Monitoring the spreading of commercial wine yeasts in the vineyard

Muller, Christo A. (2003-12)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Traditionally, wine has been produced by the spontaneous fermentation of grape juice by yeast that originate from the grapes and winery equipment. Research has shown that the population composition and dynamics of these yeasts and other microorganisms are very complex. Kloeckera and its anamorph, Hanseniaspora, dominate the yeast population found on the surfaces of grapes, although prevailing Saccharomyces cerevisiae strains complete the fermentation process. The yeast S. cerevisiae is an important factor contributing to the quality of wines and, therefore, the improvement of wine yeasts receives considerable attention worldwide. Apart from classical yeast breeding studies, genetic engineering and recombinant DNA techniques are increasingly being used in strain development research programmes. These techniques might enable the wine yeasts to produce heterologous enzymes that degrade polysaccharides, convert malic acid to lactic acid, increase glycerol production, release roam and flavour compounds, secrete antimicrobial peptides, etc. The release of recombinant yeast strains (genetically modified organisms, GMOs) is subject to statutory approval. Therefore, it is important to answer several questions prior to the use of such genetically improved yeast in the commercial production of wine. For example, will recombinant yeast strains be able to multiply and spread in nature, and will this GMO be able to out-compete the natural microflora because of its newly acquired genetic traits. Since existing commercial wine yeasts are used in the abovementioned strain development research, it is essential to determine already at this early stage to what extent these wine yeast strains survive and spread in nature and to what extent they influence the fermentations of the following vintages. This study is divided into two sections. The aim of the first section is to sample a representative number of yeast strains from various vineyards in different climatological areas, mainly in the Western Cape, South Africa. These yeast strains were identified mainly by electrophoretic karyotyping (contour-clamped homogenous electric field electrophoresis; CHEF). The second part of the study summarises the results obtained when Fourier transform infrared (FT-NIR) spectroscopy was used to differentiate commercial wine yeast strains. Sets of data, containing the spectra of the mostly used commercial wine yeast strains, were constructed and used as a reference library. The spectra of the isolated yeast strains were then compared to the reference dataset with specific FT-NIR computer software using mathematical calculations. In conclusion, the two methods used in conjunction with one another proved that the commercial wine yeast strains do not easily disperse from the cellar into the vineyard. The commercial wine yeast strains are also more likely to be found near the cellar and the places where the grape skins are dumped. Therefore, should a recombinant yeast strain be used in winemaking, it would not be dispersed into the vineyard. It therefore appears that the commercial use of genetically improved yeast does not pose a high risk in terms of dominance of the indigenous microbial population in the environment

AFRIKAANSE OPSOMMING: Wyn is tradisioneel gemaak deur die natuurlike gisting van druiwesap deur giste wat op die druiwe en keldertoerusting voorkom. Navorsing het getoon dat die samestelling en dinamika van die gispopulasie en ander mikro-organismes baie kompleks is. Kloeckera en sy anamorf, Hanseniaspora, domineer die inheemse gispopulasie op druiwedoppe, terwyl Saccharomyces cerevisiae in baie klein getalle op die druiwedoppe voorkom, maar later die fermentasie oorheers en uiteindelik voltooi. Die gis S. cerevisiae speel 'n baie belangrike rol in die kwaliteit van wyn en daarom geniet die verbetering van wyngiste wêreldwyd besondere aandag. Benewens die klassieke gistelingstudies, word genetiese manipuleringstegnieke toenemnd in navorsingsprojekte gebruik wat daarop gefokus is om wyngisrasse te verbeter. Hierdie tegnieke mag die giste in staat stelom heteroloë ensieme te produseer wat polisakkariedes afbreek, appelmelksuur afbreek, gliserolproduksie verhoog, smaak- en geurkomponente vrystel, antimikrobiese peptiede afskei, ens. Voordat sulke geneties gemanipuleerde giste het egter in kommersiële wynproduksie gebruik sal kan word, is daar heelwat wetlike vereistes waaraan voldoen sal moet word en vrae wat vooraf beantwoord sal moet word. Byvoorbeeld, sal die rekombinante giste in staat wees om vinniger te vermeerder as gevolg van die nuwe genetiese eienskappe en sodoende die natuurlike populasies onderdruk? Omdat kommersiële wyngiste in bogenoemde gisverbeteringprogramme gebruik word, is dit noodsaaklik om nou reeds die verspreiding van die kommersiële giste te monitor en te bepaal hoe geredelik hulle in die natuur kan versprei en oorleef, en hoe hulle wynfermentasies van die daaropvolgende jare beïnvloed. Die studie is in twee gedeeltes verdeel. Die doel van die eerste gedeelte was om 'n verteenwoordigende aantal gisrasse uit die wingerde van 'n aantal wynplase in verskillende klimaatstreke te isoleer, spesifiek in die Wes-Kaap, Suid-Afrika. Die gisrasse was grotendeels deur elektroforetiese kariotipering (kontoer-geklampte homogene elektriese veld; CHEF) geïdentifiseer. Die tweede deel van die navorsing was gefokus op die onderskeiding tussen die mees gebruikte kommersiële wyngiste met 'Fourier-Transform Near Infrared' (FTNIR) spektroskopie. Eerstens is 'n stel data, bestaande uit die spektrum data oor die kommersiële wyngiste opgestel om as 'n verwysingsbiblioteek te dien. Tweedens is die spektrum van data oor die geïsoleerde giste onder presies dieselfde toestande met die verwysingsbiblioteek vergelyk. Dié tegniek maak dit moontlik om tussen die kommersiële wyngiste te onderskei. As die twee metodes saam gebruik word vir identifikasie, kan die afleiding gemaak word dat kommersiële wyngiste nie maklik vanaf die kelder na die wingerd versprei nie. Die kommersiële wyngiste is ook meestal naby die kelder en die dopstortingsterreine gevind. Sou 'n rekombinante gisras dus gebruik word om wyn te maak, sal dit nie maklik versprei nie. Die kommersiële gebruik van geneties gemanipuleerde wyngiste behoort dus nie In groot omgewingsrisiko in te hou nie.

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