Glycerol-Mediated Freeze-Thaw Stabilization of Probiotic-Loaded High Internal Phase Emulsions

This study developed high internal phase emulsions (HIPEs) using glycerol to partially replace water, aiming to enhance freeze-thaw stability for Lactiplantibacillus plantarum P9 (P9). Soy protein isolate was employed as the emulsifier to construct HIPEs with varying glycerol contents (0-40% w/w). The results demonstrated that glycerol significantly improved the physicochemical properties of HIPEs: higher glycerol content reduced droplet size (from 15.13 µm to 4.8 µm), lowered water activity (from 0.92 to 0.76), and enhanced viscosity and viscoelasticity, forming a dense hydrogen-bonded network. Freeze-thaw stability analysis revealed that glycerol suppressed ice crystal nucleation and growth, as evidenced by differential scanning calorimetry showing delayed phase transition temperatures (-19.91°C for 40% glycerol vs. -13.19°C for control). HIPEs with 20% and 40% glycerol retained structural integrity after three freeze-thaw cycles, while control samples exhibited severe phase separation. Furthermore, glycerol-enriched HIPEs significantly improved the viability of encapsulated P9, with post-freeze-thaw survival rates increasing from 7.06 log CFU/g (0% glycerol) to 9.32 log CFU/g (40% glycerol). This study provides a novel strategy for designing HIPEs with superior freeze-thaw stability, facilitating the application of probiotics in frozen functional foods.