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

2. 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

3. 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 (XOS)
AXOS
AXOS (WPC)
Increase in optical density

4. 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

5. 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
...

6. 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 : 30% protein content
Proteins interfere with AXOS degradation
Competition between protein and AXOS breakdown
Too high ammonium production

7. 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

8. 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

9. 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

10. 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

11. 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)

12. 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

13. 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

14. Workpackage III Task III. 3
Workpackage III Task III.3.: Evaluation of AXOS supplementation in mixed microbial populations
Blanc
AXOS
AXOS
4. AXOS
5. AXOS
6. FOS (6)
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 6: AXOS has selective Bifidobacterium effect

15. 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
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

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

17. 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

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

19. 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

20. 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

21. 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...)

22. 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
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

23. 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