Excipient Induced\nUnusual Phase Separation in Bovine\nSerum Albumin Solution: An Explicit Role Played by Ion-Hydration

We report an instantaneous room-temperature\nphase separation\nof\n1 mM bovine serum albumin solution in the presence of (20% acetic\nacid+0.2 M NaCl), a routinely used food preservative; an opaque liquid-like\nphase (L) coexists in equilibrium with a granular gel like phase (G).\nInterestingly, neither 20% acetic acid nor 0.2 M NaCl individually\ninduces such a phase separation. Field emission scanning electron\nmicroscopy (FESEM) and high-resolution transmission electron microscopy\n(HRTEM) imaging show aggregated proteins to be dispersed in the upper\nphase, while the lower phase is composed of cross-linked fibrils (hydrogels).\nMid-IR FTIR, Raman scattering, and circular dichroism (CD) experiments\nreveal a significant increase in the β-sheet content in BSA,\nwhich confirms the formation of amyloids in the presence of the excipient.\nBoth L and G phases undergo distinct visual and microscopic changes\nupon incubation at 25 and 80 °C. It is evident that the added\nsalt plays a pivotal role in bringing about this otherwise unique\nphase behavior. We divulge the explicit role of the ion associated\nhydration using THz-FTIR measurements in the 1.5–16.7 THz (50–550\ncm<sup>–1</sup>) frequency window. Systematic alteration in\nthe ion-induced THz-active mode of water envisions the key role of\nions in shaping the various phases. Our study depicts an intriguing\nobservation of severe amyloidosis of BSA upon the addition of a food\npreservative, even at room temperature, which is expected to add new\ninsight into amyloid research. Considering the increasing number of\nindividuals suffering from several neurodegenerative disorders (Alzheimer’s,\nParkinson’s, type-2 diabetes, obesity, cancer, etc.), this\nstudy leads a caution toward critically revisiting the usage of known\nfood preservatives.