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Under specific conditions, the fermentation of whey permeate (WP) by Brettanomyces claussenii can create bioproducts with high galactose concentrations and potential functionalities. The aims of this research are to optimize the fermentation of WP by B. claussenii using response surface methodology to maximize the production of ethanol and galactose, and to characterize various products obtained with this approach. For this purpose, five fermentation factors were studied to determine their impacts on ethanol and galactose: temperature (20 - 40°C), substrate concentration (5 - 15%TS), lactase enzyme/substrate ratio (0 - 40 IU/ g lactose), inoculation level (6 - 8 log cfu/mL), and time (6 - 30 days). Linear models, containing quadratic and interaction effects, were built for the optimization of both responses. Optimal levels were predicted for the maximum obtainment of ethanol and galactose simultaneously, which utilized the following parameters: 15%TS, 37 IU / g lactose, 28°C, 7.5 log cfu/mL, and 30 days, which together were predicted to produce 4.0%v/v ethanol and 51 g/L galactose in the final product. These parameters were then applied to 18-L fermentations, and the resulting fermentates were processed via distillation and freeze-drying. As a result, four product streams were obtained: a fermented product with 3.4%v/v ethanol and 56 g/L galactose; a 45%v/v ethanol distillate; a galactose-rich drink base (63 g/L); and a galactose-rich powder (55%w/w). These results demonstrate that it is possible to maximize the production of ethanol and galactose from the fermentation of WP and to design manufacturing processes based on these optimization models, to develop novel, potentially functional bioproducts from this stream.

Consumer interest in functional beverages has been increasing recently and has led to expansion in the market. Fermented beverages containing acetic acid such as kombucha are often produced using alternative microorganisms such as lactic and acetic acid bacteria as well as non-Saccharomyces yeasts. These organisms are used to produce health-promoting compounds which make these products popular with consumers. One such alternative yeast, Brettanomyces claussenii, has been gaining recognition for its biotechnological potential. Its capabilities include fermentation of non-traditional substrates and production of valuable by-products. Specifically, this species has the ability to utilize lactose and aerobically produce acetic acid. Harnessing the potential of this yeast leads to opportunities to upcycle lactose-containing dairy by-products to acetic acid fermented beverages.

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.

The COVID-19 pandemic has had dire effects on the United States (US) food industry through impacts on workers’ health and wellbeing and supply chain disruptions. The objectives of this study were to determine what the food industry needs to be able to control COVID-19 impacts in the work environment and what mitigation strategies are being implemented. A web-based needs assessment survey was distributed from January to April 2021, via 13 food professional/trade organizations and 2 social networks, targeting management professionals at food (produce, dairy, poultry, and beef/pork) processing facilities and produce farm operations in the US. Statistical analyses evaluated patterns in self-reported adoption of mitigation strategies against COVID-19 in the participants’ facilities/operations and perceived needs of the industry regarding COVID-Responses to open-ended questions were analyzed using thematic analysis. In total 145 responses were received, of which 79 were usable, including 38 (48%) from the dairy, 17 (22%) from the fresh produce, and 24 (30%) from a mixture of other food industry sectors. Only two usable responses were from the beef/pork sector and none from the poultry sector. Findings revealed that several social distancing, biosecurity, and surveillance mitigation strategies against COVID-19 are commonly implemented in the participants’ facilities/operations, but their implementation frequency differs by the facility/operation size and industry sector. Also, findings indicated that collaboration between the food industry and government agencies, contingency plans and appropriate training, and new technologies are needed to control COVID- 19 in the food industry. Subject to limitations associated with the relatively low response rate (possible selection bias), the findings suggest that the US food industry is prepared to safeguard workers’ health and businesses in the event of a new COVID-19 variant or similar future disaster, provided that appropriate structures are put in place to ensure coordination and compliance, both before and during such an outbreak.

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.

The COVID-19 pandemic has greatly impacted the U.S. food supply and consumer behavior. Food production and processing are being disrupted as illnesses, proactive quarantines, and government-mandated movement restrictions cause labor shortages. In this environment, the food industry has been required to adopt new, additional practices to minimize the risk of COVID-19 cases and outbreaks among its workforce. Successfully overcoming these challenges requires a comprehensive approach that addresses COVID-19 transmission both within and outside the facility. Possible interventions include strategies (i) to vaccinate employees, (ii) to assure that employees practice social distancing, (iii) to assure that employees wear face coverings, (iv) to screen employees for COVID-19, (v) to assure that employees practice frequent hand washing and avoid touching their faces, (vi) to clean frequently touched surfaces, and (vii) to assure proper ventilation. Compliance with these control strategies must be verified, and an overall COVID-19 control culture must be established to implement an effective program. Despite some public misperceptions about the health risk of severe acute respiratory syndrome coronavirus 2 on foods or food packaging, both the virus biology and epidemiological data clearly support a negligible risk of COVID-19 transmission through food and food packing. However, COVID-19 pandemic-related supply chain and workforce disruptions and the shift in resources to protect food industry employees from COVID-19 may increase the actual food safety risks. The goal of this review was to describe the COVID-19 mitigation practices adopted by the food industry and the potential impact of these practices and COVID-19-related disruptions on the industry's food safety mission. A review of these impacts is necessary to ensure that the food industry is prepared to maintain a safe and nutritious food supply in the face of future global disruptions.

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.

Blue discoloration in fresh mozzarella cheese (FMC) is a defect caused by certain biotypes of Pseudomonas spp. This defect has been increasing in frequency worldwide over the last decade; instances have occurred recently in the U.S., Italy, Germany, England, and Spain. In the current study, lactose oxidase (LO) was utilized as a surface treatment to investigate its potential to inhibit blue discoloration in two types of FMC: that produced through direct acidification (DA) by addition of citric acid, and that produced by starter fermentation (SF). Cheese samples were surface-treated with one of four concentrations of LO: 0, 0.012, 0.12, and 1.2 g/L; they were then inoculated with ~4.25 log CFU of the P. fluorescens biovar IV isolate with designation “FSL W5-0203”. A negative sample (NS) was also prepared, which was treated with neither LO nor P. fluorescens. Cheese samples were stored at 6 °C for 16 days. Pictures of the cheese samples were taken during this time, and the Commission Internationale de l'Eclairage (CIE) colorimetric system was used to quantify changes in color undergone by the samples. In DA the surface application of LO at a concentration of 1.2 g/L resulted in retention of conventional color characteristics; a concentration of 0.12 g/L also performed promisingly, but with less consistency than the higher concentration. A LO concentration of 0.012 g/L did not show an inhibitory effect. Quantification of LO's effects on blue discoloration in SF revealed less consistency than that seen with DA FMC; however, based on the researchers' visual assessments, the 1.2 g/L concentration performed the best out of those investigated, with obvious blue coloration occurring in only 1 out of 6 samples. A decrease in pH was observed when LO was applied at a concentration of 1.2 g/L in both the DA and SF cheeses; this indicates that LO is oxidizing the cheese's residual lactose to produce lactobionic acid. This study demonstrates a promising clean-label approach to prevent blue color defects in DA FMC, and provides hope for further progress in combating blue color in SF FMC.

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

T7 bacteriophages (phages) have been genetically engineered to carry the lacZ operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E. coli, the enzymatic activity of the released β-gal was monitored using a colorimetric substrate. Compared with a control T7 phage, our T7lacZ phage generated significantly higher levels of β-gal expression following phage infection, enabling a lower limit of detection for E. coli cells. Using this engineered T7lacZ phage, we were able to detect E. coli cells at 10 CFU·mL–1 within 7 h. Furthermore, we demonstrated the potential for phage-based sensing of bacteria antibiotic resistance profiling using our T7lacZ phage, and subsequent β-gal expression to detect antibiotic resistant profile of E. coli strains.

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.

Dairy manufacturing generates whey by-products, many of them considered waste; others, such as whey permeate, a powder high in lactose and minerals from deproteinated whey, have unrealized potential. This study identified yeast species capable of utilizing lactose from whey permeate to produce ethanol or organic acids, and identified fungal species that reduced the acidity of whey by-products. Reconstituted whey permeate was fermented anaerobically or aerobically for 34 days, using species from Cornell University’s Food Safety Lab, Alcaine Research Group, and Omega Labs. Yeast species: Kluyveromyces marxianus, Kluyveromyces lactis, Dekkera anomala, Brettanomyces claussenii, Brettanomyces bruxellensis; mold species: Mucor genevensis and Aureobasidium pullulans. Density, pH, cell concentrations, organic acids, ethanol, and sugar profiles were monitored. Under anoxic conditions, K. marxianus exhibited the greatest lactose utilization and ethanol production (day 20: lactose non-detectable; 4.52% ± 0.02 ethanol). Under oxic conditions, D. anomala produced the most acetic acid (day 34: 9.18 ± 3.38 g/L), and A. pullulans utilized the most lactic acid, increasing the fermentate’s pH (day 34: 0.26 ± 0.21 g/L, pH: 7.91 ± 0.51). This study demonstrates that fermentation of whey could produce value-added alcoholic or organic acid beverages, or increase the pH of acidic by-products, yielding new products and increasing sustainability.

Global dissemination of antibiotic-resistant bacteria in food animals is a major public health concern. Whilst many countries have implemented prudent antibiotic use policies and surveillance systems both in clinical and veterinary settings, there are no such systems in place in Albania and little is known about the levels of antibiotic-resistant bacteria in food animals within the country. A total of 172 poultry samples were taken from six Albanian farms over a 3-month period and were tested for the presence of Enterobacteriaceae. In total, 91 bacterial isolates were obtained and were characterised by species (Escherichia coli, Salmonella spp. or other Enterobacteriaceae) and by susceptibility to 11 antibiotics. Resistance rates of E. coli and Salmonella isolates were, respectively: amoxicillin (86%, 64%); chloramphenicol (77%, 82%); ciprofloxacin (93%, 73%); cefotaxime (14%, 0%); gentamicin (12%, 0%); kanamycin (30%, 18%); nalidixic acid (91%, 73%); streptomycin (70%, 55%); sulphonamides (91%, 73%); tetracycline (95%, 73%); and trimethoprim (79%, 64%). Multidrug resistance to at least four antibiotics was observed in 95% of E. coli isolates and 82% of Salmonella. In conclusion, these data indicate that: (i) Salmonella and E. coli isolates from Albanian poultry farms exhibit high to extremely high levels of antibiotic resistance; (ii) Salmonella and E. coli isolates exhibit resistance to multiple antibiotics; and (iii) multidrug resistance profiles among Enterobacteriaceae are geographically widespread. Implementation of prudent antibiotic use policies in food animals and related surveillance will be necessary to reduce the emergence, spread and establishment of highly resistant strains across poultry farms in Albania.

Anthocyanins and norbixin are natural pigments used in food; however, they are unstable. The aim of this study was to evaluate the microencapsulation technique to protect these pigments. Elderberry extract (source of anthocyanins) and norbixin were encapsulated using a microfluidic device with palm oil as middle phase in a water-in-oil-in-water emulsion. The formulations were characterized for morphology, particle size, encapsulation efficiency, zeta potential, color release under heating, Fourier transform infrared spectrophotometry, and color stability under different conditions. Spherical, mononucleated microcapsules, with particle size of 187-190 μm (elderberry) and 164-184 μm (norbixin), and with encapsulation efficiencies values of 47.80-54.87% (elderberry) and 49.18-74.73% (norbixin) were obtained. The formulations showed high color retention, with the encapsulated elderberry extract stored at pH 3.0 being the most stable. This study shows that the microencapsulation of these pigments using a microfluidic device provided protection, and represents a new method for anthocyanins and norbixin delivery in foods.

Pulsed-field gel electrophoresis (PFGE) characterization of 335 temporally and spatially matched clinical, bovine, and human Salmonella enterica subsp. enterica isolates revealed 167 XbaI PFGE patterns. These isolates were previously classified into 51 serotypes and 73 sequence types, as determined by multilocus sequence typing. Discriminatory power of PFGE (Simpson's index, D = 0.991) was considerably higher than that of multilocus sequence typing (D = 0.920) or serotyping (D = 0.913). Although 128 PFGE types each only represented a single isolate, 8 PFGE types represented >4 isolates, including (i) three serotype Enteritidis and Heidelberg patterns that were only identified among human isolates, (ii) two PFGE patterns (each representing serotypes Bardo and Newport) that were significantly more common among bovine isolates as compared with human isolates; (iii) two PFGE types that each includes two serotypes (4,5,12:i:- and Typhimurium; Thompson and 1,7:-:1,5); and (iv) one PFGE type that includes eight Typhimurium isolates from humans and cattle. Characterization of isolates collected over multiple farm visits indicated that given specific PFGE types persisted over time on 11 farms. On an additional seven farms, isolates with a given sequence type represented multiple PFGE type, which typically only differed by

Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

Salmonella is one of the leading causes of foodborne illness in countries around the world. Treatment of Salmonella infections, in both animals and humans has become more difficult with the emergence of multidrug-resistant (MDR) Salmonella strains. Foodborne infections and outbreaks with MDR Salmonella are also increasingly reported. To better monitor and control the spread of MDR Salmonella, it is important to understand the mechanisms responsible for drug resistance and how drug resistance is transmitted to and between Salmonella strains. This review summarizes current knowledge on antimicrobial drugs used to treat Salmonella infections and provides an overview of MDR Salmonella in the United States and a discussion of the genetics of Salmonella drug resistance, including the mechanisms responsible for the transmission of drug-resistance genes in Salmonella, using data from the United States and other countries.

A collection of 179 human and 156 bovine clinicalSalmonellaisolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA,manB, andmdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specificSalmonella entericaserotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related toSalmonellaserotype Typhimurium.Salmonellaserotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number ofSalmonellaisolates carried two copies ofmanB(33 isolates) or showed small deletion events infimA(nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification ofSalmonellasurface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission ofSalmonella.

Salmonella is the leading cause of known food-borne bacterial infections in the United States, with an incidence rate of approximately 15 cases per 100,000 people. The rise of antimicrobial-resistant Salmonella subtypes, including the appearance of subtypes resistant to ceftriaxone, represents a particular concern. Ceftriaxone is used to treat invasive cases of Salmonella in children and is closely related to ceftiofur, an antibiotic commonly used to treat diseases of cattle. In order to develop a better understanding of the evolution and transmission of ceftiofur resistance in Salmonella , we characterized ceftiofur-resistant and -sensitive Salmonella isolates from seven New York dairy farms. A total of 39 isolates from these seven farms were analyzed for evolutionary relatedness (by DNA sequencing of the Salmonella genes fimA , manB , and mdh ), antibiotic resistance profiles, and the presence of bla CMY-2 , a beta-lactamase gene associated with resistance to cephalosporins. Our data indicate that (i) resistance to ceftriaxone and ceftiofur was highly correlated with the presence of bla CMY-2 ; (ii) ceftiofur-resistant Salmonella strains were geographically widespread, as shown by their isolation from farms located throughout New York State; (iii) ceftiofur-resistant Salmonella strains isolated from farms represent multiple distinct subtypes and evolutionary lineages, as determined by serotyping, DNA sequence typing, and antimicrobial-resistance profiles; and (iv) ceftiofur-resistant Salmonella strains evolved by multiple independent acquisitions of an identical bla CMY-2 allele and by clonal spread of ceftiofur-resistant subtypes.

While serotyping and phage typing have been used widely to characterize Salmonella isolates, sensitive subtyping methods that allow for evolutionary analyses are essential for examining Salmonella transmission, ecology, and evolution. A set of 25 Salmonella enterica isolates, representing five clinically relevant serotypes (serotypes Agona, Heidelberg, Schwarzengrund, Typhimurium, and Typhimurium var. Copenhagen) was initially used to develop a multilocus sequence typing (MLST) scheme for Salmonella targeting seven housekeeping and virulence genes ( panB , fimA , aceK , mdh , icdA , manB , and spaN ). A total of eight MLST types were found among the 25 isolates sequenced. A good correlation between MLST types and Salmonella serotypes was observed; only one serotype Typhimurium var. Copenhagen isolate displayed an MLST type otherwise typical for serotype Typhimurium isolates. Since manB , fimA , and mdh allowed for the highest subtype discrimination among the initial 25 isolates, we chose these three genes to perform DNA sequencing of an additional 41 Salmonella isolates representing a larger diversity of serotypes. This “three-gene sequence typing scheme” allowed discrimination of 25 sequence types (STs) among a total of 66 isolates; STs correlated well with serotypes and allowed within-serotype differentiation for 9 of the 12 serotypes characterized. Phylogenetic analyses showed that serotypes Kentucky and Newport could each be separated into two distinct, statistically well supported evolutionary lineages. Our results show that a three-gene sequence typing scheme allows for accurate serotype prediction and for limited subtype discrimination among clinically relevant serotypes of Salmonella . Three-gene sequence typing also supports the notion that Salmonella serotypes represent both monophyletic and polyphyletic lineages.

Sequential fluid delivery with minimized external equipment is vital towards a point-of-care diagnostic device. In this work, we have further developed the On-chip Electrowetting Valves concept for the sequential delivery of the reagents to the reaction site in a miniaturized capillary-driven microfluidic chip. Specifically, a disposable polymeric microfluidic device was developed containing capillary force driven microchannels. The device was fabricated using laser ablation and inkjet printing and required no external pumping equipment. The assay was conducted on the microchip containing microfluidic channels with embedded electrowetting valves and a porous membrane patterned with capture molecules and colloidal gold labels. To conduct the assay, the microchip was connected with a low voltage supply which was capable of sequentially opening the valves, delivering the sample and the rinsing reagent to generate visual results. Using T7 bacteriophage as a model, we have demonstrated the development of the device, operation of the valves and execution of the automated assay.