Structural and functional evolution of flaxseed protein during probiotic fermentation: Insights into proteolytic degradation, conformational rearrangement, and thermal property

Flaxseed protein (FP) is a promising plant-based protein source, yet its application in food systems is limited by its inherent structural characteristics. This study systematically investigated the structural and functional evolution of flaxseed protein (FP) during fermentation by Lactobacillus bulgaricus and Bifidobacterium lactis . The fermentation process induced significant time-dependent changes in particle size distribution, shifting from large aggregates (> 100 µm) to a homogenized system dominated by 10–100 µm particles, accompanied by the complete disruption of the native dense microstructure into sub‑50 µm dispersed particles. SDS‑PAGE analysis revealed progressive degradation of high‑molecular‑weight fractions (> 100 kDa) and accumulation of low‑molecular‑weight peptides (15–35 kDa), confirming extensive proteolysis. UV‑spectroscopy indicated a decrease in absorbance at 280 nm and a red‑shift in peak position, suggesting the release of aromatic amino acids and a transition toward disordered conformations. Differential scanning calorimetry demonstrated a marked reduction in denaturation temperature (from 149.27°C to 101.77°C) and an increase in enthalpy change (from 16.77 J/g to 22.80 J/g), reflecting decreased thermal stability and enhanced hydration potential. The results collectively delineate a three‑stage mechanism: initial protease‑driven hydrolysis (0–4 h), intermediate hydrophobic‑electrostatic recombination (4–8 h), and final formation of uniform particles (8–10 h). These structural modifications render fermented FP more suitable for gel‑based food applications by lowering its thermal transition requirements and improving water interaction. This work provides a theoretical foundation for the targeted fermentation design of plant proteins to tailor their techno‑functional properties.