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.

Evaluation of the efficacy of commercial protective cultures to inhibit mold and yeast in cottage cheese.

Biopreservation is defined as using microbes, their constituents, or both to control spoilage while satisfying consumer demand for clean-label products. The study objective was to investigate the efficacy of bacterial cultures in biopreserving cottage cheese against postprocessing fungal contamination. Cottage cheese curd and dressing were sourced from a manufacturer in New York State. Dressing was inoculated with 3 different commercial protective cultures-PC1 (mix of Lacticaseibacillus spp. and Lactiplantibacillus spp.), PC2 (Lacticaseibacillus rhamnosus), and PC3 (Lactic. rhamnosus)-following the manufacturer recommended dosage and then mixed with curd. A control with no protective culture was included. Nine species of yeast (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 species of mold (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum) were included in the study. Fungi strains were previously isolated from dairy processing environments and were inoculated onto the cheese surface at a rate of 20 cfu/g. Cheese was stored at 6 ± 2°C. Yeast levels were enumerated at 0, 7, 14, and 21 d postinoculation. Mold growth was visually observed on a weekly basis through d 42 of storage and imaged. Overall, the protective cultures were limited in their ability to delay the outgrowth in cottage cheese, with only 8 of the 20 fungal strains showing an effect of the cultures compared with the control. The protective cultures were not very effective against yeast, with only PC1 able to delay the outgrowth of 3 strains: D. hansenii, Tor. delbrueckii, and Mey. guilliermondii. The efficacy of these protective cultures against molds in cottage cheese was more promising, with all protective cultures showing the ability to delay spoilage of at least 1 mold strain. Both PC1 and PC2 were able to delay Pen. chrysogenum and Pho. dimorpha outgrowth, and PC1 also delayed Pen. commune, Pen. decumbens, and Pen. roqueforti to different extents compared with the controls. This study demonstrates that commercial lactic acid bacteria cultures vary in their performance to delay mold and yeast outgrowth, and thus each protective culture should be evaluated against the specific strains of fungi of concern within each specific dairy facility.

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.

Evaluation of the efficacy of commercial protective cultures against mold and yeast in queso fresco.

In this study, we evaluated the efficacy of 3 commercial protective cultures designated PC1 (Lactobacillus spp.), PC2 (Lactobacillus rhamnosus), and PC3 (Lactobacillus rhamnosus) as biopreservatives in queso fresco (QF) against 9 yeast strains (Candida zeylanoides, Clavispora lusitaniae, Debaryomyces hansenii, Debaryomyces prosopidis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia fermentans, Rhodotorula mucilaginosa, and Torulaspora delbrueckii) and 11 mold strains (Aspergillus cibarius, Aureobasidium pullulans, Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, Penicillium roqueforti, Mucor genevensis, Mucor racemosus, Phoma dimorpha, and Trichoderma amazonicum). All fungal spoilage strains were previously isolated from dairy processing environments. A positive control (C) with no protective culture was included. Fungal spoilage organisms were inoculated on cheese surfaces at an inoculum level of 20 cfu/g, and cheeses were stored at 6 ± 2°C throughout the study. For yeast enumeration, cheeses were sampled on d 0, 7, 14, and 21 postinoculation. Significant inhibition was detected for each yeast strain by comparing yeast counts for each cheese treated with protective culture against the control cheese using one-way ANOVA with Bonferroni correction performed individually at d 7, 14, and 21 postinoculation. Mold growth was visually observed and imaged weekly through 70 d postinoculation. Whereas PC3 inhibited Cl. lusitaniae, Mey. guilliermondii, and Ph. dimorpha, PC2 inhibited the outgrowth of Cl. lusitaniae, D. hansenii, and Ph. dimorpha. Protective culture 1 had the broadest spectrum of efficacy across yeast and molds, delaying spoilage caused by 4 distinct yeast strains (Cl. lusitaniae, D. hansenii, D. prosopidis, and Mey. guilliermondii), and inhibiting visible growth of 2 mold strains (P. chrysogenum and Ph. dimorpha). Results demonstrated that commercial protective cultures vary in performance, as indicated by the breadth of mold and yeast inhibition at both the genus and species level. This study suggests that manufacturers looking into using protective cultures should investigate their efficacy against specific fungal strains of concern.

Control of Listeria monocytogenes in whole milk using antimicrobials applied individually and in combination.

Dairy product recalls and dairy-related illnesses are often the result of contamination with Listeria monocytogenes, which can occur throughout the dairy production and supply chains. The use of antimicrobial compounds is one practical approach for controlling pathogen survival and growth in foods. The goal of this study was to use fluid milk as a model system to identify listeristatic or listericidal treatments that show promise for application in fluid milk and for further evaluation in other dairy products (e.g., cheese). Caprylic acid (CA), ε-polylysine (EPL), hydrogen peroxide, lauric arginate (LAE), and sodium caprylate (SC) were added individually or in combination to whole milk inoculated with L. monocytogenes at ˜4 log10 cfu/mL. Samples were stored at 7°C for 21 d, and L. monocytogenes counts were determined weekly. Inhibitory concentrations of LAE (800 mg/L) and EPL (100-400 mg/L), as well as SC and CA (3,200 mg/L each), were identified. The addition of EPL at 800 mg/L reduced L. monocytogenes counts by >3 log10 cfu/mL from initial inoculation levels after 21 d. Addition of hydrogen peroxide to milk reduced counts by >3 log10 cfu/mL from initial inoculation within 24 h (400 and 800 mg/L) or by d 7 (200 mg/L). Although the combinatory treatments of EPL + CA, EPL + LAE, and LAE + SC were characterized as indifferent, EPL + SC worked synergistically to reduce L. monocytogenes populations in milk over 21 d. Overall, these data identify potential antimicrobial treatments to control L. monocytogenes in milk and serve as a foundation for the continued development of antimicrobial controls for L. monocytogenes in dairy products.

Efficacy of Antimicrobials Applied Individually and in Combination for Controlling Listeria monocytogenes as Surface Contaminants on Queso Fresco.

Outbreaks of listeriosis are continually attributed to the consumption of Hispanic-style soft cheeses contaminated with Listeria monocytogenes postpasteurization. Once contaminated, L. monocytogenes can grow rapidly in cheeses like Queso Fresco (QF) even when stored at refrigeration temperatures. Several antimicrobials, including acidified calcium sulfate with lactic acid (ACSL), ε-polylysine (EPL), hydrogen peroxide (HP), lauric arginate ethyl ester (LAE), and sodium caprylate (SC), have demonstrated antilisterial activity in food. The objectives of this study were to determine the efficacy of these antimicrobials used individually and in combination to control L. monocytogenes as surface contaminants on QF and to identify additive and synergistic interactions. Cheeses were surface inoculated at ∼4 log CFU/g, dipped in antimicrobial solutions, vacuum packaged, and then stored at 7°C for 35 days. L. monocytogenes counts were determined 24 h after application of the antimicrobials and then weekly throughout storage. Dip treatments in a 5% (v/v) HP solution reduced L. monocytogenes counts to <0.5 log CFU/g within 24 h with no increase in counts through day 35. Dip treatments in LAE at 2 and 5% alone and in combination with EPL at 10% produced initial reductions in pathogen counts (1.5 to 1.8 CFU/g) but did not inhibit pathogen growth compared with the sterile water control. Dip applications of ACSL at 25% also produced an initial ∼1.5-log reduction in L. monocytogenes counts followed by regrowth. Application of SC at 10% alone and in combination with either EPL or LAE inhibited growth to <1 log CFU/g through 21 days of storage. The combination of ACSL+SC worked synergistically to inhibit the growth of L. monocytogenes on QF to <1 log CFU/g through 35 days. These data indicate that HP alone and treatments containing EPL, LAE, or ACSL in combination with SC are promising postlethality treatments and process controls for L. monocytogenes on QF through a 21-day shelf life.

Synergistic Antimicrobial Combinations Inhibit and Inactivate Listeria monocytogenes in Neutral and Acidic Broth Systems.

The use of antimicrobial compounds can be an effective approach to control Listeria monocytogenes in ready-to-eat foods, but it can also be limited by cost, restrictions on concentrations in foods, and potential changes to organoleptic properties. Combinatorial approaches that produce additive or synergistic effects allow for reductions in individual antimicrobial concentrations while achieving the same level of control. The present study determined the MIC and MBC of an antimicrobial compound when used alone or in binary combinations against L. monocytogenes in growth media adjusted to pH values 7.4 and 5.5 and characterized interactions as synergistic, additive, or antagonistic. Inhibitory and bactericidal concentrations were defined as changes in L. monocytogenes counts of ≤1.0 or ≥3.0 log CFU/mL compared with the starting inoculum, respectively. Individually, lauric arginate (LAE), hydrogen peroxide (HP), and ε-polylysine (EPL) inhibited L. monocytogenes growth at the lowest concentrations when applied alone in broth adjusted to pH 7.4. Similarly, LAE, EPL, and HP had the lowest MBCs in broth adjusted to both pH levels. The inhibitory efficacy of both caprylic acid and sodium caprylate (SC) increased at the lower pH, with reductions in MICs of >98%. In total, 35 and 19 additive or synergistic inhibitory and bactericidal combinations were identified at pH values 7.4 and 5.5, respectively. Combinations of acidified calcium sulfate with lactic acid (ACSL) and SC were among the most synergistic inhibitory groupings at both pH levels, whereas EPL+LAE were the most effective bactericides at pH 7.4. Combinations of SC with EPL or ACSL were also among the most effective bactericides at pH 5.5. These data serve as a foundation for developing more effective antimicrobial approaches for the control of L. monocytogenes in foods with different pH levels.