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Objective:

Workpackage III Investigation of the effect of AXOS of variable DP and DS on gastrointestinal microbial populations in model systems. WP III: Investigation of the effect of AXOS of variable DP and DS on gastrointestinal microbial populations in model systems. Objective:

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Objective:

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  1. Workpackage IIIInvestigation of the effect of AXOS of variable DP and DS on gastrointestinal microbial populations in model systems

  2. WP III: Investigation of the effect of AXOS of variable DP and DS on gastrointestinal microbial populations in model systems Objective: • Understanding the relationship between AXOS structure and growth of microbial populations, gene expression and immunomodulatory action in model systems relevant for animals and humans Tasks for LabMET (University of Gent) • Task III.1: Evaluation of AXOS supplementation in axenic bacterial cultures • Task III.3: Evaluation of AXOS supplementation in intestinal microbial populations • Task III.4: Evaluation of AXOS supplementation in the simulator of the intestinal microbial tract

  3. WP IIITask III.1.: Evaluation of AXOS supplementation in axenic bacterial cultures • Growth curves of pure cultures on AXOS • Bacteria: • Bifidobacterium longum • Bifidobacterium breve • Bifidobacterium adolescentis • Mixed bifidobacteria (SHIME) • Bacteroides vulgatus • Sugars (6 g/L): from left to right • Arabinose • Xylose • AXOS 3-0.09 (XOS) • AXOS 3-0.25 • AXOS 67-0.58 (WPC) Increase in optical density

  4. WP IIITask III.1.: Evaluation of AXOS supplementation in axenic bacterial cultures • Results: • Bifidobacteria • Variable growth on AXOS structures • Higher substitution with arabinose gives lower yield • Mixture of bifidobacteria grow well on AXOS • 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

  5. WP IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial cultures SHIME: Simulator of the Human Intestinal Microbial Ecosystem Batch tests 1: SHIME colon compartments • Incubate colon suspension with AXOS • Measure: • SCFA, NH4+, enzymatic activity • Microbial groups • ...

  6. WP IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial cultures • Increase in SCFA for AXOS: • Solely in distal colon, not in proximal colon • AXOS 67-0.58: 30% protein content • Proteins interfere with AXOS degradation • Competition between protein and AXOS breakdown • Too high ammonium production

  7. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • 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

  8. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • Take home 2: • AXOS purity plays important role • Purified AXOS compounds are used • Take home 3: • AXOS breakdown takes place in distal colon • AXOS degrading enzymes are repressed in proximal colon • Glucose inhibits e.g. xylanase

  9. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • 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

  10. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • AXOS is more bifidogenic than FOS in mixed microbiota • AXOS with higher DS generate slower bifidogenic effect Take home 4: • In presence of other intestinal bacteria, Bifidobacteria can cope with the arabinose substitution of AXOS

  11. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • 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 (this part of research is ongoing)

  12. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • INDUCTION • Incubate mixtures of bifidobacteria with mixture of glucose and AXOS (6g/L) • Glucose %: 0, 0.1, 1, 5, 10, 20, 100 % • First growth phase on glucose • Second growth phase on AXOS: only if glucose < 10% • Take home 5 • The presence of >10% glucose inhibits growth on AXOS

  13. DNA/RNA 3 types of cells Amplified fragments Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • 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

  14. Workpackage IIITask III.3.: Evaluation of AXOS supplementation in mixed microbial populations • 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 6: 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)

  15. 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) Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  16. Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  17. AXOS : SCFA production increase in transverse colon AXOS: shift towards proportionally more propionate and butyrate Inulin: primary effect in ascending colon Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  18. Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  19. Ammonium FOS: lower ammonium production temporary effect AXOS: lower ammonium production as remaining effect Cancer related enzymes: Azoreductase: significant decrease in ascending and descending colon Nitroreductase: signficant decrease in proximal colon AXOS treatment has more pronounced effects than FOS Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  20. Green rectangle = treatment AXOS breakdown occurs in distal colon compartments Enzyme repression in proximal colon Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  21. Ascending and transverse colon: no signicifant clustering Descending colon: treatment based clustering Focus on DGGE for specific groups (lactobacilli, bifidobacteria...) Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  22. Take Home 7: 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 Risk for colorectal cancer is highest in distal colon AXOS may be the first prebiotic which has beneficial and selective effects in distal colon Workpackage IIITask III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem

  23. 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 markers in distal colon WP III: Investigation of the effect of AXOS of variable DP and DS on gastrointestinal microbial populations in model systems

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