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Lesson 11 Sturing van gastro-intestinale microbiota via pre- en probiotica Dr. Ir. Tom Van de Wiele Prof. Dr. Ir. Willy Verstraete LabMET Laboratorium.

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Presentatie over: "Lesson 11 Sturing van gastro-intestinale microbiota via pre- en probiotica Dr. Ir. Tom Van de Wiele Prof. Dr. Ir. Willy Verstraete LabMET Laboratorium."— Transcript van de presentatie:

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2 Lesson 11 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

3 Lesson 72 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, bacteriële cellen vs humane cellen

4 Lesson 73 Human microbiome project (NIH) Human genome project baseparen genen Human microbiome project genen !! “Bacteria rule the world !”

5 Lesson 74 Endogene microbiota Maag: zuurtolerante bacteriën Dunne darm: galzoutresistent/facultatief anaëroob Colon: lumen: anaërobe micro-organismen wand: micro-aërofielen & facultatief anaëroben

6 Lesson 75 Published 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 dieren Evolutionair gezien een zeer oude groep bacteriën met symbiotische eigenschappen

7 Lesson 76 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

8 Lesson 77 zuigeling log KVE/g feces kinderenvolwassenenbejaarden Het GI microbieel ecosysteem Inoculatie van het GI stelsel na geboorte Stabilisatieperiode minder dan 2 jaar

9 Lesson 78 Colonmicrobiota en gezondheid Verdere vertering: % extra energie gastheer Productie van KKVZ als voeding voor colonocyten Immunostimulatie Productie van vitaminen (K, B 12...) Kolonisatieresistentie tegen pathogenen Vorming van gezondheidsbevorderende componenten uit voeding Gezondheidseffecten:

10 Lesson 79 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 !

11 Lesson 710 Disbalans tussen ‘+’ en ‘-’: ziektepatronen diverticuloseIBD inflammatory bowel disease coloncarcinoom

12 Lesson 711 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

13 Lesson 712 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

14 Lesson 713 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

15 Lesson 714 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 !!!!

16 Lesson 715 Luminale processen TNO - Intestinal Model TIM Ugent - LabMET Simulator Humaan Intestinaal Microbieel Ecosysteem SHIME Fermentatieprocessen Toxine productie Stabiliteit probiotica, prebiotica... Manifestatie pathogenen Productie biologisch actieve componenten Voorspellen biobeschikbaarheid...

17 Lesson 716 Epitheliale processen Caco2, HT29... Ussing Chambers... Adhesie van probiotica: darmepitheel of mucussecretie Transport bioactieve peptides... Immunologische respons Epitheliale enzymatische activiteit...

18 Lesson 717 LabMET : Onderzoek met de SHIME Voedingsmiddelen: Pre- en probiotica Pre- en probiotica Fyto-oestrogenen Fyto-oestrogenen Contaminanten Contaminanten Chemische analyse: HPLC HPLC GC GC IC IC … … Functionele analyse: Enzymatische activiteit Enzymatische activiteit Metabool patroon Metabool patroon 13 C 13 C … … Microbiële gemeenschap: Conventioneel: uitplatingen Conventioneel: uitplatingen Moleculair: DGGE, RT-PCR, Flow Cytometrie Moleculair: DGGE, RT-PCR, Flow Cytometrie Biologische activiteit: (bio-assays) Estrogeniciteit Estrogeniciteit Toxiciteit Toxiciteit (Anti-)carcinogeniciteit (Anti-)carcinogeniciteit … …

19 Lesson 718 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

20 Lesson 719 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

21 Lesson 720 NDO structures

22 Lesson 721 Case Study : Arabinoxylan oligosaccharides (AXOS) AXOS are derived from Arabinoxylan –Complex sugar in hemicelluloses of plants –Mainly present in cereal bran and aleuron layer

23 Lesson 722 Arabinoxylan molecule AXOS degradation by enzymes –Xylanases –Xylosidases –Arabinofuranosidase –Esterase (cross links) DP = degree of polymerization DS = degree of substitution

24 Lesson 723 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 ???

25 Lesson 724 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

26 Lesson 725 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

27 Lesson 726 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 AXOS (WPC) FOS (2

28 Lesson 727 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

29 Lesson 728 Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures Incubate colon suspension with AXOS Measure: – SCFA, NH 4 +, enzymatic activity – Microbial groups –... Batch tests 1: SHIME colon compartments

30 Lesson 729 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 Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures

31 Lesson 730 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

32 Lesson 731 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

33 Lesson 732  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

34 Lesson 733  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

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

36 Lesson 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

37 Lesson 736 DGGE all bacteria –AXOS modulate microbial community –Changes in certain Enterococcus sp. –Increase in Bifidobacterium sp. DGGE bifidobacteria –AXOS , AXOS and AXOS stimulate B. Longum Take home 5: AXOS has selective Bifidobacterium effect 1.Blanc 2.AXOS AXOS AXOS AXOS FOS (6) Task 2.: Evaluation of AXOS supplementation in mixed microbial cultures

38 Lesson 737 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 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

39 Lesson 738 Task 3.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

40 Lesson 739 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

41 Lesson 740 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

42 Lesson 741 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

43 Lesson 742 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...)

44 Lesson 743 Task 3: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem Take Home 6: AXOS –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

45 Lesson 744 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

46 Lesson 745 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...

47 Lesson 746 Probiotics: required properties

48 Lesson 747 Properties: proposed health benefits Probiotic claims:

49 Lesson 748 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

50 Lesson 749 Survival during gastrointestinal transit Survival : Bif E > Bif L Bif L: stomach lower survival than intestine Bif E: equal survival

51 Lesson 750 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 !

52 Lesson 751 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 !

53 Lesson 752 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 ?

54 Lesson 753 Contact information LabMET – Ghent University Coupure Links 653 B-9000 Gent URL: Tel:


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