Parts of this thesis have already been published: - OPUS4

203 Pages·2011·1.88 MB·English

Maillard products and coffee roasting products
activate NF-κB and Nrf2
in cell culture and intact human gut tissue ex vivo

Maillard Produkte und Röstprodukte aktivieren NF-κB und Nrf2
in verschiedenen Zelltypen und humanem Darmgewebe ex vivo

Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von
Tanja Sauer
aus Bad Dürkheim
Als Dissertation genehmigt von der
Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 20.09.2011
Vorsitzender der
Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatterin: Prof. Dr. Monika Pischetsrieder
Zweitberichterstatter: Prof. Dr. Gerald Muench
Parts of this thesis have already been published:

Publications
Tanja Sauer, Martin Raithel, Jürgen Kressel, Gerald Münch and Monika Pischetsrieder
Nuclear translocation of NF-κB in intact human gut tissue upon stimulation with coffee and
roasting products
Accepted in August 2011 in Food & Function

Tanja Sauer, Martin Raithel, Jürgen Kressel, Gerald Münch and Monika Pischetsrieder
Activation of the transcription factor Nrf2 in macrophages, Caco-2 cells and intact human gut
tissue by Maillard reaction products and coffee
Submitted in April 2011

Talk
Tanja Sauer
Coffee-induced regulation of redox-sensitive transcriptions factors - Protective mechanism
against oxidative stress associated diseases
Neuroscience Colloquium 2010, Kioloa, Australia

Poster
Tanja Sauer and Gerald Muench
Coffee roasting products as a trigger for the cellular antioxidant defense system in oxidative
stress associated diseases
Annual meeting of the Society for Free Radical Biology and Medicine (SFRBM) 2010,
Orlando, Florida (USA)
Tanja Sauer and Gerald Muench
Coffee roasting products as a trigger for the cellular antioxidant defense system in oxidative
stress associated diseases
Student Conference & Career Day 2010 – School of Medicine, University of Western
Sydney, Australia

Tanja Sauer and Monika Pischetsrieder
Einfluss von Kaffee auf zelluläre antioxidative Mechanismen in Makrophagen
39. Deutscher Lebensmittelchemikertag 2010, Stuttgart, Germany

Tanja Sauer, Gerald Muench and Monika Pischetsrieder
Coffee, coffee-related Maillard products and Alzheimer’s Disease
International Conferences for Alzheimer’s Disease (ICAD) 2010, Honolulu, Hawaii (USA)

Tanja Sauer, Gerald Muench and Monika Pischetsrieder
Biphasic effects of food-derived AGEs
Alzheimer’s and Parkinson’s Disease Symposium on Pathomechanisms in Neuro-
degeneration 2010, Sydney, Australia

Tanja Sauer and Monika Pischetsrieder
Die H O -vermittelte, zelluläre Wirkung von Maillard-Produkten auf redox-sensitive
2 2
Transkriptionsfaktoren im Modell-System und in Kaffee
38. Deutscher Lebensmittelchemikertag 2009, Berlin, Germany

Tanja Sauer and Monika Pischetsrieder
Isolierung H O -produzierender Komponenten aus einer Maillard-Mischung
2 2
59. Arbeitstagung LChG-Regionalverband Bayern der GDCh 2008, Munich, Germany
TABLE OF CONTENTS     
TABLE OF CONTENTS

1 LITERATURE OVERVIEW...................................................................................................1
1.1 Coffee.............................................................................................................................1
1.1.1 The coffee bean.......................................................................................................1
1.1.2 Processing of raw coffee beans...............................................................................2
1.1.2.1 Coffee roasting..............................................................................................2
1.1.3 The chemical composition of roasted coffee beans.................................................3
1.1.4 Brewing methods.....................................................................................................6
1.1.5 Coffee as bio-active beverage.................................................................................8
1.1.5.1 Physiological effects of coffee brew..............................................................8
1.1.5.2 Bio-active components in coffee...................................................................8
1.2 The Maillard reaction......................................................................................................9
1.2.1 The Maillard reaction scheme according to Hodge................................................10
1.2.2 Primary effects of food-derived Maillard products..................................................14
1.2.3 Secondary effects of food-derived Maillard products and advanced glycation
endproducts (AGE) in vivo......................................................................................18
1.3 Reactive oxygen species (ROS)...................................................................................23
1.3.1 Endogenous generation of ROS............................................................................24
1.3.2 Detoxification of cellular ROS................................................................................24
1.3.3 Impact of ROS on cell components and signalling pathways................................26
1.3.4 Maillard-dependent generation of ROS in a cell free system.................................27
1.4 The human gastrointestinal tract..................................................................................29
1.4.1 The histology of the intestine.................................................................................29
1.4.2 Pathology of gut associated diseases....................................................................32
1.4.3 Macrophages - immune associated cells...............................................................34
1.5 Aims of this study..........................................................................................................36
TABLE OF CONTENTS     
2 CHARACTERIZATION OF H O GENERATION IN MAILLARD TEST SYSTEMS...........37
2 2
2.1 Introduction...................................................................................................................37
2.2 Results..........................................................................................................................38
2.2.1 Generation of H O by Maillard products...............................................................38
2 2
2.2.1.1 The role of temperature in the generation of H O ......................................42
2 2
2.2.1.2 The role of pH in the generation of H O .....................................................43
2 2
2.2.2 De novo generation of H O by Maillard products..................................................44
2 2
2.2.3 Activity-guided fractionation of Maillard products...................................................48
2.2.3.1 Size exclusion chromatography (SEC).......................................................48
2.2.3.2 Ultra-filtration...............................................................................................51
2.2.4 De novo generation of H O by the active fraction of Maillard products ................52
2 2
2.2.5 Generation of H O by coffee extract.....................................................................54
2 2
2.3 Discussion....................................................................................................................56
3 NUCLEAR TRANSLOCATION OF NF-κB.........................................................................63
3.1 Introduction...................................................................................................................63
3.2 Results..........................................................................................................................65
3.2.1 Cell growth and cell viability of macrophages........................................................66
3.2.2 NF-κB activation by Maillard products....................................................................68
3.2.2.1 Stimulation of different cell types with Maillard products.............................68
3.2.3 NF-κB activation by coffee extract.........................................................................69
3.2.3.1 Stimulation of different cell types with coffee extract..................................69
3.2.3.2 Stimulation of macrophages with raw coffee extract...................................72
3.2.4 Ex vivo stimulation of intact human gut tissue.......................................................74
3.2.4.1 Establishing a method for ex vivo stimulation of intact human gut tissue...74
3.2.4.2 Stimulation of intact human gut tissue with Maillard products and coffee
extract.........................................................................................................77
3.3 Discussion....................................................................................................................78
TABLE OF CONTENTS     
4 NUCLEAR TRANSLOCATION OF NRF2..........................................................................82
4.1 Introduction...................................................................................................................82
4.2 Results..........................................................................................................................85
4.2.1 Detection of early, intermediate & late stage Maillard products
in Maillard mixtures.................................................................................................85
4.2.2 Nrf2 activation by Maillard products.......................................................................86
4.2.2.1 Stimulation of different cell types and tissue with Maillard products
during short-term incubation.......................................................................87
4.2.2.2 Stimulation of macrophages with Maillard products
during long-term incubation........................................................................89
4.2.3 Nrf2 activation by coffee extract.............................................................................93
4.2.3.1 Stimulation of different cell types and tissue with coffee extract
during short-term incubation.......................................................................94
4.2.3.2 Stimulation of macrophages with coffee extract
during long-term incubation........................................................................94
4.2.4 Involvement of Maillard-dependent H O in Nrf2 activation...................................96
2 2
4.2.4.1 The role of extracellular H O in Nrf2 activation by Maillard products.........96
2 2
4.2.4.2 The role of extracellular H O in Nrf2 activation by coffee extract............101
2 2
4.2.5 The effect of a pure H O solution on Nrf2 translocation.....................................102
2 2
4.3 Discussion..................................................................................................................103
5 EXTRA- AND INTRACELLULAR ROS DURING STIMULATION...................................109
5.1 Introduction.................................................................................................................109
5.2 Results........................................................................................................................109
5.2.1 Extracellular H O concentration during stimulation of
2 2
macrophages and human gut tissue.....................................................................109
5.2.1.1 Stimulation of macrophages with Maillard products..................................109
5.2.1.2 Stimulation of macrophages with coffee extract........................................110
5.2.1.3 Stimulation of intact human gut tissue with
Maillard products and coffee extract.........................................................112
5.2.2 Intracellular oxidative stress level........................................................................114
TABLE OF CONTENTS     
5.3 Discussion..................................................................................................................118
6 SUMMARY........................................................................................................................121
7 DEUTSCHE ZUSAMMENFASSUNG...............................................................................131
8 MATERIALS AND METHODS..........................................................................................142
8.1 Materials.....................................................................................................................142
8.1.1 Instrumentation....................................................................................................142
8.1.2 Laboratory equipment..........................................................................................143
8.1.3 Chemicals and reagents......................................................................................143
8.1.4 Buffers and solutions............................................................................................146
8.1.5 Cell lines and primary cells..................................................................................150
8.1.6 Primary antibodies...............................................................................................150
8.1.7 Secondary antibodies...........................................................................................150
8.2 Methods......................................................................................................................151
8.2.1 Preparation of Maillard reaction mixtures and control solutions...........................151
8.2.2 Preparation of roasted and raw coffee extract.....................................................151
8.2.3 Cell culture...........................................................................................................152
8.2.4 Tissue culture.......................................................................................................152
8.2.5 Detection of hydrogen peroxide (H O )................................................................153
2 2
8.2.6 De novo generation of hydrogen peroxide (H O )................................................155
2 2
8.2.7 Fractionation of Maillard products via size exclusion chromatography................155
8.2.8 Fractionation of Maillard products via ultra-filtration.............................................156
8.2.9 Detection of lysine: Ninhydrin assay....................................................................157
8.2.10 Detection of ribose: Orcinol assay.....................................................................157
8.2.11 Nuclear translocation of Nrf2 and NF-κB...........................................................158
8.2.12 Nuclear protein content in cells: Dc Protein assay.............................................162
8.2.13 Total protein content in tissue: Bicinchoninic acid (BC) assay...........................163
8.2.14 Oxidative stress level: 2’,7’-Dichlorofluorescein (DCF) assay............................163
TABLE OF CONTENTS     
8.2.15 Cytotoxicity of the Maillard reaction mixture and coffee extract.........................164
8.2.16 Cell viability of intact human gut tissue during mucosa oxygenation.................166
8.2.17 Statistical analysis..............................................................................................167
BIBLIOGRAPHY..................................................................................................................168
LIST OF ABBREVIATIONS.................................................................................................187
LIST OF FIGURES...............................................................................................................190
LIST OF TABLES................................................................................................................192
1 LITERATURE OVERVIEW 1
1 LITERATURE OVERVIEW

1.1 Coffee
Coffee is a popular and worldwide consumed beverage. In 2009, the per capita consumption
in Germany averaged 150 L coffee [1]. The coffee plant (Figure 1.1A), which is grown mainly
in Brazil and Columbia, belongs to the Rubiaceae family. There are about 80 species which
slightly differ in their composition of ingredients and thus their taste and aroma. Coffea
arabica and coffea canephora variant robusta are of most commercial importance with about
75 % and 25 % of world production respectively [2].

1.1.1 The coffee bean
Each coffee cherry (Figure 1.1B) contains two separate coffee seeds (beans). The seed is
surrounded by a spermoderm tissue, the silverskin, and is embedded into the coffee hull, the
endocarp. A layer of mucilage encloses the hull, followed by the pulp, a thick layer of spongy
cells. Reaching maturity the coffee cherry shows an intense dark red colour [2].
A BB
cccoooffffffeeeeee ssseeeeeeddd
pppuuulllppp
eeennndddooocccaaarrrppp
ssspppeeerrrmmmooodddeeerrrmmm
(((sssiiilllvvveeerrr ssskkkiiinnn)))
Figure 1.1: The coffee plant. (A) Rubiaceae – Coffea arabica [3]. (B) A longitudinal cut through the coffee cherry
(modified [2]).