Calcium and magnesium improve Lactobacillus bulgaricus survival via membrane and metabolic protection

Spray drying is widely used for probiotic powder production, but low bacterial survival limits industrial application. Improving cell tolerance to spray-drying stress remains a major challenge. The purpose of this work was to investigate the underlying mechanisms by which calcium (Ca²⁺) and magnesium ions (Mg²⁺) protect Lactobacillus bulgaricus and increase its survival during spray drying. Proteomic analysis revealed that spray drying severely impaired fatty acid metabolism and the glycolytic pathway in Lactobacillus bulgaricus. The addition of Ca²⁺ (10 mmol/L) or Mg²⁺ (20 mmol/L) led to significant up-regulation of the genes related to these pathways, concurrently increasing Ca²⁺/Mg²⁺ ATPase activity after spray drying. The maintenance of membrane integrity by these two ions was achieved through the reduction of depolarization and membrane damage, with a concomitant reduction in lipid oxidation of 18–22 %. In addition, they increased the intracellular ATP levels (1.86–2.08 times) and maintained membrane fluidity by increasing the unsaturated fatty acid content. These effects synergistically stabilize energy metabolism and the membrane structure, resulting in an increased survival rate. Overall, this study demonstrated that Ca²⁺ and Mg²⁺ enhance the spray drying tolerance of Lactobacillus bulgaricus by coordinating lipid homeostasis and bioenergetics, providing a practical strategy to improve probiotic stability.