Molecular mechanisms of plasmin-induced dense aging gels in skimmed milk from different direct UHT treatments revealed based on peptidomics and molecular dynamics simulations.

This study investigated the molecular mechanisms underlying the formation of dense aging gels in skimmed milk subjected to different direct ultra-high temperature (dUHT) treatments. The results showed that in accelerated assays, plasmin could interact with αs1-casein (αs1-CN), αs2-CN, β-CN, κ-CN, and La through van der Waals interactions, hydrophobic interactions, and hydrogen bonding. During storage, dUHT milk samples treated at 147 °C for 6 s, 142 °C for 6 s, and 142 °C for 3 s exhibited an 8 % reduction in α-helix content, along with an increase in β-fold and irregular curl content. The dUHT treatment disrupted the tertiary structure of milk protein molecules, leading to a decrease in surface hydrophobicity and surface free sulfhydryl content. Peptidomics analysis indicated that plasmin mainly hydrolyzed α-CN and β-CN. The hydrolysis sites of four casein monomers and whey proteins by plasmin were identified, and this was further confirmed by molecular docking. Peptide hydrogel modeling and molecular dynamics results demonstrated that electrostatic interactions, van der Waals forces, and hydrogen bonding were the key forces maintaining the three-dimensional mesh structure of peptide hydrogel gels. This study revealed the formation mechanism of dUHT milk aging gel, which has practical guidance for the process of dUHT milk processing.