Novel insights into the coagulation mechanism induced by Dregea sinensis protease: rheological properties, molecular and microstructural changes

This study used rheology, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and molecular docking to explore the effects of Dregea sinensis (D. sinensis) protease at different stages (pre-curd, curd, post-curd) on milk gel gelling. The results demonstrated that milk gels in the curd stage had a water retention capacity of 76.89 ± 0.69 %, significantly higher than those in the pre-curd (62.14 ± 2.28 %) and post-curd stages (69.20 ± 1.33 %). Electrostatic interaction between protease and milk protein in the curd stage led to a small particle size (172.27 ± 1.52 nm) and a high zeta potential (−56.12 ± 3.46 mV), enhancing elastic modulus (G′) and viscous modulus (G″), thereby improving its structural stability. Microstructure exhibited a uniform, dense gel network and a stable crystal structure at 2θ = 19.98°, indicating a composite milk gel. FT-IR analysis demonstrated an absorption peak at 3432.62 cm⁻¹ for the –O-H stretching vibration in the curd stage. Molecular docking further verified that hydrogen bonding and hydrophobic interactions between D. sinensis protease and casein were primarily driven by the active amino acid residues Gln21, Gln94, and Arg142, facilitating the formation of a stable gel state structure. These findings highlight the potential for novel plant rennet in food innovation.