There were fatty acid groups in Fraction resulted from Aceton crystallization (ACF) that have decreased and increased. Fatty acid groups that were decreased include PUFA which fell from 18.47% to 8.55% and cis-FA from 22.98% to 14.68%. The fatty acid groups that were increased include MUFA from 53.48% to 58.91%, SFA from 28.05% to 32.1%, and trans-FA from 48.97% to 52.78%. The crystallization method with the n-hexane solvent turned out to produce a fraction (NCF) with a fatty acid content profile that was similar to the acetone solvent (table 1). Only palmitic acid, stearic acid, and arachidic acid produced significantly different value (single-factor ANOVA with p<0.05).

The success of separating fatty acids with the winterization method using organic solvents is very dependent on the ratio between fatty acids and solvents. This is closely related to the polarity of fatty acid and solvent and the cooling temperature [34]. Long-chain SFA has two molecular groups with different polarities. Carboxylic groups have a polar property while long chains carbon are non-polar, the longer the decreasing polarity of fatty acids [22]. This makes the SFA content in acetone and n-hexane solvents were increased. The concentrations of Palmitic acid as the most dominant SFA in both fractions increased from 13.58% in WM to 22.54% in ACF and 21.29% in NCF. While stearic acid actually decreased from 8.8% in WM to 2.6% in ACF and 3.67% in NCF. Theoretically, stearic acid has a lower solubility in acetone if compared to palmitic acid and its solubility decreased with decreasing temperature. So, the palmitic acid content in ACF and NCF should decrease [19], but it did not happen. This was probably due to the diverse content of fatty acids in the oil so that it affected the behavior phase in the crystallization process which became more complicated. In addition, it also caused the decreasing in the nucleation velocity [35]. Another cause was the presence of polymorphisms of fatty acid crystals so that saturated fatty acids did not crystallize [18].

The phenomenon of the decreasing of PUFA content in both ACF and NCF was contrary to the theory in general, where the solubility of fatty acids in organic solvents tends to increase along with a large number of double bonds [19]. EPA as a PUFAs dominant component in ACF and NCF has decreased from 8.08% to 4.80% in ACF and 4.79% in NCF. At the crystallization method with a decrease in temperature, PUFA would be concentrated in the liquid fraction and separated from the crystals [19]. This data also contradicts other studies that state the concentration of PUFA increased sharply in the NCF fraction [33].

The Crystallization with urea produced a fraction (UCF) with a different profile of fatty acid groups compared to ACF and NCF. Urea crystallization was able to reduce SFA to 2.51%, MUFA to 42.97%, and trans-FAs to 36.13%. On the other hand, this method was also able to increase PUFA to 52.61% and cis-FAs to 59.45%. This result was in line with research by Guil-Guerrero and belarbi [20] where urea crystallization able to increase the composition of EPA. Urea crystallization was also known to be able to separate PUFA and cyclic fatty acids with SFA, MUFA, and trans-FAs in oil [15], so as to increase PUFA recovery [20].

The palmitic acid content in the urea crystallization fraction decreased to 1.25% and stearic acid to 0.33%. Urea crystals had a tetragonal geometry with a diameter of 5.67 Å. The existence of aliphatic long-chain compounds capable to make a binding to form a hexagonal structure with an inner diameter of 8-12 Å [19]. The more of double bonds and the presence of cyclic would reduce the inclusion with urea crystals. This means PUFA was difficult to form inclusions with urea [32]. In addition, the trans fatty acid structure was preferred to form urea complexes. Therefore, in the crystallization of urea, SFA and trans-FAs will be carried by urea to form the Urea Inclusion component (UIC). Whereas cis-FAs, PUFA, and cyclic fatty acids keep remaining in a solution known as non-Urea Complexing Fraction (NUCF) [15]. Even so, the urea crystallization fraction still contained SFA and EA. According to Wanasundara et al [19], totally eliminating SFA and trans-FAs with urea complexation may be difficult, because there some short-chain fatty acids did not interact to form complexes with urea crystals [36]. Therefore, it was very reasonable that the level of lauric acid in the urea fraction did not decrease at all. It might also be due to the limited capacity of urea so that it could not interact fully with all SFA and trans-FAs, where an increase in the amount of urea would increase the number of fatty acids trapped in UCF [36].

Omega (n) is a fatty acid-related naming system based on the position of the double bonds calculated from the end of the methyl at the fatty acid structure [22]. n-3 means that there is a double bond in the third position carbon calculated from the very end methyl group. ALA, EPA, DHA are an example of n-3 fatty acids which are most often used for health. Status of fatty acid content based on omega classification can be seen in Fig. 2.