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Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits but remains commercially underexploited due to its inability to survive during long-term storage in the dried state. Our objective was to study the effect of various protective molecules (maltodextrin, trehalose, antioxidants, and fructooligosaccharides), being efficient on other bacteria, on the freeze-dried stability of L. salivarius CECT5713. The culturability was evaluated after freezing, freeze-drying, and subsequent storage at 37 °C, as well as the biochemical composition of cells in an aqueous environment using Fourier Transform Infrared (FTIR) micro-spectroscopy. The assignment of principal absorption bands to cellular components was performed using data from the literature on bacteria. The membrane fatty acid composition was determined after freeze-drying and storage. Glass transition temperature of the liquid and freeze-dried bacterial suspensions and water activity of the freeze-dried samples were measured. The best storage stability was observed for the formulations involving maltodextrin and antioxidants. The analysis of the FTIR spectra of freeze-thawed cells and rehydrated cells after freeze-drying and storage revealed that freeze-drying induced damage to proteins, peptidoglycans of the cell wall and nucleic acids. Storage stability appeared to be dependent on the ability of the protective molecules to limit damage during freeze-drying. The inactivation rates of bacteria during storage were analyzed as a function of the temperature difference between the product temperature during sublimation or during storage and the glass transition temperature, allowing a better insight into the stabilization mechanisms of freeze-dried bacteria. Maintaining during the process a product temperature well below the glass transition temperature, especially during storage, appeared essential for L. salivarius CECT5713 storage stability. Key points L. salivarius CECT5713 highly resisted freezing but was sensitive to freeze-drying and storage  Freeze-drying and storage mainly altered cell proteins, peptidoglycans and nucleic acids  A glassy matrix containing maltodextrin and an antioxidant ensured the highest storage stability

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