Tuning lysozyme nanofibers dimensions using deep eutectic solvents for improved reinforcement ability.

Deep eutectic solvents (DESs), a novel generation of solvents, have recently been described as efficient and timesaving fibrillation agents for proteins. In this context, the present work aims at assessing the effect of the hydrogen bond donor (HBD) of cholinium chloride ([Ch]Cl):carboxylic acid based DESs on the dimensions (length and width) of lysozyme nanofibers (LNFs). Mono-, di- and tri-carboxylic acids (acetic, lactic, levulinic, malic and citric acids) were used to prepare different DES formulations, which were successfully used on the fibrillation of lysozyme. The results showed that the carboxylic acid (i.e. the HBD) plays an important role on the fibrillation efficiency and on the length of the ensuing LNFs with aspect-ratios always higher than those obtained by fibrillation with [Ch]Cl. The longest LNFs were obtained using lactic acid as the HBD with an average length of 1004 ± 334 nm and width of 31.8 ± 6.8 nm, and thus an aspect-ratio of ca. 32. The potential of these protein nanofibers as reinforcing additives was evaluated by preparing pullulan (PL)-based nanocomposite films containing 5% LNFs with different aspect-ratios, resulting in highly homogenous and transparent films with improved mechanical performance.