Quantification of Essential Fatty Acids and Assessment of the Nutritional Quality Indexes of Lipids in Tilapia Alevins and Juvenile Tilapia Fish (Oreochromis niloticus)

To determine the nutritional quality of the lipid segment in tilapia (Orechromis niloticus) during different periods of development (alevins and juveniles), the total lipids (TL), linolenic (LNA), eicosapentaenoic (EPA), docosapentaenoic (DHA), linoleic (LA) and arachidonic (AA) acids were quantified , and the lipid nutritional quality indexes were calculated for the tilapia. The lipid profile showed that the species present high indexes of monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) in both development phases. The indexes of nutritional quality of lipids, atherogenic index (AI) and thrombogenic index (TI) present low values and represent beneficial health effects, both in the alevin and juvenile fish. The quantifications of LNA, EPA, DHA, LA and AA show the greatest values (mg/g of total lipids) for the juveniles. However, regardless of the development phase in which the tilapia is, the fish may be considered a source of essential fatty acids a kind of potential and nutritional food, reflecting beneficial effects for consumer’s health.


Introduction
To achieve a healthy diet, consumers all over the world are looking for food that provides energy and offers other nutritional benefits.
In addition, the Nile tilapia provides excellent sensorial and nutritional characteristics, such as tasty meat, low amounts of fat and calories, absence of Y-shaped fishbone and good indexes for being converted into food and have its fillet used as food, which optimizes the industrialization of this species (Furtado, Resende, & Freitas 2010;Chukwu, 2009;Sklan, Prag, & Lupatsch, 2004).
In general, fish meat is considered to be a positive factor in the human diet because it is a good source of proteins, vitamins and lipids of a good quality, such as polyunsaturated fatty acids, especially Omega-3 and 6 fatty acids (Ponzoni, Hamzah, Tanan, & Kamaruzzaman, al., 2005;Köprücü & Ozdemir, 2005;Gutiérrez-Espinosa, 2011).
The aim of this study was to assess the nutritional quality of Oreochromis niloticus alevins and juveniles by quantifying the fatty acids n-3 and n-6 and nutritional quality indexes of lipids.

Sampling
Alevin and juvenile tilapia samples with an estimated age of approximately 40 or 60 days, respectively, were acquired from a recreational fishing site in the city of Maringá, Paraná State.The juvenile tilapia (20 individuals) were eviscerated, filleted and ground in a food processor to obtain a homogeneous consistency.The alevins (40 individuals) were also ground in a food processor, discarding only the viscera.The samples were stored in a nitrogen atmosphere and frozen at -18°C until the time of analysis.All of the chemical analyses were performed in triplicate.

Physical and Chemical Characterization
Moisture, ash, and protein contents were determined according to AOAC (1997).Total lipids were extracted by the Bligh and Dyer (1959) method.

Fatty Acids Composition
Fatty acid methyl esters (FAME) were prepared as described by Joseph and Ackman (1992) and analyzed using gas chromatograph 14-A (Shimadzu, Japan) with a flame ionization detector and a capillary column made of melted silica CP Sil-88, a capillary column (100 m, 0.25 mm and 0.25 mm).The temperature of the column was programmed at 2 °C/min from 180 °C to 240 °C, and the temperature of the injector was kept at 220 °C and 245 °C.The gas flux was 1.4 mL.min -1 for the carrier gas (H 2 ), 30 mL.min -1 for the make-up gas (N 2 ) and 30 mL.min -1 and 300 mL.min -1 for the H 2 and for the flame synthetic air.The ratio for the sample partition (split) was 1:100.Peak areas were determined using the CG-300 computing integrator, and the fatty acid composition was expressed as the weight percentage of each fatty acid relative to the total fatty acids (%).FAME were identified by comparison with the retention times of standards from Sigma (St. Louis, MO, USA) and equivalent chain-length values (ECL).

Quantification of Acids LNA, EPA, DHA, AA, LA
The fatty acids LNA, EPA, DHA, AA and LA were quantified in mg/g of total lipids through their internal standardization by means of those methyl esters standards for fatty acids.Because it was not present in the analyzed samples, tricosanoic acid methyl ester (23:0) was used as the internal standard, and calculations were performed using the following equation (Visentainer, 2012)

Statistical Analysis
The values for the proximate composition and the fatty acid composition were subjected to the statistics variance analysis (ANOVA).The Tukey's test was applied to the unequal variances among the average sample values, keeping the significance level in 5% in all analyses through the software Statistica, version 7.0 (2004).

Results
In The mean values of the proximate composition for samples of tilapia alevins and juveniles tilapia are shown in Table 1.The results are averages in triplicates with the respective estimates for the standard deviation.Values in the same line that are followed by the same letter do not differ between them (p> 0.05).
Table 2 presents the fatty acids composition obtained in samples of tilapia alevins and juvenile tilapias.The sum of SFA, MUFA, PUFA, n-6, n-3, the ratios of PUFA/SFA and n-6/n-3 and the nutritional quality index of lipid fraction in alevins and juvenile tilapia are shown in Table 3.
Table 4 shows the concentration in mg/g of total lipids of the fatty acids, LNA, EPA, DHA, LA and AA on the samples of alevins and Juvenile tilapia.Each value is the triplicate average of fatty acids LNA (Linolenic acid), EPA (Eicosapentaenoic acid), DHA (Docosahexanoic acid), AA (Arachidonic acid) and LA (Linoleic acid) expressed in mg/g of total lipids with respective estimates of standard deviations.Averages on the same line followed by different letters are meaningfully different at level 0.5%.

Discussion
The proximate composition of a certain type of food provides information about its nutritional potential.The knowledge of the chemical composition of fish presents a fundamental importance to the standardization of food products on the basis of nutritional criteria when it provides assistance for dietary decisions (Castagnolli, 1992).
The samples of tilapia alevins have higher percentages of moisture (78.82%) and ash (1.65%) compared to the samples of juvenile tilapia, which presented higher percentages of proteins (16.94%) and lipids (6.13%).This fact may be due to the alevins samples were in a development stage at the time of the collection, thus, the energy generated from feeding did not accumulate as fat.According to Souza and Maranhão (1998), young fish have more moisture and less fat in their meat than adult fish because they are in a developing phase and have less fat available for storage.
Ribeiro, Logato, Paula, Costa, Murgas and Freitas (2008) found moisture contents in tilapia fillets of approximately 78%, which are similar to those found in this study.Moreover, the same authors found a higher percentage of ashes (5.30%) compared to those observed in this study (1.52 to 1.65%).To Omolara and Omotayo (2008), the high moisture content is a disadvantage because it increases susceptibility to microbial spoilage and degradation of polyunsaturated fatty acids and consequently decreases the preservation time of the fishes.
The lipid content obtained by us for both alevins (2.65%) and for juveniles(6.13%)corroborates with the assessment by Beirão, Teixeira, Batista, Santo, Damian and Meinert (2004), which reported that tilapia can be considered to be a lean fish because their average fat content is below 7.0%.Furthermore, the data obtained for moisture also corroborate the values (79.16%) found by Beirão, Teixeira, Batista, Santo, Damian & Meinert (2004), who note that the moisture content of fish in general varies from 60.0 to 80.0% and that lean fish have higher moisture in their muscle structure.According to Olagunju Muhammad, Mada, Mohammed, Mohammed and Mahmoud (2012), considering their fat content, fish can be grouped into four general categories: lean fish (< 2%), low fat (2 to 4%), medium fat (4 to 8%), and high fat (> 8%).Therefore, the alevines and juveniles analyzed in this study can be considered to have a low fat and medium fat, respectively, this study was similar.
The proximate composition values of the tilapia samples analyzed in this study are similar to the values found by Simões, Ribeiro, Ribeiro, Park and Murr (2007): 77.13% of moisture, 2.60% of lipids, 19.36% of protein and 1.09% of ash in tilapia fillets.
Abimbola, Kolade, Ibrahim, Oramadike and Ozor (2010) found results of approximately 18.65% protein, 0.55% lipid, 1.30% ash and 79.50% moisture in tilapia guineensis, whereas for tilapia melanotheron, the values were 18.74%, 0.70%, 1.06% and 79.50% for protein, lipid, ash and moisture, respectively.According to Furtado et al (2010), although the fish are from the same species, differences in the physical-chemical composition of the fish meat are expected because of several factors; age can be considered the main factor.
According to Henderson (1996), the saturated and monounsaturated fatty acids in fish may be the result of a lipidic diet.In addition to the diet, the saturated fatty acids may be generated through non-lipidic carbon sources and then transformed into monounsaturated fatty acids.
The sums of PUFA and MUFA (Table 3) were not significantly different (p > 0.05) between the alevin and juvenile samples but are higher to the values (PUFA = 16.7%;MUFA = 28.2%)found by Inhamuns and Franco (2001) in a study performed with Marapá muscles and the values (PUFA=0.67%;MUFA=2.67%)found by Furtado et al. (2010) in a study performed on tilapia filets.
Concerning the SFA sum, the contents presented a difference (p < 0.05) of 38.23% in alevins and 39.59% in juveniles; such values are lower to the values found by Inhamuns and Franco (2001) in Mapará samples (50.4%).
According to Martin, Almeida, Ruiz, Visentainer, Matshushita, de Souza and Visentainer (2006), diets based on a ratio of n-6/n-3 lower than 1:1 are not recommended because they inhibit transformation of linoleic acid into long chain polyunsaturated fatty acids (PUFA-VLC).However, the results obtained in this study corroborate those of Simopoulos (1999), who recommends a ratio of n-6/n-3 between 5 and 10.
Food with polyunsaturated and saturated fatty acids ratio (PUFA/SFA) below 0.45 have been considered as undesirable by the Department of Health and Social Security (1984); this ratio coincide with recommended by Enser et al (1998) based on information from the English Health Department: foods with ratio under 0.45 have the potential to raise blood cholesterol levels.In this study, the ratio of PUFA/SFA was 0.56 and 0.51 for alevins and juveniles, respectively; such values are higher than the value (0.45)However, the index of PUFA/SFA, which was evaluated separately, has received restrictions as does not consider the metabolic effects of monounsaturated fatty acids (Menezes, Miranda, Pinheiro, Cintra, Freire, & Cabral Júnior, 2009).
The evaluation of the nutritional quality of the lipid fraction of tilapia in two different developmental phases, alevin and juvenile phases, presented similar values to the those of the atherogenic index (IA) and thrombogenic (IT), 0.60 and 0.87, respectively, which were higher for tilapia in the juvenile phase,.However, the index related to ratio between hypocholesterolemic and hypercholesterolemic fatty acids (HH) was higher in the alevins (1.63).Testi, Bonaldo, Gatta and Badiani (2006) found superior values of HH to fish varying from 2.03 to 2.46.
Low values of IA and IT are beneficial to health according to Assunção (2007), who suggested that fatty acids are more favorable to health because they prevent the emergence of coronary diseases according to Turan, Sonmez and Kaya (2007).Moreover, the higher the ratio between hypocholesterolemic and hypercholesterolemic fatty acids, the more adequate that oil or fat is for the nutrition (Santos-Silva, Bessa & Santos-Silva, 2002;Sousa Bentes, Souza, Simões, & Mendonça, 2009).The values found in this study are similar to those found by Tonial, Bravo, de Souza, Matsushita, Furuya & Visentainer (2011) in tilapia from Nile feed that were fed rations supplemented with soybean oil for a period of 90 days; these values varied from 0,41 -067 (IA); 0,90 -1,09 (IT) and 1,39 -2,10 (HH).
The polyunsaturated fatty acids LNA, EPA, DHA, AA, and LA are highly important to nutrition, especially LNA, because it is a precursor of n-3 fatty acids, and LA, because it is a precursor of n-6 fatty acids.
The fatty acids LNA, EPA, DHA, AA, and LA levels were significantly higher (p < 0.05) in the juvenile tilapia samples (5.82, 1.43, 15.20, 25.63, and 91.28 mg/g of total lipids, respectively) than in the alevin samples.
According to the study by Visentainer (2003), the average values found for these fatty acids in tilapia samples submitted to different treatments with linseed oil were between (5.52-59.29 mg/g), (0.15-2.54 mg/g) and (9.93-26.13mg/g) for LNA, EPA and DHA, respectively.
The results of this study show that despite that the analyzed tilapia were not of market size, they presented a satisfactory amount of essential fatty acids for consumer health.

Conclusion
Although tilapia were analyzed below the weight recommended for commercialization, we conclude that the percentage of PUFA and MUFA in alevin and juvenile tilapia were high, suggesting benefits to human health.
The indexes of nutritional quality of lipids IA and IT presented low values, representing beneficial health effects from both alevins and juveniles.Despite tilapia alevins presenting lower quantities (mg/g of TL) of LNA, EPA, DHA, LA, and AA compared to juveniles, the lipid fraction of tilapias evaluated in the two different development phases allows us to consider tilapia as a rich source of essential fatty acids; therefore, as these fish have good potential as a nutritional food with beneficial effects for the consumer's health.
: LNA, EPA, DHA, AA or LA (mg/g) = [(Ax x Mp x Fcx) / (Ap x MA x Fc)], where Ax = LNA, EPA, DHA, AA or LA area; Ap = internal standard area; Fcx = theoretical factor for correction of the acids LNA, EPA, DHA, AA or LA; Mp = mass of the internal standard added to the to the sample in milligrams; MA = mass of the total lipids sample in grams; and Fc = conversion Factor to present the results in mg of total fatty acids starting from the LNA, EPA, DHA, AA and LA methyl esters.

Table 1 .
Proximate composition of tilapia alevins and juvenile tilapia

Table 2 .
Fatty acid composition from the total lipids of tilapia alevin and juvenile tilapia samples The values given in the table above are standard deviation means ± SD of triplicate analysis.Different letters in the same line indicate differences (p < 0.05) by the Tukey's test.The individual fatty acids were expressed as a percentage of total fatty acids.

Table 3 .
Sum and ratios of fatty acids and nutritional quality index of lipid fraction in tilapia alevin (Orechromis niloticus) and juvenile tilapia (Orechromis niloticus)

Table 4 .
Absolute quantification of fatty acids (mg/g of total lipids) LNA, EPA, DHA, AA and LA