In this study, optimization for culture condition was performed to confirm the feasibility as a starter of Acetobacter pasteurianus, a strain that can be used as a food ingredient. The optimal temperature and pH conditions of the selected Acetobacter pasteurianus SRCM101388 were 28°C and pH 6.00, respectively. Response surface methodology (RSM) was used to optimize medium composition, and Plackett-Burman design (PBD) was used to obtain the effective selection of culture medium, resulting in that glucose, sucrose, and yeast extract had the highest effect on increasing biomass. The optimal concentration, which was performed by central composite design (CCD), were determined to be 10.73 g/L of glucose, 3.98 g/L of sucrose, and 18.73 g/L of yeast extract, respectively. The optimal concentrations of trace elements for the production of biomass were found to be 1 g/L of ammonium sulfate, 0.5 g/L of magnesium sulfate, 2 g/L of sodium phosphate monobasic, 2 g/L of sodium phosphate dibasic, and the final optimized medium was pH 6.10. From the result of incubation in 5 L jar fermenter, SRCM101388 had faster DO(dissolved oxygen) reduction at lower RPM than higher RPM, and it was able to grow even under the condition in which the aeration was maintained. The biomass was 2.53±0.12×109 CFU/mL when incubated for 18 hours at 150 rpm, 0.5 vvm, pH 6.0, and 28°C. Fed-batch culture was performed in a 300 L fermenter. Specific growth rate was faster than that of jar fermenter, and the viable cell count increased by 28 hours. The final biomass was 5.15±0.35×109 CFU/mL at 80 rpm, 1.0 vvm, 0.2 bar, pH 6.0 and 28°C in 300 L fermenter. Feeding medium was added 15.4 g/L of glucose and 5.6 g/L of sucrose. The cryoprotective agent effect and survival rate during freeze-drying were confirmed by using a food grade cryoprotective agent. The viability after freeze-drying were in the order of maltodextrin (86%), trehalose (81%), and maltose (74%). After 8 months, the viability is Maltose (11%), trehalose (9%), and maltodextrin (3%) were shown in order.