Peroxyl radical-mediated oxidative crosslinking in Faba Bean and Pea Proteins: Structural roles of di-Tyr and di-Trp for the formulation of high-moisture extruded plant-based meat analogues

Abstract: The development of plant-based meat analogues through high-moisture extrusion (HME) has intensified due to sustainability considerations; however, during HME, proteins are exposed to thermomechanical conditions that may promote oxidative reactions and potentially alter the structuring mechanisms responsible for fibrosity. Recent studies indicate that during extrusion processes lipid and protein oxidation may occur, inducing the formation of carbonyl groups and covalent bonds, which could contribute to the stabilization of the characteristic fibrous structure of meat analogues. In this context, we investigated peroxyl radical (ROO)-mediated oxidative crosslinking, as a chemically relevant model for oxidative processes in food matrices, in faba bean (Vicia faba, FP) and pea (Pisum sativum, PP) proteins and their blends, aiming to elucidate how protein composition modulates the formation of covalent bonds associated with structural protein networks in meat analogues. ROO induced oxidation (AAPH 100 mM) generated comparable levels of hydroperoxides and carbonyl groups across formulations, without detectable changes in the overall secondary structure. However, a marked protein source–dependent behavior was observed: in contrast to PP, whose oxidation led to only minor changes in molecular weight distribution, FP oxidation resulted in pronounced protein crosslinking and the formation of high-molecular-weight aggregates; in blended systems, covalent aggregation progressively decreased as the FP fraction was reduced. LC–MS/MS analysis confirmed the formation of di-tyrosine species in FP, PP, and their blends, whereas di-tryptophan formation was detected exclusively in FP, supporting a differential oxidative crosslinking mechanism associated with the major proteins (legumin ~38 kDa, and vicilin ~47 kDa). Overall, these results demonstrate that the faba bean-to-pea protein ratio allows modulation of the density and nature of oxidative crosslinking under ROO conditions. Given that the formation of anisotropic fibrous structures during HME relies on protein unfolding, alignment, and subsequent stabilization of protein–protein networks, we propose that these covalent linkages may play a structural role in consolidating the protein network during extrusion, thereby influencing final fibrosity. Future studies will correlate di-Tyr/di-Trp formation and covalent aggregation with fibrosity degree, anisotropy, and microstructure in high-moisture extruded products.