Metabolite profiling [Elektronische Ressource] : a useful tool for the investigation of induced mutants and developmental changes in crops / Thomas Frank

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: English
• Poids de l'ouvrage: 2 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Allgemeine Lebensmitteltechnologie


Metabolite Profiling –
A Useful Tool for the Investigation of Induced Mutants
and Developmental Changes in Crops


Thomas Frank



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. Th. F. Hofmann
Prüfer der Dissertation: 1. Univ.-Prof. Dr. K.-H. Engel
2. Univ.-Prof. Dr. W. Schwab


Die Dissertation wurde am 06.04.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 06.07.2009 angenommen. Acknowledgements

The work presented was carried out under supervision of Prof. Dr. Karl-Heinz Engel,
Chair of General Food Technology at the Technische Universität München. I would
like to thank Prof. Engel for the opportunity to work on this exciting subject. Many
thanks to him for his scientific support, for many fruitful discussions and for his
guidance in completing this thesis.

I am indebted to all partners involved in the EU project NOFORISK, especially to Dr.
Ib Knudsen and Prof. Dr. Angharad Gatehouse for the fruitful and helpful discussions
and for sharing valuable scientific ideas.

Particular thank to Prof. Dr. Qing-Yao Shu and Dr. Xiao-Li Shu for the excellent
collaboration and for providing the valuable crop materials.

Thanks to Dr. Renate Habernegg from the Bavarian Authority for Health and Food
Safety for performing the mineral analysis for rice and soybean. Furthermore thanks
to Rene Schneider from the Institute of Brewing Technology I at Technische
Universität München for providing barley material and for performing the micromalting
experiments.

I am greatful to have been able to work at the Chair of General Food Technology with
my collegues Andreas Barnsteiner, Hedwig Strohalm, Iulia Poplacean, Dr. Ron
Baudler, Martina Denk, Oxana Fastovskaya, Dr. Marta Dregus, Dr. Ludwig Ziegler
and Dr. Bernhard Meier. Special thanks to Dr. Andreas Miller and Richard Röhlig for
many extended and fruitful discussions.

Thanks to my sadly deceased friend and colleague Bertrand Seumo Meuleye for the
good teamwork and the fruitful discussions. I will never forget his enthusiasm in
scientific research.

Finally, I would like to thank my family, in particular my parents, my brother and my
grandmother, my parents-in-law and my brother-in-law for their amazing support
during the last years. Special thanks to my lovely wife Sylvia for her continuous
believe in me. I will always be there for you.
I
TABLE OF CONTENTS


1 INTRODUCTION AND OBJECTIVES..........................................1


2 BACKGROUND ...........................................................................3

2.1 CROPS IN AGRICULTURE AND NUTRITION ............................................ 3
2.1.1 Rice, barley and soybean ........................................................................ 3
2.1.2 Germinated brown rice............................................................................. 4

2.2 PHYTIC ACID............................................................................................... 5
2.2.1 Structure and natural occurrence............................................................. 5
2.2.2 Biosynthesis of phytic acid...................................................................... 6
2.2.3 Relevance in agriculture and nutrition...................................................... 8
2.2.4 Generation of low phytic acid (lpa) crops................................................. 9
2.2.5 Methods for determination of phytic acid ............................................... 10

2.3 METABOLOMICS ...................................................................................... 12
2.3.1 Definitions .............................................................................................. 12
2.3.2 Metabolite Profiling ................................................................................ 13
2.3.3 Metabolomics-based Applications ......................................................... 16
2.3.3.1 Safety assessment of novel foods................................................... 16
2.3.3.2 Functional Metabolite Profiling ....................................................... 19


3 MATERIALS AND METHODS21

3.1 MATERIALS............................................................................................... 21
3.1.1 Chemicals.............................................................................................. 21
3.1.2 Plant materials ....................................................................................... 22
3.1.2.1 Rice................................................................................................. 22
3.1.2.2 Soybean.......................................................................................... 24
3.1.2.3 Barley 25
3.1.3 Equipment.............................................................................................. 25 II
3.2 METHODS ................................................................................................. 26
3.2.1 Analysis of inositol phosphates.............................................................. 26
3.2.1.1 Preparation of mobile phase ........................................................... 26
3.2.1.2 Extraction procedure ....................................................................... 26
3.2.1.3 HPLC-RI.......................................................................................... 27
3.2.1.4 Quantification of inositol phosphates............................................... 27
3.2.2 Determination of divalent cations 27
3.2.3 Metabolite profiling of rice...................................................................... 28
3.2.3.1 Preparation of standard solutions.................................................... 28
3.2.3.2 Rough rice....................................................................................... 28
3.2.3.3 Germination of rice.......................................................................... 28
3.2.3.4 Extraction procedure ....................................................................... 29
3.2.3.5 Fractionation and analysis of lipids ................................................. 29
3.2.3.6 Fractionation and analysis of polar extract...................................... 30
3.2.4 Metabolite profiling of barley .................................................................. 30
3.2.4.1 Preparation of standard solutions.................................................... 30
3.2.4.2 Malting procedure............................................................................ 31
3.2.4.3 Sample preparation......................................................................... 31
3.2.4.4 Extraction, fractionation and GC analysis........................................ 31
3.2.5 Metabolite profiling of soybean .............................................................. 32
3.2.5.1 Standard solutions and sample preparation .................................... 32
3.2.5.2 Extraction, fractionation and GC analysis 32
3.2.6 Gas chromatography ............................................................................. 33
3.2.7 Gas chromatography – mass spectrometry ........................................... 34
3.2.8 Response factor..................................................................................... 35
3.2.9 Recovery................................................................................................ 35
3.2.10 Analysis of metabolite profiling data ...................................................... 35
3.2.10.1 Identification of rice, barley and soybean constituents .................... 35
3.2.10.2 Statistical assessment..................................................................... 36






III
4 RESULTS AND DISCUSSION...................................................37

4.1 ANALYSIS OF GERMINATING SEEDS .................................................... 37
4.1.1 Introduction............................................................................................ 37
4.1.2 Germinating rice seeds.......................................................................... 38
4.1.2.1 Detection and Identification of Metabolites...................................... 38
4.1.2.2 Principal Component Analysis......................................................... 43
4.1.2.3 Relative Quantification of Compounds ............................................ 46
4.1.3 Malting of barley grains 53
4.1.3.1 Identification of barley constituents ................................................. 54
4.1.3.2 Principal component analysis.......................................................... 54