Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: During wine fermentation, numerous grape must constituents serve as nutrients to wine yeast (Saccharomyces cerevisiae), which enable their growth and successful completion of alcoholic fermentation. Many of these nutritional factors, in particular nitrogen, also act as precursors for yeastderived flavour compounds such as higher alcohols, esters and volatile fatty acids. Yeast nitrogen metabolism thus plays a determining role in wine aroma and quality. Not only is the nitrogen source, concentration and supplementation timing important, but various environmental factors and the genetic constitution of the yeast strain used for fermentation will also contribute to fermentation outcomes. The main goal of this work was to explore the complex interactions between a number of contributing factors; namely nitrogen source, timing of addition, yeast strain and fermentation matrix. Broadly, this study assessed the impact of seven different nitrogen combinations, added either to the initial grape must or after the onset of fermentation, on fermentation performance and aroma compound production by nine commercial wine yeast strains. Fermentations were done in synthetic grape must, and validated for a subset of parameters in real grape must. The nitrogen treatments were designed according to the generally established order of preference of S. cerevisiae for individual amino acids as source of nitrogen under fermentative conditions, and the potential of certain amino acids to participate in metabolic pathways that produce specific aroma compounds. The results reveal that different nitrogen combinations can lead to unexpected aroma outcomes, depending strongly on the genetic background of individual yeast strains and the timing of nitrogen addition. Certain nitrogen treatments consistently resulted in significant increases or decreases in specific aroma compound concentrations in comparison to the treatment fermented on ammonium as only nitrogen source, for multiple yeast strains. These compounds were classified as nitrogen treatment dependent. Other aroma compounds were produced similarly for all nitrogen treatments and were designated as nitrogen treatment independent. The presence of specific amino acid groups (for example the branched-chain and aromatic amino acids) could be correlated to significantly altered production patterns of related (such as higher alcohols) or unrelated (diethyl succinate) aroma compounds relative to the other nitrogen treatments. Taken together, a number of interesting and novel hypotheses regarding the metabolic pathways involved could be derived from the data. Ultimately, this initial assessment of interactive effects during fermentation will contribute to practical guidelines for winemakers to allow matching grape must constituents (such as nutrients) with the intrinsic aroma production capabilities of specific yeast strains in order to modulate wine aroma, style and quality. / AFRIKAANSE OPSOMMING: Tydens wynfermentasie dien talle druiwemosbestanddele as voedingstowwe vir wyngis (Saccharomyces cerevisiae) wat hul groei bevorder en hul in staat stel om alkoholiese fermentasie suksesvol te voltooi. Baie van hierdie voedingstowwe, veral stikstof, dien ook as voorlopers vir geurkomponente afkomstig van gismetabolisme, soos hoër alkohole, esters en vlugtige vetsure. Die stikstofmetabolisme van gis speel dus ‘n bepalende rol in wynaroma en -kwaliteit. Nie net is die stikstofbron, konsentrasie en tydsberekening van stikstof toevoeging belangrik nie, maar verskeie omgewingsfaktore, asook die genetiese samestelling van die gisras aangewend vir fermentasie, sal bydra tot die fermentasie uitkomste. Die hoofdoel van hierdie werk was om die komplekse interaksies tussen ‘n aantal bydraende faktore te ondersoek; naamlik die stikstofbron, tyd van stikstof toevoeging, gisras en fermentasiematriks. Breedweg het hierdie studie die impak van sewe verskillende stikstofkombinasies, toegedien tot die druiwemos voor of na die aanvang van fermentasie, op die suksesvolle verloop van fermentasie en die produksie van aromakomponente deur nege kommersiële wyngisrasse bepaal. Fermentasies is in sintetiese druiwemos uitgevoer, en ‘n deelversameling van die fermentasies in regte druiwesap te herhaal. Die stikstofbehandelings is ontwerp in ooreenstemming met die algemeen vasgestelde voorkeurvolgorde van S. cerevisiae vir individuele aminosure as stikstofbron onder fermentatiewe kondisies, en die potensiaal van sekere aminosure om mee te doen in metaboliese paaie wat spesifieke aromaverbindings produseer. Die resultate toon dat verskillende stikstofkombinasies tot onverwagte aroma-uitkomste kan lei wat sterk afhanklik is van die genetiese agtergrond van individuele gisrasse en die tyd van stikstof byvoeging. Sekere stikstofbehandelings het konsekwent, vir veelvuldige gisrasse, tot beduidende toenames of afnames in die konsentrasies van spesifieke aromakomponente gelei in vergelyking met die behandeling wat ammonium as enigste stikstofbron bevat het. Hierdie verbindings is as stikstofbehandeling afhanklik geklassifiseer. Ander aromaverbindings is soortgelyk vir alle stikstofbehandelings geproduseer en is aangewys as stikstofbehandeling onafhanklik. Die teenwoordigheid van spesifieke aminosuurgroepe (byvoorbeeld die vertakte-ketting en aromatiese aminosure) kon gekorreleer word met beduidende veranderings in produksiepatrone van verwante (soos hoër alkohole) of onverwante (dietielsuksinaat) aromakomponente relatief tot die ander stikstofbehandelings. Alles inaggenome kon ‘n aantal interessante en nuwe hipoteses rakende die betrokke metabolise padweë van die data afgelei word. Uiteindelik sal hierdie aanvaklike bepaling van interaktiewe effekte tydens fermentasie bydra tot praktiese riglyne vir wynmakers, wat hulle in staat sal stel om druiwesapbestanddele (soos nutriënte) te strook met die intrinsieke aromaproduksie kapasiteite van spesifieke gisrasse, en sodoende wynaroma, styl en kwaliteit te moduleer. / Oenobrands and THRIP for funding this project

Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Wine is a product of the fermentation of grape juice. Alcoholic fermentation is mainly conducted by the yeast Saccharomyces cerevisiae which metabolises grape sugars to mainly ethanol, CO2 and glycerol. Aside from these primary fermentation compounds, the yeast also produces many secondary metabolic by-products that are important to wine quality and style. Malolactic fermentation (MLF) is a secondary fermentation that normally occurs after alcoholic fermentation. Lactic acid bacteria (LAB) are responsible for the conversion of malic acid to lactic acid and CO2 during MLF, which is important for wine deacidification and also contributes to microbial stability. Malolactic fermentation and LAB strains can also influence the aroma profile of wines. The main genera associated with this process are Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Oenococcus oeni is the main species associated with MLF because it is able to survive the harsh physiochemical environment of winemaking. Recently L. plantarum has also been introduced as a commercial MLF starter culture. Research has started to focus on the potential of wine yeast and LAB interactions or combinations to alter the wine aroma profile via the production and/or degradation of aroma compounds. The overriding goal of this study is to unravel the interactions between wine yeast and different LAB strains and their impact on wine aroma and flavour. The first aim was to assess LAB growth during co- and sequential inoculation strategies, the ability to complete MLF and the impact on the production of aroma compounds in combination with two different yeast strains in a medium containing full complement of nitrogen supplementation. Malolactic fermentation was successful in the different inoculation strategies and the bacterial combination (L. plantarum and O. oeni) completed MLF in the shortest time. The impact of the bacterial strains on the modification of aroma compounds was bigger in co- than sequential inoculation. A general increase in total esters (contributing to the fruity character of wines) especially ethyl lactate and ethyl acetate was observed. The production of esters, volatile fatty acids and higher alcohols proved to be dependent on either the yeast strain used and/or the LAB strains used. The second aim of the research was to assess the effect of NH4Cl (ammonium) and amino acids supplementation on yeast and LAB strains (both in co- and sequential inoculation strategies) and the impact on the aroma profile of the fermented must. Fermentations supplemented with ammonia as sole nitrogen source showed the highest total bacterial growth in terms of cell numbers. Malolactic fermentation was completed in the shortest time with O. oeni and the bacterial combination inoculums. The co-inoculated strategies in combination with amino acids supplementation showed the biggest impact on the aroma compound profiles of the different fermentation strategies and bacterial treatments. A general increase in total esters was observed for NH4Cl additions with ethyl lactate and ethyl acetate showing the highest concentrations. The concentration of esters, volatile fatty acids and higher alcohols were strongly influenced by the yeast and the single LAB strains used. The results generated from this study showed that the chemical composition of the fermentation medium and the selection of yeast and LAB strains are important because these factors have an influence on the aroma and flavour profiles of wines. / AFRIKAANSE OPSOMMING: Wyn is die produk van gefermenteerde druiwe. Die gis, Saccharomyces cerevisiae is verantwoordelik vir alkoholiese fermentasies waar druiwe suikers na hoofsaaklik etanol, CO2 en gliserol gemetaboliseer word. Die gis produseer ook sekondêre metaboliete wat ‘n belangrike bydrae lewer tot wynstyl en kwaliteit. Appelmelksuurgisting (AMG) is ‘n sekondêre fermentasie wat gewoonlik na alkoholiese fermentasie plaasvind. Melksuurbakterieë (MSB) speel ‘n sleutel rol in die omskakeling van appelsuur na melksuur en CO2 gedurende AMG. Hierdie fermentasie lei tot ‘n afname in die suurheidsgraad en verbeter die mikrobiese stabiliteit van die wyn. Appelmelksuurgisting en MSB rasse kan die aroma- en geurprofiel van wyne beïnvloed. Die belangrikste genera wat met AMG geassosieer word is Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Oenococcus oeni is die mees algemene ras wat vir AMG gebruik word omdat dit in uiterste wyn toestande kan oorleef. Mees onlangs is Lactobacillus plantarum as kommersiële aanvangskultuur vir AMG geïdentifiseer. Navorsing het onlangs meer begin fokus op gis en MSB interaksie of kombinasies as ‘n strategie om die aroma profiele van wyne te verander. Die hoofdoel van die studie is om die interaksie tussen wyngiste en verskillende MSB rasse en die effek op die aroma profile van wyne te bestudeer. Die eerste doelwit was om die impak van die twee giste op die groei en AMG vermoeë van MSB gedurende ko- en sekwensiële inokulasie praktyke en die impak op die produksie van aroma komponente, in ‘n medium wat die volledige stikstof aanvullings bevat, te bestudeer. Appelmelksuurgisting was suksesvol in die verskillende inokulasie praktyke en die bakteriese kombinasie (L. plantarum en O. oeni) het AMG in die kortste tyd voltooi. Die impak van die bakteriese rasse op die modifikasie van die aroma komponente was groter met ko- as sekwensiële inokulasies. Daar was ‘n toename in die totale esterkonsentrasies veral in etiellaktaat en etielasetaat. Die produksie van esters, vlugtige vetsure en hoër alkohole word beïnvloed deur die gisras en MSB rasse wat gebruik word. Die tweede doelwit was om die impak van NH4Cl (ammonium) en aminosure aanvullings op die gis- en MSB rasse gedurende ko- en sekwensiële inokulasie strategieë te bepaal. Melksuurbakterieë se groei was beter met die ammonium aanvulling. Appelmelksuurgisting was in die kortste tyd voltooi met O. oeni en die bakteriese kombinasie. Die ko-inokulasie praktyke in kombinasie met die kompleks aminosure aanvulling het die grootste impak op die produksie van aroma komponente gehad. Daar was weereens ‘n toename in die totale esterkonsentrasies vir die NH4Cl aanvulling, veral in etiellaktaat en etielasetaat. Die gis en MSB rasse speel ‘n rol by die produksie en konsentrasies van esters, vlugtige vetsure en hoër alkohole. Die resultate van hierdie studie bewys dat die chemiese samestelling van die fermentasie medium, die seleksie van gis- en MSB rasse is belangrik omdat hierdie faktore die aroma en geur profiele van wyne beïnvloed.