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Changes in muscle properties during postmortem storage of farmed sea

bream (Sparus aurata)

Article  in  Journal of Food Process Engineering · September 2009

DOI: 10.1111/j.1745-4530.2009.00522.x

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jfpe_522 922..946

CHANGES IN MUSCLE PROPERTIES DURING POSTMORTEM

STORAGE OF FARMED SEA BREAM (SPARUS AURATA)

M.D. SUÁREZ1, T.F. MARTÍNEZ1,3, M.I. SÁEZ1, B. ALFÉREZ1 and

M. GARCÍA-GALLEGO2
1
Departamento de Biología Aplicada
Universidad de Almería
04120 Almería, Spain
2
Departamento de Biología Animal
Universidad de Granada
Campus Universitario de Fuentenueva
Granada, Spain

Accepted for Publication April 16, 2009

ABSTRACT

This study assesses the effects of storage temperature on postmortem

changes in textural parameters (firmness, water holding capacity), and their
relationship with biochemical and electrophoretic determinations (muscle
collagen content and solubility, amount and integrity of sarcoplasmic and
myofibrillar proteins) in sea bream muscle. Storage at 1C resulted in a pro-
longation of the firmness compared with 4C, with no noticeable changes in
water holding capacity. This delay in muscle softening at 1C could be partially
explained by a limited proteolysis of some myofibrillar proteins, although
inconsistent tendency was observed for other fractions. In contrast, the rate of
muscle collagen degradation was higher at 4C compared with 1C, this becom-
ing the main contributing factor to firmness losses. The great variety of protein
fractions separated electrophoretically, and the relatively minor and contra-
dictory changes observed suggest the unfeasibility of electrophoresis as a
routine procedure for studying freshness in stored sea bream.

PRACTICAL APPLICATIONS

According to the results obtained, it is strongly recommended that the

storage of sea bream (Sparus aurata) throughout the commercial chain is

3
Corresponding author. TEL: +34-950-015267; FAX: +34-950-015476; EMAIL: tomas@ual.es

Journal of Food Process Engineering 34 (2011) 922–946. All Rights Reserved.

922 © 2009 Wiley Periodicals, Inc.
DOI: 10.1111/j.1745-4530.2009.00522.x
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 923

carried out at 1C, instead of the usual temperature of 4C. This modification
clearly delays the process of muscle softening, thus improving objective
quality in this species.
The electrophoretic separation of muscle proteins has been previously
proposed as a suitable procedure for assessing the freshness of fish during cold
storage. However, the results obtained here indicate that the attempts aimed to
the development of an electrophoretic procedure for detecting modifications in
muscle proteins that could be correlated with flesh textural properties were
unsuccessful. This is especially true if short-term deterioration is assessed.

INTRODUCTION

There is an increasing interest in the investigation related to quality

changes of cultured fish occurring during the different steps of the commer-
cialization chain. Numerous chemical and physical methods are available with
the purpose of assessing the freshness of fish during storage, and fish meat
texture is a relevant sensorial characteristic that influences decisively the
desirability of the product by consumers. Texture is determined by a variety of
factors, such as collagen content, the quantity and distribution of lipids, the
rate and intensity of postmortem pH changes and proteolysis occurring in
myofibrils (Mohr 1987).
One of the most important indexes of freshness in fish is firmness, which
diminishes rapidly after death and during cold storage (Hatae et al. 1985;
Toyohara and Shimizu 1988; Montero and Borderias 1990; Oka et al. 1990).
The knowledge of changes taking place during deterioration offers valuable
information related to the processes that cause the softening and, consequently,
might allow the development of methods aimed to prevent its early occurrence.
The physical and chemical modifications occurring in the muscle of
cultured fish from the moment of slaughtering vary considerably depending on
some factors prior to death (species, age, diet, type of capture) and others that
occur during harvest and storage (method of slaughtering, handling, conser-
vation) (Huss 1988). One of the most relevant aspects is postmortem storage
temperature, given that this factor markedly influences both the duration and
the characteristics of the ageing process in fish (Grigorakis 2007), and its
control is relatively easy. Refrigeration slows down bacterial growth, enzy-
matic breakdown and lipid oxidation, and consequently, cold storage retards
fish spoilage (Melvin et al. 2004).
Although it is commonly admitted that low temperature stalls the dete-
rioration of fish, this assumption cannot be admitted as a general tendency
(Iwamoto et al. 1990b). In this sense, in some species, rigor mortis delays as
storage temperature decreases (Iwamoto et al. 1990a), although the opposite
924 M.D. SUÁREZ ET AL.

tendency has been reported in several marine species as well like the red
snapper (Pagrus major) (Iwamoto et al. 1990a) or bastard halibut (Paralich-
thys olivaceus) (Tanaka 1991).
The range at which storage temperature can be decreased is limited
because of the possible impacts of thermal stress on meat quality, and thus,
early studies indicated negative effects of temperatures close to 0C for cod
(Gadus morhua) (Love and Elerian 1964). However, other studies have shown
that losses by spoilage were lower in superchilled fish compared with ice
storage (Kato et al. 1974; Uchiyama et al. 1978a,b; Aleman et al. 1982).
These antecedents, showing disparate results among fish species and
storage temperatures suggest the difficulty of predicting the postmortem
changes occurring in a given species. There is a lack of studies broaching these
phenomena in sea bream (S. aurata), despite the thriving economic impor-
tance of this species in the Mediterranean aquaculture (104,000 tons in 2006;
FAO Globefish, 2008).
Therefore, the aim of the present work was the assessment of the influ-
ence of two different temperatures (4C versus 1C) on postmortem changes in
S. aurata muscle during storage for up to 168 h. The parameters studied were
firmness, water holding capacity and modifications in collagen, sarcoplasmic
proteins (SPP) and myofibrillar proteins (MFP). The objectives were: (1) the
elucidation of the role of SPP, MFP and collagen proteins during the postmor-
tem period; and (2) the possible control of softening by decreasing storage
temperature from 4C (usual in fish commercial chain) to 1C, in order to extend
the commercial life of fresh fish.

MATERIALS AND METHODS

Animals and Sampling

Sea bream (S. aurata), weighing an average of 300 g each, were
obtained from an intensive floating cages fish farm (ADRAPEC, Adra, Almería,
Southeast Spain). Circular cages (16 m Ø, approximately 105,000 fish each,
17 kg/m2 culture density) were located 1.5 km offshore (25 m average depth,
41 ! 1 mg/L salinity). During the entire period of the culture, animals were fed
twice a day with an amount equivalent to 2% per day of their body weight using
a commercial feed (Dibaq, 4.5 mm diameter; 9.46% ash; 43.27% crude protein
and 21.30% crude lipid, all the values expressed on dry matter basis). After a
24-h fasting period, the animals were randomly chosen, removed from the
cages, and killed according to the requirements of the Council Directive
86/609/EEC (overdose of metacain, approved euthanasia protocol number
C.2.2 of the experimental aquarium AL/2/U, Universidad de Almería, Spain).
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 925

Once in the laboratory, whole fish (keeping viscera and skin) were
wrapped up in aluminum foil and stored at 4C (lot MS4) or at 1C (lot MS1) in
covered polypropylene containers. Samples of both lots were withdrawn at 5,
10, 24, 48, 96 and 168 h (n = 4 per sampling time and lot) after slaughtering
(hours postmortem, hpm). In each fish, muscle firmness was determined, and
then samples of muscle were removed from the dorsal anterior part of the
body. A portion was used immediately for water holding capacity (WHC)
determinations. The rest of the muscle sampled was frozen in liquid nitrogen
and stored at –80C until it is used for protein extraction, collagen determina-
tions and protein hydrolysis studies.

Firmness
Whole fish firmness was measured by compression of the anterior area to
the dorsal fin, above the lateral line, on the left lateral wall of the fish, using a
texture analyser (Mark-10 model ESM, Mark-10 Corporation, Hicksville,
NY), equipped with a 12.5-mm cylindrical probe, according to Einen and
Thomassen (1998). The probe was pressed downwards at a constant speed of
1 mm/s into the fish, until a penetration depth of 5 mm (prior to the breakage
of muscle fibers). Fishes were kept on ice during the assays.

WHC
A modification of the original method of Pastoriza et al. (1998), specially
designed for fish muscle, was used. Briefly, muscle samples were placed in a
tube inside of which another tube was fitted in such a way that the muscle sample
was never in contact with the water released, and then were slightly centrifuged
(630 g, 30 min, 10C). The amount of water lost from the muscle portion after the
centrifugation was expressed as a percentage of the initial fresh weight of the
sample. The WHC was calculated as the difference between the initial percent-
age of water in the muscle (determined by stove dehydration at 105C until
constant weight) and the percentage of water released by centrifugation.

Determination of pH
The pH of flesh samples was determined using a penetration electrode
(model GLP 21, Crison Instruments, Barcelona, Spain; sensitivity 0.01 pH
units) after carrying out a lateral incision in the dorsal muscle in order to place
the tip of the electrode deep in the muscle mass (Ginés et al. 2002).

Estimation of Protein Hydrolysis

The extent of protein hydrolysis was determined by using the colorimet-
ric trinitrobenzenosulfonic (TNBS) acid method as described by Hernandez-
926 M.D. SUÁREZ ET AL.

Herrero et al. (1999). Briefly, fish muscle samples were homogenized (0.1 g/
mL) in borax buffer (25 mM Na2B4O7 · 10H2O), containing 2% (w/v) sodium
dodecyl sulfate (SDS), pH 8.9, and then shaken in a thermostatic bath at 75C
for 15 min with the purpose of preventing proteolytic degradation. After that,
mixtures were shaken at 60C for 2 h, and thereafter, the extracts were diluted
1:25 (v/v) in 0.2 M phosphate buffer, pH 8.2. Samples (60 mL) were then
incubated (60 min, 42C) with 470 mL of 0.2 M phosphate buffer (pH 8.2) and
470 mL of 0.1% TNBS aqueous solution. After incubation, absorbance at
405 nm was measured and compared with an L-leucine standard. Results were
expressed as mmol L-leu per 100 g of fresh muscle.

Collagen Content and Solubility

Total collagen was estimated by the method of Palka (1999), based on the
measurement of the hydroxyproline (OH-Pro) content in a muscle sample
previously subjected to acid hydrolysis in 6 N HCl (0.1 g muscle/mL) at 110C
for 24 h. OH-Pro was determined in muscle samples according to the ISO
(1978) method. This procedure is based on the oxidation of OH-Pro by
chloramine-T, followed by the addition of 4-dimethyl-aminobenzaldehyde,
which generates a colored complex quantifiable spectrophotometrically at
560 nm. A factor of 11.42 was used, according to Sato et al. (1989), to trans-
form the amount of OH-Pro into collagen. Results (average of triplicate mea-
surements) were expressed as milligram of collagen per gram of fresh weight.
The separation of the different collagen fractions in muscle extracts
(ASC, acid-soluble collagen; PSC, pepsin-soluble collagen; and ISC,
insoluble collagen) was carried out according to the method described by Sato
et al. (1988). Independently of the fraction considered (ASC, PSC or ISC), the
estimation of collagen was based on the measurement of OH-Pro content in
extracts, which was carried out according to the ISO (1978) method. Prior to
OH-Pro determinations, muscle homogenates were hydrolyzed according to
the procedure described by Eckhoff et al. (1998) slightly modified by perform-
ing a preliminary extraction with cold 0.1 N NaOH to remove non-collagenous
protein. Briefly, muscle samples were homogenized in 10 volumes (w/v) of
0.1 N NaOH, the homogenates were centrifuged (10,000¥ g, 20 min, 4C)
and supernatants were collected. Pellets were resuspended in five additional
volumes of 0.1 N NaOH, stirred overnight and centrifuged again as described.
The pellets remaining after the second centrifugation were resuspended in five
volumes of 0.5 M acetic acid. The mixtures were stirred for 24 h and centri-
fuged (10,000¥ g, 20 min, 4C), and the supernatants were collected and used
as ASC fraction. PSC was rendered soluble by digestion with porcine pepsin
at an enzyme-to-substrate ratio of 1:20 (w/w) in 0.5 M acetic acid. The diges-
tion was performed at 4C for 24 h before centrifugation (10,000¥ g, 20 min,
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 927

4C). The final supernatant was the PSC, and the insoluble matter was the
ISC fraction.

Extraction of Muscle SPP and MFP Fractions

Protein extracts were prepared only from muscle fractions after 10 and
168 h postmortem, in order to maximize possible differences in muscle pro-
teins due to the time of storage. For the extraction of SPP, freeze-dried portions
of muscle were homogenized (1.5 mg muscle/mL) in chilled low ionic
strength buffer (10 mM NaHCO3, 1 mM ethylene glycol tetraacetic acid,
pH 7.2) by using a mechanical homogenizer (Polytron PT-2100, Kinematica
AG, Lucerne, Switzerland) at 10,000 rpm for 1 min. Heating of the homo-
genates was prevented by keeping samples on ice during the process. The
homogenates were centrifuged (10,000¥ g, 20 min, 4C) and supernatants were
collected. Pellets were resuspended in the same low ionic strength buffer and
re-extracted as described above. Supernatants of both consecutive centrifuga-
tions were pooled, and together constituted the SPP fraction.
Remaining pellets were again extracted twice in chilled myofibrillar
extracting buffer (8 M urea, 50 mM Tris–HCl, 2% v/v Triton X-100, 2 mM
dithiotreitol, pH 7.2) as described, and the pooled supernatants obtained after
the double centrifugation were collected as MFP fraction.

Estimation of Soluble Protein

Concentration of soluble proteins in SPP extracts was estimated by the
spectrophotometric method of Bradford (1976), measuring absorbance at
595 nm. Owing to the interference of detergents with Bradford method, con-
centration of soluble protein in MFP extracts (containing Triton X-100) was
estimated by Biuret reaction (Gornall et al. 1949) using alkaline copper sulfate
reagent and measuring the color developed at 540 nm. In both procedures,
bovine serum albumin was used as standard in order to quantify the amount of
soluble protein in extracts.

Electrophoresis of Muscle Proteins

SDS-polyacrylamide gel electrophoresis (PAGE) separation of soluble
protein fractions contained in SPP and MFP extracts was performed according
to Laemmli (1970) in a Mini Protean II electrophoresis chamber (Bio-Rad,
Richmond, CA), using 4% polyacrylamide stacking gels and 10% separating
gels.
SPP and MFP extracts were diluted (1:1) in a sample buffer (0.125 M
Tris–HCl, 20% v/v glycerol, 0.04% w/v bromophenol blue, 2% w/v SDS and
20% v/v b-mercaptoethanol) and were boiled for 2 min.Appropriate volumes of
928 M.D. SUÁREZ ET AL.

those mixtures were placed in each well in order to load 20 and 25 mg of soluble
protein per lane from SPP and MFP extracts, respectively. Five microliters of
molecular mass markers (Sigma Chem. Co., St. Louis, MO) were added to
each gel. The standard protein mixture contained myosin (200.0 kDa), b-
galactosidase (116.3 kDa), phosphorylase b (97.4 kDa), bovine serum albumin
(66.2 kDa), ovoalbumin (45.0 kDa) and carbonic anhydrase (29.0 kDa).
After the electrophoretic separation, gels were stained overnight using
0.1% (w/v) Coomassie brilliant blue (BBC R-250, Sigma Chem. Co.) in
methanol-acetic acid-water solution (40:10:50); and then destained in the same
solution without the stain.

Densitometric Analysis
Densitometry of gels was performed using specific software (Image
Analyser, Genesnap version 6.08, Synoptics, Cambridge, U.K.) with the aim
of assigning relative molecular masses to the SPP and MFP separated bands,
and to quantify the relative contribution of each individual fraction to the total
optical density of each lane, expressed as percentage.

Statistics
Data were analyzed using a one-way analysis of variance, followed by a
comparison of means (Fisher’s least significant difference procedure). Unless
otherwise specified, a significance level of 95% was considered to indicate
statistical differences (P < 0.05). When results were expressed as a percentage
(e.g., densitometry), data were normalized using the arcsine transformation of
their square root previously to statistical analysis, according to Sokal and
Rohlf (1981). All statistics were conducted using specific software (Statgra-
phics Plus 4.0, Statistical Graphics Corp., Rockville, MD).

RESULTS

Firmness, WHC, pH and Estimation of Protein Hydrolysis

Changes in firmness, WHC, pH and proteolysis (estimated by TNBS) of
sea bream muscle during the 168 h postmortem (hpm) storage period at 4 and
1C (MS4 and MS1, respectively) are shown in Fig. 1.
For MS4, the initial values of firmness increased up to a maximum at
10 hpm, and then declined first at a low rate until 48 hpm, and then abruptly at
96 and 168 hpm. In a similar trend, MS1 reached its maximum firmness
at 10 hpm, and then this parameter declined progressively up to the end of the
storage period. Although tendencies were similar, marked quantitative differ-
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 929

firmness (N)

FIG. 1. POSTMORTEM CHANGES IN MUSCLE FIRMNESS (N; A), WATER HOLDING

CAPACITY (WHC, %; B), pH (C) AND PROTEOLYSIS (nmol leu/100 G FRESH MUSCLE;
(D) DURING FISH STORAGE AT 4 AND 1C. ERROR BARS INDICATE ! STANDARD
DEVIATION
TNBS, trinitrobenzenosulfonic acid.

ences were observed between the fish stored at both temperatures along the
storage period, and thus, firmness was consistently higher in muscle stored at
1C compared with that kept at 4C (P = 0.0249). These differences disappeared
at the end of the storage period (Fig. 1A).
The initial WHC (62%) increased to 68% at 10 h, and persisted at similar
values for fish stored at both temperatures throughout the 168 hpm period.
Results showed that storage at temperatures of 1 and 4C did not affect this
parameter. On the other hand, time markedly influenced (P = 0.0017) WHC
values when the entire storage period was considered, independently of the
storage temperature.
Changes in muscle pH differed according to the storage temperature, and
thus for MS1 did not vary significantly over time, but a significant decrease at
24 h for MS4 was observed, followed by a gradual increase up to values
similar to those of MS1 around the 96 hpm. In other words, significant differ-
ences were found during the first half of the storage period, in which the lower
930 M.D. SUÁREZ ET AL.

temperature (1C) prevented the decline in pH. Nevertheless, considering the

complete period of storage, neither temperature nor storage time affected
pH values.
The evolution of protein hydrolysis, estimated by measurement of the
products released by the TNBS method, is shown in Fig. 1D. Proteolysis
varied in a similar way at both temperatures, increasing significantly
(P = 0.0040 and 0.0382 for MS1 and MS4, respectively) at 24 hpm, and then,
values practically did not change in MS1 samples, although they experienced
a secondary increase just at the end of the experimental period when fish
muscle was stored at 4C. Coming across the total period of the trials, it was
observed that the major contributing factor to the differences observed in
proteolysis was certainly the time (P < 0.001), while in relation to temperature,
storage at 4C tended to generate more proteolysis than storage at 1C, although
the differences were not significant except at point 168 hpm (P = 0.0193).

Collagen Content and Solubility

The variations in muscle collagen content (total collagen and ASC, PSC
and ISC collagen fractions) during storage are shown in Table 1. The initial
total collagen content declined as cold storage progressed, this decrease being
statistically significant at 4C (P = 0.0070) when the complete storage period
was considered. No differences in total collagen were attributable to storage
temperature.
With regard to collagen solubility, results shown in Table 1 indicate that
when studying the complete storage period, the relative influence of the three
analytical fractions of collagen varied significantly in muscle stored at 4C.
ASC (P = 0.0006) and ISC (P = 0.0048) decreased throughout the storage
period, while opposed tendency occurred for PSC (P = 0.0001). The same
trend was observed for muscle samples kept at 1C, although in a less notice-
able manner, given that differences attributable to the storage time were sig-
nificant in the ASC fraction (P = 0.0008), but not significant in PSC and ISC
fractions.
Although a significant decrease in ASC fraction was found at both storage
temperatures, differences in the moment of such losses between MS4 and MS1
were also evident. Thus, ASC had decreased significantly after 10 hpm in MS4
(P = 0.0472), but this effect was delayed up to 48 hpm in MS1 (P = 0.0250)
samples. Additionally, at 4C, ASC experienced a secondary decrease at the end
of the experimental period, reaching the lowest values at 168 hpm. In oppo-
sition, in MS1 samples, the ASC percentage persisted to be practically stable
from 48 to 168 hpm.
The effects attributable to temperature on total collagen and collagen
fractions were more noticeable in the period from 48 hpm onwards. In this
 TABLE 1.
TOTAL COLLAGEN AND RELATIVE DISTRIBUTION OF COLLAGEN FRACTIONS (ACID-SOLUBLE [ASC], PEPSIN-SOLUBLE [PSC] AND
INSOLUBLE [ISC]) EXTRACTED FROM SEA BREAM MUSCLE DURING FISH STORAGE AT 4 AND 1C

Storage Parameter Postmortem time (h)

temperature
5 10 24 48 96 168

4C Total collagen (g/kg) 5.54 ! 0.35b 5.56 ! 0.38b 5.03 ! 0.99ab 5.04 ! 0.79ab 4.08 ! 0.29a 4.04 ! 0.69a
ASC (%) 12.57 ! 1.45c 8.94 ! 2.52b 8.50 ! 1.74b 8.98 ! 0.29b 8.09 ! 1.67b,* 5.42 ! 1.64a
PSC (%) 61.53 ! 1.59a 66.23 ! 3.96ab 67.30 ! 4.17bc 69.25 ! 3.87bc 73.75 ! 5.18cd 78.41 ! 3.33d,*
ISC (%) 25.90 ! 2.84c 24.91 ! 3.88c 24.20 ! 4.07c 21.97 ! 3.65bc,* 18.38 ! 3.50ab 16.17 ! 2.71a,*
1C Total collagen (g/kg) 5.54 ! 0.35b 5.47 ! 0.93b 5.53 ! 0.55b 5.03 ! 0.23ab 4.92 ! 0.89ab 4.34 ! 0.84a
ASC (%) 12.57 ! 1.45b 11.39 ! 2.90b 12.04 ! 4.03b 6.50 ! 3.82a 3.66 ! 2.53a,* 5.60 ! 1.33a
PSC (%) 61.53 ! 1.59a 63.03 ! 4.24a,b 62.55 ! 5.00a,b 61.04 ! 6.37a 68.28 ! 3.65b 67.15 ! 1.90ab,*
ISC (%) 25.90 ! 2.84 25.58 ! 1.99 25.41 ! 4.64 31.96 ! 7.00* 27.31 ! 6.85 29.27 ! 2.35*

Within each row, values (mean ! standard deviation) with different lower-case superscripts indicate significant differences (P < 0.05) due to storage time.
Values sharing asterisks express significant differences due to storage temperature (* P < 0.05).
MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE
931
932 M.D. SUÁREZ ET AL.

FIG. 2. EFFECT OF STORAGE TIME AND TEMPERATURE ON THE AMOUNT OF

SARCOPLASMIC (SPP) AND MYOFIBRILLAR (MFP) SOLUBLE PROTEIN CONTENT IN
EXTRACTS OF SEA BREAM MUSCLE
(A) SPP. (B) MFP. Values marked with asterisks are significantly different due to temperature of
storage (*P < 0.05; **P < 0.01). Error bars indicate ! standard deviation.

sense, significant differences were observed only in PSC (P = 0.0016) and

ISC (0.0002) fractions.
In short, storage at 1C preserved muscle collagen to a higher extent than
storage at 4C, although marked differences in individual fractions and sam-
pling times could be observed.

SPP and MFP Fractions

Soluble protein contents in sarcoplasmic and myofibrillar extracts
(g/100 g of fresh muscle) of sea bream stored at 4 and 1C, after 10 and
168 hpm, are shown in Fig. 2. It is noticeable that myofibrillar soluble protein
content was much higher (fivefold to sixfold) than the concentration measured
in sarcoplasmic extracts, thus reflecting the quantitative dominance of struc-
tural proteins in the muscle fiber.
With respect to the influence of storage time and temperature on the
amount of soluble protein in extracts, no differences were found between 10
and 168 hpm in MS4 or MS1 samples for SPP. On the other hand, at 168 hpm,
a higher amount of SPP soluble protein was extracted from muscle samples
stored at 1C compared with those kept at 4C (P = 0.0034, Fig. 2A). Soluble
MFP content in extracts tended to be higher in MS1 samples compared with
MS4 (Fig. 2B), although no significant differences were detected, indepen-
dently of storage time.

SDS-PAGE Separation of Muscle Proteins

Electrophoretic profiles of SPP and MFP extracts are shown in Fig. 3.
SPP extracts contained nine principal bands after SDS-PAGE separation, with
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 933

FIG. 3. SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS

SEPARATION OF THE PROTEIN FRACTIONS CONTAINED IN SARCOPLASMIC (SPP) AND
MYOFIBRILLAR (MFP) EXTRACTS OF SEA BREAM MUSCLE

relative molecular masses of 97, 60, 51, 45, 36, 34, 27 and 25 kDa. Numerous
fractions were obtained for MFP extracts, although the most contributing to
total protein content, in terms of optical density, were the bands of 200, 110
and 45 kDa. Although these protein fractions were not specifically isolated and
characterized, according to the similarity with the values checked with previ-
ous literature, these bands were designated as myosin, a-actinin and actin,
respectively. Other individual bands were grouped in the study, because of
their similar molecular masses, in a unique fraction of 32–38 kDa, and
together were designated as tropomyosin. Similarly, several protein fractions
separated were grouped in a 51–69 kDa category, and finally, in a low molecu-
lar mass group ranging from 23 to 29 kDa.

Densitometric Quantification of Muscle Proteins

Relative optical density of each protein fraction, expressed as a percent of
total optical density of SDS-PAGE-separated proteins, is shown in Tables 2
and 3 for SPP and MFP, respectively.
 934

TABLE 2.
EFFECTS OF TEMPERATURE (4 AND 1C) AND TIME (10 AND 168 H) OF STORAGE ON SARCOPLASMIC PROTEINS EXTRACTED
FROM SPARUS AURATA MUSCLE. VALUES INDICATE THE RELATIVE OPTICAL DENSITY OF EACH PROTEIN FRACTION
(EXPRESSED AS % OF TOTAL OPTICAL DENSITY) AFTER SDS-PAGE SEPARATION

Storage conditions Protein fraction

Temperature Time 97 kDa 60 kDa 51 kDa 45 kDa 41 kDa 36 kDa 34 kDa 27 kDa 25 kDa
(h)

4C 10 17.91 ! 0.12b,* 10.38 ! 0.78a 17.82 ! 3.08 2.07 ! 0.34 9.62 ! 1.54a,** 16.80 ! 1.06a 10.34 ! 1.70a 5.05 ! 0.64* 7.58 ! 1.20
168 11.95 ! 1.84a 11.95 ! 0.20b 16.83 ! 2.38 1.97 ! 0.14 15.41 ! 2.10b 18.81 ! 0.82b 18.27 ! 1.12b 4.53 ! 0.60 7.66 ! 0.78*
M.D. SUÁREZ ET AL.

1C 10 13.73 ! 1.80* 13.73 ! 1.36 17.15 ! 1.90 1.79 ! 0.36a 15.80 ! 1.92** 15.57 ! 0.70 10.67 ! 1.80a 6.27 ! 0.68b,* 8.95 ! 0.90b
168 12.81 ! 0.88 9.17 ! 0.68 17.15 ! 1.52 2.18 ! 0.16b 16.09 ! 1.30 17.43 ! 1.80 16.17 ! 1.56b 4.05 ! 0.66a 5.94 ! 0.74a,*

Within each column, values (mean ! standard deviation) with different lower-case superscripts indicate significant differences (P < 0.05) due to storage time for a given
temperature, and values sharing asterisks show significant differences due to storage temperature (* P < 0.05; ** P < 0.01) for a given time.
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
 TABLE 3.
EFFECTS OF TEMPERATURE (4 AND 1C) AND TIME (10 AND 168 H) OF STORAGE ON MYOFIBRILLAR PROTEINS EXTRACTED FROM
SPARUS AURATA MUSCLE. VALUES INDICATE THE RELATIVE OPTICAL DENSITY OF EACH PROTEIN FRACTION (EXPRESSED AS % OF
TOTAL OPTICAL DENSITY) AFTER SDS-PAGE SEPARATION

Storage conditions Protein fraction

Temperature Time (h) 200 kDa† 110 kDa‡ 51–60 kDa group 45 kDa§ 32–38 kDa¶ LMW†† group

4C 10 35.72 ! 2.56b 2.25 ! 0.28a 1.46 ! 0.16a,* 16.61 ! 1.96a 36.83 ! 3.30a 6.89 ! 0.80
168 11.80 ! 4.56a,** 2.40 ! 0.68b 3.34 ! 1.14b 22.78 ! 2.00b 49.52 ! 1.52b 5.98 ! 2.08
1C 10 32.27 ! 1.14 3.15 ! 0.38 0.48 ! 0.40a 14.96 ! 4.06 38.17 ! 4.04 6.50 ! 1.18
168 25.60 ! 12.07** 4.94 ! 1.26 3.38 ! 0.22b 20.83 ! 3.50 38.18 ! 6.92 4.82 ! 0.48

Within each column, values (mean ! standard deviation) with different lower-case superscripts indicate significant differences (P < 0.05) due to storage time
for a given temperature, and values sharing asterisks show significant differences due to storage temperature (* P < 0.05; ** P < 0.01) for a given time.
† Protein fraction designed as myosin heavy chain in text.
‡ Protein fraction designed as a-actinin.
§ Protein fraction designed as actin.
¶ Protein fractions designed as tropomyosin group.
†† LMW: low molecular weight protein fraction, including bands ranging 23–29 kDa.
SDS -PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE
935
936 M.D. SUÁREZ ET AL.

With regard to SPP, when all the protein fractions were studied together,
neither the factor “storage temperature” nor “storage time” caused significant
differences on their relative optical densities. Nevertheless, significant differ-
ences were found for some individual bands, although no general tendency
could be observed. Thus, relative optical density of 97 kDa fraction decreased
significantly due to storage time (10 versus 168 hpm) when fish were kept at
4C (P = 0.0053), whereas this effect was not noticeable for fish stored at 1C.
Opposite trend was observed for 60 kDa fraction (P = 0.0019 for 4C) when
comparing the initial and final time sampling points. Fractions 41, 36 and
34 kDa increased their relative optical density at 4C due to storage time
(P = 0.0177, 0.0250 and 0.008, respectively), although this effect was persis-
tent only for the 34 kDa fraction at 1C (P = 0.0090).
Similar inconsistency was observed for MFP, as shown in Table 3. The
effect of temperature and storage time was not significant on the relative
optical density of the bands as a whole. Nevertheless, the effect of storage time
at 4C on the 200 kDa (decrease, P = 0.0001), 110, 51–60, 45 and 32–38 kDa
fractions was noticeable (increase, P = 0.0323, P = 0.0161, P = 0.0321 and
0.0019, respectively). This effect was not observed for the same fractions when
muscle was stored at 1C. Indeed, only a significant increase for 51–60 kDa
fraction (P = 0.0278) was observed.

DISCUSSION

The assessment of fish freshness has been a major issue in fish research
in the last decades, and consequently, a vast number of indicators are available
(e.g., chemical, physical and sensorial) with the purpose of evaluating directly
or indirectly postmortem processes taking place in fish muscle after sacrifice
(Olafsdottir et al. 1997; Delbarre-Ladrat et al. 2006; Herrero 2008).
Fish muscle firmness is an accurate index of freshness, given that this
parameter declines rapidly after slaughter, even in cold storage (Hatae et al.
1985; Oka et al. 1990). The softness implies a decrease in organoleptic
quality, and therefore, it is crucial to know the processes involved in this
deterioration in order to implement good storage practices that prevent or
delay its appearance.
Protein degradation causes changes in the rheological properties of refrig-
erated fish muscle. Although not completely ascertained, it is assumed that a
partial molecular fragmentation and/or textural relaxation of the muscle fiber
are implicated in the softness of stored fish. In this sense, several studies
indicated that the breakage and disorganization of Z lines and sarcolemma
takes place, as well as the degradation of myofibrils proteins such as dystro-
phin (Papa et al. 1996), titin and nebulin (Astier et al. 1991), or desmin
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 937

(Verrez-Bagnis et al. 1999). These degradation processes are based on the

activity of several proteases with different catalytic properties, like acid (as
cathepsin), neutral (either Ca2+-dependent, as calpain, or not) and basic pro-
teases (Makinodan et al. 1985).
On the other hand, several studies (Sato et al. 1994; Ando et al. 1995;
Bremner 1999) have also pointed out that the alterations in textural properties
of fish muscle are the result of the disintegration of the extracellular matrix,
and thus, the different collagen fractions would be involved in these processes,
possibly by the action of endogenous muscle collagenases (Bracho and Haard
1996).
According to these antecedents, it seems necessary that studies focused
on fish firmness during storage should assess the possible effects of storage
conditions on both collagen and muscle fiber proteins. From that point of view,
the present work was aimed to evaluate the relationships of firmness and water
holding capacity with the possible modifications in SPP and MFP, as well as in
collagen content in cultured sea bream muscle during cold storage at two
temperatures (4 and 1C).
The results obtained in the present study indicated that muscle stored at
1C exhibited a higher firmness in the early storage period (Fig. 1A), and a
delay in the rate at which this parameter decreases throughout the storage
period when compared with that kept at a higher temperature (4C).
On the other hand, temperature did not modify the time at which the
maximum firmness was observed (10 hpm), thus suggesting that the initiation
of rigor mortis was not delayed by lowering the storage temperature. Never-
theless, further determinations are needed with the purpose of ascertaining
specific effects of storage temperature on rigor mortis.
Differences in firmness due to storage temperature were kept until
168 hpm. At the end of the storage period, differences disappeared, likely due
to increased proteolytic activity in the muscle, as observed in Fig. 1D.
Previous studies have shown that storage temperature determines both the
initiation and the progress of the rigor mortis (Skjervold et al. 2002), and
generally low temperatures during slaughter can delay the beginning of rigor
mortis in warm water species (Lowe et al. 1993; Skjervold et al. 2001), thus
improving the freshness characteristics of stored fish (Nakayama et al. 1994;
Robb et al. 2000; Bagni et al. 2007). Nevertheless, excessive chilling could
accelerate rigor mortis in some species (Iwamoto et al. 1985). Sampling
intervals in the present study are too wide to elucidate this point, given that
actual measurements of firmness during the rigor mortis period were not
carried out.
The pattern observed for postmortem softness in the present study on
S. aurata was in accordance with previous studies on sea bass (Dicentrarchus
labrax; Poli et al. 2001), Atlantic salmon (Salmo salar; Espe et al. 2004) and
938 M.D. SUÁREZ ET AL.

trout (Salmo irideus; Montero and Borderias 1990) stored under analogous
conditions.
The WHC of the muscle is of great importance in order to define the
commercial value and consumer acceptance, as it is an accurate index of the
juiciness of the fish (Laroche et al. 1995). It is well known that WHC is
strongly influenced by the structural changes in muscle proteins, by fiber
contraction status and by both intracellular and extracellular water distribution
(Schnepf 1989). The WHC of muscle tissue is also affected by factors like
pH (Ofstad et al. 1996), protein denaturation (Ashie et al. 1997; Ashie and
Simpson 1998), the space among myotomes and the length of sarcomeres
(Ofstad 1995). WHC in the present study was not affected by storage tem-
perature (Fig. 1B), and this result could be interpreted as a consequence of
the absence of important changes in muscle cell protein structure. Thus, the
maintenance of the integrity of most SPP and MFP fractions separated by
SDS-PAGE (Tables 2 and 3), as well as the limited amount of products of
proteolysis released from muscle during storage (Fig. 1D), are indirect evi-
dences in agreement with limited protein damages.
It could be thought that lower temperature preserves the integrity of sea
bream muscle proteins, given that this effect might explain the higher values of
firmness in MS1 than those in MS4 during the 168 hpm storage period.
Nevertheless, the lack of a parallelism between firmness values (Fig. 1A) and
proteolysis (Fig. 1D) suggests that TBNS values are not enough to explain the
dissimilar values of firmness. On the other hand, there was a clear similarity
between the evolution of TNBS and the WHC (Fig. 1C,D), a fact that might
reflect the close relationship between the integrity of protein fractions and their
ability to retain water molecules.
The lack of correlation between muscle protein hydrolysis and firmness
values suggests that other factors are involved. In this sense, some studies have
shown that collagen is a muscle constituent that contributes largely to firmness
in sea bream (Hallett and Bremner 1988). In fact, our results show that the total
collagen content of sea bream muscle decreased, in agreement with previous
studies. However, given that this decrease in total collagen was not affected by
temperature, the total amount of this constituent does not explain differences
in firmness due to storage temperature (Fig. 1).
According to the modifications observed in collagen fractions due to
storage temperature (Table 1), it is suggested that not the total amount of
collagen, but the relative contribution of each collagen fraction (ASC, PSC and
ISC) could account for the differences observed in firmness. Thus, it was
remarkable that lower temperature kept the initial amount of ASC and ISC for
a longer period and, consequently, the disappearance of these fractions could
likely be responsible for the beginning of muscle softness. In a previous study,
Suárez et al. (2005) found a direct relationship between muscle ASC content
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 939

and firmness in sea bream samples stored at 4C, reporting a significant

decrease in both parameters during the first 24 hpm.
In relation to pH, it is well known that, in terrestrial animals, pH is a
major contributing factor to WHC changes during postmortem meat matura-
tion. In comparison, pH changes in fish muscle are very weak, and conse-
quently, the influence of this factor on the WHC of sea bream muscle is
probably negligible. In fact, pH modifications shown in Fig. 1C were lower
than one pH unit, independently of the storage temperature and time. No
significant differences in pH during the storage time were observed at 1C,
whereas at 4C, pH decreased significantly at 24 hpm (Fig. 1C); these results
indicate that low temperature prevented the decline of pH for up to 24 hpm,
although this effect disappeared later. From a practical point of view, the pH
value cannot be considered a valid index to evaluate the effects of storage
temperature on sea bream muscle quality, given that this parameter was unable
to discriminate between the two temperatures studied after 24 hpm of cold
storage.
The amount of SPP and MFP extracted from sea bream muscle and their
electrophoretic patterns have been studied only at 10 and 168 h of storage,
given that great modifications at intermediate times were not expected, accord-
ing to preliminary studies.
Electrophoretic patterns of SPP (Fig. 3, Table 2) showed nine groups of
bands in S. aurata, a similar profile to that described by Chéret et al. (2006) for
sea bass muscle. These authors described 12 groups, likely because they were
able to resolve better low molecular mass proteins by using 15% polyacryla-
mide gels, compared with 10% gels in the present work.
Separations of MFP gave six groups of bands (Fig. 3, Table 3), also
similar to those described by Chéret et al. (2006) for sea bass muscle. Based
on these authors and others (Michalczyk and Surówka 2007 for trout), it was
possible to assign some of these bands to specific proteins in this study.
Both SPP and MFP bands were used for the assessment of the effects of
storage time and temperature on sea bream muscle. The most noticeable effect
was the persistence of the SDS-PAGE-separated protein fractions (considered
as a whole) of both SPP and MFP between 10 and 168 h. In this sense, the
degradation of SPP or MFP as a whole does not seem capable of explaining
significant effects found in the firmness. In other words, the significant
changes detected in firmness during storage (Fig. 1A) could not be explained
by the minor changes in optical density obtained for SPP or MFP.
Previous studies have also shown that changes in the electrophoretic
profile of proteins from fish muscle during storage have been virtually non-
existent or very subtle, even after long periods. Thus, Tejada et al. (2003)
could not find changes in actomyosin in sea bream after 20 days refrigeration,
and Verrez-Bagnis et al. (2001) found slight changes in SPP in sea bass during
940 M.D. SUÁREZ ET AL.

storage (a small decrease in a 100-kDa protein after 96 h of storage, together

with a marked decline in a 16-kDa band of unknown identity). Aubourg et al.
(2005) reported a 22-kDa polypeptide of unknown identity, as a marker of
degradation process in turbot (Psetta maxima) muscle. Chéret et al. (2006)
observed the appearance of two bands of 20.5 and 30.5 kDa in sea bass muscle
after 2 days of storage. Other authors have not seen significant changes in the
electrophoretic pattern of SPP of sea bass during storage (Verrez-Bagnis et al.
2002; Ladrat et al. 2003).
Nevertheless, as it has been detailed above, several individual fractions
showed significant decreases due to temperature and/or storage time (Tables 2
and 3) in the present study. In fact, there was a certain parallelism between the
loss of optical density of some individual fractions and muscle firmness, and
thus the MFP fraction of 200 kDa (designed as myosin heavy chain, MHC)
might explain at least part of the differences observed between muscle firm-
ness at 4 and 1C, at least up to 96 hpm. In effect, the degradation of MHC band
at 4C (P = 0.0001) did not occur at 1C, and this result could reflect an effect of
temperature on the protection of this structural protein and, thereafter, in the
preservation of the firmness. However, the lack of parallelism between this fact
and the evolution of firmness after 96 hpm (Fig. 1) suggests additional causes
that determine changes in firmness in long storage periods.
Unfortunately, the lack of certainness regarding the individual contri-
bution of the different protein fractions to the firmness of the muscle, as well
as the inconsistency observed between the evolution of the optical density of
SPP and MFP fractions and firmness, limits the practical recommendation
of the electrophoretic separations of muscle proteins as predictable index of
freshness in S. aurata. This topic has to be clarified prior to any practical
application.

CONCLUSIONS

The storage of sea bream at 1C extended the firmness period compared

with 4C, with no substantial changes in muscle WHC or in muscle protein
hydrolysis.
The delay in the softness at 1C can be attributed mainly to the preserva-
tion of ASC and ISC collagen fractions, although certain contributions of MFP
cannot be fully discarded, given the persistence of the 200 kDa myofibrillar
fraction at a lower storage temperature. According to the results obtained, it is
strongly recommended that the storage of sea bream (S. aurata) throughout the
commercial chain should be carried out at 1C instead of the usual temperature
of 4C.
Although slight modifications in several individual protein fractions sepa-
rated by SDS-PAGE give valuable information related to the integrity of fish
 MUSCLE PROPERTIES OF FARMED SEA BREAM DURING STORAGE 941

muscle proteins, the results obtained in the present work for sea bream suggest
the scarce utility of electrophoretic separation of muscle proteins as a routine
procedure in the assessment of fish freshness, especially if short-term deterio-
ration is studied.

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