Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, yeasts release extracellular enzymes that significantly impact wine properties. While the extracellular proteins of Saccharomyces cerevisiae have been characterised, those of non-Saccharomyces yeasts remain largely unknown. Most of these enzymes break down sugar polymers or catalyse the liberation of glycosidically-bound molecules. Another category of enzymes of oenological interest is represented by acid proteases that are able to prevent or reduce protein haze, as reported in literature, while simultaneously increasing the assimilable nitrogen content of wine. The liberation of amino acids from peptides and proteins that serve as aroma precursors may also have an indirect effect on wine aroma. In a recent study performed at the Institute for Wine Biotechnology (IWBT), the sequences of two aspartic proteases were retrieved from non-Saccharomyces yeast species isolated from South African wines. The genes, MpAPr1 and CaAPr1, were isolated from two non-Saccharomyces species, Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384, respectively. However, no further characterization was undertaken. This study aimed to clone these two genes into a recombinant bacterial host for expression and purify the corresponding enzymes as a first step toward characterizing their kinetic properties. Considering that some non-Saccharomyces species have been shown to produce more than one acid protease, an additional aim was to identify novel acid proteases within M. pulcherrima IWBT Y1123. Cloning of the genes and transformation of the expression vectors into E. coli were achieved. Optimal conditions for induced expression were established following extensive optimization. Furthermore, while native extraction of the recombinant proteins was unsuccessful, denaturing conditions allowed their recovery, suggesting that the recombinant proteins are encapsulated into inclusion bodies. Recombinant MpAPr1 was purified by using a nickel based column system and mass fingerprinting of the purified enzyme (MpAPr1) confirmed its identity. Purification was followed by refolding experiments, but yielded poor recovery of active enzymes. Unfortunately, recombinant expression of CaAPr1 could not be observed for reasons yet to be elucidated that may include the large sequence dissimilarities between CaAPr1 and MpAPr1. Finally, Southern blot analysis on the genomes of M. pulcherrima IWBT Y1123 and C. apicola IWBT Y1384 revealed that both possess at least one additional protease other than those previously described. Further analysis of the extracellular proteome of M. pulcherrima IWBT Y1123 also confirmed the presence of at least one enzyme able to hydrolyze BSA at a low pH. Unfortunately, mass fingerprinting performed on the entire extracellular proteome and on small groups of proteins thereof did not allow the identification of these enzymes. / AFRIKAANSE OPSOMMING: Gedurende fermentasie van druiwe sap skei gis ekstrasellulêre ensieme af wat ‘n aanmerklike impak op wyn eienskappe het. Terwyl die ekstrasellulêre proteïene vanaf Saccharomyces cerevisiae al gekarakteriseer is, bly die van nie-Saccharomyces spesies grootliks onbekend. Meeste van hierdie ensieme breek suiker polimere af of kataliseer die vrystelling van glikosiediese verbonde molekules. ‘n Ander klas van ensieme wat van belang is vir oenologie word voorgestel deur proteases wat in staat is daartoe om proteïenewaas te verminder, soos voorheen geraporteer is in literatuur, terwyl dit terselfde tyd die assimileerbare stikstof inhoud kan vermeerder. Die vrystelling van aminosure vanaf peptiede en/of proteïene wat as aroma voorlopers dien mag ook ‘n indirekte effek op die wyn se aroma profiel hê. In ‘n onlangse studie wat uitgevoer is by die Instituut vir Wynbiotegnologie (IWBT) was die volgordes van twee aspartiese proteases bepaal vanaf twee nie-Saccharomyces gis spesies wat geisoleer was uit Suid-Afrikaanse wyne. Die gene MpAPr1 en CaAPr1, was afsonderlik geisoleer vanuit twee nie- Saccharomyces giste, Metschnikowia pulcherrima IWBT Y1123 en Candida apicola IWBT Y1384. Egter was daar geen verder karakterisering van hierdie ensieme nie. Die doel van hierdie studie is om die bogenoemde gene in ‘n rekombinante bakteriese gasheer te kloneer vir uitdrukking en suiwering as ‘n eerste stap tot karakterisering van hul kinetiese eienskappe. Om in ag te neem dat sommige nie-Saccharomyces spesies meer as een protease produseer was ‘n aditionele mikpunt om vir nuwe suur proteases te soek binne M. pulcherrima IWBT Y1123. Klonering van hierdie gene en transformasie van die uitdrukkings vektore in E. coli was suksesvol. Optimale kondisies vir die induksie van ekspressie was bevestig na omvattende optimalisering. Verder, terwyl inheemse ekstraksie van die rekombinante proteïene onsuksesvol was, het denatureerende kondisies toegelaat vir suksesvolle ekstraksie, wat voorgestel het dat die rekombinante proteïene geinkapsileer word in inklusie liggame. Rekombinante MpAPr1 was gesuiwer deur gebruik te maak van ‘n niekel gebaseerde kolom sisteem en massa petied fingerafdrukke van die gesuiwerde ensiem (MpAPr1) het die identiteit bevestig. Suiwering was gevolg deur hervouing eksperimente, maar het swak opbrengste gelewer van die aktiewe ensiem. Ongelukkig kon die rekombinante ekspressie van CaAPr1 nie gevisualiseer word nie vir redes wat nog bevestig moet word, maar wat mag behels dat daar groot volgorde veskille tussen MpAPr1 en CaAPr1 kan wees. Uiteindelik was Southern blot hibridiseering analises uitgevoer op die genome van albei M. pulcherrima IWBT Y1123 en C. apicola IWBT Y1384 wat voorgestel het dat albei ten minste een addisionele protease, anders as die wat voorheen geidentifiseer was, bevat. Verder analiese van die ekstrasellulêre proteoom van M. pulcherrima IWBT Y1123 het ook die teenwoordigheid van ten minste een ensiem bevestig wat die vermoë het om BSA te hidroliseer by ‘n lae pH. Ongelukkig het massa peptied vingerafdrukbepaling wat uitgevoer was op die hele ekstrasellulêre proteoom en op klein groepe protein nie identifikasie van hierdie ensieme bevestig nie.

Made available in DSpace on 2015-04-17T14:49:37Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1723546 bytes, checksum: d6ebd3071471297d43af6b6d405af8d9 (MD5) Previous issue date: 2014-02-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Yeasts of the genus Saccharomyces spp. are used as biotechnological tool in brewery and after five fermentative production processes; they are discarded by the beer industry. The cell wall of yeasts is mainly composed of β - glucan, which is a glucose homopolymer capable of changing the immune response in the host, and by manoproteins (MP), which have recognized emulsifying properties. The aim of the present study was to obtain these two cell wall fractions of discarded yeast employing an additional step to obtain MP in a process applicable to the extraction of β - glucan. The fractions obtained were characterized and MP was evaluated for potential for application as an emulsifier in mayonnaise formulations. From the cell wall of yeasts on dry basis, the MP and β glucan yield was 4.16% and 10 %, respectively. The residual moisture of β - glucan was 4.9 ± 0.1 % and MP was 3.9 ± 0.9 % and no changes in this parameter were observed during 28 days of storage in bi-oriented polypropylene packs (BOPP) at 10°C, 25°C or 40° C. The SDS-PAGE profile of MP showed proteins with molecular weight of 58 kDa and 64 kDa, and the highest content of constituent amino acids was observed for valine (8.9%), aspartic acid (8.2%) and leucine (6.9%). Each 100 g of mayonnaise prepared with MP at concentrations of 0.6%, 0.8 % and 1.0 % contained, on average, 25 ± 0.27 g moisture, 1.6 ± 0.10 g ash, 2.4 ± 0.04 g protein, 56 g ± 0.41 lipid and 14 ± 0.12 g carbohydrates, and no changes in attributes aroma, color, flavor and texture were observed in the study period (28 days). The results showed applicability of the method proposed for obtaining β -glucan and MP and suggest that MP has potential for application as bioemulsifier in foods, particularly in mayonnaise formulations. / Leveduras do gênero Saccharomyces spp. são empregadas como ferramenta biotecnológica, na produção de cerveja e após em média cinco processos fermentativos de produção, são descartadas pela indústria cervejeira. A parede celular de leveduras é formada principalmente pelas β-glucana, que são homopolímeros de glicose capazes de alterar a resposta imune no hospedeiro, e pelas manoproteínas (MP) que possuem reconhecidas propriedades emulsificantes. No presente estudo foi proposto obter estas duas frações da parede celular de levedura descartada em cervejeira empregando uma etapa adicional para obtenção da MP em um processo aplicável a extração da β-glucana. As frações obtidas foram caracterizadas e a MP avaliada quanto ao potencial para aplicação como emulsificante em formulações de maionese. A partir da parede celular de levedura em base seca, o rendimento da MP e da β-glucana foi de 4,16% e 10%, respectivamente. A umidade residual da β-glucana foi de 4,9 ± 0,1% e da MP foi de 3,9 ± 0,9% e não foram observadas alterações neste parâmetro durante 28 dias de armazenamento em embalagens de polipropileno bi-orientado (BOPP) a 10 °C, 25 °C ou 40 °C. O perfil de SDS-PAGE da MP evidenciou proteínas de peso molecular de 58 kDa e 64 kD e o maior teor de aminoácidos constituintes foi observado para a valina (8,9%), ácido aspártico (8,2%) e leucina (6,9%). Cada 100 g de maionese preparada com MP nas concentrações de 0,6%; 0,8% e 1,0% continham, em média, 25 ± 0,27g de umidade, 1,6 ± 0,10 g de cinzas, 2,4 ± 0,04 g de proteína, 56 ± 0,41 g de lipídeos e 14 ± 0,12 g de carboidratos, e não foram observadas alterações nos atributos aroma, cor, sabor, textura no período analisado (28 dias). Os resultados obtidos evidenciam aplicabilidade do método proposto para obtenção de MP e β-glucana e sugerem que a MP possui potencial para aplicação como bioemulsificante em alimentos, em especial em formulações de maionese.

Submitted by WENDER MESSIATTO DA SILVA (wendermessiatto@yahoo.com.br) on 2018-06-08T16:36:25Z No. of bitstreams: 1 silva_wm_me_Ilha.pdf: 2741794 bytes, checksum: 942a41a5e8d98fe17b8da88fec150d33 (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2018-06-11T14:05:34Z (GMT) No. of bitstreams: 1 silva_wm_me_ilha.pdf: 2741794 bytes, checksum: 942a41a5e8d98fe17b8da88fec150d33 (MD5) / Made available in DSpace on 2018-06-11T14:05:34Z (GMT). No. of bitstreams: 1 silva_wm_me_ilha.pdf: 2741794 bytes, checksum: 942a41a5e8d98fe17b8da88fec150d33 (MD5) Previous issue date: 2018-04-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os impactos ambientais decorrentes de vazamentos em postos de combustíveis têm sido preocupantes para órgãos ambientais ao redor do mundo, sobretudo por deteriorarem a qualidade das águas subterrâneas utilizadas como fonte de abastecimento, sendo os compostos benzeno, tolueno e xilenos (BTX) os de maior preocupação à saúde pública. A biorremediação é uma técnica viável do ponto de vista econômico e ecológico quando se utiliza consórcio microbiano de bactérias e fungos aliado a solução de nutrientes. Este trabalho verificou os efeitos da biorremediação sobre microambientes formados em erlenmenyers contendo solo arenoso fino de textura arenosa fina, bastante comum no noroeste paulista, contaminado por soluções sintéticas de BTX por intermédio de consórcio de microrganismos (bactérias ácido láticas, leveduras, bactérias fototróficas e actinomicetes). O desempenho de um consórcio de microrganismos foi verificado por meio de alterações de pH, teor de umidade, teor de carbono e pela de remoção de BTX do microambiente constituído de solo arenoso fino contaminado por solução sintética de BTX mediante a bioestimulação comparando-se os efeitos do ácido esteárico como surfactante. O uso de ácido esteárico como surfactante prejudicou o processo de biorremediação nos microambientes, com isso a remoção de BTX foi menor do que nos microambientes que não possuíam ácido esteárico. De acordo com resultados deste trabalho, um valor adequado para a aplicação da solução de microrganismos em campo é o de 0,02 mL de solução de microrganismos por cm³ de solo arenoso fino, pois ao se utilizar 6,25 mL de solução de microrganismos para 300 cm³ de solo arenoso fino no período de 7, 30 e 58 dias de experimento foi constatado resultados econômicos e relativamente eficientes no curto prazo. / The environmental impacts resulting to leaks at fuel stations has been worrying for environmental agencies around the world mainly for deteriorating the quality of the groundwater used as a water supply whereby the BTX compounds (benzene, toluene and xylene) the most worrisome public health. Bioremediation is a viable technique because the use of bacterial, fungal and yeast microbial consortia is economic and ecological associated with nutrient solutions. The effects of bioremediation verified through a consortium of microrganisms (lactic acid bacteria, yeasts, phototrophic bacteria and actinomycetes) on microenvironment in enlermeyers containing fine sandy texture lateritic soil, very common in São Paulo northwest region, contaminated by synthetic solutions of BTX. The performance of a consortium of microorganisms verified by pH changes, moisture content and organic carbon content of the microenvironments and the rate of BTX removal in fine sandy soil contaminated with BTX synthetics solutions by bioestimulation, comparing the effects of stearic acid as surfactant. The use of stearic acid as a surfactant impaired the bioremediation process in the microenvironments, thus the removal of BTX was lower than in microenvironments that did not possess stearic acid. According to the results of this work, a value of microorganisms per cm³ of microorganisms solution to the soil is 0.02 mL of microorganisms solution 300 cm³ of fine sandy soil in the period of 7, 30 and 58 days of experiment was confirmed and is useful in the short term.

Orientador: William Deodato Isique / Resumo: Os impactos ambientais decorrentes de vazamentos em postos de combustíveis têm sido preocupantes para órgãos ambientais ao redor do mundo, sobretudo por deteriorarem a qualidade das águas subterrâneas utilizadas como fonte de abastecimento, sendo os compostos benzeno, tolueno e xilenos (BTX) os de maior preocupação à saúde pública. A biorremediação é uma técnica viável do ponto de vista econômico e ecológico quando se utiliza consórcio microbiano de bactérias e fungos aliado a solução de nutrientes. Este trabalho verificou os efeitos da biorremediação sobre microambientes formados em erlenmenyers contendo solo arenoso fino de textura arenosa fina, bastante comum no noroeste paulista, contaminado por soluções sintéticas de BTX por intermédio de consórcio de microrganismos (bactérias ácido láticas, leveduras, bactérias fototróficas e actinomicetes). O desempenho de um consórcio de microrganismos foi verificado por meio de alterações de pH, teor de umidade, teor de carbono e pela de remoção de BTX do microambiente constituído de solo arenoso fino contaminado por solução sintética de BTX mediante a bioestimulação comparando-se os efeitos do ácido esteárico como surfactante. O uso de ácido esteárico como surfactante prejudicou o processo de biorremediação nos microambientes, com isso a remoção de BTX foi menor do que nos microambientes que não possuíam ácido esteárico. De acordo com resultados deste trabalho, um valor adequado para a aplicação da solução de microrganismos em campo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The environmental impacts resulting to leaks at fuel stations has been worrying for environmental agencies around the world mainly for deteriorating the quality of the groundwater used as a water supply whereby the BTX compounds (benzene, toluene and xylene) the most worrisome public health. Bioremediation is a viable technique because the use of bacterial, fungal and yeast microbial consortia is economic and ecological associated with nutrient solutions. The effects of bioremediation verified through a consortium of microrganisms (lactic acid bacteria, yeasts, phototrophic bacteria and actinomycetes) on microenvironment in enlermeyers containing fine sandy texture lateritic soil, very common in São Paulo northwest region, contaminated by synthetic solutions of BTX. The performance of a consortium of microorganisms verified by pH changes, moisture content and organic carbon content of the microenvironments and the rate of BTX removal in fine sandy soil contaminated with BTX synthetics solutions by bioestimulation, comparing the effects of stearic acid as surfactant. The use of stearic acid as a surfactant impaired the bioremediation process in the microenvironments, thus the removal of BTX was lower than in microenvironments that did not possess stearic acid. According to the results of this work, a value of microorganisms per cm³ of microorganisms solution to the soil is 0.02 mL of microorganisms solution 300 cm³ of fine sandy soil in the period of 7, 30 and 58 days of exper... (Complete abstract click electronic access below) / Mestre

Resumo: Os ecossistemas costeiros são considerados de grande importância sócio-econômica, política e cultural, pois possuem alta produtividade que suporta uma ampla gama de atividades econômicas e de lazer. Possuem elevado valor paisagístico, atraindo inúmeros habitantes e, assim, contribuem ao desenvolvimento das cidades litorâneas. Além disso, oferecem suporte às comunidades biológicas com manutenção de muitas espécies residentes ou que utilizam a zona costeira como berçário para reprodução, beneficiando a biodiversidade como um todo. Sendo uma região que sofre grande pressão antrópica, a Zona Costeira está sujeita a inúmeros impactos causados por atividades humanas, tais como as portuárias, industriais e de exploração de petróleo. Essas geram impactos por lançarem diversos poluentes, entre eles os hidrocarbonetos e metais que podem ter efeito deletério na biota local. No ambiente marinho, o compartimento mais afetado em relação a essa contaminação é o sedimento em função dos derivados de petróleo que atingem as águas costeiras possuírem maior densidade, nele se depositando. Atualmente existe uma grande preocupação com os impactos gerados por atividades antrópicas em ecossistemas litorâneos e muitos estudos têm sido feitos em busca de alternativas para avaliar e remediar tais impactos. Uma delas consiste no uso de micro-organismos tanto como indicadores da contaminação por poluentes quanto para a diminuição da contaminação, processo popularmente conhecido como biorremediação. Deste modo, o presente estudo teve como objetivos: 1) avaliar o efeito da presença de hidrocarbonetos na abundância de bactérias heterotróficas, cianobactérias e leveduras em sedimentos e águas estuarinas de duas regiões distintas, dos Estuários de Santos e do Rio Itanhaém, tanto no que diz respeito às características ambientais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coastal ecosystems have great socio-economic, political and cultural importance, as well as high productivity which support a wide range of economic activities and leisure. They have high landscape value thus attracting numerous people contributing to the development of coastal cities. Moreover, its high productivity supports biological communities and maintenance of many species living or using the coastal zone as a nursery for breeding, benefiting whole biodiversity. Being a region that suffers high anthropogenic pressure, the Coastal Zone is subject to numerous impacts caused by human activities such as port activities, industrial and oil exploration. These generate impacts on the environment by launching several pollutants, including hydrocarbons and metals that can have deleterious effects on local biota. The most affected marine environment is the sediment due to oil products that reach coastal waters have a higher density and end up being deposited in sediments. Nowadays there is great concern about the impacts generated by human activities on coastal ecosystems and many studies have been done in search of alternatives to evaluate and remedy such impacts. One alternative is the use of micro-organisms both as indicators of contamination by pollutants and for reduction of contamination, a process popularly known as bioremediation. Thus, this study aimed to evaluate the effect of the presence of hydrocarbons in the abundance of heterotrophic bacteria, cyanobacteria and yeast on sediment and estuarine waters of two distinct regions: The Estuaries of Santos and Itanhaém River are located in the metropolitan area of Santos, Sao Paulo State; and to isolate microorganisms with emphasis on yeasts, for further evaluation of its potential both as bioindicators of hydrocarbon contamination and bioremediation of estuarine sediments in order to contribute... (Complete abstract click electronic access below) / Orientador: Fernando Carlos Pagnocca / Coorientador: Ana Júlia Fernandes Cardoso de Oliveira / Banca: Hilda de Souza Lima Mesquita / Banca: Marcelo Antonio Amaro Pinheiro / Mestre

A suinocultura brasileira encontra-se atualmente em um nível técnico de destaque no contexto mundial. Entretanto, diversas estratégias são utilizadas para restringir a compra dos produtos brasileiros por países estrangeiros, em especial barreiras sanitárias. Os objetivos desse trabalho foram determinar a microbiota fúngica da pele de suínos sem lesões aparentes e estabelecer os possíveis agentes micóticos de importância nesta espécie. Foram obtidas 261 amostras de animais com 20 a 120 dias de idade, provenientes de 11 granjas do Rio Grande do Sul. As amostras foram obtidas apenas dos animais com ausência de qualquer patologia. Essas foram semeadas em Agar Sabouraud com cloranfenicol. Obteve-se o crescimento de 509 culturas, sendo 305 (59,92%) fungos filamentosos e 204 (40,08%) leveduras. A distribuição do número de diferentes colônias isoladas por amostra demonstra uma predominância de monocultura no grupo 1 (animais de 20 a 60 dias de idade) em relação ao grupo 2 (animais acima de 60 dias de idade). Dentre os fungos filamentosos septados, ocorreu predominância dos hialohifomicetos (43,03%) em relação aos feohifomicetos (10,41%), enquanto que observou-se apenas 6,48% de filamentosos asseptados. O Aspergillus (22,90%) foi o gênero de hialohifomiceto mais isolado, com predominância do A. flavus (5,70%). Dentre os feohifomicetos, o gênero Cladosporium foi o mais prevalente (9,04%). No que concerne os zygomicetos, o gênero mais isolado foi Mucor (4,13%). Em relação às leveduras, o gênero Candida foi o mais isolado (14,73%), sendo a distribuição de freqüência das espécies: C. albicans (13,56%), C. parapsilopsis (0,59%), C. famata (0,20%), C. tropicalis (0,20%) e outras espécies de Candida (0,20%). A reduzida quantidade de diferentes fungos isolados por amostra nos animais do grupo 1, em relação ao grupo 2, pode ter sido influenciada pela menor quantidade de grãos utilizada na fabricação de ração nos animais mais novos, maior freqüência de limpeza e desinfecção das instalações, bem como a não mistura de animais provenientes de diferentes origens. A alta prevalência do gênero Candida observada em suínos, um gênero normalmente comensal do trato gastrintestinal, se torna importante quando esses animais são acometidos por doenças imunossupressoras. Estudos clínicos longitudinais e/ou infecções experimentais talvez possam definir a importância destes agentes em suínos. / Brazilian pig production has reached a technical and techological level that stands out in international pig production. However, several strategies have been used to limit access of Brazilian pig products to international market, including health barriers. The objective of our work was to determine the fungal flora of the healthy pig skin and to determine possible micotic agents important for this animal species. 261 samples from 20-120 day old pigles were obtained from 11 pig farms from the State of Rio Grande do Sul, Brazil. All sampled animals were negative for any skin lesions. The samples were cultured in Sabouraud agar and cloranfenicol. Cultures were obtained from 509 materials, with 305 (59,95%) filamentous fungi and 204 (40,08%) yeasts. The distribution of different fungi isolated each simple shows a high prevalence on group 1 (20-60 days old) in relation to group 2 (more than 60 days old). Among septated filamentous fungi, there was a great preponderance of hialohifomycetes (43,03%) in relation to feohifomycetes (10,41%), while only 6,48% were aseptated filamentous. Aspergillus (22,90%) was the most isolated genus of hialohifomycetes and A. flavus (5,70%) was the more prevalent specie isolated. As to zygomycetes, the most isolated genus was Mucor (4,13%). Regarding yeasts, the genus Candida was the most prevalent (14,73%) and the species distribution was: C. albicans (13,56%), C. parapsilopsis (0,59%), C. famata (0,20%), C. tropicalis (0,20%) and other Candida species (0,20%). The little quantity isolated per sample could have been influenced by quantity of grains used in pig feed in these different stages of production, types of facilities, stress and environmental changes could be important to imbalance the flora, as well as favoring diffusion of mycosis in the herds. The limited number of different fungi isolated from animal flora on group 1 may have been influenced by the smaller use of grains in the feed mill in this stage, a better cleaning and disinfection of premises, as well as avoiding mixing pigs of different source. Clinical longitudinal studies and/ or experimental infections perhaps may help to define the importance of these fungi to that species.

L'utilisation des levures en industrie est indissociable de sa transformation à l'état déshydraté. Le procédé de séchage provoque un certain nombre de dysfonctionnements dans les cellules de levure qui affectent leur fonctionnalité et leur viabilité. Afin de protéger les levures au cours de la déshydratation, des additifs alimentaires sont souvent utilisés comme les émulsifiants et les antioxydants. Cependant, les levures sont capables de produire naturellement des substances protectrices, telles que le glutathion (GSH) et le tréhalose (TRE). Dans ce contexte, trois souches non-Saccharomyces (NS) appartenant aux genres et espèces Torulaspora delbrueckii, Metschnikowia pulcherrima et Lachancea thermotolerans ont été étudiées. En effet, malgré leur intérêt pour de multiples applications agro-alimentaires, leurs mécanismes de résistance à la déshydratation associés à la synthèse de GSH et de TRE sont à l’heure actuelle méconnus. Cette investigation peut permettre à terme la formulation de nouvelles souches de levure NS sous forme sèche, sans aucuns additifs. Dans un premier chapitre, l’impact de la déshydratation de la levure « modèle » Saccharomyces cerevisiae en lit fluidisé pré-pilote a été corrélé à la synthèse du GSH et du TRE. Ainsi, il a été possible d’élucider les effets de la modulation de la composition du milieu de culture afin d'optimiser la conservation des cellules avant, pendant et après la déshydratation. Dans un deuxième chapitre, les conditions précédemment définies ont été appliquées aux levures NS afin de comprendre les effets de la déshydratation sur leur fonctionnalité. Cette étude a démontré que le GSH joue un rôle important dans la protection des levures NS, en fonction des conditions de culture et de déshydratation utilisées. Dans un troisième chapitre, certains phénomènes de résistance à l'oxydation des trois souches NS ont été étudiés. Il a été clairement démontré que la sensibilité des cellules à l’attaque oxydante dépend des conditions de culture-déshydratation et est inhérente à la souche étudiée. Enfin, dans un quatrième chapitre, une étude approfondie au niveau biochimique de la souche de levure la plus sensible, L. thermotolerans, a été réalisée par la micro-spectroscopie infrarouge avec rayonnement synchrotron. Les cellules cultivées en GSM, milieu favorisant la synthèse du glutathion, en plus de présenter une meilleure viabilité, montrent une plus grande intensité dans les bandes spectrales des lipides CH2 et CH3, associées à la fluidité de la membrane plasmique. Par ailleurs, les cellules cultivées en milieu TSM, milieu favorisant la synthèse du tréhalose, présentent une dénaturation plus élevée des protéines marquée par une intensité plus élevée des pics de feuillet β, et une intensité plus élevée des groupements C=O (oxydes lipidiques) corrélée à la peroxydation lipidique. Ces deux phénomènes expliquent la diminution de la viabilité obtenue sur cette souche lors de sa déshydratation. Les connaissances fondamentales acquises dans le cadre de cette étude seront utiles pour obtenir de nouvelles souches de levures déshydratées sans additifs et à hautes performances. Elles contribuent également à l’amélioration des méthodes de formulation et de déshydratation actuellement utilisées en industrie. / The use of yeasts in industry is inseparable from their ability to be produced and dehydrated. This dehydration process causes various dysfunctions in yeast cells that affect their functionality and viability. In order to protect yeasts from dehydration, food additives are often used as emulsifiers and antioxidants. However, yeasts are able to produce naturally protective substances, such as glutathione (GSH) and trehalose (TRE). In this context, three non-Saccharomyces (NS) strains, belonging to the different genera and species Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans, were studied in this thesis. Despite the great interest aroused by their multiple agro-food applications, their dehydration resistance mechanisms associated with the synthesis of GSH and TRE, are currently unknown. This study is ultimately aimed at the formulation of new NS yeast dried strains without any food additives. In a first chapter, the impact of the “reference yeast” Saccharomyces cerevisiae dehydration in a pre-pilot fluidized bed has been correlated with the synthesis of GSH and TRE. It was possible to modulate the culture medium composition in order to optimize cell preservation before, during and after dehydration. In a second chapter, the previously defined conditions were applied to NS yeasts strains in order to understand the effects of dehydration on their microbial functionality. This study demonstrated that GSH plays an important role in NS yeasts protection, depending on the culture and dehydration conditions. In a third chapter, some oxidation resistance phenomena of the three NS strains were studied. It was clearly demonstrated that the susceptibility of cells to oxidative attack was dependent on culture-dehydration conditions and was yeast strain-dependent. Finally, in a fourth chapter, an in-depth biochemical study of the most dehydration-sensitive yeast strain, L. thermotolerans, was performed by synchrotron FTIR micro-spectroscopy. Cells grown in GSM (medium favoring the production of GSH), besides showing a better viability, showed a greater intensity in the spectral bands of lipids CH2 and CH3, associated with the plasma membrane fluidity. In addition, TSM grown cells (TSM is a medium favoring the production of TRE) exhibited a higher protein denaturation, suggested by the intensity of β-sheet peaks, and C=O (lipid oxides) bands correlated with lipid peroxidation. These data can explain the decreased of viability of this strain during production-dehydration process. The fundamental knowledge acquired in this study will be useful to obtain new dehydrated yeast strains without additives and with high performance. It will be useful also to improve the formulation and dehydration methods currently used in industry.

Orientador: Márcia Justino Rossini Mutton / Banca: Flávia Cecílio Ribeiro Bregagnoli / Banca: Francisco Vicente Gaiotto Cleto / Resumo: As indústrias sucroenergéticas têm como preocupação o controle de contaminantes da fermentação, responsáveis por afetar a viabilidade da levedura, provocando diversos transtornos no processo, comprometendo a eficiência fermentativa e o rendimento industrial. Dentre as alternativas para o controle das contaminações, destacam-se o uso de antimicrobianos sintéticos. Sua utilização continua pode favorecer o desenvolvimento de cepas resistentes, contribuindo para o incremento do custo de produção, além da possibilidade de incorporação de resíduos no produto final. Objetivou-se avaliar o efeito do biocida convencional (monensina sódica) e biocidas naturais preparados à base de própolis (Extrato Hidroalcoólico de Própolis - EHP e Extrato Oleoso de Própolis- EOP) sobre a fisiologia das leveduras, o controle dos contaminantes do processo fermentativo e composição do destilado. O delineamento experimental utilizado foi o Inteiramente Casualizado com parcelas subdivididas, com 4 repetições. Os Tratamentos Principais foram: Testemunha, EOP, EHP e monensina sódica (Kamoran WP). Os Tratamentos Secundários constituíram-se nos 10 ciclos fermentativos. Avaliaram-se as características químico-tecnológicas do caldo, mosto e vinho, parâmetros microbiológicos das leveduras e composição do destilado obtido. Os resultados obtidos demonstraram que os biocidas avaliados apresentaram efeito similar, sendo efetivos no controle dos contaminantes da fermentação, não afetando negativamente suas características fisiológicas. Não afetaram a composição a composição do destilado final obtido / Abstract: The control of fermentation contaminants is one of the sugar mills concerns. The fermentation contaminants are responsible to affect the yeast viability, generating several overturns to the process, compromising the fermentative efficiency as well the industrial yield. Among the alternatives to control contamination, the use of synthetic antimicrobials can be highlighted. Its progressed use may favor the development of resistant strains, contributing in production cost improving, besides the possibility of residues incorporation into the final product. This work aimed evaluate the effect of conventional biocides (sodic monensin) and natural ones based on propolis (Propolis Hydroalcoholic Extract - EHP and Propolis Oily Extract - EOP) under the yeasts physiology, the fermentative process contaminants control, and the distilled composition. The experimental design used was the split-plot with four replications. The main treatments were: Control, EOH, EHP, and sodic monensin (Kamoran WP). The secondary treatments were the 10 fermentative cycles. The evaluated characteristics were: juice, must, and wine chemical-technical characteristics, yeasts microbiologic parameters, and the distillated obtained composition. The results obtained showed that the evaluated biocides presented similar effect, being effectives to control the fermentation contaminants, not affecting negatively its physiologic characteristics. They did not affect the composition of the distilled obtained / Mestre

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-06-28T22:53:24Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jessica_pereira_matos.pdf: 1785661 bytes, checksum: f5dbf30e3ce913eae470d1e958272e42 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-07-18T12:52:05Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jessica_pereira_matos.pdf: 1785661 bytes, checksum: f5dbf30e3ce913eae470d1e958272e42 (MD5) / Made available in DSpace on 2018-07-18T12:52:05Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) jessica_pereira_matos.pdf: 1785661 bytes, checksum: f5dbf30e3ce913eae470d1e958272e42 (MD5) Previous issue date: 2017 / Na produ??o do bioetanol lignocelul?sico, as hexoses provenientes da celulose s?o fermentadas a etanol por muitos micro-organismos que ocorrem naturalmente. Entretanto, as pentoses, provenientes da hidr?lise da hemicelulose, como xilose e arabinose, s?o fermentadas a etanol por poucas esp?cies selvagens conhecidas e com baixos rendimentos. H?, portanto, necessidade de sele??o de estirpes de leveduras que sejam capazes de fermentar pentoses e glicose conjuntamente e de forma eficiente. Neste estudo, duas linhagens de leveduras, Candida akabanensis UFVJM-R131 e Galactomyces geotrichum UFVJM-R10, foram avaliadas na fermenta??o alco?lica da fra??o hemicelul?sica de torta de girassol solubilizada por hidr?lise com ?cido dilu?do. A hidr?lise da hemicelulose foi realizada misturando-se 31% de biomassa seca em solu??o de H2SO4 a 6%, que foi mantida por 38 minutos a temperatura de 121?C a 1 atm. A caracteriza??o cromatogr?fica do hidrolisado obtido revelou a exist?ncia de glicose (7,57 g L-1), xilose (19,53 g L-1) e arabinose (8,85 g L- 1), al?m de 5-hidroximetilfurfural (0,71 g L-1), furfural (0,05 g L-1) e ?cido ac?tico (5,27 g L- 1). Ambas as leveduras mostraram-se capazes de produzir etanol a partir do hidrolisado ?cido da torta de girassol. Os processos conduzidos com as leveduras G. geotrichum UFVJM-R10 e C. akabanensis UFVJM-R131 apresentaram valores de YP/S de 0,29 e 0,27 g etanol g-1 a??cares, respectivamente. As quantidades dos inibidores identificados no hidrolisado n?o afetaram a efici?ncia da fermenta??o alco?lica. A suplementa??o do hidrolisado com fontes de nitrog?nio e minerais aumentou a taxa de consumo da xilose e da arabinose. Os resultados obtidos permitiram concluir que as linhagens G. geotrichum UFVJM-R10 e C. akabanensis UFVJMR131 possuem potencial para a produ??o de bioetanol a partir da fra??o hemicelul?sica de biomassas vegetais. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Biocombust?veis, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / In the production of bioethanol lignocellulosic, the hexose from cellulose are fermented to ethanol by many microorganisms that occur naturally. Though, the pentoses from hemicellulose hydrolysis, as xylose and arabinose, are fermented to ethanol by few known wild strains. This fact point the need to select strains of yeasts capable of ferment pentoses and glucose together more efficiently. In the present study, two lineages of yeast, Candida akabanensis UFVJM-R131 and Galactomyces geotrichum UFVJM-R10, were evaluated on alcoholic fermentation of hemicellulosic fraction from sunflower cake solubilized by hydrolysis with dilute acid. The hydrolysis of hemicellulose was performed utilizing 31% of dry biomass in H2SO4 at 6% under 121?C and pressure at 1 atm for 38 minutes. Chromatographic characterization of the hydrolyzate obtained showed the presence of glucose (7.57 g L-1), xylose (19.53 g L-1) and arabinose (8.85 g L-1), besides 5-hydroxymethylfurfural (0.71 g L-1), furfural (0.05 g L-1) and acetic acid (5.27 g L-1). Both yeasts were able to produce ethanol from the acidic hydrolyzate. The fermentation carried out with G. geotrichum UFVJM-R10 and C. akabanensis UFVJM-R131 presented YP/S values of 0.29 and 0.27 g ethanol g-1sugars, respectively. The amounts of the inhibitors identified in the hydrolyzate did not affect the efficiency of the alcoholic fermentation. The supplementation of the hydrolyzate with nitrogen and mineral sources increased the rate of consumption of xylose and arabinose. The results obtained allowed to conclude that the strains G. geotrichum UFVJM-R10 and C. akabanensis UFVJM-R131 have potential for the production of bioethanol from vegetal hemicellulosic fraction.

A transformação do mosto de uva em vinho envolve uma série de ações combinadas de diferentes gêneros e espécies de microrganismos. A espécie Saccharomyces cerevisiae domina a fase intermediária e a fase final da fermentação alcoólica. De modo geral, as leveduras enológicas podem ser caracterizadas pela capacidade fermentativa, produção de H2S (sulfeto de hidrogênio) e seu comportamento killer. A Embrapa uva e vinho possui em sua Coleção, diversas leveduras autóctones isoladas de bagas de uvas oriundas de diversas regiões do Brasil. Entretanto, a diversidade genética destes isolados não é conhecida. Neste estudo foram avaliados a capacidade fermentativa, formação de H2S, fator killer e sensibilidade ao fator killer de 150 leveduras provenientes das cultivares Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) e Moscato Tradicional (MBTF14) todas oriundas da região de Farroupilha- RS. A capacidade fermentativa foi avaliada juntamente com a formação de H2S, inoculando as leveduras em meio mosto sulfito. Os testes ao fator killer e sensibilidade ao fator killer foram avaliados através do meio Lorena/ELNC (80:20). As linhagens com perfil para elaboração de vinhos e produtoras da toxina killer foram identificadas por amplificação da região ITS1- 5.S- ITS2 por PCR e por PCR-RFLP. Foi avaliada também a diversidade genética de 23 linhagens da espécie de Saccharomyces cerevisiae da Coleção da Embrapa Uva e Vinho, usando a técnica de PCR-RAPD. Foram empregados para detectar a variabilidade genética das leveduras os oligonucleotídeos iniciadores: (GTG)5, (GAC)5, (GACA)4 e M13. Os resultados mostraram que a maioria das linhagens apresentaram baixa velocidade fermentativa aliada à diferentes níveis de produção de H2S. Somente 3 linhagens apresentaram capacidade fermentativa adequada quando comparadas com as linhagens de referencia 1vvt/97 e K1, quais sejam, 29MBF14, 39MBTF14 e 50MBF14. Apenas a linhagem 29MBTF4 formou pequenas quantidades de H2S. Verificou-se que 64% das linhagens isoladas mostraram-se metabolicamente capazes de biossintetizar H2S. Somente 9,33% apresentaram comportamento killer e apenas 6,66% mostraram sensibilidade à proteína killer. Os resultados apresentados sugerem ter relação com as cultivares utilizadas no isolamento. Verificou-se a existência de diferenças genéticas entre as linhagens de Saccharomyces cerevisiae estudadas com todos os iniciadores utilizados. Os iniciadores que mais discriminaram linhagens de Saccharomyces cerevisiae foram (GTG)5 e (GAC)5. / Grape must conversion into wine involve combined actions of different genus and species of microorganism. The species Saccharomyces cerevisiae dominates intermediate and final stages of alcoholic fermentation. Generally, the oenological yeasts are characterized by their fermentative capacity, production of H2S (hydrogen sulfide) and killer behavior. Embrapa Grape and Wine has a Yeast Collection that encompasses many autochthonous strains isolated of grape berries from different regions of Brazil. However, the genetic diversity of these yeasts are stilling known. This study has evaluated the fermentative capacity, production of H2S, killer factor and killer factor sensibility of 150 yeasts isolated from the cultivars Malvasia Bianca (FMB14), Moscato Alexandria (FMA14) and Moscato Tradicional (MBTF14) all belonging from Farroupilha commune in Rio Grande do Sul State. The fermentative capacity has been tested along with the evaluation of H2S production by the inoculation of the yeasts in sulfite must medium. The production and detection of factor killer and the evaluation of sensitive characteristics have been measured in Lorena/ELNC (80:20) solid medium. Yeasts with optimal fermentative characteristics and the ones producing killer toxin have been identified by amplification of ITS1-5.8S-ITS2 region by PCR -RFPL. This study also has evaluated the genetic diversity of 23 yeasts strains of Saccharomyces cerevisiae, belonging to the Yeast Collection of Embrapa Grape and Wine, employing PCRRAPD technique. The primers (GTG)5, (GAC)5, (GACA)4 and M13 have been used to detect the yeasts genetic diversity. The results showed that the majority of the yeasts analyzed have demonstrated low fermentative velocity combined with different levels of H2S production. From the three cultivars analyzed, only Moscato Tradicional showed yeasts with a suitable fermentative capacity when compared to the reference yeasts EMBRAPA 1vvt97 e K1, and they were named as 29MBF14, 39MBTF14 and 50MBF14. It was verified that 84%, 76% and 36% of the isolated strains from Malvasia Bianca, Moscato Tradicional and Moscato Alexandria, respectively, were capable to biosynthesize H2S. Concerning to killer behavior, 14%, 12% and 2% of the isolated strains from Moscato Tradicional, Moscato Alexandria and Malvasia Bianca, respectively, were capable of producing killer factor. These outcomes suggest the influence of the cultivar into the microflora biodiversity. Genetic differences were also demonstrated between the strains of Saccharomyces cerevisiae for all the primers tested. Primers GTG5 and GAC5 were the most discriminative.