Extraction of orbitides from flaxseed
The goal of this project is to establish an efficient and economical industrial process for extraction of a Kaufmann and Tobschirbel orbitide (KT) mixture from flaxseed oil. KTs occur at a low level in flaxseed oil and must be concentrated at least 600 fold to produce a useful commercial concentrate. KT peptides are more polar than most lipids may be separated using solid- or liquid- phase extractants. Extraction protocols were investigated to determine a better approach for KT peptide extraction. Commercial solid-phase extraction methods would require the adaptation of bench-scale silica flash column chromatography. The first approach was to develop methods for separation of peptides using only silica, ethyl acetate and ethanol. Ethyl acetate is known to remove both oil and peptides from silica. Therefore, the ability of low temperature to decrease the peptide elution from silica was studied. The other method utilized liquid-liquid extraction. In order to measure the success of an extraction an analytical method was required to evaluate the separation of peptides from oil. An analytical procedure was developed that readily determined the relative concentration of peptide and lipid. Aqueous and anhydrous ethanol partitioning was used to extract the KT mixture from flaxseed oil. Ethanol solutions between 50 and 100% in water (v/v) were mixed with flaxseed oil. The oil and peptide content of the extracts were determined using 1H-NMR. Liquid-liquid extraction using 70% aqueous ethanol at volume ratio (solvent to oil) of 0.25:1 produced a mole ratio of 2:1 (KTs to oil) making it the optimal solvent for KT extraction. In the second part of this project, the scale of liquid-liquid extraction was increased through several 10 to 30-fold steps to establish a potential industrial extraction process for recovery of the KT mixture. The feasibility of processing the solvent containing mixed peptides was investigated. Multiple evaporation and adsorption methods were also tested, including falling film evaporation, rotary evaporation, a combination of rotary evaporation and freeze drying, and a combination of rotary evaporation and spray drying. Various experimental methods to enrich and isolate KTs from water-rich fraction were performed. At the end of this project, 3328.89 g of KT mixture was produced that was suitable for commercial purposes. The increase of extraction scale was 140,000 fold.
Orbitide, Kaufmann and Tobschirble peptides, NMR, Flash chromatography, Extraction
Master of Science (M.Sc.)
Food and Bioproduct Sciences