Dissertations / Theses on the topic 'Cocoa fermentation'

Il cacao (Theobroma cacao L.) è un’importante coltura a livello mondiale. La sua produzione è alla base dell’economia della maggior parte dei paesi produttori e dei profitti di molte industrie dolciarie. Attualmente, circa il 70% delle fave di cacao nel mondo è raccolto in Africa. Il Ghana è il secondo paese produttore dopo la Costa d’Avorio. La maggior parte del cacao proviene da piccoli agricoltori che spesso usano sistemi di coltivazione obsoleti e poco organizzati. La crescente domanda di cacao nel mercato mondiale ha aumentato l’attenzione su una produzione sostenibile attraverso una serie di miglioramenti per la coltivazione e i processi post-raccolta. Questa tesi fa parte di un progetto che ha come obiettivo a lungo termine quello di incrementare la produzione di cacao di alta qualità da parte di piccoli coltivatori in Africa occidentale. Per questo lavoro sono state esaminate fave di cacao prodotte da 30 piccoli agricoltori di sei regioni del Ghana. I campioni sono stati analizzati e comparati sotto forma di fave di cacao intere, in polvere e liquore di cacao. I risultati hanno rilevato alcuni difetti nel processo di fermentazione evidenziati dai bassi livelli dell’ indice di fermentazione e dalla presenza di Ocratossina A. Dai risultati si evince anche che le fave di cacao prodotte in Ghana hanno grandi dimensioni, alto contenuto di grassi e presentano note aromatiche fruttate.

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.

Cocoa bean fermentation is an essential but spontaneous fermentation process to obtain the necessary raw material for the production of cocoa-derived products, among which chocolate. Successful cocoa bean fermentation processes are typically dominated by three microbial groups, namely yeasts, lactic acid bacteria, and acetic acid bacteria. The use of functional starter cultures may allow to gain a better control over the fermentation process. Previously, a number of candidate functional starter cultures have been proposed for the lactic acid bacteria, namely Lactobacillus fermentum 222 and Lactobacillus plantarum 80, and for the acetic acid bacteria, namely Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848T, and Acetobacter senegalensis 108B. The metabolism of bacteria determines an important part of their physiology, and this is recently being investigated by using computational models. The aim of this PhD thesis was to develop such models for the candidate functional starter cultures for the cocoa bean fermentation process and to perform the related computational analysis. The computational models developed were genome-scale metabolic models, which constitute a comprehensive repertoire of metabolic enzymes with their concomitant reactions, and this at genome-scale. The reconstruction of such models requires a combination of high-quality genome re-annotation, comparative genomics, manual curation, and experimental validation. Genome-scale metabolic modeling together with the use of previously published experimental data under cocoa fermentation conditions allowed to contextualize the experimental data and to gain new insights into the metabolic properties of the candidate functional starter cultures. Simulations with the A. pasteurianus 386B genome-scale metabolic model revealed the metabolic roles of lactate and ethanol, the energetic properties of the strains’ aerobic respiratory chain, and the possible functional role of an NAD(P)+ transhydrogenase. Modeling the metabolite dynamics of A. ghanensis LMG 23848T under cocoa fermentation conditions revealed an alternative strategy for its diauxic growth, compared with A. pasteurianus 386B, which was related to a difference in lactate consumption rate and pyruvate overflow. For A. senegalensis 108B, it was shown that, next to lactic acid, also citric acid could sustain its growth in vitro as the sole carbon source. Furthermore, the absence of the glyoxylate cycle predicted from its genome did not correspond with its species description that reports growth on ethanol as the sole carbon source. For L. fermentum 222 and L. plantarum 80, core genome-scale metabolic models allowed to gain insight into the possible metabolic flux distributions as a function of environmental conditions. The modeling also indicated a current lack in knowledge; for example, concerning the presence and consumption of undefined substrates in the complex medium used.In summary, genome-scale metabolic modelling of candidate functional starter cultures for the cocoa bean fermentation process provided useful in silico tools to gain insight into their metabolic properties at a systemic level.
La fermentation du cacao est un processus essentiel pour obtenir la matière première nécessaire pour la production de produits dérivés du cacao, comme par exemple le chocolat. Une fermentation de cacao favorable est caractérisée par la domination de trois groupes de microorganismes :les levures, les bactéries lactiques, et les bactéries acétiques. L'utilisation de cultures de départ fonctionnelles permet un meilleur contrôle sur le processus de fermentation. En ce qui concerne les bactéries, de nombreuses cultures "starter" ont été proposées, à savoir Lactobacillus fermentum 222 et Lactobacillus plantarum 80 pour les bactéries lactiques et Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848T, et Acetobacter senegalensis 108B pour les bactéries acétiques. Le métabolisme des bactéries constitue une partie importante de leur physiologie et la recherche actuelle se concentre de plus en plus sur la modélisation du métabolisme et la simulation des flux métaboliques par ordinateur. Cette thèse de doctorat a été consacrée au développement et à l'analyse de tels modèles computationnels pour des cultures fonctionnelles "starter" proposés pour la fermentation du cacao.Les modèles qui ont été développés dans cette thèse sont des modèles métaboliques à l’échelle du génome. La reconstruction du réseau métabolique a entraîné la ré-annotation du génome, une étude de génomique comparative, la curation manuelle des annotations et la validation du modèle par des expériences in vitro. La modélisation nous a permis de contextualiser des données expérimentales déjà publiées pour en obtenir de nouvelles informations concernant les propriétés métaboliques des cultures starter. Des simulations utilisant le modèle métabolique de A. pasteurianus 386B ont clarifié les rôles métaboliques de l’acide lactique et de l’éthanol, les propriétés énergétiques de sa chaîne respiratoire, et ont permis d'assigner un rôle possible à une NAD(P)+ transhydrogénase. La modélisation de la dynamique des métabolites provenant d’un milieu de croissance de A. ghanensis LMG 23848T dans des conditions simulant la fermentation du cacao, a mis en évidence une stratégie alternative de croissance biphasique comparé à A. pasteurianus 386B. Ceci est dû à une différence dans le taux de consommation de l’acide lactique et à l’éventuelle production de pyruvate. Pour A. senegalensis 108B, les expériences ont démontré, tant pour l’acide lactique que pour l’acide citrique, que ces sources de carbone permettaient, à elles seules, la croissance de cette bactérie. L’absence du cycle du glyoxylate chez A. senegalensis 108B ne correspondait pas à la description de cette espèce, laquelle pouvant croître sur l’éthanol comme seule source de carbone. Pour L. fermentum 222 et L. plantarum 80, la modélisation de leur métabolisme du carbone a permis d’explorer les distributions de flux métaboliques en fonction des substrats consommés. Les simulations ont aussi révélé le manque de connaissance que nous avons sur ces bactéries lactiques, telle que la consommation de substrats non identifiés venant du milieu de croissance et qui pourrait influencer leur dynamique de croissance.En résumé, la modélisation métabolique à l’échelle du génome des cultures starter proposées pour la fermentation du cacao a permis le développement d’outils in silico qui peuvent être utilisés pour mieux comprendre le métabolisme global de ces souches.
Het cacaoboonfermentatieproces is een essentieel maar spontaan proces dat nodig is om de noodzakelijke grondstof, met name de gefermenteerde cacaobonen, voor de productie van cacao-afgeleide producten, waaronder chocolade, te bekomen. Succesvolle cacaoboonfermentatieprocessen worden typisch gedomineerd door drie microbiële groepen, met name gisten, melkzuurbacteriën en azijnzuurbacteriën. Om meer controle te verkrijgen over het fermentatieproces is het gebruik van functionele starterculturen aangewezen. In vorige studies werd reeds een reeks kandidaat-functionele starterculturen voorgesteld. Voor de melkzuurbacteriën zijn dit Lactobacillus fermentum 222 en Lactobacillus plantarum 80 en voor de azijnzuurbacteriën zijn dit Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848T en Acetobacter senegalensis 108B. Het metabolisme van bacteriën bepaalt in grote mate hun fysiologie, en dit wordt recent onderzocht door middel van computationele modellen. Het ontwikkelen en analyseren van zulke modellen voor de voorgestelde kandidaat-functionele starterculturen vormde het onderwerp van deze doctoraatsthesis.De computationele modellen waarvan sprake waren genoomwijde metabole modellen (GEMs), dewelke het repertoire aan metabole enzymen en de biochemische reacties die zij katalyseren in de bacteriële cellen omvat. De reconstructie van het metabole netwerk op genoomschaal vraagt om een gecombineerde aanpak van hoge-kwaliteit genoomherannotatie, comparatieve genomica en experimentele validatie. De GEMs werden gebruikt om reeds gepubliceerde experimentele data onder cacaofermentatiecondities te contextualiseren en nieuwe inzichten te verkrijgen in de metabole karakteristieken van de kandidaat-functionele starterculturen. Door middel van simulaties met het A. pasteurianus 386B GEM kon de metabole rol van melkzuur en ethanol, en de energetische karakteristieken van de aerobe respiratieketen van deze stam aangetoond worden, alsook de mogelijke metabole functie van een NAD(P)+ transhydrogenase. Het modelleren van de microbiële dynamica van A. ghanensis LMG 23848T onder cacaofermentatiecondities wees op een alternatieve strategie voor de tweevoudige groei van deze stam ten opzichte van de tweevoudige groei van A. pasteurianus 386B onder dezelfde condities, en dit omwille van een verschil in melkzuurconsumptiesnelheid en pyruvaatsecretie. Voor A. senegalensis 108B werd aangetoond dat deze stam, naast melkzuur, ook op citroenzuur als enige koolstofbron kon groeien. De afwezigheid van de glyoxylaatcyclus, voorspeld op basis van het genoom, bij A. senegalensis 108B is in tegenstelling tot de soortbeschrijving, dewelke stipuleert dat deze azijnzuurbacteriesoort in staat is tot groei op ethanol als enige koolstofbron. Voor L. fermentum 222 en L. plantarum 80 leidde de ontwikkeling van GEMs tot nieuwe inzichten in de mogelijke metabole fluxverdelingen, voornamelijk ten aanzien van substraatverbruik. Het modelleren van de microbiële dynamica wees ook op een tekortkoming aan huidige kennis over deze stammen, bijvoorbeeld met betrekking tot het gebruik van ongedefinieerde substraten in een rijk groeimedium.Samenvattend werden door middel van de ontwikkelde GEMs van de kandidaat-functionele starterculturen voor cacaoboonfermentatieprocessen nieuwe inzichten verkregen in hun metabolisme en dit op systeemniveau.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

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

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

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.

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.

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

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

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

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

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.

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

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.

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: Glaucia Maria Pastore
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: As lipases são dotadas de ação hidrolítica sobre óleos e gorduras, produzindo ácidos graxos, glicerol, monoglicerídeos e/ou diglicerídeos. Além disso, lipases produzidas por Penicillium roqueforti, Penicillium camemberti e Geotrichum candidum contribuem para a produção de aroma e sabor característicos em queijos do tipo "Blue Cheese", os quais são desenvolvidos, principalmente, pela presença de metilcetonas. Nos últimos tempos, alguns autores vêm discutindo a possibilidade do emprego de algumas espécies de Aspergillus na produção de concentrados destes compostos, para utilização em produtos alimentícios com aroma de "Blue Cheese". Este trabalho teve como objetivo otimizar a produção de metilcetonas utilizando lipase extracelular de Aspergillus sp. pré selecionada. Para produção da lipase utilizou-se um meio de cultivo com farelo de trigo e as melhores condições para esta produção foram 60% de umidade após 120 horas de incubação a 35°C. Como substratos foram testados creme de leite de vaca, creme de leite de cabra e gordura de coco sendo que, apenas o úWmo foi considerado adequado devido ao seu conteúdo de ácido láurico (C-12:0) pelo qual a lipase utilizada mostrou afinidade. Neste substrato, a maior produção de metilcetonas totais foi 4,43 mg/g, obtida em pH 5,0 após 48 horas de incubação a 30°C. Nestas condições a metilcetona produzida em maior quantidade foi a 2-heptanona equivalendo a 3,98 mg/g de óleo de coco. Este pode ser considerado um bom resultado com potencialidade para aplicação industrial
Abstract: Lipases are able to hydrolyse oi! and fats, producing fat acids, glicerol, monoglicerides and/or diglicerides. Besides, lipases from Penicillium roqueforti, Penicillium camembertiand Geotrichumcandidum contribute to the aroma and characteristic flavor production of the "blue cheese" that are developed, mainly, by the methyl ketones presence. Lately some authors have been discussing the possibility of employementof some Aspergillus strains in the production of concentrated of these compounds for obtaining of nutritious products containing "blue cheese" aroma.The aim of this work was to optimize the methyl ketones productionby a Aspergillussp strain, previouslyselected as extracellular lipase producer. For lipase roduction,wheat bran was utilized as culture medium and the best conditionswere found with 60% of humidity, after 120 h of incubation, at 35°C. For methyl ketone synthesis cow and goat cream milk and coconut fat were tested as substract and only the latter was considered appropriated due to its lauric acid (C-12:0) with which fhe used lipase showed affinity. In this substrate, the largest total methyl ketones productionwas 4,43 mg/g, obtained in pH 5,0, after 48 h of incubation, at 30°C. In that condition, 2-heptanonewas the principal methyl ketone produced rising to 3.98 mg/g of coconut oi!. This can be considered a good result with potentialityfor industrialapplication
Mestrado
Mestre em Ciência de Alimentos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Biotechnology is inserted within the search for new products and processes, thus becoming an important tool in the development of biotechnological natural aroma production processes. Flavors compounds obtained from these processes represent a very promising and viable way of production for industries mainly due to the increase of consumer preference for natural food additives and products. Many microorganisms isolated from various environments have potential to synthesize flavors compounds when cultivated in an appropriate culture medium. Fermentation processes allow the use of agro-industrial wastes in which disposal on the environment may cause serious pollution issues. The residues composition, mostly the carbon source, allows the growth of microorganisms and the production of higher aggregate value compounds. The aim of this work is the to produce and quantify the 6-pentyl-á-pyrone lactone, responsible for the coconut-like aroma, by using agro-industrial waste as support and the filamentous fungus Trichoderma harzianum. Initially three agro-industrial wastes (sugarcane bagasse, coconut shell powder and cassava bagasse) were tested concerning the aroma production. Then the gas chromatography coupled to headspace extraction technique provided the quantitative and qualitative analysis of the lactone. Through the chromatogram area, the 4040 strain of Trichoderma fungus and the cassava bagasse were selected as culture medium. The latter was studied by the physical chemistry aspect. The tests done in order to evaluate the microorganism lactone toxic level did not show a complete inhibition in cell growth, only a few spores. The total factorial design in two levels was applied to verify the influence of the nutrients concentration (C/N) and temperature in the production of 6-pentyl-á-pyrone. The largest production of aroma compound resulted in 3.78 mg/g MS at the 7th culture day. The linear and quadratic terms related to temperature were important to the proposed model at a 95% trust level for aroma maximum concentration, by setting values between 25 and 28°C. The C/N ratio effect and the interaction between these two variables were not statistically meaningful.
A biotecnologia se insere na busca de novos produtos e processos, tornando-se uma ferramenta importante no desenvolvimento de processos de produção de aromas naturais biotecnológicos. Compostos de aroma obtidos por estes processos representam uma alternativa de produção bastante promissora e viável para as indústrias, principalmente devido ao aumento da preferência dos consumidores por aditivos alimentícios e produtos naturais. Muitos microrganismos, isolados dos mais variados ambientes, possuem potencial para sintetizar compostos de aroma quando cultivados em meios de cultura adequados. Os processos fermentativos possibilitam o aproveitamento de resíduos agroindustriais, cuja disposição no meio ambiente gera sérios problemas de poluição. A composição dos resíduos, principalmente a fonte de carbono, permite o crescimento dos microrganismos e produção de compostos de maior valor agregado. O presente trabalho tem como objetivo produzir e quantificar a lactona 6-pentil-á-pirona, responsável pelo aroma característico de coco, utilizando resíduo agroindustrial como suporte e fungo filamentoso Trichoderma harzianum. Inicialmente, três resíduos agroindustriais (bagaço de cana, pó da casca de coco e bagaço de mandioca) e cinco microrganismos foram testados quanto à produção do aroma. A técnica de cromatografia gasosa acoplada à extração em headspace permitiu as análises qualitativas e quantitativas da lactona. Através da área do cromatograma, a linhagem 4040 do fungo Trichoderma e o suporte bagaço de cana foram selecionados como meio de cultivo. O resíduo bagaço de cana foi caracterizado sob o aspecto físico-químico. Os testes realizados para avaliar o nível de toxidez da lactona sobre o microrganismo não apresentaram inibição total no crescimento celular, apenas pouca presença de esporos. O planejamento fatorial completo em dois níveis foi empregado para avaliar a influência da concentração de nutrientes (C/N) e da temperatura sobre a produção do 6-pentil-á-pirona. A maior produção do composto de aroma resultou em 3,78 mg/g MS no 7º dia de cultivo. Os termos lineares e quadráticos relacionados à temperatura foram significativos no modelo proposto a um nível de confiança de 95% para a máxima concentração do aroma, sendo direcionadas para valores de 25 a 28ºC. O efeito da razão C/N e de interação entre as duas variáveis não foram estatisticamente significativos.

This paper aims to compare qualitatively and quantitatively alcoholic fermentation promoted by yeast Saccharomyces cerevisiae cells immobilized in three inert supports: fiber of dwarf coconut (Cocos nucifera) and two varieties of bamboo (Phyllostachys caniço and Guadua angustifolia). The three materials were also evaluated on their use as supports. The process of immobilization was dumping of equal volumes of materials (two liters) into a suspension of yeast cells (in order of 109 cells / mL) followed by drainage and consequent food with equal volumes of must of molasses. The pH must was adjusted with commercial sulfuric acid to around 4.20 and received addition of 3 ppm of bactericidal for control of bacterial contamination. There was also addition of 10 ppm of nutrient for fermentation in all fermentative cycles. At the end of ninety-one fermentative cycles the process was interrupted, and observed that the three materials have proved physical resistance and that the efficiency of the process and efficiency of fermentation of yeast-coconut system had values above those of yeast-bamboo systems since initial stages, showing to be the fiber of coconut support for the immobilization of cells more efficient. However, around the cycle of number seventy-four, the three systems began to provide similar efficiencies, which suggests that there has been a gradual increase in the number of yeast cells immobilized in systems yeast-bamboo during the cycles. The counting of cells in free media fermented showed that there was satisfactory cell detachment related with the figures already cited in the literature.
O presente trabalho tem como objetivo comparar qualitativa e quantitativamente fermentações alcoólicas promovidas por células de leveduras Saccharomyces cerevisiae imobilizadas em três suportes inertes: fibra de coco anão (Cocos nucifera) e duas variedades de bambu (Phyllostachys caniço e Guadua angustifolia). Os três materiais também foram avaliados quanto a seu uso como suportes. O processo de imobilização consistiu da imersão de volumes iguais dos materiais (dois litros) em suspensão de células de leveduras (da ordem de 109 células/mL) seguida de drenagem e conseqüente alimentação com volumes iguais de mosto de melaço. O mosto teve o pH corrigido com ácido sulfúrico comercial para cerca de 4,20 e recebeu adição de 3 ppm de bactericida para controle da contaminação bacteriana. Também houve adição de 10 ppm de nutriente para fermentação em todos os ciclos de fermentação. Ao final de noventa e um ciclos fermentativos, o processo foi interrompido, sendo observado que os três materiais mostraram-se resistentes fisicamente e que a eficiência do processo e a eficiência da fermentação do sistema levedura-coco apresentaram valores superiores aos dos sistemas levedura-bambu desde os primeiros ciclos, mostrando ser a fibra de coco um suporte de imobilização de células mais eficaz. Contudo, por volta do ciclo de número setenta e quatro, os três sistemas começaram a apresentar eficiências similares, o que sugere que houve um gradativo incremento do número de células imobilizadas nos sistemas leveduras-bambu no decorrer dos ciclos. As contagens de células livres nos meios fermentados demonstraram que houve desprendimento celular satisfatório com relação a valores já citados na literatura.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
The liquid of the rind of green coconut (LCCV), an effluent stream from the industrial processing of green coconut rind, is rich in sugars and is a suitable feedstock for fermentation. The first step of this study was to evaluate the potential of natural fermentation of LCCV. As the literature did not provide any information about LCCV and due to the difficulty of working with such an organic effluent, the second step was to characterize the LCCV and to develop a synthetic medium to explore its potential as a bioprocess diluent. The third step was to evaluate the influence of initial condensed and hydrolysable tannins on alcoholic fermentation. The last step of this work was divided into several stages: in particular to evaluate (1) the influence of the inoculum, temperature and agitation on the fermentation process, (2) the carbon source and the use of LCCV as diluent, (3) the differences between natural and synthetic fermentation of LCCV, in order to determine the best process conditions. Characterization of LCCV included analyses of the physico-chemical properties as well as the content of DQO, DBO and series of solids. Fermentation was carried out in bench-scale bioreactors using Saccharomyces cerevisiae as inoculum, at a working volume of 5L and using 0.30% of soy oil as antifoam. During fermentations, the effects of different initial sugars concentrations (10 - 20%), yeast concentrations (5 and 7.5%), temperatures (30 - 50?C) and agitation rates (400 and 500 rpm) on pH/sugars profiles and ethanol production were evaluated. The characterization of LCCV demonstrated the complexity and variability of the liquid. The best conditions for ethanol conversion were (1) media containing 15% of sugar; (2) 7.5% yeast inoculum; (3) temperature set point of 40?C and (4) an agitation rate of 500 rpm, which resulted in an ethanol conversion rate of 98% after 6 hours of process. A statistical comparison of results from natural and synthetic fermentation of LCCV showed that both processes are similar
A utiliza??o do l?quido da casca de coco verde (LCCV) em fermenta??o surgiu como uma alternativa ao aproveitamento de um efluente, rico em a??cares ferment?veis, liberado pelas usinas de beneficiamento da casca de coco verde. A primeira fase deste trabalho foi avaliar o potencial fermentativo do l?quido da casca de coco verde atrav?s da fermenta??o natural do l?quido. Por n?o possuir informa??o dispon?vel na literatura e pela dificuldade de se trabalhar com um efluente org?nico, a segunda fase foi realizar a caracteriza??o do l?quido e a elabora??o de um meio sint?tico, para melhor explorar seu potencial como diluente em bioprocessos. A terceira fase, estudar a influ?ncia de taninos iniciais condensados e hidrolis?veis em fermenta??o alco?lica. A ?ltima fase foi dividida em tr?s etapas, na qual se avaliou a influ?ncia da quantidade de in?culo no processo fermentativo; a influ?ncia da fonte de carbono e do uso de LCCV como diluente; a temperatura; a agita??o e, finalmente, o estudo comparativo entre o LCCV in natura e sint?tico, nas condi??es ?timas de processo. Para a caracteriza??o foram realizadas an?lises f?sico-qu?micas do LCCV, bem como os teores de DQO, DBO e s?rie de s?lidos. As fermenta??es foram realizadas em biorreator de bancada, com volume de trabalho de 5L, Saccharomyces cerevisiae e 0,30% de ?leo de soja como antiespumante. Nas fermenta??es foram avaliadas diferentes concentra??es de a??cares iniciais (10 a 20%) e de levedura (5 e 7,5%), e diferentes temperaturas (30 a 50?C) e agita??es (400 e 500rpm). Durante o processo foram analisados o perfil de pH e de a??cares, e a produ??o de etanol. A caracteriza??o do LCCV demonstrou a complexidade e variabilidade do l?quido. As melhores condi??es foram alcan?adas nos meios contendo 7,5% de levedura, 15% de a??cares, 40?C, sob agita??o de 500rpm, sendo obtida uma efici?ncia de convers?o em etanol de 98% ap?s 6 horas de processo. Durante o estudo comparativo do LCCV in natura e sint?tico, atrav?s dos par?metros avaliados durante a fermenta??o, um estudo estat?stico revelou a similaridade do meio sint?tico com o meio natural

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O Brasil é um dos principais produtores mundial de abacaxi e maracujá, com uma grande quantidade de resíduos desperdiçados que já se tornou um sério problema aos produtores de abacaxi e maracujá e as indústrias de alimentos em geral. Em países desenvolvidos, a produção agropecuária se caracteriza com baixos custos operacionais, em razão das utilizações de restos de culturas e de resíduos agroindustriais como alternativas na alimentação animal, contribuindo para minimizarem os problemas de poluição. O objetivo deste trabalho foi definir ações no campo da pesquisa na alimentação alternativa nutricional para animais , de modo a realizar o aproveitamento destes resíduos enriquecidos nutricionalmente (protéico, vitamínico, mineral e energético) utilizando a levedura Saccharomyces cerevisiae como agente da metabolização, de uma maneira tecnicamente viável, visando a disponibilidade do produto, para os produtores. Foram feitas as isotermas de dessorção dos resíduos em estudo, nas temperaturas usuais dos processos das fermentações, 25, 30, 35 e 40 0C, ajustando os dados através da aplicação de modelos matemáticos, e verificou-se que, deve-se iniciar o processo de fermentação com umidade inicial do substrato para os três resíduos acima de 84% (b.u.), o que equivale a atividade de água acima de 0,90. No estudo cinético verificou-se que o melhor tempo em que o microrganismo atingiu o maior teor de proteína bruta foi, em média, de 48 h, para todos os resíduos analisados. Utilizou-se a metodologia de planejamento fatorial mais configuração estrela para estudar as influências das variáveis de entrada sobre o processo de enriquecimento nutricional dos resíduos casca de abacaxi (Caa), coroa de abacaxi (Coa) e casca de maracujá (Cam), com concentrações de leveduras de 1, 3 e 5% e temperaturas de 30, 34 e 38 oC. Fez-se o planejamento fatorial mais configuração estrela para os três resíduos, com duas variáveis de saída (respostas: teor de proteína bruta e teor de aumento protéico). O aumento protéico (AP) encontrado para os três resíduos analisados, foi em média de 2,40 vezes em relação ao in natura. Os valores otimizados dos três resíduos casca de abacaxi (Caa), coroa de abacaxi (Coa) e casca de maracujá (Cam) (concentração Oliveira M.M.de 14 em torno de 3 – 5,8% e temperaturas de 34 – 40 oC), no tempo de 48 h, visando baratear os custos do enriquecimento e obter um teor de proteína adequado na alimentação animal foram de 24,66, 23,88 e 22,74%, respectivamente. Foram realizadas as análises de proteína bruta (PB), matéria seca (MS), cinzas (MM), matéria orgânica (MO), fósforo (P), cálcio (Ca), magnésio (Mg), potássio (K), fibra detergente neutra (FDN), fibra detergente ácida (FDA), energia bruta (EB) e digestibilidade “in vitro” (DIVMS in vitro) nos resíduos operando com as variáveis de entrada (concentração de levedura e temperatura) com valores otimizados. Conclui-se que através da bioconversão, os resíduos alcançam concentrações nutricionais que, podem ser transformados em suplemento nutricional, sendo uma alternativa alimentar para os animais, na época de escassez de alimento no semiárido.
Brazil stands out among the big world-wide producers of pineapple (Ananas comosus L. Mer) and passion fruit (Passiflora edulis Sims), with a great amount of wasted residues, which has become a serious problem for the pineapple and passion fruit producers and the aliment industries in general. In developed countries, the farming production is characterized by low operational costs, because of the use of the remaining portions of cultures and agro-industrial residues as an alternative in animal feed, which also contributes to minimizing pollution problems. The aim of this work was to define actions in the field of research of nutritional alternative feed for animals, in order to carry through the exploitation of these nutritionally enriched (protein, vitamin, mineral and energy) residues by using Saccharomyces cerevisiae yeast as an agent of metabolism, in a technical viable way, aiming at the availability of the product to the producers. Desorption isotherms of the residues in study were done at the usual temperatures of fermentation processes, 25, 30, 35 and 40 oC, fitting the data through the application of mathematical models, and it was verified that the process of fermentation must be initiated with the initial moisture content of the substratum for the three residues above of 84% (w.b), which is equivalent to the water activity above of 0.90. In the kinetic study it was verified that optimum time where the microorganism reached the highest content was, on average, 48 hours, for all the residues analyzed. Methodology of factorial design plus configuration star was used to study the influences of the entrance variable on the process of nutritional enrichment of the residues of pineapple rind (PR), crown of pineapple (CP) and rind of passion fruit (PF), with concentrations of yeast at 1, 3 and 5% and temperatures of 30, 34 and 38 °C. The factorial design plus configuration star was performed for the three residues, with two exit variables (response: gross protein content and protein increase content). The protein increase content (PI) found for the three analyzed residues was on average of 2.40 times in relation to in natura. The optimized values of the three residues of pineapple rind (PR), crown of pineapple (CP) and passion fruit rind (PFR) (concentration around 3-5.8% and Oliveira M.M.de 16 temperatures of 34-40 °C), in the time of 48 hours, aiming to lower the costs of the enrichment and to obtain an adequate protein content in the animal feed were of; 24.66, 23.88 and 22.74%, respectively. Gross protein (GP), dry substance (DS), leached ashes (LA), organic substance (OG), phosphorus (P), calcium (Ca), magnesium (Mg), potassium (K), fiber neutral detergent (FND), acid fiber detergent (AFD), rude energy (RE) and digestibility in vitro (DIVMS in vitro) analysis were performed in the residues, operating with the entrance variables (concentration of yeast and temperature) with optimized values. It can be concluded that through bioconversion, the residues reach nutritional concentrations that can be transformed into a nutritional supplement alternative, being an option to feed animals at times of food scarcity in semi-arid climates.

碩士
國立屏東科技大學
食品科學系所
105
Healthy awareness on people’s choice of food is rising, thus, the demand of pure and nature healthy foods is increasing. There are many different kinds of chocolate in the markets today because of its beneficial effect when consumed. This benefit mainly comes from the chemical composition contained within the chocolate. And chocolate by far is a popular confectionary all over the world. However in Taiwan, cocoa cultivars producing chocolates are known to have lack of special flavor and taste. Thus, chocolates fermenting process has to be established to improve the flavor and teste. In doing so, the cocoa pod mucilage initially acts a substrate for the existing microorganisms. The fermentation of cocoa beans can produce different microflora. Next, spontaneous fermentation of cocoa bean was investigated. Strains with highest acidity and lowest pH of acetic acid bacteria, and highest alcohol production of yeast, and diacetyl production of lactic acid bacteria were isolated. The first strain was identified as Saccharomyces cerevisiae. The medium containing 2％(w/v) fructose and 0.5％(w/v) yeast extract with pH 7.0 was prepared. 2％(v/v) inoculum ratio of S. cerevisiae was added into the medium and was incubated at 30℃ for 28 hours. The second strain was Acetobacter pasteriunus, medium containing 2.0％(w/v) yeast extract and 1.0％(v/v) alcohol with pH 5.0 was also prepared. Similarly, 1％(v/v) inoculum ratio of A. pasteriunus was transferred into the medium and was incubated at 30℃ (115 rpm) for 30 hours. The third strain was Lactobacillus rhamnosus. The medium containing 2％(w/v) glucose and 1％(w/v) tryptone with pH 8.0 was prepared. 2％(v/v) inoculums ratio of L. rhamnosus was added into the medium and was incubated at 30℃ for 28 hours. The fermentation of cocoa seeds are divided into two step fermentations (aerobic and anaerobic process), which involve control of the amounts of acetic acids and culture temperature. In the anaerobic stage, the growth of lactic acid bacteria and yeast was observed by dialysis culture. Dialysis culture results showed that after S. cerevisiae was cultured for 36 hours at 30℃ couldn‘t be inhibited by inoculating L. rhamnosus. This provides a basis when inoculating lactic acid bacteria in cocoa beans. This study investigated the microorganisms in Taiwan’s cocoa beans in order to obtain special flavor during fermentation. From inspection of microorganisms adding in cocoa beans, the change of the alcohol fermentation, titratable acidity and pH value were detected. After 40 hours (30℃) of fermentation of cocoa beans with S. cerevisiae(10％, v/w; 8.10 Log CFU/mL), fermented cocoa beans had the highest alcohol concentrations 1.40％. At the same time, inoculation of Lactobacillus rhamnosus(10％, v/w; 8.50 Log CFU/mL) could reduce the total phenol content and did not affect the yeast. Moreover, in according with the results of optimal culture conditions, A. pasteriunus (15％, v/w; 8.60 Log CFU/mL) inoculated into cocoa beans at 30℃ for 88 hours of fermentation, the none –mucilase backed cocoas had the lowest total phenol (11.12 μg/mL) and titratable acid (0.09％) at the end point of fermentation. Therefore, controlling the cocoa fermentation can be done through suitable inoculum concentration of starter cultures, control of fermentation time, they can produce good flavor. Finally, from the result by controlling fermentation cocoas will can set-up a stable quality and shorten the fermentation time, and producing low phenal, low acidity cocoas and good flavor cocoas.

碩士
環球科技大學
生物技術研究所
107
In recent years, cocoa beans’ trading has increased in Taiwan which indicates that the inclination of Taiwanese people’spreference in chocolate. In the early years, cocoa trees were first plantedin Pingtung, South Part of Taiwan. Through the improvement of field management, cocoa cultivation has planted at the Central Part of Taiwan.However, due to the geographical of Taiwan, causing the differences climate between Central and South part of Taiwan. These differences climate may affect the quality of cocoa especially antioxidant capacity. Therefore, the purpose of this research is to compare of the antioxidant capacity (FRAP, DPPH and TPC) before and after fermentation between the four seasons in central and southern part of Taiwan. The FRAP, DPPH and TPC tests were carried out by water extraction and methanol extraction, respectively. The results showed that DPPH scavenging activities of cocoa bean before fermentation in central and southern part of Taiwan showed no significant difference, the scavenging ability in all treatments are above 95%except fermented cocoa in Middle and South part of Taiwan has lower the highest scavenging activities on May,39.2% and 93.1%, respectively. In FRAP analysis showed that either central or southern part of Taiwan showed significant difference. In methanol extract, the highest FRAP reduction ability was found in unfermented cocoa on November (1106.6μmol/g) and fermented cocoa was found on February (417.4 μmol/g). The TPC content analysis showed that the highest content in unfermented cocoa harvested on February at central part of Taiwan(50.2μg/gDW) and the fermentedcocoa was found on February (23.06μg/gDW) in methanol extract. However, in water extract, the south part of Taiwan fermented cocoa which harvested on November was the highest (15.09μg/gDW)。

碩士
國立宜蘭大學
食品科學系碩士班
106
Soybean residue and rice bran are rich in nutrients and active components, but high moisture content of extracted residue or high lipase activity could cause rapidly decaying and hard to be application except for animal feed or fertilizer. Radio frequency (RF) can rapidly heat up water molecules or ions in food by rapid conversion of electric field; therefore, RF drying can overcome the heat and mass transfer resistances to accelerate drying processing. The objectives of this study were to dry the soybean residue and to inactive lipase of rice bran by RF heating. Then the soybean residue and rice bran were the media for Poria cocos solid-state fermentation to increase bioactive components and improve antioxidant activities. The results showed that they required 14, 22 and 30 min for reducing moisture content from 80% to 15% to dry 1, 1.5 and 2 kg of soybean residue by RF, respectively. RF drying rate was 25 times of 45℃ cold air drying, and the total energy consumption was reduced to 1/9. The total polyphenols content and DPPH free radical scavenging ability in RF soybean residue were higher than cold air dried soybean residue, but there was no significant difference in color between two different drying methods of soybean residue. RF dried soy residue contains 54.5% of carbohydrates, 27.6% of crude protein and 10.6% of crude fat. On the other hand, 1 kg rice bran was stabilized by RF with gap of 6 cm heating 2 min to inhibit lipase activity without affecting color and antioxidant activity. RF stabilized rice bran contains 48.3% of carbohydrates, 15.4% of crude protein and 19.1% of crude fat. RF really stabilized rice bran during 8 weeks storage at 4, 25, and 37℃. Finally, Poria cocos solid-state fermentation was carried out with different fermentation time periods (0, 30 and 60 days) with 500 g mixed medium (40% moisture content) with different ratios of soybean residue and rice bran (1:0, 2:1, 1:1, 1:2 and 0:1). All the contents of crude polysaccharide, crude triterpenoids, total polyphenols and flavonoids in Poria cocos solid-state fermentation product increased with the increase of the proportion of rice bran in the mixed media and fermentation time. However, considering the time cost and other factors, the suitable Poria cocos solid-state fermentation conditions: 40% of the moisture content, soybean residue and rice bran = 1:1, at 25℃ for 30 days. It took only 30, 200 sec at the RF electrode gap of 15 cm to pasteurize and reduce the moisture content below 15%; therefore, it could replace the traditional sterilization 60 min in an autoclave and cold air drying 100 min at 45℃. RF heating greatly shorten pasteurization and drying time of Poria cocos solid-state fermentation product in 3 min to complete, but also retain the appearance of Poria cocos white mycelium color, to avoid browning. The Poria cocos fermentation product after RF treatment contained 5.03% of mycelium, 9.83% of crude polysaccharide, 4.43% of crude triterpenoids, 3.54 mg gallic acid equivalent/g DW of total polyphenols, 0.38 mg quercetin equivalent/g DW of flavonoid content and good antioxidant capacity. Zebrafish animal experiments confirmed the 200 mg/L extracts from the Poria cocos solid-state fermentation product to have antioxidant activity.

碩士
大葉大學
食品工程研究所
90
Wolfiporia cocos is a medicinal fungi, its sclerotium has been long been used as traditional Chinese herb with diuretic, sedative and tonic.Pharmacology studies have proven that its avtive ingredients possess anti-inflammatory, anti-aging, immunity, anti-tumor, sedative, diuretic and anti-emetic properties. Triterpenes and pachymaran have been reported as major biochemical activities. Mycelium is cultured through fermentation and used in health food products to develop product into commercial quantities. Fermentation culture using the chemically defined media culture followed. The process not only prevented interference during the effective ingredient extraction, but also further controlled the Wolfiporia cocos growth within the condition required for the effective ingredients. Then chemically defined media was added to Wolfiporia cocos and cultured in 25℃controlled temperature oscillator to evaluate the effects of the initial pH, different carbon-nitrogen ratio, glucose concentration, nitrogen concentration and inoculation dosage of the culture medium on the mycelium biomass , reducing sugar and the mycelium extracellular polysaccharide composition, and analyzed the crude protein, crude fat, total sugar ,ash and water content of mycelia and sclerotium. Findings showed that initial pH was 3.0；carbon-nitrogen ratio was 30:1；glucose concentration was 5.0%； and the mycelium biomass content of the nitrogen concentration was 1% fermented culture mycelium biomass was higer. And extracellular polysaccharid and showed that initial pH was 3.0; carbon-nitrogen ratio was 30:1; glucose concentration was 5.0%; and the mycelium biomass content of the nitrogen concentration was 1% fermented culture was higer. A 5L fermentation tank was used to stuffy the physicochemical factor including the different temperature, different stirring speed, changes of the mycelium biomass, reducing sugar, pH in supernatant and extraceullar polysaccharide under 168-hours fermentation process. Finding showed that maximum mycelium biomass was achieved under 30℃and 200 rpm stirring speed. Extracellular polysaccharide was achieved under a 30℃, 200 rpm stirring speed.