Crystallization of SPOMFs
The crystallization of SPOMFs was performed at a controlled temperature
in a 100 mL batch reactor. In general, SPOMFs of 5 g was mixed with a
certain ratio of solvents, and then the mixture was allowed to
crystallize at a selected temperature. At the end of the
crystallization, the crystals were removed by low-temperature filtration
to obtain the liquid fraction 1 that was rich in palmitoleic acid.
Samples were obtained at regular time intervals, and solvent in the
sample was evaporated under reduced pressure. The fatty acid composition
was analyzed by GC after being diluted to 2 mg SPOMF/mL.
Several parameters were optimized, which were crystallization
temperature, solvent, substrate ratio of SPOMFs to solvent (w/v) and
crystallization time. Five solvents were used to prepare the free fatty
acid solutions, and they were hexane, methanol, ethanol, acetone and
isopropanol. Crystallization temperature was in the range of 0 °C to -40
°C. Six different SPOMFs-to-solvent ratios in the range of 1:3 to 1:18
(w/v) were used, and it was found that the best range for investigation
was 1:9 to 1:18. The crystallization time was increased from 4 h to 16
h. While one of these parameters was being optimized, the others were
held at a fixed level. After one parameter optimization was completed,
the optimal value of this parameter was used for the optimization of
other parameters. The design for these optimization experiments is
outlined in Table 1. The yield and enrichment of palmitoleic acid
content were used as the main response variables for the optimization.
As a small amount of saturated fatty acids still remained in the liquid
fraction 1, a secondary crystallization was conducted for further
concentration of palmitoleic acid. Secondary crystallization was
conducted at -40 °C by mixing 5 g liquid fraction 1 with 20 mL methanol
for a period of time. The resulting crystals were separated from the
liquid, giving liquid fraction 2. The fatty acid composition was
analyzed (see below) and the yield of palmitoleic acid was calculated.