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Open Master Thesis Hungli Wang 071720.Pdf Choose an item. The Pennsylvania State University The Graduate School CHARACTERIZATION OF MICROBIAL DYNAMICS AND VOLATILE METABOLOME CHANGES DURING FERMENTATION OF CHAMBOURCIN GRAPES IN TWO PENNSYLVANIA REGIONS A Thesis in Food Science by Hung Li Wang © 2020 Hung Li Wang Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2020 The thesis of THEHung LiPURDUE Wang was UNIVERSITY reviewed and approved GRADUATE by the following: SCHOOL Josephine Wee STATEMENT OF COMMITTEE APPROVAL Assistant Professor of Food Science Thesis Advisor Helene Hopfer Assistant Professor of Food Science Darrell W. Cockburn Assistant Professor of Food Science Robert F. Roberts Professor of Food Science Head of the Department of Food Science Approved by: Dr. ii ABSTRACT Numerous studies have indicated that the wine microbiome could generate various volatile compounds which could lead to distinguishing and different wine characteristics. However, little research regarding the wine microbiome is investigating specific microorganisms and their role within the entire microbial community under a more comprehensive sampling method. Thus, in this study we conducted direct sampling from Central and Northeast PA wineries instead of using lab-scale production to study the effect of the wine microbiome on the wine metabolome. Resulting grape and wine samples were characterized by next-generation sequencing and headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Collectively, the innovative sampling and experimental techniques provided a high-resolution picture of microbial dynamics and the resulting wine volatile profiles. Overall, we illustrated how microbial diversity and relative abundance of specific microorganisms change as fermentation progressed. Also, various wine volatile metabolites that are formed during the different fermentation stages were identified. Finally, we were able to establish a prediction model based on the microbial and volatile compositions, which could be used in the future to predict wine quality and wine characteristics in general and could also be applied in winemaking processes to help define regionality and terroir. The wine industry in PA is a relatively young industry, experiencing rapid growth that is expected to continue in the future. Besides European grapes of the Vitis vinifera genus, the environment of PA provides an ideal growing environment for the interspecific hybrid grape Vitis ssp. Chambourcin, one of the most commonly grown varieties in PA. In this study, we characterized the impact of winery-specific microorganisms on the aroma profiles of Chambourcin red wine during the fermentation process (0-20 days). Interestingly, microbial compositions in the iii wines made from grapes from different regions within PA was characterized by high fungal and bacterial diversity in the earlier fermentation stages (0-4 days). Additionally, changes in wine metabolites, such as volatile esters and alcohols that appeared in the later fermentation stages (7- 20 days) provide insights into potential regional differences of Chambourcin red wines. In addition, key discriminant features (microbes and volatile compounds) were identified between the Central and East regions of PA by partial least-squares-discriminant analysis (PLS-DA). Finally, the regularized canonical correlation analysis (rCCA) modeling analyses suggested potentially causal correlation between the microbial communities and wine volatile compounds. In summary, these results provide grape growers and wine producers with knowledge-based recommendations on how to improve wine production through an increased understanding of the impact of native microbial populations on the development of important wine aroma attributes. iv TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................................... xi LIST OF TABLES ........................................................................................................................ xx ACKNOWLEDGEMENTS ........................................................................................................ xxii Chapter 1 LITERATURE REVIEW ............................................................................................... 1 1.1. The Pennsylvania Wine Industry ..................................................................................... 1 1.2. Characteristics of Pennsylvania Chambourcin, a French-American hybrid wine grape cultivar that is important to Pennsylvania .................................................................................... 4 1.3. Microorganisms found in vineyards and wineries can impact winemaking, fermentation, and final wine characteristics. ...................................................................................................... 7 1.3.1. Recent advances in high-throughput next generation sequencing (NGS) technologies have altered the way we study microbial ecology in complex systems such as winemaking. ……………………………………………………………………………………. 12 1.4. Core aroma compounds in the grape and wine metabolome that contribute to sensory properties of final wines............................................................................................................. 14 1.4.1 The concept of terroir in winemaking and the contributing of microorganisms to shaping terroir. ....................................................................................................................... 17 v 1.4.2 Advances in Gas Chromatography – Mass Spectrometry approaches improves resolution of wine volatile profiling ...................................................................................... 19 Chapter 2 Significance, Innovation, and Approach ...................................................................... 21 2.1. Significance ................................................................................................................... 21 2.1.1. Economic impact of wine industry in Pennsylvania .............................................. 21 2.1.2. Microorganisms present on grape berries and throughout fermentation influence the chemical properties in wine ................................................................................................... 22 2.1.3. Data-driven knowledge for improvement of winemaking ..................................... 23 2.2. Innovation ...................................................................................................................... 23 2.3. Approach ....................................................................................................................... 25 2.3.1 Rationale ................................................................................................................... 25 2.3.2. Hypothesis and specific aims ................................................................................. 26 Chapter 3 Characterization of microbial dynamics and volatile metabolome changes during fermentation of Chambourcin grapes in two Pennsylvania regions ............................................. 28 Abstract ......................................................................................................................................... 28 3.1. Introduction ................................................................................................................... 30 3.2. Materials and Methods .................................................................................................. 33 vi 3.2.1 Fermentation and winemaking sample collection for microbiome-metabolome analysis .................................................................................................................................. 33 3.2.2. Preparation of total microbial DNA ....................................................................... 38 3.2.3. Amplification, and purification of target gene sequences ...................................... 38 3.2.4. Sequencing library preparation and construction ................................................... 40 3.2.5. Raw sequence data processing and phylogenetic tree construction ....................... 40 3.2.6. Taxonomic distribution and diversity analyses of microbial populations obtained from Chambourcin wine fermentations ................................................................................. 41 3.2.7. Preprocessing of microbial data for regression analyses ....................................... 44 3.2.8. Determination of cluster number for data clustering to improve taxa differentiation from Linear discriminant analysis Effect size (LEfSe) ......................................................... 45 3.2.9. Characterization of microbial taxa using LEfSe during fermentation .................... 46 3.2.10. HS-SPME-GC-MS for identification and quantification of volatile compounds throughout fermentation of Chambourcin red wine .............................................................. 47 3.2.11. Mass spectral deconvolution and preprocessing of wine metabolite data for regression analyses ................................................................................................................ 48 3.2.12. Distribution and statistical analyses of wine volatile compounds during the fermentation process .............................................................................................................. 49 vii 3.2.13. Partial least squares-discriminant analysis
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