Novel glyceryl ethers phospholipids produced by solid to solid transphosphatidylation in the presence of a food grade phospholipase D
Authors: Arranz‐Martínez Pablo , Casado Víctor , Reglero Guillermo , Torres Carlos F. 
Published: March 2017

Enzymatic transphosphatidylation between batyl alcohol and phospatidylcholine has been investigated. In situ product removal methodology and the utilization of a green biphasic medium consisting of a buffer and an organic phase comprised of generally recognized as safe flavoring additives, have been established as milestones to increase the applicability of the process. Different organic phases (limonene, pinene, ethyl isovalerate, ethyl lactate, ethyl butyrate, hexanal, and cyclopentanone) and one aqueous phase (0.1 M acetate buffer at pH = 5.5) were studied. The relative proportion of organic and aqueous phases was also investigated. Different molar ratios alkylglycerol/phosphatidylcholine (10:1, 5:1, 2:1, 1:1, and 1:2) were also evaluated. Addition of calcium chloride and the reaction temperature have been adjusted in the enzymatic transphosphatidylation. Finally, the percentage of phospholipase D and the molar concentration of the reactants were also optimized. The ratio transphosphatidylation to hydrolysis was evaluated for the different biphasic reaction mixtures tested. The best results (ca. 70% w/w of glyceryl ether phospholipid) were achieved utilizing 5% (w/w) of phospholipase D, equimolar ratio of reactants and limonene/acetate buffer at a volumetric ratio 1:3. The volumetric productivity of the process was then optimized up to 126 mM and scaled‐up to 50 g of reaction mixture with similar results in terms of composition and yield. This study reports a procedure for the production of tailor‐made phospholipids from commercial sources with high volumetric yield. These highly valuable ingredients are intended to be used as both delivery systems and bioactive lipids.

Practical applications: Solid to solid transphosphatidylation based on in situ product removal has been developed for the synthesis of a novel glyceryl ether phospholipid, incorporating batyl alcohol into the polar head of the phosphatidylcholine in the presence of a food grade PLD. Enzymatic transphosphatidylation usually reported in the literature utilize very low concentration of phospholipid and a large excess of the other reactant. In this sense, the present study evaluated different molar ratios alkylglycerol/phosphatidylcholine. Besides, regarding novel phospholipid as food for human use, the utilization of toxic organic solvents was avoided in its production, replacing them by GRAS organic solvents. Hence, this procedure readily scalable is intended to be used for obtaining this novel glyceryl ether phospholipid with capabilities to perform as both novel delivery systems and bioactive lipids. In addition, a very simple downstream process for product recovery has been also described.

Schematic representation of a solid to solid transphosphatidylation reaction between phosphatidylcholine and batyl alcohol in a biphasic reaction medium comprised of an acetate buffer and an organic solvent generally recognized as safe (limonene).