FATTY ACID PROFILE OF FROZEN FILLETS OF COBIA (Rachycentron canadum) STUNNED BY ELECTRONARCOSIS

The objective of this research was to evaluate the effect of desensitisation by electronarcosis on the changes to the fatty acid profile of cobia (Rachycentron canadum) fillets stored at low sub-zero temperature. For stunning, 50, 100 and 150 V were used for 120 s and then exsanguination was performed, followed by storage at –18 °C for 180 days. A total of 47 fatty acids were detected in cobia fillet samples, and most of the quantified fatty acids differed (p < 0.05) between treatments and storage times. The n6/n3 ratio did not differ (p > 0.05) among treatments; however, the n6/n3 ratio and Σhypocholesterolaemic fatty acids/Ʃhypercholesterolaemic fatty acids ratio were significantly affected by the storage times and their outcomes. In general, the electronarcosis intensities used to numb cobia did not promote differences during the frozen storage of the fillets for most of the analysed variables but a significant effect of the storage time was noted within the treatments, with meat quality loss observed over time.


INTRODUCTION
The cobia Rachycentron canadum is a species of pelagic, migratory fish found in tropical and subtropical seas. In the western Atlantic, it is found from southern Canada to Argentina. In Brazil, it is distributed throughout all coasts (from Amapá to Rio Grande do Sul) (Shaffer and Nakamura, 1989). Rachycentron canadum has a preference for water temperatures between 20 and 30 °C, migrating from the southern to the northern hemisphere in search of warmer water during autumn and winter. In nature, it can tolerate salinities between 8 and 44.5 ppm (Shaffer and Nakamura, 1989). In commercial fish farms, sexually mature females can reach a weight above 8 kg in 18 months of culture. Males reach sexual maturity at 1 year when the fish weighs about 7 kg (Su et al., 2000). Several cultivations of this species have recently been initiated in Brazil, and its adaptation capacity to net tanks has been a success.
Freezing is a technique used for long-term preservation of fish, allowing the maintenance of organoleptic and nutritional characteristics, by minimising enzymatic and microbial activities (Alizadeh et al., 2007). Nonetheless, the freezing method, which can be fast or slow, is a variable that interferes with the quality of the fish. Slow freezing causes the formation of intracellular ice crystals that cause cell disruption, increasing water loss, enhancing enzymatic activity and damaging the texture of the fillets (Alizadeh et al., 2007).
Electrical stunning (electronarcosis) is regarded as an appropriate method for fish desensitisation, without changing fish quality (Lines et al., 2003;Roth et al., 2012). However, the need for observations on the behaviour and welfare of fish during breeding and at the time of slaughter have been emphasised (Lines et al., 2003). In some countries, such as the UK, electronarcosis is utilised as the main method for paralysing fish, despite a lack of consensus among the scientific community and regulators.
According to Suárez-Mahecha et al. (2002), it is increasingly important to know the concentrations of fatty acids in fish from the natural environment and in culture, considering society's constant search for healthier foods. Fish is particularly enriched with long-chain fatty acids (C14-C22), which can be saturated fatty acids (SFA) or unsaturated fatty acids (USFA). The fatty acid composition varies according to the animal species, food habit, season, water temperature, diet, habitat and maturation physiological state (Ogawa and Maia, 1999;Mohanty et al., 2019).
Freezing is a technique used for long-term preservation of fish, allowing the maintenance of organoleptic and nutritional characteristics, by minimising enzymatic and microbial activities (Alizadeh et al., 2007;Sone et al., 2019). Nonetheless, the freezing method, which can be fast or slow, is a variable that interferes with the quality of the fish affecting the lipid stability due the oxidative processes that are not avoided by the low negative temperature of storage (Secci and Parisi, 2016). Slow freezing causes the formation of intracellular ice crystals that cause cell disruption, increasing water loss, enhancing enzymatic activity and damaging the texture of the fillets (Alizadeh et al., 2007;Sone et al., 2019).
The objective of the present study was to evaluate the effect of different voltages of electric shock applied to cobia fillets (R. canadum) as a method of desensitisation on the fatty acid profile and its changes during frozen storage of the fillets for 180 days.

MATERIAL AND METhODS
The experiment was carried out in Ubatuba (São Paulo, SP, Brazil), at the Clarimundo de Jesus base, belonging to the Oceanographic Institute of the University of São Paulo (Brazil), and at the Aquaculture Laboratory of the Department of Animal Science of the Faculty of Animal Science and Food Engineering (FZEA) of the University of São Paulo (USP), Pirassununga Campus (SP, Brazil). The specimens, obtained from a commercial production farm in the municipality of Ubatuba (SP, Brazil) had an average weight of approximately 2 kg.
To evaluate the effect of electronarcosis on the quality of cobia meat kept frozen for 180 days, the fish were submitted to the following three treatments: 50, 100 and 150 V, respectively, using alternating current, for 120 s. For each treatment, 120 fish were used, which were placed in a 120-L plastic box, in which the electrical conductivity of water was adjusted to 700 μS, for the correct application of the electricity. After the desensitisation, the fish were slaughtered, by cutting the branchial arches and then submerged in cold water for 3 min, for exsanguination.
Before and after the desensitisation, the water quality parameters were measured with the aid of a U-10 multi-parameter probe (Horiba, Ubatuba, SP, Brazil). The behaviour of the animals was observed during the exposure to electric shock, and the desensitisation time was considered as starting from the moment of electric current application until the complete unconsciousness, which was determined by the absence of reflexes to lateral line stimulation, and the absence of opercular movements and rotation of the eyes.
A 4×3 factorial scheme was used, with four assessment points (0, 60, 120 or 180 days at -18 °C after slaughtering) and three electric shock voltages (50, 100 or 150 V), according to the following statistical model: where Y ij = the observed value relative to time i at voltage j; m = the general average of the variable; α = the effect of treatment i (storage days); βj = the effect of treatment j (voltages of the current); and e ij = the contribution of chance associated with the effect of time i on voltage j. The whole experiment was performed with three replicates, with one fillet per replicate that were analysed immediately after death (0 Time) and after storage at low negative temperature (-18 °C), at different storage time (60, 120 and 180 days). At the different storage times, the fillets were submitted to the lyophilization to stabilize the matrix before the fatty acid profile analysis.
The total lipid content of the fish samples was analysed according to the method of Folch et al. (1957). Accordingly, 2 g of fresh 3/7 sample was reconstituted from the lyophilised sample, by the addition of 1.5 mL of distilled water per 0.5 g (average) of the lyophilised sample. The extract was filtered through filter paper into a test tube. To maintain a constant chloroform:methanol:water ratio of 8:4:3 (v/v/v), as suggested in the original method, 9.8 mL (11.3-1.5 mL) of 0.88% (w/w) aqueous KCl solution was added to the extract (30.3:15.1:11.3). The blends were held overnight at refrigerated temperature to allow separation of the two phases. Then, the solvents were removed by vacuum evaporation (Buchi Rotavapor RE111) until only the fat remained at the bottom of the tube. The fat was dried under pressurised nitrogen and dissolved in 5 mL chloroform. Another 1 mL of chloroform solution was added before methylation of the fatty acids for analysis by gas chromatography (GC).
The fatty acids were methylated, as described by Morrison and Smith (1964). For saponification, 5 mL of 0.5 M of methanolic KOH was added, and the tubes were then placed in a 90 °C water bath for 40 min, with stirring every 10 min, and finally cooled. A mixture of potassium carboxylates and glycerol was formed. One mL of distilled water and 2.5 mL of 2 M HCl were added to the saponified fatty acids. A 2.5-mL aliquot of petroleum ether (40-60 °C) was added, and the tubes were stirred to favour the extraction of fatty acids by the solvent. The ether phase was transferred twice, and the two phases were separated by the addition of 2.5 mL petroleum ether. Then, the petroleum ether containing the dissolved fatty acids was evaporated.
Esterification was carried out according to the modified method of Morrison and Smith (1964). Cyclohexane (0.5 mL) and 2 mL of 14% BF 3 -methanol were added. The samples were placed in a water bath at 90 °C for 3 min, cooled in a water bath, and 2.5 mL of distilled water and 2.5 mL petroleum ether were added. Themethylated fatty acids were extracted twice by the addition of 2.5 mL petroleum ether, the solvent was distilled, and the methylated fatty acids were dissolved in 1 mL hexane.
Each sample was transferred to a vial and sealed. GC analysis was performed using a Varian 430 gas chromatograph equipped with a Supelco Omegawax 320 capillary column (30 m × 0.32 mm i.d., 0.25 mm film thickness) and a flame ionisation detector (FID). The GC conditions were set as follows: injection volume, 1 μL; carrier gas (He) flow rate, 1.5 mL min -1 ; injector temperature, 220 °C; detector temperature, 300 °C; split ratio, 1:20. The flows of He, air and H 2 to the FID were 25, 300 and 30 mL min -1 , respectively.

Fatty acid profile
Due to the expected similar content in total lipids (4.52 ±1.15/100 g fillet), as expected since the cobia submitted to the different stunning methods were of the same group and were similarly fed before the treatment, the fatty acid profile was expressed in percentage of each fatty acid in relation to the total fatty acid content. As it is known, the quantitative expression of fatty acids poses some incertitude, from an analytical point of view, and this expression can be appropriate in the case of matrix with different lipid contents.

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Evaluation of the fatty acid profile with storage time revealed significant differences in all the detectable fatty acids. The highest values were recorded on the first day of storage, which differentiated them from the other storage times. Furthermore, the USFA contents decreased over time, and the SFA contents increased proportionally, probably due to the progressive lipid oxidation in the fillets throughout storage.

h/h index
The Σhypocholesterolaemic fatty acids/Σhypercholesterolaemic fatty acid ratio (H/H index) is strictly related to cholesterol metabolism. In the present study, the H/H index decreased (Figure 1) during storage at -18 °C, with the treatments at 50 and 150 V associated with the largest variations in this parameter, mainly in the first 60 days.
Linoleic/alpha-linolenic acid ratio (LA/ALA) Significant differences (p < 0.05) in the LA/ALA ratio among the treatments occurred during storage (Figure 2). It was possible to observe a sharp decrease in the LA/ALA ratio for the 50 and 150 V treatments, up to 60 days of storage, followed by a plateau, with no further changes throughout the experiment, in all the treatments studied.

Thrombogenic fatty acids
There were no significant differences in the thrombogenic fatty acids (p > 0.05) among the treatments (Figure 3). However, it was possible to observe significant differences related to storage time and their outcomes (p < 0.05), with an increase in the values of thrombogenic acids over storage time.

LA/ALA ratio
As stated by Price and Schweiggert (1976), reactions that occur in the live animal and after slaughter are similar, but it should be considered that after physiological death, the tissues are unable to eliminate certain metabolites. Hence, the variation in the LA/ALA ratio may be explained by the action of desaturase and elongase enzymes, which, in animals, convert LA and ALA into DPA (C22:5 n6) and DHA (C22:6 n3). As seen in Table 1, the decrease in the LA and ALA contents (also observed in Figure 2) was directly related to the increase in DPA and DHA.
In the study by Nazemroaya et al. (2009), a gradual increase in the LA/ALA ratio was observed between 0 and 5 months of frozen storage (from 1.6 to 4.5), whereas, from 5 to 6 months, this value almost doubled (from 4.5 to 9.3) for the narrow-barred Spanish mackerel samples (S. commersoni). In shark (C. dussumieri), the LA/ALA increased significantly between 0 and 2 months, from 3.3 to 34.8; after the fifth month, this index could no longer be measured because no more ALA could be detected.

Thrombogenic index
In the same study mentioned above, Nazemroaya et al. (2009) noted a gradual increase in the thrombogenicity index from 0 to 6 months of storage for both fish species (shark and mackerel), reflecting an increase in the thrombogenic fatty acids with increased storage. Although there is no ideal value for the thrombogenicity index, the lower this value, the better the nutritional/functional quality of the fat, due to the prevention of coronary diseases (Tonial et al., 2011). When working with tilapia supplemented with soybean oil, Tonial et al. (2011) found thrombogenicity values close to 0.98. Comparatively lower values were found in the current research, irrespective of the electric shock intensity.
n6/n3 ratio Again, in the study performed by Nazemroaya et al. (2009), there was a gradual decrease in the n6/n3 ratio from 4.16 to 2.43 in narrow-barred Spanish mackerel (S. commersoni) and 2.03 to 1.01 in shark meat (C. dussumieri), during frozen storage for 6 months. The London Department of Health and Social Security (Leathard, 1994) advocates that for the human diet, an n6/n3 ratio below 4 is desirable for the prevention of cardiovascular diseases. In comparison, the values found in this research ranged from 4.45 and 6.32. Among the four species of fish (barred sorubim, spotted sorubim, pacu and golden dorado) investigated by Ramos Filho et al. (2008), pacu showed the highest n6/n3 ratio (3.65) whilst the spotted sorubim showed the lowest value (0.95), yet all values were indicative of a good nutritional quality.

CONCLUSIONS
The results of this study demonstrate that the three intensities of the electricity tested can be used for cobia desensitisation, but without further research, no conclusive effects on the fatty acid profile can be established.
At all the intensities tested, electronarcosis caused a decrease in the USFA and an increase in the SFA during storage of the cobia fillets times at sub-zero temperature.
Among the treatments, the 100-V intensity better stabilised the H/H index, maintaining the fillet quality.
The different currents applied did not modify the levels of the thrombogenic fatty acids and maintained the n6/n3 ratio, which was modified by the storage time.