcium, may play an important role in conferring activity to the enzyme. It has been speculated that conformational changes in domain I and III may play critical roles in calpain activation by their potential influence on the active site conformation in domain II. The
Tenderizing Mechanisms | Enzymatic
amino acid sequence of domain III is not homologous to any other known protein, but has two sets of sequences that predict EF hand Ca2 þ -binding sites. The crystal structure of mcalpain suggests that this domain resembles the C2-domain found in several Ca2 þ -regulated proteins like protein kinase C. Domain IV is a calmodulin-like domain that has four sets of sequences that predict EF hand Ca2 þ -binding sites. The protease m-calpain is active under in vitro conditions mimicking postmortem muscle pH, ionic strength, and temperature. Although the calpain enzymes are active under postmortem conditions, their level of activity is somewhat compromised by the low pH and high ionic strength conditions that develop within the meat during storage. During postmortem storage in beef and pork, m-calpain has been shown to become increasingly associated with the myofibril. It has been suggested that this myofibril-associated m-calpain may indeed be active. Although calcium is necessary for their activation, both mand m-calpain will also autolyze (selfdegrade) when incubated with calcium. Autolysis reduces the mass of the 80-kDa subunit of m-calpain to 76 kDa, and the mass of the 80-kDa subunit of m-calpain is reduced to 78 kDa. The 28-kDa subunit of both enzymes is reduced to 18 kDa. Brief autolysis also reduces the Ca2 þ concentration required for half-maximal activity of either enzyme. Extended autolysis leads to inactivation of the enzyme. Autolysis occurs under situations that allow activity, both in living cells and in postmortem muscle, but the significance of this is not clear. Both autolyzed and unautolyzed forms of the enzymes have been shown to have activity. However, the autolyzed form of m-calpain appears to be more hydrophobic and binds tightly to subcellular organelles, including myofibrils. The presence of the autolyzed form of m-calpain in postmortem tissue has been suggested to indicate that activity has occurred. One of the tissue-specific forms of calpain, often referred to as p94 or novel calpain-1, deserves mention. This musclespecific calpain isoform was the first tissue-specific calpain identified. The messenger ribonucleic acid for p94 in muscle has been reported to be as much as 10 times that of either m- or m-calpain. The p94 peptide appears to be a single polypeptide that has a structure similar to the large catalytic subunit of m- and m-calpain. It has a predicted molecular weight of 94 000 – slightly larger than the catalytic subunit of the ubiquitous calpains. This larger size is due to three unique regions: one in the N-terminus, one in the catalytic domain, and one at the interface of domains III and IV. Unlike m- and m-calpain, which are sarcoplasmic proteins, p94 is associated with the myofibrillar fraction. More specifically, p94 appears to be closely localized if not bound to the large myofibrillar protein titin. This calpain has proven to be very difficult to study as it autolyzes rapidly during conventional extraction procedures and so it has been very difficult to ascertain its role, if any, in proteolysis/tenderization.
calpastatin molecule may inhibit at least four calpain molecules. Calpastatin plays a major role in the regulation of the expression of calpain proteolytic activity. The amount of calcium required to allow half-maximal binding of calpastatin to calpains is generally lower than that required for half-maximal activity of the unautolyzed and autolyzed forms of m-calpain and for half-maximal activity of autolyzed m-calpain. Calpastatin binding is reversible as calcium chelators can cause calpastatin to dissociate from calpain. The level of inhibitory activity of calpastatin declines during postmortem aging (Figure 1). The level of inhibitory activity of calpastatin that remains at approximately 24 h after slaughter is associated with tenderness. Calpastatin is actually degraded in postmortem muscle and this rate of degradation is related to the rate at which it loses its ability to inhibit calpain. Both degradation of calpastatin and its loss of inhibitory activity are related to the rate of proteolysis and tenderization observed in meat. There is good evidence that calpains are at least partially responsible for the degradation of calpastatin. Currently, the conditions that promote calpain degradation of calpastatin in postmortem muscle have not been defined. Even though there has been much research done on the calpain system over the years, relatively little is known about its regulation. Certainly, the endogenous inhibitor of the calpains, calpastatin, is involved, but there is evidence to suggest that other mechanisms may also be important, particularly in meat. Environmental factors in the early postmortem muscle cell can influence calpain activity and inhibition of calpain by calpastatin. These can include factors like pH and ionic strength. Therefore, it is important to examine other mechanisms that may affect calpain activity to fully explain how calpain activity is regulated in meat. Alterations in pH or ionic strengths in early postmortem meat have the potential to cause conformational changes allowing for increased hydrophobicity. This increased hydrophobicity has been hypothesized to lead to aggregation of the enzyme. Likewise, pH/ionic
3.5 Calpastatin activity (units mg−1 heated protein)
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3 2.5 2 1.5 1 0.5 0
Calpastatin
Calpastatin, the endogenous inhibitor of m- and m-calpain, has been found in all the tissues that contain calpains. Calpastatin in the skeletal muscle is a single polypeptide that contains within its structure four repeating domains that each has calpain inhibitory activity. Theoretically, then one
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Figure 1 Effect of aging time on calpastatin activity from porcine longissimus muscle. Data taken from Melody, J.L., Lonergan, S.M., Rowe, L.J., et al., 2004. Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. Journal of Animal Science 82, 1195–1205.
Tenderizing Mechanisms | Enzymatic strength changes may alter the conformation of substrate proteins and render them less susceptible to cleavage by m-calpain. A slightly accelerated pH decline has been shown to be associated with more rapid attainment of ultimate tenderness and more rapid proteolysis. However, a greatly exaggerated rate of pH decline, like the rapid pH decline that results in pale, soft, and exudative pork, seems to result in very limited aging potential. Hypothetically, a rapid pH decline would lead to an increased level of activity of catheptic enzymes and increased proteolysis; however, in most cases, this does not seem to occur. The product that has an exceptionally rapid pH decline has often been shown to also exhibit limited proteolysis of muscle proteins associated with tenderization. Low pH values have been shown to destabilize m-calpain and to promote more rapid autolysis and/or activation and subsequent inactivation in in vitro studies and may do the same in muscle tissue. Therefore, the rate of pH decline may play a very pivotal role in the attainment of ultimate tenderness.
Caspase Enzyme System Caspases are a family of enzymes that are involved in apoptosis, or programmed cells death. Cell death by apoptosis is characterized by a systematic and organized dismantling of a cell. Common hallmarks of apoptosis include shrinkage of the cell, cell membrane blebbing, chromatin condensation, damage to deoxyribonucleic acid, and the formation of apoptotic bodies without causing a generalized inflammatory response. Apoptosis is adenosine triphosphate (ATP) dependent, which may seem to be incongruous with postmortem tissue; however, in most postmortem muscle ATP can be produced for a period of time via anaerobic glycolysis, which may be different from the classical necrotic state. Necrosis is typically caused by a catastrophic loss of energy and is a passive process. It is accompanied by a loss of membrane integrity and swelling of organelles. Thus, in reality, as the loss of energy in the early postmortem cell is a gradual process, the argument could be made that early postmortem tissue resides in a ‘nether region’ between the two states of apoptosis and necrosis. The apoptotic process is choreographed by the caspases. Caspases are cysteine proteases that require their substrates to have aspartate residues. There are more than a 1000 substrates that have been identified for the caspases, and they include myofibrillar and cytoskeltetal proteins. Activation of caspases can be initiated by pathological events including ischemic/ hypoxic conditions. There are two general classes of caspases, initiatior caspases (caspases 8, 9, 10, and 12) and effector or executioner caspases (caspases 3, 6, and 7). Initiator caspases are activated when a stimulus for apoptotic events is received. Once they have been stimulated, these initiator caspases activate the executioner caspases by cleaving a linker that separates the small and large subunits of the catalytic domain. Once activated, the executioner caspases are responsible for the enzymatic cleavage of substrates that are ascribed to the caspase system. Since the early 2000s, caspases have been suggested to play a role in postomortem proteolysis related to tenderness. Many of the caspase substrates are proteins that have been shown to be at least partially degraded during the early postmortem
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period. These include (but are not limited to) actin, troponinT, desmin, and myosin light chains. The question of whether or not caspase enzymes or calpain enzymes are the predominant systems involved in early postmortem proteolysis has been hotly debated. It has proven difficult to rule out either system. Indeed, there is evidence that the two systems work together in the cell. For example, it has been shown that the calpain inhibitor, calpastatin, can be cleaved by caspases 1, 3, and 7, thereby directly influencing the activity of the calpain system. Thus, it may not be a question of which enzyme system is responsible for postmortem proteolysis, but rather, how do the two systems work together. For a more detailed discussion of relevant research on this topic, the reader is referred to the Further Reading section of this article.
Exogenous Enzymes In addition to allowing the endogenous enzymes to tenderize meat, exogenous enzymes, mostly of plant origin, have been used to augment the tenderization process. The most commonly used plant enzymes are papain (from papaya), bromelain (from pineapple), and ficin (from figs/ficus). More recently, actinidin (from kiwi) has been investigated, as has been zingibain (from ginger). Papain, bromelain, and ficin are all cysteine proteases and have a broad spectrum activity, cleaving a wide variety of bonds, thus degrading a large number of muscle proteins. These proteases are active in the pH range found in meat (papain, pH range 5.8–7; bromelian, pH range 5–7; and ficin, pH range 5–8). The ideal temperature range for these proteins is approximately 50–60 1C, making them maximally active on heating. Actinidin has a higher pH range than the aforementioned enzymes (ideal range is 7–10, but can have activity at pH 5–7) but the temperature range is similar. Zingibain is obtained from a crude extract of ginger. It has a maximum activity at pH 6–7 and a temperature of 60 1C. These plant-derived enzymes are very effective tenderizers. In addition to acting on myofibrillar proteins, most will also act very effectively on connective tissue proteins as well. In fact, one of the major challenges of using these enzymes is countering the effect of overtenderizing. However, continued research in the application of these enzymes is yielding better ways to utilize them. For further detailed information, the reader is referred to a review by Bekhit et al. (2013).
Conclusions On the basis of available data the major candidate to explain proteolysis of myofibrillar proteins and thus tenderization postrigor is the calpain protease system. The mode of action of the calpains is not yet fully defined and questions remain as to the role of m-calpain given the in vitro requirement for a Ca2 þ ion concentration exceeding that observed in postmortem muscle. The existence of the calpains in living muscle and other tissues is consistent with the involvement of these enzymes in tenderization (which reflects protein degradation) but suggests a mode of action more intricate than previously thought.
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Tenderizing Mechanisms | Enzymatic
An increase in ionic strength postmortem may assist degradation of proteins by enzymes and also lead to solubilization of proteins, but in itself is not the sole mechanism causing tenderization. Equally, changes in the binding of actomyosin (the complex of contractile proteins formed at rigor) or cleavage of myofibrillar proteins due to Ca2 þ ions do not offer plausible explanations for the mechanism that results in tenderization. It also appears that the cathepsin proteases are unlikely to have a role in early postmortem cleavage of proteins (proteolysis) and thus tenderization and this also applies to other enzyme groups such as the serine proteases, proteasomes and matrix metallopeptidases. Recent work on caspases has indicated that they may be worthy of further investigation, especially with respect to their interaction with the calpain system. The use of exogenous plant enzymes is a useful method to tenderize meat cuts beyond what can be achieved via postmortem aging alone and is a viable method to use particularly for cuts that have high amounts of connnective tissue.
See also: Carcass Composition, Muscle Structure, and Contraction. Conversion of Muscle to Meat: Aging; Glycogen; Glycolysis; Rigor Mortis, Cold, and Rigor Shortening. Tenderizing Mechanisms: Chemical; Mechanical
Further Reading Bekhit, A., Hopkins, D., Geesink, G., Bekhit, A., 2013. Exogenous proteases for meat tenderization. Critical Reviews in Food Science and Nutrition. doi:10.1080/ 10408398.2011.623247. Goll, D.E., Boehm, M.L., Geesink, G.H., Thompson V.F., 1997. What causes postmortem tenderization? In: Proceedings 50th Reciprocal Meat Conference, Iowa, pp. 60−67. Ames, IA: American Meat Science Association. Goll, D.E., Thompson, V.F., Li, H., Wei, W., Cong, J., 2003. The calpain system. Physiological Reviews 83, 731–801. Goll, D.E., Thompson, V.F., Taylor, R.G., Ouali, A., Chou, R.G., 1999. The calpain system in skeletal muscle. In: Wang, K.K., Yuen, R.W. (Eds.), Calpain: Pharmacology and Toxicology of Calcium-Dependent Protease. Philadelphia, PA: Taylor and Francis, Publishers, pp. 127–160. Hopkins, D.L., Thompson, J.M., 2001. Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels. Meat Science 59, 99–209.
Hopkins, D.L., Thompson, J.M., 2001. The relationship between tenderness, proteolysis, muscle contraction and dissociation of actomyosin. Meat Science 57, 1–12. Hopkins, D.L., Thompson, J.M., 2002. Factors contributing to proteolysis and disruption of myofibrillar proteins and the impact of tenderisation in beef and sheep meat. Australian Journal of Agricultural Research 53, 149–166. Huff-Lonergan, E., Mitsuhashi, T., Beekman, D.D., Parrish, Jr., F.C., Olson, D.G., 1996. Proteolysis of specific muscle proteins by m-calpain at low pH and temperature is similar to degradation in postmortem muscle. Journal of Animal Science 74, 993–1008. Huff-Lonergan, E.J., Lonergan, S.M., 1999. Postmortem mechanisms of meat tenderization: The roles of the structural proteins and the calpain system. In: Xiong, Y.L., Ho, C.-T., Shahidi, F. (Eds.), Quality Attributes of Muscle Foods. New York, NY: Kluwer Academic/Plenum Publishers, pp. 229–251. Kemp, C.M., Parr, T., 2012. Advances in apoptotic mediated proteolysis in meat tenderisation. Meat Science 92, 252–259. doi:10.1016/j.meatsci.2012.03.013. Kemp, C.M., Sensky, P.L., Bardsley, R.G., Buttery, P.J., Parr, T., 2010. Tenderness − An enzymatic view. Meat Science 84, 248–256. Lamare, M., Taylor, R.G., Farout, L., Briand, Y., Briand, M., 2002. Changes in proteasome activity during postmortem aging of bovine muscle. Meat Science 61, 199–204. Moldovenu, T., Hosfield, C., Lim, D., et al., 2002. Ca2 þ switch aligns the active site of calpain. Cell 108, 649–660. Monin, G., Ouali, A., 1991. Muscle differentiation and meat quality. In: Lawrie, R. (Ed.) Developments in Meat Science, vol. 5. London: Elsevier Applied Science, pp. 89–157. Ouali, A., 1992. Proteolytic and physiochemical mechanisms involved in meat texture development. Biochimie 74, 251–265. Strobl, S., Fernandez-Catalan, C., Braun, M., et al., 2000. The crystal structure of calcium free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proceedings of the National Academy of Sciences USA 97, 588–592. Takahashi, K., 1996. Structural weakening of skeletal muscle tissue during postmortem ageing of meat: The non-enzymatic mechanism of meat tenderization. Meat Science 43, s67–s80. Takahashi, K., 1999. Mechanism of meat tenderization during post-mortem ageing: Calcium theory. In: Proceedings 45th International Congress of Meat Science and Technology, pp. 230−235. Yokohama: Wageningen Academic Publishers. Uytterhaegen, L., Claeys, E., Demeyer, D., 1994. Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility. Journal of Animal Science 72, 1209–1223.
Relevant Website www.calpain.net Calpain Research Portal.
Glossary Calpains A group of enzymes, including m-calpain and m-calpain, that when activated by calcium degrade the cytoskeletal proteins. Calpastatins Endogenous inhibitor of m- and m-calpains. Cathepsins Cysteine proteases that degrade many different types of proteins. Contractile proteins Muscle proteins, such as actin and myosin, that are directly involved in the contractile process and minimally involved in tenderization.
Cytoskeletal proteins A set of structural proteins (includes titin, nebulin, and desmin) are denatured by calpains during tenderization. Endogenous proteases Proteases involved in muscle remodeling in life that take on the role of degrading cytoskeletal proteins postmortem. Exogenous enzymes Protease enzymes usually of plant material that when applied to meat degrade myofibrillar and connective tissue proteins.
Introduction
Enzymatic Tenderization
The process of meat tenderization is a complex phenomenon. It is accomplished naturally by the presence of endogenous enzymes and it can be augmented by the application of exogenous enzymes, typically purified from plant sources. In postmortem muscle, natural tenderization begins at slaughter and continues to occur while the meat is held at refrigerated temperatures over the next 2–3 weeks. This natural tenderizing process is often referred to as ‘aging’ the product. Tenderization via natural aging is done by the actions of enzymes in the muscle that function to regulate the growth and repair of living muscle, often by serving a role in the initiation of the removal of damaged proteins so that new proteins can be inserted into the appropriate muscle structure. The majority of these endogenous enzymes act on the structures of the myofibril, the main contractile organelle of the muscle cell. The tenderization that occurs via these endogenous enzymes starts at death and is essentially slowed or has ceased by 7 days postmortem in most species. Tenderization via the application of exogenous enzymes is generally less specific than endogenous enzymes. These enzymes target not only myofibrillar proteins but also the connective tissue proteins of the muscle. Most plant enzymes have the capacity to tenderize the product to a greater degree than is possible with endogenous enzymes, leading to the need for the processor to carefully monitor the process to avoid ‘overtenderizing’ the product and creating an overly soft, or even ‘mushy’ texture to the meat. Both types of enzymes are important in the production of tender meat products. An appropriate understanding of the enzyme systems being relied on for tenderization is important to allow maximal tenderness development in a given product.
Role of Endogenous Proteases
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During aging of meat, major structural changes occur in the muscle tissue. Many of these changes are associated with myofibrils, the contractile elements of muscle cells, and their linkages to the cell membrane (sarcolemma) through the cytoskeletal network. As myofibrils make up approximately 80% of the volume of the muscle cell, disruption of myofibrillar structure and, in particular, the cytoskeletal network has the greatest influence on meat tenderness during aging. Changes in the connective tissue are minimal during aging, although the amount of connective tissue that varies between different cuts influences the basic tenderness. Degradation of some proteins linking myofibrils to the sarcolemma and to each other has been observed during the early postmortem period. Other changes that are correlated with increased tenderness include breakages within the myofibrils themselves, particularly within the I-band. These breakages lead to increased fragility and fragmentation of the myofibrils. The histochemical and biochemical evidence indicates that much of the tenderization associated with postmortem aging is due to the action of the enzymes, which are known to be endogenous to the muscle. Some of the major myofibrillar and cytoskeletal proteins that are known to be degraded early during postmortem aging include (but are not limited to) titin, nebulin, desmin, and troponin-T. Interestingly, the most abundant proteins of the myofibril, actin and myosin, are not significantly degraded during postmortem aging.
Cathepsins Early research on the mechanism responsible for the development of meat tenderness during aging focused on the cathepsins. The cathepsins are endogenous proteases found in
Encyclopedia of Meat Sciences, Volume 3
doi:10.1016/B978-0-12-384731-7.00248-8
Tenderizing Mechanisms | Enzymatic lysosomes in living muscles. The most frequently studied cathepsins with respect to meat tenderness include cathepsins B, D, L, and H. The majority of the cathepsins are active at acidic pH values (usually between pH 5 and 6). These proteases were originally of interest because in living tissue lysosomes are one of the major sites of protein degradation. Additionally, these enzymes are active at acidic pH values, near the pH values found in postrigor meat.
439
be stressed that other enzyme systems like the serine proteases and the proteasomes may have a role in postmortem proteolysis. Recent studies on the proteasome endopeptidase complex (EC 3.4.25.1) in bovine skeletal muscle have suggested that it could play a role in protein degradation late in the aging process. At this stage, however, there is insufficient evidence to substantiate involvement of these other enzyme systems.
Characteristics of the cathepsins Cathepsin B (EC 3.4.22.1) is a glycoprotein that has a molecular weight of approximately 25 000. It is a cysteine protease and has been shown to have activity over the pH range of 4–6.5. Cathepsin B degrades many proteins in the muscle, including myosin and actin. Cathepsin D (EC 3.4.23.5) is an aspartyl protease with an approximate molecular weight of 42 000. This glycoprotein has been shown to have activity over the pH range 2.5–5.0. Like cathepsin B, cathepsins D and L (EC 3.4.22.15) will degrade myosin and actin. Cathepsin L has also been shown to have activity against α-actinin, troponin-T, and troponin-I and is active over the pH range 3.0–6.5. Cathepsin H (EC 3.4.22.16) is a cysteine protease with a molecular weight of approximately 25 000 and is active from pH 5.5 to 6.5. Like all of the cathepsins mentioned in this article, cathepsin H has a high specific activity against myosin. In addition to degrading many myofibrillar proteins, several of the cathepsins have the ability to hydrolyze connective tissues, especially cathepsins B and L. Cathepsin B has been shown to have activity against collagen and proteoglycans. Cathepsin L has been shown to have activity against collagen, proteoglycans, and elastin. Some studies have indicated that limited proteolytic alteration of collagen occurs especially after long periods of aging.
Cystatin Muscle also contains a family of potent cysteine-type protease inhibitors collectively known as cystatins. These cystatins are found distributed throughout the muscle cell and in the living muscle are thought to modulate the activity of cysteine proteases. In postmortem muscle, the pH should favor the activity of many of the cathepsins and because of this it might be expected that myosin and possibly actin would be among the proteins degraded. However, there is little evidence of either myosin or actin degradation in postmortem muscle. The presence of cystatins may help explain why there is little evidence of cathepsin activity in postmortem muscle. Although there is some evidence that proteins like myosin and actin can be degraded when meat is stored at relatively high temperatures or for extremely long periods of time, myosin and actin are not degraded in the first week after slaughter under normal storage conditions. Therefore, the current evidence implicating the cathepsins in the tenderization of meat during the early stages of normal postmortem aging (when most tenderization occurs) is somewhat limited. Owing to this, in recent years, a much larger effort has been focused on the calpain enzyme system, a system that seems to degrade the same proteins that are degraded in postmortem muscle under normal meat storage conditions. Unlike the cathepsins, calpains degrade the majority of proteins into relatively few fragments, which is similar to what is seen in meat. It should
Calpain System The endogenous calpain system has been implicated as playing a major role in the proteolysis of muscle proteins under postmortem conditions. Some of the proteins that have been shown to be substrates of calpains include titin, nebulin, troponin-T, desmin, synemin, talin, and vinculin. Most of these proteins have structural roles within the muscle cell. Degradation of these proteins has been associated with a weakening of the muscle cell and the myofibrillar structure and with tenderness.
Calpain enzymes The calpain system is composed of several isoforms of tissuespecific and ubiquitous calcium-dependent cysteine proteases (calpains, EC 3.4.22.17), and their specific competitive inhibitor, calpastatin. The two best-characterized isoforms of calpains are the so-called ubiquitous forms m-calpain and m-calpain. They are referred to as ubiquitous because they are found in most tissues. These proteases are named m-calpain and m-calpain in reference to the amount of calcium they require for activation in vitro. m-Calpain requires between 3 and 50-mM calcium for half-maximal activity, whereas m-calpain requires between 0.4 and 0.8-mM calcium for half-maximal activity. Both m- and m-calpain are heterodimers composed of an 80- and a 28-kDa subunit. The 28-kDa subunit is identical in both m-calpain and m-calpain. The C-terminus of this subunit has four sets of amino acid sequences that predict calcium-binding EF hand structures; however, the exact function of this subunit is not known. The 80-kDa subunits of m- and m-calpains are similar, but are encoded for by different genes. The 80-kDa subunit is composed of four domains. Domain I, the N-terminal domain, has no sequence homology to any known polypeptide. Domain II is the catalytic domain and contains a cysteine residue as well as a histidine and asparagine residue that form a catalytic triad similar to that seen in other cysteine proteinases (including papain). Recent determination of the crystal structure of m-calpain has shown that in the absence of calcium, critical regions of the catalytic domain, domain II may be misaligned. The region of domain II (referred to as domain IIa) that contains the cysteine residue and the region of domain II that contains the critical histidine and aspargine residues (domain IIb) appear to be held slightly apart and rotated, potentially rendering the protease inactive. Release of specific structural constraints, possibly triggered by calcium, may play an important role in conferring activity to the enzyme. It has been speculated that conformational changes in domain I and III may play critical roles in calpain activation by their potential influence on the active site conformation in domain II. The
Tenderizing Mechanisms | Enzymatic
amino acid sequence of domain III is not homologous to any other known protein, but has two sets of sequences that predict EF hand Ca2 þ -binding sites. The crystal structure of mcalpain suggests that this domain resembles the C2-domain found in several Ca2 þ -regulated proteins like protein kinase C. Domain IV is a calmodulin-like domain that has four sets of sequences that predict EF hand Ca2 þ -binding sites. The protease m-calpain is active under in vitro conditions mimicking postmortem muscle pH, ionic strength, and temperature. Although the calpain enzymes are active under postmortem conditions, their level of activity is somewhat compromised by the low pH and high ionic strength conditions that develop within the meat during storage. During postmortem storage in beef and pork, m-calpain has been shown to become increasingly associated with the myofibril. It has been suggested that this myofibril-associated m-calpain may indeed be active. Although calcium is necessary for their activation, both mand m-calpain will also autolyze (selfdegrade) when incubated with calcium. Autolysis reduces the mass of the 80-kDa subunit of m-calpain to 76 kDa, and the mass of the 80-kDa subunit of m-calpain is reduced to 78 kDa. The 28-kDa subunit of both enzymes is reduced to 18 kDa. Brief autolysis also reduces the Ca2 þ concentration required for half-maximal activity of either enzyme. Extended autolysis leads to inactivation of the enzyme. Autolysis occurs under situations that allow activity, both in living cells and in postmortem muscle, but the significance of this is not clear. Both autolyzed and unautolyzed forms of the enzymes have been shown to have activity. However, the autolyzed form of m-calpain appears to be more hydrophobic and binds tightly to subcellular organelles, including myofibrils. The presence of the autolyzed form of m-calpain in postmortem tissue has been suggested to indicate that activity has occurred. One of the tissue-specific forms of calpain, often referred to as p94 or novel calpain-1, deserves mention. This musclespecific calpain isoform was the first tissue-specific calpain identified. The messenger ribonucleic acid for p94 in muscle has been reported to be as much as 10 times that of either m- or m-calpain. The p94 peptide appears to be a single polypeptide that has a structure similar to the large catalytic subunit of m- and m-calpain. It has a predicted molecular weight of 94 000 – slightly larger than the catalytic subunit of the ubiquitous calpains. This larger size is due to three unique regions: one in the N-terminus, one in the catalytic domain, and one at the interface of domains III and IV. Unlike m- and m-calpain, which are sarcoplasmic proteins, p94 is associated with the myofibrillar fraction. More specifically, p94 appears to be closely localized if not bound to the large myofibrillar protein titin. This calpain has proven to be very difficult to study as it autolyzes rapidly during conventional extraction procedures and so it has been very difficult to ascertain its role, if any, in proteolysis/tenderization.
calpastatin molecule may inhibit at least four calpain molecules. Calpastatin plays a major role in the regulation of the expression of calpain proteolytic activity. The amount of calcium required to allow half-maximal binding of calpastatin to calpains is generally lower than that required for half-maximal activity of the unautolyzed and autolyzed forms of m-calpain and for half-maximal activity of autolyzed m-calpain. Calpastatin binding is reversible as calcium chelators can cause calpastatin to dissociate from calpain. The level of inhibitory activity of calpastatin declines during postmortem aging (Figure 1). The level of inhibitory activity of calpastatin that remains at approximately 24 h after slaughter is associated with tenderness. Calpastatin is actually degraded in postmortem muscle and this rate of degradation is related to the rate at which it loses its ability to inhibit calpain. Both degradation of calpastatin and its loss of inhibitory activity are related to the rate of proteolysis and tenderization observed in meat. There is good evidence that calpains are at least partially responsible for the degradation of calpastatin. Currently, the conditions that promote calpain degradation of calpastatin in postmortem muscle have not been defined. Even though there has been much research done on the calpain system over the years, relatively little is known about its regulation. Certainly, the endogenous inhibitor of the calpains, calpastatin, is involved, but there is evidence to suggest that other mechanisms may also be important, particularly in meat. Environmental factors in the early postmortem muscle cell can influence calpain activity and inhibition of calpain by calpastatin. These can include factors like pH and ionic strength. Therefore, it is important to examine other mechanisms that may affect calpain activity to fully explain how calpain activity is regulated in meat. Alterations in pH or ionic strengths in early postmortem meat have the potential to cause conformational changes allowing for increased hydrophobicity. This increased hydrophobicity has been hypothesized to lead to aggregation of the enzyme. Likewise, pH/ionic
3.5 Calpastatin activity (units mg−1 heated protein)
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Calpastatin, the endogenous inhibitor of m- and m-calpain, has been found in all the tissues that contain calpains. Calpastatin in the skeletal muscle is a single polypeptide that contains within its structure four repeating domains that each has calpain inhibitory activity. Theoretically, then one
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Figure 1 Effect of aging time on calpastatin activity from porcine longissimus muscle. Data taken from Melody, J.L., Lonergan, S.M., Rowe, L.J., et al., 2004. Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. Journal of Animal Science 82, 1195–1205.
Tenderizing Mechanisms | Enzymatic strength changes may alter the conformation of substrate proteins and render them less susceptible to cleavage by m-calpain. A slightly accelerated pH decline has been shown to be associated with more rapid attainment of ultimate tenderness and more rapid proteolysis. However, a greatly exaggerated rate of pH decline, like the rapid pH decline that results in pale, soft, and exudative pork, seems to result in very limited aging potential. Hypothetically, a rapid pH decline would lead to an increased level of activity of catheptic enzymes and increased proteolysis; however, in most cases, this does not seem to occur. The product that has an exceptionally rapid pH decline has often been shown to also exhibit limited proteolysis of muscle proteins associated with tenderization. Low pH values have been shown to destabilize m-calpain and to promote more rapid autolysis and/or activation and subsequent inactivation in in vitro studies and may do the same in muscle tissue. Therefore, the rate of pH decline may play a very pivotal role in the attainment of ultimate tenderness.
Caspase Enzyme System Caspases are a family of enzymes that are involved in apoptosis, or programmed cells death. Cell death by apoptosis is characterized by a systematic and organized dismantling of a cell. Common hallmarks of apoptosis include shrinkage of the cell, cell membrane blebbing, chromatin condensation, damage to deoxyribonucleic acid, and the formation of apoptotic bodies without causing a generalized inflammatory response. Apoptosis is adenosine triphosphate (ATP) dependent, which may seem to be incongruous with postmortem tissue; however, in most postmortem muscle ATP can be produced for a period of time via anaerobic glycolysis, which may be different from the classical necrotic state. Necrosis is typically caused by a catastrophic loss of energy and is a passive process. It is accompanied by a loss of membrane integrity and swelling of organelles. Thus, in reality, as the loss of energy in the early postmortem cell is a gradual process, the argument could be made that early postmortem tissue resides in a ‘nether region’ between the two states of apoptosis and necrosis. The apoptotic process is choreographed by the caspases. Caspases are cysteine proteases that require their substrates to have aspartate residues. There are more than a 1000 substrates that have been identified for the caspases, and they include myofibrillar and cytoskeltetal proteins. Activation of caspases can be initiated by pathological events including ischemic/ hypoxic conditions. There are two general classes of caspases, initiatior caspases (caspases 8, 9, 10, and 12) and effector or executioner caspases (caspases 3, 6, and 7). Initiator caspases are activated when a stimulus for apoptotic events is received. Once they have been stimulated, these initiator caspases activate the executioner caspases by cleaving a linker that separates the small and large subunits of the catalytic domain. Once activated, the executioner caspases are responsible for the enzymatic cleavage of substrates that are ascribed to the caspase system. Since the early 2000s, caspases have been suggested to play a role in postomortem proteolysis related to tenderness. Many of the caspase substrates are proteins that have been shown to be at least partially degraded during the early postmortem
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period. These include (but are not limited to) actin, troponinT, desmin, and myosin light chains. The question of whether or not caspase enzymes or calpain enzymes are the predominant systems involved in early postmortem proteolysis has been hotly debated. It has proven difficult to rule out either system. Indeed, there is evidence that the two systems work together in the cell. For example, it has been shown that the calpain inhibitor, calpastatin, can be cleaved by caspases 1, 3, and 7, thereby directly influencing the activity of the calpain system. Thus, it may not be a question of which enzyme system is responsible for postmortem proteolysis, but rather, how do the two systems work together. For a more detailed discussion of relevant research on this topic, the reader is referred to the Further Reading section of this article.
Exogenous Enzymes In addition to allowing the endogenous enzymes to tenderize meat, exogenous enzymes, mostly of plant origin, have been used to augment the tenderization process. The most commonly used plant enzymes are papain (from papaya), bromelain (from pineapple), and ficin (from figs/ficus). More recently, actinidin (from kiwi) has been investigated, as has been zingibain (from ginger). Papain, bromelain, and ficin are all cysteine proteases and have a broad spectrum activity, cleaving a wide variety of bonds, thus degrading a large number of muscle proteins. These proteases are active in the pH range found in meat (papain, pH range 5.8–7; bromelian, pH range 5–7; and ficin, pH range 5–8). The ideal temperature range for these proteins is approximately 50–60 1C, making them maximally active on heating. Actinidin has a higher pH range than the aforementioned enzymes (ideal range is 7–10, but can have activity at pH 5–7) but the temperature range is similar. Zingibain is obtained from a crude extract of ginger. It has a maximum activity at pH 6–7 and a temperature of 60 1C. These plant-derived enzymes are very effective tenderizers. In addition to acting on myofibrillar proteins, most will also act very effectively on connective tissue proteins as well. In fact, one of the major challenges of using these enzymes is countering the effect of overtenderizing. However, continued research in the application of these enzymes is yielding better ways to utilize them. For further detailed information, the reader is referred to a review by Bekhit et al. (2013).
Conclusions On the basis of available data the major candidate to explain proteolysis of myofibrillar proteins and thus tenderization postrigor is the calpain protease system. The mode of action of the calpains is not yet fully defined and questions remain as to the role of m-calpain given the in vitro requirement for a Ca2 þ ion concentration exceeding that observed in postmortem muscle. The existence of the calpains in living muscle and other tissues is consistent with the involvement of these enzymes in tenderization (which reflects protein degradation) but suggests a mode of action more intricate than previously thought.
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An increase in ionic strength postmortem may assist degradation of proteins by enzymes and also lead to solubilization of proteins, but in itself is not the sole mechanism causing tenderization. Equally, changes in the binding of actomyosin (the complex of contractile proteins formed at rigor) or cleavage of myofibrillar proteins due to Ca2 þ ions do not offer plausible explanations for the mechanism that results in tenderization. It also appears that the cathepsin proteases are unlikely to have a role in early postmortem cleavage of proteins (proteolysis) and thus tenderization and this also applies to other enzyme groups such as the serine proteases, proteasomes and matrix metallopeptidases. Recent work on caspases has indicated that they may be worthy of further investigation, especially with respect to their interaction with the calpain system. The use of exogenous plant enzymes is a useful method to tenderize meat cuts beyond what can be achieved via postmortem aging alone and is a viable method to use particularly for cuts that have high amounts of connnective tissue.
See also: Carcass Composition, Muscle Structure, and Contraction. Conversion of Muscle to Meat: Aging; Glycogen; Glycolysis; Rigor Mortis, Cold, and Rigor Shortening. Tenderizing Mechanisms: Chemical; Mechanical
Further Reading Bekhit, A., Hopkins, D., Geesink, G., Bekhit, A., 2013. Exogenous proteases for meat tenderization. Critical Reviews in Food Science and Nutrition. doi:10.1080/ 10408398.2011.623247. Goll, D.E., Boehm, M.L., Geesink, G.H., Thompson V.F., 1997. What causes postmortem tenderization? In: Proceedings 50th Reciprocal Meat Conference, Iowa, pp. 60−67. Ames, IA: American Meat Science Association. Goll, D.E., Thompson, V.F., Li, H., Wei, W., Cong, J., 2003. The calpain system. Physiological Reviews 83, 731–801. Goll, D.E., Thompson, V.F., Taylor, R.G., Ouali, A., Chou, R.G., 1999. The calpain system in skeletal muscle. In: Wang, K.K., Yuen, R.W. (Eds.), Calpain: Pharmacology and Toxicology of Calcium-Dependent Protease. Philadelphia, PA: Taylor and Francis, Publishers, pp. 127–160. Hopkins, D.L., Thompson, J.M., 2001. Inhibition of protease activity 2. Degradation of myofibrillar proteins, myofibril examination and determination of free calcium levels. Meat Science 59, 99–209.
Hopkins, D.L., Thompson, J.M., 2001. The relationship between tenderness, proteolysis, muscle contraction and dissociation of actomyosin. Meat Science 57, 1–12. Hopkins, D.L., Thompson, J.M., 2002. Factors contributing to proteolysis and disruption of myofibrillar proteins and the impact of tenderisation in beef and sheep meat. Australian Journal of Agricultural Research 53, 149–166. Huff-Lonergan, E., Mitsuhashi, T., Beekman, D.D., Parrish, Jr., F.C., Olson, D.G., 1996. Proteolysis of specific muscle proteins by m-calpain at low pH and temperature is similar to degradation in postmortem muscle. Journal of Animal Science 74, 993–1008. Huff-Lonergan, E.J., Lonergan, S.M., 1999. Postmortem mechanisms of meat tenderization: The roles of the structural proteins and the calpain system. In: Xiong, Y.L., Ho, C.-T., Shahidi, F. (Eds.), Quality Attributes of Muscle Foods. New York, NY: Kluwer Academic/Plenum Publishers, pp. 229–251. Kemp, C.M., Parr, T., 2012. Advances in apoptotic mediated proteolysis in meat tenderisation. Meat Science 92, 252–259. doi:10.1016/j.meatsci.2012.03.013. Kemp, C.M., Sensky, P.L., Bardsley, R.G., Buttery, P.J., Parr, T., 2010. Tenderness − An enzymatic view. Meat Science 84, 248–256. Lamare, M., Taylor, R.G., Farout, L., Briand, Y., Briand, M., 2002. Changes in proteasome activity during postmortem aging of bovine muscle. Meat Science 61, 199–204. Moldovenu, T., Hosfield, C., Lim, D., et al., 2002. Ca2 þ switch aligns the active site of calpain. Cell 108, 649–660. Monin, G., Ouali, A., 1991. Muscle differentiation and meat quality. In: Lawrie, R. (Ed.) Developments in Meat Science, vol. 5. London: Elsevier Applied Science, pp. 89–157. Ouali, A., 1992. Proteolytic and physiochemical mechanisms involved in meat texture development. Biochimie 74, 251–265. Strobl, S., Fernandez-Catalan, C., Braun, M., et al., 2000. The crystal structure of calcium free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proceedings of the National Academy of Sciences USA 97, 588–592. Takahashi, K., 1996. Structural weakening of skeletal muscle tissue during postmortem ageing of meat: The non-enzymatic mechanism of meat tenderization. Meat Science 43, s67–s80. Takahashi, K., 1999. Mechanism of meat tenderization during post-mortem ageing: Calcium theory. In: Proceedings 45th International Congress of Meat Science and Technology, pp. 230−235. Yokohama: Wageningen Academic Publishers. Uytterhaegen, L., Claeys, E., Demeyer, D., 1994. Effects of exogenous protease effectors on beef tenderness development and myofibrillar degradation and solubility. Journal of Animal Science 72, 1209–1223.
Relevant Website www.calpain.net Calpain Research Portal.