Food Hydrocolloids 24 (2010) 434443

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

A method of egg yolk fractionation. Characterization of fractions

Amanda Laca, Benjamn Paredes, Mario Daz*
Department of Chemical Engineering and Environmental Technology, University of Oviedo, C/Julian Claveria s/n., 33071 Oviedo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: A simple method of egg yolk fractionation was carried out and three fractions (granules, lipidic paste and
Received 16 June 2009 watery fraction) were obtained. The chemical and physical properties of each fraction were assessed in
Accepted 16 November 2009 order to evaluate its potential new applications. Additionally, lyophilization was evaluated as a possible
technique to increase product shelf life.
Keywords: From a structural point of view, granules stress resistance is higher than in the case of the lipidic paste.
Yolk
Cure test and differential scanning calorimetry analyses showed that granules proteins are more resistant
Fractionation
to heat denaturation than lipidic paste proteins. However, heat denaturation resistance of granules
Freeze-drying
Egg proteins is affected by freeze-drying process. Granules colour is not affected by lyophilization, while
Rheology lipidic paste colour suffers a slight modication. Granules and lipidic paste, both fresh and lyophilized,
Physical properties have high emulsifying properties at all pH studied. Particle size measurements and scanning electron
Thermal properties microscopy showed an increase in particle size of lyophilized fractions. According to their composition,
granules, lipidic paste and watery fraction seem to be suitable to be employed as potential ingredients in
food, cosmetic and biotechnological uses, respectively.
2009 Elsevier Ltd. All rights reserved.

1. Introduction approaches to the development of the egg industry is fractionation

Egg is broadly recognised as a very valuable source of proteins
for human nutrition and is known to contain many substances with
biological function beyond basic nutrition (Lopez-Fandino, Recio, &
Ramos, 2007). That is why, since the mid-1970s, numerous studies
(Bengtsson, Marklund, & Olivecrona, 1977; Burley & Valdehra, 1979;
Castellani, David-Briand, Guerin-Dubiard, & Anton, 2005; Chiou,
2003; Fichtali, Charter, Lo, & Nakai, 1993; Merkle & Ball, 2000;
Meslar & White, 1978) characterizing biophysiological functions of
egg components and seeking novel biologically active substances in
hen eggs have been conducted. Specically, in recent scientic
literature (Hatta, Kapoor, & Juneja, 2008; Miguel, Lopez-Fandino,
Ramos, & Aleixandre, 2005; Mine & DSilva, 2008; Rehault, Anton,
Nau, Gautron, & Nys, 2007) numerous biological activities of egg
white and egg yolk components are described. These include novel
antimicrobial activities, antiadhesive and antioxidant characteris-
tics, hypercholesterolemic and immunomodulatory properties, and
egg-protein-derived peptides with antihypertensive activities.
As future new applications of egg components are pursued, it is
important to continue exploring the egg products industry and its
application of new technologies (Froning, 2008). One of the main
* Corresponding author. Tel.: 34 985103439; fax: 34 985103434.
E-mail address: mariodiaz@uniovi.es (M. Daz).
of egg components and move forward with new innovative
applications.
Egg yolk contains approximately 50% solids. The major constit-
uents of the solid matter are lipids (6570% on dry basis) and
proteins (30% on dry basis), consisting of proteins in solution
referred to as livetins, lipoprotein particles including high-density
lipoproteins, HDLs, and low-density lipoproteins, LDLs, and phos-
vitin (Li-Chan, Powrie, & Nakai, 1995). Each constituent of yolk
possesses special physical and chemical characteristics responsible
for its own functional properties. Thus yolk represents a major
source of active principles usable in medical, pharmaceutical,
cosmetic and biotechnological industries.
So as to widen the applications of egg yolk, a number of different
fractionation methods have been developed, many of them based
on the use of organic solvents (Kwan, Li-Chan, Helbig, & Nakai,
1991; Sheumack & Burley, 1988). However, in most applications,
these methods are not completely innocuous due to the possible
solvent residuals in the nal products and, in addition; the frac-
tionation process could adversely affect the separated compound
activity. Another approach to isolate the egg yolk compounds has
been based on heating methods (Liot, 2002), but heat usually
causes protein denaturalization. The use of supercritical uid
extraction (Aro, 2007) has also been employed as a separation
method, nevertheless, high costs have rendered this process less
practical (Froning, 2008). In summary, individual constituents of

0268-005X/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2009.11.010
 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443
yolk are difcult to separate and only plasma and granules can be
easily fractionated from yolk at an industrial scale (Anton, Le
Denmat, Beaumal, & Pilet, 2001).
The aim of this work has been the physicalchemical charac-
terization of egg yolk fractions obtained by means of a simple and
easily scalable process. Until now, these fractions have not been
characterized from a structural and colorimetric point of view.
Additionally, the behaviour of the samples was assessed fresh and
freeze-dried reconstituted in order to evaluate lyophilization as
a possible technique to increase the fractions shelf life. According to
their properties these fractions could be potentially employed as
emulsier agent in food, ingredient in cosmetics or source to aisle
immunoglobulins.
2. Materials and methods
2.1. Fractionation of egg yolk
The fractionation method was developed modifying the proce-
dure described by Merkle and Ball (2000). Egg yolks were prepared
from fresh eggs. The shelling of the eggs and the separation of the
yolk from the albumen were performed manually. The albumen
residuals were eliminated from the yolk using a blotting paper, and
the removal of the vitelline membrane was achieved using twee-
zers. Next, the egg yolk material is mixed with water (1:1.5 v/v) to
dilute the egg yolk. Then the pH of the diluted egg yolk is adjusted
to 7 by the addition of NaOH (1 N) and it is kept overnight at 4  C
before centrifuging at 4  C and 10 000  g for 45 min to separate
into plasma (supernatant) and granule fractions (fraction 1:
precipitate).
Sodium alginate 1% (w/v) solution is added to plasma to achieve
a nal concentration of alginate of 0.1% (w/v). This mixture is
centrifuged at 20  C and 10 000  g for 15 min to obtain a lipidic
paste (fraction 2) and a waste watery fraction (fraction 3).
The general scheme for egg yolk fractionation is shown in Fig. 1.
2.2. Chemical analyses
The pH was measured at 20  C using a Crisom pH25 pH meter.
The dry matter was assessed with an HR73 Halogen Moisture
Analyzer, the amount of sample being around 1 g. Water activity
(aw) was measured with a water activity-meter (FA-st lab Prosoft,
GBX) based on the hygrometric method, also known as dew point
technology. The total lipids content was evaluated by following the
extraction by acid hydrolysis method described by Toschi, Bendini,
Ricci, and Lercker (2003). The Kjeldahl method of nitrogen analysis
was conducted to calculate the protein content. According to Jiang,
Fenton, and Sim (1991), accuracy of enzymatic methods to
Egg yolk
Dilute
Add NaOH to pH 7
Held overnight at 4C
Centrifuge
Precipitate Supernatant
(Fraction 1: Granules) (Plasma)
Add sodium alginate
Centrifuge
Lipidic paste Watery fraction
(Fraction 2) (Fraction 3)
Fig. 1. General scheme for egg yolk fractionation.
435
determine cholesterol in eggs is very similar to the one obtained by
HPLC or gas chromatography, and hence cholesterol content was
determined using a commercial enzymatic photometric test-kit
(ENZYTEC uid Cholesterol).
All analyses were carried out in triplicate.
2.3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
The protein prole of the obtained fractions was investigated by
SDS-PAGE. All samples were diluted so the amount of protein in
each gel lane was about 25 mg. Samples were diluted 3:1 (v/v) in
a dissociation buffer consisting of a 0.5 M TrisHCl pH 6.8, 0.05%
bromophenol blue, 35% glycerol, 5% b-mercaptoethanol, 8% (w/v)
SDS solution, and heated in boiling water for 5 min. Electrophoresis
were run on polyacrylamide gels (stacking: 3.5% and resolving:
12%) with a migration buffer consisting of a 0.02 M Tris(hydrox-
ymethyl)aminomethane, glycine 5 M, SDS (w/v) 0.1% solution. The
proteins were stained with the usual staining procedure (Coo-
massie blue 0.1%, methanol 50%, acetic acid 10% and water 40%).
The gels were distained in a solution containing acetic acid (10%),
methanol (40%) and water (50%). SDS-PAGE Molecular Weight
Standards, Broad Range (Bio-Rad) were used as protein standards.
2.4. Lyophilization
Fractions 1 and 2 were freeze-dried at 70  C and 0.1 mBa in
a Telstar Cryodos Lyophilizator. Samples were frozen at 80  C
previous to lyophilization. The dry extract of lyophilized samples
was approximately 97% (w/w).
2.5. Microorganisms count
Total plate count of samples from fractions 1 and 2, both fresh
and lyophilized-reconstituted were determined according to the
European Standard ISO 4833, 2003. Lyophilized fractions 1 and 2
were reconstituted with distilled water until the water content
(%w/w) of their respective fresh fractions was achieved.
2.6. Determination of emulsifying properties
Inklaar and Fortuins (1969) procedure was modied to evaluate
the emulsifying properties of the 1 and 2 obtained fractions, both
fresh and lyophilized-reconstituted (as it was explained above).
An amount of 2 mg sample (fresh or reconstituted) was diluted
in 10 mL of 0.075% (w/v) NaCl solution, and the pH was adjusted
with NaOH (0.1 N) or HCl (0.1 M) solution. Then 13 mL of cotton
seed oil were added. The mixture was stirred up for 10 s at
8000 rpm with a HEIDOLPH Diax 900 homogenizer, and nally the
sample was let rest in a measuring cylinder at room temperature
(20  C) for 1 week. After this time not emulsied oil volume was
determined and emulsifying percentage (F) was calculated as
follows:
 
F% Vadded oil  Vnot emulsified oil $100=Vadded oil
Emulsifying properties were determined at pH 3, 5 and 7 for
each sample and analyses were carried out in triplicate.
2.7. Particle size measurement
Samples of fresh fractions 1 and 2 were diluted with distilled
water (1 g/150 mL) until completely dispersed, and then were
analyzed by a particle size analyzer (Malvern Mastersizer S). The
sample solution was dispersed in distilled water at 1200 rpm until
436 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443
an obscuration rate of 2025% was obtained. Background and
sample were measured for 10 s. Optical properties of the sample
were dened as follows: refractive index 1.460 and absorption 0.00.
Droplet size measurements are reported as the volume-weighted
P 4P 3
mean diameter: d4,3 nidi / nidi , where ni is the number of
droplets of diameter di. Lyophilized fractions 1 and 2 were recon-
stituted as it was explained previously, and they were measured in
the same way described above. Each sample was measured in
triplicate.
2.8. Colour measurement
Fractions 1 and 2 fresh and lyophilized-reconstituted, as it was
described, were measured for colour in the L*, a*, b* system.
Measurements were carried out using an UltraScan VIS spectro-
photometer (HunterLab). It was standardized with a light trap and
a white title, and the green title was used to verify the instrument
long-term performance.
A xed amount of sample was poured into the measurement cell
and analyses were conducted in specular exclusion mode. This
mode is used to measure colour including the effects of gloss and
texture, so the evaluation of colour is closer to human-eye
perception.
The colour changes are expressed as DE with the colour of the
fresh fractions as a reference sample, so DE is the total colour
change due to the freeze-drying process calculated from (Santipa-
nichwong & Suphantharika, 2007):
q All samples were dehydrated in a graded ethanol series and
DE DL*2 Da*2 Db*2
Analyses were carried out in duplicate.
2.9. Differential scanning calorimetry (DSC)
The differential scanning calorimetry tests were carried out in
aluminium pans hermetically sealed. A DSC 822e equipment was
used, developing temperature ramps from 20  C to 100  C at a heating
rate of 5  C/min in a nitrogen atmosphere. The denaturation
temperatures were determined using the equipment software.
DSC measurements were performed for both fractions 1 and 2,
fresh and lyophilized; lyophilized fractions were reconstituted as it
was explained above. Analyses were conducted at least in duplicate.
2.10. Rheological measurements
Both fractions 1 and 2 were characterized reologically, fresh and
lyophilized; lyophilized fractions were reconstituted as it was
described previously. The rheological tests were carried out using
a Haake MARS II rotational rheometer with a Peltier unit to control
the temperature. To avoid slippage a serrated plate/plate measuring
system (PP35) was used, with a gap of 1 mm. All the test (except
cure tests) were carried out at 20  0.1  C and before starting any
measurements, the sample rested for at least 15 min, allowing the
stresses induced during sample load to relax. A glass hood and
silicone oil was employed to avoid sample desiccation during the
analyses. All tests were carried out at least in duplicate.
In dynamic conditions the stress sweeps were performed from
0.01 to 1000 Pa for fraction 1 and from 0.01 to 100 Pa for fraction 2
at a frequency of 1 Hz. The frequency sweeps were carried out from
10 to 0.1 Hz at a constant shear stress of 10 Pa. This stress value is in
the linear viscoelasticity range that was previously established by
the stress sweeps. Cure tests were carried out from 20  C to 100  C
at a heating rate of 5  C/min and at a frequency of 1 Hz and
a constant shear stress of 10 Pa.
The rheological parameters measured in the dynamic tests were
the complex modulus (G*), the storage (G0 ) and the loss (G00 ) moduli
and its relation (tan d G00 /G0 ).
The experimental data of all frequency sweep tests were
correlated to the following power law equation (Gabriele, de Cin-
dio, & DAntona, 2001):
G* Au1=z
where G* is the complex modulus in Pa, u the frequency in Hz, z
(dimensionless) the coordination number and A (G* in Pa at 1 Hz)
the proportional coefcient.
2.11. Scanning electron microscopy SEM
The fresh fractions samples were encapsulated in 2.5% agar at
45  C and then cut into 34 mm cubes. The gel samples were xed
overnight in 3% glutaraldehyde in 25 mM phosphate buffer (pH
6.8), rinsed in several changes of buffer and post-xed overnight in
2% osmium tetraoxide with 0.1 M imidazole. The lyophilized frac-
tions were reconstituted as it was previously explained and treated
in the same way.
Fresh and lyophilized-reconstituted fractions were coagulated
as follows: 5 g of each sample were introduced in a glass bottle and
kept at 90  C during 30 min in a drying oven. After that they were
let to cool at room temperature and xed overnight in 3% glutar-
aldehyde in 25 mM phosphate buffer (pH 6.8). Then they were
rinsed in several changes of buffer.
once 100% ethanol was achieved, they were moved in a graded
acetone series, so samples were nally in 100% acetone. The
samples were critical point dried thought CO2 in a Critical Point
Dryer Balzers CPD 030, Polaron. Dry fractions were fractured and
torn with a blade and fragments were mounted on aluminium SEM
stubs, and coated with gold in a Sputtering Balzers SCD 004. The
microscope used was the JEOL-6100 SEM.
2.12. Statistical analyses
Data were analyzed by running t-tests to compare means, at
a 5% probability level. Previously F-tests at a 5% probability level
were carried out to compare standard deviations. In case the data
presented heterogeneity, they were statistically homogenized.
Standardized skewness and standardized kurtosis were used to
assess if the samples came from normal distributions. The software
used was STATGRAPHICS Plus 3.1.
3. Results and discussion
3.1. Basic characteristics
Table 1 shows composition of the obtained fractions compared
with yolk composition. Fraction 1 constitutes 29%, fraction 2, 62%
and fraction 3, 9% (w/w) of initial yolk dry extract. As can be seen,
granules contain most egg yolk proteins, while lipidic paste
contains most egg yolk lipids and watery fraction dry matter is
mainly constituted by proteins. Granules data agree with those
presented by Li-Chan et al. (1995) who reported that granules
contain 34% lipid and 60% protein on a moisture-free basis. Anton
and Gandemer (1997) results show an amount of lipid content in
granules in accordance with Table 1 data, however, these authors
reported higher values of protein and cholesterol content in gran-
ules. It is important to point out that, as can be seen in Table 1,
major amount of egg yolk cholesterol is concentrated in fraction 2.
The values of pH were in all cases about 7 due to the fact that
 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443 437

Table 1 100
Composition of obtained fractions compared with yolk. Average values  SD are
reported.
80
Yolk Fraction 1 Fraction 2 Fraction 3
60

F (%)
(granules) (lipidic paste) (watery fraction)
Dry matter (% w/w) 51a 41.4  1.5 36.6  0.8 3.1  0.4
Proteins (% w/w) 16a 24.0  0.9 6.9  0.1 1.9  0.1 40
Total lipids (% w/w) 36a 16.6  2.8 28.8  2.2 0.27  0.19
Cholesterol 1260b 291  36 957  58 50  2 20
(mg/100g sample)
pH 66.4c 6.94  0.01 7.03  0.05 7.03  0.02 0
a
Values adapted from Powrie and Nakai (1986). 3 5 7
b
Value from Ministry of Health and Consumer Affairs database (Spain, 2009). pH
c
Value from Stadelman (1995).
Fig. 3. Emulsifying percentage (F) for fractions 1 and 2 at different values of pH, open
diluted egg yolk pH is adjusted to by the addition of NaOH during bars: fresh faction 1, solid bars: lyophilized fraction 1, dotted bars: fresh fraction 2,
the fractionation process. dashed bars: lyophilized fraction 2.
According to Manso, Cuhna, and Oliveira (2006), shelf life
indicators are microorganisms or compounds that have a notorious
proteins of the yolk are concentrated in fraction 2 (>45 kDa), while
inuence on the product safety and on quality (nutritional value,
the lightest ones are concentrated in fraction 3 (<97 kDa). Fraction
appearance or avour characteristics). The handling during the
1 contains mainly proteins between 66 and 200 kDa.
separation process results in changeable microorganism count and
All the egg yolk peptides, except 15 kDa apo-LDL, identied from
in all cases values for fresh samples were higher than those for
SDS-PAGE proles by Le Denmat, Anton, and Beaumal (2000) can
lyophilized-reconstituted ones (data not shown). Thus, the freeze-
be seen in lane Y. In lanes P and F2 clearly appears 175 kDa apo-LDL,
drying process led to be a suitable method to reduce the amount of
while 32 apo-HDL can be seen in lane F1. It is important to point out
total microorganism by one order of magnitude. Anyway,
that bands of g-livetin (6070 kDa) and b-livetin (3849 kDa) are
a pasteurization step is always necessary to assure a completely
easily identied in lane F3.
safe product for food industry applications.
It is well known that immunoglobulin Y (IgY) is the predomi-
The low values of water activity in freeze-dried fractions (around
nant fraction of g-livetin (Schade & Chacana, 2007) and this
0.50) related to those of fresh fractions (around 0.97) guarantee
immunoglobulin possesses important applications in biomedical
microbiological growth inhibition of majority of bacteria, many
uses such as an immunologic tool in the elds of medical diagnosis
yeasts and most molds, even at optimal temperatures (Beuchat,
or for passive immunization therapy (Hatta et al., 2008). Hence,
1981; Scott, 1957). In addition, these low aw values reduces the rate
fraction 3 becomes an interesting source to aisle immunoglobulin
of spoilage changes such as lipid oxidation (Homma & Masao, 1982;
for biotechnological purposes.
Seow & Goh, 1976). Obara, Obiedzinski, and Kolczak (2006) reported
that this rate, specically, the cholesterol oxidation, is even lower in
freeze-dried egg powders than in spray-dried egg powders. 3.3. Emulsifying properties
Thus, an immediate lyophilization after the fractionation process
not only reduces the amount of total microorganisms; but also, due As can be seen in Fig. 3, fractions 1 and 2, both fresh and
to the low aw of freeze-dried samples, inhibits microbiological lyophilized reconstituted, have high emulsifying power. There are
growth and also spoilage reaction rate. Therefore, freeze drying not signicant differences (p < 0.05) between samples, neither at
could result in a useful process to increase the product shelf life. pH 3 nor at pH 5; nevertheless, fresh fractions emulsifying power
was signicantly different at pH 7. Although Le Denmat et al. (2000)
3.2. Protein prole reported that at pH 7, granules have better emulsifying properties
than plasma, in this work, fraction 2 has at this pH higher values of F
SDS polyacrylamide gel electrophoresis of yolk, plasma and the than fraction 1 (granules). This is due to the fact that plasma
obtained fractions is showed in Fig. 2. As can be seen the heaviest proteins and lipids are concentrated in fraction 2 (Table 1 and
Fig. 2). Granules require a minimum ionic strength of 0.3 M NaCl to
become solubilized at pH 7, whereas plasma is solubilized at any
Std Y P F1 F2 F3
ionic strength (Li-Chan & Kim, 2008). This fact could also be
responsible of the signicantly different F values of fresh fractions
200 apo-LDL 175 at pH 7.
116 apo-LDL 137
Freeze-dried reconstituted fraction 1 emulsifying power was not
97 apo-HDL 105 signicantly different at any essayed pH and the same happens to
-livetin 83 lyophilized fraction 2. However, fresh fraction 1 emulsifying power
66 apo-HDL 79
-livetin and apo-LDL 60-70 was signicantly different at pH 3 and at pH 7 and the same
apo-HDL 53 behaviour was observed for fresh fraction 2; in both cases
45 Phosvitin 46
-livetin 38-40
Table 2
Particle size (d4,3) of fractions 1 and 2. Average values  SD are reported.

apo-HDL 32 Particle size (mm)

21
Fraction 1 Fresh 1.06  0.04
Lyophilized 47.61  2.86

Fraction 2 Fresh 149.66  4.68

Fig. 2. SDS-polyacrylamide gel electrophoresis of yolk (Y), plasma (P) and egg yolk
Lyophilized 303.18  13.18
fractions (F1, F2 and F3). Std: standard molecular weights (kDa).
438 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443

Table 3
Values of L*, a* and b* colour coordinates for fractions 1 and 2. Average values  SD are reported. The colour changes are expressed as D.

Fraction 1 Fraccion 2

L* a* b* L* a* b*
Fresh 78.45  1.07 8.18  0.18 35.22  0.75 66.64  0.71 19.38  0.45 60.52  1.81
Lyophilized 78.94  0.45 7.16  0.39 33.83  0.63 71.25  0.25 14.97  0.18 50.64  2.45
D 0.49 1.03 1.39 4.61 4.41 9.87
DE 1.80 11.76

emulsier power were higher at pH 7. This can be explained
because the increase in pH towards alkalinity imparts a negative
charge to the protein molecules, which facilitated emulsication by
promoting protein-to-fat interaction and reducing protein-to-
protein interaction through electrostatic repulsion (Wong & Kitts,
2003).
Therefore pH seems to affect more the emulsifying properties of
fresh samples than those lyophilized. Since the lyophilization
process involves a slight heating (Nail, Jiang, Chongprasert, &
Knopp, 2002), the freeze drying can produce partial denaturation of
some proteins (Arakawa, Prestrelski, Kenney, & Carpenter, 2001;
Chang, Kendrick, & Carpenter, 1996), affecting its properties and
tending to homogenize its emulsier power under different pH
values.
3.4. Particle size
As can be seen in Table 2, there are signicant differences
(p < 0.05) between fresh and lyophilized-reconstituted samples, so
lyophilization clearly modies nal particle size. In both cases the

Fig. 4. Stress sweep of fraction 1 (up) and 2 (down), fresh (square shape) and lyophilized (triangular shape). Full symbols represent storage modulus (G0 ) and empty symbols
represent loss modulus (G00 ).
 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443 439

1
tan ()

0
1 10 100 1000

Shear stress (Pa)

Fig. 5. Loss tangents (tan d) of stress sweeps of fraction samples: fraction 1 (square
shape) and fraction 2 (triangular shape). Full symbols represent fresh samples and
empty symbols represent lyophilized samples.
freeze-drying process results in a noticeable increase of the d4,3
diameter, but this increase is different for fractions 1 and 2. The
diameter value for fresh fraction 1 compared to lyophilized one
increased by 50 times, while in fraction 2 this value is only dupli-
cated. In this way, protein structure is more affected by the freeze-
drying process than lipids: as the protein content is considerably
higher in fraction 1 than 2, the different increase in the d4,3 diam-
eter is again probably due to protein structure modications during
lyophilization.
Fig. 7. Loss tangents (tan d) of frequency sweeps of fraction samples: fraction 1 (square
shape) and fraction 2 (triangular shape). Full symbols represent fresh samples and
empty symbols represent lyophilized samples.
The amount of fat may be responsible of the bigger particle size
of fraction 2 compared to 1 (Argov et al., 2008), because the amount
of lipids is higher in fraction 2 than in 1 and egg yolk lipids tend to
associate producing larger particles.
Since particle size has inuence on industrial processes such
as solid transport or heat transfer (Welti-Chanes, Vergara-Bal-
deras, & Bermudez-Aguirre, 2005), this parameter value should
be taken into account in industrial processing and equipment
design.

3.5. Colour

The lightness (L*), redness (a*) and yellowness (b*) values for
fractions 1 and 2, both fresh and lyophilized are shown in Table 3.
There are not signicant differences (p < 0.05) in the L*, a* and b*
values between fresh and lyophilized samples of fraction 1.
However, respect to fraction 2, there are not signicant differences
in the L* values, while the a* and b* values are signicantly different
between fresh and lyophilized samples. These differences were also
showed by the total colour change (DE) which value is higher in
fraction 2 than in 1.
Thus, the freeze-drying process has no effect on fraction 1 colour
parameters, while redness and yellowness values of fraction 2
suffer a decrease, being the lyophilized samples greener and bluer
in appearance than the fresh sample.
This different effect on colour caused by lyophilization could be
due to the fraction pigment content. The colour of egg yolk is
attributed to fat-soluble carotenoids (xanthophylls; including
lutein, zeaxanthin, b-cryptoxanthin and minor amounts b-caro-
tene) (Li-Chan & Kim, 2008). Since the lipids are concentrated in
fraction 2, obviously the yolk pigments appeared mainly in this
fraction, and that is why the colour parameters are different
between fresh and lyophilized samples.

Table 4
Powerlaw parameters describing frequency sweeps for fractions 1 and 2. Average
values  SD are reported.

Fresh Lyophilized
2
A (kPa) z R A (kPa) z R2
Fig. 6. Frequency sweep of fraction 1 (up) and 2 (down), fresh (square shape) and
Fraction 1 27.12  4.63 9.49  1.28 0.97 4.20  0.90 7.65  0.23 0.99
lyophilized (triangular shape). Full symbols represent storage modulus (G0 ) and empty
Fraction 2 0.83  0.16 3.00  0.80 0.99 2.06  0.13 9.37  0.40 0.99
symbols represent loss modulus (G00 ).
440 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443

100000

10000

G', G'' (Pa)

1000

100

10

1
10 20 30 40 50 60 70 80 90 100
T (C)

10000

1000
G', G'' (Pa)

100

10
10 20 30 40 50 60 70 80 90 100
T (C)
Fig. 8. Cure test of fraction 1 (up) and 2 (down), fresh (square shape) and lyophilized (triangular shape). Full symbols represent storage modulus (G0 ) and empty symbols represent
loss modulus (G00 ).

3.6. Thermal properties
DSC is a technique widely used for the study of thermal tran-
sitions of food proteins since it provides information on the
conversion from native to heat-denaturated state. This conversion
is a phenomenon that is accompanied by a signicant uptake of
heat, which leads to an endothermic peak in the DSC thermogram
(Cordobes, Partal, & Guerrero, 2004). Transition temperatures were
90.7  4.6  C and 86.6  1.2  C for fresh fractions 1 and 2 respec-
tively, and 87.4  6.7  C and 87.9  2.9  C for lyophilized-recon-
stituted fractions 1 and 2 respectively.
Granule proteins (mainly HDLs and phosvitin) have been
demonstrated as being more resistant to heat denaturation and
subsequent aggregation (Le Denmat, Anton, & Gandemer, 1999)
than plasma proteins in which LDLs have been described as heat
sensitive (Anton et al., 2001). The results reported above are in
accordance with this, transition temperature of fresh fraction 1
(granules) was higher than the fresh fraction 2 transition temper-
ature (in fraction 2 are concentrated the heaviest proteins of the
plasma, Fig. 2). However, transition temperatures of both lyophi-
lized fractions were very close one to each other (around 87  C).
Thus, the freeze-drying process seems to affect heat denatur-
ation resistance of HDLs and phosvitin, as can be drawn from the
higher transition temperature of fresh fraction 1 compared to the
lyophilized one.
3.7. Rheological characterization
3.7.1. Stress sweeps
Stress sweep results for fractions 1 and 2 are shown in Fig. 4. As
can be seen, for fraction 1 the linear vicoelastic range goes from 0.1
to 100 Pa approximately, while for fraction 2 is approximately
between 0.1 and 10 Pa, thus the structure resistance is ten times
lower in fraction 2 respect to fraction 1. These different stress
resistance are due both to the high protein content in fraction 1
(since proteins are responsible of structure support), and, also, to
the high lipid content in fraction 2 (since lipids favour the ow).
Both fractions, fresh and lyophilized reconstituted, resulted
more elastic than viscous (G0 value is higher than G00 ) for nearly all
stresses. However, while lyophilization produces a decrease in G0
value for fraction 1, for fraction 2 produces an increase in the
storage module.
As can be seen in Fig. 5, the relation between G0 and G00
(tan d G00 /G0 ) is similar in fraction 2, both fresh and lyophilized
reconstituted. Nevertheless, for fraction 1 this relation is
different in fresh and lyophilized samples. This fact is as well
 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443 441

Fig. 9. SEM micrographs of fractions: (A) fresh fraction 1, (B) fresh fraction 2, (C) lyophilized fraction 1 and (D) lyophilized fraction 2. Magnication is 5000. Bar 10m.

due to the different protein and lipid content of fractions 1 and 3.7.2. Frequency sweeps
2. Since proteins properties are mainly modied by freeze- Frequency sweep results for G0 and G00 of fractions 1 and 2 are
drying process, fraction 1 G0 and G00 relation is more affected by shown in Fig. 6. For fraction 1 both moduli increased slightly when
lyophilization. the frequency increased. It is important to take into account that

Fig. 10. SEM micrographs of coagulated fractions: (A) fresh fraction 1, (B) fresh fraction 2, (C) lyophilized fraction 1 and (D) lyophilized fraction 2. Magnication is 5000. Bar 10m.
442 A. Laca et al. / Food Hydrocolloids 24 (2010) 434443
the value of the G0 module for fresh samples was higher than for the
lyophilized-reconstituted ones, and the same happens with G00
module values, so, again, lyophilization could be responsible of the
modication of granules structure. In the case of fraction 2 the
behaviour is different: the values of the G0 moduli for fresh samples
were lower than those for the lyophilized-reconstituted ones, while
G00 module values were similar. Both moduli increased when the
frequency increased for the fresh sample, but the increase for
the lyophilized sample is much less marked.
Results showed in Fig. 7 are in accordance with those of the
stress sweeps: while the relation between G0 and G00 (tan d G00 /G0 )
is similar in fraction 2, both fresh and lyophilized, in the case of
fraction 1 this relation is different.
The coordination number z is a measure of the number of
rheological units correlated with one another in the three-dimen-
sional structure, while the parameter A is related to the strength of
the interaction between those units (Mancini, Montanari, Peressini,
& Fantozzi, 2002). Low values of z and A mean the tendency of
dispersed droplets to coalesce when the system undergoes
mechanical stress (Peressini, Sensidoni, & de Cindio, 1998).
According to that and, as can be seen in Table 4, fresh fraction 1 is
more stable than fresh fraction 2. However, lyophilization produces
a decrease in A and z values for fraction 1, while in fraction 2 the
opposite effect is observed. Thus, the freeze-drying process seems
to approximate the three-dimensional structure behaviour of
fractions 1 and 2.
3.7.3. Cure tests
The gel point occurs at the temperature when G0 and G00 cross
each other at a given frequency (Cordobes et al., 2004). The gel
point was determined in this way for fresh samples and resulted
in a value of 81.6  6.1  C for fraction 1 and 76.8  1.2  C for
fraction 2.
During heating, storage modulus G0 was nearly constant until
a certain temperature was reached, at which it rapidly increased
indicating transition from a liquid-liked state (sol) to a solid-like
state (gel). This temperature is usually taken as gelation tempera-
ture and is one of the common methods to detect the gelling point
in the absence of a crossover between G0 and G00 (Lamsal, Jung, &
Johnson, 2007). According to it, and, as can be seen in Fig. 8,
lyophilized-reconstituted fractions 1 and 2 starts the transition
from sol to gel at around 70  C. These results agree with those
shown by DSC analysis. Gel point of fresh fraction 1 (granules) was
higher than the one for fresh fraction 2, while gelication
temperature of both lyophilized samples were very similar, which
is also in agreement with DSC results.
3.8. Microstructure
Microstructures of fractions 1 and 2, both fresh and lyophilized,
can be seen in Fig. 9. Close packing structures of protein spheres are
observed in fraction 1, while irregular shapes of both lipid and
protein were found in fraction 2. SEM micrographs showed as well
bigger particle size in fraction 2 (fresh and lyophilized recon-
stituted) compared to fraction 1. These results agree with particle
size measurements (Table 2). Nevertheless, the increase of particle
size caused by the freeze-drying process is easier detected by the
particle size measurements.
Microstructures of coagulated fractions 1 and 2, both fresh and
lyophilized, can be observed in Fig. 10. Again fraction 1 shows a well
organized structure close to that of globular proteins (mainly due to
the granules HDL high content) (Anton, 2007), while fraction 2
shows an irregular structure. Fresh coagulated samples are very
similar to its corresponding lyophilized coagulated samples. Thus,
despite cure tests and DSC results have shown small differences in
coagulation temperatures, the nal structure of the samples seems
to be quite similar between fresh and lyophilized samples.
4. Conclusions
The presented fractionation process allows obtaining three egg
yolk fractions: granules, lipidic paste and watery fraction. Granules
have shown to keep good emulsifying, gelling and other properties
that make them suitable to be used in the same way as egg yolk,
specically in food applications, with the additional advantage of
its lower cholesterol content. The lipidic paste composition makes
it useful to be used as a source of bioactive lipids, being its stress
and heat resistance lower than in the case of granules. The proteins
of the watery fraction can be recovered for different biotechno-
logical purposes. To sum up, the egg yolk fractionation process
shows to be an appropriate method to obtain products with
potential uses in some interesting applications. In addition, freeze-
drying results in a suitable technique to increase the fractions shelf
life with scarcely negative effects on the characteristics of the
obtained products.
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