PURPOSE:To obtain the titled spheroplast useful for breeding a bacterium for vinegar production in high formation ratio, by treating a bacterium belonging to the genus with a cell wall digesting enzyme in the presence of EDTA and an osmotic pressure stabilizer in a solution having a specific pH. CONSTITUTION:A bacterium [e.g. Acetobacter aceti No.1,023 (FERM P-7122)] is subjected to a shaking culture in a medium, centrifuged and the cell is collected. The cell is suspended in a solution having pH <=7 such as phosphoric acid buffer solution in the presence of EDTA and an osmotic pressure stabilizer (e.g. sucrose) and then treated with a cell wall digesting enzyme (e.g. glair lysozyme) at 30 deg.C for 5min to prepare spheroplast of a bacterium belonging to the genus Acetobacter in high formation ratio.