Entwicklung Von Methoden Zum Nachweis Von Cocos Nucifera Im Hinblick Auf Kokosnussallergien Sowie Im Kontext Der Echtheitsprüfung Von Kokosblütenzucker"

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Entwicklung Von Methoden Zum Nachweis Von Cocos Nucifera Im Hinblick Auf Kokosnussallergien Sowie Im Kontext Der Echtheitsprüfung Von Kokosblütenzucker Entwicklung von Methoden zum Nachweis von Cocos nucifera im Hinblick auf Kokosnussallergien sowie im Kontext der Echtheitsprüfung von Kokosblütenzucker Dissertation zur Erlangung des Doktorgrades Doctor rerum naturalium Dr. rer. nat. Universität Hamburg Fakultät für Mathematik, Informatik und Naturwissenschaften Fachbereich Chemie Institut für Lebensmittelchemie & GALAB Laboratories GmbH, Hamburg vorgelegt von Jasmin Wrage Hamburg, 2020 Die vorliegende Arbeit wurde in der Zeit von Mai 2016 bis August 2019 unter der Leitung von Dr. Jürgen Kuballa bei der Firma GALAB Laboratories GmbH angefertigt und wissenschaftlich von Prof. Dr. Sascha Rohn des Instituts für Lebensmittelchemie der Universität Hamburg betreut. 1. Gutachter der Dissertation: Prof. Dr. Sascha Rohn 2. Gutachter der Dissertation: Prof. Dr. Bernward Bisping Tag der Disputation: 11. September 2020 Tag der Druckfreigabe: 05. Oktober 2020 _______________________________________________________________________________________________________________ Seite I Diese Arbeit wurde in Teilen im Rahmen des Projekts „FOODOMICS“ angefertigt. Die Förderung dieses Vorhabens erfolgte aus Mitteln des Bundesministeriums für Ernährung und Landwirtschaft (BMEL) aufgrund eines Beschlusses des deutschen Bundestages. Die Projektträgerschaft erfolgte über die Bundesanstalt für Landwirtschaft und Ernährung (BLE) im Rahmen des Programms zur Innovationsförderung. (FKZ 281601914) _______________________________________________________________________________________________________________ Seite III Hiermit versichere ich ehrenwörtlich, dass die vorliegende Dissertationsschrift von mir selbstständig verfasst wurde und ich keine anderen als die angegebenen Hilfsmittel benutzt habe. Die eingereichte schriftliche Fassung entspricht der auf dem elektronischen Speichermedium. Außerdem erkläre ich, dass ich keinen Promotionsversuch an einer anderen Hochschule oder Fakultät unternommen habe und ich die vorliegende Arbeit noch nicht an einer anderen Stelle in gleicher oder in wesentlichen Teilen ähnlicher Abhandlung als Prüfung eingereicht habe. Hamburg, den 16. Juni 2020 ____________________________________ Jasmin Wrage _______________________________________________________________________________________________________________ Seite V Teile der vorliegenden Arbeit wurden bereits veröffentlicht: Wrage J., Burmester (geb. Uthmann) S., Kuballa J., Rohn S., Coconut sugar (Cocos nucifera L.): Production process, chemical characterization, and sensory properties, LWT – Food Science and Technology, 112, 2019. Wrage J., Kleyner O., Rohn S., Kuballa J., Development of a DNA-Based Detection Method for Cocos Nucifera Using TaqMan™ Real-Time PCR, Foods, 9, 332, 2020. _______________________________________________________________________________________________________________ Seite VII An dieser Stelle möchte ich mich sehr herzlich für die vielfältig erfahrene Unterstützung bei der Anfertigung der vorliegenden Arbeit bedanken: Mein Dank gilt zunächst Prof. Dr. Rohn für die Möglichkeit, diese Promotionsarbeit unter seiner wissenschaftlichen Betreuung am Institut für Lebensmittelchemie der Universität Hamburg anfertigen zu können. Auch möchte ich mich bei Dr. Jürgen Kuballa für die Bereitstellung des Arbeitsplatzes und der Geräte bedanken. Ich danke ihm außerdem für die mir übertragene Verantwortung und wissenschaftliche Freiheit. Ebenso danke ich Frau Carola Biesemann und Herrn Michael Hebendanz von der Delphi Organic GmbH, Frau Dürten Möllgaard und Herrn Rudolf Meincke von Voicevale Ltd. sowie Frau Dr. Steinecke vom Palmengarten Frankfurt und Herrn Eberhardt Kirchner von Elevate Social Businesses Ltd. für die Bereitstellung der Probenmaterialien und ihre kritische Diskussionsbereitschaft. Ein großer Dank gebührt Oxana Kleyner und Petra Knies-Platte für die Einführung in das molekularbiologische Arbeiten und ihre wertvollen Ratschläge. Neben ihnen danke ich ebenso Stephanie Burmester für die Organisation und Durchführung zahlreicher sensorischer Verkostungen. Ein weiterer Dank gilt den Kollegen/innen Kathrin Hofmann, Henning Zaun, Carina Niemann, Enya Ebel, Franziska Schmelter und Christin Claassen sowie vielen anderen GALAB-Mitarbeitern für die erbrachte Unterstützung und viele schöne Momente – auch abseits der Arbeitszeit. Auch möchte ich mich bei Maike Arndt und Marina Creydt für unsere netten Treffen mit immer ausgiebigen (wissenschaftlichen) Gesprächen bedanken. Mein besonderer Dank gilt schließlich neben vielen Freunden (insbesondere Familie Teubner und Familie Cassel) meiner Familie, die mich mein gesamtes Studium hinweg immer moralisch unterstützt hat und jederzeit für mich da ist. Der größte Dank gebührt Timo Hörburger, ohne dessen unermüdlichen Rückhalt, die vielen aufbauenden Worte, wenn mal wieder alles wie verhext war, sowie das stets offene Ohr für meine Gedanken, die vorliegende Arbeit nicht möglich gewesen wäre. _______________________________________________________________________________________________________________ Seite IX Inhaltsverzeichnis Abkürzungsverzeichnis ................................................................................................................................. 1 Größen und Maßeinheiten ........................................................................................................................... 3 Kapitel 1 Vorwort .......................................................................................................................................... 5 Kapitel 2 Kurzfassung/Abstract .............................................................................................................. 7 Kapitel 3 Theoretische Grundlagen ..................................................................................................... 11 3.1 Kokospalme, Cocos nucifera .................................................................................................................... 11 3.1.1 Botanische Einordnung, Morphologie und Verwendungszweck ................................... 11 3.1.2 Herstellung von Kokosblütenzucker und rechtlicher Hintergrund .............................. 12 3.2 Lebensmittelallergie ................................................................................................................................... 15 3.2.1 Definition und rechtlicher Hintergrund ................................................................................... 15 3.2.2 Kokosnussallergie .............................................................................................................................. 17 3.3 DNA als Analyt .............................................................................................................................................. 17 3.3.1 DNA-Isolierung und -Charakterisierung .................................................................................. 19 3.3.2 Konventionelle PCR und Visualisierung der DNA-Amplifikate ....................................... 21 3.3.3 Real-time PCR und Sondendesign ............................................................................................... 23 3.3.4 DNA-Sequenzierung .......................................................................................................................... 24 3.4 Metabolite als Analyten ............................................................................................................................. 25 3.4.1 Trennung von Metaboliten mittels Flüssigkeitschromatographie ................................ 26 3.4.2 Detektion von Metaboliten mittels Massenspektrometrie ............................................... 28 3.4.3 Auswertung von non-targeted Metabolic-Fingerprinting-Experimenten .................. 30 3.5 Weitere analytische Verfahren .............................................................................................................. 32 3.5.1 ELISA ....................................................................................................................................................... 32 3.5.2 13C/12C-Stabilisotopenanalyse ...................................................................................................... 32 Kapitel 4 Methodenentwicklung zum DNA basierten Nachweis von ...................................... 35 Cocos nucifera ...................................................................................................................................................... 4.1 Zielsetzung und analytische Herangehensweise ............................................................................ 35 4.2 Material und Methoden ............................................................................................................................. 35 4.2.1 Probenmaterial ............................................................................................................................................. 35 _______________________________________________________________________________________________________________ Seite XI 4.2.2 Primer und Sonden für die PCR ................................................................................................... 36 4.2.3 Probenvorbereitung und DNA-Isolierung ............................................................................... 36 4.2.4 Reinheits- und Konzentrationsbestimmung der DNA-Isolate ......................................... 36 4.2.5 Konventionelle PCR ........................................................................................................................... 37 4.2.6
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