Beef Tenderness –

An Introduction - Myostatin, Calpain, Calpastatin – to the article (below) by

Dr. Koohmaraie “Biochemical Factors Regulating the Toughening and Tenderization Processes of Meat”

There are two distinct times when we can affect beef tenderness.

One is prior to slaughter and the other is post-mortem…

Prior to slaughter, we can design breeding programs to produce cattle that carry genetic influences on beef tenderness – such as variation in genes for myostatin and calpain.

Dr. Wheeler of the USDA and his colleagues at the US Meat Animal Reserach Center have shown that the non-functional myostatin gene as exhibited by the Piedmontese breed has the largest impact on beef tenderness of any single genetic feature researched to date.

The effects of calpain, and it’s inhibitor calpastatin, come into play post-mortem and are proven to have an effect on tenderness by degrading key muscle proteins during ageing. With genetic markers that have recently become available and others that will be discovered in the future, it may be possible to select breeding animals that carry the more positive calpain and less of the inhibitor calpastatin to create a positive effect on beef tenderness in the offspring.

However, breeding programs that introduce the non-functional myostatin gene (by using a ‘2-copy’ or homozygous sire or dam to produce ‘1-copy’ heterozygous offspring) can immediately provide improved beef tenderness, genetically, due mostly to the reduction in connective tissue in the muscle.

Dr. Koohmaraie has commented to NAPA that the Piedmontese non-functional myostatin provides a much more dramatic positive effect on beef tenderness than any other genetic feature he is aware of at this time.

Dr. Shackleford’s research indicates pre-slaughter effects on tenderness also must include feeding regimes and livestock handling, in addition to the slaughter process itself. There are many ways in which we can take a “genetically tender” beef animal and create a “tough” beef product.

During and after slaughter, we have even more opportunity to create “tough” or “tender” beef. Chilling conditions and ageing time play huge roles. Cooking methods have an effect, as well.

The following report by Dr. Koohmaraie discusses various influences on beef tenderness, from a biochemical viewpoint.

As a brief introduction to “calpain” and “calpastatin”, the following remarks may be useful.

Vicki Johnson - Exec. Director - NAPA

The Agricultural Research Service (ARS), USDA’s chief research agency, named Dr. Koohmaraie as an “Outstanding Senior Research Scientist of 2000.”

He’s now serving as Acting Center Director of the Roman L. Hruska U.S. Meat Animal Research Center (MARC) in Clay Center, Nebraska.

He has won an agency award for his research and project leadership to enhance meat quality and safety. Koohmaraie’s team not only developed the first rapid tests for detecting pathogens on beef, pork and poultry carcasses, but also provided techniques to greatly reduce or eliminate E. coli O157:H7 in red meat.

Koohmaraie’s key accomplishments include leadership in research on meat tenderness and development of a rapid tenderness-based beef classification system, a rapid method to predict saleable meat from a beef carcass and ways to reduce pathogenic bacteria on meat. He’s best known among scientists for his pioneering studies on enzymes that affect muscle growth and meat tenderness. He has authored or coauthored more than 300 publications.

Calpain, a calcium-dependent protease, has been recognized as a key player in postmortem tenderization of skeletal muscle (Koohmaraie, 1996). Calpastatin is a widely distributed endogenous inhibitor protein that specifically acts on calpain. The calpain system, and its inhibitor, calpastatin, is believed to be the primary proteolytic enzyme system involved in postmortem tenderization of aged beef (Koohmaraie et al., 1991). Koohmaraie (1996) indicated that the degradation of structural muscle proteins by calpain is responsible for meat tenderization during postmortem storage of meat. Calpastatin activity at 24 h postmortem is inversely proportional to postmortem tenderization and accounts for a greater proportion of the variation in beef tenderness than any other single variable (Koohmaraie, 1994).

Biochemical Factors Regulating the Toughening and Tenderization Processes of Meat

M. Koohmaraie

USDA-ARS, Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, NE 68933-0166, USA

Abstract

The purpose of this manuscript is to present a brief review of the biochemical basis for longissimus toughening and tenderization processes. Also, to explore the potential technologies that can be developed based on this knowledge to reduce variation in tenderness, thus, improving consumer acceptance of meat. Results suggest that after slaughter longissimus has low to intermediate shear force value (probably tender). Rigor development-induced changes increase its shear force. Maximum toughness is observed between 12 to 24 h postmortem. The toughening process seems to occur equally in all carcasses. Postmortem storage at refrigerated conditions tenderizes longissimus. Postmortem tenderization is caused by enzymatic degradation of key myofibrillar and associated proteins. The function of these proteins is to maintain the structural integrity of myofibrils. Current data indicates that u-calpain is responsible for degradation of these proteins. Unlike the toughening process, there exists a large variation in the rate and extent of tenderization which is responsible for variation in tenderness at the consumer level. Potential strategies for the control of the variation in meat tenderness are discussed.

Introduction

Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and, perhaps more importantly, the adoption of such information by the meat industry.

Eating satisfaction results from the interaction of tenderness, juiciness, and flavor. However, as outlined previously (Koohmaraie, 1995), the problem of consumer dissatisfaction will be solved only when we solve the problem of unacceptable variation in meat tenderness. The objectives of this manuscript are to briefly review the biochemical basis of meat tenderness, and to indicate the potential technologies for improving meat tenderness that could be developed using this information. This is not meant to be a comprehensive review of the meat tenderness literature.

The variation in meat tenderness either exists at slaughter, is created during postmortem storage, or is a combination of both. Certainly, meat preparation by consumers can also be a considerable source of variation in meat tenderness; however, that is not a researchable problem, but a problem that can and should be addressed through consumer education.

In an attempt to determine the source(s) of variation in meat tenderness, we conducted an experiment which demonstrated that lamb longissimus has an intermediate shear force value immediately after slaughter (5.07 kg), toughens during the first 24 h (maximum toughness was achieved at 9 to 24 h; 8.66 kg), and then becomes tender during postmortem storage at 4C (3.10 kg). Because sarcomere length (SL) decreased (from at-death length of 2.24 um to 24 h postmortem length of 1.69 um) as shear force increased, we concluded that sarcomere shortening during rigor development is the cause of lamb longissimus toughening from 0 to 24 h postmortem (Wheeler and Koohmaraie, 1994).

To test the accuracy of our conclusion regarding the cause of meat toughening, we determined shear force values at various postmortem times in the lamb longissimus that was not allowed to shorten (Koohmaraie et al., 1996b). Results indicated that shear force value does not increase during rigor development when muscle is prevented from shortening. A conservative interpretation of this data is that postmortem meat toughening is completely alleviated in the absence of shortening, but shortening is not necessarily the cause of toughening. A more direct interpretation is that since preventing sarcomere shortening prevented meat toughening, the toughening that occurs during the first 24 h of slaughter is the result of sarcomere shortening.

The conservative interpretation would suggest that factors other than sarcomere shortening are responsible for meat toughening. The question is what are these factors?

A possible candidate could be the hypothesis, proposed by Goll et al. (1995), that toughening during the first 24 h postmortem is caused by a change in the actin/myosin interaction from a weak-binding state to a strong binding state, and that this increase in toughness does not have to be, but may be accompanied by and exacerbated by shortening. The results of Koohmaraie et al. (1996b) do not support this aspect of the Goll et al, (1995) hypothesis, because no toughening occurred when shortening was prevented. The state of actin/myosin interaction hypothesis could explain the results of those studies that show no relationship between shortening and toughness when measured very early postmortem (i.e., prior to tenderization).

On the other hand, there is substantial evidence that supports sarcomere shortening as the cause of meat toughening. The best evidence is the classic work of Locker and Hagyard (1963). Nevertheless, the literature is far from unanimous on the relationship between SL and meat tenderness. There are numerous examples to demonstrate that increased toughness is associated with decreasing SL (Herring et al., 1965; Hostetler et al., 1972; Bouton et al., 1973; Davis et al., 1979) and many examples that do not support such a relationship (Culler et al., 1978; Parrish et al., 1979; Smith et al., 1979; Seideman et al., 1987; Shackelford et al., 1994; Koohmaraie et al., 1995). The question is why does this disagreement exit? Marsh and Leet (1966) demonstrated a clear-cut relationship between sarcomere length and meat tenderness in excised tissue. In excised muscle, maximum toughness was shown to develop at about 40% shortening. With shortening more or less than 40%, the meat was more tender (Marsh and Leet, 1966).It is in the case of muscle attached to the skeleton that a different relationship has been reported. The best case is the report of Smulders et al. (1990). They examined the relationship between SL and tenderness in unaged and aged meat of 67 beef carcasses.

They reported a strong relationship between SL and tenderness in unaged meat (48 h postmortem). The r = -0.50 (based on shear force) was observed for all 67 animals.

However, when they separated carcasses based on their pH value at 3 h postmortem, an entirely different picture emerged.

The correlation between shear force and SL in carcasses with pH value of 6.3 or greater was -0.84 in unaged meat and -0.80 in aged meat (i.e., between SL measured at 48 h postmortem and shear force measured after 48 h or 14 d of aging).

However, no relationship was found between these traits in carcasses whose 3 h pH value was less than 6.3. These authors concluded that

“Tenderness is very highly dependent on shortening in slow- glycolysing muscles, but it is completely independent in muscles of more rapid pH decline.” They concluded that the tenderness of fast-glycolysing carcasses is due to more rapid aging. In my opinion, assuming that there exists a significant range in SL, the relationship between SL and meat tenderness is modified by the extent of postmortem tenderization. The strongest relationship would then be expected when very little postmortem tenderization has occurred (e.g., after one day postmortem in lamb, r = - 0.52 between shear force and SL;) and the weakest relationship is expected when extensive postmortem tenderization has taken place.

Accepting the above premises, toughening during the first 24 h postmortem can then be attributed to rigor- induced sarcomere shortening.

I believe that this rigor- induced toughening occurs equally in all carcasses.

This does not mean that there is no variation in SL within the longissimus of a given carcass. Rather, a greater proportion of the variation in sarcomere length is accounted for by within- animal variation than between- animal variation.

In other words, due to a large variation in factors affecting rigor development, there exists a large variation in SL within a given longissimus. However, the overall mean SL for a given muscle from one carcass is probably the same as the mean of SL for another carcass and, thus, rigor- induced toughening is also the same. The shear force value at any given time is the balance between two opposing processes: SL shortening and tenderization (see below).

However, because the events leading to tenderization begin before the SL shortening process is completed, it may be difficult to demonstrate that all longissimus reaches the same level of rigor- induced toughening. To conclusively demonstrate this point, one must examine these events in carcasses that do not undergo postmortem tenderization. Lambs carrying the callipyge gene may prove to be a good model to test the accuracy of this hypothesis because postmortem tenderization occurs to a very limited extent in these carcasses (Koohmaraie et al., 1995, 1996a),

The Tenderization Phase

Sometime after death, an opposing process called tenderization begins and will continue for some time postmortem. To maximize the benefits of postmortem storage on meat tenderness, beef should be stored for 10-14 d, lamb for 7-10 d, and pork for 5 d.

Unlike the toughening phase, the tenderization phase does not occur equally in all animals. In fact, it is well documented that there is a large variation in the rate and extent of postmortem tenderization (for review see Koohmaraie , 1992a,b; 1995).

It is this variability in the tenderization process that results in inconsistency in meat tenderness at the consumer level. To solve this problem, we must identify the reasons for the variability in the rate and extent of postmortem tenderization so that the tenderization process can be manipulated to equalize it between carcasses and/or develop the necessary technology to identify those carcasses that will not respond to postmortem tenderization. Without this information, we will continue to have inconsistency in meat tenderness at the consumer level, and branded product (these may exist in countries other than the U.S.) and, thus, value-based marketing will not be possible.

Mechanisms of postmortem tenderization.

The mechanisms of meat tenderization during storage of carcasses at refrigerated temperatures has been researched by various laboratories (for review see Penny, 1980; Davey, 1983; Goll et al., 1983; 1991, 1995; Greaser, 1986; Koohmaraie 1988, 1992, 1994, 1995; Koohmaraie et al., 1995; Ouali, 1990, 1992,; Taylor et al., 1995a). Current evidence suggests that proteolysis of key myofibrillar and associated proteins is the cause of meat tenderization. These proteins are involved in inter- (e.g., desmin and vinculin) and intra-myofibril (e.g., titin, nebulin, and possibly troponin-T) linkages or linking myofibrils to the sarcolemma by costameres (e.g., vinculin, dystrophin), and the attachment of muscle cells to the basal lamina (e.g., laminin, fibronectin and the newly described 550 kDa protein [Hattori et al., 1995]).

The function of these proteins is to maintain the structural integrity of myofibrils (for review see Price, 1991). Degradation of these proteins would, therefore, cause weakening of myofibrils and, thus, tenderization.

Although the list of these proteins may change over the years, I believe the principle will stand the test of time; that is , proteolysis of key myofibrillar and associated proteins is responsible for postmortem tenderization.

There has been considerable debate about the specific proteases responsible for these changes.

A protease must meet certain criteria to be considered a possible candidate for involvement in postmortem tenderization (Koohmaraie, 1988). Goll et al. (1983) provided the logic for the first criteria which is that the protease must be endogenous to skeletal muscle cell.

Secondly, the protease must have the ability to reproduce postmortem changes in myofibrils in an in vitro setting under optimum conditions. Finally, the protease must have access to myofibrils in tissue. If a protease does not have these characteristics, it can not be considered as a candidate in the postmortem tenderization process.

Likewise, if a protease meets these criteria, it would be impossible to exclude its possible involvement in the tenderization process.

Of all the potential candidates (Koohmaraie 1988, 1992a,b, 1994), calpains are the only proteases that meet all of the above requirements.

Based on the results of numerous experiments reported by different laboratories, it can be concluded that proteolysis of key myofibrillar proteins by u- calpain is the underlying mechanism of meat tenderization that occurs during storage of meat at refrigerated temperatures. There is much evidence in support of proteolysis causing tenderization and that it is mediated by calpains (for review see Penny, 1980; Goll et al. 1983; 1991, 1995; Koohmaraie 1988, 1992, 1994, 1995; Koohmaraie et al., 1995; Ouali, 1990, 1992; Taylor et al., 1995a).

In spite of overwhelming evidence in support of the calpain proteolytic system as the underlying mechanism of postmortem proteolysis, there still exist some doubts. Some of these are legitimate and will have to be addressed before the calpain theory can become fully convincing. Some of these are based on the following: 1) u- calpain is so rapidly inactivated that it can not account for tenderization beyond 24 to 48 h postmortem; 2) how could u-calpain ever be active when muscle contains twice as much calpastatin as u-calpain activity?; and 3) if u- calpain is indeed involved in postmortem proteolysis, why is m-calpain not degraded during postmortem storage? Some of these questions can be answered easily by using existing data and others will require additional data.

One of the arguments is that not enough u- calpain is available to cause tenderization after 24 to 48 h (i.e., the question #1 above). This argument is based on data that uses perhaps the least sensitive methodology to quantify u- calpain activity (i.e., hydrolysis of casein and quantification of released polypeptides after TCA precipitation). Use of a more sensitive quantification method (e.g., radiolabeled casein) indicates that indeed skeletal muscles contain significant u- calpain activity, even after extended storage at 4C (about 5- 10% remains after 14 d; Koohmaraie, Arbona and Whipple; unpublished data).

Therefore, the inability to detect u- calpain activity during postmortem storage with such an insensitive method should not be used as the basis for drawing far reaching- conclusions. In fact, because u- calpain autolysis and inactivation is an intermolecular process, it will not go to completion, and, therefore, u- calpain will retain some of its activity even after extensive autolysis (Cottin et al., 1986; Inomata et al; 1988,; Edmunds et al., 1991; Nishimura and Goll, 1991; Koohmaraie, 1992).

Unlike u- calpain, m- calpain autolysis is an intramolecular process; extensive autolysis will result in complete inactivation of m- calpain (Inomata et al; 1988, Cottin et al., 1991; Edmunds et al., 1991; Nishimura and Goll, 1991; Koohmaraie, 1992).

Therefore, once m- calpain is exposed to sufficient calcium it will undergo autolysis which results in its complete inactivation.

Since even after extended storage all m- calpain activity can be recovered and because m- calpain is completely inactivated when sufficient calcium is present (infusion of carcasses with calcium chloride; Koohmaraie et al., 1989), I conclude that u- calpain and not m- calpain is responsible for meat tenderization .

Another frequently mentioned argument against calpain involvement in postmortem tenderization is that muscle contains an excess of calpastatin relative to u- calpain and, therefore, u- calpain can never be active (i.e., the question #2 above).

Firstly, this issue is species dependent. The ratio of calpastatin:u- calpain is about 4:1, 2.5:1 and 1.5:1 in beef, lamb and pork muscle, respectively (Ouali and Talmant, 1990; Koohmaraie et al., 1991). Secondly, an important clarification is needed. The data reported by Ouali and Talmant (1990) and Koohmaraie et al. (1991) and most, if not all, of the literature uses m- calpain to quantify calpastatin activity.

This is a very important point, since it takes twice as much calpastatin to inhibit u- calpain as to inhibit m- calpain (Koohmaraie, unpublished data).

Therefore, the actual ratio of calpastatin:u- calpain is only one-half of that mentioned above, i.e., about 2:1, 1.25:1, and 0.75:1 in beef, lamb. and pork muscle, respectively.

The argument regarding excess calpastatin activity is not as significant as it first appears and, in fact, at most, the ratio is only 2:1 in beef. These ratios are consistent with the rate of postmortem proteolysis and tenderization in these three species (Dransfield et al., 1981; Etherington et al., 1987; Koohmaraie et al., 1991). We are in the process of determining the ability of the calpains to use each other as substrates to help answer question #3) above.

In my opinion, current data indicates that calpains (and more specifically, u- calpain) are the only proteases that are directly involved in the events leading to meat tenderization. Because myofibrils are a poor substrate for the multicatalytic protease complex (MCP) and because MCP does not degrade the same proteins that are degraded postmortem (Koohmaraie, 1992c; Taylor et al., 1995b), MCP can not have a direct role in postmortem proteolysis that results in meat tenderization. Also, we have ruled out a primary role for MCP in postmortem calpastatin degradation (Doumit and Koohmaraie, 1996), but not a secondary role (further degradation of calpastatin fragments generated by calpain).

Because MCP is not active at pH less than 7.0, it is doubtful if they play any role in postmortem tissue (Tanaka et al., 1988). With regard to lysosomal proteases, until it is clearly documented that they are released from lysosomes (in living muscle, lysosomal proteases are normally located in lysosomes and, presumably, have to be released to have access to myofribrils) and an adequate explanation is provided for lack of actin and myosin degradation (cathepsins degrade myosin and actin efficiently, but neither are degraded during postmortem storage), no role, primary or secondary, can be assigned to these proteases.

Strategies for Eliminating Inconsistency in Tenderness

Based on the information presented, strategies for enhancing meat tenderness might include steps to prevent/minimize the toughening phase or accelerate/enhance the tenderization phase.

One method of preventing the development of toughness is to freeze carcasses immediately after slaughter and then store them at subfreezing temperatures to prevent thaw-shortening when carcasses are thawed. Koohmaraie et al. (1996b) demonstrated that meat does not toughen when sarcomere shortening is prevented.

The principal source of error in cooking of prerigor meat is excision- and heat- induced shortening. Locker (1985) stated that “Tenderness relates to cooked meat, but cooking in a prerigor state involves such a dramatic modification of the myofibrils that the resultant material is sheer artifact.”

Thus, to prevent excision- and heat- induced shortening, we developed and used a novel approach to measure tenderness of meat having prerigor sarcomere lengths (Wheeler and Koohmaraie, 1994).

The protocol involved clamping of muscle sections while on the carcass, excision, brief storage at - 30C, unclamping and storing muscle sections at - 5C for 8 d. At this temperature, glycolysis will proceed and ATP will be depleted (Moran, 1930; Smith, 1930; Marsh and Thompson, 1958); thus, shortening does not occur during thawing.

At present, we do not fully understand the biochemistry of muscle stored at - 5C, but it appears to be different than that of muscle stored at or below - 10C. Davey and Gilbert (1976) demonstrated that during storage at - 10C glycolysis does not occur, whereas, the ATP concentration declines due to enzymatic hydrolysis to levels insufficient to cause contraction during thawing. Smith (1930) detected accumulation of lactic acid in frog muscle at - 5C, but not at - 10C.

Davey and Garnett (1980) reported that rapid freezing of carcasses and subsequent extended storage at - 10C (>10 d), removes the hazards of toughness development from cold- and thaw- shortening. Bowling et al. (1987) reported that compared to carcasses stored according to conventional protocol, longissimus from carcasses stored at - 70C for 5 h, then at +16C for 4 h, and then at 1C for 15 h had longer sarcomere lengths (2.0 vs. 1.93 um), less shear force (4.26 vs. 5.03 kg), and higher sensory panel tenderness rating (5.3 vs. 4.69). With the current stage of knowledge, it would seem that it is practical to use this technology. A potential protocol to prevent the development of toughness would include the following steps: 1) electrically stimulate carcasses immediately after slaughter (to reduce ATP concentration, thereby reducing frozen storage time necessary to deplete ATP), 2) pass carcasses through a freeze tunnel for a period of time to be determined (time necessary to freeze SL in prerigor state; the goal should be conditions to produce SL of not less than 2.2 um), 3) store at - 5C (rather than - 10C to speed up ATP depletion) for a period of time to be determined (time necessary to drop pH to <5.8; thus, no thaw- shortening should occur), and 4) fabricate and distribute.

Therefore, if practical, the commercialization of this technology should consistently result in production of tender meat.

Another method which is also based on preventing development of toughness is variation in the way carcasses are hung during rigor development. Based on his observations on the relationship between contraction and toughness, Locker (1960) stated that “it should be possible to improve the quality of the longissimus for example by hanging the carcass in such a way that this muscle is stretched and prevented from shortening.” Others demonstrated a marked improvement in longissimus tenderness on longissimus when carcasses were laid horizontally or hung from the pelvis (Herring et al., 1965; Hostetler et al., 1972, Bouton et al., 1973).

One could take advantage of our knowledge of the mechanisms of postmortem meat tenderization and manipulate the systems involved to accelerate/enhance the tenderization process, such as Calcium- Activated Tenderization (Koohmaraie et al., 1988, 1989, 1990; Koohmaraie and Shackelford, 1991; Wheeler et al., 1991, 1992, 1993, 1994; Kerth et al., 1995; Lansdell et al., 1995; Miller et al., 1995a,b; Wulf et al., 1996). In spite of its well documented effectiveness, there is no evidence of its use by the industry.

The simplest and best documented method of improving, but not eliminating, the inconsistency of meat tenderness is to ensure that meat is not consumed without adequate aging. To maximize consistency in tenderness, beef, lamb, and pork should be aged for 10-14, 7-10, and 5 d, respectively. If none of the above technologies are adopted, then the surest method of ensuring consistency of meat tenderness is to classify carcasses based on tenderness. We have developed a tenderness-based classification method for beef that can operate at the chain speed of 400 carcasses per h. The system is essentially an automated version of shear force measurement that only takes about 10 min. to perform (Shackelford et al., 1996).

It is apparent that there are a variety of methodologies to eliminate the inconsistency of meat tenderness at the consumer level. The question that needs to be addressed is: “Why are these technologies not adopted by the industry?” It is, perhaps, far more urgent to answer this question rather than it is to develop more technologies.

Dr. M. Koohmaraie, USDA-ARS

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