Plant-Based Protein Bioinks with Transglutaminase Crosslinking: 3D Printability and Molecular Insights from NMR and Synchrotron-FTIR

The increasing demand for sustainable and functional plant-based foods has driven interest in 3D food printing technologies, which require bioinks with tailored rheological and structural properties. This study investigated the effects of transglutaminase (TGase) on the structure–function relationships of plant protein bioinks from fava bean, mung bean, pea, and soybean. TNBS assays showed a dose-dependent increase in crosslinking (27.46–64.57%), with soybean and pea proteins exhibiting the highest reactivity (p < 0.05). 1H-NMR confirmed protein-specific ε-(γ-glutamyl)lysine bond formation, and synchrotron FTIR revealed TGase-induced α-helix reduction and β-sheet enrichment, indicative of network formation across all proteins. SDS-PAGE analysis demonstrated TGase-mediated polymerization with high-molecular-weight aggregates, particularly pronounced in soybean, while SEM images revealed denser, more continuous protein networks compared to untreated samples. Rheological characterization showed enhanced viscoelasticity and shear-thinning behavior in all bioinks, supporting extrusion and post-printing stability. Textural analysis indicated improvements in hardness, springiness, cohesiveness, and chewiness across all proteins, with soybean and fava showing the most pronounced increases. These results demonstrate that TGase is a versatile tool for reinforcing plant protein networks, improving printability, structural integrity, and texture in 3D-printed foods, while highlighting protein-specific differences in response.