Journal of Dispersion Science and Technology

ISSN: 0193-2691 (Print) 1532-2351 (Online) Journal homepage: http://www.tandfonline.com/loi/ldis20

Ovalbumin at oil-water interfaces: Adsorption and

emulsification

Bhawna Panjwani, Sharad Gupta & Prachi Thareja

To cite this article: Bhawna Panjwani, Sharad Gupta & Prachi Thareja (2017): Ovalbumin at oil-
water interfaces: Adsorption and emulsification, Journal of Dispersion Science and Technology,
DOI: 10.1080/01932691.2017.1384387

To link to this article: http://dx.doi.org/10.1080/01932691.2017.1384387

Accepted author version posted online: 06

Oct 2017.

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 Ovalbumin at oil-water interfaces: Adsorption and

emulsification

Bhawna Panjwani

Chemical Engineering, Indian Institute of Technology, Gandhinagar, India

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Sharad Gupta

Biological Engineering, Indian Institute of Technology, Gandhinagar, India

Prachi Thareja*

Chemical Engineering, Indian Institute of Technology, Gandhinagar, India

*Address correspondence to Prachi Thareja, Chemical Engineering, Indian Institute of

Technology, Gandhinagar, India. E-mail: prachi@iitgn.ac.in

ABSTRACT

We study the adsorption of protein ovalbumin (OVA) at Corn Oil (CO), Soybean Oil

(SBO), Olive Oil (OO) and water interfaces along with the emulsification of these oils in water.

The dynamic interfacial tension (IFT) measurements show a reduction in IFT in the order SBO -

water ~ CO-water > OO-water, with OVA adsorption being dominated by the free diffusion of

OVA at the interfaces. CO-water, OO-water and SBO-water emulsions cream with time. The

cream phase consists of jammed closed packed oil droplets due to depletion induced inter-droplet

attractions with higher G′ and G′′ (~700Pa ) for emulsions with 1 wt% OVA.

1
 GRAPHICAL ABSTRACT
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KEYWORDS: emulsions, interface, ovalbumin, proteins, rheology

1. Introduction

Emulsions are thermodynamically metastable materials, which become unstable over

time depending on the properties of the bulk and dispersed phase, the nature, and concentration

of the emulsifiers, the storage and application conditions. [1] Oil-in-water emulsions are common

in foods such as mayonnaise, butter, and salad dressings. Also, oil-in-water emulsions with

vegetable oils are used to reformulate the meat products. The fat content is reduced by replacing

animal fats with vegetable oils which have a low proportion of saturated fatty acids and high

proportions of mono or polyunsaturated fatty acids.

Proteins are the most widely used emulsifiers in food grade oil-in-water emulsions. [2,3]

The adsorption of the proteins at the oil-water interface is known to stabilize the oil-in-water

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 emulsions by lowering the oil-water interfacial tension along with forming a viscoelastic layer at

the interface. [4,5] Baldursdottir et al. and Yampolskaya et al. report that the optimum

concentration of proteins leading to stable oil-in-water emulsions depends on the isoelectric

point, molecular weight, molecular structure, and flexibility of the protein molecules. [6,7]

Techniques of dynamic surface tension, interfacial dilational rheology, neutron reflectivity, and

spectroscopy have been used to understand the structure of the proteins at the interface. [8–15]
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Oil-in-water emulsions are also subjected to the instability of creaming and flocculation

of droplets when the protein concentration is lower or higher than that corresponding to the

complete interfacial coverage of the oil droplets. [16,17] At low protein concentrations, incomplete

interfacial coverage of oil droplets leads to bridging induced droplet flocculation while the high

protein concentrations result in the depletion induced flocculation of droplets. [18]

The depletion induced interaction potential is described with a model first proposed by

Asakura and Oosawa. [19] The model considers a solution of non-adsorbing macromolecules

(small hard spheres) between two plates or two large hard spheres as a function of the separation

distance. When the separation between the surfaces of the plates or large spheres decreases, the

space between the two surfaces eventually becomes smaller than the size of the macromolecules

(or small spheres). The concentration of non-adsorbed entities increases outside of the gap

resulting in an osmotic pressure gradient between the bulk solution and the interstitial space. As

a result, the colloidal spheres/plates are pushed into contact. [19,20] Experiments with colloid-

polymer mixtures have shown that depletion induced attraction can cause phase separation

leading to colloid-rich and colloid-poor phases. [15,21]

Experimental studies on the model oil-in-water emulsions demonstrate that the depletion

attractions between droplets can be induced by non-adsorbing surfactant micelles,

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 polysaccharides that are excluded from the interstitial space. [18,22–25] Also, dispersions of casein

micelles (i.e., skim milk) have been shown to exhibit depletion induced droplet flocculation due

to un-adsorbed casein or addition of various non-adsorbing polysaccharides. [26–30] These results

indicate that delicate balance between the protein and oil concentration is required to form stable

emulsions with long shelf lives.

Despite their widespread use as salad dressings and fat substitutes, limited studies have
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been published to understand the influence of dietary proteins on the stabilization, flocculation,

and creaming of droplets in vegetable oil-in-water emulsions. This work aims to investigate the

influence of globular protein ovalbumin (OVA) concentration on creaming and flocculation of

food grade oil-in-water emulsions with 10 wt% Corn Oil (CO), Soybean Oil (SBO) and Olive

Oil (OO). First, the kinetics of adsorption of OVA at the CO-water, OO-water and SBO-water

interfaces is investigated with dynamic interfacial tension measurements. The Serrien model is

used to link the measurements of dynamic interfacial tension to the diffusivity of OVA, coverage

of OVA at oil-water interfaces. Our results indicate that CO-water, OO-water, SBO-water

emulsions show creaming. The cream phase is further characterized by microscopy to understand

the influence of the concentration of OVA and the distribution of the size of droplets on the

mechanical properties of the cream phase.

2. Materials and methods

2.1 Materials

OVA is a globular protein obtained from the chicken egg white, belonging to the serpin

superfamily with 385 amino acids. It is supplied by Sigma Aldrich and is used as received (MW

44.3kDa ). CO, SBO, and OO are obtained from the local market and are used without further

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 purification. All the studies are carried out using ultrapure water from a Millipore system (18.2

MΩ.cm at 25°C). The composition and properties of the oils are summarized in Table 1:

2.2 Methods

2.2.1 Sample preparation

An OVA stock solution is prepared by dissolving OVA in water at a concentration of

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15mg /ml by stirring at 1000rpm for 3 hrs. The resulting OVA solution is then centrifuged at

5000rpm for 15min to remove any impurities. The supernatant OVA solution is then further

diluted to the desired concentration (0.1 - 1 wt %) at a temperature of 25°C.

15g of oil-in-water emulsions are prepared with 10 wt% oil in 30ml glass vials. The OVA

stock solution is diluted with water to obtain the desired OVA concentration, then 10 wt% oil is

added and then stirred with a high power homogenizer (from Wiggen Hauser) at 8000rpm for

4min.

2.2.2 Dynamic interfacial tension (IFT) measurement

The IFT measurements are carried out using the One Attension Theta Optical

Tensiometer (Biolin Scientific, Finland). The instrument uses the pendant drop method to record

the IFT data as a function of time. To calculate the IFT between oil and water in the presence

OVA, 2.5ml of oil is poured in the cuvette. A drop of aqueous OVA solution is then suspended

in the oil filled cuvette with a syringe needle. The image of the drop is captured with a USB 3

digital camera, and the profile of the shape of the drop is fitted using the Young-Laplace

equation to calculate the IFT. Each IFT measurement is performed for 1800sec.

2.2.3 Turbidimetry

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 The serum phase of the emulsion is diluted 100 times, and the absorbance (A) is

measured at 600 nm in a cuvette with a path length (l) of 1cm. The turbidity (T) of the serum

phase is calculated from [31]

2.303 A
T (1)
l

2.2.4 Optical microscopy

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The microscopic images of the emulsions are observed with an optical upright polarizing

microscope (Carl Zeiss AxioScope A1) in bright field mode. The images are recorded as soon as

the emulsions are created corresponding to no phase separation (t0 ) and after 24 hrs (t24 ). For the

emulsions showing segregation of phases, the images of the serum phase and the cream phase are

recorded separately.

2.2.5 Rheology of cream phase

The samples after the emulsification are allowed to age for 24 hrs after which they are

centrifuged at 10000rpm for 15min for the complete separation of the oil droplets (cream phase)

from the serum phase. This cream phase is then used for rheological studies. The dynamic

rheology measurements of the cream phase are carried out using stress and strain controlled

Modular Compact Rheometer (Anton Paar MCR 302) using a parallel plate geometry (diameter,

25mm ) and a gap of 0.5mm. The evolution of the storage modulus (Gʹ) and loss modulus (Gʹʹ)

of the cream phase are recorded as a function of angular frequency () in the range ω = 100 -

0.1rad /s and at an oscillatory strain of 0.5%.

3. Results and discussion

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 3.1 Adsorption kinetics of OVA at SBO-water, CO-water and OO-water

interfaces

The dynamic IFT (σ) of CO-water, OO-water and SBO-water interfaces with variable

OVA concentrations of 0.1 – 1 wt% is measured (Figure 1). As shown, σ of CO-water, SBO-

water, and OO-water interface begins to decrease instantly and rapidly falls till 200 – 400sec in

the presence of OVA. The decline in σ then slows down and reaches almost a plateau in 1800sec.
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The plateau of σ is called equilibrium IFT (σ eq). It is also evident that σ eq decreases with

increasing OVA concentration for the OO-water and SBO-water interfaces but remains almost

constant for the CO-water interface. The OO-water interface shows the minimum decrease of σ

followed by a similar decrease of σ for CO-water and SBO-water interface. Subsequently, the

surface pressure π  t    σ0   σt   increases almost linearly up to 200 – 400sec and then plateaus

at the end of the experiment. Here, σ0 is the IFT of the bare interface and σt is the IFT in the

presence of the interfacially adsorbed species. Therefore, πeq increases with increasing OVA

concentration for the OO-water and SBO-water interfaces but remains almost constant for the

CO-water interface.

The kinetics of protein adsorption at the interface has been widely reported in the

literature. [32–34] Three different protein adsorption regimes have been reported in dynamic IFT

measurements. Regime I: The lag phase is observed at a low bulk concentration of proteins (10-8

M or 10 µg/ml) in which σ remains constant or decreases slightly with time. At moderate bulk

concentrations of proteins (10-5 M or 100 µg/ml), the lag phase is not observed. [33] Regime II: A

rapid decrease in σ is observed due to the diffusion of protein molecules at the interface followed

by molecular rearrangement and reorientation. Regime III: An additional slow decrease in σ is

7
 observed which is ascribed to the interfacial gelation of protein molecules. Due to the high bulk

concentration of OVA, only regimes II and III are seen in our experiments.

Serrien and Joos report one of the first mathematical models to discuss the mechanism of

adsorption of proteins at the interface. [35,36] The model describes the reduction in σ due to the

combination of the diffusion and rearrangement of the protein molecules at the interface as given

by Equation (2).
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  4t 1  
σ   σeq   αexp (  ) 2      β  exp  kt  (2)
  πτ  

Here, α and β are parameters which give the relative importance of the diffusion

concerning the rearrangement of protein molecules at the interface; k is the rate constant for the

reorientation process, and τ is the diffusion relaxation time defined as follows:

Γeq
2

τ  (3)
4Deff c2

Γeq represents the interfacial concentration of the protein molecules forming a monolayer

or multilayer, assuming a diffusion controlled adsorption. Deff is the diffusivity and c is the

protein concentration in bulk. Ward and Tordai have further expressed the Γeq of the protein

molecules adsorbed at the interface due to diffusion at any given time (t) as [37]

Deff t
Γeq   2c (4)
π

The increase in π resulting from the interfacial adsorption of the protein molecules when

combined with the two-dimensional equation of state  a  KBT , where a is the area per

molecule, K B is the Boltzmann constant and T is the temperature is given by Equation (5):[32]

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 Deff t
π  t   2RTc (5)
π

Therefore, Equation 5 indicates that   t  t , upto the time scales when the adsorption

at the interface is dominated by the diffusion of the protein molecules from the bulk. Equation 6

is used to calculate diffusivity (Deff)[38]:

2
π  1  dσ  
  
4  RTc  dt1/2 t 0 
Deff (6)
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π against √t is plotted for the time until the data show a linear trend as shown in Figure 2.

Deff of the OVA molecules for the CO-water, SBO-water and OO-water interfaces are presented

in Table 2.

As shown in Table 2, the increase in OVA concentration results in a decrease in Deff of

OVA, which may arise from a reduction in the OVA mobility due to an increase in the number

of OVA molecules. Another possible explanation for this trend can be deduced from the

structure of the OVA. The OVA, being a globular protein undergoes denaturation and thus

unfolds in the solution. This unfolding phenomenon may restrict the diffusion of OVA molecules

due to steric hindrance and result in low diffusivity. [36,38] Data also shows that Deff of OVA is the

lowest for SBO-water interface.

Tables 3–5 enlist the parameters obtained by fitting the IFT data of the OO-water, CO-

water and SBO-water interfaces to the Serrien model.

eq and τ of OVA molecules are higher at the CO-water interface than at the OO-water

interface and the SBO-water interface. This is consistent with the low Deff of the OVA towards

the OO-water and SBO-water interfaces. τ decreases with increasing OVA concentration for the

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 three interfaces, a trend consistent with a previous study. [34] On the other hand, parameter k

shows no significant variation in the concentration range of OVA studied, suggesting that

reorientation or unfolding of OVA molecules at interfaces not be influenced by the concentration

of OVA molecules. For all three interfaces, at all OVA concentrations, k is on the order of 10-3

sec indicating a slow reorientation and rearrangement process concerning diffusion [34] .

Although no systematic variation of α and  is observed with an increase in the OVA

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concentrations for the three interfaces, the values of α are always greater than , indicating that

the diffusion dominates the kinetics of OVA adsorption.

3.2 CO-water, OO-water, SBO-water emulsions with OVA

3.2.1 Visual observations and Turbidity of the emulsions

Visually OO-water, SBO-water, and CO-water emulsions appear white shortly after the

preparation and separate into an oil and water phase very quickly. However, when OVA is added

to these emulsions, the emulsions separate into a creamy white layer on top and a translucent

serum layer at the bottom. Since OO, CO and SBO are lighter than water, large droplets of oil

quickly cream and form the creamy layer at the top. The smaller oil droplets remain in the serum

phase.

To quantify the extent of the creaming of the oil droplets in CO-water, OO-water and

SBO-water emulsions with varying concentrations of OVA, the turbidity of the serum phase is

measured. The higher turbidity of the serum phase is correlated with decreased creaming of the

oil drops. Figure 4 shows that the turbidity of the serum phase increases as the concentration of

OVA increases from 0.1 – 1 wt% for OO-water emulsions. This indicates that the creaming of

OO drops decreases with increasing OVA concentration. On the other hand, the CO-water

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 emulsions show an increase in the turbidity of the serum phase till OVA concentration of 0.8

wt% followed by a decrease in the turbidity of the serum phase at a concentration of 1 wt%

OVA. The turbidity of the serum phase in the SBO-water emulsions shows no monotonic trend

with an increase in the OVA concentration. For all OVA concentrations, the serum phase of the

CO-water emulsions shows the maximum turbidity indicating the lowest creaming of the CO

droplets. The increase in turbidity with an increase in protein concentration corresponds to the

better stability as described in the literature [39] .

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These results are consistent with the dynamic IFT data: The OO-water interface shows

the minimum πeq (~10 - 11 mN/m) at all OVA concentrations, indicating the lowest OVA

adsorption at the interface. Thus the OO droplets do not get stabilized by the OVA molecules

and therefore cream, in agreement with the low turbidity of the serum phase of the OO-water

emulsions. The CO-water and SBO-water interface shows a high πeq (14 – 15 mN/m), indicating

higher adsorption of OVA and subsequently decreased creaming of CO/SBO droplets.

3.2.2 Microscopy

(a) Serum phase. Figure 5a, b show the serum phase of OO-water, CO-water and SBO-

water emulsions with 0.1 wt% and 1 wt% OVA at t = 0 hrs and t = 24 hrs respectively. As shown

in Figure 5a, initially the serum phase contains oil droplets for all three emulsions, irrespective

of OVA concentration. However, after 24 hrs, no or a very few oil droplets are observed

indicating the creaming of the droplets.

At a lower OVA concentration of 0.1 wt% in the SBO-water emulsions, almost all large

oil droplets cream. In the serum phase of OO-water emulsions, few tiny droplets are noticeable,

while larger droplets are observed in the serum phase of the CO-water emulsions. At an OVA

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 concentration of 1 wt%, a few small oil droplets are observed in the serum phase of all three

emulsions after 24 hrs. This is also in agreement with greater turbidity of the serum phase of the

three emulsions at 1 wt% OVA than at 0.1 wt% OVA.

(b) Cream phase. The images of the cream phase of the SBO-water, OO-water and CO-

water emulsions with 0.1 wt% and 1 wt% OVA are shown in Figure 6. As shown, the cream

phase of all the three emulsions consists of jammed droplets. Nonspherical droplets are also
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observed due to the crowding of droplets in the cream phase.

As shown in Figure 7a–c, with increasing OVA concentration, the droplet size

distribution becomes narrower, and the average size of the oil droplet decreases. The

microscopic images and the analysis show that more small droplets are observed in the cream

phase of the SBO-water, OO-water and CO-water emulsions with 1 wt% OVA than in the cream

phase of emulsions with 0.1 wt% OVA. Also, OO-water emulsions show the maximum number

of smaller sized droplets.

Rapid creaming of the oil droplets of the serum phase can be attributed to two possible

mechanisms: 1) depletion flocculation due to the high concentration of OVA in the aqueous

phase or 2) bridging flocculation resulting from the adsorption of the OVA molecules on

multiple droplets causing the aggregation of droplets. However, since OVA is a globular protein

that denatures and unfolds over time at the interface, we speculate that bridging flocculation is

less likely to occur. Also, a dense packing of flocculated droplets as shown in our experiments is

a characteristic of droplet flocculation induced by depletion interactions. [18] We, therefore,

believe that the depletion flocculation resulting from the non-adsorbed OVA is the controlling

mechanism of the observed creaming of the droplets.

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 3.2.3 Rheology of the cream phase

The small amplitude oscillatory frequency sweep results of the cream phase of the

emulsions show that the Gʹ > Gʹʹ in the experimental range of ω, indicating the solid-like

character of the cream phase. Both the moduli increase with an increase in the concentration of

OVA. The results show that at OVA = 0.1 wt% and 1 wt%, Gʹ of the cream phase decreases in

the order OO-water > SBO-water > CO-water.

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A previous study by Pal and others indicate that the viscosity, yield stress and Gʹ increase

in the soft-sphere dispersions with decreasing droplet size of the dispersed phase [40,41] . Similarly,

the smaller droplet size of the cream phase of the emulsions with 1 wt% OVA explains their G′

greater than the cream phase of the emulsions with 0.1 wt% OVA. Moreover since the

concentration of non-adsorbed OVA is higher at 1 wt% OVA than at 0.1 wt% OVA, the strength

of depletion attraction is greater in the emulsions with 1 wt% OVA. It can be seen that the cream

phase of the OO-water emulsions is the densest with the largest percentage of droplets of smaller

size between 5 – 10 m and thus the greatest values of Gʹ with respect to the cream phase of the

CO-water and SBO-water emulsions.

4. Conclusions

Our results show that the adsorption of OVA at various commercial food grade oil-water

interfaces investigated in this study is manifested by a reduction in the oil-water interfacial

tension. The kinetics of OVA adsorption is dominated by diffusion and depends on the

concentration of OVA, while the rearrangement and reorientation of OVA molecules at the

interface occur at slower time scales, regardless of the concentration of OVA. The CO-water,

OO-water, and SBO-water emulsions with 10 wt % oil separate in the cream phase and the

13
 serum phase with time. The cream phase consists of densely packed droplets with smaller droplet

size in emulsions with 1 wt% OVA than in emulsions with 0.1 wt% OVA. This phenomenon

manifests as the solid-like rheological signature of the cream phase. OO-water emulsions have

the highest creaming with the largest modulus of the cream phase.

Acknowledgements

The authors thank IIT Gandhinagar for providing seed funding for this work.
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Protein Solutions: BSA, Casein and Buttermilk. Colloids Surf. 1992, 68, 219–233.
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 Table 1. Composition and physical properties of CO, SBO, and OO.

Oil % Saturated % Unsaturated % Oleic % Linoleic Oil-water Density

fatty acid fatty acid acid acid IFT (mN/m) (kg/m3 )

CO 16% 84% 31% 53% 17.7 920

SBO 15% 85% 20% 65% 26 920

OO 15% 85% 75% 10% 16.6 920

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 Table 2. Deff of OVA for CO-water, OO-water and SBO-water interfaces.

Concentration of Deff for CO-Water Deff for OO-Water Deff for SBO-water

OVA (wt %) Interface (m2 /sec) Interface (m2 /sec) Interface (m2 /sec)

0.1% 2.6×10-10 2.4×10-10 9.2×10-11

0.2% 4.8×10-11 1.1×10-10 1.6×10-11

0.4% 1.4×10-11 1.3×10-11 2.2×10-12

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0.6% 4.3×10-12 3.2×10-12 1.3×10-12

0.8% 2.6×10-12 3.4×10-12 8.9×10-13

1% 1.5×10-12 3.2×10-12 8.2×10-13

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 Table 3. Parameters obtained by fitting the IFT data to the Serrien Model for the CO-water
interface.

wt% OVA Γeq (mg/m2 ) τ (sec) k (sec) α 

0.1% 289.4 81.3 1.9 × 10-3 13.3 6.6

0.2% 241.5 77.4 1.8 × 10-3 8.5 4.6

0.4% 249.9 73.6 1.7 × 10-3 8.0 4.3

0.6% 193 61.3 1.7 × 10-3 7 3

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0.8% 184.7 53 1.5 × 10-3 8 3.2

1% 161.6 45.3 1.7 × 10-3 7.7 3.8

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 Table 4. Parameters obtained by fitting the IFT data to the Serrien Model for the OO-water
interface.

wt% OVA Γeq (mg/m2 ) τ (sec) k (sec) α 

0.1% 131.5 18.9 1.9 × 10-3 11.4 8.4

0.2% 153.1 14 2 × 10-3 16.5 8.1

0.4% 103.8 13.6 2 × 10-3 10.7 7.4

0.6% 66.9 10.1 1.9 × 10-3 8.11 6.0

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0.8% 76.1 6.8 2.6 × 10-3 10.8 7.8

1% 82.4 5.5 2.3 × 10-3 15.0 7.3

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 Table 5. Parameters obtained by fitting the IFT data to the Serrien Model for the SBO-water
interface.

Wt% OVA Γeq (mg/m2 ) τ (sec) k (sec) α 

0.1% 70 13.7 1.94 × 10-3 8.3 3.1

0.2% 54.8 12 3.8 × 10-3 5.5 2.9

0.4% 39.9 11.4 2.11 × 10-3 5.0 2.3

0.6% 41 9.6 2.37 × 10-3 5.5 2.3

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0.8% 41.9 7.9 2.06 × 10-3 6.5 2.5

1% 46.8 6.8 2.17 × 10-3 6.9 3.5

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 Figure 1. The dynamic IFT of (a) CO-water interface, (b) OO-water interface and (c) SBO-water
interface with varying concentrations of OVA. π versus t data for (d) CO-water interface (e) OO-
water interface (f) SBO-water interface. The solid red lines represent the fitting of the Serrien
model to the experimental data.
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 Figure 2. π versus  t at OVA concentrations of (0.1 – 1) wt% for (a) CO-water interface (b)
OO-water interface (c) SBO-water interface. The red lines represent the linear fits to the data.
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 Figure 3. CO-water, OO-water and SBO-water emulsions after 24 hrs of homogenization with
an OVA concentration of 0 wt%, 0.1wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8wt% and 1 wt%.
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 Figure 4. Turbidity of the serum phase of SBO-water, OO-water and CO-water emulsions after
24 hrs with an OVA concentration of 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt% and 1wt%.
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 Figure 5. Microscopy images of the serum phase of CO-water, OO-water, SBO-water emulsions
with a) 0.1 wt% OVA b) 1 wt % OVA at t = 0min and t = 24 hrs.
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 Figure 6. Microscopy images of the cream phase of CO-water, OO-water, SBO-water emulsions
with 0.1 wt% and 1 wt % OVA after t = 24 hrs of homogenization.
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 Figure 7. Droplet size distribution in the cream phase of (a) CO-water (b) OO-water (c) SBO-
water emulsions with 0.1 wt% and 1 wt% of OVA concentration.
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 Figure 8. G′ and G′′ of the cream phase of OO-water, SBO-water and CO-water emulsions with
0.1 wt% and 1 wt% of OVA at ω = 0.1-100rad /s.
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