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.

Sourdough production represents a dynamic fermentation process, involving complex interactions between microorganisms and the flour-water matrix. The quality of mature sourdoughs depends on factors such as the type of inoculum (spontaneous or starter culture-initiated), process parameters (temperature, pH, and dough yield), and ingredient variations (constituents of flour and water). Concerning the latter, the mineral availability may influence the microbial diversity and metabolite kinetics during sourdough production. Hence, in the present study, 10-day laboratory backslopped wheat sourdough productions were carried out, differing in mineral contents of the water phase, based on the screening of mineral compositions of different flours and waters collected from three Belgian bakery companies. The minerals added as chloride salts were calcium, magnesium, manganese, or sodium. A polyphasic approach, encompassing microbiological analysis (both culture-dependent and culture-independent) and metabolite target analysis, was employed. Although influences of calcium (positive) and manganese (negative) on yeast growth and of calcium (negative) on acetic acid bacteria (AAB) growth were found, no clear-cut trend concerning the lactic acid bacteria (LAB) diversity was found, except for the occurrence of a three-phase LAB evolution. Among 17 typical LAB sourdough species retrieved, Lactiplantibacillus plantarum/paraplantarum, Levilactobacillus brevis, and Limosilactobacillus fermentum prevailed toward the end of the backslopping cycles in all cases. Regarding the yeast species diversity, wheat sourdoughs supplemented with calcium chloride harbored only Saccharomyces paradoxus, whereas Maudiozyma saulgeensis and Naumovozyma castellii were only found in sourdoughs supplemented with sodium chloride. Metabolite target analysis revealed higher ethanol and glycerol concentrations in sourdoughs produced in the presence of calcium chloride and magnesium chloride compared to the others. Furthermore, sourdoughs produced in the presence of manganese chloride contained the highest lactic acid concentrations. These findings may serve as an indicator for ingredient selection for steered sourdough production, which in turn can influence the flavor of the resulting sourdough bread.

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.

The microbial consortium of Fructilactobacillus sanfranciscensis and Maudiozyma humilis is associated with complex trophic interactions that support persistence in sourdough ecosystems. However, its stability may depend on technological parameters, including dough yield, flour type, backslopping conditions and refreshment regime. This study assessed the long-term stability of this consortium in two wholemeal wheat mother sourdoughs from Belgian industrial bakeries, referred to as IB-A and IB-B. These sourdoughs differed in dough yield, namely 160 for IB-A and 200 for IB-B, and in backslopping conditions. Both sourdoughs were subjected to weekly or triweekly refreshment cycles, each combining cold storage at 4 °C with backslopping at either room temperature for 24 h or 30 °C for 16 h. Under weekly refreshment, the consortium persisted for one month in both sourdoughs. Under triweekly refreshment, Frul. sanfranciscensis and Maud. humilis remained stable only in IB-A, whereas both species were lost from IB-B after 12 and 15 weeks, respectively. In IB-B, Levilactobacillus parabrevis and Pediococcus parvulus became prevalent. In contrast, stable production of acetic acid, ethanol, and mannitol in IB-A indicated sustained metabolic activity of the Frul. sanfranciscensis–Maud. humilis consortium. These findings show that the long-term viability and metabolic fitness of this consortium require suitable combined process parameters.

Lambic and gueuze beers are Flemish artisan-style beers produced through spontaneous fermentation and maturation in wooden barrels, involving successive microbial communities of Saccharomyces and Brettanomyces yeasts, lactic acid bacteria (LAB), and acetic acid bacteria. Although these beers generate culinary enthusiasm due to their complex flavor profiles and traditional character, information on the microbial and biochemical diversity among marketed end products is limited. This study explored 15 commercially available lambic and gueuze beers from different breweries and two purchase years (2023 and 2025) to provide an overview of the viable microbial consortia still present in the bottles at the time of consumption, while examining the diversity of residual carbohydrates and flavor-affecting molecules. Most bottled commercial beers remained both microbiologically viable and biochemically diverse at the time of purchase, while inter-brand differences were consistent with the non-standardized nature of lambic beer refermentation and storage. Across the beer samples examined, the viable microbial communities were predominated by Brettanomyces and certain LAB species. The metabolite profile showed high levels of lactic acid and appreciable contributions from acetic acid and Brettanomyces-derived ethyl esters, with very low residual fermentable saccharides. One outlier among the beer brands showed markedly elevated residual saccharides (glucose and fructose), which is not a property of gueuze beers, whereas another one was characterized by a distinct metabolite signature and substantially higher organic acid concentrations, indicating a strongly acidic chemical profile. These findings contributed to a deeper understanding of artisan fermented alcoholic beverage systems and their variability across commercial products.

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.

As the most abundant nonstarch polysaccharides in cereals, arabinoxylans (AXs) contribute significantly to the global intake of dietary fiber. They play a crucial role in the breadmaking process with respect to dough rheology and texture, bread volume, and nutritional quality, especially when starting from wheat or rye flour. During (sour)dough preparation, AX-degrading enzymes from either endogenous or microbial origin are activated, causing changes in the AX composition and structure that, in turn, affect the viscosity and gas retention capacity of the bread dough. These effects lead to beneficial changes in dough handling, which may improve bread volume and crumb texture. The production of sourdough-based breads represents an interesting case. The sourdough microbiota, consisting primarily of lactic acid bacteria and yeasts, may substantially contribute to AX alteration by either producing AX-degrading enzymes or contributing to the acid-mediated activation of endogenous ones as a result of dough acidification upon fermentation of the flour-water matrix. To monitor the quantity, composition, and structure of AXs during (sourdough) bread production, various methods and analytical techniques have been developed. A better understanding of the fate of AXs during sourdough production and of the specific AX-affecting capabilities of the microorganisms that inhabit the sourdough ecosystem may allow for better control and steering of the breadmaking process, resulting in more standardized bakery products of improved quality.

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.

Arabinoxylans are constituents of wheat flour that contribute to the dietary fiber properties of wheat. They exist in water-extractable and water-unextractable forms and contribute to human health. In bakery technology, especially the water-extractable arabinoxylans (WE-AX) are important due to their impact on viscosity and dough rheology. This study provides insights into the impact of wheat flour fermentation on WE-AX during sourdough production, offering potential applications for improving sourdough bread quality and its health benefits. The production of sourdoughs is known to increase the WE-AX fraction, yet the underlying (bio)chemical mechanisms remain unclear. This study investigated the alteration of WE-AX during the fermentation of wheat flour for sourdough production using 1H Diffusion Ordered SpectroscopY (DOSY) Nuclear Magnetic Resonance (NMR) at elevated temperature to analyze the structural changes of WE-AX during wheat flour fermentation for sourdough production with different lactic acid bacteria (LAB) strains. The results confirmed that DOSY NMR at elevated temperatures greatly improved the applicability of the method for analyzing larger biomolecules. Overall, a size reduction of the WE-AX compounds with increasing fermentation time was found. This was indicated both by the occurrence of higher self-diffusion coefficients, and increased transverse relaxation times. Further research is necessary to explain deviations from the general trend.

The use of mixed-strain starter cultures (MSSCs) for sourdough production can lead to the obtention of sourdoughs and sourdough breads with novel flavour profiles. In the present study, the Type 3 sourdough productions were started with a MSSC composed of the lactic acid bacteria strain Companilactobacillus crustorum LMG 23699 and the yeast strain Wickerhamomyces anomalus IMDO 010110. The MSSC prevailed for at least 12 weeks of cold storage backslopping cycles, highlighting its potential. Also, wholemeal wheat and white wheat breads produced with a sourdough started with the MSSC that was refreshed twice before use (CcWaR) had desirable specific volumes, high lactic acid and amino acid concentrations that could be related to health-promoting characteristics, and a volatile organic compound profile characterized by esters with fruity notes. Further, triangle tests showed that the white wheat CcWaR breads differed from the breads produced using commercial baker's yeast (BY), W. anomalus IMDO 010110 as non-conventional yeast (Wa), and a sourdough started with Coml. crustorum LMG 23699 supplemented with baker's yeast (CcBY). Specifically they were described as fruitier and more sour breads. Moreover, the production of Wa breads represented new and fruity yeast-leavened bread types. Therefore, this leavening agent could be an alternative to obtain breads with a more desirable flavour than BY breads, but without the sourness of sourdough breads. Overall, the present study launched two new bread types characterized by the use of a non-conventional yeast and enhanced fruitiness.

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.

Sourdough bread consumption has been associated with improved glucose and appetite regulation thanks to the presence of organic acids produced during fermentation of the flour-water mixture. We investigated the effects of whole meal sourdough bread (WSB) rich in lactic acid on energy intake, satiety, gastric emptying, glucose, and C-peptide response compared to whole meal yeast bread (WYB). Forty-four normal-weight participants (age: 30 +/- 10 y; BMI: 23 +/- 2 kg/m(2)) participated in this double-blind, randomized cross-over trial, consisting of two study visits separated by one week. During each study visit, gastric emptying, subjective appetite, glucose, and C-peptide concentrations were measured at regular time intervals over a 4-h period. After 4 h, ad-libitum energy intake was assessed. Despite no effect of bread type on ad-libitum energy intake at the subsequent meal (p = 0.068), WSB led to lower hunger (p < 0.001), higher fullness (p < 0.001), lower desire to eat (p < 0.001), and lower prospective food consumption (p < 0.001) compared to WYB. WSB had a higher gastric half-emptying time (p = 0.002), lower glucose response between 15 and 30 min (p < 0.05) after bread consumption, and lower C-peptide response between 15 and 90 min (p < 0.05) after bread consumption, compared to WYB. These findings suggest that the consumption of WSB, rich in lactic acid, acutely enhanced satiety and improved the postprandial metabolic response. However, these effects did not result in reduced ad-libitum energy intake.

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.

Erythritol is a sugar alcohol that is occasionally produced by heterofermentative lactic acid bacteria (LAB). Although such biosynthesis has rarely been reported during sourdough production, the potential of the sourdough strains Limosilactobacillus fermentum IMDO 130101 and IMDO TC9L10 to produce erythritol was demonstrated in the context of their broader metabolic capacity both during wheat sourdough productions and in a wheat sourdough simulation medium (WSSM). Indeed, both strains produced up to 0.4 mM of erythritol during starter culture-initiated wheat sourdough productions. The strain L. fermentum IMDO 130101 was further used to carry out controlled fermentation processes in WSSM and was able to synthesize 0.53 ± 0.03 mM of erythritol. During both the wheat sourdough productions and WSSM fermentation processes, acetate was co-produced with erythritol and/or glycerol. The primary role of erythritol was likely its involvement in redox balancing. Besides, the pathways for erythritol production were probed in silico, but no genes corresponding with erythrose-producing or erythrose-converting enzymes could be identified. Therefore, it was suggested that the enzymes responsible for the production of erythritol in L. fermentum IMDO 130101, and other heterofermentative LAB species, are the same as those producing mannitol or glycerol. IMPORTANCE:Lactic acid bacteria have a limited biosynthesis capacity. Their main carbohydrate breakdown pathways enable them to produce energy and to maintain their redox balance. The latter is accomplished through the production of lactic acid in the case of homofermentative lactic acid bacteria and ethanol or acetic acid in the case of heterofermentative ones. However, under certain conditions, other branches of this pathway become active, which lead to end-metabolites that are produced seldomly. An example of such an end-metabolite is erythritol, which was already detected during sourdough production but never investigated in detail. The present study showed the production of erythritol by Limosilactobacillus fermentum inhabiting sourdough environments and its involvement in redox balancing.

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.