Lehrstuhl für Technische Mikrobiologie
Production of prebiotic exopolysaccharides by lactobacilli
Markus Tieking
Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktor - Ingenieurs
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr.- Ing. E. Geiger
Prüfer der Dissertation: 1. Univ.-Prof. Dr. rer. nat. habil. R. F. Vogel
2. Univ.-Prof. Dr.- Ing. D. Weuster-Botz
Die Dissertation wurde am 09.03.2005 bei der Technischen Universität eingereicht und durch
die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
am 27.05.2005 angenommen.
Lehrstuhl für
Technische Mikrobiologie
Production of prebiotic exopolysaccharides by lactobacilli
Markus Tieking
Doctoral thesis
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
Freising 2005
Mein Dank gilt
meinem Doktorvater Prof. Rudi Vogel für die Überlassung des Themas sowie die stete
Diskussionsbereitschaft,
Dr. Michael Gänzle für die kritische Begleitung, die ständige Diskussionsbereitschaft sowie
sein fachliches Engagement, welches weit über das übliche Maß hinaus geht,
Dr. Matthias Ehrmann für seine uneingeschränkte Bereitwilligkeit, sein Wissen auf dem
Gebiet der Molekularbiologie weiterzugeben, seine unschätzbar wertvollen praktischen
Ratschläge auf diesem Gebiet sowie für seine Geduld,
meiner lieben Frau Manuela, deren Motivationskünste und emotionale Unterstützung mir
wissenschaftliche und experimentelle „Durststrecken“ zu bewältigen halfen,
der Ernst Böcker GmbH und dem Fleischmann Fonds für die finanzielle Unterstützung,
meinen Labortisch- und Büronachbarn Clarissa Schwab, Nicoline Vermeulen und Dirk
Margosch für die freundschaftliche und humorvolle Arbeitsatmosphäre trotz chronischem
Platzmangels und zu heißen Labortemperaturen,
Georg Maier für seine Hilfsbereitschaft, seinen Fleiß und Einsatz,
all den Kollegen an der TMW für das konstruktive Miteinander und das kollegiale Verhältnis,
all den Student(in)en, die ich im Rahmen von Diplom- und Semesterarbeiten betreut habe für
die meist engagierte und interessierte Mitarbeit.
Abbreviations frequently used in this thesis:
aa amino acid
AFLP Amplified Fragment Length
Polymorphism
bp base pairs
cfu colony forming units
dNTP desoxynucleoside triphosphate
dTDP desoxythymidine diphosphate
DP degree of polymerization
EMBL European Molecular Biology
Laboratory
EPS exopolysaccharide
FOS fructooligosaccharide
GOS glucooligosaccharide
HMW high molecular weight
HoPS homopolysaccharide
HePS heteropolysaccharide
HeOS heterooligosaccharide
HPAEC-IPAD High Performance Anion Exchange
Chromatography with Integrated
Pulsed Amperometric Detection
HPLC High Performance Liquid
Chromatography
LAB lactic acid bacteria
LMW low molecular weight
ORF open reading frame
PAGE polyacrylamide gel electrophoresis
PCR polymerase chain reaction
GlcNAc N-acetyl-glucosamine
GalNAc N-acetyl-galactosamine
GPC gel permeation chromatography
RAPD Randomly Amplified Polymorphic
DNA
RI refractive index
RT-PCR reverse transcrictase PCR
SDS sodium dodecyl sulfate
UDP undecaprenylphosphate
WS-PS water soluble polysaccharides
Contents
1 Introduction 1
1.1 Lactic acid bacteria in cereal fermentations 1
1.2 Exopolysaccharides from lactic acid bacteria 1
1.3 Glucan and fructan biosynthetic enzymes 6
1.4 Application of EPS from lactobacilli: overview 8
1.5 Motivation and objectives 9
2 Materials and Methods 11
2.1 Strains, media and growth conditions 11
2.2 EPS-screening 11
2.2.1 Screening of cereal associated lactobacilli for EPS synthesis 11
2.2.2 Screening of lactobacilli isolated from ducks for EPS synthesis 13
2.2.3 Isolation of lactobacilli from pig feces and screening for EPS
production 13
2.2.4 Monosaccharide composition of EPS 14
2.2.5 Preparation of doughs for the purpose of EPS screening 14
2.3 High Performance Liquid Chromatography (HPLC)-analytics 15
2.4 Molecular techniques 16
2.4.1 General molecular techniques 16
2.4.2 Construction of primers targeting fructosyl- and glucosyltransferases 19
2.4.3 Isolation of mRNA and RT-RCR 19
2.4.4 Amplified Fragment Length Polymorphism (AFLP) 20
2.4.5 Plasmids 21
2.4.6 Nucleotide sequence analysis of the levansucrase gene sequence from L.
sanfranciscensis TMW 1.392 21
2.4.7 Nucleotide sequence analysis of the glucansucrase gene sequence from L.
animalis TMW 1.971 21
2.4.8 Cloning, heterologous expression and purification of the levansucrase 22
2.4.9 Insertional inactivation of the levansucrase gene by single crossover
integration 23
2.4.10 Isolation of RNA from dough fermented with L. sanfranciscensis TMW
1.392 and TMW 1.392∆lev, RT-RCR and amplification of a levansucrase and
phosphotransacetylase internal fragment from cDNA 25
2.5 Characterization of the sucrose metabolism of L. sanfranciscensis
TMW 1.392 and TMW 1.392∆lev in medium and doughs 26
2.6 Characterization of the levansucrase from L. sanfranciscensis 27
2.7 Partial purification and characterization of oligosaccharides from enzymatic
reactions and sourdoughs 28
2.8 Chromatographic separation of oligosaccharides 29
3 Results 30
3.1 In situ production of exopolysaccharides during sourdough fermentation
by cereal and intestinal isolates of lactic acid bacteria 30
3.1.1 Screening of sourdough lactobacilli for EPS production 30
3.1.2 PCR-screening for a levansucrase gene and sequence of the
amplification product 33
3.1.3 Comparison of the sequence of levV-levR PCR-products with other
bacterial levansucrases 34
3.1.4 Expression of the levansucrase gene in L. sanfranciscensis 36
3.1.5 In situ EPS-formation in sourdough 37
3.2 Molecular and functional characterization of a levansucrase from
Lactobacillus sanfranciscensis 40
3.2.1 Nucleotide sequence analysis of the L. sanfranciscensis TMW1.392
levansucrase gene 40
3.2.2 Analysis of the amino acid (aa)sequence and alignments with bacterial
levansucrases 41
3.2.3 Insertional inactivation of the levansucrase gene and sucrose
metabolism by the levansucrase deficient strain 43
3.2.4 Dough fermentations with the wild type strain and the levansucrase
deficient strain. 46
3.2.5 Heterologous expression and biochemical characterization of the
recombinant enzymes. 50
3.3 Evidence for formation of heterooligosaccharides (HeOS) by Lactobacillus
sanfranciscensis during growth in wheat sourdough. 56
3.3.1 Enzymatic synthesis of fructooligosaccharides (FOS) and HeOS 56
3.3.2 FOS and HeOS from sucrose or raffinose as fructosyl-donor and
–acceptor 57
3.3.3 HeOS from maltose and maltotriose as fructosyl-acceptors 58
3.3.4 HeOS from xylose or arabinose as fructosyl-acceptors 60
3.3.5 Oligosaccharides formed during sourdough fermentations with
L. sanfranciscensis 61
3.4 Extracellular homopolysaccharides and oligosaccharides from intestinal
lactobacilli 64
3.4.1 Screening of lactobacilli isolated from ducks for HoPS synthesis 64
3.4.2 Production of oligosaccharides by HoPS-producing strains 64
3.4.3 Molecular screening for genes encoding glucosyl- and
fructosyltransferases 66
3.4.4 Identification of lactobacilli from the pig feces and their ability
for HoPS formation 69
3.5 Isolation and nucleotide sequence analysis of the L. animalis strain 1.971
glucansucrase gene 72
3.5.1 Nucleotide sequence analysis 72
3.5.2 Amino acid sequence analysis 73
4 Discussion 75
4.1 Screening of cereal lactobacilli for EPS synthesis and genes coding for EPS
synthesizing genes 75
4.2 Isolation and characterization of a levansucrase from L. sanfranciscensis
TMW 1.392 79
4.3 Heterooligosaccharides in sourdough fermentations 83
4.4 Screening of lactobacilli isolated from the intestinum of ducks and pigs 86
4.5 Molecular characterization of a glucansucrase from L. animalis 88
5 Summary and concluding remarks 89
6 Zusammenfassung und Ausblick 93
7 References 97
8 List of publications 111
1
1 Introduction
1.1 Lactic acid bacteria in cereal fermentations
Following a long tradition, sourdough is used as leavening agent in bread production.
Sourdough fermentations as well as baking agents based on sourdoughs retained their
importance in contemporary baking technology because of the improve
