Comparative analysis of fermentative yeasts during spontaneous fermentation of grapes from different management systems

Bagheri, Bahareh (2014-04)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The microorganisms associated with grape berry surface can be influenced by numerous factors such as agronomic parameters. Hence, the focus of this study was comparison between three agronomic farming systems to evaluate their impact on yeast diversity. In addition, the dynamics of the yeast population throughout wine alcoholic fermentation were monitored. Three vineyards (conventional, biodynamic and integrated) were chosen and the experiment was carried out during the 2012 and 2013 vintages. A total of 600 yeast isolates including Saccharomyces and non-Saccharomyces were obtained from grape must and during different stages of fermentation including beginning, middle and end of alcoholic fermentation, from all three vineyards. Yeast species diversity in grape must and their population dynamics were evaluated by cultivating the yeasts in nutrient media and using “Polymerase Chain Reaction and sequence analysis of the ITS1-5.8S rRNA-ITS2 region. Eight, four and one species were detected from biodynamic, conventional and integrated must in 2012 vintage whereas, 2013 vintage displayed a higher diversity and 12, 11 and 9 different species were identified from biodynamic, conventional and integrated vineyard, respectively. Aureobasidium pullulans was the most frequent isolate in all three vineyards whereas Saccharomyces cerevisiae was below detection level in grape must and was only isolated in low frequencies in biodynamic must (3% of the total population) in both vintages. In general, the overlap of common yeast isolates (e.g. M. pulcherrima and H. uvarum) was observed in the musts obtained from different vineyards although unique minor species could be isolated and clearly demonstrated the distinction between the three vineyards. Moreover, biodynamic must displayed a higher degree of diversity in both 2012 and 2013 compared to the conventional and integrated vineyards. The beginning of all spontaneous fermentations was dominated by non-Saccharomyces yeast species (e.g. H. uvarum, C. zemplinina), as the fermentation proceeded, the population of non-Saccharomyces species were gradually decreased and strongly fermentative yeast S. cerevisiae dominated and completed the fermentations. The dynamics of S. cerevisiae strains was also evaluated during different stages of fermentation (beginning, middle and end), using interdelta PCR methods. A high diversity (10-18 strains per fermentation) and the sequential substitution of S. cerevisiae strains were observed throughout spontaneous fermentations. In addition, integrated vineyard displayed the highest S. cerevisiae strains compared to biodynamic and conventional vineyard.

AFRIKAANSE OPSOMMING: Die mikro-organismes wat met die oppervlak van druiwe bessies geassosieer word kan deur veskeie agronomiese faktore beїnvloed word. Gevolglik was die focus van die studie om ‘n vergelyking tussen die impak van drie verksillende boerdery sisteme op die invloed op gis diversiteit te bepaal. Die dinamiek van gis populasies tydens alkoholiese fermentasie is bykomstig bestudeer. Drie verskillende wingerde (konvesioneel, biodinamies en geïntegreerd) is gebruik vir die studie tydens die 2012 en 2013 oesjare. In total is 600 gis isolate, insluitend Saccharomyces en nie-Saccharomyces giste, verky van druiwe mos tydens verkillende fases van die fermentasie proses (begin, middle en einde) vir al drie wingerde. Die diversiteit en populasie dinamika van gis spesies in die druiwe mos is geëvalueer deur die giste in verskillendde media op te groei en ook deur die gebruik van die “polymerase ketting reaksie” (PKR) en DNS volgorde bepaling van die ITS1-5.8S rRNA-ITS2 gebied. Tydens die 2012 oesjaar is agt, vier en een afsonderlike spesies geїsoleer, in vergelyking met die 12, 11 en 9 verskillende spesies wat tydens 2013 geidentifiseer is is uit die biodinamiese, konsensionele en geïntegreerde onderskeidelik. Aureobasidium pullulans is teen die hoogste frekwensie geїsoleer in al drie wingerde, terwyl Saccharomyces cerevisiae onder die deteksie limiet was in druiwe mos en ook slegs in lae getalle in die biodinamiese mos (3% van die totale populasie) in beide oesjare. Oor die algemeen is ‘n oorvleuling tussen verwante spesies (bv. M. pulcherrima en H. uvarum) waargeneem en die mos vanaf verskillende wingerde, terwyl meer geringe spesies deurgans geїsoleer kon word en duidelik ‘n verkill tussen die drie wingerde uitgewys het. Druiwe mos uit die biodinamiese wingerd het verder ‘n hoёr graad van diversiteit en beide 2012 en 2013 vertoon as beide die konvesnionele en geïntegreerde wingerde. Die begin van alle spontane fermentasies was gedomineer deur die populasie van nie-Saccharomyces gis spesies (bv. H. uvarum, C. zemplinina), wat geleidelik afgeneem het met die verloop van die fermentasie. Die populasie van die sterk fermentatiewe, S. cerevisiae, het toegeneem tydens fermentasie en die fermentasie afgehanel as dominante gis. Die dinamika van S. cerevisiae rasse is ook geëvalueer tydens die verskillende fases van fermentasie (begin, middle en einde) deur gebruik te maak van interdelta PKR metodes. ‘n Hoё diversiteit (10-18 rasse per fermentasie) en die opeenvolgende verplasing van S. cerevisiae rasse was waargeneem deur die verloop van spontane fermentasies. Daarbenewens het die geïntegreerde wingerd die grootste getal S. cerevisiae rasse in vergelyking met die biodinamiese en konvensionele wingerde opgelewer.

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