The lactoperoxidase system (LS), an antimicrobial system naturally present in milk that is activated by H 2 O 2 , has been used to inhibit microbial outgrowth in raw milk in areas where refrigeration is not viable. This study evaluated lactose oxidase (LO) as a novel activator of the LS. Lactose oxidase oxidizes lactose and produces H 2 O 2 needed for the activation of the LS. The antimicrobial effect of different concentrations of LO with and without components of the LS, thiocyanate (TCN) and lactoperoxidase (LP), was evaluated in model systems and then applied in pasteurized milk and raw milk. In general, an increase in LO caused greater reductions of Pseudomonas fragi in the model systems and treatments were more effective at 6°C than at 21°C. At 6°C, the LO solution at 0.12 and 1.2 g/L showed significantly higher microbial reduction than the control when both added alone and combined with LS components. At 21°C, treatments with 1.2 g/L of LO solution achieved a reduction of >2.93 log cfu/mL in 24 h, but at lower levels there was not a significant reduction from the control. Higher concentrations of TCN led to a greater P. fragi reduction at both temperatures when LO was added alone but not when combined with LP. In pasteurized milk, the LO solution at 0.12 g/L caused a reduction of approximately 1.4 log of P. fragi within 24 h when added alone and a reduction of approximately 2.7 log when combined with LP and TCN. Bacterial counts remained at significantly lower levels than the control during storage, and the TCN-supplemented milk exhibited an approximately 6-log difference from the control by d 7. In raw milk, the total bacterial growth curve showed a longer lag phase when the LS was activated by LO (11.3 ± 1.4 h) compared with the control (4.0 ± 1.0 h), but it was not different from the recommended method (9.4 ± 1.0 h). However, the total bacterial count after 24 h for the sample treated with LO and TCN (5.3 log cfu/mL) was significantly lower compared with the control (7.2 log cfu/mL) and the recommended method (6.1 log cfu/ mL). Results from this study suggest that LO is an alternative source of H 2 O 2 that enhances the microbial inhibition achieved by the LS. Lactose oxidase could be used to develop enzyme-based preservation technologies for applications where cold chain access is limited. This enzymatic approach to improving the shelf life of dairy products also represents a novel option for clean label spoilage control.

The inhibitory effect of lactose oxidase on the growth of foodborne pathogens and spoilage microorganisms associated with dairy products was evaluated through an overlay inhibition assay. Lactose oxidase generates hydrogen peroxide via lactose oxidation into lactobionic acid. Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, Staphylococcus aureus, Pseudomonas fragi, and Penicillium chrysogenum were used as indicators. A commercially available solution of lactose oxidase was applied at different concentrations (0, 0.12, 1.2, and 12 g/L) in 4 types of media [brain heart infusion agar (BHI), BHI + sodium thiocyanate (NaSCN), BHI + lactose, and BHI + NaSCN + lactose] to evaluate the effect of lactose and thiocyanate on microbial inhibition. Lactose oxidase inhibited the growth of all the indicators at a concentration of 12 g/L of the enzyme solution in the presence of lactose alone and in combination with NaSCN. However, supplementation with NaSCN had no effect on the magnitude of microbial inhibition. Staphylococcus aureus was the most sensitive pathogen, and Ps. fragi was the most sensitive of all the indicators in general to lactose oxidase. Listeria monocytogenes and Ps. fragi showed higher susceptibility to the antimicrobial effect of lactose oxidase at 6°C than at their corresponding optimum growth temperature. The inhibitory effect was attributed to the generation of hydrogen peroxide from the oxidation of lactose. Findings from this study demonstrate that lactose oxidase could be used as a novel approach to inhibit the growth of mold and bacteria. It could also be applied as a label-friendly preservative in dairy foods.