Dissertations / Theses on the topic 'Fermentation du cacao'

La fermentation du cacao qui conditionne la qualité aromatique du chocolat, commence par une étape de fermentation alcoolique suivie d’une fermentation acétique, conduites par des souches sauvages de levures et de bactéries acétiques. Réalisée localement à petite échelle, en absence de tout contrôle, elle conduit à des produits finaux de qualité variable, allant du meilleur au pire. Le travail présenté ici fait partie d’un projet qui vise à maitriser la qualité aromatique du chocolat par l’utilisation d’un starter microbien associée à un mode de conduite de la fermentation. Un objectif spécifique est le développement d’un modèle de la fermentation du cacao pour disposer d’un outil permettant le choix d‘un starter et des conditions de sa mise en œuvre. La stratégie de modélisation utilisée est modulaire, pas à pas, avec le souci de construire un modèle simple capable de décrire l’essentiel des processus et des réactions qui occurrent lors de la fermentation. La première étape a été la sélection de deux souches de levures L35 (S. cerevisiae) et L36 (P. kudriavzevii) et d’une souche de bactérie acétique B17 (A. ghanensis) lors de fermentations industrielles ayant conduit à du chocolat de bonne qualité aromatique. Le modèle de la fermentation alcoolique a été développé en utilisant une souche sélectionnée LM (S. cerevisiae) puis a été adapté pour les souches L35 et L36. Le modèle de la fermentation acétique a été développé en utilisant la souche B17. Le modèle global résulte de l’intégration de ces deux sous modèles et d’un modèle permettant de décrire l’élévation de la température des fèves du fait de la production de chaleur lors de la fermentation. Le modèle global permet de décrire assez bien l’ensemble des phénomènes ayant lieu lors de fermentation du cacao : évolution des populations microbiennes, de la consommation/production de glucose, éthanol et acide acétique, et l’évolution de la température, en fonction des conditions initiales (température, concentration en sucres de la pulpe de cacao et niveau d’inoculation du starter). Les résultats de simulations ont permis d’identifier les phénomènes et paramètres clé pour le bon déroulement de la fermentation du cacao. Concernant la fermentation alcoolique, le modèle montre qu’elle est rapide, soit une journée, qu’elle s’achève généralement par l’épuisement des sucres et que la réussite de l’inoculation nécessitera de maitriser la qualité microbiologique de la pulpe de cacao qui dépend du délai entre l’écabossage et l’inoculation. La fermentation acétique avec la souche B17 est conditionnée par la température initiale et l’évolution de la concentration en éthanol qui peut paradoxalement ralentir le démarrage de cette fermentation
The fermentation of cocoa, which drives the aromatic quality of chocolate, begins with an alcoholic fermentation stage followed by an acetic fermentation, conducted by wild strains of yeasts and acetic bacteria. Realized locally on a small scale, in the absence of any control, it leads to products of variable quality, ranging from the best to the worst. The work presented here is part of a project aimed to control the aromatic quality of chocolate using a microbial starter in controlled conditions of fermentation. A specific objective is the development of a model of cocoa fermentation to have a tool allowing the choice of a starter and the conditions of its implementation. The used modeling strategy is modular, step by step, with the aim of building a simple model able of describing the major processes and reactions that occur during fermentation. The first step was the selection of two strains of yeasts, L35 (S. cerevisiae) and L36 (P. kudriavzevii), and a strain of acetic bacteria, B17 (A. ghanensis), during industrial fermentations that led to chocolate of good aromatic quality. The model of alcoholic fermentation was developed using a selected strain LM (S. cerevisiae) and was adapted for strains L35 and L36. The model of acetic fermentation was developed using the B17 strain. The overall model results from the integration of these two sub-models and a model that describes the rise in temperature of the beans due to the production of heat during fermentation. The global model makes it possible to describe quite well all the phenomena that occur during cocoa fermentation: evolution of microbial populations, consumption / production of glucose, ethanol and acetic acid, and the evolution of temperature, depending on initial conditions (temperature, sugar concentration of the cocoa pulp and level of inoculation of the starter). The results of simulations made it possible to identify the key phenomena and parameters for the smooth running of cocoa fermentation. Regarding the yeast fermentation, the model shows that it is fast, one day is sufficient, and usually ends with the exhaustion of sugars and that the success of the inoculation will require control the microbiological quality of cocoa pulp conditioned by the delay between pods opening and the inoculation. The acetic fermentation with B17 strain is conditioned by the initial temperature and the evolution of the ethanol concentration, which can paradoxically slow down the start of this fermentation

La consommation régulière du chocolat est associée à des effets bénéfiques sur la santé, en particulier dans les pathologies associées à un syndrome inflammatoire chronique. Cette inflammation met en jeu le système immunitaire et les polyphénols du cacao semblent jouer un rôle sur ces effets, via la voie de NF-κb. Les procédés de transformation du cacao et notamment la fermentation dégradent les polyphénols contenus dans les fèves. En effet, la fermentation à elle seule, diminue de 90% la teneur des polyphénols initialement présents. Il est donc important de connaitre son impact sur la teneur et / ou la composition des polyphénols afin ensuite de pouvoir les associer aux activités antiinflammatoires et immunomodulatrices du cacao. Un lot de fèves de cacao de variété Forastero a été divisés en deux lots, le premier a été fermenté et l’autre non. Après délipidation de la poudre de cacao puis extraction des polyphénols, la teneur en polyphénols totaux des extraits a été déterminée par la méthode de Folin-Ciocalteu. Les composés chimiques présents dans les extraits de cacao ont été identifiés par UPLC-MS/UV. Les flavanols majoritaires identifiés ont été quantifiés par LC/MSMS. Les activités antioxydantes du cacao ont été évaluées avec le test DPPH et ORAC. Pour déterminer les activités immunomodulatrices, les teneurs en tumor necrosis factor-alpha (TNF-α) et oxyde nitrique (NO) ont été déterminées. La viabilité cellulaire a été faite afin de d’évaluer la cytotoxicité des extraits de cacao sur les cellules J774A.1. Cette étude a montré que la fermentation a diminué la teneur en polyphénols et établi une corrélation entre cette teneur en composés phénoliques et les activités antioxydantes des extraits de cacao. Les extraits de cacao fermenté ou non ont stimulé la production de TNF-α, cependant l’étude sur les activités immunomodulatrices mérite d’être approfondie car le cacao fermenté ou non n’a eu aucun effet sur le NO produit par les cellules J774A.1. Par ailleurs, le cacao fermenté ou non n’a pas eu d’effet toxique sur les cellules J 774A.1. La fermentation a influé sur les activités immunomodulatrices du cacao selon le type de marqueurs en diminuant la production de TNF-α
Regular consumption of chocolate is associated with beneficial effects on health, particularly in pathologies associated with a chronic inflammatory syndrome. This inflammation involves the immune system and cocoa polyphenols appear to play a role in these effects via the NF-κb pathway. Cocoa processing processes and especially fermentation degrade the polyphenols contained in the beans. In fact, the fermentation alone reduces the content of the polyphenols initially present by 90%. It is therefore important to know its impact on the content and / or the composition of polyphenols in order to be able to associate them with the antioxydants and immunomodulatory activities of cocoa. A batch of Forastero variety cocoa beans was divided into two batches, the first fermented and the other not. After fat and cocoa polyphenols extraction, the total polyphenol content of the extracts was determined by the Folin-Ciocalteu method. The chemical compounds present in the cocoa extracts have been identified by UPLC-MS / UV. The majority flavanols identified were quantified by LC / MSMS. The antioxidant activities of cocoa were evaluated with DPPH and ORAC test. To determine the immunomodulatory activities, the levels of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) were determined. Cell viability was done to evaluate the cytotoxicity of cocoa extracts on J774A.1 cells. This study showed that fermentation decreased the polyphenol content and correlated this phenol content with the antioxidant activities of the cocoa extracts. The extracts of fermented cocoa or not stimulated the production of TNF-α, however the study on the immunomodulatory activities deserves to be deepened because the fermented cocoa or not had no effect on the NO produced by the J774A cells. 1. In addition, the fermented or unfermented cocoa did not have any toxic effect on J 774A.1 cells. Fermentation influenced the immunomodulatory activities of cocoa according to the type of markers by decreasing the production of TNF-α

A qualidade das matérias-primas do chocolate depende de uma fermentação eficiente das sementes de cacau, já que é nesta etapa que ocorrem transformações bioquímicas como a liberação de aminoácidos e açúcares redutores que durante a torração irão formar os precursores do sabor do chocolate. O processo fermentativo ocorre espontaneamente e a polpa que envolve os grãos, rica em carboidratos, é o substrato para o desenvolvimento dos microrganismos fermentativos, e a atividade destes resulta na remoção da polpa com produção do mel do cacau, contribuindo para a formação dos aromas e sabores. Existem outros métodos para a retirada da polpa, estando patenteados métodos mecânicos e por ação de enzimas pectinolíticas. Contudo, a utilização dos processos mecânicos existentes não é eficiente e o uso de enzimas ainda não é economicamente viável em larga escala. A melhoria da fermentação vem sendo objeto de pesquisa e se considera que a inoculação de leveduras produtoras de pectinases durante a fermentação poderia contribuir para a eficiência do processo, com obtenção de um produto mais uniforme. Com esse objetivo, nesse trabalho, leveduras de diferentes espécies com potencial produção de enzimas pectinolíticas foram selecionadas e posteriormente avaliadas durante a fermentação e na qualidade final das amêndoas. Os dados obtidos revelaram que leveduras do gênero Kluyveromyces se mostraram as mais eficientes, mas as espécies Candida utilis e Saccharomyces cerevisiae também mostraram bons resultados, enquanto que as fermentações sem a inoculação de leveduras apresentaram baixa eficiência na produção do mel de cacau. A fermentação com K. marxianus (MMIII-41), apresentou elevação de temperatura até 34°C com queda do pH em 2,9 e coloração marrom em suas amêndoas, indicando boa qualidade, enquanto que as fermentações naturais apresentaram valores de temperatura e pH de 29°C e 3,5 e coloração amarelada em suas amêndoas devido à polpa e fibras vegetais aderidas. Durante a prova de corte, a espécie S. cerevisiae mostrou a maior quantidade de amêndoas com coloração marrom, enquanto que a espécie K. marxianus que apresentou o melhor desempenho fermentativo com degradação da fração vicilina evidenciada em gel SDS-PAGE, mostrou somente 14% de amêndoas marrons. É possível concluir que a inoculação de leveduras com produção de enzimas pectinolíticas extracelulares e o revolvimento das sementes durante a fermentação, contribui para uma maior rapidez da fermentação e melhor qualidade das amêndoas. O volume do material fermentado não permitiu alcançar as temperaturas obtidas na maior escala, mesmo assim, de acordo com os resultados obtidos nas avaliações de atividade enzimática, volumes de mel drenados, aspecto externo das sementes após 192 horas, prova de corte e degradação de vicilinas, dentre as espécies pré-selecionadas para atividade pectinolítica, as leveduras K. marxianus, Kluyveromyces fragilis, C. utilis e S. cerevisiae, revelaram, nas condições de fermentação estudadas, ter condições de trazer benefícios a qualidade das amêndoas.
The quality of the raw material of chocolate depends on an efficient fermentation of cocoa beans, as it is at this stage that biochemical transformations occur as the release of amino acids and reducing sugars that during roasting will form the precursors of chocolate flavor. The fermentation process occurs spontaneously and the pulp surrounding the seeds, rich in carbohydrates, is the substrate for the development of fermentative microorganisms, and its activity results in the removal of the pulp and honey production of cocoa, contributing for the formation of aromas and flavors. There are other methods to remove the pulp, and mechanical methods being patented by the action of pectic enzymes. However, the use of existing mechanical processes is not efficient and the use of enzymes is not yet economically viable on a large scale. Improving the fermentation has been the subject of research and considered that the inoculation of yeasts producing pectinase during fermentation could contribute to the efficiency of the process, obtaining a more uniform product. With that goal in this paper, yeasts of different species with potential production of pectic enzymes were selected and then evaluated during the fermentation and the final quality of the beans. The data obtained showed that yeasts Kluyveromyces have shown the most efficient, but the species of Candida utilis and Saccharomyces cerevisiae also showed positive results, whereas the fermentation without yeast inoculation showed low efficiency in the production of honey cocoa. The fermentation with K. marxianus (MMIII-41), showed increase of temperature to 34 ° C with a pH drop to 2.9 in their brown and almonds, indicating good quality, while natural fermentations showed values of pH and temperature of 29°C and 3.5 and yellow coloring in their beans due to pulp and vegetable fibers bonded. During the test cutting, the species S. cerevisiae showed the greatest amount of almonds and brown, while the species K. marxianus which showed the best fermentation performance degradation with the vicilin fraction evidenced by SDS-PAGE, showed only 14% of brown almonds. It was concluded that inoculation with yeast production of extracellular pectic enzymes and revolving seeds during fermentation, contributing to a faster fermentation and a better quality of almonds. The volume of the fermented material is not allowed to reach temperatures obtained on the largest scale yet, according to the results obtained in the enzymatic activity, volumes of honey drained, the external appearance of the seeds after 192 hours, proof of cutting and degradation of vicilin among the species pre-selected for pectinolytic yeasts K.marxianus, Kluyveromyces fragilis, C. utilis and S. cerevisiae, revealed in fermentation conditions studied, be able to benefit the quality of the beans.

Le developpement de la fraction volatile du cacao resulte des effets combines de la variete, du traitement post-recolte (fermentation et sechage) et de la torrefaction. Les resultats analytiques ainsi que des etudes de correlation par analyse en composantes principales (acp) ont permis une approche de la part respective de ces effets. La caracterisation de la fraction volatile d'un lot de cacao sanchez au cours de la fermentation et de 9 clones et hybrides non fermentes et fermentes a ete determinee avant et apres torrefaction. Apres extraction par entrainement a la vapeur, les fractions volatiles des echantillons sont analysees par chromatographie et les composes volatils sont caracterises et identifies par couplage gc/ms. L'arome cacao est compose d'une fraction constitutive, d'une fraction fermentaire et d'une fraction additionnelle d'origine thermique. La fraction constitutive, (principalement composee d'alcools et d'esters) est peu importante, celle d'origine fermentaire est par contre determinante dans la composition de l'arome final. Elle est principalement constituee de composes d'origine microbiologique et/ou biochimique (alcools, esters, acides) qui s'accumulent tres rapidement et de composes d'origine thermique (aldehydes de strecker, pyrazines, furannes) formes essentiellement en fin de fermentation. La torrefaction conduit a la formation d'un nombre limite de composes nouveaux (pyrazines et pyrroles) mais surtout a l'augmentation de la teneur des composes volatils prealablement formes au cours de la fermentation. Une etude en systeme modele a permis de caracteriser l'influence des parametres de traitement (temperature, duree, teneur en eau) et le role (nature et teneur) des precurseurs dans le developpement de l'arome a la torrefaction. L'utilisation de systemes modeles a permis d'interpreter la formation de nombreux composes volatils mais n'a cependant pas permis de modeliser correctement la formation de l'arome cacao d'origine thermique

Dans cette thèse , nous avons étudié les polyphénols du cacao dans le but de valoriser le contenu polyphénolique des graines de cacao et d'évaluer la capacité de ces polyphénols à autoriser l'authentification des fèves en fonction de leur origine géographique et de leur état de fermentation. Un extrait polyphénolique total des graines de cacao a d'abord été préparé avec des techniques et des solvants utilisables industriellement. Puis, une méthode de fractionnement par CPC nous a permis d'isoler l'épicatéchine avec une pureté de 96 %. L'hémisynthèse de métabolites de l'épicatéchine, retrouvés in vivo, a été réalisée. Enfin, ces métabolites ont été testés sur un système de prolifération de cellules cancéreuses de lignée leucémique (K 562). Les extraits polyphénoliques totaux de fèvees de cacao ont subi une analyse par RMN à deux dimensions. Puis les résultats obtenus ont été traités par une analyse de données multidimensionnelle. La succession de ces analyses, a permis le reclassement correct (0 % d'erreur) des fèves de cacao en fonction de leur origine géographique et de leur état de fermentation au jour près. Cette thèse a vu l'émergence d'une technique efficace de purification de l'épicatéchine et d'une méthode d'authentification non équivoque de l'origine géographique des fèves de cacao.

La demande croissante pour des chocolats de qualité supérieure a généré un besoin de diversification des arômes dans les gammes de chocolats proposées par les producteurs. La qualité du chocolat découle fortement du potentiel aromatique du cacao, qui est le résultat des réactions biochimiques qui ont lieu pendant la culture, la récolte, le traitement post-récolte et la transformation des fèves de cacao. Les composés volatils et non volatils du cacao contribuent à la perception sensorielle finale du chocolat. Au cours de la transformation, les arômes caractéristiques du chocolat se développent principalement pendant la fermentation, le séchage, la torréfaction et, dans une moindre mesure, le conchage. Bien que la fermentation du cacao ait un impact important sur sa qualité, et qu'elle soit étudiée depuis plusieurs décennies, il s'agit toujours d'un processus empirique et non maîtrisé. Les conditions de torréfaction et de conchage ont également un impact significatif sur la transformation des précurseurs d'arômes obtenus au cours de la fermentation, principalement en raison des réactions de Maillard qui ont lieu au cours de ces étapes. Dans un objectif de compréhension des mécanismes de formation de la qualité aromatique et sensorielle du chocolat, des essais de fermentation, de torréfaction et de conchage, tous dans des conditions spécifiques, ont été réalisés. Les caractéristiques, chimiques et sensorielles des fèves de cacao, tout au long de leur transformation en chocolat, ont ensuite été étudiées. Une attention particulière a été portée sur l'impact de la fermentation sur le développement des composés aromatiques et de leurs précurseurs. L'étude de l'utilisation de starters levuriens pour les fermentations s'est révélée particulièrement intéressante. Elle a permis de caractériser son impact et celui du temps de fermentation sur les différences observées dans la taxonomie microbienne, fongique et bactérienne, dans la masse en fermentation. En effet, la composition du microbiote peut influencer considérablement la composition volatile et non volatile des fèves, ce qui se traduit par des différences dans les profils aromatiques perçus au cours de l'analyse sensorielle des liqueurs et des chocolats obtenus. Enfin, des modèles prédictifs ont été développés afin de prédire les arômes des produits du cacao sur la base de leur composition chimique, en tenant compte des conditions de traitement auxquelles ont été soumises les fèves. Globalement, l'étude vise à mieux comprendre la formation des arômes dans le cacao et fournit des outils importants pour la production de chocolats capables de présenter des notes sensorielles fines, uniques et très recherchées, afin de mieux répondre à la demande croissante des consommateurs
The increasing demand for high-quality chocolate creates the need to diversify the range of flavors offered by chocolate producers. Cocoa quality derives strongly from its flavor, which may in itself be seen as the result of the biochemical reactions that take place during the cultivation, harvest and post-harvest processing of the cocoa beans. Both, volatile and non-volatile compounds contribute to the final flavor perception of cocoa. During processing, the characteristic flavors of chocolate are developed mainly during fermentation, drying, roasting and, to a lower extent, conching. Although cocoa fermentation has an important impact on the homogeneity of cocoa quality and has been studied for several decades, it is still an empirical and not mastered process. Roasting and conching conditions also have a significant impact on the subsequent transformation of the flavor precursors obtained during fermentation, mainly driven by the non-enzymatic Maillard reactions that take place during these steps. By carrying out fermenting, roasting and conching trials, all under specific conditions, and by later proceeding to the analysis of the physical, chemical and sensory characteristics of cocoa beans throughout their transformation all the way into chocolate, it is hoped to better understand the mechanism of aroma formation and its link with flavor perception. In this study, an important focus has been placed on the impact of fermentation on the development of aroma compounds and their precursors. The use of yeast starter cultures during fermentation has been of special interest in this study, as well as their impact and that of fermentation time on the compositional differences in the taxonomy of fungal and bacterial microbiota present in the fermentation mass throughout the entire process. This, because the microbial composition has the potential of greatly influencing the resulting volatile and non-volatile composition of the beans, which translates into differences in the perceived flavor profiles of the liquors and chocolates obtained thereof. Lastly, predictive models have been developed in an attempt predict the flavor of the cocoa products based on their chemical composition, taking into account the processing conditions to which they had been submitted. Globally, the study aims to gain a deeper understanding of flavor formation in cocoa and provides important tools for the production of chocolates capable of displaying unique and highly sought-after flavors in an attempt to better meet the increasing demand for fine flavor cocoa and chocolate products

Orientadora : Profa. Dra. Adriane Bianchi Pedroni Medeiros
Coorientador : Prof. Dr. Luiz Alberto Junior Letti
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa: Curitiba, 04/04/2017
Inclui referências : f.53-58
Resumo: O objetivo principal deste estudo foi avaliar de forma inédita o cultivo do fungo filamentoso Rhizopus stolonifer em fermentação no estado sólido em cascas do fruto de cacau (Theobroma cacao) obtidas do estado do Pará, região norte do Brasil. Na primeira etapa foi selecionada a cepa Rhizopus stolonifer NRRL 28169 de acordo com sua capacidade de produzir moléculas com atividade antioxidante e de colonizar a casca de cacau sem nenhum suplemento nutricional. Na sequência foi realizado um estudo que determinou o método de extração de compostos com capacidade antioxidante de forma prática e econômica utilizando uma solução de etanol em 50% (v/v). A casca de cacau já possui um enorme potencial biotecnológico, com inúmeros biocompostos apresentados pelas análises de cromatografia gasosa acoplada a espectrometria de massa. O processo de fermentação em estado sólido nos permite observar uma mudança na composição presente casca, gerando novas moléculas bioativas com potencial antioxidante, chegando a 7,09 ± 0,05 ?M de equivalência em Trolox no teste DPPH e 5,170 ± 0,035 x 104 ?M de equivalência em Trolox no teste ORAC. O aumento na concentração dos compostos fenólicos durante a fermentação fica evidenciado pela quantificação de fenóis totais alcançando um concentração de 114,40 mg em equivalência de ácido gálico. Palavras-chave: Casca de cacau; Rhizopus; Fermentação em estado sólido; Atividade antioxidante; Ácido cinâmico.
Abstract: The main objective of this study was to evaluate in an unprecedented way the cultivation of the filamentous fungus Rhizopus stolonifer in solid state fermentation in the cocoa pod husk from Theobroma cacao obtained from the state of Pará, northern region of Brazil. In the first step of this study the strain Rhizopus stolonifer NRRL 28169 was selected according to its capacity to produce molecules with antioxidant activity and to colonize the cocoa pod husk without any nutritional supplementation. Consequently, a study was carried out to determine the extraction method of compounds with antioxidant capacity in a practical, economical and manageable using a 50% (v / v) ethanol solution. The cocoa pod husk already has an enormous biotechnological potential, with numerous biocomposites presented by GC-MS and HPLC. The solid-state fermentation process allows us to observe a change in the present composition of cocoa pod husk, generating new bioactive molecules with antioxidant potential, reaching 7.09 ± 0.05 ?M equivalence in Trolox in the DPPH assay and 5.170 ± 0.035 x 104 ?M in Trolox equivalence in the ORAC assay. The increase in the concentration of the phenolic compounds in during the fermentation is evidenced by the quantification of total phenols reaching a concentration of 114.40 mg in gallic acid equivalence in TPC assay. Keywords: Cocoa pod husk; Rhizopus; Solid-state fermentation; Antioxidant activity; Cinnamic acid.

Dans un contexte économique tendu pour les chocolatiers « premium », la maîtrise de la qualité organoleptique de la matière première est un impératif et un avantage compétitif. La qualité aromatique d'un chocolat est entre autre liée aux traitements post-récolte et particulièrement à l'étape de fermentation. Cette étape est essentielle au développement des caractéristiques sensorielles. Or, les outils de contrôle qualité du cacao sont basés principalement sur l'évaluation sensorielle d'échantillons. Cette méthode est onéreuse et chronophage. Le développement d'outils rapides et robustes d'analyse de la qualité devient nécessaire. Cette étude a pour objectif de caractériser le déroulement de la fermentation afin de sélectionner des marqueurs spécifiques de la qualité du cacao. L'étude s'est intéressée aux marqueurs mesurables chez le planteur (à travers l'évolution de la température et du pH), et à ceux issus des transformations biochimiques des fèves (de type azoté et polyphénolique). Ces marqueurs ont ensuite été confrontés aux données traditionnelles de qualité (cut-test et dosage de l'azote ammoniacal), et aux données sensorielles, afin de sélectionner les plus pertinents pour le pilotage de fermentation. L'analyse globale de l'ensemble des résultats a permis de faire ressortir huit bio-marqueurs analytiques et trois bio-marqueurs sensoriels, représentatifs de la qualité du cacao et en lien avec le développement de la fermentation. Par ailleurs, des recommandations de méthodes de mesure de ces bio-marqueurs à mettre en place et permettant de contrôler la qualité ont été proposés
In a difficult economic context for "premium" chocolate makers, the control of the organoleptic quality of the raw material is an imperative and a competitive advantage.The aromatic quality of a chocolate is highly related to post-harvest treatment and particularly to the fermentation. This step is essential to the development of sensory characteristics.However, cocoa quality control tools are mainly based on sensory evaluation of samples. This method is expensive and time consuming.Fast and robust tools for quality analysis are needed. This study aims at characterizing the course of the fermentation in order to select specific markers of the quality of cocoa. The study focused on markers which can be measured on plantation (through the evolution of temperature and pH), and those from biochemical transformations of the beans (nitrogenous and polyphenol type). These markers have been then compared to data from traditional quality methods (cut-test and ammonia nitrogen quantification) and to sensory data in order to select the most relevant ones for the control of fermentation. The overall analysis of the database enabled to identify eight analytical bio-markers and three sensory bio-markers, representative of the quality of cocoa and in connection with the development of fermentation. In addition, recommendations for methods of measuring these bio-markers to implement and to monitor the quality have been presented

Consumption of cocoa and dark chocolate products has been associated with positive health outcomes including reduced onset of cardiovascular disease, inflammation, diabetes, obesity, and platelet disorders. Cocoa polyphenols, putatively responsible for these beneficial activities, are highly impacted by cocoa variety, agronomic effects and processing history. However, the difference in polyphenol concentration and composition between cocoa products originating from different hybrid clones (selected for high yield) or from different fermentation conditions is not fully understood. Detailed polyphenol characterization including determination of total polyphenol and total procyanidin concentrations, and qualitative and quantitative analysis of (mean) degree of polymerization was conducted. Significant differences in total polyphenol and procyanidin concentrations were observed between five genetic clones grown by the USDA-ARS Cocoa Germplasm Repository located in Mayagüez, Puerto Rico. To facilitate cocoa fermentation research in laboratories distant from cocoa harvesting sites, a laboratory-scale cocoa fermentation model system was developed in this study. This model system used dried, unfermented, cocoa beans and simulated pulp medium as the starting material. The model system supported growth of the essential succession of cocoa fermenting microorganisms and generated similar chemical changes to those observed in on-farm cocoa fermentation. Using this model system, the impact of inoculation with proprietary yeast strains Saccharomyces cerevisiae Lev F and Saccharomyces cerevisiae Lev B on cocoa polyphenol concentration and composition was evaluated. Inoculation with both yeast strains resulted in increased fermentation rate and Lev B inoculation resulted in higher total polyphenol and procyandin contents at the end of fermentation. Overall, the present work addressed the influence of cocoa variety selection and fermentation process conditions on the composition and concentration of polyphenols. These findings will contribute to continued efforts to develop cocoa products with optimized bioactivity and maximum disease preventative effects.
PHD

Cocoa beans (Theobroma cacao) are a highly concentrated source of dietary flavanols- bioactive compounds associated with the health protective properties of cocoa. Cocoa beans undergo processing steps, such as fermentation, roasting, winnowing, grinding, pressing, etc., to produce a final product with specific desirable sensory attributes. It is well established that these processing steps, specifically fermentation and roasting, result in dramatic degradation of cocoa's native flavanols, but it is possible that these processing steps may generate compounds with novel activities, potentially preserving or enhancing bioactivity. Raw unfermented cocoa beans were processed by way of a partial factorial approach to produce cocoa powders from the same batch of raw beans using various combinations of fermentation [unfermented, cool fermented (maximum 46°C), hot fermented (maximum 60°C))] and roasting [unroasted, cool roasted (120°C), hot roasted (170°C)]. To simulate cocoa fermentation in a highly controlled environment, a pilot-scale fermentation model system was employed to eliminate many external unknowns and ensure that the differences between our cocoa powders were due to our various treatments, rather than unknown factors occurring during fermentation and roasting. Low and high molecular weight fractions (8-10 kDa cutoff) were produced from cocoa powder extracts (CPE) of each treatment to quantify Maillard reaction products (MRP). A HILIC-UPLC MS/MS method was developed to more efficiently and sensitively quantify cocoa flavanols with high degrees of polymerization (DP) produced during processing. Overall, cocoa processing significantly (p<0.05) decreased the total phenolic and total flavanol concentrations of CPEs. Hot roasting had the greatest impact on native flavanol degradation yet produced CPEs with the highest mean degree of polymerization (mDP). All CPEs dose-dependently inhibited α-glucosidase enzyme activity, with cool fermented/cool roasted cocoa powder exhibiting the best inhibition (IC50 of 62.2 µg/mL). Increasing flavanol mDP was correlated with decreasing IC50 values, suggesting that the complex flavanols produced during processing enhance cocoa's bioactivity (or their production is associated with other products that enhance bioactivity). Alternatively, high molecular weight CPE fractions were correlated with increasing IC50 values, suggesting that MRPs interfere with enzyme inhibition or are associated with other products (polyphenols, macronutrients, etc.) that interfere with enzyme inhibition. Overall, the data presented within this work indicate that the components of processed cocoa powders are promising inhibitors of α-glucosidase, despite a significant reduction in native flavanol composition induced by processing, and moreover that fermentation and roasting conditions can positively influence the bioactivity of cocoa despite losses of native flavanols.
Master of Science in Life Sciences
According to the Centers for Disease Control and Prevention, obesity-related chronic conditions such as cardiovascular disease and type 2 diabetes mellitus (T2D) are the leading cause of preventable and/or premature death, with 51% of the American population predicted to be obese by 2030. Cocoa (Theobroma cacao) is a highly concentrated source of polyphenols, and these compounds have been shown to interact with and inhibit digestive enzymes responsible for carbohydrate breakdown. By inhibiting the activity of these digestive enzymes, it is possible to slow down carbohydrate absorption after a meal and ultimately reduce large spikes in blood glucose levels, being a promising strategy in the prevention and maintenance of T2D. Cocoa beans undergo processing steps to produce a final product, such as cocoa powder, and it is known that these processing steps reduce the levels of beneficial polyphenols. Yet, how this processing-induced degradation effects the health protective activities of cocoa is still widely unknown and is the focus of this work. Through highly controlled cocoa bean processing, cocoa powders of different processing conditions were produced and used to assess how various processing parameters impacted digestive enzyme activity. Overall, processing steps did reduce levels of native polyphenols. However, these losses did not demonstrate a reduction in enzyme inhibition and certain processing conditions actually enhanced digestive enzyme inhibition. This research shows promise for the potential use of processed cocoa powder as an effective strategy in the prevention and maintenance of T2D and further work must be done to understand the mechanisms behind this relationship.

La qualità delle fave di cacao disponibili in commercio, che rappresentano la principale materia prima per la produzione di cioccolato, dipende da diversi fattori inclusi: il tipo di piantagione, le pratiche agricole ed il processo di post raccolta. Tra queste; fermentazione ed essicazzione sono generalmente considerate le più rilevanti, dal momento in cui, durante queste fasi, vengono formati e fissati i precursori degli aromi del cacao. Inoltre, esse rappresentano un step cruciale durante il quale possono verificarsi contaminazioni da parte dei funghi filamentosi. La fermentazione è caratterizzata da una successione ben definita di lieviti, batteri lattici e batteri acetici, a tal fine, lo scopo del presente lavoro di tesi è stato quello di esplorare e descrivere in modo completo le comunità batteriche e fungine coinvolte nella fermentazione delle fave di cacao e valutare se l’origine geografica ed il metodo di fermentazione potessero influenzare la loro composizione. Per ottenere tali risultati il gene 16s rRNA è stato usato come marker per descrivere la comunità batterica totale mediante High Throughput Sequencing (HTS), dimostrando come tale approccio abbia la capacità di evidenziare la totalità delle comunità batteriche a livello di specie. In un secondo approccio l’Internal Transcribed Spacer 1 (ITS1) ed il dominio D1/D2 della sub unità maggiore dell’RNA ribosomiale (26s rRNA) sono stati selezionati per descrivere la popolazione fungina. I risultati hanno evidenziato come le due regioni abbiano la capacità di descrivere la composizione generale delle popolazioni, sebbene il dominio D1/D2 sia stato in grado di analizzare più nel dettaglio la composizione. Infine gli stessi campioni sottoposti all’analisi mediante HTS sono stati analizzati mediante SPME-GC-MS per evidenziare i principali composti aromatici formatisi durante il processo di post raccolta. Complessivamente i risultati indicano chiaramente che l’approccio mediante HTS ha le potenzialità per fornire una dettagliata visione d’insieme delle comunità batteriche e fungine presenti durante le fasi di post raccolta delle fave di cacao, inoltre le analisi statistiche hanno evidenziato come l’ITS1 ed i composti volatili possano essere utilizzati per la tracciabilità geografica.
The quality of commercial cocoa beans, the principal raw material for chocolate production, depends on several factors including type of plantations, the agricultural practices and the post-harvest processing. Among these, fermentation and drying are generally considered the most relevant, since during these phases cocoa flavors precursors are formed and fixed. Furthermore, they represent crucial steps during which filamentous fungi contamination might occur. Fermentation is characterized by a well-defined succession of yeasts, lactic acid bacteria and acetic acid bacteria, so that, the aim of the described studies was to explore total bacterial and fungal communities involved in cocoa bean fermentation and to evaluate if geographical origin and fermentation method might affect their composition. To achieve these results, 16s rRNA gene was used as marker to assess the total bacterial community by using High Throughput Sequencing (HTS), indicating that this approach has the ability to provide a comprehensive view of the cocoa bean microbiota at the species level. In a second approach, Internal Transcribed Spacer 1 (ITS1) and the D1/D2 domain of the Large subunit (LSU) of the nuclear ribosomal RNA (26S rRNA) were screened to assess the total fungal community. Results revealed the ability of these two genomic regions to describe reliably the general composition, even if D1/D2domain was able to go deeper into the fungal composition resulting in a higher resolution. In the last approach the same samples subjected to HTS investigation were analyzed through SPME-GC-MS in order to underline the principal key-aroma compounds formed during the post-harvest processing. Overall, results point out clearly that HTS approach has the ability to provide a comprehensive view of the total bacterial and fungal communities, and statistical analyses have shown how analyses of ITS1 sequences and volatile compounds might be useful for the geographical traceability of the processed cocoa beans samples.

La qualità delle fave di cacao disponibili in commercio, che rappresentano la principale materia prima per la produzione di cioccolato, dipende da diversi fattori inclusi: il tipo di piantagione, le pratiche agricole ed il processo di post raccolta. Tra queste; fermentazione ed essicazzione sono generalmente considerate le più rilevanti, dal momento in cui, durante queste fasi, vengono formati e fissati i precursori degli aromi del cacao. Inoltre, esse rappresentano un step cruciale durante il quale possono verificarsi contaminazioni da parte dei funghi filamentosi. La fermentazione è caratterizzata da una successione ben definita di lieviti, batteri lattici e batteri acetici, a tal fine, lo scopo del presente lavoro di tesi è stato quello di esplorare e descrivere in modo completo le comunità batteriche e fungine coinvolte nella fermentazione delle fave di cacao e valutare se l’origine geografica ed il metodo di fermentazione potessero influenzare la loro composizione. Per ottenere tali risultati il gene 16s rRNA è stato usato come marker per descrivere la comunità batterica totale mediante High Throughput Sequencing (HTS), dimostrando come tale approccio abbia la capacità di evidenziare la totalità delle comunità batteriche a livello di specie. In un secondo approccio l’Internal Transcribed Spacer 1 (ITS1) ed il dominio D1/D2 della sub unità maggiore dell’RNA ribosomiale (26s rRNA) sono stati selezionati per descrivere la popolazione fungina. I risultati hanno evidenziato come le due regioni abbiano la capacità di descrivere la composizione generale delle popolazioni, sebbene il dominio D1/D2 sia stato in grado di analizzare più nel dettaglio la composizione. Infine gli stessi campioni sottoposti all’analisi mediante HTS sono stati analizzati mediante SPME-GC-MS per evidenziare i principali composti aromatici formatisi durante il processo di post raccolta. Complessivamente i risultati indicano chiaramente che l’approccio mediante HTS ha le potenzialità per fornire una dettagliata visione d’insieme delle comunità batteriche e fungine presenti durante le fasi di post raccolta delle fave di cacao, inoltre le analisi statistiche hanno evidenziato come l’ITS1 ed i composti volatili possano essere utilizzati per la tracciabilità geografica.
The quality of commercial cocoa beans, the principal raw material for chocolate production, depends on several factors including type of plantations, the agricultural practices and the post-harvest processing. Among these, fermentation and drying are generally considered the most relevant, since during these phases cocoa flavors precursors are formed and fixed. Furthermore, they represent crucial steps during which filamentous fungi contamination might occur. Fermentation is characterized by a well-defined succession of yeasts, lactic acid bacteria and acetic acid bacteria, so that, the aim of the described studies was to explore total bacterial and fungal communities involved in cocoa bean fermentation and to evaluate if geographical origin and fermentation method might affect their composition. To achieve these results, 16s rRNA gene was used as marker to assess the total bacterial community by using High Throughput Sequencing (HTS), indicating that this approach has the ability to provide a comprehensive view of the cocoa bean microbiota at the species level. In a second approach, Internal Transcribed Spacer 1 (ITS1) and the D1/D2 domain of the Large subunit (LSU) of the nuclear ribosomal RNA (26S rRNA) were screened to assess the total fungal community. Results revealed the ability of these two genomic regions to describe reliably the general composition, even if D1/D2domain was able to go deeper into the fungal composition resulting in a higher resolution. In the last approach the same samples subjected to HTS investigation were analyzed through SPME-GC-MS in order to underline the principal key-aroma compounds formed during the post-harvest processing. Overall, results point out clearly that HTS approach has the ability to provide a comprehensive view of the total bacterial and fungal communities, and statistical analyses have shown how analyses of ITS1 sequences and volatile compounds might be useful for the geographical traceability of the processed cocoa beans samples.

La fermentation du cacao est une fermentation spontanée qui dure de 4 à 8 jours et repose principalement sur la succession de trois groupes de microorganismes : les levures, les bactéries lactiques et les bactéries acétiques qui réalisent les fermentations alcoolique, lactique et acétique respectivement. Les fèves saines sont stériles jusqu’à l’ouverture de la cabosse. L’inoculation des fèves est réalisée le plus souvent naturellement à l’aide de l’environnement autour de l’écabossage et de la mise en fermentation. Les procédés de traitements post-récolte sont différents d’un pays à l’autre et influencent le déroulement de la fermentation. Cependant, trois espèces de bactéries lactiques et acétiques (Lactobacillus plantarum, Lactobacillus fermentum et Acetobacter pasteurianus) dominent les fermentations dans tous les pays. Par contre, leur diversité intra spécifique n’a été que rarement étudiée. Dans cette étude nous avons utilisé des méthodes moléculaires indépendantes de la culture (PCR-DGGE et metabarcoding) pour étudier les communautés bactériennes associées à la fermentation des fèves de cacao selon trois pays : le Mexique, la Côte d’Ivoire et la Guyane. La méthode de metabarcoding a également été utilisée pour identifier la contribution des surfaces liées à l’environnement pré et post récolte des cabosses de cacao lors d’une fermentation réalisée au Mexique. La dominance des genres Lactobacillus et Acetobacter au cours de la fermentation dans chaque pays a été confirmée. De plus, la présence de genres spécifiques à chaque pays a été mise en évidence lors du premier jour de la fermentation. L’ensemble des surfaces lié à l’environnement de la fermentation semble participer à l’inoculation des genres dominants. Elles agissent en tant que réservoirs bactériens. Une collection de souches de bactéries lactiques et acétiques a été constituée. L. plantarum et A. pasteurianus ont été les deux espèces isolées en majorité. La diversité intra spécifique des souches d’A. pasteurianus a été étudiée. Pour cela, leurs polymorphismes génomiques ont été analysés à l’aide d’une amplification par PCR sur des séquences répétées et leurs caractéristiques biochimiques ont été comparées dans un milieu simulant les conditions de la pulpe de cacao au 2ème jour de fermentation. Notre étude a contribué à montrer que les mêmes souches d’A. pasteurianus peuvent être présentes dans les 3 pays différents. Certaines souches se distinguent pour leur plus grande affinité pour l’acide lactique que les autres, ce qui est intéressant pour améliorer la qualité organoleptique du cacao final. Les résultats sur la diversité intra spécifique nous permettent de proposer des candidats potentiels pour l’élaboration de starters de culture pour la fermentation des fèves de cacao
Cocoa fermentation is a spontaneous fermentation that lasts 4 to 8 days. It is mainly based on the succession of three groups of microorganisms: yeasts, lactic acid bacteria and acetic bacteria that carry out respectively the alcoholic, lactic and acetic fermentation. The beans are sterile until the opening of the pod. The inoculation of the beans is usually naturally done using the environment around the pod opening and the fermentation process. Post-harvest treatment processes differ from one country to another and influence the fermentation progress. However, three species of lactic and acetic bacteria (L. plantarum, L. fermentum and A. pasteurianus) dominate the fermentations in all countries. On the other hand, their intraspecific diversity was rarely studied. In this study, we used the metabarcoding method to study the interspecific diversity of bacterial communities associated with the fermentation of cocoa beans in 3 countries: Mexico, Ivory Coast and Guyana. In addition, this method was used to identify the contribution of the surfaces related to the pre- and post-harvest environment of cocoa pods during the fermentation, which was carried out in Mexico. The dominance of the genera Lactobacillus and Acetobacter during fermentation in each country has been confirmed. In addition, the presence of country-specific genera was founded on the first day of fermentation. All the surfaces linked to the fermentation environment participate to the inoculation of the dominant genera. They act as bacterial tanks. A collection of lactic and acetic bacteria strains was produced. L. plantarum and A. pasteurianus were the most isolated species. Intra-specific diversity of A. pasteurianus strain was studied. For this, their genomic polymorphisms were analyzed using PCR amplification on repeated sequences and their biochemical characteristics were compared in a specific medium, simulating the conditions of the cocoa pulp at the 2nd day of fermentation. Our study showed that the strains of A. pasteurianus could be present in the three different countries. Some strains were distinguished for their greater affinity for lactic acid than the others, which is interesting in order to improve the organoleptic quality of the final cocoa. The results on intra-specific diversity allow us to propose potential candidates for the production of culture starters for the fermentation of cocoa beans

Les feves du cacaoyer (theobroma cacao l. ) constituent la matiere premiere pour l'elaboration du chocolat. L'amelioration de la qualite du produit fini devient une des priorites des partenaires de la filiere cacao au meme titre que certains caracteres agronomiques. La qualite resulte de l'effet combine du materiel vegetal, de l'environnement, des pratiques culturales et du traitement post-recoltes des feves. Dans ce cadre, plusieurs voies d'etudes ont ete suivies, a savoir l'evaluation de la diversite genetique indispensable pour le developpement d'un programme d'amelioration de l'espece t. Cacao, et la cartographie de qtl d'interet agronomique. Enfin, la fermentation, une des etapes importantes pour le developpement de certains precurseurs de saveur a ete etudiee via une analyse proteique des feves. L'etude de 155 genotypes par rapd et rflp n'a pas revele de reelle structuration de l'espece en groupes morphologiques ou selon leur origine geographique. Cependant, certaines classes d'individus comme les nacional, presentent une forte differenciation genetique. Parmi eux, les genotypes nacional faiblement heterozygotes du point de vue moleculaire pourraient representer les nacional de reference. Compte tenu de l'absence de groupes nettement definis, les marqueurs moleculaires se revelent utiles pour l'etablissement d'une core collection. Deux cartes genetiques ont ete elaborees a partir de deux descendances en filiation permettant a la fois la caracterisation et le suivi de qtl relatifs a trois caracteres agronomiques. Peu de qtl communs aux deux populations ont ete detectes, suggerant entre autre l'influence de l'environnement sur les caracteres quantitatifs analyses. L'electrophorese bidimensionnelle a permis de suivre le comportement des proteines de la feve durant la fermentation. Des differences significatives du contenu proteique initial des feves ont ete mises en evidence et attribuees a des degres variables de maturite des fruits a la recolte. Une forte quantite de proteines non degradees est encore observee en fin de fermentation

Le cacao est la 3ème denrée la plus commercialisée dans le monde et 23 pays sont classés selon l’ICCO comme producteurs de cacao fin, dont Madagascar. Le cacao de Madagascar est classé cacao fin à 100 % selon l’ICCO depuis 2016. Il est un des plus réputés au monde, même s'il ne représente que 0,2 % de la production mondiale. Peu d’études existent concernant la caractérisation du cacao malgache et sa fermentation. La fermentation est une étape post-récolte pour l’obtention des caractéristiques aromatiques et sensorielles d’un cacao de qualité. Ce travail a permis de déterminer les critères de qualité et de suivre les évolutions des critères organoleptiques, de la composition volatile et les micro-organismes au cours de la fermentation. Le cacao produit à Madagascar appartient principalement aux variétés Criollo et Trinitario. Le suivi de la fermentation montre qu’au niveau sensoriel, les descripteurs « végétal, terreux, astringent, amer » des fèves de cacao non ou peu fermentées (24-48 h) s’estompent pour laisser place aux descripteurs cacao, chocolat, fruité, acide pour des fèves de cacao en fin de fermentation (96 h-144 h). De même, les teneurs en composés volatils liés aux descripteurs sensoriels recherchés tels que l’acétate de 3-méthylbutyle, l’acétate d'éthyle, le benzaldéhyde, la tétraméthylpyrazine, l’acide acétique augmentent au cours de la fermentation. L’étude de la flore microbienne a permis d’identifier l’origine de la production de certains composés volatils. Notamment, la levure Hanseniaspora opuntiae présente un intérêt particulier car elle est associée à la production de 2-phényléthanol. L’étude des communautés levuriennes par voie moléculaire a confirmé la présence des levures isolées et a mis en évidence d’autres souches notamment du genre Pichia qui sont aussi productrices de molécules volatiles telles que l’acétate de 2-phényléthyle et l’acétate d'éthyle
Cocoa is the third most traded commodity in the world and 23 countries are classified as fine cocoa producers according to the ICCO, including Madagascar. The Malagasy cocoa production is classified as 100 % fine cocoa by the ICCO since 2016. It is one of the most famous in the world, even though it only represents about 0.2 % of the world production. Few studies exist concerning the characterization of Malagasy cocoa and its fermentation. Fermentation is a crucial post-harvest treatment step for obtaining the specific aromatic and sensory quality of cocoa. In this work, the quality criteria could be determined and the changes in organoleptic criteria, volatile composition and microorganisms during fermentation were monitored.Most of the cocoa produced in Madagascar belongs to the Criollo and Trinitario varieties. The monitoring of fermentation showed that the sensory negative descriptors « vegetal, earthy, astringent, bitter » of incompletely fermented cocoa beans (24-48 h) were progressively replaced by positive descriptors “cocoa, chocolate, fruity, acid” as measured in cocoa beans at the end of fermentation (96 h-144 h). Likewise, the contents of volatile compounds such as 3-methylbutyl acetate, ethyl acetate, benzaldehyde, tetramethylpyrazine, acetic acid increase during fermentation. The study of microbial flora has made it possible to identify the origin of the production of certain volatile compounds. The Hanseniaspora opuntiae yeast is of particular interest because it is associated with the production of 2-phenylethanol. The study of the yeast communities at the molecular level confirmed the presence of the yeasts identified and highlighted other strains in particular of the Pichia gender which also are potential producers of volatile compounds such as 2-phenylethyl acetate and ethyl acetate

Orientador : Marisa de Nazare H. Jackix
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O cupuaçu (Theobroma grandiflorum Schum), espécime do mesmo gênero botânico que o cacau, é um dos frutos mais importantes da região Amazônica. A comercialização de sua polpa, com sabor exótico muito apreciado e em plena expansão no Brasil, começa a despertar interesse no mercado exterior. A crescente industrialização da polpa tem disponibilizado um volume significativo de sementes, atualmente um subproduto com incipiente aproveitamento industrial. Correspondendo a 20% do peso do fruto, esta semente de composição química semelhante a do cacau têm sido objeto de pesquisas que comprovam que quando submetidas às mesmas etapas de processo que aquele, resultam num produto semelhante ao chocolate. O processo de industrialização do cupuaçu inicia-se de modo análogo ao cacau, com a fermentação das sementes. A etapa é indispensável, pois ocorrem importantes transformações que estão relacionadas com o desenvolvimento dos precursores de sabor e com a qualidade global do chocolate. Realizar um estudo comparativo das transformações que ocorrem nas sementes de cacau e cupuaçu, submetidas às mesmas condições de processo fermentativo, foi o objetivo deste trabalho...Observação: O resumo, na integra, podera ser visualizado no texto completo da tese digital
Abstract: Cupuassu (Theobroma grandiflorum SChum), specimen of the same botanical genus that cocoa, is one of the most important fruits of the Amazon area. The commercialization of its pulp, with very appreciated exotic flavor and in full expansion in Brazil, it begins to wake up interest in the external market. The growing industrialization of the pulp has made avaliable a significant volume of seeds, nowa by-product with incipient industrial use. Corresponding at 20% of the weight of the fruit, this seed of similar chemical composition of the cocoa has been object of researches that check that when submitted to the same process stages as that, they result in a product similar to the chocolate. The process of industrialization of the cupuassu begins in a similar way to cocoa, with the fermentation of the seeds. The stage is indispensable, because important transformations that are related with the development of the flavor precursors happen and with the global quality of the chocolate. To accomplish a comparative study of the transformations that happen in the cocoa seeds and cupuassu, submitted to the same conditions of fermentation process, it was the objective of this work...Note: The complete abstract is available with the full electronic digital thesis or dissertations
Mestrado
Mestre em Tecnologia de Alimentos

As principais matérias-primas do chocolate, obtidas a partir das sementes secas do fruto do cacaueiro (Theobroma cacao), são a manteiga e o líquor de cacau. Para se obter matérias-primas de alta qualidade é necessário que o processo que antecede a industrialização, no caso a fermentação, seja padronizado para que sejam formados nas sementes os precursores de aroma, sabor e cor característica do chocolate. No interior do fruto do cacaueiro são encontradas as sementes envoltas por uma mucilagem composta por: água, pectina, sacarose, glicose, frutose, proteínas, ácidos e sais. O processo fermentativo do cacau ocorre sem qualquer tipo de inóculo ou padronização. Devido a este fato, os padrões de qualidade das sementes obtidas são as mais adversas e muitas vezes a presença de compostos interferentes e não desejáveis são formados ao longo desse caminho. Visando a otimização do processo fermentativo este trabalho teve por objetivo selecionar leveduras de ocorrência espontânea presentes na fermentação do cacau, reinoculá-las no processo natural in locu e comparar com o processo de ocorrência espontânea, avaliando assim o potencial do coquetel de leveduras a ser utilizado futuramente para padronizar o processo. Para tanto, foram isoladas 367 linhagens de leveduras de ocorrência espontânea em duas fazendas no sul da Bahia. As linhagens passaram por uma seleção onde foi implementado um programa de seleção composto por três ensaios: ensaio de crescimento em pectina; análise de Açúcar Redutor Total livre (ART); e avaliação de atividade enzimática. Foi possível selecionar três linhagens de leveduras promissoras com potencial pectinolítico as quais foram testadas in locu no município de Itabuna-BA. O processo de isolamento, seleção e reintrodução das linhagens selecionadas no processo fermentativo do cacau se mostrou uma prática altamente eficaz. Os resultados obtidos com a inoculação inicial de leveduras selecionadas, antecipou os eventos como produção de etanol, ácido acético, drenagem do mel e elevação da temperatura em 24 horas em relação ao controle.
The fundamental raw material to produce chocolate, obtained from dried seeds of cocoa fruit (Theobroma cacao), are butter and cocoa liquor. In order to obtain high quality of raw materials, it is necessary standardize the procedure before industrialization, known as fermentation, so that the aroma, taste and color precursors of chocolate must be formed in the seeds. Inside the fruits exists a white mucilaginous pulp, which covers the beans, it contains water, pectin, sucrose, glucose, fructose, proteins, acids and salts. The fermentation of cocoa seeds occurs in wooden boxes or piles on the ground without any control or standardization. Due to this fact, the quality of the seeds are the most adverse, the presence are often of interfering compounds and undesirable products could be formed along the way. To optimize the fermentation process this study aimed to select pectinolytic yeasts of spontaneous occurrence from cocoa fermentation, re-inoculate them in the natural process and compare with the spontaneously occurring process. Consequently evaluate the yeast cocktail potential as a standard inoculum. Therefore, we isolated 367 yeast strains from spontaneous cocoa fermentation in two different farms in southern Bahia - Brazil. The strains were analyze to a selection-screening program, which consists of three tests: ability to grow in pectin medium; Total free Reducing Sugar Analysis (ARTL); and evaluation of enzyme activity. It was possible to select three yeast strains with promising pectinolitic potential. Those strains were tested in locu in Itabuna-BA, Brazil. The results of that program, selection and re-introduction in the fermentation process proved to be a highly effective practice. The results obtained with the initial inoculation of selected yeasts, could anticipate the fermentation events in 24 hours, such as the production of ethanol, acetic acid, sweating drainage and temperature rise when compared with the control.

Orientadores: Nelson Horacio Pezoa Garcia, Denise Calil Pereira Jardim
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As sementes de cacau (Theobroma cacao L.) da variedade Forastero são extremamente ricas em compostos fenólicos, que representam em média 15 a 20% de seu peso seco e desengordurado, sendo que 60% pertencem à classe dos flavonóides, compostos apontados atualmente como responsáveis pela prevenção de doenças coronárias, diminuição do colesterol sérico, auxiliadores do sistema imunológico, entre outros. Durante a etapa de fermentação, são perdidos, em média, 70% dos flavonóides devido a importantes reações bioquímicas que ocorrem principalmente pela diminuição do pH, aumento de temperatura (45-50°C) e atuação de certas enzimas presentes no fruto ou produzidas pelos microrganismos que participam desta etapa. Tais reações são, em parte, responsáveis pela redução do amargor e da adstringência melhorando assim o desenvolvimento do sabor do chocolate. Desta forma, o presente trabalho visou modificar a etapa de fermentação de sementes de cacau para a produção de chocolate rico em flavonóides sem prejudicar seu sabor. Para isso, procurou-se inibir as enzimas que são possivelmente as principais responsáveis pela perda dos flavonóides através da adição de inibidores químicos (bissulfito de sódio e sulfato cúprico) na etapa de fermentação. Foram realizados sete experimentos distintos: ensaios A e G (fermentações convencionais com duração de 7 e 3 dias respectivamente); ensaios B, C e F (fermentações por 7 dias, modificadas com adição de 5mg, 10mg e 5mg de bissulfito de sódio/100g de massa de sementes com polpa após 48hs, 48hs e 120hs respectivamente e ensaios D e E (fermentações por 7 dias modificadas com adição de 5mg e 10mg de sulfato de cobre/100g de massa de sementes com polpa após 48 horas do início respectivamente). Os resultados indicaram que, de uma forma geral, todos os tratamentos propostos mantiveram maior teor de compostos fenólicos em relação à fermentação convencional (ensaio A). Quanto aos compostos fenólicos totais, o ensaio D apresentou a maior retenção (62,70%) desde o início da fermentação ao término da secagem, enquanto que no ensaio A foram retidos 36,38% destes compostos. Em relação aos flavan-3-óis e procianidinas, observou-se maior retenção, para monômeros, nos ensaios D (34,27%) e G (33,72%); para dímeros, nos ensaios D (21,83%) e G (21,78%); para trímeros, nos ensaios C (22,85%), D (22,37%) e F (22,38); para quatrâmeros, nos ensaios C (25,84%), D (24,77%) e F (27,21) e para pentâmeros, nos ensaios C (35,24%), D (34,45%) e F (34,16). Observou-se que a maior perda dos compostos fenólicos estudados ocorreu entre o término da fermentação e a secagem. Verificou-se que o residual de Cobre remanescente da adição feita durante a fermentação (ensaios D e E) nos liquors e nos chocolates produzidos foi de 0,23 e 0,36mg de cobre/100g de liquor (ensaios D e E respectivamente) e 0,025 e 0,036mg de cobre/100g de chocolate (ensaios D e E respectivamente), todos valores consideravelmente inferiores ao Limite Máximo Tolerado (LMT) definido pela Agência Nacional de Vigilância Sanitária (ANVISA), correspondente a 3,0mg de cobre/100g de amostra. Os chocolates produzidos a partir dos ensaios B, C, D e E mostraram aceitabilidade sensorial igual ou melhor ao convencional (A), enquanto que os produzidos a partir dos ensaios F e G apresentaram aceitabilidade mediana e incertezas com relação a intenção de compra
Abstract: Cocoa seeds (Theobroma cacao L.) from the Forastero variety are very rich in phenolic compounds which represent 15-20% of the defatted dry weight. The principal compounds are (+)-catechin, (-)-epicatechin and 60% of procyanidins that belong to the flavonoid class. These compounds are currently been considered responsible for coronary heart disease prevention, lowering the serum cholesterol and helping the immunological system. During the fermentation, 70% of the total phenolic compounds are lost in important biochemical reactions accelerated by the reduction in pH, temperature increase (45-50°C) and action of some enzymes, present in the fruit or produced by the microorganism growing at this stage. These reactions contribute to a reduction in bitterness and astringency, improving the flavor of the chocolate. The objective of this work was to modify the fermentation stage of cocoa seeds to produce flavonoid-rich chocolates without prejudicing its flavor. This was done by the inactivation of enzymes probably responsible for flavonoid degradation, through the addition of chemical inhibitors (sodium bissulfite and cupric sulphate) during the fermentation stage. Seven experiments were carried out: Experiments A and G (conventional fermentations during 7 and 3 days respectively); experiments B, C and F (modified fermentations during 7 days, with the addition of 5mg, 10mg and 5mg of sodium bissulfite/100g of cocoa seeds with pulp after 48hs, 48hs and 120hs since the beginning of fermentation respectively) and experiments D and E (modified fermentations during 7 days, with the addiction of 5mg and 10mg of cupric sulphate/100g of cocoa seeds with the pulp after 48hs and 120hs since the beginning of fermentation respectively). The results showed that all the treatments proposed maintained higher quantities of phenolic compounds as compared with the conventional experiment (A). Considering the total phenolics, experiment D showed the highest retention (62,70%) from the beginning of the fermentation up to the end of the drying stage, while in the experiment A, the retention was 36,38%. Considering the flavan-3-ols and procyanidins, a higher retention of monomers was observed in experiments D (34,27%) and G (33,72%); of dimers in experments D (21,83%) and G (21,78%); of trimers in experiments C (22,85%), D (22,37%) and F (22,38); of tetramers in experiments C (25,84%), D (24,77%) and F (27,21) and of pentamers in experiments C (35,24%), D (34,45%) and F (34,16). It was observed that the greatest loss of the phenolic compounds studied occurred between the end of fermentation and the beginning of the drying stage. It was shown that the copper residue in the liquor and chocolate remaining from the addition during fermentation (experiments D and E) was 0,23 and 0,36mg of copper/100g of liquor (experiments D and E respectively) and 0,025 e 0,036mg of copper /100g of chocolate (experiments D and E respectively). These values are below the Maximum Tolerated Limit (MTL) defined by ANVISA for this metal (3,0mg/100g). The chocolates B, C, D and E showed equal or better sensory acceptance as compared with conventional (A), and F and G chocolates which showed average sensory acceptance and uncertainty with respect to buying intention
Mestrado
Mestre em Tecnologia de Alimentos

Chocolate is one of the most important products for the food industry, being of economic interest all over the world. The cocoa quality depends directly on the post-harvest processing, being the cocoa-pulp fermentation a crucial step for chocolate quality development. The aim of this work was to study the diversity of aroma-producing yeasts associated with cocoa beans fermentation and to select suitable yeast starter culture to cocoa flavor modulation. A total of 39 cocoa-derived yeast isolates were screened for their capacity to produce volatile aroma compounds in a cocoa pulp simulation medium. The seven highest aroma-producing yeasts were identified by ITS-rRNA gene sequencing as belonging to Pichia kudriavzevii, in spite of exhibiting different metabolic profiles. A computer-assisted analysis of rep-PCR genomic fingerprints of Pichia kudriavzevii strains clearly differentiated the upper aroma-forming yeast strains (G1 group; P. kudriavzevii LPB06 and P. kudriavzevii LPB07) from the other strains (G2 group). This demonstrates the potential of rep-PCR technique as a promising genotypic tool for rapid and reliable speciation of aromatic yeast strains. In the second stage of this study, two strains with superior aroma production, namely P. kudriavzevii LPB06 and P. kudriavzevii LPB07, were used in cocoa beans fermentation at laboratory scale. They were able to establish an accelerated fermentation process with efficient yeast growth, sugars consumption and ethanol formation compared to the spontaneous process. The resulting cocoa beans were analyzed by diverse chemical analysis methods, including SPME-GC/MS, FTIR spectroscopy and metal and colorimetric analysis. All together, the results indicated that inoculated fermentations generated cocoa beans with better color development and richer aroma composition, suggesting that cocoa-associated yeast diversity at strain level can be exploited for flavor modulation of cocoa beans.
Atualmente, o chocolate é um dos produtos mais importantes para a indústria de alimentos, sendo de interesse econômico em todo o mundo. A qualidade do cacau depende diretamente do processamento pós-colheita, sendo a fermentação da polpa um passo crucial para o desenvolvimento da qualidade do chocolate. O objetivo deste trabalho foi estudar a diversidade de leveduras aromáticas associadas à fermentação de cacau e selecionar uma cultura iniciadora com potencial para modular o flavor de chocolate. Um total de 39 leveduras foram isoladas e caracterizadas quanto à formação de compostos aromáticos. As sete melhores produtoras foram identificadas através do sequenciamento do gene ITS-rRNA como Pichia kudriavzevii, apesar de apresentarem diferentes perfis metabólicos. Análise de impressões digitais (fingerprints) dos isolados pela técnica de rep-PCR claramente distinguiu as cepas com maior produção de compostos aromáticos, demonstrando o potencial desta técnica como uma ferramenta para rápida e confiável seleção de leveduras. Na segunda etapa deste estudo, duas cepas com superior formação de aroma (P. kudriavzevii LPB06 e P. kudriavzevii LPB07) foram testadas como culturas iniciadoras para fermentações de cacau em escala laboratorial. Estas duas cepas foram capazes de estabelecer um acelerado processo fermentativo, com eficiente consumo de açúcares e formação de etanol, em comparação ao método natural. As amêndoas de cacau resultantes destes processos foram analisadas por diferentes métodos químicos, incluindo SPME-GC/MS, espectroscopia FTIR e análises de metal e calorimetria. Os resultados indicaram que as fermentações inoculadas desenvolveram amêndoas de cacau com melhor cor e composição de aroma, sugerindo que a diversidade de levedura em fermentações de cacau pode ser explorada para a modulação do flavor de chocolate.

碩士
國立高雄餐旅大學
飲食文化暨餐飲創新研究所
107
Chocolate are often made with cocoa beans. The production process is cumbersome. The most important thing in the process of making cocoa paste from cocoa beans is the fermentation step. The rise of Taiwan's cocoa industry and the control conditions are not standardized. Therefore, this study The cocoa beans of five cocoa manufacturers in Pingtung, are determined according to the cutting test method of the Ministry of Economic Affairs, and the degree of fermentation of the domestic cocoa beans in Taiwan, as well as the moisture and oil content of cocoa beans, and then the cocoa beans are made into 70%. The chocolate was measured for viscosity, pH value, color and texture to explore the current state of Taiwanese chocolate, and to establish Taiwanese chocolate flavored roulette through the results of sensory evaluation. In the sectioning test method, it was found that the cocoa manufacturers had the highest score (86.05), while the fermentation days were the longest among the five manufacturers, while the cocoa manufacturers were the lowest (33%) in the oil analysis, with significant differences (p<0.05), it is presumed that the longer the fermentation time, the higher the complete score of fermentation, but the part of the cocoa butter content is the cocoa bean cultivation, and the nutrition is more relevant. After making 70% chocolate, the taste of the big item is more frequently selected in the flavor description of the fee, sour, bitter, astringent and fermented, and the fineness of the flavor is more Illicium, licorice, Cinnamon, musty, wine and smoked, the average consumer preference was 3.82, a significant difference (p<0.05), so it is known that these flavors are less acceptable to consumers in chocolate.

碩士
國立屏東科技大學
食品科學系所
105
The composition, flavor and patterns of cocoa beans are changed after fermentation and roasting, become meticulous taste and thermoplastic after grinding, it be known as cocoa liquor. Cocoa liquor could be directly consumed, used for processing, or further processed into different forms products, i.e., cocoa butter, cocoa mass, and cocoa powder. However, those manufacturing process will direct affect components changes in products and flavors’ formation. Though, there were many studies in the past explored the effects, for the development of Taiwan's local industry, these fundamental researches are still important and must be done. In this research, cocoa cultivated in Pingtung will be chosen. Its composition changing will be analyzed at each stage of processing, i.e., moisture content, Titratable acidity, pH, Total polyphenol, Theobromine and 5-hydroxymethyl-2-furfural (HMF), in order to understand the impact of processing conditions. The study consists of three variables. First, cocoa beans will ferment at different temperature (35, 40 and 45℃). Then, roast the fermented beansat different temperature (120, 130 and 140℃) and time (15 and 25 mins). After roasting, peel it off and grind the nibs in 24, 36 and 48 h by mortar mill for difference in particle sizes. The result shows, moisture content of cocoa beans decrease after fermentation, and reduce to 3.53-7.23% by using hot air dryer for 48 h. After fermentation, Titrable acid increase to 1-1.39% and slight decrease after roasting, but total polyphenol decrease both of two process, from 23.77-54.39 mg/g to 6.81-21.48 mg/g, this shows the same situation of Theobromine. 5-hydroxymethyl-2-furfural (HMF) content shows positive correlation with roasting temperature and time. Partical size of cocoa was observed by Scanning electronic microscopy, it showed the partical size of most of cocoa nibs were less than 30μm, however, there was some large one could observed in 500 times magnification. After 36 h grinding, the partical size decrease during increase grinding time.