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FOODHYDROCOLLOIDS

Food Hydrocolloids 20 (2006) 11141123

Impact of a thermal treatment on the emulsifying properties of egg yolk.

Part 2: Effect of the environmental conditions

Fabien Guilmineau, Ulrich Kulozik

Technische Universitat Munchen, Chair for Food Process Engineering and Dairy Technology, Weihenstephaner Berg 1, 85354 Freising, Germany

Received 25 July 2005; accepted 8 December 2005

Abstract

The protein solubility and emulsifying properties of native and heat-treated egg yolk (EY) suspensions were investigated in various

environmental conditions. Four distinct conditions were tested by combining two levels of pH, namely pH 4.0 and 6.5, and two levels of

ionic strength, namely 0.15 and 0.52 M NaCl, in a model oil-in-water (O/W) emulsion containing 30% oil (v/v). Although the protein

solubility was greatly reduced by the thermal denaturation in all tested environmental conditions, the average size of oil droplets

obtained in emulsions made with heated EY was observed to be either similar or slightly smaller than that obtained with native EY,

depending on the environmental conditions. Using heat-treated EY rather than native EY led to a significant increase of the interfacial

protein concentration in all environmental conditions. This increased interfacial protein concentration was shown to have a major impact

on the flocculation behaviour of the emulsions, as well as on their rheological properties and stability to creaming. Hypotheses regarding

the mechanisms by which insoluble protein aggregates stabilise O/W emulsions at various pH and ionic strengths are discussed.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Egg yolk; Protein denaturation; Emulsifying properties; Environment

1. Introduction

Egg yolk (EY) is a reference food emulsifier used in

many applications ranging from bakery products to salad

dressings. Commercial EY is routinely pasteurised at

temperatures between 60 and 68 1C in order to ensure its

microbiological safety (Cunningham, 1995). Applying

more severe thermal treatment conditions would allow to

extend the shelf life of EY and further reduce the risk of

microbial spoilage. But the impact of an increased degree

of thermal denaturation of the heat sensitive EY proteins

on the emulsifying properties of this key functionalingredient is not well understood. In the first part of this

work we have used dilute EY suspensions to study the

impact of the heat treatment intensity on the emulsifying

properties of EY (Guilmineau&Kulozik, submitted). This

work has notably shown that the interfacial concentration

of proteins at the O/W interface was significantly increased

with an increasing degree of protein denaturation. This was

shown to allow a significant decrease of the flocculation in

O/W emulsions made with denatured EY, and significantly

impacted the rheological properties and stability of the

emulsions obtained. This work was, however, carried out

on a model emulsion using constant environmental

conditions, namely a pH of 6.5 and an ionic strength (IS)

corresponding to 0.52 M NaCl. But the many applications

of EY in industrial food products cover a much wider

range of pH and ionic strengths, varying with the

formulation of the products. The environmental conditionshave been shown to have a significant impact on the

physico-chemical and emulsifying properties of EY and its

components (Aluko & Mine, 1998; Anton & Gandemer,

1999;Le Denmat, Anton, &Beaumal, 2000; Mine, 1998).

Fresh EY has a pH of about 6.2 and an IS of about 0.17 M

NaCl (Anton, 1998). In these natural conditions, EY is

constituted of a continuous aqueous phase containing

about 80% of the total dry matter and referred to as

plasma, and insoluble denser structures with a size ranging

from 0.3 to 2 mm referred to as granules. EY plasma

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0268-005X/$ - see front matterr 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodhyd.2005.12.006

Corresponding author. Tel.: +49(0)8161 71 3535;

fax: +49(0)8161 71 4384.

E-mail address: [email protected] (U. Kulozik).
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contains globular glycoproteins known as a-, b-, and g-

livetins, as well as the low-density lipoproteins (LDL),

which apoproteins are called lipovitellenins. Granules

contain a phosphoprotein known as phosvitin, as well as

the high-density lipoproteins (HDL), which apoproteins

are called lipovitellins (Burley & Vadehra, 1989). Upon

increase of the IS to values above 0.3 M, granules dissociatebecause of the rupture of the phosphocalcic bridges

between lipovitellins and phosvitins (Causeret, Matringe,

& Lorient, 1991). This is a major change of the EY

microstructure driven by the environmental conditions.

Partial disruption of EY granules by mechanical shear

forces has also been observed, leading to the formation of

granule fragments, notably during high pressure homo-

genisation (Anton, Beaumal, & Gandemer, 2000). Most

salad dressings are prepared at a pH around 4.0 by

addition of acetic and/or citric acid, which contributes to

the products characteristic taste and inhibits growth of

most microorganisms (Ford, Borwankar, Pechak, &

Schwimmer, 2004). This makes this value of low pH a

particularly relevant one when it gets to studying the

behaviour of EY in model food emulsions.

In this work, model oil-in-water (O/W) emulsions

containing 30% (v/v) sunflower oil were prepared within

a range of pH and IS most commonly occurring in food

emulsions. The impact of two levels of pH, namely pH 4.0

and 6.5, and two levels of IS, namely 0.15 and 0.55 M

NaCl, on the emulsifying properties of EY was studied.

The objective of this work is to compare the behaviour of

non-heated (native) EY to that of heat-treated EY in the

various environmental conditions defined above. The level

of protein denaturation used for the heat-treated EY washigher than that normally reached with industrial EY

pasteurisation, and was chosen based on the results

obtained in the first part of this study (Guilmineau &

Kulozik, submitted). The solubility of EY proteins as well

as the key characteristics of the emulsions such as particle

size distribution, flocculation and interfacial protein con-

centration were investigated. Hypothesis regarding the

colloidal interactions taking place between oil droplets in

the various environmental conditions studied are discussed.

2. Material and methods

2.1. Preparation and heating of an EY suspension

Freshly laid eggs from Lohman Tradition hens were

collected from the Universitys research farm (Thalhausen)

and used within 48h after collection. A suspension

containing 20% (w/w) pure EY in an isotonic NaCl

solution (i.e. 0.17 M NaCl) is prepared as described by

Guilmineau and Kulozik (submitted): it is referred to as the

native EY20.

2.1.1. Thermal treatment of the EY20 suspension

Native EY20 was heated as described by Guilmineau

and Kulozik (submitted) with a holding time of 12 min at

74 1C: we obtained the heated EY20. Because of the

dilution of the native EY prior to heating, the heated

suspension remained fluid despite the intense heat treat-

ment. Heated EY could therefore be used just as easily as

native EY to prepare O/W emulsions.

2.2. Preparation of emulsions

2.2.1. Preparation of the continuous phase

The continuous phase was prepared by diluting EY20

(native or heated) so as to obtain a concentration of 1.12%

(w/w) EY dry matter. Dilutions were made either in an

aqueous NaCl solution (not buffered) or in a 0.01M

acetate buffer (Sodium acetate trihydrate/acetic acid) at

pH 4.0, containing NaCl so as to obtain a concentration

of either 0.15 or 0.52 M in the final continuous phase.

The continuous phases prepared without buffer were

measured to have an average pH of 6.4970.04 and the

ones made with the acetate buffer had an average pH of

4.0370.05.

All continuous phases were allowed to equilibrate under

mild agitation (magnetic stirrer) for 30 min before the

production of the emulsion. Commercial refined sunflower

oil was used to prepare the emulsions (Cereol Deutschland

GmbH, Mannheim, Germany). A pre-emulsion with a

dispersed phase volume fraction j 0:3 was prepared by

pouring 180 ml of sunflower oil in 420 ml continuous phase

while mixing with a dispersing system model Ultra Turrax

T25 (IKA Werke, Staufen, Germany) equipped with an

18 mm diameter toothed-disc dispersing tool (model

S25KR-18G) at 8000 rpm. The oil was added over the

course of 1 min and the agitation was maintained for afurther minute in order to obtain a homogeneous pre-

emulsion. The median oil droplet diameter in the pre-

emulsion was measured to be about d50,31316mm

throughout the trial period. The pre-emulsion was then

further emulsified by passing it once through the first stage

of a high-pressure homogeniser model APV 1000 (Invensys

APV, Albertslund, Denmark) at a pressure of 200 bars and

a temperature of 25 1C.

2.3. Characterisation of the EY suspension and emulsions

2.3.1. Protein solubility

The solubility of proteins in the EY samples was assessed

using the method published by Morr et al. (1985)with the

following modification. Samples of each of the four

continuous phases prepared as described in Section 2.2

(two pH and IS levels) were equilibrated under mild

agitation at 20 1C for 1 h. An aliquote of each suspension

was kept for total protein content measurement (Pt), and

the rest was centrifuged twice at 19,000gfor 20 min in order

to separate the insoluble proteins. An aliquote of the

supernatant containing the soluble proteins was also

analysed for protein content (Ps). The protein content

was determined using the colorimetric method of Mark-

well, Hass, Bieber, and Tolbert (1978). The percentage of

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solubility was determined as given in Eq. (1) below.

Solubility% Ps

Pt 100. (1)

2.3.2. Particle size distribution and flocculation

measurementAll particle size measurements were carried out using a

laser diffraction spectroscope model LS230 (Beckman-

Coulter, Germany). Details of the method used are given

byGuilmineau and Kulozik (submitted).

2.3.3. Stability to creaming

The stability to creaming is measured by following the

formation of a clear droplet-free phase at the bottom of

a sample of emulsion, using a light scattering optical

analyser model Turbiscan MA1000 (Formulaction,

France). All details regarding the determination of the

initial creaming rate and relative height of the cream layer

formed during 1 week of storage at 10 1C are given byGuilmineau and Kulozik (submitted).

2.3.4. Rheological characterisation of the emulsions

A controlled shear rate rheometer model Rheomat 115

equipped with a double-gap geometry model MS 0/115

(Contraves GmbH, Stuttgart, Germany) was used. The

details of the method used are given by Guilmineau and

Kulozik (submitted).

2.3.5. Interfacial protein concentration

The method used in this study to separate the oil droplets

from the continuous phase of the emulsions was adaptedfromPatton and Huston (1986). All details of the method

used are described by Guilmineau and Kulozik (sub-

mitted).

2.3.6. Zeta-potential measurements

Zeta potential was measured by a laser light-scattering

technique using a Zetasizer Nano-Z model ZEN 2600

(Malvern Instruments, Herrenberg, Germany). The drift

velocity of oil droplets placed in an electric field is

accurately determined from the Doppler shift of the light

scattered by the moving droplets. The emulsions were

diluted 10,000 fold (v/v) in a buffer solution corresponding

to their continuous phase (same pH and NaCl concentra-

tion). All measurements were carried out at a constant

temperature of 25 1C.

2.3.7. Statistical analysis

Three replicates were carried out on three different

weeks. Oil droplet size distribution, flocculation, rheologi-

cal properties, stability, interfacial protein concentration

and zeta-potential were measured each time twice. These

parameters were subjected to a one-way analysis of

variance using Statgraphics software (Statistical Graphics

Corporation, Rockville, MD, USA) with a confidence level

of 95% (po0:

05). A multiple range test was used to

determine which means are significantly different from

others.

3. Results

The solubility of native EY protein is significantlyimpacted by the pH and IS (Fig. 1). The protein solubility

is lowest at pH 4, independently from the IS. At pH 6.5,

virtually all proteins are soluble at an IS of 0.52 M NaCl,

whereas only about two thirds are soluble at low IS (0.15 M

NaCl). The protein solubility in heated EY is always much

lower than in native EY. The influence of the environ-

mental conditions follows the same trend with heated EY

as with the native one. Thus, protein solubility in heated

EY is also maximum at pH 6.5 and high IS, and minimal

at pH 4.

Le Denmat et al. (2000) have shown that in native EY,

plasma proteins have a very high solubility over a wide

range of pH and IS. On the other hand, granules wereshown to be insoluble at a pH of 3, and only partially

soluble in the physicochemical conditions occuring in

native EY (i.e. pH of 6.5 and IS of 0.15 M NaCl). Our

results are consistent with those presented byLe Denmat et

al. (2000)regarding the solubility of EY proteins. The low

solubility measured at pH 4 is explained by the insolubility

of granules at this low pH (Fig. 1). At a pH of 6.5 and

0.15 M NaCl, granules are mostly insoluble because their

constituents are aggregated via Ca2+ bridging between

negatively charged phosphoserine residues of HDL and

phosvitin (Causeret et al., 1991). Increasing the IS from

0.15 to 0.55 M NaCl leads to the disruption of phospho-calcic bridges resulting from the displacement of divalent

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0

20

40

60

80

100

120

Proteinsolu

bility(%)

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 1. Impact of pH and NaCl concentration on the solubility of proteins

in native (non-heated) and heated (i.e. 74 1C for 12 min.) EY suspensions.

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Ca2+ cations by an excess of monovalent Na+. This

induces the dissociation of granules, and therefore an

increased level of protein solubility.

The lower levels of protein solubility measured in heated

EY suspensions reflect the denaturation of some EY

proteins which associate to form insoluble protein aggre-

gates (see Guilmineau & Kulozik, submitted). In heatedsamples at a pH of 6.5, an increase of the IS from 0.15 to

0.55 M also leads to an increase of the level of solubility

from 25% to 37%. This indicates that some granule

proteins are still native after the heat treatment, and can

therefore still be dissociated by addition of NaCl. This is

consistent with observations that granule proteins are more

resistant to heat than plasma proteins (Anton, Le Denmat,

& Gandemer, 2000; Le Denmat, Anton, & Gandemer,

1999).

The median oil droplet diameter obtained in O/W

emulsions prepared with native and heated EY is

significantly influenced by the environmental conditions

(Fig. 2). The smallest droplets are formed at a pH of 6.5

and an IS of 0.52 M NaCl, whereas the largest are formed

at a pH of 4 and an IS of 0.15 M NaCl. The heat treatment

seems to have a rather small impact on the size of oil

droplets achieved, except at a pH of 4 and a low IS

where heated EY allows to obtain much smaller droplets as

native EY.

The interfacial protein concentration in emulsions

prepared with native EY is significantly higher at a pH of

6.5 than at a pH of 4 (Fig. 3). The IS does not seem to

influence the interfacial protein load at pH 6.5, but at pH 4

the interfacial load is lower at an IS of 0.52 M NaCl than at

an IS of 0.15 M NaCl. On the other hand, when heated EY

is used, the interfacial protein concentration is much higher

than when native EY is used, under all conditions tested.

Moreover, there does not seem to be any negative impact

of the environmental conditions on the interfacial proteinload, since the differences measured between the various

conditions tested are not significant.

The zeta-potential of oil droplets in the emulsion is

significantly impacted by the environmental conditions

(Fig. 4). Droplets carry a positive charge at a pH of 4 and a

negative one at a pH of 6.5. Increasing the concentration of

NaCl at a pH of 4 leads to a decrease of the zeta-potential,

which is due to the enhanced screening of the adsorbed

proteins positive charges by negative counter ions.

However, at a pH of 6.5, increasing the IS leads to an

increase of the negative zeta-potential, despite the increased

screening by positive ions from the dissolved NaCl. This

suggests that the negative charge density of the interface is

greatly increased by the addition of NaCl, in a way, which

more than compensates the screening effect. This may be

due to an increased adsorption of the strongly negative

phosvitin, following the dissociation of granules taking

place at high pH and IS.

A heat treatment of the EY did not have any significant

impact on the zeta-potential obtained at the oil droplet

surface, regardless of the environmental conditions.

The values of zeta-potential obtained with native EY are

consistent with measurements from Le Denmat et al.

(2000). The zeta-potential does not exceed 10 mV in all

environmental conditions tested, which is always well

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0

1

2

3

4

5

6

7

8

mediandrople

tdiameterd50.3

(m)

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 2. Impact of pH and NaCl concentration on the median oil droplet

diameter (d50.3) achieved in O/W emulsions containing native (i.e. non-

heated) and heated (i.e. 74 1C for 12 min.) egg yolk.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Interfacialproteinload[mg

.m-2]

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 3. Impact of pH and NaCl concentration on the interfacial protein

concentration measured in O/W emulsions containing native (i.e. non-

heated) and heated (i.e. 74 1C for 12 min.) egg yolk.


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below the 20 mV evaluated by Friberg (1997) to produce

effective electrostatic stabilisation. The charge of the

interface changes from a slightly positive charge at pH

4.0 to a slightly negative one at pH 6.5, which is consistent

with the expected variation of EY protein charges when

predicted from their isoelectric points.

Adsorption of heated EY proteins did not affect thecharge of the interfacial film. Similar observations were

made with caseins, for which it was reported that a change

in the geometry of the protein had relatively small effects

upon the zeta-potential of the emulsion droplets (Dalgleish,

1999). This suggests that despite the increased proportion

of denatured proteins present at the interface, the adsorbed

layer adopts a conformation, which maintains the same

charge density as the proteins in the native form.

Both the environmental conditions and a heat treatment

were measured to have a very significant impact on the

flocculation of oil droplets (Fig. 5). The flocculation factor

of emulsions prepared with native EY is always higher than

that of emulsions prepared with heated EY, at all

environmental conditions tested. With native EY, the IS

seems to drive the flocculation, whereby a high IS leads to

the largest level of flocculation, regardless of the pH. At

low IS, a pH of 6.5 leads to a significantly higher level of

flocculation than that obtained at a pH of 4. When heated

EY is used as opposed to native one, the level of

flocculation in emulsions containing 0.52 M NaCl is greatly

reduced, down to a level similar to that obtained at low IS.

The flocculation factor seems to be less dependant on the

environmental conditions, although emulsions prepared at

a pH of 6.5 tend to be slightly more flocculated than those

prepared at a pH of 4.

The consistency index (K, according to the Herschell-

Bulkley model) of the emulsions made with native EY is

driven by the IS, whereby a high IS promotes a high value

of K, regardless of the pH (Fig. 6). When heated EY is

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-5

0

5

10

15

Zetapotential(mV)

Native

Heated

pH 4

0.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 4. Impact of pH and NaCl concentration on the zeta potential of oi

droplets taken from O/W emulsions containing native (i.e. non-heated)

and heated (i.e. 74 1C for 12 min.) egg yolk.

0

1

2

3

4

5

Flocculationfactor(-)

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 5. Impact of pH and NaCl concentration on the flocculation factor

measured in O/W emulsions containing native (i.e. non-heated) and heated

(i.e. 74 1C for 12 min.) egg yolk.

0

5

10

15

20

25

ConsistencyindexK(mPa.s

n)

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 6. Impact of pH and NaCl concentration on the consistency index

(K) of O/W emulsions containing native (i.e. non-heated) and heated (i.e.

74 1C for 12 min.) egg yolk.


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used, the consistency of the product is less impacted by the

environmental conditions as when native EY is used.

All emulsions prepared have a flow index comprised

between 0.9 and 1.0, which reflects the near-newtonian flow

behaviour of these fluid emulsions. The flow index of

emulsions made with native EY is lowest at high IS

(Fig. 7), which is when the consistency index is the highest(Fig. 6). At low IS, we observe that the flow index is

significantly higher at a pH of 6.5 than at a pH of 4.0.

However, when heated EY is used, the flow index of the

emulsion is much less impacted by the environmental

conditions than when native EY is used, and averages

around a value of 0.95 in all tested environments.

For the emulsion prepared with native EY, we measured

that the creaming rate of oil droplets is much higher at pH

4 and 0.15M NaCl than in any other environmental

conditions (Fig. 8). However, in emulsions prepared with

heated EY, there is no significant impact of the environ-

mental conditions on the creaming rate. The creaming rate

is always slightly lower when heated EY is used as

emulsifier rather than native EY.

The relative cream height reported here after 1-week

storage, when the level of the cream is stabilised, reflects

the ability of oil droplets to pack in an efficient manner

under normal gravity (Fig. 9). When native EY is used, the

tightest packing of droplets is achieved at pH 4 with an IS

of 0.15 M, and the most lose packing at a pH of 6.5 and an

IS of 0.52 M. However, when heated EY is used, the final

level of creaming is not influenced by the environmental

conditions. The cream obtained is rather more compact

than that obtained with native EY, except at pH 4 and

0.15 M NaCl.

4. Discussion

The discussion aims at comparing the results of this

work to those obtained in similar conditions with native

EY by other authors and focuses on explaining the results

obtained in the present study regarding the impact of heat

denaturation of EY.

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0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Flowindex(-)

Native

Heated

pH 40.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 7. Impact of pH and NaCl concentration on the flow index of O/W

emulsions containing native (i.e. non-heated) and heated (i.e. 74 1C for

12 min.) egg yolk.

0

1

2

3

4

5

6

7

Initialcreamingrate(%

/h)

Native

Heated

pH 40.15 M

40.52 M

6.50.15 M

6.50.52 M[NaCl]

Fig. 8. Impact of pH and NaCl concentration on the initial creaming rate

(first 611 h after emulsification) measured in O/W emulsions containing

native (i.e. non-heated) and heated (i.e. 74 1C for 12 min.) egg yolk.

0

10

20

30

40

50

60

70

80

RCHafter1weekstorag

e(%)

Native

Heated

pH 4

0.15 M

4

0.52 M

6.5

0.15 M

6.5

0.52 M[NaCl]

Fig. 9. Impact of pH and NaCl concentration on the relative cream height

(RCH) reached 1 week after emulsification measured in O/W emulsions

containing native (i.e. non-heated) and heated (i.e. 74 1C for 12 min.) egg

yolk.


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4.1. Thermally aggregated EY proteins can adsorb at the

O/W interface independently from the environmental

conditions

4.1.1. Emulsifying activity (EA) of native and heat-treated

EY

Le Denmat et al. (2000) found that the size of oildroplets formed in emulsions containing native EY was

independent from the environmental conditions. The

energy used by Le Denmat et al. (2000) to disperse the

oil by high-pressure homogenisation was much higher than

the one used in the present work, and the amount of EY

proteins in the continuous phase was 25 mg/ml, which is

about 8 times more than the one we used (i.e. 3.2 mg/ml).

As could be expected,Le Denmat et al. (2000)obtained an

average oil droplet size about 10 times smaller than the one

we achieved in the present work. They, however, clearly

demonstrated a decreased EA of granules at a pH of 3,

which was attributed to the poor solubility of granule

proteins at this pH. The authors found that this did not

affect the EA of complete EY, since the same mean droplet

diameter d320:42mm was achieved in all environmental

conditions. It was concluded that the emulsifying proper-

ties of EY were driven by plasma proteins. However, in our

study, where we dispersed the oil using a comparatively

lower energy (i.e. single pass through one stage homo-

geniser at 200 bar), we observed an increased d50.3 in

emulsions prepared at a pH of 4 and 0.15M NaCl,

indicating a decreased EA (Fig. 2). This could be due to the

fact that the dispersing forces generated during emulsifica-

tion were insufficient to allow a shear-driven dissociation

of granule proteins at this low pH and IS.Anton, Beaumal,et al. (2000)showed that insoluble EY granules do adsorb

at the O/W interface, and suggested that fragments of

granules obtained during homogenisation can also play the

same role. As the intensity of disruption forces increases,

the partial dissociation of granules could lead to a more

efficient adsorption of granule fragments at the O/W

interface, and an overall better surface activity of EY. We

observed that the EA of EY in our study was high at a pH

of 4 and 0.55M NaCl (Fig. 2), despite the low protein

solubility (Fig. 1). This seems to indicate that the presence

of high concentrations of NaCl facilitates the shear-

induced dissociation of insoluble EY granules at pH 4,

even when relatively low shear forces are used.

When an EY suspension is heated, part of the granule

proteins are denatured and form heterogeneous aggregates

together with denatured EY plasma proteins. Previous

work has shown that the heat treatment applied in the

present study leads to the thermal aggregation of just over

60% of the total EY proteins (Guilmineau & Kulozik,

submitted). The forces bonding proteins within heat-

coagulated EY has been shown to be mostly hydrophobic

interactions (Kiosseoglou & Paraskevopoulou, 2005).

These interactions are quite weak and can probably be

dissociated by the shear forces present in the conditions

used for emulsification (i.e. 200 bar, one stage homogenisa-

tion). The interfacial film in emulsions prepared with heat-

treated EY could therefore be formed by the adsorption of

a multitude of minute fragments of thermally aggregated

proteins, obtained by the shear-driven disruption of much

larger protein aggregates during high pressure homogeni-

sation. This hypothesis is supported in the present work by

the fact that denatured EY proteins are adsorbed at the O/W interface, even though they form insoluble protein

aggregates in the continuous phase. This change in the

nature of the interfacial film when using heated EY can

explain the fact that the EA of EY at a pH of 4 and 0.15 M

NaCl is better when heated EY is used as opposed to native

EY (Fig. 2). This result suggests that the shear intensity

obtained during homogenisation in the conditions of this

study was sufficient to allow a thorough dissociation of

thermally aggregated EY proteins, although not sufficient

to dissociate insoluble native granules at low pH and IS.

4.1.2. Interfacial protein concentration under various

environmental conditions

The isoelectric region (pI) of apo-LDL has been

measured between pH 6.5 and 7.3 (Kojima &Nakamura,

1985; Nakamura, Hayakawa, & Sato, 1977), and that of

apo-HDL, although not characterised precisely, is assumed

to be in the enlarged neutral pH region as well (Le Denmat

et al., 2000). The increased interfacial protein concentra-

tion observed with native EY at pH 6.5 (Fig. 3) can be

explained by the proximity of this pH to the pI of apo-LDL

and apo-HDL. The decreased charge density of proteins

around their pI favours their tight re-arrangement leading

to the formation of dense interfacial films, which was

notably observed with bovine serum albumin (Das &Chattoraj, 1980).

This work shows that the interfacial film formed with

heated EY contains a greater concentration of protein than

the film formed with native proteins (Fig. 3). This reflects

the formation of a thick film of aggregated proteins in

emulsions made with heated EY. During high-pressure

homogenisation, the protein aggregates formed during

heating could be broken down by the shear forces into a

multitude of smaller aggregates, which seem to adsorb

favourably at the O/W interface. It has indeed been

reported that the turbulences obtained in high-pressure

homogenisation favour the adsorption of large protein

aggregates because the convective mass transport rate

increases with the size of molecules (Walstra, 1983).

Furthermore, the mechanical disruption of protein aggre-

gates held together by hydrophobic interactions probably

leads to the exposure of many hydrophobic sites. The

increased surface hydrophobicity of the protein fragments

could greatly contribute to their rapid adsorption at the

O/W interface. After adsorption, interactions between

neighbouring aggregates could lead to the formation of a

thick and cohesive interfacial film, possibly forming multi-

ple layers. The adsorption of protein aggregates occurs at

all environmental conditions tested, without being nega-

tively impacted by the environment.

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4.2. The adsorption of denatured EY protein aggregates

reduces the impact of the environmental conditions on the

emulsifying properties of EY

4.2.1. Decreased level of flocculation in emulsions made with

heated EY

The EY proteins forming the interfacial film are chargedpolymers, and therefore tend to stabilise emulsion droplets

against aggregation through a combination of electrostatic

and steric repulsions (Claesson, Blomberg, Fro berg, Ny-

lander, & Arnebrant, 1995). The principal difference

between these two types of interactions is their sensitivity

to pH and IS (Hunter, 2000). The electrostatic repulsion

between emulsion droplets is dramatically decreased when

the electrical charge on the droplet surface is reduced (e.g.

by altering the pH) or screened (e.g. by increasing the

concentration of electrolyte in the aqueous phase). In

contrast, steric repulsion is fairly insensitive to both

electrolyte concentration and pH (McClements, 1999).

The stability of the emulsion, and particularly the tendency

for oil droplets to flocculate, depends on the intensity of the

steric and electrostatic repulsive forces in relation to the

attractive forces (e.g. van der Waals, hydrophobic interac-

tions). The magnitude and range of electrostatic repulsion

between droplets decrease as the IS of the solution

separating them increases because of electrostatic screen-

ing. This explains the susceptibility of protein-stabilised

emulsions to flocculation when electrolyte concentration is

increased above a critical level (Demetriades, Coupland,&

McClements, 1997). Electrostatic screening can explain the

increased flocculation at a high IS (0.55 M NaCl) when

native EY is used (Fig. 5). At low IS, the lower level offlocculation observed at pH 4.0 compared to pH 6.5 is

probably due to an increased electrostatic repulsion

obtained at pH 4.0 because of the increased interfacial

charge density. As already discussed, it appears that the pI

of the proteins, which are most active at the interface is close

to pH 6, so that their charge density is higher at pH 4 than at

pH 6.5. This difference is clearly shown at low IS by the

measurement of the zeta-potential of oil droplets (Fig. 4).

The decreased level of flocculation obtained at high IS

when heated EY is used as emulsifier indicates a change in

the stabilisation mechanism. Indeed, the absence of

modification of the zeta-potential when a heat-treatment

is applied to the EY, rules out any explanation of the

flocculation behaviour based on electrostatic interactions.

However, a change of the steric interaction is supported by

the increase of interfacial protein concentration measured

when heated EY is used instead of native EY (Fig. 3). Since

the strength of steric repulsions imposed by a polymer

when adsorbed at the interface partially depends on the

amount of polymer adsorbed (Hunter, 2000), it is expected

that the contribution of the steric repulsion to the overall

repulsion forces is increased when heated EY is used. The

impact of the electrostatic screening which appears to drive

the flocculation when native EY is used seems to be

overridden by the increased steric repulsion when heated

EY is used. These results illustrate the fact that an

increased steric contribution over the electrostatic interac-

tions leads to a decreased sensitivity of an emulsions

stability to changes of environmental conditions.

4.2.2. Levelling of the rheological properties of the

emulsionsIt appears that the consistency index of the emulsion

containing native EY is high when the emulsion is

flocculated, which is the case at an IS of 0.55 M NaCl

(Fig. 6). This can be explained by an increase of the

effective volume of the dispersed phase in the flocculated

emulsion which is due to the continuous phase trapped

between oil droplets within the flocs (Dickinson &

Stainsby, 1982). The emulsions having a high degree of

flocculation at high IS also display the lowest values of flow

index, which indicates a higher level of pseudoplasticity

(Fig. 7). This reflects the deflocculation of the droplets

under the effect of increased shear rate (Bower, Washing-

ton,& Peruwal, 1997), and indicates that the flocs formed

at high IS are sensitive to shear forces.

When heated EY is used, the deflocculation of the

emulsion at high IS leads to a decrease of its consistency

index. We also measure a slight pseudoplasticity in

emulsions made with heated EY. This could be due to

the presence of EY protein aggregates in the continuous

phase, which orientation in the shear field (with possible

dissociation) leads to a decreased resistance to flow at

increased shear rate.

4.2.3. Improved creaming stability and formation of a

compact creamIn emulsions made with native EY, the initial creaming

rate seems to be correlated to the median oil droplet

diameter (Figs. 8 and 2, respectively), which is consistent

with the expected impact of the droplet size on the

creaming velocity, as described for example in Stokes

law. Given the intermediate oil droplet concentration in

our model system (j 0:3), droplet flocculation is

expected to increase the creaming velocity because the

flocs have a larger effective size than the individual

droplets. However, we did not observe any correlation

between the degree of flocculation and the creaming rate of

the emulsions. It should be noted that the emulsions were

stirred vigorously prior to the beginning of the creaming

rate measurement, which probably led to some degree of

droplet deflocculation. The initial creaming rate in emul-

sions prepared with heated EY does not seem to be

impacted by the oil droplet size as much as those prepared

with native EY. Despite significant differences in oil

droplet size with a median oil droplet diameter varying

between 3.4 and 4.3mm, the initial creaming rate of oil

droplets in emulsions made with heated EY was constant at

about 1% creaming per hour. This result shows that the

modification of the interfacial film composition and

density-taking place when heated EY is used has a positive

effect on the stability of emulsions to creaming by

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decreasing the creaming rate. This could be due to an

increased density of the oil droplets under the effect of the

increased density of the interfacial film (Fig. 3). The

adsorption of dense material at the O/W interface can

significantly increase the density of oil droplets, and

therefore slow down the creaming (Tan, 1990). This effect

is particularly significant for small oil droplets. In this work,the creaming rate was estimated by observing the kinetic

displacement of a front line separating the floating fat phase

from the sedimenting fat-free phase in a standing sample of

the emulsion. The position of the front moving from the

bottom of the tube upwards reflects the displacement of the

small droplets of the distribution, since they are the slowest

to migrate. It is therefore expected that this method would

allow to detect changes in the density of oil droplets, which

is assumed to be the case in this work.

As highlighted by McClements (1999), the thickness of

the creamed layer formed after complete creaming of the

dispersed phase depends on the effectiveness of droplet

packing and is impacted by the same factors as those

determining the structure of flocs. When native EY is used,

there seems to be a correlation between the median oil

droplet size and the compactness of the cream layer

(Fig. 9). This can be due to the fact that when the droplet

size decreases at constant oil phase volume ratio, the number

of droplets and therefore the interfacial area increases as

well, so that more continuous phase remains trapped

between the oil droplets, giving a less compact cream.

Notice that emulsions made with native EY have a tendency

to flocculate more than the ones made with heated EY,

which reflects the presence of greater attraction forces

between oil droplets. This can lead to oil droplets stickingtogether when coming in contact during creaming, and

therefore forming a cream layer with a relatively open

structure. This could explain why emulsions containing

denatured EY, which tend to be less flocculated, also tend to

form a more compact cream layer than those made with

native EY (Fig. 9). The only environmental condition in

which the cream made with native EY is more compact than

that obtained with heated EY (i.e pH 4 and 0.15 M NaCl) is

also that for which the flocculation factor is minimal, which

is consistent with the hypothesis formulated above. It

appears that when attraction forces between droplets are

weak, the droplets are able to roll around each other more

freely and can therefore pack more closely together. Visual

observations revealed that the cream formed in emulsions

prepared with native EY was cohesive and formed a lump,

which did not re-disperse easily under mild agitation. On the

other hand, the cream formed in emulsions prepared with

heated EY could easily be evenly re-dispersed by simple

manual agitation, which can be an advantage in products,

which have to be shaken just before use.

5. Conclusions

This work has shown that the emulsifying activity of

thermally denatured EY is similar or even better than that

of native EY, depending on the environmental conditions.

Using heated EY rather than native one led to a significant

increase of the interfacial protein concentration under all

environmental conditions. This demonstrates the ability for

thermally denatured insoluble EY protein aggregates to

adsorb efficiently at the O/W interface. EY protein

aggregates formed during heating are thought to bedisrupted by the high shear forces occurring in high-

pressure homogenisation. The resulting micro-particles of

aggregated proteins would constitute the interfacial film

when heat-treated EY is used as emulsifier. The adsorption

of denatured EY proteins was shown to greatly decrease

the flocculation of oil droplets, particularly at high ionic

strength. This is thought to be due to an increased

contribution of the steric repulsions between droplets when

protein aggregates are adsorbed at the interface. The

rheological properties as well as the stability of the

emulsions against creaming were shown to be much less

sensitive to variations of the environmental conditions

when heated EY was used rather than native EY. Further

work is required to study the impact of the environmental

conditions on the composition of the interfacial film

formed with both native and denatured EY.

Acknowledgement

The authors thank Jan Peter Luh and Mario Henrique

Martinez Marins for their valued contribution in the

experimental phase of this work.

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