Integrating shotgun metagenomics and metabolomics to elucidate the dynamics of microbial communities and metabolites in fine flavor cocoa fermentation in Hainan.

The aim of this study was to investigate the dynamic profile of microorganisms and metabolites in Hainan Trinitario cocoa during a six-day spontaneous box fermentation process. Shotgun metagenomic and metabolomic approaches were employed for this investigation. The potential metabolic functions of microorganisms in cocoa fermentation were revealed through a joint analysis of microbes, functional genes, and metabolites. During the anaerobic fermentation phase, Hanseniaspora emerged as the most prevalent yeast genus, implicated in pectin decomposition and potentially involved in glycolysis and starch and sucrose metabolism. Tatumella, possessing potential for pyruvate kinase, and Fructobacillus with a preference for fructose, constituted the primary bacteria during the pre-turning fermentation stage. Upon the introduction of oxygen into the fermentation mass, acetic acid bacteria ascended to dominant within the microflora. The exponential proliferation of Acetobacter resulted in a decline in taxonomic richness and abundance. Moreover, the identification of novel species within the Komagataeibacter genus suggests that Hainan cocoa may serve as a valuable reservoir for the discovery of unique cocoa fermentation bacteria. The KEGG annotation of metabolites and enzymes also highlighted the significant involvement of phenylalanine metabolism in cocoa fermentation. This research will offer a new perspective for the selection of starter strains and the formulation of mixed starter cultures.

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.

Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation.

Cocoa beans used for chocolate production are fermented seeds of Theobroma cacao obtained by a natural fermentation process. The flavors and chemical compounds produced during the fermentation process make this step one of the most important in fine chocolate production. Herein, an integrative analysis of the variation of microbial community structure, using a shotgun metagenomics approach and associated physicochemical features, was performed during fermentation of fine cocoa beans. Samples of Forastero variety (FOR) and a mixture of two hybrids (PS1319 and CCN51) (MIX) from Bahia, Brazil, were analyzed at 7 different times. In the beginning (0 h), the structures of microbial communities were very different between FOR and MIX, reflecting the original plant-associated microbiomes. The highest change in microbial community structures occurred at the first 24 h of fermentation, with a marked increase in temperature and acetic acid concentration, and pH decrease. At 24-48 h both microbial community structures were quite homogenous regarding temperature, acetic acid, succinic acid, pH, soluble proteins and total phenols. During 72-96 h, the community structure resembles an acidic and warmer environment, prevailing few acetic acid bacteria. Taxonomic richness and abundance at 72-144 h exhibited significant correlation with temperature, reducing sugars, succinic, and acetic acids. Finally, we recommend that dominant microbial species of spontaneous fine cocoa fermentations should be considered as inoculum in accordance with the farm/region and GMP to maintain a differential organoleptic feature for production of fine chocolate. In our study, a starter inoculum composed of Acetobacter pausterianus and Hanseniaspora opuntiae strains is indicated.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation.

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4-23.0 g/l of mannitol, 3.9-6.2 g/l acetic acid, and 8.6-10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4-18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51-95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

High-throughput identification of the microbial biodiversity of cocoa bean fermentation by MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI-TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI-TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (-20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI-TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial identification with MALDI-TOF MS has increased the number of samples that can be analysed in a given time, a prerequisite for high-throughput methods. This method is already widely used for the identification of clinical microbial isolates, whereas in food fermentation research, including cocoa bean fermentation, microbiota is mostly identified by time-consuming, biochemical-based phenotyping and molecular approaches. This study presents the use of MALDI-TOF MS for characterizing the microbial biodiversity of cocoa bean fermentation. The feasibility of MALDI-TOF MS identification of cocoa-specific microbiota has been shown with samples collected during on-site studies in two countries of origin, Bolivia and Brazil.

Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.

Spontaneous cocoa bean fermentation carried out in a novel-design stainless steel tank: influence on the dynamics of microbial populations and physical-chemical properties.

Spontaneous cocoa bean fermentations carried out in a novel-design 40-kg-capacity stainless steel tank (SST) was studied in parallel to traditional Brazilian methods of fermentation in wooden boxes (40-kg-capacity wooden boxes (WB1) and 600-kg-capacity wooden boxes (WB2)) using a multiphasic approach that entailed culture-dependent and -independent microbiological analyses of fermenting cocoa bean pulp samples and target metabolite analyses of both cocoa pulp and cotyledons. Both microbiological approaches revealed that the dominant species of major physiological roles were the same for fermentations in SST, relative to boxes. These species consisted of Saccharomyces cerevisiae and Hanseniaspora sp. in the yeast group; Lactobacillus fermentum and L. plantarum in the lactic acid bacteria (LAB) group; Acetobacter tropicalis belonging to the acetic acid bacteria (AAB) group; and Bacillus subtilis in the Bacillaceae family. A greater diversity of bacteria and non-Saccharomyces yeasts was observed in box fermentations. Additionally, a potentially novel AAB belonging to the genus Asaia was isolated during fermentation in WB1. Cluster analysis of the rRNA genes-PCR-DGGE profiles revealed a more complex picture of the box samples, indicating that bacterial and yeast ecology were fermentation-specific processes (wooden boxes vs. SST). The profile of carbohydrate consumption and fermentation products in the pulp and beans showed similar trends during both fermentation processes. However, the yeast-AAB-mediated conversion of carbohydrates into ethanol, and subsequent conversion of ethanol into acetic acid, was achieved with greater efficiency in SST, while temperatures were generally higher during fermentation in wooden boxes. With further refinements, the SST model may be useful in designing novel bioreactors for the optimisation of cocoa fermentation with starter cultures.

Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity.

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

Study on the microflora and biochemistry of cocoa fermentation in the Dominican Republic.

Cocoa fermentation was monitored at the IDIAF (Instituto Dominicano de Investigaciones Agropecuarias y Forestales) "Mata Larga" experimental station, in San Francisco de Macoris, Dominican Republic. The maximum average fermentation temperature reached 51 degrees C after 48 h and the pH reached 4.5 after 144 h of fermentation. A significant decrease in glucose, fructose and citric acid was seen in the pulp over the first 48 h. There was a delay of 24 h between maximum microbial growth and maximum concentrations of the respective metabolites, which occurred after 48 h for ethanol and after 72 h for acetic acid. A maximum concentration in lactic acid was found after around 120 h of fermentation. The aerobic mesophilic flora increased from 6.1x10(6) to a maximum of 4.2x10(7) CFU g(-1) of dry matter after 48 h of fermentation. Yeasts displayed maximum development after 24 h (6.1x10(7) CFU g(-1) of dry matter), whilst for lactic and acetic acid bacteria it occurred after 48 h (7.3x10(7) and 1.5x10(8) CFU g(-1) of dry matter respectively). The yeasts isolated belonged to the genera Hanseniaspora and Candida, the lactic acid bacteria to the genus Lactobacillus, and the acetic acid bacteria to the genus Acetobacter. The differences compared to other fermentation trials concerned the micropopulation from a qualitative point of view.

Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans.

Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.