Abstract: The aim of this study was to obtain high-density encapsulated Enterococcus faecalis by optimization of encapsulated fermentation medium.In this study,the viable encapsulated bacteria concentration was used as the evaluation index,Plackett-Burman(PB)test was used to analyze the main factors of encapsulated fermentation medium. Then,response surface method(RSM)was used to optimize the main factors. The stability of freeze-dried powder of Enterococcus faecalis cultured under the optimal conditions was studied. Results showed that,the optimal fermentation medium was designed as follows:Glucose 4.93%,peptone 1%,beef extract 0.8%,yeast 0.6%,ammonium citrate 0.3%,K₂HPO₄·7H₂O 0.2%,MgSO₄·7H₂O 0.06%,MnSO₄·H₂O 0.045%,NaAc·3H₂O 0.6%,Tween 80 0.1%,sodium alginate 1.87%,and nanometer calcium carbonate 3.65%.Under these conditions,the viable encapsulated Enterococcus faecalis reached 6.83×10⁹ CFU/mL,with an error of only 4.12% compared with the predicted value. Storage stability test showed that the prepared freeze-dried powder still retained more than 80% survival rate after 90 d storage at 37 ℃,indicating that the freeze-dried powder of encapsulated cultured Enterococcus faecalis had high stability.