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

Tasks for LabMET (University of Gent) 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

WP III Task 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

WP III Task 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

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

Increase in SCFA for AXOS: AXOS 67-0.58: 30% protein content WP III Task 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

Workpackage III Task III. 3 Workpackage III Task 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

Workpackage III Task III. 3 Workpackage III Task 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

Workpackage III Task III. 3 Workpackage III Task 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

Workpackage III Task III. 3 Workpackage III Task 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

Workpackage III Task III. 3 Workpackage III Task 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)

Workpackage III Task III. 3 Workpackage III Task 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

Enrichment on AXOS (6 g/L) with colon bacteria Workpackage III Task 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 DNA/RNA Amplified fragments 3 types of cells Separated fragments

Workpackage III Task III. 3 Workpackage III Task III.3.: Evaluation of AXOS supplementation in mixed microbial populations Blanc AXOS 3-0.09 AXOS 3-0.25 4. AXOS 12-0.26 5. AXOS 67-0.58 6. FOS (6) 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

1 g starch+3 g inulin or AXOS /L Workpackage III Task III.4.: 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

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

Workpackage III Task III. 4 Workpackage III Task III.4.: 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 and butyrate Inulin: primary effect in ascending colon

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

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

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

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

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