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 Lesson 1

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

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

Endogene microbiota Maag: zuurtolerante bacteriën Dunne darm: galzoutresistent/facultatief anaëroob Colon: lumen: anaërobe micro-organismen wand: micro-aërofielen & facultatief anaëroben Lesson 7

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 Lesson 7 Published by AAAS

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

Het GI microbieel ecosysteem Inoculatie van het GI stelsel na geboorte Stabilisatieperiode minder dan 2 jaar log KVE/g feces zuigeling kinderen volwassenen bejaarden Lesson 7

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 Lesson 7

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 ! Lesson 7

Disbalans tussen ‘+’ en ‘-’: ziektepatronen diverticulose IBD coloncarcinoom Left: Small colonic diverticula. Right: Larger diverticula in a complex array. Alarge-mouth diverticulum may be mistaken for the colonic lumen. In this image, the lumen is at bottom right. inflammatory bowel disease Lesson 7

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

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

In vivo dierenstudies Conventionele dieren: Voordelen: Nadelen: 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

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

Simulator Humaan Intestinaal Microbieel Ecosysteem Luminale processen Fermentatieprocessen Toxine productie Stabiliteit probiotica , prebiotica ... Manifestatie pathogenen Productie biologisch actieve componenten Voorspellen biobeschikbaarheid ... TNO - Intestinal Model TIM Ugent - LabMET Simulator Humaan Intestinaal Microbieel Ecosysteem SHIME Lesson 7

Epitheliale processen Caco2, HT29... Ussing Chambers... Adhesie van probiotica: darmepitheel of mucussecretie Transport bioactieve peptides... Immunologische respons Epitheliale enzymatische activiteit ... Lesson 7

LabMET : Onderzoek met de SHIME Voedingsmiddelen: Pre- en probiotica Fyto-oestrogenen Contaminanten Chemische analyse: HPLC GC IC … Biologische activiteit: (bio-assays) Estrogeniciteit Toxiciteit (Anti-)carcinogeniciteit … Microbiële gemeenschap: Conventioneel: uitplatingen Moleculair: DGGE, RT-PCR, Flow Cytometrie Functionele analyse: Enzymatische activiteit Metabool patroon 13C … Lesson 7

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

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

NDO structures Lesson 7

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

Arabinoxylan molecule AXOS degradation by enzymes Xylanases Xylosidases Arabinofuranosidase Esterase (cross links) DP = degree of polymerization DS = degree of substitution Lesson 7

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

Objectives and methods 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

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 Lesson 7

Sugars (6 g/L): from left to right 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 (2<DP<5) Lesson 7

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

Batch tests 1: SHIME colon compartments 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 ... Lesson 7

Ascendens < transversum < descendens Reason: Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures 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 Lesson 7

AXOS breakdown takes place in distal colon 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 Lesson 7

Batch test 2: Enrichment experiment 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 Lesson 7

AXOS is more bifidogenic than FOS in mixed microbiota 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 Lesson 7

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 Lesson 7

The presence of >10% glucose inhibits growth on AXOS 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 >10% glucose inhibits growth on AXOS Lesson 7

Enrichment on AXOS (6 g/L) with colon bacteria 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 DNA/RNA extraction PCR amplification DNA/RNA Amplified fragments 3 types of cells Separated fragments Lesson 7

AXOS modulate microbial community Changes in certain Enterococcus sp. 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 Blanc AXOS 3-0.09 AXOS 3-0.25 4. AXOS 12-0.26 5. AXOS 67-0.58 6. FOS (6) Lesson 7

1 g starch+3 g inulin or AXOS /L 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 Lesson 7

Task 3.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem Lesson 7

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 Lesson 7

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 Lesson 7

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 Lesson 7

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

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

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

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

Probiotics: required properties Lesson 7

Properties: proposed health benefits Probiotic claims: Lesson 7

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

Survival during gastrointestinal transit Survival : Bif E > Bif L Bif L: stomach lower survival than intestine Bif E: equal survival Lesson 7

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

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

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

Contact information LabMET – Ghent University Coupure Links 653 B-9000 Gent E-mail: tom.vandewiele@ugent.be URL: http://labMET.ugent.be/ www.shimetec.be www.food2know.be Tel: +32 9 264.59.76 Lesson 7