BLBS102-c13 BLBS102-Simpson March 21, 2012 13:15 Trim: 276mm X 219mm Printer Name: Yet to Come
13 Seafood Enzymes 255Heat-Stable Alkaline ProteinasesKinoshita et al. (1990b) reported the existence of up to four
distinct heat-stable alkaline proteinase (HAP) in fish muscle:
two sarcoplasmic proteinases activated at 50◦C and 60◦C, and
two myofibril-associated proteinases activated at 50◦C and 60◦C,
respectively. The distribution of the four proteinases was found
to be quite diverse among the 12 fish species that were studied.
The mechanisms activating these proteinases in vivo and their
precise physiological functions are not clear.
The participation of one or the other protease in the many
different degradation scenarios that occur has still been only par-
tially elucidated. However, various studies have found a close
relationship between protein degradation in fish muscle and the
activity of specific proteases. A high level of activity of cathep-
sin L has been found in chum salmon during spawning, a period
during which the fish exhibit an extensive softening in texture
(Yamashita and Konagaya 1990). Similarly, Kubota et al. (2000)
found an increase in gelatinolytic activity in the muscle of ayu
during spawning, a period involving a concurrent marked de-
crease in muscle firmness. Also, in the muscle of hake, a consid-
erably higher level of proteolytic activity during the prespawning
period than in the postspawning period was found (Perez-Borla
et al. 2002).
The possible existence of a direct link between protease activ-
ity and texture has been explored in situ by perfusing protease
inhibitors into fish muscle and later measuring changes in tex-
ture during cold storage. The activity of metalloproteinase and
of trypsin-like serine protease was found in this way to play
a role in the softening of flounder (Kubota et al. 2001) and of
tilapia (Ishida et al. 2003).POSTMORTEM HYDROLYSIS OF LIPIDS
IN SEAFOOD DURING FROZEN AND
COLD STORAGEChanges in the lipid fraction of fish muscle during storage can
lead to changes in quality. Both the content and the composi-
tion of the lipids in fish muscle can vary considerably between
species and from one time of year to another, and also differ
greatly depending upon whether white or red muscle fibers are
involved. As already mentioned, these two types of muscle fibers
are separated from each other, the white fibers generally consti-
tuting most of the muscle as a whole, although fish species vary
considerably in the amounts of dark meat, which has a higher
myoglobin and lipid content. In species like tuna and in small
and fatty pelagic fish, the dark muscle can constitute up to 48%
of the muscle as a whole, whereas in lean fish such as cod and
flounder, the dark muscle constitutes only a small percentage of
the muscle (Love 1970). Since triglycerides are deposited pri-
marily in the dark muscle, providing fatty acids as substrate to
aerobic metabolism, whereas the phospholipids represent most
of the lipid fraction of the white muscle, phospholipids consti-
tute a major part of the lipid fraction in lean fish (Lopez-Amaya
and Marangoni 2000b).
Not much research on lipid hydrolysis in fresh fish during ice
storage has been carried out, research having concentrated moreon changes in the lipid fraction during frozen storage. This
could be due to freezing being the most common way of storing
and processing seafood and to lipid hydrolysis playing no appre-
ciable role in ice-stored fish before microbial spoilage becomes
extreme. Knudsen (1989), however, detected an increase in free
fatty acids in cod muscle during 11 days of ice storage, indica-
tive of the occurrence of lipolytic enzyme activity, an increase
that was most pronounced during days 5 to 11. Ohshima et al.
(1984) reported a similar delay in the increase in free fatty acids
in cod muscle stored in ice for 30 days. Both results are basically
consistent with the observation of Geromel and Montgomery
(1980) of no lysosomal lipase activity being evident in trout
muscle after seven days on ice. In contrast, the authors reported
that slow freezing and fluctuations in temperature during frozen
storage were found to result in the release of acid lipase from
the lysosomes of the dark muscle of rainbow trout.
Several researchers have reported an increase in free fatty
acids during frozen storage of muscle of different fish species
such as trout (Ingemansson et al. 1995), salmon (Refsgaard et al.
1998, 2000), rayfish (Fernandez-Reiriz et al. 1995), tuna, cod,
and prawn (Kaneniwa et al. 2004). The release of free fatty acids
during frozen storage can induce changes in texture by stimula-
tion of protein denaturation and through off-flavors being pro-
duced by lipid oxidation (Lopez-Amaya and Marangoni 2000b).
Refsgaard et al. (1998, 2000) observed a marked increase in
free fatty acids in salmon stored at− 10 ◦Cand− 20 ◦C. This
increase in free fatty acid content was connected with changes
in sensory attributes, suggesting that lipolysis plays a significant
role in deterioration of the quality of salmon during frozen
storage.
Kaneniwa et al. (2004) detected a large variation in the forma-
tion of free fatty acids among nine species of fish and shellfish
stored at− 10 ◦C for 30 days. Once again, this demonstrates the
large variation in enzyme activity in seafood species. Findings of
Ben-gigirey et al. (1999) indicate that the temperature at which
fish are stored has a clear influence on the lipase activity occur-
ring in the muscle. They noted, for example, that the formation
of free fatty acids in the muscle of albacore tuna during storage
for the period of a year was considerably higher at− 18 ◦C than
at− 25 ◦C.
Two classes of lipases, the lysosomal lipases and the phos-
pholipases, are apparently involved in the hydrolysis of lipids in
fish muscle during storage. Nayak et al. (2003) found significant
differences among four fish species (rohu, oil sardine, mullet,
and Indian mackerel) in the degree of red muscle lipase activity.
This is quite in line with differences in lipid hydrolysis among
species that Kaneniwa et al. (2004) reported.
Although both the lipase activity and the formation of free
fatty acids in fish muscle are well documented, only a few stud-
ies have actually isolated and characterized the muscle lipases
and phospholipases involved. Aaen et al. (1995) have isolated
and characterized an acidic phospholipase from cod muscle, and
Hirano et al. (2000) a phospholipase from the white muscle of
bonito. Similarly, triacylglycerol lipase from salmon and from
rainbow trout has been isolated and characterized by Sheridan
and Allen (1984) and by Michelsen et al. (1994), respectively.
Knowledge of the properties of the lipolytic enzymes in the