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Dissection of the Molecular Mechanism of Hop Inhibition in Lactobacillus Brevis

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Dissection of the Molecular Mechanism of Hop Inhibition in Lactobacillus Brevis TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Technische Mikrobiologie Dissection of the molecular mechanism of hop inhibition in Lactobacillus brevis Benjamin C. Schurr 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: Univ.-Prof. Dr. W. Liebl Prüfer der Dissertation: 1. Univ.-Prof. Dr. R. F. Vogel 2. Univ.-Prof. Dr. W. Schwab Die Dissertation wurde am 27.08.2014 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 23.10.2014 angenommen. Dissection of the molecular mechanism of hop inhibition in Lactobacillus brevis Benjamin C. Schurr Doctoral thesis Freising 2014 Vorwort Die vorliegende Arbeit entstand im Rahmen eines durch Haushaltsmitteln des BMWi über die AiF-Forschungsvereinigung Wissenschaftsförderung der Deutschen Brauwirtschaft e. V geförderten Projektes (AiF 16124 N). Die Erstellung der Arbeit wurde zeitweise durch ein Stipendium aus dem Ganzenmüller-Fonds ermöglicht. Für die Bewilligung dieses Stipendiums bedanke ich mich herzlich bei dem hierüber entscheidenden Gremium und speziell bei Prof. Dr. Horst-Christian Langowski. Ferner gilt mein Dank für projektbezogene Leistungen der deutschen Wirtschaft der Simon H. Steiner Hopfen GmbH und besonders Herrn Dr. Martin Biendl. Mein persönlicher Dank gilt: Im Besonderen meinem Doktorvater Prof. Dr. Rudi F. Vogel für die Überlassung des Forschungsthemas und die Organisation der Finanzierung über die gesamte Dauer des Projektes. Danke auch für zahlreiche Anregungen, sowie das Interesse an meiner Arbeit und die Bereitschaft zur fachlichen Diskussion. Ein besonderes Dankeschön für die stete Unterstützung in allen Phasen des Projektes. Prof. Dr. Wilfried Schwab für die Begutachtung der vorliegenden Arbeit und Prof. Dr. Wolfgang Liebl für die Übernahme des Prüfungsvorsitzes. Insbesondere Dr. Jürgen Behr für die Betreuung meiner Arbeit. Danke für zahlreiche Diskussionen, Denkanstöße, Tipps und Tricks, sowie die fortwährende Unterstützung und außergewöhnliche Zusammenarbeit. Prof. Dr. Matthias Ehrmann, Prof. Dr. Ludwig Niessen, Monika Hadek und Margarete Schreiber für ihre fachliche Unterstützung. Angela Seppeur für ihre Unterstützung bei zahlreichen organisatorischen Fragen. Für ihre Unterstützung bei meiner Arbeit und die gemeinsame Zeit in Freising meinen Kollegen. Bärbel Assmann und Dr. Katherine Evert. Christian Vogt und Prof. Dr. Thomas Hofmann vom Lehrstuhl für Lebensmittelchemie und Molekulare Sensorik für die Zusammenarbeit. Dr. Hannes Hahne und Prof. Dr. Bernhard Küster vom Lehrstuhl für Proteomik und Bioanalytik für die Zusammenarbeit. In besonderem Maße meiner Familie. Danke für eure Unterstützung, euer Verständnis und den Rückhalt während meines Studiums und meiner Promotionszeit. Zuletzt einem ganz besonderen Menschen. I Abbreviations °C degree Celsius 2D two-dimensional 3-MPA 3-mercaptopropionic acid A ampere a atto (10-18) ACC acetyl-CoA carboxylase AcN acetonitrile ACP acyl carrier protein ADI arginine deiminase ADP adenosine diphosphate AmBic ammonium bicarbonate APS ammonium persulfate ATP adenosine triphosphate β-CD β-cyclodextrin BLM bilayer lipid membrane Bromophenol Blue 3′,3′′,5′,5′′-tetrabromophenolsulfophthalein sodium salt cal BC calibrated years before Christ Calcein-AM O′-diacetate tetrakis(acetoxymethyl) ester CCCP carbonyl cyanide 3-chlorophenylhydrazone cDNA copyDNA cEt crude ethanol hop extract cHex crude hexane hop extract CID collision induced dissociation CoA coenzyme A CP crossing point cTHF crude tetrahydrofuran hop extract II Da Dalton DEE diethyl ether df hop draff dH2O deionised water DMS dimethyl sulphate DNA desoxyribonucleic acid dNTP desoxy nucleoside triphosphate ΔΨ membrane potential dr hop δ-resin ds double-stranded DTT 1,4-dithio-D,L-threitol EDTA ethylenediaminetetraacetic acid EF-Tu elongation factor thermo unstable em emission er hop ε-resin ETD electron transfer dissociation EtOH ethanol ex excitation f femto (10-15) F01-11 hop ε-resin subfraction 1 – 11 FA fatty acid, formic acid FAS fatty acid synthase Fig. figure FMOC 9-fluorenylmethoxycarbonylchloride g gram GABA γ-aminobutyric acid GAD (system) glutamic acid decarboxylase (system) III GadB glutamic acid decarboxylase GadC Glu/GABA antiporter GC gas chromatograph(y) gh green/fresh hops Glc glucose Glu glutamic acid h hour, hecto (102) ha hectare (10,000 m2) hr hop hard resin k kilo (103) λ wavelength L litre L. Lactobacillus LAB lactic acid bacteria LC-ESI MS/MS liquid chromatography coupled to tandem mass spectrometry with electrospray ionisation LC-MS liquid chromatography-mass spectrometry lmw low molecular weight µ micro (10-6) m milli (10-3), meter M Mol, molar, mega (106), hop variety HALLERTAUER MAGNUM m/z mass to charge ratio MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry MEBAK Mitteleuropäische Brautechnische Analysenkommision MeOH methanol MES 2-[N-morpholino]ethanesulfonic acid mgf mascot generic format IV MIC minimum inhibitory concentration min minute mio million MPI message passing interface mRNA messenger ribonucleic acid MRS DE MAN, ROGOSA and SHARPE Mw molecular weight n nano (10-9) NCBI National Center for Biotechnology Information Nig nigericin NMR nuclear magnetic resonance spectroscopy o.n. over night OD optical density OPA ο-phthalaldehyde p pico (10-12) PAGE polyacrylamide gel electrophoresis PC phosphatidylcholine Pe hop variety PERLE pHin/ex intracellular / extracellular pH-value pl hop pellets pmf proton motive force ppm parts per million qRT-PCR quantitative real-time polymerase chain reaction RFU relative fluorescence unit rh raw hops RNA ribonucleic acid RP-HPLC reversed phase high performance liquid chromatography V s second sd standard deviation SDS sodium n-dodecyl sulphate sr hop soft resin ss single-stranded Tab. table TCA trichloroacetic acid TEMED N,N,N’,N’-tetramethylethylendiamine TFA trifluoroacetic acid THF tetrahydrofuran TMW Technische Mikrobiologie Weihenstephan Tr hop variety HALLERTAUER TRADITION tr hop total resin Tricine N-[tris(hydroxymethyl)methyl]glycine Tris tris(hydroxymethyl)aminomethane Ts hop variety HALLERTAUER TAURUS TUM Technische Universität München UV ultra violet V volt v/v volume / volume Val valinomycin w/v mass / volume w/w mass / mass ZfP Zentrallabor für Proteinanalytik, München VI Contents 1 Introduction .............................................................................................. 1 1.1 Beer and the use of hops (Humulus lupulus) ˗ a brief history............. 1 1.2 The hop plant, hop products and hop compounds ............................. 2 1.3 Spoilage susceptibility of beer and beer-spoiling microorganisms ..... 7 1.4 Antimicrobial mode of action of hop compounds ................................ 9 1.5 Hop stress tolerance mechanisms ................................................... 10 1.6 Hop stress and cytoplasmic membranes ......................................... 12 1.7 The glutamic acid decarboxylase (GAD) system ............................. 16 1.8 Objectives ........................................................................................ 19 2 Material and Methods ............................................................................ 21 2.1 Material ............................................................................................ 21 2.1.1 Devices .................................................................................... 21 2.1.2 Chemicals ................................................................................ 23 2.1.3 Hop compounds ....................................................................... 28 2.1.4 Consumables and other supplies ............................................. 31 2.1.5 Molecular biological kits and supplies ...................................... 33 2.1.6 Microorganisms ........................................................................ 34 2.2 Methods ........................................................................................... 34 2.2.1 Determination of UV-Vis absorption profiles ............................ 34 2.2.2 Determination of Distribution ratios (Dorg/aq) ............................. 34 2.2.3 Isolation of single iso-co/n/ad-humulone analogues................. 35 2.2.4 RP-HPLC analysis conditions of hop compounds .................... 36 2.2.5 Preparative RP-HPLC of hop compounds ................................ 36 2.2.6 Methylation of an iso-α-acids mixture and cis-iso-cohumulone 36 2.2.7 Preparation of imino-iso-α-acids .............................................. 37 VII 2.2.8 Preparation of humulinic acids ................................................. 37 2.2.9 Preparation of crude hop extracts ............................................ 38 2.2.10 Identification of hop derivatives via LC-ESI MS/MS and GC MS...... ............................................................................... 38 2.2.11 Assessment of electrochemical membrane characteristics via bilayer lipid membrane (BLM) techniques ................................ 39 2.2.12 Media and culture conditions ................................................... 40 2.2.13 Determination of minimum inhibitory concentration (MIC) ........ 41 2.2.14 Cluster Analysis of MIC values................................................
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