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

Open AccessPublished:July 30, 2021DOI:https://doi.org/10.3168/jds.2021-20450

      ABSTRACT

      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.

      Key words

      INTRODUCTION

      Listeria monocytogenes is a ubiquitous, gram-positive, facultative anaerobe that grows at refrigeration temperatures (
      • Berger S.
      Listeriosis global status.
      ;
      • FDA
      Listeria (Listeriosis).
      ). The pathogen 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 (
      • FDA
      Microbiological Surveillance Sampling: FY14:-16 Raw Milk Cheese Aged 60 Days.
      ). Due to its ubiquitous nature and the ready-to-eat nature of dairy products, L. monocytogenes is a pathogen of concern for dairy processors (
      • Boor K.J.
      • Wiedmann M.
      • Murphy S.
      • Alcaine S.
      A 100-Year Review: Microbiology and safety of milk handling.
      ). It can contaminate bulk tank milk samples from nonhygienic sampling of the cow udder or from the cow-milking equipment (
      • Fedio W.M.
      • Jackson H.
      On the origin of Listeria monocytogenes in raw bulk-tank milk.
      ). Listeria monocytogenes may contaminate raw milk products as observed from past outbreaks, but it can also contaminate products postpasteurization from the dairy environment where it can survive for years, making it a danger in both raw and pasteurized dairy products (
      • Boor K.J.
      • Wiedmann M.
      • Murphy S.
      • Alcaine S.
      A 100-Year Review: Microbiology and safety of milk handling.
      ).
      Ingestion of L. monocytogenes has the potential to cause the disease listeriosis among children, immunocompromised individuals, pregnant women, and the elderly. Epidemiological studies in the United States have shown that L. monocytogenes has some of the highest hospitalization and case-fatality rates of all the foodborne pathogens (
      • Mead P.S.
      • Slutsker L.
      • Dietz V.
      • McCaig L.F.
      • Bresee J.S.
      • Shapiro C.
      • Griffin P.M.
      • Tauxe R.V.
      Food-related illness and death in the United States.
      ), making it a danger to society if improper protocols are followed in food processing facilities. It was estimated by
      • de Noordhout C.M.
      • Devleesschauwer B.
      • Angulo F.J.
      • Verbeke G.
      • Haagsma J.
      • Kirk M.
      • Havelaar A.
      • Speybroeck N.
      The global burden of listeriosis: A systematic review and meta-analysis.
      that globally the burden of listeriosis was 23,150 illnesses and 5,463 deaths in 2010. In the United States, dairy products, such as fresh soft cheeses, hard cheeses, and soft-ripened cheeses were most commonly associated with L. monocytogenes contamination in the cheese category between 1986 and 2008 and have resulted in numerous deaths (
      • FDA
      Joint FDA/Health Canada Quantitative Assessment of the Risk of Listeriosis from Soft-Ripened Cheese Consumption in the United States and Canada: Report Vol. 2020.
      ). Furthermore, there have been 7 outbreaks in the United States associated with L. monocytogenes since 2011 that caused illness and death from dairy product consumption (
      • Centers for Disease Control and Prevention
      Listeria outbreaks.
      ). Globally, contamination of soft and soft-ripened cheeses with L. monocytogenes continues to occur, as these cheese types have low salt content, near-neutral pH, and high water activity (
      • Barría C.
      • Singer R.S.
      • Bueno I.
      • Estrada E.
      • Rivera D.
      • Ulloa S.
      • Fernández J.
      • Mardones F.O.
      • Moreno-Switt A.I.
      Tracing Listeria monocytogenes contamination in artisanal cheese to the processing environments in cheese producers in southern Chile.
      ). Therefore, if contaminated postpasteurization, L. monocytogenes can grow to dangerous levels (
      • Van Tassell M.L.
      • Ibarra-Sánchez L.A.
      • Takhar S.R.
      • Amaya-Llano S.L.
      • Miller M.J.
      Use of a miniature laboratory fresh cheese model for investigating antimicrobial activities.
      ;
      • Lawton M.R.
      • Jencarelli K.G.
      • Kozak S.M.
      • Alcaine S.D.
      Short communication: Evaluation of commercial meat cultures to inhibit Listeria monocytogenes in a fresh cheese laboratory model.
      ).
      In addition to fresh cheeses, L. monocytogenes may pose a threat to cheeses made with raw milk. In the United States, unpasteurized milk cheeses must be aged at a temperature of at least 1.7°C and held for 60 d before distribution (21 CFR 133.182(a);
      • FDA
      Code of Federal Regulation Title 21, Part 133.182.
      ). In a raw milk, soft-ripened cheese risk assessment, the US Food and Drug Administration (FDA) predicted that 4.7% of cheeses had L. monocytogenes contamination. According to this risk assessment, reducing contamination in the manufacture of raw milk cheese by a factor of 3 log10, 4 log10, or 5 log10 would reduce the average risk of an immunocompromised individual or pregnant woman from becoming sick from cheese contaminated with L. monocytogenes. The addition of an antimicrobial on the cheese surface that could reduce L. monocytogenes contamination by 2 log10 cfu would provide a lower risk to immunocompromised individuals than consuming cheese with no antimicrobial treatment (
      • FDA
      Joint FDA/Health Canada Quantitative Assessment of the Risk of Listeriosis from Soft-Ripened Cheese Consumption in the United States and Canada: Report Vol. 2020.
      ).
      The ubiquitous nature of L. monocytogenes and its potential to contaminate dairy products has led to the evaluation of different methods to inhibit the pathogen in milk and cheese products (
      • Boor K.J.
      • Wiedmann M.
      • Murphy S.
      • Alcaine S.
      A 100-Year Review: Microbiology and safety of milk handling.
      ). High pressure processing as a control strategy has the advantage of inactivating L. monocytogenes with minimal disruption of cheese quality (
      • Tomasula P.M.
      • Renye J.A.
      • Van Hekken D.L.
      • Tunick M.H.
      • Kwoczak R.
      • Toht M.
      • Leggett L.N.
      • Luchansky J.B.
      • Porto-Fett A.C.S.
      • Phillips J.G.
      Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco.
      ). Addition of antimicrobials including bacteriocins and other generally recognized as safe (GRAS) substances such as organic acids have been evaluated to be applied alone or in combination to reduce the amount needed for protection (
      • Van Tassell M.L.
      • Ibarra-Sánchez L.A.
      • Takhar S.R.
      • Amaya-Llano S.L.
      • Miller M.J.
      Use of a miniature laboratory fresh cheese model for investigating antimicrobial activities.
      ;
      • Kozak S.M.
      • Brown S.R.B.
      • Bobak Y.
      • D'Amico D.J.
      Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.
      ). Protective cultures, commonly lactic acid bacteria, can inhibit pathogen growth through production of acids or bacteriocins or competition for nutrients. This strategy is dependent on the specific cheese matrix and how cultures thrive in its environment due to factors such as water activity and pH (
      • Coelho M.C.
      • Silva C.C.G.
      • Ribeiro S.C.
      • Dapkevicius M.L.N.E.
      • Rosa H.J.D.
      Control of Listeria monocytogenes in fresh cheese using protective lactic acid bacteria.
      ;
      • Lawton M.R.
      • Jencarelli K.G.
      • Kozak S.M.
      • Alcaine S.D.
      Short communication: Evaluation of commercial meat cultures to inhibit Listeria monocytogenes in a fresh cheese laboratory model.
      ). Listeria monocytogenes outgrowth over time has been shown in different experiments that tested different methodology at inhibiting the pathogen (
      • Van Tassell M.L.
      • Ibarra-Sánchez L.A.
      • Takhar S.R.
      • Amaya-Llano S.L.
      • Miller M.J.
      Use of a miniature laboratory fresh cheese model for investigating antimicrobial activities.
      ;
      • Kozak S.M.
      • Brown S.R.B.
      • Bobak Y.
      • D'Amico D.J.
      Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.
      ).
      • D'Amico D.J.
      • Druart M.J.
      • Donnelly C.W.
      60-day aging requirement does not ensure safety of surface-mold-ripened soft cheeses manufactured from raw or pasteurized milk when Listeria monocytogenes is introduced as a postprocessing contaminant.
      found that L. monocytogenes still had the potential to grow even after the 60-d storage period required by the 21 CFR in both raw and pasteurized milk cheeses, meaning an effective method to combat this outgrowth is necessary.
      Listeria monocytogenes outgrowth and consumption in raw milk or contaminated cheeses can pose a serious health risk, especially as consumers move toward a trend of eating unprocessed or minimally processed food (
      • Ricchi M.
      • Scaltriti E.
      • Cammi G.
      • Garbarino C.
      • Arrigoni N.
      • Morganti M.
      • Pongolini S.
      Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing.
      ). Some consumers view common production methods and ingredients as “unnatural” or “artificial,” causing difficulties in the addition of antimicrobial treatments. There is currently no definition of a clean label by a regulatory authority. Consumers often view foods produced with an artificial ingredient as not falling under the clean-label category (
      • Asioli D.
      • Aschemann-Witzel J.
      • Caputo V.
      • Vecchio R.
      • Annunziata A.
      • Naes T.
      • Varela P.
      ).
      With consumers moving toward a trend of desiring “clean labels,” clean-label antimicrobial treatments that are effective against L. monocytogenes are needed in dairy products. Lactose oxidase (LO) is an enzyme that many consumers would categorize in the clean-label category because it is commercially produced by a strain of mold, Microdochium nivale. Enzymes made from plant or microbial products are often used as effective coagulants for cheese, so consumers may be primed to view LO as naturally occurring in dairy products (
      • Ben Amira A.B.
      • Besbes S.
      • Attia H.
      • Blecker C.
      Milk-clotting properties of plant rennets and their enzymatic, rheological, and sensory role in cheese making: A review.
      ).
      Lactose oxidase oxidizes lactose into lactobionic acid (LBA;
      • Ahmad S.K.
      • Brinch D.S.
      • Friis E.P.
      • Pedersen P.B.
      Toxicological studies on lactose oxidase from Microdochium nivale expressed in Fusarium venenatum.
      ) as it concurrently reduces O2 into H2O2 (
      • Nordkvist M.
      • Nielsen P.M.
      • Villadsen J.
      Oxidation of lactose to lactobionic acid by a Microdochium nivale carbohydrate oxidase: Kinetics and operational stability.
      ). H2O2 is GRAS and can be used as an antimicrobial agent in different production processes within the United States (
      • FDA
      CFR- Code of Federal Regulations Title 21.184.1366.
      ). In the European Union, H2O2 is only allowed as a processing aid for certain foodstuffs, such as the whitening of fish (
      • Himonides A.T.
      • Taylor K.D.A.
      • Knowles M.J.
      The improved whitening of cod and haddock flaps using hydrogen peroxide.
      ); however, the use of glucose oxidase, an enzyme which also produces hydrogen peroxide (
      • Duke F.R.
      • Weibel M.
      • Page D.S.
      • Bulgrin V.G.
      • Luthy J.
      Glucose oxidase mechanism. Enzyme activation by substrate.
      ) is allowed as an additive in certain foods (EFSA CEP Panel et al., 2019). H2O2 has been shown as an effective agent in inhibiting L. monocytogenes in milk, and its combination with thiocyanate (TCN) has been deemed to be an effective activator of the lactoperoxidase system (LPDS), a natural antimicrobial system in milk (
      • Kussendrager K.D.
      • van Hooijdonk A.C.M.
      Lactoperoxidase: Physico-chemical properties, occurrence, mechanism of action and applications.
      ; FAO, 2006). The LO inhibited L. monocytogenes both alone and in combination with TCN using an inhibition assay in a previous study (
      • Lara-Aguilar S.
      • Alcaine S.D.
      Short communication: Screening inhibition of dairy-relevant pathogens and spoilage microorganisms by lactose oxidase.
      ). Its usefulness in fluid milk and cheese products as an inhibitor of L. monocytogenes has not yet been explored.
      The purpose of this study was to evaluate LO as an antimicrobial in fluid milk inoculated with L. monocytogenes. The first part of this study explored the efficacy of LO on the growth of L. monocytogenes in UHT skim milk inoculated at 2 log cfu/mL and 4 log cfu/mL over a 21-d storage period. These results were then used to determine if increasing concentrations of LO showed improved efficacy against L. monocytogenes inoculated at 3 log cfu/mL and 5 log cfu/mL in UHT skim milk over 48 h. Last, it was investigated whether LO inhibited L. monocytogenes growth in raw milk when stored at refrigeration temperatures. Our results showed that LO has potential as a prevention strategy for L. monocytogenes outgrowth in fluid dairy products.

      MATERIALS AND METHODS

      Listeria monocytogenes Cocktail Preparation

      Five isolates of Listeria monocytogenes (Table 1), 4 isolated from fresh cheese outbreaks and 1 local laboratory strain, were obtained from the Food Safety Laboratory at Cornell University (Ithaca, NY). Each isolate was streaked onto Brain Heart Infusion (BHI) agar and incubated at 37°C for 24 h. After incubation, a sterile inoculation loop was used to transfer an individual colony of each strain into 5 mL of BHI broth. Broth cultures were grown at 37°C for 18 h to obtain cultures of optical density = 1.00 (9 log cfu/mL). A Listeria cocktail was prepared by transferring 1,000 μL of each broth into a sterile tube and vortexed to combine. The cocktail was serially diluted in PBS and plated onto BHI agar to confirm target inoculum levels.
      Table 1Strains of Listeria monocytogenes used to produce a cocktail used for inoculation of milk samples
      IdentificationOutbreakSource typeSource siteIsolate dateSerotype
      FSL-X1–0001Laboratory strain 10403S1/2a
      FSL-R9–56212012 Ricotta cheeseFoodCheese6/19/20121/2a
      FSL-R9–56232013 Semi fresh style cheeseHumanPlacenta5/29/20134b
      FSL-R9–56252014 Soft cheeseHumanBlood7/6/20144b
      FSL-R9–56242013 Queso frescoHumanBlood8/14/20131/2b

      Application of LO in UHT Skim Milk

      We used UHT-processed skim milk (Parmalat USA Corp.) to minimize background microflora. Twenty-five milliliters of milk was aseptically transferred to individual 50-mL tubes (VWR International) and treated with various levels of LO to produce final concentrations of 0.006, 0.012, and 0.12 g/L (LactoYield, Chr. Hansen). One hundred microliters of the L. monocytogenes cocktail described above were added into each treatment to obtain an approximate microbial load of 2 log cfu/mL or 4 log cfu/mL. Inoculated milk with no LO was used as a positive control. Milk with no LO that was not inoculated with L. monocytogenes was used as a negative control to confirm absence of L. monocytogenes in the commercial product. Samples were stored at 6°C for 21 d with samples for microbiological load taken on d 0, 2, 4, 7, 14, and 21. At each time point, each treatment was sampled from the same tube in duplicate for all experiments. Before plating, serial dilutions of each sample were prepared in PBS. Listeria counts were enumerated at all sampling points by spread plating in duplicate on Modified Oxford Agar (MOX), and plates were incubated at 30°C (
      • Curtis G.
      • Nichols W.
      • Falla T.
      Selective agents for Listeria can inhibit their growth.
      ;
      • Henderson L.O.
      • Cabrera-Villamizar L.
      • Skeens J.
      • Kent D.
      • Murphy S.
      • Wiedmann M.
      • Guariglia-Oropeza V.
      Environmental conditions and serotype affect Listeria monocytogenes susceptibility to phage treatment in a laboratory cheese model.
      ) for 48 h. The pH of each sample was taken in duplicate at all sampling points using established methodology (
      ). For all pH measurements, an InLab Smart Pro-ISM pH probe (Mettler Toledo) connected to an iCinac (AMS Alliance) was used. The experiment was performed in triplicate.

      High Concentration of LO in UHT Skim Milk Over 48 h

      Twenty-five milliliters of UHT skim milk (Parmalat USA Corp.) was aseptically transferred to individual tubes and treated with various levels of LO to produce final concentrations of 0.12, 1.2, and 12 g/L (LactoYield, Chr. Hansen). One hundred microliters of the Listeria cocktail were inoculated into each treatment to obtain an approximate microbial load of 3 log cfu/mL or 5 log cfu/mL. Inoculated milk with no LO was used as the positive control. Milk with no LO that was not inoculated with L. monocytogenes was used as the negative control to confirm the absence of the pathogen in the commercial product. Samples were stored at 6°C for 48 h of storage with samples for microbiological load taken at h 0, 2, 4, 6, 8, 12, 24, 36, and 48. Before plating, serial dilutions of each sample were prepared in PBS. Listeria counts were enumerated at all sampling points by spread plating in duplicate on MOX, and plates were incubated at 30°C for 48 h. The pH of each sample was taken in duplicate at all sampling points. The experiment was repeated in triplicate.

      Application of LO and TCN in Raw Milk

      Raw whole milk was collected from the Cornell University Dairy Plant 3 h after milking and stored at refrigeration temperatures for 1 d (24 h) before performing the experiment. Raw milk was shaken before sampling to ensure homogeneity. Twenty-five milliliters of milk was aseptically transferred to tubes and treated with various levels of LO and TCN (VWR International). We added LO to milk to produce final concentrations of 0.12, 0.6, and 1.2 g/L. Following the addition of LO, TCN was added to obtain a final concentration of 14 mg/L. One hundred microliters of the Listeria cocktail was inoculated into each treatment to obtain an approximate microbial load of 2 log cfu/mL or 4 log cfu/mL. One treatment of each LO concentration did not include the addition of TCN, and inoculated milk with no LO was used as the positive control. Milk without LO that was not inoculated with L. monocytogenes was used as the negative control to confirm the absence of the pathogen in the raw milk produced from Cornell. Samples were stored at 6°C for 7 d of storage with samples for microbiological load taken on d 0, 2, 4, and 7. Before plating, serial dilutions of each sample were prepared in PBS. Listeria counts were enumerated by spread plating in duplicate on MOX, and plates were incubated at 30°C for 48 h. The total aerobic count of the negative control was determined by plating the serial dilutions on Standard Plate Count (SPC) agar and incubating plates at 32°C for 48 h. The SPC counts were not taken in the UHT trials because the high heat and time of UHT pasteurization produces a microbially shelf-stable product. The pH of each sample was taken in duplicate at all sampling points. The experiment was repeated in triplicate.

      Statistical Analysis

      All statistical analyses were performed using R software (version 3.5.2, https://www.r-project.org/). We performed ANOVA and Tukey's honest significant difference tests at each time point to determine log differences in Listeria counts between all treatments and the positive control. The same tests were performed at each time point to determine differences in pH values between all treatments and the negative control.

      RESULTS AND DISCUSSION

      Application of LO in UHT Skim Milk Results in a Bacteriostatic Effect Against L. monocytogenes

      In previous work, it was shown that background microbiota could have an effect on LO efficacy (
      • Lara-Aguilar S.
      • Alcaine S.D.
      Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.
      ) on the target bacteria. To understand the interaction between LO and L. monocytogenes, we initially used UHT skim milk to have a dairy environment that was free from background microbiota. Multiple concentrations of LO were added to UHT milk, and their efficacy to control L. monocytogenes outgrowth was monitored. Four of the isolates used for the L. monocytogenes challenge cocktail were from fresh cheese outbreaks, and one was a reference laboratory strain. The treatments were challenged with both a high, 4 log cfu/mL, and a low, 2 log cfu/mL, inoculum to represent variable contamination scenarios.
      When L. monocytogenes was inoculated at a target inoculum of 2 log cfu/mL, all LO treatments resulted in a reduction in the number of L. monocytogenes when compared with the control starting at d 2 of storage (Figure 1). Within 2 d of storage, a 0.78 log cfu/mL outgrowth of the control was observed, whereas each treatment group inhibited outgrowth of L. monocytogenes. This trend continued throughout the rest of the 21-d trial (Table 2). Significant differences (P < 0.05) between each treatment and the control were shown starting on d 2 of storage and continued throughout the trial. By d 21, the control outgrowth reached 8 log cfu/mL, whereas each treatment group fell below the limit of detection (LOD).
      Figure thumbnail gr1
      Figure 1Listeria monocytogenes counts inoculated at 2 log cfu/mL in UHT skim milk treated with lactose oxidase (LO) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–d) indicate significant differences (P < 0.05) between treatments on the same day. For counts lower than the limit of detection, a value of 0.5 log10 cfu/mL was used. A horizontal line was drawn at the limit of detection to represent y = 1.0 log cfu/mL. The 0.006 and 0.012 g/L treatments were undetectable in 21 d, and the 0.12 g/L treatment was undetectable in 14 d. Error bars represent the SD.
      Table 2pH (± SD) of UHT skim milk treated with lactose oxidase (LO) stored at 6°C
      Treatment1Time (d)
      02471421
      Control6.62 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.67 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.72 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.70 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.70 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.71 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      LO
       0.006 g/L6.62 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.67 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.66 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.59 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.55 ± 0.08
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.47 ± 0.07
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       0.012 g/L6.61 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.66 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.57 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.51 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.42 ± 0.08
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       0.12 g/L6.61 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.07
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.57 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.47 ± 0.09
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.37 ± 0.14
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.17 ± 0.10
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      a–c Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      When L. monocytogenes was inoculated at a higher level of 4 log cfu/mL (Figure 2), the control differed significantly (P < 0.05) from groups treated with LO within 48 h of storage. By d 4 of storage, the L. monocytogenes outgrowth was observed in the control, whereas each treatment group remained below the initial inoculation level. By d 7, outgrowth in the control reached 7.2 log cfu/mL, whereas each treatment group fell below that in a concentration-dependent fashion (0.006 g/L = 3.6, 0.012 g/L = 3.4, 0.12 = 2.8 log cfu/mL). On d 14, the control reached levels of above 8 log cfu/mL, and reduction of L. monocytogenes was shown for each treatment group. Even at the lowest concentration of LO (0.006 g/L), the treatment fell to 2.0 log cfu/mL compared with the initial inoculation of approximately 4 log cfu/mL. At a concentration of 0.012 g/L, the count of L. monocytogenes dropped to 1.6 log cfu/mL. At the highest concentration of LO, the number of L. monocytogenes was reduced below the LOD. By d 21 of storage, all treatment groups fell below the LOD.
      Figure thumbnail gr2
      Figure 2Listeria monocytogenes counts inoculated at 4 log cfu/mL in UHT skim milk treated with lactose oxidase (LO) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–d) indicate significant differences (P < 0.05) between treatments on the same day. For counts lower than the limit of detection, a value of 0.5 log10 cfu/mL was used. A horizontal line was drawn at the limit of detection to represent y = 1.0 log cfu/mL. The 0.006 and 0.012 g/L treatments were undetectable in 21 d, and the 0.12 g/L treatment was undetectable in 14 d. Error bars represent the SD.
      These results suggested the production of H2O2 from the presence of LO in milk was sufficient to cause L. monocytogenes to fall below the LOD with no regrowth after long-term storage. A previous study (
      • Lara-Aguilar S.
      • Alcaine S.D.
      Short communication: Screening inhibition of dairy-relevant pathogens and spoilage microorganisms by lactose oxidase.
      ) screened LO against a set of dairy-relevant microorganisms, including L. monocytogenes using a well-diffusion assay. A zone of clearing was observed around the LO wells. However, when catalase was spotted next to the well, L. monocytogenes growth was observed, suggesting that the production of H2O2 was the driving cause of bacterial inhibition of LO.
      In both our contamination scenarios, L. monocytogenes outgrowth was inhibited by LO in comparison to the control starting at d 2 of storage and lasted throughout the rest of the trial. According to the FDA risk assessment, a listeriostatic process control measure shows an average increase of less than 1 log cycle over 2 or more time intervals in the number of L. monocytogenes in replicate trials with the food of interest. A listericidal process control is one that provides a reduction of 5 orders of magnitude (
      • FDA
      Joint FDA/Health Canada Quantitative Assessment of the Risk of Listeriosis from Soft-Ripened Cheese Consumption in the United States and Canada: Report Vol. 2020.
      ). According to these results, even at the lowest concentration of LO, listeriostatic inhibition was observed.
      The relative safety of H2O2 means it has many uses in the food industry (
      • Linley E.
      • Denyer S.P.
      • McDonnell G.
      • Simons C.
      • Maillard J.-V.
      Use of hydrogen peroxide as a biocide: New consideration of its mechanisms of biocidal action.
      ), such as a sanitizer on food contact surfaces to provide an antimicrobial effect (
      • Govaert M.
      • Smet C.
      • Verheyen D.
      • Walsh J.L.
      • Van Impe J.F.M.
      Combined effect of cold atmospheric plasma and hydrogen peroxide treatment on mature Listeria monocytogenes and Salmonella typhimurium Biofilms.
      ), and as a food additive to reduce microbial contamination. According to the 21 Code of Federal Regulations, H2O2 may be added to milk and other food products at a maximum concentration of 0.05% to milk intended for cheesemaking [21 CFR 184(a);
      • FDA
      Code of Federal Regulation Title 21, Part 184.
      ]. Main advantages of using H2O2 in the food industry are that it has broad-spectrum activity and lack of environmental toxicity following its complete degradation (
      • Linley E.
      • Denyer S.P.
      • McDonnell G.
      • Simons C.
      • Maillard J.-V.
      Use of hydrogen peroxide as a biocide: New consideration of its mechanisms of biocidal action.
      ). H2O2 has a long history of use worldwide for milk preservation (
      • Martin N.H.
      • Friedlander A.
      • Mok A.
      • Kent D.
      • Wiedmann M.
      • Boor K.J.
      Peroxide test strips detect added hydrogen peroxide in raw milk at levels affecting bacterial load.
      ). It has also been shown to be an effective antimicrobial in different food products. It was used as an effective antimicrobial to reduce counts of L. monocytogenes on mung bean sprouts after combination with a hot water treatment. These counts were reduced by approximately 2 log cfu/g on mung beans after storage (
      • Trzaskowska M.
      • Dai Y.
      • Delaquis P.
      • Wang S.
      Pathogen reduction on mung bean reduction of Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes on mung bean using combined thermal and chemical treatments with acetic acid and hydrogen peroxide.
      ).
      • Kozak S.M.
      • Brown S.R.B.
      • Bobak Y.
      • D'Amico D.J.
      Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.
      found that a concentration of H2O2 of 100 mg/L inhibited growth of L. monocytogenes when stored at 7°C in whole milk. Overall, there was a 2.5 log cfu/mL reduction in the number of L. monocytogenes when H2O2 was added at this concentration. Concentrations of 400 mg/L were bactericidal within 24 h of storage. Our results are consistent with the reduction of L. monocytogenes found in previous studies due to H2O2 production.
      There is little literature regarding the exact mechanism behind bacterial death via H2O2. It is an oxidative biocide, meaning it removes electrons from susceptible chemical groups, oxidizing them and then becoming reduced. Oxidative biocides may severely damage microbial structures, causing the release of intracellular compounds, which are then oxidized (
      • Finnegan M.
      • Linley E.
      • Denyer S.P.
      • McDonnell G.
      • Simons C.
      • Maillard J.-Y.
      Mode of action of hydrogen peroxide and other oxidizing agents: differences between liquid and gas forms.
      ). H2O2 may then act via the formation of hydroxy radicals that oxidize thiol groups in enzymes and proteins (
      • Russell A.D.
      Similarities and differences in the responses of microorganisms to biocides.
      ). It is currently believed that the Fenton reaction leading to the production of free hydroxy radicals is the basis of the reaction, and evidence exists for the reaction of this leading to the oxidation of DNA, proteins, and lipids in vivo (
      • Linley E.
      • Denyer S.P.
      • McDonnell G.
      • Simons C.
      • Maillard J.-V.
      Use of hydrogen peroxide as a biocide: New consideration of its mechanisms of biocidal action.
      ).
      Lactase oxidase also produces LBA at the same time as generating H2O2, and a significant difference in pH (P < 0.001) was observed between the control sample and UHT skim milk treated with the highest LO concentration (0.12 g/L) at d 4 of storage. By d 14 of storage, these significant differences remained. By d 7, all treatments were significantly lower (P < 0.05) in pH than the control. By the end of the trial, d 21, the control remained at approximately 6.7, whereas the lower LO treatment groups (0.006 and 0.012 g/L) were above 6.4, and the highest concentration of LO (0.12 g/L) was 6.2. The presence of lactic acid bacteria was likely not the driving cause of this pH reduction, as UHT skim milk was used, and the pH of the control increased throughout the trial. Therefore, in the treatment groups, the production of LBA by the LO reaction in solution likely resulted in the pH reduction.
      Lactobionic acid is obtained from the oxidation of lactose, which has metal-chelating and humectant properties. It has many potential applications in foods and pharmaceutical products (
      • Gutiérrez L.-F.
      • Hamoudi S.
      • Belkacemi K.
      Lactobionic acid: A high value-added lactose derivative for food and pharmaceutical applications.
      ). Lactobionic acid has shown inhibition of L. monocytogenes when combined with other antimicrobial compounds at 21°C in 2% milk, but has not resulted in inhibition when added to milk alone (
      • Chen H.
      • Zhong Q.
      Lactobionic acid enhances the synergistic effect of nisin and thymol against Listeria monocytogenes Scott A in tryptic soy broth and milk.
      ). Therefore, the combination of LBA with other antimicrobials such as H2O2 may show further inhibition in milk. Results from the same study showed that the addition of LBA to 2% milk significantly reduced the pH of the milk after addition by 0.44 units at a concentration of 10 mg/L (
      • Chen H.
      • Zhong Q.
      Lactobionic acid enhances the synergistic effect of nisin and thymol against Listeria monocytogenes Scott A in tryptic soy broth and milk.
      ), consistent with the results obtained from this study. The exact mechanism of action of LBA is currently not well known; however, it is thought that it is due to its chelating and oxidation properties. Furthermore, in our study, measuring a reduction in pH was an indirect measurement of LBA concentration in the milk. It is not well known exactly how much LBA is produced by this reaction; therefore, future studies should determine the titratable acidity of the milk to quantify this value.

      High Concentration With LO Inhibits L. Monocytogenes Outgrowth in UHT Skim Milk Over 48 h

      In the previously established LO treatment levels, L. monocytogenes counts did not increase between the start of the trial and d 2 (Figure 1, Figure 2). We were interested if higher concentrations of LO would reduce L. monocytogenes levels during those 48 h compared with the control. We challenged the LO with a moderate, 3 log cfu/mL, and high, 5 log cfu/mL, inoculum of L. monocytogenes. At the moderate inoculum, the control grew approximately 1 log cfu/mL in 48 h, as shown in Figure 3. The lowest concentration of LO, 0.12 g/L, was significantly different from the control (P < 0.0001) at h 8 of storage. At h 48, this concentration was 1.2 log cfu/mL below the control. At h 24 of storage, the control reached 3.5 log cfu/mL, and the 1.2 g/L LO treatment group was reduced to 2.7 log cfu/mL. This inhibition continued to h 48 of storage when the control grew out to 4.0 log cfu/mL and the treatment was further reduced to 2.3 log cfu/mL. The highest concentration, 12 g/L, showed significant differences from the control group throughout the entire period of storage. At h 12, this treatment fell to 2.4 log cfu/mL, whereas the control reached a level of 3.5 log cfu/mL. On h 48 of storage, the control reached levels of 4 log cfu/mL and the treatment group was further reduced to 1.6 log cfu/mL. Each of these treatments showed a bacteriostatic effect throughout the entire trial.
      Figure thumbnail gr3
      Figure 3Listeria monocytogenes counts inoculated at 3 log cfu/mL in UHT skim milk treated with lactose oxidase (LO) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–d) indicate significant differences (P < 0.05) between treatments on the same day. Error bars represent the SD.
      In the high inoculum challenge with 5 log cfu/mL, all treatment groups were significantly lower than the (P < 0.05) from the control from h 2 and remained significantly lower throughout the trial (Figure 4). By hour 48 of storage, the 0.12 g/L concentration of LO was 1.1 log cfu/mL less than the control. The second highest LO treatment, 1.2 g/L, was not statistically significant from the 0.12 g/L treatment throughout the entire trial. By h 48 of storage, this treatment demonstrated a 1.2 log cfu/mL reduction from the control. The highest concentration of LO, 12 g/L, showed the most efficacy toward reduction of L. monocytogenes in UHT skim milk when inoculated at 5 log cfu/mL. By h 24, this treatment was significantly different from both the control (P < 0.05) and the 0.12 and 1.2 g/L treatments (P < 0.05). At h 36, this treatment was approximately 0.8 log cfu/mL below the control. By h 48, the control grew out to almost 6 log cfu/mL, whereas the 12 g/L treatment was reduced to 4 log cfu/mL.
      Figure thumbnail gr4
      Figure 4Listeria monocytogenes counts inoculated at 5 log cfu/mL in UHT skim milk treated with lactose oxidase (LO) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–d) indicate significant differences (P < 0.05) between treatments on the same day. Error bars represent the SD.
      However, the higher levels of LO showed significant differences in the pH from the control over the 48-h trial (Table 3). By h 12, the pH of the highest concentration of LO, 12 g/L, dropped to 6.55 and was significantly different (P < 0.05) from the pH of the control. Significant differences in pH were not observed between the control and the lowest LO level of 0.12 g/L throughout the trial. By h 48 of storage, the 1.2 g/L LO concentration was significantly different (P < 0.05) from the control. The 12 g/L LO concentration reached a pH of 6.44 by the end of the 48-h storage period, a 0.23-unit drop below the control.
      Table 3pH (± SD) of UHT skim milk treated with lactose oxidase (LO) during storage at 6°C (n = 9)
      Treatment1Time (h)
      0246812243648
      Control6.62 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.63 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.63 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.64 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.66 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.67 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      LO
       0.12 g/L6.69 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.66 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.65 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       1.2 g/L6.62 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.61 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.61 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.61 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.60 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.59 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.58 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       12 g/L6.63 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.61 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.59 ± 0.00
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.57 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.56 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.55 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.52 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.48 ± 0.08
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.44 ± 0.10
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      a,b Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      The higher concentrations of LO used in the 48-h trials showed greater inhibition of L. monocytogenes. As noted earlier, these observations were likely due to greater production of H2O2 that LO produces.
      • Kozak S.M.
      • Brown S.R.B.
      • Bobak Y.
      • D'Amico D.J.
      Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.
      found that a concentration of 100 mg/L of H2O2 inhibited L. monocytogenes growth in UHT whole milk over a period of 21 d, whereas concentrations of 400 mg/L and 800 mg/L were bactericidal within 24 h of storage. Furthermore,
      • Martin N.H.
      • Friedlander A.
      • Mok A.
      • Kent D.
      • Wiedmann M.
      • Boor K.J.
      Peroxide test strips detect added hydrogen peroxide in raw milk at levels affecting bacterial load.
      concluded that the higher concentration of H2O2 in raw milk reduced total bacterial concentrations in raw milk when stored at 6°C. Our results that showed that higher concentrations of LO produced a greater antimicrobial effect were consistent with these studies. However, the large drop in pH, particularly at the highest LO concentration used, means that there is a potential effect on the milk in a way that may not be sensorially acceptable. Furthermore, LO may produce oxidative flavors that consumers may not find acceptable, which should be investigated in future studies. Future research is needed to investigate whether other methods for LO use, such as immobilization, might allow for the use of higher levels of the enzyme to produce high level of H2O2 to achieve the bactericidal effects observed in the
      • Kozak S.M.
      • Brown S.R.B.
      • Bobak Y.
      • D'Amico D.J.
      Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.
      study, and to also minimize the effect on pH by controlling exposure time of the milk to LO. If milk is to be made into cheese using starter cultures, topical application of the treatment to prevent environmental contamination of L. monocytogenes may be a way to minimize the antimicrobial effect of LO on catalase-negative starter cultures.

      Effect of LO on Raw Milk pH

      The LO treatments significantly reduced the pH of the raw milk comparison to the control by d 2 of storage (Table 4). By d 7 of storage, the 0.6 g/L LO and 0.6 g/L LO-TCN treatments had a pH approximately 0.3 units lower than the control. Furthermore, the 1.2 g/L LO and 1.2 g/L LO-TCN treatments were more than 0.3 pH units below the control on d 7 of storage. Supplementation with TCN appeared to have no effect on the pH in comparison to the LO treatment. As noted earlier, the observed pH drop was due to LBA produced by LO. The implications of the pH drop on sensory acceptance of the raw milk or of cheese made from LO-treated raw milk need further investigation.
      Table 4pH (± SD) of raw milk treated with thiocyanate (TCN) and lactose oxidase (LO) during storage at 6°C (n = 4)
      Treatment1Time (d)
      0247
      Control6.78 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.78 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.78 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.68 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      LO
       0.12 g/L6.77 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.74 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.69 ± 0.00
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.63 ± 0.06
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       0.6 g/L6.75 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.67 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.60 ± 0.09
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.40 ± 0.12
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       1.2 g/L6.73 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.51 ± 0.07
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.36 ± 0.15
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      TCN with LO
       0.12 g/L6.77 ± 0.03
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.73 ± 0.01
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.69 ± 0.00
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.62 ± 0.05
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       0.6 g/L6.72 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.67 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.55 ± 0.04
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.41 ± 0.15
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
       1.2 g/L6.73 ± 0.02
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.61 ± 0.07
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.52 ± 0.08
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      6.27 ± 0.19
      Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.
      a,b Means within a column with different superscript letters are significantly different (P < 0.05) between treatments.

      Application of LO and TCN Provide a Bacteriostatic Effect on L. monocytogenes in Raw Milk

      Supplementation with TCN assists activation of the LPDS, a natural antimicrobial system present in raw milk. The LPDS is comprised of the following 3 components: lactoperoxidase, TCN, and H2O2 (
      • Kussendrager K.D.
      • van Hooijdonk A.C.M.
      Lactoperoxidase: Physico-chemical properties, occurrence, mechanism of action and applications.
      ). Ultra-high temperature pasteurization completely inactivates the lactoperoxidase enzyme (
      • Barrett N.E.
      • Grandison A.S.
      • Lewis M.J.
      Contribution of the lactoperoxidase system to the keeping quality of pasteurized milk.
      ), leading to no possible activation of the LPDS in UHT skim milk. Also,
      • Lara-Aguilar S.
      • Alcaine S.D.
      Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.
      determined that supplementation of raw milk with TCN alone had no antimicrobial efficacy on Pseudomonas fragi counts, which reinforced the theory of TCN supplementation having a synergistic effect with LO. For these reasons, we did not test TCN supplementation in UHT skim milk or TCN supplementation alone in raw milk and its efficacy against L. monocytogenes. Furthermore, in real-world situations, L. monocytogenes contamination is more of a concern in milk that will not see a pasteurization step, and thus it is important to understand how LO works in such products.
      Similar to the UHT skim milk experiments, LO-treated raw milk was challenged with 2 inoculation scenarios with a low and high load of 2 log cfu/mL and 4 log cfu/mL, respectively. In both scenarios, L. monocytogenes exhibited a slower outgrowth in raw milk than in UHT skim milk (Figure 5, Figure 6), with low inoculum reaching 5.3 log cfu/mL (Figure 1) in UHT skim milk in 7 d, whereas it only reached 3.1 log cfu/mL in the raw milk (Figure 5). Similarly, outgrowth of the positive control with the high inoculum reached >7 log cfu/mL after 7 d (Figure 2) in the UHT milk, whereas it only reached a level of 5.8 log cfu/mL (Figure 6) in the raw milk.
      Figure thumbnail gr5
      Figure 5Listeria monocytogenes counts inoculated at 2 log cfu/mL in raw milk treated with lactose oxidase (LO) and thiocyanate (TCN) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–c) indicate significant differences (P < 0.05) between treatments on the same day. For counts lower than the limit of detection, a value of 0.5 log10 cfu/mL was used. A horizontal line was drawn at the limit of detection to represent y = 1.0 log cfu/mL. The LO treatments were undetectable in 21 d. Error bars represent the SD.
      Figure thumbnail gr6
      Figure 6Listeria monocytogenes counts inoculated at 4 log cfu/mL in raw milk treated with lactose oxidase (LO) and thiocyanate (TCN) during storage at 6°C. Numbers on the treatment label indicate the concentration of LO solution (g/L). Bars with different letters (a–d) indicate significant differences (P < 0.05) between treatments on the same day. Error bars represent the SD.
      Multiple reasons can explain the discrepancy between the growth of L. monocytogenes in raw milk versus UHT skim milk, with the background microbiota being the primary cause. Raw milk contains a variety of microorganisms, including a large lactic acid bacteria population. In descending order, this lactic acid bacteria population typically includes Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, and Enterococcus spp. (
      • Quigley L.
      • O'Sullivan O.
      • Stanton C.
      • Beresford T.P.
      • Ross R.P.
      • Fitzgerald G.F.
      • Cotter P.D.
      The complex microbiota of raw milk.
      ). Psychrotrophic bacteria, including Pseudomonas, Acinetobacter, and Aeromonas spp. are present in raw milk and thrive at cold temperatures (
      • Raats D.
      • Offek M.
      • Minz D.
      • Halpern M.
      Molecular analysis of bacterial communities in raw cow milk and the impact of refrigeration on its structure and dynamics.
      ). The background microbiota present in raw milk may make it difficult for L. monocytogenes to proliferate. A study by
      • Jia Z.
      • Bai W.
      • Li X.
      • Fang T.
      • Li C.
      Assessing the growth of Listeria monocytogenes in salmon with or without the competition of background microflora–A one-step kinetic analysis.
      concluded that L. monocytogenes growth was affected by background microbiota in commercially-produced salmon when compared with a sterile salmon sample. Furthermore,
      • Gonzales-Barron U.
      • Campagnollo F.B.
      • Schaffner D.W.
      • Sant'Ana A.S.
      • Cadavez V.A.P.
      Behavior of Listeria monocytogenes in the presence or not of intentionally-added lactic acid bacteria during ripening of artisanal Minas semi-hard cheese.
      found that L. monocytogenes could grow slightly in pasteurized milk and raw milk cheeses; however, the pasteurized milk cheese showed greater outgrowth of L. monocytogenes than raw milk cheese, likely due to a less complex background microbiota. Our results were consistent with those of other studies that show that L. monocytogenes better proliferates in environments with fewer other microorganisms present and grows to higher levels in environments with fewer microorganisms present.
      Figure 7 shows the SPC of the microbial population present in raw milk control samples with no L. monocytogenes inoculation over a 7-d storage period. The high and low L. monocytogenes inocula are included in the graphs for reference. These figures show that the initial concentration of microorganisms present in the raw milk was approximately 3.4 ± 0.5 log cfu/mL on d 0 of storage. Within 2 d of storage at refrigeration temperatures, this concentration increased to 6.0 ± 1.5 log cfu/mL. After 7 d of storage, these counts reached 8.3 ± 0.16 log cfu/mL, indicating that a complex background microbiota was present in the raw milk at this time point. The growth of microorganisms in the raw milk by d 2 of storage can help explain why L. monocytogenes exhibited a slow outgrowth within the raw milk samples. On d 2 of storage with both the 2 log cfu/mL and 4 log cfu/mL inoculation trials, L. monocytogenes exhibited only slight outgrowth, suggesting it competes poorly in the presence of other microorganisms.
      Figure thumbnail gr7
      Figure 7(A) Total bacterial growth curve in raw milk. The 2 log cfu/mL inoculum of Listeria monocytogenes in raw milk was plotted for comparison. Error bars represent the SD. (B) Total bacterial growth curve in raw milk. The 4 log cfu/mL inoculum of L. monocytogenes was plotted for comparison. Error bars represent the SD.
      At low concentrations, LO efficacy was reduced in raw milk, but the supplementation with TCN restored some efficacy. For example, the effect of an LO treatment of 0.12 g/L in raw milk on both low and high inocula of L. monocytogenes did not inhibit outgrowth in comparison to the control (Figure 5, Figure 6), though that concentration was able to inhibit growth in UHT milk (Figure 1, Figure 2). This reduction of efficacy may be due to the fact that in raw milk, there are other factors that affect H2O2 such as the presence of background microbiota that in essence divert the activity of H2O2 away from L. monocytogenes. There may also be species in the raw milk that produce a sufficient level of catalase to degrade the H2O2 produced by this low level of LO, as well as the lactoperoxidase present in the milk. However, with the supplementation of TCN at 14 mg/mL, the 0.12 g/L LO was sufficient to inhibit L. monocytogenes outgrowth (Figure 5, Figure 6).
      This was also observed with Pseudomonas fragi inhibition in raw milk (
      • Lara-Aguilar S.
      • Alcaine S.D.
      Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.
      ). H2O2 activates the LPDS, which uses H2O2 to oxidize TCN, and in turn leads to the formation of antimicrobial compounds (
      • Seifu E.
      • Buys E.M.
      • Donkin E.F.
      Significance of the lactoperoxidase system in the dairy industry and its potential applications: A review.
      ). It is possible that the level of TCN in raw milk is insufficient to produce enough antimicrobial compounds to inhibit L. monocytogenes, and the excess H2O2 produced by LO is lost. With supplementation of TCN, there is more substrate for lactoperoxidase to produce antimicrobial compounds and thus inhibit L. monocytogenes. This also suggests the mechanism of L. monocytogenes inhibition by LO in UHT milk versus LO in raw milk is different, and further research is needed to understand these differences.
      Treatments with higher levels of LO were effective at inhibiting L. monocytogenes with and without TCN (Figure 5, Figure 6). When the 2 log cfu/mL inoculum was used, concentrations of 0.6 g/L LO alone and combined with TCN and 1.2 g/L alone and combined with TCN differed from the control as well as the 0.12 g/L LO treatment by d 4 of the trial. By d 7 of treatment, the 0.6 g/L LO combined with TCN and the 1.2 g/L LO and 1.2 g/L combined with TCN reduced the level of L. monocytogenes to below the LOD. Similarly, when L. monocytogenes was inoculated into the raw milk at a concentration of 4 log cfu/mL, each of these treatments were significantly different from the control by d 4 of storage. The 0.6 LO-TCN, 1.2 g/L LO, and 1.2 g/L LO-TCN treatments all reduced levels of L. monocytogenes to below 3 log cfu/mL; a bacteriostatic effect was present using these levels. Higher concentrations of LO had more of an effect against L. monocytogenes in raw milk.

      CONCLUSIONS

      The goal of this study was to explore the possible antimicrobial effect of LO on L. monocytogenes in various milk products as a possible safety enhancement to prevent the pathogen's outgrowth. We first explored the antimicrobial effects of LO to reduce L. monocytogenes counts in UHT skim milk and then used these results for investigation of further application in other milk products. Our results showed that very low concentrations (0.006, 0.012, and 0.12 g/L LO) were effective at inhibiting L. monocytogenes inoculated at both 2 log cfu/mL and 4 log cfu/mL concentrations in UHT skim milk. Each of these treatments had minimal effect on the pH until d 4 of storage; by d 7 of storage all treatments differed significantly in pH from the control due to the production of LBA. We observed that the highest concentration of LO that proved effective in the UHT trials did not have the same effect on L. monocytogenes in raw milk, likely due to the presence of a diverse microbiota as well as the presence of the LPDS in raw milk. Therefore, we increased concentrations of LO and supplemented with sodium TCN. These concentrations proved inhibitory to outgrowth of L. monocytogenes during a 1-wk storage period, demonstrating that LO and LO supplemented with TCN are effective and could be leveraged to improve the food safety of dairy products. Future studies will evaluate the efficacy of LO as an inhibitor of L. monocytogenes and other pathogenic organisms in other dairy products, such as cheese and yogurt.

      ACKNOWLEDGMENTS

      The authors thank the New York State Milk Promotion Advisory Board (Albany, NY) for their support of this project through the New York State Department of Agriculture and Markets (Albany, NY). The authors also thank the staff and students of the Cornell Food Safety Laboratory (Cornell University, Ithaca, NY) for allowing us to use their space. The authors have not stated any conflicts of interest.

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