The autochthonous strain Latilactobacillus sakei sp. sakei ACU-2 was selected as a meat starter culture for dry sausage production. Transferring this strain from laboratory scale to industry requires an increase in biomass production, while lowering process costs. In this study, a combination of techniques was applied in order to optimize the culture medium composition to enhance biomass production of L. sakei ACU-2. One variable at a time experiments, Plackett–Burman design, and mixture design were performed to fulfill the strain nutritional requirements. Eventually, the optimized formulation contained 19.46 g/L yeast extract; 8.28 g/L whey protein concentrate; 2.26 g/L soy peptone; 30 g/L cerelose; 1 g/L Tween 80; 5 g/L sodium acetate; 0.2 g/L magnesium sulfate and 0.05 g/L manganese sulfate. When L. sakei ACU-2 was cultivated in a bioreactor using the alternative medium, an enhancement of 75.5% of biomass production was achieved, in comparison to its growth in the commercial de Man, Rogosa, and Sharpe medium. Furthermore, a reduction of 62–86% of the cost was also attained. These results support a promising large-scale application of the designed medium for high biomass yields of the starter culture at minor costs.