Enzymatic synthesis of lipids containing omega-3 fatty acids

Abstract: Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are omega-3 fatty acids, which have health promoting effects. Since they are highly prone to oxidation, they should be protected, in particular DHA. Several lipids containing DHA were evaluated with respect to the ability of the lipids to protect DHA against peroxidation using an HPLC method developed as part of this research. Furthermore, the protection of free DHA in a liposome system containing antioxidants was investigated. The phospholipids offered protection against peroxidation when DHA was incorporated at one position of either phosphatidylcholine or phosphatidylethanolamine. Furthermore, alfa-tocopherol combined with ascorbic acid offered the best protection to free DHA in a liposome system containing phosphatidylcholine. Enzymatic methods were developed to incorporate either DHA or EPA at the sn-1 position of 2-acyl-lysophosphatidylcholine. Since the enzymes utilised discriminated against these fatty acids, in particular DHA, screening for the appropriate enzyme was performed. Among the investigated lipases, those from Candida antarctica and Rhizopus arrhizus showed the best performance in incorporating DHA and EPA, respectively. The incorporation of these fatty acids could be increased by performing the reaction at a water activity of 0.22, which allowed some hydrolysis to occur simultaneously with the esterification. Since DHA and EPA were discriminated against in both reactions, the content of these fatty acids in the synthesised phosphatidylcholine was increased with reaction time. The ability of the lipases to discriminate against DHA and EPA was utilised to enrich these fatty acids in fish oil and squid oil. Furthermore, the competitive factors for both EPA and DHA were evaluated in both marine oils and a mixture of methyl esters. Hydrolysis of all substrates and ethanolysis of squid oil were performed, converting mainly other fatty acids and leaving DHA and/or EPA in the substrate. The competitive factors achieved for DHA and EPA were affected by several properties of the lipase, including the fatty acid specificity, regiospecificity, stereospecificity and triglyceride specificity. Hence, the competitive factors were influenced by the structure of the substrate, i.e. methyl esters or triglyceride. In the glyceride structure of the latter, the location of DHA also affected the competitive factors. Furthermore, the co-substrate, in this case water or ethanol, influenced the competitive factors. The lipases from Thermomyces lanuginosus, Pseudomonas cepacia and Pseudomonas fluorescens initially showed no formation of ethyl esters of DHA during ethanolysis. Neither did the Pseudomonas lipases provide any EPA ethyl esters during the early stages of ethanolysis. This could be explained by these lipases not being able to perform ethanolysis of DHA located in a triglyceride and the Pseudomonas lipases not being able to perform ethanolysis of EPA located in a triglyceride. Ethanolysis gave higher competitive factors than hydrolysis for both EPA and DHA for four of the five lipases evaluated. The lipase from Thermomyces lanuginosus gave the highest competitive factors for DHA and was considered to be the superior lipase for the enrichment of DHA due to its high DHA recovery together with high DHA enrichment.