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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Technische Mikrobiologie

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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Für Technische Mikrobiologie TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Technische Mikrobiologie Interaction between Lactobacillus hordei, Lactobacillus nagelii and Saccharomyces cerevisiae isolates from water kefir Di Xu 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. Wolfgang Liebl Die Dissertation wurde am 15.01.2019 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 18.03.2019 angenommen. ACKNOWLEDGEMENTS This thesis was carried out under the supervision of Prof. Dr. Rudi F. Vogel and Dr. Jürgen Behr at the Lehrstuhl für Technische Mikrobiologie, Technische Universität München. I am most sincerely grateful to Prof. Dr. Rudi F. Vogel, who gave me an opportunity for achieving my PhD and a great number of supports in forming scientific thinking and writing. Thus, I benefit a lot for my future career and my life. Also, I would like to gratefully thank my supervisor Dr. Jürgen Behr who helped me a lot with the research design and provided many insightful discussions and suggestions about my research project throughout the doctoral period. Many thanks to Dr. Frank Jakob for creating a cooperative study with Dr. Daniel Wefers from the Karlsruhe Institute of Technology and for their help in eventually publishing my first manuscript. I would like to thank Julia Bechtner for the cooperation with manuscripts. Many thanks to Dr. Andreas J. Geißler and Lara Eisenbach for their help in processing my data of genomics and proteomics. I would like to extend my thanks to to Prof. Dr. Ludwig Niessen, Prof. Dr. Matthias Ehrmann, all my colleagues and technical assistants. Many thanks to Angela Seppeur who is friendly to help on the daily routine. Special thanks to Jie Luo and Zhen Peng for their company when I arrived in Freising, and Roman Prechtl and Evelin Wigmann for the best company during the PhD period. Many thanks to Roman Prechtl for correcting the German summary in thesis as well. I In particular, I am most sincerely grateful to my parents for dual spiritual and financial support. It was their encouragement inspired me to achieve my PhD. Also, special thanks to Prof. Dr. Hua Wei and all my best friends, like Hui Wang, Xiang Lu, Dr. Xiuxiu Wu, Yue Li, Dr. Zhu Liu and Yuhong Mao etc. for your concern. Gratefully thanks to China Scholarship Council for providing scholarship to afford four years in accomplishing my PhD thesis. II CONTENT ACKNOWLEDGEMENTS .......................................................................................... I CONTENT .................................................................................................................. III LIST OF PUBLICATIONS INCLUDED IN THIS THESIS ...................................... V SUMMARY ................................................................................................................ VI ZUSAMMENFASSUNG ............................................................................................ IX 1. INTRODUCTION ..................................................................................................... 1 1.1 Water kefir ........................................................................................................................................ 1 1.1.1 The origin of water kefir .............................................................................................................. 1 1.1.2 The diversity of water kefir .......................................................................................................... 1 1.2 Metabolism of water kefir consortium ............................................................................................. 3 1.3 Polysaccharides of water kefir .......................................................................................................... 6 2. MOTIVATION AND APPROACH .......................................................................... 7 3. MATERIAL AND METHODS ................................................................................. 9 3.1 Strains, medium and pre-culture condition ...................................................................................... 9 3.1.1 Water kefir medium (WKM)...................................................................................................... 10 3.1.2 Sucrose-MRS medium ............................................................................................................... 10 3.1.3 Chemical defined medium (CDM) ............................................................................................. 10 3.2 Determination of cell growth and metabolites ................................................................................ 11 3.2.1 Viable cell counts ...................................................................................................................... 11 3.2.2 Chromatographic analysis of sugars and organic acids................................................................ 12 3.2.3 Chromatographic analysis of amino acids .................................................................................. 13 3.2.4 pH measurements ...................................................................................................................... 14 3.3 Structural analysis of EPS .............................................................................................................. 14 3.3.1 EPS production activity test and isolation................................................................................... 14 3.3.2 Structural analysis of EPS via methylation analysis .................................................................... 15 3.3.3 Determination of molar mass and radius by AF4-MALS-UV...................................................... 17 3.4 Setting up a hydrophilic slide model mimicking physical contact in kefir granules ...................... 18 3.5 Genomics ......................................................................................................................................... 19 3.5.1 Genome sequencing................................................................................................................... 19 3.5.2 Genome assembly...................................................................................................................... 19 3.5.3 Genome annotation and submission ........................................................................................... 20 III 3.5.4 Genomic statistics and visualization ........................................................................................... 21 3.6 Proteomics ....................................................................................................................................... 22 3.6.1 Sample preparation for proteomic analysis ................................................................................. 22 3.6.2 Liquid chromatography and mass spectrometry .......................................................................... 23 3.6.3 Identification and quantification of peptides and proteins ........................................................... 24 4. RESULTS (PUBLICATIONS BASED) .................................................................. 25 4.1 Manuscript 1: Lactobacillus hordei dextrans induce Saccharomyces cerevisiae aggregation and network formation on hydrophilic surfaces ......................................................................................... 25 4.2 Manuscript 2: Lifestyle of Lactobacillus hordei isolated from water kefir based on genomic, proteomic and physiological characterization...................................................................................... 35 4.3 Manuscript 3: Label-free quantitative proteomic analysis reveals the lifestyle of Lactobacillus hordei in presence of Sacchromyces cerevisiae...................................................................................... 46 4.4 Manuscript 4: Comparative proteomic analysis of Lactobacillus nagelii and Lactobacillus hordei in the presence of Saccharomyces cerevisiae isolated from water kefir ............................................... 87 5. MAIN FINDINGS ................................................................................................. 146 6. DISCUSSION ........................................................................................................ 147 6.1 Sugar transport and metabolism .................................................................................................. 147 6.2 Pyruvate and citrate metabolism .................................................................................................. 149 6.3 Peptide transport systems and peptidases, amino acids biosynthesis and metabolism ................ 152 6.4 Acid tolerance by ADI pathway .................................................................................................... 155 6.5 Fatty acid biosynthesis and riboflavin metabolism ...................................................................... 156 6.6 The role of L. hordei dextrans for granule formation................................................................... 157 LIST OF PUBLICATIONS DERIVED FROM THIS WORK ..............................
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