The objective of this research was to determine the concentration of lactose oxidase (LO) needed to activate the lactoperoxidase system (LPS) in skim milk and assess its ability to inhibit the growth of Pseudomonas fragi, a milk spoilage strain. LO oxidizes the lactose in milk and produces hydrogen peroxide needed for the activation of the antimicrobial system. Seven treatments were evaluated at 4 and 21 °C: the control, and three levels of LO or LPS+LO (0.012, 0.12 and 1.2 g/L). The base LPS was obtained adding 30 mg/L of bovine lactoperoxidase and 14 mg/L of NaSCN to ultra-pasteurized skim milk. Three independent trials of the experiment were performed and the microbial reduction was calculated for 1, 4 and 7 days. The effect of treatment, temperature,
time and their interactions was determined through a multi-factorial analysis. Also, for both temperatures, a one-way ANOVA was conducted separately for each day to determine the significance of the treatments followed by a Tukey’s test. The results showed that treatments were more effective at refrigeration temperature (p<0.001). At 4 °C, LO at 0.12 and 1.2 g/L showed a significantly higher reduction than the control (p<0.001) when added alone and combined with the system for every time point. An increase in the concentration of LO caused higher reductions of P. fragi at day 7, achieving a >2.93 log CFU/mL reduction for the 1.2 g/L treatments. At 21 °C, treatments with a concentration of 1.2 g/L of LO achieved a reduction of >2.93 log CFU/mL, while under the other conditions reductions were not significantly different from the reduction observed for the control (p<0.05). Results confirm that lactose oxidase can be used to inhibit the growth of Pseudomonas fragi and represents a new way to extend the shelf-life of dairy products. The application of LO serves as an opportunity to reduce food waste and for the dairy industry to benefit from a longer shelf-life while meeting the consumer’s demand for clean label products. Further research will assess the inhibition of other spoilage microorganisms in different dairy products, as well as the effect of the inoculation level and thiocyanate concentrations.