Food Microbiology

Molecular-based microbiological approaches have become valuable tools for the food industry. However, even the most advanced molecular techniques are limited in their ability to differentiate based on viability creating the potential for biased results when applied to the food industry. The objective of this study was to generate a viable microbial bio-map of a commercial ready-to-eat (RTE) meat manufacturing process and assess its utility as a diagnostic tool. Product samples were collected from a commercial RTE meat manufacturing facility at various locations throughout processing. Samples were homogenized and aliquoted for culture-based microbial isolation and 16S rRNA gene sequencing. Homogenates were split into pairs and subject to either no treatment (Control) or treated with 25 M PMAxx (PMA) to remove free and non-viable cellular DNA. Overall, PMA treatment resulted in a less rich microbial community compared to Control samples. Paired analysis revealed that the impact of PMA varied by location with the greatest effects being observed at the beginning and end of manufacturing. Both Control and PMA treated samples identified a shift in the microbial population after thermal processing; however, only PMA treated samples identified a secondary shift in the microbial population occurring after slicing. Taxonomic analysis identified Lactobacillus as a predominant genera in sliced and packaged products. These results were further confirmed by the identification of Lactobacillus sakei on packaged product using a culture-based approach. These results suggest PMA treatment provides a higher level of sequencing resolution by removing background DNA. ImportanceMicrobial bio-mapping is a valuable tool for the meat and poultry industry to assess process control and evaluate the efficacy of intervention systems. In recent years it has become more common to incorporate the use of molecular techniques, such as qPCR and 16S rRNA, to quantitatively track target pathogens and gain a more holistic understanding of the microbial community throughout processing. One major limitation we face when applying these DNA-based techniques to the food industry is their inability to differentiate between DNA from viable versus non-viable cells, which may result in the false identification of pathogenic or spoilage microorganisms and bias microbiota results. To practically apply this technology in a ready-to-eat meat manufacturing setting, it is crucial to develop and validate strategies that are capable of differentiating between viable and non-viable cellular DNA.

Listeria monocytogenes is a foodborne pathogen of utmost interest to food industry stakeholders because it persists in food processing environments. The ability to form biofilms - bacterial communities of autoaggregated cells embedded in a self-produced matrix - contributes to its persistence. While it is known that biofilm cells exhibit different gene expression than their planktonic counterparts, it remains to be elucidated whether those differences persist once cells detach from the biofilm and what their implications might be for food safety. Therefore, this study examines the differential sigB expression in biofilm-derived cells from three L. monocytogenes strains isolated from the environment within a food model subjected to varying osmotic stress over a 15-day storage period. Under our experimental conditions, biofilm-derived L. monocytogenes cells showed higher sigB expression compared to planktonic counterparts. The upregulation was strain-dependent and transient, suggesting that physiological memory may influence stress adaptation during early storage but dissipates over time. Then, the safety implications of sigB upregulation in biofilm-derived cells were assessed by evaluating cell survival under a simulated gastric environment (pH 1-3). The biofilm-derived cells showed a significant increase in survival under severe gastric conditions compared to the planktonic counterparts. Overall, our findings highlight the need to consider biofilm-derived cells in shelf-life studies and predictive models to more accurately reflect real contamination scenarios. Relying exclusively on planktonic cultures introduces a bias that may compromise risk analysis and decision-making.

Non-Typhoidal Salmonella (NTS) continues to be a leading cause of foodborne illness worldwide. Food manufacturers implement hurdle technology by combining more than one approach to control food safety and quality, including preservatives such as organic acids, refrigeration, and heating. We assessed the variation in survival in stresses of genotypically diverse isolates of Salmonella enterica to identify genotypes with potential elevated risk to sub-optimal processing or cooking. Sub-lethal heat treatment, survival in desiccated conditions and growth in the presence of NaCl or organic acids were investigated. S. Gallinarum strain 287/91 was most sensitive to all stress conditions. While none of the strains replicated in a food matrix at 4{degrees}C, S. Infantis strain S1326/28 retained the greatest viability, and six strains exhibited a significantly reduced viability. A S. Kedougou strain exhibited the greatest resistance to incubation at 60{degrees}C in a food matrix that was significantly greater than S. Typhimurium U288, S Heidelberg, S. Kentucky, S. Schwarzengrund and S. Gallinarum strains. Two isolates of monophasic S. Typhimurium, S04698-09 and B54 Col9 exhibited the greatest tolerance to desiccation that was significantly more than for the S. Kentucky and S. Typhimurium U288 strains. In general, the presence of 12mM acetic acid or 14mM citric acid resulted in a similar pattern of decreased growth in broth, but this was not observed for S. Enteritidis, and S. Typhimurium strains ST4/74 and U288 S01960-05. Acetic acid had a moderately greater effect on growth despite the lower concentration tested. A similar pattern of decreased growth was observed in the presence of 6% NaCl, with the notable exception that S. Typhimurium strain U288 S01960-05 exhibited enhanced growth in elevated NaCl concentrations. An understanding of the molecular basis of phenotypic variation in response to stress has the potential to improve process validation during food challenge tests, improve processing, and result in more reliable risk assessments in the food industry.

Plant-based cheese analogs have emerged as a novel global market trend driven by sustainability concerns for our planet. This study examines eleven soft ripened plant-based cheese analogs produced in Europe, primarily with bloomy rinds and cashew nuts as the main ingredient. First, we focused on exploring the macronutrients and salt content stated on the labels, as well a detailed fatty acid analysis of the samples. Compared to dairy cheeses, plant-based cheeses share similarities in lipid content, but their fatty acid profiles diverge significantly, with higher ratio of mono- and polyunsaturated fatty acids such as oleic and linoleic acids. We also investigated the microbiota of these analog products, employing a culture-dependent and -independent approaches. We identified a variety of microorganisms in the plant-based cheeses, with Lactococcus lactis and Leuconostoc mesenteroides being the dominant bacterial species, and Geotrichum candidum and Penicillium camemberti the dominant fungal species. Most of the species characterized are similar to those present in dairy cheeses, suggesting that they have been inoculated as culture starters to contribute to the sensorial acceptance of plant-based cheeses. However, we also identify several species that are possibly intrinsic to plant matrices or originate from the production environment, such as Pediococcus pentosaceus and Enterococcus spp. This coexistence of typical dairy-associated organisms with plant associated species highlights the potential microbial dynamics inherent in the production of plant-based cheese. These findings will contribute to a better understanding of plant-based cheese alternatives, enable the development of sustainable products, and pave the way for future research exploring the use of plant-based substrates in the production of cheese analogues.

Wine is a complex matrix resulting from a fermentation process carried out by specific microbial communities. These communities can be in competition and the development of some microorganisms, as the yeast Brettanomyces bruxellensis, can impact the fermentation process and lead to organoleptic alterations of wine. To manage this risk, microbiological diagnostic methods as microscopic observations, qPCR or flow cytometry are already used in oenology, but remain either not specific enough, or tedious. In this context, IAGE (Ingenierie et Analyses en Genetique Environnementale) has developed the first digital-PCR system enabling the detection and quantification of B. bruxellensis during the whole winemaking process. Furthermore, wine DNA extraction was optimized to enable a representative and sensitive analysis of B. bruxellensis intact cells, as well as an easy-to-implement protocol to cope with the increasing number of samples to analyze. The IAGE workflow for B. bruxellensis quantification has been proven to be successful when analyzing naturally-contaminated samples during the different steps of the winemaking process and offers a robust method to oenologists for appropriate treatments and risk management in wine cellars. HighlightsO_LIDevelopment of a dPCR method led to a highly-specific analysis of B. bruxellensis intact cells in different steps of the winemaking process. C_LIO_LIDNA extraction method has been optimized to be robust across various types of wine with varying concentrations of inhibitors, as well as throughout different stages of the wine making process. C_LIO_LIThe complete process was proven successful in analyzing a large number of naturally-contaminated samples, giving results in less than 48 hours. C_LI

Miso is a Japanese traditional fermented food with soybeans, salt and koji, and has gained attention among people for its sophisticated flavor and preservability. Koshu miso is a unique miso made by mixing two types of koji (rice and barley), and is produced primarily in Yamanashi Prefecture, Japan. We characterized the microbiota of Koshu miso at three distinct fermentation stages. Our analysis revealed that the genus Staphylococcus dominated across all miso samples. Notably, Staphylococcus sequences in the miso matched those found in rice and barley koji, indicating the influence of raw ingredients on the initial microbial community. Additionally, analysis of the manufacturing environment suggested similarities between the environmental surfaces and miso, highlighting the importance of the manufacturing environment in serving as a medium for microbial transfer. These findings underscore the critical importance of both raw ingredients and manufacturing equipment in shaping the microbial composition and evolution of miso throughout the fermentation process.

Spore-forming Bacillus species, including pathogenic Bacillus cereus and spoilage-associated Bacillus subtilis, are major contributors to foodborne illness and product degradation. Understanding their metabolic behaviour in diverse food matrices is essential for improving risk assessment, spoilage prediction, and fermentation control. This study integrates isothermal microcalorimetry and targeted metabolomics to characterize the metabolic activity of B. cereus and B. subtilis in five nutrient sources: Brain Heart Infusion (BHI) medium, oat drink, milk, pea hydrolysate, and a combined oat-pea matrix. Metabolic heat production was monitored for 24 hours at 30{degrees}C. In BHI, B. cereus exhibited a shorter lag phase (mean {+/-} sd: 4.3 hours {+/-} 0.8) than B. subtilis (7.9 hours {+/-} 1.0) but produced less total heat. Across all food matrices, B. subtilis consistently generated more heat. The oat-pea matrix supported the highest calorimetric growth rates, surpassing oat or pea alone, and showed sugar depletion and accumulation of organic acids, indicating enhanced carbohydrate metabolism. Free amino acid release was matrix- and species-specific: B. subtilis had increased levels in oat, while B. cereus did so in pea. While B. cereus was metabolically active in all matrices, cereulide levels were matrix-dependent: 47.3 {+/-} 1.7 ng/mL in oat, 3.0 {+/-} 0.1 ng/mL in oat-pea, and undetectable in pea. These findings reveal clade-specific and matrix-driven metabolic strategies. This is the first study to combine calorimetry and metabolomics to evaluate Bacillus activity in plant-based and dairy matrices. This approach enhances our understanding of microbial physiology in complex food systems and provides a foundation for developing targeted strategies to improve food safety, stability, and product design.

Cytotoxic Bacillus cereus group strains are common causes of foodborne illness including diarrhea. However, our ability to assess food safety risks associated with the exposure to cytotoxic B. cereus group strains via contaminated food is limited due to the lack of predictive tools. In this study, we experimentally quantified the growth of 17 cytotoxic B. cereus group strains, representing six phylogenetic groups, in skim milk broth and used the growth data to develop an exposure assessment model. While none of the tested strains showed detectable growth in HTST milk at 4 or 6{degrees}C, 15 of the 17 strains showed growth at 10{degrees}C, 1 of the 17 strains showed growth at 8{degrees}C, and all strains grew at [≥]14{degrees}C. Growth data for 16 strains allowed us to generate linear secondary growth models, which were then used to develop the exposure assessment model. We simulated a five-stage supply chain with up to 35 consumer storage days, as that was the timing when distinguishable variations in percent milk containers over 105 CFU/m with different B. cereus genotypes were observed. When the initial contamination level of the HTST milk is set at an average of 100 CFU/mL, the model predicts that, on consumer home storage day 21 and 35, 2.81{+/-}0.66 and 4.13{+/-}2.53 % (mean {+/-} standard deviation) of the milk containers would exceed B. cereus group concentrations of 105 CFU/mL; these data represent the average across all strains. Sensitivity analysis showed that variation in the input parameter Q0, the initial physiological state of cells, has the largest effect on models prediction for 1 of 4 group II isolates, 1 of 6 group IV isolates and both group V isolates, suggesting the need to better characterize the growth parameters of these isolates. What-if scenario analysis showed that increased mean and variability in storage temperature at the consumers home both have substantial influence on final predicted B. cereus group concentration in milk containers. This model introduces an initial tool designed to facilitate risk-based food safety decision making for products that are contaminated with low B. cereus group levels.

Climate change is causing a lack of acidity during winemaking and oenologists use several solutions to cope with such a problem. Lachancea thermotolerans, which has the potential to tolerate the harsh physicochemical conditions of wine, has emerged as a promising alternative for pH management during winemaking and, currently, it is the most valuable yeast used for acidity control in wine. In this work an amenable method for L. thermotolerans genotyping based on a multiplexed microsatellite amplification in 6 different loci was developed. This specific and sensitive method was used to distinguish between 103 collection strains obtained from different geographical and isolation sources, and then challenged against a 429 L. thermotolerans isolates from several wineries and harvests. The procedure was also tested for fermentation monitoring and strain implantation. The procedure was conceived to simplify the methodology available for L. thermotolerans genotyping, making it easy for applying in wine-related laboratories. This method can be applied to distinguish between L. thermotolerans strains in selection programs and to follow implantation of inoculated strains during winemaking with optimal results.

As a result of recent advancements in design and optimization of high-pressure processing units, the technology is gaining rapid adoption across various sectors of food manufacturing, thus requiring extensive microbiological hurdle validation studies for efficacious and feasible utilization of the technology. Commercial adoption of high-pressure processing is gaining momentum of industrial importance because of recent advances in the engineering of pressure-based pasteurization units. With tremendous ability of plethora of microorganisms to move towards fitness through vertical and horizontal gene transfer mechanisms, prevention of natural and anthropogenic pathogens of public health concern is a daunting task and a moving target. Current study discusses, Reducing the Cost Associated with High Pressure Processing: Efficacious Alternatives to the Current Standard Procedure in the Food Manufacturing Industry, with microbiological challenge studies for inactivation of the pathogen exposed to various times and intensity levels of elevated hydrostatic pressure (Pressure BioScience Inc.). Elevated hydrostatic pressure is a non-thermal procedure that exposes pathogens to pressures of up to 80,000 PSI (>550 MPa). Various times (3, 4, and 5 minutes) at pressure intensity levels of 600 MPa, (87K PSI), 550 MPa (79K PSI), 480 MPa (70K PSI), 415 MPa (60K PSI), and 345 MPa (50K PSI) of elevated hydrostatic pressure (Hub880 Explorer, Pressure BioScience Inc), were investigated at 4{degrees}C and for 45{degrees}C for inactivation of Shiga toxin-producing Escherichia coli O157:H7 (STEC) (ATCC numbers BAA 460, 43888, 43894, 35150, 43889, and 43890) respectively, Big Six non-O157 Shiga toxin-producing E. coli (nSTEC) (ATCC numbers BAA 2196, 2193, 2215, 2440, 2219, and 2192) respectively, Salmonella serovars (ATCC numbers 13076, 8387, 6962, 9270, and 14028) respectively, and Listeria monocytogenes (ATCC numbers 51772, 51779, BAA 2657, and BAA 13932). Studies were conducted in two biologically independent repetitions as a blocking factors of a randomized complete block design containing three repetitions per time/treatment within each block, analyzed statistically using GLM procedures of SAS 9.4 software at type one error level at 5% using Tukey- and Dunnett-adjusted ANOVA. A Barocycler Hub840 unit (Pressure BioScience Inc., Southeastern, MA), equipped with a water jacket and circulating water bath for precise application of hydrostatic pressure and controlled temperature was utilized. Up to 0.95 and 2.60 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli Non-O157 at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 4.42 and 5.10 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli Non-O157 at planktonic stages were achieved using application of pressure at 345 MPa and 480 MPa for 5 minutes and 4 minutes, respectively at 45{degrees}C. Up to 1.63 and 3.14 log reductions (P < 0.05) of non-habituated Listeria monocytogenes at planktonic stages were achieved using application of pressure at 345 MPa and 600 MPa for 5 minutes and 3 minutes, respectively at 4{degrees}C. Up to 4.91 and 6.37 log reductions (P < 0.05) of non-habituated Listeria monocytogenes at planktonic stages were achieved using application of pressure at 550 MPa and 480 MPa for 4 minutes, respectively at 45{degrees}C. Up to 2.87 and 5.82 log reductions (P< 0.05) of non-habituated Salmonella serovars at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 5.17 and 6.79 log reductions (P< 0.05) of non-habituated Salmonella serovars at planktonic stages were achieved using application of pressure at 415 MPa and 600 MPa for 5 minutes and 3 minutes, respectively at 45{degrees}C. Up to 0.86 and 1.35 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli O157:H7 at planktonic stages were achieved using application of pressure at 345 MPa and 480 MPa for 5 minutes and 4 minutes, respectively at 4{degrees}C. Up to 2.02 and 6.12 log reductions (P<0.05) of non-habituated Shiga toxin-producing Escherichia coli O157:H7 at planktonic stages were achieved using application of pressure at 345 MPa and 550 MPa for 5 minutes and 4 minutes, respectively at 45{degrees}C. Results of this study could be incorporated as a part of predictive public health microbiology modeling and risk assessment analysis for prevention of pathogen related disease and illness episodes.

In this study, we evaluated the expression of genes that may serve as markers of temperature- and ethanol-induced stress responses during the acclimation of Lactiplantibacillus plantarum strains of enological interest. The transcriptional responses of eight genes potentially related to stress adaptation were analyzed in two strains acclimated at 18{square}{degrees}C and 21{square}{degrees}C. Gene expression was assessed at the start (time zero) and after 48 hours of acclimation. Following acclimation, the cells were inoculated into sterile Pinot noir wine, and L-malic acid consumption was monitored over 19 days to determine whether acclimation temperature influenced cell survival and fermentation performance. Both strains exhibited similar L-malic acid consumption and survival patterns, although transcriptional responses varied depending on the temperature. Notably, gapB, glmS, and rfbB emerged as promising gene markers for future studies. These findings suggest that gene expression profiling of acclimated cells may support the selection of robust Lpb. plantarum strains for use as starter cultures in winemaking.

The impact of water addition to grape juice in winemaking, on both alcoholic and malolactic fermentation duration and outcome has been examined using commercial wine yeasts, Lalvin EC1118 and Lalvin R2 and malolactic bacteria Lalvin VP41. As expected, dilution with water did not impede fermentation, instead resulted in shortened duration, or in the case of malolactic fermentation enabled completion in these conditions. Addition of complex organic nutrient further shortened alcoholic fermentation by Lalvin R2 and in some conditions also reduced the duration of malolactic fermentation. In general, volatile compounds and some major yeast metabolites were present at lower concentrations at the end of fermentation where juices were diluted and the addition of organic complex nutrient also influenced the concentration of some compounds in wine. These findings are significant to commercial winemaking, highlighting that winemakers should consider potential impacts of juice dilution on processing efficiencies along with wine flavour and aroma. Highlights: Gardner et al. The effect of grape juice dilution on fermentationO_LIGrape juice dilution shortened both alcoholic and malolactic fermentation C_LIO_LIIn some conditions addition of commercial nutrient decreased fermentation duration C_LIO_LIIn general wine volatiles decrease with grape juice dilution C_LIO_LIIsoamyl acetate can be decreased in wine by grape juice dilution C_LI

Methanogens are detected in human gut from the first moments of life and there is a diversification of methanogens during infancy. However, the sources of acquisition of methanogens are not well elucidated. We therefore investigated 56 dairy products as potential sources of methanogens by applying molecular biology. In the presence of negative controls, we obtained an overall prevalence of methanogens in 85.7% (48/56) of samples by real-time PCR. Further PCR-sequencing identified 73.2% (41/56) of Methanobrevibacter smithii. We also found for the first time in dairy products 1.8% (1/56) of Methanobrevibacter oralis, 7.1% (4/56) of Methanobrevibacter millerae, 1.8% (1/56) of Methanobrevibacter ruminantium, 1.8% (1/56) of Methanocorpusculum sp. We observed a significant presence (p-value=0.001) of methanogens in fermented dairy products compared to unfermented dairy products. This study gives credit to the fact that dairy products could be considered as a source of methanogens for humans, especially for children.

Authentication of acai products is increasingly important due to the risk of species substitution among morphologically similar Euterpe taxa, with implications for food quality, labeling accuracy, and consumer trust. Despite advances in molecular methods, rapid and cost-effective tools for discriminating closely related Euterpe species in processed commercial matrices remain limited. This study evaluated High-Resolution Melting (HRM) analysis targeting two complementary chloroplast markers -- psbK-I and ycf1b -- as a practical approach for species-level authentication of acai (Euterpe oleracea and E. precatoria) and jucara (E. edulis) products. In silico specificity analysis confirmed that the ycf1b primer pair shows amplification restricted to the Arecaceae family, supporting the analytical robustness of the method. The combined markers enabled reliable differentiation of all target species, including closely related taxa, with a detection limit of approximately 10% in admixed samples. When applied to 50 commercial products, HRM successfully authenticated 46 samples, substantially outperforming DNA sequencing, which was limited by amplification failure and mixed chromatograms. Mislabeling was detected in one acai sorbet and three frozen acai pulps marketed as acai but molecularly identified as E. edulis, constituting a violation of Brazilian food labeling regulations. These findings demonstrate that HRM analysis provides a robust, rapid, and scalable strategy for routine species authentication in processed plant-based matrices, with potential for integration into food quality control workflows and large-scale commercial monitoring programs.

Wood-ageing of conventionally fermented beers is gaining increased attention in the production of sour beers with a noteworthy balance between sourness, wood aroma and flavour complexity. Besides the extraction of wood-derived compounds into the beer, wood-aged sours owe their layered flavour profile to the activity of a variety of wild microorganisms that reside in the barrels or that emerge from the brewing or maturation environment. However, until now wood-ageing of craft beers largely remains a process of trial and error that often generates unexpected or undesirable results. Therefore, to better understand the process and develop control strategies to improve the consistency, predictability and overall quality of the resulting beer, more insight is needed into the interactions between the wood, the microorganisms and the maturing beer. Nevertheless, as studying these interactions on an industrial scale is highly challenging, the objective of this study was to develop a reproducible and easy-to-manipulate experimentally tractable system that can be used to study wood-ageing of beer on a lab scale. Barrel-ageing was mimicked in a 0.5 liter glass jar filled with beer and closed off by a wooden disk. Furthermore, the system was equipped with a synthetic community composed of four bacterial species (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four fungal species (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that represented key microbes previously identified in wood-ageing experiments with 225-liter barrels. In order to test the hypothesis that the barrel-ageing process of beer can be replicated in the simplified in-vitro system, the system was subjected to 60 days of ageing and microbial community dynamics and beer chemistry were compared with a 38-week industrial barrel-ageing experiment using the same beer. Beer samples were collected at regular time points and subjected to both qPCR assays targeting the eight selected species and chemical analysis. Results revealed that in vitro ageing showed similar trends in the temporal dynamics of the microbial populations and beer chemistry as those observed during 38-weeks of barrel-ageing in 225-liter barrels. Furthermore, results were found to be highly reproducible. Altogether, the in-vitro system was found to be a robust and reproducible system that has great potential to perform more in-depth research about the intricate interactions between microbes, wood and maturing beer and to develop control strategies to improve the consistency, predictability and overall quality of the resulting beer.

Norwegian kveik are a recently described family of domesticated Saccharomyces cerevisiae brewing yeasts used by farmhouse brewers in western Norway for generations to produce traditional Norwegian farmhouse ale. Kveik ale yeasts have been domesticated by farmhouse brewers through serial repitching of the yeast in warm wort (>30{degrees}C) punctuated by long periods of dry storage. Kveik yeasts are alcohol tolerant, flocculant, capable of utilizing maltose/maltotriose, phenolic off flavour negative, and exhibit elevated thermotolerance when compared to other modern brewers yeasts belonging to the Beer 1 clade. However, the optimal fermentation and growth temperatures (Topt) for kveik ale yeasts and the influence of fermentation temperature of the production of flavour-active metabolites like fusel alcohols and sulfur compounds (H2S, SO2) are not known. Here we show that kveik ale yeasts have an elevated optimal fermentation temperature (Topt) when compared to commercial American Ale yeast (SafAle US-05) and that they produce fewer off-flavours at high temperatures (>30{degrees}C) when compared to commercial American Ale yeasts. The tested kveik yeasts show significantly higher maximum fermentation rates than American Ale yeast not only at elevated temperatures (>30{degrees}C), but also at typical ale fermentation temperatures (20{degrees}C-25{degrees}C). Finally, we demonstrate that kveik ale yeasts are heterogeneous in their Topt and that they attenuate standard wort robustly above their Topt unlike our control American Ale yeast which showed very poor apparent attenuation in our standard wort at temperatures >> Topt. Our results provide further support that kveik yeasts may possess favourable fermentation kinetics and sensory properties compared to American Ale yeasts. The observations here provide a roadmap for brewers to fine tune their commercial fermentations using kveik ale yeasts for optimal performance and/or flavour impact.

Although previous studies have associated feeding dogs uncooked meat with their carriage and excretion of antibiotic resistant (ABR) Escherichia coli, this practice remains popular amongst dog owners in some countries, including the UK. Uncooked meat fed to dogs is purchased from stores selling meat primarily for human consumption, or it is a brand of commercial raw dog food (RDF), which is commonly sold frozen and thawed prior to feeding. We tested 58 packets of uncooked meat (beef, chicken, lamb and pork) purchased from large chain grocery stores (15 stores) and 15 packets of thawed, chicken-based RDF (11 brands) for the presence of ABR E. coli. All samples were purchased from the city of Bristol, UK. Among grocery store-purchased meat, chicken samples were significantly more likely to be positive for E. coli resistant to amoxicillin, spectinomycin, streptomycin, and the critically important antibiotics cefotaxime and ciprofloxacin, than other types of meat. When comparing grocery store chicken to chicken-based RDF we found no significant difference in sample-level positivity for resistant E. coli, which, for RDF was 80% (amoxicillin), 80% (spectinomycin), 87% (streptomycin), 27% (amoxicillin-clavulanate), 27% (cefotaxime) and 47% (ciprofloxacin). We conclude that despite it having been purchased frozen and thawed prior to testing according to the manufacturers instructions, contamination of RDF with ABR E. coli is similar to that of uncooked meat primarily intended for human consumption after cooking, and so the same strict hygiene practices are advised when handling RDF as uncooked meat. Our findings provide a rationale to explain why feeding uncooked meat or RDF to dogs in the city of Bristol is associated with them excreting E. coli resistant to critically important antibiotics.

A detailed characterization of the microbial ecosystem involved in the production processes of fermented foods is essential. Although fermented foods are an important part of the human diet and have seen an increasing interest nowadays, some challenges still need to be solved. Specifically, yeast identification through culture-independent methodologies is still limited to the genus level. Unlike for bacterial species identifications, long-read sequencing technologies have barely been used for yeast species identification, and, to the best of the authors knowledge, it has not been validated with mock communities reflecting food fermentation processes yet. Therefore, in the current study, we present an amplicon-based metabarcoding approach targeting the full-length internal transcribed spacer (ITS) region comprising the ITS1, 5.8S rRNA gene, and ITS2 using the PacBio HiFi sequencing platform. This method was validated using mock communities composed of yeast species involved in sourdough, lambic beer, and cocoa fermentation processes. Accurate species-level identification was achieved for most of the species. However, special attention should be given to Saccharomyces-rich niches, as accurate species-level identification for this genus is still challenging. Furthermore, underestimation of the relative abundance of species with short ITS regions, such as Pichia and Brettanomyces, occurred. In addition, the method was successfully applied to describe the yeast diversity present in two sourdough and two lambic beer samples. Overall, the current method provides an unprecedented way of determining the species-level yeast composition of complex ecosystems present in fermented food products. ImportanceTo date, species-level identification of common yeasts present in food fermentation ecosystems has been difficult, if not impossible, when using short-read sequencing methods. However, species-level identification is essential when evaluating and describing the characteristics of fermented food microbiomes. The current study reports on the development and validation of an amplicon-based metabarcoding approach combined with long-read PacBio HiFi sequencing targeting the full ITS region, comprising the ITS1 and ITS2 regions as well as the 5.8S rRNA gene. The described methodology enables species-level identification of the most common yeasts present in food fermentation ecosystems. This new methodology is of importance for all researchers in the field of fermented foods. By extension, researchers in other fields of microbiology can find inspiration in this paper.

Members of the family Lactobacillaceae, which now includes species formerly belonging to the genera Lactobacillus and Pediococcus, but also Leuconostocaceae, are of foremost importance in food fermentations and spoilage, but also as components of animal and human microbiota and as potentially pathogenic microorganisms. Knowledge of the ecological distribution of a given species and genus is important, among other things, for the inclusion in lists of microorganisms with a Qualified Presumption of Safety or with beneficial use. The objective of this work is to use the data in FoodMicrobionet database to obtain quantitative insights (in terms of both abundance and prevalence) on the distribution of these bacteria in foods and food environments. We first explored the reliability of taxonomic assignments using the SILVA v138.1 reference database with full length and partial sequences of the 16S rRNA gene for type strain sequences. Full length 16S rRNA gene sequences allow a reasonably good classification at the genus and species level in phylogenetic trees but shorter sequences (V1-V3, V3-V4, V4) perform much worse, with type strains of many species sharing identical V4 and V3-V4 sequences. Taxonomic assignment at the genus level of 16S rRNA genes sequences and the SILVA v138.1 reference database can be done for almost all genera of the family Lactobacillaceae with a high degree of confidence for full length sequences, and with a satisfactory level of accuracy for the V1-V3 regions. Results for the V3-V4 and V4 region are still acceptable but significantly worse. Taxonomic assignment at the species level for sequences for the V1-V3, V3-V4, V4 regions of the 16S rRNA gene of members of the family Lactobacillaceae is hardly possible and, even for full length sequences, and only 49.9% of the type strain sequences can be unambiguously assigned to species. We then used the FoodMicrobionet database to evaluate the prevalence and abundance of Lactobacillaceae in food samples and in food related environments. Generalist and specialist genera were clearly evident. The ecological distribution of several genera was confirmed and insights on the distribution and potential origin of rare genera (Dellaglioa, Holzapfelia, Schleiferilactobacillus) were obtained. We also found that combining Amplicon Sequence Variants from different studies is indeed possible, but provides little additional information, even when strict criteria are used for the filtering of sequences.

The fermentation of cocoa beans is a key process to supply high quality ingredients for the chocolate industry. In spite of several attempts to obtain standardised microbial cultures for cocoa fermentation, it is still a spontaneous process. It has been suggested lactobacilli present potential for quorum sensing (QS) regulation in cocoa fermentation, and in the present research, laboratory scale fermentations were carried out to further elucidate possible QS influence on microbial shifts and fermented seeds quality. The experimental design comprised the 96 hours-fermentations designated as F0 (control), F1 (yeasts, lactic acid bacteria, and acetic acid bacteria), F2 (yeasts and acetic acid bacteria), F3 (yeasts only), with evaluation of the microbial succession by plate counting, determination of enzymatic activities by classical methods and qualitative evaluation of flavour compounds by gas-chromatography (GC-MS) with headspace sampling. Besides, QS was estimated by quantification of the expression of luxS genes by Reverse Transcriptase Real Time PCR analysis using selected primers. The results demonstrated that microbial successions were displayed in lab conditions, but no statistical difference in terms of microbial enumeration and -diversity metrics were observed among the experimental and control fermentations. Moreover, enzymatic activities were not correlated to the total microbiota, indicating the seeds endogenous hydrolases protagonist enzymes secretion and activity. Regarding luxS genes measuring for the species Lactiplantibacillus plantarum and Limosilactobacillus fermentum, genes were active in fermentation in the start to the end phase and to the beginning to the middle phase of fermentation, respectively. Correlation analysis among luxS expression and volatile metabolites evidenced Lp. plantarum association with detrimental compounds for fermentation quality. This data contributes to our previous research which monitored fermentations to survey enzymatic changes and QS potential along the process and sheds light of QS-related strategies of lactobacilli dominance in cocoa fermentations.