Wines are composed of numerous compounds that are complex, making them difficult to analyze. Wine evaluation and discrimination is typically done through chemical and human sensory evaluation. Unfortunately, both of these methods are time consuming and expensive. Therefore a new rapid analysis technique for wine discrimination and analysis is desired. The electronic nose has been suggested as an alternative to current wine discrimination techniques. In this study, a quartz microbalance-based electronic nose system was utilized to analyze the overall volatile components of wine. The electronic nose was optimized for Cabernet Sauvignon and Mouvèdre wine to gain maximum sensor response from the sensors. Response surface methodology was used to determine the optimum sensor response by varying three experimental parameters: sensor temperature, sample temperature and equilibrium time. The maximum sensor response occurred at an equilibrium time of 20 min for each varietal and at a sample temperature of 55ºC and 56ºC for Cabernet Sauvignon and Mouvèdre, respectively. The optimum sensor temperature selected for this study was 40ºC for both varietals. Using the optimum sensor settings, the electronic nose was used to analyze Cabernet Sauvignon wines. Grapes were treated with ethanol spray (5%, and 10%) 13 weeks post-bloom, which has been shown to affect the overall quality of the final wine product. Wine samples were evaluated using chemical analyses, human sensory evaluation and electronic nose. Significant differences between the wines were observed based on pH, percent alcohol, and color intensity only. A consumer sensory panel consisting of 81 panelists was unable to differentiate amongst sample treatments. However, the electronic nose was able to differentiate between the control group and the treated samples 100% of the time. Canonical discriminant analysis of the data placed the 5% ethanol treatment as a sub-set of the 10% ethanol treatment. The results indicate that the electronic nose can be used as a discriminatory tool for assessing wines. / Master of Science

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Wine is the result of a variety of biochemical reactions and microbial interactions which contribute to the organoleptic properties of wine. Wine aroma and flavour encapsulate the sensory experience of wine and could ultimately determine wine quality and consequently influence consumer acceptance and preference of a product. A thorough understanding of potential factors influencing wine aroma and flavour is therefore needed in order to exploit such factors. The aim of this study was to evaluate the influence of commercial malolactic fermentation (MLF) starter cultures on wine composition, aroma and flavour and the potential impact on consumer preference of experimentally produced red wines. An analytical platform was established to capture the compositional changes induced by different MLF bacteria in experimentally produced red wines. A fast gas chromatography flame ionisation detection (GC-FID) method was developed to determine 39 wine volatile compounds in less than 15 minutes per sample. A 3-fold reduction in analysis time was achieved in comparison to a conventional GC-FID method (40 minutes). Analytes quantified comprise a large boiling point and polarity range which illustrates the robustness of the method. A method was also developed for the direct quantification of carbonyl compounds including diacetyl, acetoin, 2,3-pentanedione and certain aldehydes using headspace solid phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME GC-MS). Both analytical methods showed satisfactory linearity, repeatability and limits of quantification. The contribution of four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures to the volatile composition, organic acid content and infrared spectral properties of experimentally produced South African red wines, showed significant strain-specific variations in the organic acid profiles, especially for the production of citric acid and lactic acid during MLF. Subsequently, concentrations of compounds related to citric acid metabolism, namely ethyl lactate, acetic acid, diacetyl and acetoin, were influenced accordingly. Bacterial metabolic activity increased the concentration of higher alcohols, fatty acids and esters, with a larger increase observed in ethyl esters compared to acetate esters. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, however, further investigation is needed to clarify this observation. Infrared spectral fingerprints were used to characterise the different bacteria and in addition, the prediction of MLF related compounds, diacetyl, acetoin and 2,3-pentanedione, from mid-infrared spectra was explored by partial least squares (PLS) models. Quantitative descriptive analysis (QDA) results depicted significant differences between wines fermented with different starter cultures, in terms of sensory attributes including buttery, fruity, nutty and yoghurt/buttermilk aroma as well as smoothness and mouth-feel attributes. Consumer preference testing results indicate that sensory differences imparted by different MLF bacteria could influence consumer-liking. Preference mapping revealed interesting relationships between sensory attributes and consumer-liking, that can be used for preliminary interpretative purposes. In conclusion, this study illustrated the potential impact of bacterial strains on wine aroma and flavour, resulting sensory properties and consumer preference through an integrative approach combining compositional, spectral, sensory and consumer data. The results presented in this study are of significance to the wine industry since they illustrate and reiterate the potential of different MLF starter cultures as an additional tool to contribute to wine aroma and flavour, and potentially influencing consumer preference and product liking. / AFRIKAANSE OPSOMMING: Wyn is die resultaat van ‘n verskeidenheid biochemiese reaksies en mikrobiologiese interaksies wat tot die organoleptiese eienskappe van die finale produk bydra. Wynaroma en geur vang die sensoriese ervaring van wyn vas en dit kan dus wynkwaliteit bepaal en gevolglik verbruikersaanvaarding asook voorkeur van ‘n produk beïnvloed. Die potensiële faktore wat wynaroma en geur kan beïnvloed moet dus vir hierdie rede deeglik bestudeer word ten einde sulke faktore ten volle te benut. Die doel van hierdie studie was om die invloed van kommersiële applemelksuurgisting (AMG) aanvangskulture op wynsamestelling, die gevolglike aroma en geur eienskappe en die potensiële impak op verbruikersvoorkeure te ondersoek. ‘n Analitiese platform is gevestig om die veranderings in samestelling veroorsaak deur verskillende AMG bakterieë in eksperimenteel bereide rooi wyne vas te vang. ‘n Vinnige gas chromatografiese vlam geïoniseerde deteksie (GC-FID) metode is ontwikkel vir die meting van 39 vlugtige komponente in minder as 15 minute per wynmonster. In vergelyking met ‘n konvensionele GC-FID metode (40 minute) is ’n 3-voudige vermindering in analise tyd behaal. Gekwantifiseerde komponente bestaan uit ‘n wye kookpunt- en polariteitsreeks wat die robustheid van die metode illustreer. ‘n Metode vir die direkte kwantifisering van karboniel komponente, insluitende diasetiel, asetoïen, 2,3-pentanedioon en verskeie aldehiede is ontwikkel met die gebruik van dampfase soliede fase mikroekstraksie gekoppel aan gas chromatografie massa spektrometrie (HS-SPME GC-MS). Albei analitiese metodes besit voldoende lineariteit, herhaalbaarheid en lae deteksie limiete. Die bydrae van vier kommersiële Oenococcus oeni AMG aanvangskulture tot die vlugtige samestelling, organiese suurinhoud en infrarooi spektrale eienskappe van Suid-Afrikaanse rooiwyn het beduidende ras spesifieke variasies in die organiese suur profiele, spesifiek vir die produksie van sitroensuur en melksuur gedurende AMG, vertoon. Gevolglik is die konsentrasies van komponente verwant aan sitroensuur metabolisme, naamlik etiellaktaat, asynsuur, diasetiel en asetoïen, dien ooreenkomstig beïnvloed. Bakteriese metaboliese aktiwiteit het ‘n toename tot gevolg gehad in die hoër alkohole, vetsure en algemene ester konsentrasies met ‘n groter toename in etiel-esters in vergelyking met asetaat-esters. ‘n Ras-spesifieke tendens om die totale aldehiedkonsentrasie te verminder na afloop van AMG, is waargeneem alhoewel verdere ondersoek in hierdie area nodig is. Infrarooi spektrale patrone is gebruik om verskillende bakterieë te karakteriseer asook om die voorspelling van spesifieke AMG verwante komponente soos diasetiel, asetoïen en 2,3-pentanedioon met die gebruik van mid-infrarooi spektrala parsiële kleinste kwadraat verskille (PLS) modelle te ondersoek. Kwantitiewe beskrywende sensoriese analise illustreer beduidende verskille tussen wyne wat gefermenteer is met verskillende aanvangskulture in terme van geure soos botteragtigheid, vrugtigheid, neutagtigheid, joghurt/karringmelkgeur, asook gladheid en mondgevoel eienskappe. Verbruikersvoorkeur resultate illustreer die groot invloed wat sensoriese verskille veroorsaak deur verskillende AMG bakterieë op verbruikersvoorkeure kan hê. Voorkeur kartering het interessante verhoudings tussen sensoriese eienskappe en verbruikersvoorkeure uitgelig. Hierdie studie illustreer die impak van bakteriese rasse op wynaroma en geur en verbruikersvoorkeure deur ‘n geïntegreerde benadering waarin samestellende, spektrale, sensoriese en verbruikersdata gekombineer is. Die resultate van hierdie studie is van belang vir die wynindustrie deurdat dit die potensiële bydrae van verskillende AMG kulture tot wynaroma en geur asook die potensiaal om verbruikersvoorkeure te beïnvloed, illustreer en beklemtoon.

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.

Thesis (MSc)--Stellenbosch University, 2012 / ENGLISH ABSTRACT: The aroma and flavour of wine are important aspects that form the basis for consumers’ organoleptic experience of wine. Therefore, an understanding of the chemical composition of wine aroma is of major importance, to establish possible links between wine chemistry, sensory attributes and consumer preference for a product. For this purpose analytical chemistry and multivariate techniques are indispensable tools for the metabolic profiling of wine. Chenin blanc is one of the most important South African export white wine varieties. However, despite its importance, very limited profiling of Chenin blanc aroma compounds has been done and information is restricted to isolated and dated reports on a few chemical compounds only. Therefore, the overall aim of this study was to obtain an in-depth view of the volatile chemical profile of this cultivar. The first task was to perform targeted volatile metabolic profiling of the three dry and offdry Chenin blanc styles, fresh and fruity, rich and ripe unwooded and rich and ripe wooded. To this end, a new, simple and robust liquid-liquid extraction technique using dichloromethane was developed and validated for extraction of analytes prior to gas chromatography flame ionization detection (GC-FID) analysis, to quantify 57 analytes in one rapid analytical procedure. This method was applied to profile 48 Chenin blanc wines. Very successful discrimination between the three styles, using the quantified volatile compounds, was obtained with two multivariate methods. These were partial least squares regression-discriminant analysis, (PLS-DA), as well linear-DA, using best subset selection for identifying the most important variables. According to the classification models, a higher content of maturation derived, malolactic fermentation derived and wood derived compounds were predominantly characteristic of the wooded wines. Higher content of some terpenes and ethyl esters were predominantly associated with the rich and ripe unwooded style Chenin blanc wines, while the fresh and fruity style were generally characterized by high levels of acetate esters. Secondly, untargeted analysis of 21 wines was done with gas chromatography mass spectrometry (GC-MS). Mathematical chromatography, using PARAllel FACtor analysis (PARAFAC and PARAFAC2), was applied to the GC-MS data for resolution of the complex chromatographic results by multi-way modeling, and to derive unbiased multivariate classification models of the three styles. This approach provided excellent style differentiation, without the arduous task of analysis of numerous standards and setting up of calibration curves, required by the targeted approach described above. Additionally, the data generated during this study will form part of the current South African wine aroma database, which does not contain any data regarding Chenin blanc at present. / AFRIKAANSE OPSOMMING: Die aroma en geur van wyn is belangrike aspekte aangesien dit die basis vorm van die wynverbruiker se organoleptiese ervaring van die produk. Derhalwe, is ‘n deeglike kennis van die chemiese samestelling van wynaroma baie belangrik, ten einde die korrelasies tussen wynchemie, sensoriese eienskappe en verbruikersvoorkeure te bepaal. Vir hierdie doel, is die insameling van analitiese chemiese data, tesame met multi-veranderlike tegnieke om relevante inligting uit die data te onttrek, onmisbaar vir die metaboliese profilering van wyn. Chenin blanc is huidiglik een van Suid-Afrika se belangrikste uitvoer wit wynvariëteite. Ten spyte hiervan, is daar tot hede egter baie min profilering van Chenin blanc se aromakomponente gedoen en die beskikbare inligting is beperk tot geïsoleerde en verouderde navorsingsbevindinge. In die lig van bogenoemde, is die oorkoepelende motivering vir hierdie studie dus om die vlugtige chemiese komponente se profiel in Chenin blanc wyn, in diepte te bepaal. Die eerste taak was die geteikende bepaling van die metaboliese profiel van drie droë of halfdroë Chenin blanc wynstyle, nl. vars en vrugtig, ryk en ryp ongehout, asook ryk en ryp gehout. Om dit te bereik, is ‘n nuwe eenvoudige en robuuste vloeistof-vloeistof ekstraksieprosedure met dichlorometaan ontwikkel, wat analise met gaschromatografie – vlamionisasie deteksie (GC-FID) voorafgaan, om die konsentrasies van 57 komponente in een vinnige analise te bepaal. Hierdie metode is gebruik om 48 Chenin blanc wyne te profileer. Deur gebruik te maak van multi-veranderlike data analitiese tegnieke, is die gekwantifiseerde vlugtige komponente data gebruik in diskriminant analise. Baie suksesvolle onderskeid tussen die drie style, is verkry deur gebruik te maak van twee multi-veranderlike metodes, naamlik: parsiële kleinste kwadrate regressie, diskriminant analise, asook liniêre diskriminant analise. Vir laasgenoemde analise, is die seleksie van die mees belangrike veranderlikes met beste sub-groep regressie bepaal. Volgens hierdie klassifikasie modelle is ‘n hoër inhoud van veroudering-, appelmelksuurgisting- en houtverwante aroma komponente baie kenmerkend in die houtbehandelde wyne. Hoër vlakke van sommige terpene en etiel esters was kenmerkend met betrekking tot ryk en ryp ongehoute Chenin blanc style, terwyl die vars en vrugtige style meer gekenmerk was met hoë vlakke van asetaat esters. Tweedens is ‘n seleksie van 21 wyne geanaliseer deur gebruik te maak van gaschromatografie – massa spektrometrie (GC-MS) in ‘n ongeteikende metaboliese profileringsbenadering. Wiskundige chromatografiese metodes, spesifiek, parallelle faktor analise (PARAllel FACtor analysis (PARAFAC and PARAFAC2)), was voorts gebruik om komplekse chromatogramme te prosesseer met wiskundige, multi-vlak modellering. Met hierdie nie-selektiewe benadering, is ook suksesvolle klassifikasiemodelle gegenereer vir die diskriminasie tussen die drie verskillende Chenin blanc style. Die voordeel van die ongeteikende GC-MS analise, gekoppel met die data hanterings- en prosesseringsprotokols in hierdie studie gebruik, is dat die arbeidsintensiewe taak om kalibrasiekurwes op te stel vir elke individuele komponent, soos wat vereis word in die geteikende benadering, nie nodig is nie. Die data wat ingewin is gedurende hierdie studie, sal ook bygevoeg word tot ‘n bestaande Suid-Afrikaanse aroma databasis, wat tans geen data aangaande Chenin blanc wyn bevat nie.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile. / AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2011. / Includes bibliogaphy. / ENGLISH ABSTRACT: Sauvignon blanc wines have become increasingly popular in South Africa as it is a cultivar that can be easily manipulated in the vineyard and cellar to produce a range of wine styles. These wines are usually given aroma descriptors such as green pepper, grassy and asparagus; while other more tropical aromas include passion fruit and guava. These aromas are thought to be mainly caused by methoxypyrazines and volatile thiols. These compounds are known to be character impacting compounds of Sauvignon blanc and are present in the grapes in the aromatic form (methoxypyrazines) or as non‐aromatic precursors (thiols) that can be released by the yeast during fermentation. Other aroma compounds such as esters, higher alcohols, fatty acids and monoterpenes are compounds that could potentially influence the aroma bouquet of a wine significantly. These aroma compounds exist either as precursors in the grapes (monoterpenes) or arise due to yeast metabolism during fermentation (esters, higher alcohols, fatty acids) and often display fruity, floral and pleasant aromas. In the cellar, winemaking practices can be manipulated to a certain extent to achieve the desired wine style. Winemaking tools such as temperature, skin contact, pressing conditions, oxygen (O2), sulphur dioxide (SO2) and yeast strain are only a few factors influencing the outcome of a wine. In general, South African winemakers maintain a very reductive environment during Sauvignon blanc wine production by using inert gasses, thereby causing the production costs to increase. There is sufficient evidence to support the reductive handling of white wine, however there seems to be a lack of information as to why the must should be treated reductively before fermentation. The over all goal of this study was thus to investigate the effect of different O2 and SO2 additions to Sauvignon blanc must before settling, specifically focussing on the typical aroma compounds often found in these wines. Chapter 2 gives an overview of the oxidation reactions occurring in must (enzymatic oxidation) and wine (chemical oxidation). This chapter also reports the origin of the specific Sauvignon blanc aroma compounds and their reaction to different must and wine treatments with a focus on oxidation. Chapter 3 reports research results focussing on the effect of the different must treatments on the character impacting compounds of Sauvignon blanc wines, specifically the methoxypyrazines and the volatile thiols. The effect of the treatments on the polyphenols and glutathione content in the must and wine was also investigated. Oxidation in the absence of SO2 led to a decrease in glutathione and certain phenolic compounds in the must. In general, volatile thiols were protected against oxidation by SO2, even when O2 was present in the must. Methoxypyrazines concentrations were not significantly influenced by the treatments. Chapter 4 elucidates the effect of the treatments on other yeast and grape derived aroma compounds often found in Sauvignon blanc wines, such as the esters, higher alcohols, fatty acids and monoterpenes. In general, the effect of SO2 seemed to have the greatest influence on the produced aroma compounds. The results reported in this thesis could possibly change the way South African Sauvignon blanc musts are handled in future during the winemaking process. It is clear that O2 and SO2 management in the cellar is of critical importance for the winemaker to produce wines of high quality. Future work is important to fully understand the mechanisms and evolution of important aroma compounds of Sauvignon blanc wines during the winemaking process. / AFRIKAANSE OPSOMMING: Sauvignon blanc wyn aroma word gewoonlik beskryf met terme soos groen rissie, grasagtig en aspersie terwyl ander tropiese aromas soos grenadella en koejawel ook dikwels voorkom. Die manipulasie van Sauvignon blanc in die wingerd en in die kelder tydens wynmaak, gee die wynprodusent die vryheid om ‘n wye reeks wyn style te produseer. Dit maak Sauvignon blanc baie populêr in die Suid‐Afrikaanse wynindustrie. Die bogenoemde aromas word waargeneem in die wyn as gevolg van die teenwoordigheid van sekere aroma komponente genaamd metoksipirasiene en vlugtige tiole. Hierdie komponente lewer ‘n unieke bydrae tot die aroma samestelling van Sauvignon blanc wyne en kom voor in die druiwe in die aromatiese vorm (metoksipirasiene) of as nie‐aromatiese voorlopers (tiole) wat tydens alkoholiese fermentasie deur die gis vrygestel kan word. Komponente soos esters, hoër alkohole, vetsure en monoterpene kan ook ‘n potensiële bydra lewer tot die algehele aroma van Sauvignon blanc wyne en kom voor in die druiwe (monoterpene) of ontstaan as gevolg van gis metabolisme gedurende alkoholiese fermentasie (esters, hoër alkohole, vetsure). Hierdie geur komponente word dikwels beskryf as vrugtig, blomagtig en oor die algemeen aangenaam. Tydens wynmaak kan die wyn tot ‘n mate gemanipuleer word om ‘n spesifieke wynstyl te bekom. Hulpmiddels soos temperatuur, dopkontak, pers omstandighede, suurstof (O2), swawel dioksied (SO2) en gisras is slegs ‘n paar faktore wat die algemene uitkoms van ‘n wyn kan beïnvloed. Oor die algemeen word Sauvignon blanc in Suid‐Afrika baie reduktief behandel tydens wynbereiding. Dit vereis sekere hulpmiddels, soos die gebruik van inerte gas, wat die produksiekoste dikwels verhoog. Navorsing ondersteun die reduktiewe behandeling van wit wyn, maar dit wil voorkom asof daar ‘n tekort aan navorsing is wat die reduktiewe behandeling van die sap voor fermentasie regverdig. Die algemene doel van die studie is dus om die effek van verskillende O2 en SO2 byvoegings tot Sauvignon blanc sap (voor afsak) te ondersoek met die fokus op die tipiese aroma komponente wat in die wyn voorkom. Hoofstuk 2 lewer ‘n algemene oorsig van die tipes oksidasie reaksies wat voorkom in sap (ensiematiese oksidasie) en wyn (chemiese oksidasie). Spesifieke Sauvignon blanc aroma komponente word ook ondersoek in terme van die oorsprong van die komponente asook die reaksie wat plaasvind met verskillende mos en wyn behandelings, met ‘n fokus op oksidasie. In hoofstuk 3 word die effek van die verskillende mos behandelings op tipiese Sauvignon blanc aroma komponente, spesifiek metoksipirasiene en vlugtige tiole, ondersoek. Die effek van die behandelings op die polifenole en glutatioon inhoud in die mos en wyn word ook gerapporteer. Oksidasie van die sap in die afwesigheid van SO2, het ‘n afname in glutatioon en sekere polifenol konsentrasies veroorsaak. Dit wil voorkom asof die produksie van vlugtige tiole oor die algemeen beskerm word teen oksidasie indien daar genoegsame SO2 teenwoordig is. Hierdie effek word ondervind selfs as die sap met suursof versadig word. Die effek van die behandelings op die konsentrasies van metoksipirasiene was nie beduidend nie. Hoofstuk 4 rapporteer die effek van die behandelings op ander aroma komponente soos esters, hoër alkohole, vetsure en monoterpene. Oor die algemeen wil dit voorkom asof die effek van SO2 beduidend was en waarskynlik die grootste invloed op die produksie van hierdie aroma komponente het. Na aanleiding van die resultate bevind in hierdie tesis, is daar ‘n moontlikheid dat die manier waarop Sauvignon blanc wyne geproduseer word in Suid‐Afrika, moontlik kan verander in die toekoms. Vir die wynmaker om hoë kwaliteit Sauvignon blanc wyne te produseer, is O2 en SO2 bestuur in die kelder van kardinale belang. Verdere navorsing moet steeds gedoen word om die meganisme en evolusie van belangrike aroma komponente in Sauvignon blanc wyne tydens die wynmaakproses, ten volle te verstaan.

La presente tesi di dottorato ha riguardato la valutazione di differenti campioni di bentoniti sodiche. Le attività pianificate sono state indirizzate a comprendere l’interazione dei suddetti materiali con proteine a diverso peso molecolare naturalmente contenute nei vini bianchi. Ulteriori indagini hanno riguardato l’eventuale impatto delle argille testate sui composti aromatici di origine varietale e fermentativa dei mosti e/o dei vini. I risultati sottolineano il ruolo chiave esercitato dal “fattore matrice”: oltre al tipo di vino, prima di pianificare un trattamento di fining, devono essere considerate anche la tipologia e la dose di bentonite da utilizzare. / This PhD thesis dealt with the evaluation of different sodium bentonite samples. The planned activities were aimed to understand the interactions between bentonites and different molecular weight proteins naturally contained in white wines. Further investigations were related to the impact of the clays on varietal and fermentative aroma compounds of musts and/or wines. Results underscore the key role exerted by the “matrix factors”: bentonite dose and typology and wine style have to be carefully considered before planning a fining treatment.

L'utilisation de souches de levures sélectionnées pour réaliser la FA est une pratique très répandue en œnologie. Après le développement de l'utilisation de levains de souche pure de Saccharomyces, l'innovation est aujourd'hui dans la mise en œuvre de levains mixtes de Saccharomyces et de non-Saccharomyces qui permettent de diversifier les produits finaux obtenus. La problématique réside dans l'existence d'interactions entre les souches rendant difficile la maîtrise de la fermentation. T. delbrueckii présente dans la flore indigène du mout de raisin est une des levures non-Saccharomyces les plus appropriées pour entrer dans la composition de ces levains mixtes. En effet, elle présente une bonne capacité fermentaire et peut permettre d'augmenter la complexité aromatique du vin ou encore de réduire son acidité volatile. L'objectif de ce travail était d'étudier les interactions pendant la FA entre des souches sélectionnées pour l'oenologie : une T. delbrueckii et une S. cerevisiae. Pour cela des expériences ont été réalisées dans des milieux synthétiques simulant le moût de raisins blancs. Le comportement des souches pures a tout d'abord été caractérisé. Il a été montré que la souche S. cerevisiae avait de meilleures performances fermentaires d'un point de vue cinétique que la souche T. delbrueckii. Toutefois, T. delbrueckii a montré des capacités acceptables pour épuiser les sucres et surtout a permis d'obtenir des profils aromatiques différents. Le comportement vis-à-vis de l'oxygénation des moûts de ces deux levures est assez semblable, T. delbrueckii étant cependant beaucoup plus sensible à ce paramètre que S. cerevisiae. L'interaction entre ces deux levures a ensuite était étudiée dans un bioréacteur à membrane sous anaérobie stricte dans différentes conditions : composition en azote assimilable du milieu et stratégie d'inoculation. Il a été clairement mis en évidence que T. delbrueckii était affectée par la présence de S. cerevisiae. Le type d'interaction soupçonné est celui d'amensalisme lié à l'excrétion par S. cerevisiae d'un constituant toxique pour T. delbrueckii. Dans ces conditions, la stratégie d'inoculation recommandée est l'ensemencement séquentiel des levures : T. delbrueckii en début de fermentation, puis l'ajout de S. cerevisiae 48 h après. Ceci permet à T. delbrueckii de se développer et d'exprimer son potentiel aromatique avant que S. cerevisiae ne soit introduit pour assurer une fin rapide de la fermentation. Toutefois, nous avons montré que même dans ces conditions, l'implantation de T. delbrueckii n'était pas garantie car, le moût n'étant pas stérile, une présence, même faible, de S. cerevisiae dans la flore naturelle peut inhiber sa croissance. Par ailleurs, il a été mis en évidence que dans les mouts à faible teneur en azote initial, ce constituant pouvait être épuisé au moment de l'inoculation de S. cerevisiae. Dans ces conditions, S. cerevisiae ne peut se développer et l'achèvement de la fermentation est alors problématique. / The use of the selected yeast strains to realize the alcoholic fermentation is very prevalent practice in vinification. After the development of utilization of the preparation of pure Saccharomyces cerevisiae strain, the innovation is now to apply the mixte starter cultures of Saccharomyces and non-Saccharomyces that allow to diversifying the obtained final products. The problem resides in the existence of interactions between the strains giving the difficulty to controle the fermentation. Torulaspora delbrueckii present in the indigenous flora of the grape must is one of the most appropriate non-Saccharomyces yeasts to enter in the composition of these multistater cultures. In fact, this strain has been presented a good fermentative capacity and could allow to increasing not only the aromatic complexity of wine, but also to reducing its volatile acidity. The objective of our work is to study the interactions during the alcoholic fermentation between the selected strains for enology: one T. delbrueckii and one S. cerevisiae. For this reason, the experiments were realized in the synthetic mediums simulated to the white grape must. The behaviours of the pure strains were firstly characterized. It was shown that the S. cerevisiae strain had the best fermentative performances, a critical point in comparaison with the T. delbrueckii strain. Nevetheless, T. delbrueckii showed the acceptable capacities to exhaust the sugars and especially to allow us to obtain the different aromatic profiles to that of S. cerevisiae. The behaviour via the oxygenation to the musts of these two yeasts is enough close, T. delbrueckii being however much more sensible to that parameter than . cerevisiae. The interactions between these two yeasts were then studied in a membrane bioreacteur under strict anaerobie in different conditions: composition in assimilable nitrogen of the medium and strategy of inoculation. It has been clearly demonstrated that T. delbrueckii has been affected by the presence of S. cerevisiae. The suspected type of this interaction is the amensalism one bound to a toxic compound excreted by S. cerevisiae. In these conditions, the recommended inoculation strategy is the sequential culture of these yeasts: T. delbrueckii at the beginning of the fermentation, then the addition of S. cerevisiae after 48 h. This allows T. delbrueckii to develop and express its potentiel of aromatic production before the S. cerevisiae is introduced to assure a rapid finish of the fermentation. However, we showed that even in these conditions, T. delbrueckii growth has been not guaranteed because of, since the must is not sterilized, a presence even small of S. cerevisiae in the natural flore can inhibite the croissance of the former. It has been also demonstrated that in the must with low intitial nitrogen content, this compound could be exhausted at the moment of the S. cerevisiae inoculation. In these conditions, S. cerevisiae can not develop and the achievement of the fermentation is yet problematic.

This research investigated yeast populations and diversity during pre-fermentation cold maceration and alcoholic fermentation of Vitis vinifera L. cv. Pinot noir grapes from a commercial vineyard (Dayton, OR). Fermentations were conducted at the Oregon State University research winery in 100 L tanks while grapes from the same vineyard lot were fermented at a commercial winery. Samples were taken daily during pre-fermentation maceration (9°C) and alcoholic fermentation (27°C) and plated on WL and lysine media to determine Saccharomyces and non-Saccharomyces populations and diversity. Total non-Saccharomyces populations increased from 1 x 10³ cfu/mL to 1 x 10⁵ cfu/mL during pre-fermentation cold maceration and reached a maximum of 1 x 10⁷ cfu/mL during alcoholic fermentation. Thirteen distinct yeast species were tentatively identified based on appearance on WL media and were initially screened for β-glucosidase activity using 4-methyllumbelliferyl-β-D-gluconopyranoside (4-MUG) plates. The identity of the isolates screening positive for β-glucosidase activity was determined by sequence analysis of the D1/D2 domain of the 26S rDNA gene. The five isolates identified were Metschnikowia pulcherrima, Hanseniaspora uvarum, Kluveromyces thermotolerans, and two Saccharomyces cerevisiae isolates. β-glucosidase activity was further characterized and quantified using a liquid media representing grape must conditions (pH 3.5, 20° Brix) at two temperatures (25°C and 8°C). While increasing sugar concentration suppressed the β-glucosidase activity of H. uvarum (-99%), β-glucosidase activity still remained relatively high for M. pulcherrima, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. At 8°C, β-glucosidase activity was reduced for M. pulcherrima compared to activity at 25°C, but activity increased for K. thermotolerans, S. cerevisiae isolate 1, and S. cerevisiae isolate 2. The yeast isolates possessing β-glucosidase activity were used in fermentations of Vitis vinifera L. cv. Pinot Noir grapes. The grapes were treated with high hydrostatic pressure (HHP) to inactivate naturally occurring yeast and bacteria. All yeast isolates grew during pre-fermentation cold maceration (7 days at 9°C) and populations increased 3 to 4 logs. Following pre-fermentation cold maceration, all ferments were warmed to 27°C and inoculated with S. cerevisiae RC212. Alcoholic fermentations were all complete within eight days and after pressing wines were analyzed for volatile aroma compounds by SPME-GC-MS. The presence of different yeast isolates during pre-fermentation cold maceration resulted in wines with unique aroma profiles. Ethyl ester concentrations were highest in the wine that did not undergo a pre-fermentation cold maceration, while concentrations of branch-chained esters were higher in the treatments with yeast present during pre-fermentation cold maceration. Pre-fermentation cold maceration with yeast isolates demonstrating β-glucosidase did not affect the concentration of β-damascenone or β-ionone. Wines that had undergone pre-fermentation cold maceration with S. cerevisiae isolate 1, S. cerevisiae isolate 2, and a combination of all isolates resulted in over twice the concentration of β-citronellol over wines that did not undergo a pre-fermentation cold maceration. / Graduation date: 2013