Isolation and Characterisation of Substances from Royal Jelly Andreas Stocker
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Isolation and characterisation of substances from Royal Jelly Andreas Stocker To cite this version: Andreas Stocker. Isolation and characterisation of substances from Royal Jelly. Other. Université d’Orléans; Université technique de Munich, 2003. English. tel-00008586 HAL Id: tel-00008586 https://tel.archives-ouvertes.fr/tel-00008586 Submitted on 27 Feb 2005 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE EN COTUTELLE PRESENTÉE A L’UNIVERSITÉ D’ORLÉANS POUR OBTENIR LE GRADE DE DOCTEUR-INGÉNIEUR DE L’UNIVERSITÉ D’ORLÉANS Discipline : Biophysique moléculaire PAR -STOCKER Andreas Isolation and characterisation of substances from Royal Jelly Soutenue le 26 septembre 2003 M. Rudi F. VOGEL Président de la Jury MEMBRES DU JURY : (Fonction) M. Eberhard BENGSCH Directeur de Thèse; Examinateur M. Jürgen POLSTER Directeur de Thèse; Examinateur M. André BRACK Examinateur M. Antonius KETTRUP Rapporteur M. Bernard HEUSCH Rapporteur Mme. Paule VASSEUR Rapporteur extérieur M. Friedrich LOTTSPEICH Rapporteur extérieur M. Helmut HORN Rapporteur extérieur Mme. Agnes H. HENSCHEN-EDMAN Membre invité Deutsch-Französische Doppelpromotion (Cotutelle de Thèse) Lehrstuhl für Biologische Chemie Centre de Biophysique Moléculaire Lehrstuhl für Technische Mikrobiologie Isolation and characterisation of substances from Royal Jelly Andreas Stocker Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München und der Faculté des Sciences, Université d’Orléans zur Erlangung des akademischen Grades eines Doktor-Ingenieurs genehmigten Dissertation. Vorsitzender/Président du Jury: Univ.-Prof. Dr.rer.nat.habil. Rudi F. Vogel Prüfungskommission/ Jury de Thèse de Doctorat: Prüfer/Examinateurs: 1. Univ.-Prof. Dr.rer.nat. Dr.rer.nat.habil. Jürgen Polster 1. Dr.Sc.habil. Eberhard Bengsch (Directeur de Thèse) HDR, CNRS, Centre de Biophysique Moléculaire, Orléans (Frankreich) 2. Dr.Sc.habil. André Brack (Directeur de Recherche) HDR, CNRS, Centre de Biophysique Moléculaire, Orléans (Frankreich) Gutachter/Rapporteurs: 3. Univ.-Prof. Dr.rer.nat. Dr.h.c. (RO) Antonius Kettrup 4. Dr.Sc.habil. Bernard Heusch (Directeur de Recherche) HDR, CNRS, Direction des Relations Internationales, Paris (Frankreich) Externe Gutachter/ Rapporteur extérieurs: 5. Prof. Dr.Sc.habil. Paule Vasseur, U.F.R. Sciences Fondamentales et Appliquées, Université Metz (Frankreich) 6. Dr.rer.nat. Dr.sc.agr. Helmut Horn, Universität Hohenheim-Stuttgart Landesanstalt für Bienenkunde 7. Univ.-Doz. Dr.phil. Dr.med.habil. Friedrich Lottspeich Max-Planck-Institut für Biochemie, Martinsried Membre invité: 8. Prof. Dr.med. Agnes H. Henschen-Edman Departement of Biological Sciences, Molecular Biology and Biochemistry, University of California, Irvine (USA) Die Dissertation wurde am 24.6.2003 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, sowie durch die Faculté des Sciences, Université d’Orléans am 7.8.2003 angenommen. III MRJP5 MRJP3 MRJP1 MRJP3 MRJP2 MRJP2 MRJP3 Central topic of this work: Major royal jelly proteins (MRJP’s), its polymorphism and proteolytic derivates (detailed information in fig. 6.5.1; page 147 and fig. 6.6.3; page 156). Preface IV Preface This dissertation was completed as the first contracted German-French joint research project (Cotutelle de Thèse) of the Technische Universität München and the Centre de Biophysique Moléculaire, CNRS UPR 4301 affiliated to the University of Orléans. Substances and results were produced in about equal parts in Germany and in France. The multidisciplinary structure of the dissertation demanded that the dissertation was integrated in several institutes thereby enabling a multifaceted execution of the theme. The joint research with the Centre de Biophysique Moléculaire, CNRS Orléans, permitted, among other procedures, the characterisation of peptides and proteins extracted from royal jelly by a multitude of physical-chemical and biochemical methods of separation. Particular peptides were produced by the Merryfield Synthesis for antibacterial tests and were characterised by NMR studies. The dissertation originated from a Diplomarbeit at the TU München in Weihenstephan with the title: Antibacterial effects of proteins fractions extracted from Royal Jelly. This microbiological and protein-analytical research became the point of departure for the following dissertation. At the TU München the research was mainly executed in the chairs of Technische Mikrobiologie, of Biologische Chemie and of Obstbau, and in the Institut für Mikrobiologie (FML), and with the laboratory of Proteomics. At the GSF München, in the Institut für Ökologische Chemie numerous and different tests as the analysis of trace elements and the characterisation of sequences of peptides extracted from royal jelly were achieved with NMR spectroscopy. In cooperation with the Universität Hohenheim, Stuttgart (Landesanstalt für Bienenkunde) the botanical origins of the royal jelly samples were determined by analysis of the pollen morphology. Preface V In cooperation with the Max-Planck-Institut für Biochemie, Martinsried (laboratory of Proteinanalytik) proteins were characterised with MALDI-MS and Edman- degradation. Antiviral properties of royal jelly against Coxsackie viruses were tested in the laboratory of Virusforschung of the same institute. The beginning phase of the Cotutelle de Thèse of the TU München was supported through organisation and financing by the Centre de Coopération Universitaire Franco-Bavarois (CCUFB), München. The research was also supported financially by the Université Franco-Allemande (UFA), Sarrebruck, Allemagne (Robert-Bosch-Stiftung). Content VI Content page 1. General introduction and objectives 1 2. Seasonal variation of microbiological activity and botanical origin of royal jelly samples 9 2.1 Introduction 10 2.2 Methods and Materials 11 2.3 Results 15 2.4 Various geographical and seasonal origins 38 2.5 Discussion 39 3. Microbiological effects 45 3.1 Inhibitory potential against nosocomial pathogen bacteria 45 3.2 Visualisation of flavan-3-ols in bacteria and lymphocytes - inhibitory effects of flavan-3-ols in bacteria and accumulation in lymphocytes 62 3.3 Extension of the investigated anti-microbial activities on viral infections 69 4. Contents of trace elements and homeostatic effects 73 4.1 Introduction 73 4.2 Methods and Materials 75 4.3 Results 77 4.4 Discussion 87 5. Multielement stable isotope ratio analysis – authenticity and origin 92 5.1 Introduction 92 5.2 Methods and Materials 95 5.3 Results 97 5.4 Discussion 106 6. Analysis, isolation and characterisation of proteins and peptides – detailed investigation of protein components 112 6.1 Gel filtration, ultra filtration and ion exchange chromatography 125 Content VII 6.2. Analytical C8 RP-HPLC and N-terminal sequencing (Edman degradation) 128 6.3 Analytical 2-D electrophoresis of selected C8 RP-HPLC peaks: from HPLC to 2-DE gel 136 6.4. Analysis of C8 RP-HPLC protein peaks with electrospray ionisation- mass spectrometry (ESI-MS) 141 6.5 Micropreparative 2-D electrophoresis and N-terminal sequencing (Edman degradation) 147 6.6. MALDI-MS characterisation and micropreparative 2-D electrophoresis 153 7. General discussion, final remarks and perspectives 158 8. Summary, Sommaire, Zusammenfassung 165 9. References 175 Acknowledgements Abbreviations VIII Abbreviations AU Absorbance Units APS ammonium persulphate cDNA copy desoxiribonucleic acid CF-IRMS continuous flow isotope ratio mass spectrometry CHAPS cholamidopropyl-dimethylammonio-propanesulfonate 1D one dimensional 2D two dimensional Da Dalton DMACA p-dimethyl-aminocinnamaldehyd DTT Dithiothreitol DSM Deutsche Stammsammlung für Mikroorganismen und Zellkulturen ESI-MS electrospray ionization mass spectrometry RJ Royal Jelly 10-HDA 10-hydroxy-∆2-decenoic acid HPLC High Performance Liquid Chromatography ICP-MS inductive coupled plasma mass spectrometry IEF isoelectric focussing IPG immobilized pH-gradients IPG-Dalt two dimensional electrophoresis with immobilized pH-gradients kDa kiloDalton LMW low molecular weight MIC minimal inhibitory concentration Abbreviations IX MALDI-MS matrix assisted laser desorption ionisation mass spectrometry MR relative molecular weight m/z mass/charge ratio NMR nuclear magnetic resonance spectrometry OES optical emission spectrometry pI isoelectric point ppb parts per billion ppm parts per million RT room temperature SDS Sodium n-Dodecyl Sulphate ssp. subspecies %T proportion of total acrylamide TEMED N,N,N’,N’-tetramethylendiamin TI-MS thermal ionisation mass spectrometry TMW Technische Mikrobiologie Weihenstephan TOF time of flight Tris Tris(hydroxymethyl)-aminomethan w/v mass per volume v/v volume per volume 1 General introduction and objectives 1 1 General introduction and objectives Royal jelly is a bee product from the hypopharyngeal, mandibular and postcerebral glands of young worker bees. It is produced under partial digestion of essentially