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].
The content of fatty acids in MAM based on the omega (n)
group
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.