Protective Mechanismsof Exopolysaccharide on Lacticaseibacillus rhamnosus During Freeze-Drying:Insights from Physiological Characteristics, Proteomic Analysis, andMolecular Dynamics Simulation

Abstract In this work, the protective mechanism of exopolysaccharide (EPS) on Lacticaseibacillus rhamnosus during freeze-drying was revealed through physiological, proteomic, and molecular dynamics simulation analyses. Physiological analysis suggested that the addition of EPS could enhance lactate dehydrogenase (LDH) and ATPase activities, improve cell membrane integrity, and raise the content of unsaturated fatty acids. Proteomic analysis revealed that EPS significantly upregulated the expression of LDH, regulated energy metabolism and nucleotide metabolism, and enhanced the repair ability of genetic material and the synthesis ability of proteins. Furthermore, molecular dynamics simulations indicated that EPS might form hydrogen bonds with the cell surface and inhibit ice crystal formation, thereby protecting cells from extreme freeze-drying conditions. The results presented in this study suggested that EPS might act as a physical barrier for probiotics, reducing the damage caused by freeze-drying, which would be conducive to the further application of EPS as a natural freeze-drying protective agent.