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Lifestyle and Functional Properties of Lactobacilli and Bifidobacteria in Water Kefir

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Lifestyle and Functional Properties of Lactobacilli and Bifidobacteria in Water Kefir TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Technische Mikrobiologie Lifestyle and functional properties of lactobacilli and bifidobacteria in water kefir Viktor Philipp Lantbert Eckel 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. Siegfried Scherer Prüfer der Dissertation: 1. Prof. Dr. Rudi F. Vogel 2. Prof. Dr. Wilfried Schwab Die Dissertation wurde am 13.05.2020 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 14.07.2020 angenommen. Abbreviations A7 25% apple juice at pH 7 Ab Acetobacter AF4-MALS assymetric flow field flow fractionation coupled to multi angle light scattering An 25% apple juice at native pH ANI average nucleotide identity ANIb average nucleotide identity based on BLAST+ B. Bifidobacterium BADGE Blast Diagnostic Gene finder BLAST Basic Local Alignment Search Tool BM Bifidobacterium medium CDM chemically defined medium CDM+AA chemically defined medium with all canonic amino acids CDM-AA chemically defined medium without amino acids dH2O distilled water DIC differential interference contrast DNA desoxyribonucleic acid DSM Deutsche Sammlung Mikroorganismen DSMZ Deutsche Sammlung Mikroorganismen und Zellkulturen EPS exopolysaccharide F6P fructose-6-phosphate F6PPK fructose-6-phosphate phosphoketolase Gn 25% grape juice at native pH G7 25% grape juice at pH 7 GOI gene of interest HePs heteropolysaccharide HoPs homopolysaccharide HPLC high performance liquid chromatography isDDH in silico DNA-DNA hybridization I KEGG Kyoto Enzyclopedia of Genes and Genomes LAB lactic acid bacteria Lb. Lactobacillus Lc. Leuconostoc MALDI TOF matrix assisted laser desorption and ionization coupled to time of flight mMRS modified deMan, Rogosa and Sharpe medium MRS deMan, Rogosa and Sharpe medium NCBI National Center for Biotechnology Information OD optical density orf open reading frame PCR polymerase chain reaction PTS phosphotransferase system RAPD randomly amplified polymorphic DNA RAST rapid annotation using subsystems technology RNA ribonucleic acid RPM revolutions per minute rRNA ribosomal RNA S. Saccharomyces TMW Technische Mikrobiologie Weihenstephan TP tryptone phytone medium TPY trypticase phytone yeast UV ultra violet light wk water kefir W/V weight by volume W/W weight by weight II Vorwort Die vorliegende Arbeit entstand im Rahmen des durch das Bundesministerium für Wirtschaft und Energie über die die AiF-Forschungsvereinigung Wissenschaftsförderung der Deutschen Brauwirtschaft e.V. geförderten Projekts „Innovative Getränkezutaten“ (AiF 19180N). Mein besonderer Dank gilt meinem Doktorvater Prof. Dr. Rudi F. Vogel. Danke für deinen Einsatz, dafür, dass du uns immer den Rücken freigehalten hast, und natürlich danke dafür, dass ich in einem derart angenehmen Umfeld; mit diesen Labors, Büros, Geräten und Kollegen, bei dir promovieren durfte. Außerdem möchte ich mich bei Dr. Frank Jakob bedanken, für die Unterstützung, die fachliche Expertise und die vielen Diskussionen. Mein Dank gilt auch Prof. Dr. Matthias Ehrmann, für alle Unterstützung und Hilfe. Mein Dank gilt auch Angela Seppeur, für die Übernahme aller Verwaltungsaufgaben und tatkräftiger Unterstützung, wo auch immer man sie brauchte. Danke an meine Doktorandenkollegen, Julia Bechtner, Lara Eisenbach, Jonas Schmid, Alex Lauterbach und allen anderen, für die diversen Troubleshooting-sessions, die guten Stunden, die schlechten Stunden und alle Unterstützung die ich erfahren habe. Natürlich gilt mein Dank auch allen Technikern, besonders Monika Engel, Margarete Schreiber und Andrea Pape, dafür, dass sie den Laden am Laufen halten und für alle Hilfe im Labor. Zusätzlich gilt mein Dank natürlich auch unseren Projektpartnern an der HSOWL, Prof. Jan Schneider, Sören Rossmann und Julian Huchtman für die angenehme Zusammenarbeit im Projekt und für das zur Verfügungstellen der Bilder. Danke auch an Prof. Dr. Ramon Torres- Ruiz am CALM für seine Hilfe bei den DIC-Aufnahmen. Danke an meine Studentinnen, Lisa- Marie Sattler und Swetlana Gerkhardt für die gute Zusammenarbeit. Natürlich gilt mein Dank auch meiner Frau Dr. Manuela Hollering, für die Kommentare zum Manuskript und alle sonstige direkte und indirekte Unterstützung meiner Dissertation. Aber natürlich auch für alles Andere, die vielen Unternehmungen, für ihre Toleranz und dafür, dass sie mir alles ermöglicht. Danke Jonathan, für deine gute Laune und die Freude die du uns bereitest. Danke Mama und Papa, Klara und Theo dass ihr immer für mich da seid. III Index 1 Introduction ....................................................................................................................................... 1 1.1. Fermented foods and beverages .......................................................................................... 1 1.2. LAB from foods .................................................................................................................. 3 1.3. Water kefir ........................................................................................................................... 4 1.4. LAB from water kefir .......................................................................................................... 6 1.5. Bifidobacterium spp. in water kefir ..................................................................................... 7 1.6. Other microorganisms involved in water kefir fermentation ............................................ 10 1.7. Exopolysaccharide formation in foods .............................................................................. 12 1.8. Turbidity in beverages ....................................................................................................... 14 2. Motivation and aims of this work .................................................................................................. 17 3. Material and methods .................................................................................................................... 19 3.1. General microbiological methods ...................................................................................... 19 3.2. EPS production and treatment ........................................................................................... 31 3.3. Analytical methods ............................................................................................................ 33 3.4. Molecular biological methods ........................................................................................... 34 3.5. Bioinformatic methods ...................................................................................................... 36 4. Results ........................................................................................................................................... 39 4.1. Isolation and identification of bifidobacteria from water kefir ......................................... 39 4.2. Phylogenetic placement of bifidobacterial isolates from water kefir ................................ 40 4.3. Determination of unique and potential functional properties of bifidobacteria from water kefir ................................................................................................................................... 48 4.4. Production of cloud-forming beverage additives using LAB from water kefir ................. 59 4.5. Final strain selection .......................................................................................................... 68 4.6. Optimization of fermentation conditions in fruit juice-based media ................................. 69 4.7. Production of dextrans using Lb. hordei TMW 1.1907 ..................................................... 71 4.8. Investigation of the structure-function relationship of glucans ......................................... 74 4.9. Stability of the in situ produced dextrans towards hydrolysis upon heat-treatment .......... 75 4.10. Stability of the dextran-based cloud systems towards hydrolysis during long-term storage . ........................................................................................................................................... 76 IV 5. Discussion ..................................................................................................................................... 78 5.1. Description of Bifidobacterium tibiigranuli spec. nov. ..................................................... 79 5.2. Investigation of the physiological properties, role in the consortium as well as application potential of bifidobacteria exclusively found in water kefir .............................................. 80 5.3. Production of novel beverage additives ............................................................................. 83 5.3.1. Strain selection .................................................................................................................. 83 5.3.2. Production of clouding dextrans in fruit juice-based fermentation media ........................ 85 5.3.3. Characterization of the macromolecular properties and the stability of in situ produced dextrans ............................................................................................................................. 87 6.
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