A Q U A C U LT U R E A N D S E A F O O D P R O C E S S I N G

Surimi Enzymology and Biotechnology

Shann-Tzong Jiang* and Li-Jung Yin
Abstract
The mechanisms of gelation, proteinase effects on gel softening and the nature of changes associated with surimi
quality are complex. Based on the results thus far studied,
this review will focus on the proteinases/inhibitors and
transglutaminase (TGase and MTGase) effects on the quality
of surimi. Seafood industry might be interested in how to
prevent the deterioration of protein gels and consequently
keep the quality of surimi-based products. Developments of
some economic bio-productions of natural inhibitors and
MTGase make the prevention of gel-softening on surimibased process possible. Therefore, production of MTGase
and cystatin, an inhibitor of cysteine proteinases, using biotechnology and their effects on inhibition of surimi gel softening will also be discussed.

Introduction
Surimi made from water-washed fish mince and mixed
with cryoprotectants has a unique ability to form elastic gels
through the interaction of myosin molecules (Lee et al.,
1997). Gel-forming of myosin occurs at two stages during
low temperature setting (0~45C) and high temperature
(90C) heating (Hashimoto and Arai, 1978, Boye and
Lanier, 1988). Low temperature setting is responsible for the
polymerization of myosin heavy chain (MHC) association
with the actions of TGase (Nowsad et al., 1993, 1994) and
with the oxidation of SH groups (Jiang et al., 1986). However, gel disintegration frequently occurs during heating
process (Jiang et al., 1998).
Shann-Tzong Jiang
Department of Food Science
National Taiwan Ocean University
Keelung 202
Taiwan
Email: sjiang@mail.ntou.edu.tw
Li-Jung Yin
Department of Food Science
China College of Marine Technology and Commerce
Taipei 111
Taiwan
Email: ljyin@mail2000.com.tw
th

Proceedings of the 57 American Meat Science Association

Reciprocal Meat Conference (pp. 57-65)
June 20-23, 2004, Lexington, Kentucky
www.meatscience.org

57th Annual Reciprocal Meat Conference

Most lysosomal proteinases are active at acidic pH and

calpains are easily autolyzed, they are, therefore, not considered to cause gel softening. The pH optima for cathepsins
B, H, L and L-like (pH 5.5~6.5) (Lee et al., 1993; Jiang et al.,
1994a) are close to that of surimi (pH 6.5-7.2). From the
thermostability and pH optima of these catheptic proteases,
they are considered to be involved in gel softening (modori
in Japanese). Although heat-stable alkaline proteinases
(HAP) have been considered to be the "modori-inducing" or
"gel-softening" factor in some fish (Makinodan and Ikeda
1971; Iwata et al., 1974b; Su et al., 1981a, b; Busconi et al.,
1984), gel-softening in threadfin-bream surimi was not
caused by HAP (Toyohara and Shimizu 1988). Some modori-inducing proteinases (MIP) have also been found in
sarcoplasmic fraction of fish muscle (Toyohara et al., 1990a,
b). Accordingly, leaching (washing) process has long been
employed to improve the quality of surimi (Kinoshita et al.,
1990). However, although cathepins B and H in Pacific
whiting could be removed by leaching, cathepsin L could
not (An et al., 1994). In addition, more than 80% residual
activities of calpains, cathepsins B, L and L-like remained in
frozen mackerel surimi or surimi gel after grinding process
(Jiang et al., 1996). These phenomena suggest that these
proteinases are very difficult to be removed during leaching
process. The presence of these residual proteinases was also
found to affect the surimi-based products (Masaki et al.,
1993; Toyohara et al., 1993).

Gelation (SUWARI) of surimi

Surimi gels are composed of a three dimensional protein
network formed mainly by actomyosin. For elucidating the
heat-induced gelation mechanism, actomyosin, myosin and
myosin subfragments have been extensively studied for protein-protein interactions occurring in surimi gels (Wu et al.,
1985a, b; Beas et al., 1988; Sano et al., 1988; Ogawa et al.,
1993, 1995). According to these studies, myosin is the most
important component in the formation of gel products. Sano
et al. (1988) found that the gelation of carp actomyosin took
place in two stages, i.e., at 30-41C and 51-80C as observed by dynamic viscoelastic behavior and differential
scanning calorimetric (DSC) analysis. Differential shear
modulus for natural actomyosin from Argentinean hake
(Merluccius hubbsi) also produced two transitional temperatures ranges at 36-38C and 48C (Beas et al., 1988). Sano
et al. (1990) thus proposed that the developments of gel
elasticity at the 1st and 2nd stages are due to the interactions among tails and among the myosin heads, respec57

tively. Ziegler and Foegeding (1993) further summarized

that gelation of myosin proceeds by losing its noncovalently
stabilized -helical structure due to heating and then by
increasing turbidity due to intermolecular association,
which leads to the formation of a rigid protein network
structure that is stabilized by covalent disulfide bonds and
various noncovalent interactions.
Gelation characteristics of surimi such as firmness, cohesiveness, and water-holding capacity can be increased by
incubating surimi paste below 40C (also referred to as "setting" or "suwari") (Kimura et al., 1991). Suwari can be
achieved within a short period of time (2-4 h) near 40C
(high-temperature setting) or within an extended period (1224 h) below 40C (low-temperature setting). Low temperature setting is related to transglutaminase activity, while
high temperature setting is related to the transition in
rheological properties of actomyosin observed at 36-38C
(Wu et al., 1985). -Helical structure of myosin, prevalent
in the tail portion, unfolded at 30-40C corresponding to
low temperature setting (Ogawa et al., 1993). Lanier (1986)
also observed a great increase in gel strength and, to a
smaller extent, the elasticity of Atlantic croaker and sand
trout surimi gels preincubated at 40C. Furthermore, of the
14 fish species studied, the gel strength was highly correlated with the decrease in -helicity (r=0.85), therefore,
Ogawa et al. (1995) proposed that the setting of surimi is
initiated by the unfolding of -helix.
Gel strength of myofibrillar proteins can be influenced by
factors affecting myosin structure. The gelation properties of
myosin are highly related to the length of the double
stranded -helical tail. Myosin rod (140 nm long) showed
higher rigidity than light meromyosin (80 nm long) at all salt
concentrations studied (Ishioroshi et al., 1982). Accordingly,
proteolysis of myosin was shown to lower surimi gel
strength (Morrissey et al., 1993). The native conformation of
myosin is of primary importance for proper gelation. Maximum gel strength cannot be obtained if myosin is denatured
or degraded prior to initiation of gelation (Niwa, 1992).

Softening (MODORI) of surimi-based products

Temperature plays an important role in surimi gelation.
Fish muscles from various species revealed similar responses to temperature (Shimizu et al., 1981), i.e. a structure-setting reaction for gelation below 40C (suwari) and a
structure disintegration reaction at 50-70C (modori). Low
temperature setting is associated with transglutaminase activity and formation of SS bonding (Jiang et al., 1986; Kimura et al., 1991, Tsukamasa and Shimizu, 1990), while
modori is induced by endogenous thermal stable proteinases that can degrade myosin rapidly (An et al., 1994;
Yongsawatdigul et al., 1995; Jiang et al., 1996, 1997; Wang
and Xiong, 1998).
Disintegration of surimi gels is considered to be due to
the proteases that are active at temperatures >50C and can
rapidly degrade myofibrillar proteins, particularly myosin
(Wasson et al., 1992a; Wang and Xiong, 1998; Boye and
58

Lanier 1988; Morrissey et al., 1993). Among numerous proteinases presented in muscle, endo-proteolytic cysteine proteinases have the most serious effect on the texture due to
their thermostability and the ability to cleave the internal
peptide bonds, while exopeptidase hydrolyze terminal peptide bonds (Kirshke and Barrett 1987). The most active proteinases in fish muscle which can soften the surimi gels vary
with species, but are generally categorized into two major
groups, i.e. cathepsins (Yamashita and Konagaya 1990a,
1991b; Toyohara et al., 1993; Seymour et al., 1994; Jiang et
al., 1996, 1997) and heat-stable alkaline proteinases (Makinodan et al., 1984, 1985; Boye and Lanier 1988; Wasson et
al., 1992b).
High levels of cathepsins B, H, L, and L-like have been
observed in Pacific whiting and arrowtooth flounder
(Wasson et al., 1992b; An et al., 1994), chum salmon during spawning migration (Yamashita and Konagaya 1990a),
mackerel (Lee et al., 1993) and bovine cardiac muscle
(Wang and Xiong 1998, 1999). Arrowtooth flounder softening is due to a cysteine proteinase with maximum proteolytic activity at 50-60C (Greene and Babbitt 1990). When
the Pacific whiting muscle was incubated at 60C for 30
min prior to cooking at 90C, most myosin heavy chain
(MHC) was degraded, and surimi did not form a gel (Morrissey et al., 1993). The proteolysis of muscle proteins and
connective tissue of fish is also induced by the infection of
Myxosporea which releases proteinases into the fish muscle
tissue. In Pacific whiting, these parasites have been identified as Kudoa paniformis and K. thyrsitis (Morado and
Sparks 1986), and in arrowtooth, K. thyrsitis (Greene and
Babbitt 1990). The degree and stage of the infection have
been linked to the variation in proteolytic activity between
individual fish, which consequently results in the variation
in gel strength of surimi (Morrissey et al., 1995; Toyohara et
al., 1993). The majority of proteolytic activity in Pacific
whiting muscle is due to cathepsin L (An et al., 1994; Seymour et al., 1994), while that in mackerel is due to cathepsins B, L and L-like (Jiang et al., 1996, 1997). Cathepsin L
has a high affinity for myosin and is not completely removed by leaching during surimi processing (An et al.,
1994). Purified cathepsin L from Pacific whiting and mackerel consists of a single peptide with MW of 28,800 and
30,000, and has a temperature optimum at 55C and 50C,
and pH optimum at 5.25-5.5 and 5.0, respectively (Lee et
al., 1993; Masaki et al., 1993; Porter et al., 1993; Seymour
et al. ,; 1994). According to Lee et al. (1993), the MW, optimal temperature and pH for cathepsin L-like were 58,000,
40C and 5.5, respectively.
Cathepsin L activity was also shown to be the major contributing factor to the softening of chum salmon muscle
during spawning migration but not feeding migration (Yamashita and Konagaya 1990b). The degradation of myofibrillar proteins by purified cathepsin L observed with SDSPAGE was identical to that of muscle stored at 4C for 7
days (Yamashita and Konagaya 1990b). In the extensively
softened muscle of chum salmon, substantial degradation of
MHC into fragments with MW of 160,000, and degradation

American Meat Science Association

of troponin T, C, I and myosin light chains were observed

(Yamashita and Konagaya 1991a).
Heat-stable alkaline proteinase (HAP) has often been reported as responsible for textural degradation of surimi gels.
Its activity has been found in muscles from a large number
of fish including rainbow trout, sardine, white croaker, carp,
common mackerel, cod, herring, and Atlantic salmon
(Makinodan et al., 1984; Stoknes et al., 1993, 1995). White
croaker meat paste formed a poor elastic gel when heated
around 60C, while purified actomyosin from croaker muscle did not (Makinodan et al., 1985). Alkaline proteinase
purified from white croaker was a heat-stable cysteine proteinase with temperature optimum at 60C and pH optimum
at 8.0 (Makinodan et al., 1987). It is composed of four different subunits (24) with MW ranging from 45,000 to
57,000. These complex subunits contribute to the thermostability of the enzyme (Makinodan et al., 1987). HAP was
also purified from Atlantic menhaden, which showed similar characteristics as white croaker with an optimum activity
at 60C and pH 7.5-8.0 (Boye and Lanier 1988). It is characterized by its narrow range of pH and temperature optima, and usually not detected below 50C (Makinodan and
Ikeda 1977; Folco et al., 1988).
In spite of the extensive studies cited above, the following
questions related to surimi must be answered: (1) how much
of these proteinases are left in the surimi, (2) what is the
frozen stability of these proteinases, and (3) are the cryoprotectants such as polyphosphate, sorbitol, and sucrose which
are usually used in surimi manufacturing affecting these
proteinases? To address these questions, research has been
conducted and some of the results are summarized in the
following sections.

Changes in cathepsins B, L, L-like and calpain activities during surimi processing

The activity of cathepsins B+L+L-like remaining in mackerel surimi after mincing, leaching, and NaCl-grinding processes, which was assayed with the synthetic peptide Z-PheArg-MCA, was 6.02, 5.23 and 4.07 units/g of muscle (Jiang
et al., 1997). Chang-Lee et al. (1989) reported that the protease activity of Pacific whiting muscle decreased gradually
during surimi processing. However, as much as 87% of
cathepsins B+L+L-like activity was left in mackerel surimi
after washing treatment (Jiang et al., 1997). The results indicate that cathepsins B, L and L-like are very difficult to remove during surimi processing. According to Kinoshita et
al. (1990), some softening-inducing proteinases in mackerel
muscle were not easily removed by washing. Based on our
data, cathepsins B, L and L-like are hypothesized to be the
active proteases in mackerel surimi. However, in the case of
Pacific whiting, cathepsin B activity sharply decreased after
surimi process (An et al., 1994). Therefore, in surimi processing, removal of cathepsins B, L and L-like seemed to be
species-dependent. Accordingly, a useful approach to inhibiting the gel softening may be to economically produce the
natural inhibitor cystatin or to rupture the membrane of

57th Annual Reciprocal Meat Conference

lysosomes without deteriorating the myofibrillar proteins for

easy removal of cathepsins B, L and L-like before or during
washing treatment.
After grinding with 2.5% NaCl, about 68% cathepsins
B+L+L-like activity remained in the ground surimi (Jiang et
al., 1996). According to Kinoshita et al. (1992), the proteolytic activity of sarcoplasmic-50C-MIP (modori-inducing
proteinases) from threadfin bream against actomyosin was
not affected by the treatment with 10% NaCl. Furthermore,
carp cathepsin B activity increased in the presence of
0.10.5 M NaCl (Hara et al., 1988). The loss of cathepsins
B, L and L-like activities (about 32%) after grinding with 2.5
% NaCl (Jiang et al., 1996) might be due to the denaturation
of enzymes during grinding.

Frozen stability of cathepsins B, L and L-like in

surimi
Although cathepsins B, L and L-like activities in mackerel
surimi decreased progressively during frozen storage at 40C, there was still 82% activity left after 8 weeks storage
(Jiang et al., 1996). This might be because cathepsins B and
L still existed in lysosomes that consequently protected
them from denaturation during frozen storage. Cryoprotectants, such as sucrose, sorbitol and polyphosphates, have
been used to prevent protein denaturation in frozen surimi
(Noguchi 1974). These reagents showed little effect on
cathepsin B and L that retained at least 83% of their activities after 8 weeks storage (Jiang et al., 1996). Although a
decrease in cathepsin L-like activity was much faster than
that in cathepsins B and L during 8 weeks storage at -40C,
there was still about 40% activity left. The activity of
cathepsins B and L in mackerel surimi without cryoprotectants only slightly decreased during frozen storage. This
result further supports the hypothesis that these proteinases
are important in the gel softening of surimi (Jiang et al.,
1996; Ho et al., 2000a).

Degradation of surimi proteins by purified cathepsin B, L, and L-like

Degradation of MHC in mackerel surimi with purified
cathepsin B, L and L-like during 5 hrs incubation at 40o or
55C was observed on SDS-PAGE, compared with those
containing E-64, a cathepsins B, H, L and L-like inhibitor.
Proteolysis was faster in samples with pH 6.5 than pH 7.0,
and faster in samples incubated at 55C than that at 40C
(Jiang et al., 1996). According to Jiang et al. (1996), cathepsins B, L and L-like not only degraded MHC, but also partially degraded actin at pH 6.5 or 7.0 after 5 hrs incubation
at 55C. These results suggested that the thermally unfolded
globular actin might be hydrolyzed by cathepsins B, L and
L-like. However, degradation of surimi proteins by cathepsins B, L and L-like could be effectively inhibited by E-64
(Jiang et al., 1996; Ho et al., 2000a). Some protease inhibitors from beef plasma (Hamann et al., 1990; Morrissey et
al., 1993; Park et al., 1994; Wang and Xiong 1998, 1999),
fish plasma (Toyohara et al., 1990b), potato and egg white
59

(Morrissey et al., 1993; Piyachomkwan and Penner 1995;

Reppond et al., 1995; Wang and Xiong 1998, 1999), and
legume seed extracts (Garcia-Carreno et al., 1996) were
also effective in preventing degradation of fish or bovine
cardiac myofibrillar proteins. Akazawa et al. (1993) indicated that bovine plasma powder was an effective inhibitor
in degradation of Pacific whiting surimi proteins. Plasma
factor XIIIa (an active transglutaminase) purified from pig
plasma also had very strong cross-linking ability on MHC of
mackerel surimi and this cross-linked protein gel was
strongly resistant to proteolysis (Jiang and Lee 1992).
Cathepsins B, L and L-like are active proteinases in many
fish, such as Pacific whiting (An et al., 1994), chum salmon
(Yamashita and Konagaya 1990a, b, c) and mackerel (Lee et
al. ,, 1993). Surimi gel softening has been attributed mostly
to cathepsin L (An et al., 1994). From our studies, cathepsins B, L and L-like could not be completely washed out
from the surimi and revealed myosin-degrading activity on
samples incubated at 40 and 50C, pH 6.5 and 7.0 (Jiang et
al., 1996; Ho et al., 2000a). Therefore, they might also be
important in gel softening of mackerel surimi.

Gel softening by purified cathepsin B, L and L-like

The gel properties of surimi ground with purified cathepsins B, cathepsin L and L-like (5 unit/g of meat) decreased
after 2 hrs incubation at 55C, compared to those of control
(p<0.05) (Jiang et al., 1996). These results provided direct
evidence to demonstrate that cathepsins B, L and L-like
could induce softening in fish gels. Heat-stable alkaline
proteinases (HAP) also deteriorate surimi gels (Su et al.,
1981a, b). The strength of HAP-added gel decreased to
about half that of the control gel (Makinodan et al., 1987).
However, HAP isolated from threadfin bream had no effect
on disintegration of surimi gels (Toyohara et al., 1990a, b).
These phenomena suggested the great contribution of
cathepsins B, L and L-like to softening of surimi gels. However, according to Jiang et al. (1996), E-64 could not completely inhibit the softening of mackerel protein gels, suggesting some factors, other than cysteine proteinases, causing gel softening existed in mackerel surimi.
Although Sp-50-MIP activity was not affected even in the
presence of 10% NaCl (Kinoshita et al., 1992), HAP proteolytic activity against carp actomyosin gradually decreased
with increasing concentration of NaCl (0.1~0.5M) (Iwata et
al., 1974a, b). Our previous study indicated that cathepsins
B, L and L-like could hydrolyze surimi protein in the presence of 0.6 M NaCl and result in gel softening of mackerel
surimi (Jiang et al., 1996). If the muscle proteins are not
properly stored, they will be hydrolyzed into fragments by
various proteinases depending on the species, storage temperature and pH of postmortem muscle. However, both
intact muscle proteins and their proteolytic fragments can
be gelled into different types of gels, i.e. firm network or
loose (disintegrated) gel structures. The degree of softening
varies with the extent of proteolysis. The intact or native
muscle proteins can form firm network structure gels. How-

60

ever, if the well cross-linked network structural sols is not

fixed at optimal time by heating at 85-100C, or the setting
process is over the optimal time, the network structural gels
will be disintegrated into softened gels. This phenomenon is
usually called modori (in Japanese) or disintegration. As
mentioned previously, HAP or cathepsin B, L and L-like
have high activity at 50-70C and a substantial amount of
activity is retained in frozen surimi even after NaClgrinding. Therefore, when the surimi with native muscle
proteins is set at 50-70C, it will easily form a soft gel due to
the action of these proteinases.

Prevention of the gel-softening (Modori) by recombinant cystatin

Production of recombinant cystatins
Expression of the recombinant cystatin from E. coli
Although the human salivary cystatin SN (Bobek et al.,
1994) or rat salivary cystatin S (Sharma et al., 1995) recombinant proteins carried a fusion protein of glutathione Stransferase, they were still expressed as insoluble inclusion
body in E. coli. The necessity of urea renaturation and solubilization with dialysis treatments for recovering the active/soluble recombinant proteins would greatly increase
running cost and also be time-consuming. Chen et al.
(2000) and Tzeng et al. (2001, 2002) used pGEM-T Easy
cloning vector containing the correct in-frame cystatin
cDNA sequence to construct the chicken lung cystatin and
mature carp ovarian cystatin expression vectors. The cDNA
for cystatins were ligated with pET-23a(+) expression vector
in Nde I and Xho I restriction enzyme sites and introduced
in frame to downstream of T7 promoter of pET-23a(+) vector. High level of the soluble recombinant cystatins was
successfully expressed in AD494(DE3)pLysS after 4 hr induction by IPTG (Chen et al., 2000; Tzeng et al., 2001,
2002).
Expression of the recombinant cystatin from Pichia pastoris
Chen et al. (2001) had ligated the cDNA for chicken cystatin with pGAPZC expression vector in Xho I and Xba I
restriction enzyme sites and introduced in frame to downstream of factor in pGAPZC vector. After transforming
the pGAPZC- chicken cystatin plasmid into Pichia pastoris
X-33 expression host, the expression vector was integrated
into the genomic DNA and the highest level of cystatin activity (about 6.33 units/mg) was obtained after two days
shaking cultivation (Chen et al., 2001).
Effect of recombinant cystatins on the inhibition of gel softening of surimi
The gel strength of mackerel surimi without recombinant
cystatin or with cathepsins B or L was much lower than
those with recombinant chicken cystatin after 2 h incubation at 55C. Although the recombinant cystatin could not
completely inhibit the gel softening of mackerel surimi presumably due to some proteases other than the cysteine proteinases, it still has high potential for using in improving the
seafood quality (Chen et al., 2000). Obvious degradation on
American Meat Science Association

myosin heavy chain and moderately on actin of surimi gels

preincubated at 50C for 15, 30, 60 and 90 min prior to the
fixation (30 min heating at 100C) occurred. However, only
very minor degradation was observed on samples with recombinant cystatin (Chen et al., 2000). To further investigate the inhibitory effect of recombinant cystatin on gel
softening of mackerel surimi, samples with/without recombinant cystatin were processed into surimi-based product.
The breaking force of samples without recombinant cystatin
decreased from 1025 g to 680 843 g, while that of samples with recombinant cystatin decreased to 960 858 g
after 15 90 min preincubation at 50C. Significant differences in breaking force, deformation (depth at the point of
gel breaking) and gel strength between samples with and
without recombinant cystatin added were observed
(p<0.05).

Prevention of gel-softening (Modori) during processing of surimi-based products by the addition of

transglutaminase
Production of microbial transglutaminase
Guinea-pig liver has been the sole source of commercial
TGase for decades. The limited source and complicated
separation procedures resulted in increase in the cost for
obtaining this enzyme, which consequently makes it not
possible to apply in food processing. Recently, efforts have
been made to search for TGase from microorganisms
(MTGase). MTGase was found in cultures of Streptoverticillium sp., Streptomyces sp. and Streptoverticillium ladakanum (Motoki et al., 1989; Ando et al., 1989; Tsai et al.,
1996a, b; Zeng et al., 2001). Microbial fermentation makes
it possible to achieve mass production of MTGase using
cheap substrates. A number of examples of the application
of MTGase in food processing have been announced. However, the potential for using MTGase in cosmetics, pharmaceutical products and medical treatment, remains uncertain
for commercial reasons and communication difficulties.
Some MTGase producing microorganisms were screened
using the hydroxamate assay and taxonomically classified
as a variant of Streptoverticillium mobaraense (Ando et al.,
1989; Washizu et al., 1994). These microorganisms excreted MTGase into the culture broth. The critical property
of MTGase, ability of the formation of G-L bonds among
proteins demonstrated that the produced enzyme was
MTGase (Nonaka et al., 1989). Motoki et al. (1989) reported
that other Streptoverticillium strains, such as St. griseocarneum, and St. cinnamoneum subsp. cinnanoneum also
have the ability to produce MTGase. It was also found in
Streptomyces sp. (Ando et al., 1989) and St. ladakanum
(Tsai et al., 1996a, b).
The fermentation procedure for the production of
MTGase is generally the same as those mentioned microorganisms (Ando et al., 1989; Motoki et al., 1989). Glucose,
sucrose, starch, glycerine and dextrin can be used as carbon
source. Inorganic as well as organic nitrogen sources can be
used, such as NH4NO3, (NH4)2SO4, urea, NaNO3, NH4Cl,
57th Annual Reciprocal Meat Conference

soya, rice, maize, wheat or wheat flour, bran, defatted soya

bean, maize-steep liquid, peptone, meat extract, casein,
amino acids and yeast extract. Necessary minerals and trace
elements are phosphate, magnesium, potassium, iron, copper, zinc and vitamins, etc. Non-ion surfactant and antifoam
can also be added if necessary. The culture is an aerobic
fermentation so that aeration and agitation are necessary.
The temperature for growth and product formation is between 25C and 35C, and the fermentation time is normally 2-4 days (Sakamoto et al., 1992). Since MTGase is
excreted into the culture medium, cell disruption is unnecessary. Its purification thus proves to be rather easy. Consequently, its commercialization has been accelerated.
Examples of fermentation and purification of MTGase
have been described by Ando et al. (1989) and Motoki et al.
(1989). The microorganism was activated at 30C for 2 days
and then cultured at the same temperature for 3 days under
aeration (10 l/min) and agitation (250 rpm). The broth had
an enzyme activity ranging from 0.28 U/ml to 2.5 U/ml,
dependent upon the strain used. After being separated by
centrifugation at 3000 rpm, the supernatant was concentrated with an ultra-filtration and then chromatographed on
Amberlite CG-50 and Blue Sepharose. The total recovery of
MTGase activity was about 42%. However, recently a
modified downstream process for purifying MTGase was
described by Ho et al. (2000b). After the fermentation of
Streptoverticillium ladakanum, the broth was centrifuged
and filtered. The enzyme was purified directly with a rapid
and simple stepwise chromatography method with CM
Sepharose CL-6B and Blue Sepharose Fast Flow. According
to the authors, this method is simple, rapid and has a
MTGase recovery of 81%.
Effects on gel-softening (Modori)
Seki et al. (1990) and Tsukamasa et al. (1993) found that
endogenous fish TGase caused suwari setting, hardening
fish protein paste at low temperature through crosslinking.
TGase from walleye Pollack, fish for surimi, has been purified and characterized by Kumazawa et al. (1996). There
seems to be no doubt that both endogenous fish TGase and
exogenous MTGase enable to improve the functionality of
fish raw materials by increase in cross-linking (Seki, 1992;
Jiang et al., 2000a, b). However, there are still some arguments as to whether the endogenous fish TGase is the only
factor in suwari setting (Nowsad et al., 1993). Kumazawa et
al. (1996) determined the G-L bond in several fish eggs, and
suggested that endogenous TGase may correlate with the
texture of raw and processed egg products. It seems that
TGase treatment improves and maintains the texture-quality
of fish products, which strictly depends on the freshness of
raw materials. MTGase participated in the setting process of
mackerel and hairtail surimi gels and mainly accelerated the
cross-linking of myosin heavy chain (MHC) (Jiang et al.,
1998). According to Jiang et al. (2004), the MHC of
MTGase-contained mackerel and hairtail muscle protein
(MP) decreased greatly, while the crosslinked MHC became predominant during incubation. No cross-linked

61

MHC was observed on samples without MTGase, suggesting that the MHC was a good substrate for MTGase.
The gel properties of mackerel surimi were much improved with the addition of MTGase (samples with 0.5 unit
MTGase/g were about 1.5 - 2.5 fold of that of control).
However, decrease in gel-forming ability of surimi-based
products was found in samples with excess of MTGase, due
to the over formation of the -(-glutamyl)-lysine bonds
which consequently make the protein gels fragile (Asagami
et al., 1995; Sakamoto et al., 1995; Seguro et al., 1995;
Jiang et al., 1998, 2000a, b).
Effects of combination use of MTGase and recombinant
cystatin on gel softening
Kumazawa et al. (1995) reported that TGase participated
in the setting process of surimi gels and mainly accelerated
the cross-linking of MHC. According to SDS-PAGE, MHC
intensity decreased with the increase of MTGase and high
molecular weight compounds were observed on the top of
gels (Hsieh et al., 2002a, b). These components were considered resulting from cross-linking of MHC (Nowsad et al.,
1995; Jiang et al., 2000a, b; Tsai et al., 1996). According to
our previous study (Chen et al., 2001), the recombinant
cystatin could improve the quality of mackerel surimi and
produce high quality surimi analogues. The combination
use of MTGase and recombinant cystatin substantially improve the gel forming ability of mackerel and hairtail surimi,
and the effect of recombinant cystatin on the gel-forming
ability of the mackerel surimi was more pronounced, when
the MTGase was simultaneously added (Hseih et al., 2002a,
b). These data suggested that the addition of recombinant
cystatin substantially prevented the texture softening caused
by endogenous proteases, and MTGase further enhanced
the gel property by the formation of cross-linking of MHC.

Conclusion
High levels of cysteine proteinase activities of cathepsins
B, H, L, and L-like were observed in Pacific whiting and
arrowtooth flounder (An et al., 1994; Wasson et al., 1992a,
b), chum salmon during spawning migration (Yamashita and
Konagaya 1990a) and mackerel (Jiang et al., 1997; Lee et
al., 1993). These proteases have been considered to be the
main factors that caused gel softening of surimi. Accordingly, the presence of these proteinases that found to be
stable at 50 - 60C had become a crucial problem for the
surimi processing. The degradation of protein gels of surimi
was substantially inhibited by the addition of recombinant
cystatin. The gel properties of surimi were much improved
with the addition of MTGase. When the MTGase was used
simultaneously with recombinant cystatin, the gel forming
ability of surimi was substantially improved (Hseih et al.,
2002a, b). As mentioned above, the recombinant cystatin
could inhibit the gel softening of mackerel surimi caused by
cathepsins and the MTGase could enhance the gel property
by the formation of cross-linking of MHC. They have high
potential for using in improving the seafood quality. The
recombinant chicken cystatin had biological and physical

62

properties comparable to the wild-type cystatin and the

expression system developed thus far are useful and economical in terms of producing recombinant cystatin for industrial application.

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