1 / 53

Sturing van gastro-intestinale microbiota via pre- en probiotica

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

vail
Download Presentation

Sturing van gastro-intestinale microbiota via pre- en probiotica

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 1

  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

  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 Lesson 7 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 Lesson 7

  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

  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 Gezondheidseffecten: Lesson 7

  9. Colonmicrobiota en gezondheid • Kolonisatie door pathogenen (infectie) • Vorming van toxines • Putrefactie • Vorming van (geno-)toxische componenten uit voeding (contaminanten) • Microbiota stimuleren vetopname en vetsynthese ! Gezondheidseffecten: Lesson 7

  10. Disbalans tussen ‘+’ en ‘-’: ziektepatronen diverticulose IBD coloncarcinoom inflammatorybowel disease 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 • 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 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 • 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

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

  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 (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 Batch tests 1: SHIME colon compartments • Incubate colon suspension with AXOS • Measure: • SCFA, NH4+, enzymatic activity • Microbial groups • ... Lesson 7

  29. 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

  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 Lesson 7

  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 Lesson 7

  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 Lesson 7

  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 Lesson 7

  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 >10% glucose inhibits growth on AXOS Lesson 7

  35. DNA/RNA 3 types of cells Amplified fragments 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 Separated fragments Lesson 7

  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 Blanc AXOS 3-0.09 AXOS 3-0.25 4. AXOS 12-0.26 5. AXOS 67-0.58 6. FOS (6) Lesson 7

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

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

  39. AXOS : SCFA production increase in transverse colon AXOS: shift towards proportionally more propionate Inulin: primary effect in ascending colon Task 3: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem Lesson 7

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

  41. AXOS breakdown occurs in distal colon compartments Enzyme repression in proximal colon Task 3: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem Lesson 7

  42. Ascending and transverse colon: no significant clustering Descending colon: treatment based clustering Focus on DGGE for specific groups (lactobacilli, bifidobacteria...) Task 3: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem Lesson 7

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

  44. 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 AXOS as new prebiotic candidate ! 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

More Related