Scientific Validation Of Polydextrose As A Fibre And

Geoff O’SullivanApplication Manager

September 2007

Scientific Validation of Polydextrose as a Fibre and Sustained Prebiotic for Digestive Health

With contributions from: Prof Glenn Gibson, University of Reading, Dr Nina Rautonen, Dr Artur Ouwerhand,Dr Kirsti Tiihonen, Dr Helen Mitchell, Dr Oliver Hasselwander and Dr Julian Stowell

2


• Rationale for Prebiotics• Market for Soluble Fibre Ingredients and Prebiotics• Definition of ‘Prebiotics’• Scientific Evidence for Existing Prebiotics• Metabolism and Associated Health Benefits• Methods for Prebiotic Evaluation

– Colon Simulator– Quantitative Methods for Specific Bacterial Strains– Human intervention studies

• Validation of Polydextrose as a Prebiotic• Linkage analysis• Future Directions

– New Biomarkers

• Regulatory– JECFA, FDA and EU

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Probiotics, Prebiotics, synbiotics and fibre

A probiotic is a live microbial food or feed supplement which beneficially affects the host by improving the balance of intestinal microflora.

A prebiotic is a nondigestible 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 and Roberfroid, 1995)

A Synbiotic – is a combination of pro- and prebiotics where the efficacy of the probiotic is enhanced by the inclusion of a prebiotic.

Fibre – can be defined in many ways. Physiologically, fibre is essential to regularize bowel function and it may also mediate glucose and cholesterol attenuating effects.

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Rationale for Prebiotics – Digestive Diseases

Diseases & disorders include:• Abdominal wall hernia• Constipation• Diverticulitis• Gastritis and non-ulcer dyspepsia• Haemorrhoids• Infectious diarrhoea• Irritable bowel syndrome• Inflammatory bowel disease• Lactose intolerance• Peptic ulcer• Hepatitis

All digestive diseases – USA

Prevalence>75 million by all digestive diseases (1998) – excluding 135 and 76 million non food borne and food borne infections/illnesses

Mortality >125,000 including deaths from cancer (1998)

Costs >$86 billion direct medical costs (1998)>$20 billion indirect costs (1998)lost productivity, disability, etc.

Ref: www.niddk.nih.gov/statistics.htm, The Burden of Selected Digestive Diseases in the USA, 2002, Sandler et al

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Rationale for Prebiotics – A Balanced Microflora

• Molecular studies indicate that the intestinal microflora consists of 1014 microbes from more than 1000 species.

• Little is known about the role played by many of the dominant bacteria in the gut that are believed to be benign such as Bacteroides, Eubacterium spp., Ruminococcus spp., Butyrovibrio spp.

• Bifidobacteria and lactobacilli are two species with known positive contributions to human health.

• As the microflora protect against incoming pathogenic microbes and modulate immune response, a balanced microflora increases well-being of the gastrointestinal tract.

Prebiotics can contribute to human digestive health by specifically stimulating growth of bifidobacteria and lactobacilli, two microbial species accepted to exhibit beneficial effects.

Ouwehand AC, Makelainen H, Tiihonen K and Rautonen, N (2006) - Digestive Health, pages 44-51, Part I Sweeteners and Sugar Alternatives in Food Technology, Edited by Helen Mitchell, Blackwell Publishing, UK.

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Definition of ‘A Healthy or Balanced Microflora’

Cummings JH et al defined a healthy or balance microflora in 2004:

‘A healthy, or balanced, flora is, therefore, one that is predominantly saccharolytic and comprises significant numbers of bifidobacteria and lactobacilli. The exact numbers are difficult to give at present

because a proportion of the gut flora have yet to be identified’

Cummings JH, Antoine J-M, Azpiroz F, Bourdet-Sicard R, Brandtzaeg P, Calder PC, Gibson GR, Guarner F,Isolauri E, Pannemans D, Shortt C, Tuijtelaars S, Watzl B (2004) PASSCLAIM – Gut health and immunity. Eur J Nutr; 43, Supplement 2:II/118-II/173

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Market of Soluble Fibre Ingredients and Prebiotics

19.7

35.2

41.511.2

36.0

5.6 5.6 12.2Fructo-oligosaccharide (FOS)

Inulin

Isomalto-oligosaccharide (IMO)

Resistant maltodextrin

Polydextrose

Lactulose

Resistant Starch

Others

GIRACT. Soluble Fibre Ingredients. Global Supply/Demand Patterns in Food, Feed & Supplements. 2004/5-2010 (July 2005).

World Demand – Volume (KT)Total: 167KT

Others include primarily oligosaccharides that are mainly marketed as prebiotics in Japan such as soy-oligosaccharides (SOS), galacto-oligosaccharides (GOS), xylo-oligosaccharides (XOS).

Not all of these compounds meet the criteria for prebiotic classification and some are at present mainly used as bulking agents.

Market – Prebiotic ingredients

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Definition “Prebiotics”

Gibson GR & Roberfroid MB (1995) Dietary modulation of the human colonic microbiotia:Introducing the concept of prebiotics. J Nutr; 125:1401-1412

Gibson GR et al (2004) Dietary modulation of the human colonic microbiotia:Updating the concept of prebiotics Nutr Res Rev; 17:259-275

The term ‘prebiotic’ was coined by Gibson and Roberfroid in 1995:

‘Prebiotics are non digestible food ingredients that selectively stimulate a limited number of bacteria in the colon, to improve host

health’

Since then, the concept has been further developed and in order to qualify for

prebiotic classification, a compound is required:

1. to resist gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption,

2. to be fermented by the gastrointestinal microflora,3. to stimulate selectively the growth and/or activity of intestinal

bacteria associated with health & wellbeing

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Metabolism and Associated Health Benefits

Ouwehand AC, Makelainen H, Tiihonen K and Rautonen, N (2006) - Digestive Health, pages 44-51, Part I Sweeteners and Sugar Alternatives in Food Technology, Edited by Helen Mitchell, Blackwell Publishing, UK.

Prebiotics have positive effects on several biomarkers related to health benefits.

Prebiotics may hence play a role in reducing the risk of colon cancer,

inflammatory bowel disease, gastrointestinal

infections and insustaining bone health.

Prebiotic

Not digested nor absorbedin small intestine

Colonic microbiota

Microbial metabolites

butyric acid

energy sourcecolonocytes

immune cells

Liverfat metabolism

cholesterol metabolism

propionic acid

acetic acid

antimicrobialactivity

immunemodulation water

retention

biomass

increasedfaecal output

detoxificationReduced pH

improvedCa2+ absorption

Prebiotic

Not digested nor absorbedin small intestine

Colonic microbiota

Microbial metabolites

butyric acid

energy sourcecolonocytes

immune cells

Liverfat metabolism

cholesterol metabolism

propionic acid

acetic acid

antimicrobialactivity

immunemodulation water

retention

biomass

increasedfaecal output

detoxificationReduced pH

improvedCa2+ absorption

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Methods for Prebiotic Evaluation (I)

A) Non-digestibility

• In vitro, by incubation of prebiotic candidate at pH and temperature conditions of the stomach and incubation with saliva, pancreatic and small intestinal enzymes and analysis of hydrolysis products.

• In vivo, by measuring recovery of prebiotic candidate in faeces after oral administration in germ-free rats.

• In vivo, by recovery of at least 90% of the ingested quantity of a prebiotic candidate the pouch of ileostomised subjects.

B) Fermentation

• In vitro, by studying anaerobic fermentation of the prebiotic candidate using pure bacterial populations or faecal slurry.

• In vivo, by measuring recovery of prebiotic candidate in faeces after oral administration in animals or humans and fermentation products such as gases (CO2, H2, CH4) or short chain fatty acids (acetic, propionic, butyric and lactic acid).

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Methods for Prebiotic Evaluation (II)

C) Selective stimulation of growth and/or activity of intestinal bacteriaassociated with health & wellbeing

• In vitro, by studying changes in composition of a mixed faecal slurry before and after fermentation of a prebiotic candidate in a colon simulator.

• This method is particularly useful for screening of candidate prebiotics and comparison with established prebiotics.Final proof that a candidate prebiotic can be classified as prebiotic has to be obtained in placebo-controlled dietary intervention trials in humans: Volunteers are (n= 8-20) are supplemented with prebiotic candidate (typically

5-15 g/day) for 2-12 days Stool sampling before and after the intervention to quantify changes in

microflora Quantification of selectively stimulated growth of bacteria by culture and

and requires culture techniques and nucleotide probe based techniquesVan Loo J (2005) Prebiotics: a nutritional concept gaining momentum in modern nutrition.Food Science and Technology Bulletin: Functional Foods; 2:83-100.

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Evaluation Tools – Colon Simulator

• Unique dynamic model for lower GI tract fermentation

• Automated continuous multi-stage simulator

• Strictly anaerobic, pH control

• Measured parameters include-

degradation rate-

metabolic end products- shifts in

microbial - community and selective

growth stimulation of intestinal bacteria

©

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4 Stage Colon Simulator

V1

pH 5.5 pH 6.0 pH 6.5 pH 7.0

+4°C+4°C

3 ml 5 ml 7 ml 9 ml

Freshmedium EffluentV2 V3 V4

N +NH2

N 2 N 2 N 2 N 2

3

V1

pH 5.5 pH 6.0 pH 6.5 pH 7.0

+4°C+4°C

3 ml 5 ml 7 ml 9 ml

Freshmedium EffluentV2 V3 V4

N +NH2

N 2 N 2 N 2 N 2

3

“proximal” “distal”

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Quantification Methods for SpecificBacteria Strains

Van Loo J (2005) Food Science and Technology Bulletin: Functional Foods; 2:83-100.

Method Main Advantages Main Disadvantages

Selective culturing & identification of biochemical characterisation

Inexpensive, allows large number of replicates

Time consuming, operator subjectivity, only culturable bacteria detected

Fluoresence in situ hybridisation (FISH)

Highly specific, also for unculturable bacteria

Time consuming, only probe for known bacteria

Percent-G+C profiling Robust method, qualitative picture of the total bacterial community

Does not distinguish between specific species

Polymerase chain reaction (PCR), also quantitative

High fidelity and reliability, allows placement of previously unidentified bacteria

Expensive, time consuming, some bias during PCR

Direct community analysis Culture-independent, diversity of entire sample can be elucidated

Some loss of diversity due to bias introduced by PCR

Denaturing gradient gel electrophoresis (DGGE)

Rapid, also for unculturable bacteria

Qualitative rather than quantitative, loss of diversity due to bias introduced by PCR

New techniques make use of 16SrRNA oligonucleotide probes which identify specificbacteria, genera or whole species or sequencing of 16SrRNA amplified by PCR.

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Randomly cross linked polymer of glucose. R can be hydrogen, sorbitol-bridge or more polydextrose

HO

CH2 OH O OH

HO O CH 2

OR OH

OH O CH 2 O

OH O O

OH OH O CH 2

CH 2 OH O

OH OH

O

O HO

OH OH O CH 2 OH

HO

HO OH

OH O CH 2 OH

O

HO OH

OH O CH 2 OH

CH 2 OH O

OH OH HO

O CH 2 O OH

HO O O

HO OH

OH O CH 2

O

Highly branched

complex 3D

structure

All bonds are present

1 – 6 and 1 – 4

Linkages

Polydextrose - Chemical Structure

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• The Molecular size of polydextrose is limited to an average of 12 DP units with a molecular weight range 180 – 5000

• Polydextrose is a randomly bonded condensation polymer of D-glucose with some bound sorbitol and a suitable acid

• For starch the range of DP is typically from 37 – 49 • Polydextrose - has the highest amount of branching and complexity of any carbohydrate

Polydextrose - Chemical Structure

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Polydextrose Caloric Utilization

• Structural compactness and complexity prevents hydrolysis by mammalian enzymes

• Large intestinal microorganisms only capable of partial conversation

• Radio-tracer studies in rats and humans confirm energy value(1).

• Other studies have confirmed this value(2) (3)etc.

Large Intestine Volatile Fatty acids 1 kcal/g

1. Archour L, Flourie B, Briet F, Pellier P, Marteau P and Rambaud J-C (1994) Gastrointestinal effects and energy value of polydextrose in healthy non obese men. American Journal of Clinical Nutrition: 59: 1362-13682. Figdor SK and Rennhard HH (1981) Caloric utilization and disposition of [14C]polydextrose in the rat. Journal of Agricultural and Food Chemistry: 29(6):1181-9

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What is an effective prebiotic?

• Increases the overall wellbeing of gastrointestinal tract

• Decreases risk of gastrointestinal diseases

• Is safe!

• Increases saccharolytic fermentation and reducesputrefactive fermentation

A product that:

19

0

2

4

6

8

10

12

14

16

V1 V2 V3 V4 V1 V2 V3 V4 V1 V2 V3 V4

0.5% PDX 1.0% PDX 2.0% PDX

Tot

al c

oncn

of P

DX

mg/

ml

Metabolism of polydextrose in the different

Stages of a colon simulator

• Polydextrose is fermented gradually but not completely throughout the four stages

•The complex structure may explain gradual fermentation

Fava F, Mäkivuokko H,Siljander-Rasi H,Putaala H, Tiihonen K,Stowell J,Touhy K, Gibson Gand Rautonen N(2007) British Journal ofNutrition 98, 123-133

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Polydextrose as a prebiotic - Dose Dependent Production of Butyrate

Polydextrose concentrations were chosen to represent a reasonable daily dose of polydextrose

©

Effect of Litesse on butyric acid production by colon microbes(Colon fermentation simulation)

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

0,0 % 2,0 % 4,0 % 8,0 %

Litesse concentration in colon

Bu

tyri

c ac

id c

on

cen

trat

ion

(m

M)

Butyric acid (g) = 0.0181 + 0.0673 x Litesse (g)

r2= 1.00, p<0.0001

Polydextrose concentration in colon

Effect of Polydextrose on butyric acid production by colon microbes

Butyric acid(g) = 0.0181 + 0.0673 x polydextrose

Source: unpublished data - Danisco

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Polydextrose - Sustained Fermentation in the Colon

“proximal” “distal”

Fermentation continues also in the distal colon with PolydextroseFatty acid production using a 4-stage fermentation simulation

0

10

20

30

40

50

60

70

Stage 1 Stage 2 Stage 3 Stage 4

mM

Butyric acid

Propionic acid

Acetic acid


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Volatile Fatty Acids

0

1

2

3

4

5

6

Acetate Propionate Butyrate Iso-butyrate

Iso-valerate

Sto

ol

co

nte

nt

(mg

/g)

0 g/d

4 g/d

8 g/d

12 g/d

* p<0.05, ** p<0.01 for comparison of results to baseline (0 g/d)**

**

****

*

Zhong Jie et al., American Journal of Clinical Nutrition (2000)

Polydextrose is fermented in the colon producing short chain fatty acids (butyrate, isobutyrate, acetate), which decrease pH

Faecal pH decrease can suppress production of enteric toxins

Increased butyrate promotes the growth of colonic cells (source of energy)

Source: unpublished data

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Prebiotic Effect

0

1

2

3

4

5

6

7

B. fragilis B. vulgatus B. intermedius Lactobacillus Bifidobacterium

Cou

nts

(x1

09/g

sto

ol)

0 g/d

4 g/d

8 g/d

12 g/d

Very large increase of beneficial Lactobacillus and Bifidus and a resulting decrease in toxic bacterias

Zhong et al., American Journal of Clinical Nutrition Vol 72. No 3 September 2000

Bad Bacteria - decreasing

Good bacteria - increasing

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Polydextrose fermentation does not result inthe accumulation of lactic acid in vivo

Polydextrose does not increase residual lactic acid concentrations in the lower intestine (p-value for difference > 0.10). (Hollie M. Probert,1* Juha H. A. Apajalahti,2 Nina Rautonen,2 Julian Stowell,3 and Glenn R. Gibson1, Applied and Environmental Microbiology, August 2004, p. 4505-4511, Vol. 70, No. 8)

Residual lactic acid concentration in rats

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Low -f ibre Western diet + 2% Litesse of dietm

mo

l/g+ 2% polydextrose

25

(Cummings and Macfarlane 1991(9) , Smith and Macfarlane, 1997 (10) )

• Ammonia, biogenic amines, indoles and phenols are produced in putrefaction. They are toxic in large quantities.

• Branched volatile fatty acids, such as isovaleric, isobutyric and 2-methylbutyric acid, are also produced in putrefaction. They are not toxic but serve as biomarkers for harmful putrefaction.(Bergman 1990 (11), Cummings and Macfarlane 1991 (9), Ito et al. 1993 (12),

Smith and Macfarlane 1997 (10) )

Adverse effects

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Production of branched fatty acids is decreasedby polydextrose in rats

Concentrations of residual branched fatty acids in the rat lower intestine after 4 weeks

Polydextrose reduces putrefaction in rat lower GI tract in Western low fiber diet (p < 0.0001 ****).

Caecal concentration of branched VFAs

0

0.2

0.4

0.6

0.8

1

1.2

Low-fibre Western diet + 2% Litesse of diet

mm

ol/

g

2% Polydextrose


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Faecal concentrations of branched fatty acidsare decreased by polydextrose in humans

In a clinical trial polydextrose reduced faecal branched fatty acids (p=0.04* from 0 to 3 weeks and p=0.004 ** from 0 to 6 weeks).

Faecal concentrations of branched VFAs

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 wk 3 wk 6 wk

Co

nce

ntr

atio

n o

f b

ran

ched

VF

As

(mm

ol/

g)

Control (normal diet)

Litesse (10 g/day)Polydextrose (10g/day)


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Fast- and slow-fermenting prebiotics in the colon

Polydextrose

Fast-fermenting prebiotics

Proximal colon Distal colon

1st generation

1st generation prebiotics increase fermentation in the proximal colon.

Fermentation of polydextrose continues still in the distal parts.

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Number of bacteriaPrebiotic


Saccharolytic fermentation

putrefaction

Rapidly fermented prebiotic leading to

Putrefaction

Putrefaction is associated with

health risks

Butyrate not produced in distal colon

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Sustained saccharolytic fermentation

Number of bacteriaPrebiotic


Saccharolytic fermentation

putrefaction

Butyrate production throughout the colon

Fermentation

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Polydextrose supplementation to normal Westerndiet leads to sustained fermentation in the colon

Stomach/Small intestine Large intestine

FaecesFood

Digestion&

Fermentation

Fat / Monosaccharides / Amino acids

Steady rateNon-absorbed

material

Steady production of short chain fatty acids,no uncomfortable rapid gas formation!

Absorption

proximal distal

Steady rate

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PutrefactiveSaccharolytic

ACIDSLactic ButyricAcetic Branched

Amines etc. Ammonia

pHDown Up

REDUCTION OF COLON CANCER

RISK

SHORTER TRANSIT TIME

Energy for epithelial and immune cells

Control of proliferation

ENHANCED MINERAL

ABSORPTION

PATHOGEN REDUCTION

Phenols indoles

Anti-bacterials

Faecal mass

BASES

Enhancing and inhibiting effects of polydextrose

TOXIC EFFECTS

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Polydextrose a typical DP 12 structure

HO

O

OH

O

HO

O

HOO O

HOO

HOOH O

OH

HOO

HOOH O

O

OHO OH

HO

HO

HOO

OHOH

OH

OH

O

HO

O

OH

HO

O

HO

O

OH

HOO

HO

OOH

O

HOOH

O

OHO

OHHO

O

HO

O

HO

HO

OH

HO


Terminal Group 1-6

Fully substituted CoreAll bonds 1-6,4,3,2

Double substituted groups

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Polydextrose before and after colon simulator

MALDI of Colon Simulator Sample vs. Regular Polydextrose

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Degree of Polymerization

Rel

ativ

e P

eak

Hei

gh

t

Colon Simulator Sample

Regular Polydextrose


35

Preliminary Linkages and Branching

Branching and Linkage Normalized Linkage Positions for 3% Polydextrose in Colon Simulator Vessels 1, 2, and 3

0

10

20

30

40

50

60

term

inal

nonbranch

ed

single

bra

nch

double b

ranch

triple

bra

nch

6-lin

ked

4-lin

ked

3-lin

ked

2-lin

ked

Type of Linkage or Branching

Are

a %

Litesse Vessel 1

Vessel 2 Vessel 3

Core material increases


Terminal groups decrease

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• Prebiotic

– 4 – 12 g of Ploydextrose, Zhong et al, American Journal of Clinical

Nutrition (2000), vol 72 pp. 1503 –9

– Promote growth of intestinal Lactobacillus and bifidus

– Fermentation in the large intestine yields short-chain fatty acids (including butyrate)

– Improved gastrointestinal function , no adverse effects

Polydextrose - Prebiotic Summary

Prebiotic

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• Improvement of mineral absorption

– dietary polydextrose (5%) increased calcium absorption and bone mineralisation in rats

– 21 days increased the bone calcium concentration and apparent calcium absorption when compared to control

– polydextrose has the potential to increase calcium in humans, linked to intestinal acidification, but unknown mechanisms are also involved

Ref: Hara, H et al. Ingestion of the soluble dietary fibre, polydextrose, increases calcium absoption an bone mineralization in normal and total-gastrectomized rats. British Journal of Nutrition, 200, 84:655-611

Polydextrose - Mineral Absorption

3737

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Definition – Dietary Fibre

Fibre definition according to the CODEX alimentarius:

Dietary fibre means carbohydrate polymers with a degree of polymerisation (DP) not lower than 3, which are neither digested nor absorbed in the small intestine. A degree of polymerisation not lower than 3 is intended to exclude mono- and disaccharides. It is not intended to reflect the average DP of a mixture.’

CODEX alimentarius. Document CL 2005/53 - FSDU Dec 2005.

39

• Soluble fibre– Dietary fibre is difficult to define - can use analysis,

physiology, chemistry or origin Growing acceptance of physiological definition

– Fifteen clinical studies show physiological benefits from Litesse

• Fecal– Bulking (increase)– Softening (increase)– Transit time (decrease)– Flora, i.e. prebiotic

(improve)– pH (decrease)– Short Chain Fatty Acid

(increase)– Carcinogens (decrease)

• Serum– Glucose (attenuate)– Lipids (attenuate)

• Intestinal– Physiology (growth)

Polydextrose - Soluble Dietary Fibre

Analysis :

AOAC 2000.11

40

Polydextrose Clinical Studies Summary

Polydextrose

41

Polydextrose Clinical Studies Summary

Polydextrose

42

Studies on the effects of Polydextrose intake on gastrointestinal function

• 120 subjects divided into four groups:

– 0, 4, 8 and 12 g Litesse/day for four weeks

• Improvement in colon function• No laxation problems • Decrease in faecal pH • Increase in faecal weight• Increase in SCFAs, especially butyrate• Increase in Lactobacillus and

Bifidobacterium (traditional method)

Zhong et al., 2000,

Laxative threshold for Litesse is 90 g/day (Burdock and Flamm 1999)

Decreased pH enhances mineral absorption and inhibits acid sensitive pathogens (Hara et al., 2000)

43

0

1

2

3

4

5

6

7

B. fragilis B. vulgatus B. intermedius Lactobacillus Bifidobacterium

Cou

nts

(x1

09/g

sto

ol)

0 g/d

4 g/d

8 g/d

12 g/d

Very large increase of beneficial Lactobacillus and Bifidus and a resulting decrease in toxic bacterias

Zhong et al., American Journal of Clinical Nutrition Vol 72. No 3 September 2000

Bad Bacteria - decreasing

Good bacteria - increasing

ZHONG JIE et al - AJCN, 2000 72: 1503-9

ZHONG JIE et al - AJCN, 2000 72: 1503-9

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DIGESTIVE HEALTH - DELIVERABLES

• Regularizing bowel function/laxation• Reduced inflammation/allergic reactions• Enhanced immune system • Increased saccharolytic bacteria• Reduced colonic pH discouraging growth of putrefactive bacteria• Lower risk of pathogens• Reduced production of toxic products e.g. ammonia, phenolics• Reduced cholesterol• Improved glycaemic control• Reduced risk of diabetes, cardiovascular disease etc• Increased butyrate production:

- Improves integrity of the gastric mucosa - Programmed death of cancer cells – apoptosis

• Reduced cancer risk – especially colon cancer• Enhanced mineral absorption - Reduced risk of osteoporosis

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• An ”immortal” human colon cancer cell line

• A good model of intestinal epithelial cells

• Caco-2 cells are exposed to different treatments and the effects on gene expression are measured

Modulation of Epithelial Gene Expression

Using Prebiotics

Aim: In vitro evidence of anti-inflammatory and anti-carcinogenic properties

health-promoting treatments can be identified by a “good” effect on gene expression

46

The Genes of Interest: Cyclooxygenases

• Mainly inducible, e.g. inflammatory cytokines or LPS

Membrane phospholipids

Arachidonic acid

phospholipase A2

cox-2cox-1

Prostaglandin H2

prostaglandin synthases

PGI2 PGD2 PGE2 PGF2

• Two cyclooxygenase (Cox) genes code for the Cox-1 and Cox-2 proteins.

• Cox-1 and –2 synthesize prostaglandins

47

• Cox-1 – expressed all the time – essential to normal tissue function and repair; the ”good” Cox. – inhibition of Cox-1 is the reason for the toxic side effects (bleeding) of NSAID’s

(e.g. aspirin)• Cox-2

– inducible in most tissues, including the gastrointestinal tract– equally important as Cox-1– overexpression is associated with (or is a triggering event in) various

inflammatory and malignant diseases; the ”bad” Cox– Cox-2 inhibitors have a chemopreventive effect

The Roles of Cox-1 and Cox-2 in Healthand Disease

48

One possible mechanism could be that microbial metabolites

affect epithelial cyclooxygenase expression

Soluble metabolites

Prebiotics as Modulators of EpithelialCox-gene Expression

colonic fermentation

epithelial cell

function

Supernatants from polydextrose fed simulation model were obtained from the different stages of the simulator and applied to Caco-2 cells.Cells were then exposed for 24 hrs prior to isolating the RNA.

49

1. Cell exposure for 24 hours

2. RNA purification

3. RNA measurement by quantitative RT-PCR - Cyclooxygenase-2 expression was determined

Caco-2 cell-based simulation method

http://www.imi.org.uk/dec1998/botnon.jpg

http://www.tandem2.ru/images/ab_images/7000.jpg

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Expression of cox-2

0,000,200,400,600,801,001,201,401,601,80

Relat

ive ex

pres

sion

at 24

h

Polydextrose normalizes Cox-2expression in Caco-2 cells

No fiber 1 % Polydextrose 2% Polydextrose

• Polydextrose fermentation in the proximal colon does not influence cox-2 expression• Expression of cox-2 is decreased in the more distal colon, in vessels 2-4 • This implies that pdx can reduce risk for inflammation and carcinogenecity in the distal colon by reducing cox-2 gene activity• Reduction of risk for colon cancer development has also been observed in animal models (Ishizuka S et al. 2003 Nutr Res 23, 117-122)

51

Main Conclusions from ColonSimulator Studies

• Two in vitro techniques have been combined in order to study fermentation of prebiotics and interaction with gut mucosal cells in more detail

• The results provide a hypothesis of how a prebiotic, specifically polydextrose, can influence mucosal gene expression beneficially via colon fermentation reducing risk for for colon inflammation and cancer development

• These novel tools can be used e.g. to gain more insight to the structure-function relationship of prebiotics and to characterize further the role of gut microbes on colon health

52

Polydextrose as an effective prebiotic - Summary

• Passes intact to the colon• Fermented throughout the colon - increasing saccharolytic fermentation (reduces pH)• Stimulates Bifidobacteria• Enhances butyrate production• Does not cause acidosis (no accumulation of lactic acid)• By competition, reduces putrefactive fermentation (less branched VFAs, less biogenic amines

– reduced cancer risk)• Enhances mineral absorption• Stimulates immune system without causing inflammation• Reduces inflammation – Dose dependent reduction in COX 2 expression• Well tolerated• Soluble fibre effects• Good stability and versatility in foods

53

Reduced Colon Cancer RiskSubstantial evidence from animal studies

• Reduced tumour incidence in models where cancer inducing chemicals mimic the effect of toxic metabolites of food components

• Anti-cancer properties also observed in genetic pre-determined models such as apc min mouse (protective gene switched off)

• Anti-cancer effects demonstrated in tumour implantation models where advanced states of cancers are studied

• Possible mechanisms:- Suppression of DNA damage and increased repair- Stimulation of apoptosis in colon

(Van Loo J (2005) Food Science and Technology Bulletin: Functional Foods; 2:83-100. Van Loo J and Jonkers N (2001) Nutr Metab Cardiovasc Dis; 11,Suppl to No 4:87-93)

Based on the data from experimental data, a EU funded research project: the SYNCAN project QLK1-1999-00346 was initiated to evaluate whether a combination of pre- and probiotics may reduce the risk of colon cancer in humans.

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Study design• 22 young adults (15 female/ 5 male)• Polydextrose: 5 g/day • probiotic mixture: Lactobacillus GG, L. rhamnosus LC705, Propionibacterium shermannii JS and Bifidobacterium breve Bbi• Fecal sampling after each two-week periods: run-in, probiotic mixture, probiotic mixture supplemented with polydextrose (5 g/day) and wash-out• total counts of bifidobacteria were measured by plating

Period Mean S.D. pRun-in 7,0 2,2 NSProbiotic 7,6 2,0 NSPDX+Probiotic 8,9 2,5 <.001Follow-up 8,5 1,5 <0.05

Results shown as log10. Statistical significance to run-in with pairwise t-test. Detection limit = 3 log10.

Results: Probiotic mixture supplemented with polydextrose increased cultured bifidobacteria

Bifidogenic Activity in Humans (II)

Tiihonen et al (2007) in press.

Only the mixture of Litesse® Polydextrose with probiotics increased bifodo count significantly.

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Reduction of Aberrant Crypt Foci by Ingestion

of Polydextrose (PDX) in the Rat Colorectum

PDX administration is mosteffective against1,2-dimethylhydrazine (DMH)induced aberrant crypt foci (ACF) when feeding startsone week prior to DHM injection

PDX may play a role in the prevention of colon carcinogenesis.

day-7 0 1 7 35

Fiber-free xPDX-A xPDX-B xPDX-C xPDX-D x

x = DMH injection= PDX feeding

Ishizuka et al (2003) Nutr Res 23:117-122.

0

10

20

30

40

50

60

70

80

90

Fiber-free PDX-A PDX-B PDX-C PDX-D

Nu

mb

er

of

AC

F

**p < 0.05, N = 7

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Polydextrose – Approval Information

The FAO/WHO’s Joint Expert Committee on FoodAdditives (JECFA) review of a food additive is oftenregarded as the final word in the independent safetyassessment of any particular substance

Polydextrose was evaluated by JECFA at its 31st

meeting in 1986

Following this review they gave polydextrose an ADIof "Not Specified", which represents the safest categoryinto which JECFA are able to place a food additive

(JECFA) Joint FAO/WHO Expert Committee on Food and Additives(FAO) Food and Aricultural Orgainisation of the United Nations

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Burdock and Flamm (1999)This is a very comprehensive review of the data thatwas submitted to the US FDA in the original FoodAdditive Petition for the approval of polydextrose as afood additive

The safety of polydextrose was also affirmed by the USFDA via the publication of 21 CFR 172.841 permittingthe use of polydextrose in a wide variety of applicationsfollowing the GMP/Quantum satis principle(e.g. no numerical limit on use)

The review on toleration (Flood, Auerbach and Craig)also discusses some of the relevant studies

5757


Flood, MT, Auerbach, MH and Craig, SAS (2004)A review of the clinical toleration studies of polydextrosein food. Food and Chemical Toxicology 42: 1531-1542

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Polydextrose Latest FDA position

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European Commission Scientific Committee forFood(EC SCF) in 1990, who following a full review of thesafety, toxicological and tolerance data available,approved polydextrose as a bulking agent for use infoods at Quantum satis (e.g. it can be used at GMPlevels, without numerical limit) Polydextrose was subsequently approved for use atQuantum satis in food under Annex I of theMiscellaneous Additives Directive(which represents the broadest use category forfood additives within the EU

Polydextrose has been on the market for many yearsand has a history of safe use, and that production ofpolydextrose is conducted under the strictest principlesof HACCP, and follows ISO standard 9001:2000


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Polydextrose Gastrointestinal Toleration

Tolerance Threshold for Sugar Substitutes inNon-adapted Adults and Children (figures in brackets)

Substance Single Dose (g) Daily Dose (g)

Fructose 70 >90

Mannitol 10 -20

Sorbitol 20 (10) 50 (30)

Xylitol 20 (10) 50 (30)

Lactitol 25 40

Maltitol 30 50

Isomalt 30 (20) 50 (45)

Polydextrose 50 90 (20)

R Grossklaus (1990) Gesundheitliche Bewertung der Risiken durch Lebensmittelzusatzstoffe am Beispiel der Zuckeraustauschstoffe, Bundesgesundsheitsblatt 12/90 (Safety evaluation of the risks from food additives by examples of sugar substitutes)

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• Polydextrose has low caloric utilisation because it is poorly digested• Because it is poorly digested, excessive consumption can cause laxation symptoms in

sensitive individuals• Because laxation is an osmotic effect, and polydextrose has higher molecular weight

than the polyols, polydextrose has a higher laxation threshold than the polyols• Polydextrose laxation threshold is comparable in adults and children

No laxation dose in g (g/kg bw/day) Adults 50 (0.7) Children 20 (1.0)

• JECFA, 1987: “Studies in man have demonstrated that polydextrose, when administered at very high doses, exert a laxative effect, with a mean laxation threshold of 90g per day or 50g as a single dose”

• EC/SCF, 1990: “Large doses of polydextrose exert a laxative effect with a mean laxative threshold of 90g per day or 50g as a single dose

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Pfizer Studies: Clinical Toleration Studies of Polydextrose

Investigator Site Year No of Subjects Duration Highest Dose g single/daily

Diarrhoea Episodes

Alter Pfizer 1974 20 male adults 3 weeks 50 / 150 11pdx;5placebo

Knirsch Pfizer 1974 57 male adults 10 days 24/79 2 at 35g/day

McMahon Tulane Univ. 1974 10 type 2 diabetics Single dose 50/50 4pdx;2glucose

Raphan a Pfizer 1975 21 adults –11M/10F 10 days 43/130 none

Raphan b Pfizer 1975 51 adults – 31M/20F 12 weeks 20/60 1@45+60g/day

Bunde Hill Top Res. 1975 11 children 2-3yrs 6 weeks 10/15 4@15g/day

1975 11 children 4-6 yrs 6 weeks 10/20 1@20g/day



1975 12 children 13-16 yrs 6 weeks 20/55 1@30+55g/day

Scrimshaw & Young

MIT 1977 16 adults – 11M/5F 8 weeks 20/50 none

Beer Univ TX 1989 24 male adults Single dose 58/58 none

Curtis Harris Labs 1990 200 female adults Single dose 40/40 none

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(Total 57 Countries – 23/08/2007 )

Europe, Middle East, Africa Austria Belgium (7/88)* Czech Rep’ (2/96)Denmark Egypt* FinlandFrance* Germany* GibraltarGreece (5/95) Hungary Iceland Ireland Israel Italy 5

Luxembourg5 Netherlands1 Norway1*Poland (7/94)* Portugal5 Saudi ArabiaSlovakia South Africa Spain5

Sweden1 Switzerland TurkeyUnited Arab Emirates United Kingdom*1 Croatia

Asia, Americas, Others Argentina2 (3/93)* Australia*1,2 Brazil*Cambodia Canada ChilePR China (10/93)2* Colombia Costa RicaEl Salvador Guatemala HondurasHong Kong Indonesia Japan3,4*Korea (7/89)* Malaysia Mexico*New Zealand* (10/84) Peru Philippines3

Singapore* Taiwan2* ThailandUnited States2 * Uruguay Venezuela

Polydextrose Approvals

LEGEND:

Bold Face: polydextrose can

be sold

Italics: Reduced citric acid catalysis OK

Underline: Phosphoric acid catalysis OK * Can be labeled dietary fiber

1.Specific diabetic endorsement

2. Laxation label statement required (may have usage trigger)

3. Commercially accepted and

sold but not formally approved

4. Classified as food, not food additive

5. Approved via EU MAD, not individual member state legislation

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Regulation on Health claims Regulatory situation in Europe, principles

• Today there is no regulation on health claims in Europe andthe situation is very different from country to country .

• European commission wants an harmonisation of the conditions of use for Nutrition and Health claims in Europe.– Adoption and implementation within proposed time

frame

• Claims will be related to the products for end consumer

• All claims used will have to be authorised in advance*

* Various transition measures concerning products launched on the market before the implementation of the new regulation

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Evaluation byEFSA Commission

'2years

Procedure for "Articles 13 HEALTHclaims"

January 2007Application Procedure

Member State authorityor CIAA List

Submission to EFSA Commission

January 30th, 2008

Commission Draft decision

Standing Cte opinion

Final decision

The Commission shall adopt a positive list of claims within 3 years = 2010

Geoff O’SullivanApplication Manager

September 2007


With contributions from: Prof Glenn Gibson, University of Reading, Dr Nina Rautonen, Dr Artur Ouwerhand,Dr Kirsti Tiihonen, Dr Helen Mitchell, Dr Oliver Hasselwander and Dr Julian Stowell

Scientific Validation Of Polydextrose As A Fibre And

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