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.

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.

The microbial and metabolite composition of Gouda cheese made from pasteurized milk is determined by the processing chain.

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.

Yeast strains do have an impact on the production of cured cocoa beans, as assessed with Costa Rican Trinitario cocoa fermentation processes and chocolates thereof.

The microbiological and metabolic outcomes of good cocoa fermentation practices can be standardized and influenced through the addition of starter culture mixtures composed of yeast and bacterial strains. The present study performed two spontaneous and 10 starter culture-initiated (SCI) cocoa fermentation processes (CFPs) in Costa Rica with local Trinitario cocoa. The yeast strains Saccharomyces cerevisiae IMDO 050523, Hanseniaspora opuntiae IMDO 020003, and Pichia kudriavzevii IMDO 060005 were used to compose starter culture mixtures in combination with the lactic acid bacterium strain Limosilactobacillus fermentum IMDO 0611222 and the acetic acid bacterium strain Acetobacter pasteurianus IMDO 0506386. The microbial community and metabolite dynamics of the cocoa pulp-bean mass fermentation, the metabolite dynamics of the drying cocoa beans, and the volatile organic compound (VOC) profiles of the chocolate production were assessed. An amplicon sequence variant approach based on full-length 16S rRNA gene sequencing instead of targeting the V4 region led to a highly accurate monitoring of the starter culture strains added, in particular the Liml. fermentum IMDO 0611222 strain. The latter strain always prevailed over the background lactic acid bacteria. A similar approach, based on the internal transcribed spacer (ITS1) region of the fungal rRNA transcribed unit, was used for yeast strain monitoring. The SCI CFPs evolved faster when compared to the spontaneous ones. Moreover, the yeast strains applied did have an impact. The presence of S. cerevisiae IMDO 050523 was necessary for successful fermentation of the cocoa pulp-bean mass, which was characterized by the production of higher alcohols and esters. In contrast, the inoculation of H. opuntiae IMDO 020003 as the sole yeast strain led to underfermentation and a poor VOC profile, mainly due to its low competitiveness. The P. kudriavzevii IMDO 060005 strain tested in the present study did not contribute to a richer VOC profile. Although differences in VOCs could be revealed in the cocoa liquors, no significant effect on the final chocolates could be obtained, mainly due to a great impact of cocoa liquor processing during chocolate-making. Hence, optimization of the starter culture mixture and cocoa liquor processing seem to be of pivotal importance.

Brytella acorum gen. nov., sp. nov., a novel acetic acid bacterium from sour beverages.

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.

Optimization of cacao beans fermentation by native species and electromagnetic fields.

Acid and bitter notes of the cocoa clone Cacao Castro Naranjal 51 (CCN 51) negatively affect the final quality of the chocolate. Thence, the fermentative process of cocoa beans using native species and electromagnetic fields (EMF) was carried out to evaluate the effect on the yield and quality of CCN 51 cocoa beans. The variables magnetic field density (D), exposure time (T), and inoculum concentration (IC) were optimized through response surface methodology to obtain two statistically validated second-order models, explaining 88.39% and 92.51% of the variability in the yield and quality of the beans, respectively. In the coordinate: 5 mT(D), 22.5 min (T), and 1.6% (CI), yield and bean quality improved to 110% and 120% above the control (without magnetic field). The metagenomic analysis showed that the changes in the microbial communities favored the aroma profile at low and intermediate field densities (5-42 mT) with high yields and floral, fruity, and nutty notes. Conversely, field densities (80 mT) were evaluated with low yields and undesirable notes of acidity and bitterness. The findings revealed that EMF effectively improves the yield and quality of CCN 51 cocoa beans with future applications in the development and quality of chocolate products.

The rotation of primary starter culture mixtures results in batch-to-batch variations during Gouda cheese production.

Industrial production of Gouda cheeses mostly relies on a rotated use of different mixed-strain lactic acid bacteria starter cultures to avoid phage infections. However, it is unknown how the application of these different starter culture mixtures affect the organoleptic properties of the final cheeses. Therefore, the present study assessed the impact of three different starter culture mixtures on the batch-to-batch variations among Gouda cheeses from 23 different batch productions in the same dairy company. Both the cores and rinds of all these cheeses were investigated after 36, 45, 75, and 100 weeks of ripening by metagenetics based on high-throughput full-length 16S rRNA gene sequencing accompanied with an amplicon sequence variant (ASV) approach as well as metabolite target analysis of non-volatile and volatile organic compounds. Up to 75 weeks of ripening, the acidifying Lactococcus cremoris and Lactococcus lactis were the most abundant bacterial species in the cheese cores. The relative abundance of Leuconostoc pseudomesenteroides was significantly different for each starter culture mixture. This impacted the concentrations of some key metabolites, such as acetoin produced from citrate, and the relative abundance of non-starter lactic acid bacteria (NSLAB). Cheeses with the least Leuc. pseudomesenteroides contained more NSLAB, such as Lacticaseibacillus paracasei that was taken over by Tetragenococcus halophilus and Loigolactobacillus rennini upon ripening time. Taken together, the results indicated a minor role of leuconostocs in aroma formation but a major impact on the growth of NSLAB. The relative abundance of T. halophilus (high) and Loil. rennini (low) increased with ripening time from rind to core. Two main ASV clusters of T. halophilus could be distinguished, which were differently correlated with some metabolites, both beneficial (regarding aroma formation) and undesirable ones (biogenic amines). A well-chosen T. halophilus strain could be a candidate adjunct culture for Gouda cheese production.

New insights into the role of key microorganisms and wooden barrels during lambic beer fermentation and maturation.

Belgian lambic beers are still produced through traditional craftsmanship. They rely on a spontaneous fermentation and maturation process that is entirely carried out in wooden barrels. The latter are used repetitively and may introduce some batch-to-batch variability. The present systematic and multiphasic study dealt with two parallel lambic beer productions carried out in nearly identical wooden barrels making use of the same cooled wort. It encompassed a microbiological and metabolomic approach. Further, a taxonomic classification and metagenome-assembled genome (MAG) investigation was based on shotgun metagenomics. These investigations provided new insights into the role of these wooden barrels and key microorganisms for this process. Indeed, besides their role in traditionality, the wooden barrels likely helped in establishing the stable microbial ecosystem of lambic beer fermentation and maturation by acting as an inoculation source of the necessary microorganisms, thereby minimizing batch-to-batch variations. They further provided a microaerobic environment, which aided in achieving the desirable succession of the different microbial communities for a successful lambic beer production process. Moreover, these conditions prevented excessive growth of acetic acid bacteria and, therefore, uncontrolled production of acetic acid and acetoin, which may lead to flavor deviations in lambic beer. Concerning the role of less studied key microorganisms for lambic beer production, it was shown that the Acetobacter lambici MAG contained several acid tolerance mechanisms toward the harsh environment of maturing lambic beer, whereas genes related to sucrose and maltose/maltooligosaccharide consumption and the glyoxylate shunt were absent. Further, a Pediococcus damnosus MAG possessed a gene encoding ferulic acid decarboxylase, possibly contributing to 4-vinyl compound production, as well as several genes, likely plasmid-based, related to hop resistance and biogenic amine production. Finally, contigs related to Dekkera bruxellensis and Brettanomyces custersianus did not possess genes involved in glycerol production, emphasizing the need for alternative external electron acceptors for redox balancing.

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.

Application of comparative genomics of Acetobacter species facilitates genome-scale metabolic reconstruction of the Acetobacter ghanensis LMG 23848T and Acetobacter senegalensis 108B cocoa strains.

Acetobacter species play an import role during cocoa fermentation. However, Acetobacter ghanensis and Acetobacter senegalensis are outcompeted during fermentation of the cocoa pulp-bean mass, whereas Acetobacter pasteurianus prevails. In this paper, an in silico approach aimed at delivering some insights into the possible metabolic adaptations of A. ghanensis LMG 23848T and A. senegalensis 108B, two candidate starter culture strains for cocoa fermentation processes, by reconstructing genome-scale metabolic models (GEMs). Therefore, genome sequence data of a selection of strains of Acetobacter species were used to perform a comparative genomic analysis. Combining the predicted orthologous groups of protein-encoding genes from the Acetobacter genomes with gene-reaction rules of GEMs from two reference bacteria, namely a previously manually curated model of A. pasteurianus 386B (iAp386B454) and two manually curated models of Escherichia coli (EcoCyc and iJO1366), allowed to predict the set of reactions present in A. ghanensis LMG 23848T and A. senegalensis 108B. The predicted metabolic network was manually curated using genome re-annotation data, followed by the reconstruction of species-specific GEMs. This approach additionally revealed possible differences concerning the carbon core metabolism and redox metabolism among Acetobacter species, pointing to a hitherto unexplored metabolic diversity. More specifically, the presence or absence of reactions related to citrate catabolism and the glyoxylate cycle for assimilation of C2 compounds provided not only new insights into cocoa fermentation but also interesting guidelines for future research. In general, the A. ghanensis LMG 23848T and A. senegalensis 108B GEMs, reconstructed in a semi-automated way, provided a proof-of-concept toward accelerated formation of GEMs of candidate functional starter cultures for food fermentation processes.

Phylogenomics of a Saccharomyces cerevisiae cocoa strain reveals adaptation to a West African fermented food population.

Various yeast strains have been proposed as candidate starter cultures for cocoa fermentation, especially strains of Saccharomyces cerevisiae. In the current study, the genome of the cocoa strain S. cerevisiae IMDO 050523 was unraveled based on a combination of long- and short-read sequencing. It consisted of 16 nuclear chromosomes and a mitochondrial chromosome, which were organized in 20 contigs, with only two small gaps. A phylogenomic analysis of this genome together with another 105 S cerevisiae genomes, among which 20 from cocoa strains showed a geographical distribution of the latter, including S. cerevisiae IMDO 050523. Its genome clustered together with that of a West African fermented food population, indicating a wider adaptation to West African food niches than cocoa. Furthermore, S. cerevisiae IMDO 050523 contained genetic signatures involved in sucrose hydrolysis, pectin degradation, osmotolerance, and conserved amino acid changes in key ester-producing enzymes that could point toward specific niche adaptations.

Microbiomes Associated With the Surfaces of Northern Argentinian Fruits Show a Wide Species Diversity.

The fiber, vitamin, and antioxidant contents of fruits contribute to a balanced human diet. In countries such as Argentina, several tropical fruits are witnessing a high yield in the harvest season, with a resulting surplus. Fruit fermentation using autochthonous starter cultures can provide a solution for food waste. However, limited knowledge exists about the microbiota present on the surfaces of fruits and the preceding flowers. In the present exploratory study, the microbiomes associated with the surfaces of tropical fruits from Northern Argentina, such as white guava, passion fruit and papaya were investigated using a shotgun metagenomic sequencing approach. Hereto, one sample composed of 14 white guava fruits, two samples of passion fruits with each two to three fruits representing the almost ripe and ripe stage of maturity, four samples of papaya with each two to three fruits representing the unripe, almost ripe, and ripe stage of maturity were processed, as well as a sample of closed and a sample of open Japanese medlar flowers. A considerable heterogeneity was found in the composition of the fruits' surface microbiota at the genus and species level. While bacteria dominated the microbiota of the fruits and flowers, a small number of the metagenomic sequence reads corresponded with yeasts and filamentous fungi. A minimal abundance of bacterial species critical in lactic acid and acetic acid fermentations was found. A considerable fraction of the metagenomic sequence reads from the fruits' surface microbiomes remained unidentified, which suggested that intrinsic species are to be sequenced or discovered.

Traditional Fermented Foods from Ecuador: A Review with a Focus on Microbial Diversity.

The development of early civilizations was greatly associated with populations' ability to exploit natural resources. The development of methods for food preservation was one of the pillars for the economy of early societies. In Ecuador, food fermentation significantly contributed to social advances and fermented foods were considered exclusive to the elite or for religious ceremonies. With the advancement of the scientific research on bioprocesses, together with the implementation of novel sequencing tools for the accurate identification of microorganisms, potential health benefits and the formation of flavor and aroma compounds in fermented foods are progressively being described. This review focuses on describing traditional fermented foods from Ecuador, including cacao and coffee as well as less popular fermented foods. It is important to provide new knowledge associated with nutritional and health benefits of the traditional fermented foods.

Genome-Based Characterization of a Plasmid-Associated Micrococcin P1 Biosynthetic Gene Cluster and Virulence Factors in Mammaliicoccus sciuri IMDO-S72.

Analysis of the de novo assembled genome of Mammaliicoccus sciuri IMDO-S72 revealed the genetically encoded machinery behind its earlier reported antibacterial phenotype and gave further insight into the repertoire of putative virulence factors of this recently reclassified species. A plasmid-encoded biosynthetic gene cluster was held responsible for the antimicrobial activity of M. sciuri IMDO-S72, comprising genes involved in thiopeptide production. The compound encoded by this gene cluster was structurally identified as micrococcin P1. Further examination of its genome highlighted the ubiquitous presence of innate virulence factors mainly involved in surface colonization. Determinants contributing to aggressive virulence were generally absent, with the exception of a plasmid-associated ica cluster. The native antibiotic resistance genes sal(A) and mecA were detected within the genome, among others, but were not consistently linked with a resistance phenotype. While mobile genetic elements were identified within the genome, such as an untypeable staphylococcal cassette chromosome (SCC) element, they proved to be generally free of virulence- and antibiotic-related genes. These results further suggest a commensal lifestyle of M. sciuri and indicate the association of antibiotic resistance determinants with mobile genetic elements as an important factor in conferring antibiotic resistance, in addition to their unilateral annotation. IMPORTANCEMammaliicoccus sciuri has been put forward as an important carrier of virulence and antibiotic resistance genes, which can be transmitted to clinically important staphylococcal species such as Staphylococcus aureus. As a common inhabitant of mammal skin, this species is believed to have a predominant commensal lifestyle, although it has been reported as an opportunistic pathogen in some cases. This study provides an extensive genome-wide description of its putative virulence potential taking into consideration the genomic context in which these genes appear, an aspect that is often overlooked during virulence analysis. Additional genome and biochemical analysis linked M. sciuri with the production of micrococcin P1, gaining further insight into the extent to which these biosynthetic gene clusters are distributed among different related species. The frequent plasmid-associated character hints that these traits can be horizontally transferred and might confer a competitive advantage to its recipient within its ecological niche.

High-throughput amplicon sequencing to assess the impact of processing factors on the development of microbial communities during spontaneous meat fermentation.

During spontaneous meat fermentation, diverse microbial communities develop over time. These communities consist mainly of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS), of which the species composition is influenced by the fermentation temperature and the level of acidification. Recent development and application of amplicon-based high-throughput sequencing (HTS) methods have allowed to gain deeper insights into the microbial communities of fermented meats. The aim of the present study was to investigate the effect of different fermentation temperatures and acidification profiles on the CNS communities during spontaneous fermentation, using a previously developed amplicon-based HTS method targeting both the 16S rRNA and tuf genes. Spontaneous fermentations were performed with five different lots of meat to assess inter-lot variability. The process influence was investigated by fermenting the meat batters for seven days at different fermentation temperatures (23 °C, 30 °C, and 37 °C) and in the absence or presence of added glucose to simulate different acidification levels. Additionally, the results were compared with a starter culture-initiated fermentation process. The data revealed that the fermentation temperature was the most influential processing condition in shaping the microbial communities during spontaneous meat fermentation processes, whereas differences in pH were only responsible for minor shifts in the microbial profiles. Furthermore, the CNS communities showed a great level of variability, which depended on the initial microbial communities present and their competitiveness.

The Microbiota of Modified-Atmosphere-Packaged Cooked Charcuterie Products throughout Their Shelf-Life Period, as Revealed by a Complementary Combination of Culture-Dependent and Culture-Independent Analysis.

Although refrigeration and modified-atmosphere packaging (MAP) allow for an extended shelf life of cooked charcuterie products, they are still susceptible to bacterial spoilage. To obtain better insights into factors that govern product deterioration, ample information is needed on the associated microbiota. In this study, sliced MAP cooked ham and cooked chicken samples were subjected to culture-dependent and culture-independent microbial analysis. In total, 683 bacterial isolates were obtained and identified from 60 samples collected throughout the storage period. For both charcuterie types, lactic acid bacteria (LAB) constituted the most abundant microbial group. In cooked ham, Brochothrix thermosphacta was highly abundant at the beginning of the shelf-life period, but was later overtaken by Leuconostoc carnosum and Lactococcus piscium. For cooked chicken products, Latilactobacillus sakei was most abundant throughout the entire period. Additionally, 13 cooked ham and 16 cooked chicken samples were analyzed using metabarcoding. Findings obtained with this method were generally in accordance with the results from the culture-dependent approach, yet they additionally demonstrated the presence of Photobacterium at the beginning of the shelf-life period in both product types. The results indicated that combining culture-dependent methods with metabarcoding can give complementary insights into the evolution of microorganisms in perishable foods.

A Combined Metagenomics and Metatranscriptomics Approach to Unravel Costa Rican Cocoa Box Fermentation Processes Reveals Yet Unreported Microbial Species and Functionalities.

Cocoa fermentation is the first step in the post-harvest processing chain of cocoa and is important for the removal of the cocoa pulp surrounding the beans and the development of flavor and color precursors. In the present study, metagenomic and metatranscriptomic sequencing were applied to Costa Rican cocoa fermentation processes to unravel the microbial diversity and assess the function and transcription of their genes, thereby increasing the knowledge of this spontaneous fermentation process. Among 97 genera found in these fermentation processes, the major ones were Acetobacter, Komagataeibacter, Limosilactobacillus, Liquorilactobacillus, Lactiplantibacillus, Leuconostoc, Paucilactobacillus, Hanseniaspora, and Saccharomyces. The most prominent species were Limosilactobacillus fermentum, Liquorilactobacillus cacaonum, and Lactiplantibacillus plantarum among the LAB, Acetobacter pasteurianus and Acetobacter ghanensis among the AAB, and Hanseniaspora opuntiae and Saccharomyces cerevisiae among the yeasts. Consumption of glucose, fructose, and citric acid, and the production of ethanol, lactic acid, acetic acid, and mannitol were linked to the major species through metagenomic binning and the application of metatranscriptomic sequencing. By using this approach, it was also found that Lacp. plantarum consumed mannitol and oxidized lactic acid, that A. pasteurianus degraded oxalate, and that species such as Cellvibrio sp., Pectobacterium spp., and Paucilactobacillus vaccinostercus could contribute to pectin degradation. The data generated and results presented in this study could enhance the ability to select and develop appropriate starter cultures to steer the cocoa fermentation process toward a desired course.

Application of a High-Throughput Amplicon Sequencing Method to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins.

Insight into the microbial species diversity of fermented meats is not only paramount to gain control over quality development, but also to better understand the link with processing technology and geographical origin. To study the composition of the microbial communities, the use of culture-independent methods is increasingly popular but often still suffers from drawbacks, such as a limited taxonomic resolution. This study aimed to apply a previously developed high-throughput amplicon sequencing (HTS) method targeting the 16S rRNA and tuf genes to characterize the bacterial communities in European fermented meats in greater detail. The data obtained broadened the view on the microbial communities that were associated with the various products examined, revealing the presence of previously underreported subdominant species. Moreover, the composition of these communities could be linked to the specificities of individual products, in particular pH, salt content, and geographical origin. In contrast, no clear links were found between the volatile organic compound profiles of the different products and the country of origin, distinct processing conditions, or microbial communities. Future application of the HTS method offers the potential to further unravel complex microbial communities in fermented meats, as well as to assess the impact of different processing conditions on microbial consortia.

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.

Temporal Shotgun Metagenomics Revealed the Potential Metabolic Capabilities of Specific Microorganisms During Lambic Beer Production.

Lambic beer production processes are characterized by a temporal succession of well-adapted microbial species. Temporal metagenomic analysis of a Belgian, traditional, lambic beer production process, which was examined microbiologically and metabolomically before, confirmed that the microbial diversity is limited. Moreover, it allowed to link the consumption and production of certain compounds to specific microbial groups or species. Fermentation characteristics, such as the conversion of malic acid into lactic acid and acetoin production, were retrieved and could be attributed to specific microorganisms, namely Pediococcus damnosus and Acetobacter species, respectively. Traits previously ascribed to brewery-specific Dekkera bruxellensis strains were confirmed during the lambic beer production process examined multiphasically; in particular, the higher production of 4-ethylguaiacol compared to 4-ethylphenol was further shown by mass spectrometric analysis. Moreover, the absence of phenolic acid decarboxylase in Brettanomyces custersianus was shown culture-independently and could explain its late occurrence during the maturation phase. Furthermore, the potential of maltooligosaccharide degradation could be ascribed metagenomically to not only Brettanomyces species but also Saccharomyces kudriavzevii, possibly explaining their degradation early in the lambic beer production process. Also, acetic acid bacteria (AAB) seemed to be able to consume maltooligosaccharides via their conversion into trehalose. Furthermore, these AAB possessed esterase genes, potentially capable of forming ethyl acetate, which may contribute to the flavor of lambic beer. Improved knowledge on the reasons behind certain community dynamics and the role of the different microorganisms in terms of potential functionality could improve brewery practices to assure to produce more quality-stable end-products.