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FREEZE -THAW STABILITY OF EGG YOLK AND WHITE-

STABILISED EMULSIONS

SITI NAQUIAH BINTI MOHD OMAR

Final Year Project Report Submitted inPartial Fulfilment of the Requirements for the

Degree of Bachelor of Science (Hons.) Food Science and Technology

in the Faculty of Applied Sciences

Universiti Teknologi MARA

JANUARY 2012

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This Final Year Project entitled Freeze-Thaw Stability of Egg Yolk and White-

Stabilised Emulsions was submitted by Siti Naquiah Binti Mohd Omar, in partial

fulfilment of the requirements for the Degree of Bachelor of Science (Hons.) FoodScience and Technology, in the Faculty of Applied Sciences and was approved by

______________________________________

Dr. Anida bt Yusoff

Supervisor

B. Sc. (Hons.) Food Science and TechnologyFaculty of Applied Sciences

Universiti Teknologi MARA

40450 Shah Alam

Selangor

_____________________________ _____________________________

Dr. Anida Binti Yusoff Assoc. Prof. Dr. Noorlaila Ahmad

Project Coordinator Programme CoordinatorB.Sc.(Hons) Food Science and B.Sc.(Hons) Food Science and

Technology Technology

Faculty of Applied Sciences Faculty of Applied Sciences

Universiti Teknologi MARA Universiti Teknologi MARA

40450 Shah Alam 40450 Shah AlamSelangor Selangor

Date: __________________

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ACKNOWLEDGEMENTS

In the name of Allah, The Most Merciful and The Most Gracious.

I begin in the name of Allah S.W.T, who sent the Prophets for guidance of all

Mankind. Billion of blessing in upon the last Prophet and the seal of Prophets. First of

all, I would like to express my gratitude to all those who gave me the possibility to

complete this final year project. I am deeply indebted to my supervisor Dr. Anida

Yusoff whose help, inspiring suggestions and encouragement to help me in all the time

of my study and to ensure this final year project completed successfully.

My thanks and appreciation goes also to Mrs. Norahiza Abdul Soheh and Mrs. Siti

Marhani Mardi, the laboratory staffs of Food Science and Technology for their

concern, encouragement and serving on my final year project.

It is difficult to overstate my gratitude to all my friends from semester 7 of Bachelor of

Science (Hons.) Food Science and Technology who had supported towards the success

ofthis study. I wish to extend my warmest thanks to all of them.

Finally, I owe my loving thanks to my family. Without their love, encouragement and

understanding it would have been impossible for me to finish this work. My deepest

gratitude goes to my parents, for their indefatigable love and support throughout my

life. Lastly my thanks go also to everyone for their direct and indirect assistance and

helpful discussion during my work. Thank you very much. Only Allah who can repay

all the kindness.

May Allah bless all of you, Amin.

Thank you very much.

Siti Naquiah Binti Mohd Omar.

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TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF TABLES vi

LIST OF FIGURES vii

LIST OF ABBREVIATIONS ix

ABSTRACT x

ABSTRAK xi

CHAPTER 1 INTRODUCTION1.1 Background and problem statement 1

1.2 Significance of study 3

1.3 Objectives of study 3

CHAPTER 2 LITERATURE REVIEW2.1 Emulsion 4

2.2 Emulsifier 4

2.3 Types of instability in emulsion 5

2.3.1 Coalescence 5

2.3.2 Flocculation 6

2.3.3 Creaming 6

2.3.4 Sedimentation 7

2.3.5 Oswald Ripening 7

2.3.6 Phase inversion 82.4 Egg yolk 8

2.5 Egg white 9

2.6 Palm oil 10

2.7 Freeze-thaw 11

CHAPTER 3 METHODOLOGY3.1 Materials 12

3.2 Methods 12

3.2.1 Preparation of emulsion 12

3.2.2 Freeze-thaw of egg yolk and egg white- stabilised emulsion 133.2.3 Determination of droplet size in emulsion 13

3.2.4 Determination of creaming stability 13

3.2.5 Determination of surface tension in egg yolk and egg white solution 14

3.2.6 Determination of rheology of emulsion 15

3.2.7 Determination of viscosity of emulsion 15

3.3 Statistical Analysis 16

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CHAPTER 4 RESULTS AND DISCUSSION4.1 Stability of emulsion at room and chilled temperature 17

4.2 Droplet size distribution of egg white and egg yolk-stabilised

emulsion 22

4.3 The rheology of emulsion 28

4.4 The viscosity of emulsion 324.5 Surface tension in egg yolk and egg white solution 34

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 36

CITED REFERENCES 38

APPENDICES 43

CURRICULUM VITAE 57

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LIST OF TABLES

Table Page

2.1 Density of Palm Oil at different temperature 10

4.1 Surface weight mean (D32) of egg white stabilised emulsion

at Day 0 and Day 7 23

4.2 Volume weight mean (D43) of egg white stabilised emulsion


4.3 Surface weight mean (D32) of egg yolk stabilised emulsion


4.4 Volume weight mean (D43) of egg yolk stabilised emulsion


4.5 The viscosity of egg yolk-stabilised emulsion 32

4.6 The viscosity of egg white-stabilised emulsion 33

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LIST OF FIGURES

Figure Page

2.1 Coalescence 5

2.2 Flocculation 6

2.3 Creaming 6

2.4 Sedimentation 7

2.5 Oswald Ripening 7

2.6 Phase Inversion 8

3.1 Wilhelmy Plate Method 154.1 The creaming index of 1% and 3 % concentration of egg yolk

stabilised emulsion with ratio (20:80) and (30:70) at room

and chilled temperature (4C) versus days of storage 18

4.2 The creaming index of 1% and 3% concentration of egg white

stabilised emulsion with ratio (20:80) at room and chilled

temperature (4C) versus days of storage 21

4.3 The droplet size distribution of 1% and 3% concentration of

egg white stabilised emulsion(Day 0) with ratio (20:80) versus

volume 23


egg white stabilised emulsion(Day 7) with ratio (20:80) versus

volume 23


egg yolk stabilised emulsion(Day 0) with ratio (20:80) versus

volume 25

4.6 The droplet size distribution of 1% and 3% concentration ofegg yolk stabilised emulsion(Day 7) with ratio (20:80) versus

volume 25



volume 26



volume 26

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4.9 Graph of G(storage modulus) and G(loss modulus) in the

same ratio (20:80)at different concentration of egg yolk

(1% and 3 %) at different freeze-thaw cycle versus time (s) 28

4.10 Graph of G (storage modulus) and G (loss modulus) in thesame ratio (30:70) at different concentration of egg yolk

(1% and 3 %) at different freeze-thaw cycle versus time (s) 30

4.11 Graph of G (storage modulus) and G (loss modulus) in the

same ratio (20:80) at different concentration of egg white

(1% and 3 %) versus time (s) 31

4.12 The surface tension versus concentration of egg yolk and egg white 35

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LIST OF ABBREVIATIONS

CI : Creaming Index

D32 : The Sauter mean (surface-weighted)

D43 : De Brouckere (volume-weighted)

HDL : High density lipoproteins

IR : Refractive Index

LDL : Low density lipoproteins

NaCl : Sodium chloride

O/W : Oil- in-water emulsion

SDS : Sodium dodecylsulphate

W/O : Water- in-oil emulsion

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ABSTRACT

FREEZE- THAW STABILITY OF EGG YOLK AND WHITE -

STABILISED EMULSIONS

The objective of this study was to determine the freeze-thaw stability of egg

yolk and white-stabilised emulsion by measuring the rheology and viscosity of

the emulsion after each freeze-thaw cycle up to three cycles. Comparison was

also carried out for the creaming index between the emulsion stored at room

(25C) and chill (4C) temperature. Besides that, the droplet size distribution

also conducted between the egg yolk and egg white stabilised emulsion at

different concentration and ratio of oil to water. Finally, the surface tension of

egg yolk and egg white solution was also determined in this study. In the

analysis of rheology, all the emulsions after the freeze-thaw cycles showed that

the emulsions behave like solid-like. The viscosity of emulsions showed an

increased as the concentration of egg yolk and egg white increase from 1% to

3%. But due to the freeze-thaw cycle process, the viscosity for egg yolk 1%

(20:80), 1% (30:70) and 3% (30:70) become decreased. The creaming stability

was determined by measuring the creaming index.The creaming index becomes

higher as the concentration of egg yolk and egg white were higher (3%) and theratio of oil to water was 20:80. Besides, the stability of emulsion was better if

keep at chill (4C) temperature compare to room (25C) temperature. The

droplet size distribution were bigger because of the less concentration (1%) of

egg yolk and egg white. The livetins and lipoprotein in egg yolk and ovalbumin

in egg white can altered the surface tension in egg yolk and egg white solution.

With the higher concentration of egg yolk and egg white, the surface tension

become lowered and the surface activity become increased.

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ABSTRAK

KESTABILAN PEMBEKUAN-NYAHBEKU KE ATAS EMULSI YANG

DISTABILKAN OLEH KUNING DAN PUTIH TELUR

Tujuan kajian ini adalah untuk menentukan kestabilan emulsi yang distabilkan

oleh kuning dan putih telur dengan mengukur reologi dan kelikatan emulsi

selepas setiap kitaran pembekuan-nyahbeku sehingga tiga kali kitaran.

Perbandingan turut dilakukan untuk indeks krim di antara emulsi yang disimpan

pada suhu bilik (25C) dan suhu dingin (4C). Selain daripada itu, taburan saiz

minyak juga dilakukan ke atas emulsi yang distabilkan oleh kuning dan putih

telur yang berbeza pada kepekatan dan nisbah minyak kepada air. Akhir sekali,

ketegangan pada permukaan larutan kuning dan putih telur juga ditentukan.

Dalam analisis untuk reologi, semua emulsi menunjukkan sifat seperti pepejal

selepas proses pembekuan-nyahbeku dilakukan. Kelikatan pada emulsi meningkat

apabila kepekatan kuning telur dan kuning putih meningkat daripada 1% kepada

3%. Tetapi oleh kerana disebabkan oleh pembekuan-nyahbeku proses, kelikatan

untuk kuning telur 1% (20:80), 1% (30:70) and 3% (30:70) semakin menurun.

Kestabilan krim ditentukan dengan mengukur indeks krim. Indeks krim semakin

tinggi apabila kepekatan kuning telur dan putih telur tinggi (3%) dan nisbahminyak kepada air adalah 20:80. Selain itu juga, kestabilan emulsi adalah lebih

baik jika disimpan pada suhu dingin (4C) jika dibandingkan dengan suhu bilik

(25C). Taburan saiz minyak adalah besar disebabkan oleh kurang kepekatan

(1%) kuning telur dan putih telur. Livetindan lipoproteindalam kuning telur

dan ovalbumindalam putih telur boleh mengubah ketegangan pada permukaan

larutan kuning telur dan putih telur. Dengan kepekatan kuning dan putih telur

yang tinggi, semakin rendah ketegangan permukaan dan aktiviti pada permukaan

juga semakin menurun.

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CHAPTER 1

INTRODUCTION

1.1 Background and problem statementMost of the food systems that exist in emulsion form consist of two

immiscible liquids (usually oil and water). One of the liquids dispersed in the

form of small spherical droplets. The emulsion system is typically stabilised

by several types of surface active components mainly proteins and surfactants.

The protein and surfactant will stabilise the dispersion of oil in the continuous

phase of emulsion. The surface-active of protein and surfactant are adsorbed at

the interface between oil and the aqueous phase to lower surface tension and

prevent oil droplets from coming close enough to aggregate.

In this study, emulsion will be form by using egg yolk and palm oil. Egg yolk

contains about 52.3% water, 31.8-35.5% lipids and 15.7-16.6% proteins. The

egg yolk lipid fraction contains approximately 66% triacylglycerol, 28%

phospholipids, 5% cholesterol and minor amount of other lipids (Powrie and

Nakai, 1985).Cunningham and Varadarajula (1973) reviewed the factors that

affecting emulsifying characteristic of hen egg yolk. The emulsifyingcomponents that are responsible in egg yolk are phospholipids, lipoproteins,

and proteins (livetin and phosvitin). It was reported that lipoproteins in egg

yolk are the most important substances but the emulsifying efficiency of

lipoproteins was reduced by addition of phospholipids (Cunningham, 1975).

Emulsifying capacity and heat stability in mayonnaise was improved by

preparing the emulsion with pancreatic phospholipase fermented egg yolk

(Dultilh and Groger, 1981).

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Eggs comprise egg albumin (egg white) that makes up about 2/3 of the total

mass of the egg and the rest is the egg yolk. Egg albumin contains great

number of different types of protein. Protein types such as ovalbumin,

ovotransferrin, ovomucoid, globulins, lysozyme, ovomucin, avidin and some

others are present in the white of an egg. These proteins are responsible for

different functions such as nourishment, blocking digestive enzymes, binding

iron, binds vitamins and many such functions. Although the egg white is

highly appreciated for its gelation and foaming properties, its emulsifying

potential is generally considered rather poor compared to that of yolk.

Palm oil is obtained from the palm mesocarp of the palm fruit. Palm oil is one

of the worlds most widely consumed edible oils, next only to soybean.

According to Robbelenet al. (1989) Malaysia is one of the largest producers of

palm oil and being the chief exporting country. The oil is mostly used as

shortening, margarine, vanaspati (hydrogenated fat) and frying fat. Because of

the palm oil is vegetable oil, therefore it is non-exhaustive and renewable.

Many studies had been done for the preparation of emulsion with palm oil. In

an emulsion, the large surface area that the particulate system offers in

enhanced interaction at the oil/water/substrate interface. Furthermore, palm oil

emulsion does not require any organic solvents in its preparation. An

important concern when employing palm oil emulsions in any processing is

their dispersion stability. The emulsion must be able to remain dispersed,

unaggregated, and resistant to creaming within the time frame needed (Ahmad

et al., 1996).

This main focus of this study is to investigate the effect of freeze-thaw process

on the stability of egg yolk and white stabilised emulsion. Freezethaw

process involve in temperature fluctuation or abuse that occur during

transportation, storage or consumption of oil-in-water (O/W) emulsions-based

product. Since O/W emulsions are thermodynamically unstable, the freeze-

thaw process can directly found to be detrimental to overall physicochemical

and textural quality of O/W emulsion (Srinivasanet al., 1997).

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CHAPTER 2

LITERATURE REVIEW

2.1 Emulsion

An emulsion consists of two immiscible liquids (usually oil and water) with

one of the liquids dispersed in the form of small spherical droplets in the other

liquids. Typically in most food emulsion, the diameters of the droplets are in

the range of 0.1 to 100 m (Dickinson, 1992). Emulsions can be classified

according to the distribution of the oil and the aqueous phase. A system which

consists of oil droplets dispersed in aqueous phase is called oil-in-water (O/W)

emulsion. Examples of food product that can be classified in this category of

emulsion are mayonnaise, milk, cream, soups and sauces. While for a system

which consists of water droplets dispersed in oil phase is called water-in-oil(W/O) emulsion. The food products under this emulsion are margarine, butter

and spreads. The substance that makes up the droplets in an emulsion is

referred to as the dispersed phase or internal phase. The continuous or external

phase is the substance that makes up the surrounding liquid (McClements,

1999).

2.2 Emulsifier

Emulsifiers are surface active molecules which absorb to the surface of formed

oil droplets during homogenisation. It can form a protective membrane which

prevents the droplets from coming close enough together to aggregate

(McClements, 1999). The emulsifier is an effective molecule that can promote

the formation and stabilisation of an emulsion (Hasenhuettl and Hartel, 2008).

The most important role of the emulsifiers during the homogenisation is to

decrease the interfacial tension between the oil and water phase, thereby

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reducing the amount of energy required to deform and disrupt the droplets.

Besides, they can form a protective coating around the droplets which prevents

them from coalescing with each other.

A surfactant is also a surface active molecule. It adsorbs to the surface of

emulsion droplets during homogenisation and form a protective membrane

which prevents the droplets from aggregating with each other during collision

(Walstra, 1993).Surfactant molecules adsorb to oil-water interfaces because

they can adopt an orientation in which the hydrophilic part of the molecule is

located in the water while the hydrophobic part is located in the oil. Thus the

contact area between hydrophilic and hydrophobic regions will be minimised.

The interfacial tension also can be reduced.

2.3 Types of instability in emulsion

2.3.1 Coalescence

Figure 2.1 Coalescence

In emulsion system, it consists of large amount of small droplets. Droplet size

and droplet size distribution has significant effects on the stability and texture

of final products (Dickinson, 1992). Coalescence is the process whereby two

or more liquid droplets merge together to form a single larger droplet. It is the

principal mechanism by which an emulsion moves toward its most

thermodynamically stable state because it involves a decrease in the contact

area between the oil and water phase. Coalescence can causes emulsion

droplets to cream or sediment more rapidly because of the increase in their

size. In O/W emulsions, coalescence eventually leads to the formation of a

layer of oil on top of the material, which referred to as oiling off. While in

W/O emulsions, it leads to the accumulation of water at the bottom of the

material (McClements, 1999).

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2.3.2 Flocculation

Figure 2.2 Flocculation

Flocculation is the process whereby two or more droplets come together to

form an aggregate in which the droplets retain their individual integrity.

Roland et al.,(2003) reported that flocculation is when two droplets become

attached to each other but are still separated by a thin film of liquid. When

more droplets are involved, an aggregate is formed, in which the individual

droplets cluster together but retain the thin liquids films between them. The

emulsifier molecules remain at the surface of the individual droplets during the

process. Droplet flocculation may either advantageous or detrimental to

emulsion quality depending on the nature of the food product. Flocculation

accelerates the rate of gravitational separation in dilute emulsions which is

undesirable because it reduces their shelf life (Luytenet al., 1993). In addition,

flocculation caused a pronounced increase in emulsion viscosity and lead to

formation of a gel (Demetriadeset al., 1997).

2.3.3 Creaming

Figure 2.3 Creaming

Creaming and sedimentation are both forms of gravitational separation.

Creaming can be describes as the upward movement of droplets due to the fact

that they have a lower density than the surrounding liquid (McClements,

1999).

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2.3.4 Sedimentation

Figure 2.4 Sedimentatiom

McClement (1999) reported that sedimentation is the reverse version of

creaming. It is the process where the droplets move downward as a result of

the fact that they have a higher density than the surrounding liquid.

2.3.5 Oswald Ripening

Figure 2.5 Oswald Ripening

The process whereby large droplets grow at the expense of smaller ones

because of mass transport of dispersed phase from one droplet to another

through the intervening continuous phase (Kabalnov and Shchukin, 1992). In

most food emulsions, it is negligible because the mutual solubility of

triaylglycerols and water are so low that the mass transport rate is significant

(Dickinson and Stainsby, 1982). However, it may be significant in O/W

emulsions which contain more water-soluble lipids.

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2.3.6 Phase inversion

Figure 2.6 Phase Inversion

It is the process whereby a system changes from O/W emulsion to a W/O

emulsion or vice versa. Phase inversion is usually influence by some alteration

in the composition or environmental conditions of an emulsion for example

dispersedphase volume fraction, emulsifier type, emulsifier concentration,

solvent conditions, temperature and mechanical agitation (Shinoda and

Friberg, 1986 ;Dickinson, 1992; Campbell et al.,1996).

2.4 Egg yolkHen egg yolk is commonly used in emulsion industry to form and stabilise

emulsions. For that reason, it has become a main ingredient in products such

as mayonnaise or salad dressing. Egg yolk is good emulsifiers and rated as

four times as effective as egg white as an emulsifiers. When focussing on the

emulsifiers in particular, egg yolk appears as a mixture of several proteins and

lipoproteins which include apoproteins and phospholipids. Egg yolk can be

fractioned into plasma and granules by a mild centrifugation without causing

any denaturation (McBee and Cotteril, 1979). The supernatant (plasma) which

represents 75-81% of the yolk dry matter, accounts for 52-58% of the proteins

and for 85% of the phospholipids. The precipitated granules which makes the

remaining 19-25% of the yolk dry matter, account for 42-48% of the proteins

and for 15% of the phospholipids (Anton and Gandemer, 1997; Dyer-Hurdon

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and Nnanna,1993; Causeretet al., 1991). Plasma is composed of 85% low

density lipoproteins (LDL) and 15% livetins. Granules are made of 70% high-

density lipoproteins (HDL), 16% phostivin and 12% LDL (McCullyet al.,

1962).

Several studies had been done on the emulsifying properties of egg yolk

constituents. Emulsions prepared with egg yolk matched closely those made

with plasma whereas emulsions prepared with granules exhibited distinct

properties(Anton, 1998; Anton and Gandemer, 1997; Dyer-Hurdon and

Nnanna, 1993). This suggests that the main contributors to yolk emulsifying

properties belong to plasma. Since the major composition in plasma is LDL

then it is considered to be the most important contributor to the emulsifying

properties of egg yolk (Mizutani and Nakamura, 1984).

2.5 Egg White

Egg white is a mixture of proteins that possess excellent foaming properties,

since each of its components performs a specific function. Globulins facilitate

foam formation, while the ovomucin-lysozyme complex (Cotterill and Winter,1955) confers stability to the foam, andboth ovalbumin and conalbumin are

heat-related (Yang and Baldwin, 1995). Conalbumin, lysozyme, ovomucin,

and ovomucoid alone have little or no foaming ability, but the interaction

between lysozyme and globulin is important to foam formation (Johnson and

Zabik, 1981). Ovalbumin is the largest protein in egg white and it contributes

to foam stability and formation properties in food systems where eggs are

utilised as an ingredient (Relkin et al., 1999; Hagolle et al., 2000; Du et.al.,

2002).Additionally, as Kiosseoglou and Sherman (1983) reported, the

presence of white in admixture with yolk in mayonnaise emulsions resulted in

a decrease of droplet size and a strengthening of emulsion structure,

suggesting that the white proteins might have somehow become involved in

the emulsification process.The emulsions prepared with egg white were more

strongly flocculated and has phase seperated systems than those of egg yolk

(Drakos and Kiosseoglou, 2006). Some changes in structural properties of egg

white protein may lead to changes in foaming, emulsifying and gelling

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abilities (Mine, 1996; Campbell et al., 2005; Song et al., 2009). These

functional properties play an important role in the manufacture of bakery

products. The foaming ability is related to the rate at which the surface tension

of the air/water interface decreases.

2.6 Palm Oil

Palm oil is one of the important sources of edible oil and most people in

Malaysia used in their daily cooking. It contains a lot of vitamins A and E and

has the colour ranges from pale yellow to deep orange. In palm oil, the

proportions of saturated and unsaturated fatty acids are almost equivalent.

Palmitic acid and oleic acid are the major fatty acids while linoleic acid and

stearic acid are put in to the minor fatty acids. Besides has a pleasant odour

and taste, it is also stable and resistant to rancidity. Furthermore, palm oil is

solid at ambient temperature in temperate climates and fluid in tropical and

subtropical climates with certain fractions held in crystalline form. When

cooled to 5-7 C, the separation of liquid and solid fractions can be clearly

seen. The iodine value of palm oil is lower (44-58) than the other vegetable

oils because of high proportion of saturated fatty acids. The saponificationvalue of palm oil is higher (195-205) than the other edible vegetable oils

(Salunkheet al.,1992).The density of palm oil differs with temperature is

shown by Table 2.1.

Table 2.1Density of Palm Oil at different temperature

Temperature ( C) Density of palm oil (kg / m )

50 890

75 870

100 860

200 789

Source: Gupta et al.(2008).

From Table 2.1, it is apparent that there was a significant different of density

of palm oil at different temperature. The higher the temperature, the density of

palm oil decreased.

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2.7 Freezethaw

There are many potential applications for O/W emulsions that can be frozen

and then thawed prior to use, e.g. refrigerated and frozen food or

pharmaceutical products. The O/W emulsions are thermodynamically unstable

systems and sensitive to environmental changes, as for example cooling and

freezing (Guzey and McClements, 2006). Many O/W emulsion-based

processed foods (e.g., ice-cream, mayonnaise, milk, cream soups and sauces)

are frozen to improve their long-term storage before thawing for further

processing or consumption. When an O/W emulsion is stored at low

temperature, crystallisation of both the oil and the water phase may occur.

These phase transition may lead to the destabilization of the emulsion.

Whereas thawing process plays an important role in membrane disintegration

as well as affecting sensory attributes of the O/W emulsions product (Nilsson

and Ekstrand, 1995).

The stability of all the food emulsion-based products to environmental stresses

such as heating, drying and freezing is strongly influenced by the

characteristics of the interfacial membranes that surround the oil droplets.

Consequently, food scientists can improve the stability of emulsion-based food

products to environmental stresses by engineering the properties of the

interfacial membranes, e.g., composition, electrical charge, thickness and

rheology. Because of most of O/W emulsions are highly unstable when they

are frozen and rapidly breakdown after thawing. When an O/W emulsion is

cooled, a variety of physicochemical processes can occur including fat

crystallisation, ice formation, freeze-concentration, interfacial phase

transitions and biopolymer conformational changes (McClements, 2004).

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CHAPTER 3

METHODOLOGY

3.1 Materials

Fresh eggs and palm oil were purchased from local supermarket. Eggs were

manually broken and egg white was separated from the egg yolk. The egg yolk

was rolled carefully on a filter paper (Whatman No. 4) to remove adhering

fragments of the egg white in order to obtain a pure and unspoiled egg yolk.

Afterwards the membrane of egg yolk was punctured and the liquid egg yolk

was allowed to flow in suitable container. The egg white was put on the other

container separately.

3.2 Methods

3.2.1 Preparation of emulsion

Oil-in-water emulsions using egg yolk and egg white were prepared with two

concentrations which are at 1% and 3% (w/v). The preparation of 1%

concentration of egg yolk and egg white were prepared by diluting 1g of egg

yolk and egg white separately in 100 ml of distilled water and stirred well.

Two different ratios of oil: water with 20:80 and 30:70 were used. For ratio

20:80, 20 ml of palm oil was poured into a beaker and 80 ml of the egg yolk

solution prepared previously was added. Then the emulsions were then

homogenised by using an Ultra Turrax T25 homogenizer equipped with a

S25KG-25F dispersing tool and run at 22000 rpm. The emulsions were

homogenised for 15 minutes at speed 3. The preparation was same for 3% and

ratio 30:70. For egg white, the ratio of oil to water was only 20:80. All the

preparations were conducted at room temperature.

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3.2.2 Freeze-thaw of egg yolk and egg whitestabilised emulsion

Freeze-thaw treatments of O/W emulsions prepared with egg yolk and egg

white were performed immediately after the preparation. Twenty ml of sample

was transferred to a glass test tubes covered, sealed and stored for 2 hours at

chilled temperature (4C) in refrigerator. After storage, the emulsions were

then thawed at room temperature (28C- 30C) for 2 hours. This chilling and

thawing cycle was repeated for three cycles. After each cycles, the rheology

and viscosity of emulsions was then analysed.

3.2.3 Determination of droplet size in emulsion

The droplet size distribution was measured by using a laser diffraction method

(Malvern MasterSizer 2000). The refractive index (IR) of the palm oil and

water at 25 C are 1.4652 and 1.336, respectively. All these IRs were used in

calculations. From droplet size distributions, the Sauter mean (surface-

weighted, D32) and De Brouckere (volume-weighted, D43) mean diameters

were obtained. Both values were expressed in micrometers (m).Triplicate

measurements were carried out and the results were presented in the mean ofthese triplicate. The measurement was took at day 0 (fresh emulsion) and at

day 7 (storage emulsion).

3.2.4 Determination of creaming stability

Creaming stability is to evaluate the relative stability of the emulsions

immediately after they were prepared. The emulsion samples were poured in

cylinder (diameter 15 mm) at 25 C. The cylinders were sealed to prevent

evaporation. All the emulsions were kept at room temperature and chilled

temperature and the movement of any creaming boundary was tracked visually

every two days with time over eight days. The emulsions separated into a top

cream layer and a bottom serum layer. The total emulsion height (HT) and

serum layer height (HS) were measured. A creaming index (CI) was calculated

as below (Keownmaneechai and McClements, 2002).

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