Characterization and evaluation of indigenous Saccharomyces cerevisiae strains isolated from South African vineyards

Van der Westhuizen, Theunes Johannes (1999-12)

Thesis (Ph.D.) -- University of Stellenbosch, 1999.

Thesis

ENGLISH SUMMARY: The bioconversion of grape juice into wine by simply allowing the yeasts, associated with grape berries and winery equipment, to ferment the sugars to ethanol, carbon dioxide and other minor, but important metabolites, is an ancient process. The art and science of winemaking has been extensively studied since the time when Louis Pasteur demonstrated, for the first time, the relationship between yeast and alcoholic fermentation. It is now recognized that the fermentation of grape must and production of premium quality wines is a complex ecological and biochemical process involving the sequential development of microbial species, as affected by a particular environment. This complex heterogeneous microbiological process includes the interaction of many microbial species, represented by fungi, yeasts, lactic acid bacteria and acetic acid bacteria, as well as the mycoviruses and bacteriophages affecting these grape-associated microorganisms. However, of all these different microbes and viruses, yeast represent the heart of the harmonious biochemical interaction with the musts derived from the various varieties of Vitis species which, in turn, are largely products of their respective genetic make-ups and the terroir. These yeasts are significant in winemaking because they not only conduct the alcoholic fermentation, but can also spoil wine during conservation in the cellar and after packaging, and they affect wine quality through the production of fermentation metabolites and through autolysis. A sound understanding of yeast systematics, biogeography and ecology is therefore essential to endeavours to preserve and exploit the hidden oenological potential of the untapped wealth of yeast biodiversity in our wine-producing regions. One of the main thrusts of this kind of eco-taxonomic survey is to determine the actual contribution of the indigenous strains of the so-called wine yeast (Saccharomyces cerevisiae) and wild yeasts (non-Saccharomyces species) to the sensory properties of wines and to eventually develop new starter culture strains for guided fermentations, including mixed starter cultures tailored to reflect the characteristics of a given wine region. Against this background, a comprehensive, long-term biogeographical survey and strain development programme was launched. This dissertation represents the first phase of this long-overdue research programme aimed to systematically catalogue yeasts in different climatic zones of the 350-year-old wine-producing regions of the Western Cape and to develop new yeast starter cultures that would further increase the quality of South African wine. The specific aims of this dissertation included (i) the evaluation of yeast fingerprinting techniques for their suitability to accurately and rapidly differentiate amongst S. cerevisiae strains; (ii) the isolation and characterization of S. cerevisiae strains from the coastal regions of the Western Cape; (iii) to determine the natural population dynamics of S. cerevisiae strains in selected vineyards over a four-year period; (iv) to make a preliminary determination of the possible effect that these indigenous S. cerevisiae isolates may have on wine flavour, and (v) to breed new starter culture strains with improved characteristics. Eighteen strains of S. cerevisiae used for commercial production of wine in South Africa were characterized by means of long-chain fatty acid analysis, randomly amplified polymorphic DNA (RAPD-PCR) and electrophoretic karyotyping (CHEF-DNA analysis). Variations in DNA profiles of the strains were apparent in the number, position and intensity of bands. It was found that electrophoretic karyotyping, as a single technique, seemed to be the most useful method to be used for routine fingerprinting. However, it was proposed that the combined use of these three techniques would provide the most reliable means of differentiating amongst wine yeast strains. Two of these fingerprinting techniques, CHEF-DNA and RAPD-PCR analysis, were used to determine the geographic distribution of indigenous S. cerevisiae strains isolated from local vineyards. Grapes were aseptically harvested from 13 sites in five areas in the coastal regions of the Western Cape during 1995. These sites were Groot Constantia and Buitenverwachting in the Constantia area; Jordan, Lievland, Mont Fleur and Nietvoorbij in the Stellenbosch area; Vergelegen in Somerset West; De Rust, Oak Valley, White Hall and Wildekrans in the Elgin/Bot River area; and Bouchard Finlayson and Hamilton Russel in the Hermanus area. After fermentation, 30 yeast colonies per sample were isolated and examined for the presence of S. cerevisiae. Five sampling sites yielded no S. cerevisiae strains. Electrophoretic karyotyping revealed the presence of 46 unique karyotypes in eight of the remaining sites. No dominant strain was identified and each site had its own unique collection of strains. The number of strains per site varied from two to 15. Only in four cases did one strain appear at two sites, while only one instance of a strain occurring at three sites was recorded. All sites contained killer and sensitive strains, however, killer strains did not always dominate. Commercial strains were recovered from three sites. Although commercial yeasts dominated the microflora at two sites, it appears that fears of commercial yeasts ultimately dominating the natural microflora seem to be exaggerated. As an extension of the 1995 survey samples were taken from the same locations at Groot Constantia, Buitenverwachting, Jordan, Lievland, Mont Fleur, Vergelegen, Bouchard Finlayson and Hamilton Russel during 1996 to 1998. This was done in an effort to assess how the natural population dynamics of S. cerevisiae are affected over the long term by abiotic factors. Thirty colonies per site were isolated and the S. cerevisiae strains were characterized by electrophoretic karyotyping. The identity of strains appearing at more than one site in the same, or different years, was confirmed by RAPD-PCR analysis. Strain numbers per site varied over the four-year study period. Weather conditions resulting in severe fungal infestations and heavy applications of chemical sprays during 1996 and early 1997 dramatically reduced the numbers of S. cerevisiae strains recovered during 1997. A return to normal weather patterns during mid 1997 resulted in a gradual recovery of the indigenous population as noted during the 1998 harvest. Indications are that some of the strains isolated are widespread in the study area and may represent yeasts typical of the area. Again, commercial wine yeast strains were recovered in only a few instances and the likelihood that commercial yeasts will eventually replace the natural yeast microflora in vineyards therefore seems remote. As a preliminary study to determine the possible effect of these indigenous S. cerevisiae strains on wine flavour, 33 of the indigenous yeasts were allowed to ferment Chenin blanc wine in laboratory fermentations. The juice was analyzed. The ability to form esters, fatty acids and higher alcohols was compared to that of two local commercial yeasts. None of the indigenous strains were found to be suitable for fermenting white must at 15°C. Their ability to ferment red musts at much higher temperatures still needs to be assessed. Furthermore, differences noted indicate that some of these strains show potential to be included in our extensive yeast-breeding programme as this would broaden the genetic pool. In parallel with the search to isolate and identify indigenous S. cerevisiae strains with good oenological potential, an extensive selection and breeding programme with cultures from our strain collection was undertaken. The aim of this programme was to generate new strains that are better suited to New World winemaking styles and conditions prevailing in South Africa. As a result, 145 hybrids, differentiated by elecrophoretic karyotyping and long-chain fatty acid analysis, were produced. Fifty-eight of the hybrids were able to ferment juice to dryness at 15°C in less than 21 days during microvinification trials. Five of the strains were released for commercial use after extensive industrial-scale evaluation. Based on the success of these interstrain hybridizations, the breeding programme will now be expanded to include some of the indigenous S. cerevisiae strains. In conclusion, it is only when we have a much better understanding of yeast biodiversity, biogeography, ecology and the interaction within yeast communities that we will be able to optimally harness the genetic pool in our strain development programme, aimed to benefit both the wine producer and the consumer.

AFRIKAANSE OPSOMMING: Die biologiese omskakeling van druiwesap na wyn is 'n oeroue proses waartydens giste wat met druiwekorrels en keldertoerusting geassosieer is, bloot toegelaat word om die suiker na etanol, koolstofdioksied en ander mindere, maar belangrike metaboliete te fermenteer. Die kuns en wetenskap van wynbereiding word al sedert die tyd toe Louis Pasteur die verwantskap tussen gis en alkoholiese fermentasie gedemonstreer het, bestudeer. Daar word nou aanvaar dat die fermentasieproses en die produksie van premiumkwaliteit wyn 'n komplekse ekologiese en biochemiese proses is wat die opeenvolgende ontwikkeling van mikrobiese spesies wat deur die spesifieke omgewing be"invloed word, behels. Hierdie komplekse heterogene mikrobiologiese proses sluit die interaksie van baie spesies soos swamme, giste, asynsuur- en melksuurbakteriee, asook mikovirusse en bakteriofage, wat die druifgeassosieerde mikroorganismes be'invloed, in. Giste verteenwoordig nietemin die kem van die harmonieuse biochemiese interaksie met sap afkomstig van verskillende varieteite van Vitis-spesies, wat op hul beurt weer grootliks produkte van hul individuele genetiese samestellings en die terroir is. Hierdie giste is belangrik in die wynmaakproses, want hulle speel nie slegs 'n rol in alkoholiese fermentasie nie, maar kan ook wynbederf en wynkwaliteit deur die produksie van metaboliese komponente tydens fermentasie en outolise beinvloed. 'n Deeglike beg rip van gissistimatiek, biogeografie en ekologie is dus belangrik om die wynkundige potensiaal van ons wynproduserende areas se ryke gisbiodiversiteit te bewaar en te benut. Een van die hooffokuspunte van die eko-taksonomiese ondersoeke is om die werklike bydrae van die inheemse rasse van die sogenaamde wyngis (Saccharomyces cerevisiae) en wildegiste (nie-Saccharomyces cerevisiae) met betrekking tot die sensoriese gehalte van wyn te bepaal. Dit sal dus tot die ontwikkeling van nuwe kommersiele rasse, wat die eienskappe van die area weerspieel, lei. Teen hierdie agtergrond is 'n langtermyn biogeografiese opname en gisrasontwikkelingsprogram begin. Die proefskrif verteenwoordig die eerste fase van hierdie program wat katagorisering van die inheemse giste in die verskillende klimaatsones van die 350 jaar oue wynproduserende areas van die Wes-Kaap en ontwikkeling van nuwe kommersiele gisrasse, wat die kwaliteit van die Suid-Afrikaanse wyn sal verbeter, behels. Die spesifieke doelwitte van die proefskrif sluit die volgende in: (i) die evaluering van die geskiktheid van giskarakteriseringstegnieke om akkuraat en vinnig tussen verskillende S. cerevisiae-rasse te onderskei; (ii) die isolasie en karakterisering van S. cerevisiae-rasse uit die kusstreke van die Wes-Kaap; (iii) die bepaling van die populasiedinamika van die inheemse S. cerevisiae-rasse in geselekteerde wingerde oor 'n vierjaartermyn; (iv) voorlopige bepaling van die moontlike invloed van inheemse S. cerevisiae-isolate op wynaroma; en (v) teling van nuwe kommersiele giskulture met verbeterde eienskappe. Agtien kommersiele wyngisrasse wat in Suid-Afrika gebruik word, is met behulp van langkettingvetsuuranalises, lukraak geamplifiseerde polimorfiese DNA-tegniek (RAPD-PCR) en elektroforetiese kariotipering (CHEF-DNA-analise) gekarakteriseer. Die verskille in die aantal, posisie en intensiteit van die bande van die DNA-profiele van die verskillende gisrasse was duidelik waameembaar. As enkeltegniek, was die elektroforetiese kariotipering die beste tegniek wat vir roetine-tipering gebruik kan word. Die gesamentlike gebruik van die drie tegnieke sal egter die akkuraatste resultaat lewer indien daar tussen rasse onderskei moet word. Twee van die tegnieke, CHEF-DNA- en RAPD-PCR-analises, is gebruik om die geografiese verspreiding van inheemse S. cerevisiae-rasse wat uit die wingerde geisoleer is, te bepaal. Druiwe is in 1995 asepties by 13 plekke in vyf areas van die kusstreke van die Wes-Kaap geoes. Dit was Groot Constantia en Buitenverwachting in die Constantia-area; Jordan, Lievland, Mont Fleur en Nietvoorbij in die Stellenbosch-area; Vergelegen in Somerset-Wes; De Rust, Oak Valley, White Hall en Wildekrans in die Elgin/Botrivier-area; en Bouchard Finlayson en Hamilton Russell in die Hermanus-area. Dertig giskolonies is na fermentasie per monster geisoleer en vir die teenwoordigheid van S. cerevisiae-rasse ondersoek. Vyf plekke het geen S. cerevisiae-giste gehad nie. Elektroforetiese kariotipering het die teenwoordigheid van 46 unieke kariotipes in die agt oorblywende plekke uitgewys. Geen dominante gisras is gevind nie en elke isolasieplek het sy eie unieke versameling rasse gehad. Die aantal gisrasse het van twee tot 15 per isolasieplek gewissel. Slegs in vier gevalle is een ras by twee plekke gevind, terwyl daar slegs een geval was waar een ras by drie plekke voorgekom het. Killer- en sensitiewe gisrasse is by aile plekke gevind, maar die killerrasse was nie altyd dominant nie. Kommersiele rasse is by slegs drie plekke gevind. Alhoewel kommersiele rasse die dominante giste by twee plekke was, blyk dit dat die vrese dat kommersiele gisrasse die inheemse mikroflora sal oorheers, oordrewe is. Ter aansluiting by die 1995-opname, is daar gedurende 1996 tot 1998 weer op dieselfde plekke te Groot Constantia, Buitenverwachting, Jordan, Uevland, Mont Fleur, Vergelegen, Bouchard Finlayson en Hamilton Russell monsters geneem. Die doel hiermee was om te bepaal hoe die inheemse populasiedinamika van S. cerevisiae oor die langtermyn deur abiotiese faktore be'invloed is. Dertig kolonies is per isolaat ge'isoleer en die S. cerevisiaerasse is met behulp van elektroforetiese kariotipering gekarakteriseer. Die identiteit van gisrasse wat in dieselfde, of verskillende, jare by meer as een isolasieplek verskyn het, is deur middel van RAPD-PCR-analise bevestig. Die aantal rasse per plek het oor die vierjaarstudieperiode gewissel. Nat en warm weerstoestande gedurende laat 1996 en vroeg 1997 het tot ernstige swaminfeksies gelei en die verhoogde gebruik van chemiese spuitmiddels het die aantal S. cerevisiae-rasse wat tydens 1997 geisoleer is, drasties verlaag. Die terugkeer van normale weerstoestande teen die middel van 1997 het tot die stadige herstel van die inheemse gispopulasie gelei. Daar was aanduidings dat sekere van die rasse oor groot dele van die studiegebied versprei is en dit kan dus dui op gisrasse wat kenmerkend van die area is. Daar is weereens slegs 'n paar kommersiele giste geisoleer en dit bevestig dus dat die moontlikheid dat kommersiele rasse die inheemse populasie sal oorheers, baie gering is. Tydens 'n voorlopige studie waarin die moontlike uitwerking van inheemse S. cerevisiaerasse op wynaroma bestudeer het, is 33 van die inheemse giste in laboratoriumeksperimente toegelaat om Chen in blanc-sap te fermenteer. Hierdie wyne is chemies ontleed. Die giste se vermoe om esters, vetsure en hoer alkohole te produseer, is daama met die van twee kommersiele rasse vergelyk. Daar is bevind dat nie een van die inheemse rasse geskik is vir die fermentasie van witmos by 15°C nie. Hul vermoe om rooimos by veel hoer temperature te gis, moet egter nog bepaal word. Verder is daar tog aanduidings dat hierdie giste oor genoegsame potensiaal beskik om in ons gisteelprogram opgeneem te kan word om sodoende die genepoel te vergroot. Daar is ook 'n intensiewe seleksie- en teelprogram met giste uit ons kultuurversameling onderneem wat gelyktydig met die isolasie en identifikasie van inheemse S. cerevisiae-rasse geloop het. Die doel van die program is die daarstelling van nuwe wyngisrasse aan die Suid-Afrikaanse wynbedryf, wat meer vir Nuwe Wereld-wynmaakstyle en Suid-Afrikaanse toestande geskik is. Die resultaat was 145 hibriede, wat deur elektroforetiese en langkettingvetsuuranalises onderskei kan word. Tydens kleinskaalse wynbereidingseksperimente het 58 van die hibriede daarin geslaag om sap binne 21 dae droog te fermenteer. Vyf van die giste is na intensiewe kommersiele evaluering kommersieel vrygestel. Die sukses met die hibriede het nou tot gevolg dat die teelprogram uitgebrei sal word deur sekere van die inheemse S. cerevisiae-rasse in te sluit. Die gevolgtrekking is dat ons gisbiodiversiteit, biogeografie, ekologie en die interaksies tussen gisgemeenskappe beter sal moet verstaan indien ons die genetiese poel tot ons beskikking optimaal in die teelprogram wil aanwend om sodoende die wynprodusent en verbruiker te bevoordeel.

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