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University of Huddersfield Repository Ngehnyuiy, Ngo Hansel Characterisation of Bacterial Polysaccharides Original Citation Ngehnyuiy, Ngo Hansel (2020) Characterisation of Bacterial Polysaccharides. Doctoral thesis, University of Huddersfield. This version is available at http://eprints.hud.ac.uk/id/eprint/35297/ The University Repository is a digital collection of the research output of the University, available on Open Access. Copyright and Moral Rights for the items on this site are retained by the individual author and/or other copyright owners. 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For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected]. http://eprints.hud.ac.uk/ CHARACTERISATION OF BACTERIAL POLYSACCHARIDES NGO HANSEL NGEHNYUIY, MSc A thesis submitted to the University of Huddersfield in partial fulfilment of the requirements for the degree of Doctor of Philosophy Department of Chemical and Biological Sciences School of Applied Sciences The University of Huddersfield March 2020 i ABSTRACT A number Gram-positive bacterial strains including Lactobacillus paracasei DG, Lactobacillus salivarius CCUG44481 and Bifidobacteria breve 7017 have been known to possess probiotic properties which has led to their increasing use in commercial probiotic products. A range of exopolysaccharides (EPS) and capsular polysaccharides (CPS) produced by these number of probiotic Gram-positive bacteria were studied in an attempt to determine if the EPS contributed to the biological activity of these cultures. EPSs were isolated, purified and their structures determined. Lactobacillus paracasei DG generated an EPS and a CPS, which were similar. The 1H-NMR spectrum indicated six anomeric signals confirming that the EPS contained six monosaccharides in their repeating units. The molecular weight determination indicated that a single polymer was produced with a narrow polydispersity. The combined GC-MS and HPAEC-PAD results confirmed that the structure was made up of L-rhamnose, D- galactose and D-N-acetylgalactosamine in a molar ratio 4:1:1. The nuclear magnetic resonance (NMR) as well as the linkage analysis indicated that the repeating unit contained 1,2-linked, 1,2,3-linked and 1,3-linked rhamnoses with a terminal hexose and an N-acetyl sugar. This analysis permitted the complete characterisation of the novel EPS structure. The results of this study allowed other research groups to determine the biological activity of the EPS with full knowledge that the immunotolerance observed was generated by a highly pure and well characterised polysaccharide. The 1H-NMR spectrum of the EPS recovered from Lactobacillus salivarius CCUG44481 identified three anomeric signals and these matched those observed in a commercial dextran. The NMR spectra suggested that the CCUG44481 strain produced a highly branched dextran than the commercial dextran with at least a third of the hexoses being present in the branches. The HPAEC-PAD and GC-MS results all gave a single glucose peak. The linkage analysis showed that the backbone was a repeating unit of α-(1,6)-linked glucose having multiple branches of 1,3-linked glucoses. The NMR spectra and the wet chemical analysis are all consistent with the EPS isolated from L. salivarius CCUG44481 being a highly branched dextran. Two EPSs (S1 and S2) and two CPSs (C1 and C2) were isolated from Bifidobacteria breve 7017. NMR and chemical analysis of the S1 and C1 fractions were bacterial glycogen with a medium molecular weight. For the S2 fraction, 1H-NMR spectrum indicated the presence of five anomeric peaks with one being identified as an impurity. The NMR spectra recorded for S2 changed with time and these corresponded to the slow loss of a ribitol like moiety on prolonged storage. The structure of the repeat unit of the S2 fraction was determined. The analysis of the C2 fraction identified two homoglycans: a β-D-(1,6)-galactofuranan and a β-D- (1,6)-glucan. The characterisation of the EPS requires a time-consuming linkage analysis method. As part of this research an attempt was made to develop a more rapid method for linkage analysis using HPAEC-PAD. A range of variously substituted methyl glucose were separated: mono, di and tri-substituted glucoses could be separated. However, different isomers of either mono or di-substituted glucoses could not be separated. This then meant a HPAEC-PAD linkage method was unlikely to succeed. ii ACKNOWLEDGEMENTS Firstly, I would like to thank the Creator who is the Almighty and who has given me the strength and health to be able to complete my PhD studies. My deepest sincere gratitude goes to my supervisor Prof. Andrew P. Laws who has been my mentor helping, assisting and guiding me throughout my research work. His encouragement, knowledge, constructive comments, suggestions and advice have been of great importance for me during this project. I would like to thank Dr Neil McLay who ran the NMR experiments. I will also extend a great deal of thanks to Prof. Paul Humphreys and his microbiology team who have been of great assistance to me as I carried out the fermentations. I also thank Dr. Sohaib Sadiq for his help and assistance in the chromatography laboratory. I would also like to thank my parents Mr and Mrs Ngo Pierre/Benedicta, siblings: Paul, Claire, Sherard and Delvin for their constant support and encouragement. Special thanks from the bottom of my heart also goes to my fiancée Emmanuelle Se Se, who has also been supportive and encouraging during my research. Lastly, I thank my fellow research colleagues who have also contributed in one way or the other for me to feel like a family throughout the research. iii Abbreviations AcO Acetyl ADP Adenosine Diphosphate ATP Adenosine Triphosphate B. Bifidobacteria CPS Capsular Polysaccharide CDM Chemically Defined Medium DMSO Dimethyl Sulfoxide d.p. Degree of Polymerisation D2O Deuterium Oxide EPS Exopolysaccharide EPSs Exopolysaccharides f Furanose FDA Food and Drug Administration Gal Galactose GalNAc N-acetyl-galactosamine GlcNAc N-acetyl-glucosamine Glc Glucose GRAS Generally Regarded As Safe ICH International Conference on Harmonisation IUPAC International Union of Pure and Applied Chemistry JCM Japanese Culture of Microorganisms OH Hydroxyl LAB Lactic Acid Bacteria L. Lactobacillus LPS Lipopolysaccharide Me Methyl MeI Methyl Iodide MRD Maximum Recovery Diluent MRS de Man, Rogosa and Sharpe Growth Media NaOH Sodium Hydroxide iv OMe Methoxy p Pyranose PEP Phosphoenolpyruvate Rha Rhamnose TCA Trichloroacetic Acid TFA Trifluoroacetic Acid Experimental terms 1D One-dimensional 2D Two-dimensional COSY Correlated Spectroscopy CZE Capillary Zone Electrophoresis DEPT Distortionless Enhancement by Polarization Transfer dn/dc Refractive index increment GC Gas Chromatography GC-MS Gas Chromatography – Mass Spectrometry HMBC Heteronuclear Multiple Bond Correlation HP-AEC-PAD High Performance Anion Exchange Chromatography Pulsed Amperometric Detection HPLC High Pressure Liquid Chromatography HSQC Heteronuclear Single Quantum Coherence HSQC-TOCSY Heteronuclear Single Quantum Coherence – Total Correlation Spectroscopy Hz Hertz IR Infrared LC Liquid Chromatography LC -MS Liquid Chromatography Mass Spectrometer M Molar (mol/dm3) MALLS Multi-Angle Laser Light Scattering MS Mass Spectrometer Mw Weight-average molecular weight Mn Number-average molecular weight v mV Millivolt NMR Nuclear Magnetic Resonance NOE Nuclear Overhauser Effect/Enhancement mg L-1 Milligrams per litre (equivalent to parts per million) μg mL-1 Micrograms per millilitre (equivalent to parts per million) ppm parts per million RI Refractive Index SEC Size Exclusion Chromatography TOCSY Total Correlation Spectroscopy UV Ultraviolet vi Table of contents 1. Introduction .................................................................................................................................... 1 1.1. Classification of Carbohydrates .............................................................................................. 2 1.1.1. Monosaccharides ............................................................................................................ 3 1.1.2. Derivatives of Monomeric Sugar Units ........................................................................... 7 1.1.3. Disaccharides .................................................................................................................. 9 1.1.4. Oligosaccharides ........................................................................................................... 10 1.1.5. Polysaccharides ............................................................................................................. 11 1.2. Bacteria ................................................................................................................................. 13 1.2.1. Bacterial Growth ........................................................................................................... 15 1.2.2. Bacterial Polysaccharides .............................................................................................

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