Metabolomic profiling of non- Saccharomyces yeasts in wine by Margaret Elizabeth Beckner Whitener Dissertation presented for the degree of Doctor of Philosophy at Stellenbosch University Institute for Wine Biotechnology, Faculty of AgriSciences Supervisor: Prof Maret du Toit Co-supervisor: Dr Urska Vrhovsek Co-supervisor: Dr Benoit Divol March 2016 Declaration By submitting this dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated) that reproduction and publication thereof by Stellenbosch University will not infringe on any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: 14 December 2015 Copyright © 2016 Stellenbosch University All rights reserved Summary Recent trends in wine making have led to the commercial production and use of non- Saccharomyces yeasts in wine making. Very little is understood however about how the use of these yeasts affects the final product. The purpose of this study was to evaluate the chemical and sensory characteristics of wine fermented with non-Saccharomyces yeasts using a sequential inoculation strategy. Targeted and untargeted analysis techniques were developed to help identify and quantify the volatile fraction of the wines produced. By combining this and sensory data we were able to build the most comprehensive picture to date of the volatile wine metabolome as it is influenced by various yeast species. The first step was a literature review dedicated to summarizing the current knowledge surrounding the metabolomics of the yeasts used in the subsequent chapters. Specifically, we sought to understand what is currently known about the use of non-Saccharomyces yeasts in wine. Also investigated were the technologies currently being used in the fields of food, wine, and yeast metabolomics. The goal was to provide the background necessary to understand the research in the subsequent chapters, as well as aid in the development and planning of the experiments discussed here within. Two stages of research were conducted. Not only did we want to understand the effects of non-Saccharomyces yeasts on wine aroma but we were interested in whether or not these effects were the same in both red and white wines. As such the first research stage, was a preliminary investigation of the yeast response to two different grape musts. Five different species of non- Saccharomyces yeasts, were chosen and grown in both Shiraz and Sauvignon blanc must and samples were collected for analysis just prior to the point at which Saccharomyces cerevisiae would usually be added to complete the fermentation. The fermentation rates were monitored and the chemical profile of the musts was evaluated. A solid-phase microextraction-Gas Chromatography-Mass spectrometry method that targeted 90 different compounds known to be found in wine was used to evaluate the headspace of the fermented musts. The results obtained helped shape the experimental design for the next phase of the project. The scale was increased to full wine production to evaluate how the yeasts could influence a completed wine product. Again, Sauvignon blanc and Shiraz were chosen and an untargeted chemical analysis method was developed to ensure that the widest possible range of analytes could be evaluated. The finished Sauvignon blanc wine was also subjected to sensory analysis which provided even greater insight into how these inoculation strategies can change the sensory profile of the wine. This research was undertaken in an attempt to answer the questions of ‘What will the wine smell and taste like if I use non-Saccharomyces yeasts during fermentation?’ and ‘Could it be superior to standard wines only inoculated with S. cerevisiae?’ The experiments conducted provided a great deal of insight that can help to begin answering these questions but there is much that remains unknown. In general, we were able to build a detailed volatiles chemical profile for each of the yeast treatments used in both Shiraz and Sauvignon blanc. While some treatments proved to be somewhat detrimental to the aroma and flavor of the wine, others showed promise in possibly enhancing its complexity. We were also able to demonstrate that the yeasts behave very differently in the two different musts. As comprehensive as these studies were, future work should be undertaken to improve the understanding of why and how these yeasts can make an impact on wine production. For example, our work did not include any genetic expression analysis of the yeasts used. Correlating genetic expression to quantitative chemical analysis would provide a much more complete picture of the wine yeast metabolome. Opsomming Onlangse tendense in wynbereiding het gelei tot die kommersiële vervaardiging en gebruik van nie-Saccharomyces giste in wynbereiding. Baie min word egter verstaan van hoe die gebruik van hierdie giste die finale produk affekteer. Die doel van hierdie studie was om die chemiese en sensoriese kenmerke te evalueer van wyn wat met nie-Saccharomyces giste gegis is deur gebruik te maak van ‘n opeenvolgende inentingstrategie. Geteikende en ongeteikende analise-tegnieke is ontwikkel om die vlugtige fraksie van die vervaardigde wyne te help identifiseer en kwantifiseer. Deur hierdie en die sensoriese data te kombineer, was ons in staat om die mees omvattende beeld tot op datum te bou van die vlugtige wynmetaboloom soos dit deur verskeie gisspesies beïnvloed word. Die eerste stap was ‘n literatuuroorsig gemik op die opsomming van huidige kennis oor die metabolomika van die giste wat in die opeenvolgende hoofstukke gebruik is. Ons het spesifiek gepoog om te begryp wat tans bekend is oor die gebruik van nie-Saccharomyces giste in wyn. Ons het ook die tegnologieë ondersoek wat tans in die gebied van voedsel-, wyn en gismetabolomika gebruik word. Die doelwit was om die nodige agtergrond te verskaf om die navorsing in die daaropvolgende hoofstukke te kan verstaan, sowel as om te help in die ontwikkeling en beplanning van die eksperimente wat hierbinne bespreek word. Twee stadiums van navorsing is onderneem. Nie net wou ons die effekte van nie- Saccharomyces giste op wynaroma verstaan nie, maar ons het ook daarin belanggestel om uit te vind of hierdie effekte dieselfde was in beide rooi- en wit wyne. As sulks was die eerste navorsingstadium ‘n voorlopige ondersoek na die gisrespons op twee verskillende druiwemoste. Vyf verskillende spesies van nie-Saccharomyces giste is gekies en in beide Shiraz- en Sauvignon blanc-mos gegroei en monsters vir analise is geneem net voor die punt waarop Saccharomyces cerevisiae gewoonlik bygevoeg sou word om die gisting te voltooi. Die gistingstempo’s is gemonitor en die chemiese profiel van die moste is geëvalueer. ‘n Soliede fase-mikroekstraksie– gaschromatografie massaspektrometrie metode wat 90 verskillende verbindings teiken wat daarvoor bekend is om in wyn voor te kom, is gebruik om die lugspasie van die gegiste moste te evalueer. Die resultate wat behaal is, het bygedra tot die opstel van die eksperimentele ontwerp vir die volgende fase van die projek. Die skaal is verhoog tot volledige wynproduksie om te evalueer hoe die giste ‘n voltooide wynproduk sou beïnvloed. Sauvignon blanc en Shiraz is weer gekies en ‘n ongeteikende metode van chemiese analise is ontwikkel om te verseker dat die breedste moontlike reeks analiete geëvalueer kon word. Die voltooide Sauvignon blanc wyn is ook aan sensoriese analise onderwerp wat nog groter insig verskaf het in hoe hierdie inentingstrategieë die sensoriese profiel van die wyn kan verander. Hierdie navorsing is onderneem in ‘n poging om vrae te beantwoord soos: ‘Hoe sal die wyn ruik en proe as ek nie-Saccharomyces giste tydens gisting gebruik?’ en ‘Sou dit beter as standaard wyne wees wat net met S. cerevisiae ingeënt is?’ Die eksperimente wat uitgevoer is, het ‘n groot mate van insig verskaf wat ons kan help om te begin om hierdie vrae te beantwoord, maar daar is baie wat nog onbekend is. Oor die algemeen kon ons ‘n gedetailleerde chemiese profiel van vlugtige stowwe vir elk van die gisbehandelings wat in beide die Shiraz en Sauvignon blanc gebruik is, bou. Hoewel sommige van die behandelings ietwat nadelig was vir die aroma en geur van die wyn, het ander belofte getoon om moontlik die kompleksiteit te verhoog. Ons kon ook demonstreer dat die giste baie verskillend in die twee verskillende moste opgetree het. Hoewel hierdie studies omvattend was, moet verdere werk in die toekoms gedoen word om ons begrip van hoekom en hoe hierdie giste ‘n impak op wynproduksie kan maak, te verbeter. Byvoorbeeld, ons werk het nie enige analise van die genetiese uitdrukking van die giste wat gebruik is, ingesluit nie. ‘n Korrelasie van die genetiese uitdrukking met kwantitatiewe chemiese analises sou ‘n baie meer volledige beeld van die wyngismetaboloom kon verskaf. This dissertation is dedicated to my family, without whose support it would not have been possible. Biographical sketch Margaret Elizabeth Beckner Whitener was born on December 11th, 1986 in Frankfort, Kentucky, USA. The daughter of a United States Army officer and elementary school teacher, she grew up moving frequently. At the age of 16 she was accepted into a prestigious residential high school, North Carolina School of Science and Mathematics. After completing her studies there she attended the University of North Carolina at Chapel Hill and graduated with a Bachelor of Science degree in Biology in 2009. Margaret was then accepted to the North Carolina State University’s Department of Food, Bioprocessing and Nutrition Sciences where she completed a Master’s Degree with a thesis entitled: Understanding Volatile Compound Production by S. cerevisiae and L. plantarum and their Role in Interactions between the Species. In 2012, she was awarded a scholarship in order to pursue a PhD from Fondazione Edmund Mach’s International Doctoral Programme, located in Trentino, Italy.