In vitro digestion and molecular dynamics reveal mechanisms of digestion resistance in type V resistant starch: Binary versus ternary complexes with lipids and/or proteins.

To elucidate the resistance of type V resistant starch to enzymatic digestion, this study investigated the relationship between the crystalline structure and digestibility of complexes formed with lipids or proteins. The incorporation of octadecanoic acid (OA) and rice protein (RP) facilitated the formation of type V crystalline structures in native rice starch (NRS), increasing its crystallinity in the early stages of digestion. During in vitro digestion, the short-range ordered structure and type V crystal content of the NRS-RP and NRS-OA-RP complexes progressively increased. In contrast, these parameters in the NRS-OA complex initially increased and then decreased. Molecular dynamics simulations revealed that binding with the NRS-RP and NRS-RP-OA reduced the stability of the digestive enzyme, as evidenced by increased radius of gyration (Rg), root-mean-square deviation (RMSD), and solvent-accessible surface area (SASA) values, accompanied by highly unstable fluctuations. This instability was further characterized by a reduced number of hydrogen bonds and a Gibbs free energy landscape displaying multiple energy minima. Binding free energy analysis further demonstrated that complex formation reduced the binding affinity between the enzyme and starch, with the ternary NRS-OA-RP complex exhibiting the lowest binding energy (-17.52 kJ/mol). In conclusion, these findings suggest that the enhanced resistance to digestion of various type V RS complexes arises from a dual mechanism involving the formation of structural barriers and the modulation of molecular interactions that reduce enzyme-starch binding affinity.