JFS:

Food Chemistry and Toxicology

Influence of Endogenous Proteases

and Transglutaminase on Thermal
Gelation of Salted Squid Muscle Paste
ABSTRACT: Thermal gelation of salted squid mantle muscle paste was studied in relation to endogenous proteases and transglutaminase. Myosin in the paste was preferentially degraded into 130-kDa and 90-kDa fragments at an optimum temperature of 30 C. Degradation was inhibited with EDTA or 1,10-phenanthroline,
suggesting the presence of metalloproteases. Myosin degradation was markedly reduced above 40 C. Although
10 mM Ca2+ increased cross-linking of myosin heavy chains by activating the endogenous transglutaminase,
setting effect on thermal gelation of the paste was offset by degradation induced by simultaneously activated
calpains. Ca2+ and the calpain inhibitor, E64, significantly improved the breaking strength and strain of thermal
gels preincubated at 40 C.
Keywords: squid muscle paste, gelation, transglutaminase, calpain, metalloprotease

Introduction

quid is not only consumed as a fresh marine product, but also

manufactured into processed foods such as dried, smoked,
soused, fermented, and canned forms, which are the major commercial products in Japan. However, gel-type foods such as kamaboko,
sausage, and ham produced from mollusks have not been available
for 2 main reasons: high cost and low functionality as a raw material.
Kamaboko is a homogeneous protein gel produced from fish
myofibrillar proteins, primarily myosin that has been washed to
remove the water-soluble extracts, ground with NaCl to solubilize
the actomyosin, and heated to form a gel (Suzuki 1981; Niwa 1992).
Fish mince is usually washed for purification of the proteins to improve gel formability and whiteness of kamaboko (Suzuki 1981).
Here we excluded the washing process to preserve the squid taste
and flavor in the gel. Our intention was to produce a kamaboko-like
gel with a specific taste and texture that differed from those of other fish products. This approach is advisable for squid because of
the solubility of their myofibrillar proteins, which would otherwise
reduce yield (Matsumoto 1958), and it should retain the influence
of soluble enzymes, such as transglutaminase and proteases.
Various factors have been proposed as possible causes of low
gel-forming capacity in squid muscle. In particular, the high level
of proteolytic activity is a major factor in determining its low gelforming ability together with its peculiar thermal gelation profile,
which lacks the characteristic setting effect. Several authors (Sakai
and Matsumoto 1981; Ehara and others 1992, 1994; Nagashima and
others 1992; Okamoto and others 1993; Ebina and others 1995)
have reported that squid myosin is preferentially degraded by
various types of endogenous proteases. Concerning thermal gelation at neutral pH and 0.6 M NaCl, Ehara and others (1992, 1994)
MS 20030337 Submitted 6/17/03, Revised 7/30/03, Accepted 8/26/03. Authors
Park, Kimura, Nozawa, and Seki are with the Laboratory of Marine Food
Science, Graduate School of Fisheries Sciences, Hokkaido Univ., Hakodate
041-8611, Japan. Author Cho is with the Dept. of Food Science, Kangunung
Natl. Univ., Kangwon 210-720, Korea. Author Yoshioka is with the Hokkaido
Industrial Technology Center, Hakodate 041-0801, Japan. Direct inquiries
to author Park (E-mail: sharangj@fish.hokudai.ac.jp).

2003 Institute of Food Technologists

Further reproduction prohibited without permission

pointed out that metalloprotease is the main type of active protease responsible for myosin heavy chain degradation in pastes of
the Japanese common squid (Todarodes pacificus), whereas
serineproteases and metalloproteases are involved in myosin
heavy chain degradation in the spare squid Loligo bleekeri (Nagashima and others 1992; Ebina and others 1995) and Loligo vulgaris
(Gmez-Guilln and others 2002).
To provide basic information for the future use of squid in manufacturing kamaboko, we investigated the gel formability of Japanese common squid mantle muscle and its gelation mechanisms in
relation to paramyosin (Tsuchiya and others 1980; Levine and others 1982; Sano and others 1989b) and the cross-linking of myosin
induced by an endogenous transglutaminase (Seki and others
1990; Kimura and others 1991; Kamath and others 1992; Wan and
others 1994; Nozawa and others 1999, 2001; Ni and others 2001),
with the intention of avoiding proteolytic gel deterioration.

Materials and Methods

Materials
Japanese common squid Todarodes pacificus, which was caught
off the coast of Hakodate, was procured from the local market within
12 h of capture and brought to the laboratory packed in ice. The
average weight was approximately 300 g. The squid was gutted and
skinned immediately. Mantles were placed in polyethylene bags
and chilled in iced water.
Coomassie brilliant blue R-250, monodansylcadaverine (MDC),
1,10-phenanthroline, phenylmethanesulfonyl fluoride (PMSF),
aprotinin, and molecular weight standard mixture were purchased
from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). EDTA was obtained
from Dojindo Laboratories (Kumamoto, Japan). N-N-[(L-3trans)carboxyoxiran-2-carbonyl]-L-Leu] agmatin (E64) was purchased from Peptide Inst. (Osaka, Japan). All other chemicals were of
analytical grade from Wako Pure Chemical Industries (Osaka, Japan).

Preparation of salted muscle paste

The skinned mantle muscles were dissected and mixed with a
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S. PARK, S. CHO, T. YOSHIOKA, M. K IMURA, H. NOZAWA, AND N. S EKI

Thermal gelation of squid muscle paste . . .

one-third volume of 2.5 M NaCl in 100 mM Tris-HCl (pH 7.2) and
one-third volume of H2O. The mixture was ground in a food cutter
(Natl. model MK-K7, Osaka, Japan) to produce a homogeneous
paste at 0 C. After standing for 1 h at 0 C, protein and NaCl concentrations of the paste were adjusted to 80 to 100 mg/g and 0.5 M,
respectively. Occasionally, final concentrations of 10 mM CaCl2 was
added to the muscle paste and mixed well. The paste was de-aerated by centrifugation at 1000 rpm and 5 C in vacuum (5000 Pa) for
10 min using a centrifugal vaporizer (EYELA model CVE-110, Tokyo,
Japan).

Tokyo) as reported elsewhere (Ni and others 1998). Storage (G) and
loss (G) moduli were monitored continuously at a fixed frequency
of 3 Hz as a function of temperature from 5 C to 80 C at a heating
rate of 2 C/min. Here, G represents the elastic component, and G
is the viscous component of the material. Analyses were carried out
in duplicate, and mean values were used in the figures.
Two-step heating method. The muscle paste was incubated at
40 C for up to 6 h to induce setting (suwari) before heating to
80 C or 90 C. Approximately 30 min was required at 5 C for the
procedures from the end of de-aeration to the start of heating or setting.

Thermal gelation and rheological measurements

Food Chemistry and Toxicology

Direct heating. The muscle paste was stuffed into plastic vessels
(3.7-cm dia, 2-cm height) for a rupture test. The plastic vessels
were heated in a water bath at 90 C for 20 min. Heating was applied at a constant rate of 5.56 C/min to the core of muscle paste
between 5 C and 70 C. The core temperature reached 80 C after
20 min. Heated samples were immediately chilled in iced water for
1 h, kept at room temperature for 2 h, and then removed from the
vessels. Breaking strength and strain were measured by using a
cylindrical plunger (5-mm dia) at a penetration speed of 0.5 mm/s
in a rheometer (Rheoner RF 3305, Yamaden, Tokyo) as described
elsewhere (Wan and others 1994; Ni and others 1998). Dynamic
rheological properties of the muscle pastes during thermal gelation
were measured using a Rheolograph-Sol (Toyo Seiki Seisakusho,

Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis
Samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were solubilized with an SDS-urea solution
containing 2% SDS, 8 M urea, 50 mM Tris-HCl (pH 8.0), and 2% 2mercaptoethanol by heating at 100 C for 2 min and continuous
stirring at 25 C overnight (Seki and others 1990; Ni and others
1998). SDS-PAGE was performed by the method of Laemmli (1970)
using 7.5% or 10% separating and 3% stacking polyacrylamide gels.
Gels were stained with Coomassie brilliant blue R250.

Transglutaminase activity
The activity of transglutaminase (EC 2.3.2.13) was assayed at

Figure 1Changes in SDS-PAGE pattern of squid muscle paste during incubation at various temperatures. Squid mantle
muscle paste (80 mg protein per gram of paste, 0.5 M NaCl, pH 7.2) was incubated at various temperatures from 2 C
to 60 C. MHC = myosin heavy chain (205 kDa); PA = paramyosin (100 kDa); AC = actin (42 kDa).
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Thermal gelation of squid muscle paste . . .

Protein determination
The protein concentration was determined by the biuret method
with bovine serum albumin, fraction V (Wako Pure Chemical Industries, Osaka, Japan) as a standard (Gornall and others 1949).

Results and Discussion

Effect of temperature and time on proteolysis of
squid muscle proteins in the salted paste

tease inhibitors such as EDTA and 1,10-phenanthroline. Furthermore, the protease myosinase I has been purified from mantle
muscle and hydrolyzes chicken myosin heavy chain between positions Ala-1161 and Thr-1162 to produce fragments of 130 kDa (myosin head portion) and 90 kDa (myosin tail portion). It did not hydrolyze these fragments further (Okamoto and others 1993).
Recently, Yokozawa and others (2002) have found that this protease is located in the extracellular matrix of mantle muscle cells,
suggesting that myosin is not a normal physiological substrate.

Thermal gelation of squid muscle paste by a directheating method

Thermal gelation of the squid muscle paste was monitored using dynamic viscoelastic measurements. Figure 3a shows the changes of the thermal gelation profile of squid muscle paste with an
increase in temperature from 5 C to 80 C at a heating rate of 2 C/
min by the direct-heating method. The storage modulus (G) increased with rising temperature up to 27 C and decreased considerably to a minimum of 300 Pa at 45 C. Above this temperature,
the G value sharply increased to 1500 Pa up to 80 C without showing any peak. It further increased to 1700 Pa during incubation at
80 C.
The loss modulus G increased with the rise of temperature to
27 C and then decreased to 45 C. Above 45 C, G rose gradually
until 75 C and then reached a plateau.

SDS-PAGE of squid mantle muscle showed the presence of myosin heavy chain, paramyosin (100 kDa), actin, and the minor
bands such as tropomyosin, troponin, and sarcoplasmic proteins
(Figure 1, time 0). The salted muscle paste was incubated at various temperatures between 2 C and 60 C. Degradation of myosin
was noted at 2 C within 3 h. With prolonged incubation time, a decrease in the myosin heavy chain band resulted in the increase of
2 new protein bands, 130 kDa and 90 kDa. The latter band almost
overlaid the paramyosin subunit band (100 kDa). Myosin degradation was greatly accelerated at an increase in temperature to 30 C,
where proteolysis of squid myosin in the salted paste reached an
optimum. Proteolysis of actin was not detected throughout any incubation temperature or time, whereas paramyosin degradation
was unclear due to the overlay of a 90-kDa-myosin fragment. It
should be noted that proteolysis of the myosin heavy chain was
greatly decreased at 40 C and was substantially interrupted at the
early stage of the incubation. Even after 12 h of incubation, residual
myosin heavy chain could be detected, suggesting thermal inactivation of protease(s) in the salted muscle paste. At 40 C, there was
no appreciable hydrolysis of the muscle proteins, including
paramyosin and actin. No proteolysis of salted muscle proteins was
observed above 50 C.

Effect of protease inhibitors

The effects of protease inhibitors on the degradation of myosin
in the muscle paste were investigated at 30 C. Figure 2 shows that
myosin proteolysis was completely inhibited with EDTA and 1,10phenanthroline: metalloprotease inhibitors. In contrast, other
types of inhibitors, such as 1 mM E64 (a cysteine protease inhibitor), 0.2 mM PMSF and 0.01 mM aprotinin (both trypsin inhibitors)
barely affected myosin degradation. These results suggest that an
endogenous metalloprotease is predominantly responsible for
myosin degradation in the muscle paste and agree with the results
reported by Ehara and others (1992) and Okamoto and others
(1993).
Ehara and others (1992) have already reported that mantle
muscle of the Japanese common squid contains strong myosindegrading activity, which is completely inhibited by metalloproURLs and E-mail addresses are active links at www.ift.org

Figure 2Effect of protease inhibitors on the degradation

of squid muscle proteins during incubation at 30 C. Squid
mantle muscle paste (80 mg protein per gram of paste,
0.5 M NaCl, pH 7.2) was incubated with 5 mM EDTA, 1.8
mM 1,10-phenanthrolin, 1 mM E64, 0.2 mM PMSF, or 0.01
mM aprotinin at 30 C for up to 12 h. Mw = molecular weight
standard; MHC = myosin heavy chain; PA = paramyosin;
AC = actin.
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25 C and pH 7.5 as described elsewhere (Nozawa and others

1999). One unit of transglutaminase activity was defined as the
amount of enzyme that catalyzed the incorporation of 1 nmol MDC
into succinylated casein for 1 min at 25 C.
Transglutaminase activity in the muscle paste was detected as
follows: MDC (2 mM final concentration) was added to the muscle
paste, containing 80 mg protein/g paste, 0.5 M NaCl, 10 mM CaCl2,
and 20 mM Tris-HCl buffer (pH 7.2). After standing for 1 h at 0 C,
the mixture (3 g) was incubated at 40 C for up to 30 or 60 min. A
portion of the mixture (0.2 g) was taken out at given times and then
mixed with 5 mL of SDS-urea solution to terminate the reaction. The
mixture was immediately heated at 100 C for 2 min and then
stirred overnight at 25 C. Fluorescence of MDC-bound proteins
was detected by illuminating a gel with an ultraviolet (UV) lamp
after SDS-PAGE.

Thermal gelation of squid muscle paste . . .

Food Chemistry and Toxicology

The overall profile of the changes in G of squid muscle paste as

a function of temperature was very similar to those of other squid
species (Gmez-Guilln and others 1997, 2002) and fish actomyosin paste as reported elsewhere (Sano and others 1988, 1989a; Ni
and others 1999, 2001). The temporal decrease in G value at 45 C
was weaker than that of fish (Sano and others 1989a).
An increase in G from 45 C to 75 C was observed at neutral pH,
reflecting the decrease in water-holding ability of the gel, which
became more rigid and less elastic in texture. As reported in scallop
muscle gelation by Yoshida and others (2003), the cause of G increase in the temperature range of 48 C to 65 C at neutral pH is
related to the presence of paramyosin, a muscle protein constituent peculiar to invertebrates and a coiled-coil molecule similar to
the myosin rod (Lowry and others 1963; Watabe and Hartshorne
1990). Therefore, the characteristic increase in G of squid muscle
paste during thermal gelation from 45 C to 75 C was supposed to
be caused by the gelation of paramyosin, which consists of 16% to
18% squid muscle proteins (Tsuchiya and others 1980).

Effect of setting on the thermal gelation of squid

muscle

Squid mantle muscle used contained approximately 6.4 units/

g of Ca2+-activated transglutaminase activity. To activate this,
CaCl2 was added to the squid muscle paste at a concentration of 10
mM as reported elsewhere (Nozawa and others 1999). It was, however, found that the overall profile of the thermal gelation of Ca2+supplemented muscle paste was similar to that of controls (without
Ca2+-addition) during setting and subsequent heating to 80 C.
Furthermore, the G and G values of thermal gel reached similar
levels of about 17000 Pa and 150 Pa at 80 C, respectively, regardless of heating methods. Thus, there was no setting effect on the gel
texture compared with that prepared by the direct-heating method
(Figure 3a). The same results were also obtained by gel rupture
tests, as shown in Figure 4. The breaking strength and strain of the
heated gels prepared by a 2-step heating method showed similar
values to those of directly heated gels, even though Ca2+ had been
added to the muscle paste (compare the closed and hatched columns in Figure 4). Both experimental results (Figure 3 and 4) clearly
demonstrated that no apparent setting response was obtained
during the thermal gelation of squid muscle paste.
To clarify why Ca2+-addition was ineffective, gels prepared by

Two-step heating including setting at 40 C for 60 min was performed to induce a setting effect during thermal gelation (Figure
3b). The setting temperature at 40 C was adopted to reduce proteolysis, as shown in Figure 1. The setting effect in fish muscle thermal gelation is largely caused by the increased cross-linking of
myosin heavy chains mediated by endogenous Ca2+ -activated
transglutaminase (Seki and others 1990; Kimura and others 1991;
Kamath and others 1992; Wan and others 1994). Microbial transglutaminase has been widely used in the commercial setting process for kamaboko (Seguro and others 1995).

Figure 3Changes in G (storage modulus) and G (loss

modulus) during thermal treatment of squid muscle paste.
(a) Squid mantle muscle paste (80 mg protein per gram of
paste, 0.5 M NaCl, pH 7.2) was heated at 2 C/min from
5 C to 80 C by a direct-heating method. (b) The same
muscle paste (solid line) and CaCl2-added paste (broken
line) were heated by a 2-step heating method including 60min setting at 40 C. Calcium was added to the muscle paste
at a final concentration of 10 mM just before heating.
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Figure 4Effects of setting on the thermal gel texture of

squid muscle pastes. Squid mantle muscle pastes were
heated by a 2-step heating method including setting process at 40 C for up to 120 min. The breaking strength and
strain of the thermal gels were measured using a cylindrical plunger (5-mm dia) at a penetration speed of 0.5 mm/
s in a rheometer. Each data value is expressed as mean
SD (n = 4). Control = control paste (80 mg protein per gram
of paste, 0.5 M NaCl, pH 7.2); + Ca = 10 mM CaCl 2 was
added to the control paste; + Ca + E64 = 10 mM CaCl 2 and
1 mM E64 were added to the control paste.
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the 2-step heating method were analyzed by SDS-PAGE (Figure 5).

During setting at 40 C and even after setting for 120 min, the control gel showed a slight degradation of the myosin heavy chain,
suggesting a minor contribution of metalloproteases to gel deterioration. However, Ca2+-supplemented gels exhibited a great loss of
the myosin heavy chain within only 10 min during setting at 40 C
(see also Figure 6b). Newly formed protein bands were detected at
2 distinct areas on the polyacrylamide gels, the top of the gel and a
middle area between the myosin heavy chain and actin bands. The
former bands as indicated by arrows in Figure 5 seemed to be crosslinked myosin heavy chains produced by the reaction of transglutaminase; and the latter bands, which consisted of many protein bands in the middle area, might represent the degradation
products of the myosin heavy chain.
These results suggest that cross-linking and degradation of the
myosin heavy chain occurred simultaneously during setting with
the addition of Ca2+. The setting response was therefore almost
completely lost by the degradation of myosin heavy chain by Ca2+dependent proteases, presumably calpains, but not metalloproteases. SDS-PAGE of the heated gel with 10 mM CaCl2 and 1 mM
E64 clearly demonstrated that the myosin heavy chain was crosslinked without any formation of its degradation products (Figure 5).
Paramyosin and actin were not cross-linked during setting, suggesting that myosin is a major substrate cross-linked by transglutaminase in squid muscle paste.
To distinguish the direct contribution of transglutaminase to the
setting effect from the combined effect of both enzymic actions,
E64 was added to the squid muscle paste with 10 mM CaCl2 (Figure
4, open columns). The breaking strength and strain of 2-step heated gels increased greatly during setting within 30 min at 40 C and
reached a plateau thereafter.
To confirm the transglutaminase action, MDC was added to the
muscle paste with 10 mM Ca2+ (Figure 6). This reduced the disappearance of the myosin heavy chain, suggesting that transglutaminase-induced myosin cross-linking was competitively inhibited. In
the presence of both 10 mM Ca2+ and 1 mM E64 (Figure 6e, 6f ),
more MDC was incorporated into the myosin heavy chain and its

Figure 5Effect of CaCl2 and E64 on SDS-PAGE pattern of

squid muscle paste during setting at 40 C. The thermal
gels shown in Figure 4 were analyzed using SDS-PAGE.
Control = control paste (80 mg protein per gram of paste,
0.5 M NaCl, pH 7.2); + Ca = 10 mM CaCl2 was added to the
control paste; + Ca + E64 = 10 mM CaCl2 and 1 mM E64
were added to the control paste. P = paste before heating; MHC = myosin heavy chain (205 kDa); PA = paramyosin
(100 kDa); AC = actin (42 kDa); arrows indicate myosin
heavy chain polymers.
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polymers, with lesser myosin degradation. Paramyosin was slightly

labeled with MDC, but not cross-linked as shown in Figure 5 and
6e. Actin was neither cross-linked nor labeled with MDC (Figure 5
and 6d). Therefore, transglutaminase-mediated myosin cross-linking predominantly occurred in the presence of E64 and resulted in
the improved setting response and thermal gel texture.
Sakai and Matsumoto (1981) reported that the addition of 5 mM
CaCl2 at neutral pH did not activate the autolytic activity of squid
mantle of the same species studied here. The inconsistency may be
caused by the different experimental conditions in which they
adopted low protein concentrations (12 to 25 mg/mL) at low ionic
strength (I = 0.02). The addition of calcium to the muscle paste of
spare squid (Loligo vulgaris) increased gel texture through improvement of endogenous transglutaminase activity, but produced very
little contribution of calpain to the gel deterioration (GmezGuilln and others 2002). Activation rates of calpains in squid muscle thus appear to depend on experimental conditions and speciesspecific activity levels. Food-grade calpain inhibitors or calpastatins
are therefore required to inhibit calpain activity in Japanese common squid muscle paste. However, there is little information concerning calpains and calpastatin in squid mantle muscle.
We had expected that microbial transglutaminase would be an

Figure 6Incorporation of MDC into squid muscle proteins

during setting at 40 C. Squid mantle muscle pastes were
heated by a 2-step heating method including setting at 40 C
for up to 30 or 60 min. (a), Control paste (80 mg protein
per gram of paste, 0.5 M NaCl, pH 7.2); (b), + 10 mM CaCl 2;
(c) and (d), + 10 mM CaCl2 and 2 mM MDC; (e) and (f), + 10
mM CaCl2, 2 mM MDC, and 1 mM E64; (d) and (f), fluorescence pattern of MDC-bound proteins of (c) and (e), respectively, which were detected using ultraviolet illumination.
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Food Chemistry and Toxicology

Thermal gelation of squid muscle paste . . .

Thermal gelation of squid muscle paste . . .

effective additive to induce the setting effect on squid muscle thermal gelation because it is fully active in the absence of calcium and
at higher temperatures, 40 C to 50 C. These conditions minimize
the influence of both calpain and metalloprotease activities in squid
muscle paste (Park and others, unpublished data, 2003).

Conclusions

Food Chemistry and Toxicology

major factor in the poor thermal gelation of squid muscle is myosin degradation induced by the high activities of metalloproteases during setting below 40 C and of calpains in the presence of
Ca2+. Addition of Ca2+ was essential to activate endogenous transglutaminase, which induces a setting effect to thermal gelation. A
strong thermal gel was therefore produced through 40 C setting in
the presence of Ca2+ and a calpain inhibitor. This technology will be
effectively applicable to the development of a new gel-type food
from squid mantle muscle.

Acknowledgments
This research was supported in part by a Grant-in-Aid from Hakodate City, Japan and a Grant-in-Aid from the Towa Food Research Foundation, Japan.

References
Ebina H, Nagashima Y, Ishizaki S, Taguchi T. 1995. Myosin heavy chain-degrading protease from spear squid muscle. Food Res Int 28:316.
Ehara T, Tamiya T, Tsuchiya T. 1992. Investigation of myosin heavy chain-degrading protease in decapoda muscle. Nippon Suisan Gakkaishi 58:237982.
Ehara T, Tamiya T, Tsuchiya T. 1994. Investigation of myosin heavy chain-degrading protease in squid muscle. Nippon Suisan Gakkaishi 60:5278.
Gmez-Guilln MC, Borderias J, Montero P. 1997. Salt, non-muscle proteins,
and hydrocolloids affecting rigidity changes during gelation of giant squid
(Dosidicus gigas). J Agric Food Chem 45:61621.
Gmez-Guilln MC, Hurtado JL, Montero P. 2002. Autolysis and protease inhibition effects on dynamic viscoelastic properties during thermal gelation of
squid muscle. J Food Sci 67:24916.
Gornall AG, Bardawill CJ, David MM. 1949. Determination of serum proteins by
means of biuret reaction. J Biol Chem 177:75166.
Kamath GG, Lanier TC, Foegeding EA, Hamann DD. 1992. Non-disulfide covalent cross-linking of myosin heavy chain in setting of Alaska pollock and
Atlantic croaker surimi. J Food Biochem 26:15172.
Kimura I, Sugimoto M, Toyota K, Seki N, Arai K, Fujita T. 1991. A study on the
cross-linking reaction of myosin in kamaboko suwari gels. Nippon Suisan
Gakkaishi 57:138996.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature 227:6805.
Levine R, Elfvin M, Sawyna V. 1982. Preparation and assay of paramyosin. In:

2478

JOURNAL OF FOOD SCIENCEVol. 68, Nr. 8, 2003

Frederiksen D, Cunningham L, editors. Methods in enzymology. Vol. 85. New

York: Academic Press. p 14960.
Lowry S, Kucera J, Holtzer A. 1963. On the structure of the paramyosin molecule.
J Mol Biol 7:23444.
Matsumoto JJ. 1958. Some aspects on the water-soluble proteins of squid muscle.
Bull Tokai Regional Fish Res Lab 20:6575.
Nagashima Y, Ebina H, Nagai T, Tanaka M, Taguchi T. 1992. Proteolysis affects
thermal gelation of squid mantle muscle. J Food Sci 57:9167, 922.
Ni S, Nozawa H, Seki N. 1998. Effect of microbial transglutaminase on thermal
gelation of carp actomyosin sol. Fish Sci 64:4348.
Ni S, Nozawa H, Seki N. 1999. The combined effect of transglutaminase and
protease inhibitors on the thermal gelation of actomyosin sol from carp and
salmon muscles. Fish Sci 65:60612.
Ni S, Nozawa H, Seki N. 2001. Effect of pH on the gelation of walleye pollack
surimi and carp actomyosin pastes. Fish Sci 67:9207.
Niwa E. 1992. Chemistry of surimi gelation. In: Lanir TC, Lee CM, editors. Surimi
technology. New York: Marcel Dekker. p 389427.
Nozawa H, Cho S-Y, Seki N. 2001. Purification and characterization of transglutaminase from squid gill. Fish Sci 67:9129.
Nozawa H, Mamegoshi S, Seki N. 1999. Effect of neutral salts on activity and
stability of transglutaminase from scallop adductor muscle. Comp Biochem
Physiol 124B:1816.
Okamoto Y, Otsuka-Fuchino H, Horiuchi S, Tamiya T, Matsumoto JJ, Tsuchiya T.
1993. Purification and characterization of two metalloproteases from squid
mantle muscle, myosinase I and myosinase II. Biochim Biophys Acta 1161:97
104.
Sakai J, Matsumoto JJ. 1981. Proteolytic enzymes of squid mantle muscle. Comp
Biochem Physiol 68B:38995.
Sano T, Noguchi SF, Tsuchiya T, Matsumoto JJ. 1988. Dynamic viscoelastic behavior of natural actomyosin and myosin during thermal gelation. J Food Sci
53:9248.
Sano T, Noguchi SF, Matsumoto JJ, Tsuchiya T. 1989a. Role of F-actin in thermal
gelation of fish actomyosin. J Food Sci 54:8004.
Sano T, Noguchi SF, Tsuchiya T, Matsumoto JJ. 1989b. Paramyosinmyosinactin
interactions in gel formation of invertebrate muscle. J Food Sci 54:7969, 842.
Seguro K, Kumazawa Y, Ohtsuka T, Toiguchi S, Motoki M. 1995. Microbial transglutaminase and -(-glutamyl)lysine cross link effects on elastic properties
of kamaboko gel. J Food Sci 60:30511.
Seki N, Uno H, Lee N-H, Kimura I, Toyota K, Fujita T, Arai K. 1990. Transglutaminase activity in Alaska pollack muscle and surimi, and its reaction with myosin B. Nippon Suisan Gakkaishi 56:12532.
Suzuki T. 1981. Fish and krill protein: Processing technology. London: Applied
Science Publishers Ltd. p 62114.
Tsuchiya T, Fukuhara S, Matsumoto JJ. 1980. Physico-chemical properties of squid
paramyosin. Nippon Suisan Gakkaishi 46:197200.
Yokozawa Y, Tamai H, Tatewaki S, Tajima T, Tsuchiya T, Kanazawa N. 2002. Cloning and biochemical characterization of astacin-like squid metalloprotease. J
Biochem 132:7518.
Yoshida W, Kunimi O, Fujiura M, Kimura M, Nozawa H, Seki N. 2003. Thermal gelation of salted paste from scallop striated adductor muscle. Fish Sci 69:1015-23.
Wan J, Kimura I, Satake M, Seki N. 1994. Effect of calcium ion concentration on
the gelling properties and transglutaminase activity of walleye pollack surimi paste. Fish Sci 60:10713.
Watabe S, Hartshorne D. 1990. Paramyosin and the catch mechanism. Comp Biochem Physiol 96B:63946.

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