Meat Science 89 (2011) 111–124

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Meat Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m e a t s c i

Review

Water distribution and mobility in meat during the conversion of muscle to meat and
ageing and the impacts on fresh meat quality attributes — A review
Kelly L. Pearce a,⁎, Katja Rosenvold b, Henrik J. Andersen c,d, David L. Hopkins e
a
Cooperative Research Centre for Sheep Industry Innovation, Murdoch University, Division of Veterinary and Biomedical Science, South Street, Murdoch, WA 6150, Australia
b
AgResearch MIRINZ, Private Bag 3123, Hamilton, New Zealand
c
Arla Foods amba, Corporate Research & Innovation, Viby, Denmark
d
Interdisciplinary Nanoscience Centre (iNANO), University of Aarhus, Faculty of Natural Science, Building 1521, Ny Munkegade, DK-8000 Århus C, Denmark
e
Industry and Investment NSW (Primary Industries) Centre for Red Meat and Sheep Development, PO Box 129, Cowra, NSW 2794, Australia

a r t i c l e i n f o a b s t r a c t

Article history: This paper reviews current knowledge on the distribution and mobility of water in muscle (myowater) ante-
Received 15 November 2009 and post mortem and factors affecting these in relation to fresh meat quality parameters; water-holding
Received in revised form 7 April 2011 capacity (WHC), tenderness and juiciness. NMR transverse relaxometry (T2) using bench-top Low-Field
Accepted 7 April 2011
Nuclear Magnetic Resonance (LF-NMR) has characterised myowater distribution and mobility as well as
structural features in meat which directly affect WHC. The current literature demonstrates that WHC is
Keywords:
Meat
correlated to the water located outside the myofibrillar network (extra-myofibrillar). This review identifies
Myowater the critical stages which affect the translocation of water into the extra-myofibrillar space and thus the
Nuclear magnetic resonance (NMR) potential for decreased WHC during proteolysis (the conversion of muscle to meat). This review discusses
Water-holding capacity how the intrinsic properties of the water held within the meat could contribute to juiciness and tenderness.
Tenderness Tenderness has been shown to correlate to T2, however breed and species differences made it difficult to draw
Juiciness firm conclusions. Further understanding of the inherent water properties of fresh meat and the factors
affecting water distribution and mobility using NMR technologies will increase the understanding of WHC
and tenderisation of fresh meat.
© 2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2. The structure and water distribution of muscle in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
2.1. Protein-associated water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
2.2. Immobilised water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
2.3. Free water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
3. Measurement of water distribution and mobility in muscle and meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4. Conversion of muscle to meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.1. The reduction in myofilament spacing during the conversion of muscle to meat . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5. Degradation of myofibrillar proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
5.1. Proteolysis and swelling of muscle fibres during ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
5.2. Desmin and integrin degradation and effects on drip loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5.3. Myowater distribution and protein degradation during ageing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
6. The role of intrinsic and extrinsic factors on myowater characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.1. Myowater distribution and mobility in different muscle types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.2. Effect of animal growth rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.3. Effect of slaughter age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.4. Effect of pre-slaughter handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
6.5. The effect of chilling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
6.6. Effect of cooking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

⁎ Corresponding author. Tel.: + 61 417964473.

E-mail address: k.pearce@murdoch.edu.au (K.L. Pearce).

0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2011.04.007
112 K.L. Pearce et al. / Meat Science 89 (2011) 111–124

7. The relationship between myowater and meat quality attributes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

7.1. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
7.2. Sarcomere length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
7.3. WHC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8. The influence of myowater characteristics on eating quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.1. Tenderness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
8.2. Juiciness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
9. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

1. Introduction
The distribution and mobility of water in muscle (myowater) and
meat have a profound influence on essential meat quality attributes
like juiciness, tenderness, firmness and appearance (Trout, 1988).
During the conversion of the living muscle to meat and during ageing
the myowater content, location and mobility will change as a function
of numerous mutual interacting factors of both ante (breed, muscle,
stress level and type etc.) and post mortem nature (slaughter
procedure, cooling rate, ageing time and temperature) (Honikel,
2004; Honikel, Kim, & Hamm, 1986).
Nuclear magnetic resonance (NMR) can provide direct informa-
tion about interactions between water protons and exchangeable
protons in proteins and thereby the chemical–physical state of water
in muscle and meat (See papers such as Bertram & Andersen, 2004;
Bertram et al., 2001). Furthermore, NMR transverse relaxation has
proven to be very informative with regard to meat structure as a high
correlation between sarcomere length and myofilament spacing has
been demonstrated using this technique (Bertram, Purslow, &
Andersen, 2002). Accordingly, NMR transverse relaxation has proven
an excellent tool to quantify the changes in water distribution and
mobility during the conversion of muscle to meat and during ageing
and thus help to explain how these changes are linked to meat quality.
This review will cover current knowledge obtained principally
from NMR characterisation of muscle and meat regarding the
following items: (1) how structural changes during the conversion
of muscle to meat and during ageing affect the water distribution and
mobility of meat and its ability to retain myowater during storage —
referred to as the water holding capacity (WHC), i.e. the ability of
fresh meat to retain its own water. Poor WHC results in high drip and
purge loss and this factor is of significant industry concern. High drip
and purge loss can represent significant loss of weight in carcasses and
cuts and thus can affect the yield and quality of processed meat, and
thereby becomes economically important (Savage, Warriss, & Jolley,
1990; Wright et al., 2005), (2) how intrinsic and extrinsic factors such
as animal growth rate and age, pre-slaughter stress and chilling can
affect water distribution and mobility, and (3) the relationship
between fresh meat quality attributes such as juiciness and
tenderness and muscle water distribution and mobility.
This review will also contain discussion on the contribution of
water mobility during ongoing protein disintegration, as a conse-
quence of proteolysis in the muscle. It is important that the sources,
populations and movement of water are recognised in order to
understand the potential links with the key meat quality attributes
juiciness and tenderness and the impact on meat quality.
2. The structure and water distribution of muscle in vivo
Muscle is a highly organised tissue, composed of individual cells
known as fibres, which are structured by connective tissue. The
structure of a muscle is demonstrated in Fig. 1. The order of com-
ponent breakdown is 1) muscle 2) muscle fasciculus 3) muscle fibre/
muscle cell 4) myofibril and 5) myofilaments. Each muscle fibre
consists of a high number of single strands or organelles called
myofibrils and these are comprised of myofilaments which are based
on thin and thick filaments (Fig. 2), predominantly made up of actin
(thin) and myosin (thick) (Rawn, 1989). Myosin is a large protein
made up of two heavy chains (Mw 220 kDa) and two pairs of small
subunits referred to as light chains (Mw 130 kDa) (Bandman, 1999).
There are many regulatory proteins associated with actin but the two
key proteins are tropomyosin (complex of three subunits; (Wong,
1991)) and troponin.
Myofibrils are laterally linked to the sarcolemma and to other
myofibrils in the I band region via transverse intermediate filaments,

Inter-fascicular water

Inter-myofibrillar water

Extra-fascicular water

Intra-myofibrillar water

Fig. 1. The muscle split into components and the locations of muscle water compartments: the intra-myofibrillar component and the extra-myofibrillar component which is
comprised of the inter-myofibrillar, inter-fascicular and extra-fascicular water populations (Baechle & Earle, 2008).
 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
Fig. 2. Fibrillar and molecular structure of the contractile elements of muscle. The skeletal muscle fibre in longitudinal section (A) as visualised by the light microscope; (B) and (C) by
the electron microscope; and (D), as reconstructed from x-ray crystallographic evidence (Davies & Werner Klinth, 2004).
which include such proteins as desmin, filamin, talin, and vinculin.
Desmin also connects the Z line proteins between myofibrils (Taylor
et al., 1995) and contributes to the maintenance of sarcomere
structure (sarcomere and cytoskeletal structure has been reviewed
in Small, Furst & Thorness (1992)) but its essentially a repeating unit
along the myofbril delimited by the Z Band along the length of the
myofibril. Integrin is a cell adhesion protein that links the
extracellular matrix to the cytoskeleton which is also important
for signalling pathways involved in muscle contraction. Titin or
connectin is a large protein (up to 3700 kDa; Labeit & Kolmerer,
1995) that is crucial to the orderly organisation of the sarcomere.
Along the length of titin are binding sites for other sarcomere
proteins and the carboxyl terminal region of titin is reported to bind
to myosin (Maruyama, 1997). Taylor, Geesink, et al. (1995) provide
a schematic diagram of how titin interacts with actin and myosin
and illustrates where other proteins such as desmin and vinculin are
located in the sarcomere.
Lean muscle contains approximately 75% water (Offer & Knight,
1988). The other main components include protein (approximately
20%), lipids (approximately 5%), carbohydrates (approximately 1%)
and vitamins and minerals (often analysed as ash, approximately 1%)
(Offer & Knight, 1988).
The greater part of the myowater, about 85% is located within the
protein-dense myofibrillar protein network (intra-myofibrillar) (Huff-
Lonergan & Lonergan, 2005), and 15% is located outside the myofibrillar
network (extra-myofibrillar) (Hamm, 1975; Lawrie, 1998).
113
Water in the intra-myofibrillar space is held:
• In the myofibrils in the space between the thick and thin filaments
(intra-myofibrillar water).
Water in the extra-myofibrillar space is found:
• In the sarcoplasm in the space between myofibrils (inter-
myofibrillar)
• Between muscle fibres and in the inter-fascicular space (inter-
fascicular) (Hamm, 1975; Offer and Knight, 1988; Offer et al., 1989).
• In the extra-fascicular space in between the muscle fasciculi (extra-
fascicular) (Schaefer, Knight, Wess, & Purslow, 2000).
The myowater in the muscle can be classified into one of three
fractions: 1) protein-associated water (charged hydrophilic groups on
the muscle proteins tightly bind water), 2) entrapped or immobilised
water (has less orderly molecular orientation toward the charged
group), and 3) free water (held only by capillary forces, and the
orientation is independent of the charged group). Detailed reviews of
these water populations are given by Fennema (1985), Huff-Lonergan
and Lonergan (2005) and Offer and Knight (1988).
2.1. Protein-associated water
Water being a dipole molecule is attracted to charged molecules
such as proteins. This protein bound water has highly reduced mobility
and does not move to other compartments and remains tightly bound
114 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
even during the application of severe mechanical or physical force
such as freezing and heating. Bound water will continually exchange
with surrounding water molecules, including immobilised water
(Huff-Lonergan & Lonergan, 2005; Offer & Trinick, 1983).
2.2. Immobilised water
Immobilised water accounts for up to 85% of the myowater. It is
located within the thick filaments and between the thick and thin
filaments of the myofibril (Honikel et al., 1986). The immobilised water
is bound either by steric effects of attraction between the filaments or
hydrogen bound to muscle proteins or other macromolecules. During
the conversion of muscle to meat and ageing this water population can
be mobilised due to alteration of muscle cell structure and changes in pH
(Huff-Lonergan & Lonergan, 2005; Offer & Knight, 1998). Some of the
factors that can influence the retention of immobilised water include
manipulation of the net charge of myofibrillar proteins and the structure
of the muscle cell and its components (myofibrils, cytoskeletal linkages
and membrane permeability) as well as the size of extra-myofibrillar
space within the muscle itself.
2.3. Free water
Free water exists in the sarcoplasmic area (Honikel, 1988) within
the muscle cells in long narrow passages, so called capillaries, where
the water is held by intermolecular forces between the liquid and the
surrounding matrix. The free water can be easily mobilised e.g.
through minor physical forces formed upon shrinking of myofibrils at
the time of rigour mortis (defined as the time point when there is no
ATP available for release of myosin from the actomyosin complex
(Honikel, 2004)). Mobility of water from this fraction is unimpeded. It
is not readily seen in pre rigour meat.
Since the myofibrils contain most of the myowater, structural
changes in the myofibrils present a logical proposition for explaining
the movement of water during the conversion of muscle to meat.
3. Measurement of water distribution and mobility in muscle and
meat
Changes in water distribution and mobility in muscle and meat
have been extensively researched in recent years using low-field
NMR, i.e. NMR relaxometry. In NMR relaxometry, the nuclei are
aligned in a magnetic field and subsequently perturbed from their
equilibrium state by a radiofrequency pulse, to which they will
subsequently return by a process described as relaxation following
the cessation of the pulse. This relaxation decay can be measured and
provides information about the physical/chemical nature of certain
nuclei in terms of mobility and compartmentalisation. Consequently,
NMR relaxometry is widely used to study the behaviour of water, the
most abundant substance in many biological materials, including
muscle and meat (Bertram & Andersen, 2007). The relaxation occurs
in two planes and is notated as T1 in the longitudinal plane or vector
and T2 in the transverse plane or vector (Bertram, Schafer, Rosenvold,
& Andersen, 2004; Bertram, Straadt, Jensen, & Aaslyng, 2007).
NMR transverse relaxation of myowater (T2) within muscle and
meat has been described as non-mono-exponential, which can be
separated into the relaxation decay of two or three exponential
populations (P21 and P22), representing the concentration of the
inherent water in these populations. These populations represent two
to three major water compartments in meat. The major population P21
is characterised by a fast relaxation time (the time constant is referred
to as T21) of 30–50 ms and contributes 80–95% of the transverse
relaxation. The second and slower population P22 has a relaxation
time (T22) of 100–250 ms and accounts for 5–15% of the transverse
relaxation (Bertram & Andersen, 2004). The fast-relaxing water
population is most likely to reflect water located within highly
organised protein structures, e.g. water in tertiary and quaternary
protein structures with high myofibrillar protein densities including
actin and myosin filament structures (intra-myofibrillar), whilst the
slow-relaxing water population reflects the water located outside the
myofibrillar network (essentially extra-myofibrillar) (Bertram &
Andersen, 2004). Beside these two major water populations, a third
water population is observed in muscle with a time constant of
between 0 and 10 ms. This population is speculated to refer to water
closely associated with macromolecules (Bertram & Andersen, 2004)
or to macromolecular protons located in water-plasticised structures
(Venturi et al., 2007).
NMR transverse relaxation during the early post mortem period,
enables simultaneous information about water distribution and
mobility, membrane properties and physical events to be obtained
(Bertram & Andersen, 2004). Furthermore, NMR transverse relaxation
has proven to be very informative with regard to meat structure, as a
high correlation with sarcomere length and myofilament spacing has
been demonstrated (Bertram, Purslow, et al., 2002). Accordingly, NMR
transverse relaxation would be expected to be an excellent tool to
quantify the effects of pH and ionic strength on the water distribution
and mobility in myofibrils. The relaxation decay rate will be dependent
on the probability of a water molecule interacting with surrounding
surfaces such as a membrane or proteins, which acts as relaxation sinks
(Bertram & Andersen, 2004; Bertram, Purslow, et al., 2002).
Simply, within meat, any change in the T21 time constant implies
alterations in the structural organisation of myofibrillar water.
Whereas the T22 time constant represents the post mortem reorgani-
sation of water closely associated with changes in membrane
properties and space in the extra-myofibrillar area. The following
changes in the T21 and T22 time constants and populations apply
(Bertram, Rasmussen, et al., 2002):
• An increase in the T21 population (P21) indicates an increase in the
number of protons (essentially water) within the myofibrils in the
intra-myofibrillar space.
• An increased T22 population (P22) indicates a higher number of
protons and therefore an increase in the extra-myofibrillar water
population.
• An increase in the T21 time constant represents an increased
relaxation time of water in the intra-myofibrillar space. This is due
to an increase in the spacing between the myofibrils or shortening of
the sarcomeres.
• An increase in the T22 time constant represents an increase in the
space in the extra-myofibrillar area.
4. Conversion of muscle to meat
During the conversion of muscle to meat the key biochemical
processes are directed towards the achievement of rigour mortis
(Honikel, 2004). A key biochemical process is the hydrolysis of the
ATP in the muscle cell, which is necessary to maintain a relaxed,
uncontracted sarcomere. With the post mortem progression of
glycolysis, glycogen levels drop (Immonen & Poulanne, 2000) and
ATP decreases to a critically low concentration. As post mortem
glycolysis reaches ultimate cessation and ATP levels deplete, the
muscles start to shorten to some degree, the extent to which is
dependent on pre rigour temperature (Locker & Hagyard, 1963). Once
the muscle is critically low in ATP, the myosin heads start to become
permanently bound to actin filaments, which result in formation of
the actomyosin complex (Rawn, 1989) and this causes the muscle to
become inextensible. This contraction results in a reduced space for
water to reside between the myofilaments (Lawrie, 1998).
The first stages in the conversion of muscle to meat are the changes
that occur at exsanguination. During slaughter hormonal stimulation
takes place (Honikel, 1993), which in combination with reduced
oxygen supply (anoxia) results in intracellular osmotic perturbation
 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
within muscle cells (Lambert, Nielsen, Andersen, & Ortenblad, 2001).
Consequently, the situation in muscle cells upon slaughter is
comparable with anoxia/ischemia, which from other cell systems is
known to be associated with increased ionic strength. Ionic strength is
also directly reflected in the osmolality of the cells which increases
significantly during the development of rigour mortis (Winger & Pope,
1981). The rise in ionic strength will increase muscle cell volume as a
result of (1) an increase in the amount of intracellular water (due to
the osmotic flow of water to maintain osmolality) and (2) increased
electrostatic repulsion between myofilament proteins. These concur-
rent processes will occur until there is re-regulation of cell volume to
original in vivo size (Bertram et al., 2004). NMR studies, as
demonstrated in Fig. 3, support this early post mortem increase in
cell volume of porcine muscle. Firstly, the fast relaxing water time
component (T21) increases which reflect a reduced proton sink
potential for the water molecules due to the increased area between
the myofibrils. Subsequently there is a sharp decrease in the slow
relaxing water population (P22) as a consequence of myowater
moving from the extra-myofibrillar space into the expanded intra-
myofibrillar matrix (Bertram et al., 2004).
Early post mortem the myofilament spacing increases due to a
decrease in sarcomere length as a result of rigour mortis induced
longitudinal contraction (commonly known as muscle or sarcomere
shortening). Longitudinal contraction is caused by the release of
calcium ions from the sarcoplasmic reticulum, whilst ATP is still
available. This allows the fibres to contract longitudinally, which in
combination with the falling muscle temperature make the sarco-
plasmic reticulum less efficient in sequestering calcium ions from the
myofilament spacing thereby accelerating the contraction of the
Fig. 3. Illustration of proposed mechanisms in the post mortem reorganisation of muscle fluids and its relation to observed changes in NMR T2 characteristics (Bertram et al., 2004).
115
fibres. Similarly to contracted muscle in the live animal, the
contraction involves a decreased length of the I band region whilst
the A band in the sarcomere remains constant (Rome, 1967). The
extent of the longitudinal shrinkage is dependant on glycogen levels
at the time of slaughter and post mortem muscle temperature.
The process of longitudinal shrinkage has important consequences
for water mobility in meat (Bertram et al., 2004; Tornberg, Wahlgren,
Brondum, & Engelsen, 2000). The decrease in sarcomere length
changes the characteristics of both the slow and fast-relaxing water
populations due to (1) an increase in the diameter of the fibre, (2) an
increase in the diameter of the myofibrils, and (3) an increase in the
lateral spacing between thick and thin filaments within the
sarcomere.
During the conversion of muscle to meat, the sarcomere not only
continues to shrink longitudinally (commonly known as muscle or
sarcomere shortening), but also laterally, as two simultaneous
processes take place within the myofibrillar system: (1) the
development of rigour mortis, i.e. the irreversible association of thick
and thin filaments after depletion of ATP forming the actomyosin
complex and (2) the decrease in pH which causes alterations of the
structure in the myofibrillar proteins (mainly myosin and actin)
(Honikel et al., 1986).
The pH decrease occurs due to the anaerobic conversion of
glycogen resulting in a build-up of hydrogen ions through lactic acid
formation (Lawrie, 1998; McGeehin, Sheridan, & Butler, 2001).
Depending on muscle type and glycogen concentration, the pH
decreases from 7.0 in the live muscle to pH 5.4 to 6.3 (mean around
5.5–5.6) in the meat (Honikel, 2004). This increase in hydrogen ions
reduces electrostatic repulsion between the myofibrillar proteins and
116 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
thereby decreases the repulsion between the filaments, which
contributes to lateral shrinkage of the muscle fibres.
If the post mortem pH decreases rapidly, i.e. whilst the muscle
temperature is still high, the myosin heads denature and shrink
(Offer, 1991). Denaturation of the myosin heads is also thought to
make a significant contribution to myofibrillar lateral shrinkage. This
process is also important as the ability of denatured myosin to bind
water is reduced resulting in decreased WHC, e.g. pale, soft and
exudative (PSE) meat. Previously, this condition was particularly
frequent in pig carcasses due to a high prevalence of the Halothane
gene (Briskey, 1964), which caused increased incidence of PSE pork,
but also appears to be a problem in Australian grain-fed beef due to
their slow cooling rate and fast rate of pH decline (Warner, Kearney,
Thompson, & Polkinghorne, 2009).
4.1. The reduction in myofilament spacing during the conversion of
muscle to meat
There is still speculation about the time point post mortem when
the lateral and longitudinal shrinkage contribute to the water
movements in muscle. Guignot, Vignon, and Monin (1993) showed
that the myofilament spacing begins to decrease drastically when pH
reaches a value around 5.9, whereas Diesbourg, Swatland, and
Millman (1988) found a rapid decrease in myofilament spacing
from 1 h to 3 h with a subsequent slower decrease in the following
24 h. Also observed were significant changes in intra- and extra-
myofibrillar water populations in the timeframe between 4 and 8 h
post mortem due to ongoing longitudinal and lateral shrinkage (Fig. 3).
As a consequence of the shrinkage in the spaces between the
myofilaments the tension becomes exerted on the hierarchy of the
transverse structural elements in the cytoskeleton that link the
myofibrils laterally to the sarcolemma in the I-band region and
between adjacent myofibrils (Craig & Pardo, 1983). During the
development of rigour mortis the shrinkage of the myofibrils is
transmitted to the whole cell and thereby reduces the volume of the
muscle cell itself (Huff-Lonergan & Lonergan, 2005; Swatland, 1995).
Offer and Knight (1998) showed that the cross-sectional area
decreased about 9% during the development of rigour mortis due to
lateral shrinkage of the myofibrils. This shrinkage of myofibrils
significantly influences water movement by reducing the space
available to hold the myowater. The critical consequence of the lateral
shrinkage of the myofilaments is that fluid moves from between the
myofilaments or intra-myofibrillar space into the sarcoplasm where it
becomes situated between the myofibrils of the muscle fibre; the inter-
myofibrillar space. This expulsion of water from between the intra-
myofibrillar space into the extra-myofibrillar space corresponds with an
increase in extra-myofibrillar water volume and a simultaneous
decrease in intra-myofibrillar area, which is reflected in relaxation
times of the different water populations (Bertram & Andersen, 2004).
The shrinking of the intra-myofibrillar area alone is not entirely
responsible for the changes in water distribution during the
development of rigour mortis. Disintegration of cellular membranes
during the development of rigour mortis is also required to facilitate
the transfer of intra-myofibrillar water to extra-myofibrillar spaces
and drip channels. Comparison of changes in the water populations
using NMR relaxometry and impedance characteristics (Py), as an
indicator of membrane integrity, implies that altered membrane
properties also contribute to water mobility in the post mortem
muscle. Bertram, Rasmussen, et al. (2002) reported that a decrease in
muscle impedance measures (Py) occurred simultaneously with an
increase in the extra-myofibrillar area during the development of
rigour mortis. This change responds to ‘leaky’ membranes and thereby
reveals that disintegration of cell membranes takes place post mortem.
Tornberg et al. (2000) have shown using NMR that a slow pH fall and
hence delayed onset of rigour mortis postpone the breakdown of cell
membranes for up to 8 h post mortem in meat.
The degree of water accumulation in the extra-myofibrillar space
is also determined by the interaction between the myofibrils and the
connective tissue and specific cytoskeletal proteins. For example;
Offer and Knight (1988) and Kristensen and Purslow (2001) provided
evidence to suggest that the shrinking myofibrillar area along with an
intact cytoskeleton (which consists of the desmin-rich intermediate
filaments and costameres) resulted in the detachment of fibre bundles
(muscle fasciculi) from the connective tissue epimysium allowing the
exudation of water to accumulate in the extra-fascicular space in
between the muscle fasciculi.
Based on the presumption of membrane disintegration and an
intact cytoskeleton the following process of water movement of water
within the muscle occurs (Yhe following points are referenced from:
Bertram et al., 2004; Kristensen and Purslow, 2001; Offer et al., 1989;
Schäfer, Knight, Wess, & Purslow, 2000; Tornberg et al., 2000):
1) The longitudinal and lateral shrinkage of the muscle fibres/cell is
concurrent with the shrinkage of the endomysial connective tissue
network surrounding a muscle fibre.
2) The connection between the endomysial network and the
connective tissue perimysium is broken.
Fluid accumulates in the perimysial network between the fibre
bundles. This is said to occur within the first 4–6 h post mortem.
3) At a later stage post mortem the muscle fibre shrinks within the
endomysial network.
4) Fluid then accumulates in gaps between the fibres and endomysial
network. This is observable 12 h post mortem.
In summary, the movement of water happens firstly from within
the myofibrillar compartment or the intra-myofibrillar space into the
inter-myofibrillar space and then into one of two other extra-
myofibrillar compartments, the inter and extra fascicular spaces
(Honikel et al., 1986; Penny, 1975). Many studies have shown the
extra-myofibrillar volume increases up to 24 h post mortem (Huff-
Lonergan & Lonergan, 2005) and it has been hypothesised that at 24 h
post mortem there is a 1.6 fold increase in volume in the extra-
myofibrillar space compared to the pre rigour state (Currie & Wolfe, 1983;
Heffron & Hegarty, 1974).
The end result is formation of gaps between the muscle fibres/
cells, the fibre bundles and in the perimysial network and the
development of water channels of approximately 20–50 μm that are
in close proximity to the connective tissue network, as also shown by
NMR microimaging (Bertram & Andersen, 2004). This corresponds to
the observations of Offer & Cousins (1992), who demonstrated
movement of water into the inter and extra fascicular space using light
microscopy.
The formation of these water channels resulting from the
formation of gaps between the muscle fibres is considered important
as they are suggested to be the primary place from where subsequent
drip and purge loss originate (Purslow et al., 2001). Many studies have
found a correlation between extra-myofibrillar volume post mortem
and drip loss (Guignot et al., 1993), and light microscopy analysis has
visualised this formation of channels emerging 2–4 h post mortem in
bovine muscles (Honikel et al., 1986; Offer & Cousins, 1992; Offer &
Knight, 1998; Schäfer et al., 2000). The gaps between muscle fibre
bundles have been shown to decrease slightly from 9 to 24 h post
mortem, possibly due to fluid outflow from these channels (Schäfer,
Rosenvold, Purslow, Andersen, & Henckel, 2002). In addition, these
studies have revealed that migration of the mobile water to the
surface of the meat samples becomes evident later in the post mortem
period, which is consistent with the hypothesis that the formation of
water channels in the meat is the source of drip.
5. Degradation of myofibrillar proteins
Single muscle fibres enter rigour mortis individually forming the
irreversible association of thick and thin filaments after depletion of
 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
ATP as a function of the initial glycogen level contained in the muscle
cells prior to slaughter (Jeacocke, 1984). Eventually the entire muscle
is in rigour mortis and inextensible. Importantly, the commencement
of ageing begins once meat is in rigour mortis so early rigour mortis
means early commencement of ageing and therefore the process of
tenderisation. However it should be noted that proteolysis (degrada-
tion of proteins) can commence before the whole muscle is in rigour
mortis (Hopkins & Thompson, 2001).
The rate and the extent of meat tenderisation are variable and
dependent on both intrinsic factors such as animal species, sex and
age, muscle fibre type and extrinsic factors, such as electrical
stimulation and temperature during chilling and storage type and
duration of the storage period (Ouali & Talmant, 1990; Valin & Ouali,
1992). Most of the reduction in toughness (increased tenderisation) is
attributed to changes in the myofibrillar proteins and cytoskeletal
proteins (Hopkins & Geesink, 2009). In general, connective tissue is
stable post mortem (McCormick, 1994; Purslow & Trotter, 1994)
although after extended periods of storage intramuscular connective
tissue does show signs of structural changes (Nishimura, Ojima, Liu,
Hattori, & Takahashi, 1996).
During post mortem tenderisation there are major changes in the
myofibrillar structure predominantly due to the proteolysis of key
myofibrillar and associated proteins. The proteins that are degraded
include those involved in inter- (e.g. desmin and vinculin) and intra-
myofibril (e.g. titin, nebulin) linkages or those which link myofibrils to
the sarcolemma by costameres (e.g., vinculin, dystrophin), and the
attachment of muscle cells to the basal lamina (Hattori et al., 1991).
The function of some of these proteins is to maintain the structural
integrity of myofibrils (Purslow et al., 2001) and proteolytic
degradation of these causes a weakening of myofibrils and, thus,
tenderisation (Koohmaraie, 1996; Robson et al., 1997; Taylor, Tassy, et
al., 1995). The evidence indicates that a number of proteins associated
with the thin and thick filaments and others found at the periphery of
the Z-disk are degraded under normal post mortem chiller conditions.
Contrary to earlier suggestions that Z-disk degradation was significant
during post mortem storage of muscle (Davey & Gilbert, 1969), Taylor,
Geesink, et al. (1995) demonstrated that degradation of proteins near
the Z-disk is what actually occurs.
It has generally been claimed that the major proteins actin and
myosin are not degraded during post mortem proteolysis (Hopkins &
Thompson, 2002; Koohmaraie & Geesink, 2006). However, studies
(Hwang, Park, Kim, Cho, & Lee, 2005; Lametsch & Bendixen, 2001;
Lametsch, Roepstorff, & Bendixen, 2002; Lametsch et al., 2003) have
reported degradation of actin and myosin during ageing. The results
were obtained using two-dimensional gel electrophoresis and mass
spectrometry (generally know as proteomics) which offers a better
protein separation compared to one-dimensional SDS-PAGE. It should
be noted that the importance of this degradation is still a matter of
debate, whilst Hwang et al. (2005) did contend that the extent of the
degradation was unlikely to contribute to tenderness as a link
between such degradation and tenderness has not been confirmed.
The main endogenous protease responsible for proteolysis of
myofibrillar proteins, resulting from the cleaving of peptide bonds, is
believed by a number of authors to be the calpains; μ-calpain and m-
calpain (Dransfield, 1994; Herrera-Mendez, Becila, Boudjellal, & Ouali,
2006; Koohmaraie, 1994, 1996; Ouali & Talmant, 1990) with others
such as calpains 3 and 10 (Ilian, Bekhit, & Bickerstaffe, 2004). There
are however other workers who have challenged the sole role of the
calpains in tenderisation (Herrera-Mendez et al., 2006; Hopkins &
Taylor, 2004) with a suggestion that other enzymatic systems could
contribute to tenderisation such as cathepsins and/or a multienzy-
matic process including proteasomes and caspases (Ouali et al., 2006).
Caspases, as suggested by Camou, Marchello, Thompson, Mares, and
Goll (2007) and Ouali et al. (2006) could contribute to post mortem
proteolysis given the inactivity of the calpains at a pH lower than 5.8.
Caspases have a significant role in apoptosis (Turk & Stoka, 2007) and
117
are likely to contribute to the early degradation steps. However as
recently stressed by Hopkins and Geesink (2009) there is no clear
evidence to link the activity of caspases to tenderisation and previous
studies have not supported a role for cathepsins in tenderisation
(Hopkins and Thompson, 2001). It is apparent that proteolysis of the
myofibrillar proteins is under the control of more than one enzyme
system because as demonstrated by Hopkins and Thompson (2001)
proteolysis of some proteins still occurred in the presence of specific
calpain inhibitors, although the meat did not tenderise.
5.1. Proteolysis and swelling of muscle fibres during ageing
An intact cytoskeleton is necessary to translate the shrinkage of
myofibrils to the whole cell (Offer & Knight, 1998). The lateral
shrinkage induced by rigour mortis results in a shrinkage of the whole
muscle fibre/cell and ‘squeezes’ water out (Purslow et al., 2001). This
water accumulates between muscle fibres in the inter-fascicular space
and other extra-myofibrillar spaces, e.g. the above mentioned water
channels, whereupon it becomes potential drip (Offer & Cousins,
1992).
However degradation of the cytoskeleton during ageing is
occurring and assists in minimising shrinkage and increasing WHC
of meat by removing inter-myofibrillar and costameric connections
and thereby reduce or remove the link between lateral shrinkage of
myofibrils induced by rigour mortis and shrinkage of the whole muscle
fibre. The proteolytic degradation of cytoskeletal proteins subse-
quently results in the swelling of the muscle cell and is expected to
allow the meat structure to retain water expelled from the myofibrils
during rigour mortis and thus not lost as drip (Melody et al., 2004).
Large numbers of cytoskeletal connections may have to be
degraded before cell shrinkage is stopped and extra-myofibrillar
water is able to flow back into the intra-myofibrillar spaces with a
resulting increase in WHC of the meat. However, the process of
myofibrillar breakdown also reduces the force that enables water to
flow between compartments. The pressure differences between intra-
and extra-myofibrillar water compartments which have been built up
during the contraction process are only slowly equalised by a flow of
water from intra- to extra-myofibrillar water compartments, where it
can be lost as drip. When the pressure differences have been
equalised, the flow of intra-myofibrillar water to extra-myofibrillar
water compartments is significantly reduced and is primarily thought
to be driven by osmotic differences created by ongoing proteolysis.
Fig. 4 details the primary changes (Kristensen and Purslow, 2001). The
driving force for the inflow of water to the extra-myofibrillar spaces
during the storage of meat is driven by the differences in protein
concentration between the intra- and extra-myofibrillar compart-
ments. The intra-myofibrillar space pre-rigour consists of the
approximately 60% muscle protein and 28% sarcoplasmic protein
(Pearson & Young, 1989) with most of the sarcoplasmic proteins
being located in the intra-myofibrillar space. Even though some
sarcoplasmic proteins are lost during drip formation post mortem
(Savage et al., 1990) a highly significant proportion of the muscle
proteins are still located in the intra-myofibrillar space and thereby
this remains the driving force for the inflow of inter-myofibrillar
water during storage of meat (Purslow et al., 2001).
Recently, the swelling of the myofibrils during the ageing of pork
was visualised using confocal laser scanning microscopy (Straadt,
Rasmussen, Andersen, & Bertram, 2007). This study by Straadt et al.
(2007) and that also by Bertram, Rasmussen, et al. (2002) indicated a
high proportion of unswollen fibres one day post mortem, followed by
an equal distribution of unswollen and swollen fibres four days post
mortem and a high proportion of swollen fibres 14 days post mortem.
These changes were found to resemble changes in the characteristics
of myowater during ageing; i.e. the intra-myofibrillar water popula-
tion across all myofibrils became increasingly similar in nature during
the ageing period (Bertram et al., 2007). The study by Straadt et al.
118 K.L. Pearce et al. / Meat Science 89 (2011) 111–124

Fig. 4. The changes in WHC of meat during ageing. (A) A simplified pre rigour muscle cell with three myofibrils connected to each other and to the cell membrane by the cytoskeleton.
(B) Post mortem shrinkage of single myofibrils leads to shrinkage of the whole muscle cell which causes an outflow of water from intra- to extracellular water compartment. (C) Proteolysis
of the cytoskeleton removes the myofibrillar strain on the cell membrane which results in an inflow of extra-myofibrillar water to the muscle cell. (D) Relationship between water flow rate,
time post mortem and quantity of proteolysed cytoskeletal proteins (Kristensen & Purslow, 2001).

(2007) is the first of its kind and further research is needed to quantify
the structural changes in relation to the water distribution throughout
ageing. This will also make it possible to get new insight in structure
versus water characteristics, e.g. water-binding capacity, as a function
of processing conditions.
Similarly, it has become apparent that the individual muscle fibres
disintegrate at different rates during ageing (Purslow et al., 2001).
However, additional studies are needed to confirm whether this
disintegration of the individual fibres is concomitant to the degree of
swelling, and whether this can be linked to specific fibre types.
5.2. Desmin and integrin degradation and effects on drip loss
Cytoskeletal proteins such as desmin and cell adhesion molecules
such as integrin play important roles in maintaining the integrity of
the muscle cell (Hynes, 1992). Both are known substrates of μ-calpain
(Huff-Lonergan & Lonergan, 2005; Lawson, 2004).
Several studies have shown that a high level of desmin
degradation is correlated with improved WHC during post mortem
storage (Melody et al., 2004; Purslow et al., 2001; Rowe, Huff-
Lonergan, & Lonergan, 2001). The intensity of intact desmin at two
and seven days post mortem was negatively correlated with drip loss
after 5 days of storage and purge loss after 7 days of storage post
mortem (Melody et al., 2004). Limited desmin degradation (compared
to more extensive degradation) has been proposed to result in the
shrinkage of the muscle cells which ultimately contributes to
formation of gaps between muscle cells and between muscle bundles
(Offer & Cousins, 1992). These gaps are used to channel drip and
purge loss during storage. Kristensen and Purslow (2001) contended
that the degradation of the cytoskeleton was responsible for an
increase in WHC during ageing.
In contrast, it has been speculated that integrin, which attaches the
cytoskeleton to the extracellular matrix, should have an impact on the
formation of drip channels in pork (Lawson, 2004). The resulting drip
channel may contribute to drip loss post mortem (Lawson, 2004;
Zhang, Lonergan, Gardner, & Huff-Lonergan, 2006). The inhibition of
calpain activity has been shown to reduce both integrin degradation
and drip channel formation in post mortem muscle (Lawson, 2004). In
meat with fast proteolysis the loss of cytoskeletal integrity and hence
extracellular linkages seems to reduce the opening up of large drip
channels (Bee, Anderson, Lonergan, & Huff-Lonergan, 2007). In order
to revisit the potential relationship between integrin content in the
pork and water distribution and mobility, Straadt, Rasmussen, Young,
and Bertram (2008) carried out a low-field NMR relaxation study on
pork combined with drip loss measurement, western blot analyses
and specific confocal laser scanning microscopic investigations. Even
though this study did not reveal a strong link between integrin
degradation and WHC, it did indicate that integrin degradation early
post mortem had an impact on the succeeding development in water
distribution and mobility during ageing. A significant positive
correlation was observed between integrin intensity on day one
post mortem and the fast-relaxing component T21 (which represents
intra-myofibrillar water) at day seven (R2 = 0.54). This indicates that
a higher amount of intact integrin day one post mortem will result in a
longer relaxation time of water in the intra-myofibrillar space due to
either an increase in the spacing between the myofibrils or decreased
water content between the myofibrils.
5.3. Myowater distribution and protein degradation during ageing
Higher drip loss has been shown to occur during early post mortem
storage which corresponds with decreased WHC from one day post
mortem. Kristensen and Purslow (2001) and Dolatowski, Twarda, and
Dudek (2004) both found that the WHC of pork gradually decreased
immediately post rigour followed by an increase as ageing continued.
It is not clear whether the water loss is directly associated with the
 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
cytoskeletal proteins or whether degradation of the protein contrib-
utes to the appearance of muscle water over time (Rosenvold et al.,
2008). Further research into this aspect is required.
Proteins play a critical role in immobilising water in meat. Initially
proteolysis improves WHC, but extended proteolysis has the potential
to reduce WHC and increase drip loss. Proteins play a critical role in
immobilising water in meat (Xiong, 2004). As cytoskeletal proteins such
as titin comprise approximately 10% of muscle protein (Labeit &
Kolmerer, 1995) there is considerable potential for water to become
available as the cytoskeletal protein tertiary structure breaks down.
Studies have shown that adherent to increased drip formation during
ageing the amount of bound myowater decreases, which suggests that
the mobilisation of water for potential drip is not merely a translocation
of water, but also a continual release of protein-associated water, as a
consequence of ongoing protein denaturation (Devine, Wells, & Lowe,
2004; Kristensen and Purslow, 2001; McGlone, Devine, & Wells, 2005).
As speculated by Devine et al. (2004), the cytoskeletal component of
drip is not present until post rigour as there is no substantial cytoskeletal
degradation pre rigour (Schäfer et al., 2002). It has been suggested that
drip arises from both myosin denaturation which occurs pre-rigour
(primarily responsible for WHC) as well as post rigour cytoskeletal
degradation and consequent tenderisation (Rosenvold et al., 2008).
Whilst post rigour drip is thus an inevitable consequence of tenderisa-
tion it may be partially contributed to by myosin denaturation also.
If this is correct then one needs to separate out pre rigour situations
that increase drip with the release of water during ageing. The use of
NMR technologies will assist in a greater understanding of this
differentiation.
6. The role of intrinsic and extrinsic factors on
myowater characteristics
6.1. Myowater distribution and mobility in different muscle types
An extensive number of studies have concentrated on the
characterisation of the myowater populations in different muscle
types using NMR relaxometry (Adzamli, Jolesz, Bleier, Mulkern, &
Sandor, 1989; Bertram, Rasmussen, et al., 2002; Bonny et al., 1988;
Hatakenaka, Ueda, Ishigami, Otsuka, & Masuda, 2001; Polak, Jolesz, &
Adams, 1988). Despite quantitative differences in characterisation of
myowater populations in these studies, they all come to the same
general conclusion; that slow-twitch red muscles (also referred to as
oxidative or Type I muscle) are characterised by a slow relaxing time
constant T22 or larger population P22 compared to fast-twitch Type IIb
white muscle. This difference in myowater characteristics between
the different muscle types has been speculated to be due to;
• Differences in the ratio in intra-/extra-myofibrillar myowater in the
different muscle fibre types (Bertram, Rasmussen, et al., 2002;
Bonny et al., 1988; Polak et al., 1988).
• Differences in fat and myoglobin concentrations between different
muscle fibre types.
• Differences in the hydrophobicity of the myosin isoforms in the
different muscle fibre types (Bonny et al., 1988).
Bertram, Purslow, et al. (2002) found some discrepancy in extra-
myofibrillar fluid spaces in muscles sampled at different periods post
slaughter compared to the corresponding 24 h post mortem measure-
ment revealing differences in myowater distribution between
different muscle types during the conversion of muscle to meat.
Consequently, care should be taken if a direct comparison between in
vivo and post mortem conditions is to be carried out.
6.2. Effect of animal growth rate
The longitudinal and radial growth of muscle is associated with
changes in muscle composition. The concentration of water changes
119
as does the protein content and concentration of ions (Bertram,
Rasmussen, et al., 2002; Dickerson & Widdowson, 1960; Sink & Judge,
1971). Due to ongoing protein synthesis and limited protein
catabolism during growth, proteins accumulate, which increases
myofibrillar protein density. Such a change in the intra-myofibrillar
space should be expected to influence the intra- and inter-myofibrillar
myowater characteristics. This has been confirmed in a study where
NMR transverse relaxation was used to characterise 24 h post mortem
porcine muscle samples (Bertram et al., 2007). Thus the intra-
myofibrillar myowater content did decrease with a simultaneous
increase in the inter-myofibrillar myowater content in muscles as a
function of increasing slaughter weight (25 to 150 kg). Thus the
change in the intra-myofibrillar myowater characteristics resembled
the increase in protein content/density of the muscles. Moreover, this
study indicated that an increased rate of protein deposition increased
the amount of extra-myofibrillar water, suggesting that high growth
rates give rise to a higher proportion of glycolytic fibres (Bertram,
Purslow, et al., 2002; Dransfield & Sosnicki, 1999; Fang, Nishimura, &
Takahashi, 1999) as glycolytic muscles have been shown to have
larger extra-myofibrillar fluid spaces than oxidative muscles (Polak et
al., 1988). These findings immediately raise the question of whether a
high extra-myofibrillar volume in vivo correlates with the potential
drip of the derived meat?
Considering the above findings, the establishment of an online
grading system based on NMR relaxation might be a future approach
in predicting protein density as a function of growth of an animal,
which potentially could also facilitate studies relating growth rate to
meat quality characteristics.
6.3. Effect of slaughter age
Slaughter age has been found to affect myowater characteristics in
porcine muscles, which again was reflected in the sensory properties
of the derived cooked pork (Bertram et al., 2007). This combined
sensory and NMR relaxometry study revealed that the myowater in
meat from young pigs (90 day old) was more loosely bound and gave
rise to the highest sensory score of the cooked pork compared to meat
from older pigs (140, 161 and 182 day old pigs). The higher overall
sensory score was a result of the attributes; high water release during
chewing and increased meat juiciness. Interestingly this study also
showed that the myofibrillar water population in meat from 90-day
old pigs was symmetrical, whilst the myofibrillar water population in
meat from pigs with an age between 140 and 180 days was bimodal.
This bimodal myowater distribution was suggested to reflect
structural constraints in the meat and indicates that the thermal
denaturation of myofibrillar structures is age-dependent, which
corresponds to the fact that an increase in intramuscular connective
tissue takes place during growth (Fang et al., 1999). Moreover, a
recent study has supported the view that a relationship between
juiciness and bimodal distribution of myofibrillar water exists
(Bertram et al., 2007). It is worth mentioning that the study by
Bertram et al. (2007) is an isolated study in domestic animals which
may be confounded with the effects of muscle growth and growth
path. Consequently, further studies of the effect of age across domestic
species are required if NMR technologies are to be potentially used in
processing plants that slaughter animals of a variety of ages.
6.4. Effect of pre-slaughter handling
Pre-slaughter handling includes mixing of unfamiliar animals at
the farm, loading, transport and abattoir lairage. Poor pre-slaughter
handling can induce stress either psychologically or physically, and it
has long been established that pre-slaughter stress can adversely
affect meat quality (Callow (1936) as cited by Fernandes, Smith, and
Armstrong (1979)).
120 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
In pigs, pre-slaughter stress immediately prior to slaughter can
significantly reduce the WHC of pork as it can result in low pH values
and higher temperatures early post mortem which causes myofibrillar
and sarcoplasmic proteins to denature (Bendall & Wismer-Petersen,
1962; Sayre & Briskey, 1963; Wismer-Pedersen, 1959) leading to PSE
pork. The unfavourable pH and temperature conditions leading to
inferior WHC were originally linked to pigs that were carriers of the
Halothane gene, but have also been shown to reduce WHC in pig
populations free of the Halothane gene (Rosenvold & Andersen,
2003).
Although a number of studies have shown that the denaturation of
myofibrillar and sarcoplasmic proteins is linked to PSE pork,
conflicting studies indicate other mechanisms are taking place within
the muscle. Bond and Warner (2007) studied the impact of pre-
slaughter stress on WHC of lamb, mimicked by the use of exercise
immediately before stunning. In exercised animals early post mortem,
the muscle temperatures were higher and the pH was lower resulting
in reduced WHC. Importantly the reduced WHC of the lamb meat was
not due to denaturation of either sarcoplasmic or myofibrillar
proteins. Further, the increased purge in the study of Bond and
Warner (2007) was not found to be a result of differences in
sarcomere length in meat from stressed and non-stressed animals.
Consequently, other factors affecting the spacing of the myofibrils and
the electrostatic attraction between myofibrils, such as ionic condi-
tions and proteolysis, seemed to be factors that explain the observed
results. Bertram et al. (2004) demonstrated that the loss of membrane
integrity and formation of loosely bound myowater took place
significantly faster and was much more severe in muscles from pigs
exercised pre-slaughter compared to pigs exposed to less stressful
pre-slaughter conditions. This indicates the importance of membrane
integrity and formation of loosely bound myowater on the water-
holding capacity of meat.
6.5. The effect of chilling
The temperature dependence of water distribution and mobility in
muscle tissue was well demonstrated in studies by Peemoeller and
Pintar (1980) and Bertram, Karlsson, and Andersen (2003). Different
carcass chilling rates will affect the water dynamics in post mortem
muscle by changing the water movements from the myofibrillar
lattice to the extra-myofibrillar space and thereby the subsequent
WHC and accumulation of potential drip. An increased rate of chilling
will improve WHC because water movements to the extra-myofibril-
lar space are reduced thereby limiting accumulation of potential drip
(Bertram, Karlsson, et al., 2003).
In the paper by Bertram, Karlsson, et al. (2003) the slow-relaxing
water population which represents the extra-myofibrillar water was
1.0–1.3% lower 24 h post mortem in fast-chilled muscle when
compared with slower chilled muscle. As stated previously, higher
levels of extramyofibrillar water are associated with a lower potential
drip loss of pork (Bertram, Purslow, et al., 2002).
A reduction in the rate of pH decline and hence decreased rate of
post mortem glycolysis appears to be the mechanism for a reduction in
drip in rapidly chilled muscle (Purslow et al., 2001). As quoted by
Bertram, Rasmussen, et al. (2002), a reduced rate of post mortem
glycolysis rate and thereby a decrease in the rate of the pH fall, will
reduce the initiation of membrane integrity loss, and in combination
with a later onset of rigour mortis prevent the ‘squeezing’ of water out
from the intra-myofibrillar area and into the extra-myofibrillar space
preventing a rapid increase in the slow relaxing population (P22).
Slow cooling stimulates these aforementioned events and gives rise to
the observed rapid increase in the slow population immediately post
mortem (Aaslyng, Bejerholm, Ertbjerg, Bertram, & Andersen, 2003).
The increase in the slow relaxing population and therefore a higher
amount of extra-myofibrillar water increases potential accumulation
of drip.
6.6. Effect of cooking
It is known that cooking denatures the muscle proteins, which
directly influences the structural characteristics (Tornberg, 2005) and
thereby also the water distribution of the meat (Bertram, Kohler,
Bocker, Ofstad, & Andersen, 2006). Such structural changes lead to
substantial loss of water (cooking loss) in the range of 15 to 35%.
However, the amount of cooking loss is highly dependent on cooking
method, cooking time/temperature and end-point temperature
(Aaslyng et al., 2003). Thermographic studies of meat have shown
that protein denaturation generally takes place in three steps; 1)
myosin (rod and light chain) denaturation at ~40–60 °C, 2) denatur-
ation of sarcoplasmic protein and collagen at ~60–70 °C and finally 3)
denaturation of actin around 80 °C (Deng et al., 2002; Stabursvik,
Fretheim, & Fr›ystein, 1984; Stabursvik & Martens, 1980; Wright,
Leach, & Wilding, 1977; Wright & Wilding, 1984).
Recently, Bertram et al. (2006) found that denaturation of the
specific muscle proteins resembles the change in water characteristics
during cooking. Thus the major changes in water characteristics took
place between 40 °C and 50 °C, which suggested that the denaturation
of myosin heads is of importance for subsequent cooking loss.
Moreover, the denaturation of myosin rods and light chains at ~53–
58 °C also affected the water distribution, whilst the denaturation of
actin at ~80–82 °C was correlated directly to expulsion of water from
the meat (cooking loss). These data resemble the findings of
Micklander, Peshlov, Purslow, and Engelsen (2002), who likewise
demonstrated in a NMR relaxation study that water mobility within
the meat was accelerated around 40 °C, and was hypothesised to be
due to the denaturation of myosin reducing the myofibrillar lattice
spacing, whilst the expulsion of water (cooking loss) first was
enhanced at higher temperatures.
Straadt et al. (2007) demonstrated pronounced shrinkage of fibres
upon cooking giving rise to large gaps between the cooked muscle
fibres, as well as at the level of the individual myofibrils. This took
place concomitant to water expulsion from the myofibrillar matrix
and thus adds to the mechanisms responsible for cooking loss given
by Bertram et al. (2006) and Micklander et al. (2002). The observed
changes in water distribution in pork during cooking reported by
Straadt et al. (2007) resemble those observed during cooking of
chicken meat (Shaarani, Nott, & Hall, 2006).
7. The relationship between myowater and meat quality attributes
7.1. pH
NMR relaxation measurements have been shown to correlate with
the pH characteristics in meat. Thus relaxation measurements on 24 h
post mortem meat have been correlated to both the pH progress and
ultimate pH in pork meat of differing quality (Purslow et al., 2001;
Renou, Monin, & Sellier, 1985). Interestingly, Renou et al. (1985)
found a positive correlation between loosely bound myowater and
ultimate pH, whilst Bertram, Whittaker, Andersen, and Karlsson
(2003) found a negative correlation. The reason for this discrepancy is
unknown; however, both studies showed stronger correlations
between 24 h relaxation measurements and early post mortem pH
than with ultimate pH. These studies confirmed that the pH–time
relationship during the conversion of muscle to meat is of great
importance for the mobility of myowater, which again correlated to
WHC of the meat as also reviewed by Rosenvold and Andersen (2003).
7.2. Sarcomere length
The sarcomere length in meat is found to be directly related to
both tenderness (Locker, 1960) and WHC (Honikel et al., 1986). In an
NMR relaxation study the intra-myofibrillar water population in pork
was demonstrated to highly correlate with the sarcomere length,
 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
which was negatively correlated with WHC (Bertram, Purslow, et al.,
2002). These findings clearly suggest that the WHC of pork is affected
by the structural features of the meat. Furthermore, a subsequent
study has shown that muscle shortening and changes in the
characteristics of intra-myofibrillar water during the conversion of
muscle to meat take place simultaneously (Bertram & Andersen,
2004).
7.3. WHC
As reviewed by Bertram and Andersen (2004), Renou et al. (1985)
were the first to show that both NMR longitudinal transverse
relaxometry correlate to WHC. Subsequently, first Tornberg, Andersson,
Göransson, and Von Seth (1993) and an extensive number of later
studies have demonstrated that NMR transverse relaxometry easily
distinguish between meat of different WHC characteristics spanning
from DFD (dark, firm and dry) to PSE (Bertram, Purslow, et al., 2002;
Purslow et al., 2001; Tornberg et al., 2000).
In the study of Bertram, Purslow, et al. (2002) it was found that
more mobile myowater within the meat (extra-myofibrillar water) is
to be considered the potential drip of the meat. In a subsequent study
where the drip was measured during storage of pork for up to 14 days
post mortem it was noticed that during the initial days of storage, the
WHC of the meat decreased, whilst extended storage made the WHC
of the meat increase indicative of increased myowater mobility during
storage (Bertram et al., 2007).
8. The influence of myowater characteristics on eating quality
8.1. Tenderness
In a recent study with sheep meat, Pearce et al. (2008) investigated
the relationship between shear force and NMR relaxation measure-
ments and demonstrated a moderate relationship of r = 0.78. The
study demonstrated that as tenderisation proceeded the fast-relaxing
water population (P21) increased with a decreasing relaxation time
(T21). As described previously, an increase in the number of relaxation
sinks within the myofibrils, i.e. protons in the intra-myofibrillar space,
over the ageing period will correspond with an increased concentra-
tion of the fast-relaxing water population and a decrease in the
relaxation time of the fast-relaxing water population. In addition
there is less water in the extra-myofibrillar space reflected by a
decrease in concentration of the slow-relaxing water population.
These combined results indicate either an increasing uptake of water
into the intra-myofibrillar space or alternatively the release of water
from bound proteins during the proteolysis process. These changes in
the structural organisation of intra- and extra-myofibrillar water
reflect the process of ageing and proteolysis. The important
conclusion from this study is that a high amount of intra-myofibrillar
water and a low amount of extra-myofibrillar water may be
associated with more tender meat.
Fjelkner-Modig and Tornberg (1986) investigated the water
distribution of raw and cooked meat in relation to sensory properties
of pork from the Hampshire and Yorkshire breeds. On average, the
samples of the Hampshire breed were more tender than those of the
Yorkshire breed. For the Yorkshire breed a significant relationship was
found between toughness and the relaxation time of the slow-
relaxing water population (r = 0.71) and the fast-relaxing water
population (r = −0.66). Despite the species different, this finding is in
direct contrast to the results of Pearce et al. (2008). For the Hampshire
breed, it was demonstrated that toughness was related to the
relaxation time of the fast-relaxing water population with a
correlation of − 0.97. Therefore an increased relaxation time for the
fast-relaxing water population which implies a greater myofibrillar
space (following cooking) gives more tender meat. This finding was
opposite to the finding for the Yorkshire breed.
121
The inconsistencies in the relationship between water population
and mobility and tenderness across species and within breeds could for
example reflect differences in the amount of intramuscular fat
which has been shown to affect the sensory properties of meat
(Fjelkner–Modig & Tornberg, 1986) or differences in the glycogen
content of meat. Further research to understand the relationship between
tenderness and water distribution and mobility in meat will increase the
understanding of WHC and tenderisation, but also confirm if NMR is a
suitable technology for online measurement of meat tenderness.
8.2. Juiciness
It is generally perceived that WHC is correlated to juiciness;
however many papers have measured WHC and evaluated juiciness
simultaneously, all with variable results (Honikel & Hamm, 1994).
These inconsistencies might be a result of the different methodologies
applied to measure WHC in these studies (Trout, 1988) and/or the
different cooking methods, as cooking methods specifically affect the
result of the sensory analysis (Bejerholm & Aaslyng, 2004). Likewise,
total water content of the meat and cooking loss cannot explain
juiciness of the cooked meat product (Safari, Fogarty, Ferrier, Hopkins,
& Gilmour, 2001; Toscas, Shaw, & Beilken, 1999; Young, Hopkins, &
Pethick, 2005). Hence, even though the characteristics of the
myowater in general must be expected to contribute to juiciness,
there is only limited knowledge of how these characteristics and the
temperature-induced changes in these characteristics during cooking
affect the sensory properties of meat.
Using NMR relaxation, Fjelkner-Modig and Tornberg (1986) found
in general only a weak correlation to the sensory attribute of juiciness,
even though a specific and significant relationship between juiciness
and relaxation characteristics was established in meat from Yorkshire
pigs. More recently, Bertram, Aaslyng, and Andersen (2005) demon-
strated that differences in juiciness as a result of different final
cooking temperatures could be ascribed to differences in the
myowater characteristics in pork. A reduction in juiciness and
tenderness at 75 °C as compared to 65 °C could be ascribed to changes
in the size of the pores confining the myofibrillar water within the
meat in combination with an expulsion of water. This data was
subsequently supported in another study where myowater charac-
teristics and juiciness likewise were correlated with the most mobile
myowater (extra-myofibrillar water), being the factor affecting
juiciness (Bertram et al., 2007). The mobility of the extra-myofibrillar
water was suggested to be of utmost importance for high water
release during chewing and thereby the perception of meat juiciness.
9. Summary
As evident from the above literature review, myowater character-
istics have an impact on major quality attributes such as WHC and
tenderness in fresh meat. This review has highlighted a number of
new potential research areas based on utilising NMR relaxometry in
meat quality characterisation.
In muscle, 85% of the water is contained within the myofibrils.
During the conversion of muscle to meat a relocation of the myowater
takes place. This reorganisation is related to physical processes, which
takes place during the post mortem period, i.e. longitudinal and lateral
contraction of the muscle fibre and altered membrane and structural
properties, which again are triggered by the changes in post mortem
metabolism. The longitudinal and lateral contraction of the muscle
fibre essentially ‘squeezes’ water out from in between the myofibrils
(the intra-myofibrillar space) into the extra-myofibrillar space.
Several studies have demonstrated a correlation between the
characteristics of the extra-myofibrillar myowater post mortem and
drip loss. Animal age, slaughter weight, muscle type and the rate of pH
decline all contribute to the degree of extra-myofibrillar fluid
development during the development of rigour mortis.
122 K.L. Pearce et al. / Meat Science 89 (2011) 111–124
Proteolysis leads to weakening of the myofibrils and hence
tenderisation of the meat. Increased proteolysis of cytoskeletal
proteins such as desmin and cell adhesion molecules such as integrin
will affect water distribution. Limited desmin degradation has been
proposed to result in increased shrinkage of the muscle cells, which
ultimately contributes to formation of gaps between muscle cells and
between muscle bundles. The reduced myofibrillar strain on the cell
membrane results in an inflow of extra-myofibrillar water to the
muscle cell and the swelling of the myofibrillar space increasing the
WHC. In contrast, the degradation of integrin is associated with larger
drip channels. These drip channels are used to funnel extra-
myofibrillar fluid from the meat as drip. The process of ageing is
also believed to result in the development of water due to the
degradation of water-binding proteins with this also contributing to
drip. Further understanding of the factors affecting myowater changes
during ageing using NMR technologies will increase the understand-
ing of WHC and tenderisation.
NMR transverse relaxation has been applied successfully in
characterising water and structural features in meat. The technique has
been used to determine WHC and other meat quality traits controlled by
water distribution and mobility, together with dynamic studies of
physical changes during conversion of muscle to meat. NMR transverse
relaxation is correlated to WHC, more specifically to the characteristics of
the extra-myofibrillar myowater population, hence indicating that this is
most critical for the amount of drip and hence WHC. Sarcomere length is
correlated with the characteristics of the intra-myofibrillar myowater
population, hence indicating that WHC is affected by the structural
features of meat that change with sarcomere shortening. Tenderness in
pork and sheep meat has been shown to correlate to NMR transverse
relaxation — however breed and species differences made it difficult to
draw firm conclusions as to the strength of the relationship.
It is generally perceived that WHC is correlated to juiciness;
however this is not the case. In fact there is a poor understanding of
the underlying mechanisms for juiciness and their relationship to
water distribution and mobility in fresh and cooked meat. Differences
in juiciness obtained by different cooking temperatures have been
shown to relate to differences in water distribution within the meat,
as a high juiciness score is ascribed to a larger amount of mobile water.
NMR studies have shown that the intra-myofibrillar water population
decreases as soon as heating is initiated followed by an increase in the
extra-myofibrillar water population. Cooking-induced shrinkage of
myofibrils occurs concomitantly with water being expelled from the
myofibrillar matrix upon cooking.
Whilst WHC and tenderness are often considered two indepen-
dent meat attributes, evidence reviewed in this paper indicates that
this is not necessarily true. For example, short sarcomere length
results in less tender meat, but also increases drip; proteolytic
breakdown of cytoskeletal proteins improves tenderness, but also
increases drip, although the underlying mechanisms for these
occurrences are still to be confirmed.
Acknowledgements
Meat & Livestock Australia and Meat & Wool New Zealand are
acknowledged for funding this work through their Meat Quality
Science and Technology programme.
Dr. Carrick Devine, Dr Mike North, Dr Robin Jacob and Professor David
Pethick are gratefully acknowledged for their valuable input to the review.
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