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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.

In this study, we investigated the antifungal activity of lactose oxidase (LO) as a potential biopreservative in dairy products. Our study objectives were to screen antifungal activity of LO against common mold strains, to detect the minimum inhibitory level of LO against the same strains, and to understand how LO affects the pH and lactic acid bacteria (LAB) counts in set yogurt. Five mold strains (Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium decumbens, and Penicillium roqueforti) were used throughout study. These strains were previously isolated from dairy manufacturing plants. Throughout the study, yogurts were stored at 21 ± 2°C for 14 d. Antifungal activity of LO was screened using 2 enzyme levels (1.2 and 12 g/L LO) against selected strains on the surface of a miniature laboratory set-yogurt model. For all tested strains, no visible mold growth was detected on the surface of yogurts covered with LO compared with control yogurt without LO. The minimum inhibitory level of LO against each strain was further investigated using 4 enzyme levels (0.12, 0.48, 0.84, and 1.2 g/L LO) on the miniature laboratory set-yogurt model. We detected 0.84 g/L LO as the minimum level inhibiting visible hyphal growth across strains. The minimum inhibitory level of LO varied for each individual strain. To study the effect of LO on the pH of yogurt, miniature laboratory set-yogurt models were covered with different enzyme levels (0.12, 0.48, 0.84, 1.2, and 12 g/L LO). At d 14, a difference was detected comparing pH values of treatments to control with no LO. Commercial low-fat set yogurt was used to study the effect of LO on LAB survival when yogurt surface was covered with 0.84 g/L LO under the same experimental conditions. Control with no LO was included. At d 14, 3 levels of catalase were added (0, 0.01, and 0.1%) to each treatment. To enumerate LAB, homogenized samples were plated on de Man, Rogosa, and Sharpe agar and incubated. Yogurts with 0.84 g/L LO had lower LAB counts compared with control yogurts, and catalase level did not have a significant effect on LAB counts. Our results demonstrated potential antifungal efficacy of LO against common spoilage organisms in dairy products with residual lactose and relatively low pH. Manufacturers should establish efficacy of LO against mold strains of interest and determine the effects of LO on organoleptic properties and LAB survival in set yogurt.

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.

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.

Acid whey, a by-product of strained yogurt production, represents a disposal challenge for the dairy industry. Utilization schemes are currently limited; however, acid whey contains valuable components that could be used to create value-added products. One potential scheme would be the fermentation of acid whey into an alcoholic beverage. Sour beers are gaining popularity and acid whey, which is sour to begin with, could provide a new product opportunity. However, the main sugar of acid whey, lactose, cannot be fermented by the traditional brewer's yeast, Saccharomyces cerevisiae. It has been reported that barley contains enzymes capable of hydrolyzing lactose to glucose and galactose, which are fermentable by S. cerevisiae. We investigated whether a barley-based mash resulted in detectable hydrolysis of lactose into sugars fermentable by S. cerevisiae. We demonstrated the ability to hydrolyze lactose in acid whey using a barley-based mash, resulting in the average release of 3.70 g/L of glucose. Additionally, the subsequent liquid was fermented by S. cerevisiae to an average ethanol concentration of 3.23% alcohol by volume. This work demonstrates the ability to hydrolyze the lactose in acid whey using barley and the opportunity to use acid whey as a fermentable sugar source in beer production.

The lactoperoxidase system (LS), an antimicrobial system naturally present in milk that is activated by H2O2, has been used to inhibit microbial outgrowth in raw milk in areas where refrigeration is not viable. This study evaluated lactose oxidase (LO) as a novel activator of the LS. Lactose oxidase oxidizes lactose and produces H2O2 needed for the activation of the LS. The antimicrobial effect of different concentrations of LO with and without components of the LS, thiocyanate (TCN) and lactoperoxidase (LP), was evaluated in model systems and then applied in pasteurized milk and raw milk. In general, an increase in LO caused greater reductions of Pseudomonas fragi in the model systems and treatments were more effective at 6°C than at 21°C. At 6°C, the LO solution at 0.12 and 1.2 g/L showed significantly higher microbial reduction than the control when both added alone and combined with LS components. At 21°C, treatments with 1.2 g/L of LO solution achieved a reduction of >2.93 log cfu/mL in 24 h, but at lower levels there was not a significant reduction from the control. Higher concentrations of TCN led to a greater P. fragi reduction at both temperatures when LO was added alone but not when combined with LP. In pasteurized milk, the LO solution at 0.12 g/L caused a reduction of approximately 1.4 log of P. fragi within 24 h when added alone and a reduction of approximately 2.7 log when combined with LP and TCN. Bacterial counts remained at significantly lower levels than the control during storage, and the TCN-supplemented milk exhibited an approximately 6-log difference from the control by d 7. In raw milk, the total bacterial growth curve showed a longer lag phase when the LS was activated by LO (11.3 ± 1.4 h) compared with the control (4.0 ± 1.0 h), but it was not different from the recommended method (9.4 ± 1.0 h). However, the total bacterial count after 24 h for the sample treated with LO and TCN (5.3 log cfu/mL) was significantly lower compared with the control (7.2 log cfu/mL) and the recommended method (6.1 log cfu/mL). Results from this study suggest that LO is an alternative source of H2O2 that enhances the microbial inhibition achieved by the LS. Lactose oxidase could be used to develop enzyme-based preservation technologies for applications where cold chain access is limited. This enzymatic approach to improving the shelf life of dairy products also represents a novel option for clean label spoilage control.

Shelf-stable milk is consumed worldwide, and this market is expected to continue growing. One quality challenge for UHT milk is age gelation during shelf life, which is in part caused by bacterial heat-stable proteases (HSP) synthesized during the raw milk storage period before heat processing. Some Pseudomonas spp. are HSP producers, and their ability to grow well at refrigeration temperature make them important spoilage organisms for UHT processors to control. Previous studies have shown that lactose oxidase (LO), a natural and commercially available enzyme that produces hydrogen peroxide and lactobionic acid from lactose, can control bacterial growth in raw milk. In this research, we investigated the ability of LO to control HSP producer outgrowth, and thus delay age gelation in UHT milk. Six strains of Pseudomonas spp. were selected based on their ability to synthesize HSP and used as a cocktail to inoculate both raw and sterile (UHT) milk at a level of 1 × 105 cfu/mL. Groups were treated with and without LO, stored for 4 d at 6°C, and monitored for cell count and pH. Additionally, a sample from each was tested for HSP activity via particle size analysis (average effective diameter at 90° angle and 658 nm wavelength) and visual inspection on each day of the storage period. The HSP activity results were contrasted using Tukey's HSD test, which showed that in UHT milk, a LO treatment (0.12 g/L) effectively prevented gelation as compared with the control. In raw milk, however, a concentration of 0.24 g/L of LO was needed to obtain a similar effect. This test was scaled up to 19-L pilot plant batches of raw milk where they were challenged with Pseudomonas cocktail, treated with LO for 3 d, and then UHT processed. Resulting UHT milk bottles were monitored for gelation. Significant differences in particle size between the LO-treated samples and the control were observed as early as 1 mo after processing, and gelation was not detected in the LO-treated samples through 6 mo of storage. These results demonstrated that LO can be used to delay age gelation in UHT milk induced by HSP-producing Pseudomonas spp., representing an opportunity to improve quality and reduce postproduction losses in the shelf-stable milk market sector.

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.

Traditional procedures for microbial testing typically involve a homogenizing step. These methods give valuable information on the presence or enumeration of a bacterial contaminant, but not where the contaminant was in the original sample. Spatial information could be useful in troubleshooting sources of bacterial contamination in a processing plant. For example, if the contaminant was localized on the top of a food such as cheese, this might indicate dripping condensate along a specific processing line as its source. The objective of this proof-of-concept study was to evaluate the use of a genetically engineered phage to detect bacterial contaminants on cheese to be able to visualize the contaminants without the use of magnification. In this study, a T7 bacteriophage engineered to overexpress the luciferase NanoLuc (Promega, Madison, WI) was utilized to reveal the spatial location of Escherichia coli on lysogeny broth (LB) agar and queso fresco (QF). Four scenarios were tested to explore how phage may be applied, with a blue bioluminescent signal revealing the spatial location of contaminants: (1) phage applied topically via molten soft agar to E. coli-inoculated (a) LB agar or (b) QF; and (2) phage incorporated within (a) LB agar or (b) QF and then inoculated with E. coli. Each was tested in triplicate. Cultures of E. coli BL21 grown for 18 h were serially diluted in phosphate-buffered saline and inoculated onto 8 ± 0.5 g of LB agar or QF in 6-well plates. Plates were incubated at 37°C for 8 h for condition 1a, 24 h for 1b and 2b, and 22 h for 2a. For 1a and 1b, stock phage was added to molten soft agar, applied topically, and incubated for 2 additional hours to allow for E. coli infection. After incubation, the substrate NanoGlo (Promega) was added to cover the surface of the agar or cheese and imaged immediately in a dark box using a digital camera and long exposure to capture the bioluminescent signal. Photographs captured small blue spots where the incubated colony-forming units were located. The lowest inoculum level of E. coli detected for each scenario was 1.43 × 101 ± 9.94, 1.18 × 101 ± 7.07, 5.48 × 101 ± 1.19 × 101, and 2.37 × 101 ± 1.40 × 101 cfu/well, for 1a, 1b, 2a, and 2b, respectively. These data demonstrate that the reporter phage proof-of-concept could be used as a forensic tool to visualize the spatial location of bacteria in a cheese matrix. Future work will translate this concept to dairy-relevant phage-pathogen systems.

The inhibitory effect of lactose oxidase on the growth of foodborne pathogens and spoilage microorganisms associated with dairy products was evaluated through an overlay inhibition assay. Lactose oxidase generates hydrogen peroxide via lactose oxidation into lactobionic acid. Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, Staphylococcus aureus, Pseudomonas fragi, and Penicillium chrysogenum were used as indicators. A commercially available solution of lactose oxidase was applied at different concentrations (0, 0.12, 1.2, and 12 g/L) in 4 types of media [brain heart infusion agar (BHI), BHI + sodium thiocyanate (NaSCN), BHI + lactose, and BHI + NaSCN + lactose] to evaluate the effect of lactose and thiocyanate on microbial inhibition. Lactose oxidase inhibited the growth of all the indicators at a concentration of 12 g/L of the enzyme solution in the presence of lactose alone and in combination with NaSCN. However, supplementation with NaSCN had no effect on the magnitude of microbial inhibition. Staphylococcus aureus was the most sensitive pathogen, and Ps. fragi was the most sensitive of all the indicators in general to lactose oxidase. Listeria monocytogenes and Ps. fragi showed higher susceptibility to the antimicrobial effect of lactose oxidase at 6°C than at their corresponding optimum growth temperature. The inhibitory effect was attributed to the generation of hydrogen peroxide from the oxidation of lactose. Findings from this study demonstrate that lactose oxidase could be used as a novel approach to inhibit the growth of mold and bacteria. It could also be applied as a label-friendly preservative in dairy foods.