Effects of Brassica on the human gut microbiota Lee Kellingray Institute of Food Research A thesis submitted for the degree of Doctor of Philosophy to the University of East Anglia September 2015 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. Lee Kellingray Ph.D Thesis, 2015 University of East Anglia Abstract Effects of Brassica on the human gut microbiota Brassica vegetables, such as broccoli, are characterised by the presence of sulphur- containing compounds, termed glucosinolates, which are associated with potential health benefits for humans. Glucosinolates are metabolised in the gut by members of the gut microbiota, producing biologically active breakdown products, such as isothiocyanates. The effects of consuming Brassica on the composition of the gut microbiota, and the bacterial mechanisms employed for glucosinolate metabolism, are unclear, and forms the basis of the research presented in this thesis. Culturing human faecal microbiotas in an in vitro batch fermentation model identified the bacterial-mediated reduction of glucoraphanin and glucoiberin to glucoerucin and glucoiberverin, respectively. An Escherichia coli strain was found to exhibit reductase activity on glucoraphanin and the broccoli-derived compound S-methylcysteine sulphoxide, through the reduction of the sulphoxide moiety. Within this fermentation model, the relative proportions of members of the genus Lactobacillus were found to significantly increase when the microbiota was repeatedly exposed to a broccoli leachate, and 16S rDNA sequencing identified these as L. fermentum. Metabolite analysis detected relatively high concentrations of lactate and short-chain fatty acids when faecal microbiotas were cultured in the presence of broccoli leachates, compared to a glucose control media. A human dietary study investigating the effects of Brassica on the microbiota composition revealed a significant association between dietary Brassica and changes to the relative proportions of a number of bacteria, many of which belong to the Clostridiales. Further studies are required to reveal the nature of this association, and whether the presence of glucosinolates may have been a factor. The work presented in this thesis highlights the strong connection between diet, the gut microbiota, and the potential health benefits to the host that may be derived from the bacterial metabolism of dietary compounds. ii Table of Contents Table of Contents ABSTRACT................................................................................................................................... II TABLE OF CONTENTS .............................................................................................................. III LIST OF FIGURES ..................................................................................................................... VII LIST OF TABLES ......................................................................................................................... X ABBREVIATIONS ....................................................................................................................... XI SYMBOLS................................................................................................................................... XV ACKNOWLEDGEMENTS .......................................................................................................... XVI 1. GENERAL INTRODUCTION ................................................................................................ 1 1.1 DIETARY BRASSICA VEGETABLES .................................................................................... 2 1.1.1 Glucosinolate structure ............................................................................................. 2 1.1.2 Biosynthesis of glucosinolates .................................................................................. 6 1.1.2.1 Chain elongation ............................................................................................................ 6 1.1.2.2 Core structure ................................................................................................................ 7 1.1.2.3 Side-chain modification .................................................................................................. 9 1.1.2.4 Exploiting the regulatory gene MYB28 ........................................................................... 9 1.1.3 Plant myrosinase enzymes ..................................................................................... 10 1.1.4 Glucosinolate hydrolysis products........................................................................... 11 1.1.4.1 Epithiospecifier protein ................................................................................................. 12 1.1.4.2 Thiocyanate forming protein ......................................................................................... 13 1.1.4.3 Nitrile specifier protein .................................................................................................. 13 1.2 POTENTIAL HEALTH BENEFITS OF GLUCOSINOLATES ....................................................... 14 1.3 BIOAVAILABILITY OF GLUCOSINOLATES .......................................................................... 15 1.3.1 Liberation of glucosinolates ..................................................................................... 15 1.3.2 Digestion of glucosinolates ..................................................................................... 17 1.3.3 Absorption & distribution of glucosinolates ............................................................. 18 1.3.4 Metabolism & excretion of glucosinolates and their products ................................. 19 1.4 BACTERIAL METABOLISM OF GLUCOSINOLATES .............................................................. 21 1.4.1 Bacterial reduction of glucosinolates....................................................................... 25 1.4.2 S-methylcysteine sulphoxide ................................................................................... 27 1.5 HUMAN GUT MICROBIOTA ............................................................................................... 30 1.5.1 Effects of the gut microbiota on human health ........................................................ 31 1.5.2 Community composition of the gut microbiota .................................................... 32 1.5.3 Intestinal lactic acid bacteria ................................................................................... 33 1.5.4 Dietary effects on the gut microbiota....................................................................... 35 1.5.5 Effect of pH changes on gut bacteria ...................................................................... 38 1.5.6 Short-chain fatty acid production ............................................................................. 39 1.6 BACTERIAL REDUCTASES .............................................................................................. 42 1.7 THESIS AIMS.................................................................................................................. 43 2 MATERIALS AND METHODS ........................................................................................... 44 2.1 GENERAL REAGENTS ..................................................................................................... 45 2.2 GENERATING A BROCCOLI LEACHATE ............................................................................. 47 2.3 BACTERIAL MEDIA ......................................................................................................... 48 2.4 LIQUID CHROMATOGRAPHY MASS SPECTROMETRY ANALYSIS .......................................... 49 2.4.1 Glucosinolate hydrolysis product analysis .............................................................. 49 2.4.2 S-methylcysteine sulphoxide analysis .................................................................... 50 2.5 GLUCOSINOLATE ANALYSIS ........................................................................................... 51 2.5.1 Desulphoglucosinolate extraction ........................................................................... 51 2.5.2 Analysis of desulphoglucosinolates by high-performance liquid chromatography . 51 2.6 METABOLITE ANALYSIS .................................................................................................. 52 2.6.1 Sample preparation ................................................................................................. 52 2.6.2 Proton nuclear magnetic resonance spectrometry analysis ................................... 53 2.7 GLUCORAPHANIN STABILITY ASSAY ................................................................................ 54 iii Table of Contents 2.8 CULTURING HUMAN FAECAL MICROBIOTAS ..................................................................... 55 2.8.1 Recruitment of faecal donors .................................................................................. 55 2.8.2 Preparation of broccoli leachate-containing media and glucose media .................. 57 2.8.3 Culturing human faecal microbiotas in a broccoli leachate-containing media and a glucose media ....................................................................................................................