International Journal of Food Microbiology

Table olives are produced from a large number of olive varieties subjected to different trade preparations, resulting in a highly heterogeneous family of fermented foods. To characterise the diversity of bacterial and fungal communities and its relationship with variety, ripeness, and trade preparation, we surveyed 363 samples from 40 producers across 6 countries, combining physicochemical measurements, viable counts, and amplicon-based metagenomics. This is the largest survey of table olive microbial communities to date and includes the first culture-independent characterisation of microbial communities for several Italian PDO and non-PDO varieties, most notably Oliva di Gaeta. The contrast between alkali-treated and naturally fermented olives was the dominant structuring factor, with HALAB (Halophilic and Alkalophilic Lactic Acid Bacteria) and other halophiles enriched in alkali-treated varieties and a diverse array of Lactobacillaceae and Pseudomonadota characterising naturally fermented olives. Despite these consistent signals, striking variability was observed within the same variety and even within the same producer, driven by stochastic colonization events, house microbiota, and the widespread use of small fermentation vessels. This variability obscured variety-specific microbial signatures and prevented reliable discrimination of Italian PDO varieties from similar non-PDO counterparts using amplicon-based approaches. The ecological and taxonomic complexity documented here, encompassing bacterial and fungal genera with largely untapped starter and flavour potential, provides the foundation for the development of variety-specific microbiome-based starter cultures. HighlightsO_LIWe report a metataxonomic survey of microbial communities in 363 table olive samples C_LIO_LIAlkali treatment and natural fermentation drive distinct microbial community structures C_LIO_LIHouse microbiota and stochastic colonization generate high within-variety variability C_LIO_LIMicrobiome data provide ecological foundations for olive microbiome-based starters C_LI

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

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

The flavours of foods and beverages are formed by the agricultural environment where the plants are grown. In the case of wine, the location and environmental features of the vineyard site imprint the wine with distinctive aromas and flavours. Microbial growth and metabolism play an integral role in wine production from the vineyard to the winery, by influencing grapevine health, wine fermentation, and the flavour, aroma and quality of finished wines. The mechanism by which microbial distribution patterns drive wine metabolites is unclear and while flavour has been correlated with bacterial composition for red wines, bacterial activity provides a minor biochemical conversion in wine fermentation. Here, we collected samples across six distinct winegrowing areas in southern Australia to investigate regional distribution patterns of both fungi and bacteria and how this corresponds with wine aroma compounds. Results show that soil and must microbiota distinguish winegrowing regions and are related to wine chemical profiles. We found a strong relationship between microbial and wine metabolic profiles, and this relationship was maintained despite differing abiotic drivers (soil properties and weather/ climatic measures). Notably, fungal communities played the principal role in shaping wine aroma profiles and regional distinctiveness. We found that the soil microbiome is a potential source of grape- and must-associated fungi, and therefore the weather and soil conditions could influence the wine characteristics via shaping the soil fungal community compositions. Our study describes a comprehensive scenario of wine microbial biogeography in which microbial diversity responds to surrounding environments and ultimately sculpts wine aromatic characteristics. These findings provide perspectives for thoughtful human practices to optimise food and beverage flavour and composition through understanding of fungal activity and abundance.

Hanseniaspora uvarum is consistently observed as the dominant non-Saccharomyces species in spontaneous grape juice fermentations. However, the physiological mechanisms and physicochemical variables influencing the prevalence of H. uvarum over other non-Saccharomyces species remain unclear. We tested the physicochemical parameters contributing to H. uvarum dominance by inoculating a chemically diverse set of grape juices with a mock community whose composition was defined following a comprehensive microbial survey of spontaneous fermentations. Our findings indicated that the chemical composition of grape juice had minimal impact on the microbial dynamics of fermentation, with H. uvarum emerging as the dominant non-Saccharomyces species in nearly all conditions tested. Grape juice composition primarily influenced the total yeast abundance of the mock community. Flow cytometry analysis confirmed that H. uvarum has a faster growth rate than Saccharomyces cerevisiae and several other Hanseniaspora spp.. Moreover, its growth was not affected by the presence of S. cerevisiae, explaining its rapid dominance in spontaneous fermentations. The rapid growth of H. uvarum negatively impacted the growth of S. cerevisiae, with significant implications for fermentation performance and sugar consumption. The results of this study suggest that the fast growth rate of H. uvarum enables it to quickly dominate the grape juice environment during the early stages of fermentation. This physiological advantage indicates that the initial abundance of H. uvarum may be critical to the outcome of spontaneous fermentations, as evidenced by its direct impact on the growth of S. cerevisiae and fermentation performance.

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.

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.

Fungi are known to produce many chemically diversified metabolites, yet their ecological roles are not always fully understood. The blue cheese making fungus Penicillium roqueforti thrives in different ecological niches and is known to produce a wide range of metabolites, including mycotoxins. Three P. roqueforti populations have been domesticated for cheese production and two populations thrive in other anthropized environments, i.e., spoiled food, lumber and silage. Here, we looked for differences in targeted and untargeted metabolite production profiles between populations using HPLC-HR-Q-TOF and UHPLC-Q-TOF-HR-MS/MS. The non-cheese populations produced several fatty acids and different terpenoids, lacking in cheese strains. The Termignon cheese population displayed intermediate metabolite profiles between cheese and non-cheese populations, as previously shown for other traits. The non-Roquefort population, the cheese population with the strongest domestication syndrome, produced the lowest quantities of measured metabolites, including known mycotoxins such as mycophenolic acid (MPA), andrastin A and PR toxin. Its inability to produce MPA was due to a deletion in the mpaC gene, while a premature stop codon in ORF 11 of the PR toxin gene cluster explained its absence and the accumulation of its eremofortin A & B intermediates. In the Roquefort population, we detected no PR toxin nor eremofortins A or B, but found no indel or frameshift mutation, suggesting downregulation. Our results suggest that domesticated cheese populations were selected for lower toxin production while populations from other anthropized environments maintained high metabolite diversity, the bioactivities of these compounds being likely important in these ecological niches.

Palm wine is an alcoholic beverage that has existed for centuries and has important economic and socio-culture values in many tropical and sub-tropical countries. Lesser known than other types of wines, palm wine is made by spontaneous fermentation of palm sap by naturally occurring microbial communities. The palm sap ecosystem has unique microbial composition and diversity, which determines the composition of the eventual wine and is likely affected by geographical distinctiveness. While these features are well understood in grape and rice wine, these features have not been understood in palm wine. In this review, we gather information of microbial communities and metabolite profiles from published studies, covering a wide range of methodologies and regions, to better understand the causal links between the principal microbial species and major metabolites of palm wine. We assess palm wine quality across production regions and local practices to provide general characteristics of palm wine and identify specific regional information. These will provide better understandings to the function of microbial communities and metabolite diversity, the contribution of regional variations and to ensure product quality in this important and widespread, yet overlooked, fermented beverage. One sentence summaryreview and synthesis of microbial ecology and metabolites in palm wine fermentation across geography and their contribution to cultural food systems

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.

Listeria monocytogenes can persist in niches of meat processing environments despite routine cleaning and disinfection. Its persistence may depend not only on stress tolerance but also on interactions with resident microbial communities, which may promote or inhibit survival. However, these ecological relationships remain poorly understood. We combined 16S rRNA V3/V4 amplicon sequencing, culture-based detection, and multilocus sequence typing (MLST) to characterize microbial communities in six German meat processing facilities over one year. We examined associations among community structure, sampling sites (drains and food-contact surfaces), and the occurrence of Listeria spp., including L. monocytogenes. Microbial communities were dominated by core genera typical of food-processing environments, particularly Pseudomonas spp. and Acinetobacter spp., but differed significantly among facilities (PERMANOVA, p = 0.001; pairwise R{superscript 2} = 0.023-0.079), indicating facility-specific communities. Culture-based analyses detected Listeria spp. in 51 of 370 environmental samples (13.8%), mainly from drains (44/51, 86.3%). L. monocytogenes was detected in five of six facilities, with 19 of 21 isolates originating from drains (90.5%). MLST of 74 typeable L. monocytogenes isolates revealed high diversity, comprising 21 sequence types across 15 clonal complexes, with lineage II predominating (86.5%). Overall microbial community composition was significantly associated with Listeria spp. and L. monocytogenes presence (PERMANOVA, p = 0.001; R{superscript 2} = 0.0137 and 0.0083). In drains, ASVs assigned to Acinetobacter, Rhizorhapis, and Vagococcus species showed positive associations with Listeria spp.-positive samples. Together these findings suggest that drains are key ecological niches for Listeria spp. and that associated taxa may indicate drain communities linked to Listeria spp. recovery. IMPORTANCEListeria monocytogenes is a major foodborne pathogen that can persist in meat processing environments despite routine cleaning and disinfection. Resident microbial communities may influence its survival, but longitudinal studies linking those communities with culture-based Listeria spp. detection remain limited. Here, we characterized microbial communities in six German meat processing facilities over 1 year using 16S rRNA gene amplicon sequencing and culture-based Listeria spp. detection, and MLST of L. monocytogenes isolates. We identified facility-specific microbial communities, identified floor drains as key niches for Listeria spp., and observed repeated recovery of different L. monocytogenes sequence types across facilities. In drains, ASVs assigned to the genera Acinetobacter, Rhizorhapis, and Vagococcus species were positively associated with culture-positive samples, identifying candidate taxa that may reflect microbial conditions associated with Listeria spp. recovery. These findings highlight the importance of considering not only whether Listeria spp. are detected, but also the resident microbial communities that may support their fitness.

Disrupted gut microbiota as a critical risk factor for many noncommunicable diseases is largely driven by gut microbiota-impacting drugs, especially orally administrated as well as biliary excreted antibiotics. Fermented food consumption has been encouraged to replenish disrupted gut microbiota, but its overall impact on host gut health remains to be elucidated. This study examined retail traditionally fermented foods and gut microbiota of consumers of fermented foods for antibiotic resistome. Dietary intervention by fermented foods was found leading to a surge of the antibiotic resistome in gut microbiota of most human subjects. Antibiotic resistome was further illustrated in traditionally fermented food samples, and viable antibiotic resistant (AR) bacteria were recovered and highly prevalent in retail kimchi and artisan cheeses assessed in this pilot screening. Identified AR isolates included pathogens of importance in nosocomial infections such as Klebsiella pneumoniae, Enterococcus, etc., as well as commensals and lactic acid bacteria, some exhibited extremely high minimum inhibitory concentration (MIC) against antibiotics of clinical significance. Exposing fermented food microbiota to representative antibiotics further led to a boost of the corresponding antibiotic and multidrug-resistance gene pools and disturbed microbiota. These results revealed an underestimated public health risk associated with fermented foods intervention, particularly to susceptible population with gastrointestinal tract symptoms and compromised immune functions seeking gut microbiota rescue. The findings call for more comprehensive investigation and investment on the benefits and potential safety challenges associated with traditionally fermented foods, productive intervention of foodborne antibiotic resistance, and strategic movements to mitigate unnecessary damages to the host gut microbiota.

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.

One of the biggest challenges for dairy producers is the substantial variability in final product properties caused by changes in the production environment. In cheese production, this variation is influenced by several factors, particularly the milk base and its pretreatment, which shape the microbiome throughout the process and ultimately affect the cheeses organoleptic characteristics. To examine the impact of three different pre-treatments for pasteurised milk-- microfiltration, protein fortification, and only pasteurisation (control)-- on microbiome dynamics, we generated metagenome sequencing data from 14 cheese production steps across these three production trials at a Danish dairy factory. We constructed three metagenomic co-assemblies, identifying nine high-quality metagenome-assembled genomes (MAGs). Our analysis revealed that a specific strain of Lactococcus lactis dominates the process, while other minor bacterial species persist at very low abundances (<1%), contributing non-negligibly to product properties. Notably, Clostridium tyrobutyricum, a known dairy spoilage bacterium, was present at low levels in pasteurised-only and protein-fortified milk trials but was nearly absent in microfiltered milk. To enhance our analyses, we implemented KHILL, a novel k-mer-based method applied directly to raw sequencing reads, which facilitates metagenomic co-assembly and enables early detection of unwanted microorganisms. Our findings provide industrial dairy producers with a comprehensive view of microbial dynamics during cheese production, offering insights to improve process consistency and product quality.

Hybridization between Saccharomyces cerevisiae and Saccharomyces eubayanus resulted in the emergence of S. pastorianus, a crucial yeast for lager fermentation. However, our understanding of hybridization success and hybrid vigour between these two species remains limited due to the scarcity of S. eubayanus parental strains. Here, we explore hybridization success and the impact of hybridization on fermentation performance and volatile compound profiles in newly formed lager hybrids. By selecting parental candidates spanning a diverse array of lineages from both species, we reveal that the Beer and PB-2 lineages exhibit high rates of hybridization success in S. cerevisiae and S. eubayanus, respectively. Polyploid hybrids were generated through rare mating techniques, revealing a prevalence of triploids and diploids over tetraploids. Despite the absence of heterosis in fermentative capacity, hybrids displayed phenotypic variability, notably influenced by maltotriose consumption. Interestingly, ploidy levels did not significantly correlate with fermentative capacity, although triploids exhibited greater phenotypic variability. The S. cerevisiae parental lineages primarily influenced volatile compound profiles, with significant differences in aroma production. Interestingly, hybrids emerging from the Beer S. cerevisiae parental lineages exhibited a volatile compound profile resembling the corresponding S. eubayanus parent. This pattern may result from the dominant inheritance of the S. eubayanus aroma profile, as suggested by the over-expression of genes related to alcohol metabolism and acetate synthesis in hybrids including the Beer S. cerevisiae lineage. Our findings suggest complex interactions between parental lineages and hybridization outcomes, highlighting the potential for creating yeasts with distinct brewing traits through hybridization strategies.

IntroductionThe use of Saccharomyces cerevisiae to ferment alcoholic beverages is an ancient tradition, with genetic evidence indicating origins in Neolithic Asia, although the domestication process of the species is not fully understood. Kveik is a group of traditional yeasts used in farmhouse brewing in western Norway preserved through generations of rural brewing practice. While recent studies have highlighted the distinctiveness of kveik, its precise phylogenetic position, genetic diversity, and domestication history remain unclear. ResultsWe performed whole-genome sequencing on 62 samples representing 25 unique Norwegian strains selected using cultural heritage criteria, and generated telomere-to-telomere (T2T) assemblies for representative isolates. Phylogenomic and population genetic analyses reveal that kveik forms a paraphyletic and early diverging group with respect to other domesticated S. cerevisiae strains. Most strains exhibit low within-strain diversity, strong geographic clustering, and little evidence of gene-flow or admixture. Mitochondrial genomes and Ty1 retrotransposon profiles corroborate this distinct lineage history. We further show that previously reported signals of gene flow between kveik and Asian fermentation strains are likely artifacts caused by population structure and selection. Divergence time estimates suggest that the common ancestor of beer, kveik, and other liquid-phase fermenting strains originated from ancestral populations 4,000 to 8,000 years ago. ConclusionKveik yeasts represent a relic of early S. cerevisiae domestication, shaped by ancient human practices, migrations, and the spread of agriculture. Our genomic resource sheds light on yeast evolution and domestication. They likely comprise some of the oldest domesticated lineages in continuous use until today, connecting endangered intangible cultural heritage to an early genetic origin.

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.

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.

Manual colony counting remains the rate-limiting, operator-dependent step in culture-based food microbiology quality control (QC). Automated colony analysis using machine learning (ML) offers the potential to standardise, accelerate, and improve the traceability of this process. However, systematic multi-method validation data for AI-based platforms against recognised international standards remain scarce. We conducted a prospective, multi-study validation of the Reshape Smart Incubator which is an automated imaging and ML-based colony analysis system, across eight ISO microbiological reference methods. In total, 887 plates were analysed, spanning qualitative (presence/absence) detection of Listeria spp. (ISO 11290-1) and Salmonella spp. (ISO 6579), and quantitative enumeration of total viable count (ISO 4833), Bacillus cereus (ISO 7932), Enterobacteriaceae (ISO 21528), coagulase-positive Staphylococci (ISO 6888), yeasts and moulds (ISO 21527), and lactic acid bacteria (ISO 15214). Automated results were benchmarked against the consensus of three or more trained technicians. The platform achieved 100% agreement with manual assessment for all both qualitative detection methods (ISO 11290-1, ISO 6579) with zero false positives and zero false negatives. For quantitative enumeration, agreement ranged from 92.97% (ISO 15214, n=122, using ISO-aligned {+/-}10%/>30 CFU thresholds) to 98.46% (ISO 21528, n=130). Where discrepancies occurred, they largely coincided with plates showing high inter-technician variability. Precision testing demonstrated a coefficient of variation of 5.88% and a mean standard deviation of 0.44 CFU for low-count plates. This study presents a comprehensive multi-ISO validation of an AI-based colony analysis system to date. The AI models demonstrated performance comparable to or exceeding that of trained human technicians across a broad range of microbiological targets, agar types, and colony morphologies, thereby supporting their use as a validated and traceable alternative to manual plate reading in accredited food microbiology quality control laboratories.