Linking cocoa varietals and microbial diversity of Nicaraguan fine cocoa bean fermentations and their impact on final cocoa quality appreciation.

Nicaraguan cocoa bean fermentations of several single local cocoa varieties originating from the same region (North Highlands of Nicaragua, San Jose de Bocay/El Cuá) were compared to fermentations of blended cocoa varietals from other producing regions of the country (Waslala and Nueva Guinea) making use of High Throughput Sequencing techniques, metabolite target analysis and sensory evaluation of cocoa liquor samples. A succession of the important cocoa-related yeasts Hanseniaspora uvarum/opuntiae, Saccharomyces cerevisiae and/or Pichia kudriavzevii was seen for single varietals and Nueva Guinea fermentations, while Kazachstania humilis dominated the mid and end phase of the Waslala cocoa fermentations. Tatumella species (mainly Tatumella terrea and Tatumella punctata) predominated the bacterial community at the onset of all fermentations followed by unusually late (generally 2 days into the fermentations) appearance of Lactobacillus fermentum relative to fermentations in other parts of the World. Acetobacter spp. were the main acetic acid bacteria during all fermentations, but also Gluconobacter spp. were involved in some single-variety fermentations. All fermentations proved complete as determined by metabolite analysis with bean sucrose being fully depleted and pulp sugars exhausted after 48-72 h of fermentation. From an organoleptic point of view, all Nicaraguan cocoas of this study reflected fine fruity (citrus or berry-like) flavours with distinct herbal or caramel notes. Floral notes were associated with the cases where P. kudriavzevii was involved in the later stages of fermentation. Intense citrus/fruity character was related to high pulp and bean citrate concentrations. Off-notes were found in some over-fermented batches where Bacillus spp. was detected. No relation between cut-test results and organoleptic appreciation was seen.

It's Gettin' Hot in Here: Breeding Robust Yeast Starter Cultures for Cocoa Fermentation.

Cocoa beans have to undergo post-harvest fermentation and drying to develop the typical 'cocoa flavor' associated with chocolate. Yeasts play a pivotal role during the fermentation but are generally outcompeted early in the process. Meersman and colleagues describe an elegant breeding-based approach to generate robust yeast starter cultures for cocoa fermentation.

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process.

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.

Characterization of strains of Weissella fabalis sp. nov. and Fructobacillus tropaeoli from spontaneous cocoa bean fermentations.

Six facultatively anaerobic, non-motile lactic acid bacteria were isolated from spontaneous cocoa bean fermentations carried out in Brazil, Ecuador and Malaysia. Phylogenetic analysis revealed that one of these strains, designated M75(T), isolated from a Brazilian cocoa bean fermentation, had the highest 16S rRNA gene sequence similarity towards Weissella fabaria LMG 24289(T) (97.7%), W. ghanensis LMG 24286(T) (93.3%) and W. beninensis LMG 25373(T) (93.4%). The remaining lactic acid bacteria isolates, represented by strain M622, showed the highest 16S rRNA gene sequence similarity towards the type strain of Fructobacillus tropaeoli (99.9%), a recently described species isolated from a flower in South Africa. pheS gene sequence analysis indicated that the former strain represented a novel species, whereas pheS, rpoA and atpA gene sequence analysis indicated that the remaining five strains belonged to F. tropaeoli; these results were confirmed by DNA-DNA hybridization experiments towards their respective nearest phylogenetic neighbours. Additionally, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry proved successful for the identification of species of the genera Weissella and Fructobacillus and for the recognition of the novel species. We propose to classify strain M75(T) ( = LMG 26217(T)  = CCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.

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.

On-farm implementation of a starter culture for improved cocoa bean fermentation and its influence on the flavour of chocolates produced thereof.

Cocoa bean fermentations controlled by means of starter cultures were introduced on several farms in two different cocoa-producing regions (West Africa and Southeast Asia). Two starter culture mixtures were tested, namely one composed of Saccharomyces cerevisiae H5S5K23, Lactobacillus fermentum 222, and Acetobacter pasteurianus 386B (three heaps and one box), and another composed of L. fermentum 222 and A. pasteurianus 386B (seven heaps and one box). In all starter culture-added cocoa bean fermentation processes, the inoculated starter culture species were able to outgrow the natural contamination of the cocoa pulp-bean mass and they prevailed during cocoa bean fermentation. The application of both added starter cultures resulted in fermented dry cocoa beans that gave concomitant milk and dark chocolates with a reliable flavour, independent of cocoa-producing region or fermentation method. The addition of the lactic acid bacterium (LAB)/acetic acid bacterium (AAB) starter culture to the fermenting cocoa pulp-bean mass accelerated the cocoa bean fermentation process regarding citric acid conversion and lactic acid production through carbohydrate fermentation. For the production of a standard bulk chocolate, the addition of a yeast/LAB/AAB starter culture was necessary. This enabled an enhanced and consistent ethanol production by yeasts for a successful starter culture-added cocoa bean fermentation process. This study showed possibilities for the use of starter cultures in cocoa bean fermentation processing to achieve a reliably improved fermentation of cocoa pulp-bean mass that can consistently produce high-quality fermented dry cocoa beans and flavourful chocolates produced thereof.

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis.

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.

Species diversity, community dynamics, and metabolite kinetics of the microbiota associated with traditional ecuadorian spontaneous cocoa bean fermentations.

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.

Spontaneous organic cocoa bean box fermentations in Brazil are characterized by a restricted species diversity of lactic acid bacteria and acetic acid bacteria.

Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)(5)-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil.

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library.

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.

(GTG)5-PCR reference framework for acetic acid bacteria.

One hundred and fifty-eight strains of acetic acid bacteria (AAB) were subjected to (GTG)(5)-PCR fingerprinting to construct a reference framework for their rapid classification and identification. Most of them clustered according to their respective taxonomic designation; others had to be reclassified based on polyphasic data. This study shows the usefulness of the method to determine the taxonomic and phylogenetic relationships among AAB and to study the AAB diversity of complex ecosystems.