La mise au point de techniques de fractionnement des moûts de Sauvignon blanc par chromatographie de partage centrifuge et par chromatographie Flash, a permis de mettre en évidence la présence de deux « nouvelles » formes précurseurs du 3SH (le S-3-(hexan-1-al)-glutathion et l'acide S-3-glutathionyl-hydroxyhexanesulfonique). L’ensemble de ces résultats a permis de confirmer la contribution majeure des S-conjugués au potentiel en 3SH des moûts. Le S-3-(hexan-1-ol)-L-cystéine, le S-3-(hexan-1-ol)-glutathion ainsi que les deux nouvelles formes identifiées, permettent d’expliquer près de 65% du 3SH libéré dans les vins. Par ailleurs, nous avons montré que l’ensemble des formes précurseurs appartenait à la même voie d’assimilation de la levure et que le S-3-(hexan-1-ol)-L-cystéine est un intermédiaire indispensable à la biotransformation des S-conjugués au glutathion, précurseurs du 3-sulfanylhexanol. De plus, le pourcentage de biotransformation des différents S-conjugués en 3SH semble directement lié à leur position dans le flux métabolique. / The development of fractionation techniques in Sauvignon Blanc musts such as centrifugal partition chromatography and flash chromatography, permitted to identify two precursor forms of 3-sulfanylhexanol : the S-3-(hexan-1-al)-glutathione, and the S-3-glutathionyl-hydroxy hexanesulfonic acid. These results confirmed the major contribution of S-conjugates in the potential liberation of 3-sulfanylhexanol in musts. The two newly identified forms along with S-3-(hexan-1-ol)-L-cysteine and S-3-(hexan-1-ol)-glutathione, explain up to 65% of the total release of 3-sulfanylhexanol. Futhermore, we showed that all precursor forms belonged to the same assimilation pathway of yeast and the S-3-(hexan-1-ol)-L-cysteine is an essential intermediate in the metabolism of S-glutathione conjugates, which is a precursor of 3-sulfanylhexanol . Finally, the biotransformation percentage of different S-conjugates of 3-sulfanylhexanol seems directly related to their position in the metabolic flow.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: The varietal character of Sauvignon blanc wine is mostly defined by the balance between tropical and green vegetative flavour nuances. Grape derived methoxypyrazines are the main aroma contributors towards green vegetative flavours. Methoxypyrazines are heat and light sensitive. Due to warm climatic conditions in South Africa, methoxypyrazine levels decrease during grape ripening. The addition of food flavourings to Sauvignon blanc wine, a practice known as spiking, has occurred in the past to improve the green character of the wines. Adding flavourings to wine and selling the wine as natural certified wine is illegal in South Africa. Currently, adulterated Sauvignon blanc wines are identified using gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) methods to quantify methoxypyrazines and compare levels to an established database. Although of high sensitivity, GC-MS and LCMS methods are costly and time consuming, therefore not optimal for routine screening of wines. Hence the need for the development of a fast and cost effective method for routine screening of large amounts of wines to identify adulteration. Small scale vinification practices were used to prepare experimental Sauvignon blanc wine. Flavourings were added to Sauvignon blanc grape juice before fermentation, during the preparation of experimental spiked wines. Control wines, containing no flavouring, were also prepared. Commercial wines were spiked after fermentation and bottling. Each wine was only spiked with a single flavouring. The flavourings added were the juice of homogenised fresh green peppers and commercially available flavourings for wine. The following commercial flavourings were used: green pepper, asparagus, grassy and tropical. The above mentioned wines were analyzed using Fourier transform infrared (FT-IR) spectroscopy, GC-MS, LC-MS and descriptive sensory analysis. The FT-IR techniques used were Fourier transform mid infrared (FT-MIR) transmission, FT-MIR attenuated reflection and Fourier transform near infrared (FT-NIR) reflection spectroscopy. The data was interpreted using the following multivariate statistical techniques: principal component analysis (PCA), partial least squares discrimination (PLS-D) and conformity testing. Multivariate models constructed from FT-MIR and FT-NIR data were able to discriminate between spiked and control wines. Sensory analysis results clearly showed differences between non-spiked wines and spiked wines with 3-isobutyl-2-methoxypyrazine concentrations 10 times higher than naturally occurring in wine. Differences between control and spiked wines with concentrations of 3-isobutyl-2-methoxypyrazine similar to concentrations naturally occurring in wines could not be detected to prove adulteration conducting sensory analysis. However, differences between control and spiked wines with levels of 3-isobutyl-2-methoxypyrazine similar to levels naturally occurring in wines could be detected using FT-IR data in conjunction with multivariate statistics. This study showed that, FT-IR spectroscopy in conjunction with multivariate statistical methods can be a possibility for the screening and identification of wines suspected of adulteration in terms of added flavourings. Descriptive sensory analysis also proved to be a potentially useful tool. However screening and training of potential panel members are time consuming. / AFRIKAANSE OPSOMMING: Die variëteitskarakter van Sauvignon blanc wyn word grotendeels gedefinieer deur die balans tussen tropiese en groen vegetatiewe aromas. Metoksipirasiene is die hoof aroma verbindings wat verantwoordelik is vir groen vegetatiewe aromas. Metoksipirasien is hitte- en ligsensitief. Warm klimaatsomstandighede in Suid-Afrika het tot gevolg dat metoksipirasien konsentrasies daal tydens druif rypwording. Sauvignon blanc wyne is in die verlede vervals deur middel van die byvoeging van vars groen soetrissies om die groen vegetatiewe karaktereienskappe van die wyne te bevorder. Die byvoeging van geurmiddels of plantekstrakte by wyn en verkoop van daardie wyn as gesertifiseerde natuurlike wyn is onwettig in Suid-Afrika. Tans word vervalsde wyne met behulp van gaschromatografie-massaspektrometrie (GC-MS) en vloeistofchromatografie-massaspektrometrie (LC-MS) opgespoor. Kwantifisering van metoksiepirasien konsentrasies in wyne en druiwesappe word vergelyk met konsentrasies in ‘n bestaande databasis. Alhoewel GC-MS en LC-MS hoë sensitiwiteitsmetodes is, is dit duur en tydrowende metodes, dus nie optimaal vir roetine sifting nie. Dus word ‘n koste- en tydseffektiewe roetine metode benodig om vervalsing van wyne op te spoor. Eksperimentele wyne is op klein skaal berei. Geurmiddels is voor fermentasie by die druiwesap gevoeg. Kontrole wyne waarby geen geurmiddels gevoeg is nie, is ook berei. Kommersiële wyne is gegeur na fermentasie en bottelering. Elke wyn is met ‘n enkele geurmiddel gegeur. Gehomogeniseerde vars groen soetrissie asook kommersieel beskikbare geursels vir wyn is gebruik. Die volgende kommersiële geursels is gebruik: groen soetrissie, aspersie, gras en tropiese geursel. Die volgende analitiese tegnieke is gebruik vir analisering van bogenoemde wyne: Fourier transformasie infrarooi (FT-IR) spektroskopie, GC-MS, LC-MS en beskrywende sensoriese analise. Die spesifieke FT-IR tegnieke wat gebruik is, is: Fourier transformasie mid-infrarooi (FT-MIR) transmissie, FT-MIR verswakte weerskaatsing en Fourier transformasie naby-infrarooi (FT-NIR) reflektansie. Die volgende multiveranderlike statistiese tegnieke is gebruik ter interpretasie van data: hoof komponent analise (PCA), parsiële kleinste kwadraat diskriminant analise (PLS-D) en gelykvormigheidstoetsing. Multiveranderlike modelle, bereken met behulp van FT-MIR en FT-NIR data, kon diskrimineer tussen gegeurde en kontrole wyne. Resultate wat verkry is tydens sensoriese analises het duidelike verskille uitgewys tussen gegeurde en kontrole wyne met betrekking tot 3-isobutiel-2- metoksipirasien konsetrasies waar 3-isobutiel-2-metoksipirasien konsentrasies 10 keer hoër was as wat natuurlik voorkom in wyn. Geen beduidende verskille kon waargeneem word in gevalle waar wyne vervals is met laer konsentrasies van geurmiddels deur sensoriese data te ontleed nie. Nietemin, statisitiese verskille tussen kontrole en vervalsde wyne kon waargeneem word vir lae-konsentrasie-geurmiddel vervalsde wyne deur FT-IR data met behulp van multiveranderlike statisitiese metodes te ontleed. Hierdie studie het gewys dat FT-IR in kombinasie met multiveranderlike statistiese tegnieke spesifiek hoof komponent analise (PCA) en parsiële kleinste kwadraat diskriminant analise (PLS-D) asook gelykvormigheidstoetsing bruikbare tegnieke is om te onderskei tussen kontrole (egte natuurlike) en vervalsde wyne ten opsigte van die byvoeging van geurmiddels. Beskrywende sensoriese analise kan ook nuttig gebruik word, alhoewel keuring en opleiding van paneellede tydrowend is.

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Grape must is a complex medium, and during wine production numerous biochemical pathways and metabolic reactions are taking place simultaneously to produce a specific taste and aroma. Microorganisms, specifically yeast, play a key role in the formation of metabolites formed during alcoholic fermentation. Sauvignon blanc, a well studied grape cultivar, is known to have a versatile range of aroma profiles ranging from “green” to “tropical”. It has been broadly stated that a “green” Sauvignon blanc can be created in the vineyard and a “tropical” Sauvignon blanc can be created by selecting a specific yeast strain, and that the balance between “green” and “tropical” characters is essential for the final aroma profile. Except for grape-derived varietal aromatic compounds such as methoxypyrazines (green), volatile thiols (tropical) and monoterpenes (floral), yeast derived volatile compounds including esters, higher alcohols, fatty acids and carbonyl compounds will also contribute to the final wine aroma. The main aim of this study was to assess how viticultural treatment-derived differences in grape must, can impact on aroma production when this grape must is fermented with different commercial wine yeast strains. The viticulture treatment focused on light intensity modulated through canopy treatment. Volatile aroma differences were compared for canopy and yeast treatments, specifically focusing on the fermentation derived bouquet (esters, higher alcohols, volatile fatty acids, carbonyl compounds and monoterpenes). Results showed significant differences between initial must compositions, including titratable acidity, malic acid and yeast assimilable nitrogen. The volatile aroma compounds were also significantly impacted although no noticeable effect on the overall fermentation kinetics was observed. Depending on the yeast strain differences in volatile compounds varied. A clear vintage effect is noticeable between volatile compounds affected by the treatments. Data generated in 2012 shows clear differences between ethyl- and acetate esters and could clearly be grouped according to yeast strain through multivariate analysis. Sensory evaluation results could clearly be distinguished according to canopy treatment and to a lesser degree according to yeast strain used. This indicates that although yeast has a more prominent impact on the fermentative bouquet that develops during alcoholic fermentation the overriding aroma is primarily derived from grape-derived metabolites which can be manipulated by canopy treatments. None the less the difference in fermentation bouquet does add to the complexity of the wine especially in the case of fermentation derived “tropical” aromas including guava and passion fruit. In some cases where shaded grapes had higher ester concentrations, the resultant wine also had higher aroma quality. This study has contributed to a better understanding of the complex relationships between canopy manipulation and yeast selection on aroma formation. The analysis of volatile aroma alone however is not enough to understand the final perception of wine taste and further indepth studies of the viticultural and oenological factors is needed. In particular, this project has focused on a single vineyard over only two vintages. The general validity of the conclusions derived from this study therefore will require additional data sets. / AFRIKAANSE OPSOMMING: Druiwemos is ‘n komplekse medium en tydens wynbereiding is daar verskeie biochemiese weë en metaboliese reaksies wat gelyktydig plaasvind om ‘n spesifieke smaak en aroma te produseer. Mikro-organismes, veral gis, speel ‘n sleutelrol in die vorming van metaboliete tydens alkoholiese gisting. Sauvignon blanc, ‘n goed bestudeerde druifkultivar, besit ‘n veelsydige reeks aromaprofiele wat wissel van “groen” tot “tropies”. Oor die algemeen word dit voorgehou dat ‘n “groen” Sauvignon blanc in die wingerd geskep word, terwyl ‘n “tropiese” Sauvignon blanc geskep kan word deur ‘n spesifieke gisras te selekteer, en die balans tussen “groen” en “tropiese” karakters is noodsaaklik vir die finale aromaprofiel. Behalwe vir druifafgeleide kultivarafhanklike aromatiese verbindings soos metoksipirasiene (groen), vlugtige tiole (tropies) en monoterpene (blomagtig), sal gisafgeleide vlugtige komponente, waaronder esters, hoër alkohole, vetsure en karbonielverbindings, ook tot die finale wynaroma bydra. Die hoofdoelwit van hierdie studie was om te bepaal hoe verskille in druiwemos wat afkomstig is van wynkundige behandeling ‘n impak op aromaproduksie kan hê wanneer hierdie druiwemos met verskillende kommersiële wyngisrasse gegis word. Die wynkundige behandeling het gefokus op ligintensiteit wat deur lowerbehandeling gereguleer is. Vlugtige aromaverskille is op grond van lower- en gisbehandelings vergelyk, met ‘n spesifieke fokus op die gistingsafgeleide boeket (esters, hoër alkohole, vlugtige vetsure, karbonielverbindings en monoterpene). Die resultate het beduidende verskille getoon tussen aanvanklike mossamestellings, waaronder titreerbare suurheid, appelsuur en gis-assimileerbare stikstof. Daar was ook ‘n noemenswaardige impak op die vlugtige aromaverbindings, hoewel geen merkbare effek op die algehele gistingskinetika waargeneem kon word nie. Die verskille in vlugtige verbindings het gewissel op grond van die gisras. ‘n Duidelike oesjaareffek was merkbaar tussen vlugtige verbindings wat deur die behandelings geaffekteer is. Data wat in 2012 gegenereer is, toon duidelike verskille tussen etiel- en asetaatesters en kon duidelik m.b.v. meervariantanalise volgens gisras gegroepeer word. Die resultate van die sensoriese evaluering kon duidelik volgens lowerbehandeling onderskei word, en tot ‘n mindere mate volgens die gisras wat gebruik is. Dít dui daarop dat hoewel gis ‘n meer prominente impak het op die gistingsboeket wat tydens alkoholiese gisting ontwikkel, is die oorheersende aroma hoofsaaklik afgelei van druifafgeleide metaboliete wat deur lowerbehandelings gemanipuleer kan word. Nietemin dra die verskil in gistingsboeket by tot die kompleksiteit van die wyn, veral in die geval van gistingsafgeleide “tropiese” aromas, insluitend koejawel en grenadella. In sommige gevalle waar beskadude druiwe hoër esterkonsentrasies gehad het, het die gevolglike wyn ook ‘n hoër aromakwaliteit gehad. Hierdie studie dra by tot ‘n beter begrip van die effek van die komplekse verhoudings tussen lowermanipulasie en gisseleksie op aromavorming. ‘n Analise van vlugtige aroma alleen is egter nie voldoende om die finale persepsie van wynsmaak te begryp nie en bykomende diepgaande studies van die wingerdkundige en wynkundige faktore word benodig. Hierdie projek het in die besonder gefokus op ‘n enkele wingerd oor slegs twee oesjare. Die algemene geldigheid van die afleidings wat van hierdie studie gemaak word, sal dus bykomende datastelle vereis. / The National Research Foundation and Postgraduate Merit Bursary for financial support

The effects of crop load and berry exposure on the composition of Marlborough Sauvignon blanc grapes and wine from the Brancott vineyard, Blenheim, were explored. Commercially grown, 2-cane and 4-cane Sauvignon blanc vines were used with a row orientation of north-south. Two exposure treatments were imposed in the following manner: complete leaf removal was undertaken in the fruit zone and 50% shade cloth was erected to give a uniform shading treatment to half the trial vines. Weekly thirty-berry and whole bunch samples were taken from each of the 32 plots with the exception of the veraison period when two samples per week were taken. Vine vigour was assessed using pruning and leaf area per vine data. Harvest occurred on different dates for 2-cane and 4-cane pruned vines so that fruit attained from both treatments had similar °Brix. Fruit was processed at the Lincoln University winery. Must analysis and wine analysis were undertaken. As expected, 4-cane vines had almost double the yield of 2-cane vines. Higher crop load significantly reduced leaf area per shoot and shoot thickness. Lower leaf area to fruit ratio for 4-cane berries resulted in delayed onset of veraison and slowed the rate of sugar accumulation. Crop load, which limited leaf area to fruit ratio, appeared to be the dominant factor in determining timing of grape physiological ripeness as expressed by °Brix over other factors such as fruit exposure. Malic acid, tartaric acid, IPMP (iso-propylmethoxypyrazine) and IBMP (iso-butyl-methoxypyrazine) were lower at equivalent °Brix in 4-cane compared with 2-cane berries. Significantly higher concentrations of quercetin were found in exposed compared to shaded berries. Must analysis showed a significant influence of crop load on berry titratable acidity and pH, reflecting berry ripening results. Exposure significantly increased the concentrations of nitrogenous compounds in 4-cane must yet showed no influence on 2-cane must. After wine processing lower malic acid concentrations in wines made from 100% exposed fruit became evident in lower wine titratable acidity but showed no influence on wine pH. Bentonite addition to wines had a small but statistically significant influence on wine by reducing pH, titratable acidity and alcohol. Bound sulphur concentrations were significantly higher in 4-cane versus 2-cane wines. At harvest, methoxypyrazine levels in grapes and wines were very low; IBMP concentrations where significantly lower than those normally found in Sauvignon blanc wines from Marlborough. This was attributed to the absence of basal leaves from the shoots of ripening berries. The results suggest that leaf area to fruit ratio is a powerful determinant of grape and wine quality.

ABSTRACT A core tool of sensory science is the use of trained descriptive panels. This research describes an investigation into the role of motivation in the performance of trained panels and the use of a trained panel to develop a better understanding of the perception of Sauvignon blanc wines. Substantial investment in time and money is directed towards ensuring trained panels perform optimally. Having selected a panel, the panel leader needs to ensure that panellists provide accurate, reliable data. Panellist motivation is also an important factor to consider. While performance psychology, education and sport science fields have researched motivation extensively, knowledge about panellist motivation within sensory science is limited. However, findings from existing research in these other areas - which suggest an important role for autonomy, competence and relatedness - can be applied to sensory panels in order to increase intrinsic motivation. The initial part of the research investigated the fundamental factors that affect and influence panellists’ motivation and participation. A survey (n=74) revealed that extra income and a general interest in food were the key drivers in inspiring people to become panellists, whilst enjoyment in being a panellist, interest in food, and extra income were key drivers for people to remain panellists. In a second survey, the intrinsic motivation of seven trained panels from four countries (n=108) was assessed. External panels were found to be more intrinsically motivated than internal panels. Experienced panellists had an increased perception of competence, which is a key factor for people to be intrinsically motivated. Understanding motivational frameworks currently used in other research fields and integrating them into existing panel training protocols may enhance and sustain panellists’ intrinsic motivation. A trained panel (n=14) was then used in the second part of the thesis to identify key flavours in Sauvignon blanc wines from Australia, France, New Zealand, Spain, South Africa and USA. Sixteen characteristics were identified and measured, including sweet sweaty passionfruit, capsicum, passionfruit skin/stalk, boxwood/cat’s urine, grassy, mineral/flinty, citrus, bourbon, apple lolly/candy, tropical, mint, fresh asparagus, canned asparagus, stonefruit, apple and snowpea. Principal component analysis was used to describe differences between regions and countries. Sauvignon blanc wines from Marlborough, New Zealand (NZ), were described by tropical and sweet sweaty passionfruit characteristics, while French and South African Sauvignon blanc wines were described as having flinty/mineral and bourbon-like flavors. Chemical analyses of these wines also showed that Marlborough, NZ wines had more methoxypyrazine and thiol compounds. A consumer study (n=109) showed that New Zealanders significantly prefer New Zealand style Sauvignon blanc. The final part of this research focused on using trained panellists to explore the interactions between volatile and non-volatile wine compounds and their effects on the aroma profile of New Zealand Sauvignon blanc wine. Four volatile aroma compounds that are important in New Zealand Sauvignon blanc wine were studied (isobutyl methoxypyrazine [MIBP], 3-mercaptohexanol [3MH], 3-mercaptohexanol acetate [3MHA], and ethyl decanoate). Each of these four aroma compounds were assessed in combination with three non-volatile polyphenolic compounds commonly found in Sauvignon blanc wine: catechin, caffeic acid and quercetin. Results showed each polyphenol had a unique effect when blended with a specific aroma compound, either suppressing, accentuating, or showing little effect on the perception of the aroma compounds. The perception of MIBP, 3MH, and ethyl decanoate were largely suppressed by the added polyphenols, with a few exceptions. The perception of 3MH was accentuated with the addition of caffeic acid, and the perception of 3MHA was accentuated with the addition of catechin. The interactive effects of aroma compounds with polyphenols likely reflect non-covalent associations in the wine solution that reduce the volatility of the aroma compounds. With an understanding of the interactive effects of volatile and non-volatile compounds in wine, winemakers might optimize the impact of selected volatile compounds by managing polyphenol levels, supporting their efforts to attain desirable wine aroma profiles.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.

ABSTRACT A core tool of sensory science is the use of trained descriptive panels. This research describes an investigation into the role of motivation in the performance of trained panels and the use of a trained panel to develop a better understanding of the perception of Sauvignon blanc wines. Substantial investment in time and money is directed towards ensuring trained panels perform optimally. Having selected a panel, the panel leader needs to ensure that panellists provide accurate, reliable data. Panellist motivation is also an important factor to consider. While performance psychology, education and sport science fields have researched motivation extensively, knowledge about panellist motivation within sensory science is limited. However, findings from existing research in these other areas - which suggest an important role for autonomy, competence and relatedness - can be applied to sensory panels in order to increase intrinsic motivation. The initial part of the research investigated the fundamental factors that affect and influence panellists’ motivation and participation. A survey (n=74) revealed that extra income and a general interest in food were the key drivers in inspiring people to become panellists, whilst enjoyment in being a panellist, interest in food, and extra income were key drivers for people to remain panellists. In a second survey, the intrinsic motivation of seven trained panels from four countries (n=108) was assessed. External panels were found to be more intrinsically motivated than internal panels. Experienced panellists had an increased perception of competence, which is a key factor for people to be intrinsically motivated. Understanding motivational frameworks currently used in other research fields and integrating them into existing panel training protocols may enhance and sustain panellists’ intrinsic motivation. A trained panel (n=14) was then used in the second part of the thesis to identify key flavours in Sauvignon blanc wines from Australia, France, New Zealand, Spain, South Africa and USA. Sixteen characteristics were identified and measured, including sweet sweaty passionfruit, capsicum, passionfruit skin/stalk, boxwood/cat’s urine, grassy, mineral/flinty, citrus, bourbon, apple lolly/candy, tropical, mint, fresh asparagus, canned asparagus, stonefruit, apple and snowpea. Principal component analysis was used to describe differences between regions and countries. Sauvignon blanc wines from Marlborough, New Zealand (NZ), were described by tropical and sweet sweaty passionfruit characteristics, while French and South African Sauvignon blanc wines were described as having flinty/mineral and bourbon-like flavors. Chemical analyses of these wines also showed that Marlborough, NZ wines had more methoxypyrazine and thiol compounds. A consumer study (n=109) showed that New Zealanders significantly prefer New Zealand style Sauvignon blanc. The final part of this research focused on using trained panellists to explore the interactions between volatile and non-volatile wine compounds and their effects on the aroma profile of New Zealand Sauvignon blanc wine. Four volatile aroma compounds that are important in New Zealand Sauvignon blanc wine were studied (isobutyl methoxypyrazine [MIBP], 3-mercaptohexanol [3MH], 3-mercaptohexanol acetate [3MHA], and ethyl decanoate). Each of these four aroma compounds were assessed in combination with three non-volatile polyphenolic compounds commonly found in Sauvignon blanc wine: catechin, caffeic acid and quercetin. Results showed each polyphenol had a unique effect when blended with a specific aroma compound, either suppressing, accentuating, or showing little effect on the perception of the aroma compounds. The perception of MIBP, 3MH, and ethyl decanoate were largely suppressed by the added polyphenols, with a few exceptions. The perception of 3MH was accentuated with the addition of caffeic acid, and the perception of 3MHA was accentuated with the addition of catechin. The interactive effects of aroma compounds with polyphenols likely reflect non-covalent associations in the wine solution that reduce the volatility of the aroma compounds. With an understanding of the interactive effects of volatile and non-volatile compounds in wine, winemakers might optimize the impact of selected volatile compounds by managing polyphenol levels, supporting their efforts to attain desirable wine aroma profiles.

ABSTRACT A core tool of sensory science is the use of trained descriptive panels. This research describes an investigation into the role of motivation in the performance of trained panels and the use of a trained panel to develop a better understanding of the perception of Sauvignon blanc wines. Substantial investment in time and money is directed towards ensuring trained panels perform optimally. Having selected a panel, the panel leader needs to ensure that panellists provide accurate, reliable data. Panellist motivation is also an important factor to consider. While performance psychology, education and sport science fields have researched motivation extensively, knowledge about panellist motivation within sensory science is limited. However, findings from existing research in these other areas - which suggest an important role for autonomy, competence and relatedness - can be applied to sensory panels in order to increase intrinsic motivation. The initial part of the research investigated the fundamental factors that affect and influence panellists’ motivation and participation. A survey (n=74) revealed that extra income and a general interest in food were the key drivers in inspiring people to become panellists, whilst enjoyment in being a panellist, interest in food, and extra income were key drivers for people to remain panellists. In a second survey, the intrinsic motivation of seven trained panels from four countries (n=108) was assessed. External panels were found to be more intrinsically motivated than internal panels. Experienced panellists had an increased perception of competence, which is a key factor for people to be intrinsically motivated. Understanding motivational frameworks currently used in other research fields and integrating them into existing panel training protocols may enhance and sustain panellists’ intrinsic motivation. A trained panel (n=14) was then used in the second part of the thesis to identify key flavours in Sauvignon blanc wines from Australia, France, New Zealand, Spain, South Africa and USA. Sixteen characteristics were identified and measured, including sweet sweaty passionfruit, capsicum, passionfruit skin/stalk, boxwood/cat’s urine, grassy, mineral/flinty, citrus, bourbon, apple lolly/candy, tropical, mint, fresh asparagus, canned asparagus, stonefruit, apple and snowpea. Principal component analysis was used to describe differences between regions and countries. Sauvignon blanc wines from Marlborough, New Zealand (NZ), were described by tropical and sweet sweaty passionfruit characteristics, while French and South African Sauvignon blanc wines were described as having flinty/mineral and bourbon-like flavors. Chemical analyses of these wines also showed that Marlborough, NZ wines had more methoxypyrazine and thiol compounds. A consumer study (n=109) showed that New Zealanders significantly prefer New Zealand style Sauvignon blanc. The final part of this research focused on using trained panellists to explore the interactions between volatile and non-volatile wine compounds and their effects on the aroma profile of New Zealand Sauvignon blanc wine. Four volatile aroma compounds that are important in New Zealand Sauvignon blanc wine were studied (isobutyl methoxypyrazine [MIBP], 3-mercaptohexanol [3MH], 3-mercaptohexanol acetate [3MHA], and ethyl decanoate). Each of these four aroma compounds were assessed in combination with three non-volatile polyphenolic compounds commonly found in Sauvignon blanc wine: catechin, caffeic acid and quercetin. Results showed each polyphenol had a unique effect when blended with a specific aroma compound, either suppressing, accentuating, or showing little effect on the perception of the aroma compounds. The perception of MIBP, 3MH, and ethyl decanoate were largely suppressed by the added polyphenols, with a few exceptions. The perception of 3MH was accentuated with the addition of caffeic acid, and the perception of 3MHA was accentuated with the addition of catechin. The interactive effects of aroma compounds with polyphenols likely reflect non-covalent associations in the wine solution that reduce the volatility of the aroma compounds. With an understanding of the interactive effects of volatile and non-volatile compounds in wine, winemakers might optimize the impact of selected volatile compounds by managing polyphenol levels, supporting their efforts to attain desirable wine aroma profiles.

The grape variety Sauvignon Blanc (SB) is the flagship of New Zealand’s wine industry and accounted for over 75 % of the value of total wine exports in 2008. Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical for the main varietal characters in New Zealand SB. These aromatic thiols are not present in the grape juice, but are synthesized and released by the yeast during alcoholic fermentation from non-aromatic precursors. The aim of this work was to elucidate the underlying genetics of volatile thiol synthesis in yeast (Saccharomyces cerevisiae) during alcoholic fermentation of grape juice. A gene-deletion strategy was chosen for the investigation of putative genes influencing 3MH and 3MHA release. The first part of this thesis optimized fermentation conditions in grape-juice-based media, which enabled auxotrophic laboratory strains, derived from S288C, to ferment grape juice to completion with high efficiency. Key steps to achieving this goal were the supplementation of the grape juice with higher than recommended amounts of amino acids, which increased the fermentation rate of auxotrophic yeast strains. Lysine auxotrophic strains especially benefited from this measure. In combination with the dilution of SB grape juice by 25 % with synthetic grape juice without sugars, the auxotrophic laboratory yeast BY4743 was able to metabolize all sugars in the grape juice-based media in a time frame similar to that of a commercial wine yeast. The key properties of the resulting wine were comparable to wine made with a commercial wine yeast under the same conditions. In the second part, these newly developed fermentation conditions were employed to screen 69 single-gene deletion strains in the laboratory yeast background BY4743. The list of the 69 candidate genes was compiled by combining existing knowledge about thiol production in yeast with the mining of several biological databases. Screening of the single-gene deletions revealed 17 genes which caused biologically relevant increases or decreases in volatile thiol production, but none abolished it. The majority of the 17 genes were related to the sulfur and nitrogen metabolism in yeast. A subset of these thiol-influencing genes were also deleted in a wine yeast, and were overexpressed in both wine yeast and laboratory yeast, to gain more insight in their regulatory effects. The findings confirmed that sulfur and nitrogen metabolism in yeast were important in regulating 3MH and 3MHA synthesis. Different sulfur and nitrogen sources were added to the grape must prior to fermentation and their effect on thiol release was studied. It was found that nitrogen sources urea and DAP, as well as, the sulfur compound S-ethyl-L-cysteine (SEC) increased 3MH and 3MHA concentrations in the resulting wines. The addition of cysteine to grape juice fermented with wine yeast deleted in genes CYS3 and CYS4 more than doubled total thiol production. Mapping approaches to investigate thiol production in yeast were employed in the final part of this thesis. Genetically mapped F2 progeny of a cross between a low thiol-producing yeast strain and a high-thiol producer were screened for their thiol phenotype. The 3MH and 3MHA phenotypes across 48 screened F2 progeny resembled normal distributions, indicating a quantitative trait. Subsequent mapping identified a locus on chromosome 14 with a small effect on the 3MHA phenotype, but no obvious candidate genes were evident in the region. Another approach to investigate the evolution of volatile thiols in yeast included the use of SEC, a thiol compound resembling the cysteinylated precursor of 3MH, as a sole nitrogen source in a yeast growth assay. It was found that most wine yeast, European yeast isolates and laboratory yeasts could utilize SEC as a nitrogen source, whereas various other S. cerevisiae isolates could not. Crosses between three pairs of Sec- and Sec+ yeast strains strongly indicated that this trait was monogenically inherited. However, no direct correlation between the SEC phenotype and volatile release could be observed. Genetic mapping experiments in one SEC-segregating yeast population linked this SEC phenotype to the leu2-D0 deletion in a cross between a Leu+ and Leu- yeast strain. It was shown that leucine auxotrophy most likely caused the Sec- phenotype. In a second F2 population of a cross between prototrophic Sec+ and Sec- strains, strong linkage was established to a region on chromosome 6 containing two candidate genes, DUG1 and IRC7. DUG1 was proved not to be the cause of the SEC phenotype, whereas IRC7 remains a strong candidate gene.