Designing Wet-Spun Cultivated Meat Muscle Fibers: The Impact of Faba Bean Protein Isolate Hydrolysis and Concentration in a Calcium-Alginate System

Replicating whole-muscle texture remains a central barrier for alternative proteins, with persistent gaps in soft-texture, fibrous anisotropy and moisture retention. Wet spinning offers a non-thermal, scalable route to produce aligned fibers, while enzymatic hydrolysis can tune protein physicochemical attributes to expand formulation options. This study evaluated faba bean protein isolate (FPI) at varied degrees of hydrolysis (DH 0-7.6%) and concentrations (0-6% w/w) as functional components in wet-spun calcium-alginate fibers designed for muscle mimetic analogs. Native and hydrolyzed FPI were characterized by SDS-PAGE and formol titration. Protein-alginate dispersions were assessed by confocal microscopy, ζ-potential, and rheological testing. Fibers were evaluated for morphology (SEM), cross-sectional area (CSA), Young's modulus, water-holding capacity (WHC), thermal stability (TGA), and early-stage ovine preadipocyte attachment. Hydrolysis shifted molecular weight from ∼18–100 kDa to <15 kDa and yielded dispersions with lower viscosity. Native FPI fibers (4-6% w/w) exhibited higher WHC (∼73% compared to ∼55%), elevated thermal degradation temperatures (∼295-310 °C compared to ∼242-256 °C), and significantly greater preadipocyte attachment than hydrolysate or control fibers. However, native FPI at high inclusion reduced Young's modulus (∼12 kPa) relative to hydrolysate formulations (∼20-23 kPa), potentially consistent with less uniform calcium-alginate network formation. Hydrolysate fibers maintained smooth, uniform morphology resembling alginate control. Protein physicochemical state, including native structure and degree of hydrolysis, functions as a primary design lever governing trade-offs between cell compatibility and matrix mechanics in wet-spun alginate-protein composites. These findings advance scaffold design for cultivated meat by establishing tunable protein parameters for balancing texture and cellular adhesion.