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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Mikrobielle Ökologie Analyse Der Bakteriellen Biodiversität Von Boviner Rohmil

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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Mikrobielle Ökologie Analyse Der Bakteriellen Biodiversität Von Boviner Rohmil TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Mikrobielle Ökologie Analyse der bakteriellen Biodiversität von boviner Rohmilch mittels kultureller und kulturunabhängiger Verfahren Franziska Thekla Breitenwieser 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. Ulrich Kulozik Prüfer der Dissertation: 1. Prof. Dr. Siegfried Scherer 2. Prof. Dr. Rudi F. Vogel Die Dissertation wurde am 23.03.2018 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 04.07.2018 angenommen. Inhaltsverzeichnis INHALTSVERZEICHNIS INHALTSVERZEICHNIS ..................................................................................................... I ZUSAMMENFASSUNG ...................................................................................................... V SUMMARY ....................................................................................................................... VII ABBILDUNGSVERZEICHNIS ......................................................................................... IX TABELLENVERZEICHNIS .............................................................................................. XI ABKÜRZUNGSVERZEICHNIS ..................................................................................... XIII 1. EINLEITUNG ................................................................................................................... 1 1.1 Rohmilch ...................................................................................................................... 1 1.1.1 Gesetzliche Vorgaben zur Rohmilchqualität ........................................................ 1 1.1.2 Rohmilchmikrobiota ............................................................................................. 2 1.1.2.1 Kontaminationsquellen auf die mikrobielle Zusammensetzung frisch ermolkener Rohmilch ................................................................................................. 2 1.1.2.2 Einflussfaktoren auf die Rohmilch ................................................................. 3 1.2 Kulturabhängige und kulturunabhängige Analyse der Rohmilchmikrobiota .............. 5 1.2.1 Kulturabhängige Methoden ................................................................................... 5 1.2.2 Kulturunabhängige Methoden ............................................................................... 7 1.3 Verderb von Milchprodukten .................................................................................... 10 1.3.1 Verderb durch vegetative Keime ........................................................................ 11 1.3.2 Verderb durch hitze-stabile Enzyme ................................................................... 12 1.4 Ziel der Arbeit ............................................................................................................ 14 2. MATERIAL UND METHODEN ................................................................................... 16 2.1 Probenmaterial und Probenahme ............................................................................... 16 2.1.1 Betriebsdaten ....................................................................................................... 16 2.1.2 Probenahme und Probenzeitraum der Rohmilch analysiert mit einer Stichprobengröße von 50 bzw. 500 Isolaten ................................................................ 17 I Inhaltsverzeichnis 2.1.3 Probenahme und Probenzeitraum der Rohmilch analysiert mit einer Stichproben-größe von 1500 Isolaten .......................................................................... 18 2.2 Kulturabhängige Analyse der Rohmilchmikrobiota .................................................. 18 2.2.1 Gesamtkeimzahlbestimmung und Biodiversitätsanalyse .................................... 18 2.2.2 Identifizierung der Isolate mittels FT-IR Spektroskopie .................................... 19 2.2.2.1 Probenaufbereitung und Messung ................................................................ 19 2.2.2.2 Auswertung und Identifizierung der Spektren ............................................. 20 2.2.2.3 Cluster-Analyse ............................................................................................ 20 2.2.3 Identifizierung der Isolate anhand von DNA-Sequenzen ....................................... 20 2.2.3.1 DNA-Extraktion ........................................................................................... 20 2.2.3.2 PCR Ansatz und Sequenzierung des 16S rDNA bzw. rpoB Gens ............... 21 2.2.3.3 Identifizierung der Sequenzen ...................................................................... 22 2.3 Kulturunabhängige Analyse (llumina Sequenzierung) der Rohmilchmikrobiota ..... 23 2.3.1 Aufarbeitung der Rohmilch ................................................................................ 23 2.3.1.1 Konzentrierung der Bakterienzellen ............................................................. 23 2.3.1.2 Entfernung des Caseins ................................................................................ 23 2.3.2 DNA-Extraktion .................................................................................................. 24 2.3.3 Anreicherung und Aufreinigung der bakteriellen DNA ..................................... 25 2.3.4 Bestimmung der bakteriellen DNA-Konzentration ............................................ 25 2.3.5 Library Preparation für die Illumina Sequenzierung .......................................... 27 2.3.6 Sequenzierung ..................................................................................................... 30 2.3.7 Sequenzauswertung und Identifizierung ............................................................. 30 2.4 Statistische Auswertung ............................................................................................. 31 2.5 Taxonomische Methoden ........................................................................................... 31 2.5.1 Phylogenetische Analyse .................................................................................... 31 2.5.2 Morphologische Charakterisierung ..................................................................... 32 2.5.3 Physiologische Charakterisierung ....................................................................... 33 II Inhaltsverzeichnis 2.5.3.1 Enzymatische Aktivität und Substratverwertung ......................................... 33 2.5.3.2 Wachstumsgrenzen ....................................................................................... 34 2.5.4 Chemotaxonomische Charakterisierung ............................................................. 34 2.5.4.1 Peptidoglykan-Analyse................................................................................. 35 2.5.4.2 Zelluläre Fettsäuren ...................................................................................... 35 2.5.4.3 Ganzzell-Zucker ........................................................................................... 35 2.5.4.4 G+C Gehalt ................................................................................................... 35 2.5.4.5 Quinone und Polare Lipide ........................................................................... 35 3. ERGEBNISSE ................................................................................................................. 37 3.1 Zusammensetzung der Rohmilchmikrobiota von 21 Betrieben ................................ 37 3.1.1 Einteilung der Tankmilchproben anhand der Gesamtkeimzahl .......................... 37 3.1.2 Abundanz und Prävalenz der häufigsten Gattungen ........................................... 38 3.1.3 Einflussfaktoren auf die Rohmilchmikrobiota .................................................... 41 3.1.3.1 Zusammenhang von Gesamtkeimzahl, Biodiversität und taxonomischen Großgruppen ............................................................................................................. 41 3.1.3.2 Einflussfaktor Probenalter ............................................................................ 43 3.1.3.3 Einflussfaktor Jahreszeit ............................................................................... 45 3.1.4 Stabilität und Variabilität der Rohmilchmikrobiota einzelner Betriebe ............. 46 3.1.5 Hofspezifische Mikrobiota .................................................................................. 49 3.1.6 Einfluß der Stichprobengröße auf die erfasste Biodiversität .............................. 50 3.2 Vergleich der Biodiversität zweier ausgewählter Betriebe mittels kulturabhängiger und -unabhängiger Analyse ............................................................................................. 51 3.2.1 Erfassung der Biodiversität mittels kulturabhängiger Analyse ........................... 52 3.2.1.1 Erfasste Biodiversität von Betrieb 4 ............................................................. 52 3.2.1.2 Erfasste Biodiversität von Betrieb 6 ............................................................. 55 3.2.1.3 Vergleich der Biodiversität beider Betriebe und Einfluss der Stichproben- größe auf die erfasste Biodiversität .......................................................................... 58 III Inhaltsverzeichnis 3.2.2 Erfassung der Biodiversität mittels
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