impaxos workpackage iii investigation of the effect of axos of variable dp and ds on...
Post on 21-Dec-2015
218 Views
Preview:
TRANSCRIPT
IMPAXOS
Workpackage III
Investigation of the effect of AXOS of variable DP and DS on gastrointestinal
microbial populations in model systems
IMPAXOS
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
IMPAXOS
WP III Task III.1.: Evaluation of AXOS supplementation in axenic bacterial cultures
Growth curves of pure cultures on AXOS
Bacteria:Bifidobacterium longumBifidobacterium breveBifidobacterium adolescentisMixed bifidobacteria (SHIME)Bacteroides vulgatus
Sugars (6 g/L): from left to rightArabinoseXyloseAXOS 3-0.09 (XOS)AXOS 3-0.25AXOS 67-0.58 (WPC)
Incr
ease
in o
ptic
al d
ensi
ty
IMPAXOS
WP III Task III.1.: Evaluation of AXOS supplementation in axenic bacterial cultures
Results: Bifidobacteria
Variable growth on AXOS structuresHigher substitution with arabinose gives lower yieldMixture 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
IMPAXOS
WP III Task III.3.: Evaluation of AXOS supplementation in mixed microbial cultures
SHIME: Simulator of the Human Intestinal Microbial Ecosystem
Incubate colon suspension with AXOS Measure:
SCFA, NH4+, enzymatic activity
Microbial groups ...
Batch tests 1: SHIME colon compartments
IMPAXOS
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
Ammonium
0
20
40
60
80
V1 V2 V3
increase (%)
with proteinwithout protein
IMPAXOS
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
X y l o s i d a s e a c t i v i t y i n S H I M E
0 , 0
0 , 2
0 , 4
0 , 6
0 , 8
1 , 0
1 , 2
1 , 4
1 , 6
A s c e n d e n s T r a n v e r s u m D e s c e n d e n s
IMPAXOS
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
IMPAXOS
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
IMPAXOS
Workpackage III Task III.3.: Evaluation of AXOS supplementation in mixed microbial populations
Bifidobacteria
4
5
6
7
8
9
10
AXOS 3-0.09(XOS)
AXOS 3-0.23 AXOS 15-0.27 AXOS 12-0.70 AXOS 58-0.58 FOS
Log CFU/mL
Start Transfer 1 Transfer 2 Transfer 3 Transfer 4
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
IMPAXOS
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)
IMPAXOS
Workpackage III Task III.3.: Evaluation of AXOS supplementation in mixed microbial populations
1. 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
IMPAXOS
Workpackage III Task III.3.: Evaluation of AXOS supplementation in mixed microbial populations
2. 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
IMPAXOS
Workpackage III Task 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
1. Blanc2. AXOS 3-0.09 3. AXOS 3-0.25
4. AXOS 12-0.26 5. AXOS 67-0.586. FOS (6)
IMPAXOS
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
2 weeks
4 g starch/L
IMPAXOS
Workpackage III Task III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem
IMPAXOS
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
AXOS - Ascending colon
0
10
20
30
40
50
60
Acetate Propionate Butyrate Total SCFA
production SCFA (mmol/L)
Startup
Treatment
Control
AXOS - Transverse colon
-2
0
2
4
6
8
10
12
14
16
18
Acetate Propionate Butyrate Total SCFAproduction SCFA (mmol/L)
Startup
Treatment
Control
Inulin - Ascending colon
0
10
20
30
40
50
60
Acetate Propionate Butyrate Total SCFA
production SCFA (mmol/L)
Startup
Treatment
Control
Inulin - Transverse colon
-7
-2
3
8
13
18
Acetate Propionate Butyrate Total SCFA
production SCFA (mmol/L)
Startup
Treatment
Control
IMPAXOS
Workpackage III Task III.4.: Evaluation of AXOS supplementation in the simulator of the intestinal microbial ecosystem
Inulin - ammonium
0
100
200
300
400
500
600
Ascendingcolon
Transversecolon
Descendingcolon
mg/L
Startup TreatmentControl
AXOS - ammonium
0
100
200
300
400
500
600
Ascendingcolon
Transversecolon
Descendingcolon
mg/L
Startup TreatmentControl
Azoreductase
-40
-35
-30
-25
-20
-15
-10
-5
0
Ascendingcolon
Transversecolon
Descendingcolon
% decrease during treatment
inulinAXOS
Nitroreductase
-14
-12
-10
-8
-6
-4
-2
0
Ascendingcolon
Transversecolon
Descendingcolon
% decrease during treatment
inulinAXOS
IMPAXOS
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
IMPAXOS
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
Xylanase
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
0 10 20 30 40 50Time (days)
U/(mL*h)
CA AXOS CT AXOS CD AXOSArabinofuranosidase
-2
0
2
4
6
8
10
0 10 20 30 40 50
Time (days)
U/(mL*h)
CA AXOS CT AXOS CD AXOS
Xylosidase
0
1
2
3
4
5
6
7
0 10 20 30 40 50
Time (days)
U/(mL*h)
CA AXOS CT AXOS CD AXOS
IMPAXOS
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...)
IMPAXOS
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
IMPAXOS
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
top related