Decarboxylase activity of the non-starter lactic acid bacterium Loigolactobacillus rennini gives crack defects in Gouda cheese through the production of γ-aminobutyric acid.

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.IMPORTANCECrack 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.

Refermentation and maturation of lambic beer in bottles: a necessary step for gueuze production.

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.IMPORTANCEGueuze 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.

Comparative genomic analysis of Periweissella and the characterization of novel motile species.

The genus Periweissella was proposed as a novel genus in the Lactobacillaceae in 2022. However, the phylogenetic relationship between Periweissella and other heterofermentative lactobacilli, and the genetic and physiological properties of this genus remain unclear. This study aimed to determine the phylogenetic relationship between Periweissella and the two closest genera, Weissella and Furfurilactobacillus, by the phylogenetic analysis and calculation of (core gene) pairwise average amino acid identity. Targeted genomic analysis showed that fructose bisphosphate aldolase was only present in the genome of Pw. cryptocerci. Mannitol dehydrogenase was found in genomes of Pw. beninensis, Pw. fabaria, and Pw. fabalis. Untargeted genomic analysis identified the presence of flagellar genes in Periweissella but not in other closely related genera. Phenotypes related to carbohydrate fermentation and motility matched the genotypes. Motility genes were organized in a single operon and the proteins shared a high amino acid similarity in the genus Periweissella. The relatively low similarity of motility operons between Periweissella and other motile lactobacilli indicated the acquisition of motility by the ancestral species. Our findings facilitate the phylogenetic, genetic, and phenotypic understanding of the genus Periweissella.ImportanceThe genus Periweissella is a heterofermentative genus in the Lactobacillaceae which includes predominantly isolates from cocoa fermentations in tropical climates. Despite the relevance of the genus in food fermentations, genetic and physiological properties of the genus are poorly characterized and genome sequences became available only after 2020. This study characterized strains of the genus by functional genomic analysis, and by determination of metabolic and physiological traits. Phylogenetic analysis revealed that Periweissella is the evolutionary link between rod-shaped heterofermentative lactobacilli and the coccoid Leuconostoc clade with the genera Weissella and Furfurilactobacillus as closest relatives. Periweissella is the only heterofermentative genus in the Lactobacillaceae which comprises predominantly motile strains. The genomic, physiological, and metabolic characterization of Periweissella may facilitate the potential use of strains of the genus as starter culture in traditional or novel food fermentations.

Sourdough production: fermentation strategies, microbial ecology, and use of non-flour ingredients.

Sourdough production is an ancient method to ferment flour from cereals for the manufacturing of baked goods. This review deals with the state-of-the-art of current fermentation strategies for sourdough production and the microbial ecology of mature sourdoughs, with a particular focus on the use of non-flour ingredients. Flour fermentation processes for sourdough production are typically carried out by heterogeneous communities of lactic acid bacteria and yeasts. Acetic acid bacteria may also occur, although their presence and role in sourdough production can be criticized. Based on the inoculum used, sourdough productions can be distinguished in fermentation processes using backslopping procedures, originating from a spontaneously fermented flour-water mixture (Type 1), starter culture-initiated fermentation processes (Type 2), and starter culture-initiated fermentation processes that are followed by backslopping (Type 3). In traditional recipes for the initiation and/or propagation of Type 1 sourdough productions, non-flour ingredients are often added to the flour-water mixture. These ingredients may be the source of an additional microbial inoculum and/or serve as (co-)substrates for fermentation. An example of the former is the addition of yoghurt; an example of the latter is the use of fruit juices. The survival of microorganisms transferred from the ingredients to the fermenting flour-water mixture depends on the competitiveness toward particular strains of the microbial species present under the harsh conditions of the sourdough ecosystem. Their survival and growth is also determined by the presence of the appropriate substrates, whether or not carried over by the ingredients added.

An in-depth multiphasic analysis of the chocolate production chain, from bean to bar, demonstrates the superiority of Saccharomyces cerevisiae over Hanseniaspora opuntiae as functional starter culture during cocoa fermentation.

Hanseniaspora opuntiae is a commonly found yeast species in naturally fermenting cocoa pulp-bean mass, which needed in-depth investigation. The present study aimed at examining effects of the cocoa isolate H. opuntiae IMDO 040108 as part of three different starter culture mixtures compared with spontaneous fermentation, regarding microbial community, substrate consumption, and metabolite production dynamics, including volatile organic compound (VOC) and phytochemical compositions, as well as compositions of the cocoa beans after fermentation, cocoa liquors, and chocolates. The inoculated H. opuntiae strain was unable to prevail over background yeasts present in the fermenting cocoa pulp-bean mass. It led to under-fermented cocoa beans after four days of fermentation, which was however reflected in higher levels of polyphenols. Cocoa fermentation processes inoculated with a Saccharomyces cerevisiae strain enhanced flavour production during the fermentation and drying steps, which was reflected in richer and more reproducible aroma profiles of the cocoa liquors and chocolates. Sensory analysis of the cocoa liquors and chocolates further demonstrated that S. cerevisiae led to more acidic notes compared to spontaneous fermentation, as a result of an advanced fermentation degree. Finally, different VOC profiles were found in the cocoa beans throughout the whole chocolate production chain, depending on the fermentation process.

Functional yeast starter cultures for cocoa fermentation.

The quest to develop a performant starter culture mixture to be applied in cocoa fermentation processes started in the 20th century, aiming at achieving high-quality, reproducible chocolates with improved organoleptic properties. Since then, different yeasts have been proposed as candidate starter cultures, as this microbial group plays a key role during fermentation of the cocoa pulp-bean mass. Yeast starter culture-initiated fermentation trials have been performed worldwide through the equatorial zone and the effects of yeast inoculation have been analysed as a function of the cocoa variety (Forastero, Trinitario and hybrids) and fermentation method (farm-, small- and micro-scale) through the application of physicochemical, microbiological and chemical techniques. A thorough screening of candidate yeast starter culture strains is sometimes done to obtain the best performing strains to steer the cocoa fermentation process and/or to enhance specific features, such as pectinolysis, ethanol production, citrate assimilation and flavour production. Besides their effects during cocoa fermentation, a significant influence of the starter culture mixture applied is often found on the cocoa liquors and/or chocolates produced thereof. Thus, starter culture-initiated cocoa fermentation processes constitute a suitable strategy to elaborate improved flavourful chocolate products.

Technological and Environmental Features Determine the Uniqueness of the Lambic Beer Microbiota and Production Process.

Lambic beers are beers produced through spontaneous fermentation and maturation in wooden barrels. The production process of lambic beers differs from the production processes of lagers and ales in process technology, environmental parameters, and the use of specific raw materials. Moreover, every lambic beer production process is unique in terms of microbiology and flavor formation because of its dependence on the spontaneous inoculation of microorganisms coming from the environmental air (contacting the open coolship and other brewery equipment) and the inner surfaces of the barrels. Several factors influence the inter- and intraspecies microbial successions during lambic beer wort fermentation and maturation and determine the final quality of the end products. The possibility to manually acidify the wort, the presence of species-specific metabolic traits, the environmental temperature, the co-occurrence of lactic acid bacteria and acetic acid bacteria, as well as yeasts, and the quality of the wooden barrels all determine the progress and outcome of the lambic beer production process. Further alterations in quality and flavor of lambic beers can be achieved by blending practices and additional bottle refermentations. This results in a vast array of lambic-derived beer products (e.g., gueuze) with complex taste and aroma profiles and specific characteristics, which separate them from most other commercially available beers.

Gluconacetobacter dulcium sp. nov., a novel Gluconacetobacter species from sugar-rich environments.

A phylogenomic analysis based on 107 single-copy core genes revealed that three strains from sugar-rich environments, i.e. LMG 1728T, LMG 1731 and LMG 22058, represented a single, novel Gluconacetobacter lineage with Gluconacetobacter liquefaciens as nearest validly named neighbour. OrthoANIu and digital DNA-DNA hybridization analyses among these strains and Gluconacetobacter type strains confirmed that the three strains represented a novel Gluconacetobacter species. Biochemical characteristics and MALDI-TOF mass spectra allowed differentiation of this novel species from the type strains of G. liquefaciens and other closely related Gluconacetobacter species. We therefore propose to classify strains LMG 1728T, LMG 1731 and LMG 22058 in the novel species Gluconacetobacter dulcium sp. nov., with LMG 1728T (=CECT 30142T) as the type strain.

Novel acetic acid bacteria from cider fermentations: Acetobacter conturbans sp. nov. and Acetobacter fallax sp. nov.

Strains LMG 1627T, LMG 1636T and LMG 1637 were all isolated from cider fermentations in the 1940s and 1950s. A recent study based on MALDI-TOF MS and dnaK gene sequence analyses suggested they represented novel Acetobacter species. In the present study, we determined the whole-genome sequences of these strains and analysed their phenotypic and chemotaxonomic characteristics. A phylogenomic analysis based on 107 single-copy core genes revealed that they represented a single Acetobacter lineage with Acetobacter aceti, Acetobacter sicerae, Acetobacter musti and Acetobacter oeni, Acetobacter estunensis and with Acetobacter nitrogenifigens as an outgroup to this cluster. OrthoANIu value and dDDH analyses among these and other Acetobacter type strains confirmed that these three strains represented two novel Acetobacter species, which could be differentiated from other closely related type strains of Acetobacter by different phenotypic tests, such as ketogenesis from glycerol. We therefore propose to classify strain LMG 1627T in the novel species Acetobacter conturbans sp. nov., with LMG 1627T (=NCIMB 8945T) as the type strain, and to classify strains LMG 1636T and LMG 1637 in the novel species Acetobacter fallax sp. nov., with LMG 1636T (=NCIMB 8956T) as the type strain.

Comparative genomics of Lactobacillus fermentum suggests a free-living lifestyle of this lactic acid bacterial species.

Lactobacillus fermentum is a lactic acid bacterium frequently isolated from mammal tissues, milk, and plant material fermentations, such as sourdough. A comparative genomics analysis of 28 L. fermentum strains enabled the investigation of the core and accessory genes of this species. The core protein phylogenomic tree of the strains examined, consisting of five clades, did not exhibit clear clustering of strains based on isolation source, suggesting a free-living lifestyle. Based on the presence/absence of orthogroups, the largest clade, containing most of the human-related strains, was separated from the rest. The extended core genome included genes necessary for the heterolactic fermentation. Many traits were found to be strain-dependent, for instance utilisation of xylose and arabinose. Compared to other strains, the genome of L. fermentum IMDO 130101, a candidate starter culture strain capable of dominating sourdough fermentations, contained unique genes related to the metabolism of starch degradation products, which could be advantageous for growth in sourdough matrices. This study explained the traits that were previously demonstrated for L. fermentum IMDO 130101 at the genetic level and provided future avenues of research regarding L. fermentum strains isolated from sourdough.

The metagenome-assembled genome of Candidatus Oenococcus aquikefiri from water kefir represents the species Oenococcus sicerae.

Recently, a metagenomic study of a water kefir fermentation ecosystem enabled the reconstruction of a metagenome-assembled genome (MAG) of an Oenococcus species that was different from the three species of this genus known so far. Therefore, the name Candidatus Oenococcus aquikefiri was proposed for this novel Oenococcus species. In the meantime, however, a fourth member of the genus, Oenococcus sicerae, isolated from French cider, was reported. The comparison of its genome sequence with the Candidatus O. aquikefiri MAG showed an average nucleotide identity (ANI) value of 98.53%. In addition, the 16S rRNA and pheS genes of the two species were 99.4% and 99.9% identical, respectively. As the presence of O. sicerae in a water kefir metagenome was also revealed by metagenomic recruitment plotting, it can be stated that Candidatus O. aquikefiri and O. sicerae belong to the same species. Intraspecies variations include the presence or absence of a citrate lyase operon and components of various phosphotransferase (PTS) transport systems.

Genome-scale metabolic modeling of Acetobacter pasteurianus 386B reveals its metabolic adaptation to cocoa fermentation conditions.

Acetobacter pasteurianus 386B has been selected as a candidate functional starter culture to better control the cocoa fermentation process. Previously, its genome has been sequenced and a genome-scale metabolic model (GEM) has been reconstructed. To understand its metabolic adaptation to cocoa fermentation conditions, different flux balance analysis (FBA) simulations were performed and compared with experimental data. In particular, metabolic flux distributions were simulated for two phases that characterize the growth of A. pasteurianus 386B under cocoa fermentation conditions, predicting a switch in respiratory chain usage in between these phases. The possible influence on the resulting energy production was shown using a reduced version of the GEM. FBA simulations revealed the importance of the compartmentalization of the ethanol oxidation reactions, namely in the periplasm or in the cytoplasm, and highlighted the potential role of ethanol as a source of carbon, energy, and NADPH. Regarding the latter, the physiological function of a proton-translocating NAD(P)+ transhydrogenase was further investigated in silico. This study revealed the potential of using a GEM to simulate the metabolism of A. pasteurianus 386B, and may provide a general framework toward a better physiological understanding of functional starter cultures in food fermentation processes.

Raw meat quality and salt levels affect the bacterial species diversity and community dynamics during the fermentation of pork mince.

Acidification level and temperature modulate the beneficial consortia of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) during meat fermentation. Less is known about the impact of other factors, such as raw meat quality and salting. These could for instance affect the growth of the pathogen Staphylococcus aureus or of Enterobacterales species, potentially indicative of poor fermentation practice. Therefore, pork batters from either normal or borderline quality (dark-firm-dry, DFD) were compared at various salt concentrations (0-4%) in meat fermentation models. Microbial ecology of the samples was investigated with culture-dependent techniques and (GTG)5-PCR fingerprinting of genomic DNA. Whilst Lactobacillus sakei governed the fermentation of normal meat, Lactobacillus curvatus was more prominent in the fermentation of the DFD meat variant. CNS were favoured during fermentation at rising salt concentrations without much effects on species diversity, consisting mostly of Staphylococcus equorum, Staphylococcus saprophyticus, and Staphylococcus xylosus. During fermentation of DFD meat, S. saprophyticus was less manifest than during that of normal meat. Enterobacterales mainly emerged in DFD meat during fermentation at low salt concentrations. The salt hurdle was insufficient to prevent Enterobacterales when acidification and initial pH were favourable for their growth.

Following Coffee Production from Cherries to Cup: Microbiological and Metabolomic Analysis of Wet Processing of Coffea arabica.

A cup of coffee is the final product of a complex chain of operations. Wet postharvest processing of coffee is one of these operations, which involves a fermentation that inevitably has to be performed on-farm. During wet coffee processing, the interplay between microbial activities and endogenous bean metabolism results in a specific flavor precursor profile of the green coffee beans. Yet, how specific microbial communities and the changing chemical compositions of the beans determine the flavor of a cup of coffee remains underappreciated. Through a multiphasic approach, the establishment of the microbial communities, as well as their prevalence during wet processing of Coffea arabica, was followed at an experimental farm in Ecuador. Also, the metabolites produced by the microorganisms and those of the coffee bean metabolism were monitored to determine their influence on the green coffee bean metabolite profile over time. The results indicated that lactic acid bacteria were prevalent well before the onset of fermentation and that the fermentation duration entailed shifts in their communities. The fermentation duration also affected the compositions of the beans, so that longer-fermented coffee had more notes that are preferred by consumers. As a consequence, researchers and coffee growers should be aware that the flavor of a cup of coffee is determined before as well as during on-farm processing and that under the right conditions, longer fermentation times can be favorable, although the opposite is often believed.IMPORTANCE Coffee needs to undergo a long chain of events to transform from coffee cherries to a beverage. The coffee postharvest processing is one of the key phases that convert the freshly harvested cherries into green coffee beans before roasting and brewing. Among multiple existing processing methods, the wet processing has been usually applied for Arabica coffee and produces decent quality of both green coffee beans and the cup of coffee. In the present case study, wet processing was followed by a multiphasic approach through both microbiological and metabolomic analyses. The impacts of each processing step, especially the fermentation duration, were studied in detail. Distinct changes in microbial ecosystems, processing waters, coffee beans, and sensory quality of the brews were found. Thus, through fine-tuning of the parameters in each step, the microbial diversity and endogenous bean metabolism can be altered during coffee postharvest processing and hence provide potential to improve coffee quality.

Characterization of novel Gluconobacter species from fruits and fermented food products: Gluconobacter cadivus sp. nov., Gluconobacter vitians sp. nov. and Gluconobacter potus sp. nov.

Strains LMG 1744T, LMG 1745, LMG 31484T, LMG 1764T and R-71646 were isolated from rotting fruits and fermented food products. A phylogenomic analysis based on 107 single-copy core genes revealed that they grouped in a Gluconobacter lineage comprising Gluconobacter oxydans, Gluconobacter roseus, Gluconobacter sphaericus, Gluconobacter kanchanaburiensis, Gluconobacter albidus, Gluconobacter cerevisiae, Gluconobacter kondonii and Gluconobacter aidae. OrthoANIu and digital DNA hybridization analyses demonstrated that these five strains represented three novel Gluconobacter species, which could be differentiated from the type strains of closely related Gluconobacter species by multiple phenotypic characteristics. We therefore propose to classify strains LMG 1744T and LMG 1745 in the novel species Gluconobacter cadivus sp. nov., with LMG 1744T (=CECT 30141T) as the type strain; to classify strain LMG 31484T as the novel species Gluconobacter vitians sp. nov., with LMG 31484T (=CECT 30132T) as the type strain; and to classify strains LMG 1764T and R-71646 in the novel species Gluconobacter potus sp. nov., with LMG 1764T (=CECT 30140T) as the type strain.

Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production.

Acidic beers, such as Belgian lambic beers and American and other coolship ales, are becoming increasingly popular worldwide thanks to their refreshing acidity and fruity notes. The traditional fermentation used to produce them does not apply pure yeast cultures but relies on spontaneous, environmental inoculation. The fermentation and maturation process is carried out in wooden barrels and can take up to three years. It is characterized by different microbial species belonging to the enterobacteria, acetic acid bacteria, lactic acid bacteria, and yeasts. This review provides an introduction to the technology and four fermentation strategies of beer production, followed by the microbiology of acidic beer production, focusing on the main microorganisms present during the long process used for the production of Belgian lambic beers. © 2018 Society of Chemical Industry.

Complementary Mechanisms for Degradation of Inulin-Type Fructans and Arabinoxylan Oligosaccharides among Bifidobacterial Strains Suggest Bacterial Cooperation.

Inulin-type fructans (ITF) and arabinoxylan oligosaccharides (AXOS) are broken down to different extents by various bifidobacterial strains present in the human colon. To date, phenotypic heterogeneity in the consumption of these complex oligosaccharides at the strain level remains poorly studied. To examine mechanistic variations in ITF and AXOS constituent preferences present in one individual, ITF and AXOS consumption by bifidobacterial strains isolated from the simulator of the human intestinal microbial ecosystem (SHIME) after inoculation with feces from one healthy individual was investigated. Among the 18 strains identified, four species-independent clusters displaying different ITF and AXOS degradation mechanisms and preferences were found. Bifidobacterium bifidum B46 showed limited growth on all substrates, whereas B. longum B24 and B. longum B18 could grow better on short-chain-length fractions of fructooligosaccharides (FOS) than on fructose. B. longum B24 could cleave arabinose substituents of AXOS extracellularly, without using the AXOS-derived xylose backbones, whereas B. longum B18 was able to consume oligosaccharides (up to xylotetraose) preferentially and consumed AXOS to a limited extent. B. adolescentis B72 degraded all fractions of FOS simultaneously, partially degraded inulin, and could use xylose backbones longer than xylotetraose extracellularly. The strain-specific degradation mechanisms were suggested to be complementary and indicated resource partitioning. Specialization in the degradation of complex carbohydrates by bifidobacteria present on the individual level could have in vivo implications for the successful implementation of ITF and AXOS, aiming at bifidogenic and/or butyrogenic effects. Finally, this work shows the importance of taking microbial strain-level differences into account in gut microbiota research.IMPORTANCE It is well known that bifidobacteria degrade undigestible complex polysaccharides, such as ITF and AXOS, in the human colon. However, this process has never been studied for strains coexisting in the same individual. To examine strain-dependent mechanistic variations in ITF and AXOS constituent preferences present in one individual, ITF and AXOS consumption by bifidobacterial strains isolated from the SHIME after inoculation with feces from one healthy individual was investigated. Among the 18 bifidobacterial strains identified, four species-independent clusters displaying different ITF and AXOS degradation mechanisms and preferences were found, indicating that such strains can coexist in the human colon. Such specialization in the degradation of complex carbohydrates by bifidobacteria present on the individual level could have in vivo implications for the successful implementation of ITF and AXOS, aiming at bifidogenic and/or butyrogenic effects.

Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.

Spontaneous vegetable fermentations, with their rich flavors and postulated health benefits, are regaining popularity. However, their microbiology is still poorly understood, therefore raising concerns about food safety. In addition, such spontaneous fermentations form interesting cases of man-made microbial ecosystems. Here, samples from 38 carrot juice fermentations were collected through a citizen science initiative, in addition to three laboratory fermentations. Culturing showed that Enterobacteriaceae were outcompeted by lactic acid bacteria (LAB) between 3 and 13 days of fermentation. Metabolite-target analysis showed that lactic acid and mannitol were highly produced, as well as the biogenic amine cadaverine. High-throughput 16S rRNA gene sequencing revealed that mainly species of Leuconostoc and Lactobacillus (as identified by 8 and 20 amplicon sequence variants [ASVs], respectively) mediated the fermentations in subsequent order. The analyses at the DNA level still detected a high number of Enterobacteriaceae, but their relative abundance was low when RNA-based sequencing was performed to detect presumptive metabolically active bacterial cells. In addition, this method greatly reduced host read contamination. Phylogenetic placement indicated a high LAB diversity, with ASVs from nine different phylogenetic groups of the Lactobacillus genus complex. However, fermentation experiments with isolates showed that only strains belonging to the most prevalent phylogenetic groups preserved the fermentation dynamics. The carrot juice fermentation thus forms a robust man-made microbial ecosystem suitable for studies on LAB diversity and niche specificity.IMPORTANCE The usage of fermented food products by professional chefs is steadily growing worldwide. Meanwhile, this interest has also increased at the household level. However, many of these artisanal food products remain understudied. Here, an extensive microbial analysis was performed of spontaneous fermented carrot juices which are used as nonalcoholic alternatives for wine in a Belgian Michelin star restaurant. Samples were collected through an active citizen science approach with 38 participants, in addition to three laboratory fermentations. Identification of the main microbial players revealed that mainly species of Leuconostoc and Lactobacillus mediated the fermentations in subsequent order. In addition, a high diversity of lactic acid bacteria was found; however, fermentation experiments with isolates showed that only strains belonging to the most prevalent lactic acid bacteria preserved the fermentation dynamics. Finally, this study showed that the usage of RNA-based 16S rRNA amplicon sequencing greatly reduces host read contamination.

Variability within the dominant microbiota of sliced cooked poultry products at expiration date in the Belgian retail.

Sliced cooked poultry products are susceptible to bacterial spoilage, notwithstanding their storage under modified-atmosphere packaging (MAP) in the cold chain. Although the prevailing bacterial communities are known to be mostly consisting of lactic acid bacteria (LAB), more information is needed about the potential variation in species diversity within national markets. In the present study, a total of 42 different samples of sliced cooked poultry products were collected in the Belgian retail and their bacterial communities were analysed at expiration date. A total of 629 isolates from four different culture media, including plate count agar for the total microbiota and de Man-Rogosa-Sharpe (MRS), modified MRS, and M17 agar as three selective agar media for LAB, were subjected to (GTG)5-PCR fingerprinting and identification by gene sequencing. Overall, Carnobacterium, Lactobacillus, and Leuconostoc were the dominant genera. Within each genus, the most encountered isolates were Carnobacterium divergens, Lactobacillus sakei, and Leuconostoc carnosum. When comparing samples from chicken origin with samples from turkey-derived products, a higher dominance of Carnobacteria spp. was found in the latter group. Also, an association between the dominance of lactobacilli and the presence of added plant material and lactate salts was found.

Oxygen and diverse nutrients influence the water kefir fermentation process.

Eight water kefir fermentation series differing in the presence of oxygen, the nutrient concentration, and the nutrient source were studied during eight consecutive backslopping steps. The presence of oxygen allowed the proliferation of acetic acid bacteria, resulting in high concentrations of acetic acid, and decreased the relative abundance of Bifidobacterium aquikefiri. Low nutrient concentrations resulted in slow water kefir fermentation and high pH values, which allowed the growth of Comamonas testosteroni/thiooxydans. Further, low nutrient concentrations favored the growth of Lactobacillus hilgardii and Dekkera bruxellensis, whereas high nutrient concentrations favored the growth of Lactobacillus nagelii and Saccharomyces cerevisiae. Dried figs, dried apricots, and raisins resulted in stable water kefir fermentation. Water kefir fermentation with dried apricots resulted in the highest pH and water kefir grain growth, whereas that with raisins resulted in the lowest pH and water kefir grain growth. Further, water kefir fermentation with raisins resembled fermentations with low nutrient concentrations, that with dried apricots resembled fermentations with normal nutrient concentrations, and that with fresh figs or a mixture of yeast extract and peptone resembled fermentations with high nutrient concentrations.