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The aim of this work was to use response surface methodology (RSM) approach, a statistical mathematical tool, to model effects and interactions of glucose oxidase (GOD), glucose, lactoperoxidase (LPO) and pH-values on the thiocyanate (SCN−) peroxidation, to determine the best concentrations of lactoperoxidase system (LP-s) components in order to obtain maximal SCN− peroxidation and so to enhance the LP-s antibacterial effects.Experimental design using RSM was used for modelling effects and interactions of GOD (28·5–142·5 IU l−1), glucose (0·55–11·11 mmol l−1), LPO (0–6284 IU l−1) concentrations, and pH-values (6·0–7·4) on thiocyanate peroxidation. A fixed SCN− concentration of 0·5 mmol l−1 was used. Experiments were carried out at 4 or at 25°C in 0·1 mol l−1 phosphate buffer. Optimized concentrations for both temperatures (4 and 25°C) were quite similar and were 85·5 IU l−1 for GOD, 8 mmol l−1 for glucose and 3927·5 IU l−1 for LPO at an initial pH-value of 6·5. SCN− peroxidation was more efficient at 25 than at 4°C. At 4°C, no interaction between factors occurred. At 25°C, thiocyanate peroxidation was affected by GOD/glucose, GOD/pH and LPO/pH. Thiocyanate peroxidation was mainly increased by glucose and LPO factors. The optimized system had a bacteriostatic effect on Listeria monocytogenes CIP 82110T and a strong bactericidal effect on Pseudomonas fluorescens CIP 6913T.Appropriate combinations of LPO, GOD, glucose concentrations and pH-values allowed maximal thiocyanate peroxidation and enhanced the antibacterial effect of the LP-s.This optimization by RSM approach allowed a better understanding of the LP-s functioning, the description of the component impacts on the SCN− peroxidation, and the observation of different interactions between the factors. The antimicrobial efficiency of LP-s can be enhanced by better concentration ratios of the LP-s components.