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Physiciochemical characteristics of pectin as

dietary fiber and its role in colonic health

Henk A. Schols

Laboratory of Food Chemistry

Primary cell wall - architecture

� Xyloglucan� Xylan� Galactan?

McCann et al. 1992 J. microsc., 166, 123-136

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Pectin plays an important role as:

�Cell Wall component

● Determine (partly) texture of tissues

● Change during ripening of e.g. fruits (endogenous enzymes)

● Change during processing due to depolymerization, de-esterification, solubilization, etc.

● Present in by-products from agro industry

� Ingredient for food industry

● Modification during extraction and down-stream processing

● Thickener and gelling agent

● Stabilizer in fruit and milk beverages

�Health promoting component

● Lowering cholesterol levels

● Dietary Fiber & fermentation

● Immuno modulating

Schematic structure of pectic polysaccharides

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soybean meal sugar beet pulp apple

total polysaccharide (w/w % dm) pectic substances (% of total PS)

16 59

67 40

20 42

structural element (% of pectic substances) homogalacturonan

xylogalacturonan rhamnogalacturonan II

rhamnogalacturonan backbone

arabinan arabinogalactan I

arabinogalactan II

0

21 4

15

60

0

29

<1 4

8

46 12

0

36

4 10

4

27 20

+

Occurrence and proportion of the various structural

elements of native pectin in apple, sugar beet and soybean

Same building blocks!!However, ‘always’ different fine-structure and different relative amounts!

�HM Pectin: sugar-acid gel

● Sugar lowers water activity and increase chain-chain interaction (= gel)

● Low pH reduces acidity – less interaction – more chain-chain interaction

� LM Pectin

● Calcium-pectate gel

● Low sugar jams

Pectin as food ingredient

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�Raw material used�Extraction conditions used

�Down stream modification (HM vs LM pectin, amidation)

�Molecular weight�Degree of methyl esterification

(and acetyl groups if present)

�Distribution of methyl esters(and acetyl groups if present)

�Calcium-sensitivity�Solubility

O

H

OH

OH

H

H

H

COOH

HO

O

H

OH

OH

H

H

H

COOCH3

HO

O

H

OH

OH

H

H

H

COOH

HO

O

H

OH

OH

H

H

H

COOH

HO

O

H

OH

OH

H

H

H

COOCH3

HO

O

H

OH

OH

H

H

H

COOCH3

HO

O

H

OH

OH

H

H

H

COOH

HO

O

H

OH

OH

H

H

H

COOCH3

HO

O

H

OH

OH

H

H

H

COOH

HO

O

H

OH

OH

H

H

H

COOH

HO

Parameters influencing food applications of pectins

Homogalacturonan key parameters:

Level and distribution of methyl esters

= Galacturonic acid (GalA) = methyl-GalA

Random

Sequential

Blockwise

Combi

Pectin functionalities are strongly determined

by level and distribution of the methyl esters

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Characterisation of pectins using enzymatic fingerprinting

Use of polygalacturonase (PG) and pectine lyase (PL) to degrade

pectins into diagnostic oligomers

PG: Release of saturated galacturonic acid oligomers

PL: Release of unsaturated galacturonic acid oligomers

Homogalacturonan:

Finger printing of methyl ester groups using PG

= Galacturonic acid (GalA) = methyl-GalA

Random

Sequential

Blockwise

Combi

= site of attack endo-PG

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42

53

64

62

1410

52

63

31

30

41

107

128

74

96

95

127

75

139

106

118 138

85

117

86

84

116

73

105

MALDI TOF MS of random esterified DM70 pectin digested by endo-PG

42= GalA4 2 methyl

HILIC-Iontrap-MSn method:methyl-esterified pectin oligomers

51= GalA5 1 methyl

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RT: 5.00 - 50.00 SM: 7B

5 10 15 20 25 30 35 40 45

Time (min)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

Inte

nsity

421

420200

521

410

520

411

300

410

511

621

831

611

721822

932

Unsaturated Saturated

HILIC-Iontrap-MSn method is also capable to separate

and identify complex mixtures of oligomers after PG

and PL digestion of Sugar Beet Pectin

Annotation: 410= 4 galacturonic acid unit; 1 methyl group. 0 acetyl groups

O-2

0,2A5

C4 + 1Me+ 1AcC2C1

C3 + 1Ac

Z1Z2 + 1MeZ3 + 1Me + 1AcZ4 + 1Me + 1Ac

O

HO

HOOH

COOH

O

H

HOOH

COOH

O

O

HOOAc

COOH

O

H

HOOH

COOMe

OO

O

H

HO

O-

OH

COOH

O

0,2A4 0,2A3 + 1Ac0,2A2

(365)(583)(759)

(309)(485)

(893)

(777)

(717)

(587)(369)

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lativ

e A

bu

nd

an

ce

583

365 587

777369

759893

351 485309541 935

717

Z2 + 1Me

C2

Z3 + 1Me + 1Ac

C3 + 1Ac

C4 + 1Me+ 1Ac

Z4 + 1Me + 1Ac0,2A5

[M- H]-1 – H2O

Z2 0,2A3

0,2A4

Z3 + 1Me

0,2A2

B

481291

O-3

511 (m/z 953)

Precise annotation of methyl ester and O-2 and O-3 acetyl group position by ring cleavage MS/MS fragmentation

HILIC-MS/MS fragmentation enables the structure

elucidation of SBP oligomers

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GalA O-2 Acetyl O-3 Acetyl Methylester

PG PL

DBabs

DHPG DHPL

DB does quantify unsubstituted mono-, di- and tri GalA oligomers

as released by PG

DHPG does also include PG released methyl esterified segments of the

pectin

DHPL does quantify PL released highly methyl esterified / acetylated

oligomers from the pectin

Introduction of descriptive parameters

Degree of Blockiness and Degree of Hydrolysis

Official AACC definition of dietary fibre

�Dietary fibre is the edible parts of plants or analogous carbohydrates that are resistant to digestion and adsorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fibre includes polysaccharides, oligosaccharides, lignin and associated plant substances. Dietary fibres promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation, and/or blood glucose attenuation.

Pectin is a dietary fibre

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Batch fermentation of DF

+ Simple, easy to prepare, ‘one bottle’ fermentation.+ Buffer-regulated pH+ Possibility to do fermentation with small amount of substrate.+ Fast, a lot of substrates can be tested simultaneously.

- Change of pH during fermentation- Accumulation of fermentation products- Depletion of substrates

Monitoring in vitro fermentation (human fecal inoculum)

� Optical Density

� pH curve

� Short Chain Fatty Acids

� Remaining dietary fibers (after hydrolysis)

� NEED TO ANALYSE INDIVIDUAL MOLECULES

Optical Density of various substrates Soy pectin

4.04.55.05.56.06.57.07.58.0

0 10 20 30 40 50Time (h)

pH

pH curve of soy pectin(different buffer systems)

SCFA profiles of Oligofructose

0

10

20

30

40

50

60

70

0 10 20 30 40 50Time (h)

Co

nc

en

tra

tio

n

(um

ol/m

l)

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

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In vivo piglet trial

Soluble pectin supplementation to diet

citrus pectin

wheat based diet +

Soy bean meal

+

autoclaved

soybean meal

wheat based diet

+

LMP/HMP3%

wheat AX

+ +

Soy pectin 3%

wheat AX

+

TiO20.25%

TiO20.25%

+ +

Sample and data collection

Gastrointestinal tract of pig (Stevens et al., 1998).

Analysis of :

� Dietary Fibre + fragments

� Short Chain Fatty Acids

� Digestibility marker

� Microbiota composition

Digesta present in:

� Ileum

� Proximal colon (pCol)

� Mid colon (mCol)

� Distal colon (dCol)

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Constituent monosaccharide composition

of total NSP in the digesta

Constituent monosaccharide composition

of total NSP in the digesta

� Starch remains more dominantly present in pectin diets

� Digestibility depends on pectin methyl esterification and complexity

� Different dietary fibers are fermented differently

� For quan4ta4ve data → diges4bility marker was added

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Apparent digestibility of pectin

� Pectin is highly fermentable.

� Fermentation profile is different for LM and HM pectin

� Although same soy pectin structures are present in CONT and aSBM,

pec4ns present are differently fermented → soluble vs insoluble soy pec4n

Apparent digestibility of starch and Dietary

Fibre (NSP)

� Starch digestion is slightly lower in presence of pectin

� Digestibility depends on pectin methyl esterification

� Also Dietary Fibre (NSP) fermentation is postponed to

distal part of colon in the presence of pectin

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Apparent digestibility of glucose and xylose

(for cellulose and cereal xylan respectively)

� Cellulose and Xylan fermentation is postponed to

the more distal part of colon in the presence of pectin

� Differences in apparent fermentability are smaller for the

feces!

Summary: w/w percentage of digested

starch, pectin and NSP in different sections

CONT: control diet;

LMP: low methyl-esterified pectin enriched diet;

HMP: high methyl-esterified pectin enriched diet;

aSBM: autoclaved soybean meal enriched diet.

DF Diet

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Concentration of SCFAs in digesta of

piglets fed with different diets

Relative abundance of microbiota at the

genus level

Lactobacillus

Prevotella

U(Ruminococcaceae)

Megasphaera

Microbiota pattern, typical for plant eaters

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Relative abundance of microbiota at the

genus level

Lactobacillus ↓

Prevotella ↑

U(Ruminococcaceae) ↑

Megasphaera ↑

Link between microbiota, SCFAs and fiber

digestibility

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Link between microbiota, SCFAs and fiber

digestibility

� Colonic digesta from LMP- and HMP- and

aSBM-fed pigs clustered separately from the

samples obtained from CONT-fed pigs.

� Prevotella, Dialister and unclassified microbes

in the family Ruminococcaceae correlated with

the LMP diet.

� Prevotella in the colonic digesta of LMP- and HMP-fed pigs correlated with

the production of butyrate and propionate.

� The digestibility of uronic acid correlated with the production of butyrate and

genera Prevotella, Dialister and unclassified microbes in the family

Ruminococcaceae.

Conclusions

� Pectins were rapidly fermented in intestinal tract of piglets

� Different fermentation characteristics for pectins,

depending on methyl esterification

� Solubilized soy pectin was differently fermented than

untreated/insoluble soy pectin

� Supplementation of pectins shifted the fermentation site

of other DFs to more distal parts

� Starch mainly utilized in small intestine! However,

fermentation speed may depend on other fibres present

� Pectin supplementation could be a strategy to control

fibre fermentability over the entire colon

� Pectin also have shown to influence the immune system !

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Thank you for your attention !

Acknowledgements

Animal Nutrition WUCarol Souza da SilvaGuido Bosch

Food Chemistry WUMelliana Jonathan Christiane RoschLingmin Tian Harry Gruppen

Host Microbe Interactomics WUMarjolein Meijerink Nico TaverneJerry Wells

Microbiology WUKlaudyna Borewicz Hauke Smidt

University Medical Center GroningenNeha Sahasrabudhe Leonie VogtPaul de Vos

UMCG

Pectin immune modulating properties

TLR2 TLR1

Immune

stimuli

NF-κB

Pro-inflammatory

cytokine

Cytoplasm

Nucleus

TLR1

Immune

stimuli

NF-κB

Pro-inflammatory

signals

Bioactive

component

Cytoplasm

Nucleus

TLR2

Different for different pectins

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Pectin does not activate TLR2, TLR4 and TLR5.

TLR activation represented as NF-κB activation of HEK-BlueTM TLR

Neha SahasrabudhePhD thesis University Medical Center GroningenExperimental studies on dietary fibers– Pattern recognition receptor interactions

Low DM pectin inhibits TLR2-

TLR1 by directly binding to

the TLR2 ectodomain

42

Low DM pectin reduces the

doxorubicin induced

inflammation in mice

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Increase of immune activity during the in vitro batch fermentation of sugar beet pectin

0 4 12 24 480

2

4

6

8

fermentation time (h)

mg

SB

P/

mL

dig

est

a

Amounts of soluble (black) and

insoluble (white) sugar beet pectin

present in batch fermentation

digesta

Increase of immune activity during the in vitro batch fermentation of sugar beet pectin

HPSEC polysaccharide profile of SBP

(0, 6, 12, 24, 48 h) fermentation

digesta

TNF-αcytokine production by Bone

Marrow Dendritic Cells of TLR2/4

knockout mice

FERMENTATIONTIME