The health benefits of polyunsaturated fatty acids are well known, however their fragility is a drawback as it leads to their degradation. The objective of this study was to develop innovative assemblies, using the capacity of starch to encapsulate small molecules, for the protection of polyunsaturated fatty acids like linoleic acid and linolenic acid. These assemblies were produced by hydrothermal treatment, and by high-pressure treatment (20min at 500MPa at a temperature of 40°C). They were then analyzed and characterized by X-ray diffraction (XRD), differential scanning calorimetry (DSC), nuclear magnetic resonance (13C CP/MAS NMR) and a heating cell in XRD. The results showed that amylose–ligand complexes were formed during both treatments and two crystalline types V6I and V6II were obtained. The techniques used show excellent complementarities in the determination of important structural features such as crystalline type, helical conformation and nature of the inclusion.