A rheological characterisation of liquid egg albumen

A rheological characterisation of liquid egg albumen
                a                   a                    a                     b                   a
Ruth Cardinaels , Joris Van de Velde , Wouter Mathues , Paul Van Liedekerke , Paula Moldenaers

a
  Soft Matter, Rheology and Technology, Department of Chemical Engineering, KU Leuven, Willem de Croylaan 46 - box
2423, BE-3001 Leuven, Belgium
b
  BIOSYST-MeBioS, KU Leuven, Willem de Croylaan 42 - box 2428, BE-3001 Leuven, Belgium

ABSTRACT

A substantial fraction of all produced eggs is industrially processed to egg-derived products. Automated
egg processing equipment is used for egg breaking, separation of egg white and yolk, homogenisation,
pasteurisation, etc. Process optimisation requires knowledge of the rheological properties of egg albumen
and yolk. In addition, rheology is a potential tool for the assessment of egg quality. Nevertheless, up to
now, the rheology of liquid egg has received little attention. In the present work, the rheology and surface
tension of liquid egg albumen is systematically characterized. As thin and thick egg white differ
significantly in their rheological behaviour, they are separated before characterisation. In addition, the
egg breaking, separation of the different egg fractions and sample loading is performed in such a way that
minimal damage to the protein structure occurs, thereby allowing characterisation of the native egg
white. In shear, the thin egg white behaves dominantly viscous, with a limited elastic contribution. Its
viscosity is slightly shear thinning, with values around 10 mPas. The thick egg white on the other hand is
a weak and soft gel with a rather low critical strain for the onset of non-linear behaviour. This egg white
has a yield stress and its viscosity is at least 10 times higher than that of thin egg white, with a significant
amount of shear thinning. Interestingly, by applying suitable flow protocols, the egg network structure
can be broken down, subsequently, it slowly builds up under rest conditions. Storage of eggs for a week at
ambient conditions leads to a significant reduction of the stiffness of the thick egg white whereas storage
for a week at 4 °C does not affect the dynamic moduli. The breakup of liquid threads of egg white is
studied with a capillary breakup extensional rheometer for both thin and thick egg white. The egg white
shows pronounced elastic behaviour in extensional flow, which becomes clear from, among other things,
the formation of so-called bead-on-a-string structures. Thin liquid egg filaments show a surprisingly high
resistance against breakup. In addition to the rheology, the dynamics of free surface flows of liquid egg
such as those occuring when eggs are opened and emptied, is also affected by the surface tension. The
presence of proteins in the liquid egg albumen reduces the surface tension considerably as compared to
that of water, demonstrating the interfacial activity of the albumen proteins.

1    Introduction

        The eggs of avian species consist of a shell, several membranes, yolk and egg albumen or egg white
(Shenstone 1968). The latter two fractions of the egg provide an excellent source of nutrients. In addition,
several egg proteins are known to have bioactive properties such as antimicrobial and antiviral activity
(Huopalahti et al. 2007). Hence, eggs and egg-derived products are a frequently consumed and valuable dietary
component. Globally, the amount of eggs that is consumed as processed egg products rather than shell eggs
exceeds one third of the total egg consumption and this ratio still continues to increase (Froning 2008). To
produce liquid, frozen or dried egg products, several processing operations including egg breaking, separation of
egg white and yolk, homogenisation, pasteurisation, etc. have to be performed. Optimisation of the different
processing steps requires knowledge of the rheological properties of egg albumen and yolk. The egg yolk
consists of protein granules suspended in an aqueous plasma (Huopalahti et al. 2007). The egg white can be
further separated in thin and thick egg white, which both contain soluble and insoluble egg proteins in an
aqueous matrix (Shenstone 1968). The thick egg white is a gel-like material and its consistency is crucial for the
egg quality perceived by consumers. A frequently used indicator for albumen freshness is the Haugh unit, which
is based on a measurement of the height of the thick albumen after pouring the liquid egg content on a flat
surface (Shenstone 1968). However, rheology is also a potential tool for the assessment of egg quality. In
addition to the rheology, the dynamics of free surface flows of liquid egg such as those occuring when eggs are
opened and emptied, is also affected by the surface tension.

      Several authors determined the shear viscosity of liquid egg white (Tung et al. 1970; Robinson and
Monsey 1972; Pitsilis et al. 1975; Lang and Rha 1982; Pitsilis et al. 1984; Lusicano et al. 1996; Atilgan and
Unluturk 2008; Kemps et al. 2010). These studies show that the viscosity of liquid egg albumen is shear

InsideFood Symposium, 9-12 April 2013, Leuven, Belgium                                                 1|P a g e

thinning; it decreases with increasing shear rate (Tung et al. 1970). The viscosity values found by the different
authors vary between 10 and a few hundred mPas. Variation between measurement results is most probably
caused by discrepancies in sample preparation and measurement protocol. Whereas some authors characterize
thick and thin liquid egg albumen separately (Robinson and Monsey 1972; Lang and Rha 1982; Kemps et al.
2010), others combine both egg white fractions (Tung et al. 1970; Pitsilis et al. 1975; Pitsilis et al. 1984;
Lusicano et al. 1996;Atilgan and Unluturk 2008). In addition, the egg white is often blended or mixed before
rheological characterisation (Robinson and Monsey 1972; Pitsilis et al. 1975; Pitisilis et al. 1984; Lusicano et al.
1996; Kemps et al; 2010). Finally, the applied flow protocols differ in the used shear rates, order of shear rates
and the presence of a preshear step. As it is shown that liquid egg white is a thixotropic material for which the
viscosity is time-dependent (Tung et al. 1970; Lang and Rha 1982), the measured viscosity will depend on the
applied preconditioning. Based on fits of the flow curves of liquid egg white, some authors suggest that liquid
egg white has a yield stress with a value of the order of 0.1 Pa (Tung et al. 1970; Atilgan and Unluturk 2008).
Alamprese et al. (2012) characterized the linear viscoelastic behaviour of blended thin and thick liquid egg
white in small amplitude oscillatory shear at frequencies between 1 and 10 Hz. In this region, liquid egg white
behaves dominantly elastic. In the present work, the rheology and surface tension of liquid egg albumen is
systematically characterized. As thin and thick egg white differ significantly in their rheological behaviour, they
are separated before characterisation. In addition, the egg breaking, separation of the different egg fractions and
sample loading is performed in such a way that minimal damage to the protein structure occurs, thereby
allowing characterisation of the native egg white.

2     Materials and Methods

2.1    Liquid egg albumen
       Fresh eggs, no older than three days after laying were stored at 4°C for a week before the experiments.
The eggs were manually broken. Thick egg white was collected after draining the thin egg white through a filter
with a pore size of 1 mm. Simultaneously, the egg yolk was removed from the thick egg white by suction. The
thin egg white used for the characterisation was collected by pouring the egg on a flat surface, after which the
thin egg white flows out and can be filled in a syringe.

2.2    Determination of surface tension
       The surface tension of the egg albumen was determined by means of the pendant drop method with a
CAM-200 device at ambient temperature. With this device, a droplet of liquid egg albumen hanging from a steel
needle was visualised with a camera. Evaporation of the water from the egg albumen was prevented by inserting
the droplet in a cuvette that was saturated with water vapour, as shown in Figure 1a. The droplet profile of a
pendant droplet is determined by a force balance between the interfacial tension and the gravity, leading to the
following equation for the interfacial tension :
                                                        R 2  g
                                                                                                               (1)
                                                           
With  the density difference between the liquid and the surroundings, R the radius of droplet curvature at the
apex, g the gravitational constant and  a shape factor that is defined by the Young-Laplace equation for a
pendant droplet. The density of the egg albumen was determined with a pycnometer at ambient temperature. By
analyzing the droplet profile as a function of time and fitting it with the Young-Laplace equation, the time
evolution of the surface tension was obtained from Eq. 1.

2.3    Shear rheology
       The shear rheology of the liquid egg albumen was characterized with a stress-controlled rotational
rheometer (Anton Paar MCR501). The temperature was kept constant at 25 °C with a Peltier. A double-wall
couette geometry (Dcup = 27,6 mm and Dbob = 26,7 mm) was used for the thin egg white. The thick egg white
was gently poured in a cup attached to the rheometer, after which a parallel plate geometry (diameter D = 40
mm) was gently lowered until it reached the sample. The setup for the shear rheology of the thick egg white is
shown in Figure 1b. For the thick egg white, a resting period of 1400s was applied after loading, during which a
very small increase in stiffness of the egg white was noticed. This is most probably caused by a small amount of
structure breakdown during manipulation and loading, which leads to structure regeneration in rest conditions.
After this rest period, either oscillatory or steady shear tests were performed on the egg white. The former allow
for the characterisation of the viscoelastic behaviour of the egg white whereas the latter provide the flow
behaviour and yield stress. For each test, egg white from a new egg was inserted in the rheometer.

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2.4     Capillary thinning extensional rheology
        The capillary thinning experiments were performed with a Haake Caber 1 (Thermo Scientific) at ambient
temperature. The egg white was gently filled in a syringe and then slowly applied between two concentric disks.
It was checked that the needle diameter of the syringe and the emptying speed of the syringe did not affect the
experimental results. The cylinder of fluid, initially placed between the two disks (diameter D0 = 4 mm), was
then rapidly stretched by moving the upper disk upwards in a step strain. This way, the height of the fluid
filament is increased almost instantaneously from Li = 2 mm to Lf = 6 mm, while the thickness of the filament
decreases, as schematically depicted in Figure 1c. Subsequently, the surface tension provides the driving force
for further thinning and eventual breakup of the fluid filament. The thinning filament was visualised with a high
speed camera (Photron Fastcam SA2) at a frame rate of 3000/s. The illumination was provided by a continuous
fiber optic light source. The spatial resolution of the setup is 0,1 pix/m. The thinning process is quantified by
the time evolution of the minimum filament diameter Dmin. The Matlab image processing toolbox was used to
determine the minimum filament diameter by combining a modified Marr-Hildreth edge detection algorithm
with a line detection protocol.

               (a)                                     (b)                                          (c)

Fig. 1 Setups for (a) determination of surface tension, (b) shear rheology and (c) extensional rheology.

3     Results and discussion

3.1     Surface tension
        The surface tension of the thin and thick egg white was determined as a function of time. For both types
of egg white, an equilibrium value was reached after about 600s. The thick egg white has an equilibrium surface
tension of 50,3  2,2 mN/m and the thin egg white has an equilbrium surface tension of 46,1  0,4 mN/m. The
fact that these values are considerably lower as compared to that of water, indicates that the proteins in the egg
white are interfacially active, thereby reducing the surface tension. The rather comparable values for thin and
thick egg white can be explained by the similar protein content of both types of egg white. Hence, the slightly
higher ovomucin content of the thick egg white, which is generally accepted to be responsible for its gel-like
properties (Huopalahti et al. 2007), appears to have no major effect on the surface tension. It should however be
noted that in case of the thick egg white, the effect of the yield stress on the surface tension determination by
means of the pendant drop method can not be completely excluded. This contribution will be investigated in
more detail in further work.

3.2     Shear rheology
        First, the viscoelastic behaviour of the native thin and thick egg white was characterized. Thereto, an
oscillating strain with a strain amplitude in the linear viscoelastic region was applied, leading to dynamic moduli
that are independent of the applied strain. The dynamic moduli of the thick egg white are shown in Fig. 2. Fig.
2a provides the storage or elastic modulus whereas Fig. 2b shows the loss or viscous modulus. These figures
clearly show that thick egg white rheologically behaves as a weak gel; storage and loss modulus are parallel
with a dominant elastic contribution. In addition, the gel is rather soft, as evidenced from the moderate values of
both storage and loss modulus, as compared to the typical gel stiffness of e.g. pectin gels (Ngouémazong et al.
2012). This rheological behaviour corresponds to the presence of a weak protein network, in which structural
relaxations occur over a whole range of relaxation times, including very long ones. The spread on the dynamic
moduli values is caused by egg to egg variability, which was even present in eggs that were collected from a
single hen. After storage of the eggs for 2 weeks at 4 °C, the average modulus (of 5 eggs) was not affected.
However, when the eggs were stored at ambient temperature, the elastic gel structure was liquefied to a large
extent and the stiffness of the gel became too low to be measured with the present shear rheometry setup.

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Interestingly, by applying shear, it was also possible to break down the gel structure. In this case, structure
regeneration as evidenced from a gradual increase in the storage modulus occurred (results not shown), showing
that the shear-induced structure breakdown is reversible in contrast to the irreversible liquefaction during
storage. In the frequency range of 0,1 – 10 rad/s, the thin egg white showed the characteristics of a low viscous
liquid with a minor elastic contribution (results not shown). This clearly indicates that the proteins in the thin
egg white do not organize in a network structure.

                               (a)                                                               (b)

Fig. 2 Dynamic moduli in small amplitude oscillatory shear of thick albumen: (a) storage modulus and (b) loss modulus.

        As a weak gel such as the thick egg white is expected to exhibit a yield stress, creep tests at different
stress values were performed to determine the stress level needed to induce flow of the egg white. The results
for several eggs are presented in Fig. 3. At low stress values the egg white behaves as an elastic solid, with a
deformation of on average 132% at a stress of 0,1 Pa. The transition to viscous flow occurs at a yield stress that
is situated between 0,5 and 3 Pa. This value is slightly higher as compared to that predicted in literature from
extrapolations of the flow curves (Tung et al. 1970; Atilgan and Unluturk 2008). After storage for a week at
room temperature, the yield stress was not altered, despite the decrease in stiffness of the protein network
(results not shown). The presence of such a yield stress is of importance in egg processing operations such as
collection of the liquid egg content from shell eggs or separation of egg white and yolk, as the processes should
be designed as such that the minimum stress level exceeds the yield stress in order to induce flow. The thin egg
white does not exhibit a yield stress.

Fig. 3 Creep tests to determine the yield stress of thick egg albumen. The applied stresses are indicated in the figure.

       Once the yield stress is exceeded, the thick egg white behaves as a viscous liquid. The viscosity as a
function of shear rate is shown in Fig. 4 for the thin (Fig. 4a) and the thick (Fig. 4b) egg white. The thin egg
white is a very low viscous liquid with a viscosity between 5 and 10 mPas (at 25°C). In the investigated range of
shear rates, the viscosity curves can be described by means of a power law:
                                                             . n 1
                                                     k                                                                 (2)

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The thin egg white has a power law exponent n of 0,86  0,04, which indicates that the flow behaviour is very
similar to that of an ideal Newtonian liquid. The thick egg white on the other hand has a viscosity that is several
orders of magnitude larger than that of the thin egg white. In addition, the power law exponent n is 0,600,08,
which indicates that the shear thinning is much more pronounced in the thick egg white as compared to the thin
egg white. This is most probably caused by the fact that structural breakdown occurs in the thick egg white
when the shear rate is increased. This is confirmed by the fact that the flow curves exhibit hysteresis when the
viscosity is measured while applying the shear rates in increasing order followed by decreasing order (results
not shown). A more detailed investigation of the effects of shear on the egg white structure is in progress at
present.

                              (a)                                                           (b)

Fig. 4 Viscosity versus shear rate curves for (a) thin and (b) thick egg albumen.

3.3     Capillary thinning extensional rheology
       Finally, the extensional rheology of the thin and thick egg white was studied by means of capillary
thinning of liquid egg filaments. A typical image sequence of a thinning filament of thin egg white is shown in
Fig. 5a. The evolution of the minimum diameter as a function of time is shown in Fig. 5b. Similar to the results
in shear, a considerable amount of egg to egg variability can be seen in Fig. 5b, which is not present when
samples from the same egg are compared. Both the straight edges of the thinning filament and the exponential
decrease of the minimum filament diameter indicate that the thinning dynamics is dominated by elasticity. In
this case, the minimum diameter as a function of time can be described by the following equation (Anna and
McKinley 2001):
                                                               t 
                                                 Dmin     exp                                                         (3)
                                                               3  ext 
in which ext is the dominant relaxation time of the liquid in extensional flow. For the thin egg white, a value for
ext of 7823 ms was obtained. The apparent extensional viscosity that can be extracted from the time evolution
of the filament diameter (Anna and McKinley 2001), provides a Trouton ratio (ext/) that is orders of
                        (a)                                                       (b)

Fig. 5 Extensional rheology of thin egg white: Time evolution of (a) filament profile and (b) minimum diameter.

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magnitude larger than that of Newtonian liquids. In addition, the extensional viscosity shows strain hardening
(results not shown), caused by an increasing resistance of the progressively more extended proteins. Finally, at
the later stages of filament thinning, beads-on-a-string structures were formed, which is a typical phenomenon
occuring in highly elastic low viscous threads (Bhat et al. 2010).

4   Conclusions

     In this work, a systematic characterisation of the surface tension and rheology of thin and thick egg white
has been performed. The egg breaking, separation of the different egg fractions and sample loading was done in
such a way that minimal damage to the protein structure occurs, thereby allowing characterisation of the native
egg white. The thick egg white is a soft and weak gel that has a yield stress of 0,5 – 3 Pa. Storage at room
temperature leads to an irreversible liquefaction of the gel structure. On the other hand, application of shear
leads to a reversible structure breakdown. The protein network of the thick egg white is rather shear-sensitive,
leading to strong shear thinning of the viscosity. The thin egg white on the other hand is a low viscous liquid for
which the viscosity exhibits limited shear thinning. Despite the fact that the thin egg white can be described as a
low viscous liquid in shear, the extensional thinning of a filament of thin egg white is dominated by elasticity,
which stabilizes the thin egg white threads. Further work will focus on the effects of shear on the egg white
protein structure and constitutive modelling of the rheological data of liquid egg white.

Acknowledgements

      R. Cardinaels is indebted to the Research Foundation Flanders (FWO) for a postdoctoral Fellowship. W.
Mathues acknowledges financial support from the ERC starting grant no. 203043-NANOFIB. Lodewijckx NV
(Cocovite) is thanked for providing the eggs.

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