Thesis (MScAgric) -- Stellenbosch University, 2003. / ENGLISH ABSTRACT: Cellulose is by far the most abundant carbohydrate available from plant biomass. These biopolymers are therefore an important renewable source of food, fuels and chemicals. Cellulose is embedded in a matrix of hemicellulose, lignin and pectin and is composed of repeating glucose units linked by p-1,4-glycosidic bonds. The individual molecules are held together by hydrogen bonds, forming largely crystalline fibres. The hemicellulose, which is a low molecular weight heteropolysaccharide, coats and binds the cellulose microfibrils, preventing the cellulose from becoming too crystalline. Three predominant types of hemicelluloses are recognised, namely 1,3- and 1,4-p-D-galactans, 1,4-p-D-mannans and 1,4-p-D-xylans, which are named according to the sugar type that forms the polymer backbone. Pectic substances contain rhamnogalacturonan backbones in which 1,4-linked a-D-galacturonan chains are interrupted at intervals with a-L-rhamnopyranosyl residues carrying neutral side chains. Two groups of enzymes, cellulases and pectinases, are required for the microbial utilisation of crystalline cellulose and pectin. Cellulases are multicomponent complexes that are often composed of endoglucanases, exoglucanases and cellobiases. Cellobiose is the major end product of concerted endoglucanase and exoglucanase activity. Cellobiose is then hydrolysed to glucose by p-glucosidases. The enzymatic breakdown of pectic polymers occurs by the deesterifying action of the saponifying enzymes, pectinesterase, releasing the methyl groups of the pectin molecule, and by hydrolase or lyase action of the depolymerases (pectin lyase, pectate lyase and polygalacturonase), splitting the a- 1.4-glycosidic linkages in the polygalacturonate chain. The yeast Saccharomyces cerevisiae has been used extensively in the alcoholic beverage industry for fermentations of wine, beer and other alcoholic beverages for many years. However, it is unable to produce extracellular depolymerising enzymes that can efficiently degrade polysaccharides, which are the main cause of clarification and filtration problems. Enzyme preparations have been used in the alcoholic beverage industries to degrade haze-forming polysaccharides, thereby improving the filterability and quality of products such as beer and wine. An alternative would be to develop S. cerevisiae strains that produce extracellular polysaccharidases, enabling the yeast to degrade polysaccharides without the addition of commercial enzyme preparations. These strains can also be very useful in improving the quality of wine, as well as cutting the costs of the winemaking process. The objective of this study was to investigate the effects of two transformed S. cerevisiae strains on different wine grape varieties. The following genes have been cloned and characterised previously: the Aspergillus niger endo-p-xylanase gene (xynC), the Butyrivibrio fibrisolvens endo-|3- 1.4-glucanase gene (endl), the Erwinia chrysanthemi pectate lyase gene (pelE) and the Erwinia carotovora polygalacturonase gene (p e h l). The yeast alcohol dehydrogenase I gene promoter (ADH1p), the alcohol dehydrogenase II gene terminator (ADH2j), the tryptophan synthase gene terminator (TRP5r) and the yeast mating-type pheromone a-factor secretion signal sequence (MFcrfs) were used to compile the following gene constructs: ADH1 p-MFa1 s-end1-TRP5r (designated END1), A DH1 p-xyn C-A DH2T (designated XYN4), ADH1 p-MFa1 s-peh1 -TRP5t (designated PEH1) and ADH1 p-MFa1 s-pelE-TRP5r (designated PELE). Two yeast integrating plasmids were constructed, one containing the END1 and XYN4 gene cassettes and the other containing the PEH1-PELE cassette. These two plasmids were then integrated into the URA3 locus of two separate industrial wine yeast strains of S. cerevisiae. To facilitate selection of the industrial yeast transformants in the absence of auxotrophic markers, the integrating plasmid containing the END1 and XYN4 gene cassettes was issued with the dominant selectable Geneticin G418-resistance {G f) marker. The integrating plasmid harbouring the PEH1-PELE gene cassette was issued with the dominant selectable sulphumetronmethyl resistance (SMR1) marker. The introduction of these plasmids into commercial wine yeast strains directed the synthesis of END1, XYN4, PELE and PEFI1 transcripts and the production of extracellular biologically active endo-P-1,4- glucanase, endo-(3-xylanase, pectate lyase and polygalacturonase. These recombinant yeasts were capable of extracting more colour from grape skins of certain varieties, as well as leading to more freeflow wine as a result of the more effective degradation of glucans, xylans and pectins in the skins. They also led to decreased turbidity in the wine, making it more filterable. Future work will entail further investigation of the effects of these recombinant yeasts on different white and red wine grape varieties. Another objective of this study was to screen non-Saccharomyces wine yeasts for the production of extracellular hydrolytic enzymes. The reason for this part of the thesis was to determine the types of extracellular hydrolytic enzymes that are produced and to determine which genera produce which kinds of extracellular enzymes. A total of 237 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces, were screened for the production of extracellular pectinases, proteases, (3-glucanases, lichenases, p-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts were all isolated from grapes and clarified grape juice to ensure that they were yeasts found in must during the initial stages of fermentation. This information can be used to pave the way to pinpoint the specific effects in wine of these enzymes produced by the so-called wild yeasts associated with grape must. This information can also be used to transform Saccharomyces wine yeasts with some of the genes from these non-Saccharomyces yeasts for the production of extracellular hydrolytic enzymes. However, future research will have to be done to determine the extent of the activity of these enzymes in wine fermentations and to obtain better knowledge of the physiological and metabolical features of non-Saccharomyces yeasts. / AFRIKAANSE OPSOMMING: Sellulose is verreweg die volopste koolhidraat in plantbiomassa. Hierdie biopolimere is dus ‘n baie belangrike hernubare bron van voedsel, brandstof en chemikaliee. Sellulose is in 'n matriks van hemisellulose, lignien en pektien gebed en is uit herhaalde glukose eenhede, wat deur middel van (3-1,4-glukosidiese bindings geheg is, saamgestel. Die individuele molekules word deur waterstofbindings aan mekaar geheg, wat aanleiding gee tot die vorming van kristallyne vesels. Die hemisellulose, wat 'n lae molekulere gewig heteropolisakkaried is, bedek en bind die sellulose vesels en verhoed daarmee die vorming van vesels wat te kristallyn is. Drie predominante tipes hemisellulose word herken en sluit 1,3- en 1,4-p-D-galaktane, 1,4-p-D-mannane en 1,4-p-D-xylane in, wat vernoem word volgens die suikereenhede wat die polimeerruggraat vorm. Pektiene bestaan uit 'n rhamnogalakturonaanruggraat waarin 1,4-gekoppelde a-D-galakturonaankettings periodiek met a-L-rhamnopiranosiel residue, bevattende neutrale sykettings, onderbreek word. Twee groepe ensieme, nl. pektinase en sellulase, word deur mikrobes vir die benutting van kristallyne pektinase en sellulase vereis. Sellulase is multikomponent komplekse wat dikwels uit endoglukanase, ekso-glukanase en sellobiase saamgestel is. Sellobiose is die hoof eindproduk van die saamgestelde aktiwiteit tussen endoglukanase en ekso-glukanase en word verder gehidroliseer tot glukose deur |3-glukosidases. Die ensimatiese afbraak van pektien polimere vind deur die de-esterifiserings aksie van die versepings ensiem, pektienesterase, plaas. Dit lei tot die vrystelling van die metielgroepe van die pektienmolekuul. Deur die hidrolase of liase aksie van die depolimerase (pektien liase, pektaatliase en poligalakturonase), split die a-1,4-glukosidiese verbindings in die poligalakturonaatketting. Die gis Saccharomyces cerevisiae word al vir jare ekstensief in die alkoholbedryf vir die fermentasie van verskeie produkte, veral druiwe, gebruik. S. cerevisiae besit egter nie die vermoe om ekstrasellulere depolimiserende ensieme wat vir die effektiewe degradasie van polisakkariede verantwoordelik is, te produseer nie, wat die hoof oorsaak van die verhelderings- en filtreringsprobleme in onder andere wyn en bier is. Dit veroorsaak ook dat S. cerevisiae nie oor die vermoe beskik om waasvormende polisakkariede in wyn te degradeer nie. Tans word ensiempreparate in die alkoholiese bedryf vir die degradasie van die probleem polisakkariede gebruik. Sodoende word die filtreerbaarheid en kwaliteit van wyn en bier verbeter. ‘n Goeie alternatief is die ontwikkeling van S. cerevisiae-rasse wat oor die vermoe beskik om ekstrasellulere polisakkarase te produseer en dus polisakkariede self sonder die byvoeging van eksterne kommersiele ensiempreparate te degradeer. Hierdie rasse sal baie voordelig wees vir die verbetering van wynkwaliteit, sowel as vir die vermindering van die kostes verbonde aan die wynmaakproses. Die objektief van hierdie studie is dus om die uitwerking van twee getransformeerde S. cerevisiae rasse, wat ekstrasellulere polisakkarases produseer, op verskillende wyndruifvarieteite na te vors. Die volgende gene is reeds voorheen gekloneer en gekarakteriseer: die endo-pxylanase- geen (xynC) van Aspergillus niger, die endo-p-1,4-glukanase-geen (endl) van Butyrivibrio fibrisolvens, die pektaatliase-geen (pe/E) van Erwinia chrysanthemi en die poligalakturonase-geen (p e h l) van Erwinia carotovora. Die alkoholdehidrogenase-geenpromotor (ADH1P), die alkoholdehidrogenase IIgeentermineerder (ADH2T), die gistriptofaansintase geen se termineerder (TRP5t) en die sekresiesein van die gisferomoon a-faktor (MFa1s) is gebruik om die volgende geenkonstrukte saam te stel: ADH1 p-MFa1 s-end1 -TRP5t (toekend as END1), ADH1 p-xynC-ADH2T (bekend as XYN4), ADH1 p-MFa1 s-peh1-TRP5T fbekend as PEH1), and ADH1 p-MFa1 s-pelE-TRP5T (bekend as PELE). Twee gisintegrerings plasmiede is gekonstrueer, een wat die END1- en XYN4- geenkassette bevat en die ander wat die PEH1-PELE-kasset besit. Hierdie twee plasmiede is daarna in twee aparte industriele wyngisrasse van S. cerevisiae by die URA3 lokus geintegreer. Vir die seleksie van die industriele wyngistransformante in die afwesigheid van ouksotrofiese merkers, is die dominante selekteerbare Geneticin G418 weerstandbiedende (G f) merker in die END1- en XYA/4-geenkassetbevattende plasmied geintegreer. Die dominante selekteerbare sulfumetronmetielweerstandbiedende (SMR1) merker is in die integreringsplasmied, wat die PEH1- PELE-geenkasset bevat, geintegreer vir seleksie. Transformasie van hierdie plasmiede in kommersiele wyngisrasse het tot die direkte sintese van die END1-, XYN4-, PELE- en PEH1-transkripte aanleiding gegee, sowel as tot die produksie van die biologies aktiewe ekstrasellulere endo-P-1,4-glukanase, endo-P-xylanase, pektaatliase en poligalaturonase. Tydens die wynmaakproses het bogenoemde rekombinante giste aanleiding gegee tot verhoogde kleurekstraksie uit die druifdoppe van sekere varieteite, asook tot verhoogde vryvloei wyn. Dit is verkry deur die effektiewe degradasie van die glukane, xilane en pektiene in die doppe. Die rekombinante giste het ook verlaagde turbiditeit in die wyn tot gevolg gehad, wat die wyne makliker filtreerbaar maak. Hierdie werk was net die eerste stap. In die toekoms sal verdere navorsing gedoen moet word om die presiese effekte van hierdie rekombinante giste op verskillende rooi en wit druifvarieteite te bepaal. ‘n Ander fokus van hierdie tesis was om nie-Saccharomyces wyngiste vir die produksie van ekstrasellulere hidrolitiese ensieme te selekteer. Die rede hiervoor is om te bepaal watter tipes ekstrasellulere hidrolitiese ensieme geproduseer word, asook watter ensieme deur watter genera geproduseer word, ‘n Totaal van 237 gisisolate wat tot die generas Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora en Kluyveromyces behoort, is vir die produksie van ekstrasellulere pektinase, protease, p-glukanase, lichenase, p-glukosidase, sellulase, xilanase, amilase en sulfiet reduktase-aktiwiteit getoets. Hierdie giste is almal vanaf druiwe en druiwesap geVsoleer om te verseker dat dit wel giste is wat gedurende die beginfases van fermentasie in die mos teenwoordig is. Hierdie inligting kan nou verder gebruik word om die spesifieke effekte wat hierdie ensieme, wat deur die sogenaamde wilde giste geproduseer word, tydens die beginfases van fermentasies op die mos het, te bepaal. Hierdie inligting kan ook in die toekoms gebruik word om Saccharomyces-wyngiste met gene van die ri\e-Saccharomycesgiste te transformeer om ekstrasellulere hidrolitiese ensieme vir die degradasie van die problematiese polisakkariede in wyn te produseer. Daar sal egter in die toekoms baie navorsing gedoen moet word om die omvang van hierdie ensiemaktiwiteite in wynfermentasies te bepaal, asook om meer kennis te bekom oor die fisiologiese en metaboliese samestelling van nie-Saccfraromyces wyngiste.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: ~-Glucosidases constitute a major group of biologically important enzymes that catalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosides that contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in all living kingdoms and perform a variety of functions in organisms ranging from bacteria to highly evolved mammals. Three different types of ~-glucosidases are found in humans, each with its own function: glucocerebrosidase (a deficiency causes Gaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactose intolerance) and cytosolic ~-glucosidase (responsible for the hydrolysis of ~- glucosides ingested with foods of plant and animal origin). In plants, the functions of ~-glucosidases include pigment metabolism, biomass conversion and cyanogenesis, a function it shares with insect ~-glucosidases. Microbial ~-glucosidases, as part of the cellulase enzyme system that is responsible for the hydrolysis of cellobiose and short-chain oligosaccharides into glucose, playa role in the conversion of cellulosic biomass to liquid fuel. These microbial ~-glucosidases also playa very important role in the enhancement of fruit and wine aromas through the liberation of monoterpenols. Monoterpenols play an invaluable role in the flavor and aroma of grapes and wine, and are present as free, volatile and odorous molecules, as well as flavorless, non-volatile glycosidic complexes. These complexes most often occur as 6-0-~-Dxvlopyranosyl- B-Dcqlucopyranosides, 6-0-~-D-glucopyranosyl-~-D-glucopyranosides, 6-0-a-L-arabinofuranosyl-~-D-glucopyranosides, 6-0-a-L-rhamnopyranosyl-~-Dglucopyranosides, or 6-0-~-D-apiofuranosyl-~-D-glucopyranosides of mainly linalool, geraniol, nerol, a-terpineol and hotrienol. Two mechanisms exist for the release of monoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis and enzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangement of the monoterpene aglycones, the focus has shifted towards the more efficient enzymatic hydrolysis that does not result in modifications of the intrinsic aromatic character of the wine. The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wine yeast Saccharomyces cerevisiae, exhibit very low activity towards the glycoside precursors, and thus the focus has increasingly fallen on the addition of exogenous ~-glucosidases to enhance wine flavor. Fungal, bacterial and some yeast ~- glucosidases have been indicated as effective aroma liberators, but these enzymes are not always suitable for use under the harsh conditions that prevail during winemaking (i.e. low pH, low temperatures, and high ethanol and glucose concentrations). The limited enzyme activities of the abovementioned microorganisms have resulted in a search among non-Saccharomyces yeasts for ~- glucosidases that can withstand these conditions. The ~-glucosidase activities of 20 wine-associated non-Saccharomyces yeasts were quantified, characterized and assessed to determine the efficiency with which they could liberate monoterpenols from their terpenyl-glycosides. The Debaryomyces pseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited the most suitable combination of properties in terms of functionality at wine pH, resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfur dioxide), high substrate affinity and large aglycone-substrate recognition. This yeast strain was also used, in conjunction with S. cerevisiae VIN13, for the small-scale fermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D. pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, as the concentrations of free terpenols (nerol, geraniol and citronellol) were significantly increased during fermentation. Future experimental work would include an in-depth study of the kinetic characteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibiting the highest terpenol-liberating activity under winemaking conditions. The next step would then be the cloning and expression of the most efficient l3-glucosidase gene in a commercial wine yeast. Such a recombinant wine yeast would release grapederived aroma compounds from their non-volatile precursors during single culture fermentations, thereby increasing the sensorial quality of wine. / AFRIKAANSE OPSOMMING: I3-Glukosidases vorm deel van 'n groot groep biologies belangrike ensieme wat die hidrolise van glikosidiese bindings binne l3-glukosiede,sowel as binne glikosiede wat slegs uit koolhidraatresidue bestaan, soos bv. sellobiose, kataliseer. Hierdie ensieme kom in alle koningkryke van lewende organismes voor en verrig 'n wye verskeidenheid funksies binne organismes wat wissel van bakterieë tot hoogs ontwikkelde soogdiere. Drie verskillende tipes l3-glukosidases,elk met sy eie funksie, kom in mense voor: glukoserebrosidase ('n gebrek hieraan lei tot Gaucher-siekte), laktaseflorizinhidrolase ('n gebrek hieraan gee aanleiding tot laktose-intoleransie) en sitosol l3-glukosidase (verantwoordelik vir die hidrolise van l3-glukosiede wat saam met voedsel van plant en dier oorsprong ingeneem word). Die funksies van 13- glukosidase binne plante sluit in pigmentmetabolisme, biomassa-omsetting en sianogenese, wat ook 'n funksie van insek l3-glukosidases is. Mikrobiese 13- glukosidases, as deel van die sellulase-ensiemsisteem wat verantwoordelik vir die hidrolise van sellobiose en kortketting-oligosakkariede na glukose is, speel 'n rol in die omsetting van sellulosebiomassa na brandstof. Hierdie mikrobiese 13- glukosidases speelook 'n baie belangrike rol in die verbetering van vrugte- en wynaroma deur die vrystelling van monoterpenole. Monoterpenole speel 'n belangrike rol in die geur en aroma van druiwe en wyn, en kom voor as vry, vlugtige en aromatiese molekules, asook geurlose, nie-vlugtige glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0- I3-D-xilopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-arabinofuranosiel-I3-D-glukopiranosiede, 6-0-I3-D-glukopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-ramnopiranosiel- I3-D-glukopiranosiede,of 6-0-I3-D-apiofuranosiel-I3-D-glukopiranosiedevan hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Monoterpenole kan op een van twee maniere van hul suikermolekules vrygestel word: suurhidrolise of ensimatiese hidrolise. Die hoë temperature waarby suurhidrolise plaasvind veroorsaak 'n herrangskikking van die monoterpeen aglikone, en die fokus het gevolglik verskuif na die meer effektiewe ensimatiese hidrolise wat nie verandering van die intrinsieke aromatiese karakter van die wyn tot gevolg het nie. Die endogene l3-glukosidases van druiwe (Vitis vinifera) en die wyngis Saccharomyces cere visiae , toon baie lae aktiwiteit ten opsigte van die aromatiese voorlopers, en dus word daar toenemend op die toevoeging van eksogene 13- glukosidases tot die wyn gefokus om meer geur vry te stel. Daar is bevind dat 13- glukosidases van fungiese, bakteriële en gis oorsprong effektiewe aromavrystelIers is, maar hierdie ensieme is nie altyd gepas vir gebruik in wyn nie, aangesien dit 'n omgewing is met 'n lae pH, lae temperatuur, en hoë etanol- en glukosekonsentrasies. Die beperkte ensiemaktiwitiet van bogenoemde mikroorganismes het gelei tot 'n soeke onder nie-Saccharomyces giste na l3-glukosidases wat in die wynomgewing kan funksioneer. Die ~-glukosidase-aktiwiteit van twintig wyn geassosieerde nie-Saccharomyces giste is gekwantifiseer en gekarakteriseer om te bepaal tot watter mate dit monoterpenole van hul terpeniel glikosiede kan vrystel. Die intrasellulêre ~- glukosidase teenwoordig in the selekstrak van Debaryomyces pseudopolymorphus, het die belowendste resultate getoon ten opsigte van funksionaliteit by wyn se pH, weerstand teen wyn geassosieerde inhibeerders (glukose, etanol en swaweidioksied), hoë substraataffiniteit en breë aglikoon-substraat herkenning. Hierdie gisras is ook in kombinasie met S. cerevisiae VIN13 gebruik vir die kleinskaalse fermentasie van Chardonnay sap. Die resultate het getoon dat die ~- glukosidase van D. pseudopolymorphus wel potensiaal het om wynaroma te verhoog, aangesien die konsentrasie van ongebonde terpenole (nerol, geraniol en citronellol) aansienlik tydens fermentasie toegeneem het. Toekomstige eksperimentele werk sluit in, onder meer, In in-diepte studie van die kinetiese eienskappe van die ~-glukosidases (beide sitesolies en sel-geassosieerd) wat die meeste terpenole onder wynrnaakkondisies vry stel, asook die klonering en uitdrukking van die enkele ~-glukosidasegeen met die hoogste aktiwiteit, in In kommersiële wyngis. Só In rekombinante wyngis sal die vrystelling van druifgebaseerde aromakomponente van hul nie-vlugtige, geurlose voorlopers tydens enkel-kultuur fermentasies teweeg bring.

Com o aumento da população e crescimento da produção industrial, há uma crescente quantidade de resíduos provenientes destas transformações. Neste estudo o foco são os coprodutos do processo de vinificação, conhecidos como bagaço. Anualmente são produzidas no mundo 60 milhões de toneladas de uva, das quais 80% são utilizadas no processo de vinificação, gerando toneladas de co-produtos orgânicos pouco aproveitados. Este é caracterizado por conter altas concentrações de compostos fenólicos, os quais podem ser reaproveitados como antioxidantes. Assim, o principal objetivo deste projeto foi apresentar a influência de diversos métodos de extração sobre a composição química e a atividade antioxidante destes extratos/frações. Para isso foram realizadas extrações por micro-ondas, ultrassom e líquido-líquido, com solventes de polaridades diferentes (etanol, água, metanol, hexano, clorofórmio e acetato de etila). Além disso, o extrato foi fracionado com auxílio de coluna aberta utilizando sílica como fase estacionária e como fase móvel solventes de diferentes polaridades. A caracterização química foi realizada através de Cromatografia Líquida de Alta Eficiência com detector UV-Vis (CLAE-UV) e Espectrometria de Massas de Alta Resolução (EMAR). Entre os principais compostos identificados estão os fenólicos como a rutina, o ácido gálico, a catequina, e alguns carboxilatos como o ácido linoleico e o ácido esteárico. Nas extrações realizadas podemos identificar que o solvente etanol, em todos os métodos de extração, obteve melhores resultados referente a qualificação e quantificação de compostos químicos em análise com CLAE-UV. A avaliação da capacidade antioxidante foi realizada através do método 2,2-difenil-1-picrilhidrazil (DPPH•). Os dados obtidos por extração micro-ondas mostraram uma maior porcentagem de varredura (2,44%±36,64), quando o solvente utilizado foi o etanol. Na extração por sonografia, os valores mais elevados 2,07%±74,22 foram observados utilizando-se acetato de etila. Na extração líquido-líquido o maior percentual de varredura (0,61±33,62) foi observado para o solvente acetato de etila. Em geral, os extratos obtidos por extração com micro-ondas apresentaram maior teor de compostos fenólicos (0,04±13,18 a 6,02mg±107,03 GAE/100g). Os resultados obtidos neste projeto indicam que este co-produto pode ser melhor aproveitado para a aplicação como uma fonte de ingredientes funcionais, como suplementos alimentares ou na indústria cosmética, por conterem compostos importantes (sic). / Submitted by Ana Guimarães Pereira (agpereir@ucs.br) on 2017-11-24T17:18:31Z No. of bitstreams: 1 Dissertacao Vania Pezzini.pdf: 896272 bytes, checksum: a82ee0965fb04dc09ece21930de3f4fa (MD5) / Made available in DSpace on 2017-11-24T17:18:31Z (GMT). No. of bitstreams: 1 Dissertacao Vania Pezzini.pdf: 896272 bytes, checksum: a82ee0965fb04dc09ece21930de3f4fa (MD5) Previous issue date: 2017-11-24 / With increasing population and industrial production growth, there is an increasing amount of waste coming from these transformations. The focus of this study will be on the co-products of the winemaking process, known as grape marc. Every year 60 million tons of grape are produced, 80% of which are used in the winemaking process, generating tons of underutilized organic co-products. This is characterized by its high concentrations of phenolic compounds which can be reused as antioxidants. Thus, the main goal of this project was to present the influence of different extraction methods on chemical composition and antioxidant activity of these extracts/fractions. For this, the extractions were performed by microwave, ultrasound and liquid-liquid, with different polarities solvents (ethanol, water, methanol, hexane, chloroform and ethyl acetate). Moreover, the extract was fractionated with the aid of an open column using silica as stationary phase and different polarities solvents as mobile phase. The chemical characterization was performed by High Performance Liquid Chromatography with UV-Vis detector (CLAE) and High Resolution Mass Spectrometry (HRMS). Among the main compounds identified are phenolics such as rutin, gallic acid, catechin, and some carboxylates such as linoleic acid and stearic acid. In the extracted extractions we can identify that the ethanol solvent, in all extraction methods, obtained better results regarding the qualification and quantification of chemical compounds in analysis with HPLC-UV. The evaluation of antioxidant capacity was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH •) method. The data obtained by microwave extraction showed a higher percentage of scanning (2,44%±36,64), when the solvent used was ethanol. In the extraction by sonography, the highest values 2,07%±74,22 were observed using ethyl acetate. In the liquid-liquid extraction the highest percentage of sweep (0,61±33,62) was observed for the solvent ethyl acetate. In general, the extracts obtained by extraction with microwaves presented higher levels of phenolic compounds (0,04±13,18 a 6,02mg±107,03 GAE/100g). The results obtained in this project indicate that this co-product can be better utilized for the application as a source of functional ingredients, as food supplements or in the cosmetic industry, because they contain important compounds (sic).

Com o aumento da população e crescimento da produção industrial, há uma crescente quantidade de resíduos provenientes destas transformações. Neste estudo o foco são os coprodutos do processo de vinificação, conhecidos como bagaço. Anualmente são produzidas no mundo 60 milhões de toneladas de uva, das quais 80% são utilizadas no processo de vinificação, gerando toneladas de co-produtos orgânicos pouco aproveitados. Este é caracterizado por conter altas concentrações de compostos fenólicos, os quais podem ser reaproveitados como antioxidantes. Assim, o principal objetivo deste projeto foi apresentar a influência de diversos métodos de extração sobre a composição química e a atividade antioxidante destes extratos/frações. Para isso foram realizadas extrações por micro-ondas, ultrassom e líquido-líquido, com solventes de polaridades diferentes (etanol, água, metanol, hexano, clorofórmio e acetato de etila). Além disso, o extrato foi fracionado com auxílio de coluna aberta utilizando sílica como fase estacionária e como fase móvel solventes de diferentes polaridades. A caracterização química foi realizada através de Cromatografia Líquida de Alta Eficiência com detector UV-Vis (CLAE-UV) e Espectrometria de Massas de Alta Resolução (EMAR). Entre os principais compostos identificados estão os fenólicos como a rutina, o ácido gálico, a catequina, e alguns carboxilatos como o ácido linoleico e o ácido esteárico. Nas extrações realizadas podemos identificar que o solvente etanol, em todos os métodos de extração, obteve melhores resultados referente a qualificação e quantificação de compostos químicos em análise com CLAE-UV. A avaliação da capacidade antioxidante foi realizada através do método 2,2-difenil-1-picrilhidrazil (DPPH•). Os dados obtidos por extração micro-ondas mostraram uma maior porcentagem de varredura (2,44%±36,64), quando o solvente utilizado foi o etanol. Na extração por sonografia, os valores mais elevados 2,07%±74,22 foram observados utilizando-se acetato de etila. Na extração líquido-líquido o maior percentual de varredura (0,61±33,62) foi observado para o solvente acetato de etila. Em geral, os extratos obtidos por extração com micro-ondas apresentaram maior teor de compostos fenólicos (0,04±13,18 a 6,02mg±107,03 GAE/100g). Os resultados obtidos neste projeto indicam que este co-produto pode ser melhor aproveitado para a aplicação como uma fonte de ingredientes funcionais, como suplementos alimentares ou na indústria cosmética, por conterem compostos importantes (sic). / With increasing population and industrial production growth, there is an increasing amount of waste coming from these transformations. The focus of this study will be on the co-products of the winemaking process, known as grape marc. Every year 60 million tons of grape are produced, 80% of which are used in the winemaking process, generating tons of underutilized organic co-products. This is characterized by its high concentrations of phenolic compounds which can be reused as antioxidants. Thus, the main goal of this project was to present the influence of different extraction methods on chemical composition and antioxidant activity of these extracts/fractions. For this, the extractions were performed by microwave, ultrasound and liquid-liquid, with different polarities solvents (ethanol, water, methanol, hexane, chloroform and ethyl acetate). Moreover, the extract was fractionated with the aid of an open column using silica as stationary phase and different polarities solvents as mobile phase. The chemical characterization was performed by High Performance Liquid Chromatography with UV-Vis detector (CLAE) and High Resolution Mass Spectrometry (HRMS). Among the main compounds identified are phenolics such as rutin, gallic acid, catechin, and some carboxylates such as linoleic acid and stearic acid. In the extracted extractions we can identify that the ethanol solvent, in all extraction methods, obtained better results regarding the qualification and quantification of chemical compounds in analysis with HPLC-UV. The evaluation of antioxidant capacity was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH •) method. The data obtained by microwave extraction showed a higher percentage of scanning (2,44%±36,64), when the solvent used was ethanol. In the extraction by sonography, the highest values 2,07%±74,22 were observed using ethyl acetate. In the liquid-liquid extraction the highest percentage of sweep (0,61±33,62) was observed for the solvent ethyl acetate. In general, the extracts obtained by extraction with microwaves presented higher levels of phenolic compounds (0,04±13,18 a 6,02mg±107,03 GAE/100g). The results obtained in this project indicate that this co-product can be better utilized for the application as a source of functional ingredients, as food supplements or in the cosmetic industry, because they contain important compounds (sic).

Os vinhos brasileiros, principalmente do Rio Grande do Sul, apresentam, muitas vezes, elevada acidez decorrente da maturação diferente das uvas. Esta acidez é determinada por altas concentrações de ácido málico. Para atenuar a acidez fixa nos vinhos, os mostos são submetidos à fermentação malolática durante ou seguidamente a fermentação alcoólica. Na prática enológica, essa fermentação representa problemas, ocorrendo de forma descontrolada e em muitos casos não chegando a finalizar. Ainda que muitas vezes eficiente, a fermentação malolática espontânea, dependente das bactérias presentes na uva ou na cantina, varia de fermentação para fermentação e de ano para ano, sendo hoje um dos pontos críticos no processo de vinificação. Estirpes de Oenococcus oeni e Lactobacillus sp., espécies de bactérias láticas, são predominantes nesse processo fermentativo. Os parâmetros como pH, dióxido de enxofre (SO2), conteúdo alcoólico e temperatura são os mais importantes que afetam o desempenho dessas bactérias no vinho. Outras espécies bacterianas podem adicionar características indesejáveis no produto final, como a produção de aminas bioativas, odor impertinente e bacteriocinas que inibem a fermentação malolática. Esse trabalho visou a seleção e identificação de bactérias láticas nativas de vinícolas da Serra Gaúcha. Foram isoladas 34 bactérias provenientes de amostras de vinhos que se encontravam em fase de fermentação malolática, e dessas, apenas 16 foram classificadas como láticas, pois apenas essas apresentaram características de catalase negativa, Gram positivas e imóveis. Nem todas as 16 bactérias obtiveram bom crescimento em meio líquido. O ensaio fermentativo de glicose com avaliação da produção de ácido e gás demonstraram que alguns isolados não acidificaram e nem produziram gás. Já o teste de fermentação de carboidratos, que utilizou 14carboidratos, constatou que as bactérias possuem preferência em metabolizar dissacarídeos e monossacarídeos. Nos testes fermentativos em mosto sintético, alguns isolados conseguiram alterar significativamente o pH, reduzindo a acidez, porém não apresentaram turbidez. A fermentação malolática nesse experimento foi acompanhada qualitativamente em cromatografia de papel e, mesmo com problemas de crescimento, alguns isolados conseguiram efetuar uma boa fermentação malolática. Com relação à avaliação da sensibilidade ao metabissulfito de potássio (K2S2O5), diferentes níveis de Ph e diferentes concentrações de etanol, os isolados desenvolveram–se melhor nas concentrações mais baixas de K2S2O5 e em graduação alcóolica menor. Os níveis de pH próximos a 3,5 foram os preferidos para o desenvolvimento bacteriano. A existência da crovinificação em vinho sintético com grau etanólico de 10%, apenas por quatro isolados que demonstraram boa fermentação malolática em mosto, certificou que quanto menor a concentração de álcool, menor também é a interferência na fermentação. Nesse experimento avaliou-se a degradação málica quali/quantitativamente, com utilização de cromatografia de papel e kit enzimático. Houve alteração de pH, comprovando a conversão do ácido málico em lático por todos os isolados. A classificação final dos isolados os enquadrou com as espécies Lactobacillus suebicus, Lactobacillus pentosus e Lactobacillus plantarum. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, CAPES / Brazilian wines, mainly from Rio Grande do Sul, often have high acidity due to the different maturation of the grapes. This acidity is determined by high concentrations of malic acid. In order to attenuate the fixed acidity in the wines, the musts are subjected to malolactic fermentation during or after the alcoholic fermentation. In oenological practice, this fermentation represents problems, occurring in an uncontrolled way and in many cases not coming to an end. Although often efficient, spontaneous malolactic fermentation, depending on the bacteria present in the grape or canteen, varies from fermentation to fermentation and from year to year, and is now one of the critical points in the winemaking process. Strains of Oenococcus oeni and Lactobacillus sp., Species of lactic bacteria, are predominant in this fermentation process. Parameters such as pH, sulfur dioxide (SO2), alcohol content and temperature are the most important that affect the performance of these bacteria in wine. Other bacterial species may add undesirable characteristics to the final product, such as the production of bioactive amines, impertinent odor and bacteriocins that inhibit malolactic fermentation. This work aimed at the selection and identification of native lactic bacteria from the Serra Gaúcha wineries. A total of 34 bacteria were isolated from samples of malolactic fermentation, of which only 16 were classified as lactic acid. Not all 16 bacteria obtained good growth in liquid medium. The fermentative glucose test with evaluation of acid and gas production showed that some isolates did not acidify or produce gas. Already the carbohydrate fermentation test, which used 14 carbohydrates, found that the bacteria have a preference inmetabolizing disaccharides and monosaccharides. In the fermentative tests in synthetic wort, some isolates were able to significantly alter the pH, reducing the cidity, but did not present turbidity. The malolactic fermentation in this experiment was accompanied qualitatively in paper chromatography and, even with growthproblems, some isolates were able to effect a good malolactic fermentation. With respect to the evaluation of the sensitivity to potassium metabisulphite (K2S2O5), different pH levels and different concentrations of ethanol, the isolates were better eveloped at lower concentrations of K2S2O5 and lower alcoholic strength. PH levels near 3.5 were preferred for bacterial development. The existence of microvinification in synthetic wine with 10% ethanolic degree, only by four isolates that demonstrated good malolactic fermentation in must, certified that the lower the alcohol concentration, the lower the interference in the fermentation. In this experiment, the degradation was evaluated qualitatively / quantitatively, using paper chromatography and enzymatic kit. There was a change in pH, confirming the conversion of malic acid to lactic acid by all isolates. The final classification of the isolates included the species Lactobacillus suebicus, Lactobacillus pentosus and Lactobacillus plantarum

Before 1994 the South African wine industry was largely isolated from the global market. Access to markets after 1994 meant that South Africa had to adapt its product quality, style and operating procedures to compete internationally. In recent years, labour unrest and trade regulations have hampered both the reputation and the ability of South African wine producers to penetrate the export market. Using the theoretical framework of cluster theory and generic marketing this inquiry investigates whether regional groupings and clusters could result in the South African wine industry becoming more competitive. This inquiry examines the role of regional identity, generic marketing, research and development, policy, and leadership in driving competitiveness in the wine industry and whether the potential for clusters exists through wine routes in South Africa. The study employs an exploratory research model which utilised semi structured interviews. The findings suggest that the research and development component of South Africa’s wine industry lags behind other New World wine producing nations, and that none of the three tiers of government are in the process of creating conditions which would be conducive for the formation of clusters. The results also indicate that wine routes have a positive effect on regional identity, and that generic marketing complements the activities of boutique wine farmers. / Dissertation (MBA)--University of Pretoria, 2013. / lmgibs2014 / Gordon Institute of Business Science (GIBS) / MBA / Unrestricted

Emerging Technologies Sustainability Assessment (ETSA) is a new technology assessment tool that was developed in order to compare emerging processes or technologies to existing alternatives. It utilizes infoIDlation modules, with the minimum use of resources such as time and money, in order to deteIDline if the process under development is comparatively favourable and should be developed beyond the early conceptual phase. The preliminary ETSA is vital in order to identify the gaps in the existing information and the specific methodologies to be used for data capture and analysis. The use of experimental design tools, such as Design-Expert, can facilitate rapid and efficient collection of necessary data and fits in well with the rationale for the ETSA. Wine distillery effluent (vinasse) is the residue left after alcohol has been distilled from fennented grape juice. It is an acidic, darkly coloured effluent, with a high COD and polyphenol content. The most popular method of disposal of this effluent, land application, is no longer viable due to stricter legislation and pressure on the industry to better manage its wastes. Although the ability of whiterot fungi to degrade a number of pollutants is well-known, fungal treatment of wine distillery effluent is still in the conceptual phase. The perfoIDlance of the fungal remediation system was assessed experimentally in terms of COD removal and laccase production using Design-Expert. Although Pycnoporus sanguine us was found to be most efficient at COD removal (85%) from 30% vinasse, laccase production was low (0.021 U/I). The optimum design for economically viable fungal treatment used Trametespubescens. This fungus was able to remove over 50% of the COD from undiluted vinasse while producing almost 800U/l of the valuable laccase enzyme within three days. Since the effluent from the fungal system did not meet the legal limits for wastewater disposal, a two-stage aerobicanaerobic system is suggested to improve the quality of the effluent prior to disposal. The ETSA was used to assess the fungal technology in relation to the two current methods of vinasse treatment and disposal, namely land application and anaerobic digestion. Based on the ETSA, which considered environmental, social and economic impacts, the fungal system proved to be potentially competitive and further development of the technology is suggested.

Phenolic and color parameters of Marechal Foch and deChaunac wines of 1980 through 1983 vintages were analysed to determine variety and aging effects. Centroid Mapping Optimization together with the Simultaneous Factor Shift algorithm were used to determine the HPLC operating conditions which resolved the maximum number of phenolic components of whole red wine. A ternary gradient system using 1 % acetic acid : methanol : acetonitrile was changed from 100:0:0 to 4.8:82.8:12.4 in 130.6 minutes. A flow rate of 1.1 mL/min and a temperature of 32.9 °C were used. The HPLC system was used to separate at least 50 components from each injection of wine and of an ethyl acetate extract of wine. Foch wines were found to have more catechin and epicatechin than deChaunac wines. Peak areas for the ethyl acetate extracts common to all wines, and areas of peaks in the chromatograms of the whole wine, not present in the neutral extract, were used to form a data set for multivariate analyses. Strong linear correlations were found between tristimulus and spectrophotometry measurements for each wine. The color of Foch wines was more stable and contained more brown and yellow hues than deChaunac wines, as determined by tristimulus measurements. Total pigment levels of each wine and various fractions of the total, including ionized, un-ionized, polymeric and sulfur dioxide-bound anthocyanins, were determined spectro-photometrically. Total anthocyanin levels (un-ionized and ionized) in deChaunac wines decreased significantly with increasing age but remained constant in Foch wines. Tannin levels as determined by absorbance readings at 280 nm and by the Folin-Coicalteu reagent method were highly correlated. Foch had more flavonoid and less nonflavonoids than deChaunac wines. The different color parameters, pigment and tannin fractions, as well as titratable acidity, pH and individual organic acids were used as a second data set of analytical parameters for multivariate analyses. A third data set composed of the combination of the analytical data and the HPLC peak areas was also used. Differences between the four vintages of wines, the two varieties, and between old and young wines within varieties were found using stepwise discriminant analysis (SDA). Discrimination of variety differences was more successful (100% correct classification by the jackknife procedure) and required fewer variables than classification by age. Cluster analyses, performed with variables chosen by SDA, gave similar results to the SDA. / Land and Food Systems, Faculty of / Graduate

Orientadores: Helena Teixeira Godoy, Carlos Eugenio Daudt / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T11:15:55Z (GMT). No. of bitstreams: 1 Facco_ElizeteMariaPesamosca_D.pdf: 27687746 bytes, checksum: d51d71b95043f23c20948f904cbac415 (MD5) Previous issue date: 2006 / Doutorado / Doutor em Ciência de Alimentos

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