Food Hydrocolloids 23 (2009) 13661373

Contents lists available at ScienceDirect

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

Oil-in-water emulsion properties of egg yolk: Effect of enzymatic modication by phospholipase A2

Katharina Daimer*, Ulrich Kulozik
Technische Universitat Munchen, Chair for Food Process Engineering and Dairy Technology, Weihenstephaner Berg 1, 85354 Freising, Germany

a r t i c l e i n f o
Article history: Received 2 April 2008 Accepted 2 October 2008 Keywords: Egg yolk Phospholipase A2 Environmental conditions Emulsion Granules

a b s t r a c t
Emulsifying properties of egg yolk were investigated after a treatment with phospholipase A2 (PLA2) where phospholipids (PLs) are converted into lyso-phospholipids. The resulting lyso-PLs are more hydrophilic and therefore show improved emulsifying activity in o/w-emulsions. However, so far no systematic study deals with the changes of egg yolks functionality due to enzymatic treatment. Little is known about the emulsion properties particularly in different environmental conditions. Egg yolks functional behaviour is highly dependent on pH and salt concentration used. Therefore, this study investigated four different environmental conditions. At pH 4 the pH of commercial dressings is simulated and pH 6.5 represents the pH of untreated egg yolk in its natural form. Two salt concentrations are used, where granules are in their native and their disrupted form, i.e. 0.15 and 0.52 M NaCl, respectively. Results suggest that signicant differences in the emulsifying properties of untreated and modied egg yolk not primarily derive from the existence of lyso-phospholipids but from structural changes in egg yolk granules and LDL micelles. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Hens egg yolk is an excellent food emulsier and therefore widely used in many applications ranging from bakery to production of cold sauces and salad dressings. Fresh egg yolk contains about 4850% total dry matter of which 80% is the water-soluble plasma fraction and 20% is present in the form of insoluble granules (McBee & Cotterill, 1979). Plasma is comprised of 85% low-density lipoproteins (LDL) and globular glycoproteins known as a-, b- and g-livetins. LDL, whose apoproteins are called lipovitellins, is the main constituent of egg yolk and represents about 68% of its total dry matter (Burley & Vadehra, 1989; Causeret, Matringe, & Lorient, 1991). Granules contain 16% phosvitin, a phosphoprotein, and 70% high-density lipoprotein (HDL) which apoproteins are called lipovitellins. Twelve percent low-density lipoproteins can be found in insoluble granule aggregates. The size distribution of granules is ranging from 0.3 to 2 mm. The complex between HDL and phosvitin is held together by phosphocalcic bridges. Granules dissociate at higher ionic strengths I > 0.3 M, because of the rupture of phosphocalcic bridges (Aluko & Mine, 1998; Anton, Beaumal, & Gandemer, 2000).

* Corresponding author. Tel.: 49 (0)8161 71 3939; fax: 49 (0)8161 71 4384. E-mail address: Katharina.Daimer@wzw.tum.de (K. Daimer). 0268-005X/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2008.10.002

Studies by Guilmineau and Kulozik (2006a, 2006b) showed that egg yolks emulsifying behaviour is highly dependent on environmental conditions (pH and ionic strength) and that a heat treatment of egg yolk can improve the emulsifying properties. Partially denatured proteins are able to adsorb at the oilwater interface and stabilise emulsions even better than native egg yolk proteins. Further, it was assessed that emulsions prepared with heated egg yolk are less sensitive to variations in pH and ionic strength. The positive effect is attributed to increased steric repulsion between oil droplets when denatured protein aggregates cover the oilwater interface. However, nowadays, not a targeted heat treatment is used in egg industry to enhance egg yolks functionality but an enzymatic treatment by phospholipase A2 (PLA2) (Dutilh & Groger, 1981). PLA2 cuts off the acyl group in position 2 of the triglyceride and converts the phospholipids into lyso-phospholipids, which show a higher solubility in water and therefore improve the emulsifying properties in o/w-emulsions. However, up to now little is reported about the properties of emulsions prepared with the PLA2-modied egg yolk. In accordance with the methodology of the study by Guilmineau and Kulozik (2006b), the present work deals with the emulsifying behaviour of enzyme-treated egg yolk. Characteristics of emulsions like oil droplet size, rheological behaviour, occulation factor and creaming were assessed at four different environmental conditions. No heat treatment was applied to the egg yolk prior to emulsication to investigate the effect of the enzyme alone.

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

1367

The objective of this work was to characterise emulsions prepared with enzyme-treated egg yolk in comparison to emulsions prepared with untreated egg yolk. 2. Materials and methods 2.1. Preparation of egg yolk suspension and enzymatic treatment Freshly laid eggs from Lohman Tradition hens were collected from the Universitys research farm (Thalhausen) and used within 48 h after collection. Each egg yolk was manually separated from the egg white and carefully rolled on a paper towel to remove the albumen completely from the vitellin membrane. The membrane was then cut open with a sharp blade. The content of the yolk was collected and gently homogenized by stirring with a glass rod in a beaker cooled with ice. An aliquot of the egg yolk suspension was incubated with PLA2 (trade name: Lecitase 10L, Novozymes, Bagsvaerd, Denmark) at 55  C for 3 h. At the beginning of incubation pH of the egg yolk suspension was 6.10 0.03. After incubation the pH was 5.85. The concentration of enzyme was calculated on a total dry matter basis. Egg yolk was incubated at a total dry matter of 44%, diluted by an isotonic salt solution containing 0.15 M NaCl, i.e. 0.4 ml enzyme/g egg yolk suspension (Lecitase activity 10.000 Units/ml) was added. The total dry matter and enzyme dosage used simulate the conditions in industrial practice. Upon incubation, the egg yolk suspension was immediately cooled down in an ice water bath and stored at 4  C. During the incubation 84% of all phospholipids were converted into lyso-phospholipids, as described in an earlier study by Daimer and Kulozik (2008). 2.2. Dilution of egg yolk suspensions Egg yolk suspensions (without and with enzymatic treatment) were diluted to a protein concentration of 3.2 mg/ml, corresponding to a total dry matter of 1.12% (w/w), in four different buffers. To achieve a pH of 4 and a pH of 6.5, dilutions were made in a 0.01 M acetate buffer (sodium acetate trihydrate/acetic acid) or a 0.01 M imidazol buffer, respectively. Buffers were adjusted by NaCl in order to obtain a concentration of 0.15 or 0.52 M in the nal dilution. 2.3. Measurement of protein solubility Protein solubility of egg yolk was assessed by measuring the protein content of the whole sample and the supernatant of the same sample after centrifugation. The ratio of protein concentration in the supernatant versus the concentration in the whole sample was dened as solubility. Every sample was diluted in the corresponding buffer to a protein content of 0.8 mg/ml and the dispersions were allowed to equilibrate under mild agitation for 1 h at 20  C. An aliquot of each suspension was kept for total protein content measurement (Pt), and the rest was centrifuged twice at 19,000 g for 20 min. The insoluble matter formed a sediment at the bottom of the tube. The subnatant contained the soluble matter and an aliquot was taken to analyse the protein content (Ps). The protein content was determined using the BCA protein assay kit (Bicinchoninic Acid Protein Assay Kit, Sigma-Aldrich). The percentage of solubility was determined as given in Eq. (1) below.

sunower oil was used as oil phase. A pre-emulsion (oil volume fraction of 0.3) was prepared with an Ultra Turrax (IKA Werke, Staufen, Germany) equipped with a 18 mm dispersing tool (S25KR18 G) for 1 min 15 s at a rotation speed of 8000 rpm. During the rst 15 s, the aqueous phase was mixed alone to obtain a homogeneous solution. During the next 15 s, the oil was carefully poured into the aqueous phase avoiding any foam formation. The agitation was maintained for 45 s, in order to obtain a homogeneous pre-emulsion which was immediately fed through the rst stage of a high-pressure homogeniser (APV 1000; Invensys APV, Albertslund, Denmark). The pressure was 20 MPa at an inlet temperature of 22  C. 2.5. Rheological characterisation A controlled shear rate rheometer model AR1000 from TA Instruments (Eschborn, Germany) was used, equipped with coneplate geometry (d 6 cm; angle: 2 , truncation: 55 mm). Shear rate _ g was increased linearly from 0 to 500 s1 over 2 min, then held at 500 s1 for 2 min and then decreased linearly back to 0 s1 over 2 min. Measurements were carried out at 20  C. The descending part of the ow curve was modelled using the Herschel-Bulkley model (Eq. (2)), where t is the shear stress, t0 the yield stress, K the consistency index and n the ow index.

_ s s 0 K gn

(2)

The consistency index K is used to characterise the ow behaviour. K is a characteristic criterion for the viscosity of a shear-thinning uid. Emulsions with higher viscosity are more stable because droplets are not able to cream so easily. 2.6. Particle size distribution and occulation measurement All particle size measurements were carried out using a laser diffraction particle size analyser, model LS230 (Beckman-Coulter, Germany). In this machine the sample to be measured is dispersed in a transporting uid, which allows to circulate the particles in the measuring cell: the default transporting uid is deionised water (also used to rinse the machine between samples). For the determination of the real particle size distribution, an aliquot (1 ml) of the homogeneous emulsion was dispersed in an excess of 0.5% SDS solution (40 ml) in order to dissociate occulated droplets. The transporting uid used for this measurement is deionised water. The median oil droplet size of the volume distribution is used to characterise the size of the droplets in a sample (d50,3 (SDS)). For the determination of the occulation factor, the composition of the transporting uid was altered in order to reproduce the pH and ionic strength of the continuous phase: this allows measuring a size distribution of occulated oil droplets (d50,3 (SDS)). The comparison of the d50,3-values from samples measured with (SDS) and without SDS (SDS) was used to dene the occulation factor F.

d50;3 SDS d50;3 SDS

(3)

In addition, the specic surface area S (m2/ml oil) was calculated from the average diameter of the surface distribution of the oil droplets (d32 in mm), according to Walstra (1983):

Solubility%

Ps 100 Pt

(1)

S 6=d32
2.7. Interfacial protein concentration

(4)

2.4. Preparation of emulsions The suspensions with a protein concentration of 3.2 mg/ml represent the aqueous phase of the emulsion. Commercial

The method used in this study to separate the oil droplets from the continuous phase of the emulsions was adapted from Patton

1368

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

and Huston (1986). Fresh emulsion (3 ml) was diluted with an equal volume of sucrose solution (200 mg/ml), which was prepared in each of the four environmental conditions used in the study. The dilution (3 ml) was carefully deposited at the bottom of a centrifuge tube containing 8 ml of a buffer solution corresponding to the aqueous phase of the emulsion. These tubes were centrifuged at 1000 g for 1 h at 10  C. After centrifugation, the cream had separated from the translucent subnatant. The tubes were immediately frozen at 40  C, and then cut just beneath the cream phase. The adsorbed and non-adsorbed protein concentration was measured in the cream (supernatant phase) and the subnatant phase, respectively, using the BCA protein assay kit as described above. We checked that at least 90% of the total protein was recovered. The interfacial protein load (G in mg/m2) was calculated based on the measured concentration of adsorbed protein per volume of oil:

100

without PLA2 with PLA2

Protein solubility (%)

80

60

40

20

Adsorbed protein concentrationmg=ml of oil G Specific surface area S m2 =ml of oil

(5)

0 pH [NaCl]

4 0.15 M

4 0.52 M

6.5 0.15 M

6.5 0.52 M

Fig. 1. Protein solubility at different pH and ionic strength of untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk suspensions (n 3).

2.8. Creaming The creaming behaviour was measured under normal gravity by a light scattering optical analyser model Turbiscan MA 1000 (Formulaction, France). Directly after homogenisation a 5 ml sample of the emulsions was lled in a glass tube. The samples were stored at 10  C and measured at various times during 1 week of storage. Within the measurement the backscattering light was recorded over the whole height of sample. At the beginning of the measurement most of the light was back scattered from the fresh homogeneous emulsion. Then, a transparent subnatant phase formed at the bottom of the tube allowing transmission of light and therefore backscattering was reduced. The percentage of creaming is calculated by assessing the backscattering during storage.

Creaming%

Height of clear phase 100 Total height of emulsion

(6)

PLA2, in four different environmental conditions. At pH 6.5 and 0.15 M NaCl, egg yolk is in its natural environmental conditions. Protein solubility for non-treated egg yolk and PLA2-treated egg yolk is 63% and 80%, respectively. When NaCl concentration is increased to 0.52 M NaCl at pH 6.5 proteins of both egg yolk types, non-treated and treated with PLA2, are nearly completely soluble. Most emulsions containing egg yolk have a pH around 4 and therefore this pH is relevant to reect an environment of a dressing or sauce. The lowest protein solubility is observed at pH 4 for nontreated egg yolk as well as enzyme-treated egg yolk. At this, pH granules are insoluble as described by Le Denmat, Anton, and Beaumal (2000). Solubility of granules is responsible for the solubility of whole egg yolk as plasma proteins are soluble in each condition used in this study (Sousa et al., 2007). Modied egg yolk shows higher protein solubility at all environmental conditions used. However, the solubility of enzymatically modied egg yolk still depends on pH and ionic strength. 3.2. Particle size Fig. 2 presents the median oil droplet diameter obtained from o/ w-emulsions when untreated and modied egg yolk was used, again depending on different environmental conditions. Results for

The initial creaming rate is calculated by recording the creaming for 18 h at various times. In the rst day of storage the creaming increased quite linearly per time allowing the calculation as follows:

Initial creaming rate%=h

Creamingin % after 18h 18 h

(7)

median droplet diameter d50,3 (m)

2.9. Statistical analysis Three replicates were carried out. Protein solubility, oil droplet size, occulation, rheological properties, interfacial protein concentration and creaming were measured three times for each trial. The parameters were subjected to a one-way analysis of variance using Statgraphics software (Statistical Graphics Corporation, Rockville, MD) with a condence level of 95% (P < 0.05). A multiple range test was used to determine which means are signicantly different from each other. 3. Results 3.1. Protein solubility The solubility of egg yolks proteins strongly depends on the environmental conditions. Additionally, an impact of enzymatic pre-treatment of egg yolk is observed. Fig. 1 shows the protein solubility level of egg yolk, which is not treated or treated with

6 5 4 3 2 1 0 pH [NaCl]

without PLA2 with PLA2

4 0.15 M

4 0.52 M

6.5 0.15 M

6.5 0.52 M

Fig. 2. Impact of pH and NaCl concentration on the median oil droplet diameter of oilin-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

1369

Flocculation factor [-]

egg yolk which were not treated by PLA2 are in agreement with results of Guilmineau and Kulozik (2006b). The largest droplets are found at more acidic pH and lower ionic strength where granules are insoluble. In this condition a droplet diameter close to 6 mm was found when untreated egg yolk was used. However, a remarkable decrease in droplet diameter is observed when egg yolk was treated with PLA2 prior to emulsication. Further, the droplet diameter does not change when the ionic strength is increased to 0.52 M NaCl at pH 4 and pH 6.5. Therefore, the emulsifying activity of modied egg yolk seems to be independent from ionic strength and only slightly dependent on pH. 3.3. Interfacial protein concentration The interfacial protein concentration was measured for emulsions prepared with untreated egg yolk and enzyme-treated egg yolk at the four environmental conditions as mentioned before. The result is presented in Fig. 3. At pH 4 oil droplets show a signicantly higher protein load when enzyme-treated egg yolk was used. The environmental conditions do hardly have an impact on the interfacial protein load of droplets when enzyme-treated egg yolk is used, since differences between the four conditions are not signicant. Droplets covered with untreated egg yolk show a higher protein load at pH 6.5 than at pH 4. Within the same pH the protein load increases with higher concentration of sodium chloride. 3.4. Flocculation factor The occulation factor between oil droplets was measured in the four environmental conditions when not enzyme-treated and enzyme-treated egg yolk was adsorbed at the interface (Fig. 4). Emulsions from both egg yolk types show higher occulation when NaCl concentration was 0.52 M. At pH 6.5, the level of occulation in emulsions containing enzyme-treated egg yolk is signicantly lower than in emulsions containing untreated egg yolk. However, at pH 4 and 0.52 M NaCl the difference in occulation factor is not as signicant, but a similar trend can be seen. At pH 4 and 0.15 M NaCl occulation seems to be identical for both, untreated and enzymetreated egg yolk. 3.5. Rheological properties The consistency index K of emulsions was measured and is presented in Fig. 5. While K of emulsions prepared with not

7 6 5 4 3 2 1 0 pH [NaCl] without PLA2 with PLA2

4 0.15 M

4 0.52 M

6.5 0.15 M

6.5 0.52 M

Fig. 4. Impact of pH and NaCl concentration on the occulation factor of oil-in-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

enzyme-treated egg yolk is driven by ionic strength, no impact of environmental conditions was found for emulsions formed with enzyme-treated egg yolk. Emulsions containing untreated egg yolk show a high consistency index at high ionic strength of 17 and 19 mPasn at pH 4 and 6.5, respectively. Emulsions containing modied egg yolk show a low consistency index comparable to emulsions at NaCl concentrations of 0.15 M with untreated egg yolk. 3.6. Creaming The initial creaming rate of emulsions during the rst 18 h after emulsication was assessed to measure the immediate impact of the PLA2 treatment. Creaming rates of emulsions containing not modied egg yolk were higher compared to creaming rates of emulsions containing enzyme-treated egg yolk. The highest creaming rate was measured at pH 4 and 0.15 M NaCl for the emulsion without PLA2 which is in accordance with results from Guilmineau and Kulozik (2006b). The lowest creaming rate found for emulsions without PLA2 was 1%/h at pH 6.5 and 0.52 M NaCl. Creaming rates for emulsions with PLA2 were below 1%/h regardless the pH and ionic strength used (Fig. 6).

2,0

25 without PLA2 with PLA2

without PLA2 with PLA2

Interfacial protein load (mg.m-2)

Consistency index (mPa.sn)

4 0.52 M 6.5 0.15 M 6.5 0.52 M

1,5

20

15

1,0

10

0,5

0,0 pH [NaCl]

4 0.15 M

pH [NaCl]

4 0.15 M

4 0.52 M

6.5 0.15 M

6.5 0.52 M

Fig. 3. Impact of pH and NaCl concentration on the interfacial protein load measured in oil-in-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

Fig. 5. Impact of pH and NaCl concentration on the consistency index K of oil-in-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

1370

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

7 6 without PLA2 with PLA2

Initial creaming rate (%/h)

5 4 3 2 1 0 pH [NaCl]

comparison to emulsions containing egg yolk which was not treated by enzyme. Emulsions prepared with egg yolk without PLA2 treatment have already been investigated by Guilmineau and Kulozik (2006b). In their study the inuence of environmental conditions on egg yolk is highlighted in detail. The present study subsequently points out differences between emulsions containing enzyme-treated egg yolk with respect to pH and ionic strength. 4.1. Enzyme-treated egg yolk shows higher protein solubility The higher protein solubility of modied egg yolk results from a breakdown of the highly aggregated granule structure (Daimer and Kulozik, 2008). Granule proteins can be solubilised after modication by PLA2, even when pH and ionic strength are low (Gorshkova, Menschikowski, & Jaross, 1996). At pH 6.5 and 0.52 M NaCl concentration, also proteins in unmodied egg yolk are completely soluble due to the rupture of phosphocalcic bridges. The mechanism of granules breakdown into smaller fragments is not clear yet. It may be similar to the effect of higher salt concentrations where sodium ions replace calcium ions and therefore disrupt phosphocalcic bridges between HDL and phosvitin. More likely, however, is that granules loose their dense aggregated structure due to structural changes within LDL micelles (12% in granules) after enzymatic modication by PLA2 (Hevonoja, Pentikainen, Hyvonen, Kovanen, & Ala-Korpela, 2000). Solubility level of proteins is likely to impact the emulsifying properties of egg yolk. In this study it was of special interest to highlight the difference in functionality of both egg yolks regarding the impact of a treatment by PLA2. The results presented in Fig. 1 lead to the assumption that not only lyso-phospholipids, which result from the enzymatic reaction, should alone be made responsible for the effect on functionality. Our observations support further results from Daimer and Kulozik (2008) that also egg yolks proteins are indirectly affected by PLA2, which primarily acts on phospholipids only. As it seems, the PLA2 reaction causes structural changes of granules and allows proteins to leave the complex granular structure. Hence, higher concentrations of soluble proteins in the continuous phase are available to act as interfacially active components. This, in turn, is expected to improve the emulsifying activity of the egg yolk system. Therefore, the following sections discuss the indirect effect of PLA2 on the emulsication process. 4.2. Emulsifying activity of enzyme-treated egg yolk is improved due to lyso-phospholipids and breakdown of granule structure The experiments were conducted in a way comparable to an earlier study by Guilmineau and Kulozik (2006b) using a moderate energy input (20 MPa, 1-stage homogenisation) and protein concentration. All median oil droplet diameters achieved with modied egg yolk were smaller than with untreated egg yolk. This result reects an increased emulsifying activity of PLA2-treated egg yolk. The droplet size obtained after high-pressure homogenisation can be attributed to the ability of the emulsier to reduce the interfacial tension but also to its capacity to prevent re-coalescence of newly formed droplets within and just after the valve of the homogeniser. The turbulences in the high-pressure homogeniser enhance collisions between newly formed droplets and therefore increase the probability of their re-coalescence. Further, it has been reported that the turbulences obtained in high-pressure homogenisation favour the adsorption of lager molecules, e.g. protein aggregates, because the convective mass transport increases with the size of molecules (Walstra, 1983). The smaller droplet size obtained with PLA2-treated egg yolk indicates that the egg yolk leads to a lower interfacial tension and prevents droplets more effectively from re-coalescence than untreated egg yolk. The lower

4 0.15 M

4 0.52 M

6.5 0.15 M

6.5 0.52 M

Fig. 6. Impact of pH and NaCl concentration on the initial creaming rate (18 h after emulsication) of oil-in-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

The relative cream height measured shows how tight the cream layer has become after 1 week (Fig. 7). The results reect how densely oil droplets are packed in the cream layer. A high relative cream layer therefore indicates that droplets cannot pack very close to each other; the cream layer is then less tight with some free space between droplets. Accordingly, a low relative cream height represents very tightly packed oil droplets. The results show that at low ionic strength the cream is more compact in emulsions without PLA2 than in emulsions containing enzyme-treated egg yolk. At high ionic strength no signicant difference was observed between emulsions with different types of egg yolk. However, without PLA2 the relative cream height is less at more acidic pH compared to pH 6.5 with the corresponding salt concentration. Emulsions with PLA2 showed no signicant difference between the four conditions investigated. 4. Discussion This study has its main focus on the effect of enzymatic treatment of egg yolk and the consequences on the emulsion properties. As little is known about functionality of egg yolk treated with PLA2, emulsion properties were systematically investigated in

100 90 without PLA2 with PLA2

relative cream height (%)

80 70 60 50 40 30 20 10 0 pH [NaCl] 4 0.15 M 4 0.52 M 6.5 0.15 M

6.5 0.52 M

Fig. 7. Impact of pH and NaCl concentration on the relative cream height (170 h after emulsication) of oil-in-water emulsions containing untreated (without PLA2) and enzyme-treated (with PLA2) egg yolk (n 3).

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

1371

interfacial tension may results from the existence of lyso-phospholipids in modied egg yolk after the enzymatic reaction. Lysophospholipids are more hydrophilic than phospholipids and therefore show an improved solubility in the aqueous phase. According to Bancrofts law, lyso-phospholipids are better emulsiers in oil-in-water emulsions than phospholipids due to their higher hydrophilicity. Until now, this is the only effect noted for the enhanced emulsifying properties of PLA2-modied egg yolk. However, the observed effects can be attributed also to the higher protein solubility of modied egg yolk which results from the breakdown of granule structure as discussed in Section 4.1. Anton and Gandemer (1997) reported that a higher solubilisation of granules improves the emulsifying activity. In their study the solubilisation of granules was achieved by high sodium chloride concentrations (>0.3 M). The effect of high ionic strength on dissociation of granules is well known and reported by different authors (Anton et al., 2000; Anton & Gandemer, 1997; Anton, Le Denmat, & Gandemer, 2000; Causeret et al., 1991; Kiosseoglou & Paraskevopoulou, 2005; Le Denmat et al., 2000; Le Denmat, Anton, & Gandemer, 1999; Sirvente et al., 2007). The most pronounced reduction of oil droplet size was found for pH 4 and [NaCl] 0.15 M (Fig. 2). The smaller granule fragments in PLA2-treated egg yolk can cover the interface more completely than large aggregates, e.g. granules in untreated egg yolk, what leads to a lower interfacial tension and therefore a lower droplet size is achieved. Further, this result reects that the emulsifying activity of untreated egg yolk strongly depends on the solubility and the structure of granules and highlights the role of protein monomers which have become soluble due to PLA2 treatment as emulsiers. When ionic strength is increased to 0.52 M NaCl at pH 4 no further reduction of the oil droplet size is achieved when enzyme-treated egg yolk is used, but a remarkable decrease occurs in emulsions containing untreated egg yolk. Guilmineau and Kulozik (2006b) suggested a shear stressinduced dissociation of granules in a high-pressure homogeniser which results in the increased emulsifying activity of granules at pH 4 and 0.52 M NaCl. This is in agreement with ndings of Sirvente et al. (2007) who showed major changes in granule microstructure after mechanical treatment. As granules are disrupted anyway by the pre-treatment with PLA2 no additional effect of the shear stress occurring in the high-pressure homogeniser was observed at 0.52 M NaCl. Overall, this effect leads to the conclusion that granule structure is the decisive factor for the emulsifying activity of whole egg yolk. 4.3. Interfacial protein load of untreated and PLA2-treated egg yolk The protein concentration at the interface of oilwater was measured to verify that proteins are not displaced by the competition with lyso-phospholipids and that a PLA2-induced increase of the emulsifying properties can be assigned to proteins. Egg yolk contains a large number of protein species which can be divided in two categories regarding protein functionality. LDL apoproteins are disordered exible proteins whereas HDL-apoproteins show a globular structure and are strongly aggregated in form of granules. The exible proteins form a thicker and less dense interfacial lm than the globular proteins (Dickinson, 1992). The higher protein concentration at the oilwater interface reects the formation of a dense, thick lm and a high afnity of proteins to the interface when PLA2-modied egg yolk was used (Fig. 3). The conclusion that granular fragments (in egg yolk treated with PLA2) can cover the interface more completely than granule aggregates (in untreated egg yolk) is supported by the results that the interfacial protein concentration is higher at pH 4 when PLA2-treated egg yolk is used (Fig. 3). When egg yolk without PLA2 treatment is used, LDL apoproteins preferentially adsorb at pH 4 and at higher pH the proportion of granules and LDL apoproteins is balanced (Le

Denmat et al., 2000; Mine, 1998). These results show that granules adsorb at the interface particularly when their protein solubility is higher (pH 6.5). Therefore, the higher interfacial protein load at pH 4 can be assigned to the higher protein solubility of granules when treated with PLA2. At pH 6.5 when granules are solubilised anyway because of pH conditions, the protein concentration at the interface of both emulsion types is not signicantly different. Additionally, an interesting point can be found when structural changes of LDL due to treatment with PLA2 are taken into account. Studies on human blood LDL describe a tighter packing of the surface of LDL micelles when one of the fatty acids is cut off. Lyso-phospholipids rearrange at the surface and apoproteins change their conformation (Kleinman et al., 1988). Further, it is reported that particle size of LDL is reduced after treatment with PLA2. In addition to the higher surface rigidity also the hydrophobicity of LDL micelles is increased after PLA2 enzymatic treatment (Hevonoja et al., 2000). This effect increases the afnity of LDL to the interface because particles with higher hydrophobicity adsorb preferentially at the o/w-interface (Cheftel, 1992). Additionally, the exibility of LDL apoproteins is reduced because proteins are tighter packed to the core of the micelle. In unmodied egg yolk LDL apoproteins have a very exible structure and unfold at the interface covering a big surface area with a relatively thin lm (Kiosseoglou & Sherman, 1983). It is likely, that after enzymatic treatment LDL apoproteins are not able to unfold at the interface therefore covering less surface of the oil droplet. In order to cover the whole surface more protein adsorbs at the interface. These results highlight an interesting discussion on interface composition in these emulsions and further studies concerning the adsorption behaviour of egg yolk proteins and phospholipids are required. 4.4. Level of occulation and rheological properties of emulsions made with PLA2-treated egg yolk The tendency of oil droplets to occulate depends on repulsive forces (electrostatic and steric) in relation to attractive forces (e.g. van der Waals, hydrophobic interactions). Proteins are charged polymers and tend to stabilise emulsion droplets against occulation through electrostatic and steric repulsions. The electrostatic repulsive forces are highly dependent on the pH and the electrolyte concentration in the aqueous phase. At high ionic strength the diffuse electrically charged layer around oil droplets is smaller. Therefore, protein-stabilized emulsions are susceptible to occulation when the electrolyte concentration is increased above a critical level (Demetriades, Coupland, & McClements, 1997). This explains the higher occulation factor observed for both egg yolk types at high ionic strength (0.52 M NaCl, Fig. 4). The increase in occulation factor is more pronounced when untreated egg yolk is used. This reects that the attractive forces in emulsions with PLA2treated egg yolk are weaker leading to less occulated droplets. The second stabilisation mechanism that can prevent droplets from aggregation is steric repulsion which is fairly insensitive to environmental conditions of the aqueous phase and depend on the amount of polymer, e.g. proteins, adsorbed at the interface (Hunter, 2000). In our study the interfacial protein concentration at pH 4 and 0.15 M NaCl is signicantly higher for enzyme-treated egg yolk (Fig. 3) but both emulsion types show the same occulation factor (Fig. 4). This result rules out any explanation of the low occulation factor found at pH 4 and 0.15 M NaCl based on steric repulsions. The charge density at pH 4 is very high because the isoelectric point of interfacially active proteins is far (pH 67), and therefore the adsorption of more protein is not expected to produce an additional stabilising effect. Therefore it is concluded, that at pH 4 electrostatic interactions are the main contributor to the repulsive forces in the emulsions investigated. Le Denmat et al. (2000) and Guilmineau

1372

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373

and Kulozik (2006b) have shown in similar environmental conditions (pH 7.0, 0.15 M/0.55 M NaCl and pH 6.5, 0.15 M/0.52 M NaCl, respectively) that the charge of the interface is below 20 mV, what is a critical level evaluated by Friberg (1997) necessary to produce effective electrostatic repulsion. They concluded that at pH 7.0 (and pH 6.5, respectively) steric repulsion comes more into effect than electrostatic repulsion. However, the reduction in occulation factor at pH 6.5 in our study cannot be explained with an increased steric repulsion as interfacial protein concentration is actually not signicantly different between both egg yolk types used for emulsication. Therefore, it is likely that interaction forces between oil droplets are weaker in emulsions containing enzyme-treated egg yolk. The occulated droplets can easily be separated by shear, i.e. during pumping of the emulsion through the particle size analyser. This effect, e.g. deocculation of droplets, is in agreement with the low consistency index measured in the emulsions with PLA2-treated egg yolk throughout all conditions investigated (Fig. 5). The rheological properties of oil-in-water emulsions are largely determined by the interactions between emulsion droplets which, in turn, depend on the structure and composition of the interfacial lm (Dickinson, 1998). As discussed by Guilmineau and Kulozik (2006b) the occulation factor correlates with the rheological properties of the emulsions when untreated egg yolk is used. The high consistency index at high ionic strength is due to an increase of the effective volume of the dispersed phase because the continuous phase is trapped between the occulated oil droplets. This, however, is not observed for emulsions containing enzymetreated egg yolk. When assuming that high occulation leads to a higher consistency index, this parameter would have to change the rheological behaviour of emulsions containing enzyme-treated egg yolk as well. This indicates that attractive forces between oil droplets covered with enzyme-treated egg yolk are weak and occulated droplets from enzyme-treated egg yolk can easily be deocculated during shearing in the gap of the rheological device or separated during circulating in the particle size analyser (see Section 2.6). The deocculated droplets orientate in the shear eld and lead to a low consistency index comparable to emulsions where occulation hardly occurred. 4.5. Stability of emulsions and formation of a compact cream The phenomena occulation and creaming are closely related. Usually occulation leads to enhanced creaming velocity because of the higher effective droplet size, i.e. droplet aggregates. However, in emulsions which are more or less concentrated, e.g. a dispersed oil volume fraction of j 0.3 in our study, droplets can arrange and constitute a network in the continuous phase and occulation can then retard or even prevent creaming. In the rst few hours after emulsication the biggest droplets will start to cream and are therefore responsible for the initial creaming rate. This means that the emulsions with the highest occulation should show the highest creaming rate as they have a bigger effective size than a single droplet. Anyway, in our study no correlation was found between the occulation and the creaming behaviour of the emulsions what supports the conclusion that occulated droplets are easily separated and attractive forces are weak. The initial creaming rate of emulsions containing untreated egg yolk (Fig. 6) correlates with the volume-based median droplet diameter (d50,3) found in these emulsions (Fig. 2) (r2 0.98). As described in Stokes law, the creaming velocity is directly proportional to the droplet diameter squared (d2) meaning that even a small decrease in droplet size can lead to an immense decrease in the creaming velocity, e.g. at pH 4 and 0.15 M NaCl. For emulsions containing PLA2-treated egg yolk no such correlation was found, but the volume-based median diameter is low and also the initial creaming

rate is low and seems to be independent on the environmental conditions. The results shown in Fig. 7 reect how closely droplets can approach each other and therefore give information about the effectiveness of droplet packing. If a network is formed by occulated droplets the height of the nal cream phase (measured after 1 week in this study) is larger because droplets are packed less densely. At pH 4 and 0.15 M NaCl no occulation occurred but droplets covered with enzyme-treated egg yolk are not able to pack as densely as droplets covered with untreated egg yolk (Fig. 7). This seems to be in accordance with the lower droplet size measured in PLA2-treated egg yolk at this condition. The decreased droplet size at constant oil volume fraction leads to a lager number of droplets and more continuous phase can be trapped between the droplets resulting in a less dense cream. At pH 6.5 and 0.52 M NaCl no differences could be observed in the packing of the cream phase (Fig. 7) when untreated or PLA2-treated egg yolk is used, although the occulation factor is much lower for PLA2-treated egg yolk. This supports the conclusion, that attractive forces are weak and the volume-based median droplet diameter is decisive for the creaming behaviour of the emulsions. 5. Conclusion Enzyme-treated egg yolk is an effective emulsier with improved emulsifying activity compared to non-treated egg yolk. This effect results from the existence of lyso-phospholipids after enzymatic reaction and from the higher protein solubility. The functionality of PLA2-treated egg yolk in emulsions is less dependent on the environmental conditions. It was shown that despite a higher occulation factor in emulsions containing high salt concentrations there is no subsequent impact on the emulsion properties, i.e. the rheological and creaming behaviour. Therefore, it is assumed that forces leading to occulation are weak in emulsions containing enzyme-treated egg yolk. Results clearly demonstrate that PLA2-treated egg yolk can improve emulsion properties at low pH and that this effect can be assigned to the higher protein solubility of modied egg yolk. In conditions where egg yolk proteins are completely soluble anyway (pH 6.5 and 0.52 M NaCl), emulsion stability is not further improved by an enzymatic pre-treatment of the egg yolk. Acknowledgment The authors thank Annette Bruemmer-Rolf for her valued contribution in the experimental phase of the work. This research project was supported by the German Ministry of Economics and Technology (via AiF) and the FEI (Forschungskreis der Ernahrungsindustrie e.V., Bonn). Project AiF 14041 N. References
Aluko, R. E., & Mine, Y. (1998). Characterization of oil-in-water emulsions stabilized by hens egg yolk granule. Food Hydrocolloids, 12(2), 203210. Anton, M., Beaumal, V., & Gandemer, G. (2000). Adsorption at the oilwater interface and emulsifying properties of native granules from egg yolk: effect of aggregated state. Food Hydrocolloids, 14(4), 327335. Anton, M., & Gandemer, G. (1997). Composition, solubility and emulsifying properties of granules and plasma of egg yolk. Journal of Food Science, 62(3), 484487. Anton, M., Le Denmat, H., & Gandemer, G. (2000). Thermostability of hen egg yolk granules: contribution of native structure of granules. Journal of Food Science, 65(4), 581584. Burley, R. W., & Vadehra, D. V. (1989). The avian egg: Chemistry and biology. New York: John Wiley & Sons. Causeret, D., Matringe, E., & Lorient, D. (1991). Ionic strength and pH effects on composition and microstructure of yolk granules. Journal of Food Science, 56(6), 15321536. Cheftel, J. C. (1992). Lebensmittelproteine. Hamburg: Behrs Verlag.

K. Daimer, U. Kulozik / Food Hydrocolloids 23 (2009) 13661373 Daimer, K., & Kulozik, U. (2008). Impact of a treatment with phospholipase A2 on the physico-chemical properties of hen egg yolk. Journal of Agricultural and Food Chemistry, 56(11), 41724180. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997). Physicochemical properties of whey protein-stabilized emulsions as affected by heating and ionic strength. Journal of Food Science, 62(3), 462467. Dickinson, E. (1992). Structure and composition of adsorbed protein layers and the relationship to emulsion stability. Journal of the Chemical Society-Faraday Transactions, 88(20), 29732983. Dickinson, E. (1998). Proteins at interfaces and in emulsions stability, rheology and interactions. Journal of the Chemical Society-Faraday Transactions, 94(12), 16571669. Dutilh, C. E., & Groger, W. (1981). Improvement of product attributes of mayonnaise by enzymic-hydrolysis of egg-yolk with phospholipase-A2. Journal of the Science of Food and Agriculture, 32(5), 451458. Friberg, S. E. (1997). Chapter 1: Emulsion stability. In S. E. Friberg, & K. Larson (Eds.), Food emulsions (pp. 155). New York: Marcel Dekker, Inc. Gorshkova, I. N., Menschikowski, M., & Jaross, W. (1996). Alterations in the physicochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2. A spin-label study. Biochimica et Biophysica Acta (BBA) Lipids and Lipid Metabolism, 1300(2), 103113. Guilmineau, F., & Kulozik, U. (2006a). Impact of a thermal treatment on the emulsifying properties of egg yolk. Part 1: Effect of the heating time. Food Hydrocolloids, 20(8), 11051113. Guilmineau, F., & Kulozik, U. (2006b). Impact of a thermal treatment on the emulsifying properties of egg yolk. Part 2: Effect of the environmental conditions. Food Hydrocolloids, 20(8), 11141123. Hevonoja, T., Pentikainen, M. O., Hyvonen, M. T., Kovanen, P. T., & Ala-Korpela, M. (2000). Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modied LDL. Biochimica et Biophysica Acta (BBA) Molecular and Cell Biology of Lipids, 1488(3), 189210. Hunter, R. J. (2000). Introduction to modern colloid science. Oxford, New York: Oxford University Press.

1373

Kiosseoglou, V. D., & Sherman, P. (1983). The inuence of egg-yolk lipoproteins on the rheology and stability of o/w emulsions and mayonnaise. 2. Interfacial tension-time behavior of egg-yolk lipoproteins at the groundnut oilwater interface. Colloid and Polymer Science, 261(6), 502507. Kiosseoglou, V., & Paraskevopoulou, A. (2005). Molecular interactions in gels prepared with egg yolk and its fractions. Food Hydrocolloids, 19(3), 527532. Kleinman, Y., Krul, E. S., Burnes, M., Aronson, W., Peger, B., & Schonfeld, G. (1988). Lipolysis of LDL with phospholipase A2 alters the expression of selected apoB100 epitopes and the interaction of LDL with cells. Journal of Lipid Research, 29(6), 729743. Le Denmat, M., Anton, M., & Beaumal, V. (2000). Characterisation of emulsion properties and of interface composition in o/w emulsions prepared with hen egg yolk, plasma and granules. Food Hydrocolloids, 14(6), 539549. Le Denmat, M., Anton, M., & Gandemer, G. (1999). Protein denaturation and emulsifying properties of plasma and granules of egg yolk as related to heat treatment. Journal of Food Science, 64(2), 194197. McBee, L. E., & Cotterill, O. J. (1979). Ion exchange chromatography and electrophoresis of egg yolk. Journal of Food Science, 44(3), 656660. Mine, Y. (1998). Emulsifying characterization of hens egg yolk proteins in oil-inwater emulsions. Food Hydrocolloids, 12(4), 409415. Patton, S., & Huston, G. E. (1986). A method for isolation of milk fat globules. Lipids, 21(2), 170174. Sirvente, H., Beaumal, V., Gaillard, C., Bialek, L., Hamm, D., & Anton, M. (2007). Structuring and functionalization of dispersions containing egg yolk, plasma and granules induced by mechanical treatments. Journal of Agricultural and Food Chemistry, 55(23), 95379544. Sousa, R. D. C., Coimbra, J. S. R., Garcia Rojas, E. E., Minim, L. A., Oliveira, F. C., & Minim, V. P. R. (2007). Effect of pH and salt concentration on the solubility and density of egg yolk and plasma egg yolk. LWT Food Science and Technology, 40(7), 12531258. Walstra, P. (1983). Formation of emulsions. In P. Becher (Ed.), Encyclopedia of emulsion technology: Basic theory, Vol. 1 (pp. 57127). New York: Marcel Dekker Inc.