Les protéines de raisin thaumatin-like et chitinase sont d’un intérêt majeur, tant pour la recherche viticole de par leur implication dans les mécanismes de défense de la vigne, que pour la recherche en oenologie en raison de leur présence en quantité majoritaire dans le pool protéique d’un jus de raisin. La mise au point d’un protocole de quantification par chromatographie liquide et dosage immuno-enzymatique utilisant des gammes de protéines purifiées à partir de jus de raisin a permis de caractériser la diminution de la concentration de ces deux protéines au cours du procédé de vinification. Les propriétés tensioactives des deux protéines thaumatin-like et chitinase purifiées ont été évaluées, révélant que ni l’une ni l’autre ne pouvait à elle seule expliquer la formation et la stabilisation des bulles et de la mousse d’un Champagne. Une étude structurale de la protéine thaumatin-like VVTL1 a ensuite été menée dans l’optique de mieux comprendre les mécanismes chimiques, biologiques et physiques dans lesquels elle peut être impliquée. Une structure de VVTL1 a été modélisée par homologie et l’analyse de ses modes normaux a permis de révéler un mécanisme de type mâchoire autour d’une cavité acide, site potentiel de l’activité enzymatique de la protéine. Un feuillet beta en épingle à cheveux isolé dans la structure s’est révélé être très conservé et absolument spécifique à la superfamille des protéines thaumatin-like, ouvrant peut-être la voie vers l’élucidation complète du rôle biologique de ces protéines. Dans une seconde approche, la détermination de la structure d’un peptide sélectionné dans la séquence de VVTL1 par modélisation sous contraintes RMN a posé les bases d’une étude modèle de l’adsorption des protéines à la surface de la bentonite, argile utilisée pour la clarification des vins. / Grape proteins thaumatin-like and chitinase are of major interest, as much by the vine defense mechanisms they are involved in as by their dominance over the grape juice protein pool. Liquid chromatography and immunoassays allowed both proteins to be quantified in grape juice and Champagne, showing that their concentration decreases through winemaking. The involvement of these proteins in gas/liquid interfaces was also studied on the purified fractions from grape juice previously made for quantification standards. Results clearly indicated that neither thaumatin-like nor chitinase could alone explain bubble formation and foam stabilization in Champagne. A first study of the three-dimensional structure of the main thaumatin-like protein VVTL1 using homology molecular modeling was then achieved and normal modes analysis was performed on the VVTL1 model. It revealed a jaw-like mechanism opening and closing an acidic cleft assessed to be the enzymatic binding site. An isolated beta hairpin turned out to be highly conserved and specific to the thaumatin-like superfamilly. This domain could provide a first clue to unravel the mystery of the protein biological activity in the field of plant-pathogen interactions. A second approach was set up for the structure determination of a VVTL1 peptide using molecular modeling under NMR restraints. It could lead to a model study of protein adsorption on bentonite, a clay used for wine clarification.

X-ray crystallography is used as a powerful tool to understand how a particular biomacromolecule accomplishes its various functions. In the present study, detailed atomic structure of bacterial di-heme cytochrome c4 isolated for the first time from an anaerobic organism and structure of two crystal forms of sweet protein thaumatin from the African shrub, are presented, as well as an example of how this structural knowledge of cytochrome c4 in combination with biochemical data have been used to classified the protein function. In parallel to modern high-throughput approaches in the crystallization of protein, basic research on their physico-chemical properties and molecular interactions has increasingly gained on importance in recent years. Our aim was to improve the success of any crystallization attempts significantly as well as to find alternative methods of predicting it. In this thesis two novel approaches in macromolecular crystallization are discussed. The first; newly discovered cross-crystallization method alias cross-influence procedure (CIP) with its subsequent application presented on two selected proteins. And the novelty in our second approach is the use of precise temperature-control to drive the crystallization process by keeping system in metastable zone. The understanding that the solubility and phase behavior of proteins is an essential part of the crystallization process, allows us to perform the temperature-controlled experiment (TCE), where the manipulation of the thermodynamics as well as the kinetics of the crystallization process were controlled. In order to improve crystal quality and/or morphology, the both CIP and TCE were found as useful tools in protein crystallization presented by means of crystallization of three different proteins (bacterial cyrochrome c4, glycoside hydrolase family 11 enzyme xylanase and sweet protein thaumatin). Finally, the preliminary crystallization information about three other enzymes (two haloalkane dehalogenase and one membrane protein hydrogenase) are given at the end, as a starting experiment for further X-ray diffraction analysis.

L'infection des baies de raisin par le champignon Botrytis cinerea, responsable de la pourriture grise, est fréquente et occasionne des dégâts importants. Pourtant, il semble que la baie dispose de moyens de défense parmi lesquels des protéines PR "Pathogenesis-Related". Chez le Pinot Noir, une chitinase (CHI4D) et une thaumatin-like (TL3) s'accumulent naturellement en grande quantité à partir de la véraison et présentent une activité antifongique contre B. cinerea in vitro. L'objectif de ce travail est de comprendre comment B. cinerea peut se développer sur des baies censées disposer de défenses suffisantes. Pour cela, l'expression spatio-temporelle des ARNm et des protéines CHI4D et TL3 a été suivie respectivement par hybridation in situ et immunohistolocalisation dans les baies, au cours de la maturation, à la suite d'un stress abiotique (UV-C) ou d'un stress biotique (B. cinerea). Dans des baies avant véraison (vertes), n'exprimant naturellement que très faiblement CHI4D et TL3, les ARNm et les protéines s'accumulent en grande quantité après application d'un stress abiotique (UV-C) ou biotique (infection artificielle par B. cinerea). Les protéines CHI4D et TL3 sont localisées au niveau des faisceaux conducteurs ainsi qu'au niveau des tissus proches des sites d'exposition aux UV-C (exocarpe) ou au niveau des sites d'inoculation de B. cinerea, suggérant qu'elles sont impliquées dans la défense de la baie avant véraison. Après véraison, les ARNm et les protéines sont naturellement accumulés au niveau de l'exocarpe et des faisceaux conducteurs qui correspondent à des sites potentiels d'entrée ou de propagation des agents pathogènes. Alors que l'application d'un stress UV-C sur ces baies ne provoque qu'un effet mineur sur l'expression de CHI4D et de TL3, au cours de l'infection par B. cinerea, les quantités de transcrits et de protéines diminuent. A un stade précoce d'infection, la diminution de la quantité des deux protéines est observée en avant du front de propagation du champignon, suggérant une dégradation par des protéases sécrétées par B. cinerea. A un stade d'infection plus avancé, cette diminution s'étend à l'ensemble de la baie. La production hétérologue des protéines CHI4D et TL3 nous a permis de confirmer que CHI4D pouvait être dégradée par des protéases aspartiques sécrétées par B. cinerea. La dégradation de TL3 n'a pas pu être reproduite in vitro. Des tests antigerminatifs effectués in vitro avec les protéines hétérologues n'ont pas permis de mettre en évidence d'effet antifongique malgré la présence d'une activité chitinase pour CHI4D et β-1,3,-glucanase pour TL3. Il est donc possible que ces protéines possèdent des fonctions autres que celles impliquées dans la défense. / Grape berries infection by the phytopathogenic fungus Botrytis cinerea, the causal agent of gray mold, is quite common and causes significant damage. However, it seems that berries have a mechanism of defense, among which are pathogenesis related proteins. In Pinot Noir grape berries, a chitinase (CHI4D) and a thaumatin-like (TL3) protein naturally accrue in large amounts from véraison and show in vitro an antifungal effect against B. cinerea. The aim of this work was to understand how B. cinerea can develop on grape berries that seem to have sufficient defense mechanisms. To do so, the spatio-temporal expression of CHI4D and TL3 mRNAs and proteins in berries was studied respectively by in situ hybridization and immunohistolocalization during maturation, after an abiotic stress (UV-C) or a biotic stress (B. cinerea). Before véraison (green berries) while the expression of CHI4D and TL3 is naturally low, mRNAs and proteins have accumulated in large amounts in berries after UV-C exposition or artificial infection with B. cinerea. CHI4D and TL3 proteins have accumulated around vascular bundles as well as near the sites of UV-C exposition (exocarp) or B. cinerea inoculation, suggesting that before véraison these proteins could be involved in the berry defense. After veraison, mRNAs and proteins naturally accumulate in the exocarp and around vascular bundles that correspond to potential sites of penetration or propagation of pathogenic agents. While the application of UV-C stress on these berries causes only a minor effect on the expression of CHI4D and TL3, during infection by B. cinerea, the amounts of mRNA and proteins decreased. At an early stage of infection, the less amounts of both proteins were observed around the fungus propagation area, suggesting that these proteins could be degraded by B. cinerea secreted proteases. At a more advanced stage of infection, the decrease extended to the entire berry.Production of heterologous CHI4D and TL3 proteins allowed us to confirm that CHI4D could be degraded by aspartic proteases secreted by B. cinerea whereas no degradation of TL3 could be observed in vitro. Both heterologous proteins showed no antifungal effect while a chitinase and a β-1,3-glucanase activities were observed respectively for CHI4D and TL3. It is therefore possible that these proteins have other functions than those involved in the defense.

Structural biology is a branch of science related to biochemistry, biophysics, and molecular biology that deals with the molecular structures of biological macromolecules, in particular nucleic acids and proteins. Structure-guided drug design uses three-dimensional knowledge of protein structures to design small molecules which block the action of specific proteins. When crystals of theses macromolecules and their complexes can be obtained, their crystal structures can be determined by using isomorphous differences between a native structure and a derivative structure. This allows crystallographers to determine the coordinates of a small number of heavy atoms which provide initial phases for macromolecules. The advent of synchrotron radiation allowed determination of a heavy atom substructure by use of anomalous differences using either multiple wavelengths (MAD) or a single wavelength (SAD); the latter has become the most common phasing method in crystallography and is the method used in this study. The use of SeMet has been by far the most successful method employed in SAD. However, in some cases production of SeMet proteins is not possible thus necessitating additional options, for example, xenon. Noble gases such as xenon may be used in SAD experiments by binding to various, non-specific sites. Advances in noble gas pressurization systems like the Hampton Research Xenon Chamber have greatly eased the production of noble gas derivatives, xenon itself being a prime candidate with a very strong anomalous signal when compared to lighter noble gases like krypton and argon. Investigation of the phasing properties of xenon was carried out on test proteins hen egg white lysozyme (HEWL), thermolysin, glucose isomerase, and thaumatin II. Phases were successfully determined for all four proteins including thaumatin II which did not bind xenon but was successful due to the anomalous signal from 17 native sulfurs. The three remaining proteins showed varying occupancies and numbers of sites including xenon sites in thermolysin and glucose isomerase which have not been observed previously. This document will serve as a guide for the preparation of xenon derivative crystals and provides a strategy for the collection and processing of data from xenon derivatives.

Orientadores: Gonçalo Amarante Guimarães Pereira, Jorge Mauricio Costa Mondego / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T09:29:17Z (GMT). No. of bitstreams: 1 Franco_SulamitadeFreitas_M.pdf: 9729318 bytes, checksum: 0c17aa7d5f8dfd63cbc8d23f9259fc83 (MD5) Previous issue date: 2008 / Resumo: Moniliophthora perniciosa é um fitopatógeno de grande importância econômica no Brasil, em especial na região do sul da Bahia, por causar a doença do cacaueiro conhecida como Vassoura de Bruxa. Esse fungo possui um ciclo de vida hemibiotrófico, apresentando duas fases distintas: a primeira biotrófica/parasítica e a segunda saprotrófica/necrotrófica. O banco de dados gerados pelo projeto genoma Vassoura de Bruxa vem sendo estudado com o intuito de desvendar as vias metabólicas utilizadas pelo patógeno na colonização do cacaueiro. Curiosamente, foi verificada a expressão de um gene similar a taumatina pelo fungo. As taumatinas de plantas são expressas durante a resposta hipersensitiva (HR) em interações planta-patógeno, sendo classificadas como PR-5 (pathogenesis related protein). Taumatinas possuem atividade antifúngica e são capazes de clivar ß-glucanas. Uma nova função foi descrita para uma taumatina de trigo: a de inibição de xilanase. Recentemente, as taumatinas vêm sendo estudadas também em nematódeos, artrópodos e fungos. O trabalho aqui descrito visou à clonagem do gene de M. perniciosa similar a taumatinas MpTLP1 e a expressão da proteína recombinante MpTLP1, bem como o estudo da expressão desse gene durante o desenvolvimento de M. perniciosa. Análises de Northern blot mostram que esse gene é induzido quando o fungo é incubado com extrato de cacau e ácido salicílico. Dados de Real time PCR mostram que esse gene é mais expresso na fase biotrófica do fungo. Esses resultados sugerem a participação de MpTLP1 na interação planta-patógeno. MpTLP1 foi expressa em E.coli e renaturada a partir de corpos de inclusão. A proteína renaturada não apresentou atividades antifúngica e ß-glucanolítica. Esses resultados podem ser atribuídos a sua renaturação ineficiente, ou pelas suas diferenças estruturais com relação a outras TLPs com atividade antifúngica e ß-glucanolítica. Algumas características de MpTLP1 podem indicar que essa proteína possa ser um inibidor de xilanase ou de outras enzimas que clivam parede celular. / Abstract: Moniliophthora perniciosa is a phytopatogen that has economical importance in Brazil, particularly in the south of Bahia, because this fungus causes the disease in cacao known as Witches' Broom disease. This fungus is a hemibiotrofic pathogen that has two distinct stages: a biotrophic (or parasitic) and a necrotrophic (or saprotrophic). The database generated by the Witches' Broom genome project has been studied in order to unravel the metabolic pathways used by the pathogen during the colonization of cacao. Curiously, it was detected the expression of a gene similar to thaumatins in M. perniciosa. Plant thaumatins are expressed during hypersensitive responde (HR) in plant pathogen interactions, being classified as PR-5 (pathogenicity related protein class-5). Thaumatins have antifungal and ß-glucanolitic activities. Recently, a new wheat thaumatin-like protein was described as a xylanase inhibitor. Thaumatins from nematodes, arthropods and fungi have also been characterized. This work aimed the cloning of the M. perniciosa thaumatin like-gene MpTLP1, the expression of the recombinant protein MpTLP1, as well as at the characterization of its gene expression during the development of M. perniciosa. Northern blot analysis shows that MpTLP1 expression is induced when the fungus is incubated with cacao extract and salicylic acid. Real time-PCR data show that MpTLP1 is more expressed in the biotrophic stage of the fungus. These results suggest the participation of MpTLP1 in plant-pathogen interaction. MpTLP1 was expressed in E. coli and refolded from inclusion bodies. The refolded protein did not have antifungal or ß- glucanolytic activity. These results can be attributed to an inefficient refolding or to structural differences in relation to other TLPs that have antifungal and ß- glucanolytic activity. Some MpTLP1 features may indicate that this protein inhibits xylanases or other enzymes that cleave cell walls. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientadores: Gonçalo Amarante Guimarães Pereira, Jorge Maurício Costa Mondego / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T03:53:42Z (GMT). No. of bitstreams: 1 Franco_SulamitadeFreitas_D.pdf: 3383952 bytes, checksum: e4041edb080f1a645fcbd6eacdc51378 (MD5) Previous issue date: 2014 / Resumo: A cultura do cacau é de grande importância econômica em países produtores da semente e produtores de chocolate, e move um mercado que alcança hoje ao redor de U$ 50 bilhões. As doenças fúngicas são um fator importante na redução de produção de cacau. No Brasil, o principal responsável pela perda na produção é o basidiomiceto Moniliophthora perniciosa, agente etiológico da doença conhecida como vassoura de bruxa, que arrasou a produção brasileira na década de 90. A doença tem como característica a não elicitação de resposta hipersensitiva (HR), o que permite a colonização de tecidos vegetais pelo fungo. HR é uma resposta que desencadeia a indução da expressão de proteínas denominadas PRs (pahtogenesis related), que agem contra patógenos. Entre as PRs encontram-se as PR5, conhecidas como taumatinas. Análise do genoma de M. perniciosa resultou na anotação de 13 genes que codificam proteínas Taumatina- like (MpTLPs). Apesar de serem bem descritas como proteínas antifúngicas em plantas, estudos em fungos basidiomicetos apontam seu envolvimento no remodelamento celular durante a formação cogumelos. M. perniciosa é a espécie de fungo com o maior número de TLPs descritos até o momento, tendo 6 deles organizados em clusters, indicando eventos de duplicação. Análise filogenética revelou uma maior quantidade de TLPs em basidiomicetos, quando comparado a ascomicetos, indicando sua participação na formação de cogumelos. No geral, MpTLPs são transcritas durante a fase de vassoura seca da doença, onde o galho seco está sujeito a infecção de fungos oportunistas, indicado a contribuição dessas proteínas no combate contra esses fungos, além de poder estar participando no desenvolvimento de sua parede para formação de basidiocarpo e na degradação da parede celular da planta. Além das MpTLPs, análise de transcriptoma revelou um total de 35 MpCSEPs (Proteínas Candidatas a Efetores de Patogenicidade Secretados pelo fungo) com valor de RPKM >50 foram identificadas em plantas de cacau infectadas. Esses genes são expressos preferencialmente na fase inicial da doença (Vassoura verde), onde encontramos o fungo ainda em fase biotrófica. Em geral, esses genes codificam proteínas pequenas e ricas em cisteínas que são características típicas de efetores de virulência (AVRs), podendo ser uma evidência de que esses genes codificam potenciais efetores. Após triagem, 16 desses genes foram escolhidos para a caracterização estrutural. Destes, sete foram clonados para expressão de proteína heteróloga, o que resultou ao final do estudo a cristalização de 2 proteínas (MpCSEP 5 e MpCSEP 14). Os cristais obtidos até o momento foram testados em linha de luz MX1 e MX2, mas não apresentou difração e os processos de refinamento dos cristais deverão ser continuados. As proteínas obtidas nesse estudo poderão ser utilizadas em testes enzimáticos em trabalhos futuros, para sua completa caracterização estrutural e funcional / Abstract: The cocoa cultivation have great economic importance in the seed and chocolate producing countries , moving a market that presently reaches around $50 billion. Fungal diseases are the major factor in reducing cocoa production. In Brazil, the main responsible for the production loss is the basidiomycete Moniliophthora perniciosa, known as the etiological agent of witches' broom disease , which devastated the Brazilian production in the 90's disease. The disease is characterized by not eliciting a hypersensitive response (HR), which allows the plant tissue colonization by the fungus. HR is a response that triggers the induction of the expression proteins called PRs (pahtogenesis related), which act against pathogens. Among the PRs, PR5 are known as thaumatins. M. perniciosa genome analysis resulted in the annotation of 13 genes encoding proteins Thaumatin - like (MpTLPs). Despite being well described as antifungal proteins in plants, fungi basidiomycetes studies suggest its involvement in cellular remodeling during mushroom formation. M. perniciosa fungal is the species with the highest number of TLPs described so far, with 6 of them organized in clusters, indicating duplication events. Phylogenetic analysis revealed a greater number of TLPs in basidiomycetes when compared to ascomycetes, indicating their involvement in the formation of mushrooms. Overall, MpTLPs are transcribed during the dry broom disease, where the dead branch is subject to opportunistic fungal infection, indicating the contribution of these proteins in the fight against these fungi, and can be participating in the development of your wallpaper for training basidiocarp and in the cell wall degradation of the plant. Besides MpTLPs, transcriptome analysis revealed a total of 35 MpCSEPs (candidate secreted effectors of pathogenicity protein by the fungus) with RPKM value > 50 have been identified in infected cocoa plants. These genes are preferentially expressed in the early phase of the disease (Green Broom), where we found the fungus still in biotrophic phase. In general, these genes encode small and rich in cysteine that are typical characteristics of virulence effector (AVRs) proteins, which can be evidence that these genes encode potential effectors. After screening, 16 of these genes were chosen for structural characterization. Of these, seven were cloned for expression of heterologous protein, resulting at the end of the study the crystallization of 2 proteins (MpCSEP 5 and 14). The crystals obtained so far been tested in MX1 and MX2 light line, but showed no diffraction and processes of crystals refinement should continue. The proteins obtained in this study may be used in enzymatic assays in future work to its full structural and functional characterization / Doutorado / Bioquimica / Doutora em Biologia Funcional e Molecular

Orientador: Helena Maria André Bolini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T16:30:20Z (GMT). No. of bitstreams: 1 Cadena_RafaelSilva_D.pdf: 521247 bytes, checksum: 6926f96ecbac6ea3d9d78e69251418c3 (MD5) Previous issue date: 2012 / Resumo: O consumo de frutas e vegetais tem sido associado com a baixa incidência de doenças degenerativas, pois efeitos protetores estão associados com componentes antioxidantes contidos nestes alimentos. Atrelado a isto, o mercado de sucos e néctares de frutas está sob um aumento significativo, o que tem atraído a atenção de agricultores, distribuidores e da indústria deste produto para saciar esta demanda. Entretanto, estes produtos estão associados à alta ingestão de sacarose proveniente da adição em sua elaboração, o que pode acarretar o desenvolvimento de doenças relacionadas ao consumo excessivo de calorias. A partir disto, este estudo objetivou analisar a aplicação de novos edulcorantes de em néctar de manga pelo estudo de perfil sensorial e físico-químico ao longo do tempo de estocagem. Neste estudo foram utilizadas amostras de néctar de manga elaboradas com polpa de manga congelada e água (1:1) adoçadas com sacarose, sucralose, estévia com 97% de rebaudiosídeo, neotame, blend acessulfame-K/sucralose/neotame (100:50:1) e blend taumatina/sucralose (1:1), totalizando 6 amostras. O perfil físico-químico foi determinado por meio da análise de pH, acidez titulável, cor (L*, a*, b*) e sólidos solúveis (ºBrix). A análise sensorial foi composta por testes descritivos, Análise Descritiva Quantitativa (ADQ) e Análise Tempo-intensidade, além de testes afetivos com escala hedônica e escala do ideal. Além destes, para o estabelecimento de equivalência de doçura foi utilizado o teste sensorial Estimação de Magnitude. A análise estatística foi composta por Análise de Variância (ANOVA), Teste de média de Tukey com nível de significância de 5% para as análises físico-químicas, ADQ, análise tempointensidade e teste afetivo com escala hedônica. O teste com escala do ideal foi analisado por distribuição em histogramas e os dados de estimação de magnitude utilizaram regressão linear em sua análise. Os provadores estabeleceram o valor de 6,8% de sacarose como a quantidade ideal para a intensidade de gosto doce. O neotame foi o edulcorante que apresentou maior poder adoçante, sendo 6026 mais doce que a sacarose, seguindo pela sucralose (627), blend taumatina/sucralose (549), blend acessulfame-K/sucralose/neotame (259) e estévia (134). Os testes físico-químicos e, em especial, a ADQ identificaram a sucralose como o edulcorante que melhor substituiu a sacarose, pois foi o aditivo que apresentou o menor número de diferenças em relação à amostra controle. A análise tempo-intensidade e os testes afetivos também identificaram a amostra sucralose com o melhor perfil relacionado ao estímulo gosto doce, com grande semelhança ao ocorrido com a sacarose e sendo esta a amostra com maior grau de aceitação no tempo zero e a única que se manteve desta forma após os 120 dias de estocagem. Em conclusão, a estévia apresentou melhorias em seu perfil quando comparadas a outros estudos, mas ainda abaixo do esperado para um substituinte de sacarose. A taumatina, uma proteína de origem vegetal apresentou bom perfil e é um aditivo que necessita de maiores estudos para melhoria de sua tecnologia. E, por fim, a sucralose, como já afirmado em outros trabalhos, é o edulcorante que melhor substitui a sacarose em sucos tropicais, sem sofrer alterações significativas após processamento térmico e ao longo do tempo de estocagem / Abstract: The consumption of fruits and vegetable has been associated with low incidence of degenerative diseases, because protective effects has been linked with antioxidant compounds contained in these foods. Attached to this, the consumer market of fruit juices and nectars under a significant increase, which has attracted the attention of producers of these products to satisfy this demand. However, these products are associated with the high ingestion of sucrose from de addition in the elaboration, which may result in the development of diseases associated with the high consumption of calorie. From that, this study aimed to analyze the application of high intensity sweeteners in mango nectar by the sensory and physical-chemical profile during the storage time. Mango nectar samples were prepared with unsweetened mango frozen pulp and water (1:1). The samples were sweetened with different high intensity sweeteners and sucrose. The sweeteners were: Neotame; Sucralose; Stevia with 97% of Rebaudioside; 1:1 Thaumatin/Sucralose blend; 100:50:1 Acesulfame-K/Sucralose/Neotame blend. The physical-chemical profile was determined by: pH, titratable acidity, color (L*, a*, b*), solids soluble (ºBrix). The sensory analysis was composed by descriptive analysis, Quantitative Descriptive Analysis (QDA) and Time-intensity Analysis (T-I), also consumer test with hedonic scale e just-about-right (JAR) scale. Magnitude estimation method was applied to establish the relative sweetness with sucrose. Statistical analysis was composed by analysis of variance and Tukey means test with 5% of significant level to physical-chemical, QDA, T-I analysis, and consumer test. The JAR scale test was analyzed by histograms distribution and the results of magnitude estimation method through linear regression. The ideal sweetness analysis revealed that 6.8% was the ideal concentration of sucrose in the mango nectar. The relative sweetness analysis showed that Neotame presented the highest sweetening power, being 6026 times sweeter than sucrose, followed by Sucralose (627), Thaumatin/Sucralose blend 1:1 (549), Acesulfame-K/Sucralose/Neotame blend 100:50:1 (259) and Stevia (134). The physical-chemical analysis and, in special, the QDA identified sucralose as the best sweetener which best sucrose substitutes, since this additive presented less differences in relation to control sample. T-I analysis and consumers test also identified the sample sucralose with best profile in relation to the sweet stimulus, with great similarity to control sample and being the sample with higher acceptability in initial time and the only one that has remained this way after 120 days of storage. In conclusion, the stevia presented improvements in its sensory profile when compared with others studies,although still lower than expected to a sucrose substitute. The thaumatin, a vegetal protein, presented good sensory profile and is a additive that requires more studies to improve its knowledges. The sucralose, as founded in other studies, is the sweetener that best replace sucrose in tropical fruit juice, with no significant change after heat treatment and during the storage time / Doutorado / Consumo e Qualidade de Alimentos / Doutor em Alimentos e Nutrição

In this diploma thesis I dealt with developing of separation technique LC-MS for determination of thaumatin. I dealt with isolation and determination of thaumatin in model food sample.

Almost all conifer ovules produce a liquid secretion as part of reproduction. This secretion, termed an ovular secretion, is produced during ovule receptivity and is involved in pollen capture and transport. Historically, examinations of the ovular secretion have focused on how they are part of pollination mechanisms. As a result, the chemical composition of the ovular secretion has not been examined systematically. Investigations into the constituents of the ovular secretion were limited to analyses for simple water soluble compounds such as sugars, minerals, amino acids and organic acids. More recently, the protein component of the secretion has been investigated using mass spectrometry-based proteomics. Proteins involved in processes such as carbohydrate modification, proteolysis, and defence have been identified in conifer ovular secretions. This biochemical complexity suggests a broader view of the function of the ovular secretion is warranted. However, protein identifications only provide putative information on function. Functional characterization of these proteins is needed in order to fully understand how they contribute to ovular secretion function. The research outlined in this dissertation describes the first functional characterizations of proteins found in conifer ovular secretions. Three proteins - invertase, chitinase, and thaumatin-like protein - were characterized in the ovular secretions of Douglas-fir (Pseudotsuga menziesii) and hybrid yew (Taxus × media). The Douglas-fir ovular secretion is capable of converting sucrose to glucose and fructose, confirming that invertases present in the secretion are functional. The invertase activity was maximal at pH 4.0. Activity was 77% of maximal at pH 4.5, the physiological pH. This indicates that post-secretory hydrolysis of sucrose occurs in situ in the Douglas-fir ovular secretion. Invertases in the ovular secretion are likely involved in controlling the movement of carbohydrates to developing pollen and could facilitate pollen selection. Chitinases present in the Douglas-fir ovular secretion are functional at physiological conditions. All three modes of chitinolytic activity, i.e. endochitinase, chitobiosidase and β-N-acetylglucosaminidase, were detected at physiological pH. β-N-acetylglucosaminidase activity was 80 % of maximal at physiological pH. Chitinases are pathogenesis-related proteins capable of hydrolysing chitin in fungal cell walls. These results suggest the ovular secretion is capable of defending the ovule against infection by phytopathogens. Thaumatin-like protein was immunolocalized to the cell wall and amyloplasts in Douglas-fir and yew nucellar tissue in a pattern consistent with a defensive role. It was also localized to the cell wall of fungal spores and germinating hyphae that were present in the micropyle of a yew ovule. These results provide additional evidence for an antifungal role for the ovular secretion. Functioning enzymes involved in pollen-ovule interactions and ovule defence are present in the conifer ovular secretion. The ovular secretion has functions beyond pollen capture. A revised functional model for the conifer ovular secretion is proposed. / Graduate / 2021-08-17