Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation

Detalhes bibliográficos
Autor(a) principal: Almeida, Otávio Guilherme Gonçalves de
Data de Publicação: 2022
Tipo de documento: Tese
Idioma: eng
Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
Texto Completo: https://www.teses.usp.br/teses/disponiveis/60/60141/tde-02032023-143959/
Resumo: In order to obtain processed chocolate with high standards it is mandatory the fermentation of cocoa seeds, which are the raw material for chocolate production. After cutting of cocoa fruits, the autochthonous microbiota infiltrates, and enters in contact with the seeds, contaminating them. As the seeds are surrounded by a mucilaginous pulp, which is rich in nutrients and presents high water activity, microorganisms find adequate conditions to proliferate and growth. Among the possible microorganisms related to cocoa fermentation, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) stand out, as these groups are related to a well-defined microbial succession along cocoa fermentation. This process allows the pulp drainage and stimulates the endogenous hydrolases to metabolize the stored substrates in the seeds. The microbial activity in consonance with the intrinsic metabolic activity of the seeds leads to the releasing of chocolate\'s flavour precursors. Many authors attribute to cross feeding the main role guiding and shaping microbial succession, since the yeasts depectinize the pulp releasing sugars that can be metabolized by LAB. The LAB convert these sugars into lactic acid, mannitol, and acetic acid. Mannitol is metabolized by AAB as carbon source, resulting in more acetic acid released by bacterial metabolism. Although the cross feeding explains microbial succession in cocoa fermentation, under the point of view of microbial ecology, literature on the microbial interactions in cocoa fermentation is scarce, specifically the occurrence and influence of quorum sensing (QS), for instance. It is known QS is a process of synchronization of gene expression intermediated by autoinducer molecules (AIs) in high cell densities conditions. As cocoa fermentation presents all the conditions for microbial enrichment and some studies have shown that bacteria related to QS may present competitive advantages in harsh environments, this research aimed initially to identify, by metagenomics, the QS related microbiota through the monitoring of the gene luxS along fermentation. Besides, bacterial genomes recovered from spontaneous fermentation were also investigated for the presence of this gene. The luxS gene is recognized as a universal QS marker because it characterizes the interspecific cell to cell communication. In the first Chapter of this work, it is presented a metagenomic analysis of an entire spontaneous cocoa fermentation sampled during 144h of fermentation. The data revealed the fungi were present along the entire fermentative process. Moreover, it was also demonstrated that reads related to the luxS gene are enriched as fermentation progresses, reaching a maximum at 72h of fermentation. It was also observed the genera Enterobacter, \"Lactobacillus\", Bacillus, and Pantoea were associated with luxS gene, which allowed to track the operational taxonomic units related to QS in cocoa fermentation. In the second Chapter, a comparative genomic analysis is presented. In that study, three strains of the species Lactiplantibacillus plantarum Lb2, Limosilactobacillus fermentum Lb1, and Pediococcus acidilactici P1 isolated from a spontaneous cocoa fermentation had their genomes sequenced and compared against all publicly available genomes of cognate species. The results shown the gene luxS is detected in all strains of this species and Lp. plantarum species in particular presents six luxS gene clusters, highlighting the high copy number of this gene in Lp. plantarum strains. For Lm. fermentum, there were only two gene clusters, and in P. acidilactici a single gene cluster. Phylogenetic analysis has shown the second gene cluster (named luxS_2) of Lm. fermentum was horizontally transferred by transduction from a Lp. plantarum strain to a Lm. fermentum strain, as both species present the same gene cluster and the flanking region of this cluster in Lm. fermentum was composed by IS30 transposases. In addition, luxS homologous sequences were determined by multiple alignment analysis to draw species- and clade-specific primers for rapid screening of strains to evaluate their potential related to QS and for qRT-PCR purposes. In the third Chapter, it is presented a comparative genomic analysis for AAB isolated from a spontaneous cocoa fermentation process, whose strains MRS7, GYC10, GYC12, GYC19, and GYC27 belonged to A. senegalensis species and were genotypically diverse, had their genomes sequenced and compared with public databases available for A. senegalensis strains at the time of publication. The study has shown A. senegalensis did not carry any luxS gene, but it presented genes related to intraspecific QS, such as acylhomoserine lactones (AHLs) and response regulators, as well as genes related to QS inhibition such as acylases and lactonases, corroborating the previous analyses presented in the first Chapter of this thesis. Besides, as AAB present potential to be applied in several industrial processes, such as cellulose production and vinegar fermentation, the metabolic pathways involved with bacterial adaptation to stressing conditions were investigated. The results shown these strains presented a good genetic repertoire related to bacterial adaptation to harsh environments, indicated by the presence of chaperones, alcohol dehydrogenases, and ABC proteins that confer tolerance to high concentrations of ethanol and high temperature. Additionally, the strains did not present pathogenic potential, as indicated by the absence of antibiotic resistance genes. In this way, the results suggested these strains of A. senegalensis isolated from cocoa fermentation could be applied also in other industrial processes. Finally, in the fourth Chapter the outcome of the Ph.D project is presented in a submitted manuscript showing the in situ detection of the luxS gene in lab scale fermentation. Thus, seven distinct fermentations were performed. The control fermentation (named F0) was conducted without duplicate, while the remaining fermentations, named F1, F2, and F3 were performed in duplicates, and inoculated with distinct combinations of cocktails containing yeasts, LAB, and/or AAB. The objective of that work was to compare the luxS gene expression along 96h of fermentation in each replicate and in the control (non inoculated fermentation - F0). In parallel, analyses for selective microbial enumeration, pH, and temperature monitoring, as well as metagenomics (16S rRNA and ITS), enzymatic activity dosage, and gas-chromatography coupled with mass spectrometry (GC-MS) for detection of volatile organic metabolites (VOCs) were performed to correlate QS potential with cocoa fermentation quality. The results revealed that even in laboratory conditions the microbial succession was observed for all fermentations, which was corroborated by microbial enumeration and metataxonomic analysis. However, no statistical difference was observed for presumptive microbial enumeration of the F0 and experimental fermentations (p > 0.05), which was also reinforced by the absence of significative difference for α-diversity metrics determined by metataxonomics among the fermentations. Additionally, no statistically significant differences of enzymatic activities were detected, and there were no microbial amplicon sequence variants correlated with enzymatic activities, suggesting the enzymatic activity was mainly shaped by endogenous hydrolases of the seeds and not by the microbial shifts per se. Regarding the luxS gene measurements for Lp. plantarum and Lm. fermentum species, it was observed the luxS genes of Lp. plantarum were active during all fermentation period, while Lm. fermentum luxS genes were detected during the first 72h of fermentation. The correlation between quality of fermentation and luxS gene expression evidenced a positive association of Lp. plantarum with undesirable sensorial attributes for fermented seeds. Based on the results of this work, it is possible to affirm that the luxS gene is active during cocoa fermentation, as presented in the first Chapter and there was progress in the understanding of the dynamics of cocoa fermentation, with demonstration of different enzymes acting along fermentation. In addition, the results of this research consolidate the hypothesis that Lp. plantarum is a possible protagonist in this type of fermentation, with great potential for expressing QS pathways. Future studies may apply the data collected throughout this study under field conditions.
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spelling Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentationProspecção metagenômica de bactérias relacionadas a quorum sensing durante a fermentação espontânea do cacauBactérias láticasCocoa fermentationFermentação de cacauLactic acid bacterialuxSluxSMetagenômicaMetagenomicsQuorum sensingQuorum sensingIn order to obtain processed chocolate with high standards it is mandatory the fermentation of cocoa seeds, which are the raw material for chocolate production. After cutting of cocoa fruits, the autochthonous microbiota infiltrates, and enters in contact with the seeds, contaminating them. As the seeds are surrounded by a mucilaginous pulp, which is rich in nutrients and presents high water activity, microorganisms find adequate conditions to proliferate and growth. Among the possible microorganisms related to cocoa fermentation, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) stand out, as these groups are related to a well-defined microbial succession along cocoa fermentation. This process allows the pulp drainage and stimulates the endogenous hydrolases to metabolize the stored substrates in the seeds. The microbial activity in consonance with the intrinsic metabolic activity of the seeds leads to the releasing of chocolate\'s flavour precursors. Many authors attribute to cross feeding the main role guiding and shaping microbial succession, since the yeasts depectinize the pulp releasing sugars that can be metabolized by LAB. The LAB convert these sugars into lactic acid, mannitol, and acetic acid. Mannitol is metabolized by AAB as carbon source, resulting in more acetic acid released by bacterial metabolism. Although the cross feeding explains microbial succession in cocoa fermentation, under the point of view of microbial ecology, literature on the microbial interactions in cocoa fermentation is scarce, specifically the occurrence and influence of quorum sensing (QS), for instance. It is known QS is a process of synchronization of gene expression intermediated by autoinducer molecules (AIs) in high cell densities conditions. As cocoa fermentation presents all the conditions for microbial enrichment and some studies have shown that bacteria related to QS may present competitive advantages in harsh environments, this research aimed initially to identify, by metagenomics, the QS related microbiota through the monitoring of the gene luxS along fermentation. Besides, bacterial genomes recovered from spontaneous fermentation were also investigated for the presence of this gene. The luxS gene is recognized as a universal QS marker because it characterizes the interspecific cell to cell communication. In the first Chapter of this work, it is presented a metagenomic analysis of an entire spontaneous cocoa fermentation sampled during 144h of fermentation. The data revealed the fungi were present along the entire fermentative process. Moreover, it was also demonstrated that reads related to the luxS gene are enriched as fermentation progresses, reaching a maximum at 72h of fermentation. It was also observed the genera Enterobacter, \"Lactobacillus\", Bacillus, and Pantoea were associated with luxS gene, which allowed to track the operational taxonomic units related to QS in cocoa fermentation. In the second Chapter, a comparative genomic analysis is presented. In that study, three strains of the species Lactiplantibacillus plantarum Lb2, Limosilactobacillus fermentum Lb1, and Pediococcus acidilactici P1 isolated from a spontaneous cocoa fermentation had their genomes sequenced and compared against all publicly available genomes of cognate species. The results shown the gene luxS is detected in all strains of this species and Lp. plantarum species in particular presents six luxS gene clusters, highlighting the high copy number of this gene in Lp. plantarum strains. For Lm. fermentum, there were only two gene clusters, and in P. acidilactici a single gene cluster. Phylogenetic analysis has shown the second gene cluster (named luxS_2) of Lm. fermentum was horizontally transferred by transduction from a Lp. plantarum strain to a Lm. fermentum strain, as both species present the same gene cluster and the flanking region of this cluster in Lm. fermentum was composed by IS30 transposases. In addition, luxS homologous sequences were determined by multiple alignment analysis to draw species- and clade-specific primers for rapid screening of strains to evaluate their potential related to QS and for qRT-PCR purposes. In the third Chapter, it is presented a comparative genomic analysis for AAB isolated from a spontaneous cocoa fermentation process, whose strains MRS7, GYC10, GYC12, GYC19, and GYC27 belonged to A. senegalensis species and were genotypically diverse, had their genomes sequenced and compared with public databases available for A. senegalensis strains at the time of publication. The study has shown A. senegalensis did not carry any luxS gene, but it presented genes related to intraspecific QS, such as acylhomoserine lactones (AHLs) and response regulators, as well as genes related to QS inhibition such as acylases and lactonases, corroborating the previous analyses presented in the first Chapter of this thesis. Besides, as AAB present potential to be applied in several industrial processes, such as cellulose production and vinegar fermentation, the metabolic pathways involved with bacterial adaptation to stressing conditions were investigated. The results shown these strains presented a good genetic repertoire related to bacterial adaptation to harsh environments, indicated by the presence of chaperones, alcohol dehydrogenases, and ABC proteins that confer tolerance to high concentrations of ethanol and high temperature. Additionally, the strains did not present pathogenic potential, as indicated by the absence of antibiotic resistance genes. In this way, the results suggested these strains of A. senegalensis isolated from cocoa fermentation could be applied also in other industrial processes. Finally, in the fourth Chapter the outcome of the Ph.D project is presented in a submitted manuscript showing the in situ detection of the luxS gene in lab scale fermentation. Thus, seven distinct fermentations were performed. The control fermentation (named F0) was conducted without duplicate, while the remaining fermentations, named F1, F2, and F3 were performed in duplicates, and inoculated with distinct combinations of cocktails containing yeasts, LAB, and/or AAB. The objective of that work was to compare the luxS gene expression along 96h of fermentation in each replicate and in the control (non inoculated fermentation - F0). In parallel, analyses for selective microbial enumeration, pH, and temperature monitoring, as well as metagenomics (16S rRNA and ITS), enzymatic activity dosage, and gas-chromatography coupled with mass spectrometry (GC-MS) for detection of volatile organic metabolites (VOCs) were performed to correlate QS potential with cocoa fermentation quality. The results revealed that even in laboratory conditions the microbial succession was observed for all fermentations, which was corroborated by microbial enumeration and metataxonomic analysis. However, no statistical difference was observed for presumptive microbial enumeration of the F0 and experimental fermentations (p > 0.05), which was also reinforced by the absence of significative difference for α-diversity metrics determined by metataxonomics among the fermentations. Additionally, no statistically significant differences of enzymatic activities were detected, and there were no microbial amplicon sequence variants correlated with enzymatic activities, suggesting the enzymatic activity was mainly shaped by endogenous hydrolases of the seeds and not by the microbial shifts per se. Regarding the luxS gene measurements for Lp. plantarum and Lm. fermentum species, it was observed the luxS genes of Lp. plantarum were active during all fermentation period, while Lm. fermentum luxS genes were detected during the first 72h of fermentation. The correlation between quality of fermentation and luxS gene expression evidenced a positive association of Lp. plantarum with undesirable sensorial attributes for fermented seeds. Based on the results of this work, it is possible to affirm that the luxS gene is active during cocoa fermentation, as presented in the first Chapter and there was progress in the understanding of the dynamics of cocoa fermentation, with demonstration of different enzymes acting along fermentation. In addition, the results of this research consolidate the hypothesis that Lp. plantarum is a possible protagonist in this type of fermentation, with great potential for expressing QS pathways. Future studies may apply the data collected throughout this study under field conditions.Para obtenção de chocolate de alta qualidade é necessário que a matéria-prima, isto é, as sementes de cacau, sejam fermentadas. Os microrganismos infiltram-se após o corte dos frutos de cacau e contaminam as sementes estéreis. Como a polpa mucilaginosa é rica em nutrientes e apresenta alta atividade de água, os microrganismos encontram condições propícias para sobrevivência e multiplicação. Destacam-se as leveduras, bactérias láticas (BAL) e bactérias acéticas (BAA), que nesta ordem, realizam uma sucessão microbiana bem definida ao longo do processo fermentativo. Esse processo permite a drenagem da polpa mucilaginosa que recobre as sementes e, ao mesmo tempo, estimula as hidrolases endógenas a metabolizar os substratos de reserva armazenados nas sementes. A atividade microbiana somada à atividade metabólica interna das sementes resulta na formação de precursores característicos do sabor e aroma do chocolate. Muitos autores consideram a alimentação cruzada como o fator determinante para a sucessão microbiana, pois as leveduras despectinizam a polpa liberando açúcares que podem ser metabolizados pelas BAL, em seguida as BAL convertem os açúcares em etanol, manitol e ácido acético. O manitol é metabolizado pelas BAA como fonte de carbono, resultando em ácido acético. Embora a alimentação cruzada explique a sucessão microbiana, do ponto de vista de ecologia de comunidades, pouco se conhece sobre as interações que ocorrem ao longo do processo fermentativo, como o quorum sensing (QS), por exemplo. O QS é um processo de sincronização da expressão gênica por meio da liberação de moléculas autoindutoras em altas densidades celulares. Dessa maneira, visto que a fermentação de cacau apresenta condições para que os microrganismos atinjam altas populações e que estudos têm indicado que bactérias relacionadas ao QS podem apresentar vantagem competitiva e adaptativa a ambientes estressantes, este trabalho teve por objetivo, inicialmente, identificar por meio da metagenômica, a microbiota relacionada com QS por meio da identificação in situ do gene luxS ao longo da fermentação e em genomas de bactérias isoladas de fermentação de cacau. O gene luxS é tido como um marcador universal de QS, pois caracteriza a comunicação interespecífica célula a célula. No primeiro Capítulo deste trabalho é apresentada a análise metagenômica de uma fermentação espontânea de cacau amostrada por até 144h. Os dados obtidos revelaram que os fungos estavam presentes ao longo de todo o processo fermentativo. Além disso, demonstram também que reads relacionados ao gene luxS são enriquecidos à medida que aumenta o tempo de fermentação, atingindo um pico máximo de detecção no tempo de 72h de fermentação. Foi também observado que os gêneros Enterobacter, \"Lactobacillus\", Bacillus e Pantoea foram associados aos genes luxS, evidenciando as unidades taxonômicas operacionais relacionadas a esse gene. No segundo Capítulo é apresentado um estudo genômico comparativo no qual três cepas de Lactiplantibacillus plantarum Lb2, Limosilactobacillus fermentum Lb1 e Pediococcus acidilactici P1 isoladas de fermentação espontânea de cacau tiveram seus genomas sequenciados e comparados contra todos os genomas públicos pertencentes a essas espécies. Os resultados mostraram que o gene luxS estava presente em todas as cepas dessas espécies e que Lp. plantarum apresentava seis clusters gênicos para luxS, evidenciando um alto número de cópias. Para Lm. fermentum foram observados apenas dois clusters gênicos e em P. acidilactici um cluster gênico. Análises filogenéticas sugeriram que o segundo cluster do gene luxS (denominado luxS_2) foi transferido horizontalmente via transdução de Lp. plantarum para Lm. fermentum, visto que ambas apresentaram o mesmo cluster e a região flanqueadora desse cluster em Lm. fermentum era composta de transposases IS30. Adicionalmente, após a investigação da presença desse gene nessas espécies, primers espécie- e cladoespecíficos foram desenhados para screening rápido de cepas para avaliação do potencial relacionado ao QS e para aplicação em estudos envolvendo qRT-PCR. No terceiro Capítulo é apresentado um estudo comparativo genômico para as BAA isoladas de processo fermentativo espontâneo de cacau, cujas cepas de Acetobacter senegalensis MRS7, GYC10, GYC12, GYC19 e GYC27 (com genótipos distintos), tiveram seus genomas sequenciados e comparados com genomas publicamente disponíveis de A. senegalensis. O estudo demonstrou que em A. senegalensis não havia genes luxS, mas sim genes relacionados ao QS intraespecífico como acil homoserina lactonas (AHLs) e response regulators, bem como genes envolvidos com a inibição de QS, codificando para acilases e lactonases, o que corroborou as predições realizadas no trabalho apresentado no primeiro capítulo desta tese. Além disso, visto que as BAA apresentam potencial de aplicação em outros processos industriais, como a produção de celulose e fermentação de vinagre, vias metabólicas envolvidas na adaptação bacteriana a processos fermentativos sob condições de estresse foram também investigadas. Os dados revelaram que essas cepas apresentam um bom potencial de adaptação em ambientes estressantes, com capacidade de sintetizar chaperonas, álcool desidrogenases e proteínas ABC, que conferem tolerância a altas concentrações de etanol e a altas temperaturas. Adicionalmente, as cepas de AAB não apresentaram potencial patogênico e nem genes de resistência a antibióticos. Dessa maneira, os resultados obtidos sugerem que as cepas de A. senegalensis isoladas da fermentação espontânea de cacau podem ser aplicadas também em outros processos industriais. Por fim, no quarto Capítulo há o desfecho da tese, com a apresentação de um artigo submetido para publicação, o qual trata da detecção in situ do gene luxS em fermentações conduzidas em escala de laboratório. Dessa maneira, seis fermentações foram realizadas em duplicata, sendo denominadas F1, F2 e F3, as quais foram inoculadas com diferentes combinações de coquetéis contendo leveduras, BAL e/ou BAA, com o objetivo de comparar a expressão do gene luxS ao longo de 96h de fermentação. Para comparação, foi preparado um controle não inoculado (sem inoculação de microrganismos, sem replicata - denominada F0). Para cada uma das condições estudadas, foram realizadas análises relativas à enumeração de microrganismos, medida de pH e da temperatura de fermentação, bem como análises de metataxonômica (16S rRNA e ITS), mensuração de atividade enzimática e detecção de metabolites voláteis (VOCs) por cromatografia gasosa acoplada a espectrometria de massas (GC-MS). Os resultados foram analisados para avaliar o potencial funcional microbiano relativo a QS e à qualidade do cacau fermentado. Os resultados revelaram que ocorreu uma sucessão microbiana típica da fermentação de cacau, que foi corroborado pelas análises clássicas de enumeração de microrganismos e de metataxonômica. No entanto, estatisticamente não houve diferenças significativas nas populações microbianas enumeradas na fermentação F0 e nas demais (p > 0,05), o que também foi reforçado pela ausência de diferença significativa entre os valores de α-diversidade para as diferentes fermentações, determinada por análises metataxonômica. Não houve correlação significativamente estatística entre atividade enzimática e alterações composicionais da microbiota total, indicando que, provavelmente, a principal atividade enzimática era decorrente das próprias hidrolases endógenas das sementes e não da microbiota per se. Em relação à mensuração do gene luxS, para as espécies Lp. plantarum houve atividade ao longo da fermentação, enquanto que a expressão do gene luxS para Lm. fermentum foi detectada apenas nas primeiras 72h. A correlação entre a qualidade da fermentação expressa por meio da quantidade de VOCs detectados e o padrão de expressão dos genes luxS evidenciou uma associação positiva entre a expressão desse gene por Lp. plantarum e características sensoriais indesejáveis para as sementes fermentadas. Considerando os resultados deste trabalho, é possível afirmar que o gene luxS foi ativo ao longo da fermentação de cacau, conforme hipótese do primeiro Capítulo, e houve avanço na compreensão da dinâmica da fermentação de cacau, com demonstração da participação de diferentes enzimas ao longo do processo. Além disso, os resultados dessa pesquisa indicam que Lp. plantarum é um protagonista neste tipo de fermentação, com grande potencial de expressão de vias de QS. Futuros estudos poderão elucidar em condições de campo os dados levantados ao longo deste trabalho.Biblioteca Digitais de Teses e Dissertações da USPMartinis, Elaine Cristina Pereira deAlmeida, Otávio Guilherme Gonçalves de2022-08-31info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/60/60141/tde-02032023-143959/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPReter o conteúdo por motivos de patente, publicação e/ou direitos autoriais.info:eu-repo/semantics/openAccesseng2023-03-20T13:23:33Zoai:teses.usp.br:tde-02032023-143959Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212023-03-20T13:23:33Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false
dc.title.none.fl_str_mv Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
Prospecção metagenômica de bactérias relacionadas a quorum sensing durante a fermentação espontânea do cacau
title Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
spellingShingle Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
Almeida, Otávio Guilherme Gonçalves de
Bactérias láticas
Cocoa fermentation
Fermentação de cacau
Lactic acid bacteria
luxS
luxS
Metagenômica
Metagenomics
Quorum sensing
Quorum sensing
title_short Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
title_full Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
title_fullStr Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
title_full_unstemmed Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
title_sort Metagenomic prospection of quorum sensing related bacteria during spontaneous cocoa beans fermentation
author Almeida, Otávio Guilherme Gonçalves de
author_facet Almeida, Otávio Guilherme Gonçalves de
author_role author
dc.contributor.none.fl_str_mv Martinis, Elaine Cristina Pereira de
dc.contributor.author.fl_str_mv Almeida, Otávio Guilherme Gonçalves de
dc.subject.por.fl_str_mv Bactérias láticas
Cocoa fermentation
Fermentação de cacau
Lactic acid bacteria
luxS
luxS
Metagenômica
Metagenomics
Quorum sensing
Quorum sensing
topic Bactérias láticas
Cocoa fermentation
Fermentação de cacau
Lactic acid bacteria
luxS
luxS
Metagenômica
Metagenomics
Quorum sensing
Quorum sensing
description In order to obtain processed chocolate with high standards it is mandatory the fermentation of cocoa seeds, which are the raw material for chocolate production. After cutting of cocoa fruits, the autochthonous microbiota infiltrates, and enters in contact with the seeds, contaminating them. As the seeds are surrounded by a mucilaginous pulp, which is rich in nutrients and presents high water activity, microorganisms find adequate conditions to proliferate and growth. Among the possible microorganisms related to cocoa fermentation, yeast, lactic acid bacteria (LAB), and acetic acid bacteria (AAB) stand out, as these groups are related to a well-defined microbial succession along cocoa fermentation. This process allows the pulp drainage and stimulates the endogenous hydrolases to metabolize the stored substrates in the seeds. The microbial activity in consonance with the intrinsic metabolic activity of the seeds leads to the releasing of chocolate\'s flavour precursors. Many authors attribute to cross feeding the main role guiding and shaping microbial succession, since the yeasts depectinize the pulp releasing sugars that can be metabolized by LAB. The LAB convert these sugars into lactic acid, mannitol, and acetic acid. Mannitol is metabolized by AAB as carbon source, resulting in more acetic acid released by bacterial metabolism. Although the cross feeding explains microbial succession in cocoa fermentation, under the point of view of microbial ecology, literature on the microbial interactions in cocoa fermentation is scarce, specifically the occurrence and influence of quorum sensing (QS), for instance. It is known QS is a process of synchronization of gene expression intermediated by autoinducer molecules (AIs) in high cell densities conditions. As cocoa fermentation presents all the conditions for microbial enrichment and some studies have shown that bacteria related to QS may present competitive advantages in harsh environments, this research aimed initially to identify, by metagenomics, the QS related microbiota through the monitoring of the gene luxS along fermentation. Besides, bacterial genomes recovered from spontaneous fermentation were also investigated for the presence of this gene. The luxS gene is recognized as a universal QS marker because it characterizes the interspecific cell to cell communication. In the first Chapter of this work, it is presented a metagenomic analysis of an entire spontaneous cocoa fermentation sampled during 144h of fermentation. The data revealed the fungi were present along the entire fermentative process. Moreover, it was also demonstrated that reads related to the luxS gene are enriched as fermentation progresses, reaching a maximum at 72h of fermentation. It was also observed the genera Enterobacter, \"Lactobacillus\", Bacillus, and Pantoea were associated with luxS gene, which allowed to track the operational taxonomic units related to QS in cocoa fermentation. In the second Chapter, a comparative genomic analysis is presented. In that study, three strains of the species Lactiplantibacillus plantarum Lb2, Limosilactobacillus fermentum Lb1, and Pediococcus acidilactici P1 isolated from a spontaneous cocoa fermentation had their genomes sequenced and compared against all publicly available genomes of cognate species. The results shown the gene luxS is detected in all strains of this species and Lp. plantarum species in particular presents six luxS gene clusters, highlighting the high copy number of this gene in Lp. plantarum strains. For Lm. fermentum, there were only two gene clusters, and in P. acidilactici a single gene cluster. Phylogenetic analysis has shown the second gene cluster (named luxS_2) of Lm. fermentum was horizontally transferred by transduction from a Lp. plantarum strain to a Lm. fermentum strain, as both species present the same gene cluster and the flanking region of this cluster in Lm. fermentum was composed by IS30 transposases. In addition, luxS homologous sequences were determined by multiple alignment analysis to draw species- and clade-specific primers for rapid screening of strains to evaluate their potential related to QS and for qRT-PCR purposes. In the third Chapter, it is presented a comparative genomic analysis for AAB isolated from a spontaneous cocoa fermentation process, whose strains MRS7, GYC10, GYC12, GYC19, and GYC27 belonged to A. senegalensis species and were genotypically diverse, had their genomes sequenced and compared with public databases available for A. senegalensis strains at the time of publication. The study has shown A. senegalensis did not carry any luxS gene, but it presented genes related to intraspecific QS, such as acylhomoserine lactones (AHLs) and response regulators, as well as genes related to QS inhibition such as acylases and lactonases, corroborating the previous analyses presented in the first Chapter of this thesis. Besides, as AAB present potential to be applied in several industrial processes, such as cellulose production and vinegar fermentation, the metabolic pathways involved with bacterial adaptation to stressing conditions were investigated. The results shown these strains presented a good genetic repertoire related to bacterial adaptation to harsh environments, indicated by the presence of chaperones, alcohol dehydrogenases, and ABC proteins that confer tolerance to high concentrations of ethanol and high temperature. Additionally, the strains did not present pathogenic potential, as indicated by the absence of antibiotic resistance genes. In this way, the results suggested these strains of A. senegalensis isolated from cocoa fermentation could be applied also in other industrial processes. Finally, in the fourth Chapter the outcome of the Ph.D project is presented in a submitted manuscript showing the in situ detection of the luxS gene in lab scale fermentation. Thus, seven distinct fermentations were performed. The control fermentation (named F0) was conducted without duplicate, while the remaining fermentations, named F1, F2, and F3 were performed in duplicates, and inoculated with distinct combinations of cocktails containing yeasts, LAB, and/or AAB. The objective of that work was to compare the luxS gene expression along 96h of fermentation in each replicate and in the control (non inoculated fermentation - F0). In parallel, analyses for selective microbial enumeration, pH, and temperature monitoring, as well as metagenomics (16S rRNA and ITS), enzymatic activity dosage, and gas-chromatography coupled with mass spectrometry (GC-MS) for detection of volatile organic metabolites (VOCs) were performed to correlate QS potential with cocoa fermentation quality. The results revealed that even in laboratory conditions the microbial succession was observed for all fermentations, which was corroborated by microbial enumeration and metataxonomic analysis. However, no statistical difference was observed for presumptive microbial enumeration of the F0 and experimental fermentations (p > 0.05), which was also reinforced by the absence of significative difference for α-diversity metrics determined by metataxonomics among the fermentations. Additionally, no statistically significant differences of enzymatic activities were detected, and there were no microbial amplicon sequence variants correlated with enzymatic activities, suggesting the enzymatic activity was mainly shaped by endogenous hydrolases of the seeds and not by the microbial shifts per se. Regarding the luxS gene measurements for Lp. plantarum and Lm. fermentum species, it was observed the luxS genes of Lp. plantarum were active during all fermentation period, while Lm. fermentum luxS genes were detected during the first 72h of fermentation. The correlation between quality of fermentation and luxS gene expression evidenced a positive association of Lp. plantarum with undesirable sensorial attributes for fermented seeds. Based on the results of this work, it is possible to affirm that the luxS gene is active during cocoa fermentation, as presented in the first Chapter and there was progress in the understanding of the dynamics of cocoa fermentation, with demonstration of different enzymes acting along fermentation. In addition, the results of this research consolidate the hypothesis that Lp. plantarum is a possible protagonist in this type of fermentation, with great potential for expressing QS pathways. Future studies may apply the data collected throughout this study under field conditions.
publishDate 2022
dc.date.none.fl_str_mv 2022-08-31
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
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dc.identifier.uri.fl_str_mv https://www.teses.usp.br/teses/disponiveis/60/60141/tde-02032023-143959/
url https://www.teses.usp.br/teses/disponiveis/60/60141/tde-02032023-143959/
dc.language.iso.fl_str_mv eng
language eng
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dc.publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
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reponame:Biblioteca Digital de Teses e Dissertações da USP
instname:Universidade de São Paulo (USP)
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instname_str Universidade de São Paulo (USP)
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reponame_str Biblioteca Digital de Teses e Dissertações da USP
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repository.name.fl_str_mv Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)
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