Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions.

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. KEY POINTS: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk.

Stress response modulation: the key to survival of pathogenic and spoilage bacteria during poultry processing.

The control of bacterial contaminants on meat is a key area of interest in the food industry. Bacteria are exposed to a variety of stresses during broiler processing which challenge bacterial structures and metabolic pathways causing death or sublethal injury. To counter these stresses, bacteria possess robust response systems that can induce shifts in the transcriptome and proteome to enable survival. Effective adaptive responses, such as biofilm formation, shock protein production and metabolic flexibility, require rapid induction and implementation at a cellular and community level to facilitate bacterial survival in adverse conditions. This review aims to provide an overview of the scientific literature pertaining to the regulation of complex adaptive processes used by bacteria to survive the processing environment, with particular focus on species that impact the quality and safety of poultry products like Campylobacter spp., Salmonella enterica and Pseudomonas spp.

Sporulation and Biofilms as Survival Mechanisms of Bacillus Species in Low-Moisture Food Production Environments.

Low-moisture foods (LMF) have clear advantages with respect to limiting the growth of foodborne pathogens. However, the incidences of Bacillus species in LMF reported in recent years raise concerns about food quality and safety, particularly when these foods are used as ingredients in more complex higher moisture products. This literature review describes the interlinked pathways of sporulation and biofilm formation by Bacillus species and their underlying molecular mechanisms that contribute to the bacteriums' persistence in LMF production environments. The long-standing challenges of food safety and quality in the LMF industry are also discussed with a focus on the bakery industry.

16S rRNA Based Profiling of Bacterial Communities Colonizing Bakery-Production Environments.

Conventional culture-based techniques are largely inadequate in elucidating the microbiota contained in an environment, due to low recovery within a complex bacterial community. This limitation has been mitigated by the use of next-generation sequencing (NGS)-based approaches thereby facilitating the identification and classification of both culturable and uncultivable microorganisms. Amplicon targeted NGS methods, such as 16S ribosomal RNA (16S rRNA) and shotgun metagenomics, are increasingly being applied in various settings such as in food production environments to decipher the microbial consortium therein. Even though multiple food matrices/food production environments have been studied, the low-moisture environment associated with bakery food production remains to be investigated. To address this knowledge gap, in this study, we investigated the microbiome associated with two bakery production sites (designated as A and B) located in Ireland using 16S rRNA-amplicon-based sequencing. Amplicons corresponding to a hypervariable region contained within the 16S rRNA gene were amplified from DNA samples purified from environmental swabs and ingredients collected at both sites at various stages (preparation, production, postproduction, and storage) across the bakery production chain, over three seasons (winter, spring, and summer). These amplicons were sequenced, and data were analyzed using the mothur pipeline and visualized using MicrobiomeAnalyst and a series of R packages. The top seven bacterial phyla identified at both sites were composed of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Patescibacteria, and Verrucomicrobia. In addition, the phyla Tenericutes (Mycoplasmatota) and Acidobacteria were observed only in samples taken at site B. Different bacterial compositions were identified at each stage of production. These same bacteria were also found to be present in the final processed food suggesting the influence of the environment on the food matrix. This study is the first demonstration of the utility of 16S rRNA amplicon-based sequencing to describe the microbiota associated with bakery processing environments.

Characterisation and Classification of Foodborne Bacteria Using Reflectance FTIR Microscopic Imaging.

This work investigates the application of reflectance Fourier transform infrared (FTIR) microscopic imaging for rapid, and non-invasive detection and classification between Bacillus subtilis and Escherichia coli cell suspensions dried onto metallic substrates (stainless steel (STS) and aluminium (Al) slides) in the optical density (OD) concentration range of 0.001 to 10. Results showed that reflectance FTIR of samples with OD lower than 0.1 did not present an acceptable spectral signal to enable classification. Two modelling strategies were devised to evaluate model performance, transferability and consistency among concentration levels. Modelling strategy 1 involves training the model with half of the sample set, consisting of all concentrations, and applying it to the remaining half. Using this approach, for the STS substrate, the best model was achieved using support vector machine (SVM) classification, providing an accuracy of 96% and Matthews correlation coefficient (MCC) of 0.93 for the independent test set. For the Al substrate, the best SVM model produced an accuracy and MCC of 91% and 0.82, respectively. Furthermore, the aforementioned best model built from one substrate was transferred to predict the bacterial samples deposited on the other substrate. Results revealed an acceptable predictive ability when transferring the STS model to samples on Al (accuracy = 82%). However, the Al model could not be adapted to bacterial samples deposited on STS (accuracy = 57%). For modelling strategy 2, models were developed using one concentration level and tested on the other concentrations for each substrate. Results proved that models built from samples with moderate (1 OD) concentration can be adapted to other concentrations with good model generalization. Prediction maps revealed the heterogeneous distribution of biomolecules due to the coffee ring effect. This work demonstrated the feasibility of applying FTIR to characterise spectroscopic fingerprints of dry bacterial cells on substrates of relevance for food processing.

Microbial detection and identification methods: Bench top assays to omics approaches.

Rapid detection of foodborne pathogens, spoilage microbes, and other biological contaminants in complex food matrices is essential to maintain food quality and ensure consumer safety. Traditional methods involve culturing microbes using a range of nonselective and selective enrichment methods, followed by biochemical confirmation among others. The time-to-detection is a key limitation when testing foods, particularly those with short shelf lives, such as fresh meat, fish, dairy products, and vegetables. Some recent detection methods developed include the use of spectroscopic techniques, such as matrix-assisted laser desorption ionization-time of flight along with hyperspectral imaging protocols.This review presents a comprehensive overview comparing insights into the principles, characteristics, and applications of newer and emerging techniques methods applied to the detection and identification of microbes in food matrices, to more traditional benchtop approaches. The content has been developed to provide specialist scientists a broad view of bacterial identification methods available in terms of their benefits and limitations, which may be useful in the development of future experimental design. The case is also made for incorporating some of these emerging methods into the mainstream, for example, underutilized potential of spectroscopic techniques and hyperspectral imaging.

Suprathreshold measures of taste perception in children - Association with dietary quality and body weight.

Childhood obesity is an increasing problem in the Western world, and is affected by a multitude of interacting factors. Recent evidence suggests that taste perception may differ between obese and normal weight children. Evidence also suggests that perception of sweet and bitter taste is linked to differential food liking of various foods. To date, most studies have focused on single food items or food groups, rather than an overall view of dietary quality, and mainly on bitterness. Thus it is unclear whether taste perception is associated with dietary quality in children. Our objective was to examine the link between taste perception, dietary quality and body weight in Irish school children, in conjunction with other known influences of body weight. Taste perception was measured using the gLMS for bitter, salty and sweet stimuli. Detailed dietary intake data were collected from 525 children aged 7-13 via a 3-day diet history. Energy misreporters were identified and excluded from the dietary analyses, leaving n = 483 children. Dietary quality was assessed using Healthy Eating Index. Salivary DNA was collected and analyzed for variations in the bitter receptor gene TAS2R38. Sex differences were observed whereby intensity perception of sweetness was lower in the overweight/obese males, while no association was observed for sweet taste in the females. Despite the differences in weight status, taste perception was not associated with differences in overall dietary quality, measured via HEI score, in this cohort. Prospective cohort studies in children are necessary to better understand the association between taste intensity, food intake and weight over time.

Green label marinades: A solution to salmonella and campylobacter in chicken products?

Introduction: The presence of meat-borne pathogens entering the home remains a concern for consumers, despite advances made in improving antimicrobial interventions and systems within the processing line. Naturally antibacterial food ingredients including citrus juice and essential oils have been proven to inhibit the proliferation of microbial growth with varying success. Aims: This study aims to investigate the antimicrobial and sensory effects of mixtures of essential oils, fruit juices and herbs at established Minimum Inhibitory Concentrations (MICs) for their biopreservative effect on general microbiota of chicken and against chicken challenged with selected pathogenic/surrogate microorganisms. Materials and methods: Three marinade compositions were designed for use on chicken meat; lemon juice, thyme oil and black pepper (M1), lime juice, lemongrass oil and chilli paste (M2), and olive oil, oregano oil, basil oil and garlic paste (M3). These marinades were assessed for antibacterial effects against Salmonella enterica, Campylobacter jejuni and Listeria innocua on marinaded chicken drumsticks stored in aerobic conditions at 4 °C. Consumer tasting sessions were also conducted with a small focus group using selected final marinades. Results: M1 and M2 were effective at significantly reducing initial pathogen carriage from 6 Log CFU/g to 2 Log CFU/g on refrigerated chicken meat as well as increasing the shelf-life of the product during cold-storage from 2 days to 7 days. However, consumer studies indicate that the flavours these marinades impart to treated products can be strong. Conclusion: These findings indicate that these designed marinades have shown excellent potential to improve food safety as well as shelf-life for the consumer, particularly in settings where food safety is often compromised such as barbecuing or in care settings. However, further recipe optimisation is required to make these marinades acceptable to consumers.

Investigation of differences in susceptibility of Campylobacter jejuni strains to UV light-emitting diode (UV-LED) technology.

Campylobacter jejuni remains a high priority in public health worldwide. Ultraviolet light emitting-diode technology (UV-LED) is currently being explored to reduce Campylobacter levels in foods. However, challenges such as differences in species and strain susceptibilities, effects of repeated UV-treatments on the bacterial genome and the potential to promote antimicrobial cross-protection or induce biofilm formation have arisen. We investigated the susceptibility of eight C. jejuni clinical and farm isolates to UV-LED exposure. UV light at 280 nm induced different inactivation kinetics among strains, of which three showed reductions greater than 1.62 log CFU/mL, while one strain was particularly resistant to UV light with a maximum reduction of 0.39 log CFU/mL. However, inactivation was reduced by 0.46-1.03 log CFU/mL in these three strains and increased to 1.20 log CFU/mL in the resistant isolate after two repeated-UV cycles. Genomic changes related to UV light exposure were analysed using WGS. C. jejuni strains with altered phenotypic responses following UV exposure were also found to have changes in biofilm formation and susceptibility to ethanol and surface cleaners.

Exploring the identification of multiple bacteria on stainless steel using multi-scale spectral imaging from microscopic to macroscopic.

This work investigates non-contact reflectance spectral imaging techniques, i.e. microscopic Fourier transform infrared (FTIR) imaging, macroscopic visible-near infrared (VNIR), and shortwave infrared (SWIR) spectral imaging, for the identification of bacteria on stainless steel. Spectral images of two Gram-positive (GP) bacteria (Bacillus subtilis (BS) and Lactobacillus plantarum (LP)), and three Gram-negative (GN) bacteria (Escherichia coli (EC), Cronobacter sakazakii (CS), and Pseudomonas fluorescens (PF)), were collected from dried suspensions of bacterial cells dropped onto stainless steel surfaces. Through the use of multiple independent biological replicates for model validation and testing, FTIR reflectance spectral imaging was found to provide excellent GP/GN classification accuracy (> 96%), while the fused VNIR-SWIR data yielded classification accuracy exceeding 80% when applied to the independent test sets. However, classification within gram type was far less reliable, with lower accuracies for classification within the GP (< 75%) and GN (≤ 51%) species when calibration models were applied to the independent test sets, underlining the importance of independent model validation when dealing with samples of high biological variability.

Using Cross-Cultural Consumer Liking Data to Explore Acceptability of PGI Bread-Waterford Blaa.

Waterford Blaa is one of only four Irish food products granted protected geographical (PGI) status by the European Commission. This study aimed to determine whether cultural background/product familiarity, gender, and/or age impacted consumer liking of three Waterford Blaa products and explored product acceptability between product-familiar and product-unfamiliar consumer cohorts in Ireland and the UK, respectively. Familiarity with Blaa impacted consumer liking, particularly with respect to characteristic flour dusting, which is a unique property of Waterford Blaa. UK consumers felt that all Blaas had too much flour. Blaa A had the heaviest amount of flouring and was the least preferred for UK consumers, who liked it significantly less than Irish consumers (p < 0.05). Flavour was also important for UK consumers. Blaa C delivered a stronger oven baked odour/flavour compared to Blaa A and was the most preferred by UK consumers. Irish consumer liking was more influenced by the harder texture of Blaa B, which was their least preferred product. Age and gender did not impact liking for Blaas within Irish consumers, but gender differences were observed among UK consumers, males liking the appearance significantly more than females. This is the first paper comparing Waterford Blaa liking of naïve UK consumers with Irish consumers familiar with the product.

Detection and identification of bacteria in an isolated system with near-infrared spectroscopy and multivariate analysis.

Near-infrared (NIR) transflectance spectra of Listeria innocua FH, Lactococcus lactis, Pseudomonas fluorescens, Pseudomonas mendocina, and Pseudomonas putida suspensions were collected and investigated for their potential use in the identification and classification of bacteria. Unmodified spectral data were transformed (first and second derivative) using the Savitzsky-Golay algorithm. Principal component analysis (PCA), partial least-squares discriminant analysis (PLS2-DA), and soft independent modeling of class analogy (SIMCA) were used in the analysis. Using either full cross-validation or separate calibration and prediction data sets, PLS2 regression classified the five bacterial suspensions with 100% accuracy at species level. At Pseudomonas genus level, PLS2 regression classified the three Pseudomonas species with 100% accuracy. In the case of SIMCA, prediction of an unknown sample set produced correct classification rates of 100% except for L. innocua FH (77%). At genus level, SIMCA produced correct classification rates of 96.7, 100, and 100% for P. fluorescens, P. mendocina, and P. putida, respectively. This successful investigation suggests that NIR spectroscopy can become a useful, rapid, and noninvasive tool for bacterial identification.

Contribution of starter cultures to the proteolytic process of a fermented non-dried whole muscle ham product.

Porcine longissimus dorsi muscles were cured by brine injection. Curing brine containing 15% (w/v) NaCl, 1.33% (w/v) glucose, 750 ppm sodium nitrite, and appropriate levels of either Lactobacillus sakei LAD, L. sakei LAD plus Kocuria varians FT4 (formally Micrococcus varians), L. sakei LAD plus papain and GDL (glucono-delta-lactone) plus K. varians FT4, was injected to the muscle at a pumping rate 15% w/v. The effect of these treatments on the proteolysis in the ham system was compared to a control ham, produced without starter culture and containing GDL acidulant to control pH and antibiotics to reduce the contribution of background microflora. Hydrolysis of sarcoplasmic and myofibrillar protein fractions was evaluated by SDS-PAGE and reverse phase-HPLC. Hams with different treatments were also investigated for differences in amino acid profile, protein and non-protein nitrogen level, colour, pH, water activity and moisture and microbiological evolution. There was no significant difference in the gross compositional analysis of any of the treatments compared to the control. There was no significant difference (p>0.05) in the protein content, non-protein nitrogen level, SDS-PAGE and free amino acid analysis between the control ham and ham inoculated with proteolytic starter culture. However, it was observed that hams containing starter cultures exhibited decreases in certain peptide fractions and corresponding increases in some free amino acids compared to the uninoculated control. It can be concluded that, while the principle mechanisms resulting in the proteolysis of this non-dried ham product involve the activity of endogeneous cathepsins, the addition of proteolytic starter cultures influence the amino acid profile thereby potentially enhancing the sensorial attributes of the ham.

Near-infrared hyperspectral imaging and partial least squares regression for rapid and reagentless determination of Enterobacteriaceae on chicken fillets.

Bacterial pathogens are the main culprits for outbreaks of food-borne illnesses. This study aimed to use the hyperspectral imaging technique as a non-destructive tool for quantitative and direct determination of Enterobacteriaceae loads on chicken fillets. Partial least squares regression (PLSR) models were established and the best model using full wavelengths was obtained in the spectral range 930-1450 nm with coefficients of determination R(2)≥ 0.82 and root mean squared errors (RMSEs) ≤ 0.47 log(10)CFUg(-1). In further development of simplified models, second derivative spectra and weighted PLS regression coefficients (BW) were utilised to select important wavelengths. However, the three wavelengths (930, 1121 and 1345 nm) selected from BW were competent and more preferred for predicting Enterobacteriaceae loads with R(2) of 0.89, 0.86 and 0.87 and RMSEs of 0.33, 0.40 and 0.45 log(10)CFUg(-1) for calibration, cross-validation and prediction, respectively. Besides, the constructed prediction map provided the distribution of Enterobacteriaceae bacteria on chicken fillets, which cannot be achieved by conventional methods. It was demonstrated that hyperspectral imaging is a potential tool for determining food sanitation and detecting bacterial pathogens on food matrix without using complicated laboratory regimes.

Bitter taste perception and dietary intake patterns in irish children.

BACKGROUND/AIMS: Variations in bitter receptor gene TAS2R38 affect the perception of bitter-tasting compound 6-n-propylthiouracil (PROP). The perception of PROP has been associated, in some reports, with the perception of fat and sweet tastes, and various food preferences and intakes. The aim was to investigate nutrient intakes and food patterns in a group of Irish children, using K-means cluster analysis, and compare these with TAS2R38 genotype and PROP taster status. METHODS: Dietary intake was measured via a 3-day diet history in 483 children aged 7-13 years. Children were genotyped for TAS2R38 variation, and PROP taster status was assessed. Anthropometric and socioeconomic data were also obtained. RESULTS: No differences were observed in macronutrient, micronutrient, or food group consumption between the TAS2R38 genotype and PROP taster groups. K-means cluster analysis identified two distinct dietary patterns, termed 'more healthful' and 'less healthful' clusters. The clusters did not differ in frequencies of TAS2R38 genotype nor PROP taster status groups, suggesting that dietary patterns are not influenced by bitter taste perception. CONCLUSION: Bitterness perception, as measured by either TAS2R38 genotype or PROP taster status, does not appear to exert a significant effect on patterns of dietary intakes.