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Evaluation of Levan-Producing Acetic Acid Bacteria for Their Potential in Gluten-Free Baking Applications

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Evaluation of Levan-Producing Acetic Acid Bacteria for Their Potential in Gluten-Free Baking Applications TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Technische Mikrobiologie Evaluation of levan-producing acetic acid bacteria for their potential in gluten-free baking applications Tharalinee Osen 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. Karl-Heinz Engel Prüfer der Dissertation: 1. Prof. Dr. Rudi F. Vogel 2. apl. Prof. Dr. Peter Köhler Die Dissertation wurde am 12.02.2018 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 16.05.2018 angenommen. Acknowledgments Acknowledgements First of all, I would like to express my gratitude to my PhD advisor, Prof. Dr. Rudi F. Vogel, for his patient guidance, invaluable suggestions, and useful critiques of this research work. Secondly, I would like to thank my supervisor, Dr. Frank Jakob, for his support, motivation, and help throughout my time as a PhD candidate. This PhD thesis would not have been possible without the ideas and initiation from both of them. I am particularly grateful to Prof. Dr. Peter Köhler for his time as the second examiner for my PhD defense, and also for enabling measurements with the Volscan Profiler and texture analyzer used in this work. Furthermore, I would like to thank Prof. Dr. Karl-Heinz Engel for his time as the chairman of the examination committee. I also thank my students: Dorothee Janßen, Janina Röller, and Sara Lopez-Grado Vela, for their works in supporting the preliminary studies. I wish to acknowledge the help provided by all the technical assistants, especially Monika Engel for her help during my first months, and Sabine Forster for her technical assistance in conducting the baking. In addition, I would like to thank Angela Seppeuer for her administrative support of the project and my PhD study. Additionally, I would like to acknowledge my colleagues: Dr. Andreas Geißler, Linda Höll, Ann-Sophie Schott, and Alexander Lauterbach, for the good time we had shared inside and outside of the office. I would like to offer my special thanks to Julia Brandt, my project partner, and Desideria Brignone for their genuine friendship. Finally, I wish to thank my father and my husband, Raffael, for their support and encouragement that have helped me throughout my study. And I would like to dedicate this work to my late mother, who I hope would be proud of me. i Abbreviations Abbreviations % percentage °C degree celcius × g times gravity A. Acetobacter AAB acetic acid bacteria AACC American Association for Clinical Chemistry AF4 asymmetric flow field flow fractionation ATP adenosine triphosphate B. Bacillus BLAST Basic Local Alignment Search Tool bp base pair CFU colony forming unit CMC carboxymethyl cellulose CO2 carbon dioxide DNA deoxyribonucleic acid dH2O demineralized water dL deciliter DY dough yield EPS exopolysaccharide FOS fructooligosaccharides FQ fermentation quotient g gram G. Gluconobacter Ga. Gluconacetobacter GF gluten-free h hour iii Abbreviations HePS heteropolysaccharide HoPS homopolysaccharide HPLC High Performance Liquid Chromatography HPMC hydroxypropyl methylcellulose K. Kozakia kDa kilodalton kg kilogram Kl. Klebsiella L liter L. Lactobacillus LAB lactic acid bacteria M molarity MALDI-TOF-MS Matrix-Assisted-Laser-Desorption-Ionization-Time-Of-Flight Mass Spectrometry MALS Multi-Angle Light Scattering MDa megadalton Mg magnesium min minute mL milliliter mm millimeter mmol millimole mMRS modified de Man, Rogosa and Sharpe medium Mn Number average molecular weight mN millinormality Mw Weight average molecular weight N newton N. Neoasaia NAD+ nicotinamide adenine nucleotide iv Abbreviations NaG sodium gluconate medium NaOH sodium hydroxide NCBI National Center for Biotechnology Information nm nanometer OD optical density P. Pediococcus PCR Polymerase Chain Reaction Rn geo number average geometric radius Rw geo weight average geometric radius rpm round per minute rRNA ribosomal ribonucleic acid s second S. Saccharomyces sp. species St. Streptococcus TCA tricarboxylic acid TMW Technische Mikrobiologie Weihenstephan V volt v/v volume by volume vvm volume of air (Liter) per unit of medium (Liter) per unit of time (minute) W. Weissella w/w weight by weight WSP water-soluble polysaccharide X. Xanthomonas Z. Zymomonas µg microgram v Table of contents Table of contents Acknowledgements ................................................................................................................. i Abbreviations ....................................................................................................................... iii Table of contents .................................................................................................................. vii 1. INTRODUCTION .............................................................................................................. 1 1.1. Celiac disease and gluten-free breads .......................................................................... 1 Using hydrocolloids to improve GF bread quality ............................................... 1 Sourdough technology for GF breads ................................................................... 2 1.2. Levan ........................................................................................................................... 3 Biosynthesis and bacterial levan production ......................................................... 4 Applications of levan ............................................................................................ 5 1.3. Acetic acid bacteria ..................................................................................................... 6 EPS from AAB ..................................................................................................... 7 Biotechnological and food applications of AAB .................................................. 7 1.4. Motivation and aims of this work ................................................................................ 8 Motivation and working hypotheses ..................................................................... 8 Aims and approach ............................................................................................... 9 2. MATERIAL AND METHODS ....................................................................................... 10 2.1. Strains, medium and cultivation ................................................................................ 10 AAB strains ......................................................................................................... 10 Cultivation of bacteria ......................................................................................... 10 Absorbance, cell counts, and pH determination ................................................. 10 2.2. Molecular techniques ................................................................................................. 11 16S rRNA colony PCR ....................................................................................... 11 NCBI BLAST ..................................................................................................... 11 2.3. Buckwheat sourdough fermentation .......................................................................... 12 vii Table of contents Preparation of sourdough .................................................................................... 12 Variation of molasses concentrations, inoculum sizes and dough yields ........... 12 Strain verification and identification .................................................................. 13 Determination of metabolites in buckwheat sourdoughs .................................... 13 2.4. EPS production, isolation, and analysis ..................................................................... 14 Levan production in shake flasks ........................................................................ 14 Levan production in bioreactor ........................................................................... 14 Isolation of EPS .................................................................................................. 14 Determination of sugar monomers in isolated EPS ............................................ 15 Structural analysis of levans from AAB ............................................................. 15 2.5. Baking experiments ................................................................................................... 16 Bread preparation and baking ............................................................................. 16 Sensory evaluation of sourdough breads ............................................................ 16 Instrumental analysis .......................................................................................... 17 Bread staling analysis ......................................................................................... 17 2.6. Statistical analysis ...................................................................................................... 18 3. RESULTS ........................................................................................................................ 19 3.1. Evaluation of in situ levan production by AAB in GF sourdoughs ........................... 19
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