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254 Part 2: Biotechnology and Enzymologyto the fine collagen fibers of the endomysium (see Fig. 13.3),
leads to muscle fibers being detached, which results in softening.
Studies have shown that the costameric proteins found in fish
muscle are already degraded 24 hours postmortem (Papa et al.
1997). This emphasizes the importance of early postmortem
changes.
It has been shown that changes in the collagen fraction of
the extracellular matrix and the softening of fish are related.
Collagen V, a minor constituent of the pericellular connective
tissue of fish muscle, becomes soluble when rapid softening
takes place, whereas no solubilization is observed in fish that
do not soften (Sato et al. 2002). Recent studies also indicate
a degradation of the most abundant collagen in fish muscle,
collagen I, during 24 hours of cold storage (Shigemura et al.
2004). This indicates that the degradation of collagen and the
resulting weakening of the intramuscular pericellular connective
tissue also play a role in the textural changes in fish muscle that
occur early postmortem.
A number of studies have revealed structural changes in the
myofibrillar proteins in fish muscle during cold storage. Analy-
sis of myofibrillar proteins from the muscle of salmon stored at
0 ◦C for as long as 23 days has shown that several new protein
fragments form (Lund and Nielsen 2001). Other studies suggest
that predominantly proteins of the cytoskeletal network, such
as the high molecular weight proteins titin and nebulin, are de-
graded (Busconi et al. 1989, Astier et al. 1991). The extent of
degradation of the muscle myofibrillar proteins varies among
species. It has been found, for example, that the intermediate fil-
ament protein desmin is clearly degraded during the cold storage
of turbot and sardines but that no degradation occurs during the
cold storage of sea bass and brown trout (Verrez-Bagnis et al.
1999).
As research shows, both myofibrillar and extracellular matrix
proteins in the muscle of many fish are degraded during storage,
and textural changes can be expected to result from degradation
of proteins from both structures.Proteases in Fish MuscleThe structural and biochemical changes just described can be
considered to largely represent the concerted action of differ-
ent endogenous proteolytic enzymes. Proteolytic enzymes of all
major classes have been documented in the muscle of various
fish species. An overview of the different proteases believed to
play a role in the postmortem proteolysis of seafood is presented
later. Further information on the proteases found in fish and ma-
rine invertebrates can be found in reviews by Kolodziejska and
Sikorski (1995, 1996).Matrix MetalloproteinasesMatrix metalloproteinases are extracellular enzymes involved in
the in vivo catabolism (degradation) of the extracellular matrix
(degradation of the helical regions of the collagens). They have
been isolated from rainbow trout (Saito et al. 2000), Japanese
flounder (Kinoshita et al. 2002) and Pacific rockfish (Bracho and
Haard 1995). Collagenolytic and gelatinolytic activities havealso been detected in the muscle of winter flounder (Teruel and
Simpson 1995), yellowtail (Kubota et al. 1998), ayu (Kubota
et al. 2000), salmon, and cod (Lødemel and Olsen 2003). Type
I collagen is solubilized and degraded by matrix metallopro-
teinases in rainbow trout (Saito et al. 2000), Japanese flounder
(Kubota et al. 2003), and Pacific rockfish (Bracho and Haard
1995), suggesting that these proteases can participate in post-
mortem textural changes.CathepsinsLysosomal proteinases such as cathepsins B, D, and L have
been isolated from a number of fish species, including herring
(Nielsen and Nielsen 2001), mackerel (Aoki and Ueno 1997),
and tilapia (Jiang et al. 1991). In vitro studies show that certain
cathepsins are capable of cleaving myofibrillar proteins (Ogata
et al. 1998, Nielsen and Nielsen 2001) and may also participate in
degradation of the extracellular matrix since they can cleave both
nonhelical regions of the collagen (Yamashita and Konagaya
1991) and collagen that has already been partly degraded by
matrix metalloproteinases. Since cathepsins in the muscle of
living fish are located in the lysosomes, they are not originally
in direct contact with either the myofibrils or the extracellular
matrix, although it has been shown that the enzymes leak from
the lysosomes in fish muscle postmortem, and from lysosomes in
bovine muscles postmortem as well (Geromel and Montgomery
1980, Ertbjerg et al. 1999).CalpainsCalpains have been reported in the muscle of various fish species
(Wang and Jiang 1991, Watson et al. 1992). They are only active
in the neutral pH range, although research shows that they also
remain active at the slightly acidic postmortem pH (Wang and
Jiang 1991) and are capable of degrading myofibrillar proteins
in vitro (Verrez-Bagnis et al. 2002, Geesink et al. 2000). This
suggests a participation in the postmortem hydrolysis of fish
muscle. Watson et al. (1992) found evidence for calpains being
involved in the degradation of myofibrillar proteins in tuna, lead-
ing to the muscle becoming pale and grainy, which is referred to
as “burnt tuna.”20S ProteasomeThe 20S proteasome enzyme is a 700 kDa multicatalytic pro-
teinase with three major catalytic sites. The term 20S refers to
its sedimentation coefficient. In the eukaryotic cells, the enzyme
exists in the cytoplasm, either in a free state or associated with
large regulatory complexes. In vivo, it is involved in nonlyso-
somal proteolysis and apoptosis. Research strongly indicates
that this proteasome is involved in meat tenderization in cattle
(Sentandreu et al. 2002). Although proteasomes have been
detected in the muscle of such fish species as carp (Kinoshita
et al. 1990a), white croaker (Busconi et al. 1992), and salmon
(Stoknes and Rustad 1995), their postmortem activity in fish
muscle has not been clarified.