# 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

## TL;DR

This paper introduces a new method using A4F-MALLS to study how heat treatment affects insect protein aggregation and polymerization in Tenebrio molitor larvae.

## Contribution

The novel use of A4F-MALLS coupling provides a detailed characterization of heat-induced protein aggregation and polymerization in insect proteins.

## Key findings

- Heat treatment increases protein aggregation, reducing solubility by 35%.
- Protein polymerization occurs via intermolecular S-S bonds, forming high molar mass and compact structures.
- A4F-MALLS effectively quantifies and characterizes aggregated and polymerized proteins.

## Abstract

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 Tenebrio molitor 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 (Mw > 107 g/mol, RGw >130 nm) and compact polymer structure (v ≤ 0.35) and resulting in intermolecular S-S bonds that preferentially mobilise proteins with Mw > 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.

Image 1

•Heat treatment of larvae proteins significantly promote their aggregation.•A4F-MALLS coupling allow the quantification and characterization of aggregates.•Heat treatment promote protein polymerisation by intermolecular S-S bond formation.•Molar mass distribution and conformation of polymers can be determined by A4F-MALLS.

Heat treatment of larvae proteins significantly promote their aggregation.

A4F-MALLS coupling allow the quantification and characterization of aggregates.

Heat treatment promote protein polymerisation by intermolecular S-S bond formation.

Molar mass distribution and conformation of polymers can be determined by A4F-MALLS.