i

Katholieke

Universiteit

Leuven

FACULTY OF BIOSCIENCE ENGINEERING

INTERUNIVERSITY PROGRAMME (IUPFOOD)

MASTER OF SCIENCE IN FOOD TECHNOLOGY

Major Food Science and Technology

Academic year 2014-2015

Heat Stability Evaluation of Oil-in-Water Emulsions Stabilized by Whey

Protein Isolate-Low Methoxyl Pectin Dry Heat Conjugates

by

Serveh Saeedi

Promotor: Prof. dr. ir. Paul Van der Meeren

Tutor: Arima Diah Setiowati

Master's dissertation submitted in partial fulfilment of the requirements

for the degree of Master of Science in Food Technology

i

Copyright

The author and promoter give the permission to consult and copy parts of this

work for personal use only. Any other use is under the limitations of copyrights

laws, more specifically it is obligatory to specify the source when using results

from this Master’s Dissertation.

Gent, August 2015

The promoter The author

Name: Prof. dr. ir. Paul Van Der Meeren Name: Serveh Saeedi

Signature: Signature:

ii

Dedication

I would like to lovingly dedicate this Master dissertation to my

parents. There is no doubt in my mind that without their continued

generosity, support and counsel I could not have completed this process.

iii

Acknowledgements

I would like to express my gratitude to Prof. Paul Van Der Meeren for giving

me the opportunity to work on this topic and his support and guidance throughout my

project. It is a great opportunity to study under his supervision and his support is

highly appreciated. My deepest sense of gratitude goes to Arima Diah Setiowati for

her invaluable support, academic advice and criticism during practical work and

writing of this master dissertation.

I also express my special thankfulness to my loving parents and family, for

their lofty love, constant support, motivation, and generosity. Special thanks go to my

sister and her family whose invaluable encouragements and support enabled me to

complete this work.

I would like to thank Arnout Declerck, Mathieu Balcaen and Quenten Denon

for their technical recommendations and support. My sincere thanks and appreciation

are also extended to all the staffs and research memberss of laboratory of Particle and

Interfacial Technology (PaInT).

Finally, my thanks go to all my friends and professors from the IUPFOOD

program in Gent and KULeuven universities. I would like to express my sincere

gratitude to all wonderful people I met during this master program.

iv

Table of Contents

Copyright …………………………………………………….................i

Dedication ………………………………………………………..….…ii

Acknowledgements ……………………………………………….…...iii

Table of contents …………………………………………………….....iv

List of Figures …………………………………………………...….…vii

List of Tables ………………………………………………….………..x

List of Abbreviations ………………………………………….……...xiii

Abstract ………………………………………………………………....1

Chapter 1: Literature Review...………………….………….….……...3

1.1. Introduction ……………………….……………………………...……………3

1.1.1. Overview ……………………………..……………………..….………...3

1.1.2. Research Objectives ……………..………………………….……….….4

1.2 Whey Protein ………………………………………………..…………...….....5

1.2.1 Whey Protein Components ……………..…………………………......6

1.2.1.1 β-Lactoglobulin …………………..……………………….……..6

1.2.1.2 α-Lactalbumin ………………………..………....………………8

1.2.1.3 Other Minor Proteins …………..…………..…………...………8

1.2.2 Whey Proteins Functional Properties ……………………….…….....9

1.3 Emulsions ………………………………………………..............…………....11

1.3.1 Emulsion Destabilization Mechanisms ……..………………..………12

1.3.2 Stabilization of Emulsions …………………..……………….……….15

1.4 Whey Protein as Emulsifier …………………………………..………….……16

1.4.1 Limiting Factors of WPI application in Emulsions ….......................18

v

1.5 Heat stability of WPI ………………………………………..…….…..………..18

1.5.1 Heat Induced Denaturation ………………...………….…….………19

1.5.2 Factors Influencing Heat Stability of WPI ……….……….………..21

1.5.3 Improvement of Heat Stability of Whey Protein Ingredients …..…23

1.6 protein-polysaccharide Conjugates via Covalent Bonding …………...…..…26

1.6.1 Mechanism of the conjugate formation ………………….….….…..27

1.6.2 Emulsifying Properties of Maillard Conjugates …………….……...29

1.6.3 Factors Influencing the Functionality of the Conjugate ………...…29

1.6.4 Studies on Heat Stability of Protein-Polysaccharide Conjugate ..…31

Chapter 2: Materials and Methods ……………………………..…....34

2.1. Materials ……………………………….……………………………………....34

2.2.Methods …………………………………………………………..…………….34

2.2.1. Whey Protein-Pectin Complex Formation …………………..…….34

2.2.2. Emulsion Preparation ………………………..………………..……35

2.2.3. Heat Stability Test of Emulsions …………….…………………….37

2.2.4. Emulsion Stability Test ………………………….…….….…..……39

2.2.5. Viscosity Measurement …………………………….………..….….41

2.2.6. Particle Size Measurement ……………………………..….…..…..42

2.2.7. Light Microscopic Observation ……………………………….…..43

2.2.8. Protein Load ………………………………….……………….…....43

2.2.9. Pectin Load ………………………………….…………………...…45

Chapter 3: Results and Discussion …………………………………. 47

3.1. Visual observation ……………………………….…………….…..………....47

3.2. Droplet size ………………………………………………….……………...... 49

3.3. Viscosity ……………………………………………………….…….……..…61

3.4. Emulsion Stability Analysis ………………………………….….……..……68

3.5. Protein Load ……………………………………………………..……..……74

vi

3.6. Pectin Load ……………………………………………….………..…….….79

Chapter 4: Conclusion ………………………………..…………….83

Chapter 5: References ………………………………………………87

vii

List of Figures

Figure 1.1 Schematic figures of emulsions. a. oil in water emulsion (O/W); b.

water in oil emulsion (W/O); c. Water in oil in water double emulsion

(W/O/W); d. Oil in water in Oil double emulsion (O/W/W) ………12

Figure 1.2 Schematic representation of common instability mechanism in food

emulsions; flocculation, coalescence, Ostwald ripening, creaming and

phase separation …………………….…………………………..……14

Figure 1.3 Mechanism of emulsion stabilization by proteins through (A)

electrostatic repulsion and (B) steric stabilization. Red dots are

hydrophobic parts of protein molecules positioned at the oil phase

(Lam and Nickerson, 2013) ……………………….…..……………17

Figure 2.1

Schematic presentation of setup for heat treatment ………………….38

Figure 2.2 Schematic illustration of the operational principle of the LUMIfuge

…………………………………………………………………...……39

Figure 2.3 Schematic presentation of front tracking method of transmission

profile with trigger value of 20% ……………………………….….. 40

Figure 2.4 Standard Curve for pectin measurement ………………………..……46

viii

Figure 3.1

Image of visual appearance of unheated (fresh) oil-in-water emulsion

(10% w/w) stabilized with 0.5% WPI (A), dry heated WPI day 8 (B),

and WPI-LMP conjugate (ratio 2:1) day 8 (C), at pH 5.0.

…………………………………………………………….…………47

Figure 3.2 Image of visual appearance of oil-in-water emulsion (10% w/w)

stabilized with 0.5% WPI, dry heated WPI, and WPI-LMP conjugates (

2:1), day 4 at pH 5.0 with NaCl, after heat treatment at 80°C for 10 and

20 min. ……………………………………………………………...48

Figure 3.3 Comparison of visual appearance of oil-in-water emulsion (10% w/w)

stabilized with 0.5% WPI, at pH 5.0 and pH 6.5, and emulsion

stabilized with WPI-LMP conjugate (2:1), day 0 and day 8, at pH 5.0

with NaCl, after heat treatment at 80°C for 20

min…………………………………………………………………..49

Figure 3.4 The mean volume-weighted droplet size, d43, of oil in water emulsion

(10% w/w oil), containing 0.5% (w/w) WPI and WPI-LMP, incubated

for 8 days at pH 6.5 and 5.0 in the absence and presence of 30 mM

NaCl, and corresponding micrograph of the emulsions containing WPI

(right) and WPI-LMP (D8) (left) at pH 5.0.

………………………………………………………………………51

Figure 3.5 Droplet size distribution curves of an oil-in-water emulsion (10% w/w

sunflower oil) stabilized with a 0.5% (w/w) WPI heat treated at 80°C

for 0, 10 and 20 min at pH 6.5 with NaCl. The micrograph image

shows WPI stabilized emulsions at pH 6.5 unheated (left) and heated

for 20 min (right) ……………………………………………………54

Figure 3.6 Changes in average droplet size, d43 , of oil in water emulsions (10%

w/w oil), stabilized with 0.5% (w/w) WPI, dry heated WPI (D4) or

WPI-LMP conjugates (D4), after heating at 80 °c for 10 and 20 min, at

pH 6.5 ………………………………………………………………56

ix

Figure 3.7 Comparison of droplet size distribution curves of oil-in-water

emulsions (10%w/w) stabilized with non-incubated and incubated (4, 8

and 16 days) WPI-LMP, unheated and heated at 80°C for 20 min at pH

6.5. ……………………………………………………………………57

Figure 3.8 Changes in average droplet size, d43, of oil in water emulsions (10%

w/w oil), stabilized with 0.5% (w/w) WPI-LMP conjugate (D0), after

heating at 80 °C for 10 and 20 min, at pH 6.5 and 5.0, with or without

NaCl. ……………………………………………………………..…58

Figure 3.9 Droplet size distribution curves of oil-in-water emulsions (10% w/w)

stabilized with a 0.5% (w/w) WPI, heat treated at 80°C for 0 and 10

min, with size distribution curve and the corresponding micrograph of

the heated emulsions after dispersion in 1% SDS at pH 6.5 (left : 10

min , right: 20 min). ……………………………………..……….….59

Figure 3.10 Viscosity profile of oil in water emulsion (10% o/w sunflower oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI (D8) and WPI-LMP

conjugate (D0) , at pH 6.5 after heat treatment at 80 °C for 10 and 20

min………………………………………….……….………………64

Figure 3.11

Viscosity profile of oil in water emulsion (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI-LMP conjugate, incubated for 4, 8 and

16 days, at pH 6.5 and 5.0, with and without NaCl, before and after

heat treatment at 80 °C for 10 and 20 min.

………………………….……………………………………………66

x

Figure 3.12 Evolution of transmission profile of an oil in water emulsion (10% w/w

oil), stabilized with 0.5% (w/w) WPI (A), and WPI-LMP conjugate

(2:1) (B), at pH 5.0. …………………………………………………69

Figure 3.13 Comparison of front tracking results of emulsions (10% w/w oil),

stabilized with 0.5% (w/w) WPI (A), and WPI-LMP conjugate (2:1),

incubated for 4days, at pH5.0 with 30mM NaCl. …..….………..70

Figure 3.14 Comparison of creaming rate (mm/day) of emulsions (10% w/w oil)

stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP

conjugate (2:1), incubated for 16 days, containing 30 mM NaCl, at pH

6.5 ……………………………………………………………………71

Figure 3.15 Comparison of creaming rate (mm/day) of emulsions (10% w/w oil)

stabilized with 0.5% (w/w) WPI and WPI-LMP conjugate (2:1),

incubated for 4, 8 and 16 days, at pH 5.0 containing 30 mM NaCl,

heated at 80°C for 0,10 and 20min. ………………………..………73

Figure 3.16 Protein load (kg/m2) of oil in water emulsions (10% w/w sunflower

oil), stabilized with 0.5% (w/w) WPI, and WPI-LMP conjugate (2:1),

unincubated and incubated for 8 days, heated for 0 and 20min at 80 °C

at pH6.5 and 5.0. ..................................................................................78

xi

List of tables

Table 2.1

Emulsions names and codes ………………………………………… 37

Table 3.1 Average Droplet Size d43 (µm) of oil in water emulsion (10% w/w oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP

conjugates, unincubated (Day 0) and incubated for 4, 8 and 16 days, at

pH 6.5 and 5.0, in the presence of 30 mM and 0 mM NaCl,

homogenized at 560 bar for 2 minutes and temperature of 50 oC.

……………………………………………………………………….….50

Table 3.2 Effect of heating at 80°c for 10 and 20 min on the mean volume-weighted

particle size, d 43 , of oil in water emulsions (10%w/w) containing 0.5%

(w/w)WPI, dry heated WPI and WPI-LMP conjugate (2:1) , non-

incubated and incubated for 4, 8 and 16 days at pH 6.5.

……………………………………………………………………………5

3

Table 3.3 Table.3.3. Effect of heating at 80°c for 10 and 20 min on the mean

volume-weighted particle size, d 43 (µm), of oil in water emulsions

(10%w/w) containing 0.5% (w/w)WPI and WPI-LMP conjugate (ratio

2:1) , non-incubated and incubated for 4, 8 and 16 days at pH 5.0, with

and without NaCl.

……………………………………………………………………..55

Table 3.5 Viscosity of oil in water emulsion (mPa.s) (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP

conjugate, at pH 6.5 and 5.0, with and without NaCl.

……….………………….61

Table 3.6 Effect of heating at 80°C for 10 and 20 min on consistency index of oil in

xii

water emulsions (10%w/w) containing 0.5% (w/w) WPIand WPI-LMP

conjugates (2:1) non-incubated and incubated for 8 and 16 days at pH 5.0

with and without NaCl.

…………………………………………………..62

Table 3.7 Creaming rate (mm/day) (LUMiFuge, 3000 rpm, 1 hr) of o/w emulsions

(10% w/w oil) stabilized with 0.5% (w/w) WPI and WPI-LMP conjugate

(2:1), containing 30 mM NaCl, at pH 6.5 and 5.0. …………………….69

Table 3.8 Amount of protein (μg/ml) in aqueous phase, serum phase and adsorbed

on oil droplets of heated and unheated oil in water emulsion (10% w/w

sunflower oil), stabilized with 0.5% (w/w) WPI, and WPI-LMP conjugate

(2:1), unincubated and incubated for 8 days, at pH 6.5 and 5.0.

.....................................................................................................................7

5

Table 3.9 Table 3.9. Amount of adsorbed protein (%) on oil droplets, ɸ32, and

protein load (kg/m2) of heated and unheated oil in water emulsion (10%

w/w sunflower oil), stabilized with 0.5% (w/w) WPI, and WPI-LMP

conjugate (2:1), unincubated and incubated for 8 days, at pH 6.5 and 5.0.

......................................................................................................................

76

Table

3.10

Amount of pectin(mg/100ml) in aqueous phase, serum phase and

adsorbed on oil droplets of heated and unheated oil in water emulsion

(10% w/w sunflower oil), stabilized with 0.5% (w/w) WPI-LMP

conjugates (2:1), unincubated and incubated for 8 days, at pH 6.5 and 5.0.

……………..…80

Table

3.11

Amount of adsorbed pectin (%) on oil droplets, ɸ32, and pectin load

(kg/m2) of heated and unheated oil in water emulsion (10% w/w

sunflower oil), stabilized with 0.5% (w/w) WPI-LMP conjugate (2:1),

unincubated and incubated for 8 days, at pH 6.5 and 5.0.

………………………………….80

xiii

List of Abbreviations

AO Alginate oligosaccharide

BSA Bovine serum albumin

CMC Carboxymethyl cellulose

DX Dextran

EA Emulsion activity

ES Emulsion stability

IEP Isoelectric point

Ig Immunoglobulin

LMP Low methoxyl pectin

PGWP Partially glycosylated whey protein isolate

SBPI Soybean protein isolate

WPI Whey protein isolate

WPC Whey protein concentrate

WPH Whey protein hydrolysate

WPN Whey protein nanoparticle

1

Abstract

The effect of covalent Maillard conjugates of whey protein isolate (WPI) and

Low Methoxyl pectin (LMP) on the emulsifying properties of WPI and the heat

stability of oil-in-water emulsions (O/W), containing 10 % (w/w) of sunflower oil and

0.5 % of WPI or conjugates were investigated as a function of pH (6.5 and 5.0) and

incubation time of the conjugates. WPI-LMP conjugates were prepared by dry heating

of a mixture of WPI and LMP at 60°C for time periods of 0, 4, 8 and 16 days. In a

similar way, whey protein isolate was dry heated without pectin, for comparison. The

properties of the emulsions stabilized by WPI-LMP conjugates, dry heated WPI and

WPI alone were compared before and after heat treatment in an oil bath of 80 °C for

10 and 20 min.

At pH 5.0 without and with salt (30 mM), emulsions containing dry heated

WPI and WPI alone exhibited flocculation, creaming and phase separation, while

emulsions containing WPI-LMP conjugates were stable against flocculation and

creaming. On the other hand, at pH 6.5, the emulsions stabilized by WPI-LMP

conjugates, dry heated WPI, and WPI alone were more stable and homogenous.

Upon heat treatment, emulsions stabilized by dry heated WPI, WPI alone, and

WPI-LMP conjugates (day 0) exhibited extensive flocculation at both pH 5.0 and 6.5.

Both the average droplet size and viscosity of these heated emulsions increased and

shear thinning behavior was observed. On the other hand, emulsions stabilized by

WPI-LMP conjugates which were incubated for 4, 8, and 16 days showed no

aggregation and flocculation. Correspondingly, there was no visible change in the

droplet size distribution and viscosity of the emulsions containing WPI-LMP

conjugates obtained by dry heating for 4, 8, and 16 days.

The observed results demonstrated that conjugation improved the emulsifying

properties and heat stability of WPI stabilized emulsions. The main reason for

improved stability of whey protein by conjugation with pectin is that covalent

2

bonding with pectin enhances the steric stabilization forces as a result of the presence

of the hydrophilic polysaccharide moiety which acts between protein layers adsorbed

at the surface and inhibits heat-induced aggregation of the droplets. Moreover,

conjugation with pectin increases the surface hydrophilicity of the oil droplets which

reduce droplet-droplet hydrophobic interactions and improves the stability against

aggregation, coalescence, and creaming.

3

Chapter 1: Literature Review

1.1. Introduction

1.1.1. Overview

Nowadays, whey protein products are increasingly used for a variety of

nutritional and functional applications in food products. Some of the applications

include sport beverages, liquid meat replacements, ice cream, salad dressing, bakery

products, infant foods and various other dairy products [Fitzsimons et al., 2007;

Jovanović et al., 2005]. One of the promising applications is the use of whey protein

as an emulsifier in oil in water (O/W) emulsions [Jovanović et al., 2005]. However,

the main challenge of whey protein application is heat-induced denaturation of whey

proteins which decreases their solubility and limits their application in food products,

particularly in emulsions [Demetriades et al., 1997a; Euston et al., 2000]. Heat

treatment can affect the structure and solubility of whey proteins, leading to

aggregation and precipitation of the whey proteins and destabilization of whey

protein stabilized emulsions [Sliwinski et al., 2003]. Therefore, the stability of whey

proteins during thermal processing is of utmost importance for product quality and

functionality.

Several methods for modification of whey proteins and improving their

functionality have been proposed [Akhtar and Dickinson, 2003; 2007; Ashokkumar et

al., 2009; Chandrapala et al., 2011; Gentes et al., 2010; Jimenez-Castano et al., 2007;

Asylbek Kulmyrzaev et al., 2000a; Lorenzen, 2007; Mishra et al., 2001; Sağlam et al.,

2013]. Whey proteins have been modified by means of conjugation with

polysaccharides via covalent bonding which follows the path of Maillard reaction

[Akhtar and Dickinson, 2003; 2007; Kika et al., 2007; Mishra et al., 2001; N

Neirynck et al., 2004]. These conjugates have been reported to improve the functional

properties, including solubility [Akhtar and Dickinson, 2003; Mishra et al., 2001; N.

Neirynck et al., 2004] and emulsifying properties [Akhtar and Dickinson, 2007; Kika

4

et al., 2007; Mishra et al., 2001; N Neirynck et al., 2004], foaming properties [Mishra

et al., 2001], as well as thermal stability of whey proteins [Jimenez-Castano et al.,

2007; G Liu and Zhong, 2013].

1.1.2. Research Objective

In this work, we aimed to improve the heat stability of whey protein-stabilized

oil in water (O/W) emulsions by replacing whey protein with whey protein-pectin

conjugates made by dry heating under controlled temperature and relative humidity as

well as evaluating the heat stability. We hypothesized that these conjugates would

exhibit improved emulsification activity and thermal stability compared to the WPI

alone. In this research, the effects of the incubation time, pH and low concentration of

salt on the emulsion properties and thermal stability of whey protein-pectin

conjugates were evaluated.

5

1.2. Whey Protein

Whey is the residual fluid of the cheese and casein manufacturing industry,

after almost complete removal of casein [Smithers, 2008]. Whey proteins are

nitrogenous compounds representing 18-20% of the total milk nitrogen content

which are recovered from milk serum or whey after precipitation of casein at pH 4.6

and a temperature of 20 °C by the action of chymosin or acid [Eigel et al., 1984].

Whey proteins exist as compact, globular proteins in their native state [Lee et al.,

1992]. They have high solubility as a result of the large number of surface hydrophilic

residues. However, they are highly sensitive to heat induced denaturation and

polymerization leading to the decrease of their solubility [Lee et al., 1992].

Different types of whey protein products are commercially available, for

instance whey protein concentrate, whey protein isolate and whey protein hydrolysate.

The important products are whey protein concentrates (WPC) and whey protein

isolates (WPI). WPC contains protein in the range of 34-80% with a low level of fat

and cholesterol and a greater amount of bioactive compounds and lactose, while WPI

contains more than 90% protein and lower levels of fat, lactose and bioactive

compounds [Gangurde et al., 2011].

WPI are obtained by two processing methods, namely ion-exchange (IE)

chromatography followed by ultrafiltration, and membrane filtration (MF) in which

microfiltration is followed by ultrafiltration-diafiltration [T Wang and Lucey, 2003].

Retentates obtained from the filtration process are then spray dried after further

concentration. Differences in total composition and in protein fractions have been

reported between the two isolation processes as well as between different

manufacturers [T Wang and Lucey, 2003].WPI obtained by microfiltration (MF) is

known to contain more glycomacropeptides and a lower concentration of β-

lactoglobulin than that obtained by ion-exchange [T Wang and Lucey, 2003].

Whey proteins can also be furthered processed by partial hydrolysis under

controlled conditions resulting in hydrolyzed whey proteins (HWP). This makes HWP

6

easily absorbed in the gut and suitable for development of infant products [Jovanović

et al., 2005].

1.2.1. Whey Protein Components

Whey proteins are primarily mixtures of β-lactoglobulin ( β-Lg), α-

lactalbumine (α-Lac), bovine serum albumin (BSA) and immunoglobuline (Ig) which

constitute 60%, 22%, 5.5% and 9% of the total whey protein, respectively [Bryant

and McClements, 1998]. Other minor fractions of whey proteins are proteose peptone,

lactoferrin, lactoperoxidase and lysozyme [De Wit, 1998].

1.2.1.1. β-Lactoglobulin

The major component of whey protein is β-lactoglobulin, and thereby its

properties impart great importance in the functionality and thermal behavior of whey

protein ingredients [Vardhanabhuti and Foegeding, 2008]. β-lactoglobulin has seven

genetic variants; among them, variant A and B are the most abundant variants which

are different from each other only in two amino acids [Cayot and Lorient, 1997; Eigel

et al., 1984]. Variant A is the most negatively charged at the pH of milk (6.6) [Cayot

and Lorient, 1997].

β-lactoglobulin is a small molecule with a molecular mass of about 18.3 kDa

and is soluble in dilute salt solutions [Kontopidis et al., 2004]. It represents more than

50% of the total whey proteins and 12 % of total milk proteins [Fox and McSweeney,

2003]. Its primary structure consists of 162 amino acid residues with one free thiol

group (C121) and two disulfide bonds (C106-C119 and C66-C160) [Hoffmann and

van Mil, 1999].The secondary and tertiary structure of β-lactoglobulin consist of 43-

50% of β-sheet, 10-15% of α helix and 15-20% of β-turn [Cayot and Lorient, 1997].

The monomeric form of β-lactoglobulin is conical, and forms a so-called calyx

or β-barrel with a hydrophobic pocket that can bind vitamin A and fatty acids (Cayot

& Lorient, 1997). The monomeric form appears at a pH below 3, while at room

temperature and pH between 5 and 7, it is present as a dimer constituted of two

identical subunits; by increasing the temperature, the equilibrium is changed to the

7

monomeric form [deWit and Klarenbeek, 1984; Kontopidis et al., 2004;

Vardhanabhuti and Foegeding, 2008]. This dimer transition to monomer is a

prerequisite for aggregation by heat [Hoffmann and van Mil, 1999].

It is known that β-lactoglobulin dissociation and aggregation are influenced by

pH, temperature, salt and protein concentration [Hoffmann and van Mil, 1999].

Heating β-lactoglobulin at 40°C induces a small reversible conformational change

[deWit and Klarenbeek, 1984], while heating at above 50°C induces irreversible

conformational changes in β-lactoglobulin during which buried hydrophobic and

thiol groups of the native protein become exposed. This exposure leads to sulphydril-

disulphide interaction with β-lactoglobulin itself or other thiol-containing proteins,

while the secondary structure is retained, and it is called as molten globule state

[deWit and Klarenbeek, 1984; Vardhanabhuti and Foegeding, 2008].

Therefore the thiol groups of β-lactoglobulin play an essential role in thermal

destabilization of whey protein stabilized emulsions. The exposed thiol group in the

newly formed monomers can form disulfide linkages by thiol/disulfide exchange

reactions. The C66-160 disulfide of β-lactoglobulin is the main disulfide involved in

the intermolecular exchange reaction due to its location on the external loop, whereas

the other disulfide bond, buried in the structure, is less prone to the intermolecular

exchange reaction [Hoffmann and van Mil, 1999].

In addition to the covalent disulfide bonds, non covalent interactions including

hydrophobic, ionic and Van der Waals also play a role in the β-lactoglobulin

aggregation process [Hoffmann and van Mil, 1999]. The β-lactoglobulin association

properties are pH-dependent. The dimer form is present at pH 5-8 and associates to

octamers at pH 3-5. On the other hand, the monomeric form exists at extreme pH

values (i.e. either below 2, or above 8). At pH values above 9, β-lactoglobulin will

undergo reversible denaturation [Cayot and Lorient, 1997].

8

1.2.1.2. α-Lactalbumin

α-lactalbumin is the second most dominant and the smallest whey protein

component with a molecular mass of about 14.2 kDa [Cayot and Lorient, 1997]. It

represents about 20% of the total whey proteins and about 3.5% of the total milk

proteins. It consists of 123 amino acid residues and four disulfide bridges. It has no

free thiol group and as a result, α-lactalbumin is known as the most heat resistant of

the whey protein components [deWit and Klarenbeek, 1984]. In addition, α-

lactalbumin has a calcium binding site, which promotes the heat stability and the

recovery of its native conformation (Cayot & Lorient, 1997).

The secondary structure of α-lactalbumin consists of two main domains of the

native molecule, namely α-helix (30%) and β-sheet (9%), which are connected by

calcium. The great flexibility and recovery of the native conformation of α-

lactalbumin is not only due to its Ca2+ binding properties but also due to the low

amount of ordered secondary structure [Cayot and Lorient, 1997]. By releasing

calcium at a pH below 4, flexible apo-α-lactalbumin is formed which is highly prone

to proteolysis (Cayot & Lorient, 1997).

Upon heating of α-lactalbumin up to 77°C and subsequent cooling, 90% of the

conformational change is reversible, whereas upon heating at 95°C for 15 min, 40%

of the changes are reversible [Vardhanabhuti and Foegeding, 2008]. The C6-C120

disulfide bond of α-lactalbumin is the most active disulfide group involved in

aggregation reactions due to its position at the surface of the protein [Wijayanti et al.,

2014]. α-lactalbumin is highly water soluble even at its isoelectric point due to the

presence of a high amount of hydrophilic groups, which leads to its incapability to

precipitate from milk at the isoelectric point [Swaisgood, 1982].

1.2.1.3. Other Minor Proteins

Bovine serum albumin (BSA) of milk is physically and immunologically

similar to blood serum albumin [Eigel et al., 1984]. BSA has 582 amino acid residues

with a molecular weight of about 66 kDa. It has 17 intramolecular disulfide bonds and

9

one free sulfhydryl group [Eigel et al., 1984]. Serum albumin constitutes of three

major domains which are different in hydrophobicity, net charge and ligand binding

sites [Eigel et al., 1984]. BSA can act as a carrier of nonpolar fatty acids in the blood

circulatory system [deWit and Klarenbeek, 1984]. It has been pointed out that fatty

acids provide stabilization of BSA against heat denaturation [deWit and Klarenbeek,

1984]. Furthermore, BSA is a well-known whey protein for its gelling properties.

During gelation of BSA, the amount of β-sheet, which is very low in the native

molecule, increases while the amount of the α-helix decreases. This transition is

particularly critical in the gelation of BSA [Wijayanti et al., 2014].

The other minor proteins present in whey proteins are Immunoglobulins which

are glycoproteins with antibody properties [Cayot and Lorient, 1997]. IgG, IgA and

IgM are the main Immunoglobulins in bovine milk and whey [deWit and Klarenbeek,

1984] . The most heat sensitive Immunoglobulin is IgM, whereas the most heat

resistant type is IgG [Sweeney and Fox, 2013]. In spite of the higher denaturation

temperature compared to β-lactoglobulin and α-lactalbumine, the presence of BSA

will reduce the heat stability of Immunoglobulins due to the interaction of the free

thiol group of BSA with Immunoglobulins [Cayot and Lorient, 1997].

1.2.2. Whey Proteins Functional Properties

Having unique properties, whey proteins have received most attention for their

versatile functionality and excellent nutritional value [Bryant and McClements, 1998;

De Wit, 1998; Jovanović et al., 2005; Smithers, 2008]. Whey proteins are recognized

nutritionally superior to other common dietary proteins due to their high amount of

essential amino acids and branched chain and sulphur amino acids, as well as their

high biological value (BV) and digestibility compared to other protein sources

[Smithers, 2008].

Moreover, whey proteins contain biologically active proteins and peptides

proper for medicinal application. Evidences about anti-cancer effects of lactoferrin,

lactoperoxidase and serum albumin have been found in some studies [Bounous et al.,

1991; Gill and Cross, 2000]. Anti-infectivity of immunoglobulins, antithrombotic

10

effects of glycomacropeptides and antimicrobial effects of lactoferrin have also been

reported [Farnaud and Evans, 2003; Hernández-Ledesma et al., 2006; Mehra et al.,

2006; Smithers, 2008].

Along with physiological benefits, whey proteins possess inherent functional

and sensory characteristics. The functional properties of whey proteins include

gelling, emulsification, foaming, water binding, solubility and viscosity, which

enable whey proteins to be used in various food products by taking advantage of one

or several functional properties for each application [Foegeding et al., 2002; Xiong,

1992]. As a gelling agent, WPI is useful for designing and improving textural

properties of various foods, such as dairy, meat and bakery products [Jovanović et al.,

2005]. Based on how the gels are prepared, they can be divided into heat induced gels

and cold set gels [Jovanović et al., 2005].

The functional properties of whey proteins are related not only to their

structure but also to their processing conditions [Bryant and McClements, 1998].

Intrinsic physiological properties of native proteins such as their amino acid

composition and their sequence, the ratio of hydrophobicity to hydrophilicity, charge

distribution, and flexibility influence the functional properties of whey proteins. In

addition, external factors such as processing conditions, isolation methods,

protein content, pH, temperature and ionic strength, as well as the interaction

with other food ingredients will also influence the functional properties of whey

proteins by changing their conformation [J Kinsella and Whitehead, 1989].

The most important functional property of whey protein is its high solubility

over a wide range of pH conditions, which is a principal prerequisite for its

functional properties such as emulsification [J E Kinsella et al., 1986]. In general,

whey proteins are soluble in the pH range of 2 – 9 [Damodaran et al., 2007]. The

solubility of proteins depends on their water binding capacity and physical state

[Damodaran, 1997]. The amount of water bound to the protein is a function of

intrinsic factors such as protein composition, number of exposed polar groups,

conformation, and surface polarity, as well as external factors such as pH, temperature

and ionic strength [J E Kinsella and Morr, 1984].

11

Moreover, protein solubility is governed by two main interactions, namely

hydrophobic and ionic interaction. Hydrophobic interaction decreases the solubility

by promoting protein-protein interaction, while ionic interactions enhance the

solubility by promoting protein-solvent interaction [Damodaran et al., 2007].

Solubility of whey proteins, even at their isoelectric point, is due to a large ratio of

surface hydrophilic to hydrophobic residues. By extensive heat treatment, the

solubility of whey proteins shifts to a minimum due to the rise of the surface

hydrophobicity as a result of protein unfolding [J Kinsella and Whitehead, 1989].

Whey proteins are widely known for their superior emulsification properties.

Having an amphiphilic structure and surface activity, whey proteins have the ability to

readily adsorb at the oil-water interface, reduce the interfacial tension at the oil and

water interface, and also form an interfacial membrane around oil droplets preventing

destabilization of the emulsion [J E Kinsella et al., 1986].

1.3. Emulsions

An emulsion is a mixture of two immiscible liquids in which one of the liquids

(called the dispersed phase) is dispersed in the other one (called the continuous

phase)[D J McClements, 2007]. The average diameter of the dispersed phase droplets

is in the range of 0.1 to 100 µm [Lam and Nickerson, 2013]. In the food industry,

typical emulsions are either oil in water (O/W), in which oil droplets are dispersed in

an aqueous phase, e.g. milk, ice-cream, and mayonnaise, or water in oil (W/O) which

consist of water droplets dispersed in an oil phase, such as butter and margarine. In

addition, multiple emulsions such as water-oil-water (W/O/W) or oil-water-oil

(O/W/O) can be made in advanced systems [Lam and Nickerson, 2013; D J

McClements, 2007].

Emulsions are formed by a homogenization process by which intense

mechanical shear is exerted using a homogenizer, a high pressure valve homogenizer,

a microfluidizer or an ultrasonic homogenizer [Lam and Nickerson, 2013; D J

McClements, 2007]. The force applied during homogenization breaks down fat

droplets into small sizes and prevents fat droplet separation.

12

Fig. 1.1. Schematic figures of emulsions. a. oil in water emulsion (O/W); b. water in

oil emulsion (W/O); c. Water in oil in water double emulsion (W/O/W); d. Oil in

water in Oil double emulsion (O/W/W)

1.3.1. Emulsion Destabilization Mechanisms

Emulsions are very prone to destabilization due to their high interfacial area

[Badolato et al., 2008]. Since food emulsions are lyophilic colloidal dispersions, the

contact between oil and water molecules, which leads to an increase in the interfacial

tension between dispersed and continuous phase and in the free energy of the system,

is unfavorable. As a result, emulsions are thermodynamically unstable and separation

of the two phases of an emulsion occurs for minimizing the interfacial contact area

and free energy of the system [Damodaran, 2005]. Various physiochemical

mechanisms such as gravitational separation (creaming and sedimentation),

flocculation, coalescence, partial coalescence and Ostwald ripening can cause

instability of food emulsions [D J Mcclements, 2007].

Gravitational separation is due to the density difference between the

continuous phase and the dispersed phase or oil droplets. Two types of gravitational

separation include creaming and sedimentation. Since oil droplets have a lower

density than the continuous phase (water), they rise to the top of the emulsion in oil-

in-water (O/W) emulsions and lead to creaming. In the case of water-in-oil (W/O)

a b

c d

Oil

Water

13

emulsions, the water droplets move downward leading to sedimentation.Nevertheless,

creaming is a reversible process [Damodaran, 2005; D J McClements, 2007].

On the other hand, in aggregation two or more oil droplets come close to each

other and form an aggregate due to the Brownian motion [Fredrick et al., 2010]. The

structure and properties of the flocks or aggregates is a function of the net interparticle

force and the oil volume fraction [Damodaran, 2005; D J McClements, 2007]. A weak

flock with a low number of droplets can be formed at low oil volume with weak

attraction forces. The principal non covalent interactions in the aggregation are Van

der Waals attraction and electrostatic and steric repulsion. If aggregates disintegrate

by gentle mechanical force (stirring), it is called flocculation, which is a reversible

process. On the other hand, coagulation requires a stronger force to disrupt the

aggregates [Fredrick et al., 2010].

When small oil droplets are in close contact for a long period of time, they

may merge together and form a single larger droplet; this phenomenon is called

coalescence [Badolato et al., 2008]. Consequently, creamed or aggregated emulsions

are more susceptible to coalescence in which there is only a thin film of continuous

phase between the droplets [Fredrick et al., 2010]. Partial coalescence occurs when

two or more partly crystalline droplets merge together in which solid crystals from

one droplet penetrate into the liquid part of the other droplet, and as a result,

aggregate whereby an irregular shape is formed [D J McClements, 2007].

Another instability mechanism is Ostwald ripening, which involves growth of

larger droplets due to the diffusion of molecules of the dispersed phase from small

droplets to the larger ones through capillary forces [Fredrick et al., 2010].Therefore

the particle size distribution of the emulsion shifts to the bigger size during

coalescence and Ostwald ripening, while it remains unchanged during gravitational

separation and aggregation [Badolato et al., 2008].

Interrelations between physiochemical instability mechanisms have been

pointed out. Instability due to flocculation and coalescence promote instability to

gravitational separation like creaming and sedimentation due to the increase in

14

particle size. Furthermore, the close vicinity of the particles for a long period due to

gravitational separation or flocculation promotes coalescence [D J McClements,

2007]. A schematic diagram of the most common instability mechanisms and their

interrelation has been given in Fig.1.2

Fig.1.2 Schematic representation of common instability mechanism in food emulsions;

flocculation, coalescence, Ostwald ripening, creaming and phase separation [Chung

and McClements, 2014].

Emulsion stability can also be influenced by environmental stress, including

homogenization, thermal processing, chilling, freezing, drying and mechanical

agitation, as well as by the aqueous phase composition such as pH, ionic strength and

presence of surfactants, sugar and biopolymers [D J McClements, 2004].

Emulsions stabilized with proteins are highly sensitive to the pH and ionic

strength [Asylbek Kulmyrzaev et al., 2000b]. They tend to flocculate at pH values

close to the isoelectric point of the adsorbed proteins due to the low electrostatic

repulsion between the droplets which is not strong enough to overcome the various

15

attractive interactions, e.g., van der Waals and hydrophobic interactions. Moreover,

the presence of minerals can promote flocculation by screening of the electrostatic

interaction through binding to the oppositely charged groups on the surface of the

emulsion droplets. This effect is highly influenced by the type of salt, ion valency and

solubility [AA Kulmyrzaev and Schubert, 2004]. Multivalent ions are more effective

in screening electrostatic interactions compared to monovalent ions.

Furthermore, emulsion sensitivity to ions is higher near the isoelectric point

of the proteins [Asylbek Kulmyrzaev et al., 2000b]. If the amount of emulsifier is not

sufficient to completely cover the interface of the system, a gap between the

interfacial membranes surrounding the droplets will appear, which leads to droplet

coalescence when these gaps come into close contact [D J McClements, 2004].

1.3.2. Stabilization of Emulsions

“Emulsion stability” refers to the ability of an emulsion to remain unchanged

in its physiochemical properties over the time-scale of observation [D J McClements,

2007]. To prepare a kinetically stable emulsion, over a time period, it is necessary to

use an emulsifier to minimize the interfacial tension between the continuous phase

and the dispersed phase [Fredrick et al., 2010].

Emulsifiers are surface active materials consisting of both a hydrophilic head

and a hydrophobic tail adsorbed at the interface in a way that the hydrophobic part is

oriented to the oil and the hydrophilic part is exposed to the aqueous phase.

Consequently, the emulsifier prevents droplet aggregation by reducing the interfacial

tension. Moreover, it helps in disruption of the emulsion droplets and formation of

smaller droplets during homogenization [Dickinson, 2003]. Emulsifier molecules are

either low molecular weight synthetic (e.g. Tween surfactants) or natural (e.g. egg

lecithin) surfactants, or macromolecules such as proteins [Dickinson, 2009].

Besides emulsifiers, hydrocolloids, which act as thickening agents or gelling

agents, are also able to stabilize emulsions through either enhancing the viscosity of

16

the continuous phase (thickening agent) or forming a gel network within the

continuous phase. In both cases the droplet movement is lowered and thus

aggregation is slowed down or prevented [D J McClements, 2007]. However, at low

concentrations, hydrocolloids can destabilize emulsions through either depletion

flocculation induced by non-adsorbing polysaccharides or bridging flocculation by

weakly adsorbed polysaccharides [Dickinson, 2009].

1.4. Whey Protein as Emulsifier

Whey proteins have a potential for being effective emulsifiers through their

amphiphilic nature and film forming abilities [Lam and Nickerson, 2013].The

adsorbed protein molecules undergo some level of unfolding, exposing the buried

hydrophobic amino acids to the surface. They align themselves at the surface in a

way that the hydrophobic amino acids are exposed to the oil phase, while

hydrophilic amino acids point to the aqueous phase [Dickinson, 1999].

Generally, proteins are less surface active than smaller molecular weight

surfactants due to their complex structural properties and conformational

constraints which hinder the protein to properly orient the hydrophilic and

hydrophobic groups at the interface [Damodaran, 2005]. On the other hand, protein-

stabilized emulsions are generally more stable than those stabilized by small

surfactants due to the pronounced surface rheological properties of proteins and the

formation of a viscoelastic layer around the oil droplets [Dickinson, 1999].

Furthermore, as compared to caseins, whey proteins are less surface active due to

their rigid globular structure, but they have a pronounced solubility and they can

readily adsorb at the oil droplet surface and form a stable layer around oil droplets

[Leman et al., 1989].

Whey protein adsorption onto the oil droplet surface is selective and it

depends on pH, ionic strength, temperature, and protein concentration. The pH

dependency of whey protein adsorption indicates that the conformational changes of

17

the protein and the effective hydrophobicity are pH dependent [Yamaachi et al.,

1980]. β-lactoglobulin adsorbs more readily at alkaline pH conditions, compared to

other whey proteins because of molecular expansion in this pH range, while α-

lactalbumin conformational changes occur more easily in the acidic pH range due to

the loss of bound calcium in acidic conditions [Yamauchi et al., 1980].

Fig.1.3 Mechanism of emulsion stabilization by proteins through (A) electrostatic

repulsion and (B) steric stabilization. Red dots are hydrophobic parts of protein

molecules positioned at the oil phase [Lam and Nickerson, 2013].

A viscoelastic layer can be provided through non covalent interaction between

adjacent adsorbed protein, for instance by polymerization at the interface involving

sulfhydryl-disulfide interchange reactions in the presence of amino acids containing

sulfhydryl and disulfide group [Dickinson and Matsumura, 1991]. These interactions

help the proteins to be adsorbed to the surface irreversibly and provide resistance

against mechanical stresses. Furthermore, the adsorbed proteins at the surface offer

electrostatic and steric stabilization preventing droplet aggregation and coalescence

(Fig.1.3) [Lam and Nickerson, 2013].

18

1.4.1. Limiting Factors of WPI Application in Emulsions

Whey proteins are less sensitive to pH and they can be used over a wider

range of pH than caseins. However, whey proteins are heat labile globular proteins

which unfold and aggregate upon heating at temperatures above their denaturation

temperature, leading to destabilization of whey protein-stabilized emulsions and

aggregation of oil droplets [Sliwinski et al., 2003]. Moreover, common heat

treatments such as preheating, pasteurization and sterilization are commonly applied

to foods containing whey protein ingredients to increase the safety and shelf life of the

product [deWit and Klarenbeek, 1984]. These heat treatments change the native state

of globular whey proteins by various mechanisms, such as denaturation, aggregation

and flocculation, leading to emulsion destabilization. Therefore, it is of vital

importance to obtain a comprehensive knowledge of the whey protein unfolding and

aggregation mechanisms to be able to optimize the processing conditions and develop

techniques to modify the whey protein properties in order to attain their maximum

functional and nutritional properties.

1.5. Heat Stability of WPI

Whey proteins are globular proteins with a tertiary structure stabilized by

disulfide bonds between the cysteine residues [D McClements et al., 1993].

Thermodynamically, the native structure of the whey proteins is the most stable

conformation formed under physiological conditions. This native structure is

stabilized by non-covalent forces including hydrogen bonding, as well as

hydrophobic, van der Waals and electrostatic interactions, while the structural

integrity of extracellular proteins is maintained by covalent disulfide bonding

[Damodaran et al., 2007]. Changes in environmental factors can influence these

interactions, leading to an alteration in the protein conformation. Heat treatment is one

of the factors which can affect the conformation of whey protein and cause

denaturation and loss of solubility, which can implicate a loss of protein functionality

[J Kinsella and Whitehead, 1989].

19

1.5.1. Heat Induced Denaturation

Protein denaturation is referred to as the change in spatial arrangement of the

polypeptide chain or a modification in the secondary, tertiary or quaternary structure

of the protein, without breaking the backbone of the peptide bonds in the primary

structure. Denaturation of globular proteins will lead to aggregation which can

influence their solubility and functionality [Damodaran et al., 2007; Mulvihill and

Donovan, 1987].

During thermal processing, heat-induced denaturation occurs which can be

either reversible or irreversible [deWit and Klarenbeek, 1984]. Reversible

denaturation of the protein structure involves a partial loss of the tertiary structure

which takes place at a temperature up to 60°C [deWit and Klarenbeek, 1984]. On the

other hand, heating above the denaturation temperature leads to irreversible

denaturation of the protein in which unfolded protein molecules further associate

through intermolecular interaction, mainly by intermolecular disulfide bonds, to form

aggregates [deWit and Klarenbeek, 1984]. Therefore, denaturation or unfolding of

proteins leading to the exposure of reactive groups, such as hydrophobic or sulfhydryl

groups, is a prerequisite for protein aggregation which involves further cross-links of

denatured proteins and is followed by precipitation (i.e. formation of insoluble

aggregates), coagulation (i.e. formation of soluble aggregates) or gelation of the

protein. In a coagulum and a precipitate, protein molecules randomly interact, while

in a gel an ordered three dimensional structure can be seen [Damodaran et al., 2007].

At the initial stages of aggregation, various interactions in proteins lead to the

formation of small oligomers that can persist at low protein concentration and

associate into monodispersed primary aggregates when their concentration exceeds a

critical value [Durand et al., 2002]. The Critical association concentration (CAC) is

the concentration above which small oligomers form large aggregates. Furthermore,

by increasing the protein concentration, the primary monodispersed aggregates

associate into larger polydisperse self-similar aggregates or a gel [Nicolai et al.,

2011].

20

In addition to the covalent intermolecular disulfide bonds, non-covalent

interactions such as ionic, van der Waals, and hydrophobic are also involved in the

protein aggregation process. The contribution of non-covalent bonding in aggregation

and gelation processes is determined by the environmental conditions, such as pH,

temperature and salt concentration. Electrostatic interactions play an important role in

protein precipitation at the isoelectric point [Hoffmann and van Mil, 1997]. The role

of non-covalent interactions in aggregation of β-lactoglobulin is more significant at

higher temperatures [Vardhanabhuti and Foegeding, 2008].

Thermal stability of proteins refers to the ability of proteins to maintain their

biologically active native state, and survive heat processing without detrimental

changes, such as increased turbidity, increased viscosity, phase separation,

precipitation or gelation [Vardhanabhuti and Foegeding, 2008]. The thermal

denaturation behavior of whey proteins reflects the collective response of the

component proteins. β-lactoglobulin and α-lactalbumin are the predominant whey

proteins. So, they contribute significantly to the thermal stability of whey protein

ingredients; particularly β-lactoglobulin plays a key role [Cayot and Lorient, 1997].

β-lactoglobulin is mainly present as a non-covalently bound dimer at ambient

temperature and neutral pH. However, it dissociates into monomers at higher

temperatures [Nicolai et al., 2011]. Upon further heating to above 50°C, β-

lactoglobulin undergoes reversible conformational changes including a partial loss or

change of the ternary structure. In this state, known as the molten globule state,

proteins are not completely unfolded and their native secondary structure is preserved

while some hydrophobic and thiol groups are exposed. Consequently, the free thiol

groups in the modified monomers can induce thiol/disulfide exchange reactions and

formation of aggregates [Hoffmann and van Mil, 1997; Nicolai et al., 2011;

Vardhanabhuti and Foegeding, 2008]. It was reported that heat treatment of β-

lactoglobulin at 80 °C resulted in a change in the secondary conformation, i.e. a

decrease in the α-helix content and a corresponding increase in the random coil

content [Kim et al., 2005]. Moreover, an increase in surface hydrophobicity, protein

flexibility and surface rheological properties was observed during heat treatment [Kim

et al., 2005].

21

A kinetic model based on radical-addition polymerization reactions for

denaturation and aggregation of β-lactoglobulin in low ionic strength conditions at

neutral pH was proposed [Roefs and Kruif, 1994]. The main reaction steps are

initiation, propagation and termination, and the free thiol group acts as a radical in this

process. The initiation step occurs when a folded native dimer of β-lactoglobulin

unfolds and exposes its free thiol group. In the propagation step, the reactive thiol

group reacts with one of the two intramolecular disulfide bonds of a nonreactive β-

lactoglobulin molecule and forms an intermolecular disulfide bond and releases a new

reactive free thiol group. The propagation leads to the formation of linearly linked

aggregates (polymerization). The polymerization reaction stops in the termination step

when a polymer is formed between two reactive intermediates without a reactive thiol

group [Roefs and Kruif, 1994].

1.5.2. Factors Influencing Heat Stability of WPI

The effects of heat are greatly influenced by pH, ionic strength, the rate of

heating, the protein concentration and the presence of lactose [deWit and Klarenbeek,

1984]. Moreover, the amino acid composition and sequence also influence the

structure and thermal behavior of whey proteins. The protein concentration, and the

number of reactive amino acids per molecule such as half-cystin (cys/2) and lysine-

residues are influential characteristics during heat treatments [deWit and Klarenbeek,

1984].

The thermal denaturation temperature of the three major whey proteins, β-

lactoglobulin, α-lactalbumin, and bovine serum albumin is around 78, 62, and 64°C,

respectively, while the isoelectric point of β-lactoglobulin, α-lactalbumin, and bovine

serum albumin is 5.2, 4.8-5.1, and 4.8-5.1, respectively [Bryant and McClements,

1998].

β-lactoglobulin, the most abundant whey protein, mainly defines the heat

behavior of whey proteins [Hoffmann and van Mil, 1997]. It is highly sensitive to heat

treatments due to the presence of free thiol groups involving intra and intermolecular

22

disulfide interactions. Numerous studies have focused on the role of the thiol group of

β-lactoglobulin in heat induced aggregation and gelation.

In spite of its low denaturation temperature, α-lactalbumin is described as the

most heat-stable whey protein due to the lack of free thiol group as compared to β-

lactoglobulin and BSA. α-lactalbumin tends to aggregate slowly compared to β-

lactoglobulin. However, it will readily aggregate in the presence of β-lactoglobulin

and BSA [Wijayanti et al., 2014]. α-lactalbumin is the only whey protein which is

able to renature after heating [Mulvihill and Donovan, 1987]. The thermal behavior of

α-lactalbumin is influenced by some factors, such as the presence of calcium,

temperature, ionic strength and the degree of purity [Vardhanabhuti and Foegeding,

2008].

During heat treatment, the denaturation rate of β-lactoglobulin and α-

lactalbumin is influenced by temperature, protein concentration, pH and ionic

strength [Nicolai et al., 2011]. The extent of these parameters influencing the

denaturation rate can be varied depending on the values of the other parameters. The

temperature dependency of the depletion rate of β-lactoglobulin and α-lactalbumin

can be described by the Arrhenius equation in which the depletion rate increased by

increasing temperatures above the critical temperature (90°C for β-lactoglobulin) due

to the reduction of the activation energy (Ea) [Nicolai et al., 2011].

The effect of pH on the denaturation and aggregation of β-lactoglobulin at

65°C was investigated [Hoffmann and van Mil, 1999]. It was found that the

denaturation of β-lactoglobulin was enhanced by increasing the protein concentration

and pH from 6 to 8 at 65°C.

The native β-lactoglobulin solution is stable against aggregation due to the

long range electrostatic repulsion except at a pH close to the isoelectric point, where

aggregation occurs at room temperature. Around the iso-electric point, proteins

contain the same amount of negative and positive charges and aggregation occurs due

to the interaction between opposite charges [Majhi et al., 2006]. Moreover, an

increasing ionic strength leads to an increase in the denaturation rate around neutral

23

pH, in which the effect is higher for multivalent ions such as CaCl2 than for NaCl

[Croguennec et al., 2003].

The size and the number of the aggregates formed during heat treatment

change with the heating conditions, the protein concentration , the pH and the type

and concentration of added salt [Mehalebi et al., 2008; Nicolai et al., 2011]. The rate

of aggregation increases exponentially with increasing temperature, especially at high

protein concentration which leads to precipitation of large amounts of protein. The

size and amount of the aggregates increases with heating time until a steady state is

obtained. Moreover, the size of the aggregates increases by increasing the protein

concentration until above its critical concentration (Cg) at which a gel is formed

[Durand et al., 2002].

This critical concentration depends on the type of protein and other factors,

such as ionic strength and pH. The rate of aggregation reaches a maximum at the

isoelectric point (IEP), in which the electrostatic repulsion is minimum, and it

decreases when the pH is increased or decreased away from the IEP. As it was

mentioned previously, salt addition also promotes the rate of aggregation by screening

the electrostatic interactions [Mehalebi et al., 2008; Nicolai et al., 2011].

1.5.3. Improvement of Heat Stability of Whey Protein Ingredients

Based on the literature studies, whey protein isolate has the potential to be a

good emulsifier. However, the applications of whey protein isolate are limited due to

its low heat stability. To overcome this problem, some research has been performed in

order to increase the heat stability of whey proteins.

The improvement of the functional properties, particularly the heat stability, of

proteins can be performed through physical, chemical, enzymatic and genetic

modification [Damodaran, 2005]. Physical modification of proteins can be performed

by partial denaturation of the proteins or protein unfolding which can be achieved by

exposing the proteins either to heat or to hydrostatic pressure under controlled heating

and shear conditions. Partial denaturation increases the surface hydrophobicity of the

24

proteins, which increases their solubility, functionality and heat stability. The main

drawback of this method is that the partial denaturation cannot be defined as an exact

value and partially denatured proteins are highly susceptible to many parameters such

as protein concentration, heating and shearing rate, pH, ionic strength and presence of

other food components. Furthermore, high partial denaturation can lead to

aggregation of the proteins, thereby decreasing the protein solubility and functionality

[Damodaran, 2005].

Soluble whey protein aggregate formation has been found to be an effective

method for modification of the whey protein functional properties and thermal

stability [K Ryan et al., 2012; K N Ryan et al., 2013]. Whey protein soluble

aggregates are intermediates between monomer proteins and an insoluble gel network

or precipitate which are formed by heating whey proteins at a concentration below

their critical gelling concentration under proper condition of pH, salt concentration,

protein concentration, heating time and temperature [McSwiney et al., 1994; K N

Ryan et al., 2013]. [K Ryan et al., 2012] proposed that whey protein soluble

aggregates with a high charge, a small size, a more compact structure, and a low

surface hydrophobicity are resistant to added salt and heat in beverage applications.

Microencapsulation is another method for producing heat stable whey proteins

[Zhang and Zhong, 2009; 2010]. Thermal pretreatment at 90 °C for 20 min was

applied to WPI solutions in nanometer-sized micelles of water/oil microemulsions to

form a whey protein nanoparticle (WPN) which improved the heat stability of WPI

due to irreversible physical and chemical bonds during pretreatment [Zhang and

Zhong, 2010]. Furthermore, [Zhang and Zhong, 2009] reported that cross-linking of

WPI by transglutaminase, before incorporation in the microemulsion or within the

microemulsion, before heat treatment enhances the thermal stability of WPN.

EDTA and trisodium citrate as chelating agents were used to improve the heat

stability of whey protein stabilized emulsions containing CaCl2 [Keowmaneechai and

McClements, 2006]. Since calcium is present in commercial whey protein products

and as it contributes in whey protein aggregation, using mineral chelating agents,

25

which bind calcium, is a proper method for improving the heat stability of WPI

stabilized emulsions.

Another method for improving the protein functionality and heat stability is

enzymatic modification. The most applicable enzymatic method that has been

reported is hydrolysis and polymerization. Partial enzymatic hydrolysis of proteins

using proteases such as pepsin and trypsin, as well as controlled polymerization of

protein by transglutaminase, which catalyses homopolymerization of proteins, can

improve their functional properties [Damodaran, 2005]. The transglutaminase enzyme

(TGase) has been applied for improving the thermal stability of whey proteins through

formation of covalent cross-links between reactive proteins [Truong et al., 2004; W

Wang et al., 2012; Zhong et al., 2013]. Before the cross-linking reaction, the whey

proteins should be unfolded to expose the enzyme-targeted sites. Therefore,

denaturation of whey proteins by heat or by addition of reducing agent such as DDT

before incubation with TGase can increase the cross-linking reaction [Tang and Ma,

2007]. It has been reported that preheating of WPI prior to treatment with TGase

improved the heat stability of WPI solution [Zhong et al., 2013].

Chemical modification of proteins may improve their functional properties by

changing either the structure of the proteins at the secondary, tertiary and quaternary

levels, or the hydrophobic to hydrophilic ratio [Damodaran, 2005]. Some of the

chemical methods that have been reported include acylation, phosphorylation,

alkylation, sulfitolysis and the amino-carbonyl reaction. Based on nutritional and

safety considerations, among these methods, phosphorylation and amino-carbonyl

(Maillard reaction) methods are more advisable for application in the food products

[Damodaran, 2005]. In contrast to other chemical methods, the Maillard reaction is a

spontaneous and naturally occurring reaction. As a result, it can be safely

incorporated into the food system without using undesirable chemical catalysis

[Oliver et al., 2006]. Therefore, this method is more preferable for improving the heat

stability of whey proteins.

26

1.6. protein-polysaccharide Conjugates via Covalent Bonding

In recent decades, there has been a considerable effort in developing protein-

polysaccharide complexes through two process: (1) covalent bonding between the

reducing end of a polysaccharide and the amine group of a protein, produced by dry

heating of a protein and polysaccharide mixture under controlled temperature and

relative humidity [Akhtar and Dickinson, 2007; Aoki et al., 1999; Dickinson and

Semenova, 1992; Einhorn-Stoll et al., 2005; Kato et al., 1990; Kato et al., 1992; Kika

et al., 2007]; and (2) non-covalent or electrostatic complexes between positively

charged patches of the protein with the negative charges of the polysaccharide

carboxyl groups at pH values below the isoelectric point of the protein [Gentes et al.,

2010; Weinbreck et al., 2003; Zaleska and Tomasik, 2002]. Covalent protein-

polysaccharide hybrids have been recognized as more advantageous as they are more

stable towards changes in solution conditions, such as pH and ionic strength, and

retain their molecular integrity and solubility [Dickinson and Galazka, 1991].

Glycation of proteins via the Maillard reaction has an influence on the

functional properties of proteins by changing their charge, solvation and conformation

[Nakamura et al., 1994].The functional properties of the complex are remarkably

different from the original biopolymers [Akhtar and Dickinson, 2007]. Furthermore,

it has been proven that protein glycation during the early stages of the Maillard

reaction can dramatically improve the emulsifying activity of a protein [Akhtar and

Dickinson, 2003; Aoki et al., 1999; Dickinson and Galazka, 1991; Einhorn-Stoll et al.,

2005; Jourdain et al., 2008; Kato et al., 1990; Kato et al., 1992; Kika et al., 2007; N.

Neirynck et al., 2004], its foaming properties [Dickinson and Izgi, 1996; Mishra et al.,

2001], solubility [Akhtar and Dickinson, 2007; Katayama et al., 2002; Mishra et al.,

2001; N. Neirynck et al., 2004], antimicrobial activity [Takahashi et al., 2000] and

heat stability [Aoki et al., 1999; Diftis and Kiosseoglou, 2006; Hashemi et al., 2014;

Sato et al., 2003; Shu et al., 1996]. In addition, at advanced stages of the Maillard

reaction, the formation of compounds with antioxidant activity [Nakamura et al.,

1998], anticarcinogenic and antimutagenic properties [Hosono, 1997] have been

reported.

27

1.6.1. Mechanism of the conjugate formation

The formation of protein and polysaccharide conjugates follows the path of

the Maillard reaction. In general, the Maillard reaction can be divided into three

stages: early, intermediate (or advanced) and final stages [Jimenez-Castano et al.,

2007; J Liu et al., 2012; Oliver et al., 2006]. In an early stage of the Maillard reaction,

the carbonyl group of a reducing sugar condenses with the free amino group to form a

Schiff base with the release of water. Subsequently, the Schiff base cyclizes to the

corresponding N-glycosylamine. With aldoses as the initial reactant, an Amadori

product ( 1-amino-1-deoxy-2-ketose) is formed through Amadori rearrangement of

N-glycosylamine, and in case of ketoses, the Heyn’s product (2-amino-2-

deoxyaldose) is formed by Heyn’s rearrangement [Oliver et al., 2006]. Mostly, the ԑ-

amino group of the lysine residue in the proteins is the primary source of reactive

amino groups. However, the imidazole group of histidine, the indole group of

tryptophan, and the guanidine group of arginine residues can also take part in this

reaction, albeit to a lesser amount [Ames, 1998].

The intermediate stage of the Maillard reaction begins with degradation of

Amadori/Heyn’s products which includes various pathways depending on the pH of

the system. At a pH up to 7, it undergoes 1,2-enolization with the formation of

furfural or hydroxymethylfurfural (HMF). At pH values above 7, 2,3-enolization of

the Amadori products leads to the formation of reductones and a variety of fission

products [Jimenez-Castano et al., 2007; J Liu et al., 2012]. All these products are

highly reactive and they are involved in various transformation reactions such as

oxidation, cyclization, hydrolysis, fragmentation, free radical reaction, and so on.

Although some color is formed at the intermediate stage, color development mostly

occurs at the final stage of the Maillard reaction in which “Melanoidins” , highly

colored, water soluble, nitrogen-containing polymeric compounds, are produced

[Jimenez-Castano et al., 2007; J Liu et al., 2012] .

However, extensive glycation decreases the solubility of proteins due to the

cross-linking and polymerization which occur during the advanced and final stages of

the Maillard reaction [Oliver et al., 2006]. This reaction is partly due to the presence

28

of sugar-derived dicarbonyl compounds which can attach to two lysine residues via

their bifunctional groups leading to cross-linking of protein molecules [Oliver et al.,

2006]. Therefore, to obtain conjugates with improved functionality and minimum

color and flavor changes which are suitable for food application, it is necessary to

perform the Maillard reaction under carefully controlled conditions to prevent the

later stages of the Maillard reaction since this is undesirable [Oliver et al., 2006].

Consequently, a good understanding of the key reaction parameters influencing the

glycation and side reactions as well as their influence on the protein functionality is

important for the development of superior functional food ingredients. Furthermore,

the types and products of the Maillard reaction are influenced by the reaction

conditions such as temperature, time, pH, protein to carbohydrate ratio, relative

humidity, intrinsic characteristics of the reactant, as well as the presence of oxygen

and reaction inhibitors such as sulfur dioxide [Ames, 1998].

The Maillard reaction can take place either in wet conditions or in dry

conditions. The optimum aw for the Maillard reaction is between 0.5 and 0.8 [Oliver et

al., 2006]. The dry heating method involves lyophilization of the solution of a protein

and a reducing sugar, which is followed by equilibration and incubation to the desired

aw or RH under controlled temperature for a specific time. Maillard-type protein-

polysaccharide conjugates can be prepared by freeze-drying of the protein-

polysaccharide mixtures with various molar ratios of protein to polysaccharide and

subsequent storage of the dried mixtures at 60°C for a given period of time under

either 65% or 79% relative humidity in desiccators containing a saturated KI or KBr

solution, respectively [Kato, 2002].

From the industrial point of view, the dry reaction is more preferable than the

wet reaction [Oliver et al., 2006]. This is due to the fact that this method has a higher

reaction efficiency as it requires less space and time. Furthermore, the resulting

product is easier to be handled and stored and has a longer-term stability compared to

the liquid products obtained from the wet reaction. Moreover, in wet conditions there

is a possibility of growth of microorganisms [G Liu and Zhong, 2013].

29

1.6.2. Emulsifying Properties of Maillard Conjugates

It is well known that food macromolecules, such as proteins and

polysaccharides, play a significant role in the stability and structure of food

emulsions [Tolstoguzov, 1991]. Proteins have received most attention for their

emulsifying properties through their surface activity and film forming abilities

[Dickinson, 2009].On the other hand, hydrophilic high molecular weight

polysaccharides are well known as stabilizing agents through their

hydrophilicity, gelling and thickening properties [Dickinson and Semenova, 1992].

Both compounds can work simultaneously to stabilize oil in water emulsions by

forming a macromolecular barrier between the dispersed droplets in the aqueous

medium [Dickinson and Galazka, 1991].

Based on theoretical considerations, for a biopolymer to provide an ideal steric

stabilization, it is required to not only adsorb at the interface strongly, but it should be

highly soluble in the aqueous medium as well [Dickinson and Galazka, 1991].These

properties are present in the conjugate, in which upon addition of the conjugate, the

hydrophobic residue of the protein molecule are anchored into the oil droplets and the

hydrophilic saccharides attached to the protein bind water molecules around the oil

droplets. This leads to the formation of a thick layer around the oil droplets providing

steric stabilization, thereby preventing oil droplet coalescence. Therefore, making

hybrid biopolymers composed of both proteins and polysaccharides is an ideal

combination of the excellent emulsifying properties of proteins and the good

stabilizing ability of polysaccharides [Dickinson and Semenova, 1992].

1.6.3. Factors Influencing the Functionality of the Conjugate

One of the most important physiochemical properties influencing the

functional properties and heat stability of conjugates is the glycation extent which can

be changed by reaction conditions such as temperature, time and pH [J Liu et al.,

2012]. The reaction time is a critical factor influencing the functionality of the

protein-polysaccharide conjugates. After a certain time of heating, the emulsifying

properties (EP) are expected to reach a steady state. The time needed to reach this

30

condition differs depending on the reactant and reaction conditions:as an example,

globular proteins will need a longer time to form Maillard conjugates than other

proteins, because of their compact conformation [Oliver et al., 2006]. The early stages

of the Maillard reaction can improve the functional properties and solubility.

Accordingly, the heat stability increases as the solubility improves. On the other hand,

the advanced stages of the Maillard reaction lead to color formation and

polymerization which is detrimental to solubility and heat stability of the conjugates.

In this context, the reaction conditions should be selected in a way which restricts the

advanced stages of the reaction [J Liu et al., 2012].

One of the critical factors in the mechanism of increasing the emulsifying

properties of the protein-polysaccharide conjugates is the balance between

hydrophobic and hydrophilic parts [Oliver et al., 2006]. The initial protein-

carbohydrate mole ratio of the conjugate has a major influence on the emulsifying

properties of the complex. When the proportion of sugar reaches a certain maximum

value , the availability of the protein to adsorb at the interface will decrease, thereby

the EP also decreases [Oliver et al., 2006]. However, the presence of free or weakly

complexed polysaccharides in the protein-polysaccharide composite should be

avoided since this leads to emulsion instability through depletion flocculation and

bridging flocculation, respectively [Dickinson and Galazka, 1991].

The type of carbohydrate and its molecular weight is another factor which

impacts the protein-carbohydrate conjugate functionality [Kato, 2002]. On

comparison of polysaccharides with mono- and oligosaccharides, during the Maillard

reaction the polysaccharides attach to a limited number of lysyl residues compared to

that of mono- and oligosaccharides, due to the high steric hindrance exerted by the

polysaccharides. Moreover, side reactions and color development during the Maillard

reaction are low for polysaccharides as compared to small carbohydrates [Kato,

2002]. In contrast, mono- and oligosaccharides have a higher reactivity and low steric

hindrance, resulting in the involvement in more side reactions, and formation of

insoluble aggregates and excessive color development, which are undesirable [Kato,

2002].

31

Studies have shown that both anionic and cationic polysaccharides improve

the EP of protein–polysaccharide conjugates [Oliver et al., 2006]. Charged oligo- and

polysaccharides are preferable over shorter chain length saccharides, in order to avoid

discoloration and protein polymerization during advanced Maillard reaction [Oliver et

al., 2006]. Hence, polysaccharides are more preferable than mono- and

oligosaccharides for the improvement of the functional properties of food proteins, as

well as preventing the nutritional loss by having less lysine destruction [Kato, 2002].

Moreover, based on Kato et al., [1990], branched polysaccharides such as

dextran and galactomannan have more influence on improving the functional

properties of protein-polysaccharide Maillard conjugates than straight chain

polysaccharides. This might be because branched chain polysaccharides provide more

steric hindrance than the straight chain polysaccharides, thus exhibiting a better

capacity in preventing coalescence of fat droplets [Kato et al., 1990]. However, the

structural differences among branched sugars also has an influence on the EP of their

Maillard conjugates [Kato et al., 1992].

1.6.4. Studies on Heat Stability of Protein-Polysaccharide Conjugate

During recent decades, extensive investigations have been performed to study

the emulsifying properties of protein-polysaccharide covalent complexes, produced

by dry-heating of protein and polysaccharide mixtures under controlled conditions.

However, much less work has focused on the thermal stability evaluation of protein-

polysaccharide conjugates. It is now well-recognized that impressive improvements in

thermal stability of proteins can be achieved via covalent bonding with

polysaccharides. The improved heat stability of proteins by conjugation with

polysaccharides is due to the increased steric repulsion forces formed by the

polysaccharide when glycated with protein molecules [Akhtar and Dickinson, 2007].

Upon adsorption of protein–polysaccharide hybrids at the droplet surface, heat-

induced aggregation effects are inhibited due to the increase of the droplet surface

hydrophilicity, resulting in the reduction of droplet–droplet interactions.

32

Jiménez-Castaño et al., [2005] investigated the effect of conjugate formation

between β-lactoglobulin and dextran on the heat stability of β-lactoglobulin. They

found that the glycated β-lactoglobulin had a lower stability to heat at pH 7.0 than the

native β-lactoglobulin, but its thermal stability was higher at pH 5.0 at temperatures

above 85 °C. The same authors also reported that glycosylation of individual whey

proteins, β-lactoglobulin, α-lactalbumin and bovine serum albumin (BSA), by

Maillard reaction using dextran of different molecular mass resulted in an improved

heat stability of β-lactoglobulin and BSA, the most thermally unstable whey proteins,

upon heating at different temperatures (50–95°C) for 15 min [Jimenez-Castano et al.,

2007].

Liu and Zhong, [2013] studied the thermal aggregation properties of whey

protein glycated with glucose, lactose, sucrose and maltodextrin after heating at 88 °C

for 2 min, with 7% w/v protein, at pH 3-7 and in the presence of 0-150 mM NaCl or

CaCl2. It was found that glycation decreased the isoelectric point and increased the

thermal denaturation temperature (Td) of all conjugates, although glycation did not

eliminate aggregation completely, particularly at the IEP of the protein [G Liu and

Zhong, 2013]. Glucose showed the least heat stability which is related to its smaller

chain length compared to that of lactose and maltodextrin as the strength of steric

repulsion is a function of the number and chain length of the saccharides [G Liu and

Zhong, 2013]. This shows that the type of the polysaccharide employed has an

influence on the properties of the resulting conjugates as it was previously mentioned.

Some studies on the impact of conjugation through dry heat treatment on the

heat stability of other types of proteins have also been reported. Sato et al., [2003]

observed an improved heat stability of carp myofibrillar protein by conjugation with

alginate oligosaccharide (AO) through Maillard reaction. Heat treatment at 80 °C for

2 h showed to have no effect on the solubility of the Mf-AO conjugate at different

NaCl concentrations and pH values. An improved stability against heat-induced

aggregation of oil-in-water emulsions prepared with complexes of soy protein isolate

with dextran, formed by dry-heating under controlled condition, could be observed

during heat treatment at 100 °C up to 30 min [Diftis and Kiosseoglou, 2006].

33

Hashemi et al., [2014] reported an improved heat stability of lysozyme by

glycosylation with xanthan gum as less turbidity was observed for the conjugate upon

heat treatment from 50-90°C.

34

Chapter 3: Materials and Methods

2.1 Materials

BiPro Whey Protein Isolate (WPI) was supplied by Davisco Foods

International Inc. (Le Sueur, MN, USA). Based on the manufacturer’s analysis, this

WPI contains approximately 92.6% protein (on dry weight basis). The WPI of this

supplier was previously analyzed by Monahan and his coworker (1996) and they

found that the protein of BiPro WPI contains 68% β-lactoglobulin, 19% α-

lactalbumin, 6% Bovine Serum Albumin and 7% Immunoglobulins. Low Methoxyl

Pectin (LMP) (OB700) was provided by Cargill (Ghent, Belgium). According to the

supplier, Unipectine OB700 is a low methoxyl apple pectin with a Dextrose

Equivalence value between 33 and 38% and a dry matter content of 89.6%. Refined

sunflower oil was purchased from a local supermarket and it was used without further

purification. All other chemical reagents, including NaCl, Na-azide (NaN3),

imidazole, Na-acetate and acetic acid were purchased from Sigma-Aldrich.

2.2 Methods

2.2.1 Whey Protein-Pectin Complex Formation

The Maillard type conjugates of WPI and LMP were prepared by

lyophilization of the mixture and subsequent dry heat treatment of the samples.

Details of the complex preparation are explained below.

Stock solutions of 5% (w/v) protein and 1% (w/v) LMP were prepared by

dissolving these components separately in distilled water. In order to prepare the stock

solutions, a correction of the protein content and the dry matter content of WPI and

LMP, respectively, was performed. Since the WPI contains 92.6% protein, to obtain

200 ml of 5% WPI stock solution, 10.799 g of WPI was required. Similarly, 5.580 g

35

of pectin was required to prepare 500 ml of 1% LMP stock solution as it contains

89.6% dry matter. The stock solutions were stirred for 1-2 hours to ensure that the

powder was well dissolved and the solution was homogenous. Prior to volume

adjustment, the pH of the stock solutions was adjusted to 7 by adding 1 N HCl/NaOH

to avoid the formation of ionic complexes which might form at lower pH upon mixing

of the stock solutions. Afterwards, the stock solutions were kept overnight in a

refrigerator in order to fully hydrate the powders. Prior to mixing, the stock solutions

were allowed to stand at room temperature to equilibrate the temperature. Mixtures of

WPI and LMP with a mass ratio (dry basis) of 2:1 and 1:0 (WPI:LMP) were prepared

at room temperature. This step was then followed by freeze drying the mixtures to

obtain dry mixtures of WPI and LMP. Depending on the amount of the water needed

to be removed from the samples, this process may take 3-4 days.

The dried mixtures were then brought into a desiccator containing a saturated

NaCl solution and incubated at 60 oC. At 60 oC, the saturated NaCl solution will

create a RH of about 74.5% [Greenspan, 1977]. In order to prepare this saturated

NaCl solution, 400 g of NaCl was dissolved in 1 L of distilled water. The desiccators

containing the saturated salt solution were then incubated at 60oC for 24 hours prior to

the incubation of dried mixtures to reach an equilibrium state. To maintain the RH

inside the desiccators, it is highly important to maintain the temperature constant

during the incubation period. The incubation was performed up to16 days and

conjugates were taken at 0, 4, 8, and 16 days. Conjugates were then stored at room

temperature for further experiments.

2.2.2 Emulsion Preparation

In this research oil in water (o/w) emulsions were prepared. The emulsions

were stabilized by WPI as the reference and by conjugates of WPI and LMP. O/W

emulsions were prepared at two different pH values, namely pH 6.5 and 5.0. As for

pH 5.0 two buffers, with and without NaCl, were prepared.

Imidazole buffer and Acetic acid/Na-Acetate buffer were used for emulsion

preparation at pH 6.5 and 5.0, respectively. The composition of the imidazole buffer

36

was 20 mM Imidazole, 30 mM NaCl and 1.5 mM NaN3, which was equal to 1.36 g/L

Imidazole, 1.75 g/L NaCl, and 0.097 g/L NaN3. While the Acetic acid/Na-Acetate

buffer was prepared to have a concentration of 20 mM and contained 1.5 mM of

NaN3, 30 mM of NaCl was added into the acetate buffer to obtain a buffer of pH 5.0

with a low salt content. The Acetic acid/Na Acetate buffers were prepared by

dissolving 0.417 ml/L Acetic acid, 1.044 g/L Na-acetate, and 0.097 g/L NaN3 in

distilled water. As for the Acetic acid/Na-Acetate buffer with salt, 1.75 g/L NaCl was

also added. NaN3 was used as an antimicrobial agent. The pH of the buffer was

adjusted by gently adding either 1 N NaOH or 1N HCl.

0.5% (w/w) WPI or conjugates was dissolved in buffer and used as the

aqueous phase. The aqueous phase solutions were stirred for 1 h and kept overnight in

the fridge to ensure complete hydration of the WPI powder and conjugates.

Oil-in-water (o/w) emulsions were prepared at room temperature by adding

sunflower oil into the aqueous phase solution which had been prepared the day before

to achieve an oil concentration of 10% (w/w). The mixtures were then pre-

homogenized at 24000 rpm for 1 minute using an IKA Ultra-Turrax TV45 (Janke &

Kunkel, Staufen, Germany). Subsequently, the pre-homogenized emulsions were

homogenized using a Microfluidizer 110S (Microfluidics Corporation, Newton, MA,

USA) for 2 minutes. The temperature of the microfluidizer was set at 55°C by

immersing the heat exchange coil of the microfluidizer in a water bath, while the

pressure of the microfluidizer was set at 4 bar of compressed air pressure, which

corresponds to a liquid pressure of 560 bar.

According to the manufacturer, the microfluidizer helps to decrease the size of

the particles and to achieve a monodispersed particle size distribution by applying

very high shear rates. When material is pumped into the inlet reservoir, it flows to the

cooling coil by the tubing. Subsequently, it reaches the interaction chamber, in which

it is accelerated at a very high velocity resulting in high shear rates near a boundary

layer in turbulent flow. The size of the droplets decreases in the dispersion as a result

of two processes, namely cavitation and impact. Cavitation is caused by the pressure

drop in the interaction chamber due to the high velocity leading to the formation of

37

bubbles which implode by restoring the pressure. Impact is a result of the collision of

particles together or with a solid object. The amount of reduction in particle size is a

function of the shear rate applied. After emulsification, the emulsions were allowed to

cool down to room temperature before heat treatment.

Table 2.1 Emulsions names and codes

Emulsifier Incubation time (Day) Sample code

WPI - WPI

Conjugate

1:0

0 1:0 D0

4 1:0 D4

8 1:0 D8

16 1:0 D16

Conjugate

2:1

0 2:1 D0

4 2:1 D4

8 2:1 D8

16 2:1 D16

In this research, different emulsions stabilized by either WPI or conjugates

were prepared. Table 2.1 shows the names and codes of the emulsions which were

studied in this research. The properties of the emulsions, such as emulsifying

properties and especially heat stability, were then evaluated.

2.2.3 Heat Stability Test of Emulsions

To study the heat stability of emulsions stabilized by WPI and conjugates,

emulsions were heat treated at 80 °C using the method optimized by Kasinos et al.

[Kasinos et al., 2015]. First, 9 ml of the emulsions were transferred into 20 ml vials

(75.5 ×22.5 mm, 1st hydrolytic class, Grace, Deerfield, IL, USA) and hermetically

sealed with aluminum caps (caps: 20 mm combination seal: aluminum cap, plain, with

center hole, silicone transparent blue/PTFE white, 35 shore, 3.0 mm; Grace,

Deerfield, IL, USA). The vials were then placed in a metallic twelve-place holder and

transferred in an oil bath (Fritel turbo SF®, 5 L capacity, Vanden Borre, Gent,

38

Belgium). The amount of the heating oil should be enough to completely immerse the

samples in the vial.

In order to have a uniform temperature inside the oil bath during heat

treatment, an IKA RW20 stirrer with 3-bladed metallic propeller stirrer of 5 cm

diameter (Janke & Kunkel) was placed and used at the left corner of the oil bath. The

rotational speed of the stirrer was set at 225 rpm. To monitor the temperature change

inside the oil bath, an electronic digital thermometer (Agilent 34970A, Diegem,

Belgium) with an attached thermocouple (type T) was placed in the oil. To double

check the temperature of the oil bath, a thermometer was also placed in the corner of

the oil bath. Emulsions were heated at 80 °C for 10 and 20 minutes. Subsequently,

heated emulsions were cooled down by air at room temperature. Visual observations

of the emulsions after emulsion preparation and heat treatment were performed. The

texture, appearance, homogeneity, phase separation (also indicated as syneresis) and

gelation were among the factors considered.

Fig.2.1 .Schematic presentation of set up for heat treatment.

39

2.2.4 Emulsion Stability Test

A LUMiFuge®116 (L.U.M., Berlin, Germany) was used for evaluating the

gravitational stability of the emulsions. The LUMiFuge stability analyser is an

analytical centrifugation system which works based on a centrifugal force. It basically

centrifuges the sample at a high speed, which provides an accelerated simulation of

the gravitational force of the earth. The principle of the LUMiFuge is based on a

measurement of NIR light transmission through the specimen over the total length of

the measurement cell. The resulting transmission profile shows the intensity of the

light transmitted as a function of position and time. By following the changes in light

transmission at a certain range of the sample position or by tracing the movement of

any phase boundary (interface between oil and serum phase), the separation behavior

and the creaming or sedimentation velocity of the sample can be obtained.

Fig. 2.2. Schematic illustration of the operational principle of the LUMIfuge

For emulsions at pH 5.0, with and without salt, 400 μl of the liquid samples

were transferred into rectangular polycarbonate cells, with a depth of 2.2 and width of

8.0 mm. On the other hand, for emulsions at pH 6.5, 2 ml of the samples were

transferred to a glass tube with an inner diameter of 11.5 mm. The samples were

centrifuged at 3000 rpm (corresponding to 1200 g) for a total of 3600 seconds. The

transmission profiles of the emulsions were recorded by the software every 60

seconds. 8 emulsions were analyzed simultaneously. The transmission profile of the

40

emulsion samples and the subsequent data analysis was performed using the

SEPView4 Software (LUM, Germany). The front tracking method was applied to

analyze the transmission profiles of the emulsions. By front tracking, it was possible

to obtain a slope from the phase separation profile curve which represented the

creaming velocity of the emulsions. The front tracking was set at a trigger value of

20%.

Fig.2.3 . Schematic presentation of front tracking method of transmission profile

with trigger value of 20%

In this experiment, the rotational speed was set at 3000 rpm which

corresponded to 1200 g or 1200 times higher than the gravity of earth (1g). Therefore

the creaming velocity that was obtained from the LUMiFuge corresponded to the

creaming velocity of emulsions at 1200g. To estimate the creaming velocity at the

normal gravity force of the earth, the creaming velocity can be rescaled using

Equation (2.1) :

Slope

1200

µm

s ×

0.001

1

mm

µm×

8600

1

s

day ₌ Creaming rate (mm

day) (eq2.1)

Trigger

41

2.2.5 Viscosity Measurement

The rheological properties of the samples before and after heating were

measured by a programmable LV-DV-II Viscometer (Brookfield, USA). The spindle

is driven by a motor through a calibrated spring. The viscometer measures the torque

required to rotate an immersed spindle in a sample at a given shear rate or rotational

speed. The shear stress is obtained from the torque value. The Brookfield Rheocalc®

software was used for collecting data from the instrument and performing the

mathematical analysis of the collected data.

Before starting the measurement, the appropriate spindle and speed were

selected by trial and error. The goal is to obtain a Viscometer dial or display (%

torque) reading between 10 and 100%. If the torque reading is under 10%, a higher

speed or a larger spindle is selected and vice versa. For liquid emulsions, spindle SC4-

18 with a rotational speed of 180 to 200 rpm was applied, whereas for highly viscous

or gel-like emulsions, spindle SC4-34 with a rotational speed of 40 to 200 rpm was

used. 8 ml of the emulsions were transferred to the sample holder and the spindle

was immersed in the center of the cylindrical sample. For highly viscous and gel-like

emulsions, measurement was carried out in the glass vial to prevent the breakdown of

the structure upon transferring the sample into the sample holder, which can influence

the value obtained from the viscometer. The measurements were performed at 21 (±1)

°C.

The results were displayed both numerically and graphically. Two graphs

were obtained for each measurement; a plot of viscosity versus shear rate and a plot of

shear stress as function of shear rate. The power law model was used as a base for

analyzing the data (Eq 1), in which shear stress (τ) (in mPa.s) is a function of the

consistency index (K) and the shear rate (γ) (in s -1) raised to the power of n (flow

index).

τ = K . γn (eq 2.2)

42

2.2.6 Particle Size Measurement

The particle size distribution of the emulsions was determined using a laser

light scattering analyzer (Mastersizer 3000, Malvern Instruments Ltd., Malvern,

Worcestershire, U.K.). According to the manufacturer, the Mastersizer 3000 uses the

technique of laser diffraction which allows to measure a particle size distribution

between 10 nm and 3.5 mm. In the laser diffraction method, after dispersion of the

sample through the measurement area of the optical bench, a laser beam passes

through a dispersed particulate sample and the angular variation in intensity of the

scattered light is measured. Large particles scatter light at small angles relative to the

laser beam and small particles scatter light at large angles. The angular scattering

intensity data are then analyzed to calculate the size of the particles that creates the

scattering pattern using the Mie theory of light scattering.

The Mastersizer 3000 software relayed the information of the analysis to the

computer to plot a graph representing the particle size distribution as a function of

droplet size and volume percentage. Prior to the measurement, the emulsions were

diluted 10 times with distilled water to avoid multiple scattering effects. Dilution of

the emulsion in 1% SDS solution was also performed for flocculated or aggregated

samples in order to differentiate between flocculation and coalescence. SDS will

break the disulfide bond if any and disintegrates the flocks while the droplets that

have undergone coalescence will not be affected. Emulsions diluted with 1% SDS

solution should be allowed to rest at room temperature for 1 hour prior to

measurement. The measurement was set at a refractive index of 1.47, and absorption

index of 0.01, and a density value of 1 g/cm3. Samples were gently shaken before

measurement to achieve a homogenous sample. The 10 times diluted emulsions were

added dropwise to the dispersion unit until an obscuration within the range between 2

and 6% was reached. The sample was passed through the dispersion system with a

speed of 1500 rpm. Each emulsion was analyzed three times and the data were

presented as an average volume-weighted mean diameter (d43). This diameter was

used for droplet size characterization as it is the most sensitive diameter to

flocculation and coalescence [Neirynck et al., 2007].

43

2.2.7 Light Microscopic Observation

Emulsions were diluted 10-fold with distilled water or 1% SDS prior to

microscopic observation. As it was mentioned previously, emulsions diluted with

SDS were left undisturbed for 1 hour at room temperature before further observation.

A drop of the diluted sample was placed on a microscope slide and was covered by a

cover slip. Immersion oil was added on top of the cover slip as an aid to increase the

resolution of the microscope. Microscopic images of the samples were taken using an

Olympus light microscope CX40 (Europe GmbH, Hamburg, Germany) equipped with

aAxiocam ERc5s camera (ZEISS, Germany). The magnification of the instrument

was set at 100 X.

2.2.8 Protein Load

The amount of protein adsorbed at the oil-water interface of an emulsion has a

substantial impact on its stability. The amount of protein adsorbed to the emulsion

particles is determined from the difference between the total protein initially present

in the emulsion (aqueous phase) and the amount of protein present in the serum phase.

To determine the amount of protein adsorbed, heated and unheated emulsions were

centrifuged at 40000 rpm for 105 minutes at 10 oC in a Beckman L7-55

ultracentrifuge using a SW 40 Ti rotor. The cream layer was removed and the serum

layer was used for protein measurement analysis.

The protein content was measured by the Kjeldahl method. This method is

based on the wet combustion of the sample by heating it with concentrated sulphuric

acid in the presence of metallic catalysts to effect the reduction of organic nitrogen in

the sample to ammonia, which is retained in solution as ammonium sulphate.

1 gram of sample was placed in a Kjeldahl flask. Catalyst (Kjeldahl tablet) and

10 ml H2SO4 were then added into the tube. The destruction was carried out in a

destruction block until a complete breakdown of all organic matter was obtained, and

a bright green colour appeared, which takes about 1.5 hour. After cooling the tubes

44

containing the digest, they were placed in the Kjeldahl distillation unit. In this unit,

the digest is diluted with distilled water and alkali-containing sodium thiosulfate is

added to neutralize the sulfuric acid. The ammonia formed is distilled into a boric acid

solution containing the indicators which are methylene blue and methyl red. This step

was then followed by titration using standardized HCl. The Amount of the protein can

be calculated based on the following formula:

% P =V×T×14×C×100

W (eq 2.3)

%P = Percentage protein by weight

V = Amount of HCl required for titration (L)

T = Normality of HCl

C = Conversion factor (6.25)

W = Weight of the sample (g)

Furthermore, protein recovery, which is the ratio between protein found in the serum

phase and total protein content of the emulsions, and protein load of the emulsions

was obtained by the following formula:

Protein Load = (1−protein recovery) ∗ C wpi ∗ C pro

SSA ∗ C oil (eq 2.4)

SSA (specific surface area) (m2/kg ) = 6

ϕ 32∗ ρ oil (eq 2.5)

Protein recovery = % protein in serum

% protein in emulsion (eq 2.6)

In above formula, C WPI, C protein and C oil represent the WPI content of the

emulsions (0.5 % for emulsions with WPI and 0.33% for emulsions with WPI-LMP),

protein fraction in the WPI (92.6%), and the the oil fraction in the emulsion (10%) ,

respectively.

45

2.2.9 Pectin Load

The pectin load was analyzed based on the method by Kintner and Buren

(1982), with a slight modification as described by Thibault [Thibault, 1979]. The

standard curve method was used to obtain pectin concentration of the samples.

galacturonic acid was used to prepare a standard solution series with concentration of

0 to 0.075 mg/ml. The serum phase was diluted 25 times before the measurement to

fit into the standard curve. 1 ml of diluted serum phase was brought into test tubes

equipped with caps and placed in an ice-water bath to cool for 5 minutes. 6 ml of

H2S04 solution (96%) was then added to each of the tubes in the ice/water bath and

each was mixed carefully using a Vortex at moderate speed for about 3 seconds,

which was repeated 3-4 times. The samples should be homogeneous to minimize the

standard deviation. The tubes were then heated in a water bath at 90o C for precisely 6

minutes and immediately placed in an ice-water bath until the temperature reached

room temperature (5-10 minutes). Subsequently, 0.1 ml of 0.15% m-hydroxydiphenyl

was added to the samples to obtain color development. For the reagent blank (0 µg/ml

uronic acid), the addition of 0,1 ml of m-hydroxydiphenyl was replaced by addition of

1 ml 0.5% NaOH. This reagent blank was used to zero the spectrophotometer. This

will be then followed by thoroughly mixing the samples using vortex. Samples were

poured into plastic cuvettes for determining the absorbance value. Because of a haze

of tiny gas bubbles formed when the m-hydroxydiphenyl and 0.5% sodium hydroxide

are added, each cuvettes was allowed to stay 20 minutes at room temperature to allow

the bubbles to dissipate. Absorption measurements were then taken at 520 nm. The

standard curve was obtained by using standard solution series of galacturonic acid

with concentration of 0 to 75 μg/ml (Fig.2.4). Then concentration of the pectin was

determined by interpolation on the standard curve for both aqueous phase and serum

phase. The pectin load was obtained with the same formula as described for protein in

section 2.2.8. In this case pectin recovery and pectin fraction were used.

46

Fig.2.4 Standard Curve for pectin measurement

y = 8,17E-03xR² = 9,97E-01

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 20 40 60 80

Ab

sorb

ance

Concentration (μg/ml)

47

Chapter 3: Results and Discussions

The emulsifying and emulsion stabilizing properties of dry heat WPI-LMP

complexes was compared to native or dry heated WPI. Besides visual

observation, also particle size analysis was performed. In addition, the stability

upon heating of these emulsions was evaluated from particle size as well as

viscosity determination.

3.1. Visual Observation

Fig.3.1. Image of visual appearance of unheated (fresh) oil-in-water emulsions

(10% w/w) stabilized with 0.5% WPI (A), dry heated WPI day 8 (B), and WPI-

LMP conjugate (ratio 2:1) day 8 (C), at pH 5.0, homogenized at 560 bar for 2

minutes, at 50 °C

Visual observation of the appearance and texture of the fresh emulsions

stabilized with 0.5% WPI, dry heated WPI, and WPI-LMP conjugates at pH 5.0 (with

and without 30 mM of NaCl) and 6.5 were performed. As it is depicted in Fig. 3.1,

emulsions containing WPI and dry heated WPI were not stable at pH 5.0 which is

near the isoelectric point of WPI and exhibited flocculation and phase separation after

B C A

WPI Dry heated

WPI, D8

WPI-LMP,

D8

48

preparation of the emulsions, while the emulsions containing WPI-LMP were stable

and remained homogenous. However, at pH 6.5 all fresh emulsions were found to be

homogenous and fluid-like without phase separation except for unincubated (day 0)

WPI-LMP conjugate (ratio 2:1) which exhibited limited phase separation at the

bottom of the emulsions the day after the emulsion preparation.

Fig.3.2. Image of visual appearance of oil-in-water emulsions (10% w/w)

stabilized with 0.5% WPI, dry heated WPI, and WPI-LMP conjugates ( 2:1),

day 4 at pH 5.0 with NaCl, after heat treatment at 80°C for 10 and 20 min.

Visual observation of the emulsions after heating at 80°C for 10 and 20 min

and subsequent cooling at room temperature for 90 minutes showed that emulsions

containing WPI and dry heated WPI were unstable toward heating. They exhibited

coagulation and gel-like structure at both pH 5.0 and 6.5. At pH 6.5 the structure of

the gel was homogeneous without appreciable (visible) phase separation/syneresis and

appeared to be harder after 20 min of heating compared to that after 10 min of

heating, whereas at pH 5.0 (with and without NaCl) extensive phase

separation/syneresis was observed. On the other hand, emulsions containing WPI-

LMP conjugates, incubated for 4, 8 and 16 days were stable and remained liquid after

heat treatment. In the case of emulsions containing unincubated (day 0) WPI-LMP

49

conjugate, gelation occurred at pH 6.5 and 5.0 with NaCl. However, they retained

their fluid-like consistency at pH 5.0 without salt.

Fig.3.3. Comparison of visual appearance of oil-in-water emulsions (10% w/w)

stabilized with 0.5% WPI, at pH 5.0 and pH 6.5, and emulsions stabilized with

WPI-LMP conjugates (2:1), day 0 and day 8, at pH 5.0 with NaCl, after heat

treatment at 80°C for 20 min.

3.2. Droplet size

The size of oil droplets in an emulsion has a significant influence on the

emulsion stability (e.g. gravitational separation, flocculation, etc.), rheology and

sensory properties of the emulsion. Knowledge about the particle size distribution of

an emulsion gives information about the size of all particles present and provides an

idea about the nature and origin of instability of the emulsion [D J McClements,

2007].

The data of the volume weighted mean diameter (d43) of unheated emulsions at

pH 6.5 and 5.0 are presented in Table 3.1. The result shows that the mean droplet size

of emulsions prepared with WPI-LMP conjugates was lower than that of emulsions

D0

pH 5 D8

pH 5

WPI

pH 5

WPI

pH 6.5

50

stabilized with WPI and dry heated WPI at both pH 5.0 (in the presence and absence

of NaCl) and 6.5.

From Table 3.1 and Fig. 3.4 we can clearly see that at pH 5.0, a substantial

increase in the droplet size of the emulsions containing WPI and dry heated WPI was

observed. The average droplet size (d 43) of the emulsion stabilized with WPI was

about 25.8 μm at pH 5.0, while at pH 6.5 it was 0.92 μm. The increase in the droplet

size of the emulsion stabilized with WPI at pH 5.0, which is close to the isoelectric

point of whey proteins (5.2), is attributed to the aggregation of whey protein as there

is less intramolecular electrostatic repulsion leading to protein aggregation and

instability of the emulsions [Demetriades et al., 1997b], as it was observed visually.

Microscopic images also further confirmed the aggregate formation at pH 5.0.

Table 3.1. Average Droplet Size d43 (µm) of oil in water emulsions (10% w/w

oil), stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP conjugates,

unincubated (Day 0) and incubated for 4, 8 and 16 days, at pH 6.5 and 5.0, in the

presence of 30 mM and 0 mM NaCl, homogenized at 560 bar for 2 minutes and

temperature of 50 °C.

pH WPI Dry Heated WPI WPI LMP Conjugates

D0 D4 D8 D16 D0 D4 D8 D16

6.5,NaCl 0.92 0.96 1.02 0.90 0.58 0.74 0.68 0.61 0.58

5.0,NaCl 25.8 - - - - 1.04 0.6 0.55 0.54

5.0 27.1 24.3 23.4 30.2 16.5 0.92 0.56 0.45 0.49

*the experiment on emulsions stabilized by dry heated WPI at pH 5.0 in the presence of NaCl was

not conducted

51

Fig.3.4. The mean volume-weighted droplet size, d43, of oil in water emulsions

(10% w/w oil), containing 0.5% (w/w) WPI and WPI-LMP, incubated for 8

days at pH 6.5 and 5.0 in the absence and presence of 30 mM NaCl, and

corresponding micrographs of the emulsions containing WPI (right) and WPI-

LMP (D8) (left) at pH 5.0.

Nevertheless, the average droplet size of emulsions containing WPI-LMP

conjugates (2:1) at pH 5.0 were found to be comparable with that at pH 6.5 which

reveals that no flocculation occurred at this pH (Fig 3.4). Similarly, micrograph

0

20

40

60

80

100

0,01 0,1 1 10 100 1000 10000

Vo

lum

e F

ract

ion

(%

)

Particle diameter (μm)

WPI, pH 5.0 + NaCl WPI-LMP D8, pH 5.0 + NaCl

WPI, pH 6.5 + NaCl WPI-LMP D8, Ph 6.5 + NaCl

52

images showed individual oil droplets which were homogenous in size (Fig.3.4). This

suggests that conjugation of WPI with LMP prevents aggregation of WPI by

increasing the solubility and hydrophilicity of the protein at the surface of the oil

droplets, as well as enhancing the steric forces leading to reducing the droplet- droplet

interactions and emulsion stability [Dickinson and Galazka, 1991].

As far as incubation days are concerned, on emulsions stabilized by WPI-LMP

conjugates day 0, or unincubated mixtures of WPI and LMP, no appreciable reduction

of droplet size was observed. The particle size was bigger than that of emulsions

stabilized by WPI-LMP conjugates day 4, 8, and 16. This showed that the

unincubated mixture had lower emulsifying activity which can be due to the fact that

no covalent bond/conjugate between WPI and LMP was formed. From the data it was

also found that there was no considerable difference in the emulsification property of

WPI-LMP conjugates incubated for 4, 8 and 16 days in terms of droplet size reduction

of the emulsion.

Comparing the droplet size of the emulsions prepared with WPI-LMP

conjugates and dry-heated WPI alone (1:0) in table 3.1, it can be seen that dry heating

of WPI in the absence of pectin has no influence in decreasing the droplet size of the

emulsion. Furthermore, the average droplet size of emulsions stabilized by dry heated

WPI and non-heated WPI was similar. These results suggest that dry-heating of a

mixture of WPI with LMP at 60°C and relative humidity of 74% for a certain period

of time leads to the formation of Maillard type conjugates between WPI and LMP

molecules. The formed conjugates can be adsorbed to the droplet surface leading to

emulsion stabilization and formation of emulsions with smaller droplet sizes

compared to the emulsions stabilized with non-conjugated WPI.

On the other hand, dry heating of WPI, without LMP, may induce denaturation

and unfolding of WPI which can modify its functional properties. If subsequent

aggregation of denatured protein occurs, this leads to a loss of solubility and

functionality of the heated WPI. Our results are in agreement with the findings of

Dickinson and Semenova, [1992] who reported that dry heating of BSA and 11S

53

globulin without dextran showed no improvement in droplet size of emulsions, while

dry heating of these proteins with dextran resulted in a smaller droplet size as

compared to the droplet size of emulsions stabilized by unheated proteins which was

due to the formation of conjugates between the protein and polysaccharide providing

steric stabilization of the emulsions. Furthermore, aggregation of denatured proteins

which occurs in dry heating of protein without polysaccharide is prevented by placing

polysaccharides in between denatured proteins [Dickinson and Semenova, 1992]

The effect of heating in an oil bath of 80°C, for a time period of 10 and 20 min,

on the particle size distribution of emulsions, at pH 6.5 and 5, with and without salt

was investigated. The data is presented in table 3.2 and 3.3. It can be derived from

table 3.2 and Fig. 3.5 that upon heating of the emulsions prepared with WPI at pH 6.5,

the droplet size of the emulsion increased. Thus the droplet size distribution curves of

the emulsions shifted to the right as a function of heating time. The mean volume

weighted diameter (d43) for unheated WPI-stabilized emulsion increased from 0.92

μm, to 10.2 μm and 19.3 μm at pH 6.5, after heating the emulsions at 80°C for 10 and

20 min, respectively. The same trend was obtained for emulsions stabilized with dry

heated WPI. The microscopic images further confirmed the presence of aggregated

particles in the heated WPI stabilized emulsions as compared to the individually

dispersed particles in unheated emulsions (Fig.3.5).

Table.3.2. Effect of heating at 80°C for 10 and 20 min on the mean volume-weighted

particle size, d43, of oil in water emulsions (10% w/w) containing 0.5% (w/w) WPI,

dry heated WPI and WPI-LMP conjugate (2:1), non-incubated and incubated for 4, 8

and 16 days at pH 6.5.

Time

(min) WPI

Dry heated WPI WPI-LMP conjugate

D0 D4 D8 D16 D0 D4 D8 D16

0 0.92 0.96 1.03 0.89 0.58 0.74 0.68 0.61 0.58

10 10.2 9 11.4 15 10.8 8.53 0.62 0.62 0.53

20 19.4 15.7 17.4 21.5 19.5 16.1 0.75 0.64 0.44

54

Fig.3.5. Droplet size distribution curves of an oil-in-water emulsions (10% w/w

sunflower oil) stabilized with a 0.5% (w/w) WPI heat treated at 80°C for 0, 10 and 20

min at pH 6.5 with NaCl. The micrograph images show WPI stabilized emulsions at

pH 6.5 unheated (left) and heated for 20 min (right)

Furthermore, it was previously found that at pH 5.0 (with and without NaCl)

even before heating, emulsions prepared with WPI and dry heated WPI (without

NaCl) already had large droplet size (Table 3.1) due to the aggregation during

emulsion preparation which was further enhanced after heating at 80°C for 10 and 20

0

2

4

6

8

10

12

0,01 0,1 1 10 100 1000 10000

Volu

me

Fra

ctio

n (

%)

Log. Particle Size (μm)

0 min 10 min 20 min

55

min (Table 3.3). In the case of emulsions prepared with dry heated WPI no

experiment was conducted at pH 5.0 in the presence of NaCl since these emulsions

were already found to be highly unstable even without NaCl at pH 5.0.

Table.3.3. Effect of heating at 80°C for 10 and 20 min on the mean volume-weighted

particle size, d43 (µm), of oil in water emulsions (10% w/w) containing 0.5% (w/w)

WPI and WPI-LMP conjugate (ratio 2:1), non-incubated and incubated for 4, 8 and 16

days at pH 5.0, with and without NaCl.

pH Time

(min) WPI

Dry heated WPI WPI-LMP conjugate

D0 D4 D8 D16 D0 D4 D8 D16

5.0,

with

NaCl

0 25.86 - - - - 1.04 0.60 0.55 0.54

10 27.8 - - - - 9.58 0.60 0.56 0.54

20 43.1 - - - - 11.00 0.59 0.56 0.53

5.0

0 18.4 24.3 23.4 30.2 16.5 0.92 0.56 0.45 0.49

10 25.1 24 27.3 27.7 25.1 1.08 0.53 0.57 0.48

20 35.4 31.2 29.4 30.5 32.3 1.03 0.49 0.45 0.49

*experiment on emulsions stabilized by dry heated WPI at pH 5.0 in the presence of NaCl was not

conducted

Fig.3.6 compares the droplet size enhancement of the emulsions prepared with

WPI, dry heated WPI (D4) and WPI-LMP conjugates (D4) when subjected to 80 °C

for 10 and 20 min. It is clear that the emulsion with WPI-LMP conjugates (D4) was

the most heat stable as no noticeable difference was found in its droplet size compared

to the WPI and dry heated WPI (D4) stabilized emulsions. In general, emulsions

containing WPI-LMP conjugates (2:1), incubated for 4, 8 and 16 days were found to

be stable against heating as they were able to retain their droplet size during heating at

80°C, for 10 and 20 min at pH 6.5 and 5.0 (Table 3.2 and Table 3.3).

56

Fig.3.6. Changes in average droplet size, d43, of oil in water emulsions (10% w/w oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI (D4) or WPI-LMP conjugates (D4),

after heating at 80 °C for 10 and 20 min, at pH 6.5

Furthermore, from Fig.3.7 and table 3.2 it can be derived that emulsions

prepared with unincubated WPI-LMP conjugates (day0) showed a dramatic droplet

size increase during heating at pH 6.5, as compared to the emulsions prepared with

incubated WPI-LMP for 4, 8 and 16 days. A similar trend was observed at pH 5 with

NaCl, while it remained the same size at pH 5.0 without NaCl after heating for 10 and

20 min at 80°C (Table 3.3). Comparison of the droplet size increase following heating

of the WPI-LMP (day0) stabilized emulsions as function of pH and ionic strength has

been given in Fig.3.8. Based on the results it can be implied that a mixture of WPI and

LMP (day 0) was able to maintain the stability of emulsions upon heating at pH 5.0.

Nevertheless, with addition of a low amount of salt, it lost its functionality and could

not maintain the stability of the emulsions. Increase in droplet size distribution of an

emulsion containing salt upon heating is due to the shielding of the electrostatic

repulsive forces by the counter-ions in the salt which leads to droplet flocculation

[Demetriades et al., 1997b]. The better stability at the lower ionic strength is

attributed to less screening of the charge around the adsorbed polyelectrolyte layer on

0

5

10

15

20

25

WPI Dry heated WPI WPI-LMP conjugate

d4

3 (μ

m)

0 min 10 min 20 min

57

each droplet which causes longer-range electrostatic repulsion between the dispersed

droplets [Dickinson and Semenova, 1992].

Additionally, in emulsions with unincubated WPI-LMP, the presence of the

small amount of free pectin in the continuous phase, which leads to droplet

flocculation, can provoke heat-induced aggregation of the emulsion droplets. As

reported by Kika et al. (2007) upon conjugation of WPC with carboxymethylcellulose

(CMC), the heat stability of the WPC improved markedly. No remarkable change in

the droplet size of emulsions prepared with incubated WPC-CMC was observed,

when subjected to 100 °C, in boiling water, while in emulsions containing un-

incubated WPC-CMC, the droplet size increased. Improved heat stability of the

incubated emulsions was explained by the protective effect of CMC which upon

adsorption at the droplet surface will increase the steric stabilization forces and

diminish the droplet flocculation phenomenon due to the decrease in the amounts of

free CMC in the continuous phase. This can also be used to explain our findings.

Fig.3.7. Comparison of cumulative droplet size distribution curves of oil-in-water

emulsions (10% w/w) stabilized with non-incubated and incubated (4, 8 and 16 days)

WPI-LMP, unheated and heated at 80°C for 20 min at pH 6.5.

0

20

40

60

80

100

120

0,01 0,1 1 10 100 1000 10000

Vo

lum

e F

ract

ion

(%

)

Log.Particle Size (μm)

D0, 0 min D0, 20 min D4, 0 min D4, 20 min

D8, 0 min D8, 20 min D16, 0 min D16, 20 min

58

Fig.3.8. Changes in average droplet size, d43, of oil in water emulsions (10% w/w oil),

stabilized with 0.5% (w/w) WPI-LMP conjugate (D0), after heating at 80 °C for 10

and 20 min, at pH 6.5 and 5.0, with or without NaCl.

The particle size of the aggregated emulsions diluted in 1% SDS was also

determined to confirm the aggregation phenomenon which was suspected to occur in

the heated emulsions. As it is depicted in figure 3.9, the droplet size distribution

curves of heated emulsions stabilized with WPI shifted to the left after dispersion in

SDS. This phenomenon is due to the ability of the SDS to break the disulfide bridges

between droplets thus breaking the aggregates. This showed that aggregation was

responsible for the rise of the droplet size of heated emulsions. However, the droplet

size of the emulsions measured in SDS solution did not fit to the original size,

especially after 20min of heating, indicating that part of droplet size increase is either

due to the coalescence which cannot be eliminated by SDS or the droplets were

strongly aggregated. As it is shown in Fig.3.9, the presence of the aggregates even

after dispersion with SDS in heated WPI stabilized emulsions was further confirmed

by microscopic images.

0

2

4

6

8

10

12

14

16

18

pH 5.0 pH 5.0 + NaCl pH 6.5 + NaCl

d 4

3(μ

m)

0 min 10 min 20 min

59

Fig 3.9. Droplet size distribution curves of oil-in-water emulsions (10% w/w)

stabilized with a 0.5% (w/w) WPI, heat treated at 80°C for 10 and 20 min, with size

distribution curve and the corresponding micrograph of the heated emulsions after

dispersion in 1% SDS at pH 6.5 (left: 10 min , right: 20 min).

0

20

40

60

80

100

0,01 0,1 1 10 100 1000 10000

Vo

lum

e F

racti

on

(%

)

Log.Particle size (μm)

WPI, 0 min

WPI, 10 min

WPI, 10 min+SDS

WPI, 20 min

WPI, 20 min+ SDS

60

The aggregation of emulsion droplets at temperatures higher than the

denaturation temperature of the whey protein is due to the unfolding of protein

molecules at the oil-water interface. The unfolding of proteins leads to an increase in

the hydrophobicity of the surface of the oil droplets which promotes interdroplet

interaction and aggregation. This aggregation is a function of temperature and heating

time [Sliwinski et al., 2003]. The effect of heating on the stability of whey protein

stabilized emulsions has been studied by several researchers [Demetriades et al.,

1997a; b; deWit and Klarenbeek, 1984; Euston et al., 2000; Sliwinski et al., 2003]. In

most of the studies, the highest aggregation of whey protein stabilized emulsions has

been reported at temperatures between 65-80°C. [Sliwinski et al., 2003] observed a

maximum droplet size (d32) and viscosity enhancement of soybean oil in water

emulsions (25% w/w) stabilized with whey protein (3% w/w), after heating for 45 min

at 75°C, and 6-8 min at 90°C. At higher heating temperatures, the maximum value

was reached more rapidly [Sliwinski et al., 2003].

In the case of emulsions prepared with conjugates of WPI-LMP (2:1), the

considerable stability against heat-induced aggregation and retention of small droplet

size are related to the formation of Maillard conjugates between WPI and LMP which

increases the thickness of the adsorbed layer at the surface of the oil droplets and

subsequently enhances the steric stabilization forces between the droplets.

Furthermore, in the emulsions stabilized with covalent WPI-LMP conjugates, the

proteins adsorbed at the surface of the droplets are less available for interaction with

non-adsorbed proteins of aqueous phase as they are bound to polysaccharides which

results in improved heat stability of an emulsion [Diftis and Kiosseoglou, 2006]. In

our experiments, it was found that by forming a conjugate with LMP, WPI was heat

stable at 80 °C for up to 20 minutes.

61

3.3. Viscosity

The data of the viscosity measurement of the emulsions before and after heat

treatment have been represented in Table 3.5. Emulsions prepared with WPI, dry

heated WPI and WPI-LMP conjugates at pH 6.5 exhibited Newtonian like behavior

before heat treatment. The viscosity was around 1.65 mPa.s for emulsions containing

WPI and dry heated WPI and about 2.5 mPa.s for emulsions containing WPI-LMP

conjugates independent of shear rate, with a flow index around 1. At pH 5.0, the

emulsions stabilized with WPI and dry heated WPI, which exhibited phase separation

and flocculation, had a higher viscosity compared to that of pH 6.5 (4-6 mPa.s), while

the emulsions stabilized with WPI-LMP conjugates showed no increase in viscosity.

Table 3.5. Viscosity of oil in water emulsions (in mPa.s) (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP conjugate, at pH 6.5

and 5.0, with and without NaCl.

pH WPI

Dry heated WPI WPI-LMP conjugate

D0 D4 D8 D16 D0 D4 D8 D16

6.5 1.69 1.64 1.64 1.65 1.7 2.88 2.26 2.4 2.01

5* 5.42 4.5 - 6.26 4.38 2.09 2.15 2.01 1.97

5,NaCl 5.9 - - - - 3.69 2.36 2.02 2.04

*: At pH 5, flocculation was observed for WPI and dry heated WPI.

At pH 6.5, where all emulsions were stable and no flocculation occurred, the

viscosity of emulsions with WPI-LMP, incubated for 4, 8 and 16 days was higher as

compared to the native and dry heated WPI. This higher viscosity of the emulsions

prepared with WPI-LMP conjugates is an important factor in improved stability of the

emulsions. As reported by Dickinson and Galazka, [1991], the surface rheological

properties of the adsorbed film are relevant to the coalescence stability, as they

observed a higher apparent viscosity for globulin-dextran conjugates than for the pure

globulin film.

62

The effect of heating on the rheological properties of the emulsions was also

investigated by measuring the viscosity of the emulsions after heat treatment and

subsequent cooling. Since the heated emulsions possess different types of consistency,

the results were expressed as consistency coefficient (mPa.s) (obtained using the

power law equation) to be able to compare the results. In case of phase separation and

syneresis upon heat treatment, which was observed for some of the heated emulsions

prepared with WPI and dry heated WPI at pH 5.0, no measurement was performed.

Table.3.6. Effect of heating at 80°C for 10 and 20 min on the consistency index of

oil in water emulsions (10% w/w) containing 0.5% (w/w) WPI and WPI-LMP

conjugates (2:1) non-incubated and incubated for 4, 8 and 16 days at pH 5.0 with

and without NaCl.

pH

Time

(min) WPI

Dry Heated WPI WPI-LMP Conjugate

D0 D4 D8 D16 D0 D4 D8 D16

6.5

0 1.69 1.64 1.64 1.65 1.7 2.88 2.26 2.41 2.01

10 2314 985 780 1371 949 2024 2.23 2.33 2.18

20 2924 757 1875 969 1305 2509 2.19 2.29 2.12

5.0,

+

NaCl

0 5.9 - - - - 3.69 2.36 2.02 2.04

10 2456 - - - - 1400 2.23 2.14 1.95

20 - - - - - 1512 2.23 2.14 1.95

5.0,

0 5.42 4.47 - 6.26 4.38 2.09 2.15 2.01 1.97

10 - - - - - 2.24 2.09 1.9 2.1

20 - - - - - 2.64 2.05 1.96 2.02

*: in columns with (-) sign, measurements were not possible due to syneresis.

63

Heating of the emulsions stabilized with WPI and dry heated WPI at pH 6.5

and 5.0 resulted in a considerable increase in consistency coefficient and by

increasing the heating time the consistency coefficient increased extensively; in some

conditions, a gel network was even formed and syneresis was observed. As it is

shown in Fig.3.10, heating of the emulsions stabilized with WPI resulted in a shear

thinning behavior in which the measured viscosity of the heated emulsions decreases

by increasing the shear rate. Moreover, as it is depicted in Table 3.6, the consistency

coefficient of unheated WPI stabilized emulsion was increased from 1.69 (mPa.s) to

2314 and 2924 (mPa.s) after heating of the emulsion at 80 °C for 10 and 20 min,

respectively, at pH 6.5. As it was previously stated, the heat stability of emulsions

stabilized by WPI-LMP conjugates was also tested at pH 5.0 in the presence of a low

concentration of NaCl (30 mM) and compared to that stabilized by WPI. The results

are presented in table 3.6. The increase in the viscosity of the emulsions by heat

treatment can be correlated to the particle size increase. As the particles become

bigger due to the aggregation, the viscosity increases.

It was reported that following heating of 20 wt% corn oil-in-water emulsions

stabilized with 2 wt% WPI at a temperature between 30 and 90°C, a significant

increase in the droplet aggregation and viscosity was observed in the temperature

range between 65-80°C at pH 7 in the presence of salt [Demetriades et al., 1997a]. In

whey protein stabilized emulsions, upon heating at above 65°C β-lactoglobulin and α-

lactalbumin, the major whey proteins, undergo conformational changes at the surface

of the oil droplets with exposure of reactive amino acid residues at the surface which

promotes protein-protein interactions via hydrophobic and thiol-disufide interchanges.

In the initial stage of aggregation, hydrophobic interactions are formed to bring

protein molecules together and then disulfide bonds are formed. In the case of

intradroplet disulfide interaction, the viscoelasticity of the surface increases, while by

interdroplet interaction flocculation occurs [Demetriades et al., 1997a]. Upon heating

of whey protein-stabilized emulsions, firstly flocculation occurs which is a loose

aggregation of droplets formed due to the large distance between particles, and after

prolonged heating, these aggregates fall apart and rearrange to compact and smaller

aggregates due to the closer contact of particles [Sliwinski et al., 2003].

64

Figure 3.10. Viscosity profile of oil in water emulsions (10% sunflower oil),

stabilized with 0.5% (w/w) WPI, dry heated WPI (D8) and WPI-LMP conjugate (D0),

at pH 6.5 after heat treatment at 80 °C for 10 and 20 min.

Upon heat treatment of the emulsions stabilized with WPI-LMP, incubated for

4, 8 and 16 days, no viscosity enhancement was observed after 10 and 20 min of

heating time and they remained liquid-like, which confirmed the results of the droplet

size measurement as no noticeable change in the particle size was observed. As it is

depicted in Fig.3.11, the heated emulsions stabilized with WPI-LMP at 80°C for 10

and 20 min showed a Newtonian viscosity profile just as unheated emulsions; no

noticeable viscosity change by changing the shear rate was observed. Moreover, no

substantial change in the consistency coefficient was found and the flow index

0,00

50,00

100,00

150,00

200,00

250,00

300,00

0,00 10,00 20,00 30,00 40,00 50,00 60,00

Vis

cosi

ty(m

Pa.

s)

Shear rate (s-1)

WPI, 10 min WPI, 20 min

Dry Heated WPI, D8, 10 min Dry Heated WPI, D8, 20min

WPI-LMP, D0, 10 min WPI-LMP, D0, 20 min

65

remained around 1 indicating that the emulsions remained stable and no aggregation

occurred upon heat treatment.

These results are in agreement with the finding of Diftis and Kiosseoglou,

[2006], who observed a stability of the emulsions containing dry-heated soy protein

isolate-dextran against heat-induced aggregation and increase in viscosity. According

to this author, the presence of dextran at the droplet surface through covalent bonding

with soy protein isolate prevents interaction between proteins at the interface and

aggregated protein in the continuous phase. Moreover, adsorption of the protein-

polysaccharide conjugates at the interface increases the steric stabilization forces

between the droplets by the bulky hydrophilic polysaccharide moiety. In this case,

LMP was able to improve the heat stability of WPI, and thus an improved heat

stability of the emulsions was obtained.

The higher thermal stability of WPI-LMP conjugates compared to the WPI

and dry heated WPI is attributed to their better solubility and increase in surface

hydrophilicity resulting in resistance to aggregation. Wang and Ismail, [2012] used

WPI and dextran to create Maillard conjugates which were found to bring about

enhanced solubility and thermal stability over a wide range of pH, including the pH

around the isoelectric point (pI) of the whey protein. The PGWP (partially

glycosylated WPI) remained soluble at all pH conditions even after heating at 80 °C

as opposed to WPI which exhibited a low solubility at pH 4.5 to 5.5. The enhanced

solubility and thermal stability of PGWP was explained by the reduction in

intermolecular interactions due to the decrease in surface hydrophobicity, and the

reduced exposure of sulfhydryl groups and shifting of the IEP to a more acidic pH .

66

p

H 6

.5 +

NaC

l

pH

5.0

+N

aC

l

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 40 min10 min20 min

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 80 min10 min20 min

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 160 min10 min20 min

67

pH

5.0

Fig.3.11. Viscosity profiles of oil in water emulsion (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI-LMP conjugates, incubated for 4, 8 and 16 days, at

pH 6.5 and 5.0, with and without NaCl, before and after heat treatment at 80 °C for

10 and 20 min.

In the case of unincubated WPI-LMP (day 0), droplet flocculation is due to the

presence of free polysaccharide (LMP) in the aqueous phase. According to [Dickinson

and Galazka, 1991], in a system containing dry-heated protein and polysaccharides,

instability of an emulsion is caused by either depletion flocculation, or bridging

flocculation. Depletion flocculation is due to the presence of non-adsorbed

polysaccharides, while bridging is a result of weak adsorption of polysaccharides to

the surface. Consequently, the amount of polysaccharide and the incubation time are

critical factors in the stability of emulsions containing covalent protein-

polysaccharide hybrids. Correspondingly, in the case of unincubated WPI-LMP (day

0), there is no time for Maillard reaction to occur between protein and polysaccharide.

As a result, the presence of free LMP in the continuous phase of the emulsion

promotes droplet flocculation and a subsequent increase in the droplet size and

viscosity.

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 40 min

10 min

20 min

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 80 min

10 min

20 min

1

10

240 250 260

Vis

cosi

ty (

mP

a.s)

Shear rate (s-1)

Day 160 min10 min20 min

68

3.4. Emulsion Stability Analysis

The results of the LUMiFuge stability analyzer were used to evaluate the

separation behavior and the stability of the emulsions against creaming. The

transmission profile of the emulsions and data analysis was obtained using the

SEPView4 Software. The front tracking method was applied to analyze the

transmission profile of the emulsions and to obtain a slope from the phase separation

curve which represents the creaming velocity of the emulsions. The trigger value in

front tracking was set at 20%. Since the creaming velocity obtained from the

LUMiFuge corresponded to the creaming velocity of emulsions at 1200g, the

creaming velocity at 1g was then calculated.

Fig. 3.12 depicts the evolution of transmission profile of emulsions at pH 5.0.

From tracing the movement of the interface it is clear that emulsions containing WPI

exhibited significant creaming and instability at pH 5.0. Creaming was very fast in the

first 5 minutes of the centrifugation and became very slow afterwards. As a result, the

creaming velocity of the highly unstable emulsions was determined from the first 5

minutes of the front tracking results (Fig.3.13), while for the emulsions containing

WPI-LMP conjugates (Fig. 3.13) incubated for 4, 8 and 16 days and other emulsions

which exhibited higher stability against creaming, creaming velocities were

determined within 60 minutes of the front tracking results (Fig. 3.13).

69

Fig.3.12. Evolution of transmission profile of an oil in water emulsion (10% w/w oil),

stabilized with 0.5% (w/w) WPI (A), and WPI-LMP conjugate (2:1) (B), at pH 5.0.

Table 3.7. Creaming rate (mm/day) (LUMiFuge, 3000 rpm, 1 hr) of o/w emulsions

(10% w/w oil) stabilized with 0.5% (w/w) WPI and WPI-LMP conjugate (2:1),

containing 30 mM NaCl, at pH 6.5 and 5.0.

pH WPI

Dry Heated WPI WPI-LMP Conjugate

D0 D4 D8 D16 D 0 D 4 D 8 D 16

pH 6.5, 0.14 0.13 0.13 0.12 0.1 0.22 0.07 0.08 0.06

pH 5.0,

NaCl 0.45 - - - - 0.47 0.12 0.12 0.11

pH 5.0 0.47 - 1.13 0.42 1.17 0.20 0.10 0.11 0.11

70

Fig.3.13. Comparison of front tracking results of emulsions (10% w/w oil),

stabilized with 0.5% (w/w) WPI (A), and WPI-LMP conjugates (2:1), incubated for

4 days, at pH 5.0 with 30 mM NaCl.

Based on the data of table 3.7, it is clear that emulsions containing WPI and

dry heated WPI are highly unstable towards creaming at pH 5.0 as compared to those

at pH 6.5, as they have a higher creaming rate. A similar trend was obtained for the

emulsions prepared with unincubated WPI-LMP. At pH 6.5, the creaming rate for

emulsions containing WPI was 0.14 mm/day, while it was 0.45 at pH 5.0 with 30 mM

NaCl, and 0.47 mm/day at pH 5.0 without NaCl. The lower stability of emulsions

containing WPI and dry heated WPI at pH 5.0, which is near the IEP of the WPI, is

attributed to the limited electrostatic repulsion between emulsion droplets, resulting in

98

98,5

99

99,5

100

100,5

101

101,5

102

102,5

0:00:00 0:14:24 0:28:48 0:43:12 0:57:36 1:12:00

Inte

rfac

e P

osi

tio

n (

mm

)

Time

107

108

109

110

111

112

113

114

0:00:00 0:14:24 0:28:48 0:43:12 0:57:36 1:12:00

Inte

rfac

e p

osi

tio

n (

mm

)

Time

71

fast creaming and phase separation of the emulsion. On the other hand, at pH 6.5,

which is further away from the IEP of WPI, the electrostatic repulsion forces are

strong enough to prevent a high creaming rate of the emulsions as compared to what

was observed at pH 5.0. As far as salt is concerned, the emulsions prepared with

unincubated WPI-LMP creamed extensively at pH 5.0 in the presence of NaCl, while

in the absence of NaCl the creaming rate was slower. This can be explained by the

effect of salt in screening the electrostatic repulsion of the adsorbed proteins on the

droplet surface resulting in lower stability against aggregation and subsequent

creaming. Demetriades et al., [1997b] observed maximum creaming and droplet

flocculation of the emulsions prepared with WPI at a pH of 4 to 6, which is near the

isoelectric point of the whey proteins, especially at higher salt concentrations.

Fig.3.14. Comparison of creaming rate (mm/day) of emulsions (10% w/w oil)

stabilized with 0.5% (w/w) WPI, dry heated WPI and WPI-LMP conjugate (2:1),

incubated for 16 days, containing 30 mM NaCl, at pH 6.5.

106

107

108

109

110

111

112

113

0:00:00 0:14:24 0:28:48 0:43:12 0:57:36 1:12:00

Inte

rfac

e P

osi

tio

n (

mm

)

Time

WPI WPI-LMP, D16 Dry Heated WPI, D16

72

On the other hand , emulsions containing WPI-LMP conjugates (2:1),

incubated for 4, 8, and 16 days, were highly stable against creaming as compared to

the emulsions containing WPI and dry heated WPI at both pH 6.5 (Fig.3.14) and 5.0,

in the presence and absence of NaCl.

These results are consistent with the findings of Diftis and Kiosseoglou,

[2003], who observed an improved stability of emulsions against creaming upon using

a dry-heated mixture of soybean protein isolate (SBPI) with sodium carboxymethyl

cellulose, and a low stability of emulsions prepared with SBPI alone. It was also

reported that the polysaccharide molecular weight, molar ratio of protein and

polysaccharide, and time of incubation are among the factors which affect the

creaming stability. According to Kato, [2002] polysaccharides provide more steric

hindrance than mono and oligosaccharides, as a result of the fact that during the

Maillard reaction they can attach to a limited number of lysyl residues as compared to

mono- and oligosaccharides, thus leading to a better functionality of the formed

conjugates, while in the case of low molecular weight saccharides, the functionality of

the protein decreases by masking most of the lysyl residues. Additionally, side

reactions and color development during the Maillard reaction are low for

polysaccharides as compared to small carbohydrates. The higher creaming stability of

the emulsions prepared with WPI-LMP (2:1) is attributed to the formation of covalent

complexes between WPI and LMP following dry heating of the mixture which

inhibits creaming and phase separation of the emulsion as a result of the presence of

polysaccharide molecules covalently linked to the adsorbed protein molecules and

hence increased steric stabilization forces through protruding into the continuous

phase. The creaming rate of heated emulsions was also analyzed. However, heated

emulsions which underwent syneresis or strongly aggregated were excluded. Since

the emulsions stabilized with WPI-LMP conjugates (2:1), incubated for 4, 8 and 16

days, remained fluid after heating at 80°C for 10 and 20 min, it was expected that

there would be no enhancement of the creaming rate. As can be derived from

Fig.3.15, no enhancement in creaming rates of the emulsions stabilized with WPI-

LMP conjugates (2:1), incubated for 4, 8 and 16 days, at pH 5.0 in the absence and

73

presence of NaCl was noted even after heat treatment of the emulsions at 80° C for 10

and 20 min compared to that of the fresh emulsions, indicating a high heat stability of

emulsions stabilized with WPI-LMP conjugates (2:1). This stability is due to the

formation of covalent bonds between WPI and LMP by dry heating of WPI with LMP

under controlled temperature and relative humidity which increases the steric

stabilization forces by increasing the effective thickness of the adsorbed layer which

prevents droplet aggregation upon heat treatment [Dickinson and Semenova, 1992].

Zhu et al., [2010] observed that covalently linked conjugates of whey protein isolate

and dextran retained their emulsifying properties even after 30 min of heating at 80

°C, as compared to the native WPI, which was attributed to the improved heat stability

of WPI-DX, as a result of enhanced steric stabilization forces, increased oil droplet

surface hydrophilicity and improved adsorptive ability of the conjugates.

Fig.3.15. Comparison of creaming rate (mm/day) of emulsions (10% w/w oil)

stabilized with 0.5% (w/w) WPI and WPI-LMP conjugate (2:1), incubated for 4, 8

and 16 days, at pH 5.0 containing 30 mM NaCl, heated at 80°C for 0, 10 and 20 min.

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

WPI WPI-LMP (D0) WPI-LMP (D4) WPI-LMP (D8) WPI-LMP(D16)

Cre

amin

g ra

te (

mm

/day

)

0 min 10 min 20 min

74

In the case of emulsions with WPI and dry heated WPI, their creaming

stability was deteriorated when subjected to heat treatment as they exhibited extensive

heat-induced aggregation and phase separation, in opposite to the emulsions with

WPI-LMP conjugates. The results of emulsion stability analysis are in harmony with

the results of particle size and viscosity measurements in which a bigger droplet size

and a higher viscosity of the emulsions stabilized with WPI and dry heated WPI lead

to a lower stability of an emulsion against creaming, while emulsions stabilized with

WPI-LMP conjugates with smaller droplet size and lower viscosity are more stable

against creaming.

3.5 Protein Load

The protein and pectin load of the fresh and heated emulsions was determined

to evaluate the effect of WPI-LMP dry heating on physiochemical properties of the

emulsions before and after heat treatment. The protein load of emulsions stabilized by

WPI and WPI-LMP conjugates day 0 and 8 at pH 6.5 and pH 5.0 with 30 mM of

NaCl before and after heat treatment for 20 minutes was performed.

As it is depicted in table 3.9, the protein load of the fresh emulsions prepared

with WPI is higher than that of emulsion containing WPI-LMP conjugates. The

difference in the protein content of these emulsions, to some extent is related to the

amount of the original protein used as an emulsifier in the emulsion preparation. In

the case of emulsions prepared with WPI-LMP conjugates from 0.5% conjugates, 1/3

out of it was pectin. Consequently, the lower protein load of the emulsions containing

WPI-LMP conjugates can be due to the replacement of part of the WPI with LMP

from the conjugate at the droplet surface. Nonetheless, at pH 5.0, this difference was

highly pronounced, as the protein load of the emulsions prepared with WPI is 5.55

mg/m2, while it was 1.44 mg/m2 for emulsions prepared with WPI-LMP conjugates

(D8) (Table 3.9).

75

Table 3.8. Amount of protein (%) in the aqueous phase, serum phase and adsorbed on

oil droplets of heated and unheated oil in water emulsion (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI, and WPI-LMP conjugates (2:1), unincubated and

incubated for 8 days, at pH 6.5 and 5.0.

pH Sample Aqueous

phase

Serum phase cream

Unheated Heated Unheated Heated

pH 6.5

WPI 0.48 0.19 0.06 0.29 0.42

WPI-LMP(D0) 0.37 0.17 0.07 0.2 0.3

WPI-LMP(D8) 0.30 0.05 0.05 0.25 0.25

pH 5.0,

NaCl

WPI 0.42 0.12 0.04 0.3 0.37

WPI-LMP(D0) 0.32 0.08 0.02 0.24 0.3

WPI-LMP(D8) 0.30 0.06 0.01 0.24 0.29

In whey protein stabilized emulsions, the protein is distributed between the

aqueous phase and the adsorbed layer at the surface of the oil droplets [Euston et al.,

2000]. The proportion of these two fractions is important in stability of an emulsion.

From the data (Table 3.9), it follows that in the emulsions prepared with WPI-LMP

conjugates, incubated for 8 days, an appreciable proportion of the protein had

adsorbed at the droplets surface, e.g. 82.52% at pH 6.5, while the amount of non-

adsorbed protein (in the serum phase) was marginal as compared to the emulsions

containing unincubated WPI-LMP and WPI. The higher amount of unadsorbed

protein in the emulsions containing WPI and unincubated WPI-LMP can be linked to

the stability of the emulsions, as it was found in the previous experiments that the

creaming stability of these emulsions was lower as compared to the emulsions

prepared with WPI-LMP conjugates (D8). It has been proved that the concentration of

the protein used as an emulsifier in emulsion preparation is critical for emulsion

stability. According to Dickinson and Golding, [1997], at a protein concentration

higher than that required for saturation surface coverage of the droplets, the stability

of emulsions with respect to creaming and flocculation decreases, which is due to the

76

increase in the amount of unbound protein in the continuous phase and following

self-association of these proteins and depletion flocculation in the system.

The protein load of the emulsions prepared with WPI and un-incubated WPI-

LMP was higher at pH 5.0 as compared to that at pH 6.5 (Table 3.9). As it was found

from the previous results, at pH 5.0 these emulsions had a lower stability against

creaming and flocculation. The higher protein load in this case is related to the

flocculation phenomena and accumulation of proteins at the surface of the oil droplets

which are stuck together. On the other hand, in emulsions with WPI-LMP conjugates

incubated for 8 days, the difference between the protein load at pH 5.0 and 6.5 was

not remarkable.

Table 3.9. Percentage of adsorbed protein (%) on oil droplets, ɸ32 (in µm), and protein

load (mg/m2) of heated and unheated oil in water emulsions (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI, and WPI-LMP conjugates (2:1), unincubated and

incubated for 8 days, at pH 6.5 and 5.0 in the presence of 30 mM NaCl.

pH Sample % adsorbed in cream ɸ32

protein load

(mg/m2)

Unheated Heated Unheated Heated

pH 6.5

WPI 60.79 87.52 0.54 2.33 3.35

WPI-LMP(D0) 54.65 80.2 0.46 1.19 1.74

WPI-LMP(D8) 82.52 82.05 0.39 1.53 1.52

pH 5.0,

NaCl

WPI 71.78 89.71 1.09 5.55 6.93

WPI-LMP(D0) 74.05 93.63 0.65 2.28 2.88

WPI-LMP(D8) 80.23 95.33 0.38 1.44 1.71

As it is shown in table 3.9 and Fig.3.16, generally by heating of the emulsions

the protein load increased specially for emulsions stabilized with WPI. Heating of the

emulsions prepared with WPI, at pH 6.5, for 20 min at 80°C resulted in an increase of

the protein load from 2.33 to 3.35 (mg/m2). At the same time by the increase of the

77

protein load and protein adsorbed in the cream phase, the non-adsorbed protein

concentration in the serum phase declined.

Similar trends have been reported by Sliwinski et al., [2003], who observed

an increase in the adsorbed amount of protein with an increase in the heating

temperature. In whey protein stabilized emulsions, the protein content present in the

aqueous phase and the adsorbed layer at the surface of the oil droplets are essential for

heat-induced aggregation of whey protein stabilized emulsions [Euston et al., 2000].

The rise in the adsorbed amount of protein upon heating of the emulsions is due to the

increase in the rate of denaturation of proteins [Sliwinski et al., 2003]. According to

Sliwinski et al., [2003], by increasing the heating temperature and time, the amount of

adsorbed protein increases, and the non-adsorbed protein fraction decreases; the

maximum values of the adsorbed protein were reached after 10 min of heating at 75

°C and after 5 min of heating at 90 °C. The higher concentration of the non-adsorbed

protein leads to the higher aggregation of the emulsion droplets, as the main

aggregation mechanism involves protein-droplet interaction in which the aggregates

of emulsion droplets are held together by the aggregates of non-adsorbed whey

protein molecules in the continuous phase of the system which act as a glue between

the oil droplets [Euston et al., 2000].

On the other hand, generally, for the emulsions prepared with WPI-LMP

conjugates, incubated for 8 days, no remarkable increase in the protein load was

observed following heating of the emulsions compared to the emulsions containing

WPI and WPI-LMP (D0). The decline in the protein load increase upon heating of the

emulsions prepared with WPI-LMP conjugates (D8) was attributed to the presence of

WPI-LMP conjugates (D8) which improve the heat stability of the emulsions and

prevent protein load enhancement due to aggregation of denatured protein from the

unadsorbed protein with the adsorbed protein at the surface of oil droplets. These

results are in line with the results of the viscosity and droplet size measurements, in

which using WPI-LMP conjugates (incubated for 4, 8 and 16 days) in emulsions

could intensively prevent an undesirable increase in the droplet size and viscosity of

78

the emulsions during heating and thus improve the stability of the emulsions against

creaming and flocculation.

Fig.3.16. Protein load (mg/m2) of oil in water emulsions (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI, and WPI-LMP conjugate (2:1), unincubated and

incubated for 8 days, heated for 0 and 20 min at 80 °C at pH 6.5 and 5.0 in the

presence of 30 mM NaCl.

The stabilizing effect of WPI-LMP conjugates is attributed to the replacement

of some of the protein at the droplet surface with WPI-LMP conjugates, and

preventing protein interaction by placing the WPI-LMP conjugates between protein

molcules. Furthermore, placing WPI-LMP conjugates at the oil droplet surface

provides repulsive steric stabilizing forces operating between the emulsion droplets,

by the presence of the polysaccharide moiety of the conjugate at the droplet surface.

The increased steric barrier brought about by the hydrophilic polysaccharide of the

0

1

2

3

4

5

6

7

8

Pro

tein

Lo

ad (

mg/

m2

) Unheated

Heated

79

conjugate has been proved by several researchers. As reported by Zhu et al., [2010],

the radii of gyration (Rg) of the WPI-DX conjugate was about 30 nm while the Rg of

the WPI was about 3-5 nm, suggesting that the thickness of the adsorbed film around

the oil droplets stabilized by the WPI-DX conjugates was about 6 times bigger than

that of the oil droplets stabilized by the WPI alone.

The results of the protein load measurement are in agreement with the

observations of Kasinos et al., [2014] who reported a reduction in the protein load

enhancement upon heating of the recombined evaporated milk emulsions upon

addition of phospholipid enriched dairy by-products. This phenomenon was correlated

to the heat stabilizing effect of phospholipid enriched dairy by-products which

decreases interactions between proteins upon heating.

3.5. Pectin Load

Tables 3.10 and 3.11 represent data of the pectin content of the emulsions

prepared with WPI-LMP conjugates. It can be derived that in the emulsion prepared

with un-incubated WPI-LMP at pH 6.5, an appreciable proportion of the pectin had

been adsorbed at the surface of the oil droplets which was higher than in the emulsion

with WPI-LMP, incubated for 8 days, while at pH 5.0, the main proportion of pectin

in the emulsions prepared with un-incubated WPI-LMP was present as unadsorbed in

the serum phase. Subsequently, the pectin load at pH 5.0 (0.1834 mg/m2) was lower

than that at pH 6.5 (0.4081 mg/m2). In contrast, for the emulsions stabilized with

WPI-LMP, incubated for 8 days, the pectin load at pH 6.5 was lower than that at pH

5.0.

80

Table 3.10. Amount of pectin (mg/100ml) in the aqueous phase, serum phase and

adsorbed on the oil droplets of heated and unheated oil in water emulsion (10% w/w

sunflower oil), stabilized with 0.5% (w/w) WPI-LMP conjugates (2:1), unincubated

and incubated for 8 days, at pH 6.5 and 5.0 in the presence of 30 mM NaCl.

pH Sample Aqueous

phase

Serum Phase Cream

Unheated Heated Unheated Heated

6.5 WPI-LMP(D0) 98.7 60.4 99.0 38.3 -0.3

WPI-LMP(D8) 91.4 61.1 63.6 30.3 27.8

5.0,

NaCl

WPI-LMP(D0) 95.3 83.5 95.2 11.8 0.1

WPI-LMP(D8) 98.1 52.6 65.5 45.5 32.6

Table 3.11. Percentage of adsorbed pectin (%) on oil droplets, ɸ32 (in µm), and pectin

load (mg/m2) of heated and unheated oil in water emulsion (10% w/w sunflower oil),

stabilized with 0.5% (w/w) WPI-LMP conjugate (2:1), unincubated and incubated for

8 days, at pH 6.5 and 5.0 in the presence of 30 mM NaCl.

pH Sample

% adsorbed in cream

ɸ32

Pectin Load (mg/m2)

Unheated Heated Unheated Heated

6.5 WPI-LMP(D0) 38.8 -0.33 0.46 0.4081 0.0000

WPI-LMP(D8) 33.18 30.44 0.39 0.2966 0.2722

5.0,

NaCl

WPI-LMP(D0) 12.34 0.12 0.65 0.1834 0.0018

WPI-LMP(D8) 46.36 33.26 0.38 0.4028 0.2890

81

Dry heating of WPI and LMP under controlled temperature and relative

humidity for a certain period of time leads to the formation of covalent bonds between

WPI and LMP which can be adsorbed at the droplet surface after emulsion

preparation. In the case of WPI-LMP conjugates, incubated for 8 days, upon

emulsification pectin could adsorb at the droplet surface via WPI-LMP conjugates,

whereas in un-incubated WPI-LMP, since no dry heating was applied no conjugate

was formed between WPI and LMP, so adsorption of pectin at the droplet surface via

conjugates was not possible. Hence, pectin adsorption to the interface might be

through other interactions. Based on the theoretical consideration, pectin adsorption

onto the oil droplet surface at pH 6.5 via electrostatic interactions with proteins is less

likely since at this pH, which is above the IEP of protein, both protein and pectin,

which is an anionic polysaccharide, carry net negative charges. However, as it can be

derived from table 3.11, the pectin load in the emulsion containing un-incubated WPI-

LMP at pH 6.5 was considerable which implies that there was interaction between

pectin and protein at the interface. As reported by Dalgleish and Hollocou, [1997],

pectin bound to the droplet surface of the emulsion stabilized with sodium caseinate

even at pH values above the protein isoelectric point, at which both polymers are

negatively charged. This author relates this phenomenon to the weak repulsive forces

between negatively charged protein and pectin which cannot prevent at least some of

the pectin binds to the surface of the protein. However, Dickinson et al., [1998]

reported the opposite observation as they found no interaction between pectin and

casein at the interface of casein-based emulsions at pH 7.0 which is above the IEP of

the casein. In this case, more analysis can be done in the future to further confirm this

result.

As a further consequence, the presence of the pectin at the oil droplet surface

could also be attributed to the emulsifying properties of the pectin. Leroux et al.,

[2003] found that citrus and beet pectin can reduce the interfacial tension between oil

and water and act as an efficient emulsifier. It was reported that the emulsifying

properties of pectin could be influenced by the molecular weight, protein associated

with the pectin, and acetyl contents.

82

Furthermore, from table 3.11 it can be derived that following heating of the

emulsions prepared with unincubated WPI-LMP at 80 °C for 20 min, a substantial

reduction in the pectin load occurred, at both pH 5.0 and 6.5. It is evident that all the

pectin had been removed from the droplet surface at pH 5.0, and at pH 6.5 there is

dramatic decrease in pectin load. As from table 3.10 it is clear that after heating pectin

mainly was present as unadsorbed pectin in the serum phase of the emulsion (99.0

mg/100ml at pH 6.5, and 95.2 mg/100ml at pH 5.0) (Table 3.10).

On the other hand, in case of emulsions prepared with WPI-LMP, incubated

for 8 days, the change in the pectin load upon heating of the emulsion was not notable

at both pH 6.5, and at pH 5.0. The difference in the pectin load of the emulsions

containing WPI-LMP conjugates, unincubated and incubated for 8 days, arose from

the influence of dry heating on the interaction between WPI and LMP. As in the case

of WPI-LMP, incubated for 8 days, covalent bonds between WPI and LMP were

formed upon prolonged dry heating of the mixture of the two polymers. Subsequently,

the pectin molecules could be adsorbed to the droplet surface indirectly through the

protein to which they were covalently bound. This covalently bound pectin did not

seem to be removed from the droplet surface upon heating of the emulsions as

compared to the pectin in unincubated WPI-LMP, and correspondingly, it enhanced

steric stabilization forces between the droplets. Moreover, as the increase in the

amount of unadsorbed pectin was negligible upon heating of the emulsion, the

stability of the emulsion against depletion flocculation that intensifies heat-induced

aggregation, arising from the unadsorbed pectin, would be remarkable [Kika et al.,

2007]. On the other hand, the presence of free pectin molecules in the continuous

phase of the emulsions containing unincubated WPI-LMP reduces the emulsion

stability against depletion flocculation, and it enhanced heat-induced droplet

aggregation.

83

Chapter 4: Conclusion

Whey protein applications as food ingredients have shown a strong growth

during recent decades, due to their high nutritional and functional properties. Since

whey proteins are sensitive to heat-induced aggregation, the stability of whey proteins

during thermal processing is an essential factor in maintaining their functionality.

Several methods have been developed to improve the thermal stability of whey

proteins. Developing protein-polysaccharide complexes through covalent bonding has

been recognized as an appropriate method for improving the functionality and thermal

stability of proteins in food applications because of its safety and stability towards

environmental changes. Therefore, our study was designed to make whey protein-low

methoxylpectin conjugates by dry heating under controlled temperature and relative

humidity, and to evaluate the heat stability and emulsifying properties of the

conjugates in oil-in-water (o/w) emulsions, as a function of pH and incubation time.

Prolonged dry-heating of WPI with LMP resulted in appreciable enhancement

of its emulsifying properties, particularly at pH 5.0 which is near the IEP of the whey

proteins. At pH 5.0, emulsions containing incubated WPI-LMP conjugates were

stable against flocculation and creaming, and they retained their small droplet size and

Newtonian rheological behavior, while emulsions with WPI and dry heated WPI

exhibited flocculation, creaming and phase separation, as well as a dramatic increase

in particle size and viscosity. No improvement in emulsifying properties of emulsions

with unincubated WPI-LMP was noted, and incubation of WPI-LMP for 4, 8 and 16

days was found to have similar effects on the emulsifying properties.

The primary positive effect of this WPI conjugation with LMP was the

dramatic improvement in the thermal stability of the heated emulsions, which was the

main aim of this research. Upon heat treatment, emulsions prepared with WPI-LMP

conjugates, incubated for 4, 8, and 16 days, were found to be stable against

aggregation and flocculation, and no apparent change in the droplet size and viscosity

84

was observed at both pH 5.0 and 6.5. However, the opposite results were obtained for

emulsions prepared by WPI, or dry heated WPI. In the case of emulsions with

unincubated WPI-LMP (day 0), no aggregation at pH 5.0 in the absence of NaCl was

observed while heating in the presence of 30 mM NaCl resulted in heat-induced

aggregation. The observed trend for emulsions with WPI-LMP (day0) hints in the

direction that the mixture of freeze dried WPI and LMP can improve the heat stability

of an emulsion at pH conditions close to the IEP of WPI even without incubation due

to the influence of pectin on the rheological properties of the emulsion, whereby the

presence of salt can eliminate this stabilizing effect due to the shielding of the

electrostatic repulsion forces. As emulsions with incubated WPI-LMP conjugates

remained unchanged by heat treatment at pH 5.0 even in the presence of NaCl, it is

inferred that the incubation time is a critical factor for preparing Maillard conjugates

with improved functionality and heat stability. In our experiments, the minimum of 4

days was enough for achieving this purpose.

Possible explanations for the observed improved thermal and emulsifying

stability are increased hydrophilicity and steric stabilization forces brought about by

the polysaccharide moiety of the conjugate. Upon conjugation of WPI with LMP, the

WPI is adsorbed at the surface of oil droplets and the hydrophilic LMP portion

protruded into the aqueous medium which leads to the formation of a highly solvated

layer near the interface, which enhances steric repulsion forces between neighboring

oil droplets and retards the creaming and coalescence processes and prevents heat-

induced aggregation.

On the basis of the above-mentioned conclusions, this study accepts the

hypothesis of the thesis which was that these conjugates would exhibit improved

emulsifying activity and thermal stability compared to the WPI alone.

To conclude on the importance of the polysaccharide-protein complexes, it is

worth to mention several interesting fields of their applications in food and non-food

products. Polysaccharide-protein complexes can be used for interfacial and thermal

stabilization of oil in water emulsions (o/w) or double emulsions (w/o/w), food

product texturization and fat or meat replacement. Another area of application in food

85

products is microencapsulation to protect food bioactive molecules that are sensitive

to external stresses such as pH, temperature, etc., and to control release of aroma and

flavor. Some of the areas of non-food applications of protein-polysaccharide

conjugates include microencapsulation for the pharmaceutical industry, and

biomaterial synthesis in tissue engineering to replace the extracellular matrix system

(EMS). Furthermore, protein-polysaccharide complexes can serve as a carrier for

bioactive molecules in gene therapy such as the delivery of plasmid DNA.

In spite of the considerable potential for protein-polysaccharide complexes to

be used in different areas, such conjugates have not been industrialized yet. This is

because of the limitation in the preparation method of these conjugates, as the current

method of the dry Maillard conjugate preparation including freeze-drying followed by

equilibration to desired aw in a controlled atmosphere is not feasible on an industrial

scale due to the prolonged time needed for the Maillard reaction to occur and the cost

of the freeze-drying method. Therefore, the future direction of research in this area

would be the optimization of the reaction conditions and developing new technologies

which are more industrially practical and cost-effective.

86

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