Zhang et al 2015 journal of food science

Bin Zhang, Shang-gui Deng, Meng Gao, and Jing Chen

Abstract: The effect of slurry ice on the quality of Skipjack tuna (Katsuwonus pelamis) during chilling storage was
investigated and compared to flake ice. Slurry ice-treated samples showed significantly higher springiness and chewiness
variables than the blank and flake ice-treated samples (P < 0.05). The growth of microorganisms in tuna muscle treated
with slurry ice was also down significantly (P < 0.05), and the total aerobic counts didn’t reach higher scores than 5.0 log
CFU/g during the whole chilling storage. Additionally, the myofibrillar protein, Ca2+ -ATPase activity, and total sulfydryl
(SH) content in muscle treated with slurry ice were all significantly higher than the blank and flake-iced samples (P <
0.05). This was probably due to the faster cooling, subzero final-temperature, and larger heat exchange derived from
slurry ice. Standard error of mean and sodium dodecyl sulfate–polyacrylamide gel electrophoresis results also confirmed
that slurry ice treatment could effectively retard the degradation of myofibrillar proteins and showed a positive effect on
the stability of tissue structures.
Keywords: Ca2+ -ATPase, myofibrillar protein, SDS-PAGE, SEM, Skipjack tuna, slurry ice, texture, total SH

Slurry ice has been reported to slow down microbial growth and provides a significantly increased
shelf-life for a broad variety of marine species, such as salmon, seabream, horse mackerel, and pink shrimp. There are
few reports focused on the effect of slurry ice on functional properties of proteins of marine species. This work provides
information on preservation of slurry ice on functional properties of proteins related to quality loss during Skipjack tuna
chilling storage. The present study may provide a deep understanding about potential applications of slurry ice, with
special attention being paid to myofibrillar functional changes during the storage.

Practical Application:

Introduction

and storage, the degree of which are influenced by many factors,
such as pretreatment processes, freezing rate and temperature, storage temperature and time, storage methods, and so on (Yamashita
and others 2003; Jiang 2014). Further, the breakdown or denaturation of muscle proteins is considered to be the main cause of the
degradation of fish/muscle jelly, surimi, or ready-to-eat seafoods,
and would lead to the deterioration of product qualities.
Slurry ice has been proven to be an economically feasible way of

preserving marine foods. Its reported advantages over traditional
fresh-water ice (such as flake, tube, and block ice) include its lower
temperature, faster chilling (due to a more rapid heat exchange),
and lower rate of physical damage (due to its spherical microscopic
particles) (Bellas and Tassou 2005; Kauffeld and others 2010). Additionally, slurry ice has also been reported to slow down microbial
growth and provide a significantly increased shelf-life for a broad
variety of species, such as salmon (Oncorhynchus kisutch) (Rodr´ıguez
and others 2008), seabream (Sparus aurata) (Tejada and Huidobro
2002), seabass (Dicentrarchus labrax) (Cakli and others 2006), bigeye snapper (Priacanthus tayenus) (Riebroy and others 2007), horse
mackerel (Trachurus trachurus) (Losada and others 2005), and pink
shrimp (Parapenaeus longirostris) (Huidobro and others 2002). Similar inhibitory effects on microbial development were obtained
when slurry ice was used for the on-board storage of hake (Merluccius merluccius), angler (Lophius piscatorius), and ray (Raja clavata)
MS 20141562 Submitted 9/18/2014, Accepted 1/1/2015. Authors are with Zhe(Barros-Vel´azquez and others 2008). In addition, combinations of
jiang Provincial Key Laboratory of Health Risk Factors for Seafood, College of Food and
Pharmacy, Zhejiang Ocean Univ., Zhoushan, Zhejiang province, 316000, China. processing technologies have also been studied. These include an
ozone-slurry ice combined refrigeration system used for the storDirect inquiries to author Zhang (E-mail: ).
age of farmed turbot (Psetta maxima) (Campos and others 2006)

Skipjack tuna (Katsuwonus pelamis) as one of the most important pelagic fishery resources has a global distribution in tropical
and subtropical oceans, which is mainly used for canning and
sashimi (fresh muscle) productions. In recent years, the consumption of tuna has been increasing steadily, and the Skipjack tuna
currently holds the most important proportion within tuna markets (Williams and Reid 2011). However, this species is also highly
perishable after the catch, due to the microbial contamination,
large amounts of nonprotein nitrogen compounds, and autolytic
enzymes in tissues, which deteriorate rapidly postmortem resulting
in formation of an obvious off-taste and soften texture. Significantly, the functional properties of the proteins (such as myofibrillar, sarcoplasmic, and connective tissue proteins) play important
roles in affecting the quality and freshness deterioration of muscle
during the process and storage, since they influence the quality
of the products directly, such as tenderness, water holding capacity, juiciness, and flavor (Xia and others 2010). Some studies have
indicated that the muscle proteins of marine products can be significantly degraded by endogenous proteases, such as cathepsins

and serine proteinases (Cao and others 2000; Godiksen and others
2009). They also indicate that they can be denatured by freezing

C 2015 Institute of Food Technologists R
doi: 10.1111/1750-3841.12812

Further reproduction without permission is prohibited

Vol. 80, Nr. 4, 2015 r Journal of Food Science C695

C: Food Chemistry

Effect of Slurry Ice on the Functional Properties
of Proteins Related to Quality Loss during Skipjack
Tuna (Katsuwonus pelamis) Chilled Storage


Effect of slurry ice on Skipjack tuna . . .

C: Food Chemistry

and sardines (Losada and others 2004a) and slurry ice combined
with melanosis inhibitors to prevent browning reactions in shellfish
species such as pink shrimp (Parapenaeus longirostris) (Huidobro and
others 2002) and Norway lobster (Nephrops norvegicus) (Aubourg
and others 2007).
Although these advantages and applications of slurry ice are well
known, few reports have focused on the effects of slurry ice on
the quality of Katsuwonus pelamis or the functional properties of
proteins (such as myofibrillar proteins) as they relate to loss of

quality of edible muscle. Therefore, with the aim of having a deep
understanding of the potential applications of slurry ice, this study
was used to compare the effects of slurry ice and flake ice treatment on the texture, chemical, and microbiological parameters of
Skipjack tuna (Katsuwonus pelamis), with special attention paid to
functional changes in proteins during the chilling storage.

18.0 and 25.0 cc/m2 /day atm. for the transmission for O2 and
CO2 , the air was removed from the bags) individually. Next, the
packaged samples were divided into 3 batches randomly. One of
the batches (30 bags, blank) was stored in an isothermal cold room
(4 °C) directly. The remaining counterpart batches were immersed
in flake ice (30 bags) and slurry ice (30 bags), respectively, before
being stored in the same isothermal cold room. The packaged
muscles were surrounded by either flake ice or slurry ice at a muscle:ice ratio of 1:5, and stored for up to 18 d. The used flake ice
and slurry ice mixtures were renewed every 12 h of storage. At 0,
2, 4, 6, 8, 10, 12, 14, 16, and 18 d of storage, the samples were
subjected to texture, microbial, and chemical analysis.

Texture profile analysis (TPA)
TPA was performed by a texture analyzer (TMS-PRO, FTC,
Virginia, USA). The dorsal muscle was placed on the platform and
Material and Methods
a P/50 cylindrical Perspex probe (50 mm diameter) simulated the
chewing process. TPA was performed under the following conPreparation and properties of flake and slurry ice
ditions: constant test speed, 1.0 mm/s; sample deformation, 30%;
A prototype system (RF-1000-SP, Nantong Refriend Ice Sysand holding time between cycles, 3 s. Texture analysis parameters
tems Co., Ltd., Nantong, China) was used to prepare the slurry
were calculated using FTC-PRO software from the force-time
ice in this work. The composition of slurry ice binary mixture was
curves generated from each sample as described by Bourne (1978).

80% ice and 20% water, all prepared from filtered seawater with
3.5% salinity. The average temperature of the immerged species in
Microbiological analyses
slurry ice was in the range of –1.0 to –1.8 °C.
During the storage intervals, the total aerobes were investigated
Flake ice was prepared using nonsalted water with an Industrial
on
a pour plate, using the plate count agar method (Harrigan and
Flake Ice Maker (LT-10000W, Nantong Refriend Ice Systems Co.,
McCance
1976). First, the samples (10.0 ± 0.2 g) were collected
Ltd., Nantong, China). The average temperature of the submerged
aseptically and then separately blended (T18 ULTRA–TURRAX,
species in flake ice was in the range of +0.4 to +1.0 °C.
sterile saline soTube ice and block ice were prepared with Ice Tube Machine IKA, Staufen, Germany) with 90 mL of 0.85%
−1
−2
lution
for
2.0
min.
Next,
serial
dilutions
(10
,
10
, and 10−3 )
(LZ-1000W) and Direct Cooling Freezing Block Ice Machine
(LW-2000A), respectively, which were both from Nantong Re- were prepared with 0.85% sterile saline solution. Subsequently,

0.1 mL of each dilution was spread on plate count agar (Difco,
friend Ice Systems Co., Ltd. (Nantong, China).
The electron micrograph of flake and slurry ice particles was Detroit, MI, USA) for total aerobes measurement. After incubated
obtained by using a light microscopy (OP 26, OLYMPUS (China) at 37 °C for 48 h, the total aerobes counts were calculated as log
Co., Ltd., Beijing, China). The analyses were made on the images CFU/g muscle.
using Image Pro-Plus software (Media Cybernetics, MD, USA).
The core temperature of the species (kept at 35 °C for 30 min Determination of extractable myofibrillar protein content
Preparation and determination of extractable myofibrillar probefore the measurements) immersed in flake and slurry ice was
determined with a multichannel temperature monitor (JK-24U, tein was carried out as described previously (Xia and others 2009)
Changzhou JAL Electronic Technology Co., Ltd., Changzhou, with some modifications. Each 5.0 g dorsal muscle was minced
China), and the cooling curves of the species were also recorded. and homogenized (10000 rpm) with 10 volumes of ice-cold buffer
(pH 7.0, 20 mM Tris-maleate, containing 0.05 M KCl) using a
blender (T18 ULTRA–TURRAX, IKA, Staufen, Germany) for
Fish material, processing, and sampling
Skipjack tuna (Katsuwonus pelamis) of approximately 550 to 60 s at approximately 0 to 4 °C. The resulting homogenate was
600 mm body length and approximately 5.2 to 6.5 kg body weight centrifuged (CR7, Hitachi, Japan) at 10000 g for 15 min (approxwere obtained at Zhejiang Industrial Group Co., Ltd. (Zhoushan, imately 0 to 4 °C), and the supernatant was discarded. Then the
China), which were caught in the East China Sea in January 2014. sediment was collected, resuspended in the same buffer, and exThe experimental frozen samples (approximately 30 fish, K-value tracted once more. After 2 repeated cycles of homogenization and
determined as approximately 6.5% to 6.8% by the reverse phase centrifugation, the sediment was added to 10 volumes of ice-cold
high performance liquid chromatography procedure reported by buffer (pH 7.0, 20 mM Tris-maleate, containing 0.6 M KCl). The
Oca˜no-Higuera and others (2011)) were immersed in slurry ice mixture was homogenized and centrifuged at 6000 g for 15 min at
4 °C. The resulting supernatant was regarded as total myofibrillar
and transported to the laboratory within 10 min.
Preparation of tuna dorsal muscle was carried out in an approx- protein solution, the concentration of which was determined after
imately 0 to 4 °C cold room. First, the fish packed in vacuum proper dilution by the method of Lowry and others (1951), with
plastic bags were thawed (for 12 h in a refrigerator at 4 °C), be- bovine serum albumin as a standard.
headed, cut into fillets from the backbone, and the collarbone and
pin bones were removed using a sterile scalp and forceps. After Determination of Ca2+ -ATPase activity
being thoroughly washed with distilled water, the obtained dorsal
According to the method of Ooizumi and Xiong (2004) with
muscles (90 pieces, length approximately 12 cm, width approxi- minor modifications, Ca2+ -ATPase activity of myofibrillar protein

mately 12 cm, and height approximately 12 cm) were packaged was assayed in medium of pH 7.0, 0.50 M Tris-maleate buffer,
into food-preservation bags (20.0×25.0 cm, 150 µm thickness, containing 0.10 M CaCl2 , 20 mM adenosine 5’-triphosphate
C696 Journal of Food Science r Vol. 80, Nr. 4, 2015


(ATP), and approximately 1.0 to 2.0 mg/mL proteins. The reaction mixture was incubated for 5 min at 30 °C in a water bath
(SHA-B, Guohua Electronic Appiance Co., Ltd., Changzhou,
China) and terminated by adding 1.0 mL of chilled 15% (w/v)
trichloracetic acid (TCA) solution. The reaction mixture was subsequently centrifuged at 4000 g for 5 min (LD5-2A, Beijing Lab
Centrifuge Co., Ltd., Beijing, China). The quantity of inorganic
phosphate liberated in the supernatant was assayed according to
the method of Fiske and Subbarow (1925). Ca2+ -ATPase activity was expressed as µmoles inorganic phosphate (Pi) released/mg
protein/min (µmolPi/mg·min). A blank solution was prepared by
adding chilled TCA prior to addition of ATP.

Determination of total SH content
Total SH content was measured using the method of Ellman (1959) as modified by Sompongse and others (1996). The
myofibrillar protein solution (1.0 mL, approximately 1.0 to 2.0
mg/mL) was added to 9.0 mL of pH 6.8, 0.2 M Tris-HCl buffer,

containing 8 M urea, 2% (w/v) sodium dodecyl sulfate, and 10
mM EDTA. Thereafter, 0.4 mL of 0.1% (w/v) DTNB solution
was added into 4.0 mL of the mixture and further subjected to
incubation for 25 min at 40 °C. Then, the absorbance of the
mixture was measured at 412 nm using a spectrophotometer (UV
2102 PC, UNICO, Shanghai, China). A blank was prepared by replacing the sample with pH 7.0, 0.6 M KCl. The total SH content
was calculated from the absorbance using the molar extinction of
13600 M−1 cm−1 and was expressed as 10−5 mol/g protein.

Standard error of mean and sodium dodecyl

sulfate–polyacrylamide gel electrophoresis
The cross-section of tuna muscle was chemically prefixed with
2.5% (v/v) glutaraldehyde in 0.1 M, pH 7.2 PBS (Phosphate
Buffered Saline) at 4 °C overnight. The samples were then postfixed with 1.0% (w/v) osmium tetroxide at room temperature for
1 h. After washing 3 times in 50 mM, pH 7.2 PBS, the fixed muscle
was dehydrated for 15 min in a graded series of ethanol (30%, 50%,
Figure 1–Schematic diagram of cooling curves
of tuna muscle (size as 12×12×12 cm) under
different ice treatments. Inset photographs of
slurry ice (A) and flake ice (B) particles
magnified 100 times by the microscope.

Figure 2–Changes in springiness (A) and chewiness (B) values of tuna muscle during storage in blank, flake ice, and slurry ice. Mean values of 3
determinations; vertical bars denote standard deviation.
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Effect of slurry ice on Skipjack tuna . . .


Effect of slurry ice on Skipjack tuna . . .

C: Food Chemistry

TPA analysis
Springiness indicates the elasticity of muscle that can be
stretched and returned to its original length. Chewiness is the
tactile sensation of labored mastication due to sustained elastic
resistance from the shrimp’s muscle, defined as the product of

hardness×cohesiveness×springiness, which showed trends similar
to that of tuna muscle springiness. From the results (Figure 2), the
obvious variations were that the springiness and chewiness values
of tuna muscle all decreased dramatically over time for the blank,
flake, and slurry iced samples (P < 0.05). However, the slurryiced samples showed significant differences in the variables (P <
0.05), losing 33.98% and 44.23% after 18 d of storage. Such losses
were significantly lower when compared with the blank (62.40%
and 83.93%) and the flake-iced (46.80% and 65.86%) samples. Accordingly, a significant inhibitory effect of slurry ice on changes
in the texture (springiness and chewiness) of tuna muscle could be
concluded for the slurry ice group but not for the blank or flake
ice group.
Statistical analysis
The above-mentioned undesirable springiness and chewiness
All above analyses were performed in triplicate. Statistical analchanges in blank and flake iced samples might be caused by
yses were performed with the SPSS package (SPSS 13.0 for winthe high activity of autolytic enzymes (for example, collagenase,
dows, SPSS Inc., Chicago, IL, USA). A Duncan’s test was used to
determine the significance at P < 0.05. Data represent the means
± SD of measurement for 3 replicates.
60%, 70%, 80%, 90%, 95%, and 100%). Next, the dehydrated muscle was dried with a critical-point drier using liquid CO2 (SFD,
Thar Instruments, Inc., PA, USA), treated with gold-coater (Eiko
IB-3, JEOL Ltd., Tokyo, Japan) for 5 min, and observed with a
scanning electron microscope (JSM-6390LV, JEOL Ltd., Tokyo,
Japan).
SDS-PAGE analysis was carried out essentially as described by
Laemmli (1970) on 12% resolving gel with 5% stacking gel. The
loading volume was 15 µL in each lane. Gels were stained with
0.1% (w/v) Coomassie Brilliant Blue R-250 in a solution containing 80% methanol and 20% acetic acid and destained in a
solution containing 50% methanol and 10% acetic acid. The relative molecular weight (MW) standards of the polypeptide bands
were determined by comparing it with a standard protein (approximately 10 to 200 KDa) solution (Shanghai Bogoo Biotechnology
Co., Ltd., Shanghai, China).

Results and Discussion
Characteristics of flake and slurry ice
A microscope was used to study the shape and surface of flake
and slurry ice particles. The 2 images inserted in Figure 1 showed
2 different types of ice particles. The slurry ice (Figure 1, inset
A) referred to a homogeneous mixture of globular smooth particles (0.15 to 0.75 mm in diameter, assayed by Image Pro Plus,
Media Cybernetics, MD, USA). Removing the surface roughness
of the individual slurry ice particle (unlike the sharp, jagged ice)
allowed the highly loaded ice particles to slip past one another
without tangling or agglomerating, which also provided greater
surface contact and faster cooling than other conventional forms
of ice, such as flake, block, and tube ice. The counterpart flake
ice (Figure 1, inset B) presented dendritic particles, which formed
large entangled clusters causing rough surfaces (images of block
and tube ice, the same as flake ice, were not shown). Additionally,
the flake ice clusters were easily broken, and formed sharp edges
and corners, which could easily result in the immersed species
damage, such as bruises, cuts, and scrapes, and simultaneously the
microbiological contamination from the damage.
The cooling curves of tuna muscle (size as 12×12×12 cm)
immersed in flake, tube, block, and slurry ice are also showed
in Figure 1. The slurry iced muscle cooled rapidly to less than
0 °C within 10 min and to –1.5 °C within 20 min, whereas
the flake iced treatment took 20 min to reach +1 °C, the tube
iced muscle to +5 °C within 25 min, and the block iced muscle
to +9 °C within 30 min. These 3 conventional ices conducted
the heat exchange with the immersed species through the air,
while the round shape of slurry ice particles enabled them to flow
freely around the entire muscles, filling all air pockets to uniformly

maintain direct contact and the desired low temperature, due to
the very large heat of fusion of ice particles (Davies 2005). In
addition, the rapid cooling of the muscle by slurry ice could also
significantly suppress the enzymatic degradation and respiratory
activity, as well as slow (or inhibit) the growth of decay-producing
microorganisms and their metabolites (Kauffeld and others 2010).
C698 Journal of Food Science r Vol. 80, Nr. 4, 2015

Figure 3–Changes in total aerobic counts of tuna muscle during storage in
blank, flake ice, and slurry ice. Mean values of 3 determinations; vertical
bars denote standard deviation.

Figure 4–Changes in extractable myofibrillar protein content in tuna muscle during storage in blank, flake ice, and slurry ice. Mean values of 3
determinations; vertical bars denote standard deviation.


ATPase, cathepsins, and so on) (Godiksen and others 2009) hydrolyzing the myofibrillar proteins and breaking down the connective tissues, which allowed further, rapid multiplication of spoilage
microorganisms and promoted the progress of spoilage (Hattula
and others 2001). Nevertheless, in comparison with blank and
flake iced samples, the tuna muscle stored in slurry ice showed a
significant maintenance of texture stability even during advanced
periods of storage. This was significantly better than in blank and
flake-iced samples (P < 0.05). This was probably due to the double
effects caused by slurry ice immersion, namely, more inhibition
(or inactivation) on spoilage microorganisms and the activity of
autolytic enzymes, resulting in the comparatively stabilization of
myofibrillar protein fraction during the whole storage. In addition, slurry-iced treatment had been reported to have a significant
inhibitory effect on the lipid oxidation in fish muscle (Rodr´ıguez
and others 2006), which might also have a relationship with the
maintenance of the muscle texture properties.

Microbiological analysis
The evolution of aerobic growth in tuna muscle along the storage in blank, flake and slurry ice batches was evaluated in the
study (Figure 3). The initial microbiological quality of tuna muscle used in this study was good, as indicated by the low initial
bacterial counts (1.77 log CFU/g). However, the blank samples
showed a notable increase in the microbial population, the total
aerobic counts reaching figures of approximately 4.10 log CFU/g
after 18 d of storage. By contrast, the microbial growth was significantly slower in the slurry-iced batch, the total aerobic counts
after 18 d being only 2.61 log CFU/g, and meanwhile significantly
(P < 0.05) lower than the flake iced batch as 3.02 log CFU/g.
The fresh muscle was highly vulnerable to postmortem texture
deterioration due to the bacterial growth, as a result of the production of off-odors and off-flavors (Kamalakanth and others 2011).
The texture deterioration also allowed further, rapid multiplication of spoilage microorganisms and promoted spoilage. The above

Figure 5–Changes in Ca2+-ATPase activity (A) and total SH content (B) in tuna muscle during storage in blank, flake ice, and slurry ice. Mean values
of 3 determinations; vertical bars denote standard deviation.
Figure 6–SEM images of cross-section of tuna
muscles storage in blank, flake ice, and slurry
ice. (A) Fresh muscle after 0-d storage at 4 °C,
(B) blank muscle after 14-d storage at 4 °C, (C)
flake ice-muscle after 14-d storage at 4 °C, (D)
slurry ice-muscle after 14-d storage at 4 °C.

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C: Food Chemistry

findings clearly indicated a significantly slower growth of aerobic
groups investigated in tuna muscle subjected to storage in slurry
ice as compared with flake ice treatment. The total aerobic counts
did not reach counts higher than 5.0 logCFU/g in all groups,
suggested that the microbial spoilage might not represent the limiting factor to the acceptability of tuna muscle (Aubourg and others
2007). These results were quite in agreement with previous results
obtained from ray (Raja clavata) (M´ugica and others 2008), horse
mackerel (Rodr´ıguez and others 2005), and turbot (Psetta maxima)
(Rodr´ıguez and others 2006), where different marine species were
comparatively stored under slurry and flake ice.

Extractable myofibrillar protein content
The content of extractable myofibrillar proteins in tuna muscle decreased significantly (P < 0.05) as chilling storage went on,
although 2 different change stages were observed in all samples
(Figure 4). The first stage was from approximately 0 to 2 d for the
3 batches with relatively smooth and steady changes. The 2nd stage
was from approximately 2 to 18 d, in which significant decrease occurred over the time. In the experiment, the initial extractable myofibrillar content of fresh muscle was 46.59 mg/g. Subsequently,
the extractable myofibrillar content of samples stored in blank and
flake ice decreased to 10.61 mg/g and 25.23 mg/g on the 18th
d, respectively. However, the slurry ice treated samples were in
good condition and the extractable myofibrillar content remained
at 32.86 mg/g after 18 d of storage.
According to the reports, with the decreased extractability of
myofibrillar proteins in fish muscle, deterioration in texture, and
decrease in water holding capacity, off-odors and off-flavors have
also been shown to occur (Lu and others 2012). Moreover, the

degradation of muscle myofibrillar and intramuscular connective
tissue was also probably caused by proteases such as cathepsins, as

well as calcium dependent proteases (Okitani and others 1980). In
several fish species, cathepsins B, D, and L were considered as the
enzymes playing the most important role in postmortem muscle
softening (Ladrat and others 2003). The release of these proteolytic enzymes from the lysosomes might cause an acceleration of
myofibrillar proteins decrease and muscle degradation (Bahuaud
and others 2008). In the current study, the slurry ice treatment
reduced the rate of decrease of extractable myofibrillar proteins in
muscle during storage to a significantly greater extent than blank
or flake ice samples. These variables also showed the same trend
as the texture assessments. These results could be explained on
the basis of a faster cooling that leads to a lower temperature during the slurry ice treatment. As a result, a large number of small
ice crystals formed only in muscle nuclei, preventing the irreversible destruction of the myofibrils by large ice crystals. More
importantly, the slurry ice particles uniformly contacted with the
immerged muscle and provided very large heat of ice fusion and
high heat exchange rates for the samples (4 times higher than those
of flake ice; Pi˜neiro and others 2004), which might play an important role in suppressing the release of the proteolytic enzymes
and further their enzymatic degradation.

Ca2+ -ATPase activity and total SH content analysis
Ca2+ -ATPase activity and total SH content of tuna muscle
treated with blank, flake, and slurry ice were all dramatically
decreased during the current 18-d storage (Figure 5). The initial Ca2+ -ATPase activity and SH content of fresh muscle (0 d)
were 1.17 µmolPi/mg·min and 5.59×10−5 mol/g, respectively;
both parameters significantly decreased to 0.27 µmolPi/mg·min
and 0.59×10−5 mol/g, respectively, for the blank samples (P <
0.05) after 18 d of storage. As a contrast, the values of flake
ice treated samples reduced their scores to 0.64 µmolPi/mg·min

Figure 7–SDS-PAGE analysis of muscle total proteins storage in blank, flake ice, and slurry ice. M: molecular weight marker, A: fresh muscle after 0-d
storage at 4 °C, B: blank muscle after 14-d storage at 4 °C, C: flake ice-muscle after 14-d storage at 4 °C, D: slurry ice-muscle after 14-d storage at 4 °C.

C700 Journal of Food Science r Vol. 80, Nr. 4, 2015


and 2.39×10−5 mol/g, while slurry-iced samples remained at
0.78 µmolPi/mg·min and 3.47×10−5 mol/g, respectively. Thus,
significantly higher (P < 0.05) values of Ca2+ -ATPase activity
and total SH content were observed in slurry-iced samples than in
blank and flake-iced samples.
Myofibrillar Ca2+ -ATPase activity has been widely used as
a measure of actomysin integrity and to monitor postmortem
changes in fish during iced or frozen storage. Any small microstructural change produced in the integrity of myofibrillar protein can lead to decreases in the activity of Ca2+ -ATPase (Reza
and others 2009). In the present results, the Ca2+ -ATPase activity of slurry-iced samples was significantly higher than that
of blank and flake-iced samples (P < 0.05). The rapidly loss of
Ca2+ -ATPase activity of blank and flake iced samples was likely
to be associated with the conformational changes of myosin globular head as well as the aggregation of this portion. In addition,
the rearrangement of protein via protein–protein interactions was
also presumed to contribute to the loss in Ca2+ -ATPase activity
(Reza and others 2009). However, the slurry-iced samples maintained a comparatively higher activity of Ca2+ -ATPase, the reasons of which perhaps were due to the stabilization effect on the
myofibrillar protein fraction, indicating larger yields during the
storage and processing.
The decrease in total SH content in muscle was reported to be
due to the formation of disulphide bonds through oxidation of
SH groups or disulphide interchanges (Riebroy and others 2007).
The stabilization of protein microstructure was also controlled to
a certain extent by the covalent disulfide bonds and some noncovalent interactions of the side-chain groups (Hossain and others
2004). In the study, the elevated temperature (at 4 °C of blank and

at approximately 0.4 to 1.0 °C of flake-iced samples) most likely
resulted in the enhanced oxidation of sulphydryl groups with the
accompanied formation of disulphide bonds. In this way, the blank
and flake iced samples showed a rapid decrease of total SH content
during the storage. Contrary, the slurry ice treatment allowed the
muscle to reach subzero temperatures rapidly, and simultaneously
slowed down the oxidation and enzymatic breakdown reactions.
SH content, myofibrillar protein content, and Ca2+ -ATPase activity all have produced better scores in samples from the slurry
ice batch.

SEM and SDS-PAGE analysis
The microstructures of tuna muscles stored in blank, flake, and
slurry ice were showed in Figure 6. From the results, considerable
differences were observed both in myofibrils and as in intramuscular connective tissues. The blank (Figure 6B) muscle myofibrils
were less distinct and tight, and the intramuscular connective tissues were ruptured obviously and showed many cracks after 14-d
storage. Meantime, changes in myofibrils and tissues in flake-iced
muscle (Figure 6C) after 14-d storage were considerably smaller
than in the blank batch, but many ruptures and inhomogeneous
structures also occurred. These changes observed in blank and
flake-iced samples might be induced by the activity of autolytic
enzymes above-mentioned and the weakening of the integrity of
muscle myofibrils. As a contrast, the myofibrils structure of slurryiced samples (Figure 6D) was still very regular, and the neighboring
myofibrils and connective tissues were also adhered to each other
tightly, which were similar to that of fresh muscle (Figure 6A).
Therefore, it could be concluded that slurry ice treatment for tuna
muscle had a positive effect on the cold-storage stability of tissue
structures, which was consistent with the above presented analyses

concerning texture, myofibrillar changes, Ca2+ -ATPase activity
and total SH results.

SDS-PAGE patterns of total proteins from tuna muscle were
shown in Figure 7. The electrophoretic profiles of muscle revealed
a broad band at approximately 200 kDa, which was assigned
to the myosin heavy chain (MHC). Paramyosin (approximately
90 kDa) was also present, as well as actin (approximately 40 kDa).
Additional bands corresponded to tropomyosin (approximately
38 kDa) and troponin T (approximately 34 kDa) (Li and others
2014). There was a considerable decrease in the intensity of
MHC, paramyosin, actin, tropomyosin, and troponin T in the
blank sample (Figure 7B) after 14 d of storage, compared with
the fresh initial, flake- and slurry-iced samples (Figure 7A, C,
and D). This significant decrease in MHC content in blank muscle
was presumed to be originated as a result of proteolysis during
the 14-d storage (Losada and others 2004b). Both indigenous
and microbial proteases might contribute to the degradation
of muscle proteins, such as cathepsins, calpains, and serine
proteinases. Although it is more resistant to degradation, less
actin was observed in the blank sample compared with the fresh
initial and treated samples. No obvious differences between the
bands of tropomyosin and troponin T in flake- (Figure 7C) and
slurry-iced (Figure 7D) samples were observed in SDS-PAGE.
However, the slurry ice treatment maintained the integrity of
the muscle proteins, significantly better than that of the blank
samples. Based on the current results and previous reports, it could
be concluded that slurry ice treatment could effectively retard
the degradation of muscle proteins during the chilling storage.
These findings also support the observations regarding texture,
extractable myofibrillar proteins, and Ca2+ -ATPase activity. In
addition, previous reports have proposed that certain polypeptides
could be employed as protein spoilage biomarkers on the basis

of the proteolytic processes happened in the myofibrillar (Morzel
and others 2000; Losada and others 2004b). Nevertheless, these
observations did not agree with the results obtained in this work,
the reasons of which were perhaps caused by different tested fish
species, muscle freshness, and storage conditions.

Conclusion
Slurry ice, a biphasic mixture of small spherical ice-particles
immersed in saline water at subzero temperature, was evaluated
for changes in the quality of skipjack tuna (Katsuwonus pelamis)
during the chilling storage. When compared with its counterpart
batch stored in flake ice, slurry ice treatment exerted a significantly inhibitory effect on relevant deterioration mechanisms,
such as growth of microorganisms, degradation of myofibrillar
proteins, decreases of Ca2+ -ATPase activity and total SH content,
and maintenance of the integrity of physical structure of tuna muscle. The findings showed that the reduced protein functionalities
could be related to changes in the chemical and physical properties of muscle proteins. Taken together, the effects of slurry ice
on shelf-life of tuna muscle are of considerable interest and might
have wide application to extend the commercialization of fresh
fish with better guarantees of quality and safety.

Acknowledgments
This study was funded by Program of Intl. S&T Cooperation,
China (Grant Nr. 2012DFA30600), Natl. Natural Science Fund
project, China (Grant Nr. 31201452), and Public Technology
Applied Research Projects, Zhejiang Province, China (Grant Nr.
2012C33081).
Vol. 80, Nr. 4, 2015 r Journal of Food Science C701

C: Food Chemistry

Effect of slurry ice on Skipjack tuna . . .


Effect of slurry ice on Skipjack tuna . . .

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