In this thesis it is performed the measurement of electrons from heavy-flavor hadron decays, with the use of the ALICE detector, at LHC. Two analysis are performed. The first one consists in the measurement of the invariant cross section of electrons from open heavy-flavor (charm or beauty) hadron decays in pp collisions at s = 13 TeV, the largest center of mass energy generated by LHC up to now, in the transverse momentum interval 0.5 < pT < 4 GeV/c. The experimental cross-section is compared to the pQCD-based prediction given by FONLL, and it sits on the upper edge of the theoretical uncertainty band over the full momentum range. The ratios between this experimental result and the equivalent measurements performed at lower center of mass energy have the potential to bring constrains to the FONLL calculation. The second analysis consists in the measurement of the nuclear modification factor, RAA , of electrons from beauty-hadron decays in the 10% most central Pb-Pb collisions at sNN = 5.02 TeV, in the interval 2 < pT < 8 GeV/c. Electrons from beauty-hadron decays are extracted from the inclusive electron yield via fits to the impact parameter distribution using templates of electrons from different sources, obtained from Monte Carlo simulations. This measurement allows to probe the energy loss of beauty quarks when traversing the Quark-Gluon-Plasma (QGP), a color-deconfined medium produced in relativistic heavy-ion collisions. A suppression of about 40-50% is observed on the RAA for pT > 3 GeV/c and it is consistent, over the full momentum range and within experimental uncertainties, with the prediction of pQCD-based models that describe a mass-dependent energy loss of the heavy-quarks while traversing the QGP, interacting with its constituents via radiative and collisional processes. This analysis brings significant improvement of the experimental uncertainties with respect to the equivalent measurement performed with the run 1 data and consists in an important step in the study of the mass-dependence of the energy loss of heavy quarks inside the QGP at low pT . / Nessa tese é feita a medida de elétrons provenientes do decaimento de hadrons que contêm quarks pesados (charm ou beauty), com o uso dos detectores do experimento ALICE, no LHC. Duas análises são realizadas. A primeira consiste na medida da seção de choque invariante de elétrons provenientes do decaimento de hadrons que contêm quarks charm ou beauty em colisões pp a s = 13 TeV, a maior energia de centro de massa gerada pelo LHC até então, na região de momento transversal 0.5 < pT < 4 GeV/c. Esse resultado é comparado com a previsão dada pelo FONLL (calculo baseado em QCD perturbativa) e se situa no limite superior da incerteza teórica, sobre toda a faixa de momento. As razões entre este resultado experimental e medidas equivalentes realizadas em mais baixa energia podem trazer informações importantes para tornar o cálculo teórico mais preciso. A segunda análise consiste na medida do fator de modificação nuclear, RAA , de elétrons provenientes do decaimento de hadrons que contêm quarks beauty, em colisões centrais de Pb-Pb a sNN = 5.02 TeV, no intervalo 2 < pT < 8 GeV/c. Elétrons provenientes de hadrons que contêm quarks beauty são extrados do conjunto de elétrons inclusivos a partir do ajuste da distribuição do parâmetro de impacto com templates de elétrons de diferentes fontes, extrados de simulações de Monte Carlo. Essa medida permite estudar a perda de energia dos quarks beauty ao atravessarem o Plasma de Quarks e Glúons (QGP), produzido em colisões entre ons pesados em altas energias. É observada uma supressão de 40-50% no RAA medido para pT > 3 GeV/c e esta é consistente, sobre toda a faixa de p T , com a previsão de modelos baseados em pQCD, que descrevem a perda de energia dos quarks pesados via processos elásticos e inelásticos com os constituintes do QGP, de maneira dependente da massa. Este resultado possui incertezas experimentais significativamente menores com relação à medida realizada com dados do run 1 e consiste num importante passo no estudo da dependência em massa da perda de energia dos quarks pesados no QGP em baixo pT .

The lepton flavor violating interactions are interesting in the sense that they are sensitive the physics beyond the standard model and they ensure considerable information about the restrictions of the free parameters, with the help of the possible accurate measurements. In this work, we investigate the lepton flavor violating H+ ! W+l and the lepton flavor conserving H+ ! W+l decays in the general two Higgs doublet model and we estimate decay widths of these decays. After that, we analyze lepton flavor violating decay ! i in the same model and calculate its branching ratio. We observe that the experimental results of the processes under consideration can give comprehensive information about the physics beyond the standard model and the existing free parameters.

Orientador: Maria Aparecida A. P. da Silva / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T15:54:43Z (GMT). No. of bitstreams: 1 Almeida_SelmaBergara_D.pdf: 1651470 bytes, checksum: a5ee60dbc52c3dc1aeab330822f4380b (MD5) Previous issue date: 2006 / Resumo: Para a recuperação do aroma e sabor de fruta fresca ao suco industrializado, subprodutos do processamento do suco de laranja, como óleo essencial e essências aquosa e oleosa, são normalmente reincorporados ao produto processado. A adição de cada um desses subprodutos confere ao produto final qualidade e estabilidade sensoriais distintas. O presente trabalho teve por objetivo avaliar a qualidade e estabilidade sensoriais do suco de laranja reconstituído, produzido com laranjas de variedades brasileiras e aromatizado com quatro diferentes aromas naturais de laranja, quais sejam: I - óleo essencial; II - frações destiladas de essência oleosa (oil phase); III - aroma formulado contendo frações destiladas de óleo essencial e de essência oleosa; e, IV - aroma formulado contendo frações destiladas de óleo essencial, frações destiladas de essência oleosa e de essência aquosa (water phase). Assim, esta pesquisa consistiu em quatro etapas, a saber: avaliação do impacto dos quatro aromas naturais sobre a aceitação e perfil sensorial do suco de laranja pronto para beber em condições pré-processamento térmico; avaliação da estabilidade sensorial dos aromas naturais ao processamento térmico do suco; avaliação do impacto dos aromas naturais sobre a estabilidade sensorial do suco pronto para beber durante o armazenamento da bebida e, finalmente, a identificação de compostos voláteis odoríferos presentes em um dos aromas mais estáveis - Aroma III. Quatro formulações de suco de laranja reconstituído foram elaboradas, adicionando-se os aromas I, II, III e IV, processadas e envasadas assepticamente em garrafas PET. Os perfis sensoriais, através de Análise Descritiva Quantitativa, e a aceitação das quatro formulações de suco de laranja pronto para beber foram avaliados antes e após o processamento térmico, bem como durante o armazenamento da bebida por 20, 40, 60 e 90 dias. Índices de escurecimento e teores de vitamina C foram determinados durante o armazenamento. Adicionalmente, amostras comerciais de suco de laranja pronto para beber foram avaliadas. Os dados foram analisados por ANOVA, teste de média Tukey ou t-Student, Mapa de Preferência, Análise de Componentes Principais, análise de correlação de Pearson e análise de regressão linear dos parâmetros sensoriais em função do tempo de armazenamento da bebida. O aroma III foi avaliado por cromatografia gasosa (CG), CG-olfatometria (Osme) e espectrometria de massas. Os resultados obtidos na primeira etapa da pesquisa sugeriram que a reincorporação dos compostos voláteis provenientes da essência oleosa contribuem menos para aumentar a aceitação do suco de laranja do que aqueles provenientes do óleo essencial. Em contrapartida, as amostras comerciais tiveram aceitação modesta entre os consumidores indicando que, no Brasil, os sucos de laranja comerciais podem ainda ter sua qualidade sensorial melhorada pela adição de novos aromas naturais. Na segunda etapa, a avaliação da aceitação das amostras pré e pós-processamento indicou boa estabilidade dos aromas naturais estudados ao processamento térmico. Os perfis sensoriais das formulações sugeriram que: i) a reincorporação ao suco de frações destiladas dos três subprodutos do processamento da laranja, resulta em maior estabilidade térmica do que aromas derivados apenas do óleo essencial; e, ii) a presença de voláteis provenientes da essência aquosa no aroma natural de laranja, mesmo que em proporções minoritárias, realça no suco notas de sabor de suco natural e suaviza as notas de aroma e sabor de casca de laranja. Na terceira etapa, durante o armazenamento da bebida, a formulação contendo apenas frações de essência oleosa apresentou, notadamente, menor estabilidade sensorial do que as demais formulações, contendo compostos voláteis provenientes do óleo essencial. Possivelmente essa ocorrência seja devida à presença de agentes antioxidantes naturais nas frações derivadas do óleo essencial. Adicionalmente, os resultados sugeriram que a adição ao suco de laranja de frações destiladas de óleo essencial resulta em melhor estabilidade sensorial à bebida do que a adição ao suco do óleo essencial na sua forma integral. Em todas as amostras, ao longo do período de armazenamento, ocorreu queda dos teores de vitamina C e concomitante escurecimento dos sucos. Finalmente, na última etapa, foram gerados 55 picos de odores, os quais apresentaram, predominantemente, notas de aroma associadas à laranja. Beta-mirceno, butanoato de etila, a-pineno, hexanal, citral e decanol foram os compostos voláteis de maior importância odorífera identificados. Os compostos ß-terpineol, citronelal, undecanal, acetato de a-terpenila, acetato de nerila e a-cariofileno foram identificados como compostos de alto poder odorífero e, limoneno, linalol, a-terpineol e decanal, como compostos de baixo poder odorífero / Abstract: To restore the natural orange aroma and flavor to industrialized juices, processors add natural flavorings obtained from three orange juice by-products: the cold-pressed oil, the oil phase and the water phase. The addition of each of these by-products results in juices with distinct sensory characteristics and acceptability. The present work aimed at evaluating the sensory quality and stability of reconstituted orange juice from Brazilian varieties, flavored with four different orange natural aromas, which are: I ¿ essential oil; II ¿ distilled fractions of the oil phase; III - formulated aroma containing distilled fractions of the essential oil and of the oil phase e, IV ¿ formulated aroma containing distilled fractions of the essential oil, distilled fractions of the oil phase and of the water phase. Thus the present research was divided into four stages: evaluation of the impact of four natural aromas (I, II, III, IV) on the acceptability and sensory profile of ready-todrink orange juice before thermal treatment; evaluation of the sensory stability of the natural aromas to the thermal treatment of the juice; evaluation of the natural aromas impact to the sensory stability of ready-to-drink orange juice during storage; the identification of odor volatiles compounds in one of the most stable aromas ¿ Aroma III. Four reconstituted orange juices formulations were elaborated, by adding the above mentioned flavorings (I, II, III, IV), processed and packed in PET bottles. The sensory profiles, by using Quantitative Descriptive Analysis, and the acceptability of the four ready-to-drink orange juices were evaluated before and after the processing, and also during their 20, 40, 60 and 90 day-storage. In addition, other commercial samples of ready-to-drink orange juice were included in this study. Determinations of the ascorbic acid and the browning index of formulated juices were done as well. Data were analyzed by ANOVA, Tukey or t-Student mean test, Principal Component Analysis, Preference Mapping, Pearson¿s correlation analysis and linear regression analysis of sensory parameters as a function of the storage time. The aroma III was evaluated by gas chromatography (GC), GC-olfactometry (OSME) and by mass spectrometry. The results obtained from the first research stage suggested that reintroduction of the volatiles compounds from oil phase contribute less to increase the acceptability of orange juice than those from the essential oil. On the other hand, all the commercial samples presented moderate acceptability by consumers, indicating that the commercial orange juices in Brazil may have their sensory quality improved by adding new natural aromas. In the second research stage, the evaluation of the pre and post-processing samples acceptability indicated that the studied natural aromas presented good stability to the thermal treatment. The formulations sensory profiles suggested that: i) the reintroduction of the distilled fractions of the three orange processing by-products to the juice results in higher thermal stability than that caused by the aromas derived from the essential oil; ii) the presence of volatiles from distilled fractions of the water phase in the natural orange aroma, although in very small proportions, enhances natural juice flavor and softens orange peel flavor and aroma. In the third research stage, during the drink storage the formulation containing only distilled fractions of the oil phase presented much less sensory stability than the other formulations, containing volatile compounds from the essential oil. A possible explanation for this occurrence is the presence of natural antioxidants in those fractions derived from essential oil. Further, the results suggested that the addition of distilled fractions of essential oil to orange juice provides better sensory stability to the drink than the addition of the integral essential oil. During storage all samples showed decrease in vitamin C and simultaneous browning of juices. Finally, in the last research stage, Fifty-five odor peaks were generated, which presented aroma notes associated mostly to orange. Beta-myrcene, ethyl butanoate, a-pinene, hexanal, citral and decanol were identified as the highest aroma impact compounds. The compounds ß-terpineol, citronellal, undecanal, a-terpinyl acetate, neryl acetate and a-cariophylene were identified as high odor power compounds and, limonene, linalool, a-terpineol and decanal, as low odor power compounds / Doutorado / Doutor em Tecnologia de Alimentos

En juillet 2012, le CERN a annoncé la découverte du boson de Higgs qui est la dernière particule manquante du Modèle Standard (MS). Le boson de Higgs a été observé dans différents canaux. La mesure précise de ses propriétés est maintenant très importante pour rechercher des déviations par rapport au SM. Cette thèse présente une recherche du boson de Higgs produit en association avec une paire de quarks top et se désintégrant en une paire de b quark, le canal ttH(H → bb) , en utilisant les données de collisions proton-proton à √s = 13 TeV, collectées avec le détecteur ATLAS en 2015 et 2016. Ce document détaille en particulier la reconstruction du systéme ttH et la séparation du signal du bruit de fond principal tt + jets. Des études récentes montrent qu'il existe une fraction importante du bruit de fond ttbb avec des jets contenant deux hadrons b. Un nouvel algorithme a été développé pour séparer ces jets des jets contenant un seul b-hadron. La description de cet outil est présenté dans cette thèse. / In July 2012, CERN announced the discovery of the Higgs boson, the last missing piece of the Standard Model (SM). The Higgs boson was observed in different channels. Precise measurement of its properties is now very important to investigate for possible deviations from the SM. This thesis presents a search for the Higgs boson produced in association with top quarks and decaying to a b quark pair, the ttH(H→bb) channel, using proton-proton collisions at √s = 13 TeV, collected with the ATLAS detector in 2015 and 2016. This document details in particular the full reconstruction of the ttH(H→bb) system and the discrimination between signal and the main background, tt+jets. The ttbb is a subset of the tt+jets backgrounds recent studies show that there is a large fraction of ttbb events with jets containing two b-hadrons. A new algorithm has been developed to discriminate such jets from single b-hadrons jets. The description of this tool is presented in this thesis.

Das IceCube Neutrino Observatorium am Südpol hat die Existenz eines diffusen astrophysikalischen Neutrinoflusses nachgewiesen. Die Flavor-Zusammensetzung astrophysikalischer Neutrinos trägt Informationen über Orte kosmischer Teilchenbeschleunigung und Auswirkungen potenzieller neuer Physik auf die Neutrinoausbreitung. Zur seiner Bestimmung ist die Beobachtung von Tau-Neutrinos nötig. Ab einer Energie von ~O(100 TeV) kann deren Wechselwirkung über geladene Ströme eine Doppelkaskaden-Topologie ergeben, bei der die zwei Energiedepositionen am Tau-Entstehungs- und Tau-Zerfallsvertex aufgelöst werden können. Diese wird zusammen mit den bereits bekannten Topologien Einzel-Kaskade und Spur zur Messung der Flavor-Zusammensetzung auf der Erde benutzt. In dieser Arbeit werden im Detektorvolumen von IceCube anfangende Ereignisse mit hohen Energien algorithmisch in drei Topologien klassifiziert. Im Datensatz mit einer Lebensdauer von 7.5 Jahren werden zum ersten Mal zwei Doppelkaskaden identifiziert; diese sind Kandidaten für Tau-Neutrinos. Die Eigenschaften der zwei Tau-Neutrino-Kandidaten werden in einer a-posteriori Analyse im Detail studiert. Die statistische Methode wird durch einen Log-Likelihood-Quotienten-Test mit multi-dimensionalen Wahrscheinlichkeitsdichten verbessert. Eine der Doppelkaskaden ist konsistent mit dem Szenario einer misklassifizierten Einzelkaskade, während für die zweite Doppelkaskade die Wahrscheinlichkeit eines nicht-Tau-Neutrino Szenarios auf nur 3% bestimmt wird. Die gemessene Flavor-Zusammensetzung ist konsistent mit der Annahme von astrophysikalischen Neutrinos sowie mit bisher veröffentlichen Resultaten. Die Messung ergibt einen astrophysikalischen Tau-Neutrino Fluss von dPhi / dE=3.0 (-1.8,+2.2) (E / 100TeV)^(-2.87) 10^(-18) GeV^(-1) cm^(-2) s^(-1) sr^(-1), was dem ersten positiven Ergebnis für die Tau-Normalisierung entspricht. Die Nichtexistenz eines astrophysikalischen Tau-Neutrino Flusses wird mit einer Signifikanz von 2.8 sigma abgelehnt. / The IceCube neutrino observatory at the South Pole has confirmed the existence of a diffuse astrophysical neutrino flux. The flavor composition of astrophysical neutrinos carries information on the environments at the sites of cosmic particle acceleration as well as potential imprints of new physics acting during neutrino propagation. To measure the flavor composition the observation of the long-elusive tau neutrinos is required. Starting at an energy of ~O(100 TeV) a tau neutrino charged current interaction can produce a double cascade topology, where the two energy depositions from the tau creation and the tau decay vertices are resolvable. This topology together with the well-established track and single cascade topology is used to measure the flavor composition on Earth. In this work, high-energy events starting in IceCube's detector volume are classified algorithmically into the three topologies. In the dataset with a livetime of 7.5 years, two events are classified as double cascades for the first time, yielding multi-TeV tau-neutrino candidates. The properties of the two tau-neutrino candidates are investigated in an a-posteriori analysis. The statistical method is improved by performing a log-likelihood-ratio test using multi-dimensional probability densities. One of the double cascades is consistent with being a misclassified single cascade, while the second double cascade is found to have a misclassification probability of only 3%. The measured flavor composition nu_e:nu_mu:nu_tau = 0.20:0.39:0.42 is consistent with astrophysical neutrinos and with previously published results. The astrophysical tau-neutrino flux is measured to dPhi / dE=3.0 (-1.8,+2.2) (E / 100TeV)^(-2.87) 10^(-18) GeV^(-1) cm^(-2) s^(-1) sr^(-1) with spectral index gamma=2.87 (-0.20,+0.21), yielding the first non-zero results for the tau normalization. The absence of an astrophysical tau-neutrino flux is disfavored at 2.8 sigma.

This study examined the effects of wet aging on water-soluble flavor compounds, descriptive flavor, consumer acceptability, and willingness-to-pay of beef strip steaks. Twenty USDA Select boneless beef loins (NAMP #180) were dorsally divided into four equal portions, which were randomized to receive either 0, 7, 14, or 21 d of wet aging. Short-chained peptide concentration increased from 4.11 mg/g on d 0 to 5.12 mg/g on d 7 (P = 0.011) and subsequently increased to 7.14 mg/g on d 21 (P < 0.001). Cooked beef contained 1.85 mg/g less short-chained peptides than raw beef (P < 0.001). Wet aging for 21 d increased the concentrations of amino acids, which are precursors for the Maillard reaction, including methionine, threonine, cystine, serine, tryptophan, and phenylalanine (P < 0.001). Wet aging decreased the nucleotide concentrations (P ≤ 0.003) and increased hypoxanthine concentration, a compound that imparts bitter flavor (P < 0.001). Flavor intensity of steaks was greater on d 14 than on d 21 (P = 0.009). Steaks that were not aged (d 0) had a more intense umami taste than those aged for 7 and 21 d (P ≤ 0.042); whereas d 7 steaks had less off-flavor, described as metallic, oxidized, and cardboard, than steaks from other aging treatments (P ≤ 0.038). Demand analysis indicated that 14-d steaks would be sold at 5.2, 5.2, and 6.7 units (0.454kg/unit) less (P < 0.001) than steaks aged for 0, 7, and 21 d if holding price constant at $14/0.454kg. Wet aging alters the content of water-soluble flavor compounds in beef, which contributes to flavor development during cooking. Wet aging for 21 d decreased umami taste and flavor intensity. However, amino acid concentrations increased during aging, which contributes to meaty and browning flavors that may be more prevalent in other direct heating methods such as grilling. In addition, results indicated that wet aging for 14 d was not sufficient to provide the flavor and tenderness improvements that were apparent after 21 d of aging since aging beef for 14 d decreased consumer acceptance and demand.

No description available.

Doctor of Philosophy / Department of Human Nutrition / Delores H. Chambers / Vegetables are popular among consumers because of their versatility of preparation, unique sensory characteristics, and exceptional health benefits. Trends such as organic farming and breeding to increase nutrition and functional health components have increased interest in understanding the flavor of vegetables, such as leafy greens. A lexicon of thirty-two flavor attributes was created to help describe the flavor of fresh leafy vegetables. This lexicon includes five “green” attributes; mouth feel characteristics such as pungent, bite, tooth-etch, and heat/burn; fundamental tastes including bitter and umami; seven terms that describe unique flavors related to specific vegetables such as cabbage, celery, lettuce, spinach, parsley, beet, and radish leaves; and a group of other terms including citrus, piney, woody, water-like, musty/earthy, floral, sulfur, metallic, soapy, petroleum-like, and overall sweet. In addition, our study encompassed a series of sensory tests which will aid in better understanding the effects of several production variables on the sensory characteristics of pac choi and tomato. Variables evaluated were production systems (i.e. organic and conventional), fertilizer amount (i.e. high, low, and no fertilizer), environment (i.e. field and high tunnel), maturity level (i.e. 2.5, 4.5, and 6.5-week old plants at the time of harvest), and shelf life (i.e. 1, 4, 9, 18 days of refrigerated storage). Samples were grown at the Kansas State University Horticulture Research Center located in Olathe, Kansas. Highly trained descriptive panelists from the Sensory Analysis Center at Kansas State University evaluated the samples. There do not appear to be major sensory differences between organic and conventional products specific to the crops and seasons studied. Furthermore, when differences were present, they generally were quite small and showed no clear trends or patterns favoring one production system over the other even after refrigerated storage. However, it is suggested that differences in flavor and volatile composition between organic and conventional pac choi may be more evident at early stages of growth.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Monoterpene alcohols (monoterpenols) play an important role in the flavour and aroma of grapes and wine. This is especially applicable to wines of a muscat variety, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement other varietal flavours and aromas. These monoterpenols can be found in grapes and wine as free, volatile and odorous molecules, as well as in flavourless, nonvolatile glycosidic complexes. These complexes most often occur as 6-0-a-L-arabinofuranosyl-p-D-glucopyranosides (vicianosides), 6-0-P-D-xylopyranosyl- P-D-gluco-pyranosides (primverosides), 6-0-P-D-glucopyranosyl-p-D-glucopyranosides (gentio-biosides ), 6-0-a-L -rhamnopyra nosyl-p-D-g lucopyra nos ides (rutinos ides), or 6-0-p-D-apiofuranosyl-p-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. These precursors are, however, hydrolyzed only to a limited extent by endogenous glycosidases during the fermentation process, as they exhibit very low activity in wine conditions. The monoterpenols can be released from their sugar moieties by one of two methods: either an acid or an enzymatic hydrolysis. The enzymatic hydrolysis mechanism is fully understood, and the process functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by an a-L-arabinofuranosidase, an a-L-rhamnosidase, a p-D-xylosidase, or a p-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a p-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. As the endogenous grape glycosides of Vitis vinifera and the yeast Saccharomyces cerevisiae show very low activity towards these aromatic precursors during the handling of the juice and winemaking processes, the focus has increasingly fallen on introducing exogenous p-glucosidases to wines and juices. Genes encoding p-glucosidases and a-L-arabinofuranosidases have been cloned from various organisms, including bacteria, fungi and yeasts. However, the activities and properties of these enzymes are not always suitable for exploitation under winemaking conditions, where a low pH, low temperatures, and high ethanol and glucose concentrations prevail. A genetically engineered wine yeast strain of S. cerevisiae that expresses glycosidases that are active in these conditions would be useful in improving the flavour and aroma of wines, thereby adding to the complexity and value of the wine. Two p-glucosidase genes, BGL 1 and BGL2 from Saccharomycopsis fibufigera, were subcloned into two Escherichia coli-yeast shuttle vectors. A dominant selectable marker gene (SMR1) was also inserted onto these plasmids. These plasmids were designated pBGL 1 (containing the BGL 1 gene) and pBGL2 (containing the BGL2 gene) respectively. Introduction of the two plasmids into two strains of S. cerevisiae then followed. A laboratory strain, L1278, was transformed to confirm the effective secretion of the expressed protein. An industrial yeast strain, VIN13, was subsequently transformed by making use of the selectable marker (resistance against sulfometuron). Enzyme assays with the synthetic substrate p-nitrophenol-j3-D-glucopyranoside (pNPG) were performed to determine the activity of the j3-glucosidases over a period of days, as well as at certain temperatures and pH values. The stability of the enzymes was also investigated. These recombinant yeasts were able to degrade the pNPG efficiently. They showed promising results concerning pH optima, with a substantial amount of activity found at the pH levels as found in the wine environment. There was also a slight increase in specific activity at lower temperatures. The recombinant yeast strains were also tested in smallscale fermentations. Three wines were made, of which two were from white cultivars (Chenin blanc and GewOrtztraminer) and one from red (Pinotage). Results obtained from micro-extraction from the finished wines showed that the terpenol content did increase, although this was not the only wine component influenced. Other flavour compounds also showed increases, especially the esters. This also played a role in the flavour increase in the wine. Future work would include optimizing the available results. This would entail the addition of another glycosidic enzyme, such as a-L-arabinofuranosidase, to the genome of the wine yeast to aid the further breakdown of glycosidic bonds. The cloning or engineering of a j3-glucosidase enzyme that is more active at low temperatures would also yield better results and release even more of the aroma of the wine. / AFRIKAANSE OPSOMMING: Monoterpeenalkohole (monoterpenole) speel 'n belangrike rol in die geur en aroma van druiwe en wyn. Dit is veral van toepassing op wyne van Muskaat-varieteite, maar hierdie geurkomponente is ook teenwoordig in ander nie-Muskaat druifsoorte, waar dit bydra tot die varieteitsqeur en aroma. Hierdie monoterpenole kom voor in druiwe as vry, vlugtige en aromatiese molekules, of as geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0-a-L-arabinofuranosiel-~-D-glukopiranosiede, 6- O-~-D-xilopiranosiel-~-D-glukopiranosiede (primverosiede), 6-0-~-D-glukopiranosiel-~-Dglukopiranosiede (gentiobiosiede), 6-0-a-L-ramno-pyranosiel-~-D-glukopiranosiede (rutinosiede), of 6-0-~-D-apiofuranosiel-~-D-glukopirano-siede van hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Hierdie geurvoorlopers word egter slegs tot In beperkte mate tydens die proses van fermentasie deur die endogene glikosidase ensieme gehidroliseer, aangesien hulle baie min aktiwiteit toon onder wynbereidingstoestande. Die monoterpenole kan op een van twee wyses van hul suikermolekules vrygestel word: 'n suurhidrolise, of ensiematiese hidrolise. Die ensiematiese hidroliseproses word baie goed begryp en behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur In a-L-arabinofuranosidase, In a-L-ramnosidase, In ~-D-xilosidase, of 'n ~-D-apiosidase gebreek. In die tweede stap word die monoterpeenalkohol deur In ~-glukosidase vrygestel. Hierdie ensiematiese afbraakproses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos wat met suurhidrolise die geval is nie. Omdat die endogene glikosidases van Vitis vinifera en die van die gis Saccharomyces cerevisiae baie lae aktiwiteit ten opsigte van die aromatiese voorlopers gedurende die hantering van die druiwesap en wynmaakprosesse toon, val die fokus al hoe meer op die inkorporering van eksogene ~-glukosidases in wyn en sappe. Gene wat vir ~- glukosidases en a-L-arabinofuranosidases kodeer, is al vanuit verskeie organismes gekloneer, insluitende bakteriee, fungi en giste. Die aktiwiteite en kenmerke van hierdie ensieme is egter nie altyd wenslik vir hul gebruik in wyn nie, aangesien dit In omgewing is met 'n lae pH, lae temperatuur, en hoe etanolvlakke en glukose-konsentrasies. In geneties veranderde wyngis van S. cerevisiae wat in staat is om glikosidases uit te druk wat onder hierdie kondisies aktief is, sal baie handig te pas kom in die verbetering van die geur en aroma van wyne, om daardeur die kompleksiteit en die waarde van die wyn te verhoog. Twee ~-glukosidasegene, BGL 1 en BGL2 vanaf die gis Saccharomycopsis fibuligera , is in twee afsonderlike Esccherichia coli-gis-pendelplasmiede gesubkloneer. In Dominante selekteerbare merkergeen (SMR1) is ook in hierdie plasmiede gekloneer. Hierdie plasmiede word onderskeidelik pBGL 1 (met die BGL 1-geen) en pBGL2 (bevattende die BGL2-geen) genoem. Hierdie twee plasmiede is hierna apart na twee rasse van S. cerevisiae getransformeer. Eerstens is 'n laboratoriumras, L1278, getransformeer om te bevestig dat effektiewe sekresie en uitdrukking van die proteTen plaasvind. Hierna is 'n industriele gisras, VIN13, getransformeer deur gebruik te maak van die selektiewe merker (bestandheid teen sulfometuron). Ensiem-bepalings met behulp van die sintetiese substraat p-nitrofeniel-p-O-glukopiranosied (pNPG) is gedoen om die aktiwiteit van die p-glukosidqses oor 'n aantal dae te bepaal, asook om die aktiwiteit by sekere temperature en pH-vlakke te meet. Die stabiliteit van die ensieme is ook bepaal. Hierdie rekombinante giste was in staat om pNPG effektief af te breek. Hulle het belowende resultate betreffende die pH-optima getoon, met 'n aansienlike hoeveelheid aktiwiteit by die pH-vlakke soos dit in die wynomgewing voorkom. Daar was ook 'n effense verhoging in die ensieme se aktiwiteite by laer temperature. Die rekombinante gisrasse is ook in kleinskaalse wynfermentasies gebruik. Drie verskillende wyne is gemaak, waarvan twee wit kultivars was (Chenin blanc en GewOrtztraminer) en een 'n rooi kultivar (Pinotage). Resultate wat deur die mikro-ekstraksie van die voltooide wyne verkry is, het getoon dat die terpenolinhoud wei verhoog het, alhoewel dit nie die enigste wynkomponente was wat beinvloed is nie. Ander geurkomponente het ook 'n verhoging in konsentrasie getoon, veral die esters. Hierdie verbindings het ook 'n rol in die verhoging van geur in die wyne gespeel. Toekomstige werk sal die beskikbare resultate verder optimaliseer. Dit sal insluit die byvoeging van nog 'n glikosidiese ensiem, soos a.-L-arabinofuranosidase, tot die genoom van die wyngis, om verdere afbraak van glikosidiese verbindings teweeg te bring. Die klonering of verandering van 'n p-glukosidase-ensiem met verhoogde aktiwiteit by laer temperature sal ook beter resultate toon en meer geur in die wyn kan vrystel.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Yeasts produce a broad range of aroma-active volatile esters and higher alcohols during alcoholic fermentation. Some of these esters and higher alcohols are important for the fruity flavors and therefore the final quality of wine and other fermented beverages. Esters are produced and hydrolyzed by alcohol acetyltransferases and esterases, respectively. In yeast, ester-synthesizing activities are represented by two alcohol acetyltransferases encoded by the ATFI and ATF2 genes, and by an ethanol hexanoyl transferase encoded by the EHTI gene. Atfl p and Atf2p appear responsible for the production of ethyl acetate and isoamyl acetate, while Ehtl p synthesizes ethyl hexanoate from ethanol and hexanoyl-CoA. Although a fair amount of information is available regarding the ATF 1 gene, limited information is available on the remaining alcohol acetyltransferases. Only two genes that code for esterases have been identified in yeast, namely lAHI and TIPI. It has also been shown that the balance between alcohol acetyltransferases and esterases is important for the net rate of ester accumulation. Higher alcohols are synthesized from the a-keto-acids in the branched-chain amino acid metabolic pathway by decarboxylation and reduction. The transamination of the amino acid to the respective a-keto-acid is catalyzed by mitochondrial and cytosolic branched-chain amino acid transferases, which are encoded by the BATI and BAT2 genes, respectively. In recent years, a strong scientific and industrial interest in the metabolism of flavoractive compounds has emerged, but information regarding the roles of specific enzymes and the physiological relevance of their metabolism remains limited. The aim of this project was to investigate the physiological and metabolic consequences of changes in the expression levels of some of the key enzymes involved in aroma compound production. The consequences of these changes on the chemical composition and the fermentation bouquet of wines and distillates were also investigated. The first part of the section on the results in this dissertation reports on the role and relative importance of the Saccharomyces cerevisiae enzymes involved in ester metabolism, namely Atflp, Atf2p, Ehtlp, Iahlp and Tiplp. The corresponding genes were overexpressed in a laboratory strain of S. cerevisiae, BY4742, and in a widely used commercial wine yeast strain, VIN13. Table wine and base wines for distillation were prepared with these VIN13 transformed strains. The ester concentrations and aroma profiles of the wines and distillates were analyzed and compared. The data indicated that the overexpression of ATF 1 and ATF2 increased the concentrations of ethyl acetate, isoamyl acetate, 2-pheylethyl acetate and ethyl caproate, while the overexpression of JAHI resulted in a significant decrease in the concentrations of ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHTI resulted in a marked increase in the concentrations of ethyl caproate, ethyl caprylate and ethyl caprate, while the overexpression of TJP1 did not decrease the concentrations of any of the esters. In most cases, there was a correlation between the increase in esters and the decrease in higher alcohols. The data suggest that yeast balances the amount of different esters produced through alcohol acetyltransferases and esterases, and that, in some cases, these enzymes appear to overlap in function and/or influence each other's activity. In the second part of the results section, the consequences of the deletion and the overexpression of two genes, BATl and BAT2, which encode transaminases that contribute to the metabolism of higher alcohols, were investigated. The genes were both disrupted in a S. cerevisiae BY4742, and overexpressed in both this laboratory strain and in the VIN13 wine yeast strain. The effects of these modifications on the general physiology of the corresponding yeast strains and on higher alcohol metabolism were assessed in a range of growth conditions, including aerobic and anaerobic growth conditions, in the presence of glucose or raffinose as sole carbon source and growth in the presence of various concentrations of amino acids. Table wine and base wines for distillation were prepared with the modified industrial strains and the concentrations of the higher alcohols and the aroma profiles of the wine and distillates were analyzed and compared. Batl deletion seemed to be lethal under the conditions that were created, and therefore only the bat2!:!.strain, together with the BATI and BAT2 overexpression strains, were investigated. These modifications did not appear to significantly affect the general physiology of the strains. The results obtained indicated that the overexpression of BATI increased the concentrations of isoamyl alcohol and isoamyl acetate, and, to a lesser extent, the concentrations of isobutanol and isobutyric acid. The overexpression of the BAT2 gene resulted in a substantial increase in the levels of isobutanol, isobutyric acid and propionic acid production, and a modest increase in the level of propanol and isovaleric acid. Interestingly, the overexpression of BAT2 led to a decrease in isoamyl alcohol and isoamyl acetate concentrations. Sensory analyses indicated that the wines and distillates produced with the strains in which the BATl and BAT2 genes were overexpressed had more fruity characteristics (peach and apricot aromas) than the wines produced by the wild-type strains. This study offers new prospects for the development of wine yeast starter strains with optimized ester and higher alcohol-producing capability that could assist winemakers in their efforts to consistently produce wine to definable specifications and styles and a predetermined flavor profile. / AFRIKAANSE OPSOMMING: Gedurende fermentasie produseer giste 'n wye verskeidenheid vlugtige aromatiese esters en hoër alkohole. Sommige van hierdie esters en hoër alkohole is belangrik vir die vrugtige geure en dra dus by tot die finale kwaliteit van wyn en ander gefermenteerde drankies. Esters word onderskeidelik deur alkoholasetieltranferases en esterases geproduseer en gehidroliseer. In giste word die ester-sintetiserende aktiwiteite deur twee alkoholasetieltransferases verteenwoordig wat deur die ATFI-en ATF2-gene, asook 'n etanolheksanoïeltransferase wat deur die EHTl-geen, gekodeer word. Dit blyk dat ATFlp en ATF2p verantwoordelik is vir die produksie van etielasetaat en isoamielasetaat, terwyl Ehtl p-etielheksanoaat vanaf etanol en heksanoïel-CoA sintetiseer. Alhoewel daar 'n redelike hoeveelheid inligting t.o.v die ATF I-geen beskikbaar is, is daar weinig inligting oor die res van die aloholasetieltransferases. Slegs twee gene wat vir esterases kodeer, is in gis geïdentifiseer, naamlik IAHI en TIPI. Daar is ook bewys dat 'n balans tussen die alkoholasetieltransferases en esterases baie belangrik is vir die netto-tempo van ester-akkumulasie. Hoër alkohole word gesintetiseer vanaf a-keto sure in die vertakte-ketting aminosuur metaboliese pad deur dekarboksilasie en reduksie. Die transaminasie van die aminosuur na die onderkeidelike a-ketosuur word deur vertakte-ketting aminosuur transferases, geleë in die mitochondrion en sitosol, en gekodeer deur BATl- en BAT2-gene, gekataliseer. In die laaste paar jare het daar 'n sterk wetenskaplike, asook industrïele, belangstelling in die metabolisme van aroma-aktiewe komponente te voorskyn gekom, maar inligting in verband met die rol van spesifieke ensieme en die fisiologiese belangrikheid van hul metabolisme is egter beperk. Die doel van hierdie projek was om die fisiologiese en metaboliese gevolge van veranderinge in die ekspressievlakke van sommige sleutelensieme betrokke by aromakomponent-produksie te ondersoek. Die gevolge van hierdie veranderinge op chemiese vlakke, asook hoe die fermentasie-aroma van die wyne en distillate beïnvloed word, is ook bestudeer. Die eerste gedeelte van die resultate rapporteer oor die rol en relatiewe belangrikheid van die Saccharomyces cerevisiae-ensieme betrokke by estermetabolisme, naamlik Atfl p, Atf2p, Ehtlp, Iahlp en Tiplp. Die gene was ooruitgedruk in 'n laboratoriurnras van S. cerevisiae, BY4742, asook in 'n kommersïele wyngisras, VIN13. Tafelwyne en basiswyne vir distillasie is gemaak met die getransformeerde VIN13-rasse. Die esterkonsentrasies en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Die data het gewys dat die ooruitdrukking van ATFI- en ATF2-gene 'n verhoging in etielasetaat, isoamielasetaat, 2-fenieletielasetaat en etielkaproaat veroorsaak het, terwyl ooruitdrukking van !AHI 'n betekenisvolle afname in etielasetaat-, isoamielasetaat-, heksielasetaat- en 2-fenieletielasetaat-konsentrasies veroorsaak het. Die ooruitdrukking van EHTI het 'n duidelike verhoging in etielkaproaat, etielkaprilaat en etielkapraat veroorsaak en die ooruitdrukking van TIPIhet geen van die esterkonsentrasies verander nie. In die meeste gevalle was daar nie 'n korrelasie tussen die toename in esters en afname in hoër alkohole nie. Die data stelook voor dat die gis 'n balans tussen die verskillende esters handhaaf deur middel van die alkoholasetieltrasferases en esterases, en in sommige gevalle blyk dit dat die ensieme dieselfde funksies het en/of mekaar se aktiwiteit beïnvloed. In die tweede gedeelte van die resultate is die oorsake van delesie en ooruitdrukking van twee gene, BAT1 en BAT2, wat kodeer vir transaminases wat tot hoër alkohol metabolisme bydra, bestudeer. Die gene is uitgeslaan in S. cerevisiae BY4742 en ooruitgedruk in BY4742 en in die wyngisras VIN13. Die effekte van hierdie modifikasies op die algemene fisiologie van die verskillende gisrasse en op hoëralkoholmetabolisme is onder 'n verskeidenheid kondisies bestudeer, naamlik aërobies en anaërobiese groeikondisies, in die teenwoordigheid van glukose of raffinose as die enigste koolstofbron, asook in die teenwoordigheid van 'n verskeidenheid konsentrasies aminosure. Tafelwyne en basiswyne vir distillasie is gemaak met die gemodifiseerde industrïele rasse en die konsentrasies van die hoër alkohole en aromaprofiele van die wyne en distillate is ontleed en vergelyk. Bat1-delesie was dodelik onder die kondisies, daarom is slegs die batlts-tes tesame met die BAT1 en BAT2 wat in die rasse ooruitgedruk is, bestudeer. Die modifikasies het nie 'n beduidende effek op die algemene fisiologie van die rasse getoon nie. Die data het wel getoon dat die ooruitdrukking van BAT1 'n verhoging in isoamielalkohol- en isoamielasetaatkonsentrasies, en tot 'n mindere mate isobutielalkohol- en isobottersuur-konsentrasies, veroorsaak het. Die ooruitdrukking van BAT2 het 'n beduidende toename in isobutanol-, isobottersuur- en propioonsuurkonsentrasies en 'n kleinere toename in propanol- en isovaleriaansuur veroorsaak. Die ooruitdrukking van BAT2 het ook gelei tot 'n afname in isoamielalkohol- en isoamielasetaatkonsentrasies. Sensoriese analises het getoon dat die wyne en distillate wat geproduseer is met die rasse waarin die BAT1 en BAT2 gene ooruitgedruk is meer vrugtige eienskappe (perske- en appelkoos-aromas) getoon het as die wyne wat deur die wildetipe rasse geproduseer is. Die studie lewer nuwe vooruitsigte vir die ontwikkeling van wyngiste met geoptimiseerde ester en hoër alkohol produserende eienskappe wat die wynmakers in staat kan stelom wyne te produseer met gedefinieerde spesifikasies en style en 'n voorafbepaalde aromaprofiel.