sturing van gastro-intestinale microbiota via pre- en probiotica
DESCRIPTION
Sturing van gastro-intestinale microbiota via pre- en probiotica. Dr. Ir. Tom Van de Wiele Prof. Dr. Ir. Willy Verstraete. LabMET Laboratorium Microbi ële Ecologie & Technologie. Humane gastro-intestinale microbiota. Planeet: 55 verschillende divisies Bacteria, 13 divisies Archaea - PowerPoint PPT PresentationTRANSCRIPT
Lesson 1 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
Lesson 7 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• >1000 species, • 1014 bacteriële cellen vs. 1013 humane cellen
Lesson 7 3
Human microbiome project (NIH)
• Human genome project• 3.109 baseparen• 25 000 genen
• Human microbiome project• 4.106 genen !!
“Bacteria rule the world !”
Lesson 7 4
Endogene microbiota
Maag: zuurtolerante bacteriën
Dunne darm: galzoutresistent/facultatief anaëroob
Colon:• lumen: anaërobe micro-organismen
• wand: micro-aërofielen & facultatief anaëroben
Lesson 7 5Published by AAAS
Representation of the diversity of bacteria in the human intestine
CFB: Cytophaga-Flavobacterium-Bacteroides Firmicutes
Samen 60% van bacteriën
CFB: meestal verwant met dierenEvolutionair gezien een zeer oude groep bacteriën met symbiotische eigenschappen
Lesson 7 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
Lesson 7 7
zuigeling
log KVE/g feces
kinderenvolwassenen bejaarden
Het GI microbieel ecosysteem
• Inoculatie van het GI stelsel na geboorte• Stabilisatieperiode minder dan 2 jaar
Lesson 7 8
Colonmicrobiota en gezondheid
• 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
GezondheidseffecGezondheidseffecten:ten:
Lesson 7 9
Colonmicrobiota en gezondheid
GezondheidseffecGezondheidseffecten:ten: • Kolonisatie door pathogenen (infectie)
• Vorming van toxines• Putrefactie• Vorming van (geno-)toxische componenten uit
voeding (contaminanten)• Microbiota stimuleren vetopname en vetsynthese !
Lesson 7 10
Disbalans tussen ‘+’ en ‘-’: ziektepatronen
diverticulose
IBD
inflammatory bowel disease
coloncarcinoom
Lesson 7 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
Lesson 7 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
Lesson 7 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
Lesson 7 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 !!!!
Lesson 7 15
Luminale processen
TNO - Intestinal Model TIM
Ugent - LabMETSimulator Humaan Intestinaal Microbieel Ecosysteem
SHIME
• Fermentatieprocessen• Toxine productie• Stabiliteit probiotica , prebiotica ...
• Manifestatie pathogenen• Productie biologisch actieve
componenten• Voorspellen biobeschikbaarheid• ...
Lesson 7 16
Epitheliale processen
• Caco2, HT29... Ussing Chambers...
• Adhesie van probiotica: darmepitheel of mucussecretie• Transport bioactieve peptides...• Immunologische respons• Epitheliale enzymatische activiteit• ...
Lesson 7 17
LabMET : Onderzoek met de SHIME
VoedingsmiddelVoedingsmiddelen:en:• Pre- en Pre- en probioticaprobiotica• Fyto-Fyto-oestrogenenoestrogenen• ContaminantenContaminanten
Chemische Chemische analyse:analyse:
• HPLCHPLC
• GCGC
• ICIC
• … …
Functionele Functionele analyse:analyse:
• Enzymatische Enzymatische activiteitactiviteit
• Metabool patroonMetabool patroon
• 1313CC
• … …
Microbiële Microbiële gemeenschap:gemeenschap:
• ConventioneeConventioneel: l: uitplatingenuitplatingen
• Moleculair: Moleculair: DGGE, RT-DGGE, RT-PCR, Flow PCR, Flow CytometrieCytometrie
Biologische Biologische activiteit:activiteit:
(bio-assays)(bio-assays)
• EstrogeniciteitEstrogeniciteit
• ToxiciteitToxiciteit
• (Anti-)carcinogeni(Anti-)carcinogeniciteitciteit
• … …
Lesson 7 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
Lesson 7 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
Lesson 7 20
NDO structures
Lesson 7 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
Lesson 7 22
Arabinoxylan molecule
• AXOS degradation by enzymes– Xylanases– Xylosidases– Arabinofuranosidase– Esterase (cross links)
• DP = degree of polymerization
• DS = degree of substitution
Lesson 7 23
Health effects
• Arabinoxylan
– Stimulation of lactobacilli
– Production of propionic acid => 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 ???
Lesson 7 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
Lesson 7 25
• 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
Research tasks
Lesson 7 26
Task 1.: Evaluation of AXOS supplementation in axenic bacterial cultures
• Growth curves on AXOS
Bacteria:Bifidobacterium longumBifidobacterium breveBifidobacterium adolescentisMixed culture BifidobacteriaBacteroides vulgatus
Sugars (6 g/L): from left to rightArabinoseXyloseAXOS 3-0 (XOS)AXOS 3-0.23AXOS 58-0.58 (WPC)FOS (2<DP<5)
Lesson 7 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
Lesson 7 28
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
• Incubate colon suspension with AXOS• Measure:
– SCFA, NH4+, enzymatic activity
– Microbial groups– ...
Batch tests 1: SHIME colon compartments
Lesson 7 29
• AXOS degrading enzymes (Xylanase, Arabinofuranosidase and Xylosidase) in SHIME:– Ascendens < transversum < 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 X y l o s i d a s e a c t i v i t y i n S H I M E
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A s c e n d e n s T r a n v e r s u m D e s c e n d e n s
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 30
• Take home 2:– AXOS breakdown takes place in distal colon– AXOS degrading enzymes are repressed in proximal
colon– Glucose inhibits e.g. xylanase
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 31
• 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
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 32
Bifidobacteria
4
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10
AXOS 3-0.09(XOS)
AXOS 3-0.23 AXOS 15-0.27 AXOS 12-0.70 AXOS 58-0.58 FOS
Log CFU/mL
Start Transfer 1 Transfer 2 Transfer 3 Transfer 4
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
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 33
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
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 34
1. 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 >10% glucose inhibits growth on
AXOSBifidobacteria: glucose effect
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Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 35
2. 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
3 types of cells
DNA/RNA
PCR amplification
Amplified fragments
DNA/RNA extraction
Separated fragments
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 36
• 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. Blanc2. AXOS 3-0.09 3. AXOS 3-0.25
4. AXOS 12-0.26 5. AXOS 67-0.586. FOS (6)
Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures
Lesson 7 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 starch+3 g inulin or AXOS /L
WASH OUT
2 weeks
4 g starch/L
Lesson 7 38
Task 3.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem
Lesson 7 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
AXOS - Ascending colon
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production SCFA (mmol/L)
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AXOS - Transverse colon
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Inulin - Ascending colon
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Acetate Propionate Butyrate Total SCFA
production SCFA (mmol/L)
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Inulin - Transverse colon
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Acetate Propionate Butyrate Total SCFA
production SCFA (mmol/L)
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Lesson 7 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
Inulin - ammonium
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AXOS - ammonium
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Lesson 7 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
Xylanase
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CA AXOS CT AXOS CD AXOSArabinofuranosidase
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Xylosidase
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Lesson 7 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...)
Lesson 7 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
Lesson 7 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
Lesson 7 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• ...
Lesson 7 46
Probiotics: required properties
Lesson 7 47
Properties: proposed health benefits
• Probiotic claims:
Lesson 7 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
Lesson 7 49
Survival during gastrointestinal transit
• Survival : Bif E > Bif L• Bif L: stomach lower survival than intestine• Bif E: equal survival
Lesson 7 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 !
Lesson 7 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 !
Lesson 7 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 ?
Lesson 7 53
Contact information
LabMET – Ghent UniversityCoupure Links 653B-9000 Gent
E-mail: [email protected]
URL:
http://labMET.ugent.be/
www.shimetec.be
www.food2know.be
Tel: +32 9 264.59.76