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 (<i>p</i> < 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.