Abstract
High purity polyunsaturated fatty acids (> 95%) are essential for the synthesis of specialized pro-resolving lipid mediators (SPMs), such as protectins, resolvins, and maresins, which are used for clinical application. To date, high purity (> 95%) eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) have been produced through various manufacturing steps using fish oil. In this study, we optimized preparative high performance liquid chromatography (HPLC) process to purify high-purity DHA ethyl ester (DHAee; > 98%) from oleaginous microalgae Shizochytrium sp. SH103 containing at least 34% DHA content. The purity and yield of DHA were determined by reverse phase chromatography with changing the mobile phase velocity, loading amount, and mobile phase composition. On a semi-preparative scale, optimal DHA separation in isocratic elution was obtained with a mobile phase velocity of 0.5 mL/min, a loading amount of 10 mg/mL, and mobile phase composition of methanol/water (96:4, v/v), wherein the purity of DHA was 98.5%. This separation was scaled up to a preparative column, resulting in 99.0% DHA fraction with a yield of 79.8%. This result suggests that a large amount of high purity DHA can be produced from microalgae when scaling up a preparative column to an industrial column.
Highlights
The intake of polyunsaturated omega-3 fatty acids, including eicosapentaenoic acid (EPA; C20:5 ω-3) and docosahexaenoic acid (DHA; C22:6 ω-3), has been shown to benefit for human health through a variety of clinical studies
gas chromatography (GC) analysis indicated that the lipid was completely converted to fatty acid ethyl ester (FAEE) by acid-catalyzed transesterification, and the DHA content was 313 mg/g
SH 103 were DHA 34.96%, docosapentaenoic acid (DPA, C22:5n6) 9.12% and palmitic acid (C16:0) 49.84%. These values were slightly different from the results of Ju et al [24] in DPA and DHA contents, which was thought to be due to differences in the culture method and the culture time
Summary
The intake of polyunsaturated omega-3 fatty acids, including eicosapentaenoic acid (EPA; C20:5 ω-3) and docosahexaenoic acid (DHA; C22:6 ω-3), has been shown to benefit for human health through a variety of clinical studies. Since esterified oil contains other impurities as well as target fatty acids, and methods have been proposed to increase the purity of the specific fatty acids by removing impurities These methods include molecular distillation, low-temperature crystallization, urea adduct formation, simulated moving bed chromatography (SMB) and supercritical fluid chromatography [13]. Molecular distillation requires high temperature under vacuum to remove undesirable impurities, which creates the risk of oxidation, polymerization and production of trans-isomers of omega-3 fatty acids [17]. This indicates that chromatographic method is suitable for obtaining pharmaceutical levels of high purity omega-3 fatty acids. Repeated production of high purity DHA was carried out using largescale HPLC to confirm the reproducibility of established separation conditions
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