Os efluentes vinícolas apresentam concentração de matéria orgânica que varia entre 1.200 mg.L-1 a 92.000 mg.L-1 de DQO. O pH é ácido, variando entre 3,5 a 5,0. A concentração de compostos fenólicos varia entre 41 a 1450 mg.L-1 C6H5OH. Para nitrogênio amoniacal a concentração varia entre 12 a 208 mg.L-1 já para sulfetos entre 50 e 500 mg.L-1S. Com base nestas informações, foi proposto um sistema de tratamento de efluentes físico-químico em batelada, composto por oxidação avançada do tipo Fenton e duas colunas de adsorção preenchidas com adsorventes alternativos: uma com resíduos de olarias (restos de cerâmicas) e outra com resíduos sólidos da construção civil (britados em recicladora). O efluente vinícola foi caracterizado através dos seguintes parâmetros: pH, turbidez, DQO, sólidos, nitrogênio, fósforo e fenol total. Os materiais adsorventes citados foram comparados ao adsorvente convencional, carvão ativado, e caracterizados no que diz respeito a: pH, densidade aparente e relativa, teor de umidade, teor de material volátil e teor de cinzas. No ensaio final para a oxidação do efluente bruto, foi aplicada a concentração de peróxido de hidrogênio de 16 g.L-1 na relação com a H2O2/DQO denominada de “z” igual a 3,36, o catalisador da reação foi o sulfato ferroso na proporção de 1:15 (FeSO4.7H2O:H2O2). O tempo de reação foi de quatro horas a 30°C em mesa agitadora a 150 rpm. Após a oxidação por Fenton, o efluente foi submetido à adsorção em coluna de cerâmica na taxa de 5m3.m2.dia-1, sequencialmente o efluente foi inserido na coluna de resíduos sólidos da construção civil na taxa de aplicação de 4 m3.m2.dia-1. O desempenho do sistema foi avaliado em função de DQO, turbidez, nitrogênio, sólidos, fenol total e cor. Ao final do tratamento a eficiência na remoção de DQO foi de 92,25%, atingindo valores de DQO de 369 mg.L-1. Além disso, o sistema de tratamento aplicado se apresentou eficaz na remoção da cor (100%), fenol total (100%), turbidez (100%), sulfatos (98%) atingindo uma concentração de 40,5 mg.L-1, nitrogênio (10%) com 17,25 mg.L-1 e na remoção de sólidos: ST (95% com 2,29 mg.L-1), STF (71% com 0,92 mg.L-1), STV (97% com 1,37 mg.L-1), SST (95% com 0,08 mg.L-1), SSF (96% com 0,01 mg.L-1), SSV (91% com 0,09 mg.L-1), SDT (95% com 2,21 mg.L-1), SDF (67% com 0,9 mg.L-1) e SDV (97% com 1,27 mg.L-1). A vantagem da aplicação de Fenton no efluente bruto consistiu em aproveitar as características ácidas do efluente, condição necessária para que o sulfato de ferro não precipitasse, exercendo assim a função de catalisador da reação. Posteriormente a oxidação, foi necessário neutralizar o pH para precipitação do ferro, condição que ocorreu nas colunas de adsorção com os resíduos sólidos da construção civil, possibilitando a precipitação, filtração e remoção do ferro. A combinação deste sistema facilitou e minimizou o uso de reagentes químicos, possibilitando a aplicação prática em vinícolas de produção sazonal e que não possui grandes áreas para a instalação de sistemas maiores e mais complexos. Conclui-se que, o sistema físico-químico proposto apresenta relevante eficiência quanto à remoção de matéria orgânica, cor, sólidos, nitrogênio, sulfatos e fenol total, possibilitando a disposição em corpos hídricos ou rede pública de esgoto. / The organic matter present on those winery effluents varies from 1.200 mg.L-1 to 92.000 mg.L-1 of COD. The pH is acidic, varying from 3,5 to 5,0. The concentration of phenolic compounds ranges between 41 to 1450 mg.L-1 C6H5OH . For ammonia the concentration varies from 12 to 208 mg.L-1. Sulfates have to between 50 and 500 mg.L-1 S. Based on these information, a physical-chemical treatment system was proposed, analyzed into batches, which consisted of the advanced Fenton ́s Oxidation and two adsorption columns filled with alternated adsorbents: one with pottery residues and the other with residues from building (concrete) sites ( crushed on crushing machines). The winery effluent characterization was establish according to the following parameters: pH, turbidity, COD, solids, nitrogen, phosphate and phenols. The absorbents resources were characterize pH, bulk and relative densities, moisture content, volatile material, ash content. The assays begun with the oxidation of the raw effluent, followed by the addition of 16 g.L-1 of hydrogen peroxide related to a COD of z=3,36 (H2O2/DQO). Ferrous sulfate catalyzed the reaction at 1:15 portion (FeSO4.7H2O:H2O2), on an agitated platform at 150 rpm for four hours at 30oC. Once Fenton’s oxidation was concluded, the effluent was applied to the pottery absorption column, at 5m3/m2.day-1, and subsequently applied to the building sites residues column at 4m3/m2.day-1. The system’s performance was evaluate according to the COD, turbidity, nitrogen, solids, phenols and color. The COD efficiency removal obtained was 92,25%, reaching COD values of 369 mg.L-1. In addition, the chosen applied treatment revealed to be efficient on the elimination of color (100%), phenols (100%), turbidity (100%), sulfates (98%) reaching a concentration of 40.5 mg.L-1, nitrogen (10%) with 17.25 mg.L-1, and on solids removals: ST (95% with values of 2,29 mg.L-1), STF (71% and 0,92 mg.L-1), STV (97% and 1,37 mg.L-1), SST (95% and 0,08 mg.L-1), SSF (96% and 0,01 mg.L-1), SSV (91% and 0,09 mg.L-1), SDT (95% and 2,21 mg.L-1), SDF (67% and 0,9 mg.L-1) e SDV (97% and 1,27 mg.L-1). The advantage of the Fenton application on the raw effluent consisted on taking benefit of the acidic environment of the effluent, environment that gave the right condition to avoid the precipitation of the iron sulfate, becoming the reaction catalyst. Once the oxidation was completed, it was necessary to neutralize de pH to allow the iron precipitation, which took place into the building residues adsorption columns, allowing iron the precipitation, filtration and removal. These methods combined enabled and minimized the use of chemical reagents, allowing it to be easily mounted in wineries with seasonality production and wineries without a large area to install bigger and more complex systems. In conclusion, the physical – chemical system proposed revealed to be efficient on the removal of the organic mass, colors, solids, nitrogen, sulfates and phenols, allowing the discharge of effluent disposal.

Orientador: Marcelo Alexandre Prado / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T19:36:01Z (GMT). No. of bitstreams: 1 Mazon_ElaineMarradeAzevedo_D.pdf: 2626954 bytes, checksum: 7cc1b7c8e92de797976754431156c2af (MD5) Previous issue date: 2013 / Resumo: O consumo de vinhos no Brasil aumentou cerca de 20% na última década, e sua comercialização vem se difundindo, principalmente, pelo aumento da oferta, aumento do poder aquisitivo da população brasileira, aumento da produção e pelos benefícios à saúde que lhe são atribuídos. A análise de elementos traço nos vinhos é de grande importância para o controle da qualidade, autenticidade, biodisponibilidade de metais e estudo da toxicidade, visando garantir a segurança alimentar para os consumidores. Este trabalho descreve o desenvolvimento de um método empregando a espectrometria de absorção atômica com forno de grafite para a determinação de arsênio (As), cádmio (Cd), cobre (Cu) e chumbo (Pb) em amostras de vinhos brasileiros. A digestão ácida preliminar não foi necessária no preparo de amostra. O programa de aquecimento, empregado no forno de grafite foi otimizado para os diferentes tipos de vinhos e as amostras foram diluídas na proporção de 1:3 com ácido nítrico (65% v/v) e peróxido de hidrogênio (30% m/m). A curva analítica foi preparada em meio aquoso e alguns modificadores foram investigados para cada elemento. As determinações foram realizadas nas seguintes faixas lineares 10-100 µg L-1 Cu; 5,0 - 60 µg L-1 Pb; 0,5 - 2,0 µg L-1 Cd; 10 - 60 µg L-1 As. Os limites de quantificação obtidos foram 11,5; 2,5; 0,22 e 5,0 µg L-1, respectivamente. As recuperações obtidas variaram de 92-103% (vinho tinto) e 99-106% (vinho branco) para o Cu, 97-107% (vinho tinto) e 95-101% (vinho branco) para o Pb, 89-102% (vinho tinto) e 98-106% (vinho branco) para o Cd, e 103-106% (vinho tinto) e 99-107% (vinho branco) para o As. Foram analisadas 45 amostras de vinhos nacionais adquiridos no comércio de Campinas/SP de diversas regiões do Brasil. Foram determinados os teores de As, Cd, Cu e Pb nas amostras de vinho e os valores encontrados estavam abaixo dos teores estabelecidos pela legislação brasileira. Foram calculadas as contribuições dos vinhos estudados na dieta de um adulto, com relação a ingesta destes elementos e os valores demonstraram que os vinhos estudados apresentaram baixa contribuição. Também foram determinados os valores de pH e os teores de polifenóis totais nos vinhos, as amostras apresentaram valores compatíveis com os relatados na literatura com valores médios para polifenóis em vinhos de mesa de 2046,0 mg L-1, vinhos tinto 1443,0 mg L-1 e espumantes 1606,0 mg L-1. Os valores de pH variaram de 2,8 a 3,8 entre os vinhos. Foi feito o estudo da acessibilidade e da fração dialisável pelo método in vitro dos elementos As, Cd, Cu e Pb nos vinhos e os resultados demonstraram para todos os elementos que a fração solúvel foi maior que 37% e a dialisável, maior que 43%, indicando alta capacidade de acessibilidade. Quanto aos dizeres de rotulagem foi observada uma necessidade de complementação da atual legislação específica para vinhos, com a finalidade de melhorar a qualidade das informações oferecidas ao consumidor através dos rótulos dos vinhos / Abstract: Wine consumption in Brazil has been increasing in 20% in the last decade, and its sales have been disseminating mainly due to offer and production increase, and to health benefits attributed to it. The analysis of trace elements in wine is of great importance for quality control, authenticity, metal bioavailability and toxicity study, aiming at granting safety to consumers. This paper describes the development of a method which employs atomic absorption spectrometry with graphite oven in order to determine arsenic, cadmium, copper and lead in Brazilian wines samples. A preliminary acid digestion has not been necessary in the sample preparation. The heating program employed to the graphite oven has been optimized for the different types of wine and the samples have been diluted in proportion of 1:3 with nitric acid (65% v/v) and hydrogen peroxide (30% m/m). The analytical curve has been prepared in aqueous medium and some modifiers have been investigated for each element. The determinations have been performed in the following linear ranges 10-100 µg L-1 Cu; 5,0 - 60 µg L-1 Pb; 0,5 - 2,0 µg L-1 Cd; 10 - 60 µg L-1 As. The qualification limits obtained were 11,5; 2,5; 0,22 e 5,0 µg L-1, respectively. The recoveries obtained ranged from 92-103% (red wine) and 99-106% (white wine) for Cu, 97-107% (red wine) and 95-101% (white wine) for Pb, 89-102% (red wine) and 98-106% (white wine) for Cd, and 103-106% (red wine) and 99-107% (white wine) for As. Brazilian wines, about 45 samples from Campinas/SP local market have been analyzed at several Brazilian regions. The contents of As, Cd, Cu and Pb have been determined in the samples and the amounts found out were below the contents established by the Brazilian legislation. The contribution of those elements on the diet of an adult have been calculated, and the amounts have shown that the studied wines presented low contribution. The pH levels and the total polyphenols contents have also been determined, the samples presented compatible rates according to the literature, with average rates for polyphenol in table wines at 2046,0 mg L-1, red wines 1443,0 mg L-1 and sparking 1606,0 mg L-1. The pH rates varied from 2,8 to 3,8 among the wines. The accessibility and the dialysis fraction through the in vitro method have also been studied for the elements As, Cd, Cu and Pb in the wines. The results show that for all elements soluble fractions greater than 37% and higher than 43% dialyzable indicating high capacity accessibility. As for labeling was observed a need complement the current legislation for wine in order to improve the quality of information offered to consumers through wine labels / Doutorado / Ciência de Alimentos / Doutora em Ciência de Alimentos

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The wine yeast Saccharomyces cerevisiae has the ability to utilize several different nitrogenous compounds to fulfill its metabolic requirements. Based upon different growth rates of the yeast in a particular nitrogen source, nitrogen compounds have been classified as either good or poor nitrogen sources. In an environment which contains different quality nitrogen sources, such as grape must, the yeast first utilizes good and then the poor nitrogen sources. This discrimination between good and poor nitrogen sources is referred to as nitrogen catabolite repression (NCR). Examples of good nitrogen sources are ammonia, glutamine and asparagine. Nitrogen sources such as allantoin, y-aminobutyrate (GABA), arginine and proline are poor quality nitrogen sources. Several regulatory proteins, Ure2p, Gln3p, Da180p,Gat1pand Deh1p, mediate NCR in S. cerevisiae. These trans-acting factors regulate transcription of NCR sensitive genes. All these proteins, except Ure2p, bind cis-acting elements in the promoters of genes that are responsible for degradation of poor nitrogen sources. Gln3p is an activator of NCR sensitive genes in the absence of good nitrogen sources. The predominant mechanism by which NCR functions is by using Ure2p to inactivate the activator Gln3p in the presence of a good nitrogen source. Several research groups have studied the Ure2p, mainly due to its prion-like characteristics. The Ure2p has two domains: a prion inducing domain located in the N-terminal region and a NCR regulatory domain located in the C-terminal domain. The aims of this study were (i) to determine the part of the C-terminal domain which is responsible for NCR, (ii) to establish if ure2 deletion mutants produce less ethyl carbamate during wine fermentations and (iii) if NCR functions in industrial yeast strains. Nested deletions of the URE2 gene revealed that the NCR regulatory domain resides in the last ten amino acids of the Ure2p. This was established by Northern blot analysis on the NCR sensitive genes DAL5, CAN1, and GAP1 genes. Ethyl carbamate in wine is produced by spontaneous chemical reaction between urea and ethanol in wine. Urea is produced by S. cerevisiae during the metabolism of arginine. Arginine is degraded to ornithine and urea by arginase, the product of the CAR1 gene. Degradation of urea by S. cerevisiae is accomplished by urea amidolyase, a bi-functional enzyme and product of the DUR1,2 gene which is subject to NCR. This study investigated if a ure2 mutant strain produced less ethyl carbamate during wine fermentations. Wine fermentations were conducted with diploid laboratory strains: a ure2 mutant strain and its isogenic wild type strain. GC/MS analysis of the wine revealed that the ure2 mutant produced less ethyl carbamate but more ethanol than the wild type strain when arginine, di-ammoniumphosphate, asparagine or glutamine were added as nitrogen sources, in combinations and separately. There was no significant difference between the wild type fermentation and the ure2 mutant fermentation when no nitrogen was added. It was found that a combination between the deletion of URE2 and the addition of a good nitrogen source resulted in lower levels of ethyl carbamate. High density micro array analysis done on an industrial strain wine yeast in Chardonnay grape must revealed that the GAP1, CAN1, CAR1 and DUR1,2 genes, responsible for transport and metabolism of arginine and degradation of urea, are NCR sensitive. These data strongly suggest that NCR functions in industrial yeast strains. / AFRIKAANSE OPSOMMING: Die wyngis Saccharomyces cerevisiae kan verskillende stikstofbronne gebruik om in sy stikstofbehoeftes te voldoen. Stikstofbronne word as goeie of swak stikstofbronne geklassifiseer op grond van die groeitempo van die gis op die betrokke stikstofbron. 'n Goeie stikstofbron laat die gis vinniger groei as wat dit op 'n swak stikstofbron sou groei. In omgewings soos druiwemos waar daar 'n verskeidenheid van stikstofbronne teenwoordig is, sal die gis eers die goeie bronne en daarna die swak bronne benut. Stikstofbronne soos ammonium, asparagien en glutamien word geklassifiseer as goeie bronne. Allantoïen, y-amino-butaraat (GABA), prolien en arginien word as swak stikstofbronne geklassifiseer. Die meganisme waarmee S. cerevisiae tussen die stikstofbronne onderskei, staan as stikstof kataboliet onderdrukking (NCR) bekend. Die proteïene wat vir verantwoordelik is NCR naamlik Ure2p, Gln3p, Gat1 p, Dal80p en Deh1 p, bind met die uitsondering van Ure2p, almal aan cis-werkende elemente in die promoters van NCR-sensitiewe gene. Die trans-werkende faktore reguleer die transkripsie van NCR-sensitiewe gene. NCR werk hoofsaaklik deur die inhibering van Gln3p deur Ure2p in die teenwoordigheid van 'n goeie stikstofbron. Die oorgrote meerderheid NCR-sensitiewe gene word deur Gln3p in die afwesigheid van 'n goeie stikstofbron geaktiveer. Heelwat navorsing is op die prionvormings vermoë van Ure2p gedoen. Ure2p het twee domeine: 'n N-terminale domein wat vir prionvorming verantwoordelik is en die C-terminale domein waar die NCR funksie van Ure2p gesetel is. Die doel van die studie was (i) om te bepaal waar in die C-terminale domein van Ure2p die NCR regulering geleë is, (ii) of ure2 delesie mutante minder etielkarbamaat tydens wynfermentasies produseer en (iii) of NCR in industriële gisrasse funksioneel is. Delesie analises van URE2 het getoon dat die NCR regulerings domein in die laaste tien aminosure gesetel is. Dit is vas gestel m.b.v. noordlike klad tegniek analises op die OALS, CAN1 en GAP1 gene.Etielkarbamaat in wyn word deur die spontane chemiese reaksie tussen ureum en alkohol geproduseer. Ureum word gedurende die metabolisme van arginien in S. cerevisiae geproduseer. Arginien word deur arginase, produk van die CAR1 geen, na ornitien en ureum afgebreek. Die bi-funksionele ureum amidoliase, gekodeer deur die DUR1,2 geen, breek ureum na CO2 en NH/ af. As gevolg van die NCRsensitiwiteit van dié gene is ondersoek ingestel na In ure2 mutant se vermoë om minder etielkarbamaat tydens wynfermentasies te produseer. Chardonnay druiwemos is met In diploiede laboratorium ras en die isogeniese ure2 mutant gefermenteer. GC/MS analise op die wyn het getoon dat die ure2 mutant minder etielkarbamaat, maar meer alkohol in vergelyking met die wilde tipe gis produseer, as arginien, di-ammoniumfosfaat, asparagien en glutamien, afsonderlik of gesamentlik byvoeg is. Daar was egter nie In merkwaardige verskil tussen die fermentasies waar geen stikstof bygevoeg is nie. Dit dui daarop dat In kombinasie van In URE2 delesie en die byvoeging van stikstof etielkarbamaat vlakke verlaag. Mikro-skyfie analise van In industriële gis in Chardonnay mos het getoon dat die GAP1, CAN1, CAR1 en DUR1,2 gene wat verantwoordelik is vir die transport en metabolisme van arginien en degradasie van ureum, wel NCR-sensitief is. Dit dui daarop dat NCRwel in industriële gisrasse funksioneel is.

Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in the production of a variety of fermented products. Many of these bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically closely related to the producer cell. Since many of these target bacteria include foodbome pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the practical importance of these peptides as food preservatives has been well documented and, in the case of nisin and pediocin PA-I, commercially explored. The increased demand from health conscious consumers for foods with no chemical preservatives is putting renewed pressure on the producer to supply a "clean and green" product, but with the same or even an extended shelf life. Various research groups are screening lactic acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds. Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence in the N-terminal region. Little is known about the production and mode of activity of pediocin PD-I. In this study, production of pediocin PD-I was significantly increased by optimizing the growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone (1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH during fermentation stimulated pediocin PD-I production and the level of organic acids produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the latter medium. Under these conditions the specific bacteriocin activity increased by a factor of approximately six after 55 h of fermentation. The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin removed the biofilms from the surfaces and reduced the biomass either completely, as in the case of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively. These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified Chardonnay must medium. To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being very effective in controlling the cell numbers of free-living cells of 0. oeni, the three bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the production and activity levels ofpediocin PD-I could be increased without genetically modifying the producer strain is an added advantage. / AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer. Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik. Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer, maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten einde Gram-negatiewe bakterieë, giste en swamme te inhibeer. Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met 'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor die meganisme van werking van hierdie bakteriosiene. In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween 80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die bakteriosien met 'n faktor van ongeveer ses verhoog. Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum 423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50% van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000 AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem. Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.

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 (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the quality and wholesomeness of the final product. Yeasts are primarily responsible for alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is conducted by lactic acid bacteria. These bacteria are important in winemaking and can have a positive or negative effect on the wine quality. Biogenic amines are one of the compounds produced by lactic acid bacteria, which affect the hygienic quality and wholesomeness of the wine negatively and directly pose a health risk to the consumer. The demand of consumers for higher quality and healthier foods has led to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide variety of food products, such as cheese, dried sausage, sauerkraut, fishery products, chocolates, wine and beer. This thesis focussed on the presence of biogenic amines in wine. The first objective of the study was to determine the ability of lactic acid bacteria isolated from South African wine to produce biogenic amines, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. The results obtained showed that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be the lactic acid bacteria responsible for tyramine and putrescine production and that it can contribute significantly to the overall biogenic amine content in wines. The results also suggest that amine production is strain dependent and not species specific. None of the lactic acid bacteria tested had the ability to produce histamine or cadaverine. It is important to remember that the ability of the lactic acid bacteria to produce biogenic amines has only been investigated in synthetic media and that it does not necessarily imply similar behaviour in wine. Wine represents a complex environment with a wide number of factors influencing microbial growth and decarboxylase activity and, thus, further investigation is necessary to determine if these amine-producing bacteria behave similarly in wine conditions. In addition, the polymerase chain reaction (PCR) amplification method was used for the identification of the tyrosine decarboxylase (TOe) gene in some of the tyramine-producing lactic acid bacteria. This was followed by the sequencing of the amplified products, which are partial TOe gene sequences, of two L. brevis strains and of a L. hilgardii strain. Only one tdc gene sequence has been described for bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis lOEB 9809 was described. An amino acid sequence alignment of the three TOe gene fragments, obtained in this study, with the known TOe gene fragment of L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of relatedness and conserved regions. To meet consumer demands, procedures are necessary to prevent the formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species involved in the winemaking process. Another possible way would be to develop a rapid detection method for bacteria carrying amino acid decarboxylase genes. The results of this study provide knowledge about which lactic acid bacteria in the winemaking process could contribute to the production of biogenic amines and the sequencing of additional partial TOe genes could possibly assist in the development of a rapid detection method for tyramine-producing lactic acid bacteria in food products. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê. Biogeniese amiene is een van die komponente wat deur melksuurbakterieë geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade, wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in wyn. Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid- Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde gedrag in wyn sal toon. Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809 en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van amiene in voedselprodukte voorkom word. Een manier van voorkoming is om amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte bydra.

Thesis (MScAgric)--Stellenbosch University, 1964. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (PhD(Agric) (Viticulture and Oenology))--University of Stellenbosch, 2006. / The winemaking process involves different complex chemical and biochemical reactions, which include those of oxygen (O2). Oxygen can come into contact with the wine through various winemaking procedures and can be used by the winemaker to enhance the quality of red wine. In wine, the main substrates for oxidation are phenolic molecules, which form quinones. These can influence the sensory characteristics of the wine. O2 can be used in fresh must to remove oxidisable phenolic molecules through a process called hyper-oxidation and can also be added to fermenting must to enhance the fermentation performance of yeast. Controlled O2 additions during ageing can lead to the wine’s colour being increased and the astringency of the wine decreased. This is due to the formation of acetaldehyde from the oxidation of ethanol, which induces the polymerisation of tannin and anthocyanin molecules. The addition of too much O2 to wine can, however, lead to unwanted over-oxidation, with certain off-odours being formed. It can also enhance the growth of unwanted spoilage microorganisms, such as Brettanomyces and acetic acid bacteria. Although research on O2 in wine was started many years ago, many questions still remain. These include the general effect of O2 on the sensory and phenolic profile of red wine especially and the microbiology of wine during ageing. An effective way of measuring oxidation, especially in red wine must also be developed. In the first part of this study, the effects of O2 and sulfur dioxide (SO2) additions on a strain of Brettanomyces bruxellensis (also known as Dekkera bruxellensis) and Acetobacter pasteurianus were investigated. Epifluorescence microscopy and plating revealed that the A. pasteurianus strain went into a viable but non-culturable state in the wine after prolonged storage under relative anaerobic conditions. This state, however, could be negated with successive increases in culturability by the addition of O2, as would happen during the transfer of wine when air is introduced. The A. pasteurianus strain was also relatively resistant to SO2, but the B. bruxellensis strain was more sensitive to SO2. A short exposure time to molecular SO2 drastically decreased the culturability of the B. bruxellensis strain, but bound SO2 had no effect on the culturability or viability of either of the two types of microorganisms. Oxygen addition to the B. bruxellensis strain also led to a drastic increase in viability and culturability. It is thus clear that SO2 and O2 management in the cellar is of critical importance for the winemaker to produce wines that have not been spoiled by Brettanomyces or acetic acid bacteria. This study should contribute to the understanding of the factors responsible for the growth and survival of Brettanomyces and acetic acid bacteria in wine, but it should be kept in mind that only one strain of each microorganism was used. This should be expanded in future to include more strains that occur in wine. The second part of this study investigated the effect of micro-oxygenation on four different South African red wines. It was found that the micro-oxygenation led to an increase in the colour density and SO2 resistant pigments of the two wines in which micro-oxygenation was started just after the completion of malolactic fermentation. In one of these wines, a tasting panel preferred the micro-oxygenation treated wines to the control. In the other two red wines, in which the micro-oxygenation was started seven months after the completion of malolactic fermentation, very little colour increase was observed. One of these two wines was also matured in an oak barrel, where the change in phenolic composition was on par with the treated wines. A prolonged period of micro-oxygenation, however, led to this wine obtaining an oxidised, over-aged character. Micro-oxygenation and maturation in an oak barrel also enhanced the survival of acetic acid bacteria and Brettanomyces in this wine. Micro-oxygenation can hence be used by the wine producer on young red wines to enhance the quality of the wine, but should be applied with care in older red wines. Future research into micro-oxygenation should focus on whether it can simulate an oak barrel. More research into the effect of micro-oxygenation on the sensory profile of the wine is needed. As mentioned, the addition of O2 can lead to oxidative degradation of wine. The brown colour in wine is often used as an indication of oxidation, but oxidative aromas can be perceived before a drastic increase in the brown colour has been observed in red wine. The third part of this study was to assess the possible use of Fourier Transform Infrared Spectroscopy (FTIR) to measure the progression of oxidation in Pinotage red wines. Three wines were used in this study and clear separation between the control and aerated wines was observed by using Principle Component Analysis (PCA). Sensory analysis of these wines confirmed this observation, with a reduction especially in berry fruit and coffee characters and an increase first in potato skin and then acetaldehyde aroma characters as the oxidation progressed. PCA analysis also revealed that in certain wines the visible spectrum of light did not indicate the progression of oxidation as sensitively as with the use of FTIR. This also correlated with the inability of the panel to observe a drastic colour change. FTIR should be further investigated as a possible means of monitoring oxidation in wine and this study should be expanded to wines made from other cultivars as well.

Thesis (PhD(Agric) Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2009. / The main goal of this project was to elucidate the underlying genetic factors responsible for the different fermentation phenotypes and physiological adaptations of industrial wine yeast strains. To address this problem an ‘omic’ approach was pursued: Five industrial wine yeast strains, namely VIN13, EC1118, BM45, 285 and DV10, were subjected to transcriptional, proteomic and exometabolomic profiling during alcoholic fermentation in simulated wine-making conditions. The aim was to evaluate and integrate the various layers of data in order to obtain a clearer picture of the genetic regulation and metabolism of wine yeast strains under anaerobic fermentative conditions. The five strains were also characterized in terms of their adhesion/flocculation phenotypes, tolerance to various stresses and survival under conditions of nutrient starvation. Transcriptional profiles for the entire yeast genome were obtained for three crucial stages during fermentation, namely the exponential growth phase (day 2), early stationary phase (day 5) and late stationary phase (day 14). Analysis of changes in gene expression profiles during the course of fermentation provided valuable insights into the genetic changes that occur as the yeast adapt to changing conditions during fermentation. Comparison of differentially expressed transcripts between strains also enabled the identification of genetic factors responsible for differences in the metabolism of these strains, and paved the way for genetic engineering of strains with directed modifications in key areas. In particular, the integration of exo-metabolite profiles and gene expression data for the strains enabled the construction of statistical models with a strong predictive capability which was validated experimentally. Proteomic analysis enabled correlations to be made between relative transcript abundance and protein levels for approximately 450 gene and protein pairs per analysis. The alignment of transcriptome and proteome data was very accurate for interstrain comparisons. For intrastrain comparisons, there was almost no correlation between trends in protein and transcript levels, except in certain functional categories such as metabolism. The data also provide interesting insights into molecular evolutionary mechanisms that underlie the phenotypic diversity of wine yeast strains. Overall, the systems biology approach to the study of yeast metabolism during alcoholic fermentation opened up new avenues for hypothesis-driven research and targeted engineering strategies for the genetic enhancement/ modification of wine yeast for commercial applications.

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.