what really is a prebiotic and why?

What really is a prebiotic and why?

ISAPP Cork 2012

Discussion Group 1

George Fahey

Paul Blatchford

Jose Garcia Mazcorro

Bob Hutkins

Arland Hotchkiss

Yong Jun Goh

Christophe Lacroix

Koen Venema

Douwe Van Sinderen

Margriet Schoterman

Juliet Ansell

Glenn Gibson

Kevin Whelan

Raylene Reimer

Bob Rastall

Which of the following is not a good idea

for a first time visitor to Ireland?

Leave the presentation preparation up to

Gibson and Rastall (in the pub)

Beer-drinking contest with an Irishman

Look to the left before crossing a busy street

Discussion themes

Definition history

Does structure determine function?

Evaluating prebiotics

Bioactivites of prebiotics, including

extraintestinal (and extrahuman) effects

Concluding thoughts: What makes a prebiotic a prebiotic

The original prebiotic concept from 1995 to ISAPP

ISAPP Cork. 2012.

Discussion Group 1

A non-digestible food ingredient that beneficially affects the host

by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the

colon, and thus improves host health

Gibson, G. R., Roberfroid, M. B. J. Nutr. 125, 1401-1412, 1995

Prebiotic definition

A dietary prebiotic is 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 ISAPP definition, IFIS Functional Foods Bulletin, 2010

A prebiotic is a non-viable food

component that confers a health

benefit on the host associated with

modulation of the microbiota Maya Pineiro, Nils-Georg Asp, Oscar Brunser, Sandra Macfarlane,

Lorenzo Morelli, Gregor Reid and Kieran Tuohy. FAO

Technical Report November 2007, Rome

A prebiotic is a non-viable food component, ingredient or supplement

that selectively modulates the microbiota of the digestive ecosystems, thus conferring benefits upon host well-

being and health Marcel Roberfroid, Francisco Guarner, Sandra Macfarlane, Satoshi

Kudo, Bernd Stahl and Bob Rastall. ILSI Expert Group on the Working Definition of Prebiotics, 17th April 2008, Brussels

The EFSA NDA panel holding an expert consultation...

Does structure affect function?

ISAPP Cork. 2012.

Discussion Group 1

Pectin – a polysaccharide with multiple functional groups

Glucuronic acid Galactose KDO Apiose

Arabinose Fucose Galcturonic acid Aceric acid

RG I XGA HGA RG II

Xylose DHA Rhamnose Acetyl- methyl-

6

6.5

7

7.5

8

8.5

9

9.5

10 0

5

10

24

0

5

10

24

Log 1

0 c

ells

/g

Bifidogenic effects of pectic oligosaccharides

Batch culture, 1% CHO, pH 6.8, bacteriology by FISH

FOS POS

Manderson et al (2005),

Applied and Environmental

Microbiology 71; 8383-8389

Manderson et al (2005),

Applied and Environmental

Microbiology 71; 8383-8389

Prebiotic potential




RG I XGA HGA RG II

Evaluation of prebiotics

ISAPP Cork. 2012.

Discussion Group 1

Freitag, 5.

Oktober

2012 13

Human gut - systems biology analyses

Molecular and

mechanistic

studies

In vitro human

intestinal cell

models

In vivo

animal

studies

In vivo

human

studies

Metagenomics

Proteomics

Transcriptomics

Metabolomics

Systems biology tools

In vitro gut

fermentation

models

C. Lacroix - LFB - ETH Zurich

Pro- and Prebiotics Impact Gut Functions & Health

Payne et al 2012 Trends Biotechnol 30:17

Prebiotic effects and the gut: testing

First experiments in humans with 13C-lactose!!

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

-50

0

50

100

150

200

250

individual 213C-CO2

H2

13C-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 213C-CO2

H2

13C-glucose

-30 0 30 60 90 120 150 180 210 240-30 0 30 60 90 120 150 180 210 240

delt

a p

lasm

a g

lucose

delt

a b

reath

CO

2

breath

hy

drogen

(pp

m)

- no 13C-glucose in plasma

- increase in 13C-CO2 in breath

- increase in 13C-acetate in plasma

- increase in breath H2

fermentation of 13C-lactose in colon

In vitro models of digestive ecosystems:

Perspectives and future directions?

• Reliable-efficient to study mechanistic effects, e.g. cross feedings

• Use of in vitro models with “omics” techniques • New models: miniaturisation, automation and

high throughput • Combine in vitro fermentation models and cell

models to add host-microbe interactions • Need to avoid studying models for the sake of

the models

Assessing prebiotic actitivity by functional genome analysis

UCC2003 is specialised to metabolise host & plant-derived glycans

Starch/Glycogen/

Amylopectin/Pullulan

Cellodextrin

Palatinose

Palatinose

Ribose

Lactose/Lactulose

Stachyose/Raffinose/Melibiose

Galactan/

Galacto-oligosaccharides

Fructo-oligosaccharides/

Sucrose

Fructose

Sialic Acid

N-linked Glycans

Lacto-N-biose

Kojibiose

Fucose

Melezitose

Functional genome analysis

• Comparison of gene clusters to show transporters, regulators, permeases, hydrolases, etc. • For example LacS in most GI lactobacilli • GOS gene cluster upregulated by bile • Confirmed uptake of trisaccharides by

bifidobacteria • Demonstration of diversity of prebiotic

utilising pathways in lactobacilli

Oligosaccharide/H+ symporter

L. ruminus

ABC transporter

L. acidophilus P

cell wall-anchored BfrA

Cell wall-anchored BfrA/Fructose-PTS

L. paracasei

Intracellular BfrA

P

Sucrose-PTS

L. plantarum

glucose fructose

(O’Donnell et al., 2011)

(Goh et al., 2006)

(Barrangou et al., 2003)

(Saulnier et al., 2007)

Diversity of FOS utilisation pathways in lactobacilli

Pathogenic E. coli (Schouler et al., 2009)

Metabolic diversity of oligosaccharides utilization by L. acidophilus

Andersen et al., PLOS ONE 2012

ABC

PTS

MFS

Isomaltose Isomaltulose

Panose Polydextrose

Cellobiose -glucan oligomers

(-1,4)

Gentiobiose (-1,6)

Polydextrose

Raffinose Stachyose

GOS Lactitol

Lactulose?

-Glu -Glu -Gal -Gal

Maltose 6-P glucosidase

Phospho--glucosidase II

Phospho--glucosidase I

-Galactosidase

Sucrose phosphorylase

-Galactosidase

Maltose phosphorylase

FOS

Evaluating prebiotics from the consumer perspective

Q1: should the definition of a prebiotic relate to the treatment of disease as well as the maintenance or improvement of health?

Q2: thinking of the definition(s) of prebiotic… can we call a compound prebiotic if it does not change the numbers of microbiota nor treat disease?

Q3: should the term prebiotic relate to a specific compound in a specific product in a specific dose in a specific disease (Sanders)?

Q4: are patients able to access prebiotics? Q5: how can patients access reliable information on prebiotics?

Do people use probiotics and prebiotics?

Hedin et al, Inflamm Bowel Dis 2010; 16: 2099-2108

Have you ever used... for your health

Crohn’s (n=131)

UC (n=103)

Controls (n=100) P value

Probiotics 43% 51% 21% <0.001

Prebiotics

Do people use probiotics and prebiotics?


Have you ever used... for your health

Crohn’s (n=131)

UC (n=103)


Probiotics 43% 51% 21% <0.001

Prebiotics 4% 2% 1% 0.358


What is a… Crohn’s (n=131)

UC (n=103)


Probiotic, mean score 0.96 1.23 0.71 0.001

1) bacteria, bug, microbe etc 41% 52% 26% 0.001

2) health benefit, treat disease 28% 41% 31% 0.114

3) name of a strain or product 27% 30% 15% 0.030

Prebiotics, mean score 0.05 0.02 0.05 0.473

1) food substance, fibre, etc 3% 1% 0% -

2) increase bacteria, increase activity 2% 1% 1% -

3) health benefit, treat disease 1% 0% 0% -

Q5: how can patients access reliable information on prebiotics?

People don’t know much about prebiotics

Bioactivities of prebiotics

ISAPP Cork. 2012.

Discussion Group 1

Extraintestinal effects

• Atopic diseases • Respiratory infections • Vaginal effects • Oral disease • Liver disease • Skin effects • Adiposity

Most implicate gut microbiota modulation as an explanation of effect

Prebiotics reduce adiposity

Lipoprotein lipase (LPL) promotes fat storage in adipose tissue ----- prebiotics reduce LPL

Adipocyte fatty acid binding protein (aP2) promotes adipocyte differentiation. Fat mass increases by increasing size and the formation of new adipocytes from precursor cells ---- prebiotics reduce differentiation

G-protein coupled receptor 43 (GPR43) reduces lipolysis and stimulates lipogenesis ---- prebiotics reduce GPR43

Prebiotics and the canine: meta-analysis • 15 published studies (1998-2007)

– 65 dietary treatments

– 418 observations

• Evaluated the effects of prebiotics on:

– Nutrient digestibility

– SCFA concentrations

– Bacterial populations

– Serum immunoglobulin concentrations

Patra, 2011

Beynen et al. (2002) Diez et al. (1998) Flickinger et al. (2003) Flickinger et al. (2003) Grieshop et al. (2002) Grieshop et al. (2004) Hesta et al. (2003) Middelbos et al. (2007a) Middelbos et al. (2007b) Propst et al. (2003) Strickling et al. (2000) Swanson et al. (2002a) Swanson et al. (2002b) Swanson et al. (2002c) Verlinden et al. (2006) Zentek et al. (2002)

– Compositional analysis of potential prebiotics

• Monomeric composition

• Chain length

• Linkages

• Branching

• Side chains

– Prebiotic activity of natural foods

• Soybean products

• Beet fibre

• Whole grains and co-products

Future of prebiotics for companion animals

• Microbiota beyond bifidobacteria

– Detailed composition – 16S rDNA pyrosequencing

– Metabolic function – metagenomics approach

• Need to study microbiome-indices of health relationships

• How do prebiotics achieve benefits for disease?

Future of prebiotics for companion animals

Saulnier et al;., 2009

Prebiotic oligosaccharides: beyond bifidogenicity

Adhesins, Ligands, and Sticktoitiveness

Prebiotics as anti-adhesive agents

0

10

20

30

40

50

60

70

80

90

100

* *

* * * *

*

*

Adhesio

n r

ela

tive t

o c

ontr

ol (%

)

Rhoades et al (2008) Journal of Food Protection 71: 2272-2277

Antiadhesive pectic oligosaccharides




RG I XGA HGA RG II

Bioactivity in pectins

Exploiting anti-adhesive prebiotics

1. Increase the avidity of adhesin for ligand

• Increase ligand valency • Increase ligand affinity

2. Mixtures of anti-adhesins to counter phase variation

3. Isolation of anti-adhesin agents from natural sources

Lee et al. 2012. Microbial Cell

Factories

5. Synthesis of natural/novel anti-adhesin agents

2-fucosyllactose

4. Prevention of extra-intestinal infections

•

•

•

•

Prebiotic activity in whole foods: kiwifruit as an example

0

20

40

60

80

100

0h 24h 48h

Pe

rce

nta

ge s

eq

ue

nce

ab

un

dan

ce

Donor 1

0h 24h 48h

Donor 2

0h 24h 48h

Donor 3

Ba

cter

oid

etes

Firm

icu

tes

Act

ino

ba

cter

iaP

rote

ob

act

eria

Rhodospirillaceae

Enterobacteriaceae

Alcaligenaceae

Bifidobacteriaceae

Coriobacteriaceae

Veillonellaceae

Lachnospiraceae

Bacillaceae

Streptococcaceae

Ruminococcaceae

Rikenellaceae

Porphyromonadaceae

Bacteroidaceae

Prevotellaceae

Measured intermediate SCFA

Propionate pathway intermediate metabolite (not measured)

Measured end-point SCFA

Propionate pathway intermediate metabolite (measured)

Intermediate metabolite (not measured)

Bacteria

LEGEND Hexoses Galactose

Mannose

Glucose

Pentoses Arabinose

Xylose

Ribose

Deoxy hexoses Fucose

Rhamnose

Pyruvate

Acetyl-CoA

Acetate

Succinate

Propionate

Formate

Lactate Butyrate

Oxaloacetate

Acrylyl-CoA

Propane-1,2-diol

DHAP + lactaldehyde

Pentose phosphate pathway

Roseburia inulinivorans Roseburia/Eubacterium group Faecalibacterium prausnitzii

Bacteroidetes Proteobacteria

Ruminococcus spp.

Bacteroidetes

Lachnospira/Ruminococcus spp. Lactobacillus/Enterococcus spp.

Veillonellaceae Actinobacteria Bacteroidetes Proteobacteria

Roseburia/Eubacterium group Faecalibacterium prausnitzii

all

all

Roseburia inulinivorans Bacteroidetes Proteobacteria

Glycolytic Pathway

Actinobacteria

Phosphoenolpyruvate


Roseburia inulinivorans Pseudobutyrivibrio



Lachnospira/Ruminococcus spp. Lactobacillus/Enterococcus spp.

Actinobacteria Proteobacteria

Veillonellaceae Veillonellaceae


Lachnospira/Ruminococcus spp. Actinobacteria Proteobacteria

Adapted from:

Louis et al. (2007) J. Appl. Micro. 102, 1197-1208;

Macfarlane & Marfarlane (2003) Proc. Nutr. Soc. 62, 67-72.

W. J. Kelly (AgResearch, NZ, pers. comm., 2012)

CH4, CO2, H+

Propane-1,2-diol

Roseburia inulinivorans Bacteroidetes Proteobacteria

Pseudobutyrivibrio

Other excreted end point metabolites

CH4, CO2

A “simplified” view of prebiotics and the functional network in the gut

Conclusions: What makes a prebiotic a prebiotic (and how do

you know)?

ISAPP Cork. 2012.

Discussion Group 1

Who is there?

• Integrated approach to microbiota characterization

• Genomic sequencing, metagenomics, FISH, qPCR, DGGE, etc.

• Need to test with the best technology available

What are they doing?

• In vitro SCFA and other organic acids

• Biotransforming enzyme activities

• Urinary, blood, faecal metabolites

• Immunology

• Blood lipids

• Patient symptoms

• No gas no glory...

What should we be measuring?

What really is a prebiotic and why? Now:

• FOS and GOS - structure, size, host enzymology

• Focus on "probiotics" - bifidobacteria and lactobacilli

• Simple view of metabolism - SCFA

In the future:

• Model systems will become more sophisticated

• New target microorganisms - will need new prebiotics

• Multiple functionality

• Understanding carbon flux through the microbiome, its impact on microbial activity and consequent impact on health

what really is a prebiotic and why?

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