Characterization of zein assemblies by ultra-small-angle X-ray scattering

Zein, a major corn protein, has an amphiphilic molecule capable of self-assembling into distinctly different structures, i.e., rods, sheets, and spheres. In this work, ultra-small-angle X-ray scattering (USAXS) was applied to investigate the formation of self-assembled zein structures in binary solvent systems of ethanol and water. The study included observing structural changes due to aging. Three distinctive regions, each corresponding to different co-existing structures having a hierarchical organization, were observed in zein-solvent systems. Rod shaped (R-g = 1.5-2.5 nm, P = 1) primary structural units were identified, believed to be molecular zein. Two-dimensional sheet-like structures (R-g = 80-200 nm, 2 < P < 3) were observed, believed to be formed by primary units first assembled into one-dimensional fibers and then into 2D sheet structures. Also, large three-dimensional spherical aggregates were observed (Rg > 1000 nm, P = 4), believed to have assembled from two-dimensional sheet structures. Aging did not change the size or the shape of the primary units, but USAXS detected changes in Rg and P values of the intermediate structures, pointing to a further level of self-assembly where proteins develop a more regular and organized structure. The viscoelastic moduli (G ' and G ''), the consistency index (K) and the flow behavior index (n), were also measured to investigate the effect of zein structural development by self-assembly on rheological behavior. Samples became more solid-like with aging. Raman spectra suggested that zein underwent secondary structure transformations from alpha-helix to beta-sheets, which influenced the size and morphology of molecular assemblies and ultimately the rheological properties of zein solutions.