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Thermal processing of packaged fruit and vegetable products is targeted at eliminating microbial contaminants (related to spoilage or pathogenicity) and extending shelf life using microbial inactivation or/and by reducing enzymatic activity in the food. The conventional process of thermal processing involves sterilization (canning and retorting) and pasteurization. The parameters used to design the thermal processing regime depend on the time (minutes) required to eliminate a known population of bacteria in a given food matrix under specified conditions. However, due to the effect of thermal exposure on the sensitive nutrients such as vitamins or bioactive compounds present in fruits and vegetables, alternative technologies and their combinations are required to minimize nutrient loss. The novel moderate thermal regimes aim to eliminate bacterial contaminants while retaining nutritional quality. This review focuses on the “thermal” processing regimes for fruit and vegetable products, including conventional sterilization and pasteurization as well as mild to moderate thermal techniques such as pressure-assisted thermal sterilization (PATS), microwave-assisted thermal sterilization (MATS) and pulsed electric field (PEF) in combination with thermal treatment as a hurdle approach or a combined regime.

The thermal processing of food relies heavily on determining the right time and temperature regime required to inactivate bacterial contaminants to an acceptable limit. To design a thermal processing regime with an accurate time and temperature combination, the D-values of targeted microorganisms are either referred to or estimated. The D-value is the time required at a given temperature to reduce the bacterial population by 90%. The D-value can vary depending on various factors such as the food matrix, the bacterial strain, and the conditions it has previously been exposed to; the intrinsic properties of the food (moisture, water activity, fat content, and pH); the method used to expose the microorganism to the thermal treatment either at the laboratory or commercial scale; the approach used to estimate the number of survivors; and the statistical model used for the analysis of the data. This review focused on Bacillus cereus, Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, and Clostridium perfringens owing to their pathogenicity and the availability of publications on their thermal resistance. The literature indicates a significant variation in D-values reported for the same strain, and it is concluded that when designing thermal processing regimes, the impact of multiple factors on the D-values of a specific microorganism needs to be considered. Further, owing to the complexity of the interactions involved, the effectiveness of regimes derived laboratory data must be confirmed within industrial food processing settings.

(1) Background: Limited evidence exists addressing the action of antimicrobial visible light against Cronobacter sakazakii. Here, we investigated the antimicrobial effects of blue-LED (light emitting diode) at 405 nm against two persistent dairy environment sourced strains of C. sakazakii (ES191 and AGRFS2961). (2) Methods: Beside of investigating cell survival by counts, the phenotypic characteristics of the strains were compared with a reference strain (BAA894) by evaluating the metabolic rate, cell membrane permeability, and ROS level. (3) Results: The two environment isolates (ES191 and AGRFS2961) were more metabolic active and ES191 showed dramatic permeability change of the outer membrane. Notably, we detected varied impacts of different ROS scavengers (catalase &gt, thiourea &gt, superoxide dismutase) during light application, suggesting that hydrogen peroxide (H2O2), the reducing target of catalase, has a key role during blue light inactivation. This finding was further strengthened, following the observation that the combined effect of external H2O2 (sublethal concentration) and 405 nm LED, achieved an additional 2–4 log CFU reduction for both stationary phase and biofilm cells. (4) Conclusions: H2O2 could be used in combination with blue light to enhance bactericidal efficacy and form the basis of a new hurdle technology for controlling C. sakazakii in dairy processing plants.

Transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs through respiratory droplets passed directly from person to person or indirectly through fomites, such as common use surfaces or objects. The aim of this study was to determine the virucidal efficacy of blue LED (405 nm) and far-UVC (222 nm) light in comparison to standard UVC (254 nm) irradiation for the inactivation of feline infectious peritonitis virus (FIPV) on different matrices as a model for SARS-CoV-2. Wet or dried FIPV on stainless steel, plastic, or paper discs, in the presence or absence of artificial saliva, were exposed to various wavelengths of light for different time periods (1–90 min). Dual activity of blue LED and far-UVC lights were virucidal for most wet and dried FIPV within 4 to 16 min on all matrices. Individual action of blue LED and far-UVC lights were virucidal for wet FIPV but required longer irradiation times (8–90 min) to reach a 4-log reduction. In comparison, LED (265 nm) and germicidal UVC (254 nm) were virucidal on almost all matrices for both wet and dried FIPV within 1 min exposure. UVC was more effective for the disinfection of surfaces as compared to blue LED and far-UVC individually or together. However, dual action of blue LED and far-UVC was virucidal. This combination of lights could be used as a safer alternative to traditional UVC.

Shiga toxin-producing Escherichia coli (STEC) are a major group of human pathogens and may persist on both abiotic and biotic surfaces. In this report, two blue-light prototypes were used to evaluate the antimicrobial efficacy against STEC on food processing surfaces (stainless steel and polyoxymethylene plastic). Investigation using a light-bulb prototype (Prototype 1 at 405 nm, 26 mW/cm2) showed significant antimicrobial effects in nutrient deficient condition but not in nutrient rich condition, demonstrating that the presence of organic matters from rich nutrient medium was thought to be light-absorptive and reduce the bactericidal efficacy of blue light, as evident from the lack of bacterial reduction when suspended in cooked meat broth. An advanced (surface-mounted-diode) light panel, Prototype 2 with high light intensity (405 nm; 50 mW/cm2) was able to inactivate a cocktail of seven STEC strains (from seven major serotypes O26, O45, O103, O111, O121, O145 and O157) on type 304 stainless steel (1.66 log10 CFU) and polyoxymethylene plastic (4.25 log10 CFU) at light dosages of 720 and 45 J/cm2, respectively when cells were illuminated in a nutrient-deficient medium (M9 broth). Post-treatment, no STEC cells were recoverable from plastic, both when tested on plates (agar or petrifilms) and by polymerase chain reaction (PCR). In contrast, surviving colonies were identified on samples taken from stainless steel, albeit only four strains could be detected by PCR analysis – those belonging to serotypes O26, O45, O103 and O157 – which indicated that the susceptibility of STEC to blue light varied across the tested strains.

Plasma-activated water (PAW) is generated by treating water with cold atmospheric plasma (CAP) using controllable parameters, such as plasma-forming voltage, carrier gas, temperature, pulses, or frequency as required. PAW is reported to have lower pH, higher conductivity, and higher oxygen reduction potential when compared with untreated water due to the presence of reactive species. PAW has received significant attention from researchers over the last decade due to its non-thermal and non-toxic mode of action especially for bacterial inactivation. The objective of the current review is to develop a summary of the effect of PAW on bacterial strains in foods as well as model systems such as buffers, with a specific focus on fruit and vegetables. The review elaborated the properties of PAW, the effect of various treatment parameters on its efficiency in bacterial inactivation along with its usage as a standalone technology as well as a hurdle approach with mild thermal treatments. A section highlighting different models that can be employed to generate PAW alongside a direct comparison of the PAW characteristics on the inactivation potential and the existing research gaps are also included. The mechanism of action of PAW on the bacterial cells and any reported effects on the sensory qualities and shelf life of food has been evaluated. Based on the literature, it can be concluded that PAW offers a significant potential as a non-chemical and non-thermal intervention for bacterial inactivation, especially on food. However, the applicability and usage of PAW depend on the effect of environmental and bacterial strain-based conditions and cost-effectiveness.

Blue light primarily exhibits antimicrobial activity through the activation of endogenous photosensitizers, which leads to the formation of reactive oxygen species that attack components of bacterial cells. Current data show that blue light is innocuous on the skin, but may inflict photo-damage to the eyes. Laboratory measurements indicate that antimicrobial blue light has minimal effects on the sensorial and nutritional properties of foods, although future research using human panels is required to ascertain these findings. Food properties also affect the efficacy of antimicrobial blue light, with attenuation or enhancement of the bactericidal activity observed in the presence of absorptive materials (for example, proteins on meats) or photosensitizers (for example, riboflavin in milk), respectively. Blue light can also be coupled with other treatments, such as polyphenols, essential oils and organic acids. While complete resistance to blue light has not been reported, isolated evidence suggests that bacterial tolerance to blue light may occur over time, especially through gene mutations, although at a slower rate than antibiotic resistance. Future studies can aim at characterizing the amount and type of intracellular photosensitizers across bacterial species and at assessing the oxygen-independent mechanism of blue light—for example, the inactivation of spoilage bacteria in vacuum-packed meats.

Cattle are an established reservoir of the foodborne bacterial pathogen Campylobacter jejuni. Our six-month study aimed to evaluate sources and pathways governing long-term presence of C. jejuni in a pasture-based dairy herd. C. jejuni was detected in all sample types (soil, pasture, stock drinking water, bird, rodents and cow faeces). It was persistently detected from cow (54%, 49/90 samples) and bird (36%, 77/211) faeces. Genetic comparison of 252 C. jejuni isolates identified 30 Multi-Locus Sequence Types (ST). ST-61 and ST-42 were persistent in the herd and accounted for 43% of the cow isolates. They were also detected on pasture collected from fields both recently and not recently grazed, indicating that grazed pasture is an important pathway and reservoir for horizontal transmission among cows. ST-61 accounted for 9% of the bird isolates and was detected at four of the six sampling events, suggesting that bird populations might contribute to the cycling of ruminant-adapted genotypes on-farm. Overall, the results indicated that management of grazed pasture and supplementary feed contaminated by bird droppings could be targeted to effectively reduce transmission of C. jejuni to dairy herds, the farm environment and ultimately to humans.

In this study, novel spore pouches were developed using mashed potato as a food model inoculated with either Geobacillus stearothermophilus or Clostridium sporogenes spores. These spore pouches were used to evaluate the sterilization efficiency of Coaxially induced microwave pasteurization and sterilization (CiMPAS) as a case study. CiMPAS technology combines microwave energy (915 MHz) along with hot water immersion to sterilize food in polymeric packages. The spore pouches were placed at pre-determined specific locations, especially cold spots in each food tray before being processed using two regimes (R-121 and R-65), which consisted of 121 &deg, C and 65 &deg, C at 12 and 22 kW, respectively, followed by recovery and enumeration of the surviving spores. To identify cold spots or the location for inoculation, mashed potato was spiked with Maillard precursors and processed through CiMPAS, followed by measurement of lightness values (*L-values). Inactivation equivalent to of 1&ndash, 2 Log CFU/g and &gt, 6 Log CFU/g for Geobacillus stearothermophilus and Clostridium sporogenes spores, respectively was obtained on the cold spots using R-121, which comprised of a total processing time of 64.2 min. Whereas, inactivation of &lt, 1 and 2&ndash, 3 Log CFU/g for G. stearothermophilus and C. sporogenes spores, respectively on the cold spots was obtained using R-65 (total processing time of 68.3 min), whereas inactivation of 1&ndash, 3 Log CFU/g of C. sporogenes spores was obtained on the sides of the tray. The results were reproducible across three processing replicates for each regime and inactivation at the specific locations were clearly distinguishable. The study indicated a strong potential to use spore pouches as a tool for validation studies of microwave-induced sterilization.

The transfer of blown pack spoilage causing Clostridium spores from the farm to the meat plant is of growing concern to the meat industry. This study investigated the environmental niches of these Clostridium spp., specifically Clostridium estertheticum and Clostridium gasigenes in the beef and sheep farm environments in New Zealand. Faecal, soil, grass, drinking water, puddle water and feed (fodder beet, hay, bailage and silage, where available) samples were collected on five beef and sheep farms during Winter and Spring in 2018, in North and South Island, respectively. Beef and sheep farm samples were tested for C. estertheticum and C. gasigenes using enrichment plus PCR, qPCR and direct plating. C. estertheticum was detected in bovine faecal (4%), soil (2–18%) and grass (0–12%) samples at concentration of up to 2.0 log10 cfu/g. C. gasigenes were found in 18–46% of faecal, 16–82% of soil, 12–44% of grass, 0–44.4% of drinking water and 0–58.3% of puddle water samples tested and the direct counts ranged from 2.4 log10 cfu/ml in puddle water to 3.4 log10 cfu/g in soil. C. estertheticum were detected by qPCR in sheep farms in ovine feces (2.3%), soil (2.3%) and fodder beet (10%). All other sample types (grass, drinking water, puddle water, baleage, hay, silage and fodder beet) were negative using direct and enrichment plus PCR methods. In contrast C. gasigenes was detected in of faecal (22.7–38.6%), soil (22.7–84.1%), grass (17.5–34.1%) drinking water (35.7–78.6%), puddle water (33.3–40%), hay baleage (57%), silage (2%) and fodder beet (10%) at concentrations of up to 3.7 log10 cfu/g/ml. It was concluded that C. estertheticum and C. gasigenes were common on beef and sheep farms with the latter having higher incidence and mean concentration.

The model food in this study known as mashed potato consisted of ribose (1.0%) and lysine (0.5%) to induce browning via Maillard reaction products. Mashed potato was processed by Coaxially Induced Microwave Pasteurization and Sterilization (CiMPAS) regime to generate an F0 of 6&ndash, 8 min and analysis of the post-processed food was done in two ways, which included by measuring the color changes and using hyperspectral data acquisition. For visualizing the spectra of each tray in comparison with the control sample (raw mashed-potato), the mean spectrum (i.e., mean of region of interest) of each tray, as well as the control sample, was extracted and then fed to the fitted principal component analysis model and the results coincided with those post hoc analysis of the average reflectance values. Despite the presence of a visual difference in browning, the Lightness (L) values were not significantly (p &lt, 0.05) different to detect a cold spot among a range of 12 processed samples. At the same time, hyperspectral imaging could identify the colder trays among the 12 samples from one batch of microwave sterilization.

Clostridium estertheticum-like strain FP3 was isolated from vacuum-packaged refrigerated spoiled lamb. This bacterium is psychrotrophic, Gram positive, spore-forming, and a strict anaerobe. Here, we report the generation and annotation of the 5.6-Mb draft genome sequence of C. estertheticum-like strain FP3.

Antimicrobial resistance (AMR) is a growing global concern and has called for the integration of different areas of expertise for designing robust solutions. One such approach is the development of antimicrobial surfaces to combat the emerging resistance in microbes against drugs and disinfectants. This review is a compressive summary of the work done in the field of material science, chemistry, and microbiology in the development of antimicrobial materials and surfaces that are inspired by examples in nature. The focus includes examples of natural antimicrobial surfaces, such as cicada wings or nanopillars, dragonfly wings, shrimp shells, taro leaves, lotus leaves, sharkskin, gecko skin, and butterfly wings, along with their mechanism of action. Techniques, compositions, and combinations that have been developed to synthetically mimic these surfaces against bacterial/viral and fungal growth in food-processing areas have also been discussed. The applications of synthetic mimics of natural antimicrobial surfaces in food-processing environments is still a naïve area of research. However, this review highlights the potential applications of natural antimicrobial surfaces in the food-processing environment as well as outlines the challenges that need mitigations.

Shiga toxin-producing Escherichia coli (STEC) are a leading cause of foodborne outbreaks of human disease, but they reside harmlessly as an asymptomatic commensal in the ruminant gut. STEC serogroup O145 are difficult to isolate as routine diagnostic methods are unable to distinguish non-O157 serogroups due to their heterogeneous metabolic characteristics, resulting in under-reporting which is likely to conceal their true prevalence. In light of these deficiencies, the purpose of this study was a twofold approach to investigate enhanced STEC O145 diagnostic culture-based methods: firstly, to use a genomic epidemiology approach to understand the genetic diversity and population structure of serogroup O145 at both a local (New Zealand) (n = 47) and global scale (n = 75) and, secondly, to identify metabolic characteristics that will help the development of a differential media for this serogroup. Analysis of a subset of E. coli serogroup O145 strains demonstrated considerable diversity in carbon utilisation, which varied in association with eae subtype and sequence type. Several carbon substrates, such as D-serine and D-malic acid, were utilised by the majority of serogroup O145 strains, which, when coupled with current molecular and culture-based methods, could aid in the identification of presumptive E. coli serogroup O145 isolates. These carbon substrates warrant subsequent testing with additional serogroup O145 strains and non-O145 strains. Serogroup O145 strains displayed extensive genetic heterogeneity that was correlated with sequence type and eae subtype, suggesting these genetic markers are good indicators for distinct E. coli phylogenetic lineages. Pangenome analysis identified a core of 3,036 genes and an open pangenome of >14,000 genes, which is consistent with the identification of distinct phylogenetic lineages. Overall, this study highlighted the phenotypic and genotypic heterogeneity within E. coli serogroup O145, suggesting that the development of a differential media targeting this serogroup will be challenging.

Antimicrobial resistance continues to rise on a global scale, affecting the environment, humans, animals and food systems. Use of natural antimicrobials has been favoured over synthetic molecules in food preservation owing to concerns over the adverse health effects of synthetic chemicals. The continuing need for novel natural antimicrobial compounds has spurred research to investigate natural sources, such as bacteria, for antimicrobials. The antimicrobial-producing potential of bacteria has been investigated in numerous studies. However, the discovery of antimicrobials has been biased towards aerobes and facultative anaerobes, and strict anaerobes such as Clostridium spp. have been largely neglected. In recent years, genomic studies have indicated the genetic potential of strict anaerobes to produce putative bioactive molecules and this has encouraged the exploration of Clostridium spp. for their antimicrobial production. So far, only a limited number of antimicrobial compounds have been isolated, identified and characterised from the genus Clostridium. This review discusses our current knowledge and understanding of clostridial antimicrobial compounds as well as recent genome mining studies of Clostridium spp. focused at identification of putative gene clusters encoding bacterial secondary metabolite groups and peptides reported to possess antimicrobial properties. Furthermore, opportunities and challenges in the identification of antimicrobials from Clostridium spp. using genomic-guided approaches are discussed. The limited studies conducted so far have identified the genus Clostridium as a viable source of antimicrobial compounds for future investigations.

Food security and environmental issues have become global crises that need transformative solutions. As livestock production is becoming less sustainable, alternative sources of proteins are urgently required. These include cultured meat, plant-based meat, insect protein and single-cell protein. Here, we describe the food safety aspects of these novel protein sources, in terms of their technological backgrounds, environmental impacts and the necessary regulatory framework for future mass-scale production. Briefly, cultured meat grown in fetal bovine serum-based media can be exposed to viruses or infectious prion, in addition to other safety risks associated with the use of genetic engineering. Plant-based meat may contain allergens, anti-nutrients and thermally induced carcinogens. Microbiological risks and allergens are the primary concerns associated with insect protein. Single-cell protein sources are divided into microalgae, fungi and bacteria, all of which have specific food safety risks that include toxins, allergens and high ribonucleic acid (RNA) contents. The environmental impacts of these alternative proteins can mainly be attributed to the production of growth substrates or during cultivation. Legislations related to novel food or genetic modification are the relevant regulatory framework to ensure the safety of alternative proteins. Lastly, additional studies on the food safety aspects of alternative proteins are urgently needed for providing relevant food governing authorities with sufficient data to oversee that the technological progress in this area is balanced with robust safety standards.

Bacterial species belonging to the genus Clostridium have been recognized as causative agents of blown pack spoilage (BPS) in vacuum packed meat products. Whole-genome sequencing of six New Zealand psychrotolerant clostridia isolates derived from three meat production animal types and their environments was performed to examine their roles in BPS. Comparative genome analyses have provided insight into the genomic diversity and physiology of these bacteria and divides clostridia into two separate species clusters. BPS-associated clostridia encode a large and diverse spectrum of degradative carbohydrate-active enzymes (CAZymes) that enable them to utilize the intramuscular carbohydrate stores and facilitate sporulation. In total, 516 glycoside hydrolases (GHs), 93 carbohydrate esterases (CEs), 21 polysaccharide lyases (PLs), 434 glycosyl transferases (GTs) and 211 carbohydrate-binding protein modules (CBM) with predicted activities involved in the breakdown and transport of carbohydrates were identified. Clostridia genomes have different patterns of CAZyme families and vary greatly in the number of genes within each CAZy category, suggesting some level of functional redundancy. These results suggest that BPS-associated clostridia occupy similar environmental niches but apply different carbohydrate metabolism strategies to be able to co-exist and cause meat spoilage.

The aim of our study was to determine the occurrence and diversity of economically important spore‐forming bacteria in New Zealand dairy farm systems. Farm dairy effluent (FDE) collected from Waikato dairy farms were tested for the presence of spore‐forming bacteria, using a new culture‐based methodology followed by genomic analysis. An enrichment step in which samples were inoculated in cooked meat glucose starch broth under anaerobic conditions, aided in the differential isolation of Bacillus and Clostridium species. Furthermore, the use of molecular methods such as ERIC genotyping, 16S rRNA gene sequence analysis identified different spore‐forming bacteria present in FDE. C. sporogenes signature PCR gave further information on the phylogenetic relationship of the different Clostridium spp. isolated in this study. In total 19 Bacillus spp., 5 Paenibacillus spp. and 17 Clostridium spp. were isolated from farm dairy effluent. Sequence types similar to economically important food spoilage bacteria viz: C. butyricum, C. sporogenes and members of the Paenibacillus Genus were isolated from all four farms, whereas, sequence types similar to potential toxigenic, B. cereus, C. perfringens, C. butyricum, and C. botulinum were found on at least three of the farms. Sampling of farm dairy effluent provides a good indicator of farm level prevalence of bacterial load as it is used to irrigate dairy pasture in New Zealand. This study highlights the presence of various spore‐forming bacteria in dairy waste water and indicates the implementation of good hygienic farm practices and dairy waste effluent management.

Aims: To determine possible preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats. Methods and Results: Molecular methods based on the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments were used to detect the presence of Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens in a total of 357 samples collected from ten slaughter stock supply farms, slaughter stock, two lamb-processing plants, their environments, dressed carcasses and final vacuum-packed meat stored at –0·5°C for 5½ weeks. Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations prior to fleece removal, but all these micro-organisms were detected in only 4 out of 26 cooling floor and chiller environmental samples. One out of 42 boning room environmental samples tested positive for the presence of C. gasigenes and C. estertheticum, but 25 out of 42 of these samples were positive for C. algidicarnis/C. putrefaciens. Nearly all of the 31 faecal samples tested positive for the presence of C. gasigenes and C. estertheticum; however, only two of these samples were positive for C. algidicarnis and/or C. putrefaciens. Clostridial species that were subject to this investigation were frequently detected on chilled dressed carcasses. Conclusions: The major qualitative and quantitative differences between the results of PCR detection obtained with the primers specific for ‘blown pack’ -causing clostridia (C. gasigenes and C. estertheticum) and those obtained with primers specific for C. algidicarnis and C. putrefaciens suggest that the control of meat spoilage caused by different groups of meat clostridia is best approached individually for each group. Significance and Impact of the Study: This paper provides information significant for controlling meat spoilage-causing clostridia in the meat-processing plants.

The use of lactic acid bacteria (LAB) as protective cultures in vacuum-packed chill-stored meat has potential application for assuring and improving food quality, safety and market access. In a study to identify candidate strains suitable for evaluation in a meat model, agar-based methods were employed to screen 181 chilled meat and meat process-related LAB for strains inhibitory to pathogens and spoilage organisms of importance to the meat industry. Six meat-derived strains, including Lactobacillus sakei and Lactococcus lactis, were found to be inhibitory to one or more of the target strains Listeria monocytogenes, Brochothrix thermosphacta, Campylobacter jejuni and Clostridium estertheticum. The inhibitory agents appeared to be either cell-associated or molecules released extracellularly with bacteriocin-like properties. Variations detected in the antimicrobial activity of LAB associated with changes to test parameters such as substrate composition underlined the importance of further in situ evaluation of the inhibitory strains in stored meat trials.

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

The spoilage potential of Brochothrix campestris and Brochothrix thermosphacta was investigated in vacuum-packed lamb. Striploins (n=338) were inoculated and stored for twelve weeks at temperatures -1.5, 0, 2 and 7 °C. Growth around 5-6 log10 CFU/cm(2) was recorded after six weeks at 0, 2 and 7 °C, and ~3 log10 CFU/cm(2) after nine weeks at -1.5 °C. B. campestris was shown to cause spoilage by nine weeks at temperatures above 0 °C by the presence of green drip and unacceptable odours. Molecular based assays for the detection and differentiation of B. thermosphacta and B. campestris were developed and validated. A TaqMan assay was designed to target a unique single-nucleotide polymorphism in the Brochothrix 16s rRNA gene with a sensitivity of

A new real-time PCR assay was developed targeted to the psychrotolerant spoilage bacteria, Clostridum estertheticum, a causative agent of ‘blown-pack’ spoilage of vacuum packaged meats during chilled storage. Further, a robust validation of the sensitivity and specificity in different meat processing related matrices was carried out. Results show that real-time PCR is a valid method for the detection of C. estertheticum spores as long as consideration is given to the matrix being tested and the sensitivity of detection required. For meat, hide, blood/drip and environmental swabs it was possible to detect low numbers of C. estertheticum spores (approx 3 spores per ml) by direct real-time PCR (without pre-enrichment of the samples). For faeces and soil matrices, a cold temperature enrichment step was required prior to DNA extraction and real-time PCR analysis to increase the ability to detect samples containing C. estertheticum spores; this was particularly important when the samples contained low numbers of spores (less than 3 spores per ml). For matrices with high levels of PCR inhibitors such as soil, it was necessary to dilute the extracted DNA sample 100 fold especially for detection of high levels of contamination (greater than 103 per ml) otherwise a pre-enrichment was required.

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log 10 and 6 log 10 CFU g − 1 .

The abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against several target organisms in the flora of product during 12 weeks storage of vacuum-packaged lamb and beef was investigated. L. sakei strains were generally found capable of developing dominant populations on both beef and lamb. L. lactis 75 grew poorly on lamb did not inhibit co-inoculated Brochothrix thermosphacta. Lamb inoculated with the Sakacin-A producer L. sakei Lb706 had lower Listeria monocytogenes populations than lamb inoculated with a bacteriocin-negative variant. In beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. Campylobacter jejuni inoculated onto beef was recovered from fewer packs when it was co-inoculated with 3000 CFU cm(-2) of L. sakei strain 27, 44 or 63. Observed inhibition did not always correlate with inhibition observed in earlier media-based studies, supporting the view that functionality identified using simple media-based screening methods may not be replicated in the complex environment of stored foods, and vice-versa. These findings further define a set of L. sakei strains with potential for the extended bio-preservation of minimally processed fresh beef and lamb.

The diversity of microflora associated with peroxyacetic acid (POAA) treated and untreated beef was investigated by 16S rDNA gene cloning, DGGE analysis and conventional bacterial cultivation. Following vacuum packaging, POAA treated and untreated meat samples were stored for up to 18 weeks at -1.5 degrees C. Each culture independent method showed Carnobacterium spp. to predominate on both POAA treated and untreated meat. However, 16S rDNA gene analysis also detected the presence of psychrotolerant Clostridium spp. in the POAA-treated beef. Culture-dependent analysis did not distinguish Carnobacterium spp. from Lactobacilli. Although culture-dependent analysis showed an increase in the ratio of Enterobacteriaceae to lactic acid bacteria from weeks 6-18 in the POAA treated compared with the untreated meat, the numbers of Enterobacteriaceae were significantly less on POAA treated than on untreated meat. The combination of data collected by culture-dependent and independent techniques provided the most robust approach for elucidating the efficacy of chemical sanitization of chilled vacuum-packaged beef. If conventional cultivation is used for monitoring bacterial spoilage of vacuum-packaged chilled meats it is recommended that culture methods specific for Carnobacterium and Clostridium spp. should be included in order to provide a more complete indication of microbial diversity.

Recent investigations of blown pack spoilage in New Zealand chilled vacuum-packaged meats have found moderate to high numbers of Enterobacteriaceae in the spoilage flora, but no clostridia, such as C. estertheticum and C. gasigenes, that are usually associated with blown pack spoilage. This study showed that pyschrotolerant Enterobacteriaceae produced gas in a lamb homogenate model under anaerobic conditions and that these organisms could cause blown pack spoilage of vacuum-packaged chilled meats. Significant gas production was observed with the majority of the psychrotolerant Enterobacteriaceae strains tested including presumptive species of Enterobacter, Serratia, Hafnia and Rahnella. However, no gas was produced in lamb homogenates inoculated with presumptive species of Ewingella americana or Yersinia enterocolitica. Gas production was also confirmed in vacuum-packaged lamb shoulders stored at 4 degrees C for 21 days after being inoculated with individual representative Enterobacteriaceae isolates. Biochemical characterisation proved to be more useful than genotype-based typing of 16S rRNA genes for discriminating different psychrotolerant Enterobacteriaceae from naturally contaminated meat microflora.

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.

The efficacy of a peroxyacetic acid formulation (POAA) at reducing Escherichia coli O157:H7 contamination on external carcass surfaces of hot-boned beef and veal with a commercial spray apparatus was determined. Hot-boned external carcass surfaces were inoculated with either a high dose (10(6) CFU/cm2) in fresh bovine feces or with a low dose (10(3) CFU/cm2) in diluent of laboratory-cultured E. coli O157:H7. Treatments included a water wash, a POAA (180 ppm) wash, or a water plus POAA wash. Samples were extracted from the external carcass surface with a cork borer to determine the numbers of viable E. coli O157:H7 remaining on the carcass surface after treatment. Although a water wash alone resulted in a 1.25 (94.4%) and a 1.31 (95.1%) mean log reduction on veal and beef inoculated with a high dose of E. coli O157:H7, the POAA treatment resulted in a substantially greater mean log reduction of 3.56 and 3.59 (>99.9%). The water wash only resulted in a 33.9% reduction on veal and 62.8% on beef inoculated with a low dose of E. coli O157:H7, whereas POAA treatment greatly improved pathogen reduction to 98.9 and 97.4% on veal and beef, respectively. The combination of a water wash followed by a POAA treatment resulted in a similar E. coli O157:H7 reduction to that achieved by POAA treatment alone. In conclusion, POAA treatment significantly reduced viable E. coli O157:H7 numbers on experimentally contaminated beef and veal carcasses, which justifies its use as a chemical intervention for the removal of this human pathogen.