Asymmetrical flow field-flow fractionation and multi-angle laser light scattering: A new analytical approach for the characterisation of insect protein aggregation/polymerisation after heat treatment of Tenebrio molitor larvae

Understanding the structural modifications of insect proteins during the transformation processes used for extract preparation is essential for optimising their functionalities and obtaining high added-value proteins. From this perspective and in addition to classical analytical approaches, we developed an original methodology based on the implementation of Asymmetrical Flow Field-Flow Fractionation and Multi-Angle Laser Light Scattering (A4F-MALLS) coupling to quantify and characterise the aggregation/polymerisation phenomena of <i>Tenebrio molitor</i> larvae proteins after heat treatment (from 65 to 95 °C). Applied to heat-treated larvae proteins in conjunction with the evaluation of intrinsic fluorescence, surface hydrophobicity and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the AF4-MALLS method enabled us to quantify and characterise the aggregated proteins (forms dissociable after urea denaturation), determining the polymer/monomer (P/M) ratio. Heat treatment significantly affects solubility (-35 %), which is due to the amplification of aggregation phenomena, as demonstrated by the increase in the P/M ratio ( × 10). Moreover, the method enabled us to quantify and characterise the polymerised protein (forms dissociable after chemical reduction of intermolecular bonds), identifying the elements by molar mass and size distribution and conformation. Proteins with cysteine groups can be polymerised under heat, causing a thiol-disulphide exchange reaction and forming a strong (M_w > 10⁷ g/mol, R_Gw >130 nm) and compact polymer structure (<i>v</i> ≤ 0.35) and resulting in intermolecular S-S bonds that preferentially mobilise proteins with M_w > 80 kDa. Given its performances, the AF4-MALLS method is a real opportunity to understand the effects of processing methods, such as thermal and non-thermal treatments, to optimise protein functionalities.