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

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


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


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


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


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


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


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


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


IMPAXOS


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



Startup

Treatment

Control

Inulin - Transverse colon

-7

-2

3

8

13

18



Startup

Treatment

Control

IMPAXOS


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


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


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


Ascending and transverse colon: no signicifant clustering Descending colon: treatment based clustering Focus on DGGE for specific groups (lactobacilli,

bifidobacteria...)

IMPAXOS


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

Documents

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