Raw milk obtained from cows infected with H5N1 requires to be treated to inactivate the virus prior to disposal. In this context, a hurdle approach was evaluated to inactivate Phi6 bacteriophage (potential H5N1 surrogate) in raw milk as a risk management option for dairy farms. The treatment involved adding hydrogen peroxide (0.1, 1, or 10 mM) and sodium thiocyanate (0.2, 2.4, or 24 µM) to raw milk, which served as substrates for the lactoperoxidase system to produce hypothiocyanite. The substrates were also a source of free radicals via UV-C mediated photo-degradation in an Advanced Oxidation Process (AOP). The raw milk was inoculated with Phi6 bacteriophage (7-8 log PFU/mL) in the presence of thiocyanate and/or hydrogen peroxide, held for 10 min at 23 °C, before passing through a spiral UV-C reactor. The infective Phi6 phages were determined on a cell lawn of the Pseudomonas host cell. It was found that the lactoperoxidase system, in the absence of UV-C, supported a 3.01 ± 0.07 log PFU/mL reduction of phage when 10 mM hydrogen peroxide and 24 µM sodium thiocyanate were applied to raw milk. Hydrogen peroxide or thiocyanate alone supported <1.2 log reduction of Phi6. When UV-C (137 mJ/cm2) was applied in the presence of added hydrogen peroxide (10 mM) and thiocyanate (24 µM), the log reduction of phages increased to 4.49 ± 0.50 log PFU. The flow pattern of milk also influenced the efficacy of treatment, suggesting that homogenous mixing during passage through the reactor was an important factor. The Phi6 inactivation was partly supported by the direct action of UV photons, the generation of free radicals (hydroxyl-radical and thiocyanate), and lactoperoxidase. When the lactoperoxidase was thermally inactivated, the log reduction was increased to 5.63 ± 0.10 log PFU, suggesting competition for hydrogen peroxide and thiocyanate between the enzyme and AOP. The study demonstrated proof of principle for a nonthermal process for inactivating viruses in raw milk.

A gas phase hydroxyl-radical process was evaluated for inactivating coliforms (Enterobacter aerogenes), Listeria monocytogenes and Vibrio parahaemolyticus introduced on, within and under the water glaze of frozen clams. The hydroxyl-radical process operating parameters were 3% hydrogen peroxide delivered at different temperatures (4, 25 or 50°C), ozone gas introduced at 20 ppm and UV-C dose varied between 120 – 500 mJ/cm2. For surface inoculated clams, E. aerogenes were reduced from 7.10±0.09 Log CFU to below the level of enumeration (<1.0 Log CFU) by applying 240 mJ/cm2 and hydrogen peroxide temperature of 4 or 50°C. The same trend was found for E. aerogenes under the water glaze. In contrast, the log reduction of E. aerogenes inoculated within the glaze was dependent of UV-C dose but independent of the temperature in which the hydrogen peroxide was introduced. When the same treatments were applied to clams inoculated with V. parahaemolyticus, the pathogen was decreased from 4.56 – 5.01 log CFU to below the level of enumeration (<1.0 log CFU) irrespective of the method of inoculation. L. monocytogenes was more tolerant to hydroxyl-radicals with a log reduction on the surface being 2.00±0.18, within glaze, 2.16±0.18 and under glaze; 2.13±0.44 log CFU with a treatment of 3% v/v hydrogen peroxide, 20 ppm ozone and UV-C dose of 240 mJ/cm2. The clams remained frozen throughout the process with no quality issues after 30-day frozen storage. The hydroxyl-radical process represents a risk management approach to enhance the food safety of frozen seafood although hydrogen peroxide residues require to be addressed.

The study aimed to optimize the performance of a gas phase hydroxyl radical process to inactivate Salmonella and Campylobacter jejuni associated with raw poultry meat. Chicken parts were inoculated with Salmonella or C. jejuni before passing through a hydroxyl-radical reactor operating with different concentrations of hydrogen peroxide mist (1-6 % v/v) and UV-C dose (112-546 mJ/cm2) with ozone feed remaining constant. The surviving populations were enumerated and meat quality assessed. It was found that the log CFU reduction of Salmonella was limited to 1 log CFU and independent of hydrogen peroxide and UV-C dose A treatment of 1 % v/v hydrogen peroxide, 12 ppm ozone and a UV-C dose of 112 mJ/cm2 supported a 0.73 +/- 0.13 log CFU of C. jejuni and 1.08 log CFU Salmonella without negatively affecting meat quality. Hydroxyl-radical treatment of naturally contaminated samples reduced the carriage of Salmonella to 10 % when applying RT-PCR and down to 6 % with culture methods. In comparison, C. jejuni was decreased to 2 % by RT-PCR and non-detectable by culture-based testing. The treatment did not result in an extension of shelf-life. In conclusion, the gas-phase hydroxyl radical process presents an additional hurdle for reducing the carriage of Salmonella and Campylobacter on poultry meat.

Hatcheries have been identified as a significant source of Salmonella within poultry production. Consequently, there is a need for effective egg disinfection methods that can reduce the pathogen burden while preserving the egg integrity and embryo. The metrics for a successful egg disinfection method are typically a reduction in Total Aerobic Count (TAC) while retaining hatching rates. In this study, a gas phase hydroxyl-radical process was validated and verified as a hatchery egg disinfection method. The process is based on applying a hydrogen peroxide mist in combination with ozone gas and UV-C to generate antimicrobial hydroxyl radicals. The treatment (2 % hydrogen peroxide, 20 ppm ozone and 19 mJ/cm2 UV-C; designated as HR) for inactivating Salmonella (serotypes Enteritidis and Typhimurium) inoculated onto eggs could eliminate the pathogen (>5 log CFU/egg reduction) but left residual TAC (1.53 log CFU/egg reduction). Surface sterilization was achieved by a pre-treatment of eggs with the photo-catalyst riboflavin (13.75 mM) followed by 3 % hydrogen peroxide delivered at 70 °C prior to the hydroxyl-radical treatment (3 % hydrogen peroxide, 20 ppm ozone and 114 mJ/cm2 designated HRS). The surface sterilization of eggs coincided with the removal of the cuticle layer with the HRS treatment but not HR. The cuticle layer was also compromised by formaldehyde treatment. When the different treatments were applied to fertile hatchery eggs (n=50 eggs per treatment group), there was no significant difference in hatchery rate (64-74 %), with hatch to fertility being higher for disinfected eggs (89-97 %) compared to the non-treated control (80 %). The seven-day mortality (0 - 2 birds) and feed conversion ratio (1.59 - 1.75 kg/kg feed) did not significantly differ between the treated vs controls. The HR treatment could eliminate Enterococcus faecium, Escherichia coli (>5 log CFU/egg reduction) although HRS was required to inactivate Pseudomonas aeruginosa (>5 log CFU/egg reduction) and reduce Aspergillus niger spores (3.08±2.25 log CFU reduction). The study has provided treatment options for hatchery egg disinfection and alternative to formaldehyde treatment.

Objectives The paper describes designing and developing an online food safety toolbox that aims to elevate the food safety knowledge of food business operators, competent authorities, and trainers.Materials and Methods The material within the food safety toolbox was based on the Codex Alimentarius (Codex) General Principles of Food Hygiene (GPFH), an internationally recognized primary food safety standard. The GPFH provides a guide to elements that should be considered when establishing good hygienic practices (GHPs), which are subsequently managed through hazard analysis and critical control point (HACCP). To support the understanding of how to apply the principles of GHPs and HACCP, the online food safety toolbox was developed. This toolbox was designed to enable users to access the principles quickly as a reminder for better understanding of more complex matters and conceptualizing, building, and maintaining food safety management systems. The learning approaches applied in the design of the toolbox were mapping, chunking (grouping topics into a logic sequence to enable an incremental approach to learning), and learning-by-asking. The self-directed learning approach collectively enables the user to understand, categorize, and contextualize food safety information for practical use. Mapping was performed to identify the different elements within the GPFH that formed the basis of the online platform and the categories in which basic information was provided for each.Results The material progresses into greater depth in the final toolbox platform and includes links to detailed descriptions of the underlying science. This user-centric design was chosen to address different users' needs and reduce the entry barrier for contextually applying the presented GHPs and HACCP.Conclusions The GHP and HACCP Toolbox for Food Safety should be regarded as a reference resource rather than a training program to empower the user and ultimately enhance food safety practices.

The following reports on the efficacy of a gas‐phase hydroxyl radical‐based process for decontaminating shredded lettuce on a laboratory and simulated commercial scale. The process is based on the ultraviolet light at 254 nm UV‐C‐mediated degradation of hydrogen peroxide mist and ozone gas to generate antimicrobial hydroxyl radicals. Escherichia coli K12 was applied as a surrogate for E. coli O157:H7, and at laboratory scale, the hydroxyl‐radical process (1.5% vol/vol H2O2 delivered at 40 ml/min, UV‐C dose 114 mJ/cm2, 20 ppm ozone, 29°C chamber temperature, and 30 s residence time) could support a 1.63 ± 0.61 log CFU reduction. This is compared to the 0.57 ± 0.18 log CFU reduction obtained for a chlorine‐based wash. In scale‐up, batches (2‐10 kg) of E. coli inoculated romaine lettuce were passed through sequential hydroxyl‐radical reactors. Here, the units were elevated to create a cascade effect, with the hydrogen peroxide mist being introduced as an intermister between the reactors. It was found that the three units placed in sequence with intermisters supported a 2.05 ± 0.10 log CFU reduction of E. coli, thereby verifying that homogenous treatment had been achieved. Additional trials operated the hydroxyl‐radical process at 4°C without loss of performance. The hydroxyl‐radical process was not negatively affected by applying a pretreatment wash. The study has demonstrated that the hydroxyl‐radical process can be applied as an alternative to postharvest wash to enhance the food safety of romaine lettuce.

Fresh produce accounts for the majority of foodborne illness outbreaks and >30% of food waste attributed to premature spoilage. To date, pathogen control has been focused on the pre-harvest stage, in the form of testing and Good Agricultural Practice, given the limited efficacy of post-harvest washing to remove field-acquired contamination. However, the open nature of production systems and unreliability of testing have led to increased attention on post-harvest interventions as a type of ‘pasteurisation’ step. Attempts to improve the washing process involve identifying and maintaining sufficient free chlorine in wash tanks to prevent cross-contamination events. Alternative sanitisers to chlorine have also been evaluated although at best, minimize cross-contamination rather than support decontamination. This has led to the development of aqueous-free produce decontamination methods with gas-phase, gas plasma and hydroxyl-radical treatments showing promise. The development of a standard validation method to compare technologies is discussed along with future directions.

GES (Guiana Extended Spectrum) carbapenemases belong to "minor class A carbapenemases" and its prevalence could be underestimated due to the lack of specific tests. The aim of this study was to develop an easy PCR method to differentiate between GES β-lactamases with or without carbapenemase activity, based on an allelic discrimination system of SNPs that encode E104K and G170S mutations, without need of sequencing.Two pair of primers and Affinity Plus probes, labeled with different fluorophores; FAM/IBFQ and YAK/IBFQ, were designed for each one of the SNPs.This allelic discrimination assay allows to detect in real time the presence of all type of GES- β-lactamases, being able to differentiate between carbapenemases and extended-spectrum β-lactamase (ESBL), through a quick PCR test that avoid costly sequencing approaches and could help to decrease the current underdiagnosis of minor carbapenemases that scape of phenotypic screenings.

Salicylaldehyde (SAL) is a natural flavoring agent with broad-spectrum antimicrobial activities but susceptible to degradation. To enhance its stability, SAL was conjugated with branched polyethyleneimine to form a SAL precursor (SALP) for controlled release applications. SAL molecules formed covalent bonds with BrPEI via imine linkages and imidazolidine rings, as confirmed by 1D and 2D NMR and FTIR spectroscopies. SALP was metastable under dry conditions but susceptible to acid-hydrolysis, triggering the release of SAL vapor, as revealed by the 1H NMR analysis. To facilitate end-use applications, SALP was encapsulated in poly(lactic acid)/poly(ethylene oxide) (PLA/PEO) nonwovens prepared by a free-surface electrospinning process. The release of SAL vapor from SALP-loaded nonwovens was studied using a citric acid (CA) solution of different concentrations. Additionally, a tri-layer nonwoven comprised of a SALP-loaded nonwoven sandwiched between two layers of CA-loaded fibers was developed. When exposed to 100 % RH, the composite nonwoven released SAL at a level of 0.046 +/- 0.011 mg SAL/mg nonwoven in 4 h. The composite nonwoven exhibited antimicrobial properties against Escherichia coli K12, showing an 8-log reduction after a 4 h exposure to SAL vapor released from a 3 x 3 cm2 nonwoven. The antimicrobial nonwoven could be promising for limiting the proliferation of spoilage and pathogenic microorganisms to enhance the safety and reduce the wastage of food products.

Wheat kernels harbor a diverse microflora that can negatively affect the suitability of the grains for further processing. To reduce surface microflora, a kernel disinfection method is required that does not affect grain functionality. Three different versions of gas phase hydroxyl-radical processes were compared with the common method for grain disinfection, that is, a bleach treatment. The gas phase hydroxyl-radicals are generated by the UV-C mediated degradation of hydrogen peroxide and/or ozone in a near water-free process. It was found that treating kernels with a bleach solution could reduce total aerobic count (TAC) and fungal count to below the level of enumeration. In comparison, the gas phase hydroxyl-radical treatment, that is, H2 O2 -UV-ozone treatment, could support a 1.3 log count reduction (LCR) in TAC and a 1.1 LCR in fungal count. The microbial load reduction for the wholemeal samples was less pronounced as endophytic microorganisms were less affected by all treatments, hinting at a limited penetration depth of the treatments. Despite reducing the microbial load on the kernel surface through the bleach and H2 O2 -UV-ozone treatments, none of these treatments resulted in a reduced microbial count on grains that underwent sprouting after the treatments. No negative effect on germination power or development of the seedling was observed for any of the treatments. The gluten aggregation behavior and xylanase activity of the wholemeal also remained unchanged after the gas phase hydroxyl-radical treatments. Our findings suggest that UV-H2 O2 -ozone treatment shows promise for dry-kernel disinfection, but further optimization of the processing parameters is required.

Eggs represent a significant vehicle for Salmonella Enteritidis with the pathogen being transferred to chicks in the hatchery, or to consumers via table eggs. In the following, the efficacy of a gas-phase hydroxyl-radical process for decontaminating hatchery and table eggs was evaluated. Recovery of Salmonella was maximized through holding eggs in tryptic soy broth containing 20% w/v glycerol for 1 h prior to plating. By using this technique, it was possible to recover 63% of the theoretical Salmonella inoculated onto eggs. The continuous hydroxyl-radical reactor consisted of a bank of UV-C lamps (254 nm) that generated hydroxyl-radicals from the degradation of hydrogen peroxide (H2O2) mist and ozone gas. The optimal treatment was defined as that which supports a 5 log CFU/egg reduction of Salmonella without negatively affecting egg quality or leaving H2O2 residues. A process of 2% v/v H2O2 delivered at 30 mL/min with a UV-C dose of 19 mJ/cm(2) and ozone (20 ppm) with a total treatment time of 10s was selected. The egg quality metrics (Haugh value, yolk index, albumin pH, yolk pH) did not negatively differ over a 35-day shelf-life at 4 or 25degree celsius compared to washed eggs or nontreated controls. The cuticle layer of eggs remained intact following hydroxyl-radical treatment. Fertilized eggs (n = 61) treated with the hydroxyl-radicals exhibited the same hatchery rate (75%) as nontreated controls (71-79%) with no defects (unhealed navels or red hocks) being observed. The same hydroxyl-radical treatment could be applied to table eggs to support >5 log CFU/egg reduction of Salmonella and was compatible with egg washing regimes practiced in industry. In comparison, the egg washing process based on sodium hydroxide and chlorine supported a 2.76 +/- 0.38 log CFU/egg reduction of Salmonella. The hydroxyl-radical treatment represents a preventative control step to reduce the carriage of Salmonella on hatchery and table eggs.

The emergence of antibiotic-resistant bacteria due to the overuse or inappropriate use of antibiotics has become a significant public health concern. The agri-food chain, which serves as a vital link between the environment, food, and human, contributes to the large-scale dissemination of antibiotic resistance, posing a concern to both food safety and human health. Identification and evaluation of antibiotic resistance of foodborne bacteria is a crucial priority to avoid antibiotic abuse and ensure food safety. However, the conventional approach for detecting antibiotic resistance heavily relies on culture-based methods, which are laborious and time-consuming. Therefore, there is an urgent need to develop accurate and rapid tools for diagnosing antibiotic resistance in foodborne pathogens. This review aims to provide an overview of the mechanisms of antibiotic resistance at both phenotypic and genetic levels, with a focus on identifying potential biomarkers for diagnosing antibiotic resistance in foodborne pathogens. Furthermore, an overview of advances in the strategies based on the potential biomarkers (antibiotic resistance genes, antibiotic resistance-associated mutations, antibiotic resistance phenotypes) for antibiotic resistance analysis of foodborne pathogens is systematically exhibited. This work aims to provide guidance for the advancement of efficient and accurate diagnostic techniques for antibiotic resistance analysis in the food industry.

A continuous Photo-Fenton Advanced-Oxidation-Process (AOP) for reducing the chlorine-demand of spent lettuce wash water was developed based on the generation of hydroxyl-radicals from the UV-C degradation of hydrogen peroxide in the presence of ferric-catalyst. It was found that an interaction between UV-C and hydrogen peroxide or ferric-catalyst concentration was associated with high hydroxyl-radical generation as determined from the oxidation of methylene blue. The optimal AOP treatment was identified as 320 mJ/cm2 UV-C dose, 9.6 mg/L H2O2, and 9 mg/L ferric-catalyst. When the treatment was applied to simulated lettuce spent wash water (6.6 g romaine lettuce per liter of distilled water containing 100 mg bentonite; pH 6.9) the chlorine demand was reduced from 150 ppm to 130 ppm. The chlorination of AOP treated water did not result in a greater log reduction of pathogens (Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella) on lettuce but did reduce cross-contamination between batches during washing. The chlorinated byproducts formed in AOP treated water exhibited higher antimicrobial activity compared to untreated controls. Although the treatment was successful in reducing cross-contamination of lettuce batches the cytotoxicity of disinfection byproducts requires to be assessed.

The following reports on the generation of hydroxyl-radical activated water prepared by passing a hydrogen peroxide solution containing Fe(III) catalyst through a UV-C reactor. The activated water was subsequently evaluated for antimicrobial activity against Escherichia coli O157:H7 in suspension or when inoculated onto mung beans. Hydroxyl-radical generation was assessed through the oxidation of methylene blue when reacted with activated water prepared from solutions of different pH (4-10), UV-C dose (32-128 mJ/cm(2)), hydrogen peroxide (0-1000 mg/L) and Fe(III) concentration (0-100 mg/L). Methylene blue oxidation was associated with high concentrations of each reactant with a positive correlation with Fe(III) concentration. Inactivation curves of E. coli O157:H7 in activated water were diphasic with an initial slow rate that increased after 15 min contact time. In contrast to the methylene blue assay, the antimicrobial action of activated water was associated with high hydrogen peroxide (500 mg/mL) and low Fe(III) catalyst (1 mg/L) with no significant interaction with UV-C dose. Evidence would suggest that the mode-of-inactivation was through a radical propagation reaction that is rate-limited by the reduction of Fe (III) to Fe (II). Here, the initial activation process via UV-C illumination results in photo-reduction of Fe(III) and propagates the formation of hydroxyl-radicals. Fe(III) to Fe(II) cycling continues with oxidation of cell structures that ultimately leads to loss of viability due to accumulation of cellular damage. When activated water was used to soak mung beans inoculated with E. coli O157:H7 a 1 log reduction was obtained with a 19% increase in germinated beans and 8.5% higher sprout yield relative to controls. The oxidation reduction potential decreased from 477 mV to 288 mV and pH increased from 3.97 to 5.47, over the 24 h mung bean soak period. The reduction of Salmonella and Listeria monocytogenes on mung beans soaked in activated water was < 1 log CFU/g with all three pathogens growing back over the sprouting period. From the results it can be concluded that activated water can enhance the germination of mung beans along with sprout yield but has limited capacity when applied alone as a seed disinfection method.

This chapter provides an overview of current and emerging approaches to decontaminate raw meat. The introduction of Hazard Analysis Critical Control Points has been the key motivator for introducing interventions with meat processing and stimulated R&D to develop pathogen controls based on physical, chemical, or biological approaches. To this end, a broad range of decontamination methods has been evaluated on the laboratory scale with a number now being applied commercially. The advantages and disadvantages of different interventions applied alone or in combination will be described. Recent advances in meta-analysis to provide an assessment on different combinations of interventions within meat processing will be provided. The underlying reasons why laboratory-based studies typically overestimate the efficacy of interventions when applied on a commercial scale will be discussed. Finally, knowledge gaps and future research directions will be described.

The animals we use for meat may sometimes carry various bacteria, parasites, or viruses, which can contaminate meat and, when consumed, can cause disease in humans. This chapter covers the most important foodborne pathogens associated with the consumption of meat products. The presence of different foodborne pathogens is variable and may depend on the type of animal, geographical region, and production systems. Foodborne pathogens vary in how they are transmitted and can survive through the food chain. It is critical that good hygienic practices during animal slaughter, handling, and preparation of meat are maintained to limit contamination. Appropriate cooking of meat is also important for limiting the transmission of pathogens to humans. Ongoing efforts to minimize the transmission of foodborne pathogens from meat consumption will need to focus on the whole food chain from farm production through to consumption to reduce the levels of pathogens at all stages.

The success of high-pressure processing (HPP) implementation by a large number of food companies demonstrates HPP is an effective safety measure that can be applied to mitigate risk for a variety of foods and beverages and to extend shelf life of refrigerated products. The HPP commercialization has led to a high demand from food manufactures to apply HPP despite the lack of a full understanding of factors that affect the efficacy of the process or associated risks linked to specific food products. Indeed, there is no standard methodology for validating HPP or established surrogates to verify process performance. Yet, a scientifically sound validation process is a critical component in assessing the efficacy of HPP as a microbial-control measure. This chapter will focus on HPP validation used for nonthermal pasteurization that reduces pathogen levels and support the reduction of vegetative microbes to extend shelf life of foods and beverages. The fundamental principles of HPP along with essential practical considerations in terms of critical product and process parameters, packaging, microbial challenge studies, and other testing and scaling up issues are discussed in detail to assist HPP end users to accelerate process innovation.

With the pressure to reduce antibiotics use in poultry production, cost-effective alternative products need to be developed to enhance the bird's immunity. The present study evaluated the efficacy of cranberry fruit by-products to modulate immunity in broiler chickens. Broiler Cobb 500 chicks were fed a control basal diet, basal diet supplemented with bacitracin (BACI, 55 ppm), cranberry pomace at 1% and 2% (CP2), or cranberry pomace ethanolic extract at 150 and 300 ppm (COH300) for 30 d. Blood sera were analyzed at days 21 and 28 of age for Ig levels by ELISA. The innate and adaptive immune-related gene expression levels in the liver and bursa of Fabricius were investigated at 21 d of age by quantitative polymerase chain reaction arrays. At day 21, the highest IgY level was found in the blood serum of the CP2-fed birds. In the liver, 13 of the 22 differentially expressed genes were downregulated across all treatments compared with the control. Expression of genes belonging to innate immunity such as caspase 1 apoptosis–related cysteine peptidase, chemokine receptor 5, interferon gamma, myeloid differentiation primary response gene 88, and Toll-like receptor 3 were significantly downregulated mainly in BACI- and COH300-fed birds. In the bursa, 5 of 9 genes associated with the innate immunity were differentially expressed. The expression of anti-inflammatory IL-10 gene was upregulated in all treatment groups in bursa compared with the control. The expression of transferrin gene was significantly upregulated in livers of birds fed COH300 and in bursa of birds fed BACI, indicating feeding practices and organ-dependant modulation of this gene in broiler. Overall results of this study showed that cranberry product feed supplementation modulated the innate immune and suppressed proinflammatory cytokines in broilers, providing a platform for future investigations to develop berry products in poultry feeding.