LWT - Food Science and Technology 53 (2013) 184e190

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LWT - Food Science and Technology

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Effects of fish sarcoplasmic proteins on the properties of myofibrillar protein gels

mediated by microbial transglutaminase
Bung-Orn Hemung, Koo Bok Chin*
Department of Animal Science and Functional Food Research Institute, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 500-757, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: Fish sarcoplasmic protein (SP) was extracted and lyophilized to obtain the SP powder. Fish myofibrillar
Received 21 July 2012 protein (MP) was mixed with SP powder (0, 0.1, 0.5, and 1.0 g/100 g) in 1.8 and 2.6 g/100 g NaCl in the
Received in revised form presence of 0.5 g/100 g microbial transglutaminase (MTG) at 4
C for 6 h. Shear stress of MP mixture
26 January 2013
decreased with increasing SP concentrations. High thermal stability of MP mixture, assessed by differ-
Accepted 5 February 2013
ential scanning calorimetry, at either 1.8 or 2.6 g/100 g NaCl, was observed when SP (1 g/100 g) was
added. The myosin heavy chain partially disappeared, suggesting the formation of cross-linked proteins.
Keywords:
The gel strength of MP was not affected by the addition of 0.1 g/100 g SP (P > 0.05) whereas it started to
Fish sarcoplasmic proteins
Microbial transglutaminase
decrease when SP was added up to 0.5 g/100 g, regardless of the NaCl concentration. The cooking loss of
Water holding capacity the MP gel was reduced efficiently when SP was added, even at a low concentration (0.1 g/100 g). A
Red sea bream further reduction of cooking loss was observed when SP concentration was increased. The smooth
NaCl concentration microstructure of the gel surface was observed in samples containing SP, showing the lower cooking loss
of fish myofibrillar protein gel.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction surimi (Piyadhammaviboon & Yongsawatdigiul, 2009). The gel

Fish sarcoplasmic proteins (SP) are a large family of proteins that
include myogens and enzymes, and are soluble in water or low
ionic strength solution. These components are normally washed
out from surimi processing since they can lead to deterioration
during surimi storage. The washing step is necessary for improving
the quality of surimi gel, especially gel strength and whiteness. In
the washing process, a high amount of water is used and vast
quantities of washed water are generated as waste, which contains
soluble SP. Lin, Park, and Morrissey (1995) reported that 1.7 kg of
protein could be recovered for every 100 kg of surimi produced. The
utilization of SP from washed water generated by surimi plants has
been investigated to utilize the by-product as food ingredients. In
addition, it would be an alternative way to reduce waste.
SP have been reported to inhibit the gelation process of the
myofibrillar protein (MP) counterpart (Hashimoto, Katoh, Nozaki, &
Arai, 1985). On the other hand, the gel strength of mackerel proteins
increases with the addition of SP (Morioka & Shimizu, 1990). SP
from threadfin bream improved textural properties of lizard fish
* Corresponding author. Tel.: þ82 62 530 2121; fax: þ82 62 530 2129.
E-mail address: kbchin@chonnam.ac.kr (K.B. Chin).
weakening effect, modori, of myofibrillar protein from milkfish was
restricted by the addition of its SP (Ko & Hwang, 1995). The gel
strength of threadfin bream surimi improved by the addition of SP
extracted from carp (Jafarpour & Gorczyca, 2009). SP from rockfish
positively contributed to the gelation of MP (Kim, Yongsawatdigul,
Park, & Thawornchinsombut, 2005). There was no absolute agree-
ment on the role of SP for the gelation of MP. However, the effects of
SP on protein gelation mediated by microbial transglutaminase
(MTG), the cross-linking enzyme, have not yet been investigated.
MTG has been isolated from Streptoverticillium spp. and is a
Ca2þ-independent enzyme (Ando et al., 1989). It catalyzes the acyl
transfer reaction between the glutamine-bound peptides/proteins
to the primary amine or lysine-bound peptides, resulting in the
formation of the 3-(g-glutamyl)-lysyl isopeptide bond. MTG has
been widely used to improve textural properties of food protein gel
including surimi gel (Cardoso, Mendes, Vaz-Pires, & Nunes, 2010).
However, high cooking loss of meat protein gels was observed
when MTG was applied (Hong & Chin, 2010). Non-meat proteins
from legume and milk (soy proteins isolate, sodium caseinate, and
whey protein concentrate) have been used successfully to over-
come this problem (Uresti, Tellez-Luis, Ramirez, & Vazquez, 2004).
For the water holding ability of SP powder from threadfin bream, it
has been reported a value of 0.43 g/g sample (Yongsawatdigul &
Hemung, 2010). However, application of SP from fish as water

0023-6438/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.02.008
 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190
holding agent has not been performed. Therefore, the objective of
this study was to investigate the effects of fish SP on the properties
of MP paste/gel mediated by MTG.
2. Materials and methods
2.1. Materials
The farmed fish (red sea bream, Pagrus major) of 0.030e0.035 m
length from Gunnae-ri, Dolsan-eup, Yeosu-si, Jeollanam-do, Korea,
were killed and transported to the Meat Science Laboratory,
Chonnam National University, Korea, in foam box covered with ice
within 20 min. Fish fillets were prepared after manual eviscerating
and filleting. All samples were packed under vacuum and stored
at 70
C until required. MTG (TG-S) containing 1 g/100 g of crude
MTG was obtained from Ajinomoto (Seoul, South Korea) and ac-
tivity was about 100 units of MTG/g. All other chemicals were of
analytical grade.
2.2. Methods
2.2.1. Sarcoplasmic protein (SP) preparation
Frozen fish fillets were allowed to thaw in the cold room over-
night before homogenizing with 3 volumes of de-ionized water (DI-
water) in a blender for 2 min. The homogenate was centrifuged at
1000  g for 15 min before supernatant collection. This process was
repeated once more. The collected supernatant (SP solution) was
used to determine for its patterns using sodium dodecyl sulfate gel
electrophoresis (SDS-PAGE) and the left solution was kept overnight
in the freezer (70
C) before lyophilizing using a freeze-dryer
(IlShin Lab. Gyeonggi-do, South Korea). The obtained powder was
used as SP powder sample. The protein content in the SP powder
was estimated by Kjeldahl method according to AOAC (2000) pro-
cedure. The protein content was about 74.4  0.22 g/100 g sample.
The same batch of SP preparation was used throughout the
experiment.
2.2.2. Myofibrillar protein (MP) extraction
Fish fillets were thawed overnight at 4
C before performing MP
extraction according to the method described by Xiong (1993), with
slight modifications. Briefly, fish samples were blended with 4 vol-
umes of washing buffer (100 mmol L1, NaCl, 50 mmol L1 phos-
phate buffer, pH 6.25) for 2 min. The homogenate was centrifuged
for 15 min at 1000  g and only pellets were collected. The process
was repeated twice. The obtained pellet was mixed with 8 volumes
of 100 mmol L1 NaCl (adjusted pH to 6.25) before filtering through
2 layers of gauze to remove connective tissue. The filtrate was
centrifuged at 1000  g for 15 min and the pellet was used as MP. The
protein concentration in pellet was determined by Biuret method
using bovine serum albumin as a standard. The protein pattern of
isolated MP was determined using SDS-PAGE technique. In addition,
the moisture content in the MP was also determined by AOAC
method in order to calculate the total solid content.
2.2.3. MP gel preparation
The MP gel was prepared at 4 g/100 g (based on total solid and
protein content using Biuret method) and the reaction mixture was
controlled at 1.8 and 2.6 g/100 g NaCl, pH 6.25. MTG was added at
0.5 g/100 g, while the concentration of SP was varied (0, 0.1, 0.5, and
1.0 g/100 g). The MP mixture was poured in to a glass vial with a
diameter of 12 mm and incubated for 6 h at 4
C. All samples were
heated in the water bath heating (WB-22, Daihan Scientific Co.,
Seoul, Korea) at the rate of about 3
C/min from 5 to 80
C
(z40 min). The cooked samples were cooled rapidly on ice and
stored overnight at 4
C (cold room).
185
2.2.4. Rheological analysis
The rheological properties of fish MP mixtures incubated at 4
C
for 6 h were tested by a rotational rheometer (RC30, Rheotec
Messtechnik GmbH, Berlin, Germany). The bob (120
) and cup with
radii of 7 and 7.59 mm, respectively, were used as a probe. Shear
stress values were recorded, while the chilled sample was sheared
up to 1000 s1.
2.2.5. Differential scanning calorimetry (DSC)
A differential scanning calorimeter (DSC S-650, Scinco Co. Ltd.,
Seoul, Korea) calibrated with indium standard was used to test for
thermal properties of MP paste. The sample (z15 mg) was encap-
sulated in the aluminum pan before heating at the rate of 10
C/min
from 25 to 100
C. A reference pan was prepared by encapsulating
the empty pan. The endothermic peaks of MTG mediated MP
mixture incubated with and without SP (1 g/100 g) was recorded.
2.2.6. SDS-PAGE
MP isolated from red sea bream was dissolved in NaCl solution
(3.5 g/100 g) at the ratio of MP:NaCl solution of (1:9) in order to
obtain the soluble MP. The soluble MP was determined for protein
concentration by Biuret method before mixing with treatment
buffer, containing SDS and b-mercaptoethanol (BME) prior deter-
mination of protein pattern using SDS-PAGE according to Laemmli
(1970). The protein sample (20 mg) was loaded onto the gel made
from 4 g/100 g acrylamide as stacking gel and 10 g/100 g acrylamide
as running gel. The samples were separated using a mini Protean II
unit (Bio-Rad Laboratories Inc., Richmond, Calif., USA). The protein
patterns of SP were also analyzed as above except the running gel
was 12.5 g/100 g acrylamide.
MP mixture (incubated at 4
C for 6 h) was mixed with 10 times
of SDS (5 g/100 g) solution before boiling for 10 min. The solution
was centrifuged at 2000  g for 15 min before centrifuging and
collected the supernatant as the soluble protein extract. Protein
concentration in the supernatant was estimated by the Biuret
method. The soluble protein was mixed with treatment buffer
containing BME in order to break down the disulfide linkages. The
pattern of soluble protein was analyzed by SDS-PAGE.
2.2.7. MP gel characterization
2.2.7.1. Cooking loss. The MP gels were brought from the cold room
to incubate at room temperature for approximately 4 h. The exu-
dates from the gel were recorded. The cooking loss of the MP gel
was expressed as a percentage of the original weight, which was
considered 100%.
2.2.7.2. Color measurement. The color of MTG mediated fish MP gel
prepared with different SP and NaCl contents were measured using
colorimeter (CR-10, Minolta Co. Ltd., Japan). The color value were
reported as hunter L, a, and b values.
2.2.7.3. Breaking force. A puncture test was applied to determine
the breaking force, representing the gel strength, using an Instron
Universal testing machine (Instron Corporation, Canton, MA, USA).
The puncture probe with a diameter of 9 mm was used. The fish MP
gels were compressed with the head speed of 50 mm/min and the
penetration length was controlled at 12 mm. The first peak was
recorded as the breaking force, which can be represented the gel
strength.
2.2.7.4. Microstructure. The cubic shape fish MP gels were pre-
pared before fixing with 2.5 g glutaraldehyde/100 g of
100 mmol L1 sodium phosphate buffer (pH 7) overnight at 4
C.
Thereafter, the samples were washed with washing buffer
(100 mmol L1 sodium phosphate buffer pH 7) for 10 min and post
186 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190
Fig. 1. Rheological properties of MP paste incubated at 4
C for 6 h with MTG and
different SP concentrations at 1.8 g/100 g (A) and 2.6 g/100 g (B) NaCl. (B) MP þ MTG,
(,) MP þ MTGþ0.1 g/100 g SP, (6) MP þ MTGþ0.5 g/100 g SP, (*) MP þ MTGþ1.0 g/
100 g SP.
fixed with 1% osmium tetraoxide (OsO4) at room temperature for
5 h. The obtained samples were washed with washing buffer 3
times for 10 min each. Subsequently, an ethanol concentration at
50, 60, 70, 80, 90, and to 100% was applied for 10 min each and a
final 100% acetone was applied for 10 min. The dried samples were
Fig. 2. SDS-PAGE patterns of MP only (A), SP only (B), and MP mixture incubated at 4
C for 6 h with MTG and different SP concentrations (C).
lyophilized overnight before gold coating (15 nm) and micro-
structures at 2000 magnification were obtained by a scanning
electron micrometer (FE-SEM, JSM-7500F, JEOL Ltd., Tokyo, Japan)
at an accelerating voltage of 15 kV.
2.2.7.5. Statistical analysis. The confidential level of 95% (P < 0.05)
was set to determine statistically significant mean values, based on
the ANOVA analysis using the PASW Statistics 18 (SPSS Inc., Chicago
IL, USA). The data was analyzed statistically with two-way analysis
of variance. If the interaction between the two factors (NaCl and SP
concentration) was not observed, data were pooled for further
analysis to obtain the effect of each factor.
3. Results and discussion
3.1. Rheological properties of MP
The shear stress of MTG-mediated fish MP paste in combination
with SP was observed as shear thinning behavior (Fig. 1). The yield
stress values were found at about 50 Pa, regardless of the NaCl
concentrations. The shear stress values of samples prepared at 1.8
and 2.6 g/100 g NaCl were similar. This suggested that a reduction
of NaCl level from 2.6 to 1.8 g/100 g did not affect the shear stress of
fish MP paste. At 1.8 g/100 g NaCl level, the maximum shear stress
value of samples decreased with increasing SP concentrations
(Fig. 1A). A reduction of shear stress upon adding SP was clearly
observed at 2.6 g/100 g NaCl (Fig. 1B). Kim et al. (2005) also re-
ported that the storage modulus of surimi paste mixed with rock-
fish SP was lower than without SP at approximately 10
C. It has
been reported that the molecular interactions of hydrated proteins
mainly contributed to an increased viscosity of concentrated pro-
tein suspension (Bonisch, Huss, Weitl, & Kulozik, 2007). Our results
also suggested that adding of SP into MP resulted in the formation
of more compact protein complexes, which might not be easily
destructed by shearing force. Therefore, a reduction of shear stress
upon adding SP was observed.
3.2. SDS-PAGE
The patterns of MP only showed that band of myosin heavy chain
(MHC) and actin at the Mw of 200 and 43 kDa, respectively (Fig. 2A).
There were more band which might be the myosin light chains
 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190 187

(z20 kDa) and tropomyosin complex (z38 kDa). These patterns

suggested that the obtained sample contained the major protein
belonging to the MP, which were suitable for the test to understand
effect of cross-linking catalyzed by MTG in the present of SP.
The protein component in SP solution was obtained for 10 major
bands as shown in Fig. 2B. In addition, the majority proteins
showed the molecular weight around 30e40 kDa. This result was
similar to that observed for SP from 16 fish species, which 10 pro-
tein bands were obtained (Nakagawa, Watabe, & Hashimoto, 1988).
Moreover, the distribution of water soluble protein was not related
to fish species as noted by Morioka and Shimizu (1993). They also
reported that the gel strength of SP will be increase when the
proportion of protein band at Mw of 94 was increased. However,
protein bands showing high intensity was found at Mw of 48, 40,
and 38 (Fig. 2B). This suggested that the gel enhancing might hardly
observed.
Protein patterns of MTG-mediated MP mixture prepared at 1.8
and 2.6 g/100 g NaCl were not different (Fig. 2C). These results also
supported the fact that 1.8 g/100 g NaCl was enough to solubilize
fish MP, thus ensuring gel formation as well as providing readily
accessible substrate for the MTG. Therefore, reduction of NaCl to
1.8 g/100 g would be possible without affecting the gelation pro-
cess. This would be the alternative way to reduce the salt content in
fish protein gel.
The degradation of MP incubated with SP might have occurred
when SP contained proteases. This may be seen by changes in MP
pattern, particularly the MHC. However, MHC was observed at the
same intensity among treatments, regardless of the different con-
centrations of SP. This suggested that protein degradation was not
observed and SP samples contained less active proteases. Park and
Park (2007) reported that there were no changes in MP patterns
when surimi was incubated with fish SP. This suggested that the gel
weakening effect up on addition of SP may not be observed.
High molecular weight polymers accumulating in the stacking
gel were also found (Fig. 2C). These results supported that MTG
catalyzed the cross-linking reaction during incubation of the pro-
tein samples at 4
C. When SP concentration was increased, the
major SP band at about 50 kDa increased as well. This suggested
that fish SP did not inhibit cross-linking of MP, even though it could
not serve as the substrate for MTG. The MHC cross-linking reaction
could not be completed even though an incubation time of 6 h was
applied. This may be due to the MTG being outside its optimal
temperature of 50
C (Ando et al., 1989). Based on the SDS-PAGE
results, the different rheological properties (Fig. 1) may not be
due to the cross-links catalyzed by MTG.

3.3. DSC

Transition temperatures of MP at 1.8 g/100 g NaCl were observed

at approximately 53 and 75
C (Fig. 3). These corresponded to the
transition temperatures of myosin and actin, respectively
(Thorarinsdottir, Arason, Geirsdottir, Bogason, & Kristbergsson,
2002). When SP (1 g/100 g) was added, the endothermic peaks
were increased to be 54 and 78
C, respectively. Chaijan, Benjakul,
Visessanguan, Lee, and Faustman (2008) reported that myoglobin,
a sarcoplasmic protein, started to interact with the myosin tail of MP
after incubation at 4
C for 6 h. These results suggested that the
interactions between fish SP and MP resulted in an increased ther-
mal stability. In addition, the most stable MP system was found to be
the fish MP at 2.6 g/100 g NaCl with the addition of SP (1 g/100 g), as
seen by the peak temperature at 60 and 85
C. The addition of SP into
fish MP led to an increased denaturation temperature of rockfish
surimi (Kim et al., 2005). It has been reported that interactions be-
tween fish myoglobin and MP was more pronounced at a high ionic
strength rather than that at a low ionic strength (Chaijan, Benjakul,
Fig. 3. Differential scanning calorimetry thermogram of MP paste incubated with MTG
at 4
C for 6 h with MTG and different SP concentrations at 1.8 g/100 g (A) and 2.6 g/
100 g (B) NaCl.
Visessanguan, Lee, & Faustman, 2007). The higher ionic strength at
2.6 g/100 g NaCl could further facilitate MP solubilization, promot-
ing the strongest interactions between SP and MP. This would
explain why samples with SP at 2.6 g/100 g NaCl showed higher
thermal stability than those at 1.8 g/100 g NaCl (Fig. 3). Proteins with
low thermal stability are susceptible to unfold and interact with
each others. The higher thermal stability of MP mixed with SP at
2.6 g/100 g NaCl would retard the protein unfolding and limit the
188 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190

intermolecular interactions. This may explain why the shear stress Table 2
of MP with SP at 2.6 g/100 g NaCl was observed at the lower value Gel strength (g force) of MTG mediated MP gels prepared at different NaCl and SP
concentrations.
than those at 1.8 g/100 g NaCl (Fig. 1). In addition, the difference in
rheological properties may not be due to disulfide bonds or iso- NaCl (g/100 g) SP concentration (g/100 g)
peptide bonds, but due to the interactions between sarcoplasmic 1.8 2.6 0 0.1 0.5 1.0
proteins, such as, myoglobin and myofibrillar proteins, as reported 690  73A 571  88B 715  77a 685  108a 607  70b 515  64c
by Chaijan et al. (2007).
Mean  SE was calculated based on 10 replicates.
A,B
Different letters among NaCl concentrations are statistically different (P < 0.05).
3.4. MP gel color a,b
Different letters among SP concentrations are statistically different(P < 0.05).

Color of fish MP gels was evaluated by the Hunter colorimeter

and the results are shown in Table 1. The lightness (L value) of the
gel increased when SP (1 g/100 g) was added. However, it has been
reported that the L value of surimi gels decreased after adding SP
(Jafarpour & Gorczyca, 2009; Kim et al., 2005). Our results were
different from previously reported studies possibly because of
different sample preparation. In our study, since the MP gel was
measured for cooking loss before determining color value, moisture
content in the sample between this and the previous study may not
be the same. Moreover, the sample containing the highest L value
was observed to show lowest value in cooking loss (Table 3). This
suggested that high water holding capacity resulted in a whiter
appearance of samples. A decrease in L value due to moisture loss in
sausage fermentation has also been reported (Perez-Alvarez, Sayas-
Barbera, Fernandex-Lopez & Aranda-Catala, 1999). The blueness
(negatively b value) of the samples seemed to be increased with
increasing SP concentration up to 1 g/100 g. This was in agreement
with those of surimi color after adding 2% rockfish SP (Jafarpour &
Gorczyca, 2009). The redness (a value) of the gel decreased by
adding 1 g/100 g SP. Ramirez, Velazquez, Echevarria, and Torres
(2007) reported that the redness of surimi gel decreased when
the insoluble solid from surimi washed water was added up to 5%.
Based on the color results, addition of SP into a fish MP gel system
would be possible. Since the L value of the gel increased, addition of
SP may not greatly affect color perception.
3.5. Breaking force
MP samples prepared at a concentration of 4 g/100 g could be
successfully induced to gelation, even at 1.8 g/100 g NaCl, while
those without MTG could not produce viscoelastic gels (data not
shown). This suggested that the cross-linking of fish MP could be
catalyzed by MTG although the salt content was lowered.
When different NaCl levels were considered, the strength of the
MP and SP mixture gel prepared at 1.8 g/100 g NaCl was a higher
value than that at 2.6 g/100 g NaCl. Based on the gel strength data, a
reduction of NaCl concentration would not only reduce NaCl con-
tent in protein gel, but also improve textural properties (Table 2).
Ramirez, Angel, Uresti, Valazquez, and Vazquez (2007) reported
that reducing salt content from 20 to 10 g/kg did not affect the
textural properties of restructured products prepared from striped
mullet when MTG was added. This phenomenon may be explained
by the lower thermal stability of MP mixture at 1.8 g/100 g NaCl, as
assessed by DSC (Fig. 3A). Normally, proteins showing low thermal
stability tend to be promptly unfolded, facilitating the gel forma-
tion. Low thermal stability of MP mixture from cold water fish
species resulted in better textural properties when compared to
that from tropical species, which have higher thermal stability
(Hemung, Li-Chan, & Yongsawatdigul, 2008).
Cardoso et al. (2010) reported that the high textural properties
of gels prepared from sea bass could be formed at about 1 g/100 g
when 0.5 g/100 g of MTG was added. Moreover, textural properties
of the low-salt restructured fish products improved by the presence
of MTG (Uresti et al., 2004). Based on this information, MTG would
be the promising ingredient to induce gelation of fish protein even
the salt content was reduced.
The breaking force of fish MP was not affected by the addition of
0.1 g/100 g SP (P > 0.05), but increasing SP concentration from 0.5
to 1.0 g/100 g resulted in significantly decreased (P < 0.05)
(Table 2). Since the changes of MP patterns on SDS-PAGE to be the
smaller proteins were not observed when SP was added (Fig. 2C),
degradation of MP due to endogenous proteases was not occurred.
Therefore, a reduction of gel strength may not be due to the action
of protease residues.
It has been reported that addition of sardine myoglobin (0.2 g/
100 g) into Pacific whiting surimi resulted in a reduction of
breaking force of surimi gel (Park & Park, 2007). They also found
that the enthalpy of the system, determining by DSC, was increased
when myoglobin was added into surimi. The interactions between
fish myoglobin and fish MP were also proved by a reduction of
Ca2þ-ATPase activity of MP upon adding myoglobin. Such in-
teractions were promoted by increasing ionic strength and tem-
perature (Chaijan et al., 2007). Chaijan et al. (2008) reported that
disulfide bonding did not contribute to myoglobin and MP in-
teractions. It has been reported that fish SP contain extremely high
surface hydrophobicity when compare to other proteins
(Yongsawatdigul & Hemung, 2010). This suggested that the hy-
drophobic interactions between SP and MP may affect thermal
stability, ultimately affecting protein denaturation. Therefore, a
reduction of breaking force of MTG-mediated MP gel in the pres-
ence of SP was observed in this study.

Table 1
The Hunter color values of MTG-mediated MP gels prepared at different NaCl and SP concentrations.

Hunter color NaCl (g/100 g) SP concentration (g/100 g)

0 0.1 0.5 1.0

L 1.8 83.6  0.26Ad 84.1  0.42Ac 83.2  0.25Ab 85.9  0.17Aa
2.6 83.8  0.13Ab 83.1  0.45Bb 83.7  0.42Ab 83.9  0.16Ba
a 1.8 0.83  0.05Aa 0.75  0.06Aa 0.33  0.05Ab 0.40  0.08Ab
2.6 0.78  0.10Aa 0.53  0.10Bb 0.43  0.05Bb 0.25  0.06Bc
b 1.8 4.65  0.17Ab 4.43  0.17Ab 4.55  0.13Ab 2.23  0.75Aa
2.6 4.85  0.10Ac 4.98  0.38Ac 4.33  0.29Ab 3.13  0.10Aa

Mean  SE was calculated from 5 replicates.

A,B
Different letters in the same column for L, a, and b values are statistically different (P < 0.05).
a,b
Different letters within the same row are statistically different (P < 0.05).
 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190 189

Table 3 3.6. Cooking loss

Cooking loss (%) of MTG-mediated MP gels prepared at different NaCl and SP
concentrations.
The cooking loss value of MTG-mediated MP gel prepared at
NaCl SP concentration (g/100 g) 1.8 g/100 g NaCl was higher than at 2.6 g/100 g NaCl, regardless of
(g/100 g) the presence of SP. High salt content may promote the water
0 0.1 0.5 1.0
1.8 38.77  1.73Aa 35.81  1.14Ab 32.64  0.22Ac 28.08  0.07Ad holding ability of the system due to the ionic interactions between
2.6 31.92  1.76Ba 28.86  0.72Bb 26.91  0.68Bc 25.11  0.23Bd Naþ or Cl ions with water. Moreover, an increase salt content could
Mean  SE was calculated based on 5 replicates.
also facilitate protein solubility and thus, soluble protein could hold
A,B
Different letters in the same column (salt concentrations) are statistically more water in the structure. Increasing salt concentration
different (P < 0.05). augmented the water holding capacity of gels prepared from sea
a,b
Different letters in the same row (SP concentrations) are statistically different bass in the presence of MTG (Cardoso et al., 2010).
(P < 0.05).
When fish SP was added into fish MP, cooking loss of the MP and
SP mixed gels gradually decreased even at the low concentration of
Yongsawatdigul and Piyadhammaviboon (2007) reported that 0.1 g/100 g. Increasing SP concentrations resulted in further
gel strength of lizard surimi improved after adding SP extracted reduction of cooking loss at either 1.8 g/100 g or 2.6 g/100 g NaCl.
from tilapia. This was partially due to the activity of tissue trans- Ramirez, Velazquez, et al. (2007) reported that expressible water
glutaminase (TG), a Ca2þ-independent enzyme, presented in SP from surimi paste was reduced upon adding insoluble solids, which
samples since Ca2þ was added. Since Ca2þ was not added in this were recovered from washed water derived from surimi produc-
experiment, the effect of endogenous TG may not be accounted tion. These results suggested that fish SP had high potential for
whether it is presented in the system or not. being the water holding agent in food protein gels, in particular the
It has also been reported that addition of common carp SP up to MTG-mediated protein gel. In this system, the excessive cross-
35 g/100 g into threadfin bream surimi resulted in an increase gel linking reaction promotes more proteineprotein interactions,
strength (Jafarpour & Gorczyca, 2009). Textural properties of MP gel resulting in a reduction of proteinewater interaction (Jaros, Jacob,
from rockfish could be improved when its SP was added at the level Otto, & Rohm, 2010).
of 1e5% (Kim et al., 2005). Insoluble solids from surimi washed Application of natural substances as water holding agents is
water were found to increase gel strength of Pacific whiting surimi needed. Whey protein, which is a by-product from cheese pro-
grade A (Ramirez, Velazquez, et al., 2007). According to the study, duction, was successfully applied into fish protein gel mediated by
the interactions between SP and MP may not be absolutely MTG under low salt conditions (Ramirez, Angel, et al., 2007). In
addressed. Their effects will be shown differently, depending on the addition, sodium caseinate was used to improve cooking yield of
fish species. protein gels (Pietrasik, Jarmoluk, & Shand, 2007). Since fish SP

Fig. 4. Microstructure at 2000 magnification of MTG mediated MP gels prepared with/without SP (1%) at 1.8 g/100 g and 2.6 g/100 g NaCl. (A) ¼ 1.8 g/100 g NaCl/without SP,
(B) ¼ 1.8 g/100 g NaCl/with SP, (C) ¼ 2.6 g/100 g NaCl/without SP, and (D) ¼ 2.6 g/100 g NaCl/with SP.
190 B.-O. Hemung, K.B. Chin / LWT - Food Science and Technology 53 (2013) 184e190
showed promising water holding potential, it would be an alter-
native source of ingredients for being the water holding agent in
fish protein. However, fish SP addition at low concentration (1 g/
100 g) was recommended to avoid a reduction of gel strength.
3.7. Microstructure
Microstructures of MTG-mediated MP gels without SP, regard-
less of the NaCl levels were similar and showed a compact and
globular structure (Fig. 4A and C). These results suggested that the
three dimensional network of MP gel prepared at either 2.6 or
1.81.8 g/100 g NaCl could be formed effectively. This also showed
that a reduction of salt content could be possible in fishery prod-
ucts. It is clear that a smoother structure was observed when 1.0 g/
100 g fish SP was added to fish MP at 1.8 g/100 g NaCl (Fig. 4B) when
compared to that without SP (Fig. 4A). Moreover, addition of 1.0 g/
100 g SP into fish MP dissolved in 2.6 g/100 g NaCl resulted in the
smoothest structure (Fig. 4D).
The smoothest structure of MP gel was corresponded with the
lowest cooking loss values, which were 28.08 and 25.11% at 1.8 and
2.6 g/100 g NaCl, respectively. Thus, it could be hypothesized that
the smooth structure is capable of holding more water, which will
ultimately reduce cooking loss of the protein gel. Jafarpour and
Gorczyca (2009) reported that the thick, strong, and smooth gel
structures were observed when SP was added into surimi gel. This
may be due to the water soluble fish SP being miscible to produce
the gel network formed by MP, which results in increased water
holding capacity.
4. Conclusions
Cooking loss of fish MP gel mediated by MTG at 1.8 g/100 g NaCl
was higher than at 2.6 g/100 g NaCl. However, it could be reduced
effectively by the addition of fish SP. When fish SP was incorporated
with fish MP, the lightness and blueness of MP gel increased, while
the redness reduced. Gel strength of MP mixed with SP at 1.8 g/
100 g NaCl was higher than at 2.6 g/100 g NaCl. The gel strength was
not affected by the addition of 0.1 g/100 g SP, whereas a reduction of
gel strength was observed for SP contents higher than 0.5 g/100 g.
In conclusion, application of fish SP at low concentration (1 g/
100 g) could reduce the cooking loss of fish protein gel without
negative effect on gel strength.
Acknowledgments
The authors gratefully acknowledge financial support from the
Brain Korea 21 project from Ministry of Education and Human Re-
sources Development, Republic of Korea. In addition, this study was
financially supported by the Chonnam National University 2010.
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