High uptake of citrus essential oil-loaded zein particles into E. coli tuned by the wet-crosslinked folic acid

Zein particles are increasingly used for encapsulation and delivery of hydrophobic bioactive. Conjugation of zein with multi-carboxylic acids by the alkali-catalysed and wet amidation process causes zein crosslinking and enables tuning the characteristics of zein particles. However, little is known about how the conjugation is associated with the crosslinking mode, i.e., intra and/or intermolecular crosslinks. In this study, folic acid (FoA) was conjugated with zein via the wet method, and citrus essential oil (CEO) was encapsulated within the constructed zein-FoA particles. Infra-red spectroscopy and nuclear magnetic resonance analysis indicated amide bond formation between zein and FoA showing that the content of primary amino groups in zein particles decreased due to conjugation. The mole fraction of FoA in the particles (χFoA) was measured as an index of conjugation extent and even though it did not exceed ∼0.1, conjugation influenced several characteristics of the particles. The contents of α-helix and β-turn structures decreased, and that of β-sheets increased with increasing χFoA. We hypothesized based on ζ-potential and hydrodynamic diameter results, as well as gel electrophoresis that conjugation with FoA caused formation of only intramolecular crosslinks at χFoA ≤ 0.035, but it additionally resulted in intermolecular crosslinks at χFoA ≥ 0.037. Conjugation with FoA at χFoA 0.091 resulted in an improvement of the zein particles biological performance. It caused an increase (∼30%) in the particle uptake by E. coli cells, as well as the magnitude of CEO delivery into the bacteria cells compared with non-conjugated particles.