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Quality changes of farmed blackspot seabream
(Pagellus bogaraveo) subjected to slaughtering and
storage under flow ice and ozonised flow ice
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Victoria Álvareza, Xesús Feásb, Jorge Barros-Velázquezb, and
Santiago P. Aubourga,*
a
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Department of Food Technology; Institute for Marine Research (CSIC), C/ Eduardo
Cabello 6, 36208-Vigo (Galicia, Spain)
b
Department of Analytical Chemistry, Nutrition and Food Science, School of
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Veterinary Sciences, University of Santiago de Compostela, 27002-Lugo
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(Galicia, Spain)
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*
Correspondent: Fax: +34 986 292762; e-mail: saubourg@iim.csic.es
1
SUMMARY
2
3
Flow ice combined with ozone (OFI condition) was evaluated for slaughter and storage
4
of farmed blackspot seabream (Pagellus bogaraveo) as compared to flow ice alone (FI
5
condition). When processed in either OFI or FI conditions, this species exhibited slow
6
biochemical and microbiological spoilage mechanisms as compared to other
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commercial fish species. The presence of ozone slowed down microbial development
8
(1.00-3.53 and 1.60-4.04 log CFU g-1 for fish mesophile counts from OFI and FI
9
conditions, respectively) and trimethylamine formation, so that fish kept under OFI
10
condition was still acceptable at the end of the experiment (day 16), while its
11
counterpart fish treated with FI was rejectable. In contrast, a small pro-oxidant effect
12
could be assessed by means of the ozone presence; however, oxidation values (peroxide
13
value and thiobarbituric acid index) reached at day 16 by individuals treated under OFI
14
conditions (8.34 and 0.19, respectively) can not be considered specially high.
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Keywords: Blackspot seabream, farming, slaughtering, chilling storage, ozone, quality
19
Running Title: Quality of iced farmed blackspot seabream
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1
INTRODUCTION
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3
Marine products provide important constituents for the human diet such as nutritional
4
and digestible proteins, lipid-soluble vitamins (A and D, namely), microelements (I, F,
5
Ca, Cu, Zn, Fe, Se and others) and -3 polyunsaturated fatty acids (Piclet, 1987;
6
Simopoulos, 1997), among others. However, marine species are highly perishable food
7
products whose freshness and quality rapidly decline post-mortem (Pigott & Tucker,
8
1990; Whittle et al., 1990). In order to slow down the mechanisms involved in quality
9
loss, the fish specimens should be refrigerated immediately after capture. Therefore, fish
10
has traditionally been cooled and stored in either flake ice (Nunes et al., 1992),
11
refrigerated sea water (Kraus, 1992), or preserved by exposure to chemical agents
12
(Hwang & Regenstein, 1995).
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Recent studies have reported the advantages of advanced chilling strategies. One of
14
such technologies is flow ice (FI), a biphasic preservation system consisting on an ice-
15
water suspension at subzero temperature. Two relevant characteristics of FI are its faster
16
chilling rate, which is a consequence of its higher heat-exchange capacity, and the
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reduced physical damage caused to aquatic food products by its microscopic spherical
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particles as compared with the damage elicited by traditional flake ice. Complete
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coverage of the fish surface by FI mixture also affords a better protection of the fish
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material respect to degradative events. The versatility of the FI technique should also be
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highlighted; FI can be pumped, thereby improving hygienic handling, and may be
22
combined with other agents, such as microbial, oxidation and melanosis inhibitors. The
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application of FI-based technologies has led to important inhibitions of autolytic
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mechanisms, microbial activity and lipid oxidation mechanisms in different commercial
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fish species (Losada et al., 2004a; Rodríguez et al., 2004; Losada et al., 2005).
3
1
Ozone is a powerful antimicrobial agent that is suitable for application in food in the
2
gaseous and aqueous states leading to significant increases in sensory quality and shelf-
3
life of fish (Kim et al., 1999). Molecular ozone and its decomposition products
4
inactivate microorganisms rapidly by reacting with intracellular enzymes, nucleic
5
material and other components. In spite of its advantages as a food additive, the pro-
6
oxidant behaviour of ozone on fish food constituents may denote a considerable
7
drawback. Thus, some previous research has shown a detrimental effect on
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phospholipid classes, polyunsaturated fatty acids and membrane proteins (Fukunaga et
9
al., 1991; Takigi-Endo et al., 2002).
10
In recent years, the fishing sector has suffered from dwindling stocks of traditional
11
species as a result of marked changes in their availability. This has prompted fish
12
technologists and the fish trade to pay more attention to aquaculture techniques as a
13
source of fish and other aquatic food products (Stickney, 1990). One of such fish
14
species is blackspot seabream (Pagellus bogaraveo) (Shaw & Curry, 1992). This high
15
value commercial species has long attracted a great interest because of its firm and
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flavourful
17
commercialisation as a farmed product (Silva et al., 2006; Palmegiano et al., 2007).
18
However, previous studies have considered farming conditions, but not the investigation
19
of quality loss mechanisms during its commercialisation as a refrigerated product.
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Accordingly, the present work focuses on the investigation of quality loss in farmed
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blackspot seabream during its chilled storage. For it, FI was applied as slaughtering
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medium and as chilling storage system. With a view to achieve a potential extension of
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its shelf life, a combined refrigeration system consisting of flow ice and ozone (OFI)
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was also evaluated in parallel. Sensory, microbiological and chemical analyses were
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carried out in both batches to assess the quality changes during 16 days of icing
flesh.
Recently,
remarkable
efforts
have
been
focused
on
its
4
1
treatment. Special attention was paid to lipid oxidation events, with a view to evaluate
2
the potential pro-oxidant effect of ozone on this important fish component.
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4
5
MATERIALS AND METHODS
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Refrigeration systems
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FI was prepared using a FLO-ICE prototype (Kinarca S.A.U., Vigo, Spain). The
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composition of the FI binary mixture was 40% ice and 60% water, prepared from
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filtered seawater (salinity: 33.0 g kg-1). The temperature of the FI mixture was -1.5ºC.
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When required, the injection of ozone in the FI mixture was accomplished with a
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prototype provided by Cosemar Ozono (Madrid, Spain), the redox potential being
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adjusted to 700 mV (0.20 mg ozone l-1). In this batch, the ozone concentration was
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constantly monitored by checking the redox potential in the liquid phase.
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Fish material, slaughtering and chilling storage
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Seventy-two specimens of blackspot seabream (Pagellus bogaraveo) (weight range:
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0.43-0.63 kg; length range: 30-34 cm) were obtained from an aquaculture facility (Isidro
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de la Cal, La Coruña, Spain) and were sacrificed at the farm by immersion in either FI
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(36 specimens) or OFI (36 specimens). In both systems, fish specimens were
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surrounded by FI or OFI at a 1:1 fish-to-ice ratio and transported during 2 h at 0ºC to
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the laboratory. Then, the fish specimens were maintained in their corresponding icing
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medium and directly placed in an isothermal room at 0ºC.
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On the next day (day 1), six specimens from each icing batch were taken for analysis.
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Analyses included the investigation of the sensory, microbiological and chemical
5
1
parameters described below. Specimens from each icing condition were divided into
2
three groups (two individuals in each group) that were studied separately (n = 3). Once
3
fish specimens had been subjected to sensory analyses, the white muscle was separated
4
under sterile conditions and employed for microbiological and biochemical analyses.
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Fish sampling was then continued at days 3, 6, 9, 13 and 16 of refrigerated storage,
6
according to the same sampling design (n = 3).
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All solvents and chemical reagents used in the experiments were reagent grade (Merck,
8
Darmstadt, Germany).
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Sensory analysis
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Sensory analysis was conducted by a sensory panel consisting of five experienced
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judges, according to guidelines concerning fresh and refrigerated fish (Table 1) (DOCE,
13
1989). Four categories were ranked: highest quality (E), good quality (A), fair quality
14
(B) and unacceptable quality (C). The panellists involved in this study had been
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involved in sensory analysis of different fish species during ten years. Previously to the
16
present experiment, the panellists were specially trained with fresh blackspot seabream.
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Sensory assessment of the fish included the examination of the following parameters:
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skin, eyes, external odour, gills, consistency and flesh odour. At each sampling time, the
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fish specimens were presented to panellists and were scored individually. The panel
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members shared samples tested.
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Quantitative microbiological analyses
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Samples of 5 g of fish muscle were dissected from skinned chilled fish specimens,
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mixed with 45 ml of peptone water (1 g l-1), and homogenised in a stomacher (Seward
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Medical, London, UK). Serial dilutions from the skin or muscle microbial extracts were
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1
prepared in peptone water (1 g l-1) (Oxoid Ltd., London, UK). Total aerobic and
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psychrotrophic bacteria were investigated in Plate Count Agar (PCA, Oxoid) after
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incubation at 30C for 48 h or at 7-8C for 10 days, respectively, as described elsewhere
4
(Rodríguez et al., 2004). Microorganisms exhibiting a proteolytic phenotype were
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investigated in casein-agar medium, as previously described (Ben-Gigirey et al., 2000).
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Microorganisms exhibiting a lipolytic phenotype were investigated in tributyrine-agar
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medium (Ben-Gigirey et al., 2000). The histaminogenic phenotype was investigated in
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Niven medium (Niven et al., 1981).
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Results obtained for each microbial group were expressed as log CFU g-1 fish muscle.
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Molecular identification of bacteria involved in histamine production in blackspot
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seabream
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Selected microorganisms were picked from Niven medium and subjected to a
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preliminary microbiological study by means of the Gram stain, oxydase and catalase
15
tests. Genetic identification was carried out by PCR targeted to 16S rRNA coupled to
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DNA sequencing. Briefly, total bacterial DNA was isolated from the pellets of 1.5 ml of
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overnight cultures after spinning at 7500 rpm for 10 min, as previously described
18
(Campos et al., 2006). Total DNA was purified from each extract by means of the
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DNeasy tissue minikit (QIAGEN Inc., Valencia, CA, USA), based on the use of
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microcolumns. The concentration of purified DNA extracts was determined by
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measuring the fluorescence developed after mixing with Hoechst 33258 reagent (Sigma,
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St. Louis, MO, USA) on a LS50 fluorimeter (Perkin Elmer, Wellesley, MA, USA). PCR
23
amplification was performed with the highly conserved 16S rDNA primer pair, p8FPL
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and p806R (Relman et al., 1992), which allowed the amplification of a 834 bp PCR
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product. All amplification assays comprised 100 ng of template DNA, 25 l of a master
7
1
mix (BioMix, Bioline Ltd., London, UK) –this including reaction buffer, dNTPs,
2
magnesium chloride and Taq DNA polymerase–, bi-distilled water (Genaxis, Montigny
3
le Bretonneaux, France), and 25 pmol of each oligonucleotide primer to achieve a final
4
volume of 50 l. Amplification conditions were as follows: a previous denaturing step
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at 94ºC for 7 min was coupled to 30 cycles of denaturation (94ºC for 60 s), annealing
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(55ºC for 60 s), and extension (72ºC for 60 s), and to a final extension step at 72ºC for
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15 min. All PCR assays were carried out on a MyCycler Thermal Cycler (BioRad
8
Laboratories, Hercules, CA, USA). PCR products were processed in 2.5% horizontal
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agarose (MS-8, Pronadisa, Madrid, Spain) gels. Prior to sequencing, the PCR products
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were purified by means of the ExoSAP-IT kit (GE Healthcare, Uppsala, Sweden).
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Direct nucleotide sequencing was performed with BigDye Terminator v3.1 Cycle
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Sequencing Kit (Applied Biosystems). The same primers used for PCR were considered
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for the sequencing of both strands of the PCR products, respectively. Sequencing
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reactions were analysed in an automatic sequencing system (ABI 3730XL DNA
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Analyser, Applied Biosystems). SNP events in DNA sequences were carefully reviewed
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by eye, using the Chromas software (Griffith University, Queensland, Australia).
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Alignment of sequences was carried out using the CLUSTALW software. Homologies
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of the sequences were searched with the GenBank BLAST tool (National Centre for
19
Biotechnology Information).
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Composition analyses
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Moisture content was determined by the difference between the weight of fresh
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homogenised muscle (1-2 g) and the weight recorded after 24 h at 105 ºC. Results were
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expressed as g water kg-1 muscle.
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Lipids were extracted by the Bligh and Dyer (1959) method. Quantification results were
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expressed as g lipid kg-1 muscle.
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NaCl content in fish muscle was determined by a modification of the Volhard method,
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which included boiling in HNO3, neutralization of NaCl meq with excess of AgNO3,
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and final determination of the excess of AgNO3 meq by reverse titration with NH4SCN
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(AOAC, 1990). Results were expressed as g NaCl kg-1 muscle.
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Nucleotide degradation analysis
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Nucleotides were obtained by extraction with a 6% (w/v) perchloric acid solution and
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analysed by HPLC according to Aubourg et al. (2005). Standard curves for adenosine
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5’-triphosphate (ATP) and each compound involved in its degradation pathway,
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adenosine 5’-diphosphate (ADP), adenosine 5’-monophosphate (AMP), inosine 5’-
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monophosphate (IMP), inosine (INO) and hypoxanthine (HX), were constructed in the
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0-1 mM range.
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Results obtained for each degradation compound were calculated as mmol kg-1 muscle.
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The K value was determined according to the following concentration ratio: K value
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(%) = 100 x (INO + HX) / (ATP + ADP + AMP + IMP + INO + HX).
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Assessment of volatile amines and trimethylamine oxide
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Total volatile base-nitrogen (TVB-N) content was measured by a steam distillation
21
method. For it, fish muscle (10 g) was extracted with 6% (w/v) perchloric acid and
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brought up to 50 ml, determining the TVB-N content –after steam-distillation of the
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acid extracts rendered alkaline to pH 13 with 2% (w/v) NaOH– by titration of the
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distillate with 10 mM HCl. The results were expressed as mg TVB-N kg-1 muscle.
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1
Trimethylamine-nitrogen (TMA-N) content was determined by means of the picrate
2
method, as previously described (Tozawa et al., 1971). This involves the preparation of
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a 5% (w/v) trichloroacetic acid extract of fish muscle. The results were expressed as mg
4
TMA-N kg-1 muscle.
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Dimethylamine-nitrogen (DMA-N) content was determined by employing the specific
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reaction of secondary amines with carbon sulphur in the presence of a copper salt
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(Dowden, 1938). This involves the preparation of a 5% (w/v) trichloroacetic acid extract
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of fish muscle. The results were expressed as mg DMA-N kg-1 muscle.
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Trimethylamine oxide-nitrogen (TMAO-N) content was determined by previous
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reduction with titanium (III) chloride (Parking & Hultin, 1982) and further assessment
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of TMA-N content according to Tozawa et al. (1971). The results were expressed as mg
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TMAO-N kg-1 muscle.
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Assessment of pH value and formaldehyde
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The evolution of pH values in blackspot seabream muscle was determined by means of
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a 6-mm diameter insertion electrode (Crison, Barcelona, Spain).
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Extraction of formaldehyde (FA) from fish muscle and further spectrophotometric
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assessment was carried out as described elsewhere (Rey-Mansilla et al., 2001). FA
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content was expressed as mg kg-1 muscle.
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Lipid damage assessment
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Free fatty acid (FFA) content was determined by the Lowry and Tinsley (1976) method
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based on complex formation with cupric acetate-pyridine. Results were expressed as g
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FFA kg-1 lipids.
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1
The peroxide value (PV) was determined according to the ferric thiocyanate method
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(Chapman & McKay, 1949). Results were expressed as meq oxygen kg-1 lipids.
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The thiobarbituric acid index (TBA-i) was determined according to Vyncke (1970).
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Results were expressed as mg malondialdehyde kg-1 muscle.
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Formation of fluorescent compounds was determined by measurements at 393/463 nm
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and 327/415 nm, as previously described (Aubourg et al., 2005). The relative
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fluorescence (RF) was calculated as follows: RF = F/Fst, where F is the fluorescence
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measured at each excitation/ emission maximum, and Fst is the fluorescence intensity of
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a quinine sulphate solution (1 µg ml-1 in 0.05 M H2SO4) at the corresponding
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wavelength. The fluorescence ratio (FR) was calculated as the ratio between the two RF
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values: FR = RF393/463 nm/ RF327/415 nm. The FR value was determined in the lipid
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extract.
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Statistical analyses
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Data corresponding to the two icing conditions were subjected to one-way analysis of
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variance to assess significant (p<0.05) differences between FI and OFI batches (Statsoft,
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1994); the effect of the icing time was also analysed (p<0.05). The SPSS 11.5 software
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for Windows (SPSS Inc., Chicago, IL, USA) was used to explore the statistical
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significance of the results obtained, including multivariate contrasts and multiple
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comparisons by the Scheffé and Tukey tests. Factor analysis (principal components)
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was carried out with all parameters measured; a Varimax normalised rotation was
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employed for factor rotation (Arvanitoyannis et al., 2005). A confidence interval at the
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95% level was used in all cases.
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RESULTS AND DISCUSSION
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Sensory analysis
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Progressive decreases in average score were observed in specimens from both batches
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as icing time progressed (Table 2). However, a general good quality was maintained for
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all OFI and FI specimens up to day 9, this representing a result of remarkable
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commercial value, considerably better than those reported for captured fish species
8
subjected to FI chilled storage (Losada et al., 2004a; Rodríguez et al., 2004; Losada et
9
al., 2005).
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Fish specimens slaughtered and stored in FI exhibited a shelf life of 13 days, while their
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counterpart specimens processed in OFI exhibited an extended shelf life of 16 days.
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Throughout the icing time, some differences were observed between specimens
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processed in FI and OFI. Remarkably, better scores for gills (days 3 and 9), external
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odour (day 6) and consistency (day 16) were observed in fish specimens treated under
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OFI system. Thus, a profitable effect of the presence of ozone was concluded, according
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to previous research concerning a captured species (Campos et al., 2005) and a farmed
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one (Rodríguez et al., 2006).
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Microbiological analyses
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One-way ANOVA was initially carried out considering aerobes, lipolytic, proteolytic
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and histaminogenic bacteria as dependent variables, and time as the factor. Table 3
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shows the most relevant results concerning microbial growth in blackspot seabream
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stored in FI or OFI.
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In the case of the counts of total aerobes, statistically significant differences were
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determined on days 1 and after 9 days of storage, this revealing a more limited
12
1
microbial growth in the OFI batch at advanced storage periods. In global terms, the
2
average difference determined for both batches up to day 16 was 0.56 log units.
3
Although the total bacterial counts did not reach levels which are considered to be
4
required for the spoilage of fish stored aerobically (Gram & Huss, 1996), storage of
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blackspot seabream in the OFI elicited a significant reduction. These results are in
6
agreement with the significantly lower counts determined for turbot (Rodríguez et al.,
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2006) and sardine (Campos et al., 2005) stored in OFI as compared with a counterpart
8
FI batch. Accordingly, as in the above fish species the storage of blackspot seabream in
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OFI implies a significantly slower growth of total aerobes with respect to storage in FI.
10
With respect to the psychrotrophic microorganisms in blackspot seabream, the counts in
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the OFI batch proved to be lower with respect to the FI batch, although such differences
12
were only significant on days 1, 3 and 9. Globally, the average difference between
13
batches throughout refrigerated storage was 0.46 log units. As in the case of the total
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aerobes, the growth of psychrotrophic bacteria in blackspot seabream muscle proved to
15
be very low. However, these results are in agreement with previous studies in which the
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combination of flow ice and ozone provided an additional reduction of psychrotrophic
17
bacteria (Campos et al., 2005; Rodríguez et al., 2006).
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The average counts of lipolytic bacteria in blackspot seabream muscle stored in the FI
19
and OFI batches, respectively, were not statistically different at the p<0.05 level.
20
Moreover, with respect to the initial counts at day 1 (2.00-2.38 log CFU g-1 range), the
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average counts obtained for lipolytic bacteria did not increase until day 13. In global
22
terms, the average difference between batches was 0.37 log units. According to these
23
results, the contribution of lipolytic bacteria to the development of hydrolytic rancidity
24
in blackspot seabream does not seem to be relevant, although this bacterial group
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exhibited a slight increase in their numbers at advanced storage times.
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1
It is well known that microbial metabolites such as peptides or amino acids, derived
2
from protein hydrolysis, contribute significantly to undesirable sensory changes in
3
seafood products (Makarios-Laham & Lee, 1993). Accordingly, we investigated the
4
comparative evolution of proteolytic bacteria in FI and OFI batches. As can be observed
5
in Table 3, statistically significant lower counts of proteolytic bacteria were observed in
6
the OFI batch than in the FI batch only on day 16. Globally, the average difference
7
between the counts of proteolytic bacteria determined for both batches was 0.46 log
8
units, this indicating a slight decrease in the growth of this bacterial group in blackspot
9
seabream muscle in the OFI batch. These results confirm previous works in which the
10
combination of flow ice and ozone provided an additional reduction of proteolytic
11
bacteria (Campos et al., 2005; Rodríguez et al., 2006).
12
Finally, the counts of histamine-producing bacteria were determined. Although the
13
average counts throughout storage were 0.30 log units lower in the OFI batch than in
14
the FI batch (Table 3), no significant differences were observed between batches.
15
However, on advanced storage times, mean value counts of the histamine-producing
16
bacteria were higher in the FI batch. In all cases, the levels of histamine-producing
17
bacteria isolated in the screening medium were not relevant in quantitative terms.
18
19
Identification of histamine-producing bacteria isolated from blackspot seabream
20
Histamine formation has not been studied in blackspot seabream up to now. Likewise,
21
no histamine-forming bacterial strain has been investigated in this fish species to date.
22
As stated above, the numbers of histamine-producing bacteria isolated in Niven’s
23
screening medium were not high. However, from the qualitative point of view, the
24
identification of such strains would expand the knowledge on the microbiological
25
spoilage and safety of this farmed fish species. Accordingly, selected colonies giving a
14
1
positive reaction in Niven medium and exhibiting different colony morphology were
2
picked and subjected to phenotypic and genotypic analysis. Table 4 compiles the results
3
of the identification study. Thus, from the 13 strains isolated only two of them resulted
4
to be Gram(+), both strains being classified as Bacillus cereus. Remarkably, 11 of the
5
13 strains were Gram(–) and most of them were proteobacteria belonging to the
6
Pseudomonas and Stenotrophomonas genera. Finally, a strain of Shewanella balticus
7
was also identified. The role of Stenotrophomonas maltophilia and Pseudomonas spp.
8
in the formation of biogenic amines in fish species has been previously reported (Ben-
9
Gigirey et al., 2000; Kim et al., 2003). Interestingly, none of the strains isolated from
10
blackspot seabream has been previously classified as prolific histamine formers such as
11
Morganella morganii, Enterobacter aerogenes or Hafnia alvei, among others. However,
12
this study confirms that blackspot seabream may harbour bacterial strains able to
13
produce biogenic amines, with the subsequent risks on fish quality and safety if the cold
14
chain is broken.
15
16
Proximate analyses
17
The moisture, lipid and NaCl contents ranged from 735-758, 7.5-11.5 and 2.7-3.9 g kg-1
18
muscle, respectively. Values for these constituents did not provide significant
19
differences (p>0.05) as a result of the icing system nor as a result of the storage time.
20
According to the lipid content in white muscle, blackspot seabream can be considered a
21
relatively low-fat-content fish species. Thus, the moisture content was found higher than
22
that reported for fatty fish species (Losada et al., 2004a), but lower than for lean fish
23
(Losada et al., 2004b), in accordance with the widely accepted inverse ratio between
24
moisture and lipid matter (Piclet, 1987).
15
1
The presence of NaCl in FI and OFI systems led to higher average values in fish
2
specimens than those reported for the untreated white muscle of fish species (0.5-1.0 g
3
kg-1 muscle) (Losada et al., 2004a; Losada et al., 2005). Nonetheless, the NaCl
4
concentrations determined in this study resulted to be considerably lower than those
5
determined in fish preserved in chilled seawater (Smith et al., 1980) or salted (Srikar et
6
al., 1993).
7
8
Nucleotide degradation analysis
9
The K value (%) (Figure 1) was calculated according to the concentration of the six
10
molecules included in the nucleotide degradation pathway. Very low values could be
11
observed for the slaughtered fish (day 1), this being in agreement with the sensory
12
assessment scores (Table 2). Both icing systems exhibited slow K value increases as the
13
storage time progressed. However, a significant increase (p<0.05) of the K value was
14
observed at the end of the storage in the FI batch, and at day 13 in counterparts stored in
15
OFI. According to these results, a higher (p<0.05) K value was concluded for specimens
16
stored in OFI as compared to their counterparts in FI, although all values can be
17
considered relatively low.
18
Thus, considerable differences have been reported for the evolution of the K values
19
along the chilled storage of fish species in flake ice. While fast increases (reaching K
20
values included in the 80-90 range) with storage time were reported for rockfish
21
(Mendes et al., 2001) and Atlantic salmon (Erikson et al., 1997), K values below 80
22
were determined in rejected specimens of turbot (Aubourg et al., 2005) and European
23
sea bass (Kyrana & Lougovois, 2002). Concerning chilling in FI, previous results prove
24
that farmed blackspot seabream undergoes slow autolytic degradation mechanisms,
16
1
exhibiting K values lower than hake (Losada et al., 2004b), horse mackerel (Losada et
2
al., 2005) and sardine (Losada et al., 2004a).
3
4
Trimethylamine oxide degradation, amine and formaldehyde formation and pH
5
evolution
6
Higher average values of TMAO-N were obtained for individuals slaughtered and kept
7
in OFI as compared to their counterparts under FI conditions (Table 5). Differences
8
were found to be significant at the end of the storage. No significant effect (p>0.05) of
9
the storage time on the TMAO-N content was determined in the OFI batch; on contrast,
10
a significant decrease (p<0.05) in the TMAO-N content was observed in the FI
11
specimens at the end of the storage time.
12
The highest TMAO-N contents have been described in elasmobranches and squids
13
(750-2500 mg TMAO-N kg-1 muscle), while gadoid fish species have shown values of
14
ca. 830 mg TMAO-N kg-1 muscle. Present results prove that TMAO-N content in
15
farmed blackspot seabream is very low, being similar to those reported for flat and
16
pelagic fishes (Gallardo et al., 1990; Huss, 1995).
17
In spite of the relatively low TMAO-N values, the TVB-N contents (Table 5) were
18
found to be relatively high when compared to other fish species (Rodríguez et al., 2004;
19
Campos et al., 2006). This result confirms that a wide variety of basic and volatile
20
compounds are included under the denomination of total volatile base compounds, not
21
only arising from the trimethylamine oxide (TMAO) breakdown. However, no
22
significant (p>0.05) differences in the TVB-N contents were observed among
23
specimens, regardless the storage time or icing system.
24
Very low TMA-N values (Table 5) were observed for specimens on day 1 in both icing
25
systems, this being in agreement to both the high quality scores obtained in the sensory
17
1
analyses (Table 1) and the low TMAO concentrations determined. As storage time
2
passed, a progressive TMA-N formation in both FI and OFI batches were obtained. In
3
all cases, higher average values were determined in FI specimens as compared to OFI
4
specimens, being these differences significant at days 3 and 9. However, TMA-N values
5
attained at advanced storage times of blackspot seabream proved to be remarkably
6
lower than those reported for captured fish species (Whittle et al., 1990; Rodríguez et
7
al., 2004) and similar to the ones obtained for farmed turbot (Rodríguez et al., 2006).
8
Dimethylamine and FA formation was determined in order to elucidate if the blackspot
9
seabream is a FA-producer fish species. Values determined fell in the following ranges:
10
0.1-0.5 (DMA-N, mg kg-1 muscle) and 190-240 (FA, mg kg-1 muscle). At the end of the
11
storage time, both metabolites exhibited relatively low values as compared to those
12
reported for gadoid fish species kept under flake ice (Botta et al., 1982) or FI (Losada et
13
al., 2004b). Moreover, no significant (p>0.05) differences in the DMA-N and FA
14
contents were observed in blackspot seabream specimens in this study, regardless the
15
storage time or icing system.
16
With respect to pH, a starting (day 1) average value of 6.10 was determined in the
17
muscle of blackspot seabream specimens slaughtered in FI or OFI. Throughout the
18
chilled storage, pH values fell in the range of 6.10-6.40, no significant (p>0.05)
19
differences being determined regardless storage time or icing system.
20
21
Lipid hydrolysis
22
Throughout the initial 9 days of icing treatment (slaughter and storage), the
23
development of lipid hydrolysis events did not provide significant (p>0.05) differences
24
between FI and OFI batches (Figure 2). Icing time neither provided significant
25
differences (p>0.05) among specimens of each batch up to day 13. At this sampling
18
1
time, a significant increase (p<0.05) in the lipid hydrolysis rate was determined in both
2
batches, although no significant (p>0.05) differences between batches were observed.
3
This increase in the lipid hydrolysis rates in blackspot seabream on day 13 coincided
4
with higher average counts of lipolytic bacteria (Table 3), this suggesting a potential
5
contribution of such microflora to the development of hydrolytic rancidity in this fish
6
species.
7
FFA formation has been reported to be produced during the first stages of the chilling
8
process (up to day 9, approximately) (Pigott & Tucker, 1990; Whittle et al., 1990) as a
9
result of endogenous enzyme (namely, lipases and phospholipases) activity. Later on,
10
microbial activity gains importance, so that FFA formation is then mostly produced as a
11
result of bacterial catabolic processes. Present results on FFA formation in blackspot
12
seabream are in agreement with this profile, in which the microbial contribution is
13
limited to advanced storage periods (13-16 days).
14
While the formation of FFA itself does not lead to nutritional losses, its assessment is
15
deemed important when considering the development of rancidity. Thus, a pro-oxidant
16
effect of FFA on lipid matter has been proposed and explained on the basis of a
17
catalytic effect of the carboxyl group on the formation of free radicals by the
18
decomposition of hydroperoxides (Aubourg, 2001).
19
20
Lipid oxidation
21
Lipid oxidation was measured by the peroxide value (primary oxidation), the
22
thiobarbituric acid reactive substances (TBARS) formation (secondary oxidation) and
23
by the assessment of interaction compounds produced between primary and secondary
24
lipid oxidation compounds and nucleophilic molecules (namely, protein-like molecules;
25
tertiary oxidation) (Table 6).
19
1
Peroxide formation exhibited a progressive increase with icing time in FI and OFI
2
batches. Higher average values were attained for individuals stored in OFI as compared
3
to FI, being such differences significant at days 3, 6 and 16. A pro-oxidant effect of
4
ozone can be concluded according to the primary oxidation formation, although values
5
obtained at the end of the experiment can not be considered especially high. During the
6
chilled storage of sardine (Losada et al., 2004a), a higher peroxide formation was also
7
obtained for specimens stored under OFI conditions than in their counterparts under FI
8
system; however, PV attained at day 12 for both OFI and FI conditions were above the
9
10 mark.
10
The development of secondary lipid oxidation events in slaughtered and stored
11
blackspot seabream was found to be considerably low. Only at the end of the
12
experiment significant (p<0.05) secondary oxidation was determined in both batches.
13
Throughout storage, lower values (days 1, 6 and 13) were obtained for FI specimens.
14
Again, a pro-oxidant effect of ozone presence can be concluded from the actual results.
15
During the chilled storage of sardine (Losada et al., 2004a), a higher TBARS formation
16
was also obtained for specimens chilled under ozone presence; thus, TBA-i values
17
attained at day 5 were above the 1.0 mark.
18
The formation of fluorescent compounds did not provide significant (p>0.05)
19
differences with icing time during the nine initial days in any of the two batches.
20
Afterwards, significant (p<0.05) increases in tertiary oxidation events were determined,
21
this being in agreement with the higher primary and secondary oxidation rates described
22
above for the final storage period. Differences between both icing conditions were not
23
obtained.
24
Among the different lipid damage parameters studied in the present experiment,
25
secondary lipid oxidation compounds are known to be the most closely related to the
20
1
formation of oxidised flavours (White, 1994). In the present work, the low values found
2
for TBARS content were in agreement to the negligible scores concerning rancid odour
3
development accounted from the sensory analyses.
4
5
Multivariate analysis
6
In order to segregate the different parameters (chemical, microbiological and sensory
7
indices) into different factors, principal component analysis (PCA) was carried out. For
8
blackspot seabream treated under OFI condition, 70.46 % of the variability of the
9
variables under study could be explained with two factors. Following a Varimax
10
rotation (Table 7), it was found that factor 1 alone accounted for 57.08 % of the
11
variability. Factor 1 had relatively high loadings (> 0.83) for all sensory attributes and
12
four chemical indices (K value, FFA, FR and TMA-N). Factor 2 accounted only for
13
13.38 % of the variability. The results obtained by PCA indicated that two sensory
14
attributes (external odour and consistency) and three chemical indices (FFA, FR and
15
TMA-N) would be the most accurate tools to check the grading decrease of the present
16
fish species when chilled under OFI condition.
17
In the case of the FI icing system, 70.85 % of the variability of all parameters under
18
study could be explained with two factors. Following a Varimax rotation (Table 7), it
19
was found that factor 1 alone accounted for 61.08 % of the variability. Factor 1 had
20
relatively high loadings (> 0.82) for all sensory attributes, lipolytic counts and four
21
chemical indices (FFA, PV, FR and TMA-N). Factor 2 accounted for only 9.77 % of the
22
variability. In this case, two sensory attributes (eyes and consistency) and TMA-N
23
would be the most accurate indices for assessing the damage produced in blackspot
24
seabream during its slaughtering and chilling storage under FI system.
25
21
1
FINAL REMARKS
2
3
This work provides the first study to date of the refrigeration quality loss mechanisms of
4
a promising farmed fish species. Thus, farmed blackspot seabream, when slaughtered
5
and stored in FI or OFI conditions, exhibits remarkably slow spoilage mechanisms
6
according to sensory evaluation, microbial development and assessment of biochemical
7
changes related to quality loss. Moreover, an increase of FA and DMA-N contents was
8
not observed, these allowing us to conclude that this species can not be considered a
9
FA-producer. According to multivariate analysis (PCA), consistency and TMA-N
10
content analyses showed to be the most accurate tools to be employed for the quality
11
damage assessment after the slaughtering and chilling storage under both OFI and FI
12
conditions.
13
The presence of ozone as preservation agent has provided marked benefits such as the
14
slowing down of microbial activity, this contributing to extend the shelf life of this fish
15
species. In contrast, a small pro-oxidant effect (primary and secondary compounds) was
16
detected; however, oxidation values reached by blackspot seabream individuals
17
slaughtered and stored under OFI conditions can not be considered specially high.
18
According to current demands on the quality of farmed fish species, it is concluded that
19
flow ice, either alone or in combination with ozone, represent a valuable slaughter and
20
storage method for keeping the quality of blackspot seabream, and for providing a safe
21
and high quality product.
22
23
22
1
FIGURE LEGENDS
2
3
4
5
Figure 1: Comparative K value (%) assessment in blackspot seabream muscle subjected
6
to slaughter and chilled storage in flow ice (FI) and ozonised flow ice (OFI),
7
respectively*.
8
9
10
* Average values of three (n = 3) independent determinations. Standard deviations are
indicated by brackets.
11
12
13
14
Figure 2: Comparative free fatty acid (FFA) formation in blackspot seabream muscle
15
subjected to slaughter and chilled storage in flow ice (FI) and ozonised flow ice
16
(OFI), respectively*.
17
18
19
* Average values of three (n = 3) independent determinations. Standard deviations are
indicated by brackets.
20
23
1
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16
ACKNOWLEDGEMENTS
17
The authors wish to thank Kinarca S.A.U. (Vigo, Spain) for providing the ozonised
18
slurry ice equipment and Isidro de la Cal (La Coruña, Spain) for providing the fish
19
specimens. This work was supported through a project grant by the Secretaría Xeral de
20
I+D from the Xunta de Galicia (Project PGIDIT 05 TAL 00701CT). The authors also
21
thank Mr. Marcos Trigo and Mrs. Carolina Gil for their excellent technical assistance.
22
23
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