How Gellan gum gelation is affected by acyl substituents: from hierarchical organization to simple viscoelastic behavior.

Gellan gum, a linear anionic exopolysaccharide, is widely employed as a gelling agent owing to its biocompatibility and tunable rheological properties. Its degree of acylation determines high acyl (HA-Gg) and low acyl (LA-Gg) forms, yielding hydrogels with different chemical-physical properties. Rheological measurements show a distinct gelation mechanism: LA-Gg forms rigid, ionically crosslinked networks (G' ∼25 kPa; critical strain ≈2%) exhibiting two-step yielding, whereas HA-Gg produces softer elastic gels lacking hierarchical organization (G' ∼1 kPa; LVR extends up to ∼500% strain). Despite their wide use, the molecular mechanism by which acyl substituents affect Gellan gum gelation is still lacking. Here a comparison between HA-Gg and LA-Gg, combining spectroscopy, rheology, and atomistic molecular dynamics (MD) simulations within a single framework is performed, providing a molecular-level interpretation of these differences. In dilute regime, circular dichroism measurements reveals distinct behavior for LA-Gg and HA-Gg, in coil and double-helix conformations, respectively. MD simulations explain the observed features, showing that acylation enhances intra-helix hydrogen bonding, stabilizing the double-helix structure, while hindering calcium-mediated inter-helix associations. Overall, acylation exerts a dual effect: it strengthens local structural units, but weakening supramolecular connectivity. This interplay governs macroscopic mechanical response, enabling rational design of Gellan gum hydrogels with tailored properties.