Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.
ABSTRACT: BACKGROUND:Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. RESULTS:The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. CONCLUSIONS:Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.
Project description:Acetobacter pasteurianus 386B is a candidate functional starter culture for the cocoa bean fermentation process. To allow in silico simulations of its related metabolism in response to different environmental conditions, a genome-scale metabolic model for A. pasteurianus 386B was reconstructed. This is the first genome-scale metabolic model reconstruction for a member of the genus Acetobacter. The metabolic network reconstruction process was based on extensive genome re-annotation and comparative genomics analyses. The information content related to the functional annotation of metabolic enzymes and transporters was placed in a metabolic context by exploring and curating a Pathway/Genome Database of A. pasteurianus 386B using the Pathway Tools software. Metabolic reactions and curated gene-protein-reaction associations were bundled into a genome-scale metabolic model of A. pasteurianus 386B, named iAp386B454, containing 454 genes, 322 reactions, and 296 metabolites embedded in two cellular compartments. The reconstructed model was validated by performing growth experiments in a defined medium, which revealed that lactic acid as the sole carbon source could sustain growth of this strain. Further, the reconstruction of the A. pasteurianus 386B genome-scale metabolic model revealed knowledge gaps concerning the metabolism of this strain, especially related to the biosynthesis of its cell envelope and the presence or absence of metabolite transporters.
Project description:Microbial strains for starter culture-initiated sourdough productions are commonly isolated from a fermenting flour-water mixture. Yet, starter culture strains isolated from matrices other than sourdoughs could provide the dough with interesting metabolic properties and hence change the organoleptic properties of the concomitant breads. Furthermore, the selection of sourdough starter cultures does not need to be limited to lactic acid bacteria (LAB), as other food-grade microorganisms are sometimes found in sourdoughs. Therefore, different strains belonging to LAB, acetic acid bacteria (AAB), and coagulase-negative staphylococci (CNS) that originated from different fermented food matrices (fermenting cocoa pulp-bean mass, fermented sausage, and water kefir), were examined as to their prevalence in a wheat sourdough ecosystem during 72-h fermentations. Limosilactobacillus fermentum IMDO 222 (fermented cocoa pulp-bean mass isolate) and Latilactobacillus sakei CTC 494 (fermented sausage isolate) seemed to be promising candidates as sourdough starter culture strains, as were the AAB strains Acetobacter pasteurianus IMDO 386B and Gluconobacter oxydans IMDO A845 (both isolated from fermented cocoa pulp-bean mass), due to their competitiveness in the wheat flour-water mixtures. Wheat breads made with G. oxydans IMDO A845 sourdoughs were significantly darker than reference wheat breads.
Project description:Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.
Project description:Spontaneous cocoa bean fermentations performed under bench- and pilot-scale conditions were studied using an integrated microbiological approach with culture-dependent and culture-independent techniques, as well as analyses of target metabolites from both cocoa pulp and cotyledons. Both fermentation ecosystems reached equilibrium through a two-phase process, starting with the simultaneous growth of the yeasts (with Saccharomyces cerevisiae as the dominant species) and lactic acid bacteria (LAB) (Lactobacillus fermentum and Lactobacillus plantarum were the dominant species), which were gradually replaced by the acetic acid bacteria (AAB) (Acetobacter tropicalis was the dominant species). In both processes, a sequence of substrate consumption (sucrose, glucose, fructose, and citric acid) and metabolite production kinetics (ethanol, lactic acid, and acetic acid) similar to that of previous, larger-scale fermentation experiments was observed. The technological potential of yeast, LAB, and AAB isolates was evaluated using a polyphasic study that included the measurement of stress-tolerant growth and fermentation kinetic parameters in cocoa pulp media. Overall, strains L. fermentum UFLA CHBE8.12 (citric acid fermenting, lactic acid producing, and tolerant to heat, acid, lactic acid, and ethanol), S. cerevisiae UFLA CHYC7.04 (ethanol producing and tolerant to acid, heat, and ethanol), and Acetobacter tropicalis UFLA CHBE16.01 (ethanol and lactic acid oxidizing, acetic acid producing, and tolerant to acid, heat, acetic acid, and ethanol) were selected to form a cocktail starter culture that should lead to better-controlled and more-reliable cocoa bean fermentation processes.
Project description:Forastero hybrid cocoa bean fermentations have been carried out in a box (B) and in a heap (H), with or without the inoculation of Saccharomyces cerevisiae and Torulaspora delbrueckii as starter cultures. The bacteria, yeasts, and microbial metabolites (volatile and nonvolatile organic compounds) were monitored during fermentation to assess the connection between microbiota and the release of metabolites during this process. The presence of starter cultures was detected, by means of culture-dependent analysis, during the first 2 days of both fermentations. However, no statistical difference was observed in any of the physicochemical or microbiological analyses. Plate counts revealed the dominance of yeasts at the beginning of both fermentations, and these were followed by acetic acid bacteria (AAB) and lactic acid bacteria (LAB). Hanseniaspora opuntiae, S. cerevisiae, Pichia pijperi, Acetobacter pasteurianus, and Lactobacillus fermentum were the most abundant operational taxonomic units (OTUs) during both fermentation processes (B and H), although different relative abundances were observed. Only the diversity of the fungal species indicated a higher level of complexity in the B fermentations than in the H fermentations (P < 0.05), as well as a statistically significant difference between the initially inoculated starter cultures (P < 0.01). However, the microbial metabolite analysis indicated different distributions of the volatile and nonvolatile compounds between the two procedures, that is, B and H (P < 0.05), rather than between the inoculated and noninoculated fermentations. The box fermentations showed faster carbohydrate metabolism and greater production of organic acid compounds, which boosted the formation of alcohols and esters, than did the heap fermentations. Overall, the microbial dynamics and associations between the bacteria, yeasts, and metabolites were found to depend on the type of fermentation.IMPORTANCE In spite of the limited effectiveness of the considered inoculated starter strains, this study provides new information on the microbial development of box and heap cocoa fermentations, under inoculated and noninoculated conditions, as we coupled yeast/bacterial amplicon-based sequencing data with microbial metabolite detection. The information so far available suggests that microbial communities have played an important role in the evolution of aroma compounds. Understanding the pathways that microorganisms follow during the formation of aromas could be used to improve the fermentation processes and to enhance chocolate quality.
Project description:Chocolate production suffered a vast impact with the emergence of the "witches' broom" disease in cocoa plants. To recover cocoa production, many disease-resistant hybrid plants have been developed. However, some different cocoa hybrids produce cocoa beans that generate chocolate with variable quality. Fermentation of cocoa beans is a microbiological process that can be applied for the production of chocolate flavor precursors, leading to overcoming the problem of variable chocolate quality. The aim of this work was to use a cocktail of microorganisms as a starter culture on the fermentation of the ripe cocoa pods from PH15 cocoa hybrid, and evaluate its influence on the microbial communities present on the fermentative process on the compounds involved during the fermentation, and to perform the chocolate sensorial characterization. According to the results obtained, different volatile compounds were identified in fermented beans and in the chocolate produced. Bitterness was the dominant taste found in non-inoculated chocolate, while chocolate made with inoculated beans showed bitter, sweet, and cocoa tastes. 2,3-Butanediol and 2,3-dimethylpyrazine were considered as volatile compounds making the difference on the flavor of both chocolates. Saccharomyces cerevisiae UFLA CCMA 0200, Lactobacillus plantarum CCMA 0238, and Acetobacter pasteurianus CCMA 0241 are proposed as starter cultures for cocoa fermentation.
Project description:The growth of filamentous fungi during the spontaneous cocoa bean fermentation leads to inferior cocoa bean quality and poses a health risk for consumers due to the potential accumulation of mycotoxins. We recently developed anti-fungal cultures with the capacity to inhibit the growth of mycotoxigenic filamentous fungi on cocoa beans. However, it is not clear how these anti-fungal cultures affect the fermentation process and cocoa bean quality. For that, the anti-fungal co-cultures, Lactobacillus fermentum M017-Saccharomyces cerevisiae H290 (A) and Lb. fermentum 223-S. cerevisiae H290 (B), were applied to 180-kg box fermentations in Honduras in three time-independent replications each including a spontaneous control fermentation. The comparison of inoculated and spontaneous fermentation processes revealed that the co-cultures only marginally affected the fermentation process and cocoa bean quality. Microorganisms reached maximal levels of 6.2-7.6 log CFU/g of yeasts and acetic acid bacteria and 7.9-9.5 log CFU/g of lactic acid bacteria during all fermentations and led to maximal metabolite concentrations in bean cotyledons of 4-12 mg/g ethanol, 2-6 mg/g lactic acid and 6-14 mg/g acetic acid. The fermentation and drying processes resulted in 38-90 mg epicatechin equivalents/g in the cotyledons of dried beans. However, the co-cultures led to up to ten times higher mannitol levels in cotyledons of inoculated beans compared to beans during spontaneous fermentation, and caused a slower fermentation process, detectable as up to 8-12 °C lower temperatures in the centre of the fermenting pulp-bean mass and up to 22% lower proportions of well-fermented beans after drying. Co-culture B-with Lb. fermentum 223 -led to improved cocoa bean quality compared to co-culture A-with Lb. fermentum M017 -, i.e. cocoa beans with 0.5-1.9 mg/g less acetic acid, 4-17% higher shares of well-fermented beans and, on a scale from 0 to 10, to 0.2-0.6 units lower astringency, up to 1.1 units lower off-flavours, and 0.2-0.9 units higher cocoa notes. Therefore, the anti-fungal co-culture B is recommended for future applications and its capacity to limit fungal growth and mycotoxin production during industrial-scale cocoa bean fermentation should be investigated in further studies.
Project description:Acetobacter pasteurianus, an acetic acid resistant bacterium belonging to alpha-proteobacteria, has been widely used to produce vinegar in the food industry. To understand the mechanism of its high tolerance to acetic acid and robust ability of oxidizing ethanol to acetic acid (> 12%, w/v), we described the 3.1 Mb complete genome sequence (including 0.28 M plasmid sequence) with a G+C content of 52.4% of A. pasteurianus Ab3, which was isolated from the traditional Chinese rice vinegar (Meiguichu) fermentation process. Automatic annotation of the complete genome revealed 2,786 protein-coding genes and 73 RNA genes. The comparative genome analysis among A. pasteurianus strains revealed that A. pasteurianus Ab3 possesses many unique genes potentially involved in acetic acid resistance mechanisms. In particular, two-component systems or toxin-antitoxin systems may be the signal pathway and modulatory network in A. pasteurianus to cope with acid stress. In addition, the large numbers of unique transport systems may also be related to its acid resistance capacity and cell fitness. Our results provide new clues to understanding the underlying mechanisms of acetic acid resistance in Acetobacter species and guiding industrial strain breeding for vinegar fermentation processes.
Project description:Cocoa bean fermentation relies on the sequential activation of several microbial populations, triggering a temporal pattern of biochemical transformations. Understanding this complex process is of tremendous importance as it is known to form the precursors of the resulting chocolate's flavour and taste. At the same time, cocoa bean fermentation is one of the least controlled processes in the food industry. Here, a quantitative model of cocoa bean fermentation is constructed based on available microbiological and biochemical knowledge. The model is formulated as a system of coupled ordinary differential equations with two distinct types of state variables: (i) metabolite concentrations of glucose, fructose, ethanol, lactic acid and acetic acid and (ii) population sizes of yeast, lactic acid bacteria and acetic acid bacteria. We demonstrate that the model can quantitatively describe existing fermentation time series and that the estimated parameters, obtained by a Bayesian framework, can be used to extract and interpret differences in environmental conditions. The proposed model is a valuable tool towards a mechanistic understanding of this complex biochemical process, and can serve as a starting point for hypothesis testing of new systemic adjustments. In addition to providing the first quantitative mathematical model of cocoa bean fermentation, the purpose of our investigation is to show how differences in estimated parameter values for two experiments allow us to deduce differences in experimental conditions.
Project description:Cocoa pulp fermentation is a spontaneous process during which the natural microbiota present at cocoa farms is allowed to ferment the pulp surrounding cocoa beans. Because such spontaneous fermentations are inconsistent and contribute to product variability, there is growing interest in a microbial starter culture that could be used to inoculate cocoa pulp fermentations. Previous studies have revealed that many different fungi are recovered from different batches of spontaneous cocoa pulp fermentations, whereas the variation in the prokaryotic microbiome is much more limited. In this study, therefore, we aimed to develop a suitable yeast starter culture that is able to outcompete wild contaminants and consistently produce high-quality chocolate. Starting from specifically selected Saccharomyces cerevisiae strains, we developed robust hybrids with characteristics that allow them to efficiently ferment cocoa pulp, including improved temperature tolerance and fermentation capacity. We conducted several laboratory and field trials to show that these new hybrids often outperform their parental strains and are able to dominate spontaneous pilot scale fermentations, which results in much more consistent microbial profiles. Moreover, analysis of the resulting chocolate showed that some of the cocoa batches that were fermented with specific starter cultures yielded superior chocolate. Taken together, these results describe the development of robust yeast starter cultures for cocoa pulp fermentations that can contribute to improving the consistency and quality of commercial chocolate production.