The increasing demand for flavors in the world market requires new strategies for obtaining these compounds which can be produced via fermentation processes using low cost substrates such as agroindustrial residues. This turns out to be a viable process and the aroma compounds generated are classified as natural. Large volumes of waste in the form of bagasse, husks, kernels or seeds including from the processing of fruit pulp are generated and these can be used as substrates in these fermentations. Thus the main purpose of this work was to use agro-industrial residues for the production of volatile aroma compounds by fermentation processes. In this regard, we investigated the potential of the production of volatile compounds by submerged fermentation using guava, passion fruit and umbu processing waste as substrates using endophytic microorganisms isolated from guava, passion fruit and umbu as well as by using the pure cultures (Aspergillus niger, Kluyveromyces marxianus and Kluyveromyces lactis) to produce volatile compounds characteristic of fruity or floral aroma. Initially the fruit pulp and wastes from the processing of guava, passion fruit and umbu were chemically characterized (moisture, ash, fiber, proteins, lipids, sugars, titratable acidity, Aw, pH and soluble solids). Fermentation with different microorganisms in synthetic medium was performed and the fermented products analyzed for aroma note. From the results of this sensory analysis, the guava residue and the yeast Kluyveromyces lactis were selected since these constituents in the medium produced fruity and floral aromas. The volatile compounds produced were captured by two headspace techniques (SPME and Purge & Trap) and analyzed in a system of gas chromatography coupled to mass spectrometry. Sixty-two microorganisms were isolated and out of these six were selected. The fermented products were analyzed for pH, soluble solids and reducing sugars as well as to establish the kinetics of the fermentation process at every 24 hours for five days. The main aroma descriptors in the fermented products were fruity, honey, fruity green, plum, guava, banana and alcohol type notes. Forty-three volatile compounds were detected in the fermented product obtained by using the Guava residue in the medium. Among the compounds identified were: Ò {caryophyllene, 2-phenylethyl acetate, × n- humulene, bisabolene, humulene, 3-methyl butanol, bergamoteno, × n-cadinene, caryophyllene oxide and 2-phenylethanol. The production of volatile compounds was quite rapid and in most cases, this production occurred on the first day of fermentation and had achieved its maximum concentration between 24-48h of fermentation. According to the response surface method analysis, the conditions of the manufacturing process were optimized. Increased production of 2- phenylethyl acetate was achieved at concentrations varying from 25 and 35g/L of yeast extract, 40 to 60 g/L glucose. Ò-caryophyllene was obtained at concentrations varying between 120 and 140g/L of guava waste, and an agitation performed between 160 and 180 rpm. The production of 2-phenylethanol was obtained at agitation varying from 228rpm and 5g/L glucose. From these optimized conditions, scale-up of fermentation was performed in a bioreactor wherein 6 times larger concentration of 2-phenylethanol and Ò-caryophyllene compounds, and 1.5 times higher concentration of 2-phenylethyl acetate were obtained. This study reveals that guava residue can be used to produce aroma compounds of great importance for the flavor and fragrance industry, the compounds being 2-phenylethyl acetate, 2-phenylethanol and Ò -caryophyllene. / A crescente demanda de aromas no mercado mundial exige novas estrategias de obtencao desses compostos. A obtencao via processos fermentativos utilizando substratos de baixo custo e uma alternativa economicamente viavel e o aroma gerado e considerado como natural. Grandes volumes de residuos na forma de bagacos, cascas, carocos ou sementes sao gerados pelo processamento de frutas, e estes podem ser utilizados como substratos nesses processos fermentativos. Diante desse contexto, esse trabalho teve como principal finalidade utilizar residuos agroindustriais para a producao de compostos volateis de aroma atraves da fermentacao submersa. Neste sentido, foi verificada a potencialidade da producao de compostos volateis atraves da fermentacao submersa utilizando residuos de goiaba, maracuja e umbu como substratos utilizando microrganismos endofitos da goiaba, maracuja e umbu de cultura puras (Aspergillus niger, Kluyveromyces marxianus e Kluyveromyces lactis) para produzir compostos volateis de aroma frutal ou floral. Inicialmente a polpa e os residuos de goiaba, maracuja e umbu foram caracterizados quimicamente (umidade, cinzas, fibras, proteinas, lipideos, acucares, acidez titulavel, Aw, pH e solidos soluveis). Foram realizadas fermentacoes com os diferentes microrganismos em meio sintetico, onde os produtos fermentados foram analisados sensorialmente. A partir dos resultados obtidos da analise sensorial, o residuo de goiaba e a levedura Kluyveromyces lactis foram selecionados uma vez que estes apresentaram a melhor capacidade de producao de aromas. Os compostos volateis produzidos foram capturados por duas tecnicas de headspace (MEFS e Purge & Trap) e analisados por cromatografia gasosa acoplada a espectrometria de massas. Foram isolados 62 microrganismos, dentre estes seis foram selecionados e os produtos fermentados foram analisados por cromatografia gasosa como tambem as caracteristicas de pH, solidos soluveis e acucar redutor para estabelecer a cinetica do processo fermentativo a cada 24h durante cinco dias. As principais descricoes de aroma para o fermentado de residuo de goiaba foram frutal, mel, fruta verde, ameixa, goiabada, banana, fermento e alcool. Foram detectados 43 compostos volateis no produto fermentado obtido pelo uso do residuo de goiaba. Dentre os compostos identificados destacam-se: Ò-cariofileno, acetato de 2-feniletil, ×-humuleno, bisaboleno, humuleno, 3-metil butanol, bergamoteno, ×-cadineno, oxido de cariofileno e 2-feniletanol. A producao dos compostos volateis foi bastante rapida, e na maioria dos casos, essa producao aconteceu a partir do primeiro dia de fermentacao e teve o seu maximo entre 24-48h de fermentacao. Segundo a analise da superficie de resposta, o processo de producao foi otimizado, onde a maior producao do acetato de 2-feniletil foi obtida em concentracoes entre 25 a 35g/L de extrato de levedura, entre 40 a 60 g/L de glicose; do Ò-cariofileno foi obtido em concentracoes entre 120 a 140g/L de residuo de goiaba, e agitacao entre 160 e 180 rpm enquanto a producao do 2-feniletanol foi obtida na agitacao entre 228rpm e 5g/L de glicose. A partir dessas condicoes otimizadas foi possivel realizar um escalonamento da producao desses compostos no biorreator e foi possivel obter uma producao 6 vezes maior para os compostos 2-feniletanol e Ò-cariofileno, e 1,5 vezes para o acetato de 2-feniletil. Desta forma, neste trabalho verifica-se que o residuo de goiaba e leveduras isoladas de frutas podem ser utilizados para produzir compostos de aroma de grande importancia para a industria de aromas e fragrancias, como o acetato de 2-feniletanol, o 2-feniletanol e o Ò-cariofileno.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-05T15:49:15Z (GMT). No. of bitstreams: 1 Mattoso_Eduardo_M.pdf: 1490484 bytes, checksum: afbf4ecf4ea37d17252e5d09173a2db0 (MD5) Previous issue date: 2005 / Mestrado / Quimica Organica / Mestre em Química

Orientador: Helena Teixeira Godoy / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T18:38:45Z (GMT). No. of bitstreams: 1 Sganzerla_Marla_D.pdf: 11263877 bytes, checksum: 32ac84fea7f4793868e0e58797653653 (MD5) Previous issue date: 2015 / Resumo: As pimentas do gênero Capsicum apresentam ampla aplicação em função dos atributos sensoriais de cor, pungência e aroma, decorrentes da presença de carotenoides, capsaicinoides e voláteis. No entanto, variações no conteúdo desses metabólitos secundários dependem de fatores como sazonalidade, ritmo circadiano, desenvolvimento da planta, características edafo-climáticas, ataque de patógenos. Neste estudo foi realizada a caracterização da composição de capsaicinoides e fração volátil de frutos de Capsicum chinense. A caracterização foi realizada através do uso de métodos analíticos cromatográficos (UPLC, UHPLC-MS e GC-MS). Com a execução desta pesquisa, foi possível caracterizar os frutos de C. chinense quanto à composição de metabólitos relacionados aos seus principais parâmetros de qualidade, fornecendo subsídios científicos para o conhecimento e melhoria da qualidade em termos de produção, aplicação industrial, compreensão de rotas metabólicas e melhoramento genético, além de gerar informações a respeito de substâncias potencialmente benéficas, devido a suas ações no organismo humano. Inicialmente foram desenvolvidos e validados os métodos, usando ferramentas estatísticas. De acordo com a etapa de otimização para a extração dos capsaicinoides, 100% de metanol combinado com o uso do banho de ultrassom por 10 minutos foram empregados. O método analítico desenvolvido através do uso de cromatografia em fase líquida de ultra eficiência permitiu a separação de 8 capsaicinoides em 4 minutos de análise cromatográfica e mostrou-se eficiente de acordo com a validação realizada. 9 acessos de pimentas C. chinense foram analisadas. Os teores de capsaicina e dihidrocapsaicina obtidos estavam nas faixas de 156 - 1442 ?g.g?1 e 26 - 478 ?g.g?1 (peso de fruto fresco), respectivamente. A caracterização do perfil de voláteis em pimentas 'Habanero', através do uso de HS-SPME (Headspace Solid-Phase Microextraction), foi realizada após etapa de otimização. Delineamento composto central e ferramenta de desejabilidade foram usadas a fim de avaliar, simultaneamente, duas respostas: soma total da área dos picos e número de compostos extraídos. O resultado maximizado para as duas respostas usando a fibra tripla (DVB/Car/PDMS) indicou o uso das condições de temperatura de extração de 40 °C e 30 min de extração. 82 compostos foram tentativamente identificados na fração volátil da pimenta 'Habanero' e os ésteres foram os compostos mais abundantes. O 2,3-dimetilciclohexanol e longifoleno foram identificados pela primeira vez em pimentas 'Habanero'. Além disso, pimentas 'Habanero' submetidas à diferentes condições de disponibilidade de água durante seu cultivo foram avaliadas quanto a composição de capsaicinoides e voláteis. Maiores quantidades de capsaicina e dihidrocapsaicina foram encontradas na pimentas maduras, alcançando valores entre as faixas de 2,85 - 3,33 mg.g-1 para capsaicina e 1,06 - 1,71 mg.g-1 para dihidrocapsaicina. Os teores de compostos voláteis totais foram maiores nas pimentas verdes (195,47 - 298,94 mg.kg-1), independente do tratamento de estresse hídrico que receberam. Os resultados obtidos com o PCA realizado possibilitaram, inicialmente, separar as pimentas 'Habanero' em função do grau de maturação. Enquanto, PCA aplicado, separadamente, nas pimentas verdes e maduras demonstrou uma separação evidente em função da data de colheita. Isso se deve ao fato de que influências do grau de maturação e época da colheita se sobressaíram no estudo. As pimentas foram agrupadas de acordo com o tratamento de irrigação recebido, mas para uma melhor compreensão dos efeitos dos tratamentos de estresse hídrico sobre a composição das amostras, seria necessário uma estratégia de estudo com monitoramento mais frequente dos frutos formados além de outras investigações sensoriais, como o uso de olfatometria. / Abstract: The Capsicum peppers are largely used due their sensorial properties as color, pungency and aroma. These characteristics occur by the presence of determinate classes of compounds including carotenoids, capsaicinoids and volatiles. These secondary metabolism derivatives compounds are influenced by two factors: genetics and plant-environment interactions and are susceptible to water availability conditions. This study had a proposal of the characterization of the capsaicinoids and volatile composition of the Capsicum chinense pepper fruits. The analysis were carried out by using of chromatographic methods (UPLC, UHPLC-MS and GC-MS). The results obtained and the characterization of the C. chinense fruits regarding the mainly compounds which influenced in the quality of the peppers provide the scientific subsidies for improvement of general quality, agricultural practices, industrial applications and breeding programs and also, the knowledge about substances potentially beneficial to the human organism. Initially, was developed and validated a fast, efficient and reproducible method to analyze capsaicinoids in Brazilian Capsicum chinense fruits by means the use of an optimization strategy to extracted the capsaicinoids. The extracts were obtained following the condition 100% of methanol and 10 min on ultrasound assisted extraction. The analytical method developed in an ultra high performance liquid chromatographic system coupled to a mass spectrometer permits the separation of 8 capsaicinoids in 4 min of time analysis expending only 2 mL of solvent as mobile phase. The method achieved was fully validated and show the effectiveness and satisfactory performance to answer the analytical needs of this research area. Different accessions of C. chinense fruits were analyzed, the contents of capsaicin and dihydrocapsaicin were in the range of 156 - 1442 ?g.g?1 and 26 - 478 ?g.g?1 of fresh fruit, respectively. Characterization of aroma profile of Capsicum chinense peppers 'Habanero' type, using Headspace Solid-Phase Microextraction (HS-SPME), was performed after an optimization step by GC-MS. Central composite design and Derringer's desirability function strategies were used for this optimization, in order to evaluate simultaneously the 'total sum peak areas' and 'number of extracted compounds' responses. The maximized results for both responses were obtained using PDMS/Car/DVB fiber, temperature of 40 °C and extraction time of 30 min. Eighty-two compounds were tentatively identified in the volatile fraction of the 'Habanero' pepper and the most abundant were hexyl isovalerate, cis-hexenyl isovalerate, hexyl 3-methylbutanoate, 3,3-dimethylcyclohexanol, longifolene, and 2-methyl-1-tetradecene. The compound 2,3-dimethylcyclohexanol and longifolene were reported for the first time in 'Habanero' pepper. In addition, Brazilian 'Habanero' peppers grown under different condition of water availability were evaluated regarding their capsaicinoids and volatiles composition. The higher values of capsaicin and dihydrocapsaicin were found in the mature peppers, achieving ranges from 2.85 - 3.33 mg.g-1 for capsaicin and 1.06 - 1.71 mg.g-1 for dihydrocapsaicin. Total volatile compounds presented higher values for the peppers in green stage of maturity with ranges from 195.47 - 298.94 mg.kg-1, whereas in the mature samples the range was from 68.74 - 118.50 mg.kg-1. According the results obtained with the PCA performed was possible separe the 'Habanero' peppers in function of the degree of maturity. While, PCA apllied, separately, for green and mature peppers showed a clear separation in function of the harvest date. This fact can be justified by the large influence of maturity degree and harvest date on the peppers composition. The peppers were clustered in function of the water availability treatments received, but to better understand the effects of water availability on the peppers composition, another strategy of evaluation, with more frequent monitoring would be interesting, as well as the inclusion of sensorial studies, as the olfactometry technique. / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

La carencia de nitrógeno es uno de los principales problemas encontrados en la elaboración del vino, especialmente relacionados con fermentaciones lentas e incompletas. En condiciones de vinificación, bajos niveles iniciales de nitrógeno limitan el crecimiento y la producción de biomasa, provocando una baja tasa de fermentación. Los compuestos nitrogenados presentes en el mosto también influyen en la producción de metabolitos volátiles y no volátiles que son los que regulan el perfil sensorial y la calidad del vino. Actualmente, el método más común utilizado para tratar carencias de nitrógeno en la fermentación, es la adición de nitrógeno. Sin embargo, el efecto de estas adiciones se rige por los requerimientos específicos nitrogenados de cada cepa y de las condiciones de fermentación. Esta tesis doctoral estudia el metabolismo nitrogenado de cuatro cepas vínicas comerciales, ampliamente utilizadas en la industria vínica española, especialmente en lo referente al crecimiento celular y la actividad fermentativa, así como la producción de metabolitos. Este estudio se ha centrado en la importancia de la cantidad y la calidad de nitrógeno presente en el mosto, con el fin de lograr un rendimiento óptimo de la fermentación. A través de este trabajo la eficiencia de diferentes marcadores ha sido probada con el fin de encontrar un biosensor que pueda ser utilizado en la detección de condiciones limitantes de nitrógeno durante el proceso de fermentación. Este trabajo se ha realizado utilizando diferentes técnicas bioquímicas, analíticas y moleculares. Así, algunas variaciones genéticas implicadas en la utilización del nitrógeno se han detectado, permitiendo en el futuro la posibilidad de mejorar el rendimiento fermentativo de estas cepas. / Gutiérrez Linares, A. (2013). Análisis del metabolismo nitrogenado durante la fermentación alcohólica en levaduras vínicas (Nitrogen metaolism in wine yeast during alcoholic fermentation: effect on growth, fermentation activity and aroma production) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/30774 / TESIS

Farroupilha, localizado na Serra Gaúcha, é o município que possui a terceira maior produção de uvas para processamento do Brasil e o maior produtor de uvas moscatéis, representando quase a metade da produção brasileira. Nos últimos dez anos, a produção e comercialização de espumantes moscatéis no Brasil aumentaram significativamente, demonstrando um crescente consumo anual. Esse produto vem conquistando diversas medalhas em concursos internacionais e agrada muito o paladar do brasileiro devido seus aromas frescos e frutados. Outro aspecto importante foi a conquista em 2015, da Indicação de Procedência de vinhos finos moscatéis Farroupilha, contribuindo ainda mais para o destaque desses produtos no mercado brasileiro e no exterior. Dentre as principais variedades cultivadas e utilizadas para a elaboração de vinhos finos tranquilos e espumantes moscatéis estão a Moscato Branco, Moscato Giallo, Moscato R2 e Malvasia de Candia. O conhecimento mais aprofundado das características sensoriais, aromáticas e qualitativas destes produtos, são importantes pois permite caracterizar o perfil dos vinhos e espumantes dessa região. O objetivo desse trabalho foi: determinar a composição aromática dos espumantes moscatéis de Farroupilha; sua evolução durante o armazenamento; caracterizar os vinhos elaborados com variedades moscatos e determinar a influência das leveduras Saccharomyces cerevisiae e Torulaspora delbrueckii na composição química e sensorial de vinhos Moscato Branco. Foram realizadas análises físico-químicas, cromatográficas e sensoriais. Os resultados permitem atribuir aos espumantes com menor tempo de armazenamento aromas cítricos e frutados e aos espumantes com maior tempo, notas de maçã, trufas, amêndoas, rosas e mel. Cada variedade de uva com a qual estes espumantes foram elaborados aportam diferentes aromas para os mesmos, contribuindo para uma grande compexidade aromática. A variedade Moscato Giallo aportou aos vinhos aromas que remetem a frutas como pera e pitanga, e ervas de quintal como alecrim. Os vinhos da variedade Moscato R2 destacaram-se pelas notas de frutas cítricas e alecrim, e os vinhos da variedade Moscato Branco apresentaram notas cítricas, pera e pitanga. As leveduras utilizadas também influenciaram na composição aromática. Os vinhos elaborados com leveduras puras de T. delbrueckii apresentaram maiores teores de acetato de isoamila (banana), hexanoato de etila (frutas, anis), 2-metil-1-propanol (alcoólico) e 2-feniletanol (rosas). Essas concentrações tendem a diminuir nas fermentações mistas. Vinhos produzidos com S. cerevisiae 100% EC 1118 se destacaram pelos maiores teores de etanal (maçã verde), 1-propanol (pungente), octanoato de etila (abacaxi, pera) e linalol (rosas). Dessa forma, este estudo demonstra que apesar da tendência do consumo de espumantes moscatéis e vinhos moscatos serem no período de 12 a 18 meses, esses produtos têm potencial para serem consumidos com maior tempo. Verificou-se também que os espumantes moscatéis de Farroupilha apresentaram uma qualidade geral equivalente aos espumantes elaborados na região de Asti, região de Denominação de Origem tradicionalmente conhecida com quase cem anos de história. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES / Farroupilha, located in the Serra Gaúcha region, is the county that owns the third largest production of grapes for processing in Brazil and the largest producer of muscat grapes, representing almost a half part of Brazilian production. In the last ten years, the production and commercialization of sparkling wines muscat in Brazil increased significantly, demonstrating a growing annual consumption. This product pleases the palate of the Brazilian and also of consumers from other countries, because every year sparkling Brazilian muscat win several medals in international competitions. Another important aspect was the achievement in 2015, of the Geographical Indication of Origin of wines from Farroupilha, contributing even more to the highlight of these products in the Brazilian and abroad market. Among the main varieties cultivated and used for the elaboration of quiet wines and sparkling wine muscat are Moscato Branco, Moscato Giallo, Moscato R2 and Malvasia de Candia. The more detailed knowledge of the sensorial, aromatic and qualitative characteristics of these products are important as they allow to characterize the profile of the wines and sparkling wines of this region. Therefore, the objective of this work was to determine the aromatic composition of the sparkling Farroupilha muscat; their evolution during storage; characterize wines elaborated with moscato varieties and determine the influence of yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii on the chemical and sensorial composition of Moscato Branco wines. Physicochemical, chromatographic and sensorial analyzes were performed. The results allow to attribute to the sparkling wines with less time of storage citrus and fruity aromas and to the sparkling ones with more time, notes of apple, truffles, almonds, roses and honey. Each grape variety provides different aromas for each sparkling wines, contributing to a higher aromatic complexity. The Moscato Giallo variety attributes to the wine aromas that refer to fruits such as pear and pitanga, and herbs such as rosemary. The wines of the variety Moscato R2 stood out for the notes of citrus fruits and rosemary, and the wines of the variety Moscato Branco presented citric notes, pear and pitanga. The yeasts used also influenced the aromatic composition. The wines elaborated with pure yeasts of T. delbrueckii presented higher levels of isoamyl acetate (banana), ethyl hexanoate (fruits, anise), 2-methyl-1-propanol (alcoholic) and 2-phenylethanol (roses). These concentrations tend to decrease in mixed fermentations. Wines produced with S. cerevisiae 100% EC 1118 showed the highest levels of ethanal (green apple), 1-propanol (pungent), ethyl octanoate (pineapple, pear) and linalool (roses). Thus, this study demonstrates that, although the consuption tendency of muscat wine and muscat sparkling wine being between 12 and 18 months, these products have the potential to be consumed with greater storage time, without losing their typical characteristics. It was also verified that the sparkling wines of Farroupilha presented similar quality to the sparkling wine produced in the region of Asti, region of Denomination of Origin traditionally known with almost one hundred years of wine history.

Les arômes fermentaires (alcools supérieurs, esters) jouent un rôle important dans le profil organoleptique des vins jeunes. Leur production dépend à la fois de la souche de levure utilisée et des paramètres environnementaux. Pour comprendre le rôle de trois facteurs clés (température, azote, lipides) sur la production des arômes fermentaires, nous avons réalisé une étude comparative entre une souche évoluée aromatique Affinity™ ECA5 et sa souche ancestrale Lalvin EC1118®. Dans un premier temps, les effets combinés des trois paramètres environnementaux ont été étudiés grâce à un plan expérimental de Box-Behnken. L'azote est le facteur ayant la plus grande influence sur la majorité des composés volatils; cependant son effet est différent selon la molécule ciblée et les interactions entre facteurs sont importantes. Par ailleurs, si les deux souches répondent dans le même sens aux changements de conditions de fermentation, les intensités de réponse sont très différentes. Pour avancer dans la compréhension des mécanismes mis en jeu et en particulier dans l'analyse de l'interaction azote / lipides, nous avons combiné des études de suivi cinétique précis de la production des arômes fermentaires et une approche transcriptomique. Ces études ont révélé des différences entre les deux souches dans la chronologie de formation des arômes, suggérant des modifications dans la régulation de leur synthèse. En particulier, le rendement de conversion entre alcools supérieurs et esters d'acétate est largement supérieur chez Affinity™ ECA5, mais diminue fortement après addition de phytostérols, en lien avec une répression des gènes de la voie de synthèse des stérols. Ces résultats montrent une gestion différente des lipides par la souche évoluée et suggèrent qu'une plus grande disponibilité en acétyl-CoA pourrait être à l'origine des propriétés aromatiques de cette souche. Enfin, une approche quantitative basée sur la filiation isotopique de sources d'azote marquées 13C a été réalisée. Cette étude confirme le rôle clé de l'acétyl-CoA dans une gestion différentielle du pool d'α-cetoacides responsable de la surproduction des alcools supérieurs chez Affinity™ ECA5. Globalement, ce travail a permis d'identifier les paramètres les plus efficaces pour orienter le métabolisme fermentaire vers la production d'arômes ainsi que certains mécanismes impliqués. Il a également permis de mieux caractériser le métabolisme de la souche évoluée Affinity™ ECA5. Les connaissances acquises constituent une avancée importante pour une amélioration de la gestion de la nutrition en fermentation œnologique. / Fermentative aromas (higher alcohols, esters) have a major role in the organoleptic profile of young wines. Their production depends on both the used yeast strain and environmental parameters. To better understand the role of three key factors (temperature, nitrogen, lipids) on the synthesis of fermentative aromas, we compared an aromatic evolved strain Affinity™ ECA5 and its parent strain Lalvin EC1118®. First, the combined effects of these three parameters were investigated through an experimental Box-Behnken design. Nitrogen greatly impacted the majority of the volatile compounds but differently depending on the targeted molecule and in interaction with the other factors. Overall, both strains showed similar responses to changes in fermentation conditions but with different intensities. To improve the understanding of the involved mechanism, particularly in the study of nitrogen / lipids interaction, several approaches were combined. A precise on-line monitoring of the production kinetics of fermentative aromas coupled with transcriptomic analysis revealed differences between the two strains in the chronology of aroma synthesis, suggesting changes in the regulation of their production. In particular, the conversion yield between a higher alcohol and its acetate ester differed greatly between the two strains. We have shown that this difference was due to a different lipid management by the strain Affinity™ ECA5. Finally, a quantitative approach based on the isotopic filiation of 13C labelled nitrogen sources was performed. This study confirms the key role of acetyl-CoA in a differential management of α-ketoacid pool responsible for the overproduction of higher alcohols by Affinity ™ ECA5.Overall, this project identified the most effective parameters to guide the fermentative metabolism toward the production of aromas and to better understand mechanisms involved. It also better characterized the metabolism of the evolved strain Affinity™ ECA5. These results are crucial to improve the management of nutrition in wine fermentation.

Made available in DSpace on 2015-04-14T13:58:44Z (GMT). No. of bitstreams: 1 435352.pdf: 1328330 bytes, checksum: 1ab78f3333dabdd8c83b32bc552e53f2 (MD5) Previous issue date: 2011-12-05 / Achyrocline satureioides, also known as Marcela is an aromatic herb widely used in Brazil and other South American countries, due to its medicinal properties. The marcela presents in its composition phenolic substances which are attributed antioxidant activity and volatile components. In this work we studied the processes of distillation by steam distillation and supercritical extraction, to evaluate the use of these techniques in obtaining the non-volatile and volatile extracts of marcela from its aerial parts, in order to obtain new products with aromatic notes. The extraction of essential oil of marcela was performed by employing the technique of steam distillation in five pressures (1.0, 1.5, 2.0, 2.5 and 3.0 bar), and is performed for each experimental condition the determination of the yield curve of essential oil versus time. For the extracts obtained by this technique, essential oils, the analysis was performed on the chemical composition of the extract through chromatography coupled with mass spectrometry (GC/MS). In relation to non-volatile extracts, they were obtained through the use of supercritical extraction process in four pressures (90, 100, 110 and 120 bar) at two temperatures (303.15 and 313.15 K) and each condition also yield curves were generated versus time. The extracts were assessed for their aromatic potential by assessing olfactometric. For both processes was carried out mathematical modeling of the curves, using models based on mass transfer consistent with the extraction technique that successfully represented the curves for both techniques. Regarding the assessment olfactometric both extracts volatile and non volatile aromatic potential presented, highlighting the sample extracted by steam distillation at 2.5 bar. / Achyrocline satureioides, tamb?m conhecida como marcela ? uma ervaarom?tica muito usada no Brasil e em outros pa?ses da Am?rica do Sul, devido as suas propriedades medicinais. A marcela apresenta em sua composi??osubst?ncias fen?licas as quais se atribuem atividade antioxidante e componentes vol?teis. Neste trabalho foram estudados os processos de destila??o por arraste a vapor e extra??o supercr?tica, visando avaliar o uso destas t?cnicas na obten??o dos extratos vol?teis e n?o vol?teis da marcela a partir de suas partes a?reas, a fim de se obter produtos com novas notas arom?ticas. A extra??o do ?leo essencial de marcela foi executada atrav?s do emprego da t?cnica de arraste a vapor em cinco press?es (1,0; 1,5; 2,0; 2,5 e 3,0 bar), sendo realizada para cada condi??o experimental a determina??o da curva de rendimento de ?leo essencial versus tempo. Para os extratos obtidos por essa t?cnica, os ?leos essenciais, foi realizada a an?lise da composi??o qu?mica do extrato via cromatografia gasosa acoplada com o espectr?metro de massas (CG/EM). Em rela??o aos extratos n?o vol?teis, os mesmos foram obtidos atrav?s do uso do processo de extra??o supercr?tica em quatro press?es (90, 100, 110 e 120 bar) em duas temperaturas (303,15 e 313,15 K)e para cada condi??o tamb?m foram geradas curvas de rendimento versus tempo.Os extratos obtidos foram avaliados quanto ao seu potencial arom?tico atrav?s de uma avalia??o olfatom?trica. Para ambos os processos foi realizada a modelagem matem?tica das curvas, utilizando modelos baseados na transfer?ncia de massa condizente com a t?cnica de extra??o que representaram com ?xito as curvas para ambas as t?cnicas. Em rela??o ? avalia??o olfatom?trica, tanto os extratos vol?teis como os n?o vol?teis apresentaram potencial arom?tico, destacando a amostra extra?da por arraste a vapor a 2,5 bar.

La fermentation malolactique permet une désacidification et une amélioration de la qualité du vin. Elle est réalisée par des bactéries lactiques principalement de l’espèce Oenococcus oeni : cette espèce est privilégiée pour son efficacité et ses intérêts organoleptiques. La capacité de O. oeni à résister au stress du vin et à garder son activité fermentaire est un sujet d’intérêt majeur. Les prélèvements des fûts de chêne ont montré que O. oeni adhère au bois et est capable de persister plusieurs mois dans le vin. Or, dans la majorité des habitats naturels, les microorganismes se développent attachés à un support, au sein d’un écosystème structuré appelé biofilm. Sous cette forme de vie, les cellules bénéficient d’une résistance accrue au stress. Ces deux propriétés que sont l’adhésion à une surface ainsi que la résistance au stress ont donc été étudiées pour O. oeni. Des observations par microscopie électronique à balayage ont ainsi permis de mettre en évidence la formation de biofilms par O. oeni. Puis, nous avons évalué la capacité de résistance au stress du vin de O. oeni sous forme de biofilm. L’intérêt pour le biofilm est croissant dans les industries agroalimentaires et biotechnologiques, en raison de la conservation de son activité métabolique en milieu stressant. C’est pourquoi la fermentation malolactique avec un biofilm de O. oeni a été suivie dans le vin. Enfin, l’influence du biofilm de O. oeni sur les transferts de composés aromatiques entre le bois et le vin a été étudiée. Cette étude est la première caractérisation du biofilm de O. oeni, de sa résistance au stress du vin et de son potentiel fermentaire / Malolactic fermentation improves wine quality, mainly by decreasing acidity. It is carried out by lactic acid bacteria, mainly Oenococcus oeni. This species is favored for its efficiency and its organoleptic outcome. O. oeni ability to withstand wine stress and to keep its fermentation activity is a subject of major interest.Samples of oak showed that O. oeni adheres to wood and is able to persist for several months in wine. However, in the majority of natural habitats, microorganisms grow attached to a surface, within a structured ecosystem called biofilm. In this form of life, cells benefit from an increased stress resistance.These two properties, adherence to a surface and stress resistance, were studied for O. oeni. Observations by scanning electron microscopy have highlighted biofilm formation by O. oeni. Then, we evaluated the wine stress resilience of O. oeni biofilm. Biofilm is a growing interest in food and biotechnology industries, due to the conservation of its metabolic activity in stressful environment. Therefore, malolactic fermentation with an O. oeni biofilm was monitored in wine. Finally, the influence of O. oeni biofilm on transfers of aromatic compounds between wood and wine was studied.This study is the first characterization of O. oeni biofilm, its wine stress resistance and its fermentation potential.

La famille aromatique du végétal dans les vins rouges recouvre une large diversité de nuances. Nos travaux révèlent que celles-ci n’ont pas toujours de lien direct avec les composés connus (2-méthoxy-3-alkylpyrazines, alcools en C6). Aussi, après avoir été définis sensoriellement, les déterminants moléculaires de cette famille d’odeurs ont été recherchés en s’appuyant, à partir de vins représentatifs, sur différentes techniques de fractionnement (CLHP, distillation sous vide) puis MDCPG-O-SM. Une première origine de ces nuances a été développée, celle associée à la maturité des raisins. La présence du 1,8-cinéole (eucalyptol), avec des notes « vertes », selon une origine variétale en lien avec la maturité des raisins a pu être attestée dans les vins de Cabernet Sauvignon. La capacité d’un cépage à en produire des quantités significatives (Fer Servadou) ainsi qu’une origine exogène associée à la présence d’une espèce d’armoise au vignoble (Artemisia verlotiorum) sont aussi établis. Une autre origine d’apparition des nuances végétales a été considérée : le pressurage des marcs. Cette étude montre le lien étroit entre la catégorie aromatique des vins de presse et la famille du végétal. En outre, elle révèle la présence de divers marqueurs odoriférants, en particulier le (Z)-4-heptenol dont l’évolution est corrélée au niveau de pression et la contribution sensorielle établie, à l’inverse de la plupart des alcools en C6 ; de même qu’elle suggère l’implication de plusieurs composés carbonylés à l’odeur caractéristique des vins de presse. L’influence des paramètres technologiques (inertage, pression, SO2, FML) est aussi précisée. La présence du salicylate de méthyle comme marqueur de défense de la plante contre plusieurs maladies cryptogamiques (mildiou, black rot, esca) et comme contributeur à l’odeur végétale de certains vins rouges a enfin pu être démontrée. / The green aromas family includes in red wines a large diversity of nuances. First, this study revealed that these nuances are not directly related to known compounds (e.g. 2-méthoxy-3-alkylpyrazines, C6 alcohols). After a sensory definition, the molecular determinants of these aroma nuances were investigated based on fractionation strategies including HPLC and vacuum distillation then MDGC-O-MS. A first source of nuances associated with grapes ripeness was developed. The presence of 1,8-cineole (eucalyptol) with a varietal origin was evidenced in Cabernet Sauvignon wines. The ability of a grape variety to produce significant amounts (Fer Servadou) and an exogenous origin associated with the presence of Artemisia species in vineyards (Artemisia verlotiorum) were also highlighted. Another origin, related to the pressing of grape marc was then considered. This study shown the connection between the aromatic category of press wines and the green aromas. Furthermore, it revealed the presence of odoriferous molecules including (Z)-4-heptenol, with a sensory contribution, associated to the increase of pressure in the press tank, but not for the majority of C6 alcohols. It also suggested the involvement of several carbonyl compounds in the characteristic odor of press wines. The influence of technological parameters (without oxygen, pressure, SO2, malolactic fermentation) was also specified. Finally, this study revealed the presence of methyl salicylate as a plant defense marker against several fungal diseases (downy mildew, black rot, esca) and as a contributor to fresh green aromas of certain red wines.

Afin d’optimiser la production d’un composé d’intérêt tout en évitant les conséquences néfastes sur la viabilité cellulaire, un défi majeur de l’ingénierie métabolique est d’obtenir un équilibre entre les flux métaboliques endogènes de la cellule et le flux consommé par une nouvelle voie de biosynthèse. Dans ce contexte d’optimisation, les stratégies combinatoires génèrent une diversité de voies métaboliques et de modes de régulation rassemblant de précieuses informations quant aux choix d’orientations stratégiques à faire. Notre étude s’inscrit dans un projet visant à produire les molécules responsables de l’arôme, de la couleur et du parfum du safran (Crocus sativus) chez Saccharomyces cerevisiae. Une approche combinatoire a été adoptée pour moduler les niveaux d’expression de trois gènes menant à la synthèse de leur précurseur commun : la zéaxanthine. Cette stratégie nous a permis de décrire des biais inattendus dans la régulation des niveaux d’expression des gènes exprimés sur plasmide. Nous avons détecté une forte interférence transcriptionnelle entre les gènes dans notre système, ainsi qu’une influence de la nature des séquences codantes. Ces éléments, identifiés comme critiques, imposent sur les niveaux d’expression des trois gènes une régulation plus importante que la force des promoteurs qui les contrôlent. Afin de poursuivre le projet vers son objectif final, la réaction de clivage de la zéaxanthine menant à la synthèse des composés d’intérêt du safran a fait l’objet d’une analyse fonctionnelle détaillée. Une absence d’activité de l’enzyme décrite dans la littérature comme responsable de cette réaction nous a conduits à proposer des perspectives d’ingénierie pour atteindre l’objectif final du projet / To optimize the production of a value added compound while avoiding toxic consequences on the cell viability, a challenge in the metabolic engineering field is to balance the endogenous metabolic fluxes and the newly consumed fluxes. In this optimization context, combinatorial strategies can generate several variants of synthetic metabolic pathways. This strategy gives precious strategic information on the right combinations of function and regulation choices to be made in the ultimate pathway reconstruction. Our study aimed at the production of the molecules responsible for aroma, dye, and fragrance of saffron (Crocus sativus) in Saccharomyces cerevisiae. A combinatorial approach was chosen to modulate expression levels of three genes involved in their common precursor biosynthesis: zeaxanthin. This strategy allowed us to describe some unexpected bias in the regulation of the plasmid-encoded genes expression levels. We detected strong transcriptional interference between the different genes in our system, and the ORF nature also seemed to influence the expression levels. These critical factors imposed a stronger regulation of the three genes expression levels than the promoter strength initially chosen to control them. The project was continued toward its final objective by making a detailed functional analysis of the zeaxanthin cleavage reaction leading to the molecules of interest synthesis. This reaction was described to be catalyzed by a specific enzyme, but no activity was observed in our experiments. This result led us to propose some tools to reach the final goal of the project