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Entwicklung Und Optimierung Von Methoden Zur Identifizierung Und Differenzierung Von Getränkerelevanten Hefen

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Entwicklung Und Optimierung Von Methoden Zur Identifizierung Und Differenzierung Von Getränkerelevanten Hefen Technische Universität München Lehrstuhl für Technologie der Brauerei II Entwicklung und Optimierung von Methoden zur Identifizierung und Differenzierung von getränkerelevanten Hefen Mathias Hutzler Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihen- stephan für Ernährung, Landnutzung und Umwelt der Technischen Univer- sität München zur Erlangung des akademischen Grades eines Doktor – Ingenieurs genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. R. F. Vogel Prüfer der Dissertation: 1. Univ.-Prof. Dr. E. Geiger (i. R.) 2. Univ.-Prof. Dr. D. Weuster-Botz 3. Univ.-Prof. Dr. Dr. h.c. H. Parlar Die Dissertation wurde am 01.07.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Wei- henstephan für Ernährung, Landnutzung und Umwelt am 09.11.2009 an- genommen. _________________________________________________ Danksagung Danksagung Die vorliegende Arbeit entstand in der Zeit von Oktober 2005 bis März 2009 wäh- rend meiner Tätigkeit am Lehrstuhl für Technologie der Brauerei II der Techni- schen Universität München in Weihenstephan. An erster Stelle möchte ich meinem Doktorvater Herrn Prof. Dr.-Ing. Eberhard Geiger für die Bereitstellung des Themas und die anregenden und angenehmen Diskussionen bedanken. Ich bedanke mich bei Prof. Dr. rer. nat. Rudi F. Vogel für die Übernahme des Prü- fungsvorsitzes. Bei Prof. Dr.-Ing. Dirk Weuster-Botz und bei Prof. Dr. Dr. Harun Parlar bedanke ich mich, dass sie die Co-Referate übernommen haben. Der Wissenschaftsförderung der deutschen Brauwirtschaft danke ich für die Fi- nanzierung der Projekte R 398 und B 95. Teile der Projektergebnisse flossen mit in die vorliegenden Arbeit ein; in diesem Zusammenhang ein ganz besonderes Dankeschön an Frau Dr. Erika Hinzmann und den wissenschaftlichen Beirat. Mein Dank gilt auch allen Mitarbeitern und Kollegen des Lehrstuhls für die gute Zusammenarbeit und die jederzeit vorhandene Unterstützung. Der freundschaft- liche und kollegiale Geist am Lehrstuhl sorgten stets für die nötige Motivation und für ein angenehmes und unbeschwertes Arbeiten. Besonders hervorheben möchte ich hierbei den Einsatz von Ana Lilia Nowak und Jacqueline Lorenz, ohne deren Unterstützung die Vielzahl an Versuchen nicht durchführbar gewesen wäre. Ich danke den Mitarbeitern der Abteilung Mikrobiologie und der Abteilung Chemi- sche Analytik des Forschungszentrums Weihenstephan für Brau- und Lebensmit- telqualität, die mich mit praktischen Ratschlägen und der Bereitstellung von Pro- ben, Verbrauchsmaterialien und Geräten unterstützten. Für den unterstützenden wissenschaftlichen Austausch und diverse Anregungen möchte ich mich bei Herrn Dr. Rainer Springer, Dr. Herbert Vogel, Dr. Sandra Rai- nieri, Dr. Mareike Wenning, Dr. Jana Felbel, Dr. Christoph Tenge, Dr. Jutta Schön- ling, Dr. Oliver Goldenberg, Dr. Georg Stettner, Dr. Marc Kusche, Dr. Friedrich Fel- senstein, Dr. Nicole Büchl, Bernhard Jaser, Wolfgang Mers, Erich Schuster, Markus _________________________________________________ Danksagung Danne und meinen „Doktorandenkollegen“ Dr. Moritz Pöschl, Sven Schönenberg, Henning Kötke, Dr. Urs Wellhöner, Dr. Andreas Brandl bedanken. Für den reibungslosen Ablauf von bürokratischen Formalitäten und finanziellen Abläufen bin ich Frau Christel Volkhart und Frau Hildegard Giebels zu großem Dank verpflichtet. Die im Rahmen meiner Versuche durchgeführten Semester-, Bachelor-, Master- bzw. Diplomarbeiten von Tanja Frickmann, Claudia Fiebig, Stephan Niebauer, Hannes Enders, Arie Susanto, Michael Hofstätter, Matthias Noack, Damian Mer- bach, Manuel Trujillo und Fei Qian hatten ebenfalls Anteil am Entstehen der vor- liegenden Arbeit. Ich danke all meinen Freunden, die mir trotz der wenigen gemeinsamen Zeit der letzten Jahre die Treue gehalten haben. Ein besonderer Dank an meinen besten Freund Martin Beckstein. Meinen Geschwistern Christoph und Iris Hutzler und meinen Eltern Irmgard und Franz Hutzler gilt ein besonders inniger Dank für die immerwährende Unterstüt- zung und dafür, dass ich mich immer auf sie verlassen kann. Meiner lieben Verlobten Sandra Müller danke ich von Herzen für die Unterstüt- zung während des Entstehungszeitraumes der Arbeit und das stete Verständnis. Die Arbeit möchte ich meinem Freund Florian Marxreiter widmen, der leider am 31.03.2008 spurlos verschwand. Im Zeitraum der Erstellung dieser Arbeit konnte ich leider zu wenig Zeit für unsere Freundschaft aufbringen. _________________________________________________ Zusammenfassung Zusammenfassung Hefen sind in der Getränkeindustrie einerseits als Schadorganismen und anderer- seits als Starterkulturen für alkoholische Getränke von Bedeutung. Die Identifika- tion von Schadhefen lässt eine Einschätzung des Verderbnispotentials und der Herkunft der Schadhefeart zu. Alkoholische fermentierte Getränke können durch Einzel- oder Mischkulturen hergestellt werden. Mischkulturen sind oft nicht defi- niert, d. h. deren Identifizierung auf Artebene kann Aufschlüsse über deren Gär- eigenschaften und mögliche Hefe-Hefe Interaktionen bringen. Einzelstarterkultu- ren bedürfen der regelmäßigen Kontrolle auf Art- und Stammebene, um einen einwandfreien Prozess zu gewährleisten. In dieser Arbeit wurden Real-Time PCR Assays, PCR-DHPLC-, Sequenzierung- und FT-IR Spektroskopie-Methoden entwickelt, optimiert bzw. auf die getränkemikro- biologische Analytik übertragen, um getränkerelevante Hefen zu identifizieren und zu differenzieren. Es wurden ein Screening Real-Time PCR Assay für geträn- kerelevante Hefe, verschiedene Real-Time PCR Identifizierungs-Assays für Nicht- Saccharomyces und Saccharomyces Hefearten – darunter auch die industriell genutzten Hefearten S. cerevisiae und S. pastorianus (syn. carlsbergensis ) – entwickelt und evaluiert. Durch sie ist es erstmals möglich, S. cerevisiae Konta- minationen in untergärigen Brauereikulturhefen sensitiv zu detektieren und S. sensu stricto Kontaminationen artspezifisch in Betriebshefen nachzuweisen. Die Real-Time PCR Systeme sind kompatibel gestaltet und können simultan ange- wendet werden. Ein Transfer auf ein Mikrochip Real-Time PCR Format war mög- lich. Die PCR-DHPLC Analyse eines DNA-Abschnittes der IGS2-rDNA ermöglichte eine hochspezifische Differenzierung der industriell genutzten Stämme der Hefe- arten S. cerevisiae und S. pastorianus (syn. carlsbergensis ). Die FT-IR Spektro- skopie und die Sequenzierungsmethoden konnten erfolgreich auf die Identifizie- rung von getränkerelevanten Hefearten übertragen werden. Durch eine Kombination der Sequenzierungsmethoden, der Real-Time PCR Assays und der FT-IR Spektroskopie konnten 363 Hefestämme identifiziert werden und diese 53 verschiedenen Hefearten zugeordnet werden. Darunter befanden sich Isolate aus Brauereien, Getränkebetrieben und Starterkulturen. Erstmals konnte die Art S. kudriavzevii aus dem Brauereiumfeld identifiziert werden. Zur Vorkultivierung getränkerelevanter Hefen wurde neben YM basierten Differentialmedien das Uni- I _________________________________________________ Zusammenfassung versalmedium YM+Bromphenol+Coumarsäure mit differenzierenden nicht- inhibierenden Eigenschaften entwickelt. II ________________________________________________ Abstract Abstract In beverage industries yeasts are considered as spoilage yeasts on the one hand and on the other hand they are of importance as starter cultures for fermented alcoholic beverages. An identification of spoilage yeasts on species level allows an evaluation or estimation of their spoilage potential and their origin. Alcoholic fermented beverages can be produced by starter cultures using single species or mixed species cultures. Frequently mixed cultures are not defined. Therefore identification on species level can clarify fermentation characteristics and yeast- yeast interactions of participated yeast species. Single species starter cultures require a continuous control of their identity on species and strain level to ensure a proper and efficient fermentation process. In this study real-time PCR assays, PCR-DHPLC methods, sequencing methods and FT-IR spectroscopy methods were being developed, optimised and trans- ferred to the microbiological analysis of beverages with the aim to identify and differentiate beverage-relevant yeasts. A screening real-time PCR assay for the detection of beverage-relevant yeasts, a variety of real-time PCR identification- assays for non-Saccharomyces and Saccharomyces species were being devel- oped and evaluated. The Saccharomyces species included the industrial yeast species S. cerevisiae und S. pastorianus (syn. carlsbergensis ). For the first time these assays enable to detect contaminations of S. cerevisiae in bottom- fermenting brewing yeasts and to detect S. sensu stricto contaminations species- specific in starter cultures. The fluorescent probes and the temperature protocols of the real-time PCR assays were designed compatible and the assays can be performed simultaneously. A transfer of the assays to a microchip real-time PCR format was successful. PCR-DHPLC analyses of an IGS2-rDNA-fragment allowed a specific differentiation of industrial strains of S. cerevisiae und S. pastorianus (syn. carlsbergensis ). FT-IR Spectroscopy and sequencing methods were being transferred successfully to identify beverage-relevant yeasts. A combination of sequencing methods, real-time PCR assays and FT-IR spectroscopy methods en- abled the identification (on species level) of 363 isolated strains from 53 species. The isolates originated from breweries, beverage companies and starter cultures. For
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