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In this study, the Single Factor Test (SFT) was used to optimize the pre-freezing conditions of L. plantarum KLDS1.0391 (KLDS1.0391). Further, the Freeze-Drying Protective Agents (FDPA) of KLDS1.0391 was optimized by Response Surface Methodology (RSM). The optimum pretreatment conditions were as the follows: initial concentration of KLDS1.0391 was 1011 CFU ? mL-1 and KLDS1.0391 was pre-freezed at –80℃ for 8 h to achieve the survival rate of 46.21%. The main components of FDPA were skim milk, sucrose, sodium glutamate and Tween-80. And the influence of four factors on the survival rate of KLDS1.0391 in freeze-drying was in order as the follows: skim milk>sucrose>Tween-80>sodium glutamate. The optimal FDPA composition was skim milk 11.3% , sucrose 9.8% , sodium glutamate 5.1% and Tween-800.2%. Under the above conditions, the survival rate of the cells was 82.98%. Comparing the predicted values, the relative error was 0.37% and the difference was not significant, which indicated that the established model could effectively reflect the actual protection of FDPA to KLDS1.0391.