| Summary: | Compared with the commonly used technique of freeze-drying, spray drying has lower energy costs. However, spray drying also has a fatal disadvantage: a lower survival rate. In this study, the survival of bacteria in a spray-drying tower decreased as the water content was reduced. The water content of 21.10% was the critical point for spray drying <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i> (<i>L. bulgaricus</i>) sp1.1 based on sampling in the tower. Based on the relationship between the moisture content of spray drying and the survival rate, the water content of 21.10% was also the critical point for the change in the survival rate during spray drying. Proteomic analysis was used to investigate the reasons for <i>L. bulgaricus</i> sp1.1 inactivation during and after spray drying. Gene Ontology (GO) enrichment revealed that differentially expressed proteins were mainly associated with the cell membrane and transport. In particular, proteins related to metal ion transport included those involved in the transport of potassium, calcium and magnesium ions. The protein–protein interaction (PPI) network revealed that Ca<sup>++</sup>/Mg<sup>++</sup> adenosine triphosphatase (ATPase) may be a key protein. Ca<sup>++</sup>/Mg<sup>++</sup> ATPase activity decreased substantially during spray drying (<i>p</i> < 0.05). Supplementation with Ca<sup>++</sup> and Mg<sup>++</sup> significantly increased the expression of ATPase-related genes and enzyme activity (<i>p</i> < 0.05). The Ca<sup>++</sup>/Mg<sup>++</sup> ATPase activity of <i>L. bulgaricus</i> sp1.1 was enhanced by increasing the intracellular Ca<sup>++</sup> or Mg<sup>++</sup> concentration, thus increasing the survival of spray-dried LAB. Bacterial survival rates were increased to 43.06% with the addition of Ca<sup>++</sup> and to 42.64% with the addition of Mg<sup>++</sup>, respectively. Ca<sup>++</sup>/Mg<sup>++</sup> ATPase may be the key to the damage observed in spray-dried bacteria. Furthermore, the addition of Ca<sup>++</sup> or Mg<sup>++</sup> also reduced bacterial injury during spray drying by enhancing the activity of Ca<sup>++</sup>/Mg<sup>++</sup> ATPase.
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