The use of mixed-strain starter cultures in sourdough production can result in distinct flavour profiles, leading to new sourdough-containing baked products. Recently, a newly discovered mixed-strain starter culture composed of the lactic acid bacterium (LAB) Companilactobacillus crustorum LMG 23699 T and the yeast Wickerhamomyces anomalus IMDO 010110 has been successfully used for sourdough productions. Knowledge about the interactions among the strains involved will allow for the further optimization of sourdough production. In the present study, a differential gene expression analysis was performed to reveal transcriptional responses of the strains as a result of cocultivation, including those related to their metabolism, cross-feeding interactions, and production of esters, the latter imparting fruity notes. The same approach was used to characterize the transcriptional response of the LAB strain to the presence of ester precursor molecules. The main changes for the LAB strain included the upregulation of genes related to the D-alanylation of lipoteichoic acids, the upregulation of nucleotide biosynthesis, and the downregulation of fatty acid biosynthesis, indicating a stress response and possible cross-feeding or scavenging interactions with the yeast. The yeast strain upregulated genes related to ammonium formation, respiration, gluconeogenesis, fatty acid biosynthesis, and ethyl acetate biosynthesis. Further, this analysis defined the estC gene of the Coml. crustorum strain as an ester biosynthesis gene involved in ethyl lactate production. In conclusion, the present study showed the added value of RNAseq-based transcriptomics in investigating interactions in a mixed-strain starter culture and the potential effects of using this starter culture for sourdough production.

Fermented foods are culturally significant and increasingly recognised for their potential health benefits, yet scientific data on household fermentation practices remain limited. We launched a co-designed citizen science (CS) initiative within the HealthFerm project to collect information about the diversity of sourdough fermentation practices implemented at the household level across Europe, how these practices vary by location and baker demographics, and their relationship with baking motivations and attitudes. This manuscript describes the citizen science survey results, as a resource for evaluating sourdough baking practices and motivations across Europe. Over 1000 participants from 33 countries registered, with 671 samples submitted. Participants also completed standardised at-home experiments and sensory evaluations, generating a dataset linking baking habits with physicochemical and sensory profiles. Distinct patterns emerged: professional bakers used older, more frequently refreshed starters and fermented at higher temperatures than household bakers. Ingredient choices and motivations varied by country, shaped by perceived health benefits. Beyond fermentation practice data collection, this initiative established a microbial biobank and harmonised metadata resource, while offering practical insights into co-design, logistics, and public engagement. The resulting framework provides a transferable model for participatory research in microbiology and food systems science.

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 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 ITS1, the 5.8S rRNA gene, and ITS2, using the PacBio HiFi sequencing platform. This method was validated using DNA-based 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 internal transcribed spacer (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 provides an important tool not only for the investigation of fermented foods but also for other fields engaged in complex microbial community analysis.

This study characterized fourteen Propionibacterium freudenreichii strains previously isolated from dairy environment for their growth, metabolic performance, enzyme activities, and vitamin B12 biosynthesis in yeast extract-lactate (YEL) and faba bean food-like media (FBM), complemented by genomic and pangenomic analyses to elucidate genetic and functional diversity. In YEL, all strains exhibited mild acidification (pH 6.9-7.1) with significant strain-dependent differences in growth (optical density 0.02-0.70). Eight strains were further selected for FBM fermentation, showing intensive acidification (pH 4.8-5.0) and a 1.9-2.9 log increase in cell density. Fermentation depleted lactate and produced propionate and acetate, while simple sugars (e.g., glucose) were completely utilized and raffinose-family oligosaccharides remained unchanged. Enzyme assays revealed pronounced strain-dependent variation: strain 282 displayed high β-glucosidase and Leu-pNA-hydrolyzing aminopeptidase activity, whereas strain AS9 showed strong Pro-pNA-hydrolyzing but low Leu-pNA-hydrolyzing activity; both lacked detectable phytate-hydrolyzing activity. FBM resulted in higher levels of cyanocobalamin, with 282 producing the highest concentration (131.0 ng/mL), while strains AS9, AS5, and J117 yielded substantial amounts (61.9-65.3 ng/mL). In contrast, YEL was associated with higher levels of pseudovitamin B12. Phenotypic microarray profiling of strains 282 and AS9 indicated broad metabolic capacity in YEL but reduced activity in FBM, with a notable acetate-utilization shift in AS9. Comparative genomics of 282, AS5, AS9, and J117 revealed a large, conserved core genome encoding a redundant intracellular peptidase repertoire, β-glucosidase, and anaerobic cobalamin biosynthesis pathways, while structural variations and accessory genes contributed to strain-specific diversity. Overall, the phenotypic traits of P. freudenreichii strains are governed primarily by quantitative regulatory variation within a conserved genomic framework rather than by the gain or loss of metabolic pathways.

Adjunct starter cultures are of interest in pasteurized cheese production as they provide additional flavor, ensure reproducible quality, and enable faster ripening. In the current study, two adjunct culture strains were selected, namely Lacticaseibacillus paracasei LP46, selected out of 49 isolates from mature Gouda cheese, and Tetragenococcus halophilus TH63, selected out of 244 isolates from a Gouda cheese brine. To date, the use of a Tetragenococcus strain in cheese production has not been reported. Both strains were applied in pilot-scale Gouda cheese productions, and the cheeses were investigated up to 32 weeks of ripening by a multiphasic approach encompassing culture-dependent and culture-independent microbiological analysis, meta-metabolomics, and organoleptic evaluations. In the case of the Lacc. paracasei strain, three batches with each time a different primary starter culture mixture were produced to investigate the effect of the primary starter culture mixture on the adjunct starter culture. The adjunct starter culture strains were able to become abundant in the cheeses. Furthermore, both adjunct starter culture strains seemed to repress the Leuconostoc strains from the primary starter culture mixtures. The Lacc. paracasei adjunct starter culture was associated with higher concentrations of acetoin and cadaverine, despite showing no biogenic amine production during the screening process. The T. halophilus adjunct starter culture increased the total amino acid concentration by 55% and also resulted in higher concentrations of acetoin and 2,3-butanedione. However, the organoleptic evaluation could not indicate a significant difference between the negative controls and the cheeses with adjunct starter cultures. Nevertheless, this first application of a Tetragenococcus strain in Gouda cheese showed that T. halophilus seems a promising cheese adjunct starter culture.

The story of Belgian lambic beers goes that they can only be produced around the Senne river since the valley of this river harbors essential yeasts that inoculate the lambic beer wort during its cooling in a coolship, a metal vessel that is open to the environmental air. However, in-depth studies highlighted the role of the wooden barrels, in which the fermentation and maturation of these lambic beer production processes take place, for the inoculation of the wort. The present study took a deeper look into all possible inoculation sources of the lambic beer wort by sampling the raw materials, environmental air, and brewing surfaces and equipment, and performing two lambic beer production processes carried out with two traditional wheat landraces (Limburgse Rode and Witte van Vlaanderen). All samples were analyzed in both a culture-dependent and culture-independent way, and with respect to their metabolites. The results revealed that the wooden barrels contributed the most to the inoculation of the lambic beer wort, since they were responsible for the subsequent growth and activity of Saccharomyces and Brettanomyces yeast, lactic acid bacteria and acetic acid bacteria species, which all played their roles during lambic beer production processes. In addition, the house microbiota was identified as an inoculation source, mainly responsible for the contribution of microorganisms that play a role during the initial fermentation phase of lambic beer production processes.

Sourdough is a flour-water mixture fermented by lactic acid bacteria (LAB) and yeasts. The addition of starter culture strains allows steering the fermentation process toward sourdoughs with tailored properties. However, the rational selection of candidate starter culture strains for sourdough production is of crucial importance, yet challenging. This review aimed to (i) describe the microbial diversity of non-starter culture-inoculated sourdoughs as source for starter culture strains for future use, (ii) examine LAB and yeast metabolism (carbohydrate, amino acid, and ester metabolism) and sourdough bread characteristics (flavor, rheology, nutritional and other health properties) as a target to screen and select strains, (iii) explore the interactions between LAB and yeasts and their implications for the use of starter cultures, and (iv) discuss how the different process parameters (raw material, oxygen, fermentation and storage duration and temperature) should be adapted for different starter culture strains. Furthermore, the techniques available for strain selection and starter culture strain monitoring are summarized, highlighting their current use, their challenges, and perspectives. Overall, a successful starter culture-initiated sourdough production process requires rational strain selection encompassing multiple complementary techniques and optimized process conditions. In addition, mixed-strain starter cultures, composed of LAB and yeast strains, show potential for enhancing sourdough quality.

Backslopped sourdoughs represent complex microbial ecosystems traditionally characterised by a stable coexistence of lactic acid bacteria (LAB) and yeasts. However, knowledge about the underlying parameters is limited. This study employed a multiphasic approach — combining sourdough metadata, culture-dependent techniques, amplicon-based metabarcoding, and metabolomics — to analyse 109 sourdoughs collected via a citizen science initiative in Belgium, France, the Netherlands, and Spain. Whereas the LAB and yeast species diversity largely aligned with previous sourdough analyses, three species previously unknown to the sourdough environment were found, namely, Fructilactobacillus vespulae, Geotrichum pandrosion, and Lachancea thermotolerans. Furthermore, this study provided a large-scale characterisation of acetic acid bacteria (AAB) in sourdough. AAB were found in up to 80.7% of the samples examined, with Acetobacter cerevisiae identified as a core species and 13 AAB species reported in sourdough for the first time. Integrated analysis revealed a complexity in sourdough that cannot be described by a single parameter, despite some trends being present. Metabolite profiling further underscored these complexities and suggested that erythritol production may be more widespread among LAB species than previously reported, specifically correlating with Levilactobacillus brevis and Lactiplantibacillus plantarum. These results emphasized that the sourdough ecosystem remains a reservoir of untapped biodiversity and highlighted the necessity of multi-angle microbiological analyses to capture the interplay between artisan practices and microbial community dynamics.

Abstract Romanian borş, a traditional fermented wheat bran beverage, is produced through spontaneous fermentation and represents a complex microbial ecosystem. Despite its cultural importance and presumed health benefits, its microbial ecology and functional potential remain poorly characterized. The present study aimed to elucidate the microbial community structure of borş and link it to functional traits relevant to fermentation performance and food functionality by integrating culture-independent sequencing with culture-dependent isolation and functional characterization. A total of 32 borş samples (12 commercial and 20 homemade) were analyzed. Amplicon-based sequencing revealed a microbiome dominated by lactic acid bacteria (LAB), with lactobacilli accounting for the majority of the bacterial communities and Lactobacillus amylolyticus being identified as the most prevalent and abundant species. The yeast communities were mainly composed of fermentative taxa, including Pichia kudriavzevii and Kluyveromyces marxianus . Lactobacillus amylolyticus and P. kudriavzevii were also the most frequently isolated species among bacteria and yeasts, respectively. These results highlighted a strong adaptation of the microbial isolates to starch-rich cereal substrates and underscored the central role of these microorganisms in wheat bran fermentation for borş production. Whereas the sequencing-based analyses showed no significant differences in overall diversity between the commercial and homemade borş samples, the cultivation-based results indicated a higher bacterial richness in the commercial products. Notably, the culture-dependent method captured substantially fewer taxa, highlighting the complementary nature of the two approaches. Of a total of 101 bacterial strains (88 LAB and 13 acetic acid bacteria) isolated, many exhibited rapid growth and strong acidification capacity, reaching pH values below 4.5 within 12 h. A functional screening revealed that 21 % of these strains displayed α-amylase activity, 65 % phytase activity, and 50 % β-glucosidase activity, highlighting their capacity to metabolize cereal substrates and enhance the nutrient availability of borş. All strains showed antibacterial activity against at least one indicator bacterium tested, with a universal inhibition of Listeria monocytogenes . Overall, Romanian borş harbored a lactic acid bacteria-dominated core microbiome with a significant functional diversity. These findings underscored its potential as a rich source of functional and technologically important strains for application in starter and protective culture development.

Considering the nutritional profile of whole oats and the potential health-enhancing effect of fermentation, oat fermentation offers significant potential for developing nutritious foods. However, fermenting oat wholemeal flour, in particular with lactic acid bacteria, proves challenging. We hypothesise this to be related to the kilning step, a heat-treatment typically applied to intact oat groats, which inactivates oat endogenous enzymes, enhancing stability against lipid oxidation and rancidity. However, kilning may also limit enzyme-induced biochemical changes of the food matrix and the enzymatic release of fermentable substrates during fermentation, thereby potentially impeding microbial growth and acidification. To investigate this, oat wholemeal flour suspensions derived from kilned and non-kilned oat groats were fermented for maximum 24h using a strain of Lactiplantibacillus plantarum as a starter culture. The ferments were comparatively evaluated microbiologically (i.e., microbial growth and starter culture prevalence) and biochemically (i.e., acidification, metabolite content, soluble fibre content, soluble protein content, and phytate reduction) over time. Fermentation proceeded faster in non-kilned oat wholemeal flour suspensions, resulting in more rapid acidification and more pronounced biochemical changes than in kilned suspensions. These results highlight the importance of active oat endogenous enzymes to provide nutrients for the fermenting lactic acid bacteria and to achieve desired biochemical changes of the matrix during oat fermentation.

Industrial production of Gouda cheeses is still affected by batch-to-batch variations, partly attributed to the rotational use of different starter culture mixtures. The current study aimed to unravel other variations by following two industrial Gouda cheese productions, made with the same starter culture mixture, up to 100 weeks. A culture-dependent analysis was combined with a culture-independent analysis through amplicon-based high-throughput sequencing (HTS) of the full-length 16S rRNA gene and meta-metabolomics, for both the cheese cores and rinds. Both cheese productions differed significantly in microbial counts throughout ripening. Lactococcus cremoris and Lactococcus lactis were the main species in the cheese cores. Additionally, Leuconostoc pseudomesenteroides was abundantly present, whereas Lacticaseibacillus paracasei was only abundant in one production, the cheeses of which had higher concentrations of free amino acids up to 26 weeks of ripening, and higher concentrations of D-lactic acid. The company's house microbiota differed from the cheese microbiota, with the exception that Loigolactobacillus rennini and Tetragenococcus halophilus were found in the brine and the cheeses. The former species caused high biogenic amine concentrations in the rinds during ripening. The microbial development of a new brine, including the fungal evolution by applying amplicon-based HTS of the whole fungal rRNA transcribed unit, went fast. However, the cheeses brined in the new brine did not contain Loil. rennini and the associated biogenic amines.

ABSTRACT The production of fruity esters by sourdough lactic acid bacteria (LAB) and yeasts has not been explored in detail. Moreover, the biosynthesis of esters by LAB species under conditions similar to those occurring during sourdough production is still questionable. Concerning yeasts, a genome mining of 75 genomes revealed a strain dependency of the presence of seven specific ester biosynthesis genes. Accordingly, PCR assays to detect these acetate ( ATF1 and ATF2 ) and ethyl ester ( EHT1 and EEB1 ) biosynthesis genes were developed and used to screen 91 strains of yeast species. Concerning LAB, a genome mining of 401 genomes revealed a species dependency of the presence of three esterase-encoding genes ( estA , estB , and estC ). A phenotypic analysis carried out with a selection of 10 strains of the LAB species Companilactobacillus crustorum , Companilactobacillus nantensis , Companilactobacillus paralimentarius, Fructilactobacillus sanfranciscensis , Lactiplantibacillus xiangfangensis , Levilactobacillus zymae , and Limosilactobacillus fermentum in a wheat sourdough simulation medium (WSSM) supplemented with ester precursor molecules ([higher] alcohols and fatty acids) revealed that their ester biosynthesis capacity was limited by the precursor concentrations. Ethyl acetate and ethyl lactate were produced by all strains, except for those of Frul. sanfranciscensis . These results suggested that one of the esterase-encoding genes considered could be implicated in the ethyl acetate and/or ethyl lactate biosynthesis. Overall, the ester biosynthesis capacity by LAB is of great interest in view of fruity flavor formation during sourdough and sourdough bread productions. IMPORTANCE The present study gave insights into the production of esters, which impart fruity flavors to fermented foods, by not only sourdough yeasts but also lactic acid bacteria. It showed that some lactic acid bacteria species can synthesize the esters ethyl acetate (sweet notes) and ethyl lactate (creamy notes) under specific conditions. The information gathered during the present study will enable sourdough bakers and companies from the bakery sector to get more information on how to produce sourdoughs that can add fruity notes to the final products after a rational screening and selection of potential starter culture strains.

Metagenomic studies of the Gouda cheese microbiota and starter cultures are scarce. During the present study, short-read metagenomic sequencing (Illumina) was applied on 89 Gouda cheese and processed milk samples, which have been investigated before concerning their metabolite and taxonomic composition, the latter applying amplicon-based, high-throughput sequencing (HTS) of the full-length 16S rRNA gene. Selected samples were additionally investigated using long-read metagenomic sequencing (Oxford Nanopore Technologies, ONT). Whereas the species identified by amplicon-based HTS and metagenomic sequencing were identical, the relative abundances of the major species differed significantly. Lactococcus cremoris was more abundant in the metagenomics-based taxonomic analysis compared to the amplicon-based one, whereas the opposite was true for the non-starter lactic acid bacteria (NSLAB). This discrepancy was related to a higher fragmentation of the lactococcal DNA compared with the DNA of other species when applying ONT. Possibly, a higher fragmentation was linked with a higher percentage of dead or metabolically inactive cells, suggesting that full-length 16S rRNA gene amplicon-based HTS might give a more accurate view on active cells. Further, fungi were not abundantly present in the Gouda cheeses examined, whereas about 2% of the metagenomic sequence reads was related to phages, with higher relative abundances in the cheese rinds and long-ripened cheeses. Intraspecies differences found by short-read metagenomic sequencing were in agreement with the amplicon sequence variants obtained previously, confirming the ability of full-length 16S rRNA gene amplicon-based HTS to reach a taxonomic assignment below species level. Metagenome-assembled genomes (MAGs) were retrieved for 15 species, among which the starter cultures Lc. cremoris and Lactococcus lactis and the NSLAB Lacticaseibacillus paracasei, Loigolactobacillus rennini, and Tetragenococcus halophilus, although obtaining MAGs from Lc. cremoris and Lc. lactis was more challenging because of a high intraspecies diversity and high similarity between these species. Long-read metagenomic sequencing could not improve the retrieval of lactococcal MAGs, but, overall, MAGs obtained by long-read metagenomic sequencing solely were superior compared with those obtained by short-read metagenomic sequencing solely, reaching a high-quality draft status of the genomes.

Cereal vinegars have been used for thousands of years, especially in Asian countries. These vinegars are still produced in a traditional way by a spontaneous, consecutive, alcoholic and acetic acid fermentation process in open vats under non-sterile conditions, which can lead to an unstable and inconsistent flavor and quality. The present study characterized the microbial diversity of complete, traditional Korean rice vinegar productions at two producers (A and B), from steamed rice to rice vinegar, applying high-throughput amplicon-based and shotgun metagenomic sequencing, in combination with meta-metabolomic analysis. Functional analysis based on metagenome-assembled genomes provided insights into the genetic potential of the different microorganisms involved. Producer A used nuruk, a traditional starter, and seed vinegar to start the alcoholic and acetic acid fermentation phases, respectively, which resulted in highly controlled productions even when different fermentation vessels were used. Producer B used only nuruk to start the vinegar productions, and the spontaneous inoculation of acetic acid bacteria did fail in one of the productions. The addition of nuruk resulted in a simultaneous rice starch saccharification and alcoholic fermentation phase characterized by producer-specific moulds, yeasts, and lactic acid bacteria (LAB). During the acetic acid fermentation phase at both producers (a) novel Acetobacter species, related to A. pasteurianus was found. The simultaneous presence of several LAB species made it hard to link them with the production of specific metabolites. Also, the species contributing to ester formation, important for the flavor, was not clear and requires further research.

As defined by law, at least 30 % of the total amount of starch- or carbohydrate-containing raw materials used for the production of Belgian lambic beers should be unmalted wheat, since this is important for the longevity of the lambic beer production process. Whereas lambic beer brewers in the 19th and 20th century depended on local wheat landraces, supplied by the local farmers, they nowadays use modern wheat varieties that are mainly cultivated for their baking quality and available on the global market. However, it could be assumed that lambic beers made with traditional wheat varieties were characterized by different flavor profiles. To know the impact of those wheat varieties on the growing microbiota and metabolites produced throughout a lambic beer production process, three different productions were assessed as a function of fermentation and maturation time. Two of them were performed with a traditional wheat landrace, namely, Witte van Vlaanderen and Limburgse Rode, and one control was performed with current standard wheat. A multiphasic analysis approach revealed that the impact of the wheat variety on the microbiology and metabolome was limited, although some metabolic differences, such as the concentrations of volatile phenolic compounds and esters, could be linked with the use of the Limburgse Rode wheat landrace. However, the most impacting factor remained the spontaneous origin of the prevailing microorganisms and the environmental conditions, such as temperature, pH, and dissolved oxygen concentration, that are encountered during lambic beer production. Finally, this work allowed to discover a new species, namely, Acetobacter zythi.

ABSTRACT Ten Gouda cheese wheels with an age of 31 weeks from six different batch productions were affected by a crack defect and displayed an unpleasant off-flavor. To unravel the causes of these defects, the concentrations of free amino acids, other organic acids, volatile organic compounds, and biogenic amines were quantified in zones around the cracks and in zones without cracks, and compared with those of similar Gouda cheeses without crack defect. The Gouda cheeses with cracks had a significantly different metabolome. The production of the non-proteinogenic amino acid γ-aminobutyric acid (GABA) could be unraveled as the key mechanism leading to crack formation, although the production of the biogenic amines cadaverine and putrescine contributed as well. High-throughput amplicon sequencing of the full-length 16S rRNA gene based on whole-community DNA revealed the presence of Loigolactobacillus rennini and Tetragenococcus halophilus as most abundant non-starter lactic acid bacteria in the zones with cracks. Shotgun metagenomic sequencing allowed to obtain a metagenome-assembled genome of both Loil. rennini and T. halophilus . However, only Loil. rennini contained genes necessary for the production of GABA, cadaverine, and putrescine. Metagenetics further revealed the brine and the rennet used during cheese manufacturing as the most plausible inoculation sources of both Loil. rennini and T. halophilus . IMPORTANCE Crack defects in Gouda cheeses are still poorly understood, although they can lead to major economic losses in cheese companies. In this study, the bacterial cause of a crack defect in Gouda cheeses was identified, and the pathways involved in the crack formation were unraveled. Moreover, possible contamination sources were identified. The brine bath might be a major source of bacteria with the potential to deteriorate cheese quality, which suggests that cheese producers should regularly investigate the quality and microbial composition of their brines. This study illustrated how a multiphasic approach can understand and mitigate problems in a cheese company.

Gouda cheeses of different production batches and ripening times often differ in metabolite composition, which may be due to the starter culture mixture applied or the growth of non-starter lactic acid bacteria (NSLAB) upon maturation. Therefore, a single Gouda cheese production batch was systematically investigated from the thermized milk to the mature cheeses, ripened for up to 100 weeks, to identify the main bacterial species and metabolites and their dynamics during the whole production and ripening. As this seemed to be starter culture strain- and NSLAB-dependent, it requested a detailed, longitudinal, and quantitative investigation. Hereto, microbial colony enumeration, high -throughput full-length 16S rRNA gene sequencing, and a metabolomic approach were combined. Culture -dependently, Lactococcus lactis was the most abundant species from its addition as part of the starter culture up to the first two months of cheese ripening. Afterward, the NSLAB Lacticaseibacillus paracasei became the main species during ripening. The milk was a possible inoculation source for the latter species, despite pasteurization. Culture -independently, the starter LAB Lactococcus cremoris and Lc. lactis were the most abundant species in the cheese core throughout the whole fermentation and ripening phases up to 100 weeks. The cheese rind from 40 until 100 weeks of ripening was characterized by a high relative abundance of the NSLAB Tetragenococcus halophilus and Loigolactobacillus rennini, which both came from the brine. These species were linked with the production of the biogenic amines cadaverine and putrescine. The most abundant volatile organic compound was acetoin, an indicator of citrate and lactose fermentation during the production day, whereas the concentrations of free amino acids were an indicator of the ripening time.

ABSTRACT The production of gueuze beers through refermentation and maturation of blends of lambic beer in bottles is a way for lambic brewers to cope with the variability among different lambic beer batches. The resulting gueuze beers are more carbonated than lambic beers and are supposed to possess a unique flavor profile that varies over time. To map this refermentation and maturation process for gueuze production, a blend of lambic beers was made and bottled, whereby one of them was produced with the old wheat landrace Zeeuwse Witte . Through the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-throughput sequencing of bacterial and fungal amplicons, in combination with metabolite target analysis, new insights into gueuze production were obtained. During the initial stages of refermentation, the conditions in the bottles were similar to those encountered during the maturation phase of lambic beer productions in wooden barrels, which was also reflected microbiologically (presence of Brettanomyces species, Pediococcus damnosus , and Acetobacter lambici ) and biochemically (ethanol, higher alcohols, lactic acid, acetic acid, volatile phenolic compounds, and ethyl esters). However, after a few weeks of maturation, a switch from a favorable environment to one with nutrient and dissolved oxygen depletion resulted in several changes. Concerning the microbiology, a sequential prevalence of three lactic acid bacterial species occurred, namely, P. damnosus , Lentilactobacillus buchneri , and Lactobacillus acetotolerans , while the diversity of the yeasts decreased. Concerning the metabolites produced, mainly those of the Brettanomyces yeasts determined the metabolic profiles encountered during later stages of the gueuze production. IMPORTANCE Gueuze beers are the result of a refermentation and maturation process of a blend of lambic beers carried out in bottles. These gueuze beers are known to have a long shelf life, and their quality typically varies over time. However, knowledge about gueuze production in bottles is scarce. The present study provided more insights into the varying microbial and metabolite composition of gueuze beers during the first 2 years of this refermentation and maturation process. This will allow gueuze producers to gain more information about the influence of the refermentation and maturation time on their beers. These insights can also be used by gueuze producers to better inform their customers about the quality of young and old gueuze beers.

BACKGROUND:Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated.RESULTS:The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms.CONCLUSIONS:Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix.

Polyphasic taxonomic and comparative genomic analyses revealed that a series of lambic beer isolates including strain LMG 32668T and the kombucha isolate LMG 32879 represent a novel species among the acetic acid bacteria, with Acidomonas methanolica as the nearest phylogenomic neighbor with a valid name. Overall genomic relatedness indices and phylogenomic and physiological analyses revealed that this novel species was best classified in a novel genus for which we propose the name Brytella acorum gen. nov., sp. nov., with LMG 32668T (=CECT 30723T) as the type strain. The B. acorum genomes encode a complete but modified tricarboxylic acid cycle, and complete pentose phosphate, pyruvate oxidation and gluconeogenesis pathways. The absence of 6-phosphofructokinase which rendered the glycolysis pathway non-functional, and an energy metabolism that included both aerobic respiration and oxidative fermentation are typical metabolic characteristics of acetic acid bacteria. Neither genome encodes nitrogen fixation or nitrate reduction genes, but both genomes encode genes for the biosynthesis of a broad range of amino acids. Antibiotic resistance genes or virulence factors are absent.