Encapsulation of Aspartic Protease in Nonlamellar Lipid Liquid Crystalline Phases

Encapsulation of proteins within lipid inverse bicontinuous cubic phases (Q₂) has been widely studied for many applications, such as protein crystallization or drug delivery of proteins for food and pharmaceutical purposes. However, the use of the lipid sponge (L₃) phase for encapsulation of proteins has not yet been well explored. Here, we have employed a lipid system that forms highly swollen sponge phases to entrap aspartic protease (34 kDa), an enzyme used for food processing, e.g., to control the cheese-ripening process. Small-angle x-ray scattering showed that although the L₃ phase was maintained at low enzyme concentrations (≤15 mg/mL), higher concentration induces a transition to more curved structures, i.e., transition from L₃ to inverse bicontinuous cubic (Q₂) phase. The Raman spectroscopy data showed minor conformational changes assigned to the lipid molecules that confirm the lipid-protein interactions. However, the peaks assigned to the protein showed that the structure was not significantly affected. This was consistent with the higher activity presented by the encapsulated aspartic protease compared to the free enzyme stored at the same temperature. Finally, the encapsulation efficiency of aspartic protease in lipid sponge-like nanoparticles was 81% as examined by size-exclusion chromatography. Based on these results, we discuss the large potential of lipid sponge phases as carriers for proteins.