Fatty acid composition and influence of temperature on the lipid stability of Arapaima gigas meat

The nutritional quality and lipid stability of Arapaima gigas fillets were investigated. A total of 27.32 kg of A. gigas fillets were obtained and the proximate composition and fatty acid profile immediately determined. In addition, the lipid oxidation parameters were analyzed during 15 and 90 days at 4 °C and –20 °C, respectively. The A. gigas fillets presented high protein (> 15%) and low lipid (< 2%) contents with elevated polyunsaturated fatty acid (PUFAs) contents (43.97%). The nutritional quality indices were the atherogenicity index (0.35), thrombogenicity index (0.28) and the hypocholesterolemic acid/ hypercholesterolemic acid ratio (2.37). Overall, an increase followed by a decrease was observed in the peroxide index (PI) and malondialdehyde content (MDA) results at both storage temperatures (p < 0.05). The lipid profile exhibited great nutritional quality, however new conservation methods should be investigated for this matrix due to increased lipid oxidation during refrigerated and frozen storage.


Introduction
Fish is considered to be a cheap source of nutrients with high biological quality, mainly due to the long-chain polyunsaturated fatty acid (PUFAs) contents (Buzzi et al., 1997), representing a great nutritional alternative compared to other types of meat matrix (Martínez-González et al., 2002).Freshwater fish are considered as important healthy products associated with a decrease in risk factors such as cardiovascular diseases, blood pressure, type 2 diabetes and chronic obstructive pulmonary disease (Simopoulos, 2009).
In general, the PUFA composition can vary depending on different factors such as diet, age, gender, environmental conditions and method of capture or fish removal (Huynh & Kitts 2009).Freshwater fish contain less eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) than marine fish.Moreover, in general freshwater fish show large amounts of the n-6 PUFAs, mainly linoleic acid (LA, C18:2n-6) and arachidonic acid (AA, C20:4n-6) (Hunter & Roberts, 2000).LA serves as a barrier against water permeability in the skin, and is the origin of AA, which is the main precursor of the eicosanoid substances (prostaglandin, thromboxanes, leucotrienes) which act on the regulation of several physiological processes such as vasoconstriction, platelet aggregation and inflammation (Sanders, 2016;Simopoulos, 2009).
Nonetheless, the high concentration of PUFAs in this type of meat matrix leads to high lipid oxidation susceptibility due to double bond instability (Shichiri et al., 2014), resulting in degradation of the PUFAs, particularly of EPA and DHA (Gómez-Estaca et al., 2014), and the formation of detrimental compounds such as malondialdehyde (MDA) which increases the cardiovascular risk factors (Zaki et al., 2014).
Arapaima gigas is a native fish species from Brazil and presents potential farming characteristics, representing an economically viable alternative for aquaculture (Carani et al., 2008) mainly due to its weight gain and carcass yield (Fogaça et al., 2011).
Previous studies already investigated the production parameters (Fogaça et al., 2011;Imbiriba, 2001), proximate composition (Fogaça et al., 2011;Martins et al., 2015;Oliveira et al., 2014) and protein deterioration compounds during storage (Oliveira et al., 2014).However, very little research has focused on the nutritional quality of this specie, especially on the lipid portion, concerning which, to the best of the authors' knowledge, no study has been carried out up to the present moment.Therefore, the aim of the present study was to determine the proximate composition, characterize the lipid profile and investigate variations in the lipids during the refrigerated (4 °C) and frozen (-20 °C) storage of Arapaima gigas fillets.

Experimental design
Three specimens of Arapaima gigas were obtained from a fish farm located in a rural area of the state of Goias, Brazil (16°3'59.823"S50 o 38'4.114"W), and processed in a fishery company located near an urban area of the same state (16°36'52.890"S48°59'56.239"W).The fishes were fasted for 2 days according to commercial practice, slaughtered in an ice bath, headed, eviscerated and filleted in the processing area.
A total of 27.32 kg of A. gigas fillets were obtained with mean weight and length of 4.6 ± 0.6 kg and 85.9 ± 8.3 cm, respectively.The fish samples were then packed, frozen (-20 °C) and transported in an ice box (0 °C) to the laboratory, where they were randomly divided for storage at two different temperatures.One group was thawed at 4 °C overnight and the proximate composition and fatty acid profile immediately evaluated.In addition, lipid oxidation was examined in fish samples at six specific time points during refrigerated storage (4 °C) for 15 days.The other group was maintained frozen (-20 °C) and lipid oxidation evaluated at seven specific time points during 90 days of frozen storage.The present experiment was repeated three times (n = 3), totaling 87 experimental units.

Proximate analysis
The moisture, protein and ash contents were determined according to Association of Official Agricultural Chemists (2012).For the moisture determination, the samples were dried at 105 °C to constant weight, the protein content was estimated using the Kjeldahl method (N × 6.25) and the ash content was evaluated by incineration in a muffle furnace (550 °C).Moreover, the total lipid content was cold-extracted as described by Bligh & Dyer (1959), extracting the total lipids from the fish samples (5 g) using a chloroform/methanol (1:2 v/v) mixture.The carbohydrate content was calculated using the following equation % carbohydrate = 100% − (% moisture + % protein + % ash + % lipid), and the energy value was determined by multiplying the percentage of protein, lipid and carbohydrate contents by the respective energy values of 4, 9 and 4 kcal (Merrill & Watt, 1973).All analyses were carried out in triplicate.

Lipid oxidation
In order to better understand lipid oxidation during refrigerated storage (4 °C), the fish samples were evaluated every other day up to the 15 th day.To evaluate lipid storage during frozen storage (-20 °C), samples were taken every other week up to the 90 th day.The primary lipid oxidation products were determined according to the peroxide index (PI) by a colorimetric method using potassium iodide oxidation (Association of Official Agricultural Chemists, 2012) with a slight modification (3:1 acetic acid:chloroform solution, v/v).The results were expressed in milliequivalents of peroxide per kilogram of fish meat.The secondary lipid oxidation products were analyzed according to the thiobarbituric acid reactive substance (TBARS) method according to Tarladgis et al. (1960), as modified by Monteiro et al. (2012).The malondialdehyde (MDA) contents were calculated and the results expressed as mg MDA/kg fish sample.All analyses were carried out in duplicate.

Fatty acid profile
The total lipids in the fish samples were cold-extracted according to Bligh & Dyer (1959).Briefly, 5 g of fish sample were extracted using a mixture of methanol: chloroform (2:1 v/v).The fatty acids were acid methylated (Chin et al., 1992;Conte-Junior et al., 2007;Kishino et al., 2002) and the fatty acid methyl esters (FAME) analyzed using gas chromatography coupled to a flame ionization detector (Perkin Elmer, Waltham, MA, USA).An Omegawax-320 column (polyethylene glycol, 30 m long, 0.32 mm internal diameter and 0.25 mm in film thickness) (Sulpeco, USA) and helium as the carrier gas (10 psi) were used yo separate the FAMEs.The injector (split of 1:20) and detector were maintained at 260 °C and 280 °C, respectively.The initial column temperature was set at 110 °C, increased at a rate of 40 °C/minute to 233 °C, and held at this temperature for 2 min.The temperature was then raised to 240 °C at 1°C/minute and held at this temperature for 21 min.The FAMEs present in the fish samples were identified by a comparison of their retention times with a commercial standard containing 37 fatty acid methyl esters (Sigma-Aldrich, St. Louis, MO, USA).All analyses were carried out in triplicate.

Statistical analyses
One-way ANOVA was carried out to evaluate the proximate composition, fatty acid profile and lipid oxidation (during the storage time) in the Arapaima gigas samples, using the XLSTAT version 2012.6.08 (Addinsoft, Paris, France).Significant differences between the mean values were determined using Tukey's test at a 95% confidence level (p < 0.05).

Proximate composition
The proximate composition results are presented in Table 1.According to Haard (1992), fish are commonly classified into groups according to their total lipid content: lean (<2%), low-fat (2% to 4%), medium-fat (4% to 8%) and high-fat (>8%).In addition, 15% of protein content can be considered as a high level (Memon et al., 2011).Thus, the present findings indicate that the A. gigas fillets can be considered as lean meat with a high protein content, resulting in a low energy value (99.44 kcal/100g), results similar to those of previous studies with A. gigas fillets (Fogaça et al., 2011;Oliveira et al., 2014).Regarding the proximate composition of the farmed A. gigas, similar total lipid (Oliveira et al., 2014), protein (Fogaça et al., 2011;Martins et al., 2015), moisture (Martins et al., 2015) and ash (Martins et al., 2015;Oliveira et al., 2014) contents were previously reported in the literature.Nonetheless, Martins et al. (2015) reported a higher total lipid content (2.60%) whereas Oliveira et al. (2014) observed a lower protein content (16.83%) in A. gigas meat.The variation in the proximate data observed as compared to aformentined studies can be explained by the high variability of fish composition depending on the species, season, environment, diet, age and sex (Martins et al., 2015).In general, the lipid and protein contents are the most variable components in farmed fish, which depend on the feeding and fish muscle activity (Thammapat et al., 2010).

Lipid oxidation at different storage temperatures
To the best of the authors' knowledge, there are no data regarding the lipid stability of A. gigas meat during refrigerated (4 °C) and frozen (-20 °C) storage.Lipid oxidation was evaluated by determining the PI and MDA during 15 days under refrigerated storage and 90 days under frozen storage (Tables 2 and 3, respectively).During refrigerated storage (4 °C) the PI increased on day 9 (p < 0.05) but decreased on day 15 (p < 0.05), whereas under frozen storage (-20 °C), the PI increased on day 30 and decreased on day 45 (p < 0.05).A similar pattern for PI was observed in Lates calcarifer during 12 days at 4 °C (Arfat et al., 2015) as well as in Atlantic halibut during 12 months of frozen storage (Abreu et al., 2011).Values are expressed as the mean ± standard derivation (n = 3).PI: peroxide index; mEq: milliequivalents; MDA: malondialdehyde.Different letters in the same column represent significant difference (p < 0.05).
Regarding the MDA values, in general the A. gigas fillets demonstrated a decrease (p < 0.05) on day 12 during refrigerated storage.According to Monteiro et al. (2012), Intarasirisawat et al. (2014) and Santos et al. (2018), a similar trend was observed for MDA formation in Nile tilapia, fish (Clarias macrocephalus) emulsion sausage and Arapaima gigas fillets stored under refrigeration.Regarding the frozen samples, the A. gigas fillets generally exhibited higher MDA values on day 45 than on days 15 and 30 (p < 0.05), but nevertheless a decrease was observed on day 60 (p < 0.05).A similar trend was observed by Abreu et al. (2011) for Atlantic halibut stored at -20 °C.
Peroxides are the primary products of lipid oxidation whereas aldehydes, ketones, alkanes and other products are secondary compounds of lipid oxidation (Guéraud et al., 2010;Guyon et al., 2016;Kumar et al., 2015).Thus a decrease in the peroxide level might be attributed to their degradation as a result of secondary oxidation reactions (López-de-Dicastillo et al., 2012).Moreover, the decrease observed in the MDA values can be attributed to a loss of secondary oxidation products due to their volatility, or to the ability of malondialdehyde to form covalent bonds with alkaline compounds from the degradation process (Intarasirisawat et al., 2014) in agreement with the results of the present study in which when the PI values were low, the MDA values were high.
In addition, an increase in the MDA values decreases the paraoxonase (PON1) level, leading to greater cardiovascular risk factors, such as atherosclerosis, coronary heart disease, hyperlipidemia, diabetes and hypertension (Zaki et al., 2014).Furthermore, increases in the PI and MDA values were also observed during frozen storage, potentially due to endogenous enzyme activities such as lipoxygenase, which, in turn, can be active at low temperatures and be considered a limiting factor for the storage period and fish commercialization (Abreu et al., 2011).
Of the SFAs, the most abundant fatty acid was palmitic (C16:0), which is an important energy source during fish growth (Henderson et al., 1984).Of the MUFAs, the main fatty acid observed in A. gigas was oleic acid (C18:1n-9), a fatty acid related to the type of fish diet (Ackman, 1989).Similar findings were reported for Labeo rohita, Cirrhinus mrigala, Catla catla (Memon et al., 2011) and Salmo trutta macrostigma (Ateş et al., 2013) species, for which C16:0 and C18:1n-9 were the major fatty acids present representing the SFA and MUFA contents, respectively.In addition, erucic acid (22:1n-9) was determined in this fish specie.In agreement with the present findings, Jankowska et al. (2003) studied Sander lucioperca raised on a farm and taken from wild life, and only detected 22:1n-9 in the lipid fraction of the farmed species, suggesting that this MUFA came from the farmed fish feed.
Table 5 shows the PUFA/SFA, n-6/n-3 and n-3/n-6 ratios.For a healthy human diet, the values recommended for the PUFA/SFA, n-6/n-3 and n-3/n-6 ratios are a minimum value of 0.45, a maximum value of 4.0 (Department of Health and Social Security, 1984) and a range from 1/1 to 1/5 (Zuraini et al., 2006), respectively.Based on the aformentioned limits, A. gigas can be considered a food matrix with a balanced fatty acid profile.Similar PUFA/SFA results were observed in Mastacembelus simack (Harlioglu & Yilmaz, 2011), Labeo rohita and Cirrhinus mrigala (Memon et al., 2011) ranging from 1.35 to 1.40.In agreement with this, Volpe et al. (2015) found n-6/n-3 and n-3/n-6 ratios within the specified limits for Onchorynchus mykiss.Afkhami et al. (2011) studied different freshwater fish species (Cyprinus carpio and Ctenopharyngodon idella) and demonstrated values for the n-3/n-6 ratio similar to the present results.Furthermore, the EPA+DHA value, another important index for cardiovascular health was calculated in A. gigas (Table 5).In agreement with the values found, other freshwater species also obtained low values for the aformentioned sum (Afkhami et al., 2011).

Indices of lipid quality
The fatty acid composition permits one to evaluate the nutritional quality of the lipid content by calculation of the atherogenicity index (AI), thrombogenicity index (TI) and hypocholesterolemic to hypercholesterolemic (H/H) ratio (Table 5).A high quality lipid fraction should have low AI and TI values but a high H/H ratio (Bentes et al., 2009).In the present research, the AI and TI values were 0.35 and 0.28, respectively.Nieminen et al. (2014) also reported AI and TI values below 1 in Acipenser baerii.According to Ulbricht & Southgate (1991) low AI and TI values can inhibit the aggregation of platelets, preventing the appearance of coronary diseases, considered beneficial for human health.The H/H ratio is related to the cholesterol metabolism where greater H/H values are more desirable, representing a positive effect on human health (Fernandes et al., 2014).The A. gigas fillets exhibited an H/H ratio of 2.37.A similar value for H/H was demonstrated by Testi et al. (2006) for Oncorhynchus mykiss (2.43).

Conclusions
Arapaima gigas can be classified as a great protein source and a lean fish with high lipid quality, containing high long-chain PUFA levels and good nutritional indices within the freshwater fish group, emphasizing the importance of this specie on human health, mainly in the prevention of cardiovascular diseases.Nevertheless, the refrigerated and frozen storage temperatures were not capable of preventing lipid oxidation during the storage of A. gigas, probably due to large amount of PUFAs present in this specie.Further studies should focus on new conservation methods for this type of food matrix such as vacuum packaging, modified atmosphere packaging, oxygen scavenger packaging and antioxidant packaging.
*Values are expressed as the mean ± standard derivation (n=3).

Table 2 .
Lipid oxidation in Arapaima gigas fillets stored at 4 °C for 15 days.

Table 4 .
Fatty acid composition of the muscle tissue of the Arapaima gigas fillets, expressed as percentages of fatty acids (%).

Table 5 .
Nutritional quality indices for the lipid fraction of Arapaima gigas fillets.