Lactose oxidase: An enzymatic approach to inhibit Listeria monocytogenes in milk.

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 H2O2. 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 concentration-dependent 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 L. monocytogenes proliferation in fresh cheese and other dairy products. This enzymatic approach is a novel application to control the foodborne pathogen L. monocytogenes in dairy products.

Short communication: Evaluation of commercial meat cultures to inhibit Listeria monocytogenes in a fresh cheese laboratory model.

Control of Listeria monocytogenes in queso fresco and other fresh cheeses continues to be a challenge in the United States. These cheese types are particularly challenging due to their high moisture and high pH, which provide favorable conditions for the growth of L. monocytogenes. Protective cultures (i.e., viable strains of lactic acid bacteria that inhibit other microorganisms) have been investigated in foods such as meat as an alternative, clean-label control strategy for L. monocytogenes. However, the efficacy of protective cultures can vary by food matrix. In this study, we were interested in whether protective cultures used to control L. monocytogenes in meats could be applied to control the pathogen in queso fresco. We selected 4 commercially available bacterial cultures used for the control of L. monocytogenes in meat: Lactobacillus curvatus, Lactobacillus sakei, Pediococcus acidilactici, and Leuconostoc carnosum. We incorporated these cultures into batches of queso fresco during manufacturing and evaluated them for their ability to inhibit the growth of surface-applied L. monocytogenes at levels of 1 × 102 and 1 × 104 cfu/g. We stored the queso fresco at 6 and 21°C for up to 21 d. After 14 d, Listeria was able to grow to 1 × 107 cfu/g on the cheese. Our data show that the bacterial cultures did not significantly inhibit the growth of L. monocytogenes in queso fresco. The results from this study highlight the complexity of antagonistic bacterial interactions and their potential variability across food matrices. Protective cultures represent an important, clean-label tool for the control of L. monocytogenes in foods, but each strain must be evaluated in the food environment it is intended for to ensure its efficacy.

Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider.

A strain of lactic acid bacteria, designated 159469T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA-DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).

Evaluation of Lactose Oxidase as an Enzyme-Based Antimicrobial for Control of L. monocytogenes in Fresh Cheese.

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.