Lipase-catalyzed production of structured triacylglycerols

Abstract: Vegetable oils and fats are major energy sources but also play an essential part in the physical and chemical properties of food products. The current change in the climate is affecting harvest yields, leading to a shortage and increased prices of raw materials that are difficult to replace. In addition, there is an increasing demand for new products with positive health effects.Triacylglycerols (TAGs) with specific properties can be produced using synthesis methods controlling which fatty acids are present in each position of the molecule. These are called structured triacylglycerols (STAGs) and enzymatic transesterification is a good method to produce them. Immobilized lipases are the catalyst of choice for STAG production as they can selectively tailor the fatty acid composition in the triacylglycerol to obtain the desired properties. Few industrial processes have been developed despite the positive aspects of lipase-catalyzed synthesis and the continuously growing application areas. Further implementation of industrial processes requires highly efficient and stable catalysts and optimized reactor systems to overcome the limitations of too high production costs. This dissertation attempts to improve the production of STAGs by the development of a comprehensive lipid analysis method and effective lipase catalysts, and optimization of reaction conditions.Analysis of reaction samples is a central part of research and quality control. There is thus a great need for efficient and cost-effective methods for the analysis of the composition of vegetable oils and fats. In this work, a high-performance liquid chromatography method with charged aerosol detection was developed for complete analysis of lipid samples. Within 80 minutes the method manages to separate free fatty acids, fatty acid esters, monoacylglycerols, diacylglycerols (DAGs) and TAGs, including several regioisomers. The developed method facilitated the performance of the succeeding research.The properties of an immobilized preparation will depend on the combination of lipase and support. In this work, it was found that the immobilization conditions and support material had profound effects on the catalyst activity. Immobilization by adsorption on highly hydrophobic supports resulted in significantly higher immobilization yield and activity, especially when compared to covalent binding. We managed to develop immobilized preparations with activities equal to commercially available preparations and even higher regioselectivity. This shows the possibility of STAG synthesis with high productivity and purity.Transesterification for the synthesis of STAGs is a multistep reaction with DAGs formed as intermediate products. Many factors influence the initial reaction rate, product yield and quality. The effects of water activity, temperature, substrate ratio and ethanol concentration were studied in the reaction between high oleic sunflower oil and ethyl stearate producing 1,2-stearin-3-olein (SOS) using immobilized lipase from Rhizopus oryzae (Lipase DF “Amano” IM). It was found that the factors influencing the enzymatic activity (water activity, ethanol addition, substrate ratio, and temperature) were highly important initially, whereas factors affecting the thermodynamic equilibrium (water activity, ethanol addition, and substrate ratio) dominated later. This emphasizes the importance of controlling the reaction parameters. The product quality was negatively affected by temperature, as an increased temperature promoted acyl migration.A water activity control system based on dry/wet nitrogen gas sparging was developed and implemented in a SpinChem® rotating bed reactor to control the water activity in non-aqueous media during enzymatic reactions. With a stepwise change in water activity from high (0.65) to low (drying of reaction solution), it was possible to increase the initial DAG and product formation while reducing the final DAG content to 3.1%. In addition, a final yield corresponding to 78% of the theoretical maximum was obtained using 0.5% (w/w) enzyme and 7 h of reaction. Excellent regioselectivity of Lipase DF “Amano” IM and minimized acyl migration resulted in only 0.11% undesired incorporation in the sn-2-position.