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Sturing van gastro-intestinale microbiota via pre- en probiotica

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Title: Microbe-gastheer interfase processen Author: Tom Van de Wiele Last modified by: Tom Van de Wiele Created Date: 11/5/2007 7:40:04 PM Document presentation format – PowerPoint PPT presentation

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Title: Sturing van gastro-intestinale microbiota via pre- en probiotica


1
Sturing van gastro-intestinale microbiotavia
pre- en probiotica
  • Dr. Ir. Tom Van de Wiele
  • Prof. Dr. Ir. Willy Verstraete

LabMET Laboratorium Microbiële Ecologie
Technologie
2
Humane gastro-intestinale microbiota
  • Planeet 55 verschillende divisies Bacteria, 13
    divisies Archaea
  • GI kanaal slechts 8 divisies vertegenwoordigd,
    waarvan 5 zeldzaam (evolutionaire reden ?)
  • Slechts 1 archae Methanobrevibacter smithii
  • Zeer selectieve omgeving nutriënten, adhesie aan
    gastheer, bacteriofagen, immuun systeem
  • gt1000 species,
  • 1014 bacteriële cellen vs. 1013 humane cellen

3
Human microbiome project (NIH)
  • Human genome project
  • 3.109 baseparen
  • 25 000 genen
  • Human microbiome project
  • 4.106 genen !!

Bacteria rule the world !
4
Endogene microbiota
  • Maag zuurtolerante bacteriën
  • Dunne darm galzoutresistent/facultatief anaëroob
  • Colon
  • lumen anaërobe micro-organismen
  • wand micro-aërofielen facultatief anaëroben

5
Representation of the diversity of bacteria in
the human intestine
CFB Cytophaga-Flavobacterium-Bacteroides
Firmicutes Samen 60 van bacteriën CFB
meestal verwant met dieren Evolutionair gezien
een zeer oude groep bacteriën met symbiotische
eigenschappen
Published by AAAS
6
Belangrijkste microbiële groepen
  • Bacteroides, Eubacterium, Clostridium,
    Bifidobacterium, Streptococcus, Lactobacillus,
    Peptostreptococcus, Peptococcus, Ruminococcus,
    Fusobacterium, Veillonella, Enterococcus,
    Propionibacterium, Actinomyces,
    Methanobrevibacter, Desulfovibrio, Helicobacter,
    Porphyromonas, Prevotella, Escherichia,
    Enterobacter, Citrobacter, Serratia, Candida,
    Gemella and Proteus

7
Het GI microbieel ecosysteem
  • Inoculatie van het GI stelsel na geboorte
  • Stabilisatieperiode minder dan 2 jaar

log KVE/g feces
zuigeling
kinderen
volwassenen
bejaarden
8
Colonmicrobiota en gezondheid
Gezondheidseffecten
  • Verdere vertering 10-15 extra energie gastheer
  • Productie van KKVZ als voeding voor colonocyten
  • Immunostimulatie
  • Productie van vitaminen (K, B12...)
  • Kolonisatieresistentie tegen pathogenen
  • Vorming van gezondheidsbevorderende componenten
    uit voeding

9
Colonmicrobiota en gezondheid
Gezondheidseffecten
  • Kolonisatie door pathogenen (infectie)
  • Vorming van toxines
  • Putrefactie
  • Vorming van (geno-)toxische componenten uit
    voeding (contaminanten)
  • Microbiota stimuleren vetopname en vetsynthese !

10
Disbalans tussen en - ziektepatronen
diverticulose
IBD
coloncarcinoom
inflammatory bowel disease
11
Sturen van microbiële balans functional foods
  • Probioticum levende bacteriën die de gezondheid
    positief beïnvloeden (melkzuurbacteriën)
  • Lactobacillus spp.
  • Bifidobacterium spp.
  • ...
  • Prebioticum voedingsmiddelen die endogene
    positieve bacteriën in situ stimuleren
  • Inuline (vb. chicorei)
  • Xylo-oligosaccharides
  • Galacto-oligosaccharides
  • ...
  • Synbioticum combinatie van pro- en prebioticum

12
In vivo humane studies
  • Voordelen
  • Representatief
  • Integratie van alle fysiologische parameters
  • Nadelen
  • Complexe proefopzet double-blind, placebo
    controled, cross-over
  •  black box  (geen bemonstering mogelijk)
  • Geen mechanistische studies
  • Ethische bezwaren
  • Tijds- en arbeidsintensief ? hoge kostprijs
  • Pro- en prebiotica grotere toegang tot humane
    interventiestudies

13
In vivo dierenstudies
  • Conventionele dieren
  • Voordelen
  • Integratie fysiologische parameters
  • Bemonstering is mogelijk
  • Nadelen
  • Niet altijd representatief voor mensen
  • Tijds- en arbeidsintensief
  • Ethische bezwaren
  • Kiemvrije of gnotobiotische dieren
  • Meer representatief
  • Heel tijds- en arbeidsintensief
  • hoge kostprijs

14
In vitro simulatietechnologie
  • Voordelen
  • eenvoudig
  • Hoger reproduceerbaarheid
  • Staalname mogelijk tijdens elke stap
  • Mechanistische studies mogelijk
  • Representatief voor bepaald proces
  • Geen ethische bezwaren
  • Nadelen
  • Ontbreken van fysiologische omgeving
  • Onvoldoende basis voor claims
  • SAMENHANG MET IN VIVO STEEDS NODIG !!!!

15
Luminale processen
  • Manifestatie pathogenen
  • Productie biologisch actieve componenten
  • Voorspellen biobeschikbaarheid
  • ...
  • Fermentatieprocessen
  • Toxine productie
  • Stabiliteit probiotica , prebiotica ...

TNO - Intestinal Model TIM
Ugent - LabMET Simulator Humaan Intestinaal
Microbieel Ecosysteem SHIME
16
Epitheliale processen
  • Caco2, HT29... Ussing Chambers...
  • Adhesie van probiotica darmepitheel of
    mucussecretie
  • Transport bioactieve peptides...
  • Immunologische respons
  • Epitheliale enzymatische activiteit
  • ...

17
LabMET Onderzoek met de SHIME
  • Voedingsmiddelen
  • Pre- en probiotica
  • Fyto-oestrogenen
  • Contaminanten
  • Chemische analyse
  • HPLC
  • GC
  • IC
  • Microbiële gemeenschap
  • Conventioneel uitplatingen
  • Moleculair DGGE, RT-PCR, Flow Cytometrie
  • Biologische activiteit
  • (bio-assays)
  • Estrogeniciteit
  • Toxiciteit
  • (Anti-)carcinogeniciteit
  • Functionele analyse
  • Enzymatische activiteit
  • Metabool patroon
  • 13C

18
Manipulation of the GI microbiota Prebiotics
  • non-digestible food ingredients that
    beneficially affect the host by selectively
    stimulating the growth and/or activity of one or
    a limited number of bacteria in the colon
  • Required properties
  • Resist hydrolysis and absorption in the upper GIT
  • Fermentable by only one or a limited number of
    potentially beneficial bacteria
  • Induce an alteration in the microbial composition
    towards more healthy one
  • Induce beneficial effects towards host

19
non-digestible oligosaccharides (NDO)
  • Carbohydrate chains
  • DP (degree of polymerization) 2-60
  • ß-glycosidic bond that are primarily taken down
    by bacterial enzymes
  • Specific enzymatic activity

20
NDO structures
21
Case Study Arabinoxylan oligosaccharides (AXOS)
  • AXOS are derived from Arabinoxylan
  • Complex sugar in hemicelluloses of plants
  • Mainly present in cereal bran and aleuron layer

22
Arabinoxylan molecule
  • AXOS degradation by enzymes
  • Xylanases
  • Xylosidases
  • Arabinofuranosidase
  • Esterase (cross links)
  • DP degree of polymerization
  • DS degree of substitution

23
Health effects
  • Arabinoxylan
  • Stimulation of lactobacilli
  • Production of propionic acid gt cholesterol
    lowering effect
  • Better absorption of calcium and magnesium in
    rats
  • Reduction in postprandial glucose and insulin
    respons in humans
  • Xylo-oligosaccharides ( AXOS without arabinose
    side chains)
  • Bifidogenic effect (even more than
    fructo-oligosaccharides)
  • Lower risk for colon cancer
  • AXOS prebiotic effects ???

24
Objectives and methods
  • Objective
  • Investigation of the effect of AXOS of
    variable DP (degree of polymerisation) and DS
    (degree of substitution) on gastrointestinal
    microbial populations in model systems
  • Methods
  • Monocultures of intestinal bacteria growth
    curves
  • Mixed cultures batch and SHIME

25
Research tasks
  • Task 1 Evaluation of AXOS supplementation in
    axenic bacterial cultures
  • Task 2 Evaluation of AXOS supplementation in
    intestinal microbial populations
  • Task 3 Evaluation of AXOS supplementation in the
    simulator of the intestinal microbial tract

26
Task 1. Evaluation of AXOS supplementation in
axenic bacterial cultures
  • Growth curves on AXOS
  • Bacteria
  • Bifidobacterium longum
  • Bifidobacterium breve
  • Bifidobacterium adolescentis
  • Mixed culture Bifidobacteria
  • Bacteroides vulgatus
  • Sugars (6 g/L) from left to right
  • Arabinose
  • Xylose
  • AXOS 3-0 (XOS)
  • AXOS 3-0.23
  • AXOS 58-0.58 (WPC)
  • FOS (2ltDPlt5)

27
Task 1. Evaluation of AXOS supplementation in
axenic bacterial cultures
  • Results
  • Probiotic bacteria (Lactobacillus,
    Bifidobacterium)
  • Variable growth on arabinose
  • Bifidobacteria substitution with arabinose gives
    lower yield
  • Bacteroides no problems with arabinose
    substitution
  • Take home 1
  • In pure cultures, several Bifidobacteria do not
    benefit from AXOS
  • Need more relevant conditions for in vivo
    situation mixed microbial cultures

28
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
Batch tests 1 SHIME colon compartments
  • Incubate colon suspension with AXOS
  • Measure
  • SCFA, NH4, enzymatic activity
  • Microbial groups
  • ...

29
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • AXOS degrading enzymes (Xylanase,
    Arabinofuranosidase and Xylosidase) in SHIME
  • Ascendens lt transversum lt descendens
  • Reason
  • Glucose is preferentially taken up and can also
    inhibit certain AXOS-degrading enzymes
  • Glucose (from starch hydrolysis) is present in
    the proximal parts of the colon

30
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • Take home 2
  • AXOS breakdown takes place in distal colon
  • AXOS degrading enzymes are repressed in proximal
    colon
  • Glucose inhibits e.g. xylanase

31
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • Batch test 2 Enrichment experiment
  • Enrich specialist bacterial groups in AXOS
    breakdown
  • Incubate descending colon suspension
  • AXOS degrading enzymes are induced
  • Sugar depleted SHIME-feed 6 g/L AXOS
  • AXOS is dominant carbon source
  • Setup
  • Plate counts Bifidobacteria, Bacteroides,
    Clostridia, total anaerobes

32
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • AXOS is more bifidogenic than FOS in mixed
    microbiota
  • AXOS with higher DS generate slower bifidogenic
    effect
  • Take home 3
  • In presence of other intestinal bacteria,
    Bifidobacteria can cope with the arabinose
    substitution of AXOS

33
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • Possible mechanisms
  • 1) INDUCTION The absence of glucose makes it
    possible that the AXOS degrading enzymes are
    induced in Bifidobacteria
  • 2) SPECIES Specific Bifidobacterium species
    capable to use the AXOS
  • 3) COOPERATION Cooperation of the Bifidobacteria
    with other intestinal bacteria (Bacteroides)
    emproves them to grow on AXOS

34
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • INDUCTION
  • Incubate mixture of bifidobacteria with mixture
    of glucose and AXOS (6g/L)
  • Glucose 0, 0.1, 1, 5, 10, 20, 100
  • Take home 4
  • The presence of gt10 glucose inhibits growth on
    AXOS

35
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • SPECIFIC SPECIES
  • Enrichment on AXOS (6 g/L) with colon bacteria
  • DGGE Denaturating Gradient Gel Electrophoresis
  • Allows separation of DNA fragments based on
    sequence
  • 1 band roughly corresponds to 1 species

PCR amplification
DNA/RNA extraction
Separated fragments
36
Task 2. Evaluation of AXOS supplementation in
mixed microbial cultures
  • DGGE all bacteria
  • AXOS modulate microbial community
  • Changes in certain Enterococcus sp.
  • Increase in Bifidobacterium sp.
  • DGGE bifidobacteria
  • AXOS 3-0.09, AXOS 15-0.26 and AXOS 67-0.58
    stimulate B. Longum
  • Take home 5 AXOS has selective Bifidobacterium
    effect
  1. Blanc
  2. AXOS 3-0.09
  3. AXOS 3-0.25

4. AXOS 12-0.26 5. AXOS 67-0.58 6. FOS (6)
37
Task 3 Evaluation of AXOS supplementation in the
simulator of the intestinal microbial ecosystem
  • What happens over a longer time frame ?
  • Where does AXOS degradation take place ?
  • Twin-SHIME
  • Same feed, pancreatine, temperature
  • Same fecal inoculum!
  • 2 different treatments Inulin and AXOS 12-0.26
  • Time scedule
  • Samples
  • Plate counts (2 times/week)
  • SCFA (3 times/week)
  • Ammonium (3 times/week)
  • Enzymes (3 times/week)
  • DGGE (1 time/week)

STABILISATION 2 weeks 4 g starch/L TREATMENT 3 weeks 1 g starch3 g inulin or AXOS /L WASH OUT 2 weeks 4 g starch/L
38
Task 3. Evaluation of AXOS supplementation in
the simulator of the intestinal microbial
ecosystem
39
Task 3 Evaluation of AXOS supplementation in the
simulator of the intestinal microbial ecosystem
  • AXOS SCFA production increase in transverse
    colon
  • AXOS shift towards proportionally more
    propionate
  • Inulin primary effect in ascending colon

40
Task 3. Evaluation of AXOS supplementation in
the simulator of the intestinal microbial
ecosystem
  • Ammonium
  • FOS lower ammonium production temporary effect
  • AXOS lower ammonium production as remaining
    effect
  • Enzymatic activity
  • Cancer related enzymes azoreductase,
    nitroreductase
  • Significant decrease during AXOS treatment,
    especially in descending colon

41
Task 3 Evaluation of AXOS supplementation in
the simulator of the intestinal microbial
ecosystem
  • AXOS breakdown occurs in distal colon
    compartments
  • Enzyme repression in proximal colon

42
Task 3 Evaluation of AXOS supplementation in
the simulator of the intestinal microbial
ecosystem
  • Ascending and transverse colon no significant
    clustering
  • Descending colon treatment based clustering
  • Focus on DGGE for specific groups (lactobacilli,
    bifidobacteria...)

43
Task 3 Evaluation of AXOS supplementation in
the simulator of the intestinal microbial
ecosystem
  • Take Home 6 AXOS 12-0.26
  • AXOS degrading enzymes only produced in distal
    colon
  • AXOS selects for more saccharolytic conditions
    (and SCFA production) in distal colon
    compartments
  • Proportional shift towards propionate lowers
    cholesterol levels in blood
  • AXOS lowers ammonium as a remaining effect
  • AXOS lowers cancer related enzymes, especially in
    descending colon
  • Risk for colorectal cancer is highest in distal
    colon
  • AXOS is the first prebiotic which beneficially
    affects several endpoints in distal colon

44
AXOS as new prebiotic candidate !
  • CONCLUSIONS
  • Pure cultures no selective effect from AXOS
    towards bifidobacteria
  • Mixed cultures selective effect towards
    bifidobacteria
  • AXOS breakdown primarily takes place in distal
    colon
  • Extra propionate production would lower
    cholesterol
  • AXOS decreases cancer related endpoints in distal
    colon
  • In vivo validation with human intervention trial
    ongoing

45
Manipulation of the GI microbiota Probiotics
  • Living microbial food supplements that
    beneficially affect the host by improving its
    intestinal microbial balance
  • Lactobacillus sp.
  • Bifidobacterium sp.
  • Lactococcus lactis subsp.
  • Enterococcus faecium
  • Streptococcus termophilus
  • Saccharomyces cerevisiae
  • ...

46
Probiotics required properties
47
Properties proposed health benefits
  • Probiotic claims

48
Case study Bifidobacterium longum
  • Encapsulation of probiotic Bifidobacterium
    longum
  • Increased survival during gastrointestinal
    transit ?
  • Prolonged colonization in colon compartments ?
  • Bif L lyophilized strain, not encapsulated
  • Bif E encapsulated strain
  • Materials and Methods
  • Survivability tests in gastric acidity and
    intestinal bile salts
  • Batch test experiments
  • Modulation of colon microbial community and
    colonization
  • SHIME run

49
Survival during gastrointestinal transit
  • Survival Bif E gt Bif L
  • Bif L stomach lower survival than intestine
  • Bif E equal survival

50
Modulation of colon microbial community
  • No significant changes from neither probiotic
    formulations
  • Functional stability
  • Stable microbial community composition
  • No important interfering effects from the
    probiotics !

51
PCR-DGGE of colon microbial community
  • Week 6 addition of Bif L
  • Week 7 washout of Bif L
  • Week 8 addition of Bif E
  • Week 9 no addition of Bif E, but Bif E maintains
    its presence in the colon !

52
ALGEMENE CONCLUSIE
  • Sturen van gastro-intestinale microbiële ecologie
  • Microbial Resource Management (cfr. Human
    resource man.)
  • In vitro Mechanistische studies onderbouwing
    van in vivo
  • In vivo fysiologische studie validatie van in
    vitro
  • Probiotica
  • Overleving maag-dunne darm transit is cruciaal
  • Vestiging tussen colon microbiota is delicaat
  • Moleculaire detectiemethodes !
  • Prebiotica
  • Stabiliteit maag-dunne darm opvolgen hydrolyse
  • Mechanisme van afbraak enkel door
    gezondheidsbevorderende of samenwerking met
    andere organismen ?

53
Contact information
  • LabMET Ghent University
  • Coupure Links 653
  • B-9000 Gent
  • E-mail tom.vandewiele_at_ugent.be
  • URL
  • http//labMET.ugent.be/
  • www.shimetec.be
  • www.food2know.be
  • Tel 32 9 264.59.76
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