The beneficial effects of lactic acid bacteria (LAB) have been intensively investigated in recent decades with special focus on modulation of the host intestinal microbiota. Numerous discoveries of effective probiotics are driven by a significantly increasing demand for dietary supplements. Consequently, technological advances in the large-scale production and lyophilization are needed by probiotic-related industries for producing probiotic LAB for commercial use. Our study had a dual

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... to determine the optimum growth medium composition and to investigate appropriate cryoprotective additives (CPAs) for Lactobacillus salivarius, and compare its responses with other Lactobacillus species. The one-factor-at-a-time method and central composite design were applied to determine the optimal medium composition for L. salivarius cultivation. The following composition of the medium was established (per liter): 21.64 g maltose, 85 g yeast extract, 1.21 ml Tween 80, 6 g sodium acetate, 0. g MgSO 4 •7H 2 O, 0.02 g MnSO 4 •H 2 O, 1 g K 2 HPO 4 , 1.5 g KH 2 PO 4 , 0.01 g FeSO 4 •7H 2 O, and 1 g sodium citrate. A cryoprotective additive combination comprising 10% (w/v) skim milk and 10% (w/v) sucrose supplemented with 2.5% (w/v) sodium glutamate was selected for L. salivarius, and its effectiveness was confirmed using culture-independent methods in the freeze-dried cells of the Lactobacillus strains. In conclusion, the optimized medium enhanced the species-specific cultivation of L. salivarius. On the other hand, the cryoprotective effects of the selected CPA mixture may also be dependent on the bacterial strain. This study highlights the necessity for precise and advanced processing techniques for large-scale production of probiotics in the food and feed industries.