| Summary: | <i>Lactobacillus acidophilus</i> strains have limiting factors such as low cell density and complex nutritional requirements in industrial production, which greatly restricts their industrial application. In this study, fermentation conditions for <i>L. acidophilus</i> were optimized and transcriptomic analysis used to understand growth mechanisms under high-density fermentation conditions. We found that <i>L. acidophilus</i> IMAU81186 has strong tolerance to gastrointestinal juice. In addition, its optimal culture conditions were 3% inoculum (<i>v</i>/<i>v</i>); culture temperature 37 °C; initial pH 6.5; and medium composition of 30.18 g/L glucose, 37.35 g/L soybean peptone, 18.68 g/L fish peptone, 2.46 g/L sodium citrate, 6.125 g/L sodium acetate, 2.46 g/L K<sub>2</sub>HPO<sub>4</sub>, 0.4 g/L MgSO<sub>4</sub>·7H<sub>2</sub>O,0.04 g/L MnSO<sub>4</sub>·5H<sub>2</sub>O, 0.01 g/L serine, and 0.3 g/L uracil. After optimization, viable counts of IMAU81186 increased by 7.03 times. Differentially expressed genes in IMAU81186 were analyzed at different growth stages using transcriptomics. We found that a single carbon source had limitations in improving the biomass of the strain, and <i>ter</i>P and <i>bfr</i>A were significantly down-regulated in the logarithmic growth period, which may be due to the lack of extracellular sucrose. After optimizing the carbon source, we found that adding 12 g/L sucrose to the culture medium significantly increased cell density.
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