Dissertations / Theses on the topic 'Alcoholic fermentation'

La melatonina és una indolamina que és produïda pel llevat a partir de l'aminoàcid triptòfan durant la fermentació alcohòlica. En aquesta tesi, estudiem la producció de melatonina en most sintètic en diferents condicions ambientals i nutricionals pels llevats, així com l'efecte d'aquesta molècula bioactiva en la dinàmica de poblacions d'una fermentació. Com a resultats, observem que la detecció estava desfasada entre la producció intracel·lular, la qual va ocórrer durant la fase d'adaptació al medi i la secreció al medi extracel·lular durant la fase exponencial o estacionària. Així mateix, la presència de melatonina millorava el desenvolupament de la fermentació i augmentava la presència de llevats no-Saccharomyces a final de fermentació. A més, vam analitzar la interacció d'aquesta molècula amb proteïnes durant la fermentació. Els resultats van mostrar que la melatonina s'unia a enzims glicolítics en llevats que tenien una alta capacitat fermentativa, reforçant la idea que la melatonina podria actuar com una molècula senyal durant la fermentació alcohòlica. Finalment, es va optimitzar un nou mètode de detecció per fluorescència utilitzant una línia cel·lular que presenta el receptor humà de melatonina per a mostres procedents de begudes fermentades. Els resultats van mostrar que aquest nou mètode disminueix el límit de detecció i és una bona alternativa en comparació amb el mètode de detecció de melatonina basat en cromatografia. No obstant, es necessita una extracció de la mostra per a realitzar l'anàlisi de melatonina.
La melatonina es una indolamina que es producida por la levadura a partir del aminoácido triptófano durante la fermentación alcohólica. En esta tesis, estudiamos la producción de melatonina en mosto sintético en diferentes condiciones ambientales y nutricionales por las levaduras, así como el efecto de esta molécula bioactiva en la dinámica de poblaciones de una fermentación. Como resultados, observamos que la detección estaba desfasada entre la producción intracelular durante la fase de adaptación al medio y la secreción al medio extracelular durante la fase exponencial o estacionaria. Así mismo, la presencia de melatonina mejoraba el desarrollo de la fermentación y aumentaba la presencia de levaduras no-Saccharomyces a final de fermentación. Además, analizamos la interacción de esta molécula con proteínas durante la fermentación. Los resultados mostraron que la melatonina se unía a enzimas glucolíticas en levaduras que tenían una alta capacidad fermentativa, reforzando la idea de que la melatonina actuaría como una molécula señal durante la fermentación alcohólica. Finalmente, se optimizó un nuevo método de detección por fluorescencia utilizando una línea celular que presenta el receptor humano de melatonina en muestras procedentes de bebidas fermentadas. Los resultados mostraron que este nuevo método disminuye el límite de detección y es una buena alternativa en comparación con el método de detección de melatonina basado en cromatografía. Sin embargo, se necesita una extracción de la muestra para realizar el análisis de melatonina.
Melatonin is an indolamine, which is produced by yeast from the aromatic amino acid, tryptophan, during alcoholic fermentation. Here, we study the production of melatonin in synthetic must in different environmental and nutrition conditions by wine yeast species as well as the effect of this bioactive molecule on the fermentation performance. The results showed that the melatonin detection was delayed between intracellular production, which occurred during lag phase, and extracellular secretion, at the exponential or stationary phase. Additionally, melatonin presence improved fermentation performance and survival of non-Saccharomyces yeasts. We also studied melatonin interactions with proteins during fermentation. Consequently, we found that melatonin was bound to glycolytic enzymes and this interaction is related to yeasts with high fermentative capacity. This reinforces the idea of melatonin acting as a signal molecule during alcoholic fermentation. Finally, we optimized a novel fluorescence method, based on a cell line that presents the human melatonin receptor, in fermented samples. We observed that this new method decreased the limit of detection and was a good alternative in comparison with a chromatographic method, although, an extraction was needed for melatonin analysis.

La fermentació alcohòlica la realitza una comunitat microbiana complexa, on els llevats vínics juguen un paper important. En els darrers anys, hi ha hagut un creixent interès per millorar complexitat del vi mitjançant fermentacions controlades quer utilitzaven no només S. cerevisiae sinó també soques seleccionades de llevats no-Saccharomyces. La investigació sobre la viabilitat dels llevats i les interaccions tenen un paper fonamental per entendre la diversitat de llevats en la fermentació mixta. En aquesta tesi, les tècniques independents de cultiu que es van aplicar per a l'anàlisi directa de mostres de vins inclouen la seqüenciació massiva, fluorescència per hibridació in situ (FISH) en combinació amb microscòpia i citometria de flux, RT-qPCR i EMA-DGGE. Aquestes tècniques independents de cultiu permeten una ràpida identificació i / o quantificació dels diferents llevats del vi. Aquestes tècniques han estat utilitzades en fermentacions espontànies a la regió del Priorat, i H. uvarum i Starm. bacillaris van ser les dues principals espècies de llevats no-Saccharomyces aïllades. H. uvarum o Starm. bacillaris perden gradualment la seva cultivabilitat quan els mostos es van inocular amb S. cerevisiae, però es podien quantificar cèl•lules de llevats en estat viable però no cultivable. La pèrdua de cultivabilitat dels no Saccharomyces va ser induïda principalment per alguns metabòlits secretats per S. cerevisiae, però els canvis en altres metabòlits principals també van influir. Aquesta interacció de llevat no Saccharomyces amb S. cerevisiae era específica d’espècie i soca.
La fermentación alcohólica es llevada a cabo por una comunidad microbiana compleja, donde las levaduras del vino juegan un papel importante. En los últimos años, ha habido un creciente interés para mejorar la complejidad del vino en fermentaciones controladas utilizando no sólo S. cerevisiae sino también algunas cepas seleccionadas de levaduras no-Saccharomyces. La investigación sobre la viabilidad de las levaduras y las interacciones tienen un papel fundamental para entender la diversidad de levaduras en fermentaciones mixtas. En esta tesis, se aplicaron técnicas independientes de cultivo para el análisis de muestras de vinos directa incluyendo la secuenciación masiva, fluorescencia por hibridación in situ (FISH) en combinación con microscopía y citometría de flujo, RT-qPCR y EMA-DGGE. Estas técnicas independientes de cultivo permiten una rápida identificación y / o cuantificación de las distintas levaduras del vino. Estas técnicas han sido utilizadas para el análisis de fermentaciones espontáneas en la región del Priorat, siendo las especies H. uvarum y Starm. bacillaris las dos principales especies de levaduras no-Saccharomyces. H. uvarum o Starm. bacillaris pierden gradualmente su cultivabilidad cuando los mostos se inocularon con S. cerevisiae, pero las levaduras se pudieron cuantificar en estado viable pero no cultivable. La pérdida de cultivabilidad de las especies no Saccharomyces fue inducida principalmente por algunos metabolitos secretados por S. cerevisiae, pero los cambios en otros metabolitos principales también influyen. Esta interacción entre levaduras no Saccharomyces con S. cerevisiae es especie y cepa dependiente.
Alcoholic fermentation is driven by complex microbial community, where wine yeasts play an important role. In recent years, there has been growing interest to enhance wine complexity by controlled fermentations using not only S. cerevisiae but also together with some selected non-Saccharomyces yeast strains. Research on yeast viability and interaction has a fundamental role to understand the diversity of yeast in mixed fermentations. In this thesis, culture-independent techniques were developed and applied for direct wine sample analysis including massive sequencing, fluorescence in situ hybridization (FISH) combined with microscopy and flow cytometry, RT-qPCR and EMA-DGGE. These culture-independent techniques enable fast identification and/or quantification of different wine yeasts. These techniques have been used during spontaneous fermentation in Priorat region, and H. uvarum and Starm. bacillaris where the two main non-Saccharomyces yeast species detected. H. uvarum or Starm. bacillaris gradually lost their culturability when musts were inoculated with S. cerevisiae, but quantifiable yeast cells existed in viable but non-culturable state. The culturability loss of non-Saccharomyces was mainly induced by some metabolites secreted from S. cerevisiae, but changes in other main metabolites also had some effect. This interaction of non-Saccharomyces yeast with S. cerevisiae showed the specificity of species and strains.

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL
Wine is an alcoholic beverage obtained from the fermentation of grape juice. Alcoholic fermentation is the main process to obtain the final product but of course other reactions take place during the transformation of grape juice into wine. Yeasts are one of the fundamental microorganisms to realize the alcoholic fermentation, that leads sugars to be transformed in ethanol and to development of other compounds. Yeasts as known are living organisms so they need nutrients for their reproduction and often the lack of some important nutrients can lead to a stuck or slug in their metabolic activity. One of the most important nutrient for yeasts growth is Nitrogen, main component of aminoacids and proteins, that constitute the basic structures of the microorganism.In winemaking, both in the cellar and in the vineyards, the addition of nitrogen under different circumstances is a common practice. Nitrogen seems to influence not only fermentation rate and yeast growth but many of the fermentation product such as ethanol, acetic acid, glycerol, and of course residual sugars and the production of some off flavours,such as hydrogen sulphide. Although each yeast, species and strains,showed different behaviour both in the fermentation rates, growth and production of different metabolites. Not only the quantity and kind of Nitrogen source but also the timing of the addition seem to influence all these aspects in slightly different way between different strain. The nitrogen compounds especially apparently ammino acids showed also a particular relationship with the production of esters of higher alcohols and other compounds but the pattern of ester production is still uncertain. In this study there will be considered the main aspects of nitrogen nutrition, how it affects the yeasts ecology and the different conditions on which it is used and also different strains and species requirements

The vast majority of chemical and bio-chemical process plants are normally characterized by large number of measurements and relatively small number of manipulated variables; these thin plants have more output than input variables. As the number of manipulated variables restricts the number of controlled variables, thin plant has presented a daunting challenge to the engineers in selecting which measured variables to be controlled. In general, this is an important problem in modern process control today, because controlled variables selection is one of the key questions which must be carefully addressed in order to effectively design control strategies for process plants. While the issue relating to controlled variables selection has remained the key question to be resolved since the articulation of CSD problem by Foss in 1970s, the work described in this thesis points out to another equally important question in CSD, that is, what is the sufficient number of controlled variables required? Thinking over this question leads one to the necessity for gaining a rational understating of the governing principle in partial control design, namely the variables interaction.In this thesis, we propose a novel data-oriented approach to solving the control structure problem within the context of partial control framework. This approach represents a significant departure from the mainstream methods in CSD, which currently can be broadly classified into two major categories as the mathematical-oriented and heuristic-hierarchical approaches. The key distinguishing feature of the proposed approach lies in its adoption of technique based on the Principal Component Analysis (PCA), which is used to systematically determine the suitable controlled variables. Conversely, the determination of the controlled variables in mathematical-oriented and heuristic-hierarchical approaches is done via the mathematical optimization and process knowledge/engineering experience, respectively. It is important to note that, the data-oriented approach in this thesis emerges from the fusion of two important concepts, namely the partial control structure and PCA. While partial control concept provides the sound theoretical framework for addressing the CSD problem in a systematic manner, the PCA-based technique helps in determining not only the suitable controlled variables but also the sufficient number of controlled variables required.Since the classical framework of partial control is not amendable to a systematic way in the identification of controlled variables, it is necessary to develop a new framework of partial control in this thesis. Within this new framework the dominant variable can be clearly defined, and which in turn allows the incorporation of PCA-based technique for the systematic identification of controlled variables.The application of the data-oriented approach is demonstrated on a nonlinear multivariable bioprocess case study, called the two-stage continuous extractive (TSCE) alcoholic fermentation process. The system consists of 5 interlinked units: 2 bioreactors in series, a centrifuge, vacuum flash vessel and treatment tank. The comparison of the two-stage design with that of single-stage design reported in literature shows that: (1) both designs exhibit comparable performance in term of the maximum allowable trade-off values between yield and productivity, and (2) two-stage design exhibits stronger nonlinear behaviour than that of single-stage. Thus, the design of control strategies for the former is expected to be more challenging.Various partial control strategies are developed for the case study, such as basic partial control strategy, complete partial control strategies with and without PID enhancement technique and optimal size partial control strategy. Note that, this system consists of 16 output variables and only 6 potential manipulated variables, which has approximately 4,000,000 control structure alternatives. Therefore, the application of mathematical approach relying on optimization is not practical for this case study – i.e. assuming that evaluation of each alternative takes 30 seconds of optimization time, thus, complete screening will require almost 4 years to complete.Several exciting new insights crystallize from the simulation study performed on the case study, where two of them are most important from the perspective of effective design of partial control strategy: 1) There is an optimal size of partial control structure where too many controlled variables can lead to the presence of bottleneck control-loop, which in turn can severely limit the dynamic response of overall control system. On the other hand, too few controlled variables can lead to unacceptable variation or loss in performance measures. 2) The nature of variables interaction depends on the choice of control structure. Thus, it is important to ensure that the nature of open-loop variables interaction is preserved by the implementation of a particular control strategy. When this is achieved, then we say that this control system works synergistically with the inherent control capability of a given process – i.e. achieving the synergistic external-inherent control system condition.The proposed approach has been successfully applied to the case study, where the optimal partial control structure is found to be 3x3 i.e. 3 controlled variables are sufficient to meet all 3 types of control objectives: overall (implicit) performance objectives, constraint and inventory control objectives. Finally, the proposed approach effectively unifies the advantages of both mathematical-oriented and heuristic-hierarchical approaches, and while at the same time capable of overcoming many limitations faced by these two mainstream approaches.

Thesis (PhDAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Stuck alcoholic fermentations are a major enological problem for the international winemaking industry. Incomplete wine fermentations are frequently characterized by high residual fructose concentrations and the near-absence of residual glucose, a fact that is due to the glucophilic character of the wine yeast Saccharomyces cerevisiae. Wines with high contents of post fermentation sugar are very susceptible for microbial spoilage since residual fructose and/or glucose can be metabolized by bacteria and yeast to undesired by-products such as volatile acid and off-flavours, resulting in wine spoilage and considerable economic losses. It has been reported that stuck fermentations are usually caused by several synergistically acting inhibition factors, and the glucose to fructose ratio (GFR) is thought to play an important role in this context. This study is aimed at contributing towards a better understanding of this industrial problem, and at finding industrially applicable solutions. In a first part, this study describes the isolation of two appropriate strains of the fructophilic yeast Zygosaccharomyces bailii from the natural microflora of grapevine, followed by trials in small scale test fermentations using stuck industrial fermentations as model media. These experiments were expanded to also investigate large scale industrial fermentations. As a result, a strategy for the treatment of stuck fermentations was developed and successfully applied in several wineries with fermentation problems. This methodology represents an entirely novel and industrially applicable solution to high residual fructose levels. In a second part, the data contributes to elucidating the molecular nature of the fructophilic phenotype of Z. bailii by characterizing some of the genes and proteins that may be responsible for the fructophilic character. In particular, the investigation focused on the first two steps of hexose metabolism, the transport of sugar into the cell by permeases and sugar phosphorylation by hexokinases, which combined are thought to be primarily responsible for sugar preference. One result of this study was Fructoferm W3©, a dry yeast product which is commercially available. Fructoferm W3 was awarded with the innovation medal for enological products at Intervitis/Interfructa, Stuttgart, Germany in 2007.
AFRIKAANSE OPSOMMING: Die voorkoms van steek alkoholiese fermentasies is ‘n ernstige problem in die internasionale wyn industrie. Onvolledige fermentasies word dikwels gekenmerk deur hoë residuele fruktose konsentrasies en die veitlike afwesigheid van residuele glukose. Die kenmerke kan meestal toegeskryf word aan die glukofilliese kakakter van die wyngis Saccharomyces cerevisiae. Wyne met ‘n hoë suiker inhoud na die afloop van fermentasie is vatbaar vir mikrobiese bederf aangesien residuele fruktose en/of glukose gemetaboliseer kan word deur bakterië en gis om ongewenste byprodukte soos vlugtige sure en bygeure te vorm wat kan lei tot wyn bederf en aansienlike ekonomies verlies. Dit is vasgestel dat steek fermentasies gewoonlik veroorsaak word deur verskeie sinergisties werkende inhibisie faktore, waartoe die glukose/fruktose verhouding ‘n noemenswaardiege bydrae lewer. Die mikpunt van hierdie studie was om ‘n bydrae te lewer tot die begrip van steek fermentasies en die daarstelling van moontlike industriële oplossings. Die eerste deel van die werk beskryf die isolasie van twee rasse van die gis Zygosaccharomyces baillie uit die natuurlike wingerd mikroflora, gevolg deur steekproewe in die vorm van kelinskaalse fermentasies met steek industriële fermentasies gebruik as model media. Hierdie ekserimente is vervolgens uitgebrei om grootskaalse industriële steek fermentasies te bestudeer. Die uitkoms van hierdie werk het gelei tot die ontwikkeling van ‘n strategie vir die behandeling van steek fermentasies wat susksesvol toegepas is in verskeie wynmakerye. Die metodiek bring ‘n nuwe en industrieel toepasbare oplossing vir hoë residuele fruktose vlakke. Die data aangebied in die tweede afdeling dra by tot die verheldering van die molekulêre natuur van die fruktofilliese fenotipe van Z. baillie deur die tipering van gene en protiëne wat moontlik verantwoordelik is vir die fruktofilliese karakter van die gis. Die ondersoek het spesifiek op die eerste twee stappe van heksose metabolisme, naamlik die invoer van suiker in die sel deur permeases en suiker fosforilering deur heksokinases, gekonsentreer. Die kombinasie van die twee prosesse is vermoedelik verantwoordelik vir die regulering van suiker voorkeur. ‘n Gevolg van die studie was die ontwikkeling van ‘n droë gisproduk, Fructferm W3©, wat kommersieel beskikbaar gestel is. Fructoferm W3 is in 2007 toegeken met die innovasie medalje vir wynkundige produkte by Intervittis/Interfructa in Stuttgart, Duitsland.

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Este estudo tem como objetivo avaliar a matéria-prima e sua influência isolada no desempenho do rendimento fermentativo de uma linhagem padrão da levedura CAT-1 testada em 31 meios de cultivos provenientes do processo de fermentação alcoólica da Usina Virgolino de Oliveira – Unidade Catanduva. Os meios foram amostrados e compostos a cada quinzena durante toda as safras 2012 e 2013. O trabalho também tem como finalidade identificar a dinâmica populacional das leveduras do mesmo processo fermentativo, através da diferenciação das linhagens por cariotipagem. As cepas isoladas foram testadas em um meio de cultivo padrão para obtenção das características tecnológicas industriais através da metodologia da Capacidade Fermentativa. Os experimentos de fermentação foram realizados nos laboratórios da Usina Virgolino de Oliveira - Unidade Catanduva em escala reduzida sempre acompanhados de um ensaio padrão utilizando meio de cultivo sintético. O primeiro ponto de estudo consistiu na caracterização da matéria-prima, mosto, e sua capacidade isolada de perturbar o Rendimento Fermentativo. Enquanto que no segundo, onde onze diferentes cepas de levedura foram identificadas ao longo das duas safras, testadas em um mesmo meio de cultivo padrão para obtenção de parâmetros industriais tecnológicos como rendimento em produto (Yp/s), rendimento em célula (Yx/s), Velocidade de consumo do substrato (Vcs), Produtividade (PROD) e Conversão (CONV) e estudada a influência no Rendimento Fermentativo. O terceiro ponto de estudo foi a comparação entre os rendimentos fermentativos obtidos experimentalmente e os rendimentos fermentativos industriais da Planta. O impacto da presença das cepas com maior rendimento em etanol foi estudado em relação aos valores de rendimento fermentativo industrial. Os resultados mostraram diferenças de desempenho da Cepa Padrão na maioria das quinzenas testadas, o que significa que há variação da matéria-prima ao longo da safra e entre as safras capazes de afetar rendimento fermentativo. Diferenças de rendimento também foram observadas entre as onze cepas nativas testadas, porém com oscilações menores e menos consideráveis do que com a matéria-prima. Os resultados obtidos em escala reduzida com base nos balanços de massa se apresentaram valores semelhantes em relação aos números de referência o que sugere que a metodologia usada para avaliar a capacidade fermentativa das cepas e a qualidade da matéria-prima foi adequada. Apesar da forte influência dos fatores estudados, não foi possível afirmar, através destes experimentos, qual deles teve papel determinante no impacto do Rendimento Fermentativo. Isso sugere que outros fatores não estudados neste trabalho estão diretamente relacionados que são capazes de influenciar o Rendimento Fermentativo.
The purpose of this study is to evaluate the sucrose mash and its influence isolated on the performance of the Fermentative Yeld of a standard strain from CAT-1 yeast tested in 31 different culture medium formulation from the Virgolino de Oliveira Plant – Catanduva Unit alcoholic fermentation process. The culture medium formulation were sampled and composed every fortnight during the whole 2012 and 2013 harvest. The work also has as purpose to evaluate the yeasts population dynamics of the same fermentative process, through the differentiation of the strains by karyotyping. The isolated strains were tested in a standard culture medium to obtain the industrial technological characteristics through the Fermentative Capacity methodology. Fermentation experiments were carried out in mill’s laboratories on a reduced scale always accompanied by a standard assay using synthetic culture medium formulation. The first point of study consisted in the characterization of the raw material, sucrose mash, and its isolated capacity to disturb Fermentative Yield. In the second, eleven different strains of yeast identified during the two harvests, they were tested in the same culture medium to obtain industrial technological parameters as Yield in product (Yp/s), Yield in cell (Yx/s), Substrate consumption velocity (Vcs), Productivity (PROD) and Conversion (CONV) and studied their influence on the Fermentative Yield. The third point of study was the comparison between the fermentative yields obtained experimentally and the industrial fermentative yields of the Plant. The impact of the presence of strains with higher Yp/s was studied in relation to industrial fermentation yield values. The results showed differences in performance of the Standard Strain in most of the fortnight tested, which means that there is variation of the raw material during the harvest and between the crops capable of affecting fermentative yield. There were also Yeld difference observed among the eleven native strains tested, but with smaller and less considerable oscillations than with the raw material. The results obtained on a reduced scale based on the mass balances were within the range expected in relation to the reference values, which suggests that the methodology used to evaluate the fermentative capacity of the strains and the quality of the raw material was adequate. Despite the strong influence of the studied factors, it was not possible to prove, through these experiments, which one had a determinant role in the Fermentative Yield impact. This suggests that other factors not studied in this work are directly related that are able to influence Fermentative Yield.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2016.
This study focused on alcoholic fermented fruit beverages that were produced from various types of fruit, value addition and thus potentially increasing the diversity of commercially available fruit wines. Non-grape alcoholic fermented fruit beverages is a complex mixture of water, alcohol, and other components, that are either initially present in the fruit, or are formed during the fermentation process. The evaluation of wine and similar fermented products quality is important for manufacturers and consumers. The routine analysis of alcoholic fermented fruit beverages acts as an important tool that is useful for wine classification, quality control and sensory evaluation. Therefore, the aims of this study were (1) to measure methanol, ethanol, titratable acidity, objective colour, total soluble solids and sensory profile as a function of yeast strain and percentage pulp in order to adapt existing technologies toward producing new fermented fruit beverage products using plums, an under-utilized agricultural produce; and (2) to measure methanol, ethanol, titratable acidity, objective colour, total soluble solids and sensory profile as a function of yeast strain, pulp percentage and sugar levels in order to adapt existing technologies toward producing new fermented fruit beverages based on red and white wine styles, while applying the technology developed in the first part of the study using red-fleshed plums, blueberries and blackberries. The independent variables (ID) were yeast strains (1) Saccharomyces cerevisiae VIN13, (2) Saccharomyces cerevisiae NT116, and (3) Saccharomyces bayanus N96, with formulations containing percentage pulp concentrations at (40%, 50% and 60%). The dependent variables (DV) constituted key quality parameters for white and red wine style, namely methanol, ethanol, titratable acidity, objective colour, total soluble solids, pH and sensory profile were measured. The optimal combination of independent variables was ascertained and in terms of the overall consumer response, for the red-fleshed plum beverage sample treatment N 96, 60% pulp showed the highest preference amongst consumers. In terms of the other dependent variables, namely methanol, ethanol, titratable acidity, objective colour, total soluble solids, pH and sensory profiles of alcoholic fermented fruit beverages based on white and red wine styles. The processing conditions developed and applied in this study towards the development of alcoholic fermented beverages utilizing plums and selected berries demonstrated ways of improving the utilization of fruit commodities by developing niche products. Hence, the development of alcoholic fermented beverages utilizing (plums and selected berries) showed potential for micro agro-industries, as well as the impact on its potential role in employment creation and income generation.

Aroma is one of the most important attributes involved in wine quality. Current trend in winemaking consists of producing wines with different aroma nuances to offer variety of wines to a developing market. Several studies have demonstrated that low temperature fermentations favours aroma synthesis and retention. In this background, new wine yeasts able to perform fermentation at low temperatures improving wine aroma while maintaining good fermentation rates are necessary. This doctoral thesis explores the oenological traits of different Saccharomyces species and hybrids relevant for present-day wine industry, especially regarding aroma production, as well as the molecular bases underneath. This exploration has been possible using different biochemical, analytical chemistry and molecular techniques to perform enzymatic activity detection, aroma profile determination and transcriptome analysis in wine fermentations. Through this doctoral thesis the abilities of different Saccharomyces species and hybrids regarding primary aroma release and secondary aroma production, especially at low temperatures, has been elucidated in order to know the different possibilities that these yeasts offer to create new wines with different aromatic nuances. One of the general conclusions of this doctoral thesis is that production and release of aromas in winemaking depends on the strain carrying out the fermentation process. Nevertheless, sometimes there was a species tendency. On the other hand, the fact that fermentation temperature affects aroma synthesis but not always in the direction to aroma increase has been demonstrated.
Gamero Lluna, A. (2011). Study of the production and release of aromas during winemaking carried out by different Saccharomyces species and hybrids [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/10741
Palancia

Dans les procédés classiques de fermentation éthanolique, l'amélioration des productivités se heurte à de nombreux problèmes et les coûts de production restent élevés. Parmi ces problèmes, l'existence de concentrations maximales en microorganismes ainsi que les effets inhibiteurs, notamment du produit de substrat, restent prépondérants. Ces problèmes ont orienté les travaux de recherche vers la mise en œuvre de procédés nouveaux de fermentation qui associent un fermenteur fonctionnant en continu à un procédé de filtration et de concentration de la biomasse. L'amélioration des productivités par ce type de technologie repose sur trois points fondamentaux qui sont la levée des inhibitions, la maitrise de l'apport des nutriments nécessaires aux besoins des microorganismes et l'obtention d'une concentration optimale de biomasse active dans le fermenteur. La partie expérimentale de notre étude s'est articulée autour d'une double approche, l'approche du procédé discontinu de fermentation éthanolique et l'approche du procédé continu. La partie modélisation et simulation de notre étude a permis d'utiliser les résultats obtenus dans l'approche expérimentale. La modélisation dynamique des procédés discontinu et continu a été étudiée en utilisant le logiciel de simulation speedup et un modèle dynamique du procédé continu a été validé.

Orientador: Silvio Roberto Andrietta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: Este trabalho teve por objetivo estudar o processo de fermentação alcoólica operando em batelada-alimentada, analisando o efeito da forma de alimentação do reator, da temperatura e da concentração de inóculo sobre o rendimento fermentativo, a produtividade e a cinética do sistema, encontrando a forma mais adequada de operação das unidades que trabalham com esse tipo de processo. Os ensaios foram realizados em reator de bancada utilizando meio sintético a base de sacarose e extrato de levedura. O microrganismo utilizado foi a cepa de levedura Y904 (Saccharomyces cerevisae). As concentrações de substrato e etanol foram determinadas pelo método colorimétrico em espectrofotômetro, e as concentrações de células foram determinadas pelo método gravimétrico. Para avaliação cinética, utilizou-se um modelo proposto por LEE et. al. 1983 modificado e os parâmentros cinéticos foram ajustados por um programa computacional desenvolvido em Delphi. Através de uma análise estatística realizada no Software Statistica 5.0 foi possível determinar quais parâmetros cinéticos apresentaram variação significativa em relação às variáveis estudadas. Como resultado, observou-se que nas condições estudadas, o rendimento em etanol obteve significância com a temperatura e a concentração de inoculo, sendo que valores elevados foram obtidos em temperaturas extremas e altas concentrações de inoculo. O rendimento em células foi afetado pela temperatura, obtendo valores elevados a baixa temperatura... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: Many fermentative processes industrial are realized in the fed-batch way, where the feeding containing substract and/or nutrients are fed the bioreactor. This work has as objective to study the process of alcoholic fermentation operating in fed-batch, analyzing the effect of feeding way the bioreactor, temperature and concentration of yeast about the fermentative yield, the productivity and the kinetic of the system, finding the more appropriate way of operating the units that work with this kind of process. The essays were realized in the bench bioreactor using synthetic substract with sucrose and yeast extract. The microorganism used is the strain of yeast Y904 (Saccharomyces cerevisae). The concentration of substract and ethanol were determined by metric color method in espectrofotometer, and the concentrations of biomass were determined by gravity methody. For the kinetic evaluation was used the proposed model by LEE et. al. 1983 modified and kinetics parameters were adjusted by a software developed in Delphi. Through a statistica analyse in the Software Statistica 5.0 it was possible to determine the kinetic parameters that presented significant variation regarding the variables studied. As a result, it was notice that in the conditions studied, the yield of ethanol obtained significant with the temperature and concentration of yeast, considering that high values were obtained in extreme temperature and high concentration of yeast... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Mestrado
Engenharia Química
Mestre em Engenharia Química

Alcoholic and malolactic fermentations are two important conversions that are involved in wine fermentation. Changes in wine microorganisms from these inoculations can modify the final wine characteristics, thus monitoring changes in microorganisms during these fermentations is necessary. Next-generation sequencing (NGS) is a technology that uses a culture independent technique to identify different species of fungi and bacteria from a sample. At present, when using this technique, it is difficult to distinguish between live and dead cells. The chemical, PMA, has been shown to bind DNA of dead cells, which prevents it from being amplified. In Chapter 2, the use of PMA were optimized, as a precursor to NGS, for accurate identification and quantification of yeast and bacterial species. In Chapter 3, the living microbial community were monitored in fermentations that differed in their yeast (S. cerevisiae) inoculation method (inoculated versus spontaneous) and their timing of bacterial (Oenococcus oeni) inoculation (inoculated during the same time of S. cerevisiae inoculation, post alcoholic fermentation, and uninoculated, which resulted in a spontaneous inoculation). Using this 2 x 3 factorial design, the effect of these factors on the strain and species relative abundance, diversity, and composition of yeast and bacteria were explored . A successful implantation (>80% relative abundance) of the S. cerevisiae inoculum were found, which resulted in changes in relative abundance for both yeast and bacterial populations and community composition as compared with spontaneous alcoholic fermentation (AF). The inoculation of the O. oeni MBR31 strain affected both bacterial communities and O. oeni strain composition with no apparent effect on yeast strain and species relative abundance, diversity and composition. An interactive effect was found, where the bacteria in spontaneous alcoholic fermentations (AF) as compared with those in inoculated AF were more easily influenced by O. oeni inoculations. Sensorial profiles indicated that inoculation of both O. oeni and S. cerevisiae changed final wine sensorial attributes. Different timing of O. oeni inoculation treatments resulted in a smaller change in the bacterial community and wine attributes compared with adding or not adding S. cerevisiae. Results suggest that fermentations without an addition of yeast inoculum had higher yeast species and strain diversity, which correlated with higher positive sensorial attributes of the wine, such as body, long finish, tropical fruit flavors as well as butter and vanilla aromas. Different timing of O. oeni inoculation affect O. oeni strain composition and bacterial species diversity and composition with no apparent correlation between microbial changes and the final wine sensorial profiles.
Irving K. Barber School of Arts and Sciences (Okanagan)
Biology, Department of (Okanagan)
Graduate

Il existe une forte demande de l'industrie pour des technologies permettant de réduire la teneur en alcool des vins. Bien que des levures à faible rendement alcool aient été développées par ingénierie génétique, les approches non-OGM sont aujourd'hui largement privilégiées. Nous avons mis en œuvre et comparé différentes stratégies d'évolution adaptative, à partir d'une souche œnologique commerciale, afin de réorienter le flux carboné vers la formation de glycérol aux dépens de l'éthanol. Après 200 générations en conditions de stress salin, nous avons obtenu des souches évoluées présentant une augmentation de la production de glycérol de 50 à 70 %, capables de diminuer de 0,45 à 0,80 % (v/v) la teneur en alcool de vins naturels ou synthétiques. Cette réorientation s'accompagne d'une accumulation de succinate et de 2,3-butanediol et d'une réduction de la vitesse fermentaire. Les mutants ont une survie accrue en conditions de stress salin et carence en glucose. Afin d'identifier les mécanismes sous-jacents, nous avons réalisé des analyses du transcriptome, du métabolome (exo- et endo-) et du génome des souches évoluées. Nous avons montré que le phénotype des mutants n'est pas dû à une dérégulation ou à des mutations des gènes de la voie de synthèse du glycérol mais à de larges modifications du métabolisme carboné, énergétique et redox. Le génome des souches évoluées présente des pertes d'hétérozygotie qui pourraient contribuer au phénotype observé. L'étude génétique d'une souche évoluée montre une origine multigénique des traits métaboliques. Une analyse de cartographie de QTL en utilisant une approche « bulk sequencing » a été initiée pour identifier les mutations impliquées dans le caractère fort glycérol/faible éthanol. Ces travaux ont ainsi permis de développer et caractériser des souches œnologiques faibles productrices d'alcool et de fournir un cadre pour l'identification des bases moléculaires impliquées.Mots-clefs : S. cerevisiae, fermentation œnologique, évolution adaptative, éthanol, glycérol, transcriptome, métabolome, génome
There is a strong demand from the industry for technologies to reduce the alcohol content of wine. Although low-alcohol yield yeasts have been developed by genetic engineering, GMO-free approaches are now widely preferred. We have implemented and compared different strategies for adaptive evolution to redirect the carbon flux towards glycerol formation at the expense of ethanol. After 200 generations salt stress conditions, we obtained evolved strains with glycerol production increased by 50 to 70%, able to decrease from 0.45 to 0.80% (v/v) the alcohol content of natural or synthetic wines. This shift is accompanied by an accumulation of succinate and 2,3-butanediol and a reduced fermentation rate. Mutants also exhibit a better survival under salt stress and glucose restriction conditions. To identify the underlying mechanisms, we analysed the transcriptome, metabolome (endo- and exo-) and genome of the evolved strains. We showed that the evolved phenotype is not due to deregulation or mutations of genes of involved in the glycerol synthesis pathway but to major changes in carbon, energy and redox metabolism. The genome of the evolved strains revealed loss of heterozygosity which could contribute to the observed phenotype. The genetic study of an evolved strain shows that the metabolic traits are under multigenic control. A QTL mapping analysis using a "bulk sequencing" approach was initiated to identify mutations involved in the high glycerol/low ethanol trait. This work has enabled the development and characterization of low alcohol wine yeast strains and has provided a framework for the identification of the underlying molecular bases.Key-words: S. cerevisiae, wine fermentation, adaptative evolution, ethanol, glycerol, transcriptome, metabolome, genome

Moët & Chandon est le leader mondial incontesté dans le secteur des vins mousseux de haute qualité. La sélection des différentes gammes de vins de qualité se fait sur la base de la dégustation de vin, après la fermentation malolactique par la dégustation des jurys d'experts. Une partie importante du volume vinifié ne peut être classé parmi les vins les plus qualitatifs à cause d'un caractère organoleptique appelé «trait lactique». Cette caractéristique est due à la formation de métabolites volatils au cours de la fermentation du vin en particulier diacétyle, acétoïne et de 2,3-pentanedione. Le diacétyle, également connu sous le nom de 2,3-butanedione, est le principal marqueur associé à l'apparition de notes beurrées. Moët & Chandon souhaite commencer une thèse pour contrôler l'apparition de trait lactique dans ses vins de bases. Bien que la production de diacétyle se produit à la fois pendant la fermentation alcoolique (FA) et la fermentation malolactique, la thèse se concentrera sur la gestion de la FA. Une approche multi-factorielle basée à la fois sur l'ingénierie microbiologique et chimique sera mis en œuvre. Dans un premier temps, le travail visera à mieux comprendre le fonctionnement et la régulation des voies métaboliques impliquées dans la formation de diacétyle. De cette information, nous allons développer des stratégies de contrôle de la fermentation afin de minimiser le caractère lactique de vins. Dans ce travail, le rôle et l'impact des paramètres de fermentation sera particulièrement évalué. Le projet commencera par la mise en œuvre des outils d'analyse nécessaires au suivi des marqueurs de trait lactique (diacétyle, acétoïne, 2,3-pentanedione, ...). Deuxièmement, sur un support synthétique, le projet permettra de caractériser le processus de fermentation. Surtout, les équilibres azotés seront contrôlés et les cinétiques de synthèse des composés responsables de trait d'arôme lactique seront déterminées. Une étude de l'impact des acides aminés précurseurs permettra de mieux comprendre le rôle du métabolisme de l'azote. Enfin, l'étude de l'effet de divers facteurs tels que la nature des substances nutritives, température de fermentation et l'apport en oxygène devrait conduire à l'élaboration de stratégies de contrôle rendant possible la diminution du caractère lactique dans les vins destinés à la fermentation secondaire. Dans la dernière partie de projet, les différentes stratégies développées sur moûts synthétiques seront évaluées dans des conditions de vinification réelles
Champagne company Moët & Chandon is the undisputed worldwide leader in the high quality sparkling wines sector. The selection of different ranges of quality wine is done on the basis of the wine tasting, after malolactic fermentation by tasting expert juries. A significant part of the vinified volume cannot be valued among the most qualitative wines because of a particular feature called « lactic trait ». This trait is attributable to the formation of volatile metabolites during wine fermentation particularly diacetyl, acetoin and 2,3-pentanedione. Diacetyl, also known as 2,3-butanedione, is the main marker associated with the appearance of milky-buttery notes. Champagne company Moët & Chandon wishes to initiate a thesis to control the onset of lactic trait in its wines. Although the production of diacetyl occurs both during alcoholic fermentation (AF) and malolactic fermentation, the thesis will focus on the management of AF. A multi-factorial approach based on both microbiological and chemical engineering will be implemented. At first, the work will aim to better understand the functioning and regulation of metabolic pathways involved in the formation of diacetyl. From this information, we will develop some strategies of fermentation control to minimize the lactic trait of wines. In this work, the role of fermentation parameters will be particularly evaluated. The project will start by the implementation of the analytical tools needed to monitor markers of lactic trait (diacetyl, acetoin, 2,3-pentanedione, ...). Secondly, on synthetic media, the project will allow to characterize the fermentation process. Especially, nitrogen balances will be performed and the kinetics of synthesis of the aroma compounds responsible of lactic trait will be determined. A study of the impact of the precursor amino acids will enable to better understand the role of the nitrogen metabolism. Finally, the study of the effect of various factors such as the nature of nitrogen nutrients, fermentation temperature and oxygen intake should lead to the development of control strategies making possible the decrease of the lactic trait in wines intended for the secondary fermentation. In the final part of the work, the different strategies developed on synthetic must will be assessed in real winemaking conditions

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL / Universitá degli Studi di Torino
In recent years, New Zealand in terms of production has expressed a certain respect and attention towards Sauvignon Blanc for its organoleptic chemical characteristics. In this project seven different amino acids were used, two blend of amino acids and the DAP (di-ammonium phosphate) used in wineries, with the aim of investigating the effect of different levels of nitrogenous source of musts from the two different vineyards ( Omaka Vineyard and Gifford's Creek Vineyard), on the progress of fermentation and the impact on the aromatic component, with the use of the "New Zealand Grape and Wine Research Program" winemaking protocols, with all the processes being reduced to improve the aromatic quality of Sauvignon Blanc in the Marlborough region of New Zealand. This experimental thesis project with the title "Influences of amino acids during alcoholic fermentation and the development of aromatic compounds in Sauvignon Blanc", observes the single amino acids added starting from two musts, with different initial nitrogenous source, and examines the reaction from the yeast during alcoholic fermentation, with reference to the type of free aromatic compounds and their concentration
N/A

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O etanol combustível produzido no Brasil apresenta alguns problemas tecnológicos que afetam a sua produção em larga escala, entre estes destacamos a contaminação do processo por microrganismos indesejáveis, e a fermentação de mosto com baixa concentração de açúcares. Neste trabalho estudou-se aspectos relacionados com leveduras contaminantes do processo industrial, e o desempenho fermentativo de linhagens industriais utilizadas em mostos concentrados e suplementados com fontes estruturalmente complexas de nitrogênio. Foram utilizados dois meios básicos, um constituido da base nitrogenada YNB, e outro a base de caldo de cana, no qual foi adicionado diferentes concentrações de sacarose. Estes meios foram suplementados com fonte de nitrogênio que variaram de simples sal inorgânico como sulfato de amônio, e preparações comerciais de hidrolizados enzimáticos (peptona) e ácidos de proteínas (casaminoácidos). Três linhagens industriais foram inoculadas em mosto a base de caldo de cana contendo sacarose 22%, 30% e 35% (p/v), e suplementados com fontes de nitrogênio. A 30°C, em cultivos agitados e não agitados, verificou-se que a utilização completa de sacarose só ocorreu no meios suplementados com uma fonte de nitrogênio. No geral a suplementação com peptona foi a que propiciou fermentação mais eficiente, quando comparada com sulfato de amônio, maior acúmulo de biomassa e manutenção da viabilidade celular. Os estudos mostraram que nas condições de cultivo e com inoculação de baixa densidade celular, é necessária a suplementação para completa utilização da sacarose. No meio com sacarose 35% (p/v), atingiu-se um nível de 14 % (v/v) de etanol no cultivo não agitado e suplementado, sugerindo ser possível a obtenção de vinhos com maior quantidade de etanol, desde que...
The fuel ethanol industry in Brazil has some technological problems that affect its production on a large scale, among those are contamination by undesirable microorganisms, and wine with low ethanol concentration. We study aspects of microbial contamination of industrial processes, and the fermentation performance of industrial strains of concentrated musts supplemented with structurally complex nitrogen sources. We used two basic media, one consisting of a nitrogen base YNB, and another based on sugar cane juice, which was added increasing concentrations of sucrose. These media were supplemented with nitrogen sources varying from simple inorganic salt such as ammonium sulfate, and commercial preparations of enzymatic hydrolysates (peptone) and acidic protein (casaminoacids). Three industrial strains were inoculated into based sugar cane juice containing 22% sucrose, 30% and 35% (w/v) and supplemented with nitrogen sources. At 30°C, in shaken cultures, it was found that the efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general supplementation with peptone was the one that led to more efficient fermentation compared with ammonium sulfate, with higher biomass accumulation and maintenance of cell viability. Studies have shown that in conditions and with low cell density nitrogen supplementation was required for almost complete sucrose utilization. In sucrose 35% (w/v), it was achieved a level of 14-15% (v/v) ethanol in nitrogen supplemented cultures, suggesting the possibility of producting wines with higher amounts of ethanol in working conditions approaching the limit of yeast alcohol tolerance. The YNB medium was more appropriate to study the demand for nitrogen for different industrial strains. In this medium was also observed that supplementation with... (Complete abstract click electronic access below)

Mestrado Vinifera Euromaster - Instituto Superior de Agronomia - UL
In the last few decades both beer and wine world started to innovate themselves, aiming to produce higher quality beverages, this brought life to a virtuous circle in which the consumers are becoming more informed and the producers invest money for the development of high quality and innovative beverages. The main objective of the work was to produce a fermented drink that conjugates beer and wine world. In order to do achieve this goal, we developed, optimized and characterized a fermented drink produced from grape must and beer wort using non-conventional yeast strains. The beer wort is produced with Braumeister (machinery that is able to maintain the temperature stable, thus having a high quality product) from Pilsner malt and blended with equal amount of Grape juice. The fermentation of the mix was performed using two strains of Torulaspora delbrueckii, one isolated from a beer media and one from a wine media, a strain of Brettanomyces bruxellensis and three different strains of S. cerevisiae¸ one used to ferment wine (ISA1000) and the two beer yeast with very opposite characteristics, US-05 is used to ferment ales style and it should be very neutral while WB-06 is used to ferment wheat beer and it gives an estery aroma to the beverage. Fermentation was monitored analysing the difference in the refraction index on the Brix° scale and composition analysed daily by HPLC. In order to choose the most suitable beverage, each drink was analysed by a trained panel aiming to describe the analytic profile of the beverage, and by a non-trained panel aiming to evaluate the degree of preference of these new beverages. Based on a compromise between technological issues and consumers preferences, the best beverage was the one obtained with S.cerevisiae WB-06.

Moët & Chandon est le leader mondial incontesté dans le secteur des vins mousseux de haute qualité. La sélection des différentes gammes de vins de qualité se fait sur la base de la dégustation de vin, après la fermentation malolactique par la dégustation des jurys d'experts. Une partie importante du volume vinifié ne peut être classé parmi les vins les plus qualitatifs à cause d'un caractère organoleptique appelé «trait lactique». Cette caractéristique est due à la formation de métabolites volatils au cours de la fermentation du vin en particulier diacétyle, acétoïne et de 2,3-pentanedione. Le diacétyle, également connu sous le nom de 2,3-butanedione, est le principal marqueur associé à l'apparition de notes beurrées. Moët & Chandon souhaite commencer une thèse pour contrôler l'apparition de trait lactique dans ses vins de bases. Bien que la production de diacétyle se produit à la fois pendant la fermentation alcoolique (FA) et la fermentation malolactique, la thèse se concentrera sur la gestion de la FA. Une approche multi-factorielle basée à la fois sur l'ingénierie microbiologique et chimique sera mis en œuvre. Dans un premier temps, le travail visera à mieux comprendre le fonctionnement et la régulation des voies métaboliques impliquées dans la formation de diacétyle. De cette information, nous allons développer des stratégies de contrôle de la fermentation afin de minimiser le caractère lactique de vins. Dans ce travail, le rôle et l'impact des paramètres de fermentation sera particulièrement évalué. Le projet commencera par la mise en œuvre des outils d'analyse nécessaires au suivi des marqueurs de trait lactique (diacétyle, acétoïne, 2,3-pentanedione, ...). Deuxièmement, sur un support synthétique, le projet permettra de caractériser le processus de fermentation. Surtout, les équilibres azotés seront contrôlés et les cinétiques de synthèse des composés responsables de trait d'arôme lactique seront déterminées. Une étude de l'impact des acides aminés précurseurs permettra de mieux comprendre le rôle du métabolisme de l'azote. Enfin, l'étude de l'effet de divers facteurs tels que la nature des substances nutritives, température de fermentation et l'apport en oxygène devrait conduire à l'élaboration de stratégies de contrôle rendant possible la diminution du caractère lactique dans les vins destinés à la fermentation secondaire. Dans la dernière partie de projet, les différentes stratégies développées sur moûts synthétiques seront évaluées dans des conditions de vinification réelles
Champagne company Moët & Chandon is the undisputed worldwide leader in the high quality sparkling wines sector. The selection of different ranges of quality wine is done on the basis of the wine tasting, after malolactic fermentation by tasting expert juries. A significant part of the vinified volume cannot be valued among the most qualitative wines because of a particular feature called « lactic trait ». This trait is attributable to the formation of volatile metabolites during wine fermentation particularly diacetyl, acetoin and 2,3-pentanedione. Diacetyl, also known as 2,3-butanedione, is the main marker associated with the appearance of milky-buttery notes. Champagne company Moët & Chandon wishes to initiate a thesis to control the onset of lactic trait in its wines. Although the production of diacetyl occurs both during alcoholic fermentation (AF) and malolactic fermentation, the thesis will focus on the management of AF. A multi-factorial approach based on both microbiological and chemical engineering will be implemented. At first, the work will aim to better understand the functioning and regulation of metabolic pathways involved in the formation of diacetyl. From this information, we will develop some strategies of fermentation control to minimize the lactic trait of wines. In this work, the role of fermentation parameters will be particularly evaluated. The project will start by the implementation of the analytical tools needed to monitor markers of lactic trait (diacetyl, acetoin, 2,3-pentanedione, ...). Secondly, on synthetic media, the project will allow to characterize the fermentation process. Especially, nitrogen balances will be performed and the kinetics of synthesis of the aroma compounds responsible of lactic trait will be determined. A study of the impact of the precursor amino acids will enable to better understand the role of the nitrogen metabolism. Finally, the study of the effect of various factors such as the nature of nitrogen nutrients, fermentation temperature and oxygen intake should lead to the development of control strategies making possible the decrease of the lactic trait in wines intended for the secondary fermentation. In the final part of the work, the different strategies developed on synthetic must will be assessed in real winemaking conditions

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

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Capes
0 objetivo do presente trabalho foi desenvolver um sistema micro-industrial de processamento da vagem da algaroba, visando à obtenção de uma aguardente bidestilada e outra bidestilada e pré-envelhecida em barris de carvalho. Para atingir este objetivo foi necessário estudar: a) a concentração do mosto entre 10o e 20°Brix, no sentido de obter o Brix ideal para fermentação; b) a fermentação natural e fermentação com o uso de fermento selecionado (Sacharomyces cerevisiae) monitorando-se de hora em hora o °Brix, pH, grau alcoólico e temperatura; c) o processo de destilação monitorando-se o grau alcoólico a temperatura do alambinque e na saída do condensador; d) determinar durante o processo fermentativo as análises físico-químicas do teor de sólidos solúveis (°Brix), Grau alcoólico, açúcares redutores, açúcares totais e pH; e) Determinar o rendimento industrial do processo; f) Após obtenção do produto bidestilado, determinar os parâmetros relacionados com a qualidade do produto (acidez total, ésteres, aldeídos, álcoois superiores, cobre e metanol; g) Análise sensorial do produto bidestilado e bidestilado pré- envelhecido em barris de carvalho. O processo consistiu em executar as etapas: 1) recepção e seleção, 2) pesagem, 3) corte da vagem, 4) trituração da vagem, 5) filtração e prensagem, 6) preparação do mosto, 7) fermentação (fermento industrial e fermento natural), 8) destilação, 9) Bidestilação, 10) envelhecimento e 11) acondicionamento. Os resultados obtidos foram: a) Na fermentação observou-se que o melhor teor de sólidos solúveis médio foi de 19°Brix e um valor residual não fermentável de 2°Brix; b) Para o processo de extração o melhor resultado foi a uma pressão de 50 kgf/cm2 com prensagem manual ou 250 kgf/cm2 com prensa automática a uma diluição 1:2(1 kg de vagem/ 2 kg dágua); c) Para o processo de fermentação natural o rendimento foi de 99,7% na fermentação e um teor alcoólico de 10,5°GL. d) O melhor rendimento industrial foi de 52% obtido no experimento 6, onde os rendimentos da extração, fermentação e destilação foram de 72%, 92% e 80,3%, respectivamente; e) as aguardentes bidestilada e bidestilada pré- envelhecida encontram-se dentro dos padrões recomendados pela legislação; f) Na análise sensorial o sabor não difere significativamente entre as duas aguardentes (bidestilada e bidestilada pré-envelhecida), no entanto existe diferença significativamente em relação ao seu aroma.
The objective of the present work was to develop a personal computer-industrial system of processing of the bean of the algaroba, seeking to the obtaining of a liquor bidestilada and other bidestilada and pré-aged in oak barreis. To reach those objective it was necessary to study: the) The concentration of the must between 10 and 20 °Brix, in the sense of obtaining ideal Brix for fermentation; b) The natural fermentation and the fermentation with use of selected ferment (Sacharomyces cerevisiae) being monitored of hour in hour Brix, pH, alcoholic degree and temperature; c) The distillation process being monitored the alcoholic degree the temperature of the still and in the exit of the condensador; d) To determine during the process fermentativo the physical-chemical analyses of the tenor of soluble solids (°Brix), alcoholic Degree, Sugars reducers, total Sugars and pH; and) To determine the industrial revenue of the process; f) After the obtaining of the product bidestilado, to determine the parameters related with the quality of the product (total acidity, ésteres, aldehydes, superior alcohols, copper and methanol; g) Sensorial analysis of the product bidestilado and pré-aged bidestilado in oak barreis. The process consisted of executing the stages: 1) Reception and Selection; 2) Pesagem; 3) It cuts of the bean; 4) Trituração of the bean; 5) Filtration and prensagem; 6) Preparation of the must; 7) Fermentation (I ferment industrial and I ferment natural); 8) Distillation; 9) Bidestilação; 10) Aging and 11) Acondicionamento. The obtained results were: a) In the fermentation it was observed that the best tenor of soluble solids médium was of 19 "Brix and a value residual non fermentável of 2 °Brix; b) Para the extraction process the best result was the a pressure of 50 kgf/cm with manual prensagem or 250 kgf/cm with automatic press to a dilution of 1:2 (1 kg of vagem/2 kg of water); c) Para the process of natural fermentation the revenue was of 99,7% in the fermentation and an alcoholic tenor of 10,5 °GL; d) THE best industrial revenue was of 52% obtained in the experiment 6, where the revenues of the extraction, fermentation and distillation were of 72%, 92% and 80,3%, respectively; and) The liquors bidestiladas and pré-aged bidestilada are inside of the patterns recommended by the legislation; f) In the sensorial analysis the flavor doesn't differ significantly among the two liquors (bidestilada and pré-aged bidestilada), however significant difference exists in relation to your aroma.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Fermentation is a complex process in which raw materials are transformed into high-value products, in this case, grape juice into wine. In this modern and economically competitive society, it is increasingly important to consistently produce wine to definable specifications and styles. Process management throughout the production stage is therefore crucial to achieve effective control over the process and consistent wine quality. Problematic wine fermentations directly impact on cellar productivity and the quality of wine. Anticipating stuck or sluggish fermentations, or simply being able to foresee the progress of a given fermentation, would be extremely useful for an enologist or winemaker, who could then take suitable corrective steps where necessary, and ensure that vinifications conclude successfully. Conventional methods of fermentation monitoring are time consuming, sometimes unreliable, and the information limited to a few parameters only. The current effectiveness of fermentation monitoring in industrial wine production can be much improved. Winemakers currently lack the tools to identify early signs of undesirable fermentation behaviour and to take preventive actions. This study investigated the application of Fourier transform mid infrared (FT-IR) spectroscopy in transmission mode, for the quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The major research objectives were firstly to establish a portfolio of quantitative calibration models suitable for quantification of the major quality determining parameters in fermenting must. The second major research objective focused on a pilot study aimed at exploring the use of off-line batch multivariate statistical process control (MSPC) charts for actively fermenting must. This approach used FT-IR spectra only, for the purpose of qualitative monitoring of alcoholic fermentation in industrial wine production. Towards these objectives, a total of 284 industrial-scale, individual, actively fermenting tanks of the seven major white cultivars and blends, and nine major red cultivars, of Namaqua Wines, Vredendal, South Africa, were sampled and analysed with FT-IR spectroscopy and appropriate reference methods during vintages 2007 to 2009. For the quantitative strategy, partial least squares regression (PLS1) calibration models for determination of the classic wine parameters ethanol, pH, volatile acidity (VA), titratable acidity (TA) and the total content of glucose plus fructose, were redeveloped to provide a better fit to local South African samples. New PLS1 models were developed for the must components glucose, fructose and yeast assimilable nitrogen (YAN), all of which are frequently implicated in problem fermentations. The regression statistics, that included the standard error of prediction (SEP), coefficient of determination (R2) and bias, were used to evaluate the performance of the redeveloped calibration models on local South African samples. Ethanol (SEP = 0.15 %v/v, R2 = 0.999, bias = 0.04 %v/v) showed very good prediction and with a residual predictive deviation (RPD) of 30, rendered an excellent model for quantitative purposes in fermenting must. The models for pH (SEP = 0.04, R2 = 0.923, bias = -0.01) and VA (SEP = 0.07 g/L, R2 = 0.894, bias = -0.01 g/L) with RPD values of 4 and 3 respectively, showed that the models were suitable for screening purposes. The calibration model for TA (SEP = 0.35 g/L, R2 = 0.797, bias = -0.004 g/L) with a RPD of 2, proved unsatisfactory for quantification purposes, but reasonable for screening purposes. The calibration model for the total content of glucose plus fructose (SEP = 0.6.19 g/L, R2 = 0.993, bias = 0.02 g/L) with a RPD of 13, showed very good prediction and can be used to quantify total glucose plus fructose content in fermenting must. The newly developed calibration models for glucose (SEP = 4.88 g/L, R2 = 0.985, bias = -0.31 g/L) and fructose (SEP = 4.14 g/L, R2 = 0.989, bias = 0.64 g/L) with RPD values of 8 and 10 respectively, also proved fit for quantification of these important parameters. The new calibration models of ethanol, total glucose plus fructose; and glucose and fructose individually, showed an excellent relation to local South African samples and can be easily implemented by the wider wine industry. Two calibration models were developed to determine YAN in fermenting must by using different reference methods, namely the enzyme-linked spectrophotometric assay and Formol titration method, respectively. The results showed that enzyme-linked assays provided a good quantitative model for white fermenting must (SEP = 14.10 mg/L, R2 = 0.909, bias = -2.55 mg/L, RPD = 6), but the regression statistics for predicting YAN in red fermenting must, were less satisfactory (data not shown). The Formol titration method could be used successfully in both red- and white fermenting must (SEP = 16.37 mg/L, R2 = 0.912, bias = -1.01 mg/L, RPD = 4). A minor, but very important finding was made with respect to the storage of must samples that were taken from tanks, but that could not immediately be analysed with FT-IR spectroscopy or reference values. Principal component analysis (PCA) of frozen samples showed that must samples could be stored frozen for up to 3 months and still be used to expand the calibration sample sets when needed. Therefore, samples can be kept frozen to a later stage if immediate analyses are not possible. For the purpose of the pilot study that focused on the use of FT-IR spectroscopy for qualitative off-line monitoring of alcoholic fermentation, a total of 21 industrial-scale fermentation tanks were monitored at 8- or 12-hourly intervals, from the onset of fermentation to complete consumption of the grape sugars. This part of the work excluded quantitative data, and only used FT-IR spectra. MSPC charts were constructed on the PLS scores of all the FT-IR spectra taken at the various time intervals of the different batches, using time as the y-variable. The primary aim of this research objective was to evaluate if the PLS batch models could be used to discriminate between normal and problem alcoholic fermentations. The models that were constructed clearly showed the variations in patterns over time, between red- and white wine alcoholic fermentations. One Colombar tank that was fermented at very low temperature in order to achieve a specific wine style, was characterised by a fermentation pattern that clearly differed form the rest of the Colombar fermentations. This atypical fermentation was identified by the batch models constructed in this study. PLS batch models over all the Colombar fermentations clearly identified the normal and problem fermentations. The results obtained in this study showed that FT-IR spectroscopy showed great potential for effective quantitative and qualitative monitoring of alcoholic fermentation during industrial wine production. The work done in this project resulted in the development of a portfolio of calibration models for the most important quality determining parameters in fermenting must. The quantitative models were subjected to extensive independent test set validation, and have subsequently been implemented for industrial use at Namaqua Wines. Multivariate batch monitoring models were established that show good discriminatory power to detect problem fermentations. This is a very useful diagnostic tool that can be further developed by monitoring more normal and problem fermentations. Future work in this regard, will focus on further optimisation and expansion of the quantitative and qualitative calibration models and implementation of these in the respective wineries of Namaqua Wines.
AFRIKAANSE OPSOMMING: Fermentasie is ‘n komplekse proses waartydens rou material getransformeer word na produkte van hoë waarde, in hierdie geval, druiwesap na wyn. In die huidige ekonomies-kompeterende samelewing, is dit al hoe meer belangrik om volhoubaar wyn te produseer wat voldoen aan definieerbare spesifikasies en style. Goeie prosesbestuur tydens die wynproduksie stadium is baie belangrik om herhaalbaarheid en gehaltebeheer te verseker. Problematiese wynfermentasies het ’n direkte impak op beide kelderproduktiwiteit en wynkwaliteit. Die voorkoming van slepende- of steekfermentasies, of selfs net om probleme te voorsien, sou uiters bruikbaar wees vir ‘n wynkundige of wynmaker, wat dan die toepaslike regstellende stappe kan neem waar nodig, om te verseker dat die wynbereiding suksesvol voltooi word. Konvensionele metodes van monitering van alkoholiese fermentasie is tydrowend, soms onbetroubaar en die inligting beperk tot ‘n paar parameters. Die huidige effektiwiteit van fermentasie monitering in industriële wynproduksie kan heelwat verbeter word. Wynmakers ervaar tans ’n behoete aan tegnologië wat die vroeë tekens van ongunstige fermentasiepatrone kan identifiseer, en hul doeltreffendheid om moontlike regstellende aksies te neem, is dus beperk. Hierdie studie het die toepassing van Fourier transformasie mid-infrarooi (FT-IR) spektroskopie in transmissie, ondersoek met die oog op kwantitatiewe en kwalitatiewe monitering van alkoholiese gisting tydens industriële wynproduksie. Die vernaamste navorsingsdoelwitte was eerstens om ’n portefeulje van kwantitatiewe kalibrasiemodelle te vestig, wat geskik is om die belangrikste kwaliteitsbepalende parameters in gistende mos te kwantifiseer. Die tweede hoofnavorsingsdoelwit was ’n loodsstudie wat ondersoek ingestel het na die opstel van multiveranderlike statistiese proseskontrole grafieke van aktief-gistende mos, met die oog op aflyn-kwalitatiewe monitering van alkoholiese gisting in industriële wynproduksie. Hiervoor is slegs FT-IR spektra gebruik. Vir die doel van hierdie studie is monsters van ’n totaal van 284 individuele, aktief-gistende tenke van die sewe hoof wit kultivars en hul versnydings en nege hoof rooi kultivars van Namaqua Wyne, Vredendal, Suid Afrika, geneem. Al die monsters is met toepaslike chemiese metodes en FT-IR spektroskopie analiseer tydens die parsseisoene van 2007 tot 2009. Vir die kwantitatiewe strategie is parsiële kleinste kwadraat (PKK1) kalibrasiemodelle vir die bepaling van die klassieke wynparameters etanol, pH, vlugtige suur (VS), titreerbare suur (TS) en die totale konsentrasie van glukose plus fruktose herontwikkel, om beter te pas op plaaslike Suid-Afrikaanse monsters. Nuwe PKK1 kalibrasiemodelle is ontwikkel vir die komponente glukose, fruktose en gis-assimileerbare stikstof, aangesien hierdie komponente gereelde aanduidings van probleemgisting is. Die regressiestatistieke het die standaardvoorspellingsfout (SVF), bepalingskoëffisiënt (R2) en sydigheid ingesluit en was gebruik om die prestasie van die herontwikkelde kalibrasiemodelle vir plaaslike Suid-Afrikaanse monsters te evalueer. Etanol (SVF = 0.15 %v/v, R2 = 0.999, sydigheid = 0.04 %v/v) het baie goeie regressiestatistiek getoon en met ‘n relatiewe voorspellingsafwyking (RVA) van 30, was dit ‘n uitstekende model vir kwantifisering in gistende mos. Die modelle vir pH en VS met RVA waardes van 4 en 3 onderskeidelik, is geskik vir semi-kwantitatiewe toepassings. Die kalibrasiemodel vir TS met ‘n RVA waarde van 2, was nie geskik vir akkurate kwantifisering nie, maar wel vir semikwantitatiewe analises. Die kalibrasiemodel vir die totale glukose plus fruktose inhoud in gistende mos, met ‘n RVA waarde van 13, het uitstekende regressiestatistiek gegee en is geskik vir akkurate kwantifiseringsdoeleindes. Die nuut-ontwikkelde kalibrasiemodelle vir glukose en fruktose, met RVA waardes van onderskeidelik 8 en 10, is geskik vir akkurate kwantifisering van hierdie belangrike parameters. Die kalibrasiemodelle vir etanol, totale glukose plus fruktose, en glukose en fruktose afsonderlik, het uitstekende korrelasies getoon met plaaslike Suid-Afrikaanse monsters en is gereed om toepassing te vind in die wyer wynindustrie. Twee kalibrasiemodelle is ontwikkel om gis-assimileerbare stikstof in gistende mos te bepaal, deur gebruik te maak van verskillende verwysingsmetodes van analise; hierdie metodes was ‘n ensiem-gekoppelde spektrofotometriese toets en die Formoltitrasie metode. Resultate het getoon dat goeie regressiestatistiek vir FT-IR spektroskopie-gebaseerde kalibrasiemodelle waar data wat met die ensiem-gekoppelde toetse verkry is, as verwysingwaardes gebruik is, in wit gistende mos (SVP = 14.10 mg/L, R2 = 0.909, sydigheid = -2.55 mg/L, RVA = 6), maar nie in rooi gistende mos nie. Die Formoltitrasie metode as verwysingsmetode, was geskik vir die ontwikkeling van goeie kalibrasiemodelle in beide rooi- en wit gistende mos (SVP = 16.37 mg/L, R2 = 0.912, sydigheid = -1.01 mg/L, RVA = 4). ’n Sekondêre, maar baie belangrike bevinding is gemaak met betrekking tot die stoor van mosmonsters wat geneem is van tenke, maar wat nie dadelik met die verwysingsmetodes en FT-IR spektroskopie analiseer kon word nie. Multiveranderlike hoofkomponentanalise op vars en gevriesde sapmonsters het getoon dat gevriesde monsters gebruik kan word om die kalibrasie datastel uit te brei, wanneer benodig. Dus, sapmonsters kan gevries word tot ’n later stadium as onmiddelike analises nie moontlik is nie. Vir die doel van die tweede navorsingsdoelwit van die studie, naamlik kwalitatiewe af-lyn monitering van alkoholiese fermentasie met FT-IR spektroskopie, is ‘n totaal van 21 industriëlegrootte fermentasietenks ge-monitor deur sapmonsters met 8- tot 12-uurlikse intervalle te trek, vanaf die begin van fermentasie, totdat al die druifsuiker gemetaboliseer is. Vir hierdie deel van die werk is die kwantitatiewe data nie gebruik nie; slegs die FT-IR spektra. Multiveranderlike statistiese proseskontrole grafieke is opgestel op grond van die PKK tellings wat bereken is op al die FT-IR spektra wat gemeet is by die verskillende tydsintervalle. Vir hierdie analise is tyd as y-veranderlike gebruik. Die vernaamste doel van hierdie ondersoek was om te evalueer of die PKK-gebaseerde modelle kon onderskei tussen normale en slepende gistings. Die modelle wat verkry is, het die variasie oor tyd in die fermentasiepatrone tussen wit- en rooiwyn fermentasies tydens alkoholiese gisting, duidelik uitgewys. Een Colombar tenk wat teen baie lae temperatuur gefermenteer is om ‘n spesifieke wynstyl te verkry, se fermentasiepatroon het aansienlik verskil van die ander Colombar tenks wat gemonitor is, en hierdie atipiese patroon is ook deur die kwalitatiewe modelle identifiseer. ‘n PKK model oor al die Colombar fermentasies kon duidelik tussen normale en slepende gistings onderskei. Die resultate wat in hierdie studie verkry is, het getoon dat FT-IR spektroskopie baie goeie potensiaal toon vir die aanwending van kwantitatiewe en kwalitatiewe monitering van alkoholiese fermentasie tydens industriële wynproduksie. Die werk wat in hierdie projek gedoen is, het gelei tot die vestiging van ‘n portefeulje van kalibrasiemodelle vir die belangrikste kwaliteitsbepalende parameters in fermenterende mos. Die kwantitatiewe modelle is baie deeglik getoets met onafhanlike toets datastelle, en daarna is die kalibrasiemodelle geimplementeer vir industriële gebruik by Namaqua Wyne. Multiveranderlike statistiese proseskontrole grafieke wat baseer is op data wat vanaf 21 verskillende fermentasietenks verkry is, het baie goeie potensiaal getoon om probleemfermentasies vroeg te identifiseer. Dié grafieke is ‘n baie nuttige diagnostiese hulpmiddel wat verder ontwikkel kan word om verskillende tipes probleemfermentasies te monitor. Toekomstige navorsing in hierdie konteks, sal toegespits word op die optimisering en uitbreiding van die kwantitatiewe en kwalitatiewe modelle, sowel as toepassing van die tegnieke in die onderskeie kelders van Namaqua Wyne.

Thesis (MSc)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: During fermentation, nitrogenous compounds serve as nutrients for the yeasts, which enable their growth, functioning and maintenance of the yeasts cells. From a winemaking perspective, a certain amount of nitrogen is required for the yeasts in order to avoid sluggish or stuck fermentation. Moreover, nitrogen metabolism leads to the production of aroma compounds such as higher alcohols, fatty acids and esters which contribute positively to overall sensory characteristics of wine. Nitrogen metabolism (uptake of ammonium and amino acids) have been extensively studied in Saccharomyces cerevisiae. Nonetheless, the fairly great variances observed between strains in terms of preference for certain nitrogen sources and metabolism thereof are not so well understood. Additionally, these mechanisms nitrogen metabolism of non- Saccharomyces yeasts are even vaguer and simply assumed to be globally similar to those of S. cerevisiae. This study aimed to investigate the uptake of nitrogen compounds (ammonium and individual amino acids) by selected non-Saccharomyces yeasts (Lachancea thermotolerans IWBT Y1240, Torulaspora delbrueckii Biodiva TD291, Pichia kluyveri FrootZen, Metschnikowia pulcherrima IWBT Y1123 and Metschnikowia pulcherrima Flavia) to assess the impact of fermentation kinetics and the production of aroma compounds during sequential fermentations with S. cerevisiae under different initial YAN concentrations, with 300 mg/L, 150 mg/L and 75 mg/L, respectively). Fermentations were performed in a synthetic grape juice medium with pure and sequential fermentations. The data showed that the assimilation of nitrogen compounds were species specific. For example, L. thermotolerans preferred alpha amino nitrogen above ammonia, where the opposite hold true for T. delbrueckii. Notable differences could also be identified for the uptake of certain single amino acids. Irrespective of the initial YAN concentrations during sequential fermentations, the yeasts only assimilated about half of the initial YAN. The non-Saccharomyces yeasts did not influence fermentation performance during sequential fermentations. However, a low initial YAN (75 mg/L) had a strong influence on the fermentation kinetics and aroma compound production. The higher uptake (compare to S. cerevisiae) of specific single amino acids by non-Saccharomyces yeasts (especially L. thermotolerans), can be tentatively correlated with certain aroma compounds produced at the end of fermentation. The results also revealed that agitation could impact overall fermentation performance and aroma compound production. This study contributes to an improved understanding of how different initial nitrogen concentrations affect growth, fermentation performances and aroma compound production of wine-related yeasts under fermentative conditions. Moreover, the uptake of single amino acids by selected non-Saccharomyces yeasts had also been identified, which is a good starting point to better understand non- Saccharomyces yeasts nitrogen requirements which may be used for the optimization of nitrogen source addition, during alcoholic fermentation, when used in mixed fermentations in order to ensure a complete alcoholic fermentation. To the best of our knowledge, the uptake of single amino acids and YAN consumption by selected non-Saccharomyces yeasts under fermentation conditions tested, have never been studied before.
AFRIKAANSE OPSOMMING: Tydens wynfermentasies dien talle stikstof komponente as voedingstowwe vir wyngis wat hul groei, funksie en onderhoud bevorder. Van `n wynmaak perspektief word daar `n sekere hoeveelheid stikstof benodig deur die wyngis om te verhoed dat slepende of onvolledige fermentasies plaasvind. Verder lei stikstofmetabolisme na die produksie van aroma verbindings, soos hoër alkohole, vlugtige vetsure en esters wat positief bydra tot die sensoriese karaktereienskappe van wyn. Die stikstofmetabolisme (opneem ammonium en aminosure) is deeglik nagevors in die wyngis Saccharomyces cerevisiae, maar die klein variasies waargeneem tussen die gisras in terme van die voorkeur van sekere stikstof komponente is egter nog onduidelik. Daarbenewens is die stikstofmetabolisme nog meer onbekend in nie- Saccharomyces wyngis en word dit oor die algemeen aanvaar dat die werking van die stikstofmetabolisme dieselfde is as in S. cerevisiae. Hierdie studie het gestreef om die opneem van stikstof komponente (ammonium en aminosure) te ondersoek van uitverkiesde nie-Saccharomyes gis (Lachancea thermotolerans IWBT Y1240, Torulaspora delbrueckii Biodiva TD291, Pichia kluyveri FrootZen, Metschnikowia pulcherrima IWBT Y1123 and Metschnikowia pulcherrima Flavia) deur te bepaal wat die impak is op die groei-kinetika en op die produksie van aroma komponente gedurende gemengde kultuur fermentasies met S. cerevisae onder verskillende aanvangs assimileerbare stikstof (300 mg/L, 150 mg/L en 75 mg/L). Fermentasies is in sintetiese druiwemos uitgevoer vir beide enkel en gemengde kultuur fermentasies. Die resultate demonstreer dat die assimilasie van stikstof ras spesifiek was. Byvoorbeeld, L. thermotolerans verkies alfa amino stikstof bo ammonium waar die teenoorgestelde waar is vir T. delbrueckii. Beduidende verskille is ook waargeneem vir die opneem van sekere individuele aminosure. Die wyngis het steeds net die helfte van die assimileerbare stikstof opgeneem gedurende gemengde kultuur fermentasies ongeag die aanvangsstikstof konsentrasies. Die nie-Saccharomyces gis het nie die fermentasie kinetika beïnvloed tydens gemengde kultuur fermentasies nie. Daar was egter ook waargeneem dat `n lae assimileerbare stikstof (75 mg/L) `n negatiewe invloed op die fermentasie kinetika sowel as aroma produksie gehad het. Die hoër opname (vergelyking met S. cerevisiae) van sekere aminosure deur nie-Saccharomyces gis, kan tydelik gekoppel word aan die produksie van spesifieke aroma verbindings aan die einde van fermentasies. Die resultate het ook gewys dat die toepassing van skud `n impak het op die fermentasie kinetika sowel as die produksie van aroma komponente. Die studie dra by om beter te verstaan van hoe verskillende aanvangsstikstof die groei, fermentasie kinetika en aroma produksie beïnvloed onder fermentasie kondisies. Die opneem van sekere aminosure deur nie-Saccharomyces gis word ook beskryf, wat `n goeie beginpunt is om beter te vertaan wat die stikstof vereistes vir die geselekteerde wyngis is, wat gebruik kan word vir die optimisering van stikstofaanvullings, sodat die risiko van probleemfermentasies verlaag sal word. So ver as wat ons kennis strek is die opneem van aminosure en die gebruik van assimileerbare stikstof deur nie-Saccharomyces wyngis onder fermentasie kondisies nog nie ondersoek nie.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Scotch whiskey is of two main types, namely Scotch malt whiskey, made from malted barley alone, or Scotch grain whiskey, made from cereals, such as wheat or maize, together with malted barley. In both processes, the enzymes from the barley are responsible for starch conversion and should always be derived entirely from the malted barley. No exogenous enzymes are allowed to be added to any mashing. The enzymes involved in the conversion process to fermentable sugars, are the aand p-amylases, limit dextrinase and p-glucosidase. Maize, on the other hand, contains no enzyme activity, therefore enzymes need to be added when producing whiskey from maize alone. In other whiskey-producing countries where maize is freely available and cheaper than barley, the use of exogenous enzymes are allowed in the mashing process and is crucial for the formation of fermentable sugars from complex carbohydrates. The cost of the enzymes, however, can push the production cost of whiskey to higher levels. Saccharomyces cerevisiae does not have any amylolytic activity, but is an excellent fermenter and produces favourable organoleptic notes, which makes it very suitable for producing potable spirit. Efforts have been made to genetically improve industrial strains, relying on classical genetic techniques followed by the selection of broad traits, such as ethanol tolerance, absence of off-flavours and carbohydrate/starch utilisation. No strain has thus far been selected for total starch degradation during the fermentation of whiskey mash. Over the last decade, considerable progress has been made in the development of genetically improved strains for the distilling, wine, brewing and baking industries. The expression of heterologous genes introduced a new dimension in approaches to the genetic improvement of industrial strains. It would therefore be cost-effective to use a yeast strain that can produce active and sufficient enzymes to ferment raw starch efficiently to alcohol without lowering the quality of the end product. No such strain has been developed to date, but the continuous improvement of starch-utilising strains has made this goal more achievable. Two a-amylase genes, namely LKA 1 and LKA2, were previously isolated from Lipomyces kanonenkoae. In this study, we selected 4 strains on the basis of criteria that are important for whiskey-specific strains. The selected strains were transformed with LKA 1, as well as with a combination of LKA 1 and LKA2 genes. The wine yeast VIN13 was included in the transformation of LKA1 and LKA2 because of its rapid fermentation rate. The genes were integrated into the genomes of the yeast strains and were stable after many generations. Assays showed that a significant increase in enzyme activity was induced in the whiskey strains, compared to the untransformed strains. The strains also showed good fermentation ability in whiskey fermentations, although optimum alcohol production was still not achieved.
AFRIKAANSE OPSOMMING: Skotse whiskey bestaan uit 2 tipes, nl. mout whiskey, gemaak slegs van mout d.w.s. gars wat die mout proses ondergaan het, en graan whiskey wat gemaak word van gewasse soos mielies of koring, waarby mout gevoeg word. Die ensieme afkomstig van die mout is verantwoordelik vir die omsetting van stysel na fermenteerbare suikers en geen eksogene ensieme mag by die gars- of graanmengsel gevoeg word nie. Die ensieme wat betrokke is by die omsetting van stysel, is die a- en ~- arnitases, limiet dekstrinase en ~-glukosidase. Mielies bevat geen ensiemaktiwiteit nie, dus moet ensieme by die proses gevoeg word indien slegs mielies vir die vervaardiging van whiskey gebruik word. In whiskey produserende lande waar mielies vryelik beskikbaar is en goedkoper is as gars, word eksogene ensieme by die graanmengsel gevoeg vir die vrystelling van fermenteerbare suikers vanaf komplekse koolhidrate. Die hoë koste van die ensieme kan egter die produksiekoste van whiskey verhoog. Saccharomyces cerevisiae besit geen amilolitiese aktiwiteit nie, maar is 'n uitstekende fermenteerder en produseer gewensde organoleptiese geure. Om hierdie redes is S. cerevisiae baie geskik vir die produksie van drinkbare etanol. Navorsingspogings om industriële rasse geneties m.b.v. klassieke genetiese metodes te verbeter, kom wydverspreid in die literatuur voor. Dit sluit in die seleksie van rasse met 'n verskeidenheid van eienskappe soos etanol toleransie, die afwesigheid van afgeur produksie en koolhidraat/stysel benutting. Geen ras is egter tot op hede geselekteer vir totale stysel afbraak gedurende fermentasie nie. Groot vordering is gedurende die laaste dekade gemaak in die ontwikkeling van genetiese verbeterde rasse vir die wyn- stokery- en brouers industrieë. Die uitdruk van heterogene gene in gisrasse gee 'n nuwe dimensie aan die genetiese verbetering van industriële rasse. Die gebruik van 'n gisras wat aktiewe en genoegsame ensieme produseer om rou stysel te fermenteer, sonder om die kwalitiet van die eindproduk nadelig te beïnvloed, kan die produksiekoste van whiskey aansienlik verminder. Geen gisras met hierdie eienskap is tot op hede ontwikkel nie, maar die voortdurende verbetering van rasse om stysel af te breek maak hierdie doel meer bereikbaar. Twee a-amilase gene, nl. LKA 1 en LKA2 is voorheen uit Lipomyces kononenkoae geïsoleer. In hierdie studie is 4 gisrasse geselekteer op grond van die kriteria wat nodig is vir whiskey giste. Die geselekteerde rasse is getransformeer met LKA 1 sowel as 'n kombinasie van LKA 1 en LKA2 gene. Die wyngis VIN13 is ingesluit by die transformasie met die LKA1 en LKA2 gene, omrede VIN13 bekend is as 'n vinnige fermenteerder. Die gene is geïntegreer in die genoom van die verskillende gisrasse en is stabiel na vele generasies. Die getransformeerde rasse het 'n betekenisvolle verhoging in ensiemaktiwiteit teenoor die nie-getransformeerde rasse getoon. AI die transformante het ook goeie fermentasie vermoë getoon in whiskey fermentasie proewe. Optimum alkoholproduksie is egter nie verkry nie.

A espécie Dekkera bruxellensis tem sido detectada como a principal levedura contaminante em diversos processos fermentativos, dentre eles o de produção de etanol combustível, apresentando uma surpreendente capacidade de crescimento e adaptação naqueles substratos. No entanto, pouco se conhece sobre suas características de crescimento em condições de estresse e comportamento fermentativo. Desta forma, este trabalho objetivou avaliar a tolerância ao estresse e as características fermentativas exibidas por três linhagens de D. bruxellensis isoladas da fermentação alcoólica, além do efeito da contaminação em mosto de caldo de cana sobre os parâmetros fermentativos, buscando informações que possam contribuir para o manejo da fermentação alcoólica quando contaminada com esta levedura. Foram realizados testes de caracterização em meio YEPD sólido e líquido em condições estressantes para as três linhagens de D. bruxellensis e uma linhagem de S. cerevisiae (PE-02). O efeito do tratamento ácido associado ao etanol sobre a viabilidade das quatro linhagens em situação de agitação e sem agitação foi também avaliado. Em seguida, testes fermentativos em meio sintético (sem reciclo celular) e em meio de caldo de cana (com reciclo celular e utilizando-se ou não tratamento ácido) foram conduzidos para verificar as características fermentativas das linhagens de D. bruxellensis em comparação com S. cerevisiae, simulando-se uma contaminação por 103 células/mL da linhagem CCA155 em meio de caldo de cana. As três linhagens de D. bruxellensis apresentaram crescimento invasivo em meio YEPD sólido, possivelmente um mecanismo de sobrevivência da levedura em condições estressantes. Observou-se uma variação na resposta das linhagens às situações de estresse (baixo pH e alta concentração de etanol). Em condições não estressantes, a linhagem PE-02 apresentou melhor desenvolvimento, no entanto, em situações de estresse de pH, concentrações de açúcar e etanol, as linhagens de D. bruxellensis desenvolveram-se melhor. O controle eficiente do crescimento destas leveduras poderia ser obtido com um tratamento combinado de baixo pH (1,5) e etanol (9%), porém houve também prejuízo significativo à levedura S. cerevisiae, embora em menor extensão. Em sistema de batelada sem reciclo celular em meio sintético, verificou-se que a agitação influenciou significativamente a produção de etanol e ácidos por D. bruxellensis. O teor alcoólico foi maior quando se utilizou glicose como fonte de carbono ao invés de sacarose. Em sistema de batelada com reciclo celular em meio de caldo de cana, foram obtidos melhores resultados quanto à produção de etanol, menor teor de açúcar redutor total residual e maior eficiência fermentativa quando se utilizou o tratamento ácido do fermento (pH 1,5), assim como melhor controle do crescimento da linhagem CCA155 quando em cultura mista com S. cerevisiae (PE-02). O tratamento ácido utilizado teve efeito não só sobre o crescimento da levedura contaminante, mas também beneficiou a levedura do processo, resultando assim na minimização do efeito da contaminante sobre a fermentação conduzida em meio de caldo de cana sob dez ciclos fermentativos de 12 horas.
The species Dekkera bruxellensis has been considered as the main contaminant yeast in several fermentative processes, including the fuel alcohol production, showing a surprising growth capacity and adaptation in those substrates. However, a little is known about their growth characteristics in stressing conditions and fermentative profile. In this context, this work aimed to evaluate the stress tolerance and fermentative characteristics exhibited by three strains of D. bruxellensis isolated from the alcoholic fermentation, besides the effect of the contamination in sugar cane juice over the fermentative parameters, searching for information that could contribute to the management of the alcoholic fermentation when the medium is contaminated with this yeast. Characterization tests in YEPD medium (solid and liquid) under stressing conditions for the D. bruxellensis and S. cerevisiae (PE-02) strains were carried out. The effect of the acid treatment associated with ethanol over the cell viability of the four strains in shaken and non-shaken flasks was also evaluated. Following, fermentative tests in synthetic medium (without cell recycle) and in sugar cane juice (with cell recycle and with/without acid treatment) were carried out to verify the fermentative characteristics of the strains of D. bruxellensis comparing to S. cerevisiae, simulating a contamination by 103 cells/mL of the strain CCA155 in sugar cane juice. All the strains of D. bruxellensis showed invasiveness in YEPD agar medium, probably a survival mechanism in stressful conditions. A variation in the response of the strains to the stressing conditions (low pH and high ethanol concentration) was observed. In nonstressing situations, the strain PE-02 showed better growth; however, in stressing conditions of pH, ethanol and sugar concentrations, the strains of D. bruxellensis had better growth performance. The effective control of their growth could be obtained with a combined treatment of low pH (1.5) and ethanol (9%), however, a significant harmful effect to the S. cerevisiae strain was also verified, but in a lower extension. In batch system without cell recycle in synthetic medium, it was verified the influence of agitation over the ethanol and acid production by D. bruxellensis. The alcohol content was significantly higher when glucose was utilized instead of sucrose. In batch system with cell recycle in sugar cane juice, the best results for ethanol production, lower residual total reducing sugar and higher fermentative efficiency were obtained with the acid treatment of the ferment at pH 1.5, as well as a better growth control of the strain CCA155, in mixed culture with S. cerevisiae (PE-02). The acid treatment utilized in this work had effect not only over the growth of the contaminant yeast, but also benefited the yeast S. cerevisiae, resulting in the minimization of the effect of the contaminant over the fermentation developed in sugar cane juice medium in 12-hour ten fermentative cycles.

No presente trabalho, primeiramente avaliou-se o desempenho fermentativo de 10 cepas de leveduras isoladas de frutas, de mel centrifugado e de resíduo do processamento de mel após a etapa de sedimentação (borra do mel), em tubo de ensaio contendo mosto constituído de Mel 15 °Brix suplementado, individualmente, com diferentes nutrientes, a saber: extrato de levedura comercial (10,0 g/L), farelo de arroz (20,0 g/L), mistura (1,0 g/L) constituída de farelos de milho, arroz e soja na proporção 5:2:5 e nutriente comercial (D&R) (0,15 g/L). Os parâmetros avaliados coexistiram da determinação do número de células em câmara de Neubauer e das concentrações de açúcares e etanol por HPLC, o que permitiu a determinação dos parâmetros rendimento, eficiência de fermentação e produtividade. Estes resultados sustentaram a seleção do mosto suplementado com extrato de levedura comercial e da melhor cepa de levedura que foram posteriormente avaliados em escala piloto. A cepa selecionada foi a levedura isolada a partir da borra de mel, a qual foi posteriormente identificada como Saccharomyces cerevisiae e nomeada Saccharomyces cerevisiae EEL 2009. A avaliação da produção de aguardente de mel em escala piloto foi realizada nas instalações do Alambique da Associação Rural de Canas, Canas-SP e o destilado obtido armazenado em tonel de carvalho de 200 L por 6 meses. Amostras foram coletadas a cada 30 dias para caracterização físico-química em conformidade com os parâmetros estabelecidos na Legislação Brasileira para aguardente de frutas. Em paralelo, as respectivas amostras foram devidamente avaliadas sensorialmente por 120 provadores não treinados, por meio de testes de aceitação, em escala hedônica, considerando os quesitos aparência, aroma, sabor, corpo e impressão global, além da atitude de compra. Estes resultados foram analisados estatisticamente por ANOVA e teste de Tukey, sendo os resultados de aceitação em relação à impressão global, analisados por meio da análise multivariada o que permitiu traçar o Mapa de Preferência Interno - MDPREF. Os resultados referentes à caracterização físico-química das respectivas amostras demonstraram que todas apresentaram os parâmetros de avaliação em conformidade com os padrões estabelecidos pela legislação. Os resultados da análise sensorial revelaram que o tempo de armazenamento de 180 dias em tonel de carvalho não foi suficiente para a ocorrência de reações desejadas, o que influenciaria nas características sensoriais da bebida, tornado-a mais agradável e suave. No entanto, apesar do pouco tempo de armazenamento, a aguardente de mel apresentou uma boa aceitação por parte dos provadores, cuja maioria manifestou sua aceitação em termos de \"gostei ligeiramente\" e \"não gostei, nem desgostei\". No tocante ao MDPREF, este revelou que a amostra referente a 180 dias de armazenamento foi preferida por um maior número de provadores. Em relação à atitude de compra, as amostras armazenadas por 60 (79,17%) e 180 dias (75,83%) apresentaram os melhores resultados. Diante do exposto, conclui-se que o mel se apresenta como uma alternativa viável para a formulação de mosto para produção de aguardente no período de entre safra de cana-de-açúcar, contribuindo para melhor aproveitamento das instalações e como fonte alternativa de renda para o produtor rural.
In this work, first it was evaluated the fermentation performance of 10 yeast strains isolated from fruits, centrifuged honey and residue of honey processing after the sedimentation (sludge honey) step. This experiment was carried out in test tube containing wort made from honey 15 °Brix supplemented individually with different nutrients, namely: commercial yeast extract (10.0 g/L), rice bran (20.0 g/L), mixture (1.0 g/L) consisting of bran corn, rice and soy in the ratio 5:2:5 and commercial nutrient (D&R) (0.15 g/L). The parameters evaluated included the cell number determination in Neubauer chamber, and sugars and ethanol concentrations by HPLC, which allowed to determine the fermentation parameters as yield, fermentation efficiency and productivity. These results supported the selection of the wort supplemented with commercial yeast extract and the best yeast strain, which were subsequently evaluated in a pilot scale. The selected strain was the yeast isolated from the sludge honey, which was later identified as Saccharomyces cerevisiae and named Saccharomyces cerevisiae EEL 2009. The production evaluation of honey spirit on a pilot scale was conducted at the Canas Rural Association Pilot Plant for cachaça production - Canas-SP and distillate was stored in oak barrel of 200 liters per 6 months. Samples were collected every 30 days for physic-chemical characterization in accordance with the guidelines established in the Brazilian Legislation for fruit spirit. Beyond that, the respective samples were properly sensory evaluated by 120 untrained consumers regarding to acceptance testing employing a hedonic scale, considering characteristics as appearance, aroma, flavor, and overall impression, and the attitude of purchase, as well. These results were statistically analyzes by ANOVA and Tukey`s test and the results of acceptance regarding to the overall impression, were analyzed by multivariate analysis which allowed tracing the Internal Preference Map - MDPREF. The results concerning the physic-chemical characterization of the respective samples showed that all presented the evaluation parameters in accordance with standards established by the legislation. The results of sensory analysis revealed that the storage time of 180 days in oak barrel was not enough for the occurrence of desired reactions, which influence the sensory characteristics of the beverage, making it nicer and smoother. However, despite short time storage, honey spirit showed good acceptance by the consumers. Most of them expressed acceptance in terms of \"liked slightly\" and \"not liked nor disliked\". Regarding the MDPREF results, these proved that the sample relating to 180 days of storage was preferred by majority of consumers. Regarding the attitude of purchase, the samples stored for 60 days (79.17%) and 180 days (75.83%) showed the best results. Therefore, we conclude that honey can be considered as a viable alternative for wort formulation for the production of spirit, mainly in the period between harvests of sugar cane, contributing to better utilization of the facilities and as alternative source of income for farmers.

As leveduras Dekkera/Brettanomyces estão envolvidas na deterioração de vinhos após o término das fermentações alcoólicas e maloláticas, tendo se apresentado como agente contaminante de processos contínuos de produção de etanol industrial. São caracterizadas pela morfologia celular típica (células alongadas e ogivais), alta produção de ácido e crescimento lento, porém de difícil identificação. Embora muitos trabalhos já tenham sido publicados acerca do seu papel na fermentação do vinho, pouco se conhece sobre o seu comportamento no processo de fermentação para produção de álcool combustível. Desta forma, este trabalho objetivou selecionar, identificar e caracterizar linhagens de Dekkera e Brettanomyces isoladas de processos fermentativos, através de testes de taxonomia clássica, moleculares (PCR e sequenciamento) e de biotipagem através do sistema killer, visando a avaliação de fermentações mistas de Saccharomyces cerevisiae e Dekkera bruxellensis, a última em níveis de contaminação variando de 101 a 103 células/mL, em meio de caldo de cana, em processo de fermentação em batelada com reciclo celular (14 ciclos de 12 horas) para produção de etanol. Os testes morfológicos e fisiológicos/bioquímicos de oito linhagens selecionadas pela alta produtividade de ácido a partir da glicose e morfologia celular característica, apontaram os gêneros Dekkera ou Brettanomyces, sem possibilidade de identificação em nível de espécie devido à ambigüidade dos resultados dos testes fisiológicos. O sequenciamento da região ITS (Internal transcribed spacer) do DNA ribossomal confirmou somente três linhagens como Dekkera bruxellensis, sendo as demais predominantemente pertencentes à espécie Pichia guillermondii. O tamanho da região ITS, incluindo o gene 5,8S, variou de 400 500 pb entre as três linhagens. A utilização do sistema killer como método de biotipagem para leveduras Dekkera mostrou-se inviável devido ao fenótipo predominantemente neutro apresentado pelas linhagens. Somente a levedura killer CCA510 (Kluyveromyces marxianus) apresentou atividade inibitória contra as três linhagens de D. bruxellensis. Os ensaios fermentativos realizados com a linhagem de D. bruxellensis (CCA059) em fermentações puras e mistas com S. cerevisiae (CCA193, PE-02), mostraram que a levedura contaminante foi capaz de crescer em meio de caldo de cana, independentemente do tamanho do seu inóculo inicial (101 a 103 células/mL), impactando negativamente a fermentação etanólica, causando a diminuição da viabilidade de S. cerevisiae, diminuindo o pH do meio, decréscimo na produção de etanol e eficiência fermentativa, possivelmente devido à produção de ácido acético a partir do ART do meio de fermentação. Extrapolando-se os resultados obtidos em escala de laboratório para a escala industrial de uma destilaria de médio porte, a contaminação por Dekkera bruxellensis acarretaria uma perda de 6 milhões a 15 milhões de litros de álcool na safra, que deixariam de ser produzidos, dependendo do nível de contaminação.
Dekkera/Brettanomyces yeasts are found to be either contaminants in wine after completion of alcoholic and malolactic fermentations and in continuous fermentations for fuel alcohol production. They are characterized by typical cell morphology (elongated and ogival cells), high acid production and slow growth, however not easily identified. Although their role in wine fermentations is well-defined, a little is known about their behavior during fermentation for ethanol production. This work aimed the screening, identification and characterization of Dekkera and Brettanomyces strains isolated from fermentative processes, through classical taxonomic tests, molecular analysis (PCR and DNA sequencing) and biotyping by killer system. Following fermentation essays were carried out to evaluate mixed fermentations of Saccharomyces cerevisiae and Dekkera bruxellensis, the last one in contamination levels varying from 101 to 103 cells/mL, in sugar cane medium, using batch fermentation process with cell recycle (fourteen 12-hour cycles) for fuel ethanol production. Morphological and physiological/biochemical tests involving eight strains selected for their high acid production from glucose and typical cell morphology, pointed out to the genera Dekkera or Brettanomyces, without possibility of species identification due to the variability and ambiguity of physiological tests. DNA sequencing of the ITS (Internal transcribed spacer) region belonging to ribosomal DNA confirmed only three strains as Dekkera bruxellensis, the others were predominantly Pichia guilliermondii. The ITS region, including the 5,8S gene, varied from 400 to 500 bp among the three strains. The use of killer system as a biotyping method for Dekkera strains might not be applied because the strains were predominantly neutral to killer toxins. Only the killer strain CCA510 (Kluyveromyces marxianus) showed to have inhibitory activity against the strains of D. bruxellensis. The fermentative essays using a strain of D. bruxellensis (CCA059) in mixed and pure fermentations with S. cerevisiae (CCA193, PE-02), have shown that the contaminant yeast was able to grown in sugar cane juice, regardless of the initial inoculum size ((101 to 103 cells/mL), impairing the bioethanol fermentation, causing diminished S. cerevisiae viability, pH decrease, lower ethanol production and fermentative efficiency, mainly due to the acetic acid production from reducing sugar present in fermentation medium. Extrapolation of the results obtained in laboratory scale to industrial scale in a medium-sized distillery, have revealed that contamination by Dekkera bruxellensis would result in a alcohol loss of 6 millions to 15 millions of liters, which would not be produced, depending on the contamination level.

Yeasts are the main protagonists of the alcoholic fermentation and strongly influence the final characteristics of wine and other alcoholic beverages. In winemaking, during spontaneous fermentation, this process is carried out by indigenous yeasts present on the grape berry and there is a progressive growth pattern of different yeast species, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. From a technological point of view spontaneous grape juice fermentations sometime become stuck or sluggish. This lack of reproducibility and predictability has favored, in the past, the use of yeast starters, generally composed of single strains of S. cerevisiae. Selected S. cerevisiae strains predominate during must fermentation, ensure rapid and reliable grape juice fermentation and, as consequence, consistent and predictable wine quality. However, there has been increasing recognition that wines made with Saccharomyces starter cultures are less complex, producing standardized wines. In fact, during spontaneous fermentation many other species (the so call non–Saccharomyces yeasts) are present in the grape must and traditionally they were considered deleterious as responsible for some wine off-flavors. Recently, the role of non-Saccharomyces species has been re-evaluated as some of them has been proposed as co-starter to simulate natural must fermentation and to confer greater complexity and specificity to the wine. In this context, the thesis project was developed in order to select new non-Saccharomyces yeast strains from fermenting musts of overripe grape berries. This stage of grape ripening is characterized to be more susceptible to infection of fungal pathogens such as Botrytis cinerea, whom yeasts present on the berry surface have to compete with. Thirty-six isolates were identified as Starmerella bacillaris isolated from destemmed dried grape of Raboso Piave variety, grown on Bagnoli DOC (Appellation of Origin) area (North-East of Italy). This species is recently proven to be of oenological interest mainly for its high glycerol production. Among them, 14 strains were phenotypically characterized and, for the first time, the antifungal activity of S. bacillaris species against B. cinerea and P. expansum were demonstrated. Moreover, fermentation performances of S. bacillaris strains were evaluated in synthetic, grape and apple musts (for the first time in cider production). When tested in sequential fermentations with S. cerevisiae this yeast improved the quality of wine and cider. The obtained results indicate that S. bacillaris can be proposed as biocontrol agent on grape in vineyard and during postharvest on stored apples and its presence on fruit surface positively influences the following juice fermentation. Therefore the released of this yeast in the vineyard can increase the sustainability of the production process and the final wine quality. The obtained results provide a solid basis for a new management of yeast used for winemaking and other alcoholic beverage production and open new prospects for a more integrated strategy to increase wine quality. Then, in this thesis, new S. bacillaris characteristics were evaluated in order to improve alcoholic beverages quality, such as mannoproteins and glutathione production. The quantification of these compounds was determined at the end of fermentation in synthetic must. S. bacillaris was proved to produce different mannoproteins with functional characteristics related to wine stability. Mannoproteins extracted from S. bacillaris cultures presented great potential in increasing the protein stabilization, while if they were produced during sequential fermentation with S. cerevisiae, enhanced the tartaric stabilization. Moreover, in this thesis, a very high glutathione content was demonstrated in S. bacillaris cells after fermentation. Therefore, yeast lees obtained from S. bacillaris fermentations can be proposed as as glutathione source. In the last part of this thesis the aim was to understand the genomic determinants of S. bacillaris technological traits described in the previous parts. Therefore. the whole-genome sequence of two relevant S. bacillaris strains was performed. By means of genome comparisons between the two S. bacillaris strains and between S. bacillaris, S. cerevisiae and other oenological yeasts, specific genes and metabolic pathways involved in technologically-relevant traits were identified and studied.
I lieviti sono i principali protagonisti della fermentazione alcolica e influenzano fortemente le caratteristiche finali del vino e di altre bevande alcoliche. Nella vinificazione, durante una fermentazione spontanea,questo processo viene effettuato da lieviti autoctoni presenti sulla bacca di uva. Alla fine della fermentazione la specie dominate tra i ceppi è Saccharomyces cerevisiae. Questa specie è universalmente conosciuta come il 'lievito di vino' ed è ampiamente preferita per iniziare fermentazioni del vino. Da un punto di vista tecnologico, le fermentazioni spontanee di succo d'uva si sono spesso bloccate. In passato, questa mancanza di riproducibilità e prevedibilità non era favorevole per una produzione di vino di qualità. L’uso di ceppo di lievito, generalmente composti da singoli ceppi come ad esempio S. cerevisiae aiuta a controllare questo problema. I ceppi selezionati di S. cerevisiae predominano durante la fermentazione del mosto, assicurando una rapida e affidabile fermentazione e, di conseguenza, una qualità del vino coerente e prevedibile. Tuttavia, si è accresciuto la convinzione che i vini ottenuti con culture di starter Saccharomyces siano meno complesse, producendo vini standardizzati. Infatti, durante la fermentazione spontanea, nel mosto d'uva sono presenti molte altre specie (i cosiddetti lieviti non Saccharomyces) e tradizionalmente sono stati considerati come responsabili di alcuni sapori no aggradevoli all’assaggio del vino. Recentemente, il ruolo delle specie non Saccharomyces è stato rivalutato poiché alcune di esse sono state proposte come co-avviatore per simulare la fermentazione dei mosti naturali e per conferire maggiore complessità e specificità al vino. In questo contesto, il progetto di tesi è stato sviluppato per selezionare nuovi ceppi di lievito non Saccharomyces da mosti fermentativi di uva passito. Questa fase di maturazione dell'uva è caratterizzata per essere più suscettibile all'infezione di patogeni fungini come Botrytis cinerea. I trentasei isolati sono stati identificati come Starmerella bacillaris , isolati da uve secche della varietà Raboso Piave, coltivate in zona Bagnoli DOC (denominazione d'origine) (nord-est dell'Italia). Questa specie è recentemente dimostrata di interesse enologico soprattutto per la sua elevata produzione di glicerolo. Tra questi, 14 ceppi sono stati caratterizzati fenotipicamente e, per la prima volta, sono stati dimostrati l'attività antifungina delle specie S. bacillaris contro B. cinerea e P. expansum. Inoltre, le prestazioni di fermentazione dei ceppi di S. bacillaris sono state valutate nei mosti sintetici, uve e mele (per la prima volta nella produzione di sidro). Durante la sperimentazione, questo lievito ha migliorato la qualità del vino e del sidro in fermentazioni sequenziali con S. cerevisiae. I risultati ottenuti indicano che S. bacillaris può essere proposto come agente biocontrollo sull'uva nel vigneto e durante la post-raccolta sulle mele conservate: la sua presenza sulla superficie di frutta influenza positivamente la seguente fermentazione dei succhi. Pertanto, il rilascio di questo lievito nel vigneto può aumentare la sostenibilità del processo produttivo e la qualità finale del vino. I risultati ottenuti forniscono una solida base per una nuova gestione del lievito utilizzato per la produzione di vino e altre bevande alcoliche e aprire nuove prospettive per una strategia più integrata per aumentare la qualità del vino. Sono state valutate le nuove caratteristiche di S. bacillaris al fine di migliorare la qualità delle bevande alcoliche, come le manoproteine e la produzione di glutatione. La quantificazione di questi composti è stata determinata alla fine della fermentazione in mosto sintetico. S. bacillaris ha dimostrato di poter produrre mannoproteine diverse, con caratteristiche funzionali legate alla stabilità del vino. Le manoproteine estratte dalle colture di S. bacillaris presentavano un grande potenziale per aumentare la stabilizzazione delle proteine, mentre se venivano prodotte durante la fermentazione sequenziale con S. cerevisiae, aumentavano la stabilizzazione tartarica. Inoltre, in questa tesi, è stato rilevato un contenuto di glutatione molto elevato in cellule S. bacillaris dopo la fermentazione. Pertanto, le fecce ottenute dalle fermentazioni di S. bacillaris possono essere proposte come fonte di glutatione. Nell'ultima parte di questa tesi si intendeva comprendere le determinanti genomiche delle caratteristiche tecnologiche di S. bacillaris descritte nelle precedenti parti. Per questo è stata eseguita la sequenza intero-genoma di due importanti ceppi S. bacillaris. Con i confronti genomici tra i due ceppi di S. bacillaris e tra S. bacillaris, S. cerevisiae e altri lieviti enologici, sono stati identificati e studiati geni specifici e i percorsi metabolici coinvolti in tratti tecnologicamente rilevanti.

Orientador: Silvio Roberto Andrietta
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica
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Resumo: Este trabalho teve por objetivo analisar o comportamento cinético da cepa de levedura (Y904-Mauri do Brasil) em nove méis com diferentes níveis de esgotamento, provenientes de unidades produtoras de açúcar e álcool. Foi avaliada a relação entre a velocidade específica de produção de etanol e a velocidade específica de crescimento microbiano. Os ensaios foram conduzidos em reator de bancada dotado de sistema de agitação e temperatura controlada. O inóculo utilizado foi uma cepa de levedura comercial encontrada na forma seca. Foram estudadas as cinéticas de produção de etanol, células e de consumo de substrato, assim como o desempenho da cepa em cada matéria-prima no que diz respeito à produtividade e rendimento em etanol. Os parâmetros avaliados foram: concentração de substrato, etanol e células. Para avaliação cinética, utilizou-se seis modelos do tipo não estruturados considerando apenas uma fonte de substrato (ART), sendo eles o de GHOSE & THY AGI, SEVEL Y et alI, JIN et alI., LEE et alI., LEVENSPIEL, GHOSE modificado e um modelo considerando duas fontes de substratos (glicose e frutose) baseado no modelo de GHOSE modificado. Foi desenvolvida uma rotina de ajuste exclusivamente para a determinação dos parâmetros adequados. Os resultados mostraram diferenças de comportamento cinético da cepa para as diferentes matérias primas testadas, assim como diferenças também nos valores dos rendimentos obtidos. Os modelos de GHOSE modificado e JIN et. all., utilizando um único substrato, foram os que mais se adequaram aos dados experimentais, mostrando que a inibição pelo substrato é significativa. Apesar do bom ajuste destes modelos, foi observado valores muito elevados para o parâmetro (Ks) que, para o modelo de GHOSE modificado, representou uma incoerência por ser este maior que o valor de (KJ). Estas incoerências foram eliminadas quando foi utilizado o modelo de GHOSE modificado que levou em consideração dois substratos (glicose e frutose). Em todos os ensaios realizados, observou-se que a velocidade específica de produção de etanol está vinculada a velocidade específica de crescimento microbiano até determinada fase da fermentação, sendo que após esta, observou-se uma diminuição da velocidade de crescimento microbiano, sem que isto ocorresse com a velocidade específica de produção de etanol mostrando que, nesta fase, as mesmas não estão mais associadas. A cinética de produção de glicerol mostrou-se similar ao do etanol, variando somente nos valores de rendimento, onde o do etanol foi 7 a 8 VEZES maior que o do glicerol
Abstract: The purpose of this work was to analyze the kinetic behavior of the yeast strain (Y904­Mauri do Brasil) using nine molasses with different exhaustion levels coming from sugar and alcohol producing units. The relationship was evaluated between the product production specific rate and the microbial growth specific rate. The essays were made in a bench bioreactor endowed with agitation system and controlled temperature. The used inoculum was a dry commercial yeast strain. The kinetics of ethanol production, cells consumption and substratum consumption, as well as the strain performance with each raw material, were studied in concern to the ethanol productivity and yield. The evaluated parameters were: substratum, ethanol and cell concentration. For kinetic evaluation, six non-structural models considering only one substratum source (ART) were used. The models were the one of GHOSE & THY AGI, SEVEL Y et alI, ]IN et alI, LEE et alI, LEVENSPIEL, the modified GHOSE and another model considering two sources of substratum (glucose and fructose) which is based on the modified GHOSE model. An adjustment routine was developed exclusively to the appropriate parameters determination. The results showed differences in the strain kinetic behavior with the different raw materials tested as well as differences in the obtained yield values. The modified GHOSE and ]IN et alI models using only one substratum have adapted the most to the experimental data, showing that the substratum inhibition is significant. In spite of the good adjustment of these models, it was observed very elevated Ks values that, in the modified GHOSE model, appeared as an incoherence since it was larger than the KI value. These incoherencies were eliminated when the modified GHOSE model was used considering the influence of the two substrata (glucose and fructose). In all the accomplished rehearsals, it was observed that the ethanol production specific rate was linked to the microbial growth specific rate until a certain fermentation phase and, after this, a decrease in the microbial growth rate was observed. This behavior didn't happen to the ethanol production specific rate showing that, at this phase, these rates are not associated anymore. The glycerol production kinetics showed to be similar the ethanol kinetics diverging only in the yield values - the ethanol yield was 7 to 8 times larger than the glycerol yield
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestre em Engenharia Química

Orientador: José Roberto Fernandes
Banca: Daniela Alonso Bocchini Martins
Banca: João Atilio Jorge
Resumo: O etanol combustível produzido no Brasil apresenta alguns problemas tecnológicos que afetam a sua produção em larga escala, entre estes destacamos a contaminação do processo por microrganismos indesejáveis, e a fermentação de mosto com baixa concentração de açúcares. Neste trabalho estudou-se aspectos relacionados com leveduras contaminantes do processo industrial, e o desempenho fermentativo de linhagens industriais utilizadas em mostos concentrados e suplementados com fontes estruturalmente complexas de nitrogênio. Foram utilizados dois meios básicos, um constituido da base nitrogenada YNB, e outro a base de caldo de cana, no qual foi adicionado diferentes concentrações de sacarose. Estes meios foram suplementados com fonte de nitrogênio que variaram de simples sal inorgânico como sulfato de amônio, e preparações comerciais de hidrolizados enzimáticos (peptona) e ácidos de proteínas (casaminoácidos). Três linhagens industriais foram inoculadas em mosto a base de caldo de cana contendo sacarose 22%, 30% e 35% (p/v), e suplementados com fontes de nitrogênio. A 30°C, em cultivos agitados e não agitados, verificou-se que a utilização completa de sacarose só ocorreu no meios suplementados com uma fonte de nitrogênio. No geral a suplementação com peptona foi a que propiciou fermentação mais eficiente, quando comparada com sulfato de amônio, maior acúmulo de biomassa e manutenção da viabilidade celular. Os estudos mostraram que nas condições de cultivo e com inoculação de baixa densidade celular, é necessária a suplementação para completa utilização da sacarose. No meio com sacarose 35% (p/v), atingiu-se um nível de 14 % (v/v) de etanol no cultivo não agitado e suplementado, sugerindo ser possível a obtenção de vinhos com maior quantidade de etanol, desde que... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The fuel ethanol industry in Brazil has some technological problems that affect its production on a large scale, among those are contamination by undesirable microorganisms, and wine with low ethanol concentration. We study aspects of microbial contamination of industrial processes, and the fermentation performance of industrial strains of concentrated musts supplemented with structurally complex nitrogen sources. We used two basic media, one consisting of a nitrogen base YNB, and another based on sugar cane juice, which was added increasing concentrations of sucrose. These media were supplemented with nitrogen sources varying from simple inorganic salt such as ammonium sulfate, and commercial preparations of enzymatic hydrolysates (peptone) and acidic protein (casaminoacids). Three industrial strains were inoculated into based sugar cane juice containing 22% sucrose, 30% and 35% (w/v) and supplemented with nitrogen sources. At 30°C, in shaken cultures, it was found that the efficient sucrose utilization occurred only in media supplemented with a nitrogen source. In general supplementation with peptone was the one that led to more efficient fermentation compared with ammonium sulfate, with higher biomass accumulation and maintenance of cell viability. Studies have shown that in conditions and with low cell density nitrogen supplementation was required for almost complete sucrose utilization. In sucrose 35% (w/v), it was achieved a level of 14-15% (v/v) ethanol in nitrogen supplemented cultures, suggesting the possibility of producting wines with higher amounts of ethanol in working conditions approaching the limit of yeast alcohol tolerance. The YNB medium was more appropriate to study the demand for nitrogen for different industrial strains. In this medium was also observed that supplementation with... (Complete abstract click electronic access below)
Mestre

Au Mexique l’état de Tamaulipas est susceptible de devenir l'un des plus grands producteurs de mezcal. Contrairement à d’autres boissons alcoolisées, la mycoflore responsable de la fermentation alcoolique du mezcal n'a pas été pleinement identifiée ou caractérisée métaboliquement. Il est d'un grand intérêt industriel de savoir si les levures indigènes impliquées dans la fermentation de moûts d’agave ont une forte nature fructophilique qui serait induite par la composition naturelle riche en fructose du moût. De plus, la caractérisation cinétique des levures et au niveau moléculaire des transporteurs impliqués dans la consommation de fructose par cette flore est pertinente étant donné que des études sur l'utilisation des hexoses par Saccharomyces cerevisiae n’ont pas montré que les transporteurs de glucose et de fructose étaient différents. Dans ce travail, la population levurienne étudiée était de 17 isolats, à partir de 103 initialement isolés de la mezcaleria "El Palmar" Emilio Lozoya, situé à San Carlos (Tamaulipas, Mexique) au niveau de différentes étapes. Seules les levures de l’espèce Saccharomyces cerevisiae ont été retenues et étudiées. En particulier de polymorphismes des 2 gènes des transporteurs principaux de sucres associes au fructose consume HXT1 et HXT3, a été évalué en comparaison à une souche de référence. Les résultats obtenus dans cette étude suggèrent la nécessité d'une analyse approfondie afin de préciser les liens entre la fructophilie et l'expression et / ou la structure des gènes des transporteurs de sucres lors de la fermentation alcoolique
The Mexican state of Tamaulipas has the potential to become one of the main producers of mezcal. But contrary to what is known about other alcoholic beverages, the mycoflora involved in this alcoholic fermentation has not been completely identified nor characterised from the metabolic point of view. There is an increasing industrial interest on knowing if the native yeasts associated to agave must fermentations possess a fructophilic behaviour, favoured by the natural high fructose composition of the agave musts. Moreover, the kinetic characterisation of these yeasts and the molecular analysis carried out on the hexose transporters genes reported to be involved in the glucose and fructose consumption is pertinent, and several studies on Saccharomyces cerevisiae have demonstrated a differential preference for each hexose. In this study, a set of 17 yeast isolates were chosen from an original collection of 103 yeast isolated at the mezcalería El Palmar Emilio Lozoya, which is located at San Carlos (Tamaulipas, Mexico) from different stages of the fermentation. Only those belonging to S. cerevisiae specie were studied, mainly concerning the polymorphisms found on their two main hexose transporter genes associated to a preferential consumption of fructose, HXT1 and HXT3, and results compared to a control strain. The results obtained showed that there is no a direct link between the fructophilic phenotype and/or the structure of these genes and that more studies are needed to establish such relationships

Les programmes de flowsheeting sont intensément utilisés dans les industries chimiques et pétrolières pour le développement et l'optimisation de procédés. Avec l'augmentation de la puissance de calcul des ordinateurs et des capacités des logiciels, les bilans matière et énergie pour des ateliers complexes incluant des centaines d'équations peuvent être résolus maintenant. Parce qu'ils sont conçus pour traiter de nombreux types d'unîtes industrielles, les programmes généraux de flowsheeting sont considérés comme des outils puissants dans le développement de modèles de procédés agro-alimentaires. Cette étude à pour objet la simulation de procédés biotechnologiques à l'aide du simulateur de procédés chimiques existant PROSIM. Elle a été appliquée en particulier à une unité industrielle de fermentation alcoolique par Saccharomyces cerevisiae, à partir de mélasse de betterave. Un modèle mécanistique non-structuré a été construit sur des réactions élémentaires dont les lois cinétiques sont simples, qui satisfont aux contraintes stœchiométriques et qui sont largement applicables, y compris pour la simulation de sous-produits tels les alcools lourds. Ce modèle a été intégré dans un module de simulation de PROSIM. L’extension de ce module à la simulation de fermenteurs aérés a été réalisée en décrivant un bilan complet avec transfert gaz-liquide et réaction dans la phase liquide. Le fonctionnement de l'atelier a été simulé avec succès en utilisant le même formalisme en régime continu et semi-continu. Le comportement des constituants dans les fermenteurs diphasiques a été étudié

Historicamente o Brasil tem marcado importante presença no setor sucroalcoleiro devido a fatos como o Programa Nacional do Álcool (Pró-álcool) e mais recentemente a tecnologia dos carros flex. Esses fatos somados aos problemas da utilização de uma matriz energética não renovável, cara e muito poluente como o petróleo, trazem a importância de aperfeiçoar a atividade no país. No entanto, diversos fatores afetam a fermentação e reduzem o rendimento fermentativo, como a entrada de contaminantes no processo e a pemanência deles, através de problemas como a qualidade da matéria prima e práticas como o processo de \"Melle-Boinot, respectivamente. Estudos anteriores revelam que grande parte desses contaminantes são do grupo chamado de bactérias láticas e que essas apresentam duas maneiras distintas de metabolizar os açúcares (homofermentativas e heterofermentativa), sendo que o tipo presente influencia em outros fatores importantes como a produção de glicerol por parte das leveduras. Além disso, acredita-se que as heterofermentativas sejam favorecidas pelas condições impostas na dorna fermentativa. Nesse contexto, o presente trabalho objetiva fazer um levantamento do tipo metabólico predominante das bactérias láticas contaminantes de destilarias brasileiras, além de entender melhor as relações existentes entre esses microrganismos e as leveduras. Para isso, foram isoladas 221 bactérias de destilarias e testadas quanto ao tipo metabólico. Adicionalmente, 2 bactérias foram escolhidas (I8 e I9), identificadas e testadas em ensaios de fermentação em conjunto com as leveduras PE-2 e Mauri, sob condições que simulam o processo industrial. Foram testados cinco tratamentos (PE-2; PE-2+I8; PE-2+I9; Mauri; Mauri+I8 e Mauri+I9), com 3 repetições cada e um total de 5 reciclos fermentativos. Ao final de cada reciclo fermentativo, amostras foram retiradas para análises químicas e microbiológicas. Os resultados revelam que, embora as hetero sejam mais agressivas e predominem em relação às homo quando simulamos o processo industrial dentro do laboratório, diferentemente do que se pensava, não ocorre essa predominância dentro da destilaria, já que 51,3% dos isolados eram homo e 48,69% eram hetero. Além disso os resultados também mostraram que todas as bactérias homo identificadas pertenciam à espécie Lactobacillus plantarum, enquanto as hetero à espécie L. fermentum, o que de acordo com dados anteriores da literatura sugerem que possa haver uma divisão de tipos metabólicos por espécie. Ao analisarmos os metabólitos de ambos os microrganismos vemos que houve menor produção de glicerol por parte das leveduras quando elas estavam em cultivo conjunto com as bactérias homofermentativas, mesmo em comparação com o controle (sem contaminação) o que pode ser explicado pelo fato de ter havido produção de succinato exclusivamente nos tratamentos com a I8 (homo). Ou seja, como as bactérias homo produzem quantidades muito superiores de ácido lático se compararmos com as heterofermentativas, as leveduras foram estimuladas a excretarem mais ácido succínico, desviando o metabolismo da produção massiva de glicerol.
Brazil has been historically very present in ethanol industry because some facts as National Alcohol Program (\"Pro-Alcohol\") and more recently the flex fuel car technology. These facts, added to the problems of using petroleum that is a non-renewable energy source, very expensive and polluting, bring the importance of improving brazilian activity. However, several factors affect the fermentation and reduce alcoholic yields, as the entry of contaminants into the process and their permanency because, for example, the quality of raw materials and everyday practices as \"Melle-Boinot\" process, respectively. Previous studies have shown that many of these contaminants are called lactic acid bacteria, which have two distinct ways to metabolize sugars (homofermentative and heterofermentative), and the type present could influence important factors such as glycerol production by yeasts. Additionally, it is believed that the hetero bacteria are favored by the fermentative industrial conditions. In this context, this work aims to survey the predominant metabolic type of lactic acid bacteria contaminants in brazilian distilleries, and understand the relationship between this microorganisms and yeasts. For that, we isolated 221 bacteria from distilleries and tested their metabolic type. In addition, two strains were chosen (I8 and I9), identified and tested with PE- 2 and Mauri yeasts under industrial process simulating conditions. By this mean, we have 6 treatments (PE-2; PE-2+I8; PE-2+I9; Mauri; Mauri+I8 and Mauri+I9) with 3 replicates each one and a total of 5 cell recycling fermentation. At the end of each recycle, samples were taken for chemical and microbiological analysis. The results showed that, although the heterofermentative are more aggressive and dominant in relation to homo when we simulate the industrial process in the laboratory, unlike previously thought, we can not find this predominance in distilleries, since 51,3% of the isolates were homo and 48,69% were hetero. In addition, the results also showed that all the homo bacteria identify was a Lactobacillus plantarum, while all the hetero identify was a L. fermentum, which according to previous literature, suggests that should exist a division of metabolic types by species. When analyzing the metabolites of both microorganisms we see that there was less glycerol production by the yeast when they were growing together with homofermentative bacteria, even in comparison with the control (without contamination) which might be explained by the fact that there was only succinate production in treatments with homo (I8). In other words, homofermentative bacteria produce much higher amounts of lactic acid when compared with hetero, so yeast cells were stimulated to excrete more succinic acid and take the metabolism away from glycerol mass production.

O presente estudo relata a avaliação da metodologia de microplaqueamento em gotas como uma forma alternativa para contagem da bactéria Lactobacillus fermentum CCT 1407 em cultura isolada, ou em cultura mista com Saccharomyces cerevisiae, utilizando o meio de cultivo MRS e actidiona como inibidor da levedura. A análise dos resultados revelou que a metodologia de microplaqueamento em gotas foi equivalente ao plaqueamento em profundidade, o qual é a metodologia rotineiramente utilizada. Este trabalho também relata o comportamento das linhagens Fleischmann, PE-2 e M-26 quando em cultivo misto com a bactéria L. fermentum CCT 1407 durante seis reciclos fermentativos realizados com mosto composto de 30% de melaço e 70% caldo de cana, incubados à temperatura de 32 o C. Foi utilizada a análise de variância para analisar as variáveis e análise sob o esquema parcelas subdivididas no tempo em delineamento blocos ao acaso. Os resultados obtidos mostram que ocorreram reduções de viabilidade nos tratamentos realizados com a linhagem Fleischmann promovendo um estímulo à multiplicação bacteriana, porém, os tratamentos com a linhagem PE-2 apresentaram menores reduções de viabilidade e o estímulo à multiplicação bacteriana foi menor. Ao comparar a o comportamento das linhagens M-26 e PE-2 durante a fermentação em cultivo misto com L. fermentum CCT 1407 não houve diferença estatística significativa, através do teste F, para os seguintes parâmetros: viabilidade celular, rendimento fermentativo, teor alcoólico, massa microbiana e glicerol, sendo detectada diferença significativa para os parâmetros contaminação bacteriana e pH do vinho delevurado. Para o início do experimento comparativo entre as linhagens M-26 e PE-2 foi inoculada a bactéria (1,5.10 6 UFC.mL -1 ) e ao final do sexto ciclo as contaminações foram 3,3.10 7 UFC.mL -1 e 6,0.10 7 UFC.mL -1 nos tratamentos realizados com as linhagens M-26 e PE-2, respectivamente. O aumento da contaminação bacteriana estimulou a formação de glicerol. O teor de trealose apresentou relação positiva com a viabilidade sendo considerado um indicador das condições fisiológicas da levedura, independentemente da linhagem avaliada.
The present study reports the evaluation of pour plate method in drops as an alternative to count the bacteria L. fermentum CCT 1407 in isolated culture, or in mixed culture with Saccharomyces cerevisiae, using the means of cultivation MRS and actidiona as inhibitor of yeast. The analysis of the results revealed that the pour plate method was equivalent to the in depth plate method, which is the methodology most commonly used. This study also presents the behavior of the strain Fleischmann, PE-2 and M-26 when in mixed culture with the bacteria L. fermentum CCT 1407 for six fermentative re cycles accomplished with composed must of 30% of molasses and 70% cane broth, incubated to the temperature of 32 o C. The variance analysis was used to analyze the variables. The obtained results show viability reductions in the treatments accomplished with the strain Fleischmann promoting bacterial multiplication, even so, the treatments with the strain PE-2 presented smaller viability reductions and the incentive to the bacterial multiplication was also smaller. When comparing the behavior of the both strains, M-26 and PE-2, during the fermentation in mixed cultivation with L. fermentum CCT 1407 there was no significant statistical difference, according to the results of the test F, for the following parameters: cellular viability, fermentative output, alcohol content, microbial mass, and glycerol. Significant difference was just detected for the parameters of bacterial contamination and pH of the wine. For the beginning of the first fermentative cycle it was inoculated 1,5.10 6 UFC.mL -1 and at the end of the sixth cycle the contamination was 3,3.10 7 UFC.mL -1 and 6,0.10 7 UFC.mL -1 in the treatments accomplished with the strains M-26 and PE-2, respectively. The increase of bacterial contamination stimulated the glycerol formation and the trehalose content-presented positive relationship with the viability being considered an indicator of the physiologic conditions of the yeast, regardless the appraised yeast strains.

As microalgas são conhecidas pela sua elevada capacidade fotossintética e pela produção de compostos de alto valor agregado. Nutrientes essenciais como nitrogênio e carbono, a intensidade da luz e a temperatura são responsáveis por mudanças substanciais na produção de biomassa e compostos de interesse. Este trabalho teve como objetivo estudar os fatores como concentração de nitrogênio, variação da temperatura, regime de iluminação e regime de operação do fotobiorreator (batelada, batelada repetida e contínuo) que afetam a formação e a composição da biomassa (proteínas, lipídeos, carboidratos, ácidos graxos e carotenoides) da microalga Heterochlorella luteoviridis e desenvolver um sistema de cultivo para a utilização do CO2 formado durante a fermentação alcoólica na produção de vinho. Primeiramente foram avaliados os efeitos da temperatura (22 °C a 32 °C) e concentrações de nitrogênio (75 mg L-1 a 375 mg L-1 NaNO3) sobre a formação de biomassa H. luteoviridis e sua composição química. A concentração de nitrogênio e a temperatura influenciaram no teor de carboidratos, carotenoides e proteínas, contudo não afetaram o perfil qualitativo de carotenoides e ácidos graxos. A maior concentração de biomassa (3,35 g L-1) foi obtida com a maior concentração de nitrogênio testada. Mantendo-se a temperatura fixa (27 °C) realizaram-se cultivos com concentração de nitrogênio entre 300 mg L-1 e 750 mg L-1 NaNO3. A máxima produção de biomassa (5,26 g L-1) e carotenoides (2,06 mg g-1) foi obtida com concentração de nitrogênio de 650 mg L-1 NaNO3. As cinéticas de crescimento e consumo de nitrogênio em batelada foram ajustadas a modelos fenomenológicos. O modelo matemático proposto neste trabalho mostrou o melhor ajuste para biomassa (R2 = 0,994) e consumo de nitrogênio (R2 = 0,996). A partir deste modelo foram realizadas simulações de cultivo em modo de operação batelada repetida e contínuo com diferentes taxas de diluição e os cultivos com as maiores produtividades de biomassa preditas foram validados experimentalmente. As culturas em batelada repetida apresentaram produtividade de biomassa menor que a predita, enquanto que a contínua apresentou produtividade similar à predita pelo modelo (1,09 ± 0,01 g L-1 d-1). Os cultivos em batelada-repetida e contínuo apresentaram biomassa com maiores teores de proteínas, ácidos graxos poli-insaturados ômega 3 e carotenoides quando comparados à batelada. O cultivo contínuo foi repetido utilizando CO2 proveniente da fermentação alcoólica da produção de vinho como fonte de carbono, o qual apresentou comportamento similar ao cultivo com a CO2 comercial. Por fim, avaliou-se os efeitos da intensidade e dos comprimentos de onda da luz no crescimento e na composição da microalga H. luteoviridis. As culturas iluminadas por LED brancos com intensidade máxima de 1500 μmol m-2 s-1 apresentaram maiores produtividades de biomassa (0,29 g L-1 d-1). Comparando culturas iluminadas por LEDs vermelhos e/ou azuis, os primeiros estimularam o crescimento da biomassa enquanto que os últimos estimularam a biossíntese de carotenoides, lipídeos e proteínas.
Microalgae are known for their high photosynthetic activity and by the production of high-value products. Essential nutrients, like nitrogen and carbon, light intensity, and temperature are responsible for substantial changes in biomass and compounds production. This work aimed to study the parameters as concentration of nitrogen, temperature variation, lighting regime and operation regime of the photobioreactor (batch, repeated-batch and continuous) that affect biomass production and composition (proteins, lipids, carbohydrates, fatty acids and carotenoids) of the microalga Heterochlorella luteoviridis, and to develop an integrated culture system to use the CO2 from alcoholic wine fermentation to grow the microalga. Firstly, the effects of temperature (22 °C to 32 °C) and nitrogen concentration (75 mg L-1 to 375 mg L-1 NaNO3) were evaluated. Both parameters affected carbohydrates, carotenoids and protein contents in the biomass but did not affect the qualitative profile of carotenoids and fatty acids. The highest biomass concentration (3.35 g L-1) was achieved at the highest nitrogen concentration. New cultures were performed using nitrogen concentration between 300 mg L-1 and 750 mg L-1 NaNO3, and keeping temperature fixed at 27 °C. The maximal biomass and carotenoid production, 5.26 g L-1 and 2.06 mg g-1, were achieved using 650 mg L-1 NaNO3. The biomass growth and nitrogen consumption were adjusted by phenomenological models. The model proposed in this work showed the highest adjust for biomass (R² = 0.994) and nitrogen (R² = 9.996) data. This model was used to simulate repeated-batch and continuous cultures using several dilution rates. The cultures with the highest predicted biomass productivities were experimentally validated. The repeated-batch cultures showed lower biomass productivities than the predicted one, while the continuous culture showed biomass productivity similar to the one predicted by the model (1.09 ± 0.01 g L-1 d-1). The biomass resultant from these cultures had higher contents of proteins, omega-3 polyunsaturated fatty acids and carotenoids, compared to the biomass from batch cultures. The continuous culture was repeated using CO2 from wine fermentation as a carbon source. The results were similar to the ones using commercial CO2, showing the feasibility of the integrated process. Finally, the effects of light intensity and wavelength were evaluated on the growth and composition of H. luteoviridis biomass. Cultures illuminated with white LEDs at maximum intensity of 1500 μmol m-2 s-1 showed the higher biomass productivity, 0.29 g L-1 d-1. Comparing cultures illuminated with red and/or blue LEDs, the former stimulated biomass growth and the latter the carotenoid, lipids and protein biosynthesis.

A atual demanda mundial para redução nas emissões de dióxido de carbono vem aumentando e o uso de etanol como combustível tem sido considerado como uma boa alternativa. Entretanto as condições de cultivo da levedura obrigam o micro-organismo a lidar com diferentes estressores, sendo as altas temperaturas e elevadas concentrações de etanol exemplos dos mais limitantes durante o processo produtivo. Assim, o presente estudo teve como objetivo encontrar mutantes de Saccharomyces cerevisiae que apresentassem maior resistência a altas concentrações de etanol e altas temperaturas concomitantemente. Para isso, utilizamos como modelo a coleção YKO (Invitrogen®), que contém 4,828 linhagens mutantes para genes não essenciais na levedura. Primeiramente, estabelecemos as condições de estresse (concentração de etanol e temperatura) as quais se mostrassem limitantes para o crescimento da linhagem isogênica selvagem (BY4741). Foram estabelecidas as condições de estresse em 10% de etanol em conjunto com aumento de temperatura para 37ºC, que foram então utilizadas para testar a tolerância das linhagens mutantes agrupadas em 53 pools. Foram identificadas as linhagens mutantes para as ORFs UBP15, THI12, CLA4, DIT1 e MTC7 que suportaram tais condições, se mostrando possíveis candidatos para melhora genética em linhagens industriais como PE2 e CAT1.
The current world demand for reduced emissions of carbon dioxide is increasing the use of ethanol as a fuel has been considered as a good alternative. However culture conditions requiring the yeast micro-organism to cope with different stressors, as the high temperatures and high concentrations of ethanol the most lititant examples during the production process. Thus, the present study aimed to find Saccharomyces cerevisiae mutants that having greater resistance to high ethanol concentrations and high temperatures concurrently. For this, we use a model collection YKO (Invitrogen®) which contains 4.828 mutant strains for non-essential genes in yeast. First, we establish the stress conditions (ethanol concentration and temperature) which would prove limiting to the growth of the isogenic wild type (BY4741). Were established stress conditions in 10% ethanol together with temperature rise to 37ºC, which were then used to test the tolerance of mutant strains grouped into 53 pools. The mutant lines were identified for UBP15, THI12, CLA4, DIT1 and MTC7 ORFs who endured such conditions are showing possible candidates for genetic improvement in industrial strains as PE2 and CAT1.

The transformation of sugar (glucose) into ethanol and CO2 involves 11 reactions in orderly sequence, each catalyzed by a specific enzyme from yeast producing ethanol, for example, Saccharomyces cerevisiae. These yeast require a source of carbon, which can be another glucose or sugar, but also require vitamins and other nutrients such as nitrogen, phosphorus, sulfur, potassium, magnesium, calcium, zinc, manganese, copper, iron, cobalt, iodine and other elements in small quantities. The nutritional needs of yeast during alcoholic fermentation influence the proliferation and cell growth and efficiency of transformation of sugar into alcohol. This study aimed to investigate the effect of supplementation of nutrients in grape juice from the sugar cane to 14º Brix and mash of molasses to 15º Brix on the process of alcoholic fermentation in batch simple, using the commercial formulations A, B and C. These formulations commercial showed in their compositions P2O5, N, MgSO4, MnSO4 and ZnSO4 in different doses for each of the three nutrients trade. The fermentations were conducted in erlenmeyers and shaker type Shaker with agitation (200 rpm) and temperature (± 32°C) controlled with weighing of an hour in hour of erlenmeyers to determine the end of fermentation. The analyses used to evaluate the efficiency of the fermentation process were: "Reducing sugars Totals (ART), sulfuric acidity, pH and alcohol content. The concentrations of nutrients tested were determined trade based on deficiencies of musts. To complement the mash of molasses, it was found that concentrations of nutrients that commercial maximizes the efficiency and productivity in the fermentation process were: 0.20 grams of A / litre of must, 0.50 grams of B / litre of must and 0,30 g of C / liter of juice. And in the mash of broth of cane were: 0.50 grams of A / litre of must, 1 g of B / litre of must and 2 grams of C / liter of juice.
Fundação de Amparo a Pesquisa do Estado de Alagoas
A transformação de açúcar (glicose) em etanol e CO2 envolve 11 reações em seqüência ordenada, cada qual catalisada por uma enzima específica das leveduras produtoras de etanol, como por exemplo, a Saccharomyces cerevisiae. Essas leveduras exigem uma fonte de carbono, que pode ser glicose ou outro açúcar, mas também exigem vitaminas e outros nutrientes tais como nitrogênio, fósforo, enxofre, potássio, magnésio, cálcio, zinco, manganês, cobre, ferro, cobalto, iodo e outros elementos em quantidades diminutas. As necessidades nutricionais das leveduras durante o processo de fermentação alcoólica influenciam na multiplicação, crescimento celular e eficiência de transformação do açúcar em álcool. O presente trabalho teve por objetivo estudar a influência da complementação de nutrientes em mosto de caldo de cana a 14ºBrix e mosto de melaço a 15ºBrix sobre o processo de fermentação alcoólica em batelada simples, utilizando as formulações comerciais A, B e C. Essas formulações comerciais apresentaram em suas composições P2O5, N, MgSO4, MnSO4 e ZnSO4 em doses diferentes para cada um dos três nutrientes comerciais. As fermentações foram conduzidas em Erlenmeyers e em agitador tipo Shaker com agitação (200 rpm) e temperatura (± 32ºC) controladas, com pesagem de hora em hora dos Erlenmeyers para determinar o final da fermentação. As análises utilizadas para avaliação da eficiência do processo fermentativo foram: Açúcares Redutores Totais (ART), acidez sulfúrica, pH e teor alcoólico. As concentrações testadas dos nutrientes comerciais foram determinadas com base nas deficiências dos mostos. Para complementar o mosto de melaço, estabeleceu-se que as concentrações dos nutrientes comerciais que maximizariam a eficiência e produtividade no processo fermentativo foram: 0,20 g de A/litro de mosto, 0,50 g de B/litro de mosto e 0,30 g de C/litro de mosto. E no mosto de caldo de cana foram: 0,50 g de A/litro de mosto, 1 g de B/litro de mosto e 2 g de C/litro de mosto.

Nous avons mis au point un fermenteur continu multi-étagé (MSCF) dans le but de reproduire les conditions de la fermentation alcoolique en conditions œnologiques. Ce bioréacteur permet de maintenir les levures dans un milieu stable et contrôlé tout en découplant la croissance et la phase stationnaire. Le système offre donc la possibilité d'obtenir des levures non croissantes dans un milieu de composition stable. Dans un premier temps, nous avons validé la pertinence du MSCF pour reproduire les conditions de fermentation du batch, par approche intégrée (des paramètres cinétiques, des métabolites intra et exo cellulaire et de l'expression génique). Nous avons ensuite utilisé ce bioréacteur pour étudier les mécanismes d'adaptation métabolique des microorganismes suite à un ajout d'azote, pratique largement répandue en œnologie. Plusieurs résultats originaux ont été obtenus concernant, notamment, la réorganisation du cycle TCA, le transport des sources azotées et la synthèse des alcools supérieurs et esters. La fiabilité de l'outil mis au point et l'originalité des données obtenues ouvrent des perspectives à l'utilisation du MSCF pour la compréhension du métabolisme et des mécanismes d'adaptation des levures
We set up a multi-stage continuous fermentor (MSCF) to mimic the conditions of alcoholic fermentation. In this bioreactor, the yeasts are in a steady and well controlled state representative of the growth and stationary phases of the batch. The ability of the MSCF to reproduce batch fermentation was assessed using an integrated approach (measurement of kinetic parameters, intra and exo-cellular metabolites and gene expression). We then used the MSCF to study the impact of nitrogen supplementation performed during the stationary phase, on yeasts metabolism. Several original results were obtained, concerning the TCA cycle, the transport of nitrogenous sources and the synthesis of higher alcohols and esters. This work points out the interest of using the MSCF to assess the effect of medium perturbations during alcoholic fermentation, especially during the stationary phase. More generally, the accuracy of the MSCF and the originality of the data obtained open new prospects for a better understanding of yeasts metabolism and regulation mechanisms

O objetivo deste trabalho foi estudar a influência de bactérias dos gêneros Bacillus e Lactobacillus, bem como de seus produtos metabólicos, na redução da viabilidade celular Saccharomyces cerevisiae, quando em cultura mista de levedura e bactérias ativas e tratadas. Também foi avaliado um meio alternativo de cultivo de bactérias e leveduras, constituído de caldo de cana diluído a 5o brix e suplementado com extrato de levedura e peptona. As bactérias Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum e Lactobacillus plantarum foram cultivadas em associação com Saccharomyces cerevisiae (cepa Y-904) por 72 h a 32oC, sob agitação. A viabilidade celular, a taxa de brotamento e a população de células da levedura, a acidez total, a acidez volátil e o pH do meio foram determinados às 0, 24, 48 e 72 h do cultivo misto. Também foi determinada a população inicial e final dos microrganismos através de plaqueamento em profundidade, em meio de cultivo tradicional (PCA para os Bacillus, MRS-agar para os Lactobacillus e YEPD-agar para S. cerevisiae) e no meio constituído de caldo de cana. As culturas de bactéria foram tratadas através do calor (esterilização em autoclave a 120oC por 20 minutos), de agente antimicrobiano (Kamoran HJ na concentração de 3,0 ppm) ou da irradiação (radiação gama, com doses de 5,0 kGy para os Lactobacillus e 15,0 kGy para os Bacillus). Os resultados mostraram que apenas os meios de cultivo mais acidificados (com maiores concentrações de acidez total e volátil, e menores valores de pH), contaminados com as bactérias ativas L. fermentum e B. subtilis, provocaram diminuição da viabilidade celular da levedura. Excetuando a bactéria B. subtilis inativada por radiação, as demais bactérias tratadas pelos diferentes processos (calor, radiação e antimicrobiano) não causaram diminuição da viabilidade celular e da população das leveduras, indicando que a presença isolada dos metabólitos celulares dessas bactérias não foi suficiente para reduzir a porcentagem de células vivas e a densidade populacional da levedura. Para todos os microrganismos, as contagens obtidas com o cultivo em meio à base de caldo de cana foram semelhantes às obtidas nos meios tradicionais, provavelmente porque o meio alternativo simula a composição do mosto de caldo de cana-de-açúcar, do qual as bactérias foram isoladas do processo industrial de produção de etanol. Sendo assim, o meio de cultivo à base de caldo de cana-de-açúcar pôde substituir os meios tradicionais de cultivo da levedura e das bactérias testadas neste trabalho.
The aim of this work was to study the influence of the bacteria Bacillus and Lactobacillus, as well as their metabolic products, in reduction of cellular viability of Saccharomyces cerevisiae, when in mixed culture of yeast and active and treated bacteria. Also was to evaluated an alternative medium (MCC) for the cultivation of bacteria and yeast, constituted of sugarcane juice diluted to 5o Brix and supplemented with yeast extract and peptone. The bacteria Bacillus subtilis, Bacillus coagulans, Bacillus stearothermophilus, Lactobacillus fermentum and Lactobacillus plantarum were cultivated in association with yeast Saccharomyces cerevisiae (strain Y-904) for 72 h on 32oC, under agitation. The cellular viability, budding rate and population of S. cerevisiae, the total acidity, volatile acidity and pH of culture were determined from 0, 24, 48 e 72 h of mixed culture. Also were determined the initial and final of microorganism population across the pour plate method, in traditional culture medium (PCA for Bacillus, MRS-agar for Lactobacillus and YEPD-agar for yeast S. cerevisiae) and in medium constituted of sugarcane juice. The bacteria cultures were treated by heat sterilization (120oC for 20 minutes), antibacterial agent (Kamoran HJ in concentration 3,0 ppm) or irradiation (radiation gama, with doses of 5,0 kGy for Lactobacillus and 15,0 kGy for Bacillus). The results of the present research showed that just the culture mediums more acids (with higher concentrations of total and volatile acidity, and smaller values of pH), contaminated with active bacteria L. fermentum and B. subtilis, caused reduction on yeast cellular viability. Except the bacteria B. subtilis treated with radiation, the others bacteria treated by different procedures (heat, radiation e antibacterial) did not cause reduction on yeast cellular viability and population, indicating that the isolated presence of the cellular metabolic of theses bacteria was not enough to reduce the percentage of the yeast live cells and a density population. For all microorganisms, the counts obtained with the cultivation medium constituted of sugarcane juice were similar obtained in traditional mediums, probably because the alternative medium simulate the composition of sugarcane must, that the bacteria were isolated in industrial process of ethanol yield. However, the culture medium constituted of sugarcane juice could be replacing traditional culture mediums of yeast and bacteria tested in this work.

There is growing interest in the role of indigenous knowledge (IK) in science education in many parts of the world. In South Africa, this comes against the backdrop of a long history of cultural alienation, neglect and suppression of the IK of the indigenous people by colonial governments. Hence, the first post-independence National Curriculum Policy Statement (Curriculum, 2005) and its subsequent modified versions emphasised the need to redress the imbalances of the past so as to make science accessible to learners from diverse cultural backgrounds. However, literature reveals that the efforts to implement an IK-based curriculum are constrained by the lack of clarity, knowledge and skills on how to effectively make use of IK in the classroom situation so as to bring about effective teaching and learning. Against this background, this study sought to understand how the incorporation of IK enables or constrains the teaching and learning of alcoholic fermentation. Underpinned by an interpretive paradigm, a qualitative case study was conducted at a high school in Mthatha District in the Eastern Cape Province of South Africa. The study comprised of a sample of ten Life Sciences teachers from four neighbouring high schools, two Grade 11 Life Sciences teachers and their classes and two focus groups of six learners from each class. Convenience and purposive sampling were used to select the participants and the research site. The study made use of a questionnaire, document analysis, lesson observation, stimulated recall interviews and focus group interviews to generate data. The questionnaire sought to get an overview of Life Sciences teachers’ attitudes, opinions and experiences on incorporating IK in science teaching. The data gathered were then used as baseline information to inform the main study. The lesson observations, stimulated recall interviews and the focus group interviews sought to understand how the incorporation of IK enables or constrains the teaching and learning of alcoholic fermentation. Document analysis, lesson observation, stimulated recall interviews and focus group interviews were conducted to understand the experience of incorporating IK in a real life classroom situation. The study was informed by Vygotsky’s socio-cultural constructivism and Shulman’s pedagogical content knowledge (PCK) theory. From Vygotsky’s theory I borrowed the idea of mediated learning, scaffolding (Bruner, 1986), zone of proximal development (ZPD), as well as the use of language and cultural artefacts as tools of analysis of the classroom interaction between the teacher and the learners during the teaching-learning process. Shulman’s theory of PCK was also used to analyse how teachers apply IK in teaching Science. The data obtained were coded inductively and presented in tables, graphs and thick descriptive texts to make it easy to understand. The findings of this research show that incorporating IK improved the teaching-learning process by arousing learners’ interest in science, increasing learners’ participation and motivation, meaning making, language use, questioning and engagement in the learning process. Teaching became learner-centred, for it was directed by learners’ questions. Collaborative learning through group discussions, debates, arguments and brainstorming emerged to be an effective way of engaging learners in learning. The teachers used probing to encourage critical thinking before scaffolding learners. However, even though teachers generally accept IK as valuable in their teaching they lack the pedagogical content knowledge to effectively incorporate it into meaningful teaching and learning. Furthermore, teachers viewed cultural diversity as a constraint to their efforts to incorporate IK in teaching science. On the contrary, their learners held the view that having cultural diversity in classrooms created an opportunity to learn from other people’s cultures. Variables such as experience and difference in the cultural background of learners and teachers alike, tended to affect the teachers’ ability to incorporate IK.

L'achèvement de la fermentation alcoolique est corrélé au niveau de formation de biomasse, qui varie selon les souches de levure S. cerevisiae. La maîtrise de cette fermentation passe par une meilleure compréhension des liens entre métabolisme azoté et formation de biomasse ainsi que des mécanismes contribuant aux variations de formation de biomasse entre souches. L'analyse des cinétiques de consommation de 18 composés azotés a montré que l'ordre de consommation des sources d'azote est similaire pour 14 souches et s'explique principalement par les caractéristiques cinétiques et le mode de régulation des perméases impliquées dans leur transport. Par contre, des variations sont observées dans les profils de consommation de l'azote, qui mettent en jeu des mécanismes différents suivant la disponibilité en azote du milieu. En présence d'un excès d'azote, les souches « faibles productrices » présentent une capacité limitée d'assimilation de l'ammonium ; lorsque l'azote est présent en faible quantité, elles métabolisent plus efficacement l'arginine stockée dans la vacuole pendant la phase de croissance. Afin de déterminer la distribution des flux d'azote dans la cellule, nous avons développé une approche quantitative basée sur la filiation isotopique de sources d'azote marquées 13C ou 15N. Cette étude a révélé une incorporation limitée des acides aminés exogènes dans la biomasse au profit de la synthèse de novo, à l'exception de la leucine majoritairement intégrée dans la biomasse et de l'arginine stockée dans la vacuole. Enfin, cette étude a confirmé expérimentalement que les capacités d'assimilation de l'ammonium et de remobilisation de l'arginine sont des éléments clés des différences de production de biomasse entre souches. Cette étude apporte un nouvel éclairage sur l'efficacité d'utilisation et l'allocation de sources complexes d'azote pendant la fermentation œnologique et sur les mécanismes impliqués dans la variabilité de production de biomasse chez S. cerevisiae
The completion of alcoholic fermentation is correlated with the level of biomass formation, which varies depending on the S. cerevisiae yeast strain. Achieving a better control of the fermentation requires a better understanding of the relationship between nitrogen metabolism and biomass formation and of the mechanisms contributing to variation in biomass formation between strains. The analysis of the kinetics of consumption of 18 nitrogen compounds showed that the order of consumption of nitrogen sources is similar for 14 strains and is mainly due to the kinetic characteristics and mode of regulation of permeases involved in their transport. By cons, variations are observed in the patterns of nitrogen consumption, which involve different mechanisms depending on the availability of nitrogen in the medium. In the presence of excess nitrogen, "low producing" strains have a limited capacity for assimilation of ammonium; when nitrogen is present in small quantities, they metabolize more efficiently arginine stored in the vacuole during the growth phase. To determine the intracellular distribution of nitrogen fluxes, we developed a quantitative approach based on isotopic filiation of 13C or 15N labeled nitrogen sources. This study revealed a limited incorporation of exogenous amino acids in biomass in favor of de novo synthesis, with the exception of leucine mostly integrated in the biomass and arginine stored in the vacuole. Finally, this study confirmed experimentally that the capacity of ammonium assimilation and remobilization of arginine are key determinant governing the differences of biomass production between strains. This study sheds new light on the efficient use and allocation of complex nitrogen sources during wine fermentation and on the mechanisms involved in the differences in biomass production within the S. cerevisiae species

Dentre as leveduras selvagens mais comumente encontradas na fermentação alcoólica, destaca-se o gênero Saccharomyces apresentando colônias rugosas com células dispostas em cachos (pseudohifas). Este biótipo de levedura tem sempre sido associado com problemas na indústria, ocasionando queda no rendimento fermentativo. Sendo assim, o presente trabalho teve por objetivo realizar a caracterização genética, morfo-fisiológica e da resistência ao estresse de linhagens de Saccharomyces cerevisiae que apresentam colônias rugosas e mucosas, buscando alternativas que possam contribuir para o manejo dessas leveduras selvagens (rugosas) na fermentação alcoólica. Foram realizados testes de caracterização para crescimento invasivo, atividade killer, temperatura, pH, concentração de etanol e açúcar, presença de actidione, floculação e capacidade fermentativa, utilizando-se 22 linhagens de leveduras (11 rugosas e 11 mucosas) selecionadas previamente por seqüenciamento da região ITS do DNA ribossomal. O efeito do tratamento ácido em diferentes valores de pH sobre o crescimento de duas linhagens (52 rugosa e PE-02 mucosa) foi também avaliado, seguido do monitoramento do crescimento em meio de caldo de cana após tratamento ácido. Em seguida, testes fermentativos em meio de caldo de cana foram conduzidos em culturas puras e mista dessas linhagens. Os testes de caracterização morfofisiológica mostraram que a invasividade em meio YEPD Agar ocorreu em baixa freqüência dentre as 22 leveduras testadas; dez dentre onze leveduras rugosas apresentaram taxa de floculação expressiva, e entre as mucosas a floculação foi praticamente inexistente; nenhuma das linhagens apresentou produção de toxina killer; as linhagens com colônias rugosas apresentaram capacidade fermentativa significativamente inferior às linhagens com colônias mucosas, em sistema de batelada com ciclo único de 48 horas em meio de caldo de cana, demonstrando velocidade mais lenta de fermentação. Quanto à resistência ao estresse por temperatura, pH, etanol e concentração de açúcar, as linhagens mucosas tiveram maior resistência à acidez do meio, enquanto as linhagens rugosas foram mais resistentes às concentrações elevadas de etanol e açúcar. Nenhuma levedura foi resistente ao actidione. A análise genética, através dos loci microssatélites, revelou a presença de dois grupos principais relacionados geneticamente, sendo o primeiro ramo constituído principalmente de colônias rugosas (67%), contendo, no entanto, a linhagem PE-02, apresentando linhagens com alta taxa de floculação e tolerância às altas concentrações de açúcar e etanol; o outro grupo (com três subgrupos) compreendeu principalmente colônias mucosas, apresentando pouca resistência às situações estressantes aqui estudadas. A levedura com colônia rugosa (linhagem 52) foi bastante afetada pelo tratamento ácido em valores de pH 1,0 e 1,5, ao contrário da levedura com colônia mucosa (PE-02). A fermentação em sistema de batelada com reciclo celular e tratamento ácido conduzido em pH 1,5 teve efeito sobre o crescimento da levedura rugosa quando em cultura mista com a levedura mucosa, não resultando em prejuízo à eficiência fermentativa, quando comparada com a cultura pura da PE-02. Em cultura pura da levedura rugosa, constatou-se eficiência fermentativa por volta de 60%, confirmando o baixo desempenho destas leveduras. O conhecimento das respostas das leveduras rugosas 12 às situações estressantes pode ajudar a manejar a fermentação alcoólica para minimizar o efeito da levedura contaminante.
Among the wild yeasts more commonly found in the alcoholic fermentation, wrinkled colonies with pseudohyphal morphology belonging to Saccharomyces genus are highlighted. This yeast biotype has been associated with industrial problems, resulting in the decrease of the fermentative yield. In this context, this work aimed to perform the genetic, morphological/physiological characterization and stress tolerance of Saccharomyces cerevisiae strains which exhibited wrinkled and mucous colonies, searching for alternatives that could contribute to the management of these wild yeasts (wrinkled colonies) in the alcoholic fermentation. Characterization tests for invasiveness in Agar medium, killer activity, temperature, pH, ethanol and sugar concentration, actidione, flocculation and fermentative capacity were carried out with 22 strains (11 wrinkled and 11 mucous colonies), which were screened previously by the sequencing of the ITS region of the ribosomal DNA. The effect of the acid treatment using different pH values over the growth of two strains (52 wrinked and PE-02 mucous) was also evaluated, following the growth monitoring in sugar cane juice after acid treatment. Fermentative tests in sugar cane juice were carried out with pure and mixed cultures of these strains. The morphological/physiological tests showed that the invasiveness in YEPD Agar medium occurred in low frequency among the 22 strains tested; ten out of eleven wrinked yeasts exhibited expressive flocculation, and among the mucous, the flocculation was near zero; none of the strains showed killer activity; the wrinkled colonies presented lower fermentative capacity comparing to mucous colonies, in a 48- hour cycle in batch system using sugar cane juice, with slower fermentation rate. Concerning the resistance to temperature, pH, ethanol and sugar concentration, the mucous colonies were more resistant to low pH, while the wrinkled colonies were more resistant to the elevated ethanol and sugar concentrations. None of the yeasts were resistant to actidione. The genetic analysis by microsatellite loci revealed the presence of two main genetic related groups, the first branch comprised mainly of wrinkled colonies (67%), containing however the PE-02 strain, showing strains with high flocculation rate and tolerance to high concentration of sugar and ethanol; the other group (with three subgroups) comprised mainly mucous colonies, showing lower resistance to the stressing conditions here studied. The yeast with wrinkled colony (strain 52) was severely affected by the acid treatment in pH values of 1.0 and 1.5, but the same did not occur with the mucous colony (PE-02). The batch fermentation with cell recycle and acid treatment in pH 1.5 had effect over the wrinkled yeast growth when in mixed culture with the mucous yeast, and did not impair the fermentative efficiency comparing to the pure culture of PE-02. In pure culture with the wrinkled colony, a fermentative efficiency as low as 60% was observed, confirming the low performance of these yeasts. The knowledge of the response to stressful conditions exhibited by the yeasts with wrinkled colonies can help to manage the alcoholic fermentation in order to minimize the effect of the contaminant yeast.

A cachaça é uma bebida típica brasileira produzida a partir da destilação do caldo de canade- açúcar fermentado principalmente por Saccharomyces cerevisiae. Grande parte da produção nacional é artesanal, e não há uma preocupação por parte dos produtores quanto ao controle microbiológico da fermentação. Este trabalho objetivou caracterizar a comunidade bacteriana contaminante do fermento utilizado na produção de cachaça. Foram coletadas quatro amostras em um alambique artesanal. A primeira (NA) foi coletada um ano anterior às outras três e utilizada como controle. As restantes foram coletadas ao final do primeiro dia de fermentação (NP), após quinze (NS) e trinta dias (NT) utilizando o mesmo fermento. Um total de 587 seqüências de 16S rDNA foram analisadas, sendo 81 da amostra NA, 177 da amostra NP, 159 da amostra NS e 170 da amostra NT. As análises das seqüências revelaram a presença de 17 gêneros e 27 espécies, além de 27 bactérias não conhecidas. Cento e setenta unidades taxonômicas operacionais (UTOs) foram identificadas utilizando o software DOTUR com uma distância evolucionária de 0.03. Quarenta e três UTOs foram identificadas na amostra controle (NA), 38 na NP, 57 na amostra NS e 38 na terceira amostra (NT). Das 170 UTOs identificadas, apenas dezessete foram identificadas duas vezes em diferentes amostras e somente uma, identificada como Lactobacillus hilgardii, foi identificada três vezes, evidenciando uma grande dinâmica populacional bacteriana durante o processo fermentativo. Análises estatísticas utilizando o software S-LIBSHUFF indicaram diferenças significativas na composição bacteriana entre amostras. Foram encontradas espécies/gêneros ainda não descritas na literatura em fermentos de cachaça, como Weissella cibaria, Leuconostoc citreum e algumas espécies de Lactobacillus. Além destas, várias bactérias não conhecidas também foram identificadas. Os resultados revelaram que a comunidade de bactérias contaminantes do processo fermentativo é muito mais complexa do que se conhecia, com características heterofermentativas e produção de congêneres que provavelmente refletem na qualidade da bebida. Este é o primeiro relato da utilização do método de seqüenciamento do gene 16S rDNA para determinar contaminantes bacterianos em fermentados de cana-de-açúcar para produção de cachaça.
Cachaça is a typical Brazilian spirit made from sugar cane fermented mainly by Saccharomyces cerevisiae. The production is mostly artisanal, and there is no microbiological control during the fermentation process. The objective of this study was to assess the bacterial community associated with the ferment used in the production of cachaça. Four ferment samples were collected from an artisanal still. The first one (NA), was collected one year previously to the other three and constituted a control reference. The remaining three samples were collected at the end of the first day of fermentation (NP), and fifteen (NS) and thirty days (NT) after using this same ferment. A total of 587 16S rDNA sequences were analyzed, being 81 sequences from the NA sample, 177 from NP, 159 from NS, and 170 from the NT sample. Sequence analyses revealed the presence of 17 genus and 27 species plus 27 unknown species. One hundred and seventy operational taxonomic units (OTUs) were identified using the DOTUR software using a cut-off evolutionary distance of 0.03. Forty three were identified in the control (NA) sample, 38 in the NP, 57 in NS, and 38 in the third (NT) sample. Of the 170 OTUs, only seventeen were detected twice in different samples and only one, identified as Lactobacillus hilgardii, was identified thrice, indicating a dynamic nature of the bacterial community during the fermentation process. Statistical analysis using the software S-LIBSHUFF indicated significant differences in the bacterial composition among samples. Our analyses also allowed to identify several bacteria not yet described as contaminants of the cachaça ferment, such as Weissella cibaria, Leuconostoc citreum and some Lactobacillus species. In addition, several unknown bacteria were also detected. The results revealed a much larger bacterial contaminant community present in the fermentative process of cachaça than previously reported with heterofermentative ability to produce secondary compounds which probably influence the quality of the final beverage. This is the first report on the utilization of the 16S rDNA sequencing method to assess the bacterial diversity of sugar cane fermentation for the production of cachaça.

Parte do malte pode ser substituído por adjuntos, que podem ser à base de cereais, ou de carboidratos de origem vegetal. A cana de açúcar constitui-se de uma matéria prima favorável à fermentação alcoólica por ser rica em carboidratos e micronutrientes, e pode ser utilizada como um adjunto do malte. Estudos de fermentação do mosto com este adjunto, na forma de caldo e de melado de cana, foram realizados na Planta Piloto de Bebidas da EEL-USP, e a partir daí, foram feitas análises físico-química, microbiológicas e sensoriais da cerveja obtida, com diferentes proporções de adjunto. As fermentações foram realizadas inicialmente em escala de bancada (1 L), utilizando mostos preparados com caldo ou melado de cana com ou sem diferentes tratamentos de clarificação, em duas concentrações diferentes (25% e 50% de caldo ou melado), além do mosto puro malte. Análises de concentração e viabilidade celular, pH, consumo de extrato e formação de etanol foram realizadas ao longo das fermentações e, também foi efetuado o cálculo de produtividade e rendimento. Os ensaios sem tratamento do caldo ou melado foram escolhidos para uma produção em uma escala de 5 L, para que análises físico-químicas e sensorial fossem realizadas. A cerveja elaborada com 25% de melado de cana sem tratamento foi a cerveja mais aceita e foi escolhida para a produção em escala piloto (200 L). Análises físico-químicas e de envelhecimento foram realizadas, e essa cerveja também foi avaliada sensorialmente juntamente com duas cervejas de mercado, onde novamente foi declarada como a mais aceita, mostrando que o melado de cana é um adjunto adequado na fabricação de cervejas, mostrando ser um potencial no mercado de bebidas. Finalmente, cervejas com melado de cana foram elaboradas na forma ale e lager em uma infra-estrutura bem equipada em um laboratório em uma universidade belga.
Part of malted barley can be replaced by adjuncts, which can be from cereals, or carbohydrates of vegetable origin. Sugarcane is a raw material favorable to the alcoholic fermentation to be rich in carbohydrates and micronutrients, and can be used as a adjunct. The wort fermentation studies with this adjunct in the form of sugarcane juice and sugarcane syrup, were performed in the Beverage Pilot Plant EEL-USP, and from there, it were made physical-chemical, microbiological and sensorial analysis with beers with different proportion of adjuncts. The fermentations were initially carried out in laboratory scale (1 L) prepared with sugarcane juice or sugarcane syrup in different treatments and in two different concentrations (25% and 50%), and also 100% of malt wort. Analysis of cell concentration and viability, pH, extract consumption and ethanol formation were held throughout the fermentations and also calculation of the productivity and yield. Assays with sugarcane juice and syrup without treatment were selected for production on a slightly larger scale (5L) and physico-chemical and sensorial analyzes were performed. Beer made with 25% of sugarcane syrup without treatment was a beer more accepted and it was chosen for the production on a pilot scale (200 L). Physico-chemical and aging analyzes were performed, and that beer was also evaluated by sensory analyzis compared with two beer market, where again it was declared as the most accepted, showing that the sugarcane syrup is a very interesting adjunct in the manufacture of beer, showing a potential in the beverage market. Finally, beers with sugarcane syrupo were prepared in ale and lager form in a well-equipped infrastructure in a laboratory in a Belgian university.

Este trabalho tem como objetivo comparar o desempenho fermentativo de estirpes de leveduras vínicas quando utilizadas em sistema celular livre ou imobilizadas em diferentes suportes (alginato de cálcio, loofa e tufo vulcânico), recorrendo a microvinificações à escala laboratorial de mosto sintético e mosto de uva. Todos os sistemas de imobilização mostraram capacidade para conduzir a fermentação alcoólica dos mostos, obtendo-se taxas de consumo de glucose e produção de etanol compatíveis com as obtidas para as mesmas estirpes em sistema livre. A dinâmica das comunidades microbianas durante a fermentação alcoólica do mosto de uva foi monitorizada através de sequenciação massiva de nova geração (NGS). Este estudo demonstrou a existência de diferenças claras entre as populações que dominam as fermentações espontâneas e as fermentações induzidas pela inoculação de leveduras, e verificou-se que a inoculação de leveduras livres também origina dinâmicas populacionais diferentes da utilização de sistemas de leveduras imobilizadas; ABSTRACT: Comparative study of the behavior of free and immobilized yeasts during alcoholic fermentation of must - the dynamics of involved population This study aims to compare the fermentative performance of wine yeast strains when used in a free cell system or immobilized on different supports (calcium alginate, loofah and volcanic tuff), using laboratory scale microvinification of synthetic and grape must. All immobilization systems showed the ability to conduct the alcoholic fermentation of musts, obtaining glucose consumption and ethanol production rates compatible with those obtained for the same strains in free cells system. The dynamics of microbial communities during alcoholic fermentation of grape must was monitored through new generation high-throughput sequencing (NGS). This study demonstrated the existence of clear differences between the populations that dominate spontaneous fermentation and the fermentations induced by yeast inoculation, and it was found that the inoculation of free yeast also originates different populational dynamics than the use of immobilized yeast systems.

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A busca por linhagens de leveduras fermentadoras de pentoses ? uma demanda do processo de produ??o de etanol a partir de substratos lignocelul?sicos. A D-xilose, proveniente dos processos de hidr?lise da hemicelulose n?o ? metabolizada por linhagens de leveduras, como as da esp?cie Saccharomyces cerevisiae, convencionalmente utilizadas na maioria dos processos industriais para obten??o de ?lcool de primeira gera??o. Neste contexto, o presente trabalho teve por objetivo isolar leveduras naturalmente ocorrentes em biomassas vegetais e avaliar sua capacidade para converter D-xilose em etanol. O isolamento foi conduzido em meio s?lido contendo D-xilose como fonte de carbono. Os isolados foram triados por teste de produ??o de g?s em meio contendo D-xilose e os que apresentaram resultado positivo foram testados para produ??o de etanol. Dos 202 isolados de leveduras capazes de assimilar D-xilose para o crescimento, quatro linhagens apresentaram habilidade em fermentar este a??car. O potencial fermentativo das leveduras selecionadas foi avaliado em meios sint?ticos, onde foram monitorados o crescimento celular e consumo dos a??cares. O teor alco?lico foi determinado ao fim de cada ensaio e as vari?veis do processo foram calculadas. As linhagens isoladas foram identificadas como Galactomyces geotricum e Candida akabanensis, n?o havendo nenhum registro de aplica??o biotecnol?gica para produ??o de biocombust?veis a partir destas linhagens. A produ??o de etanol foi de 4,7 a 14,0 g L-1 e a efici?ncia fermentativa de at? 69%. O processo de bioconvers?o da xilose, contida em hidrolisados hemicelul?sicos, a etanol surge como perspectiva potencial para eventual aplica??o das linhagens de leveduras selecionadas.
Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016.
The screening for new strains of pentose fermenting yeast is a demand for ethanol production process from lignocellulosic substrates. D-xylose from processes of hemicellulose hydrolysis is not metabolized by yeast strains conventionally used in industrial processes for obtaining first generation ethanol, such as Saccharomyces cerevisiae species. In this context, this study aimed to isolate naturally occurring yeasts in vegetal species or its parts and assess their ability to convert D-xylose to ethanol. The isolation was carried out in solid medium containing D-xylose as only carbon source. The isolates were screened by gas production test in medium containing D-xylose and those who had positive results were tested for ethanol production. Of the 202 isolates of yeasts capable of assimilating D-xylose for growth, four strains also showed ability to ferment this sugar. The potential of fermentative selected yeasts has been reported in synthetic media, in which the cell growth and consumption of sugars were monitored. Alcohol content was determined after each run and some process variables were calculated. The isolated strains were identified as Geotricum galactomyces and Candida akabanensis, not having any biotechnological application record for production of biofuels from these species. Ethanol production achieved 4.7 to 14.0 g L-1 and the fermentative efficiency up to 69%. The bioconversion process of xylose (contained in hemicellulose hydrolysates) to ethanol appears as natural perspective for eventual application of selected yeasts.

La ricerca di microrganismi fermentanti in grado di migliorare le proprietà microbiologiche, fisiche, chimiche, sensoriali e organolettiche delle bevande alcoliche fermentate come vino, birra, idromele, sidro, è considerata ad oggi un punto chiave per lo sviluppo del settore. Negli ultimi anni, l'aumento della domanda al consumo di nuove bevande fermentate ha causato un enorme bisogno di tecnologie di produzione innovative al fine di ottenere diverse tipologie di prodotti caratterizzati da proprietà sensoriali peculiari. Per raggiungere tale obiettivo, l’impiego di nuovi microrganismi, sia fermentanti che non fermentanti, in grado di conferire al prodotto caratteristiche organolettiche peculiari ed uniche, può rappresentare una valida alternativa ai classici ceppi attualmente in commercio. Il settore vitivinicolo-enologico risulta quello in cui la ricerca ha sviluppato conoscenze sufficienti ad applicare nuove specie e nuovi ceppi di lieviti su scala industriale. La ricerca di lieviti non convenzionali applicabili alle bevande alcoliche che nell’Europa meridionale sono considerate secondarie rispetto al vino, ovvero birra, sidro ed idromele, risulta essere ancora poco investigata, essendo scarsi gli studi disponibili sulla fermentazione di queste bevande. In particolare, il settore birrario sta riscoprendo un rinnovato interesse nei confronti di lieviti isolati da matrici territoriali, considerati responsabili del miglioramento e della creazione di bouquet particolari, che consentono di ottenere produzioni legate al territorio e differenziate su un mercato in forte espansione. Analogamente alla produzione di birra artigianale, la produzione di idromele sta attualmente vivendo un interesse a livello amatoriale e professionale in tutto il mondo. Sebbene non sia una bevanda molto popolare, il suo consumo in Europa è in costante crescita. Anche in questo settore la letteratura scientifica risulta limitata, specialmente in ambito microbiologico, dato che per condurre le fermentazioni alcoliche sono spesso usati ceppi di S. cerevisiae di origine enologica. Diversamente dal mosto d’uva, il mosto-miele risulta carente in fattori nutrizionali che limitano la crescita microbica. Per tale motivo, la ricerca di lieviti appropriati in grado di crescere e fermentare queste matrici risulta essere un punto cruciale per mantenere un elevato standard qualitativo di questa bevanda. Sulla base di queste premesse, durante il triennio di Dottorato di ricerca è stata condotta uno studio inerente: l’ecologia microbica di matrici zuccherine; l’isolamento, la caratterizzazione genotipica e tecnologica di lieviti, sia Saccharomyces che non-Saccharomyces; la loro applicazione, sia singolarmente che in combinazione, durante le fermentazioni sperimentali di mosto di birra e mosto-miele; la valutazione della cinetica di fermentazione, metaboliti secondari, composti organici volatili ed analisi sensoriale dei prodotti ottenuti.