Immobilized Pseudomonas fluorescens Lipase on Eggshell Membranes for Sustainable Lipid Structuring in Cocoa Butter Substitute

Abstract

As the supply of cocoa becomes increasingly volatile, biotechnological innovations such as lipid engineering with lipases play a crucial role in supporting more stable, ethical, and sustainable chocolate production systems. This study explores the potential of Pseudomonas fluorescens lipase immobilized on eggshell membrane-based carriers for the synthesis of a cocoa butter substitute (CBS). The carriers were prepared by treating eggshells with different acids to generate chemically distinct support materials. Lipase immobilization was performed using both adsorption and covalent binding techniques. All resulting biocatalysts were characterized and compared to the free enzyme with respect to pH and temperature optima, as well as thermal and solvent stability. Immobilization caused shifts in the enzyme’s optimal operating conditions and significantly improved its stability at elevated temperatures and in the presence of organic solvents. Among the tested systems, the lipase immobilized by adsorption onto a hydrochloric acid-treated carrier exhibited the best performance. Using this biocatalyst, a CBS containing 93.54 ± 0.16% of the target triacylglycerols (POP, POS, and SOS) was successfully synthesized and reused over five consecutive synthesis cycles without significant loss of activity. These findings demonstrate the potential of waste-derived biomaterials for the development of efficient, stable, and reusable biocatalysts in the enzymatic production of functional lipids.