Sturing van gastro-intestinale microbiota via pre- en probiotica

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Lesson 1 1 Sturing van gastro-intestinale microbiota via pre- en probiotica Dr. Ir. Tom Van de Wiele Prof. Dr. Ir. Willy Verstraete LabMET Laboratorium Microbiële Ecologie & Technologie

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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 Presentation

Transcript of Sturing van gastro-intestinale microbiota via pre- en probiotica

Page 1: Sturing van gastro-intestinale microbiota via pre- en probiotica

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

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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

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Human microbiome project (NIH)

• Human genome project• 3.109 baseparen• 25 000 genen

• Human microbiome project• 4.106 genen !!

“Bacteria rule the world !”

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Endogene microbiota

Maag: zuurtolerante bacteriën

Dunne darm: galzoutresistent/facultatief anaëroob

Colon:• lumen: anaërobe micro-organismen

• wand: micro-aërofielen & facultatief anaëroben

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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

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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

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zuigeling

log KVE/g feces

kinderenvolwassenen bejaarden

Het GI microbieel ecosysteem

• Inoculatie van het GI stelsel na geboorte• Stabilisatieperiode minder dan 2 jaar

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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:

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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 !

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Disbalans tussen ‘+’ en ‘-’: ziektepatronen

diverticulose

IBD

inflammatory bowel disease

coloncarcinoom

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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

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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

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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

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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 !!!!

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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• ...

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Epitheliale processen

• Caco2, HT29... Ussing Chambers...

• Adhesie van probiotica: darmepitheel of mucussecretie• Transport bioactieve peptides...• Immunologische respons• Epitheliale enzymatische activiteit• ...

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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

• … …

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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

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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

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NDO structures

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Case Study : Arabinoxylan oligosaccharides (AXOS)

• AXOS are derived from Arabinoxylan– Complex sugar in hemicelluloses of plants– Mainly present in cereal bran and aleuron layer

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Arabinoxylan molecule

• AXOS degradation by enzymes– Xylanases– Xylosidases– Arabinofuranosidase– Esterase (cross links)

• DP = degree of polymerization

• DS = degree of substitution

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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 ???

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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

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• 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

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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)

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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

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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

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• 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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Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures

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• 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

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• 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

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Bifidobacteria

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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

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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

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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

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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

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• 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

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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

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Task 3.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

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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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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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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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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...)

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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

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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

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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• ...

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Probiotics: required properties

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Properties: proposed health benefits

• Probiotic claims:

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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

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Survival during gastrointestinal transit

• Survival : Bif E > Bif L• Bif L: stomach lower survival than intestine• Bif E: equal survival

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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 !

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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 !

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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 ?

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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