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Influence of Grape-Associated Filamentous Fungi and Their Exoproteome on the Gushing in Sparkling Wine

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Influence of Grape-Associated Filamentous Fungi and Their Exoproteome on the Gushing in Sparkling Wine TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Influence of grape-associated filamentous fungi and their exoproteome on the gushing in sparkling wine Elisabeth Ingeborg Vogt-Hrabak Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Karl-Heinz Engel Prüfer der Dissertation: 1. Prof. Dr. Rudi F. Vogel 2. Prof. Dr. Siegfried Scherer Die Dissertation wurde am 16.10.2017 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 20.12.2017 angenommen. Danksagung Ich möchte mich bei meinem Doktorvater Herrn Prof. Dr. Vogel für die Möglichkeit bedanken meine Dissertation am Lehrstuhl für Technische Mikrobiologie anzufertigen. Danke, Rudi, für all die Freiheiten in der Gestaltung meiner Doktorarbeit, für die anregenden Gespräche und die zahlreichen Fortbildungen, die ich besuchen durfte. Besonderer Dank gilt auch meinem Mentor Herrn apl. Prof. Dr. Niessen. Ludwig, vielen Dank für alle Tipps und Tricks in der Mykologie, für eine gute, angenehme, aber auch oft lustige Zusammenarbeit und für die unzähligen Diskussionen über Pilze, Gushing und Schaumwein. Auch Herrn apl. Prof. Dr. Ehrmann möchte ich herzlich für die gefühlt stundenlangen Debatten über die Wahl der richtigen Klonierungsstrategie danken. Matthias, vielen Dank! Es war immer interessant und hat Spaß gemacht. Ei gaz dikes Dakeshö geht a ei Büo „ West – Vroni, Marion, Meike. Danke für das Unterstützen, Aufbauen, „Reflektieren und immer Dasein wenn es nötig war. Natürlich möchte ich mich auch bei allen anderen Doktoranden, aber insbesondere bei Thomas, Tomas, Maik, Ann-Sophie, Jule, Andi, Max und Chris, bedanken, die die Zeit am Lehrstuhl unvergesslich für mich gemacht haben. Weiterhin will ich mich bei den Technischen Assistentinnen bedanken, allen voran Andre Pape, für die gute Arbeitsgemeinschaft und die stets hervorragende Erledigung all meiner Aufträge. Natürlich gehen auch sonnige Grüße und ein großes Danke an Angela Seppeur für die Unterstützung in allen Verwaltungsangelegenheiten. Ein Dankeschön geht auch an all meine Studenten, die theoretische oder praktische Semesterarbeiten unter meiner Betreuung angefertigt haben. Mein Dank geht ferner an den Forschungskreis der Ernährungsindustrie (FEI) und den Verband Deutscher Sektkellereien e. V. für die Unterstützung dieser Arbeit. Außerdem möchte ich Herrn Prof. Ulrich Fischer (Weincampus Neustadt), Frau Prof. Doris Rauhut (Hochschule Geisenheim), Herrn Prof. Armando Venâncio (Universidade do Minho), Herrn Dr. Giancarlo Perrone (ISPA/CNR), Herrn Dr. Enric Sebastian (INCAVI), Herrn Dr. Dietrich Blankenhorn (Staatliche Lehr- und Versuchsanstalt Weinsberg), Herrn Dipl.-Päd. Erhard Kührer (BoKu Wien) sowie der Bayrischen Landesanstalt für Weinbau für die Bereitstellung von verschiedenem Probenmaterial zu Forschungszwecken danken. Großer Dank geht auch an meine Freunde und meine Familie. Gesondert möchte ich meinen Eltern für die stetige Unterstützungen während meines gesamten Studiums danken. Dazu widme ich meine Dissertation in liebevoller Erinnerung meinem Vater Dr. Klaus Vogt, der mir schon als kleines Mädchen den Floh ins Ohr gesetzt hat auch einen naturwissenschaftlichen Doktor zu erlangen. Ganz besonderer Dank geht an Max für seine schier unmenschliche Geduld mit mir in allen Lebenslagen. Danke für dein Verständnis, deine Unterstützung und dafür, dass du mit mir durchs Leben tanzt. Table of Contents Table of Contents 1 Introduction ................................................................................................................................... 1 1.1 Wine ....................................................................................................................................... 1 1.1.1 Basic procedure of winemaking ...................................................................................... 2 1.1.2 Methods for sparkling wine production ......................................................................... 3 1.1.3 Microbiota in the vineyard with emphasis on filamentous fungi ................................... 4 1.2 Gushing of carbonated beverages .......................................................................................... 6 1.2.1 Definition and classification ............................................................................................ 6 1.2.2 Basic principles of bubble formation .............................................................................. 8 1.2.3 Suggested mechanisms for primary gushing in beer ...................................................... 9 1.2.4 Surface-active proteins involved in beer gushing ......................................................... 10 1.2.5 Prevention strategies for gushing in beer ..................................................................... 12 1.2.6 Factors inducing secondary gushing in carbonated beverages ..................................... 14 1.2.7 Comparison of beer and sparkling wine ....................................................................... 15 1.3 Motivation, hypotheses and objectives ................................................................................ 16 2 Materials and methods ................................................................................................................ 19 2.1 Materials .............................................................................................................................. 19 2.1.1 Chemicals...................................................................................................................... 19 2.1.2 Equipment .................................................................................................................... 26 2.1.3 Proteins ........................................................................................................................ 27 2.1.4 Consumables ................................................................................................................ 28 2.1.5 Oligonucleotides and plasmids ..................................................................................... 29 2.1.6 Organisms ..................................................................................................................... 30 2.1.7 Nutrients ....................................................................................................................... 31 2.1.8 Software and databases ............................................................................................... 33 2.2 Methods ............................................................................................................................... 34 2.2.1 Protein chemical methods ............................................................................................ 34 2.2.2 Molecular biological methods ...................................................................................... 37 2.2.3 Mycological methods .................................................................................................... 41 2.2.4 Bioinformatics............................................................................................................... 43 3 Results .......................................................................................................................................... 47 3.1 Development of a new technique for the preparation of proteins from (sparkling) wine.... 47 3.2 Comparison of gushing and non-gushing sparkling wines .................................................... 50 3.3 Influence of external factors on the protein composition of single-variety wines ............... 52 I 3.4 Screening of filamentous fungi isolated from grapes ........................................................... 55 3.4.1 Screening for protease activity ..................................................................................... 55 3.4.2 Screening for surface activity........................................................................................ 56 3.4.3 Secretion of low molecular weight proteins ................................................................. 56 3.4.4 Induction of gushing in sparkling water as model system ............................................ 57 3.5 Gushing test with culture supernatants of Penicillium oxalicum .......................................... 59 3.5.1 Influence of culture conditions and foam fractions on the gushing potential .............. 59 3.5.2 Effect of temperature and pH of the sparkling water on gushing ................................. 60 3.5.3 Applicability of sparkling wine in gushing tests ............................................................ 61 3.6 Investigation of the exoproteome of Penicillium oxalicum................................................... 62 3.7 Identification of proteins from the exoproteome of P. oxalicum ......................................... 64 3.8 Cloning approaches of PDE_07106 and PDE_04519 in Escherichia coli ................................ 66 3.9 Establishment of immunochemical methods for the detection of PDE_07106 and PDE_04519 ....................................................................................................................................... 69 3.9.1 Antigen design and
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