Use of Ionic Compounds Infusion to Improve Meat Tenderness:
Dipartimento di Scienze Veterinarie, Universitą di Camerino,
Via Circonvallazione 93, 62024 Matelica (MC), Italy
WORDS: Meat tenderization, ionic compounds, calpain
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.
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 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.
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
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.
of Meat Tenderization
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
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
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
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
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
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
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
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.
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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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
Tuli 158 5.71 2.94-12.38 34
5.67 2.37-11.91 31 4.74 2.41-8.30
Blue 144 5.82 2.52-10.57 42
Boran 138 6.58 3.15-11.79 55
7.30 3.43-12.50 63 6.05 2.66-11.03
breeds 767 5.95 2.37-12.50 42
TABLE 2. Changes
in Skeletal Muscle During Postmortem Storage of Carcasses
Table 3. Experimental Evidence Showing the Role of Proteolysis in Postmortem
* 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
* 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
General Properties of the Calpain System
found in all vertebrate cells that have been examined.
Name Polypeptides Ca2+- required for half maximal activity
Name Polypeptides Tissue
p94, calpain 3 94,
82-kDa skeletal muscle, rat
80-kDa stomach muscle
43-kDa stomach muscle
calpains that have been isolated in protein form are cysteine proteases
with optimal pH of 7.2-8.2
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.
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
2. The known cathepsins all degrade myosin
* 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
* There is very little
proteolysis of muscle proteins during post mortem storage at 2˚
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
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
Ionic Compounds Injected During Postmortem Aging in Carcasses of Meat
Species Animals, n Ionic
compounds injected Study
Koohmaraie et al81
Koohmaraie et al82
Koohmaraie et al82
Koohmaraie et al65
Beef 12 Calcium chloride
Koohmaraie et al65
Koohmaraie and Shackelford84
Beef 10 Calcium chloride
Morgan et al66
St. Angelo et al85
Beef 90 Maltose, dextrose,
Farouk et al86
Beef 25 Calcium chloride
Wheeler et al87
Beef 20 Calcium chloride
Wheeler et al88
Beef 10 Calcium chloride
Diles et al89
Farouk and Price90
Beef 12 Calcium chloride,
Geesink et al36
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
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