Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase

Abstract

The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4°C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid+docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36h, at 40°C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

Keywords: Immobilization; Lipase; Polyunsaturated fatty acids; Soybean oil.

Fig. 1

Stability of lipases during 6 months of storage at 4 °C. ■ free AN lipase; □ immobilized AN lipase; ● free RJ lipase; ○ immobilized RJ lipase. ★ and ♦ represent the total n-3 PUFAs content on soybean oil after acidolysis using immobilized AN and RJ lipases. Each data point represents the average of three experiments and the error bars show the standard deviation. Operational conditions: 13 g of sardine-FFA + soybean oil (3:1), 166 mg of AN lipase or 100 mg of RJ lipase, 12 h. AN, Aspergillus niger; RJ, Rhizopus javanicus.

Fig. 2

TAG profiles obtained by EASI(+)-MS for original soybean oil (A) and soybean oil modified by the acidolysis reactions catalyzed by Aspergillus niger (B) and Rhizopus javanicus (C) lipases.