The development of polysaccharide degrading wine yeast strains

Louw, Campbell (Campbell Trout) (Stellenbosch : University of Stellenbosch, 2004-12)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: The polysaccharides that are present in wine originate from the grapes, the fungi that grow on the grapes and from other microorganisms that come into contact with the must during winemaking. The grape-derived polysaccharides of most concern in winemaking are pectin, glucan and xylan that can be enzymatically degraded by pectinases, glucanases and xylanases, respectively. These are the main structural polysaccharides of the cell wall of the grape cell. Degradation of the cell walls will result in the separation and rupture of the grape cells, and cell wall-bound compounds will be released into the must. Treating the must with pectinase and macerating enzyme preparations can result in an increase in free-flow juice, an improvement in must clarification and filtration, and an increased extraction of phenols and tannins. The tannins that are extracted polymerise with anthocyanins in red wine during ageing, resulting in increased colour intensity and stability. Wine aroma is also influenced by enzyme treatment. The degradation of the cell wall contributes to the release of glycosidically-bound terpene or alcohol precursors from the berries. The hydrolysis of these precursors during fermentation can result in an improvement in aroma. It can thus be seen that it is possible to improve wine quality and processing by supplementing the endogenous enzymes that are present in the fermentation with commercial enzyme preparations. Commercial enzymes are typically crude fungal preparations. The majority of commercial pectinase and glucanase preparations are derived from Aspergillus and Trichoderma, respectively. Since the endogenous polysaccharase activity of Saccharomyces cerevisiae is very limited, the heterologous expression of specific polysaccharase genes in an industrial yeast strain can improve the winemaking process, resulting in a higher quality wine without the addition of expensive commercial enzyme preparations. Since only the desired enzymes are secreted by the recombinant strain, there will be no undesired sideactivities, which can be detrimental to wine quality. Several pectinase-, glucanaseand xylanase-encoding genes, cloned from a variety of organisms, have been expressed successfully in laboratory strains of S. cerevisiae. Attempts have also been made to construct industrial wine yeast strains that express these polysaccharase genes and secrete the encoded enzymes. Fermentation with some of these strains resulted in a decrease in total phenolics and turbidity, an increase in juice extraction, and alterations in the colour and aromatic profile of the resulting wines. In this study, four polysaccharide-degrading, recombinant wine yeast strains were constructed. The endo-β-1,4-xylanase gene, XYN2, and the endo-β-1,4-glucanase gene, end1, were previously cloned from the soft rot fungus Trichoderma reesei and the rumen bacterium Butyrivibrio fibrisolvens, respectively. These genes were subcloned into different expression cassettes which were used to construct the four integration plasmids. The recombinant plasmids contained the following gene cassettes: TEF1P-XYN2-ADH2T (plasmid pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmid pDLG30) ADH1P-MFα1S-end1-TRP5T and ADH2P-XYN2-ADH2T (plasmid pDLG33), ADH1P-MFα1S-end1-TRP5T and YG100PXYN2- ADH2T (plasmid pDLG39). These four plasmids were then separately integrated into the ILV2 locus of the commercial wine yeast strain S. cerevisiae VIN13. Wine was made with the four strains constructed in this study, a pectolytic strain, VIN13[pPPK], a glucanase- and xylanase-secreting strain, VIN13[pEX], an untransformed VIN13 strain, and an untransformed strain with the addition of the commercial enzyme preparation Rapidase EX Colour. Microvinification experiments were carried out on Pinot noir, Ruby Cabernet and Muscat d’Alexandria wines. Fermentation with the polysaccharide-degrading strains resulted in significant improvements in juice extraction, colour intensity and stability, and in alterations in the aromatic profiles of the wines produced. Subject to the approval by the regulatory authorities and eventual consumer acceptance of the use of genetically modified organisms (GMOs) in fermented foods and beverages, it might be required that the GM status of the yeast that is used appears on the label. Currently, there is no robust technique available with which the use of GM yeast can be revealed in a finished wine because the yeast cells and their DNA are removed from or denatured in the wine during filtration and processing. One way with which the undeclared use of a GM yeast in winemaking could be exposed would be to compare the chemical profile of a suspect wine with that of non-GM wine. In order to explore this concept further, a secondary aim of this study was to investigate whether Fourier Transformation Infra Red (FT-IR) spectroscopy coupled with multivariate data analysis could distinguish between wines fermented with transgenic and non-transgenic yeast strains, or between wines fermented with different transgenic strains. The results showed that this method could be used to classify wines fermented with different yeast strains if fermentation with the strain resulted in a unique chemical profile in the resulting wine. This was a preliminary study and these findings were summarised as an addendum to the thesis.

AFRIKAANSE OPSOMMING: Die polisakkariede wat in wyn teenwoordig is, is afkomstig van die druiwe, die swamme wat op die druiwe groei en vanaf ander mikroörganismes wat tydens die wynmaakproses met die mos in aanraking kom. Die belangrikste druifpolisakkariede in wynbereiding is pektien, glukaan en xilaan, wat onderskeidelik deur pektinases, glukanases en xilanases afgebreek kan word. Hierdie is die vernaamste strukturele polisakkariede van ‘n druifsel se selwand. Die afbreking van die selwande veroorsaak dat die druifselle skei en skeur, met die gevolg dat die selwandgebonde verbindings in die mos vrygelaat word. Die behandeling van die mos met pektinase en versappingsensiempreparate kan tot ʼn toename in vry-afloopsap lei, sowel as ʼn verbetering in mosverheldering en -filtrasie en ʼn verhoogde ekstraksie van fenole en tanniene. Die tanniene wat geëkstraheer word, polimeriseer in rooiwyn tydens veroudering, en dit lei tot verhoogde kleurintensiteit en -stabiliteit. Wynaroma word ook deur ensiembehandeling beïnvloed. Die afbreking van die druifselwand dra by tot die vrylating van glikosidiesgebonde terpeen- en alkoholvoorlopers uit die korrels. Die hidrolise van hierdie voorlopers tydens gisting kan lei tot ʼn verbetering van die aroma. Dit is dus duidelik dat dit moontlik is om wynkwaliteit en wynbereiding te verbeter deur die endogene ensieme wat in die gisting teenwoordig is met kommersiële ensiempreparate te supplementeer. Kommersiële ensiempreparate is tipies ongesuiwerde swampreparate. Die meerderheid kommersiële pektinase- en glukanasepreparate word onderskeidelik vanaf Aspergillus en Trichoderma verkry. Aangesien die endogene polisakkaraseaktiwiteit van Saccharomyces cerevisiae baie beperk is, kan die heteroloë uitdrukking van spesifieke polisakkarase-gene in ʼn industriële gisras die wynbereidingsproses verbeter en lei tot ʼn hoër kwaliteit wyn sonder die byvoeging van duur kommersiële ensiempreparate. Omdat die verkose ensieme deur die rekombinante ras uitgeskei word, sal daar geen ongewenste newe-effekte teenwoordig wees wat ʼn nadelige effek op wynkwaliteit kan hê nie. Verskeie mikrobiese gene wat vir pektinases, glukanases en xilanases kodeer, is reeds voorheen uit ‘n wye verskeidenheid van organismes gekloneer en suksesvol in laboratoriumrasse van S. cerevisiae uitgedruk. Pogings is ook aangewend om industriële wyngisrasse te konstrueer wat hierdie polisakkarasegene uitdruk en hul enkodeerde ensieme uitskei. Gisting met sommige van hierdie rekombinante gisrasse het gelei tot ʼn afname in totale fenoliese verbindings en troebelheid, ʼn verhoging in sapekstraksie, en veranderings in die kleur en aromatiese profiel van die gevolglike wyne. In hierdie studie is vier polisakkaried-afbrekende, rekombinante wyngisrasse gekonstrueer. Die endo-β-1,4-xilanasegeen, XYN2, en die endo-β-1,4- glukanasegeen, end1, is voorheen reeds onderskeidelik vanaf die sagte vrotswam, Trichoderma reesei, en die rumenbakterium, Butyrivibrio fibrisolvens, gekloneer. Hierdie gene is in vier integrasieplasmiede in verskillende ekspressiekassette gesubkloneer. Die plasmiede het die volgende geenkassette bevat: TEF1P-XYN2- ADH2T (plasmied pDLG29) ADH1P- MFα1S -end1-TRP5T (plasmied pDLG30) ADH1PMFα1S- end1-TRP5T and ADH2P-XYN2-ADH2T (plasmied pDLG33), ADH1P-MFα1S end1-TRP5T and YG100P-XYN2-ADH2T (plasmied pDLG39). Hierdie vier plasmiede is toe afsonderlik in die ILV2-lokus van die kommersiële wyngisras, S. cerevisiae VIN 13, geïntegreer. Wyn is met hierdie vier gekonstrueerde gisrasse gemaak, die pektolitiese gisras, VIN13[pPPK], die glukanase- en xilanase-afskeidende gisras, VIN13[pEX], die ongetransformeerde VIN13-ras, en met ʼn ongetransformeerde VIN13 gis waarby die kommersiële ensiempreparaat, Rapidase EX Colour, bygevoeg is. Mikro-wynbereidingseksperimente is op Pinot noir-, Ruby Cabernet- en Muscat D’Alexandria wyne uitgevoer. Gisting met die polisakkaried-afbrekende gisrasse het gelei tot ʼn noemenswaardige verbetering in sapekstraksie, kleurintensiteit en kleurstabiliteit, asook in veranderinge in die aromatiese profiele van die geproduseerde wyne. Indien die gebruik van geneties gemodifiseerde organismes (GMOs) in gefermenteerde voedsel en drank deur die reguleringsowerhede goedgekeur en uiteindelik deur die verbruiker aanvaar sou word, sou dit vereis kon word dat die GMstatus van die wyngisgis op die etiket van die wynbottel aangebring word. Verpligte etikettering van GM-wyn sal metodes vereis waarmee die ‘nalentskap’ van GMgisselle in die finale produk geïdentifiseer en gemoniteer kan word. Tans is daar geen robuuste tegnieke beskikbaar waarmee die gebruik van GM-giste openbaar kan word nie, aangesien die gisselle en hul DNA tydens filtrasie en prosessering verwyder word. Een wyse waarop die onverklaarde gebruik van ‘n GM-gis in wynbereiding blootgestel sou kno word, is om die chemiese profiel van die verdagte wyn met dié van ‘n nie-GM-wyn te vergelyk. Ten einde hierdie konsep verder te ondersoek was ‘n sekondêre doelwit van hierdie studie om te bepaal of FT-IR (Fourier-transformasie-infrarooi) spektroskopie tesame met meervariante dataanalise gebruik kan word om te onderskei tussen wyne wat met transgeniese en nietransgeniese gisrasse gegis is, of tussen wyne wat met verskillende transgeniese rasse gegis is. Die resultate het aangedui dat hierdie metode gebruik kan word om wyne wat met verskillende gisrasse gegis is, te klassifiseer indien die betrokke gisras ʼn unieke chemiese profiel in die uiteindelike wyn veroorsaak het. Dit was egter ʼn voorlopige ondersoek en is as ʼn byvoegsel tot die tesis geskryf.

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