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In the present investigation, a low-cost fermentation medium was designed to achieve the maximum production of lactase from the Bacillussp. VUVD101 strain through the screening of different nutritional and physical variables, using the Plackett-Burman design. Fourteen variables of the fermentation process were selected: incubation time, temperature, pH, RPM, DO, inoculum size, inoculum age, MgSO4, l-Cysteine, KH2PO4, CaCl2, K2PHO4, corn steep liquor and lactose. The selected variables were evaluated through statistical analysis, based on their significance, coefficient value and standard effect plot. The results suggested that six variables, namely, corn steep liquor, lactose, MgSO4, temperature, pH and RPM, had influence with high confidence levels, while the remaining eight variables did not show a significant effect on production. The analysis of the variance value R2(0.96) also showed the model used for prediction to be significant (p less than 0.05). The plot for the standard effect for each component and its traits provided accurate data by which to select well-suited variables for further optimization. In comparison with the basal medium, 68% higher enzyme activity was achieved from the model of the optimized medium, and lactase activity was found to be 18.31 U/ml.