TOWARDS A BETTER U�DERSTA�DI�G OF THE PECTI� STRUCTURE-

FU�CTIO� RELATIO�SHIP I� BROCCOLI PUREES

Katholieke

Universiteit

Leuven

FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME

MASTER OF FOOD TECHNOLOGY (IUPFOOD)

Promotor: Prof.Dr.ir. M. Hendrickx

Department of Microbial and Molecular Systems.

Centre for Food and Microbial Technology.

Laboratory of Food Technology.

JUNE 2011

Master dissertation submitted in partial

fulfillment of the requirements for the Degree

of Master in Food Technology.

By: Baiye Mfortaw Mbong Victor

This dissertation is part of the examination and has not been corrected for eventual errors

after presentation. Use as a reference is only permitted after consulting the promoter, stated

on the front page.

TOWARDS A BETTER U�DERSTA�DI�G OF THE PECTI� STRUCTURE-

FU�CTIO� RELATIO�SHIP I� BROCCOLI PUREES

Katholieke

Universiteit

Leuven

FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME

MASTER OF FOOD TECHNOLOGY (IUPFOOD)

Promotor: Prof.Dr.ir. M. Hendrickx

Department of Microbial and Molecular Systems.

Centre for Food and Microbial Technology.

Laboratory of Food Technology.

JUNE 2011

Master dissertation submitted in partial

fulfillment of the requirements for the

Degree of Master in Food Technology.

By: Baiye Mfortaw Mbong Victor

ACK�OWLEDGEME�T

I would like to express my sincere and deepest gratitude to Prof. Marc Hendrickx and Prof.

Ann Van Loey for their advices and support and for providing me with all the necessary

facilities necessary to carry out my master thesis in the Laboratory of Food Technology.

My profound gratitude goes to Stefanie Christiaens for her supervision and tolerance. She was

more a friend than a supervisor. Even though she was very busy doing other things, she was

always present to share her knowledge by giving helpful suggestions and above all, she seems

never angry when something went wrong with the experiment. Thanks for your patience.

I would like to appreciate the administrative and technical staff, together with Doctoral

students and Post-doctoral researchers of the Laboratory of Food Technology, for their

constant encouragement and moral support throughout my study period.

Special thanks to the entire staff of the Department of Food safety and food quality of Ghent

University, and my colleagues for their support.

My sincere appreciation to the Flemish Interuniversity Council (Vlir-UOS) for granting the

fellowship to do a Master study in Food Technology in Belgium.

I would like to acknowledge my loving family and friends far and near for their unfailing love

and constant prayers so that my study in Belgium should be a success.

.

i

ABSTRACT

The role of pectin in the consistency of vegetable purées, an important quality attribute of

these plant-based food products, is largely unknown. Therefore, the structure-function

relationship of pectin was investigated in broccoli (Brassica oleracea L. cultivar italica)

purées. The effects of pretreatments, low temperature blanching (LTB) and high temperature

blanching (HTB), high pressure homogenization (HPH), and cooking on the macroscopic

properties of broccoli purée were investigated. The macroscopic characteristics of the

differently treated broccoli purées were expressed in terms of their consistency and particle

size distribution. These attributes were linked to the chemical structure of broccoli pectin.

Pectin was fractionated according to its solubility in water-soluble pectin (WSP), chelator-

soluble pectin (CSP), sodium carbonate-soluble pectin (NSP) and quantified. The degree of

methyl-esterification (DM) of pectin, WSP and CSP was determined and the molar mass

distribution of the different pectin fractions was investigated.

Syneresis (the spontaneous separation of the solid and liquid components of a purée) of the

broccoli purée did not occur when the purée was high pressure homogenized or when a large

amount of WSP was induced. Generally, the consistency of the broccoli purées increased after

cooking due to increased solubilization of pectin. After HPH, the consistency of the

differently high pressure homogenized purées decreased due to the formation of more small

particles. LTB resulted in a lower DM of the pectin, more Ca2+cross-linked pectin (CSP) and

an increased amount of large particles. When the purées were subjected to HTB, a small

increase in the solubility of the pectin was observed. Also, an increase in the consistency and

a decrease in the particle size distribution were often observed after HTB.

Anti-pectin antibodies were used to perform in situ (microscopy) and ex situ (immunodot

assays) analyses on the differently treated broccoli purées. It was established that water-

soluble pectin consisted of unbranched pectin which was highly esterified whereas the pectic

polymers that were less-esterified had abundant side chains that were less esterified. Ionically

cross-linked pectin, on the other hand, contained pectin with a rather broad range in DM, with

the low- and high esterified pectins occuring in either highly branched or unbranched domains

of pectin. The in situ visualization of pectin in the differently treated broccoli samples showed

ii

the importance of the regions of the cell wall lining intercellular spaces. Pectin with a broad

range in the DM and also Ca2+ cross-linked pectin were observed in this cell region.

iii

Table of contents

ABSTRACT .............................................................................................................................................. i

Part I: Literature review ........................................................................................................................ xvi

1 Pectins .................................................................................................................................................. 1

1.1 Introduction ................................................................................................................................... 1

1.2 Chemical structure of pectin ......................................................................................................... 1

1.2.1 Homogalacturonan ................................................................................................................. 2

1.2.2 Rhamnogalacturonan-I .......................................................................................................... 4

1.2.3 Rhamnogalacturonan-II ......................................................................................................... 5

1.3 Mechanisms of pectin structural modifications (pectin conversion reactions) ............................. 5

1.3.1. Enzymatic pectin conversions ............................................................................................... 6

1.3.1.1 Enzymatic de-methoxylation ...................................................................................... 7

1.3.1.2 Enzymatic depolymerization ...................................................................................... 9

1.3.2 on-enzymatic pectin conversions....................................................................................... 11

1.3.2.1 Chemical de-methoxylation ...................................................................................... 11

1.3.2.2 Chemical depolymerization ....................................................................................... 11

1.4 Cross-linking mechanisms of pectin ........................................................................................... 14

1.4.1 Ca2+

cross-linked pectin ..................................................................................................... 14

1.4.2 Borate-ester cross-linked pectin .......................................................................................... 15

1.4.3 Uronyl ester cross-linked pectin. ......................................................................................... 16

1.5 Anti-pectin antibodies ................................................................................................................. 17

2 Texture/rheology of plant-based foods ............................................................................................... 21

2.1 Cell-wall composition in relation to mechanical properties ........................................................ 21

2.1.1 The plant cell ....................................................................................................................... 22

iv

2.1.2 Cell wall components .......................................................................................................... 23

2.1.2.1 Cellulose ................................................................................................................... 23

2.1.2.2 Hemicellulose ........................................................................................................... 23

2.1.2.3 Pectin ........................................................................................................................ 23

2.1.2.4 Proteins and glycoproteins ....................................................................................... 24

2.1.2.5 Water ........................................................................................................................ 24

2.2 Textural/rheological characteristics of (processed) fruits and vegetables ................................... 24

2.3 Microstructure and rheological properties of plant based foods ................................................. 26

2.4 Pectin structure-function relationship ......................................................................................... 27

2.5 Objective of this thesis ................................................................................................................ 28

Part II: Experimental work .................................................................................................................... 29

3 Materials and methods ....................................................................................................................... 30

3.1 Experimental set-up..................................................................................................................... 30

3.2 Vegetable material: broccoli ....................................................................................................... 31

3.3 Preparation of broccoli purées ..................................................................................................... 31

3.3.1 Pretreatments........................................................................................................................ 31

3.3.1.1 Low temperature blanching (LTB) ............................................................................. 31

3.3.1.2 High temperature blanching (HTB) ............................................................................ 31

3.3.2 High pressure homogenization (HPH) ................................................................................. 31

3.3.3 Cooking ................................................................................................................................. 32

3.4 Bostwick rheometry .................................................................................................................... 33

3.5 Particle size distribution (wet sieving) ........................................................................................ 33

3.6 Extraction of alcohol insoluble residue (AIR) ............................................................................ 34

3.7 Determination of the degree of methyl-esterification (DM) ....................................................... 35

v

3.7.1 Determination of galacturonic acid (GalA) content ........................................................... 35

3.7.2 Determination of methyl-ester groups ................................................................................ 36

3.8 Fractionation of AIR ................................................................................................................... 37

3.9 Molar mass distribution ............................................................................................................... 38

3.9.1 Lyophilization (freeze-drying) and dialysis ........................................................................ 38

3.9.2 High Performance Size-Exclusion Chromatography (HPSEC) .......................................... 38

3.10 Immunodot assays ..................................................................................................................... 40

3.11 Microscopic analysis with anti-pectin antibodies ..................................................................... 40

4 Results and discussion ........................................................................................................................ 42

4.1 Bostwick consistency of broccoli purées .................................................................................... 42

4.2 Particle size distribution of broccoli purées ................................................................................ 43

4.3 Degree of methyl-esterification (DM) of broccoli purées ........................................................... 46

4.4 Changes in pectin solubility ........................................................................................................ 49

4.4.1 Changes in water soluble pectin (WSP) ............................................................................... 49

4.4.2 Changes in chelator soluble pectin (CSP) ........................................................................... 50

4.4.3 Changes in sodium carbonate soluble pectin ( SP) ............................................................ 51

4.4.4 Changes in residue pectin .................................................................................................... 52

4.4.5 Fractionation yield ............................................................................................................... 52

4.4.6 Changes in pectin solubility: summary ................................................................................ 53

4.5 Degree of methyl-esterification (DM) of WSP and CSP fractions ............................................. 54

4.6 Changes in the molar mass (MM) distribution of different pectin fractions ................................ 55

4.7 Immunodot assays of WSP and CSP fractions ............................................................................. 60

4.7.1 Binding of anti-HG antibodies to WSP of broccoli purées .................................................. 60

4.7.2 Binding of anti-HG antibodies to CSP of broccoli purées ................................................... 62

vi

4.8 In situ immunolabeling of pectin with anti-pectin antibodies ..................................................... 64

4.8.1 Immunolabeling of non-cooked broccoli purées .................................................................. 64

4.8.2 Immunolabeling of cooked broccoli purées ......................................................................... 66

4.8.3 Localization of Ca2+

cross-linked pectin .............................................................................. 68

General conclusion ................................................................................................................................ 70

REFERENCES ...................................................................................................................................... 72

vii

LIST OF FIGURES

Figure 1.1: Schematic representation of (A) the conventional structure of pectin and (B) the

alternative structure of pectin (Vincken et al., 2003; Willats et al., 2006).

Figure 1.2: The structure of HG (Ridley et al., 2001).

Figure 1.3: Schematic presentation of possible pectin (only homogalacturonan) conversion

reactions in plant-based foods and possible routes for tailoring quality parameters: PME =

pectinmethylesterase, Ca2+ = calcium cross-linking, PG = polygalacturonase, PL = pectate

lyase, T = temperature (Sila et al., 2009).

Figure 1.4: Blockwise de-esterification pattern produced by plant PME (Limberg et al.,

2000).

Figure 1.5: Random distribution of de-esterified GalA by fungal PME (Markovic et al.,

1984).

Figure 1.6: Schematic representation of enzymatic digestion of pectin with endo- PG from

Kluveromyces fragilis (Daas et al., 1999).

Figure 1.7: Completely random de-methoxylation pattern by alkaline saponification

(Limberg et al., 2000).

Figure 1.8: β-eliminative depolymerisation reaction mechanism (Bemiller et al., 1972).

Figure 1.9: Schematic representation of Ca2+ binding to polygalacturonate sequences: ‘egg

box’ dimer (Vincken et al., 2003).

Figure 1.10: The borate 1,2-diol ester that cross-links two monomeric units of RG-II (Ridley

et al., 2001).

Figure 1.11: The uronyl ester cross-linked pectin (Vincken et al., 2003).

Figure 1.12: The antibody structure (Mian et al., 1991).

viii

Figure 1.13: Anti-HG antibodies. (I) Schematic representation of epitopes of some anti-HG

antibodies. (II) Type, antigen and primary references. (Willats et al., 2006, Christiaens et al.,

2011).

Figure 1.14: Immuno-dot-assays of JIM5, JIM7, LM18, LM19, LM20 and PAM1 binding to citrus

pectins with defined DE and DBabs. Highly methyl-esterified citrus pectin (DE=94%) was de-esterified

with carrot PME (P-series), Aspergillus aculeatus PME (F-series) and NaOH (C-series).

Figure 2.1: The hierarchy of structures (Waldron et al., 2003).

Figure 2.2: The plant cell structure.

Figure 2.3: The morphology of a plant cell (Van Buggenhout et al., 2009).

Figure 3.1: Experimental setup; LTB = low temperature blanching for 40 minutes at 60oC;

HTB = high temperature blanching for 5 minutes at 95oC; HPH = high pressure

homogenization (100 bar).

Figure 3.2: High pressure homogenizator.

Figure 3.3: Bostwick consistometer.

Figure 3.4: Wet sieving device.

Figure 3.5: Principle of HPSEC (Lathe et al., 1956).

Figure 4.1: Overview of the Bostwick consistency of the differently treated broccoli purées.

Mean values (n=2) with standard deviations are shown.

Figure 4.2: Particle size distribution of the blended broccoli purées.

Figure 4.3: Particle size distribution of the blended and cooked broccoli purées.

Figure 4.4: Particle size distribution of the high pressure homogenized broccoli purées.

Figure 4.5: Particle size distribution of the high pressure homogenized and cooked broccoli

purées.

Figure 4.6: Standard curve for determining the GalA content: absorbance at 520 nm and 25oC

as a function of the concentration of GalA (µg /ml H2O).

ix

Figure 4.7: Standard curve for determining the amount of methyl-ester groups: absorbance at

412 nm and 25oC as a function of the concentration methanol (µg/ml phosphate buffer).

Figure 4.8: Degree of methyl-esterification (DM) of differently treated broccoli purées. Mean

values (n=6) with standard deviations are shown.

Figure 4.9: Changes in the water soluble pectin fraction. The percentage of WSP is defined

as the GalA content of WSP compared to the GalA content in WSP, CSP, NSP and residue.

Mean values (n=6) with standard deviations are shown.

Figure 4.10: Changes in the chelator soluble pectin fraction. The percentage of CSP is

defined as the GalA content of CSP compared to the GalA content in WSP, CSP, NSP and

residue. Mean values (n=6) with standard deviations are shown.

Figure 4.11: Changes in the sodium carbonate soluble pectin fraction. The percentage of NSP

is defined as the GalA content of NSP compared to the GalA content in WSP, CSP, NSP and

residue. Mean values (n=6) with standard deviations are shown.

Figure 4.12: Changes in the residue fraction. The percentage of residue is defined as the

GalA content of residue compared to the GalA content in WSP, CSP, NSP and residue. Mean

values (n=6) with standard deviations are shown.

Figure 4.13: Fractionation yield (%) calculated by the percentage of GalA in WSP, CSP,

NSP and residue compared to the total amount of GalA in AIR.

Figure 4.14: DM (%) of WSP and CSP fractions relative to the DM (%) of the AIR. Mean

values (n=6) with standard deviations are shown.

Figure 4.15: Comparison of the molar mass (MM) distribution of WSP, CSP and NSP of the

raw blended broccoli purée. Elution times of pullulan standards are indicated to allow for a

rough estimation of the MM.

Figure 4.16: Comparison of the molar mass (MM) distribution of the WSP of A) the blended

broccoli purées and B) the high pressure homogenized broccoli purées. Elution times of

pullulan standards are indicated to allow for a rough estimation of the MM.

x

Figure 4.17: Comparison of the molar mass (MM) distribution of the CSP of A) the non-

cooked broccoli purées and B) the cooked broccoli purées. Elution times of pullulan standards

are indicated to allow for a rough estimation of the MM.

Figure 4.18: Immunodot assays showing the binding of the anti-HG antibodies JIM5, JIM7,

LM18, LM19, LM20 and PAM1 to the WSP fraction of the differently treated broccoli

purées.

Figure 4.19: Immunodot assays showing the binding of the anti-HG antibodies JIM5, JIM7,

LM18, LM19, LM20 and PAM1 to the CSP fraction of the differently treated broccoli purées.

Figure 4.20: Representative pictures showing the binding of the anti-HG antibodies JIM5,

JIM7, LM18, LM19 and LM20 to the 80 – 125 µm size fraction of the differently non-cooked

blended broccoli purées. Scale bars = 50 µm.

Figure 4.21: Representative pictures showing the binding of the anti-HG antibodies JIM5,

JIM7, LM18, LM19 and LM20 to the 80 - 125 µm size fractions of the differently cooked

blended broccoli purées. Scale bars = 50 µm.

Figure 4.22: Representative pictures showing the binding of the anti-HG antibody 2F4 to the

80 - 125 µm size fraction of the differently non-cooked and cooked blended broccoli purées.

Scale bars = 50 µm.

xi

LIST OF TABLES

Table 1: Classification of textural characteristics (Waldron et al., 2003).

xii

LIST OF ABBREVIATIO�S

AIR = alcohol insoluble residue

CSP = chelator-soluble pectin

DB = degree of blockiness

DBabs = absolute degree of blockiness

DM = degree of methyl-esterification

FITC = fluorescein isothiocyanate

GalA = galacturonic acid

HG = homogalacturonan

HPH = high pressure homogenization / high pressure homogenized

HPSEC = high-performance size exclusion chromatography

HTB = high temperature blanching / high temperature blanched

LTB = low temperature blanching / low temperature blanched

MAbs = monoclonal antibodies

MM = molar mass

MPBS = phosphate-buffered saline containing milk powder

NSP = sodium carbonate-soluble pectin

PBS = phosphate-buffered saline

PL = pectin lyase

xiii

PG = polygalacturonase

PME = pectin methylesterase

PMEI = pectin methylesterase inhibitor

RG-I = rhamnogalacturonan-I

RG-II = rhamnogalacturonan-II

WSP = water-soluble pectin

xiv

GE�ERAL I�TRODUCTIO�

A daily intake of vegetables of around 600 g per meal is recommended by different health

organizations; however, few people manage to consume this amount. Led by consumer

demand, the food industry has shown an increased interest in the manufacture of healthier and

more natural vegetable food products, such as purées. The vegetable tissue, broccoli (Brassica

oleracea L. Var. italica) is frequently used in these applications, and was selected for this

thesis study as a model system with high nutrient content and interesting plant physiology.

The rheological and textural properties of this kind of plant-based food products are important

quality attributes because they determine consumer demand and acceptability to a large

extent. Therefore, understanding the influence of processing on microstructure and rheology

of this material will help to design food products with improved organoleptic.

Processes aiming to alter the texture and structure of processed vegetables often focus on

pectin. This cell wall polysaccharide plays an important role in this context because

rheological and textural changes taking place during vegetable processing can be (partially)

ascribed to enzymatic and/or chemical conversions of pectin. Therefore, in-depth insight into

the structure-function relationship of pectin is thus necessary to tailor the rheological and

textural properties of plant-based food products.

Assessing the pectin structure-function relationship has, so far, predominantly been performed

using ex situ analysis techniques. However, several recent advances in pectin analysis now

make it possible to establish the in situ localization of specific pectin epitopes by means of

anti-pectin antibodies. In this thesis study, both methods will be combined to evaluate the

impact of processing on pectin in broccoli purées.

This thesis has been divided into four chapters. The first two chapters give an overview of the

relevant literature. The first chapter gives a detailed explanation of pectin and its chemical and

enzymatic conversions during processing of plant-based food products. A brief explanation of

the various anti-pectin antibodies is also included in the first chapter. The second chapter

deals with the relationship between the texture and rheology of plant-based foods and the

plant structure (cell wall and cell wall components), together with pectin structure-function

relationship. Chapters three and four contain the experimental part of this thesis. An attempt

xv

was made to better understand the pectin structure-function relationship in broccoli purées. In

chapter three, all materials and methods used to perform the experiments were discussed.

Chapter four contains the results and a discussion of these results.

xvi

Part I: Literature review

Chapter 1: Pectins

1

1 Pectins

1.1 Introduction

Pectin is the most structurally complex family of polysaccharides in nature, making up to

approximately 35% of primary walls of dicots and non-graminaceous monocots, 2-10% of

grass and other commelinoid primary walls and up to 5% of walls of woody tissue (Mohnen

et al., 2008). Pectin is abundant in walls that surround growing and dividing cells, walls of

cells in the soft part of the plant, and in the middle lamella and cell corners (Waldron et al.,

2003).

A variety of lines of evidence indicate a role for pectin in plant growth, development,

morphogenesis, defense, cell wall adhesion, wall structure, signaling, cell expansion, wall

porosity, binding of ions, growth factors and enzymes, pollen tube growth, seed hydration,

leaf abscission, and fruit development (Mohnen et al., 2008). Pectin is also used as a gelling

and stabilizing agent in the food and cosmetic industries. Pectin is also used in the production

of a variety of multiple specialty products including edible and bio-degradable films,

adhesives, paper substitutes, foams and plasticizers, surface modifiers for medical devices,

materials for biomedical implantation, and for drug delivery (Sila et al., 2009).

In this thesis, the focus will lay on pectin’s role in the texture or theological properties of

plant-based food products.

1.2 Chemical structure of pectin

Pectin is a family of complex polysaccharides that contains α-(1–4) linked-D-galacturonic

acid (GalA) residues. Three pectic polysaccharides have been isolated from primary cell walls

and were structurally characterized. These are: homogalacturonan (HG), which constitutes the

backbone of pectin (Bemiller et al., 1986), and rhamnogalacturonan-I (RG-I) and

rhamnogalacturonan-II (RG-II), which make up the side chains (Albersheim et al., 1960;

O’Neill et al., 1990) according to the conventional model (Figure 1.1A).

Chapter 1: Pectins

2

Vincken et al. (2003) however presented an alternative structure in which RG-I is the

‘backbone’ of pectin polymers (Figure 1.1B). Nowadays, the conventional model of pectin

structure has been readopted (Caffall et al., 2009).

Figure 1.1: Schematic representation of (A) the conventional structure of pectin and (B) the

alternative structure of pectin (Vincken et al., 2003; Willats et al., 2006).

1.2.1 Homogalacturonan

Homogalacturonan (HG) is a polymer of α-(1–4) linked-D-GalA residues with a maximum

length of approximately 72 to 100 GalA residues (Thibault et al., 1993; Van Buggenhout et

al., 2009). These GalA residues can be methyl-esterified at C-6 and/or acetylated on O-2

and/or O-3 (Mohnen et al., 1999; Vincken et al., 2003) (Figure 1.2). HG is referred to as the

‘smooth region’of pectin (Schols et al., 1995).

Chapter 1: Pectins

3

Some substitutions of HG are important structural features which define the functional

properties of pectin (Voragen et al., 1995). The amount of methyl-ester groups per 100

galacturonic acid residues is expressed as the degree of methyl-esterification (DM). Although

the DM has been considered to be the main structural feature of pectin, the pattern of methyl-

ester distribution has recently been found to be of great relevance in the functionality of the

polymer (Daas et al., 2000; Korner et al., 1998; Limberg et al., 2000). Other structural

features of pectin include the molar mass distribution profile, degree of acetylation and degree

of branching.

Naturally, the amount of methyl-esters present on C-6 of GalA units in HG varies from one

plant to another. Based on the DM, pectins are classified in two categories: high-methyl

pectin and low-methyl pectin. The latter encompasses pectins with a DM < 50% while the

former only includes pectins whose DM is > 50%. High-methyl pectins are formed naturally

in the golgi-apparatus (Ridley et al., 2001).

At intra-chain level, the degree of blockiness (DB) is used to elaborate the distribution of non-

methoxylated GalA residues on the HG chain (DeVries et al., 1986) and enables comparison

of methoxylation pattern between different pectin samples (Guillotin et al., 2005; Ström et al.,

2007). It expresses the total amount of non-esterified GalA liberated by endo-

polygalacturonase as a percentage to the total number of non-esterified GalA present in pectin

(Daas et al., 1999, Ström et al., 2007). The absolute degree of blockiness (DBabs) relates the

amount of non-methyl-esterified GalA residues liberated by endo-polygalacturonase to the

total number of non-esterified and esterified galacturonic acid residues in pectin (Daas et al.,

1999).

Chapter 1: Pectins

4

1.2.2 Rhamnogalacturonan-I

Rhamnogalacturonan–I (RG-I), contains a backbone of the repeating disaccharide [→4)–α–

D–GalA–(1→2)–α–L–Rha–(1→]. GalA residues may be O-acetylated on C-2 and/or C-3

(Komalavilas et al., 1989). However, there is no conclusive chemical evidence that the GalA

residues in RG-I are methyl-esterified (Rihouey et al., 1995).

Depending on the plant source and method of isolation, 20-80% of the rhamnosyl residues are

substituted at C-4 with neutral oligosaccharide side chains (O’Neill et al., 1990). Although

their relative proportions and chain lengths may differ depending on the plant source, the

predorminant side chains contain linear and branched α-L-arabinofuranosyl, and/or β-D-

galactopyranosyl residues.

RG-I is believed to play an important role in determining both the structural and biological

functions of the cell wall (Oomen et al., 2002). However, the structure-function relation of

RG-I in processed foods is not well known (Ridley et al., 2001; Willats et al., 2001).

Figure 1.2: The structure of HG (Ridley et al., 2001).

Chapter 1: Pectins

5

1.2.3 Rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a substituted galacturonan present in all higher plant

primary cell walls analyzed to date (O’Neill et al., 1990). The demonstration that wine and

other fruit juices contain relatively high amounts (20-150 mg/l) of RG-II (Doco et al., 1997),

that RG-II binds heavy-metals (Pellerin et al., 1997; Pellerin and O’Neill, 1998; Szpunar et

al., 1999; Tahiri et al., 2000), and that RG-II has immune-modulating activities (Shin et al.,

1998) has led to a greater interest in the structure of RG-II and to the role of this enigmatic

pectic polysaccharide in human nutrition and health.

In contrast to HG and RG-I, which are notable for their heterogeneity, RG-II is highly

conserved with little or no structural diversity. It has a backbone composed of 9 GalA

residues that are α-(1-4)-linked on which 4 heteropolymeric side chains are attached (Ridley

et al., 2001; Vidal et al., 2000). These side chains contain eleven different monosaccharides

(O’Neill et al., 1996; Vidal et al., 2000). Reports have shown that in vivo; RG-II exists as a

cross-linked dimer covalently linked through borate – diol esters (Kobayashi et al., 1996;

O’Neill et al., 2001; Vidal et al., 2000). This feature is important in the formation of a

macromolecular pectic network, thus improving some mechanical properties of plant cells.

1.3 Mechanisms of pectin structural modifications (pectin conversion reactions)

Pectin conversion mechanisms are of high interest to food scientists (Sila et al., 2009), in

order to better understand biochemical and physiological changes in plant-based foods,

including possible structure–function tailoring. Pectin can be degraded in planta by

endogenous and/or exogenous (pathogenic) enzymes, as well as by postharvest storage and/or

processing dependent non-enzymatic conversion reactions. Only the well-known HG

conversion mechanisms are indicated in Figure 1.3, despite the possibility of many other

pectin conversion reactions.

Chapter 1: Pectins

6

1.3.1. Enzymatic pectin conversions

A wide range of endogenous and exogenous enzymes can synergistically modify and degrade

pectin smooth and hairy regions. In the smooth region, the enzymes involved can either be

esterases and/or depolymerases. In the hairy region, the enzymes catalyzing the degradation

of the highly branched rhamnogalacturonan regions of pectin are, for example,

Figure 1.3: Schematic presentation of possible pectin (only homogalacturonan) conversion

reactions in plant-based foods and possible routes for tailoring quality parameters: PME =

pectinmethylesterase, Ca2+ = calcium cross-linking, PG = polygalacturonase, PL = pectate

lyase, T = temperature (Sila et al., 2009).

Chapter 1: Pectins

7

rhamnogalacturonase, rhamnogalacturonan lyase, β-galacturonase and α-arabinosidase

(Vincken et al., 2003). The hairy region degrading enzymes can be endo- and exo-acting.

Only enzymatic de-esterification and depolymerization of HG will be discussed.

The yield of enzymatic conversion depends on enzyme activity and substrate accessibility.

Extrinsic and processing factors such as temperature, salt concentration, pH and pressure also

play an important role (Duvetter et al., 2006; Jurnak et al., 1996; Sila et al., 2007a; Verlent et

al., 2004).

1.3.1.1 Enzymatic de-methoxylation

Pectin methylesterase (PME, E.C. 3.1.1.11) is a cell-wall-bound enzyme which catalyzes the

de-esterification of HG, converting the methyl-esterified carboxyl groups in negatively

charged carboxyl groups, releasing both protons and methanol (Limberg et al., 2000) (Figure

1.3). The de-methoxylated HG can:

1. cross-link with divalent ions (such as Ca2+, Mg2+) forming supra-molecular assemblies

and/or gels, important for engineering texture and rheological properties,

2. form a substrate for pectin de-polymerizing enzymes, associated with

texture/viscosity loss. This is useful in increasing juice extraction yields and in

controlling cloud stability.

PME exists in all higher plants, but is particularly abundant in citrus fruits (Johansson et al.,

2002; Voragen et al. 1995). This enzyme is also produced by a number of bacteria and fungi,

some of which are pathogenic to plants (Giovane et al., 2004; Johansson et al., 2002). PME

can thus be either endogenous or exogenous in origin. The distribution of free carboxylic

groups along HG is quite diverse as the action mechanism of PME depends on its origin.

In general, enzymatic conversions of polysaccharides can be described by three modes of

action namely, “single chain”, “multiple chain” and “multiple attack” (Greenwood et al.,

1968). Therefore the modes of action of PMEs are often described in terms of these

mechanisms. In the case of a single chain mechanism, PME demethoxylates a sequence of

adjacent GalA residues. When the end of the chain or a non-esterified residue is reached, the

Chapter 1: Pectins

8

enzyme dissociates from its substrate. The multiple chain mechanism involves a completely

random de-esterification, the enzyme-substrate-complex dissociates after each enzymatic

conversion. In the case of the multiple attack mechanism, a limited average number of

subsequent residues is converted per enzymatic attack. This number is described as the

“degree of multiple attack” (Catoire et al., 1998; Grasdalen et al., 1996).

It is important to note that the mode of action of PME may be influenced by other factors,

such as pH (Cameron et al., 2008; Catoire et al., 1998; Denes et al., 2000). It has been

described that PMEs with an alkaline pl (mostly plant PMEs) de-esterifies ‘blockwise’,

generating long sequences of non-esterified GalA residues (Figure 1.4), whereas PMEs with

acidic pl (mostly microbial PME) de-esterify more randomly (Figure 1.5) (Markovic et al.,

1984; Limberg et al., 2000; Ralet et al., 2001; Cameron et al., 2008), though the resulting

pattern of methyl-esterification is different, more ‘ordered’, than the pattern after chemical

demethoxylation (Denes et al., 2000; Limberg et al., 2000; Ralet et al., 2001; Ralet et al.,

2003; Duvetter et al., 2006).

In plant tissue, PMEs occur as a large set of multigene isoenzymes and are highly regulated in

specific manner (Goldberg et al., 2001). The optimum pH for plant PME activity varies

between 6.0 and 8.0 and its optimum temperature ranges from 40°C to 60°C depending on its

source (Christensen et al., 1998; Ly-Nguyen et al., 2002; Savary et al., 2003). Metal cations

can stimulate the activity of plant PME in food processing (Bordenave et al., 1996).

Inactivation of plant PMEs generally occurs above 70oC, depending on the type of plant

(Waldron et al., 2004). During a high pressure treatment (200- 500 MPa), they are not

completely inactivated (Sila et al., 2004).

Figure 1.4: Blockwise de-esterification pattern produced by plant PME

(Limberg et al., 2000).

Chapter 1: Pectins

9

Figure 1.5: Random distribution of de-esterified GalA by fungal PME

(Markovic et al., 1984).

1.3.1.2 Enzymatic depolymerization

There are three enzymes that depolymerize the HG domain in pectin. These include:

polygalacturonase, pectate lyase and pectin lyase.

a) Polygalacturonase

Polygalacturonase (PG) is an enzyme that catalyses the hydrolytic cleavage of glycosidic α-

(1,4) linkages between GalA residues in the HG domain of pectin. The activity of the enzyme

decreases gradually with increasing DM, since PG can only catalyse the hydrolytic cleavage

between non-esterified GalA residues (Rexova-Benkova et al., 1976; Benen et al., 2003). PG

can be endo-acting (E.C. 3.2.1.15), resulting in a major decrease in molar mass of pectin

(important for softening of fruits during ripening), or exo-acting, releasing monoGalA (E.C.

3.2.1.67) or diGalA (E.C. 3.2.1.82) from the non-reducing end of the chain. The last

mentioned enzyme has not been reported in plants although (Benen et al., 2003b).

The exact number of GalA residues cleaved depends on the origin of the enzyme. Endo-PG

from Kluveromyces fragilis, for example, depolymerizes pectin when at least four adjacent

non-esterified GalA residues are present on the molecule (Daas et al., 1999; Daas et al., 2000)

(Figure 1.6).

Chapter 1: Pectins

10

The textural properties of processed products, like tomatoes, where the endogenous PG

activity level is high, are a result of chemical breakdown of the cell wall material, but also of

transformations facilitated by PME and PG. The role of PME hereby is creating the PG-

substrate, de-esterified pectin. In fruits and vegetables with negligible activity of endogenous

PG such as broccoli, thermal stimulation of cell-wall-bound PME within the optimal range of

PME catalytic activity reduces the vulnerability of the products to thermal softening (Van

Buggenhout et al., 2009).

During processing of fruits and vegetables, the activity of PG decreases with increasing

pressure and complete inactivation of PG activity occurs at pressure in the pressure range

500-600 MPa (Verlent et al., 2006). Also, thermal inactivation of PG occurs at temperatures

of about 60oC (thermo-labile) or 80oC (thermo-stable) (Duvetter et al., 2009).

b) Lyases

Lyases are classified according to their mode of action and the substrate they act upon.

Several pectinolytic lyases exist that catalyze the cleavage of α-(1,4) linkages in the HG

domain of pectin by the mechanism of β-elimination (Rexova-Benkova et al., 1976). The

activity of endo-pectin lyase (E.C. 4.2.2.10) increases with increasing DM (Benen et al.,

2003; Yadav et al., 2009), while endo-pectate lyase (E.C. 4.2.2.2) and exo-pectate lyase (E.C.

Figure 1.6: Schematic representation of enzymatic digestion of pectin with endo- PG

from Kluveromyces fragilis (Daas et al., 1999).

Chapter 1: Pectins

11

Figure 1.7: Completely random de-methoxylation pattern by alkaline saponification

(Limberg et al., 2000).

4.2.2.9) preferentially cleave intermediate or low DM pectin (Benen et al., 2003; Benen et al.,

2003a).

1.3.2 !on-enzymatic pectin conversions

Pectins are most stable in aqueous solutions around pH 3.5, their pKa value. At higher or

lower pH, they are subjected to non – enzymatic degradation reactions.

1.3.2.1 Chemical de-methoxylation

Non-enzymatically, pectins are de-esterified rapidly in an alkaline medium (pH > 5.0)

producing a completely random pattern (Figure 1.7) of de-esterified pectin (Limberg et al.,

2000; Massiot et al.,1992, Ralet et al., 2001). Alkaline conditions result in the saponification

of ester groups with the liberation of methanol. The saponification of pectin accelerates with

increasing pH and temperature (Voragen et al., 1995).

1.3.2.2 Chemical depolymerization

Pectin may undergo either acid or base catalyzed depolymerization. First and most important

is the base-catalysed splitting of pectin chains through the β-elimination reaction, a process

that takes place in parallel with the de-esterification of pectin and proceeds even when pectin

is heated at neutral or weakly acidic pH (Keijbets et al., 1974). Most plant-based foods have a

pH of above 4.5 and are processed at 80oC or higher, making them susceptible to the β-

elimination reaction (Sila et al., 2005).

Chapter 1: Pectins

12

The second mechanism leading to pectin degradation is acid hydrolysis (pH < 3.0), whereby

in acid conditions, pectin with low DM hydrolyses faster (Krall et al., 1998). Acid hydrolysis

is of less importance during regular food processing than β-elimination.

Finally, pectin can be degraded through hydroxyl radical-mediated scission of the polymer

(Liszkay et al., 2003; Scopfer et al., 2002), which is thought to predominate in postharvest

fruit softening (Fry et al., 2002).

β-elimination and acid hydrolysis will now be discussed in more detail.

a) β-elimination

β-elimination is known to proceed via an E1cB mechanism (Bemiller et al., 1972) (Figure

1.8). The hydrogen at C-5 is removed by a hydroxyl ion resulting in an unstable, intermediary

anion, which is stabilized by the cleavage of the glycosidic linkage in the β-position to the

carboxyl function, leading to the formation of a glycosyl anion and an unsaturated bond

between C-4 and C-5 (Albersheim et al., 1960; Neukom et al., 1958). A prerequisite is the

presence of a methyl ester group at C-6, because its electron-withdrawing effect makes the

proton at C-5 sufficiently acidic (Bemiller et al., 1972; Keijbets et al., 1974; Neukom et al.,

1958). As a result, pectin with a high DM is more susceptible to β-elimination than pectin

with a low DM (Albersheim et al., 1960; Diaz et al., 2007; Krall et al., 1998; Sajjaanantakul

et al., 1989; Sajjaanantakul et al., 1993). The DM is more important than the distribution of

the methoxyl-esters in pectin (Fraeye et al., 2007). During the heating of pectin, simultaneous

chemical demethoxylation can occur, generating pectin with a lower DM. As the DM of the

pectin decreases, the β-elimination rate also decreases (Albersheim et al., 1960; De Roeck et

al., 2009; Kravtchenko et al., 1992).

Chapter 1: Pectins

13

Figure 1.8: β-eliminative depolymerisation reaction mechanism (Bemiller et al., 1972).

The reaction rate increases with an increase in pH because hydroxyl groups initiate the

reaction (Diaz et al., 2007; Kravtchenko et al., 1992; Krall et al., 1998; Sila et al., 2006). β-

elimination is also stimulated in the presence of cations, whereby the extent of the cleavage

increases roughly with salt concentration and with valency of the ions. The suggestion is that

the ions may interact with the free carboylic groups on the pectin, resulting in an overall

decrease in the negative charge, which may facilitate the approach of the hydroxyl ions

needed to initiate the β-elimination reaction (Keijbets et al., 1974; Sajjaanantakul et al.,

1993). However, Sila et al. (2006) reported a decline in the extent of the β-elimination when

the concentration of Ca2+ ions exceeded 0.1M. This effect was ascribed to the calcium cross-

linking of the pectin chain. β-elimination is also stimulated by organic anions and other

possible proton acceptors (Keijbets et al., 1974).

The rate of β-elimination increases with an increase in temperature. Activation energies

ranging from 83 to 136 kJ/mol have been reported (De Roeck et al., 2009; Diaz et al., 2007;

Sila et al., 2006). These high values indicate that the reaction rate constant depends strongly

Chapter 1: Pectins

14

on temperature. Therefore, the kinetic information on the β-elimination mechanism is of vital

importance to elucidate and model the mechanical properties of processed plant-based foods.

b) Acid hydrolysis

Pectin with a low DM is susceptible to acid hydrolysis at pH < 3.0. The three steps of this

reaction mechanism can be described as follows: (i) protonation of the glycosidic oxygen to

give the conjugated acid, (ii) an unimolecular heterolysis of the conjugated acid with the

formation of a non-reducing end group and a carbonium-oxonium ion, (iii) addition of water

to the carbonium-oxonium ion with formation of a reducing end group and a proton

(Smidsrod et al., 1966).

Pectin with high DM is more stable in acidic conditions compared to pectate (Diaz et al.,

2007). The lower the DM, the faster pectin hydrolyses (Krall et al., 1998). Slow acid

hydrolysis of pectin has been measured at pH 6.0 and reaction rates increase with a decrease

in pH, especially in neutral sugar side chains (Diaz et al., 2007; Krall et al., 1998; Smidsrod

et al., 1966).

1.4 Cross-linking mechanisms of pectin

There are three important types of cross-links between pectic substances: Ca2+ cross-linking,

borate-diol ester and uronyl ester cross-linking (Ridley et al., 2000; Vincken et al., 2003;

Mohnen, 2008). Hydrophobic interactions between methyl ester groups and hydrogen

bonding are also possible. Pectins isolated from sugar beet, in contrast to those isolated from

example broccoli and tomato, contain significant amounts of ferulic acid. In the family

Chenopodiaceae, ferulic acid cross-links occur in the arabinan and galactan side chains of

pectin (Oosterveld et al., 1997).

1.4.1 Ca2+

cross-linked pectin

Cross-linking of HG with Ca2+ is promoted by the presence of long chains of consecutive

non-esterified galacturonic acid residues and only small amounts of hairs. This mechanism is

mainly known as the ‘egg-box model’ (Grant et al., 1973) (Figure 1.9).

Chapter 1: Pectins

15

The mechanism involves junction zones created by the ordered, side-by-side associations of

galacturonans, whereby specific sequences of GalA monomers in parallel or adjacent chains

are linked intermolecularly through electrostatic and ionic bonding of carboxyl groups. Ca2+

cross-linking of demethoxylated pectin is exploited in texture engineering, whereby low-DM

pectin is being associated with higher thermal resistance in solid plant-based foods (Sila et al.,

2004; Smout et al., 2005; Vu et al., 2004). Also, gelation is dictated by the amount and

distribution of methoxyl ester groups over the entire pectin molecule (Capel et al., 2005;

Strom et al., 2007; Thibault et al., 1985).

1.4.2 Borate-ester cross-linked pectin

There is accumulating evidence that borate ester cross-linking of RG-II is required for the

formation of a macromolecular pectic network within the plant cell wall (Matoh et al., 1998).

A borate-diol ester, which cross links two molecules of RG-II and one boron molecule

(Willats et al., 2006), only occurs at the apiofuranosyl residues of the 2-O-methyl-D-Xyl-

containing side chains. HG chains are also sensitive to this cross-linking since they function

as the backbone of RG-II (Figure 1.10).

Figure 1.9: Schematic representation of Ca2+ binding to polygalacturonate sequences: ‘egg

box’ dimer (Vincken et al., 2003).

Chapter 1: Pectins

16

1.4.3 Uronyl ester cross-linked pectin.

Uronyl ester cross-links are formed between HG and other wall polysaccharides (Vincken et

al., 2003) (Figure 1.11). This type of cross-linking results from a trans-esterification reaction

which uses methyl-esterified HG as a donor substrate and a wall polysaccharide as an

acceptor substrate.

Figure 1.10: The borate 1,2-diol ester that cross-links two monomeric units of RG-II

(Ridley et al., 2001).

O

O

HG

C O

O Wall polysaccharide

OH O

HG

Figure 1.11: The uronyl ester cross-linked pectin (Vincken et al., 2003).

Chapter 1: Pectins

17

1.5 Anti-pectin antibodies

An antibody, also called an immunoglobulin, is a glycoprotein molecule that is produced by

plasma cells in response to an immunogen. Antibodies bind specifically to one or a few

closely related antigens (Mian et al., 1991). Each antibody actually binds to a specific

antigenic determinant. The unique part of the antigen recognized by an antibody is called the

epitope. These epitopes bind with their antibody in a highly specific interaction, called

induced fit that allows antibodies to identify and bind only their unique antigen in the midst of

the millions of different molecules. The valency of antibody refers to the number of antigenic

determinants that an individual antibody molecule can bind. The valency of all antibodies is at

least two and in some instances more. Antibodies are typically made of basic structural units,

each with two large heavy chains and two small light chains and form, for example,

monomers with one unit, dimers with two units or pentamers with five units (Mian et al.,

1991) (Figure 1.12) .

Five different antibody isotypes or classes are known in mammals, which perform different

roles, and help direct the appropriate immune response for each different type of foreign

object they encounter. These include IgA, IgD, IgE, IgG, IgM (Mian et al., 1991).

Figure 1.12: The antibody structure (Mian et al., 1991).

Chapter 1: Pectins

18

Technologies and molecular probes capable of placing the chemistry of pectin and its

variations in the context of biological functions at the tissues level and cells are largely

derived from the use of antibody probes. In this respect, monoclonal antibodies (mAbs) can

be used for the analysis of pectin in plant science. This is because they allow defined

structural domains to be precisely localized in the domain of intact cell wall architecture.

In recent years, the number of anti-pectin antibodies has increased significantly and

monoclonal antibodies with specificities for numerous side chains and backbone domains are

now available (Willats et al., 2003). Moreover, several antibodies to HG have been generated

and these tools have, for instance, indicated that the methyl-esterification of HG is highly

varied in relation to cell development.

The current set of anti-HG antibodies, which have different specificities in relation to HG

methyl-esterification, include JIM5, JIM7, LM7, LM18, LM19, LM20, PAM1 and 2F4

(Figure 1.13).

mAb Antigen Primary references

JIM5 HG VandenBosch et al. (1989); Knox et al. (1990)

JIM7 HG Knox et al. (1990)

LM18 HG Verhertbruggen et al. (2009)

LM19 HG Verhertbruggen et al. (2009)

LM20 HG Verhertbruggen et al. (2009)

PAM1 de-esterified HG blocks Willats et al. (1999); Manfield et al. (2005)

2F4 Ca2+-linked HG dimers Liners et al. (1989)

(I)

(II)

Figure 1.13: Anti-HG antibodies. (I) Schematic representation of epitopes of some

anti-HG antibodies. (II) Type, antigen and primary references. (Willats et al., 2006,

Christiaens et al., 2011).

Chapter 1: Pectins

19

According to Christiaens et al., (2011), JIM7 can be used as a general anti-pectin probe. As

shown in Figure 1.14, the binding strength of JIM7 decreases with a decrease in DM.

However, both the degree and pattern of methyl de-esterification play a role in the binding

strength of JIM7.

In Figure 1.14, it can be seen that JIM5 binds to all pectic samples tested, except to the parent

citrus pectin (DM = 94%). The binding strength of JIM5 seems to increase with decreasing

DM, but becomes weaker again when the DM falls down below ± 20% (Christiaens et al.,

2011; Willats et al., 2000). Therefore, the epitope recognized by JIM5 probably contains both

methyl-esterified and non-methyl-esterified GalA residues (Willats et al., 2000).

LM18 and LM19 bind preferentially to low methyl-esterified pectin. From Figure 1.14, it can

be seen that the binding strength of LM19 is greater than that of LM18. The binding strength

of LM18 increases with decreasing pectin DM and LM18 has a preference for F-series (and

C-series) pectin (Verhertbruggen et al., 2009). It seems that a stretch of un-esterified GalA

residues is necessary for the binding of LM19.

LM20 exhibits similar characteristics as JIM7, but it does not bind to F - and C - series with a

very low DM pectin (Christiaens et al., 2011) (Figure 1.14). LM20 requires high methyl-

esterified GalA residues for binding.

In contrast to the above mentioned anti-HG antibodies, PAM1 is the most specific antibody.

From Figure 1.14, it can be seen that PAM1 preferentially binds to plant PME de-esterified

pectin (Christiaens et al., 2011; Manfield et al., 2005). The distribution of methyl groups has

a greater influence on the binding capacity of PAM1 than the DM. Thus, PAM1 is, due to its

specificity, ideally suited to visualise the zone of pectin methyl de-esterification.2F4 binds

specifically to HG that is cross-linked through Ca2+ (Willats et al., 2001a; Liners et al., 1989).

Chapter 1: Pectins

20

Figure 1.14: Immuno-dot-assays of JIM5, JIM7, LM18, LM19, LM20 and PAM1 binding to

citrus pectins with defined DE and DBabs. Highly methyl-esterified citrus pectin (DE=94%) was

de-esterified with carrot PME (P-series), Aspergillus aculeatus PME (F-series) and NaOH (C-

series).

Chapter 2: Texture/rheology of plant-based foods

21

2 Texture/rheology of plant-based foods

2.1 Cell-wall composition in relation to mechanical properties

The hierarchy of structures conceptually links the molecular composition of cell-wall

components to the mechanical properties of the range of plant organs supported by cell walls,

and hence to cell-wall-dependent quality characteristics (Waldron et al., 2003). The hierarchy

comprises 5 main levels of structure: the cell wall polymers, the cell wall, the plant cell, the

tissue, and the plant organ. The cell-wall-dependent characteristics of the plant organ, “food

quality”, will depend on the interacting properties of the different levels of structure (Waldron

et al., 2003) (Figure 2.1). The parenchyma cell and cell wall components will be briefly

discussed in this section.

Figure 2.1: The hierarchy of structures (Waldron et al., 2003).

Chapter 2: Texture/rheology of plant-based foods

22

2.1.1 The plant cell

The major parts of a parenchyma cell are the cell wall, plasma membrane, cytoplasm, nucleus, and

vacuole (Figure 2.2). The plant cell wall is a dynamic structure built up of cellulose microfibrils

imbedded in a continuous matrix of pectic substances, hemicelluloses, proteins, lignins, lower

molecular weight solutes and water (Van Buren, 1979) (Figure 2.3).

Figure 2.3: The morphology of a plant cell (Van Buggenhout et al., 2009).

Figure 2.2: The Plant cell structure.

Chapter 2: Texture/rheology of plant-based foods

23

Up to three strata or layers may be found in plant cell walls:

1. The middle lamella, which is a layer rich in pectins. This outermost layer forms the

interface between adjacent plant cells and glues them together. Cellulose fibrils are missing.

2. The primary cell wall which is generally a thin, flexible and extensible layer formed while

the cell is growing. It consists mainly of pectic polysaccharides, hemicelluloses and cellulose

in similar amounts. While cellulose’s function is giving rigidity, pectic substances and

hemicelluloses contribute plasticity to the cell wall.

3. The secondary cell wall is a thick layer formed inside the primary cell wall after the cell is

fully grown. It is not found in all cell types, only in woody plants. Secondary cell walls

contain a wide range of additional compounds that modify their mechanical properties and

permeability.

2.1.2 Cell wall components

2.1.2.1 Cellulose

Cellulose is the single most abundant polysaccharide component of vegetables (Waldron et

al., 2003). Its glucan chains interact closely through hydrogen bonding, excluding water to

produce areas of crystallinity which impart considerable tensile strength. The naturally-

occurring crystalline structure is known as Cellulose I. However, several other forms (II, III,

and IV) can be produced as a result of thermal or mechanical treatments.

2.1.2.2 Hemicellulose

Hemicelluloses, unlike pectic polysaccharides, are usually solubilized only by treatments that

disrupt the hydrogen bonds which link them strongly to cellulose microfibrils. Such

treatments include increasing strengths of alkali. Hemicelluloses are found in both primary

and secondary cell walls of both monocotyledonous and dicotyledonous plant tissues. They

can differ greatly in different cell types, and in different species (Waldron et al., 2003).

2.1.2.3 Pectin

This family of complex polysaccharides was extensively discussed in chapter 1.

Chapter 2: Texture/rheology of plant-based foods

24

2.1.2.4 Proteins and glycoproteins

Structural proteins (1-5%) are found in most plant cell walls. They are classified as

hydroxyproline-rich glycoproteins, arabinogalactan proteins, glycine-rich proteins, and

proline-rich proteins. Each class of glycoproteins is defined by a characteristic, highly

repetitive protein sequence (Waldron et al., 2003). Most proteins are glycosylated, contain

hydroxyproline and become cross-linked in the cell wall. These proteins are often

concentrated in specialized cells and in cell corners. The relative composition of

carbohydrates, secondary compounds and proteins varies between plants and between the cell

type and age.

2.1.2.5 Water

Water is an important component of the primary cell wall and has four major functions

(Northcote et al., 1972). It acts a structural component in the matrix gel, as a transport

medium, a stabilizing and /or wetting agent.

2.2 Textural/rheological characteristics of (processed) fruits and vegetables

Textural and rheological properties of fruits and vegetables are important quality attributes

used in the food industry. Food texture is mostly used in reference to solid foods, whereas

food rheology is mostly used in reference to liquid foods (McKenna et al., 2003), but some

foods will exhibit a solid- or liquid-like behaviour.

Texture can be defined as “the sensory and functional manifestation of the structural,

mechanical, and surface properties of foods detected through the senses of vision, hearing,

touch, and kinesthetics” whereby kinesthetics comprises the sensation of presence, movement,

and position as resulting from nerve-ending stimulation (Szczesniak et al., 2002). This

definition conveys important concepts such as:

A. Texture is a sensory property and, thus, only a human being can perceive and describe it.

The so-called texture testing instruments can detect and quantify only certain physical

parameters which then must be interpreted in terms of sensory perception;

Chapter 2: Texture/rheology of plant-based foods

25

B. It is a multi-parameter attribute, evidenced by the large number of words described in

Table 2.1 below.

C. It derives from the structure of the food (molecular, microscopic or macroscopic); and

D. It is detected by several senses, the most important ones being the senses of touch and

pressure.

Processing of fruits and vegetables alters the initial mechanical properties. Therefore in

texture/rheology engineering, processing can be viewed as a controlled effort to preserve,

transform, destroy, and/or create structure/texture changes. This brings us to the definition of

the term ‘engineered texture’, which connotes a controlled transformation of a native structure

into a desirable structure worthy of consumption as a recognizable food (Aguilera et al.,

1985).

Table 2.1: Classification of textural characteristics (Waldron et al., 2003).

Chapter 2: Texture/rheology of plant-based foods

26

2.3 Microstructure and rheological properties of plant based foods

Based on the type of vegetable, different processes can lead to microstructures with different

rheological properties. Particle size distribution, morphology and phase volume are important

parameters used to explain the complex relationship between rheology and microstructure of

plant-based foods (Barnes et al., 1989). The resulting microstructures differ in the manner of

cell separation: either breaking across the cell wall or through the middle lamella (Lopez-

Sanchez et al., 2010).

After mechanical processing, plant food dispersions are a combination of i) a liquid phase

containing pectic materials and organic acids and ii) a dispersed phase formed of all insoluble

plant solids such as the cell wall materials. Therefore, the rheological properties are expected

to depend on both the solids in the serum phase and the particle volume fraction of insoluble

solids. Also, the effective particle volume fraction depends on particle parameters such as the

size, morphology, hardness and inter-particle forces (Aguilera et al., 2000).

Studies that relate particle size to rheological properties are often contradictory. Tomato with

intermediate particle sizes and wide particle size distributions was shown to have a high

viscosity and yield stress. This was attributed to an interplay between surface area

enhancement and efficient particle packing to create a more cohesive network. In contrast to

this, it was found that the viscosity of tomato purées increased with reduced particle sizes by

other authors (Yoo et al., 1994). They explained this as due to the shorter interaction distance

between small particles. However, it was recently showed that a smaller particle size can lead

to reduced viscoelastic moduli (Sanchez et al., 2003). It was pointed out that it is incorrect to

explain the changes of rheology through the particle size without taking into account other

parameters such as the total concentration of insoluble solids which has an effect on the

viscoelastic properties of the systems. Systems with similar particle size and increased pulp

content result in an enhancement of the viscoelastic properties (Den Ouden et al., 2002).

Furthermore, particle hardness and cell structure has been shown to have an influence on the

rheological properties. Denser and less deformable cells will present a lower volume fraction

for the same weight concentration leading to lower viscosity and yield stress (Lopez-Sanchez

et al., 2002).

Chapter 2: Texture/rheology of plant-based foods

27

High pressure homogenization has been used to modify not only the particle size and

morphology but also the particle interactions, thus changing rheological properties such as

viscosity (Lopez-Sanchez et al., 2010).

2.4 Pectin structure-function relationship

The quality attributes, particularly textural and rheological properties, of many plant-based

foods are very dependent on the pectin content and composition in combination with the type

of postharvest handling processes and/or (pre-) processing steps applied. The susceptibility

and suitability of pectin for many applications is governed by its structural features such as

molar mass, neutral sugar content, proportions of smooth and hairy regions, ferulic acid

substitution, amount of methoxyl and acetyl esters, and the distribution of the esters groups on

the polymer (Braccini et al., 1999; Daas et al., 1999).

Today, the best known and the most exploited pectin structure–function relationship is the

participation of HG in calcium-mediated gel formation, qualifying it as a thickening,

stabilizing, gelling, and/or texturizing agent (Sila et al., 2009). Pectin is used as an ingredient

in jams, jellies, confectioneries, desserts, and yogurts.

Processes aiming to alter the texture and structure of processed fruits and vegetables often

take into account the way pectin changes in the cell wall (Sila et al., 2006; Van Buren, 1979;

Waldron et al., 2003). This is because pectin changes are very important in relation to

processing-texture changes (see Figure 1.3).

The following methods can be used for the reduction of process-induced texture degradation

during processing of fruits and vegetables (for example, broccoli): activation of endogenous

PME, infusion of exogenous PME and stimulation of ionically cross-linked pectin by addition

of calcium,.

Decreasing DM through stimulation of PME activity leads to the limitation of β-elimination.

PME is stimulated by the application of thermal treatments at 50oC – 600C for 35 minutes or

more. This is called a low temperature blanching (LTB) pretreatment and its application to

fruit and vegetable tissues, for example broccoli, can help reduce softening, during subsequent

high-temperature processing (Waldron et al., 2004; Fraeye et al., 2009). Thus, PME plays a

Chapter 2: Texture/rheology of plant-based foods

28

key role in lowering DM either to limit availability for β-elimination or enhance possibilities

of an ionic cross-linking between pectin and divalent cations (Waldron et al., 2004).

The availability of endogenous calcium ions can influence the effect of PME activity (Fraeye

et al., 2009). A good way to improve texture is combination of a PME-stimulating

pretreatment and calcium addition.

2.5 Objective of this thesis

The role of pectin on the consistency of vegetable purées is largely unknown. This thesis

therefore aims to gain a better insight into the pectin structure-function relation of vegetable

purées. In this study, broccoli (Brassica oleracea L. Var. italica) was selected as a relevant

case study from which broccoli purées were obtained.

The effect of different pretreatments, high pressure homogenization and cooking was

evaluated. Chemical, macroscopic and microscopic analyses were performed to gain an

insight into the pectin structure-function relationship. Anti-pectin antibodies were, for

example, used for in situ investigation of pectin.

29

Part II: Experimental work

Chapter 3: Materials and methods

30

3 Materials and methods

3.1. Experimental set-up

A schematic overview of the experimental set-up is given in figure 3.1.

Twelve different broccoli purées were prepared (Figure 3.1). Broccoli purées were either

pretreated for 40 minutes at 60oC (low temperature blanching) or for 5 minutes at 95oC (high

temperature blanching) or not pretreated. After the pretreatment, samples were either blended

or blended followed by high pressure homogenization at 100 bar. In a final step, samples were

either cooked at 120oC for 20 minutes or not cooked.

Figure 3.1: Experimental setup; LTB = low temperature blanching for 40

minutes at 60oC; HTB = high temperature blanching for 5 minutes at 95oC;

HPH = high pressure homogenization (100 bar).

Chapter 3: Materials and methods

31

3.2. Vegetable material: broccoli

Broccoli (Brassica oleracea L. Var. italica) was bought in a local shop in Belgium and stored

in the fridge at 4oC before processing and analysis.

3.3 Preparation of broccoli purées

After cutting the broccoli florets and stems into small pieces, the pieces were vacuum packed

in plastic bags and subjected to one of the pretreatments (no, LTB or HTB). The pretreated

samples were placed in a blender with the addition of deionized water (150 g broccoli + 200 g

water) and then blended. This procedure was whether or not followed by a HPH and/or by a

cooking step.

3.3.1 Pretreatments

3.3.1.1 Low temperature blanching (LTB)

After cutting the broccoli florets and stems into small pieces, they were vacuum packed in

plastic bags in order to have a good heat transfer during the thermal pretreatment. The bags

were completely immersed in a water bath at 60oC for 40 minutes, after which they were

immediately removed from the water bath and cooled down in ice water to ambient

temperature.

3.3.1.2 High temperature blanching (HTB)

Similarly, the cutted brocolli pieces were vacuum packed in plastic bags. The bags were then

completely immersed in a water bath at 95oC for 5 minutes, after which they were

immediately removed from the water bath and cooled down in ice water to ambient

temperature.

3.3.2 High pressure homogenization (HPH)

A picture of the high pressure homogenization machine used is shown in figure 3.2.

Homogenization is a mechanical process that involves the subdivision of particles or droplets

into micron sizes to create a stable dispersion or emulsion for further processing. The process

occurs in a special homogenizing valve, the design of which is the heart of the homogenizing

Chapter 3: Materials and methods

32

equipment. The broccoli purée passes through a minute gap in the homogenizing valve. This

creates conditions of high turbulence and shear, combined with compression, acceleration,

pressure drop, and impact causing the disintegration of particles and dispersion throughout the

product.

After homogenization, the particles are of a uniform size, depending on the operating

pressure. The homogenizer is the most efficient device for particle and droplet size reduction.

The actual properties of the product vary with pressure and product type in a complex

relationship. In general, higher processing pressure produces smaller particles, down to a

certain limit of micronization (Schultz et al., 2004).

In practice, the sample funnel was first filled with deionized water and a pressure of 100 bars

was built up by turning the wheel of the homogenizing valve. The system was cooled to a

temperature of 4oC by a cryostat. After adding the broccoli purée into the funnel, the pressure

was adjusted at regular intervals since the viscosity of the sample influenced the

homogenization pressure. The homogenized sample was then collected and water was again

added into the sample funnel.

3.3.3 Cooking

Metal tubes were filled with broccoli purées, with each tube having a capacity of about 24 ml.

The tubes were first equilibrated for 20 minutes at 40oC in a water bath. The tubes were then

Figure 3.2: High pressure homogenizator.

Chapter 3: Materials and methods

33

placed in an oil bath at 120oC for 20 minutes for the actual cooking process. The samples

were removed and cooled in ice water to ambient temperature.

3.4 Bostwick rheometry

A picture of the Bostwick consistometer is shown in figure 3.3. 100 g of broccoli purée was

weighed down for this experiment. 30 seconds was started immediately after filling and

opening the chamber of the Bostwick consistometer. After the chamber was opened, the purée

flowed through the device. The pulp and liquid fraction were read respectively after the 30

seconds. This procedure was repeated two times for each sample.

3.5 Particle size distribution (wet sieving)

Sieve analysis is a simple but proven method of separating bulk materials of all kinds into size

fractions and to obtain the particle size and distribution through weighing of the size fractions.

Wet sieving was used to separate the purée into different size fractions : > 1 mm, 1 mm – 500

µm, 500 µm – 250 µm, 250 µm - 125 µm, 125 µm – 80 µm, 80 µm – 40 µm, < 40 µm (rest

water fraction) (Figure 3.4). 200 g of the sample was used. To start with the wet sieving

procedure, deionized water was turned on with always same flow rate. During the sieving, an

amplitude of 1.5 mm and a running time of 2 minutes were set. After this time, the water at

the outlet was always clear. The sieves were then weighed and the weights of the different

fractions were determined. The weight of the rest water was determined as the difference

Figure 3.3: Bostwick consistometer.

Chapter 3: Materials and methods

34

between the purée weight (200 g) and the sum of the weights of the different size fractions (>

40 µm).

3.6 Extraction of alcohol insoluble residue (AIR)

Alcohol insoluble residue (AIR) was extracted from broccoli purées following the procedure

described by McFeeters and Armstrong (1984). Approximately 15.0 g of broccoli purée was

weighed and completely homogenized in 96 ml of 95% ethanol using a mixer (Buchi mixer

B-400, Flawil, Switzerland). The suspension was filtered over a MN615 filter paper

(Machery-Nagel Ø 90mm) and the residue rehomogenized in 48 ml of 95% ethanol and

filtered again. The residue was homogenized again in 48 ml of acetone and allowed to stir for

10 minutes in the fridge at 4oC before final filtration. The AIR was dried overnight at 40°C on

a large petri dish. The AIR was ground using a mortar and pestle and stored in a desiccator

Figure 3.4: Wet sieving device.

Chapter 3: Materials and methods

35

over P2O5 until analysis. For each condition, a stock of AIR was collected prior to subsequent

characterization and fractionation experiments.

3.7 Determination of the degree of methyl-esterification (DM)

The degree of methyl-esterification of all broccoli purées was determined. This was done by

the determination of both the galacturonic acid (GalA) content as well as the methyl-ester

content.

3.7.1 Determination of galacturonic acid (GalA) content

A standard curve for the GalA analysis was established. MonoGalA monohydrate was used as

a stock solution to make a dilution series from 0 to 110 µg GalA.H2O/ml water. The resulting

concentration of GalA (µg/ml) was eventually converted into mol/g AIR for the different

samples. It should be noted that the standard curve was repeated each time new solutions were

prepared and used.

Secondly, the GalA of the different samples was determined quantitatively by the colorimetric

hydroxyl phenyl phenol method.

Pectin in the AIR was subjected to an acid hydrolysis with concentrated sulfuric acid to

liberate free GalA residues according to the method described by Ahmed and Labavitch

(1977). A beaker containing a magnetic stirrer and 10 mg of AIR was placed in an ice bath on

a magnetic stirring plate after which 8 ml of H2SO4 (98%) was added. 2 ml deionized water

was added 2 times drop by drop whereby the sample was stirred for 5 minutes. Subsequently,

the sample was stirred for 1 hour to complete the hydrolysis. Depending on the expected

amount of GalA, the sample was diluted to 25 or 50 ml with deionized water. The hydrolysis

of each sample was replicated two times.

After hydrolysis, the concentration of GalA was determined using the spectrophotometric

method according to Blumenkrantz and Asboe-Hansen (1973). 3.6 ml of cold sulphuric acid –

sodium tetraborate (0.0125 M sodium tetraborate in 98% H2SO4) was added to 0.6 ml of the

hydrolyzed samples in an ice bath. The mixture was heated for 5 minutes in an oil bath at

100oC after vortexing. The samples were cooled down in a sink and placed in an ice bath. 60

Chapter 3: Materials and methods

36

µl of m-hydroxydiphenyl-solution (0.15% 3-phenylphenol in 0.5% NaOH) was added, which

resulted in a colorimetric reaction. For the blank, 60 µl 0.5% NaOH was used instead of the

m-hydroxydiphenyl-solution. The absorbance was measured with a UV - Vis

spectrophotometer (Ultraspec 500 pro UV/Visible Spectrophotometer, GE Healthcare,

Uppsala, Sweden) at 520 nm and 25oC after 1 minute of vortexing and 1 minute of waiting.

The analysis was repeated three times.

3.7.2 Determination of methyl-ester groups

A standard curve for the methanol analysis was established. The standard curve was obtained

by performing a dilution series in 0.0975 M phosphate buffer pH 7.5 with 0 to 20 µg

methanol/ml phosphate buffer. The concentration of methanol was converted to mol

methanol/g AIR for the different samples. It should also be noted that the standard curve was

repeated every time new solutions were prepared and used.

Pectin in the AIR was subjected to an alkaline hydrolysis according to the method described

by Ng and Waldron (1997). Approximately 20 mg of AIR was weighed, and 8 ml deionized

water was added. The mixture was sonicated for 10 minutes to suspend the AIR. 3.2 ml of 2

M NaOH was added to hydrolyze the samples for 1h at 20oC. The samples were then

neutralized by adding 3.2 ml of 2 M HCl followed by an equilibration at 25oC for 15 minutes.

Subsequently, the samples were adjusted to 50 ml or 25 ml with 0.0975 M phosphate buffer

pH 7.5. This step was performed in duplicate for each sample.

The amount of methanol released was determined in triplicate by a colorimetric reaction

according to the method described by Klavons and Bennett (1986). To 1 ml of the hydrolyzed

samples, alcohol oxidase was added. One unit of alcohol oxidase activity is defined as the

amount of alcohol oxidase that catalyzes the oxidation of 1 µmol methanol to formaldehyde

per minute at pH 7.5 and 25oC. After gentle shaking, the samples were incubated for 15

minutes at 25oC. This was followed by a reaction of the formed formaldehyde with 2 ml

pentanedione solution consisting of 2 M ammonium acetate, 0.05 M acetic acid and 0.02 M

2,4–pentanedione. This resulted in a yellow-colored product, 3,5-diacetyl-1,4-dihydro-2,6-

dimethylpyridine. For the blank, 2 ml deionized water was used. The samples were incubated

at 58oC for 15 minutes and cooled to room temperature. The absorbance of the yellow-colored

Chapter 3: Materials and methods

37

product was measured with a spectrophotometer at 412 nm (Ultraspec 1100 pro UV/Visible

Spectrophotometer, GE Healthcare, Uppsala, Sweden).

Finally, the DM was estimated by taking the ratio of moles of methanol to the moles of

galacturonic acid and expressed as a percentage.

3.8 Fractionation of AIR

AIR was fractionated into water soluble pectin (WSP), chelator soluble pectin (CSP), and

sodium-carbonate soluble pectin (NSP).

According to the method described by Braga et al. (1998), WSP was extracted with hot water.

In this method, approximately 0.25 g of AIR from broccoli purées was weighed in a Schott

bottle of 100 ml. The samples were homogenized by stirring in 45 ml of hot water (100°C) for

5 min. The resulting suspension was cooled in a sink, and then filtered using the filter paper

MN 615 Ø 90 mm. The volume of the filtrate was adjusted to 50ml. The filtrate was labeled

WSP. The residue was collected and used in subsequent analysis.

For the extraction of the CSP, the residue was incubated in 45 ml of 0.05 M cyclohexane-

trans-1, 2-diamine tetra-acetic acid (CDTA) in 0.1 M potassium acetate (KAc) pH 6.5 for 6

hours at 28°C. To improve the suspension of the residue, the mixture was first stirred for 15

minutes at ambient temperature, before incubating it in a shaking water bath according to the

method described by Chin et al. (1999). The suspension was filtered using the filter paper MN

615 Ø 90mm and the filtrate was adjusted to 50ml with CDTA + KAc. The residue was taken

to the next fractionation step. The filtrate was labeled CSP.

For the extraction of the NSP, the residue was incubated in 45 ml of 0.05 M Na2CO3

containing 0.02 M NaBH4 for 16 h at 4 °C, with constant stirring, followed by re-incubation

for another 6 h at 28 °C in a shaking water bath according to the method described by Chin et

al. (1999). The resulting mixture was filtered using the filter paper MN 615 Ø 90mm and the

filtrate was adjusted to 50 ml with Na2CO3 + NaBH4. The filtrate was designated NSP. The

residue of the fractionation (predominantly cellulose and hemicellulose) was dried at 40oC

overnight and stored.

Chapter 3: Materials and methods

38

All of the pectin fractions (WSP, CSP, and NSP) were analyzed for their GalA content. There

was a difference in the determination of the GalA content in the pectin fractions compared to

the method described in section 3.7.1. Because the pectin fractions are liquid, 2 ml of the

fraction was hydrolyzed by adding it drop by drop to 8 ml of concentrated H2SO4. After 5

minutes stirring, 2 ml of deionized water was added. The rest of the procedure was similar to

the one described for AIR.

The methyl-ester content of the WSP and CSP fractions was also determined. Together with

their respective GalA content, their DM could be established. The DM of NSP could not be

determined because of the saponification of methyl-esters that occurred during the extraction

of the NSP fractions. For the determination of the methyl-ester groups, 8 ml of the WSP and

CSP fractions was used. The procedure then proceeded as described in section 3.7.2.

3.9 Molar mass distribution

3.9.1 Lyophilization (freeze-drying) and dialysis

To get a more concentrated product, lyophilization of the WSP, CSP and NSP fractions was

done using a freeze-dryer (Christ Alpha 2–4 LSC) and the dry powder was kept in a

desiccator until subsequent analysis.

5 mg of the WSP and 150 mg of the NSP lyophilized samples were dissolved in 2 ml of

deionized water respectively and extensively dialyzed against deionized water. In the case of

the CSP fractions, 30 mg of the samples was dissolved in 2 ml of deionized water and

extensively dialysed against 0.1 M NaCl followed by dialysis against deionized water. This

process of dialysis was accomplished with the aim of removing low molecular weight solutes

by using a dialysis membrane with a molecular weight cut-off between 12 and 14 kDa.

3.9.2 High Performance Size-Exclusion Chromatography (HPSEC)

Changes in the molar mass distribution of broccoli purée pectin were studied using High

Performance Size-Exclusion Chromatography (HPSEC).

The principle of HPSEC is based on the separation of particles according to particle sizes. The

column is filled with polymer beads with pores of different sizes. Small particles can

Chapter 3: Materials and methods

39

penetrate all the pores, while the larger particles only migrate between the polymer beads

(Figure 3.5). This result in a smaller time required for large particles to travel through the

column and they elute quicker than the smaller particles which are longer retained in the

column. It should be noted that the HPSEC analysis is performed under elevated pressure.

The analysis was performed using an ÄKTA purifier equipped with a mixed-bed column of

TSK-gel (GMPWxl, 300 mm x 7.8 mm, pore size = 100 – 1000 Å, particle size = 13 µm,

theoretical plates/column ≥ 70000, pH range = 2 – 12, maximum pressure = 300 Pa; Tosch

Bioscience, Stuttgart, Germany) in combination with a TSK guard column (PWxl) and data

were analyzed with UNICORN software. Deionized water supplied by a Simplicity Millipore

water purification system (Milli Q water) was used to prepare the eluents. The eluent used

was a solution of 0.05 M NaNO3 to avoid non-specific interactions between the sample and

the matrix (GE Healthcare – Gel Filtration). 20 µl of samples was injected in the system after

filtration (Millex-HV, Millipore filter) (Millipore, Carrigtwohill, Co. Cork, Ireland). Elution

was executed at 35 °C with 0.05 M NaNO3 buffer, pH 6.9, at a flow rate of 0.7 ml/min. The

eluent was monitored using a Shodex R101 refractive index detector (Showa Denko, K.K.,

Tokyo, Japan). At pH 6.9, using 0.05M NaNO3, the electrostatic expansions of

Figure 3.5: Principle of HPSEC (Lathe et al., 1956).

Chapter 3: Materials and methods

40

polygalacturonic acid are suppressed and the hydrostatic characteristics closely match those of

pullulan. For the standard curve, pullulan standards (MW range 188 - 788 000 Da), which

have structure and hydrodynamic characteristics similar to those of polygalacturonic acid,

were used. Monogalacturonic acid was used daily to equilibrate the system.

3.10 Immunodot assays

The WSP and CSP fractions were used for immunodoting. One µl of the antigen was dotted

onto a piece of nitrocellulose (Hybond ECL, GE Healthcare, Uppsala, Sweden, 0.45 µm pore

size) in a ten-fold dilution series. After 30 minutes of air-drying, the nitrocellulose membrane

was blocked with phosphate-buffered saline (PBS) ( 140 mM NaCl, 8.0 mM Na2HPO4.2H2O,

2.7 mM KCl, 1.5 mM KH2PO4, pH 7.3) containing 5% milk powder (MPBS) (Applichem,

Darmstadt, Germany) for one hour, followed by an incubation with primary antibody for 1

hour and 30 minutes with shaking at room temperature. The following dilutions of primary

antibody in 1% MPBS were used: hybridoma supernatant of JIM5 and JIM7 were diluted 1:12

and 1:10, respectively, hybridoma supernatant of LM18, LM19 and LM20 were diluted 1:20

and PAM1 was diluted 1:10. After washing, the membranes were incubated with secondary

antibody for 1 hour. An anti-rat peroxidase conjugate antibody (Nordic Immunology, Tilburg,

The Netherlands) was used as secondary antibody for JIM5, JIM7, LM18, LM19 and LM20

and was diluted 1/1000 in 1% MPBS. For PAM1, an anti-polyhistidine peroxidase antibody

(Sigma-Aldrich, St. Louis, United States), diluted 1/2000 in 1% MPBS, was used. In a final

step, a color reagent (25 ml PBS, 5 ml chloronaphthol (4 CN), and 16.7 µl 27% (v/v) H2O2)

was added which stained positive blots blue-purple after 40 minutes of development.

3.11 Microscopic analysis with anti-pectin antibodies

Initially, the different purée size fractions were fixed in 70% (v/v) ethanol and stored at 4oC.

Prior to immunolabeling, the ethanol was removed by centrifugation (Microfuge 22R

Centrifuge, Beckman Coulter, Germany) for 5 minutes at 22oC and 3000 g. After this the

pellet was washed two times with 1 ml phosphate buffer saline (PBS) for 5 minutes at 3000g.

50 µl of each sample was used for microscopic analysis.

Chapter 3: Materials and methods

41

Firstly, incubation with primary antibody (JIM5, JIM7, 2F4, LM18, LM19, LM20), which

was diluted in 3% milk-PBS (MPBS) to block non-specific labeling, was carried out for 1h

and 30 minutes at ambient temperature. The following dilutions were made: JIM5 and JIM7

were diluted 1:5 in 3% MPBS; LM18, LM19, LM20 and 2F4 were diluted 1:10 in 3% MPBS.

After incubation with the primary antibody, a washing step followed. Samples were washed

three times for 15 minutes in 1 ml PBS at 22oC and 400 g. A secondary anti-rat antibody

coupled to fluorescein isothiocyanate (FITC) (Nordic Immunology, Tilburg, The Netherlands)

was used to visualize JIM5, JIM7, LM18, LM19 and LM20. The antibody was diluted 1:20 in

3% MPBS. In the case of 2F4, an anti-mouse secondary antibody coupled to FITC diluted

1:50 was used. After washing with PBS three times for 15 minutes in the dark, sections were

examined using an Olympus BX-41 microscope (Olympus, Optical Co. Ltd, Tokwo, Japan)

equipped with epifluorescence illumination (X-CiteR Fluorescence Illumination, Series 120Q,

EXFO Europe, Hants, United Kingdom). Micrographs were taken using image analysis

software (cell*, Soft Imaging System).

In the case of PAM1, the primary antibody was diluted 1:5 in 5% MPBS. As secondary

antibody, an anti-poly-histidine antibody was used. The antibody was diluted 1:1000 in 5%

MPBS. After incubation with the secondary antibody, a tertiary antibody, anti-mouse

antibody coupled to FITC was used. The antibody was diluted 1:50 in 5% MPBS. Sections

were also examined using an Olympus BX-41 microscope equipped with epifluorescence

illumination.

Chapter 4: Results and discussion

42

4 Results and discussion

4.1 Bostwick consistency of broccoli purées

The Bostwick consistency of the differently treated broccoli purées is shown in figure 4.1.

For the blended non-cooked purées, it can be observed that a clear syneresis (syneresis is the

spontaneous separation of the solid and liquid components of a purée) occurs in two broccoli

purées (raw blended purée and LTB blended purée). When broccoli was HTB before

blending, no syneresis occurred. For the blended and cooked purées, a clear syneresis only

occurs in one broccoli purée (the LTB blended cooked purée). This purée also has the lowest

consistency compared to the raw and HTB blended cooked purées. The consistency of the raw

blended cooked purée was in the same range as that of the HTB blended cooked purée. When

broccoli purées were subjected to HPH, the consistency was generally lower. For the HPH

Figure 4.1: Overview of the Bostwick consistency of the differently treated

broccoli purées. Mean values (n=2) with standard deviations are shown.

Chapter 4: Results and discussion

43

purées that were not cooked, the consistency was the lowest when the broccoli was LTB. The

consistency of the HTB and the raw HPH samples was almost in the same range. For the HPH

purées that were cooked, there were some differences observed in the consistency of the

differently treated broccoli purées. After cooking, the HTB purée had the lowest consistency,

while the LTB purée had the highest consistency. This result was opposite from the one

obtained for the HPH purées that were not cooked. In general, cooking increases the

consistency of the differently treated broccoli purées except for the HTB HPH purée.

4.2 Particle size distribution of broccoli purées

A comparison of the particle size distributions of the differently treated broccoli purées is

shown in figures 4.2 – 4.5. From these figures, it can be observed that the 125 µm – 250 µm

and 250 µm – 500 µm size fractions are generally the most important size fractions in the

differently treated broccoli purées. The rest water, containing the liquid fraction of the purée

and particles smaller than 40 µm, also makes a large contribution to the particle size

distribution of the differently treated broccoli purées.

From figure 4.2, it can be seen that the LTB blended broccoli purée has more large particles

than the raw and HTB blended purées.

Figure 4.2: Particle size distribution of the blended broccoli purées.

Chapter 4: Results and discussion

44

From figure 4.3, it can be observed that, after cooking of the blended purées, there is a

significant difference in the particle size distribution of the differently treated broccoli purées.

There are more large particles in the raw and the HTB blended cooked purées compared to the

LTB blended cooked purée. The weight fraction of the LTB blended cooked sample in the

rest water fraction (< 40 µm) is markedly higher compared to the raw and HTB blended

cooked samples.

After HPH of the differently treated broccoli purées (Figure 4.4), the 125 µm – 250 µm size

fraction became the most important one. Also, the effect of the pretreatment conditions was

somewhat smaller compared to the blended samples. After LTB HPH, there were generally

somewhat larger particles compared to the raw and HTB HPH broccoli purées, but the

differences were smaller than when the samples were just blended.

Figure 4.3: Particle size distribution of the blended and cooked broccoli purées.

Chapter 4: Results and discussion

45

There was a substantial difference in the particle size distribution of the differently treated

broccoli purées after HPH and subsequent cooking (Figure 5.5). The raw and LTB HPH

cooked purées have more large particles compared to the HTB HPH cooked purée. There was

generally less rest water compared to the HPH purées that were cooked (except for the raw

HPH cooked sample was the value was similar).

Figure 4.5: Particle size distribution of the high pressure homogenized and cooked

broccoli purées.

Figure 4.4: Particle size distribution of the high pressure homogenized broccoli purées.

Chapter 4: Results and discussion

46

4.3 Degree of methyl-esterification (DM) of broccoli purées

A standard curve was required for both analyses (GalA and methyl-ester content) for the

determination of the DM of pectin. Whenever new solutions were used, standard curves were

repeated.

For the determination of the GalA content, a typical standard curve is shown in figure 4.6.

x = Concentration of galacturonic acid (µg/ml H2O).

y = Absorbance at 520 nm and 25oC.

y = 0.0122x + 0.0288; R2 = 0.9982

For the methyl-ester groups, a typical standard curve is shown in figure 4.7.

Figure 4.6: Standard curve for determining the GalA content: absorbance

at 520 nm and 25oC as a function of the concentration of GalA (µg /ml

H2O).

Chapter 4: Results and discussion

47

x = Concentration of methyl-ester groups (µg methanol/ml phosphate buffer).

y = Absorbance at 412 nm and 25oC.

y = 0.0412x + 0.0272; R2 = 0.9971

An overview of the DM of the differently treated broccoli purées is shown in figure 4.8.

There are substantial differences in the DM of the differently treated broccoli purées. For the

blended non-cooked purées, the DM ranges from 60% (raw blended non-cooked) to 39%

when the broccoli purée was LTB. The DM of the HTB blended purée is about 55%, higher

than that of the LTB blended purée but lower than that of the raw blended sample. After

cooking of the different blended purées, there is generally a decrease in the DM. The DM was

about 49% for the raw blended cooked purée; 34% for the LTB blended cooked purée; and

45% for the HTB blended cooked purée. After HPH, there was generally no shift in the DM

as compared to the blended purées.

Figure 4.7: Standard curve for determining the amount of methyl-ester groups:

absorbance at 412 nm and 25oC as a function of the concentration methanol (µg/ml

phosphate buffer).

Chapter 4: Results and discussion

48

From the above mentioned observations, it can be concluded that after LTB, there was a

significant decrease in the DM of the purées. The LTB pretreatment boosted endogenous

PME activity, leading to an increased demethoxylation. According to Sila et al. (2009), this

favours the formation of a pectin-calcium complex. This phenomenon has previously been

demonstrated by different authors (Canet et al., 2005; Ni et al., 2005; Sila et al., 2004; Wu

and Chang, 1990). This conclusion can be used to explain the observation of more large

particles in LTB broccoli purées (see section 4.2). The decrease in DM after cooking of the

differently treated broccoli purées was due to chemical de-esterification (saponification).

From the data, it can also be concluded that HPH has no effect on enzymatic and/or chemical

de-esterification of pectin.

Figure 4.8: Degree of methyl-esterification (DM) of differently treated

broccoli purées. Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

49

4.4 Changes in pectin solubility

4.4.1 Changes in water soluble pectin (WSP)

The changes in WSP content are as shown in figure 4.9. To assess changes in WSP, the GalA

content of WSP is compared to the sum of the GalA content in WSP, CSP, NSP and residue.

Generally, the percentage of WSP is higher in the cooked samples than in the non-cooked

samples. This can be seen by the increase in the WSP fraction from about 36.6% (raw + blended)

and 34.4% (raw + HPH) to about 66.7% (raw + cooked) and 69.8% (raw + HPH + cooked),

respectively. For the blended, HPH, cooked and non-cooked broccoli purées, the WSP fraction is

always the lowest in the LTB samples. It can also be seen that HTB sometimes resulted in a small

increase in WSP compared to the corresponding raw purées.

Figure 4.9: Changes in the water soluble pectin fraction. The percentage of WSP is

defined as the GalA content of WSP compared to the GalA content in WSP, CSP, NSP and

residue. Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

50

4.4.2 Changes in chelator soluble pectin (CSP)

The changes in CSP content were evaluated by comparing the amount of GalA of the CSP

fraction to the sum of the GalA content in WSP, CSP, NSP and residue, and are shown in

figure 4.10. Raw blended broccoli purée contains approximately 25% CSP, which is

comparable to the amount of CSP in the raw blended cooked purée. HPH resulted in an

increase in CSP for the raw non-cooked sample, while a decrease was observed for the

cooked sample.

Generally, the percentage of CSP was the highest for the LTB (cooked or non-cooked blended

or HPH) samples, which are also characterized by the lowest amount of WSP. For the HTB

samples that were cooked, a low value of CSP (± 18%) can be observed. For the non-cooked

HTB samples, a large difference between the blended and the HPH purée is seen. This large

difference may be due to the low extraction yield for the HPH sample (see section 4.4.5).

Figure 4.10: Changes in the chelator soluble pectin fraction. The percentage of CSP is

defined as the GalA content of CSP compared to the GalA content in WSP, CSP, NSP and

residue. Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

51

4.4.3 Changes in sodium carbonate soluble pectin (!SP)

The changes in NSP content were evaluated by comparing the amount of GalA in the NSP

fraction to the sum of the GalA content in WSP, CSP, NSP and residue, and are shown in

figure 4.11. Generally, it can be seen that the amount of NSP in the different broccoli purées

decreases tremendously after cooking. For the raw samples, the amount of NSP ranges from

about 30.7% (raw + blended) and 23.7% (raw + HPH) to about 1.6% (raw + blended +

cooked) and 3.3% (raw + HPH + cooked). After LTB, the amount of NSP was the highest

(cooked or non-cooked, blended or HPH broccoli purées). These samples were also

characterized by the lowest amount of WSP and the highest amount of CSP. The amount of

NSP was lowest in the HTB purées.

Figure 4.11: Changes in the sodium carbonate soluble pectin fraction. The percentage of

NSP is defined as the GalA content of NSP compared to the GalA content in WSP, CSP,

NSP and residue. Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

52

4.4.4 Changes in residue pectin

The changes in pectin content of the residue were evaluated by comparing the amount of

GalA in the residue to the sum of the GalA content in WSP, CSP, NSP and residue. Figure

4.12 shows that some GalA was still present in the residue after the AIR was fractionated. It

can be seen that the percentage of residue was higher in the non-cooked blended samples

compared to the cooked blended samples. Also, the percentage of residue was higher in the

HPH cooked samples compared to blended cooked samples.

4.4.5 Fractionation yield

The fractionation yield for each of the various samples was calculated to evaluate how much

pectin was recovered from the AIR (Figure 4.13). The fractionation yield can be defined as

the percentage of GalA in WSP, CSP, NSP and residue compared to the total amount of GalA

Figure 4.12: Changes in the residue fraction. The percentage of residue is defined as the

GalA content of residue compared to the GalA content in WSP, CSP, NSP and residue.

Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

53

in AIR. A fractionation yield between 56% (LTB blended sample) and 100% (HTB HPH

cooked sample) was observed. In general, the fractionation yields were rather low. This was

probably because there was some loss of GalA during the fractionation. From figure 4.13, it

can be observed that the cooked (both the blended and the HPH) samples resulted in a higher

fractionation yield compared to the non-cooked samples.

4.4.6 Changes in pectin solubility: summary

A significant change in the proportions of pectin fractions, together with their respective

residues was noted upon the various treatments of broccoli purées. Raw blended broccoli

purée contained predominantly water soluble pectin (about 35%), a substantial amount of

chelator soluble pectin (about 25%), and a high amount of sodium carbonate soluble pectin

(about 30%). LTB resulted in the transformation of WSP into insoluble pectin (CSP and

Figure 4.13: Fractionation yield (%) calculated by the percentage of GalA in

WSP, CSP, NSP and residue compared to the total amount of GalA in AIR.

Chapter 4: Results and discussion

54

NSP). The extent of the transformation was strongly related to the DM of AIR (Sila et al.,

2009). A decrease in DM lowers the solubilization rate of pectin in water and provides a

larger opportunity for pectin-Ca2+ cross linking. Interestingly, during cooking, insoluble

pectin (especially NSP) was converted into water soluble pectin. This is clearly evident from

the increasing proportions of the WSP fraction after cooking, which are paralleled by

declining proportions of NSP. This dynamic conversion of pectin was less pronounced in the

CSP fraction. The phenomenon of increasing WSP content during thermal processing has

been reported in some plant-based foods (Van Buggenhout et al., 2009). These results

indicate that a substantial solubilization and degradation of matrix polysaccharides occurs

during cooking. Nevertheless, it is unlikely that all of the observed changes in the Bostwick

consistency of the differently treated broccoli purées can solely be explained by the changing

solubility patterns of pectin fractions.

4.5 Degree of methyl-esterification (DM) of WSP and CSP fractions

The DM of the pectin fractions, WSP and CSP, relative to the DM of AIR is presented in

Figure 4.14. As discussed earlier, the DM of the NSP fraction was not determined because of

the saponification of methyl-esters by Na2CO3 during fractionation.

There was a significant difference in the DM of the WSP and CSP fractions. The DM of the

WSP fractions was quite high, ranging from 45% to 75% while the CSP fractions had a lower

DM, ranging from 6% to 47%. This indicates that WSP contains high-esterified pectin while

CSP contains low-esterified pectin. From figure 4.14, it can be observed that the DM of the

WSP fraction almost follows the same trend as the DM of the AIR. Also, there were very low

DM values for some CSP fractions especially after cooking and/or HPH.

Chapter 4: Results and discussion

55

4.6 Changes in the molar mass (MM) distribution of different pectin fractions

The high molar mass polymers in the broccoli purée pectin fractions have homogeneous

molar mass distribution patterns, with each fraction characterized by a distinct molar mass

average. This is demonstrated for the raw blended non-cooked sample (WSP124KDa, CSP558KDa,

and NSP776KDa) in Figure 4.15. The WSP fraction has the lowest MM compared to the NSP

and CSP fractions. When looking at the width of the MM distributions, it is clear that NSP

displayed the narrowest distribution, whereas WSP and CSP consisted of a diverse set of

pectic polymers with a large range in MM.

Figure 4.14: DM (%) of WSP and CSP fractions relative to the DM (%) of the AIR.

Mean values (n=6) with standard deviations are shown.

Chapter 4: Results and discussion

56

The comparison between the differently treated broccoli purées in molar mass (MM)

distribution of WSP and CSP fractions is shown in figures 4.16 and 4.17.

From figure 4.16(A), it can be observed that the WSP molar mass distribution for the

differently pretreated blended non-cooked samples almost follows the same pattern. The same

observation could be assigned for the high pressure homogenized non-cooked samples

(Figure 4.16(B)). After the cooking process, there were pronounced changes in the MM

distribution patterns. The WSP fractions showed increasing concentrations of solubilized

Figure 4.15: Comparison of the molar mass (MM) distribution of WSP, CSP and NSP

of the raw blended broccoli purée. Elution times of pullulan standards are indicated to

allow for a rough estimation of the MM.

Chapter 4: Results and discussion

57

polymers characterized by a polydisperse MM range that covered the MM spectrum of all

pectin fractions (WSP, CSP, and NSP). The MM distribution of the cooked samples showed

three unresolved peaks. A possible explanation for this is that the early eluting peaks represent

the proportions of the NSP fractions (and a little bit of the CSP fractions) which are thermo-

solubilized, whereas the last eluting peaks illustrate transformations in the WSP fractions due

to β-eliminative depolymerization (shifts towards low molar mass fragments). The middle

peaks illustrate the original peaks of the WSP fractions. This clearly demonstrates a dynamic

change in WSP during cooking. Thermo-solubilization of pectin was greatly influenced by the

type of pre-treatment condition used. The intensity of the transformations diminished after

LTB.

For the CSP fraction (Figure 4.17(A)), it can be observed that the CSP MM distribution of the

blended and HPH non-cooked samples almost follows the same pattern. There was however

an increase in the right hand side of the peak for the cooked samples (Figure 4.17(B)). This

observation can be attributed to the fact that pectin, originally present in the WSP fraction,

may be de-esterified during the cooking and may turn into CSP due to a higher mobility of

calcium and pectin at high temperature and by which pectin-Ca2+ cross links can be formed.

The results for the MM distribution of NSP for the differently treated broccoli purées are not

shown in this thesis. This is because there were no changes in the MM distribution of NSP

upon the treatments used.

Chapter 4: Results and discussion

58

Figure 4.16: Comparison of the molar mass (MM) distribution of the WSP of A) the

blended broccoli purées and B) the high pressure homogenized broccoli purées. Elution

times of pullulan standards are indicated to allow for a rough estimation of the MM.

B)

A)

Chapter 4: Results and discussion

59

A)

Figure 4.17: Comparison of the molar mass (MM) distribution of the CSP of A) the non-

cooked broccoli purées and B) the cooked broccoli purées. Elution times of pullulan

standards are indicated to allow for a rough estimation of the MM.

B)

Chapter 4: Results and discussion

60

4.7 Immunodot assays of WSP and CSP fractions

4.7.1 Binding of anti-HG antibodies to WSP of broccoli purées

The immunodot assays in figure 4.18 show the binding of the anti-HG antibodies JIM5, JIM7,

LM18, LM19, LM20 and PAM1 to the WSP fraction of the differently treated broccoli

purées. The immune profile of heterogeneous pectic samples when applied to nitrocellulose

and probed with mAbs is related to the relative mobilities of different pectic components

within the sample away from the point of application on the nitrocellulose. Unbranched HG

regions (outer ring) migrate further than branched domains (central dot) (Willats et al., 1999).

From figure 4.18, it can be seen that JIM5 binds very weakly to the non-cooked LTB and

HTB treated samples. These samples were resolved predominantly on the nitrocellulose as

smaller diameter dots, with very limited visibility. There was no binding of JIM5 after

cooking to the broccoli purées. The binding strength of JIM7 was quite different from that of

JIM5. It can be seen that JIM7 binds to all of the WSP samples of the different broccoli

purées. Both the blended and the HPH samples showed a similar degree of binding capacity.

After cooking, the binding capacity of JIM7 was lower. The outer rings of the various cooked

samples were less intensely labeled with JIM7. This disappearance of unbranched highly-

esterified WSP may be linked to the conversion of WSP into CSP that was observed in the

MM distribution of CSP of the cooked samples (explained in Section 4.6). It can be observed

that LM18 binds very weakly to the LTB pretreated samples that were not cooked. These

PME de-esterified samples exhibited no labeling of the outer ring on the nitrocellulose paper.

LM19 binds to all samples (non-cooked or cooked, blended or HPH). Also, like LM18, the

binding strength of LM19 is higher for the LTB non-cooked samples. It can be observed that

the outer rings of the various samples (both uncooked and cooked) were less intensely labeled

with LM19 antibody. LM20 binds specifically to the non-cooked blended and HPH samples

with almost the same binding strength. After cooking of the blended samples, the binding

strength of LM20 was almost the same as for the non-cooked samples. This observation was

different for the high pressure homogenized samples. After cooking of the HPH samples, the

binding strength of LM20 decreased. The non-cooked samples showed some labeling of the

outer ring. After cooking, there was no labeling of the outer rings. Long stretches of non-

methyl-esterified GalA residues are necessary for PAM1 to bind. But from figure 4.18, there

Chapter 4: Results and discussion

61

was only very weak binding of the PAM1 antibody visible to the LTB and HTB non-cooked

samples.

From the above observations, it can be concluded that the overall binding intensity of the anti-

HG antibodies, JIM5, LM18, and PAM1 to the different WSP fractions was weak, which can

be attributed to the preferential binding of these antibodies to pectins with a low (LM18, and

PAM1) or medium range DM (JIM5) (Verhertbruggen et al., 2009). Antibodies, JIM7 and

LM20, both are able to recognize highly methyl-esterified pectin bound to the WSP fraction

of all samples. LM19, recognizing medium to low methyl-esterified pectin, preferentially

Figure 4.18: Immunodot assays showing the binding of the anti-HG antibodies

JIM5, JIM7, LM18, LM19, LM20 and PAM1 to the WSP fraction of the

differently treated broccoli purées.

Chapter 4: Results and discussion

62

bound to the central dot of the applied samples. This suggests that WSP consists of

unbranched pectin which is highly-esterified whereas the pectic polymers that are less-

esterified have abundant side chains.

4.7.2 Binding of anti-HG antibodies to CSP of broccoli purées

The immunodot assays in figure 4.19 show the binding of the anti-HG antibodies JIM5, JIM7,

LM18, LM19, LM20 and PAM1 to the CSP fraction of the different broccoli purées.

From figure 4.19, it can be seen that JIM5 binds to all CSP fractions but the binding was more

pronounced for the LTB samples. This may be due to the combination of a high amount of

CSP and a low DM. Only the outer ring of the LTB samples was clearly labeled. The binding

strength of JIM7 was much larger than that of JIM5. Being a general anti-pectin probe, it can

be seen that JIM7 binds to all of the CSP samples of the differently treated broccoli purées.

The binding strength of JIM7 for the high pressure homogenized samples was a little bit

higher than for the blended samples. Also, both the outer ring and central dot were labeled for

all CSP fractions. The labeling of LM18 to the CSP fractions was quite similar to the labeling

of JIM5. LM18, having preference for lowly esterified pectins (Verhertbruggen et al., 2009),

displayed the highest labeling intensity for the LTB (non-cooked or cooked, blended or HPH)

samples. The outer ring and central dot of the LTB samples were both labeled with LM18.

The binding strength for antibody LM19 was generally comparable to that of JIM7.

However, there was no difference in the binding strength of LM19 between the blended and

the HPH samples. LM20 binds specifically to the blended and HPH samples with almost the

same binding strength. After cooking of the blended samples, the binding strength of LM20

was lower than for the uncooked samples, with the low temperature blanched sample being

more labeled. The same observation could be done for the high pressure homogenized

samples. The labeling of both the outer ring and central dot with LM20 was visible for both

the non-cooked and cooked samples. PAM1 finally had a very high binding specificity for the

LTB pretreated broccoli purées. This is due to the blockwise de-esterification of the GalA

residues of the pectin by PME during LTB.

It can generally be concluded that the binding strength of the different anti-HG antibodies was

greater for the CSP fraction of the differently treated broccoli purées than for the WSP

Chapter 4: Results and discussion

63

fractions. Binding of all the anti-HG antibodies indicates that CSP contains pectin with a

rather broad range in DM. Also for the CSP fraction, the low- and high esterified pectins

occur in either highly branched or unbranched domains of pectin.

Figure 4.19: Immunodot assays showing the binding of the anti-HG antibodies

JIM5, JIM7, LM18, LM19, LM20 and PAM1 to the CSP fraction of the

differently treated broccoli purées.

Chapter 4: Results and discussion

64

4.8 In situ immunolabeling of pectin with anti-pectin antibodies

As mentioned previously, the in situ visualization of pectin with anti-HG antibodies has been

considered as a powerful tool that can be used to investigate pectin conversions throughout

the cell wall. In this study, the immuno-fluorescence labeling of monoclonal antibodies

(JIM5, JIM7, PAM1, LM18, LM19, LM20, 2F4) was examined. It should be noted that the in

situ immunolabeling of the anti-HG antibodies was done for the 80 - 125 µm weight fraction

of the differently treated broccoli samples. Due to time limitations, only the blended samples

were analyzed.

4.8.1 Immunolabeling of non-cooked broccoli purées

Figure 4.20 shows the binding of the anti-HG antibodies JIM5, JIM7, LM18, LM19 and

LM20 to the 80 - 125 µm weight fraction of the differently non-cooked blended broccoli

purées. For the raw non-cooked sample, the epitopes of JIM5 were distributed mostly across

the cell junctions but sometimes points of the cell wall were labeled as well. A different

phenomenon was noticed with JIM7. Specifically, the epitopes of JIM7 were distributed

across the entire cell wall and this in a more dot-like manner. Also, the region of the cell wall

lining the intercellular spaces was labelled with JIM7. JIM7 labeling was also observed at

regions of cell breakage. Together with the dot-like distribution of epitopes, this indicates the

effectiveness of JIM7-epitopes in cell-cell adhesion of the cells. The epitopes of LM19 were

evenly distributed across the entire cell wall, with a rather low labeling intensity. A different

phenomenon was noticed with LM20 that mainly recognizes pectin in the region of the cell

wall lining the intercellular spaces at the cell junctions but also results in a discontinuous

labeling of the cell wall adjacent to the plasma membrane. The occurrence of epitopes of the

antibodies, LM19, LM20, JIM5 and JIM7, at regions of the cell wall lining intercellular

spaces suggests that these regions contain pectin with a broad range of DM. The labeling

intensity of PAM1 antibody was not clear enough for conclusions to be done and LM18

showed no labeling at all (results not shown). These observations for PAM1 and LM18 were

also applicable for the other samples.

Chapter 4: Results and discussion

65

Figure 4.20: Representative pictures showing the binding of the anti-HG antibodies

JIM5, JIM7, LM18, LM19 and LM20 to the 80 – 125 µm size fraction of the differently

non-cooked blended broccoli purées. Scale bars = 50 µm.

Chapter 4: Results and discussion

66

LTB pretreatment of the blended non-cooked sample influenced the labeling pattern of some

antibodies. Specifically, JIM5 epitopes at the regions of cell junctions were more pronounced.

The binding and labeling intensity of JIM7 for the LTB-treated non-cooked sample was

higher than for the raw non-cooked sample. JIM7 specifically binds to the entire cell wall as

well as to the region of the cell wall lining the intercellular spaces as the raw non-cooked

sample, but in a more uniform and homogenous pattern. This may be due to higher epitope

accessibility. Also, the regions of cell breakage were clearly labeled with JIM7. LTB induces

almost the same labeling pattern for LM19 as the raw non-cooked sample. The epitopes of

LM19 were also evenly distributed across the entire cell wall, with rather low labeling

intensity. The labeling pattern of LM20, after LTB, was similar to the raw non-cooked

sample, only the amount of discontinuous labeling was less. Clear observations pointing at

PME activity were not done. However, Christiaens et al. (2011) previously showed that PME

de-esterification in broccoli predominantly takes place in the cell junctions.

After HTB, JIM5 epitopes were, like after LTB, preferentially distributed in the regions of the

cell wall lining intercellular spaces at cell junctions. JIM7 also followed the same pattern of

labeling as the LTB-treated sample. The labeling of LM19 was, on the other hand, not

uniform. Some regions of the cell wall and the cell junctions experienced very pronounced

labeling, whereas in others, the labeling was very weak. The labeling of LM20 was only

slightly observed at some cell junctions of the broccoli cells.

4.8.2 Immunolabeling of cooked broccoli purées

Figure 4.21 shows the binding of the anti-HG antibodies JIM5, JIM7, LM18, LM19 and

LM20 to the 80 - 125 µm size fractions of the differently cooked blended broccoli purées. The

binding of JIM5 for the raw cooked sample was quite similar to the raw blended non-cooked

sample where it mainly labeled the regions of the parenchyma cell walls lining the

intercellular spaces at cell junctions and sometimes parts of the cell wall were labeled as well.

The epitopes of JIM7 were evenly distributed across the entire cell wall and the cell junctions.

This result was different from the raw blended non-cooked sample in that the binding

specificity of JIM7 in the latter sample occurred in a more dot-like pattern. The binding

Chapter 4: Results and discussion

67

specificities of LM19 and LM20 were similar to that of JIM5 for the raw blended cooked

sample.

For the LTB cooked sample, the binding specificity of the JIM5 epitope was somewhat

similar to that of the cooked raw sample as well as the blended non-cooked sample. The same

observation could be noticed for JIM7. This therefore indicates cooking of the LTB sample

did not affect the binding pattern and specificity of JIM5 and JIM7 antibodies. The epitopes

of LM19 were evenly distributed across the entire cell wall in the LTB cooked sample. LM20

showed preferential labeling of the cell wall lining the intercellular spaces.

For the cooked HTB sample, the labeling of JIM5 was similar to that of the HTB non-cooked

sample. Also, JIM7 mainly labeled the cell wall, with exception of the middle lamella. This

was mainly visible for cells with swollen cell walls. LM19 and LM20 epitopes were

predominantly distributed at the cell junctions, similar to the result obtained for the raw

cooked sample. It can also be concluded that the binding specificities of LM19 and LM20

were not affected by the cooking process.

Chapter 4: Results and discussion

68

4.8.3 Localization of Ca2+

cross-linked pectin

Localization of Ca2+-cross-linked pectin is possible with monoclonal antibody 2F4 (Liners et

al., 1989) (Figure 4.22). It can be seen that the epitopes of 2F4 are predominantly located in

the regions of the cell wall lining intercellular spaces for the non cooked samples. This

observation leads to a conclusion that pectin cross-linked with endogenous Ca2+ is located in

these domain regions. After cooking, there occurred a more dot-like distribution of the 2F4

antibody.

Figure 4.21: Representative pictures showing the binding of the anti-HG antibodies

JIM5, JIM7, LM18, LM19 and LM20 to the 80 - 125 µm size fractions of the

differently cooked blended broccoli purées. Scale bars = 50 µm.

Chapter 4: Results and discussion

69

Figure 4.22: Representative pictures showing the binding of the anti-HG

antibody 2F4 to the 80 - 125 µm size fraction of the differently non-

cooked and cooked blended broccoli purées. Scale bars = 50 µm.

70

General conclusion

In this thesis, the structure-function relationship of pectin in broccoli purées was investigated.

Syneresis (that is the spontaneous separation of the solid and liquid components of a purée)

occurred when raw broccoli was just blended to create a purée. This phenomenon did not

occur when a high amount of WSP was induced or when the purée was high pressure

homogenized.

Generally, the consistency of the broccoli purées increased after cooking due to the high

amounts of the WSP. Pectin solubilization and β-eliminative depolymerization were

visualized in the molar mass distribution of the WSP fractions of the cooked purées. Also, a

decrease in DM after cooking of the differently treated broccoli purées was observed due to

chemical de-esterification.

After HPH, a lot of small particles were formed (smaller that 40 µm) and were collected in the

rest water fraction after wet sieving. This generally led to a decrease in consistency of the

broccoli purées. No clear syneresis was observed after HPH, probably because of the more

small particles formed. HPH generally had no effect on enzymatic and/or chemical de-

esterification of pectin or on the molar mass distribution of the pectin fractions.

LTB led, in some of the purées, to a decrease in consistency and an increase in more large

particles which may be linked to increased pectin-calcium cross linking. This was reflected in

a decrease in pectin solubilization in water and corresponding high amounts of CSP (and

NSP). LTB diminished the intensity of thermosolubilization after cooking. The lower DM for

LTB samples, due to PME-stimulation during LTB, was also noticed in immunodot assays by

more labeling with anti-HG antibodies binding to low-esterified pectin. The binding of the

Ca2+ ions to the blocks of unesterified GalA residues of the pectin after LTB was

demonstrated with the anti-HG antibody, 2F4.

When the purées were subjected to HTB, a small increase in the solubility of pectin (WSP)

was observed. Also, an increase in the consistency and a decrease in the particle sizes were

often observed. No syneresis occurred in any of the HTB broccoli purées.

71

In conclusion, the rheological properties of broccoli purées can be linked to a combination of

particle size distribution and pectin solubilized in the serum phase.

In the future, it will be interesting to develop some models to predict and evaluate

quantitatively the effects of processing on the physical functionalities (rheological flow

behaviour) of vegetable purées. Another possibility is to do research on the influence of

structure-enabling and preservation processes on the microstructure of vegetable-based

(multi-phase) systems (e.g. sauces, soups, purées). Finally, research aiming to gain insight in

the effect of processing conditions on the flavour profile of (multi-phase) vegetable-based

products, including the understanding of the relations between flavor perception and food

(micro-) structure can be done. The research should focus on the correlation of the chemical

flavour profile of processed vegetable-based food with sensory analysis.

72

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