Journal of Food Engineering 68 (2005) 303–308

www.elsevier.com/locate/jfoodeng

Production of fish-protein products (surimi) from small pelagic

fish (Sardinops pilchardusts), underutilized by the industry
Christos A. Bentis, Anastasioss Zotos *, Dimitrios Petridis
Thessaloniki Educational Institute (TEI), Department of Food Technology, School of Food Technology & Nutrition,
54101 Thessaloniki, P.O. Box 14561, Greece

Received 24 November 2003; accepted 9 June 2004

Abstract

A sardine surimi product was studied, based on three experimental factors: (a) control of pH-value (2 levels) (b) improvement of
protein solubility (2 levels) and (c) cryoprotectants (3 levels). Samples were frozen at 20
C and kept at this temperature for 25
days. All samples were then partially thawed, mixed with 2 g kg 1 sodium chloride and heated at 90
C for 90 min. The combination
of sorbitol (40 g kg 1) with the salt mixture (sodium chloride 0.45 g kg 1 calcium chloride 0.3 g kg 1 and ammonium chloride 1.25
g kg 1) led to the hardest and more elastic products. Protein loss during the processes was relatively small (6.9% on a dry weight
basis), and its solubility remained at high levels. The overall product recovery was 200 g kg 1. The use of different batches led in
statistically significant differences among the final products. The concentration of eicosapentaenoic and docosahexaenoic fatty acids
showed that under-utilized sardine could be an important source of polyunsaturated fatty acids and MaxEPA products.
 2004 Elsevier Ltd. All rights reserved.

Keywords: Surimi; Sardine; MaxEPA; Under-utilized fish

1. Introduction ity surimi, similar to that produced from the fore-

An effort has begun to exploit some fish species that
are either in abundance or underutilized. This effort
has led in the production of frozen fish-protein products
widely known as surimi mainly because access to the
Alaska pollock sources has been limited (Chen, Chiu,
& Huang, 1997).
Alaska pollock (Theragra chalcogramma) is the most
common fish for such product (Lanier, 1992; Suzuki,
1981). Research has shown that besides Alaska pollock,
and after the appliance of specific chemical methods,
other species may also be used and produce a high qual-
*
Corresponding author. Fax: +30 2310 791360.
E-mail address: zotos@food.teithe.gr (A. Zotos).
mentioned species (Trondsen, 1998).
However, despite serious attempts to use species such
as mackerel and sardine, the control of the factors that
influence the large-scale production of such products re-
main difficult (Chen et al., 1997). These factors are the
high lipid content, the water-soluble proteins, as well
as the pigment and trimethylamine oxide (TMAO) in
dark-fleshed fish mince. Therefore adequate washing is
required to prepare high quality surimi (Shimizu, Toyo-
hara, & Lanier, 1992).
The colour of surimi can be improved by increasing
the washing cycles (Kim et al., 1996), washing time and
water quantity (Chen et al., 1997). Long period washing
would result in high hydration of mince and degradation
of myofibrillar proteins, making the subsequent dehy-
dration process more difficult and could repress the gel-
forming ability. The colour of dark-fleshed fish surimi

0260-8774/$ - see front matter  2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jfoodeng.2004.06.003
304 C.A. Bentis et al. / Journal of Food Engineering 68 (2005) 303–308
could be improved by leaching of mince with hydroper-
oxide or sodium percarbonate or by adding some fat/
casein material to mask the colour (Chen et al., 1997).
In Greece fish (mainly small fish) are processed by
salting, drying, smoking, canning, etc. However, the fish
are often too small for processing and thus have no com-
mercial value and are usually dumped. Thus in Greece
the lack of utilization of small fish may also have an ad-
verse effect on the environment.
Thus, the aim of this investigation is to introduce an
application for the Greek industry to use such fish spe-
cies. Therefore, the possibility of producing surimi prod-
ucts from small pelagic fish, such as sardine, was studied
by leaching the fish flesh-mince, modifying the pH-
value, enhancing the gel forming ability and finally
stabilising the product using sugar solutions as cryopro-
tectant agents during freezing and frozen storage.
2. Materials and methods
Approximately 21 kg (three batches, 7 kg at three dif-
ferent times) of fresh sardines (Sardinops pilchardus)
were purchased, 2–3 h after catching, from the local fish
market of Thessaloniki. All fish had an average length of
13 ± 2.13 cm. They were iced for up to 6 h before
processing (immediately after purchase). The whole
process was performed by hand (due to the lack of the
necessary equipment) at the laboratories of the Thes-
saloniki Institute of Technology, Department of Food
Technology.
2.1. Frozen surimi
Samples of raw sardine from each batch, were
washed, gutted, head and bones removed, minced and
then split into two parts (A & B). Part A was washed
with water (0–5
C) while part B was washed with a 5
g kg 1 solution of sodium bicarbonate in order to stabi-
lize the pH value of the mince (Suzuki, 1981; Lanier,
1992). The duration of both washings was 30 min. After
adequately dewatering the mince, both parts were
washed with a 100 g kg 1 solution of hydrogen peroxide
for 5 min at 0–5
C. Then, parts A & B were split in half
(A1–A2 & B1–B2). A1–B1 were washed with a 2 g kg 1
sodium chloride solution while A2–B2 was washed with
2 g kg 1 salt-mixture solution (consisting from 0.45
g kg 1 NaCl, 0.3 g kg 1 CaCl2 and 1.25 g kg 1 NH4Cl)
to investigate the interaction of a transglutaminase en-
hancer (CaCl2) and inhibitor (NH4Cl) (Morales, Rami-
rez, Vivanco, & Vazquez, 2001). After dewatering the
mince again, all four fore-mentioned parts were once
again split into three parts (C, D, E). Each part was
washed with a 40 g kg 1 sorbitol solution, 40 g kg 1 mal-
tose solution and 40 g kg 1 sorbitol-maltose (1:1) solu-
tion, respectively. A small amount 0.2 g kg 1 of
sodium tripolyphosphate was added to all sugar solu-
tions in order to optimise the cryoprotection of the pro-
tein matrix (Suzuki, 1981; Lanier, 1992). Surimi was
packed into polyethylene bags (350 g), frozen down to
25
C and stored at the same temperature for 25 days.
2.2. Surimi gel preparation
The 350 g surimi sample was partially thawed at
room temperature, cut into small pieces and chopped
with an ordinary domestic cutter for 4 min with 20
g kg 1 sodium chloride. The paste was stuffed in stain-
less steel tubes, 2.5 cm in diameter and 10 cm in length
and was heated at 90
C for 60 min.
2.3. Moisture
Moisture content was determined by the CEC (Com-
mission of European Communities) recommended
method ISOR 1442 (CEC, 1979).
2.4. Protein analysis
The method used for salt soluble protein (SSP) was
that of Cowie and Mackie (1968). In the procedure
adopted, salt soluble nitrogen, non-protein nitrogen
(NPN), and total nitrogen (TN) were determined. SSP
was calculated as follows (SSN-NPN/TN-NPN · 100).
2.5. Lipid extraction and analysis
The lipid content was determined by the Bligh and
Dyer (1959) method as modified by Hanson and Olley
(1963). The fatty acid profile was performed according
to a simple and quick method of Humberside Polytech-
nic as described in Zotos (1991). Peroxide value (PV)
was determined according to the method of AOAC
(1984).
2.6. Instrumental analysis
The instrumental analysis was performed using an In-
stron UTM analyser, model 1140 (Instron Ltd. UK)
with a flat probe of 6 cm diameter. The 12 different sam-
ples of surimi were cut in a cylinder form with an ana-
logue device (2.25 cm in height and diameter).
Firmness, elasticity and juiciness were measured by a
compression test using a 10 kg weight, at 10 mm/min
speed and a compression limit of 70%. Juiciness was
measured by placing the samples on a dried and pre-
weighed filter paper. A pressure of 10 kg was then
applied for 1 min. After accurately re-weighing the fil-
ter paper, the loss of water from each sample was
calculated.
 C.A. Bentis et al. / Journal of Food Engineering 68 (2005) 303–308
2.7. Experimental design
The factors studied in this investigation were the
washing of the mince with water or sodium bicarbonate
solution (2 levels), the washing with different salt solu-
tions (2 levels: sodium chloride 2 g kg 1 and a mixture
of sodium chloride, calcium chloride and ammonium
chloride, 0.45, 0.3 and 1.25 g kg 1, respectively) and fi-
nally the washing with cryoprotectants for product sta-
bilization (3 levels: 40 g kg 1 sorbitol, 40 g kg 1 maltose
and 40 g kg 1 mixture of sorbitol and maltose 1:1). The
combination of every level, which represents a different
treatment, is shown in Table 1.
2.8. Sensory evaluation
Sensory evaluation was performed using 13 experi-
enced members of the academic staff for testing the 12
(2 · 2 · 3) treatments of the experiment and the design
was increased with a crab-like surimi sample from
Atlantic Pollock purchased from the market, in order
to conform to the particular Plan 13.17a described by
Cochran and Cox (1957). The panellistsÕ experience on
sensory assessment was acquired through regular partic-
ipation for at least 4–5 years in similar kinds of projects
concerning fish and meat products. Four sensory varia-
bles were examined: firmness, elasticity, juiciness and
surface colour. The intensity of the four variables was
recorded in a 15 cm unstructured scale. The lineÕs left
end was marked at 0 cm for all variables as extremely
non-firm, non-elastic, dry and gray, whereas the right
end was marked at 15 cm as extremely firm, elastic, juicy
and white. Samples were presented to the panellists
through a particular precedence, carried out by the use
of Minitab Statistical Software Package (Ver. 13.1).
After the samples (coded with 3-digit random numbers)
were left for 30 min at room temperature, 4 segments of
5 cm diameter were placed in an odourless plastic con-
tainer, covered with a watch glass, and served to the
panellists. The evaluation occurred in individual booths
equipped with white lights. De-ionized water and crack-
ers with unsalted tops were provided to clean the palate
between samples.
2.9. Statistical analysis
A balanced incomplete block design (BIBD) was ap-
plied including t = 13 treatments, n = 4 replicates per
Table 1
Encoding and analysis of the three experimental factors
Water (P1)
Sodium chloride (L1) S1
Salt mixture (NaCl, Ca2Cl & NH4Cl) (L2) S1
Code S1 stands for washing the mince with 4% sorbitol solution, S2 with 4% maltose and S3 with 4% sorbitol:maltose 1:1 mixture. A 0.02% sodium
tripolyphosphate solution was also included in every treatment.
305
treatment, b = 13 panellists and k = 4 treatments per
panellist (k < t). Adjusted sensory mean scores were de-
duced for the 13 treatments and at that point, the pur-
chased surimi was excluded from further investigation.
All surimi products, as handled at the various levels
of the three factors under investigation, with all varia-
bles considered, were statistically analyzed using two
different approaches: (a) A 3-way analysis of variance
(ANOVA) with no replicates for the factors and their
interaction terms on the whole set of sensory and physic-
ochemical variables. Statistically significant differences
were tested using the SNK (Student-Newman–Keuls)
test for comparison of level mean values (Zar, 1984).
(b) The ANOVA procedure was further enhanced by
the appliance of Principal Component Analysis (PCA)
in order to investigate the combined effects of all four
factors (including the raw material studied afterwards
as a factor) on the profile of the final product (Dunt-
eman, 1989; Jollife, 1986).
All statistical analysis was carried out on Minitab
Statistical Software Package (Ver. 13.1) and Statistica
Software Package (Ver. 6.0).
3. Results and discussion
3.1. Proximate analysis
Both lipid and protein concentrations were quite low
while moisture, inversely related to the lipid content,
was quite high (Table 2). It is well known that moisture
and lipid are highly dependent in a fatty fish such as sar-
dine, the higher the moisture the lower the lipid and vice
versa. It can be observed from Table 2 that both moisture
and proteins were maintained at significantly high levels
(836 and 134 g kg 1, respectively), indicating a non-
significant loss of proteins (6.9% on a dry weight basis,
probably due to some loss of the sarcoplasmic fraction).
It was also observed that proteins were exhibited high
functionality since they retained a significant amount
of water (836 g kg 1). The mean salt soluble protein
(SSP) of the raw material was 79.8% ± 3.8% (±95% Con-
fidence Intervals) while that of the final product was
61.7% ± 11.7%. Indicating a high solubility even after
the whole process. The flesh yield after removing skin,
head, gut and bones was, as expected, quite low (286
g kg 1), while a 700 g kg 1 product yield was observed
due to processing, with an overall recovery of 200 g kg 1.
Sodium bicarbonate (P2)
S2 S3 S1 S2 S3
S2 S3 S1 S2 S3
306 C.A. Bentis et al. / Journal of Food Engineering 68 (2005) 303–308

Table 2
Proximate analysis of sardine flesh and surimi found in this investigation as well as results from other study
Raw material-product Species Moisture Protein Protein Lipid Lipid
g kg 1 g kg 1 g kg 1 (on dry weight basis) g kg 1 g kg 1 (on dry
weight basis)
Sardine Sardinops pilchardus 833.0 (23.0) 147.0 (22.7) 880.0 (102.0) 19.7 (4.0) 118.0 (21.1)
(present investigation)
Sardine surimi Sardinops pilchardus 836.0 (23.1) 134.4 (23.2) 819.5 (138.1) 17.7 (2.8) 107.9 (25.9)
(present investigation)
Sardine Sardinops ocellata 760–800 160 12
surimi (Morales et al., 2001)
Values are means of triplicate determinations. Standard deviations are shown in parenthesis.

3.2. Factor effects analysis 3.3. Effect of different batches

The results of the ANOVA (Table 3) revealed that
washing the mince with sodium bicarbonate (P2) re-
duced the weight loss during the heating process (208
g kg 1) with a simultaneous reduction of sensory (5.7
cm), instrumental firmness (0.45 Nt), increase of pH-
value (7.57) and moisture content (853 g kg 1) of the fi-
nal product. The salt mixture solution (L2) increased the
weight loss during the heating process (331 g kg 1), the
pH-value from its initial level to 7.5 as well as the PV
to 10.7 meq O2/kg lipid. Samples treated with sorbitol
solution (S1) (cryoprotecting agent) were firmer prod-
ucts than all others as assessed by the panellists (8.4
cm). A similar result has also been reported by Suzuki
(1981). Furthermore, a synergistic effect was observed
between sorbitol (S1) and the mixture of salts (L2) lead-
ing to even firmer surimi products (10.8 cm). The inter-
action of the salt mixture (L2) and sodium bicarbonate
(P2) also indicated a synergistic effect on the pH-value,
the PV and moisture. These variables presented their
maximum values at that particular combined level
(P2L2).
During the surimi production process it was observed
that fish originated from various batches, differentiated
in size and spawning period. This observation was con-
firmed by the proximate analysis of the three different
batches used in this investigation. The particular inter-
batch differentiation (as in moisture, protein and lipid
content, Table 4) had an important effect on the forma-
tion and the quality of the final product. Sardines from
batches 1 and 2 were probably captured during the pre-
spawning period, while those from batch 3 were fished
during the post-spawning period, considering that
they were caught 1 week after the first two batches.
Kurokawa (1983) has found that sardine muscle origi-
nating from samples during the post-spawning period
had a lower gel forming ability than the pre-spawning
ones, an event that was also confirmed in this investiga-
tion. Samples produced from samples of the 3rd batch,
showed (a) low levels of sensory firmness: 2nd batch
0.76 Nt = 1st batch 0.58 Nt > 3rd batch 0.37 Nt, (b)
higher pH-values 3rd pH 8.03>1st pH 7.08=2nd pH
6.93, (c) low levels of SSP 1st 71.30%=2nd

Table 3
ANOVAÕs statistically significant results of factors on the variables under study
Factor Affected variable F-Value p-Value Levels
1 2 3
Washing (P) Weight loss (WL) (g kg 1) 59.70 0.016 309.5 207.9
PH 37.29 0.026 7.00 7.57
Moisture (g kg 1) 61.49 0.016 818.6 853.4
Sensory firmness (cm) 63.38 0.015 9.0 5.7
Instrumental firmness (Nt) 6.38 0.030 0.69 0.45
Salt (L) WL (g kg 1) 121.47 0.008 186.3 331.2
PH 48.58 0.049 7.08 7.48
PV (meq O2/kg lipid) 37.91 0.025 4.67 10.73
Sugar (S) Sensory firmness (cm) 21.48 0.044 8.4 5.7 8.2
Combined levels Combined levels
Salt · sugar (L2 · S1) Sensory firmness (cm) 28.57 0.034 10.8
Washing · salt (P1 · L2) WL (g kg 1) 103.8 0.009 449
Washing · salt (P2 · L2) pH 33.03 0.029 8.03
Moisture (g kg 1) 26.47 0.036 868.9
PV (meq O2/kg lipid) 41.69 0.023 15.94
 C.A. Bentis et al. / Journal of Food Engineering 68 (2005) 303–308 307

Table 4
Proximate analysis of sardines from the three batches
1 1 1 1
Batch Species Moisture g kg Protein g kg Protein g kg (on dry weight basis) Lipid g kg Lipid g kg 1
(on dry weight basis)
1st Sardinops pilchardus 825.9 (10.9) 154.5 (11.5) 887.9 19.4 (5.3) 111.5
2nd Sardinops pilchardus 812.5 (6.1) 165.8 (6.2) 883.8 21.5 (0.8) 116.2
3rd Sardinops pilchardus 867.5 (6.5) 113.1 (7.2) 860.7 18.3 (2.6) 139.3
Values are means of triplicate determinations, except from 1st batch which is six fold. Standard deviations are shown in parenthesis.

66.70% > 3rd 37.70% and (d) higher susceptibility to li-
pid oxidation PV: 3rd 15.94 meq/kg lipid > 2nd 5.51
meq/kg lipid=1st 4.69 meq/kg lipid.
It was also observed that the samples produced from
sardines of batch 2 lost the greater amount of water dur-
ing heating: water loss: 2nd 157.17 g kg 1 > 3rd 74.70
g kg 1=1st 65.20 g kg 1.
3.4. Correlation of sensory and instrumental variables
Sensory and instrumental firmness correlated fairly
(Pearson correlation r = 0.74, p = 0.006) while sensory
and instrumental elasticity correlated sufficiently
(r = 0.85, p < 0.001). A strong correlation was observed
between instrumental elasticity and firmness (r > 0.97,
p < 0.001).
3.5. Principal component analysis (PCA)
Variables that showed statistically significant differ-
ences from the ANOVA, as well as those, which mostly
contributed to the formation of the first three major
component axes were studied. The 1st major axis ex-
plains 61.4% of the total variance, the 2nd axis explains
16.5% and the 3rd one 13.8%, summing to a total of
90.7%. Variable effects are represented with arrows in
Fig. 1, commencing from the centre of the three-dimen-
sional space. Variable correlations are represented by an-
gles formed between each couple of arrows. An oblique
angle between two variables reflects a positive action,
an obtuse angle shows a negative (opposing) effect and
a vertical angle shows no correlation. Treatments that
are close to a variable exert a strong influence on it.
PCA (Fig. 1) reveals that raw material (three different
batches) is a very important parameter on the profile
formation of the product, as also noticed by other scien-
tists (Okada, 1992; Suzuki, 1981). Treatments 10, 11 and
12 from the 3rd batch are characterized by high mois-
ture content, pH-value and peroxide value (PV), and
low values of total protein (TP), salt soluble protein
(SSP), weight loss (WL) and functional properties (such
as instrumental firmness –IF and elasticity –IE) (Fig. 1).
Treatments of that particular batch were produced with
the combination of factor levels L2 and P2 (Table 1).
The 2nd batch (treatments 7, 8 & 9) revealed high
functional properties (apart from treatment 8) and low

Fig. 1. Principal component analysis showing the first three major axes.
308 C.A. Bentis et al. / Journal of Food Engineering 68 (2005) 303–308
Table 5
Fatty acid profile of sardine, surimi and cod-liver oil
Fatty acids % Fresh sardine Sardine surimi Cod-liver oil
C20: 5x-3 (EPA) 11.3 9.8 22.1
C22: 6x-3 (DHA) 24.5 28.7 14.5
Saturated 40.1 38.9 21.0
Monounsaturated 13.9 11.1 9.9
Polyunsaturated 46.0 50.0 69.1
moisture (Fig. 1), and was used to produce surimi with a
combination of factor levels L2 and P1 (Table 1). Max-
imum percentage of SSP was observed for treatment 8
(S2L2P1). The latter product had a pH-value of 7.0. It
is known that the solubility of proteins in solutions of
stable ionic strength increases along with the pH-value
increase within the range of 5.0–7.5.
Finally, all treatments from 1st batch (treatments 1–
6) occupy a central position in the figure, thus indicating
that there was little influence on the surrounding varia-
ble pattern. The only common characteristic of these
treatments is their washing with sodium chloride (L1).
3.6. Fatty acid methyl-esters
The fatty acid profiles of sardine, surimi and cod-liver
oil were also studied. As shown in Table 5, there were
significant differences among the fatty acid profiles of
the raw material (fresh sardine) and that of purchased
pharmaceutical oil from cod (Gadus morrhua) liver. Pol-
yunsaturated fatty acids contained in the cod liver oil
reach 69% of the total amount of the fatty acids, while
those of sardine do not exceed 50%. Eicosapentaenoic
acid (EPA) is also found at 50% greater concentrations
in cod liver oil than in the oil deriving from sardine flesh.
Even so, when taking under consideration that a signif-
icant portion of sardine was not used (such as head, tail
and internal organs), we can assume that under-utilized
sardine could be used as a source of polyunsaturated
fatty acids and they might be pharmaceutically utilized
in producing MaxEPA products.
4. Conclusions
The use of sorbitol, in this investigation as a cryopro-
tective agent along with washing the fish mince with a
mixture of salts, resulted in a harder and more elastic
surimi gel.
Maximum weight loss during cooking was observed
for the treatments washed with water (without attempt-
ing to stabilize the pH-value). Washing the minced fish
meat with the mixture of salts enhanced this event.
Protein denaturation did not show any statistically
significant difference from the ongoing procedure, but
it was influenced by the quality and condition of the
raw samples. The condition of sardine samples influ-
enced the general profile of the final product.
Lipid content of sardine-flesh was highly rich in n-3
polyunsaturated fatty acids and especially in EPA and
DHA. Thus it could lead to another direction of utiliza-
tion of this fish.
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