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Use of Ionic Compounds Infusion to Improve Meat Tenderness:
A  Review

 

Paolo Polidori

Francesco Fantuz

 

Dipartimento di Scienze Veterinarie, Universitą di Camerino, Via Circonvallazione 93, 62024 Matelica (MC), Italy

 

KEY WORDS: Meat tenderization, ionic compounds, calpain

Abstract

The present work is an attempt to review and update what is known about the effects of ionic compounds injection of carcasses of meat animals, including the effects on meat tenderness and meat sensorial characteristics. Pre-rigor ionic compound injection to change the rate of glycolysis, rate and state of contraction, and rate of proteolysis appears to be a feasible method of postmortem meat tenderization.

Introduction

Consumers consider meat tenderness the most important palatability trait of meat quality,1 and its variability is an area of major concern in the meat industry.2-4 Although the practice of storing meat after the death of an animal to improve its texture was probably established long ago, its beneficial effects were not recognized until the beginning of this century.5 Later, proteolyisis of muscle proteins was proposed and has since been considered the primary mechanism of meat tenderization.6

Meat tenderness varies considerably among species, among animals within a species, and among different muscles held for different times post mortem.7 Although the connective tissue content is responsible for some of this variation, the virtual lack of change in this component during postmortem storage while considerable tenderization occurs has led to the conclusion that the proteins in the muscle myofibril primarily control meat texture.8 The changes in myofibrillar proteins after death are believed to be mainly proteolytic in nature; most efforts to date have focused on identifying the proteases involved and the substrates altered.5,9,10

Postmortem proteolysis of the major myofibrillar proteins has been found to be negligible. The disappearance of the troponin T band and the appearance of a 30,000 Da band on sodium dodecyl sulphate polyacrylamide gel electrophoreisis (SDS-PAGE) has been documented, and the extent of this change has been shown to correlate to meat tenderness.11-13 However, although postmortem aging normally improves muscle tenderness, in some muscles the 30,000 Da component does not develop during aging. For this reason, Greaser and Fritz8 stated that the degradation of this protein is a primary event that results in the weakening of the myofibril structure but rather a marker for some other change.

Desmin, an intermediate filament protein believed to link the Z-lines of adjacent myofibrils, decreases rapidly after death. This process has been suggested as the reason for the reduction in sarcomere alignment of adjacent myofibrils that occurs after death.14

This review focuses on the effects of ion injection on meat tenderness. First, the enzymatic mechanisms of meat tenderization are summarized; second, some properties of the calpain system are discussed; and finally, the results obtained in previous experiments regarding the effect of ion injection on tenderizing meat are described.

Meat Tenderization

Consumers consider meat tenderness to be the most important palatability trait of meat quality.15 Different factors have been reported to affect meat tenderness, such as animal age, muscle pH and temperature, sarcomere length, amount and type of collagen, and muscle fiber type and size.4,5,16-18 With regard to these factors, some authors19,20 found that the intensity and rate of these modifications are not only species dependent but also, within a given species, muscle dependent.

It is well known that meat tenderness increases gradually during postmortem storage (Table 1), and it is generally accepted that degradation of myofibrillar proteins and structure disruption by endogenous proteases are responsible for this postmortem tenderness improvement.5,9,10,21-23 During postmortem storage of carcasses, numerous changes occur in skeletal muscle, some of which result in the loss of tissue integrity that translates into improved meat tenderness (Table 2).

To further substantiate the argument that proteolysis is the principal reason for meat tenderization during postmortem storage (Table 3), some authors24,25 have also found that the major reason for the observed differences in meat tenderness between Bos taurus (tender) and Bos indicus (tough) breeds of cattle is the reduced rate of myofibrillar protein degradation during postmortem storage. Also, differences in the rate of postmortem tenderization and proteolysis in skeletal muscle from pigs, sheep, and cattle were apparently because of the differences in the rate of myofibrillar protein degradation. The results obtained in these studies clearly indicate that proteolysis of key myofibrillar proteins is the principal reason for the ultrastructural changes in skeletal muscle that result in tenderization.

Enzymatic Mechanisms
of Meat Tenderization

In living muscles, intracellular protein degradation is mediated by a number of different endogenous proteolytic enzymes.5 Because most changes occurring in the course of meat tenderization are currently believed to be the result of proteolysis, every proteinase located inside muscle cells could be a potent contributor to meat aging and must be considered in this context.

In the proteolytic systems detected in skeletal muscle, 2 proteinases have been noted that are able to degrade the myofibrillar proteins. These proteinases are calcium-dependent cysteine proteinases located in the cellular cytosol, calpain I and calpain II. They are active at micromolar (50 to 70 µmol) and millimolar (1 to 5 mmol) concentrations of Ca2+ respectively.26 The calcium-dependent proteases, calpain I and calpain II, also referred to as µ-calpain and m-calpain,9,27 have an optimum pH of 7.528 and have been shown to degrade Z-disk, troponin-T, and desmin. The calpain proteolytic system also includes a tissue-specific calpain, calpain 3 or nCL-1.29 Although calpain 3 has 10-fold more mRNA in muscle cells than calpain I or calpain II, the calpain 3 enzyme has never been identified.30 Other proteinases have been isolated, such as cathepsins B, H, and L,31,32 but their importance to meat tenderization is not completely clear.33,34

Because endogenous protein inhibitors may constitute a powerful regulatory system for muscle proteinases, interest in their identification and characterization has increased markedly. A specific calpain inhibitor called calpastatin has been isolated from muscles of various animal species.35-38 Some properties of the calpain system are listed in Table 4.

Calpain Activity and
Postmortem Tenderization

After incubating muscle strips in a Ca2+-containing solution, some authors discovered the total degradation of Z-disks.39,40  From that observation, efforts were begun to identify the Ca2+-activated factors (called CAF at that time) responsible for this degradation. However, even before CAF had been completely purified, it was shown that muscles with more CAF activity underwent a greater degree of postmortem tenderization than muscles with less CAF activity.41 Both CAF treatment and postmortem storage resulted in the appearance of several polypeptide fragments migrating in the 30,000-Da range in SDS-PAGE.

Since these early observations, a great deal of evidence has accumulated indicating that the calpains have an important role in postmortem tenderization (Table 5), and many studies have focused attention on this role.9,22,42-46 This evidence has led to the prevailing view that the calpains are responsible for 90% or more of the tenderization that occurs during postmortem storage,43 and that, furthermore, this tenderization is the direct result of calpain's ability to degrade Z-disks in skeletal muscle.

Skeletal muscle calpains, which have similar native molecular weights as 110 kDa,47 dissociate into 2 subunits of 80 and 30 kDa by SDS-PAGE. The active site containing a cysteine residue is located in domain II of the 80-kDa subunit, but the function of the 30-kDa unit is unknown.48 Cloning and sequencing studies have shown that calpain I and calpain II originate from different genes, with approximately 52% homology between their amino acid sequences.49 There are no clearly documented major differences in the catalytic properties of the calpains other than their respective calcium requirements; they cleave the same proteins and have similar cleavage site specifities.47

The enzymes involved in postmortem proteolysis have been the subject of much debate; however, it is now generally accepted that calpain I (also called µ-calpain) is the major enzyme involved.28,50 The calcium concentration in postmortem muscle could reach 150 µmol,51 which is sufficient to activate calpain I but insufficient for calpain II activity.9 The activity of calpain I is mostly regulated by calpastatin, its endogenous inhibitor.50 Levels of calpastatin in muscle vary considerably among species,52,53 breeds,24,54,55 and muscles.56,57 The ratio of calpastatin:calpain I is about 4:1, 2.5:1, and 1.5:1 in beef, lamb, and pork muscle, respectively.4

An obstacle to establishing the role of calpains in meat tenderization is that the activity of the calpain-calpastatin system is measured in vitro but the in situ activity is what dictates the rate of proteolysis and tenderization.58 Unfortunately, there is no method of measuring in situ activities. To circumvent this problem, Dransfield51,59 developed a computer model that estimates the postmortem changes in the in situ activities of the calpains and the associated changes in meat tenderization. The principle of the model is to calculate the accompanying pH and calcium ions profiles from the postmortem temperature profiles of meat. These profiles are subsequently used to estimate the in situ activities of calpains and calpastatin based on their in vitro activities.

Variations in calpain activity, rather than in sarcomere length, are believed to cause toughness variation in thaw rigor muscle.60 Reduced proteolysis is therefore a factor that should be considered in understanding the mechanisms of cold-shortening toughness.61

Meat Tenderization Via Prerigor Injections of
Ionic Compounds

Both Webster's New World Dictionary of the American Language62 and Dorlands's Illustrated Medical Dictionary63 define infusion similarly as the introduction of a solution into the body, specifically into a vein, and perfusion as the act of pouring (such as a liquid) over or through an organ or a tissue. The American Heritage Dictionary of the English Language64 specifically defines infusion as "the introduction of a solution into a vein" and defines perfusion as "the injection of a fluid into an artery in order to reach tissues." When citing other works in this article, we have used their original term, although we believe that infusion should refer to the introduction of a solution via a vein and perfusion should refer to the introduction of a solution via an artery.

Infusion or perfusion of compounds to change the rate of glycolysis, rate and state of contraction, and rate of proteolysis appear to be feasible methods of manipulating the postmortem tenderization process in meat.37,65-67 It was reported in the mid1950s that infusion of bovine carcasses with salt solutions caused a considerable improvement in tenderness.36 Salts generally influence the functional properties of meat products68 and are believed affect contraction and shortening, protein-protein interactions, protein solubility, proteolytic enzyme activity, and lattice swelling.69

Beekman et al70 and Geesink et al36 reported a positive effect of sodium chloride (NaCl) or NaCl and phosphate treatment on meat tenderness. Consequently, it was suggested that NaCl injection may improve tenderness in different ways, such as dissociation of actomyosin or solubilization of proteins from myofilaments.

Stevenson-Barry and Kauffman71 reported that injection of either NaCl or sodium pyrophosphate (NaPPi) into hot-boned muscles reduces cold-induced toughening. They suggested that the mechanism did not involve inhibition of contraction because sarcomere lengths were not changed. Furthermore, because both NaCl and NaPPi injections into muscles improved tenderness, the authors suggested that ionic strength was one of the important reasons for improving tenderness.

Polyphosphates, including pyrophosphate, triphosphate, and metaphosphate, have high buffering capacity at neutral to alkaline pH (pH 6.5 to 9.0) due to their polyanionic characteristics.72 Injection or infusion of pyrophosphates increased meat pH and improved meat quality by assisting solubilization of myosin and increasing uptake and retention of water.73

Sodium chloride is a useful compound for meat because it is a well-known food additive, has long been used for its positive effects on preservation and flavor of meat, and is not considered an unfamiliar chemical additive such as NaPPi or calcium chloride (CaCl2). Moreover, unlike CaCl2 and other salts, NaCl does not cause bitterness.37 In fact, CaCl2 is not only salty, it also has a metallic, bitter flavor when used at the same level as NaCl. It is not surprising that trained sensory panelists can detect it in meat.74 However, Offer and Knight73 suggested that a combination of NaCl and NaPPi produced a synergistic effect at lower salt concentrations.

The results obtained in a recent study75 indicated that infusion of beef carcasses with CaCI2 accelerated postmortem tenderization. The direct action of calcium ions was described by Takahashi.76 Calcium ions have a dual function in postmortem muscle; the rise of sarcoplasmic calcium ion concentrations to 3 to 5 mmol induces rigor contraction, and the further rise of the calcium ion concentration to 0.1 mmol weakens the structures of myofibrils, desmin intermediate filaments, and probably the endomysium and perimysium, thereby tenderizing the meat. However, Table 6 summarizes the different ionic compounds injected before rigor in carcasses of meat animals in previous experiments. The salt most frequently used has been calcium chloride, and the most common salt concentration was 0.3 mol. Calcium chloride infusion was recently shown77 to be an effective method of reducing toughness also in pork meat.

Other studies1,78,79 have been performed to establish the conditions required to improve meat tenderness by using calcium chloride marination, and consequently to reduce the time required for the postmortem tenderization. The results obtained by Gonzalez et al1 showed that calcium chloride marination was effective in increasing tenderness and in reducing the postmortem storage time necessary to achieve an acceptable level of tenderness in beef muscle Cutaneus trunci. The results obtained by Whipple and Koohmaraie79 showed that the improvement in tenderness was due by the activation of calpain II, also called m-calpain.

Conclusions

Many methods have been suggested in the past to change the pre-rigor properties of muscles in an attempt to improve meat quality. The simplest and best documented method of improving, but not eliminating, the inconsistency of meat tenderness is to ensure that meat is not consumed before an adequate aging period. To maximize consistency in tenderness, beef, lamb, and pork should be aged for 10 to 14, 7 to 10, and 5 days, respectively.4 However, use of ionic compound injection by the meat industry would also reduce the variation found in meat tenderness, reducing the need to depend on an adequate aging period as the primary factor for reducing meat toughness.

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96. Wulf DM, Morgan JB, Tatum JD, Smith GC: Effects of animal age, marbling score, calpastatin activity, subprimal cut, calcium injection, and degree of doneness on the palatability of steaks from Limousine steers. J Anim Sci 74:569-576, 1996.

97. Yancey EJ, Dikeman ME, Addis PB, et al: Effects of vascular infusion with a solution of saccharides, sodium chloride, and phosphates with or without vitamin C on carcass traits, Warner-Bratzler shear force, flavor-profile, and descriptive-attribute characteristics of steaks and ground beef from Charolais cattle. Meat Sci 60:341-347, 2002.

Recommended Reading

Ouali A: Proteolytic and physiochemical mechanisms involved in meat texture. Biochimie 74:251-265, 1992.

 

 

 

Table 1. Effect of Length of Postmortem Storage on Beef Shear Force Values (kg/cm2)

 

                                                                   

               7 days    14 days % > 6 kg/     Correlation % > 6 kg day 7
Breed       n aging mean aging range cm2  mean   range   /cm2 to day 14

 

Angus              102       5.11            2.57-9.30          22        4.05      2.48-9.04         5             0.58

 

Tuli                    158       5.71           2.94-12.38         34        4.58      2.33-9.24         8             0.65

 

Hereford           106       5.67           2.37-11.91         31        4.74      2.41-8.30        12            0.66

 

Belgian Blue     144       5.82           2.52-10.57         42        4.82      2.64-8.41        14            0.61

 

Boran               138       6.58           3.15-11.79         55        5.14     2.84-11.25       26            0.76

 

Brahman           119       7.30           3.43-12.50         63        6.05     2.66-11.03       34            0.80

 

All breeds         767       5.95           2.37-12.50         42        4.86     2.33-11.25       17            0.72


Source: Koohmaraie.4

 

 

TABLE 2. Changes in Skeletal Muscle During Postmortem Storage of Carcasses

 

Table 3. Experimental Evidence Showing the Role of Proteolysis in Postmortem Meat Tenderization

 

* Z-disk weakening and/or degradation, which leads to fragmentation of myofibrils

* Degradation of desmin, which leads to fragmentation of myofibril, probably through disruption of transverse crosslinking between myofibrils

* Degradation of titin; titin filaments connect myosin filaments, along their length, from the M-line to the Z-disk; therefore, degradation of titin during post mortem storage would cause weakening of myofibril strength and, consequently, improvement in meat tenderness

* Degradation of nebulin; because of the location of nebulin in myofibrils (I-band), it is not clear how nebulin degradation will affect meat tenderness

* Disappearance of troponin-T and simultaneous appearance of polypeptides with molecular weight 28-32 kDa; This is the most noticeable and reported change that occurs during postmortem storage

* Appearance of a polypeptide with molecular weight of 95 kDa; neither the origin nor its significance to meat tenderness is known

* Perhaps the most significant observation is that the major contractile proteins, myosin and actin, are not affected, even after 56 days of postmortem storage

 

Source: Koohmaraie.10 

 

* Incubating muscle slices with calcium chloride induces proteolysis of myofibrillar proteins and fragmentation of myofibrils; however, incubation of muscle slices with calcium chelators (EDTA and EGTA) prevents both degradation of myofibrillar proteins and myofibril fragmentation

* Infusion of carcasses with calcium chloride accelerates postmortem changes (degradation of myofibrillar proteins, tenderness) in skeletal muscle so that postmortem storage to ensure meat tenderness is no longer necessary

* Infusion of carcasses with zinc chloride inhibits all postmortem changes measured (degradation of myofibrillar proteins, myofibril fragmentation, tenderization)

* Muscle from b-adrenergic agonist (BAA)-fed lambs, which does not undergo postmortem proteolysis (no detectable degradation of myofibrillar proteins and myofibril fragmentation during postmortem storage), is tougher than muscle from untreated lambs. However, calcium chloride infusion of carcasses from BAA-fed lambs induces degradation of myofibrillar proteins and eliminates their meat toughness

 

Source: Koohmaraie.9

 

 

Table 4. General Properties of the Calpain System

 

Occurrence: found in all vertebrate cells that have been examined.

Ubiquitous Calpains

Name                      Polypeptides Ca2+- required for half  maximal activity

Calpain I (µ-calpain)                                 80, 28-kDa                                       3-50 µmol

Calpain II (m-calpain)                                80, 28-kDa                                    400-800 µmol



Tissue-specific calpains

Name                      Polypeptides               Tissue

skm-calpain, p94, calpain 3                      94, 82-kDa                            skeletal muscle, rat lens

n-calpain-2, nCL-2                                      80-kDa                                     stomach muscle

n-calpain-3, nCL-3                                      43-kDa                                     stomach muscle

All calpains that have been isolated in protein form are cysteine proteases with optimal pH of 7.2-8.2


Calpastatin

Multiheaded protein inhibitor that inhibits only the calpains; expressed in several different isoforms that have one, three, or four inhibitory domains and different N-terminal sequences.


Cellular distribution

The calpains and calpastatins studied thus far are located exclusively intracellularly; various proportions of the calpains are associated with subcellular organelles, which are primarily myofibrils in skeletal muscle, but may include the plasma membrane, mitochondria, and nuclei.

Source: Goll et al.80

 

 

Table 5. Role of Calpain System in Postmortem Tenderization

 

* Very little degradation of myosin and actin occurs during postmortem storage at 2˚ to 4˚C for 72 h, even though most of the total postmortem tenderization occurs during that period

    1. The calpains are unique among the known proteolytic enzymes in that they do not degrade undenaturated myosin and actin

    2. The known cathepsins all degrade myosin and actin

* Very little degradation of a-actinin, the major protein in skeletal muscle Z-disks, occurs during postmortem storage at 2˚ to 4°C for 72 h

    1. Several of the cathepsins and many other proteases (ie, trypsin) degrade the Z-disk structure, and this degradation is accompanied by degradation of a-actinin; the calpains, however, are unique in that they degrade the Z-disk structure, but they release a-actinin from this structure without degrading it to small fragments

    2. Degradation of myosin, actin and a-actinin occurs during post mortem storage at 37˚C, and this degradation may be due to the cathepsins

* There is very little proteolysis of muscle proteins during post mortem storage at 2˚ to 4°C

    1. The sarcomere structure remains largely intact

    2. Functional actomyosin and a-actinin can be isolated from skeletal muscle even after 13 days of postmortem storage at 2˚ to 4˚C

* Several studies have shown that increasing Ca2+ concentration in muscle results in increased tenderness

* A number of studies have shown that tenderness increases to a greater extent during postmortem storage in those muscles that have higher calpain (especially calpain I) or lower calpastatin activities than in other muscles; low calpastatin activity especially seems highly associated with increased postmortem tenderization

 

Source: adapted from Taylor et al.45

 

 

 

Table 6. Ionic Compounds Injected During Postmortem Aging in Carcasses of Meat Animals

 

Species Animals, n Ionic compounds injected Study

Lambs              24              Calcium chloride                      Koohmaraie et al81

Lambs              20              Calcium chloride                      Koohmaraie et al82

Lambs              18              Sodium chloride                      Koohmaraie et al82

Lambs              24              Calcium chloride                      Koohmaraie et al65

Beef                 12              Calcium chloride                      Koohmaraie et al65

Lambs              12              Zinc chloride                           Koohmaraie83

Lambs              32              Calcium chloride                      Koohmaraie and Shackelford84

Beef                 10              Calcium chloride                      Morgan et al66

Lambs              25              Calcium chloride                      St. Angelo et al85

Beef                 90              Maltose, dextrose,                  Farouk et al86

                                           polyphosphate and glycerin

Beef                 25              Calcium chloride                      Wheeler et al87

Beef                 20              Calcium chloride                      Wheeler et al88

Beef                 10              Calcium chloride                      Diles et al89

Lambs              24              Maltose, dextrose,                  Farouk and Price90

                                           polyphosphate, glycerin

                                           plus calcium chloride

Beef                 12              Calcium chloride,                     Geesink et al36

                                           Sodium chloride,

                                           Zinc chloride

Beef                 16              Calcium chloride                      Boleman et al91

Beef                 12              Calcium chloride                      Kerth et al92

Beef                 22              Calcium chloride                      Lansdell et al93

Beef                 22              Calcium chloride                      Miller et al94

Beef                 20              Calcium chloride                      Wheeler et al95

Beef                114             Calcium chloride                      Wulf et al96

Beef                 20              Calcium chloride                      Wheeler et al74

Beef                 12              Sodium pyrophosphate           Lee et al37
                                           plus sodium chloride              

Lambs              36              Calcium chloride                      Polidori et al38

Beef                 48              Calcium chloride                      Polidori et al75

Beef                 36              NaCl and phosphates             Yancey et al97

Pork                 36              Calcium chloride                      Rees et al77

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