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

Discipline: contamination; Keywords: Listeria, lactose oxidase, milk, enzyme, lacto-peroxidase.

Listeria monocytogenes is a pathogen which may cause listeriosis with sometimes grave consequences. It is a ubiquitous, gram-positive, facultative anaerobe that grows at refrigeration temperatures and is found throughout the environment, specifically in soil and water. Listeria monocytogenes may survive in food that has a relatively high acid and salt content and can tolerate high and low temperatures. Due to its ubiquitous nature and the ready-to-eat nature of dairy products, L. monocytogenes is a pathogen of concern for dairy processors. It can contaminate bulk tank milk samples from non-hygienic sampling of the cow udder or from the cow-milking equipment. Listeria monocytogenes may contaminate raw milk products as observed from past outbreaks, but it can also contaminate products post-pasteurization from the dairy environment where it can survive for years, making it a danger in both raw and pasteurized dairy products. Apart from heat, antibiotics in dairy products is a potential way to control L. monocytogenes, thereby enhancing the safety of such products. However, consumers are increasingly critical to the use of antibiotics and therefore other options should be investigated. One option is microbial-based enzymes such as lactose oxidase (LO). It is a microbial-derived enzyme with antimicrobial properties. It oxidizes lactose into lactobionic acid and reduces oxygen, generating H2O2. The study of Dr B T Flynn and colleagues reported here investigated the effects of LO in UHT skim milk using different L. monocytogenes contamination scenarios. Their results were published in the Journal of Dairy Science, Volume 104 of 2021, page 10594 to 10608, the title being: Lactose oxidase: An enzymatic approach to inhibit Listeria monocytogenes in milk.

Following the study on UHT skim milk, the same 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 per litre LO each reduced L. monocytogenes counts to below the limit of detection between 14 and 21 days of refrigerated storage, dependent on the concentration of LO. In the 48-hour trials in UHT skim milk, LO treatments were effective in a concentration-dependent fashion. The highest concentration of LO in the 21-day trials, 0.12 g per litre, did not show great inhibition over 48 hours, so concentrations were increased in these experiments. In the lower inoculum, after 48 hours, a 12 g per litre LO treatment reached levels of 1.7 log cfu per mL, a reduction of 1.3 log cfu per mL from the initial inoculum, whereas the control grew out to approximately 4 log cfu per mL, an increase of 1 log cfu per mL from the inoculum on day zero. When a higher challenge inoculum of 5 log cfu per mL was used, the 0.12 g per litre and 1.2 g per litre treatments reduced the levels by 0.2 to 0.3 log cfu per mL below the initial inoculum and the 12 g per litre 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 per mL, 1.2 g per litre LO alone and combined with sodium thiocyanate reduced ~0.8 log cfu per mL from the initial inoculum on day 7 of storage, whereas the control grew out to >1 log cfu per mL from the initial inoculum. Furthermore, in the higher inoculum, 1.2 g per litre LO combined with sodium thiocyanate reduced L. monocytogenes counts from the initial inoculum by >1 log cfu per mL, whereas the control grew out 2 log cfu per 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.