Listeria monocytogenes is a ubiquitous pathogen that can cause morbidity and mortality in immunocompromised individuals. Growth of L. monocytogenes is possible at refrigeration temperatures due to its psychrotrophic nature. The use of antimicrobials in dairy products is a potential way to control L. monocytogenes growth in processes with no thermal kill step, thereby enhancing the safety of such products. Microbial-based enzymes offer a clean-label approach for control of L. monocytogenes outgrowth. Lactose oxidase (LO) is a microbial-derived enzyme with antimicrobial properties. It oxidizes lactose into lactobionic acid and reduces oxygen, generating H 2 O 2 . This study investigated the effects of LO in UHT skim milk using different L. monocytogenes contamination scenarios. These LO treatments were then applied to raw milk with various modifications; higher levels of LO as well as supplementation with thiocyanate were added to activate the lactoperoxidase system, a natural antimicrobial system present in milk. In UHT skim milk, concentrations of 0.0060, 0.012, and 0.12 g/L LO each reduced L. monocytogenes counts to below the limit of detection between 14 and 21 d of refrigerated storage, dependent on the concentration of LO. In the 48-h trials in UHT skim milk, LO treatments were effective in a concentrationdependent fashion. The highest concentration of LO in the 21-d trials, 0.12 g/L, did not show great inhibition over 48 h, so concentrations were increased for these experiments. In the lower inoculum, after 48 h, a 12 g/L LO treatment reached levels of 1.7 log cfu/mL, a reduction of 1.3 log cfu/mL from the initial inoculum, whereas the control grew out to approximately 4 log cfu/mL, an increase of 1 log cfu/mL from the inoculum on d 0. When a higher challenge inoculum of 5 log cfu/mL was used, the 0.12 g/L and 1.2 g/L treatments reduced the levels by 0.2 to 0.3 log cfu/mL below the initial inoculum and the 12 g/L treatment by >1 log cfu/mL below the initial inoculum by hour 48 of storage at refrigeration temperatures. After the efficacy of LO was determined in UHT skim milk, LO treatments were applied to raw milk. Concentrations of LO were increased, and the addition of thiocyanate was investigated to supplement the effect of the lactoperoxidase system against L. monocytogenes. When raw milk was inoculated with 2 log cfu/mL, 1.2 g/L LO alone and combined with sodium thiocyanate reduced ~0.8 log cfu/mL from the initial inoculum on d 7 of storage, whereas the control grew out to >1 log cfu/ mL from the initial inoculum. Furthermore, in the higher inoculum ,1.2 g/L LO combined with sodium thiocyanate reduced L. monocytogenes counts from the initial inoculum by >1 log cfu/mL, whereas the control grew out 2 log cfu/mL from the initial inoculum. Results from this study suggest that LO is inhibitory against L. monocytogenes in UHT skim milk and in raw milk. Therefore, LO may be an effective treatment to prevent L. monocytogenes outgrowth, increase the safety of raw milk, and be used as an effective agent to prevent...

Listeria monocytogenes is a ubiquitous pathogen that can cause morbidity and mortality in the elderly, immune compromised, and the fetuses of pregnant women. The intrinsic properties of fresh cheese—high water activity (aW), low salt content, and near-neutral pH—make it susceptible to L. monocytogenes contamination and growth at various points in the production process. The aim of this study was to investigate the ability of lactose oxidase (LO), a naturally derived enzyme, to inhibit the growth of L. monocytogenes in fresh cheese during various points of the production process. Lab-scale queso fresco was produced and inoculated with L. monocytogenes at final concentrations of 1 log CFU/mL and 1 CFU/100 mL. LO and LO sodium thiocyanate (TCN) combinations were incorporated into the milk or topically applied to the finished cheese product in varying concentration levels. A positive control and negative control were included for all experiments. When L. monocytogenes was inoculated into the milk used for the cheese-making process, by day 28, the positive control grew to above 7 log CFU/g, while the 0.6 g/L treatment (LO and LO + TCN) fell below the limit of detection (LOD) of 1.3 log CFU/g. In the lower inoculum, the positive control grew to above 7 log CFU/g, and the treatment groups fell below the LOD by day 21 and continued through day 28 of storage. For surface application, outgrowth occurred with the treatments in the higher inoculum, but some inhibition was observed. In the lower inoculum, the higher LO and LO-TCN concentrations (0.6 g/L) reduced L. monocytogenes counts to below the LOD, while the control grew out to above 7 log CFU/g, which is a >5 log difference between the control and the treatment. These results suggest that LO could be leveraged as an effective control for L. monocytogenes in a fresh cheese.