RESUMO
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 2â¯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 48-72â¯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.
Assuntos
Bactérias/metabolismo , Cacau/microbiologia , Chocolate/microbiologia , Fermentação/fisiologia , Fungos/metabolismo , Ácido Acético/metabolismo , Acetobacter/metabolismo , Bactérias/isolamento & purificação , Reatores Biológicos/microbiologia , Enterobacteriaceae/metabolismo , Fungos/isolamento & purificação , Gluconobacter/metabolismo , Hanseniaspora/metabolismo , Limosilactobacillus fermentum/metabolismo , Pichia/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
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) (â=âLMG 26217(T) â=âCCUG 61472(T)) as the type strain of the novel species Weissella fabalis sp. nov.
Assuntos
Cacau/microbiologia , Microbiologia de Alimentos , Leuconostocaceae/classificação , Filogenia , Weissella/classificação , Composição de Bases , Brasil , DNA Bacteriano/genética , Equador , Fermentação , Genes Bacterianos , Leuconostocaceae/genética , Leuconostocaceae/isolamento & purificação , Malásia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Peptidoglicano/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Weissella/genética , Weissella/isolamento & purificaçãoRESUMO
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.
Assuntos
Biodiversidade , Cacau/metabolismo , Cacau/microbiologia , Microbiologia de Alimentos , Leveduras/classificação , Leveduras/genética , Brasil , Análise por Conglomerados , Côte d'Ivoire , Primers do DNA/genética , DNA Fúngico/genética , DNA Ribossômico/genética , Eletroforese em Gel de Gradiente Desnaturante , Equador , Fermentação , Variação Genética , Genótipo , Malásia , Reação em Cadeia da Polimerase , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Bactérias/metabolismo , Cacau/metabolismo , Cacau/microbiologia , Fermentação , Ácido Acético/metabolismo , Bactérias/isolamento & purificação , Brasil , Eletroforese em Gel de Gradiente Desnaturante , Ácido Láctico/metabolismoRESUMO
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.
Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodiversidade , Cacau/microbiologia , Fermentação , Bactérias/classificação , Bactérias/genética , Brasil , Côte d'Ivoire , Dados de Sequência Molecular , FilogeniaRESUMO
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.