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The Pennsylvania State University The Graduate School MICROBIAL AND CHEMICAL ANALYSIS OF WILD YEASTS FROM CHAMBOURCIN HYBRID GRAPES FOR POTENTIAL USE IN WINEMAKING A Thesis in Food Science by Chun Tang Feng Ó 2020 Chun Tang Feng Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science December 2020 ii The thesis of Chun Tang Feng was reviewed and approved by the following: Josephine Wee Assistant Professor of Food Science Thesis Advisor Edward G. Dudley Professor of Food Science Ryan J. Elias Professor of Food Science Robert F. Roberts Professor of Food Science Head of the Department of Food Science iii ABSTRACT Native microbial populations present on grape berries in the vineyard and in winery environments can influence final wine quality. Previous studies on wild yeasts isolated from Vitis vinifera grapes have been reported to enhance wine flavor complexity. Although commercial Saccharomyces cerevisiae has been historically used for winemaking due to its efficiency and reliability in alcoholic fermentation, the role of other fungal populations on fermentation and physicochemical properties of final wines are not well characterized. Chambourcin, a French-American hybrid grape, is the most abundant hybrid grape variety grown in Pennsylvania and is relatively more resistant to cold temperatures and fungal diseases compared to Vitis vinifera grapes. In this study, we isolated and identified wild yeasts from three regional wineries that grow and produce Chambourcin to explore their potential to enhance complexity of final wines. We selected five candidate yeasts, Hanseniaspora uvarum NV192410, H. opuntiae NV192404, Pichia kluyveri NV192402, P. kudriavzevii SM192402 and Aureobasidium pullulans SM190002 and characterized their ability to tolerate varying concentrations of sulfite (0~100mg/L sodium metabisulfite, pH=3) and ethanol (0~12%v/v). We further developed a laboratory scale fermentation system that allowed analysis of non-volatile and volatile compounds derived from inoculated fermentations using candidate wild yeasts using Ultra High-Performance Liquid Chromatography and Gas Chromatography Mass Spectrometry. One hundred and twenty yeast isolates were obtained from three regional vineyards which comprised of 29 unique yeast species. Two wild yeast strains H. opuntiae NV192404 and P. kudriavzevii SM192402 demonstrate tolerance when grown in 8-10 % ethanol and iv are able to convert sugars to ethanol at a level comparable with control strain S. cerevisiae BY4742 (0.5 g ethanol/g sugar). Concentration of wine important non-volatile compounds were conserved among wild yeasts. Of interest in winemaking, H. opuntiae NV192404 was positively correlated to acetoin and linalool (pleasant buttery and flowery odor) and P. kudriavzevii SM192402 had positive correlation with 1-butoxy-1-ethoxyethane and ethyl 2-hexenoate (fruity aroma). In summary, microbial and chemical analysis of candidate wild yeasts can play a role in fermentation and in wine flavor complexity. Future work will focus on sensory and consumer studies to determine whether the differences of wine flavor can be detected and appreciated. v TABLE OF CONTENTS LIST OF FIGURES ................................................................................................... vii LIST OF TABLES ..................................................................................................... x ACKNOWLEDGEMENTS ...................................................................................... xi Chapter 1 1.1. Statement of Issue .......................................................................................... 1 1.2. Non-Saccharomyces yeasts on grapes ........................................................... 4 1.3. Characterization of important yeast physiological properties related to winemaking ................................................................................................... 9 1.4. Core non-volatile and volatile metabolites as the indicators of wine flavor .............................................................................................................. 13 Chapter 2 2.1. Significance ................................................................................................... 18 2.2. Hypothesis and Objectives ............................................................................ 21 2.3. Important considerations to experimental design .......................................... 22 2.3.1. Combination of early stage fermentation and addition of sulfite are two important aspects to capturing diverse wild yeasts populations ..... 22 2.3.2. Development of a laboratory scale fermentation using sterile Chambourcin juice inoculated with candidate yeast strains for wine chemical profile analysis ........................................................................ 26 Chapter 3 3.1. Introduction ................................................................................................... 30 3.2. Materials and Methods .................................................................................. 34 3.2.1. Grape sampling and juice collection ................................................... 34 3.2.2. Growth media and fungal isolation ..................................................... 35 3.2.3. Molecular identification of fungal isolates .......................................... 37 3.2.4. Physiological characterization of non-Saccharomyces yeasts ............. 39 3.2.4.1. Tolerance assays ........................................................................ 39 3.2.4.2. Laboratory scale fermentation ................................................... 40 vi 3.2.5. Analysis of flavor compounds of fermented Chambourcin juice ........ 42 3.2.5.1. Optimization of Ultra High-Performance Liquid Chromatography (UHPLC) protocol .............................................. 42 3.2.5.2. Analysis of non-volatile compounds by UHPLC ...................... 44 3.2.5.3. Analysis of volatile compounds by Gas Chromatography Mass Spectrometry .......................................................................... 46 3.2.6. Statistical Analysis .............................................................................. 48 3.3. Results ........................................................................................................... 48 3.3.1. Isolation and identification of fungal diversity on spontaneous fermentation of Chambourcin grape must .............................................. 48 3.3.2. Physiological characterization of non-Saccharomyces yeasts ............. 62 3.3.2.1. Candidate Hanseniaspora strains tolerate lower sulfite concentration compared with S. cerevisiae BY4742 ...................... 62 3.3.2.2. Variable ethanol tolerance of candidate wild yeast strains ....... 66 3.3.2.3. Fermentation kinetics of candidate wild yeast strains in sterile Chambourcin juice ............................................................... 71 3.3.3. Chemical composition of wines fermented by non-Saccharomyces yeasts ...................................................................................................... 73 3.3.3.1. Core non-volatile compounds as fermentative performance contributed by candidate wild yeast strains .................................... 73 3.3.3.2. Distinct volatile profile of inoculated fermentations with yeast strains ..................................................................................... 83 3.4. Discussion ...................................................................................................... 95 3.4.1. Diverse wild yeast populations in the early stages of fermentation represent unique microbial terroir across three PA vineyards ............... 95 3.4.2. Filamentous fungal populations in grape must could be an indicator of grapevine health and wine quality ...................................... 99 3.4.3. Characterization of physiological properties in wild yeast provides insights into potential for winemaking ................................................... 101 Chapter 4 4.1. Major findings and conclusion ...................................................................... 107 4.2. Future Study .................................................................................................. 109 References ................................................................................................................... 110 vii LIST OF FIGURES Figure 1-1. Importance of selection and timing of adding non-Saccharomyces yeast strains in winemaking to produce wine with increased flavor complexity and microbiological control. .............................................................. 3 Figure 1-2. Alcoholic fermentation and glycolytic pathways in yeast. ....................... 14 Figure 2-1. Significant decrease in wild yeast diversity was observed after 24 hours of spontaneous fermentation of Isabella grape must. ................................. 23 Figure 2-2. Decrease of yeast colony forming capacity (cells/ml) at different sulfite concentrations depending on time of incubation (min). ............................ 25 Figure 2-3. Wine associated compounds are categorized into two major groups, non-volatile and volatile metabolites. ................................................................... 28 Figure 2-4. Analysis of non-volatile and volatile compounds for identifying mouthfeel and flavor profile contributed by non-Saccharomyces yeast species during laboratory scale fermentation. ..................................................................
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