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.

Fruit beers, beers with or without a co-fermentation step with fruits.

Belgium is known for its traditional lambic beer productions, obtained through spontaneous fermentation and maturation in wooden barrels. Lambic beer is also used to make fruit lambic beers, such as Kriek beer. Despite fruit beer being an old beer type, dating back to the second half of the seventeenth century, no research has been performed on lambic beer-fruit co-fermentation processes. Further, these beers get competition from market-driven, sweet, (fruit-)flavored ones without the co-fermentation step. This paper will first discuss a new, general fruit beer classification, going from sour fruit beers produced through co-fermentation to sweet ones without a co-fermentation step. Second, a state-of-the-art of the scarce literature on the microbiology and metabolomics of lambic beer-fruit co-fermentation processes will be given.

Wheat Sourdough Breadmaking: A Scoping Review.

Using sourdough in breadmaking can enhance bread's shelf-life and flavor compared to exclusive baker's yeast use and is believed to increase its nutritional quality and healthiness. Previous research established insight into the microbial ecology of sourdough, but the link between leavening agent use, processing, and bread quality remains elusive. However, such knowledge is key for standardization, research on the health benefits, and the definition of sourdough bread. In this systematic scoping review, we analyzed 253 studies and identified large variations in the type and amount of leavening agent, fermentation conditions, and bread quality (specific loaf volume and acidification). The interrelation between these elements and their effect on the extent of fermentation is discussed, together with issues preventing proper comparison of breadmaking procedures. With this review, we want to contribute to the dialogue concerning the definition of sourdough-type bread products and the research into the health benefits attributed to them.

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.

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.

Various cold storage-backslopping cycles show the robustness of Limosilactobacillus fermentum IMDO 130101 as starter culture for Type 3 sourdough production.

Type 3 sourdoughs, which are starter culture-initiated and subsequently backslopped, are less studied than other sourdough types. Yet, they can serve as a model to assess how competitive starter culture strains for sourdough production are and how the microbial composition of such sourdoughs may evolve over time. In the present study, Limosilactobacillus fermentum IMDO 130101 was used to produce Type 3 sourdoughs, prepared from wheat and wholemeal wheat flours. Therefore, an initial fermentation of the flour-water mixture was performed at 30 °C for 48 h. This was followed by cold storage-backslopping cycles, consisting of refreshments (50 %, v/v), fermentation steps of 16 h, and storage at 4 °C each week, every three weeks, and every six weeks. The microbial dynamics (culture-dependent and -independent approaches) and metabolite dynamics were measured. In all sourdoughs produced, starter culture strain monitoring, following an amplicon sequence variant approach, showed that Liml. fermentum IMDO 130101 prevailed during one month when the sourdoughs were refreshed each week, during 24 weeks when the sourdoughs were refreshed every three weeks, and during 12 weeks when the sourdoughs were refreshed every six weeks. This suggested the competitiveness and robustness of Liml. fermentum IMDO 130101 for a considerable duration but also showed that the strain is prone to microbial interference. For instance, Levilactobacillus brevis and Pediococcus spp. prevailed upon further cold storage and backslopping. Also, although no yeasts were inoculated into the flour-water mixtures, Kazachstania unispora, Torulaspora delbrueckii, and Wickerhamomyces anomalus were the main yeast species found. They appeared after several weeks of storage and backslopping, which however indicated the importance of an interplay between LAB and yeast species in sourdoughs. The main differences among the mature sourdoughs obtained could be explained by the different flours used, the refreshment conditions applied, and the sampling time (before and after backslopping). Finally, the metabolite quantifications revealed continued metabolite production during the cold storage periods, which may impact the sourdough properties and those of the breads made thereof.

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.

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.

Impact of drought and salt stress on galactinol and raffinose family oligosaccharides in common bean (Phaseolus vulgaris).

Due to climate change, farmers will face more extreme weather conditions and hence will need crops that are better adapted to these challenges. The raffinose family oligosaccharides (RFOs) could play a role in the tolerance of crops towards abiotic stress. To investigate this, we determined for the first time the importance of galactinol and RFOs in the roots and leaves of common bean under drought and salt stress conditions. Initially, the physiological characteristics of common bean under agronomically relevant abiotic stress conditions were investigated by measuring the growth rate, transpiration rate, chlorophyll concentration and membrane stability, allowing to establish relevant sampling points. Subsequently, the differential gene expression profiles of the galactinol and RFO biosynthetic genes and the amount of galactinol and RFO molecules were measured in the primary leaves and roots of Phaseolus vulgaris cv. CIAP7247F at these sampling points, using RT-qPCR and HPAEC-PAD, respectively. Under drought stress, the genes galactinol synthase 1, galactinol synthase 3 and stachyose synthase were significantly upregulated in the leaves and had a high transcript level in comparison with the other galactinol and RFO biosynthetic genes. This was in accordance with the significantly higher amount of galactinol and raffinose detected in the leaves. Under salt stress, raffinose was also present in a significantly higher quantity in the leaves. In the roots, transcript levels of the RFO biosynthetic genes were generally low and no galactinol, raffinose or stachyose could be detected. These results suggest that in the leaves, both galactinol and raffinose could play a role in the protection of common bean against abiotic stresses. Especially, the isoform galactinol synthase 3 could have a specific role during drought stress and forms an interesting candidate to improve the abiotic stress resistance of common bean or other plant species.

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.

A phylogenomic and comparative genomic analysis of Commensalibacter, a versatile insect symbiont.

BACKGROUND: To understand mechanisms of adaptation and plasticity of pollinators and other insects a better understanding of diversity and function of their key symbionts is required. Commensalibacter is a genus of acetic acid bacterial symbionts in the gut of honey bees and other insect species, yet little information is available on the diversity and function of Commensalibacter bacteria. In the present study, whole-genome sequences of 12 Commensalibacter isolates from bumble bees, butterflies, Asian hornets and rowan berries were determined, and publicly available genome assemblies of 14 Commensalibacter strains were used in a phylogenomic and comparative genomic analysis. RESULTS: The phylogenomic analysis revealed that the 26 Commensalibacter isolates represented four species, i.e. Commensalibacter intestini and three novel species for which we propose the names Commensalibacter melissae sp. nov., Commensalibacter communis sp. nov. and Commensalibacter papalotli sp. nov. Comparative genomic analysis revealed that the four Commensalibacter species had similar genetic pathways for central metabolism characterized by a complete tricarboxylic acid cycle and pentose phosphate pathway, but their genomes differed in size, G + C content, amino acid metabolism and carbohydrate-utilizing enzymes. The reduced genome size, the large number of species-specific gene clusters, and the small number of gene clusters shared between C. melissae and other Commensalibacter species suggested a unique evolutionary process in C. melissae, the Western honey bee symbiont. CONCLUSION: The genus Commensalibacter is a widely distributed insect symbiont that consists of multiple species, each contributing in a species specific manner to the physiology of the holobiont host.

Meat fermentation at a crossroads: where the age-old interplay of human, animal, and microbial diversity and contemporary markets meet.

Despite being part of the now often unfavourably perceived category of processed meats, fermented meats remain of substantial nutritional, economic, and cultural importance in today's foodscapes. This translates into a vast assortment of different products. Fermentation is driven by microorganisms (e.g. in fermented sausages), although the terminology is sometimes used to also designate products in which microbial contributions are less dominant and that depend primarily on the activity of endogenous meat enzymes (e.g. in raw hams). A summary is given of the main microbial groups that characterize various types of meat and, in particular, their fermented derivatives. Moreover, it is argued that producers of fermented meat products struggle to adapt to a contemporary dietary context of change. On the one hand, they wish to reassure consumers by reaffirming the position of fermented meat products as traditional strongholds. On the other hand, producers are trying to alleviate some of the perceived concerns through technological innovation, for instance related to the impact of processing on food safety and health. This review raises the point that these sometimes contradictory trends can affect the choice of meat type, ingredients, and processing parameters, and how these choices, in turn, can affect microbial diversity.

Impact of process parameters on the specific volume of wholemeal wheat bread made using sourdough- and baker's yeast-based leavening strategies.

The final quality of wholemeal wheat bread is determined by the process parameter settings and leavening strategy. We hypothesise that the used leavening strategy may influence the optimal process parameter settings and, as such, the specific volume of the bread loaf. To analyse this interaction, bread was leavened with (i) a type 1 sourdough (SB), (ii) a type 1 sourdough combined with baker's yeast (YSB), or (iii) baker's yeast (YB). For each leavening strategy, the specific volume of bread, in response to variations in mixing time (4-10/4-14 min), water absorption (60-85 %), and proofing time (1-7/1-3 h), was analysed using an I-optimal response surface experimental design. Data modelling identified a substantially lower maximal specific volume of SB (2.13 mL/g), compared to YSB (3.30 mL/g) and YB (3.26 mL/g). The proofing time and water absorption mostly influenced the specific volume of the SB and YSB, respectively. However, the mixing and proofing times mainly affected the specific volume of YB. The type 1 sourdough reduced the mixing time and water absorption required for an optimal specific volume of bread compared to baker's yeast. These results challenge the idea of yielding higher volumes upon using sourdough compared to baker's yeast and highlight the importance of optimisation of bread dough formulations and breadmaking processes.

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.

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.

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.

Acetic Acid Bacteria in Sour Beer Production: Friend or Foe?

Beer is the result of a multistep brewing process, including a fermentation step using in general one specific yeast strain. Bacterial presence during beer production (or presence in the beer itself) is considered as bad, since bacteria cause spoilage, produce off-flavors, and/or turbidity. Although most problems in the past related to lack of hygiene and/or cleaning, bacteria do still cause problems nowadays. Despite this negative imago, certain bacteria play an irreplaceable role during fermentation and/or maturation of more unique, funky, and especially refreshing sour beers. The term sour beers or sours is not restricted to one definition but covers a wide variety of beers produced via different techniques. This review proposes an uncluttered sour beer classification scheme, which includes all sour beer production techniques and pays special attention to the functional role of acetic acid bacteria. Whereas their oxidation of ethanol and lactate into acetic acid and acetoin usually spoils beer, including sour beers, organoleptically, a controlled growth leads to a desirable acidic flavor in sour beers, such as lambic-style, lambic-based, and red-brown acidic ales.