Understanding prebiotics and scientific discoveries furthering
their development Karen P. Scott
Microbiology Group
Rowett Institute of Nutrition and Health
What are synbiotics, prebiotics, probiotics The gut microbiota Can we use prebiotics to improve health? Are Prebiotics the most effective approach to improving health?
CONTENT
Definitions
• Probiotics
live microorganisms which, when administered in adequate amounts confer a health benefit on the host (FAO/WHO 2001)
• Prebiotics
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. (Gibson et al. 2010)
Synbiotics
mixtures of probiotics and prebiotics that beneficially affect the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinal tract (Andersson et al 2001)
Definitions
• Probiotics
Consumption of live bacteria
• Prebiotics
Food for bacteria that are already in large intestine
Synbiotics
mixtures of live bacteria and substrate that can help them (and resident bacteria) grow and survive
Prebiotics
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.
Must reach the large intestine
undigested by host enzymes
Must only be fermented by
one/few colonic
bacteria, not by all bacteria
OR
Must change the balance of
fermentation products
Must be a ‘measurable’
health benefit
Definition Description Example
Fructooligosaccharides
(FOS)
Increases numbers
of Bifidobacteria
Increases butyrate
production
Eg increases Ca
absorption
Prebiotics
Accepted examples of prebiotics
Fructo-oligosaccharides (FOS)
Galacto-oligosaccharides (GOS)
Inulin
Candidate prebiotics
Lactulose
Human milk oligosaccharides
(HMOs)
Arabinoxylan
Resistant starch
Occurrence
FOS/inulin occur naturally in some foods
Chicory, garlic, artichoke, leek
Increase amounts by supplementation
cereals, bread, yoghurts,etc
Use in infant formula/foods
(FOS and GOS)
Target (adult) intake: 2 – 20g/day
Health benefits
Change bacterial growth/activity
Stool bulk/transit time
BUT current lack of real biomarker for
GI health
Garlic (9-16%) Leeks
(3-10%)
Onion (2-6%)
What are synbiotics, prebiotics, probiotics The gut microbiota Can we use prebiotics to improve health? Are Prebiotics the most effective approach to improving health?
CONTENT
Human Gut Microbiota
Colon
1010 - 1012/ ml gut contents (large intestine)
Outnumber human cells in the body by 10 : 1
Complex and diverse bacterial community
>500 hundred different bacterial species colonise each individual
Most are highly oxygen sensitive, but can be cultured
Molecular approaches (mainly based on 16S ribosomal genes) allow analysis of bacterial communities without cultivation
Until recently >70 % of ‘phylotypes’ belonged to no known cultured species
Faecalibacterium prausnitzii
Eubacterium rectale
Colinsella aerofaciens
Clostridium clostridioforme
Bacteroides vulgatus
Anaerostipes hadrus
Ruminococcus bromii
Eubacterium hallii
Blautia wexleri
Bacteroides dorei
Roseburia faecis
Dorea longicatena
Subdoligranulum variabile
Bacteroides uniformis
Ruminococcus obeum
Bacteroides ovatus
Blautia luti
Parabacteroides distasonis
sp nov A2-166
sp nov SR1/5
Lachnospira pectinoschiza
sp nov 80/3
Dialister invisus
Roseburia inulinivorans
Ruminococcus callidus
othersData from 6 healthy, obese individuals; converted from Walker et al 2011, ISME J
Dominant bacterial phylotypes
~50% of sequences correspond to 25 most abundant species We have cultured representatives of all of them
XIVa, E.rectale/Roseburia group
Bacteroidetes
IV, Ruminococcus group
295 additional
phylotypes
8% 5%
2%
0.6%
IV, F.prausnitzii group
major
butyrate
producers
Major propionate
producers
Major lactate
utilisers
(into butyrate)
Top 25 range of products
Food
Small intestine* large intestine
Digestion of dietary carbohydrate,
starches, sugars, fat, protein
Resistant Starch, Fibre (NSP), prebiotics
SCFA, gases, phytochemicals,
other metabolites, minerals
Blood stream
faeces
Monosaccharides, fat, aminoacids,
soluble phytochemicals
absorption
Undigested carbohydrate, Lignin, unabsorbed nutrients
Proximal Distal
SCFA concentration gradient (acetate, butyrate, propionate)
[Adapted from Topping & Clifton, 2001 Physiol Rev. 81; 1031-1064]
Fate of dietary products in the GIT
Transported in blood to
peripheral tissues,
used in lipogenesis
Important for
gluconeogenesis
in the liver;
satiety signalling
Major energy source
for human colonocytes;
Protects against cancer
Disease Microbial association Therapy target
or substrate
Scientific evidence
IBS Altered diversity
(inc Clostridium;
Dec Bifido, Fprau)
Inc or dec fibre
intake
(eg inc starch)
variable
IBD Reduced diversity (inc proteo/enterobacteria;
dec Lachnospiraceae,
cluster IV)
Colorectal cancer Inc Fusobacterium, E.
coli
dietary fibre
prebiotics
Alter fermentation, faecal
bulking, reduced transit time
Coronary heart
disease
Lower serum
cholesterol;
Inc β-glucan
Reduce cholesterol, BP
Obesity Reduced diversity Appetite regulation ?
Diabetes Inc opportunist pathogens,
dec butyrate producers prebiotics Modify glycemic and insulin
response
Osteoporosis Inc colonic pH Prebiotics Lower pH, increase Ca
absorption
Diseases where dysbiosis of the gut microbiota has
been noted (and opportunities for prebiotics)
The microbial community and disease : IBS
Irritable bowel syndrome (IBS) – complex disease, different manifestations
Increased fibre intake helps some sufferers, increases symptoms in others
more variability between IBS-C individuals than between healthy volunteers.
PCA of FISH data for background samples Significantly less Roseburia and Bifidobacteria species in IBS:healthy
Chassard et al Aliment Pharm Ther. 2012, 35; 828-838
7.5
8
8.5
9
9.5
10
10.5
0.5 1 1.5 2 2.5 3 3.5
1=Back; 2=Dextrose; 3=Starch
log
Rre
c
5
5.5
6
6.5
7
7.5
8
8.5
9
9.5
10
0.5 1 1.5 2 2.5 3 3.5
1=Back; 2=Dextrose; 3=Starch
log
Bif
E.rectale/Roseburia group p=0.006
Blue – healthy Red – IBS
Bifidobacteria p=0.029
baseline dextrose starch
baseline dextrose starch
What are synbiotics, prebiotics, probiotics The gut microbiota Can we use prebiotics to improve health? Are Prebiotics the most effective approach to improving health?
CONTENT
Health benefits attributed to prebiotics
Improved gut function
stool bulking and faster gut transit
Management of Inflammatory bowel disease
reduction in inflammatory markers
Prebiotics may reduce the risk of colon cancer
reduced DNA damage and cell proliferation, faster gut transit
Prebiotic (GOS/FOS) supplementation of infant formula increases
bifidobacteria numbers
Prebiotics (FOS/Inulin) increase calcium absorption and bone health
pH effect
Consumption of prebiotics instead of sugars can lower glycaemic index induce a lower blood glucose rise after meals compared to those containing sugars
Rodent studies indicate that prebiotics reduce food intake and fat mass
SCFA stimulated secretion of gut peptides
(PYY, GLP-1, reduced ghrelin) more work required
?
Jan 2014 – EFSA preliminary approval of health claim
Inulin/FOS can lower glycaemic index
“consumption of foods containing non-digestible carbohydrates instead of
sugars induces a lower blood glucose rise after meals compared to those
containing sugars”
Jan 2014 – EFSA preliminary approval of health claim
Increased consumption of Native chicory inulin can
increase stool frequency
Galacto-oligosaccharides (GOS)
GOS are produced from lactose
Gal Glu Gal Glu Gal Glu n
Glu
β-galactosidase
Lactose Lactose Galacto-
Oligosaccharides (n = 1 – 7)
Glucose
GOS stimulate growth of Bifidobacteria
9 studies in healthy adults between 1990 – 2004
2 studies also showed increase in Lactobacilli
8 studies in infants comparing GOS formula with normal formula
2 studies also showed increase in Lactobacilli
Applications – improve infant formula to be more representative of breast milk
- use to improve constipation and increase Bifidobacteria
populations in geriatrics
More recent studies using GOS prebiotic
supplementation
Prevents decrease in bifidobacteria population and butyrate production
otherwise shown following amoxicillin treatment
Ladirat SE et al BJN 2014
Reduced colic incidence in formula fed infants, increased bifidobacteria
(mimics effect of human milk)
Giovannini et al J Am Coll Nut 2014
Increases Ca absorption and bifidobacteria counts in adolescent girls
Whisner et al 2013 BJN
Increases bifidobacteria in healthy adults – but response is individual-specific
Responders and non-responders
Davis LMG 2011 PLosOne
- Bacterial effects mainly focus on Bifidobacteria
Dietary supplementation with ‘traditional’ prebiotics
Many studies have shown that fructo-oligosaccharides (FOS) and inulin
stimulate numbers of Bifidobacteria = Bifidogenic
BUT Bifidobacteria produce lactate, and prebiotics stimulate butyrate
production
HOW?
2 possible mechanisms
1. additional direct stimulation
of butyrate butyrate producers
2. Bacterial cross-feeding
Fructans, β(1-2) fructose polymer
FOS n= 2-8 (DP)
inulin 9<n<60
0
10
20
30
40
50
Bact Cl XIVa Rrec2 Rrec1 Ehal Cl IV Fprau Rum CoAT Bifids
% o
f u
niv
ers
al 1
6S
rR
NA
ge
ne
co
pie
s
background
control
inulin
Effect of prebiotics (inulin) on the faecal microbiota
Cluster XIVa Cluster IV
P = 0.005
P = 0.027
Q-PCR quantification of dominant groups of the human gut microbiota
Stimulation of bifidobacteria and Faecalibacterium prausnitzii –
produces butyrate Fuller et al, BJN (2007); Ramirez et al, BJN (2009)
Commensal anaerobes use prebiotic substrates for growth
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
YCFA basal
Starch
P95 FOS
Synergy
HP
Dahlia inulin
XOS
0
0.2
0.4
0.6
0.8
1
1.2
Bi. infantis E. rectale R.inulinivorans
R. faecis
P95 FOS
synergy
HP
dahlia
As complexity of substrate
No. of bacteria utilising it
Structure of prebiotic important
Scott KP et al FEMS Micro Ecol 2014
Starch/FOS
Acetate L-Lactate Formate
Eubacterium
hallii
Bacterial cross-feeding
Acetate
Lactate
Formate
Butyrate
0
2
4
6
8
10
12
18
20
Bif. adol.
E. hallii
16
14
SC
FA
co
nce
ntr
ati
on
[m
M]
Duncan SH et al, AEM, 2004
Bifidobacterium
adolescentis
Gut bacteria exist in a complex mixed ecosystem
Starch/FOS
Acetate L-Lactate Formate
Eubacterium
hallii
Butyrate
cross-feeding
Acetate
Lactate
Formate
Butyrate
0
2
4
6
8
10
12
18
20
Bif. adol. Bif. adol.
E. hallii E. hallii
16
14
SC
FA
co
nce
ntr
ati
on
[m
M]
(Duncan et al, AEM, 2004;
Belenguer et al, AEM 2006)
Bifidobacterium
adolescentis
Bacterial cross-feeding
Gut bacteria exist in a complex mixed ecosystem
Duncan SH et al, AEM, 2004
Belenguer et al, AEM 2006
Increase in Bifidobacteria
(Resistant starch) FOS INULIN
Direct utilisation by butyrate producers
[Butyrate]
Mechanisms by which prebiotics increase butyrate
production?
[Lactate] [Acetate]
Utilisation of intermediate
products: CROSS-FEEDING
Prebiotics and butyrate – 2 routes for production
Synbiotic = probiotic plus prebiotic
Combination critical - Probiotic bacterium should be able to use prebiotic as
a growth substrate – can help survival and establishment
Is a prebiotic best given along with a probiotic bacterium
- as a synbiotic?
Lactitol and Lb. acidophilus in elderly Increase in Lb. acidophilus and total Bifidobacteria
synergy 1(FOS) plus B. animalis Increased population of total bifidobacteria,
Mix of 4 bacteria plus FOS reduced upper GIT infection in intensive care patients
Different mix of 4 bacteria with 4 prebiotics –reduced post-operative infections following liver transplants (3%
compared to 48%)
Synbiotic therapy (GOS + B. breve + Lb. casei) significantly improved numbers of beneficial bacteria (Bifids
and Lactobacilli), and decreased the number of harmful bacteria in elderly patients undergoing
gastroenterological surgery
Synbiotic consumption (B. longum plus Synergy1) improved clinical symptoms in Crohns disease patients
Distinguishing responders : non-responders is an important factor for
consideration in all prebiotic/probiotic/synbiotic studies
Volunteers with the lowest initial levels of bifidobacteria gave the
maximum increase in bifidobacteria numbers after inulin
supplementation
Ch
an
ge
in
bifid
ob
acte
ria
(lo
g1
0 c
ells
/g fa
ece
s)
aft
er
tre
atm
ent
Bifidobacteria (log10 cells/g faeces)
at start of study
Prebiotic effect of fruit and vegetable shots containing Jerusalem artichoke
inulin: a human intervention study P. Ramnani, et al BJN (2010), 104, 233–240
Background levels of host bacteria critical in level of response
Greatest change in
Bifidobacteria numbers in
individuals with lowest starting
populations
Increasing carbohydrate content of diet has a prebiotic effect –
Increases numbers of Roseburia group and stimulates
butyrate production
- is this selective fermentation ?
Increasing RS III component of diet has a prebiotic effect –
selective fermentation, increasing numbers of
R. bromii and E. rectale
- what is benefit on host health ?
What makes a prebiotic? – not always easy to answer
Prebiotics
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. (Gibson et al. 2010)
Identifying new prebiotics
Is there a role for new prebiotics to specifically modify the composition of the gut microbiota and improve health?
Targeted approach :
Microbiome studies identify bacteria lacking in disease states Genomic studies identify substrate degradation capabilities In vitro testing to confirm bacterial growth on novel ‘prebiotic’ In vitro testing to confirm increased population In vivo confirmation – microbiota composition and health markers
Ultimate requirement: Personalised nutrition based on individual microbial profile
Prebiotics can help boost the numbers of key species
ALREADY THERE
Novel prebiotics
Acknowledgements
Jenny Martin Sylvia Duncan Petra Louis Alan Walker Professor Harry Flint Everyone in the microbial ecology group - including numerous visitors and summer students Everyone in the Human Nutrition Unit Genomics section (Gill Campbell, Pauline Young) BioSS (Claus-Dieter Mayer, Grietje Holtrop)
Annick Bernalier-Donadille, Christophe Chassard