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Heat-Stable Alkaline Proteinases
Kinoshita 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 partially 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 cathepsin 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 decrease in muscle firmness. Also, in the muscle of hake, a considerably 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 activity and texture has been explored in situ by perfusing protease
inhibitors into fish muscle and later measuring changes in texture 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 STORAGE
Changes in the lipid fraction of fish muscle during storage can
lead to changes in quality. Both the content and the composition 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 constituting 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 primarily 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 constitute 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 more


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on 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 appreciable 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, indicative 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 stimulation of protein denaturation and through off-flavors being produced 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◦ C and −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 formation 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 occurring 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 phospholipases, 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 studies 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


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muscle in seafood and of the responses the enzymes show to
various processing parameters, however, is sparse. Extensive
reviews of work done on the lipases and phospholipases in
seafood has been presented by Lopez-Amaya and Marangoni
(2000a,b).

ENDOGENOUS ENZYMATIC REACTIONS
DURING THE PROCESSING OF SEAFOOD
During seafood processing such as salting, heating, fermentation, and freezing, endogenous enzymes can be active and contribute to the sensory characteristics of the final product. Such
endogenous enzyme activities are sometimes necessary in order
to obtain the desired taste and texture.

Salting of Fish
Salting has been used in many countries for centuries as a means
of preserving fish. Today, the primary purpose of salting fish
is no longer only to preserve them. Instead, salting enables
fish products with sensory attributes that are sought after, such
as salted herring and salted cod, as produced on the northern

European continent and in Scandinavia. Enzymatic degradation
of muscle proteins during salting is a factor that contributes to
the development of the right texture and taste of the products.

Ripening of Salt-Cured Fish
Spiced sugar–salted herring in its traditional form is made by
mixing approximately 100 kg of headed, ungutted herring with
15 kg of salt and 7 kg of sugar in barrels, usually adding spices
as well. After a day or two, after a blood brine has been formed,
saturated brine is added, after which the barrels are stored at
0–5◦ C for up to a year. During this period, a ripening of the
herring takes place, and it achieves its characteristic taste and
texture (Stef´ansson et al. 1995).
During the ripening period, both intestinal and muscle proteases participate in the degradation of muscle protein, contributing to the characteristic softening of the fillet and liberating free
amino acids and small peptides that help create the characteristic flavor of the product (Nielsen 1995, Olsen and Sk˚ara 1997).
Studies have shown that intestinal trypsin- and chymotrypsinlike enzymes migrate into the fillet, where they play an active role in the degradation of muscle proteins during storage
(Engvang and Nielsen 2000, Stef´ansson et al. 2000). Furthermore, Nielsen (1995) shows that muscle amino peptidases are
also active in the salted herring during storage.
In southern Europe, a similar product based on the use of
whole sardines or anchovies is produced. In contrast to the salted
herring from Scandinavia, these salted sardines and anchovies
are stored at ambient temperature, which can vary between 18◦ C
and 30◦ C (Nunes et al. 1997). Nunes et al. (1997) found that
proteases from both the intestines and the muscles participated in
the ripening of sardines (Sardina pilchardus). Hernadez-Herrero
et al. (1999) reported an increase in proteinase activity as well
as in protein hydrolysis during the storage of salted anchovies

(Engraulis encrasicolus) and found a close relationship between
proteolysis and the development of the sensory characteristics

of the product.

Production of Fish Sauce and Fish Paste
Fish sauce and fish paste are fermented fish products produced
mainly in Southeast Asia, where they are highly appreciated food
flavorings. Fish sauce is the liquefied protein fraction, and fish
paste is the “solid” protein fraction obtained from the prolonged
hydrolysis of heavily salted small pelagic fish. Production takes
place in closed tanks at ambient tropical temperatures during a
period of several months (Gildberg 2001, Saishiti 1994). The
hydrolysis represents the combined action of the fishes’ own
digestive proteases and of enzymes from halotolerant lactic acid
bacteria (Saishiti 1994). Orejani and Liston (1981) concluded,
on the basis of inhibitor studies, that a trypsin-like protease is one
of the enzymes responsible for the hydrolysis. Vo et al. (1984)
detected a high and stable level of activity of intestinal amino
peptidase during the production of fish sauce. Del Rosario and
Maldo (1984) measured the activity of four different proteases in
fish sauce produced from horse mackerel. During a four-month
period of measurement, they found the activity of cathepsins
A, C, and B to be stable and that of cathepsin D to decrease.
Raksakulthai and Haard (1992) found that cathepsin C obtained
from capelin was active in the presence of 20–25% salt, suggesting that this enzyme is involved in the hydrolysis of capelin
fish sauce. These studies indicate that several different proteases
need to be active and that their concerted action is necessary to
achieve the pronounced hydrolysis required for production of
such fish sauces.
It is notable that the similarly high storage temperatures
present in southern Europe do not lead to a solubilization of
salted sardines and anchovies. Ishida et al. (1994) reported that

at 35◦ C salted Japanese anchovies (Eriobotrya japonica) degraded to a marked degree, whereas at this temperature salted
anchovies from southern Europe (E. encrasicolus) were structurally stable. Also, they detected a thermostable trypsin-like
proteinase in the muscle of both salted Japanese anchovies and
European anchovies, but its activity was much higher in the
Japanese anchovies. An explanation of the difference between
the European and the Asian products might be a large difference
between the hydrolytic enzyme activity of the respective raw
materials.

Production of Surimi
Surimi is basically a myofibrillar protein concentrate that forms
a gel due to cross-linking of its actomyosin molecules (An et al.
1996). It is made from minced fish flesh obtained mainly from
pelagic white fish of low fat content, such as Alaskan pollack
and Pacific whiting. The mince is washed several times with
water to remove undesired elements such as connective tissue
and lipids. The particulate is then stabilized by cryoprotectants
before being frozen (Park and Morrisey 2000).
Surimi is used as a raw material for the manufacture of various
products, such as imitation crabmeat and shellfish substitutes.


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The manufacture of surimi products involves the use of a slow
temperature-setting process, which can be in the temperature
range of 4–40◦ C, followed by a heating process involving temperatures of 50–70◦ C, which results in the gel strength being
enhanced (Park 2000). Surimi-based products are very important fish products in the Asian and Southeast Asian countries,
where Japan has the largest production and marketing of surimibased products. The quality and the price are closely dependent
on the gel strength (Park 2000).
The enzyme transglutaminase plays an important role in the
gelation process through catalyzing the cross-linking of the actomyosin (An et al. 1996). Some of the differences in gelling
capability among different species are due to the properties and
levels of activity of muscle transglutaminase (An et al. 1996,
Lanier 2000). Studies have shown that the addition of microbial transglutaminase can increase the gel strength obtained in
fish species having low transglutaminase activity (Perez-Mateos
et al. 2002, Sakamoto et al. 1995). The effect of transglutaminase on the processing of surimi has been reviewed extensively
by An et al. (1996) and by Ashie and Lanier (2000).
A softening of the gel during the temperature-setting and
heating processes can occur due to auto-lysis of the myosin
and actomyosin through the action of endogenous heat-stable
proteinases. Whether or not this occurs is partly a function of
the species used for the surimi production. Two groups of proteinases have been identified as being responsible for the softening: cysteine cathepsin and HAP.
The presence of HAP in the muscle of different fish species
used for surimi production and the effects it has on degradation
of the fish gel during the heating process have been taken up in a

number of studies (Makinodan et al. 1985, Toyahara et al. 1990,
Cao et al. 1999). The finding of Kinoshita et al. (1990b) that the
fish species differ in the amount of HAP in muscle, could partly
explain why softening of the gel is more pronounced in some
fish species than in others.
A study by An et al. (1994) indicated that the cysteine proteinase cathepsin L contributes to degradation of the myofibrils
in surimi at a temperature of about 55◦ C during the heating
process, a result substantiated by Ho et al. (2000), who also
measured the softening of mackerel surimi upon the addition of
mackerel cathepsin L.

TECHNOLOGICAL APPLICATIONS OF
ENZYMES FROM SEAFOOD
Utilization of enzymes from seafood as technical aids in both
seafood processing and other areas of food and feed processing
has been an area of active research for many years. There are
two factors that have provided the primary motivation for such
research: (1) the cold-adaptation properties of seafood enzymes
and (2) the increasing production of marine by-products used
as potential sources of enzymes. Although the results have been
promising, many of the potential technologies are still in their
initial stages of development and are not yet fully established
industrially.

257

Improved Processing of Roe
Roe is considered by many to be a seafood delicacy. Russian
caviar, produced from the roe of sturgeon, is the form best
known, although roe produced from a variety of other species,

such as salmon, trout, herring, lumpfish, and cod, has also
gained wide acceptance. The roe is originally covered by a twolayer membrane (chorion) termed the roe sack. In some species,
mechanical or manual separation of the roe from the sack results
in damage to the eggs and in yields as low as 50% (Gildberg
1993). Pepsin-like proteases isolated from the intestines of
seafood species, as well as collagenases from the hepatopancreas of crabs, have been shown to cleave the linkages between
the sac and the eggs without damaging the eggs. Such enzyme
treatment has been reported to increase the yield from 70% to
90% (Gildberg et al. 2000, and references therein).

Production of Fish Silage
Fish silage is a liquid nitrogenous product made from small
pelagic fish or fish by-products mixed with acid. It is used as a
source of protein in animal feed (Aranson 1994, Gildberg 1993).
In its manufacture, the fish material is mixed with 1–3.5% formic
acid solution, reducing pH to 3–4. This is optimal for the intestinal proteases and aspartic muscle proteases contained in the
fish material, allowing the solubilization of the fish material to
proceed as an autolytic process driven by both types of protease
(Gildberg et al. 2000). Gildberg and Almas (1986) have reported
the existence of two very active pepsins (I and II) in silage manufactured from cod viscera. They were able to show that fish
by-products having low protease activity could be hydrolyzed
and used for silage by adding protease-rich cod viscera.

Deskinning and Descaling of Fish
Deskinning fish enzymatically can increase the edible yield
as compared with that achieved by mechanical deskinning
(Gildberg et al. 2000). It also provides the possibility of utilizing alternative species such as skate, the skin of which is
very difficult to remove mechanically without ruining the flesh
(Stef´ansson and Steingrimsdottir 1990). It has been shown that
herring can be deskinned enzymatically by use of acid proteases

obtained from cod viscera (Joakimsson 1984). Enzymatic removal of the skin of other species has been reported as well.
Kim et al. (1993) have described removal of the skin of filefish
by use of collagenase extracted from the intestinal organs of the
fish. Crude protease extract obtained from minced arrowtooth
flounder has been found to be effective in solubilizing the skin
of pollock (Tschersich and Choudhury 1998).
Removing squid skin can be a difficult task. Skinning machines only remove the outer skin of the squid tubes, leaving the
tough rubbery inner membrane. Strom and Raa (1991) reported
a gentler and more efficient enzymatic method of deskinning
the squid, using digestive enzymes from the squid itself. Also,
a method for deskinning the squid by making use of squid liver
extract has been developed by Leuba et al. (1987).


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For certain markets, such as Japanese sashimi restaurants
and fresh fish markets, skin-on fillets without the scales are
demanded. Also, fish skin is used in the leather industry, where
descaling is likewise necessary. Obtaining a prime quality
product requires gentle descaling. Mechanical descaling can be
difficult to accomplish without the fish flesh being damaged,
especially in the case of certain soft-fleshed species, where
enzymatic descaling results in a gentle descaling (Svenning
et al. 1993). Digestive enzymes of fish have proved to be useful
for removing scales gently (Gildberg et al. 2000).

Improved Production of Fish Sauce
As already indicated, the original process for manufacturing fish
sauce is carried out at high ambient temperature, involving the
use of an autolytic process catalyzed by endogenous proteases.
The rate of the hydrolysis depends on the content of digestive enzymes in the fish. There has been an obvious interest,
however, in shortening the time required for producing the fish
sauce, and use has also been made of other fish species, such
as Arctic capelin and Pacific whiting. Arctic capelin is usually
caught during the winter, when the fish has a low feed intake and
its digestive enzyme content thus is low. Research has shown,
however, that supplementing Arctic capelin with cod intestines
or squid pancreas, both of which are rich in digestive enzymes,
allows an acceptable fish sauce to be produced during the winter
(Gildberg 2001, Raksakulthai et al. 1986). Tungkawachara et al.
(2003) showed that fish sauce produced from a mixture of Pacific
whiting and surimi by-products (head, bone, guts, and skin from
Pacific whiting) has the same sensory quality as a commercial

anchovy fish sauce.

Seafood Enzymes Used in Biotechnology
The poor temperature stability of seafood enzymes is a useful
property that has led to the production of enzymes useful in
gene technology, where only very small amounts of enzymes
are needed. Alkaline phosphatase from cold-water shrimp (Pandalus borealis) is more heat labile than alkaline phosphatases
from mammals and can be denaturated at 65◦ C for 15 minutes
(Olsen et al. 1991). The heat-labile enzyme is therefore more
suitable as a DNA-modifying enzyme in gene-cloning technology, where higher temperatures can denaturate the DNA. The
enzyme is recovered for commercial use from shrimp-processing
wastewater in Norway. Other seafood enzymes with heat-labile
properties, such as Uracil-DNA N-glycosylase from cod (Lanes
et al. 2000), and shrimp nuclease, are likewise produced commercially as recombinant enzymes for gene-cloning technology.

Potential Applications of Seafood Enzymes
in the Dairy Industry
Research has shown that digestive proteases from fish, due to
their specificity, can be useful as rennet substitutes for calf chymosin in cheese making (Brewer et al. 1984, Tavares et al. 1997,
Shamsuzzaman and Haard 1985). Due to their heat lability, they
can be useful for preventing oxidized flavor from developing

in milk (Simpson and Haard 1984). Simpson and Haard (1984)
found that cod and bovine trypsin are equally effective in preventing copper-induced off-flavors from developing in milk. But
the cod enzyme has the advantage of being completely inactivated after pasteurization at 70◦ C for 45 minutes, whereas 47%
of the bovine trypsin is still active. These studies show that coldadaption properties of marine enzymes can be an advantage in
the processing of different foods.

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