Association between casein nanostructure and the freeze-dried survival rate of Streptococcus thermophilus

The viability of lactic acid bacterium, such as Streptococcus thermophilus, is often compromised during freeze-drying. While skim milk is a common cryoprotectant, the mechanism by which metal ions enhance its protective effect remains unclear. This study investigated how sodium and potassium ions influence the cryoprotective efficacy of a skim milk-based formulation by modulating the aggregation state and surface interactions of casein micelle. The interactions between ions and casein were characterized using fluorescence spectroscopy, circular dichroism, and transmission electron microscopy. Our results demonstrated that sodium ions (Na+) were associated with the formation of casein nanostructures with a larger hydrodynamic radius, whereas potassium ions (K+) led to smaller aggregates. This structural divergence was linked to alterations in protein secondary structure and surface hydrophobicity. Crucially, a strong positive correlation was observed between the hydrodynamic size of casein nanostructures and the survival rate of S. thermophilus 937, with sodium salts (e.g., sodium glutamate) yielding a significantly higher survival rate (87.56%) than their potassium counterparts (57.09%). Furthermore, ultrasound-assisted treatment with sodium glutamate was shown to further enhance survival by optimizing protein conformation and solubility. We conclude that the ion-specific modulation of casein nanostructure is a key factor governing cryoprotection, operating within a multifactorial mechanism that includes anion effects and direct ion-cell interactions. This provides a theoretical foundation for designing advanced protective formulations in the starter culture industry.