handouts presentatie seminar Buik en brein

1. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 1
Het effect van het microbioom op gedrag –
van wetenschap naar praktijk (en weer terug)
Koen Venema
Beneficial Microbes Consultancy, Wageningen, The
Netherlands, koen.venema@outlook.com

2. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 2
Logo Yakult
Vingerafdruk

3. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 3
• initiator and organisor of the
Beneficial Microbes Conference series
• editor-in-chief of the journal
Beneficial Microbes
• founder and CEO of
Beneficial Microbes Consultancy
2008
2010
2012
next: 10-12 Oct. 2016
2015

4. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 4
Waar gaan we het vandaag over hebben?

5. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 5
Tijdens deze gast lezing zal ik een uitleggen wat microbiota en
probiotica zijn en doen en tevens uitleggen waarom het nodig is om in
multidisciplinaire teams de rol van de microbiota en probiotica op
moderne ziektes en aandoeningen, zoals overgewicht, allergiën,
inflammatoire ziektes, autisme, gedrag, depressie, etc., te onderzoeken.
Verwacht een combi van een klein beetje hard-core science met enkele
interessante feiten en af en toe wat humor.
“If you don't like bacteria, you're on the wrong planet.”
― Stewart Brand
“Wanneer je al het meercellige leven van de aarde zou
wegvagen, zou het microbiële leven waarschijnlijk een
klein beetje verschuiven. Verdwijnt het microbiële
leven, dan betekent dat de onmiddelijke dood voor
onze planeet.”
― Carl Woese

6. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 6
Als kinderen geboren worden, komen ze uit
een relatief steriele omgeving van de
baarmoeder in een wereld vol met bacteriën…

7. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 7
Script van deel 1 van de presentatie
 Microbiota
 aandacht in de wetenschap
 rol van microbiota bij ziekte en gezondheid
 hoe ziet onze microbiota er nu uit?
 ontwikkeling microbiota van jong tot oud
 probiotica
 prebiotica
 feces transplantatie (repoopulate)
 niet alle probiotica zijn hetzelfde
 Take home messages deel 1

8. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 8
Microbiota in de (wetenschappelijke) pers

9. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 9

10. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 10

11. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 11
.... want de microbiota is betrokken bij:
- colon kanker
- inflammatory bowel disease (IBD)
- allergie (huid, luchtwegen)
- overgewicht
- type I en II diabetes
- levensduur
- prikkelbaar darm syndroom (IBS)
- angst en depressie
- brein ontwikkeling en cognitie
- autisme
- Parkinson’s
- Alzheimer’s
- .....
Probiotics and Prebiotics
Current Research and Future Trends
2015. Eds. K. Venema & A.P. do Carmo

12. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 12
Het aantal bacteriën in de darm is 10x zo veel als onze
eigen lichaamscellen
Volwassenen kunnen ~ 1 kg darmbacteriën met zich
meedragen
We deponeren per jaar ons eigen gewicht aan bacteriën
in het toilet
Als we 70 zijn komt dat overeen met 12 olifanten
> 50% van feces is bacteriën (natgewicht)
Elk persoon heeft meer darm bacteriën dan het aantal
mensen dat er ooit op deze planeet geleefd heeft
400 - >1000 species
“Soundbites”
Courtesy of Roderick Mackie / Glenn Gibson

13. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 13

14. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 14
“Exposed to a world of micro-organisms”
• Totale oppervlak van de darm is de grootte van een tennis court
• Dus dit moet goed verdedigd worden tegen indringers

15. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 15
Dit is waar uw lunch
nu is, met uw levende
bacteriele gasten

16. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 16

17. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 17
Bacteriën strijden om de aanhechting aan darm-epitheel

18. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 18
Als kinderen geboren worden, komen ze uit
een relatief steriele omgeving van de
baarmoeder in een wereld vol met bacteriën…

19. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 19

20. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 20
Moedermelk als een bron van gezonde bacteriën
Probiotica

21. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 21

22. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 22
microbiota
verstoring
microbiota
verstoring
ontsteking

23. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 23
Ziekte van Crohn
gereduceerde microbiele
genen diversiteit

24. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 24
Microbiota en overgewicht/obesitas

25. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 25
microbiota
verstoring
microbiota
verstoring
overgewicht

26. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 26
Kunnen we (een aantal van) die ziektes managen?

27. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 27
Probiotics: definition
live microorganisms which, when administered in
adequate amounts, confer a health benefit on the host
FAO/WHO 2001

28. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 28
De term probiotica mag sinds 14 Dec 2013
niet meer gebruikt worden

29. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 29
The “Health Claim Games”

30. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 30
The “Health Claim Games”

31. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 31
De wetenschappelijke wereld gebruikt de term
nog steeds per de definitie van FAO/WHO2001
De term probiotica mag sinds 14 Dec 2013
niet meer gebruikt worden

32. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 32

33. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 33
Hoe voorkom je
bijvoorbeeld
reizigers diarhee?

34. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 34

35. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 35
<105
106
107
108
109
CFU/ml
Niet alle probiotica zijn gelijk! Slide van Ger Rijkers
#55
#22
#36
0 1 2 3 4 5 6
Time (hrs)
Lactobacillus acidophilus

36. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 36
Hoe werken probiotica?
Hoe kunnen op het brein inwerken als ze in de darm zitten?
barrier/
mucosal
integrity
lumen/
resilience
immune system
dendritic cell
epithelial
cell
microbiota
probiotics

37. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 37

38. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 38

39. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 39
Hoe kies je dan het juiste probiotica?
Courtesy of Ger Rijkers

40. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 40
Hoe blijf je op de hoogte?
Welke publicaties zijn van goede kwaliteit?
Wat voor type bewijs is voldoende?

41. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 41
Graad A
Graad B
Graad C
Graad D
Aanbeveling op basis van bewijs
Systematische review of meta-analyse
(minstens) 1 RCT
(minstens) 1 goed opgezette en
gecontroleerde studie
(minstens) 1 cohort studie
Goed opgezette beschrijvende
studies, case control, case series
Verslagen van expert comitees,
klinishe ervaring van experts

42. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 42
Zijn DBPCRT altijd nodig?
Double-Blind Placebo-Controlled Randomized Trials
Courtesy of Eric Claassen

43. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 43
Zijn DBPCRT altijd nodig?
Double-Blind Placebo-Controlled Randomized Trials
DBPCRT leveren graad A bewijs en zijn het meest overtuigend....
.... als ze op de juiste manier uitgevoerd worden!

44. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 44

45. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 45
Prebiotics: definition
a selectively fermented ingredient *
that results in specific changes in
the composition and/or activity of
the gastrointestinal microbiota, thus
conferring benefit(s) upon host
health
* largely restricted to carbohydrates until now
Blatchford, Venema and others 2014, Intl J Probiotics Prebiotics 8(4)

46. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 46

47. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 47
Effect van probiotica op maagdarm ziektes

48. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 48
Effect van soort probiotica n

49. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 49

50. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 50
■ genome stability
Sybesma et al. AEM 2013

51. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 51
Antibiotica gebruik & C. difficile
C. difficile
bacterie

52. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 52
• C. difficile vaak ongevoelig voor antibiotica
• dat is juist de reden dat na een antibioticum kuur C.diff
uitgroeit
• en is tegelijk de reden dat een volgende antibioticum kuur niet
goed helpt
• veel patienten hebben recidiverende C.diff infecties
• soms/vaak zijn die levensbedreigend
• vaak is dan de enige optie nog .....
• FECES TRANSPLANTATIE
• cure-rate van ~90% !!
• geen medicijn is zo goed !
Antibiotica gebruik & C. difficile

53. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 53
Feces transplantaties.....
....

54. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 54
Graad A
Graad B
Graad C
Graad D

55. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 55
Do you (have to) believe what a company tells you?
Is makkelijker opschrijven in ENG dan NL 
Wanneer geen probiotica?
immunocompromised?
kritiek zieke mensen?
zeer jong kinderen?
ouderen met een verminderde
weerstand?
zwangere vrouwen?
In veel van deze gevallen worden probiotica al
gebruikt.

56. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 56

57. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 57

58. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 58
understatement
Bij veel van de hedendaagse problemen kunnen probiotica een
gezonde bijdrage leveren

59. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 59
Wrap up and
Take home messages

60. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 60
Conclusies Deel 1
• Probiotica werken! Maar het maakt
wel uit welke stam (of mengsel) je
kiest.

61. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 61
Script van deel 2 van de presentatie
 gut-brain axis
 rol van microbiota
 preclinische aanwijzingen
 klinische studies
 niet alle probiotica zijn hetzelfde
 Take home messages deel 2

62. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 62
.... want de microbiota is betrokken bij:
- colon kanker
- inflammatory bowel disease (IBD)
- allergie (huid, luchtwegen)
- overgewicht
- type I en II diabetes
- levensduur
- prikkelbaar darm syndroom (IBS)
- brein ontwikkeling en cognitie
- autisme
- .....
control rat autistic rat

63. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 63
Anxiety
BALB/c SPF mice w/o antibiotics
ATM = neomycin 5 mg/mL, bacitracin 5 mg/mL, and pimaricin 1.25 μg/mLAfter ATM: more explorative

64. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 64
Anxiety
BALB/c SPF mice w/o antibiotics
After ATM: biochemical
changes in the brain.
BDNF: Brain-derived neurotrophic factor,
a protein which is active in areas vital to
learning, memory, and higher thinking

65. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 65
Anxiety – microbiota transplant

66. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 66
Anxiety – microbiota transplant

67. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 67
Birth delivery mode alters anxiety-like behaviour (8-9 wks)
Original setup
Natural birth Cross-fostered C-section
marble bury test

68. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 68
Stress alters the
gut-brain axis
coritsol
cytokines
adrenocorticotropic hormone
serotonin (5-HT)
barrier function of the gut
Increased permeability
translocation of endotoxin
increased colonic transit
mucosal inflammation
changes in colonic morphology
etc.
Altered levels and metabolism of:
Altered:

69. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 69
Immune
O’Mahony et al. Psychopharmacology (2011)
Endocrine
Visceral Pain
Open Field Increased colonic transit, intestinal
permeability, mucosal inflammation, colonic
morphology (O’Malley et al., 2010, Stress)

70. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 70
Autism Spectrum Disorders
control rat autistic rat + PA
Single intracerebroventricular (ICV)
infusions (4 µL of 0.26 M solution over
4 min) of propionic acid (PA)

71. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 71
http://apc.ucc.ie
Desbonnet et al 2014

72. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 72
Social deficit after propionate infusion
control rat autistic rat + PA
Single intracerebroventricular (ICV)
infusions (4 µL of 0.26 M solution over
4 min) of propionic acid (PA)

73. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 73
Odds Ratio: 1.23
(1.07, 1.40)
1,191,920
participants,
12,897 of which
had ASD

74. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 74
Parkinson’s disease
Parkinson’s disease is the second most common
neurodegenerative disease and is characterized by region-
specific loss of dopaminergic neuromelanin-containing
neurons in the brain
Parkinson’s disease patients suffer from gastrointestinal
discomfort
nearly half of all Parkinson’s disease patients report long-
term constipation
Parkinson’s disease patients had a significantly increased
intestinal permeability
Future studies might elucidate further links between gut and
brain function in Parkinson’s disease, and should preferably
comprise investigations of the gut microbiota.
Pro- and/or prebiotics that strengthen the epithelial barrier
function might be beneficial to these patients

75. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 75
Amyloid protein precursor (APP) is a pivotal protein in the
formation of the plaque-inducing amyloid-β protein, which
causes neuronal degeneration in the brain of Alzheimer’s
disease patients
systemic administration of LPS in mice has been shown to
increase APP expression and caused neuronal cell death
significantly increased plasma levels of LPS in Alzheimer’s
disease patients compared with healthy controls were
detected, which might suggest that Alzheimer’s disease
patients suffer from elevated intestinal permeability
Pro and/or prebiotics that have an anti-inflammatory effect or
reduce bacterial and/or LPS translocation might be beneficial
for these patients
Alzheimer’s disease

76. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 76
Probiotic Alters GABA
Receptors in various
brain areas
Bravo et al., PNAS Sept 2011
L. rhamnosus JB-1 reduces anxiety and
behavioural despair

77. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 77
Defensive Marble Burying
Elevated Plus Maze
174 1205
Esc = escitalopram

78. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 78
Log10[Latency](s)
W
ater/Sal
W
ater/LPS
B
G
O
S/Sal
B
G
O
S/LPS
0.0
0.5
1.0
1.5
2.0
*
*
*
Timeinlight(s)
W
ater/Sal
W
ater/LPS
B
G
O
S/Sal
B
G
O
S/LPS
0
25
50
75
100
*
*
*
Prebiotic BGOS feeding is anxiolytic
less anxious

79. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 79
[BDNF](pg/mgprotien)
frontal cortex hippocampus
0
50
100
150
200 *water
FOS
GOS
Anxiolytic changes coincided with
changes in brain biochemistry, and....

80. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 80
0.0
0.2
0.4
0.6 *
*
[TNFa](pg/mgprotein)
0
2
4
6
8
** *
[IL1b](pg/mgprotein)
0.00
1.75
3.50
5.25
7.00
*
[IL6](pg/mgprotein)
0.0
3.5
7.0
10.5
14.0
[IL10](pg/mgprotein)
.... a reduction in inflammatory cytokines

81. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 81
Human trials

82. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 82
Human trials (1)
in order of publication date
• 124 healthy volunteers; mean age 61.8 years
• Double-blind, placebo (milk) controlled study
• Lactobacillus casei Shirota in milk-drink for 3
weeks
• Mood and cognition were measured at baseline,
and after 10 and 20 days of consumption.

83. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 83
Human trials (1); LcS
POMS = profile of mood state
• An unexpected finding was that
the consumption of probiotics
resulted in a slightly-poorer
performance on two measures
of memory.

84. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 84
• 31 chronic fatigue syndrome patients
• RDBPC study
• Lactobacillus casei Shirota in sachet-format for 8
weeks
• Beck Depression Inventory (BDI) and the Beck
Anxiety Inventory (BAI) at baseline and after 8 w.
Human trials (2)

85. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 85
Human trials (2); LcS
• there was a significant improvement in anxiety among those taking the
active LcS compared to the placebo. The differences as assessed by the
BDI did not reach statistical significance.
BDI: a 21-question multiple-choice self-report inventory, one of the most widely used psychometric tests for
measuring the severity of depression
BAI: is a 21-question multiple-choice self-report inventory that is used for measuring the severity of anxiety in
children and adults

86. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 86
• 55 volunteers with a score of ≤12 in the HADS-
anxiety subscale and in the HADS-depression
subscale and ≤ 20 in the HADS total score on initial
examination
• RDBPC study
• L.helveticus R0052 + B. longum R0175 in a stick
format, or placebo (xylitol, maltodextrin, plum
flavour and malic acid) for 30 days
• Tests: HSCL-90; HADS; PSS, CCL
• Urinary cortisol
Human trials (3)

87. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 87
Human trials (3); L.h R0052 / B.l R0175
Hopkins Symptom Checklist-90.The HSCL-90 is a 90-item self-reported
multidimensional questionnaire screening a broad range of psychopathological
disorders
Hospital Anxiety and Depression Scale: The HADS, is a 14-item self-assessment
instrument, often applied and convenient for measuring psychological distress in
subjects with somatic or psychosomatic disorders
Perceived Stress Scale: The PSS is a 14-item self-reported questionnaire
assessing the degree to which recent life situations are appraised as stressful
Coping Checklist: The CCL is a validated 29-item questionnaire measuring five
types of coping strategies when confronting an adverse event: ‘problem solving’;
‘avoidance with wishful thinking’; ‘seeks social support’; ‘positive re-evaluation’;
‘self-blamed’.

88. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 88
Human trials (3); L.h R0052 / B.l R0175
Probiotics-treated subjects had a lower global severity index of the HSCL-90 over
time, due to lower values for somatisation, depression and anger–hostility.
The potential usefulness of the probiotics as an anti-stress/anti-anxiety agent was
supported by diminished HADS global scores over time.
Taken together, probiotics appears to show a beneficial effect on general signs of
anxiety and depression, but this did not generalise to the PSS, although all three
tests comprise self-reported measures.
In the CCL test the two groups differed in emotional reactivity, with subjects
administered probiotics reducing their self-blame score.
L. helveticus R0052 and B. longum R0175 taken in combination display
beneficial psychological effects in healthy human volunteers

89. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 89
• Same study as the previous one, but now in a
subpopulation of 25 subjects with urinary free
cortisol (UFC) levels less than 50 ng/ml at baseline
• More scores were beneficially affected by the
probiotics in this subpopulation
Human trials (3a)

90. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 90
• 36 healthy women, age 30 y +/- 10.4
• RDBPC study
• B. animalis subsp lactis DN-173 010, Streptococcus
thermophilus CNCM I-1630, 2 strains of L. bulgaricus,
CNCM I-1632 and CNCM I-1519 and Lactococcus lactis
subsp lactis CNCM I-1631fermented milk, non-
fermented milk or no intervention for 4 weeks
• functional magnetic resonance imaging before and after
the intervention to measure brain response to an
emotional faces attention task and resting brain activity.
Human trials (4)

91. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 91
Human trials (4)
A fermented milk product with probiotic resulted in robust alterations in the
response of a widely distributed brain network to a validated task probing
attention to negative context, which have been associated with the dampening
of emotional and sensory responsiveness.

92. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 92
• 44 university students who performed strenuous
exercise daily
• RDBPC study
• Lactobacillus gasseri OLL2809, OLL2809 + α-
lactalbumin, or placebo for 4 weeks
• Profile of Mood States (POMS) questionnaire
Human trials (5)

93. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 93
Human trials (5)
LG2809 ingestion was effective elevating mood from a depressed state.

94. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 94
• 224 healthy volunteers
• RDBPC study
• yogurt containing L. gasseri SBT2055 and B.
longum SBT2928, or control yoghurt for 12 weeks
• levels of two stress-induced hormones,
adrenocorticotrophic hormone (ACTH) and cortisol
Human trials (6)

95. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 95
Human trials (6)
These results suggest that the probioticcontaining test yogurt confers health
benefits by alleviating stress.

96. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 96
• 50 healthy participants, mean age 23.22 years
• RDBPC study
• two strains of L. acidophilus CUL60 and CUL21,
B.lactis CUL34 and B.bifidum CUL20 in a capsule, or
placebo for 6 weeks
• anxiety levels decreased in the probiotic condition
whilst increasing in the placebo condition (p=0.042).
• ‘continuity of attention’ increased in response to the
probiotic treatment and decreased with the placebo
• group (p=0.035)
Human trials (7)
Abstract

97. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 97
• 75 infants
• RDBPC study
• L. rhamnosus GG or placebo during the first 6 months
of life
• attention deficit hyperactivity disorder (ADHD) and
Asperger syndrome (AS)
• At the age of 13 y, ADHD or AS was diagnosed in 6/35
(17.1%) children in the placebo and none in the
probiotic group (P = 0.008).
Human trials (8)

98. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 98
• 40 healthy participants without current mood disorder
• RTBPC study
• a multispecies probiotic containing B. bifidum W23,
B.lactis W52, L.acidophilus W37, L.brevis W63,
L.casei W56, L.salivarius W24, and 2 strains of
Lactococcus lactis W19 and W58 in a sachet, or
placebo
• cognitive reactivity to sad mood was assessed using
the revised Leiden index of depression sensitivity
scale
Human trials (9)

99. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 99
Human trials (9)
The LEIDS-r is a self-reported
questionnaire with 34 items that
assesses to what extent
dysfunctional thoughts are
activated when experiencing mild
dysphoria
(i.e., it measures cognitive
reactivity to sad mood, also
referred to as vulnerability to
depression).
Rumination is the compulsively focused attention on the
symptoms of one's distress, and on its possible causes
and consequences, as opposed to its solutions.

100. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 100
• 30 patients with laryngeal cancer
• RDBPC study
• Unknown strain of Clostridium butyricum for 2
weeks, twice a day in a capsule, or placebo
• Serum corticotropin-releasing factor
• The degree of anxiety assessed by the Hamilton
Anxiety Scale (HAMA)
Human trials (10)

101. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 101
Human trials (10) m
Probiotics can ameliorate the clinical anxiety and biochemical features
of stress in patients scheduled for laryngectomy (and other surgery?)

102. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 102
• 45 healthy medical students under academic
examination stress
• RDBPC study
• L.casei Shirota fermented milk, or placebo for 8
weeks prior to taking national exam
• Subjective anxiety scores
• Salivary cortisol levels
• The presence of physical symptoms
Human trials (11)
[Epub ahead of print]

103. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 103
Human trials (11)
LcS may prevent hypersecretion of cortisol and physical symptoms (not shown
in this presentation) under stressful conditions, possibly through vagal afferent
signaling to the brain and reduced stress reactivity in he brain (the latter based
on a parallel rat study)

104. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 104
• 45 healthy volunteers with no history of psychiatric
disorders
• Double-blind, placebo (maltodextrin) controlled
study
• FOS, Bimuno®-galacto-oligosaccharides, or
maltodextrin, daily for 3 weeks
• Awakening salivary cortisol measured on day 0 and
day 21
• Emotional test battery on day 21
Human trials (12); prebiotic

105. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 105
Dot-probe task:
Attention to positive
stimuli is increased
BGOS reduces awakening cortisol and increases attention to positive
stimuli in healthy volunteers
Human trials (12); prebiotic

106. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 106
Bepalen onze bacteriën in de darm wat we in de supermarkt uit de
schappen halen?

107. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 107
Other things that could not be
covered in this presentation
The gut microbiota produces other neuromodulatory molecules, such as
serotonin, GABA, melatonin
The gut microbiota is involved in brain development and cognition
The gut microbiota affects the blood brain barrier
Since pro- & prebiotics modulate the gut microbiota composition and
activity, they may also be involved in happiness, trustworthiness,
depression, sleeping, etc. … **
… in addition to anxiety, stress and autism
There is still room for mouse-models to study these gut microbiota-brain
interactions
** time for an anekdote?

108. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 108
Wrap up and
Take home messages

109. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 109
Conclusions Part 2
• The gut microbiota is involved in brain functioning
• Pro-, pre- (and synbiotics) modulate the gut microbiota and
thereby can influence brain functioning as well
• Although mostly evidenced in animal studies, some clinical trials
show very promising results

110. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 110
Script van deel 3 van de presentatie
 dynamische, computer gestuurd in vitro model
van het maag-darm kanaal
 TIM-1
 TIM-2
 modulatie van de microbiota samenstelling en
activiteit
 mogelijkheid om te kijken naar parameters,
microorganismen, dieet componenten die gedrag
beinvloeden
 Take home messages deel 3

111. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 111
A model to simulate the GI tract ?
requirements for such models
• simulating successive dynamics
conditions
• sampling in time at different intestinal
segments
• validation in vitro versus in vivo
In the late 1980’s, TNO started the development of the TNO
dynamic in vitro models of the GI tract, nick-named TIM

112. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 112
Successive dynamic conditions in the GI tract
peristalsis, gastric acid, salivary and gastric
enzymes, gastric emptying
secretion of digestive enzymes, bile, intestinal
transit time
absorption of (digestion) products and water
cecum and colon transit
dense active microbiota, microbial metabolites
biotransformation enzymes

113. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 113
TIM-1
gastric-small-intestinal model
1. stomach 6. gastric secretion
2. duodenum 7. intestinal
secretion
3. jejunum
4. ileum
5. ileo-caecal valve
8. pH electrodes
9. prefilter
10 absorption system
1
2
3
4
5
9
10
9
10
7
6
8
8
8
8

114. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 114
Schematic view of TIM-1 with sampling ports

115. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 115
Peristaltic movements Gastro-intestinal transit

116. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 116
Stomach and ileal delivery and pH curves
0
20
40
60
80
100
120
0 1 2 3 4 5 6
time (h)
pH
adult liquid
adult semi-solid
baby
0
2
4
6
8
10
0 60 120 180 240 300 360
time (min)
gastric pH
duodenal pH
jejunal pH
ileal pH
mealdelivery(%ofintake)

117. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 117
TNO’s in vitro model of the colon (TIM-2)
1. colon compartment; 2. inlet ileum effluent;
3. level sensor; 4. pH control; 5a.dialysate;
5b. membrane; 6. sampling / colon effluent;
7. nitrogen flush.

118. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 118
Schematic view of the large-intestinal model

119. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 119
Physiological concentrations of metabolites
0
100
200
300
400
0 1 2 3 4 5 6 7 8 9
SCFA(mmol/L)
Time (hours)
dialysis on
dialysis off
Physiological
range
TIM-2 dialysis system – unique and required

120. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 120

121. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 121
human models: babies, adults, elderly
healthy vs. patients
average vs. extremes
pig model
pre-ruminant calf model
dog model (FIDO)
TIM-systems available at UM

122. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 122
human models: babies, adults, elderly
healthy vs. patients
average vs. extremes
pig model
pre-ruminant calf model
dog model (FIDO)
TIM-systems (soon) available at UM

123. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 123
Validation of the TIM-systems for food studies
TIM-1 has been validated with respect to
protein digestion
carbohydrate digestion
fat digestion
survival of probiotic microorganisms
bioaccessability of fat-soluble vitamins
TIM-2 has been validated with respect to
the composition of the microbiota
the metabolic acitivity of the microbiota

124. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 124
Validation survival LAB during GI passage
in vitro (i.v.) versus humans (h) J. Dairy Sci. 80:1031, 1997
0
5
10
15
20
25
30
35
0 60 120 180 240 300 360
time (min)
cumulativedelivery(%ofintake)
Bif. (i.v.)
Bif. (h)
L.ac (i.v.)
L.ac (h)

125. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 125
Mäkivuokko et al., MEHD 2012 - 23: 28-34
TIM-1
Stomach and small intestine
model

126. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 126

127. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 127concept of intestinal flora (microbiota) is old, but little is known about it
Hieronymous Bosch (ca.1450–1516) - Garden of earthly delights
painted in 1503-1504; on display in Museo del Prado in Madrid

128. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 128
Saccharolytic and proteolytic activity
• Saccharolysis leads primarily to SCFA and gasses
• SCFA: acetate, propionate, butyrate
• lactate -> only accumulates when there
is a fast fermentation
• CO2, CH4, H2
• Proteolysis in addition leads to toxic metabolites
• BCFA: iso-butyrate, iso-valerate
• ammonia
• phenolics: phenol, indol, p-cresol, skatol
• H2S, CH3SH, etc

129. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 129
Colonic metabolites
some examples
• Butyrate:
• 70% of the fuel of colonocytes
• lack -> epithelial atrophy
• reduces cancer promotion in transformed tissue
• possibly reverses transformation by induction of
differentiation
• H2S, CH3SH:
• block butyrate oxidation
• ammonia:
• liver cirrhosis
• etc.

130. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 130
Prolonged carbohydrate fermentation
proximal colon: primarily saccharolytic
fermentation
transverse colon: combination of saccharolytic
and proteolytic fermentation
distal colon: primarily proteolytic fermentation
distal colon: colon cancer and IBD

131. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 131
Experiments were done in TNO’s in vitro model of the colon (TIM-2):
• healthy adult microbiota
• average parameters in the proximal colon
• standardized conditions

132. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 132
BCFA production
BCFA control
0
2
4
6
8
0 20 40 60 80 100 120
time (hrs)
mmol
i-butyrate
i-valerate
BCFA inulin
0
2
4
6
8
0 20 40 60 80 100 120
time (hrs)
mmol
BCFA control + Clostridium difficile
0
2
4
6
8
0 20 40 60 80 100 120
time (hrs)
mmol
BCFA inulin + Clostridium difficile
0
2
4
6
8
0 20 40 60 80 100 120
time (hrs)
mmol
Van Nuenen et al., MEHD 2003 15: 137-144

133. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 133
Phenolics production
0
200
400
600
phenol p-cresol indol skatol
p-cresol/indol(μmol/l)
10*phenol(μmol/l)
control
inulin
Van Nuenen et al., MEHD 2003 15: 137-144

134. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 134
DNA micro-array (I-chip)

135. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 135
TIM-2 results; FOS/inulin
TNO I-Chip close to 400 probes
5-40 fold
Maathuis et al., AJCN 2010; Rose et al., BJN 2010

136. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 136
• Many unknown Oligosaccharides gradually degraded in 6 hours
2.2 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 52.8
Time [min]
-17.5
-15.0
-10.0
-5.0
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
46.7
min
nC
Response
TIM t 0h
TIM t 15min
TIM t 2h
TIM t 6h
TIM t 24
Quite slow
fermentable fiber,
thus may reach
the distal colon.
Oligosaccharide profile of TIM-2 fermentation
Fibersol-2® over 24 hours – with HPAEC

137. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 137
Oligosaccharide profile of TIM S11 (beta-glucan)
over 24 hours – with high performance anion exchange
chromatographie
TIM t0
TIM t 0:15
TIM t1
TIM t2
TIM t4
TIM t6
TIM t24
Oligosaccharide profile of TIM-2 fermentation of
beta-glucan over 24 hours – with HPAEC

138. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 138
Fermentation of beta-glucan leads to increase in
immunomodulation – BMDCs from TLR2/4 KO mice

139. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 139
pectin
branched arabinan linear arabinan
IL-10
IL-4
IL-12
MCP-1
LA BA -C +C
Fermentation of pectin leads to immunomodulatory
structures – from BA to LA

140. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 140
How to study the gut microbiota

141. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 141
Limited accessibility of the colon

142. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 142
Tool 2: Carbohydrates labeled with stable isotopes
Stable (not radioactive) isotope 13C
Substrates were uniformly labeled (each 12C -> 13C)
Different isotopes follow the same biochemical route
with no preference for one isotope over the other *
Due to differences in mass between the different
isotopes, they can be traced in microbial metabolism
* We have some evidence that this may not be entirely true
isotope:
natural
abundance:

143. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 143
Incubation
13C-labeled
substrate
13C ends up in
microbial metabolites
13C ends up in
microbial biomass
(incl. DNA/RNA)
Principle: Use of stable isotopes
De Graaf and Venema, AMP 2008
using various 13C-labeled
substrates: inulin, starch,
lactose, GOS, 6’-SL

144. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 144
[U-13C]Glc
CO2
intracellular extracellular
Protein
& Fatty
acids
VEMP
VFHL
VWLP
VLDH
VACK
VLACt
VFORt
VACt
VPYR
VACCOA
Pyr
AcCoA
AcAcetylCoA
Lactate
Acetate
CO2
Butyrate
Formate
CO2
Acetate
Lactate
Formate
Acetate
Butyrate
VPFL
H2
VPDH
VBUK
VBUt
VLACex
VFORex
VACex
VBUex
89.1 mmol/min
82.7
16.0
54.5
209.0
48.4
162.6
35.799.3
176.4
208.2
99.3
155.1
7.5
209.0
48.4
97.2
Unlabeled
substrates
13C labeled
substrate
> 90% reversibility
130
Computer model:
Flux within intestinal microbiota

145. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 145
Baby microbiota:
- No propionate (C3) on FOS
and GOS
- No butyrate (C4) on GOS
- Succinate (Succ) on both
substrates
Elderly microbiota:
- No Succ on either substrate
- Alanine (Ala) on both
Both microbiotas:
- Ethanol (EtOH) production
- Differences in the amount of
acetate (C2), butyrate (C4)
and lactate (Lac)
Summer Course Glycosciences (12th European Training Course on Carbohydrates) 3-7 June 2012 Groningen, The Netherlands © TNO 145
FOS/GOS Comparison - 2D NMR t=4h

146. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 146
Incubation
13C-labeled
substrate
13C ends up in
microbial metabolites
13C ends up in
microbial biomass
(incl. DNA/RNA)
Principle: Use of stable isotopes
De Graaf and Venema, AMP 2008

147. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 147
16S rRNA Stable Isotope Probing (SIP)
Incubation RNA
extraction
Density gradient
ultracentrifugation
(CsTFA)
13C-labeled
substrate
Gradient
fractionation Molecular analyses heavy
vs. light fractions
 16S rRNA - based molecular
fingerprinting of microbial
diversity
 Cloning and sequencing of
16S rRNA
De Graaf and Venema, AMP 2008

148. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 148
1
3
5
7
t0
t1
t2
t4
t8
0
25
50
ratio(%)
fractiontime(h)
Bifidobacterium bifidum
t0
t1
t2
t4
t8
SIP after 13C-GOS feeding
Maathuis et al., J Nutr, in press
micro-array analysis

149. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 149
Determination of caloric value
of fermented carbs
• 1 gram of inulin has a caloric value of 2.05 kcal
• similarly, 1 gram of lactose has a caloric value of 2.41 kcal
1 gram of starch 1.69 kcal
• Therefore, depending on the fermentable substrate, the
energy-harvest of the body is different ----> link of
microbiota with obesity
Venema (2010) Curr Opin Clin Nutr Metab Care
- implication for obesity

150. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 150
Obesity and microbiota - enterotypes
Current TIM-2
pooled microbiota
TIM-2 of the future
1. Microbiota of lean en
obese individuals
2. Microbiota of 3
enterotypes

151. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 151
L = lean
O = obese
Not in all cases obese (O) ↑ than lean (L)
GOS: energy extraction equal, despite clear differences in SCFA and lactate production
Lactulose: highest production, plus O ↑ L
Pectins: L ↑↑ than O
Adult microbiota
874 kJ/mol 1536 kJ/mol 2192 kJ/mol 1364 kJ/mol

152. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 152
[U-13C]Glc
CO2
intracellular extracellular
VEMP
VFHL
VWLP
VLDH
VACK
VLACt
VFORt
VACt
VPYR
VACCOA
Pyr
AcCoA
AcAcetylCoA
Lactate
Acetate
CO2
Butyrate
Formate
CO2
Acetate
Lactate
Formate
Acetate
Butyrate
VPFL
H2
VPDH
VBUK
VBUt
VLACex
VFORex
VACex
VBUex
89.1mmol/min
82.7
16.0
54.5
209.0
48.4
162.6
35.799.3
176.4
208.2
99.3
155.1
7.5
209.0
48.4
97.2
13C labeled substrate
130
microbiota host
Host – microbe interaction :
relevance for (gut) health

153. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 153
diet
fermentation by
gut microbiota
microbial
metabolites
tissue and organ
metabolism
host physiological
response

154. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 154
human feeding trials @
Maastricht University hospital
Substrate delivery
via a nasal tube
Colon sampling back
through catheter
Plasma sampling
Breath sampling
GC and LC-MS
analyses
13C

155. Yakult seminar “Gut – brain – microbiota” – Almere – 09-Jun-2016 © Beneficial Microbes Consultancy 155
First experiments in humans with lactose!!
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240
-50
0
50
100
150
200
250
individual 213
C-CO2
H2
13
C-glucose
-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240
deltaplasmaglucose
deltabreathCO2
breathhydrogen(ppm)
- no 13C-glucose in plasma
- increase in13C-CO2 and H2 in breath
- increase in 13C-acetate in plasma
fermentation of 13C-lactose in colon

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Wrap up and
Take home messages

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Limitations of the in vitro models
no feed-back mechanism
except pH and volume
no mucosal cells
no local or humoral immune system

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Combination with biological models
intestinal segments in transport chambers
various gastrointestinal areas
various species, including humans
cultured mucosal cells e.g. Caco-2, HT29
cultured immunocompetent cells

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Conclusions Part 3
• Validated in vitro models of the gastrointestinal tract are
excellent tools to study the functionality of pro- & prebiotics,
and effects of these and other dietary factors on the gut
microbiota.
• For more information:
koen.venema@outlook.com
k.venema@maastrichtuniversity.nl

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Acknowledgements
• Henrike Hamer
• Steven Vanhoutvin
• Freddy Troost
• Daisy Jonkers
• Robert-Jan Brummer
• Tao He
• Roel Vonk
April 20, 2011TIFN Satiety
screenings
study
160
Dank u hartelijk voor uw aandacht!