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.