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13 Seafood Enzymes 251implications of this are minor, since it is only after the quality of
the fish has declined beyond the level of acceptability that these
reactions take place on a large scale.
The relation between the accumulation of breakdown products
of ATP and the postmortem storage period of seafood products
has resulted in various freshness indicators being defined. Saito
et al. (1959) defined a freshness indicatorKas a simplified way
of expressing the state of nucleotide degradation by a single
number:K= 100 ×[Inosine]+[Hypoxanthine]
(
[ATP]+[ADP]+[AMP]+[IMP]
+[Inosine]+[Hypoxanthine])HigherKvalues correspond to a lower quality of fish. TheK
value often increases linearly during the first period of storage on
ice, but theKvalue at sensory rejection differs between species.
An alternative and simplerKivalue correlates well with theK
value of wild stock fish landed by traditional methods (Karube
et al. 1984):Ki= 100 ×[Inosine]+[Hypoxanthine]
[IMP]+[Inosine]+[Hypoxanthine]
BothKvalues reflect fish quality well, provided the numerical
values involved are not compared directly with the values from
other species. Still,Kvalues necessarily remain less descriptive
than the concentrations of the degradation products themselves.
ATP is an important regulator of biochemical processes in
all animals and continues to be so during the postmortem pro-
cesses. One of the most striking postmortem changes is that of
rigor mortis. When the concentration of ATP is low, the con-
tractile filaments lock into each other and cause the otherwise
soft and elastic muscle tissue to stiffen. Rigor mortis is thus a
direct consequence of ATP depletion. The biochemical basis and
physical aspects of rigor mortis are reviewed by Hultin (1984)
and Foegeding et al. (1996).
ATP depletion and the onset of rigor mortis in fish are highly
correlated with the residual glycogen content at the time of death
and with the rate of ATP depletion. The onset of rigor mortis
may occur only a few minutes after death or may be postponed
for up to several days when rested harvest techniques, gentle
handling, and optimal storage conditions are employed (Azam
et al. 1990, Lowe et al. 1993, Skjervold et al. 2001). The strength
of rigor mortis is greater when it starts soon after death (Berg
et al. 1997). Although the recommended temperature for chilled
storage is generally close to 0◦C, in some fish from tropical
and subtropical waters, the degradation of nucleotides has been
found to be slower, and the onset of rigor mortis to be further
delayed when the storage temperature is elevated to 5◦C, 10◦C,
and even 20◦C (Saito et al. 1959, Iwamoto et al. 1987, 1991).
Rigor mortis impedes filleting and processing of fish. In the
traditional fishing industry, therefore, filleting is often postponed
until rigor mortis has been resolved. In aquaculture, however, it
has been shown that rested harvesting techniques can postpone
rigor mortis sufficiently to allow prerigor filleting to be carried
out (Skjervold et al. 2001). Fillets from prerigor filleted fish
shorten, in accordance with the rigor contraction of the muscle
fibers, by as much as 8%. Prerigor filleting has few problems withgaping, since the rigor contraction of the fillet is not hindered by
the backbone (Skjervold et al. 2001). Gaping, which represents
the formation of fractures between segments of the fillets, is
described further in the Section Postmortem Proteolysis in Fresh
Fish.
All enzyme reactions are virtually stopped during freezing at
low temperatures. The ATP content of frozen prerigor fish can
thus be stabilized. During the subsequent thawing of prerigor
fish muscle, the leakage of Ca^2 +from the organelles results in
a very high level of myosin Ca^2 +–ATPase activity and a rapid
consumption of ATP. This leads to a strong form of rigor mortis
called thaw rigor. Thaw rigor results in an increase in drip loss, in
flavor changes, and in a dry and tough texture (Jones 1965) and
gaping (Jones 1969). Thaw rigor can be avoided by controlled
thawing, holding the frozen products at intermediate freezing
temperatures above− 20 ◦C for a period of time (Mcdonald and
Jones 1976, Cappeln et al. 1999). During this holding period,
ATP is degraded at moderate rates, allowing a slow onset of rigor
mortis in the partially frozen state.
Rigor mortis is a temporary condition, even though in the
absence of ATP, the actin-myosin complex is locked. The reso-
lution of rigor mortis is due to structural decay elsewhere in the
muscular structure, as will be discussed later.ENZYMATIC DEGRADATION OF
TRIMETHYLAMINE-N-OXIDEThe substance TMAO is found in all marine seafood species
but occurs in some freshwater fish as well (Anthoni et al. 1990,
Parab and Rao 1984, Niizeki et al. 2002). It contributes to cellu-
lar osmotic pressure, as previously described, but several other
physiological functions of TMAO have also been suggested.
TMAO itself is a harmless and nontoxic constituent, yet it forms
a precursor of undesirable breakdown products. In seafood prod-
ucts, TMAO can be degraded enzymatically by two alternative
pathways as described later.The Trimethylamine-N-Oxide
Reductase ReactionMany common spoilage bacteria reduce TMAO to trimethy-
lamine (TMA) by means of the enzyme TMAO reductase (EC
1.6.6.9):CH 3 CH 3
O=NCH 3 NADH→NCH 3 NAD H 2 O
CH 3 CH 3.
TMA has a strong fishy odor, and TMAO reductase activ-
ity is responsible for the typical off-odor of spoiled fish. Since
TMAO reductase is of microbial origin, the formation of TMA
occurs primarily under conditions such as cold storage that al-
low microbial growth to take place. TMA is thus an important
spoilage indicator of fresh seafood products. The formation of