Interfacial and emulsification properties of sono-emulsified grape seed oil emulsions stabilized with milk proteins

Journal Pre-proofs Interfacial And Emulsification Properties Of Sono-Emulsified Grape Seed Oil Emulsions Stabilized With Milk Proteins Mayumi Silva, Bogdan Zisu, Jayani Chandrapala PII: DOI: Reference:

S0308-8146(19)31889-8 https://doi.org/10.1016/j.foodchem.2019.125758 FOCH 125758

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Food Chemistry

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11 August 2019 14 October 2019 20 October 2019

Please cite this article as: Silva, M., Zisu, B., Chandrapala, J., Interfacial And Emulsification Properties Of SonoEmulsified Grape Seed Oil Emulsions Stabilized With Milk Proteins, Food Chemistry (2019), doi: https://doi.org/ 10.1016/j.foodchem.2019.125758

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INTERFACIAL AND EMULSIFICATION PROPERTIES OF SONO-EMULSIFIED GRAPE SEED OIL EMULSIONS STABILIZED WITH MILK PROTEINS

Mayumi Silvaa, Bogdan Zisub, Jayani Chandrapalaa* aSchool

of Science, RMIT University, Bundoora, VIC 3083, Australia

bSpraying

Systems, Fluid Air, Melbourne, Australia

*Corresponding author Tel.: + 61 (0) 3 9925 2130 E-mail address: [email protected]

Abstract Emulsions were designed under low frequency ultrasound (20 kHz) at energy densities of 11.7 – 117.0 J/mL using grape seed oil and milk protein solutions containing different casein to whey protein ratios of 80:20, 60:40, 50:50 and 40:60. An increase in energy densities produced emulsions with a smaller droplet size and narrow size distribution at all milk protein ratios. However, the minimum sono-energy density required to produce stable emulsions varied depending on the ratio of caseins (CN) and whey proteins (WP) in the continuous phase. In addition, the composition of the interfacial layer was dependent on the composition of the milk proteins in the continuous phase. The interfacial layer was predominantly covered by the CN and CN-WP aggregates in the presence of equal or greater amounts of caseins than whey proteins (80:20, 60:40 and 50:50), while WP aggregates and CN-WP aggregates were the primary constituents of whey protein-rich emulsions (40:60).

Keywords Sono-emulsion, milk proteins, interfacial tension, emulsifying ability, grape seed oil

1. Introduction Caseins and whey proteins have excellent but different emulsifying characteristics due to the differences in their molecular structures (Parkinson & Dickinson, 2004). Ultrasound is used as an emulsification technique in order to improve the emulsifying properties of milk proteinbased emulsions

(O'Sullivan, Arellano, Pichot, & Norton, 2014; Shanmugam &

Ashokkumar, 2014). However, low frequency ultrasound influences the physicochemical, thermal, and structural properties of milk protein solutions depending on the protein composition and ultrasonic conditions in use (Silva, Zisu, & Chandrapala, 2018). Research has been carried out to understand the effect of sonication on skim milk (Shanmugam, Chandrapala, & Ashokkumar, 2012), micellar caseins (Chandrapala, Martin, Zisu, Kentish, & Ashokkumar, 2012; Chandrapala, Martin, Kentish, & Ashokkumar, 2014) ,whey proteins (Chandrapala J, 2012; Chandrapala, Zisu, Palmer, Kentish, & Ashokkumar, 2011) and mixtures of caseins and whey proteins (Silva, Zisu, & Chandrapala, 2018). A previous study conducted by Silva, Zisu, and Chandrapala (2018) showed that the sonication-induced physicochemical and structural modifications of milk proteins highly depended on the ratio of caseins and whey proteins present in the milk solutions. The application of high intensity low frequency ultrasound induces the dissociation and denaturation of β-lactoglobulin while unfolding the dimeric form of β-lactoglobulin into random coils leading to aggregation via βsheet intermolecular crosslinking. Milk protein solutions consisting of mostly whey proteins produced large amounts of disulphide linked aggregates while the casein-rich systems produced more hydrophobically mediated aggregates. These composition-dependent structural changes in milk proteins may influence the emulsifying characteristics. Emulsifying properties of caseins and whey proteins have been widely studied individually in terms of processing conditions and processing techniques (Dickinson & Golding, 1998; Pichot, 2012; Ranadheera, Liyanaarachchi, Chandrapala, Dissanayake, & Vasiljevic, 2016).

A previous study showed that the emulsifying properties of casein and whey protein mixtures strongly depended on the ratio of caseins and whey proteins present, initial protein concentration and temperature during emulsification by high pressure homogenization (Surel, Foucquier, Perrot, Mackie, Garnier, Riaublanc, et al., 2014). Moreover, the composition of the interfacial layer and the adsorption behaviour of proteins at the oil-water interface are also influenced by the state of the proteins in the bulk solution (Dalgleish, 1995; Dickinson, 2001; Dickinson & McClements, 1995). However, almost no studies have been carried out to understand the interfacial and emulsifying behaviour of milk systems with varying casein to whey protein ratios during ultrasonication. Therefore, a fundamental understanding of sonication-induced interfacial and emulsifying behaviour of milk protein systems containing varying levels of caseins and whey proteins will be beneficial to improve the physicochemical, functional and storage properties of the respective emulsions containing GSO. Apart from the emulsification technology and emulsifying agents, the oil phase plays an important role on the physicochemical, nutritional and structural aspects of the food emulsions (McClements, 2015b). Grape seed oil (GSO) which is a by-product of wine manufacturing, contains high amount (85-90%) of polyunsaturated fatty acids (PUFA), mainly linoleic acid, tocopherols and tocotrienols which are isomers of vitamin E (Fernandes, Casal, Cruz, Pereira, & Ramalhosa, 2013; Madawala, Kochhar, & Dutta, 2012). The present study examined the effect of low-frequency ultrasound (20 kHz) on the interfacial and emulsifying properties of milk protein (3.3% w/v) systems with varying casein to whey protein ratios of 80:20, 60:40, 50:50, 40:60, pure milk protein isolate (MPI) and pure whey proteins isolate (WPI) during the processing of primary emulsions containing 10% (w/w) grape seed oil. 2. Materials and methods 2.1.1. Materials

Milk Protein Isolate (MPI) and Whey Protein Isolate (WPI) powders were purchased from Tatura milk industries limited (Victoria, Australia). The MPI and WPI powders contained a total protein content of 85.5% (w/w) and 88.8% (w/w) respectively. Pure grape seed oil (GSO) (Azalea Brand, NSW, Australia) was purchased from a local supermarket. All the chemicals and filters used were obtained from Sigma Aldrich Pty Ltd (Castle Hill, NSW, Australia) or Bio-Rad Laboratories Pty Ltd (Gladesville, NSW, Australia). Ultra-pure (MilliQ) water was used in all experiments. 2.1.2. Charcoal treatment for GSO The interfacial tension of the emulsions depends on the oil properties such as the origin, purity, polarity of the major lipid molecules (e.g., triglycerols or terpenes) and the presence of surface active components like free fatty acids, monoacylglycerols, diacylglycerol or phospholipids (McClements, 2015b). These surface active substances may interfere with the adsorption behaviour and will result in obtaining inaccurate and inconsistent data (Dopierala, Javadi, Krägel, Schano, Kalogianni, Leser, et al., 2011). Thus, GSO was purified using an activated charcoal treatment to remove the surface active impurities. For that purpose, GSO was mixed with grounded charcoal in 10:1 (w/w) ratio and stirred overnight using a magnetic stirrer. Then the oil was centrifuged (Beckman Coulter, United States) at 20 °C and the supernatant (oil portion) was filtered through a Whatman No.40 filter paper and a 0.45 µm PTFE syringe filter (Acrodisc® syringe filters, Sigma Aldrich Pty Ltd, NSW, Australia). The treatment was carried out three times and ensured the absence of surface active materials using DIFT data. 2.2.

Methods

2.2.1. Preparation of the aqueous phase Milk protein solutions with different casein to whey protein (C:W) ratios of 80:20, 60:40, 50:50 and 40:60 were prepared by dissolving the required amount of MPI and/or WPI

powders in MilliQ water at 40 °C while keeping the total protein concentration at 3.3% (w/v). Pure MPI and WPI solutions were used as controls. Initially the required amount of MPI was added to pre-heated (40 °C) MilliQ water and stirred for 15 min. Then, the WPI powder was added and stirred for another 45 min to ensure complete mixing. The solution was kept overnight at 4 °C for further hydration. On the following day, the pH of the solutions was adjusted to 6.7 using 1M HCl and 1M NaOH. 2.2.2. Preparation of emulsions Oil-in-water primary emulsions were prepared with grape seed oil (GSO) and milk protein solutions with an oil volume fraction of 10% (w/w). Fifty millilitre aliquots of samples were prepared by adding 5 mL of GSO into 45 mL of milk protein solution. Then the two phases were emulsified using a 20 kHz low frequency ultrasonic unit with a 500 W ultrasonic horn (Q Sonica, Bandelin electronic, Berlin, Germany) at 60% amplitude for different time periods of 1, 3, 5, 7 and 10 min. The ultrasonic horn tip was positioned approximately 5 mm from the surface of the sample near the oil and water interface. The temperature of the solutions was maintained at 20±2 °C using an ice bath. The applied energy densities were 11.7 J/mL, 35.1 J/mL, 58.5 J/mL, 81.9 J/mL and 117.0 J/mL for time periods of 1, 3, 5, 7 and 10 respectively. 2.2.3. Determination of dynamic interfacial tension (DIFT) The effect of sonication on the DIFT at GSO-milk interface were determined with a drop profile tensiometer (PAT-1TM, Sinterface Technologies, Germany) using the pendent drop method. In order to obtain sonicated milk systems, 50 mL aliquots of milk protein solutions were sonicated using a 20 kHz high frequency ultrasonic unit (Q Sonica, Bandelin electronic, Berlin, Germany) at 60% amplitude for 1, 3, 5, 7 and 10 min. The drop volume was kept constant at 20 µL and the measurements were conducted for 200 s. The temperature of the measuring cell chamber was maintained at 25 °C using a water bath (Julabo, Germany). The

calibration of the tensiometer was conducted with a sphere (1.5 mm of radius) at 25 °C. The density of the GSO and protein solutions were measured using a densitometer (METTLER TOLEDO, PortableLabTM) at 25 °C. According to our experimental results, the equilibrium interfacial tension at grape seed oil-water interface was 20.08 ± 0.75 mN/m at 25 °C. 2.2.4. Determination of the percentage of adsorbed proteins, interfacial protein concentration and surface protein composition The adsorbed protein percentage (AP%) and interfacial protein concentration (Г) were determined using a method described Ye (2008). Emulsions were centrifuged (Beckman Coulter, United States) at 15000 g for 40 min at 20 °C. After centrifugation, the oil droplets and adsorbed proteins were separated as a cream layer at the top of the centrifuge tube and the aqueous phase was settled to the bottom. Then the aqueous layer was filtered through a 0.22 µm filter (Sigma Aldrich Pty Ltd, NSW, Australia). The cream layer was dispersed in MilliQ water and re-centrifuged at 15000 g for 40 min to obtain the washed cream. The protein contents in aqueous phases of both centrifuged emulsions and protein solutions were determined by Bradford assay. In brief, the aqueous phases were diluted 40 fold with MilliQ water and 20 µL of the diluted aqueous phase was mixed with 1 mL of Bradford reagent (Bio-Rad Laboratories Pty Ltd, NSW, Australia). Then the mixture was incubated for 5 min at room temperature and the absorbance was determined at 595 nm using a UV-VIS Spectrophotometer (PerkinElmer, Lambda 35, Australia). The standard curve (R2 = 0.9972) was developed using Bovine serum albumin (BSA) in the concentration range of 250 – 1250 µgmL-1. The adsorbed protein content (AP%) and interfacial protein concentrations (Г, expressed in mg/m2) were determined using following equations (Equation 01 and 02): 𝐀𝐏% =

(𝐂𝐬 ― 𝐂𝐟) × 𝟏𝟎𝟎 𝐂𝟎

Equation 1

Where, Cs is the protein concentration in the supernatant of initial milk protein solution, Cf is the protein concentration in the filtrate of emulsion, C0 is the initial protein concentration of the milk protein solution applied for the emulsion preparation (3.3 g/100 mL). Г=

(𝐂𝐬 ― 𝐂𝐟) × 𝐃(𝟑,𝟐) 𝟔𝛟

Equation 2

Where, D(3,2) is the surface-average diameter of the emulsion obtained from the Mastersizer and ϕ is the oil volume fraction of the emulsion. The composition of the proteins at the adsorbed layer was determined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions as described by Ye and Singh (2000). Briefly, the cream was mixed with SDS sample buffer and β-Mercaptoethanol followed by heating at 95 °C for 5 min in a boiling water bath. In order to remove the fat, heated samples were centrifuged at 2500 g for 30 min. Ten microliter aliquots of prepared samples were injected to precast 4–20% mini-protean gels (Bio-Rad Laboratories Pty Ltd, NSW, Australia) and the gels were run for 45 min at 200 V. Casein , whey protein and molecular weight standards (10–250 kDa) were run simultaneously. Gels were then rinsed with MilliQ water, stained with Coomasie stain and destained using MilliQ water overnight as described by Silva, Zisu, and Chandrapala (2018). Later, the gels were scanned and the intensities of the protein bands were determined using ImageJ 1.52e software (Wayne Rasband, USA). The percentage composition of whey proteins and caseins were determined as a fraction of the sum total. 2.2.5. Emulsion activity and stability indexes The emulsion activity index (EAI) and emulsion stability index (ESI) of the emulsions were measured by a turbidimetric method as described by Pearce and Kinsella (1978). Immediately after sonication, the emulsions were diluted 100 folds with 0.1% (w/v) sodium dodecyl sulphate (SDS) and vortexed for 10 s. The absorbance of the diluted emulsion was measured

using a UV-VIS Spectrophotometer at 500 nm wavelength. The emulsion activity index (EAI, expressed in m2g-1) was calculated using the following equation (Equation 03): 𝐄𝐀𝐈 =

𝟐 × 𝟐.𝟑𝟎𝟑 × 𝐀𝟎 × 𝐃 𝐂 × 𝛟 × 𝐋 × 𝟏𝟎𝟎𝟎

Equation 3

Where, A0 is the initial absorbance, D is the dilution factor (100), C is the concentration of protein in the milk protein solutions (33 mgmL-1), Φ is the oil volume fraction and L is the light path length (0.01 m). The emulsions were stored at 4 °C and absorbance was re-measured after 24 h following the same procedure. The emulsion stability index (ESI, expressed in hours) was calculated using the following equation (Equation 04): 𝐄𝐒𝐈 =

𝐀 × ∆𝐭 ∆𝐀

Equation 4

Where, A is the absorbance after 24 h, Δt is the time interval (24 h) and ΔA is the change in absorbance during 24 h (A0 – A). 2.2.6. Size of emulsion Size of the emulsion globules were determined by laser diffraction method using a Mastersizer 3000 attached to a hydroMV sample handling unit (Malvern Instruments, Worcester, UK). Emulsions were directly added to the dispersion unit (containing water) until 15 – 20% obscuration level was reached and the measurements were taken at a rotating speed of 1250 rpm at 25 °C. The refractive indexes of GSO and milk protein solutions used were 1.471 and 1.336 respectively. The D(4,3), D(3,2), D10, D50 and D90 values were recorded as an average of five measurements. 2.2.7. Surface charge of emulsion Emulsions were diluted 200 times using MilliQ water and the zeta potential measurements were obtained using the Zetasizer (Nano series, Malvern Instruments, Worcester, UK) at 25

°C. The refractive indexes for GSO and milk protein solutions were considered as 1.471 and 1.336 respectively. Three replicate measurements were made for each sample. 2.2.8. Statistical analysis All the experiments were at least duplicated and reported as the average with standard deviation. One-way ANOVA was used to determine the significant differences between and within the experiments at 95% confident level. Pearson correlation coefficient (r) was calculated to analyse the relationship between emulsifying and physicochemical properties. All the statistical analysis was performed with SPSS version 25 software (SPSS, IBM).

3. Results 3.1.

Dynamic interfacial tension (DIFT)

The ease of the interfacial layer formation between the oil phase and the aqueous phase is denoted by the dynamic interfacial tension (DIFT) at oil-water interface (McClements, 2015a). As shown in figure 1, the DIFT between purified GSO and sonicated milk protein solutions prepared with different caseins to whey protein (C:W) ratios of 60:40, 50:50 and 40:60 were independent from the applied energy density. However, DIFT at C:W ratio of 80:20 was slightly increased at 11.7 J/mL energy density compared to the untreated solution followed by a slight reduction on prolong sonication (117.0 J/mL) simultaneously showing the highest DIFT among all ratios under all energy densities. In addition, the decrease in DIFT was observed for sonicated MPI and WPI. Interestingly, milk protein solutions stabilized with MPI and other ratios attained its equilibrium interfacial tension value immediately after the oil drop formation (~20 s) in comparison to WPI which took a relatively longer time (~180 s) for attaining the equilibration.

3.2.

Percentage of adsorbed proteins, interfacial protein concentration and surface protein composition

The adsorbed protein percentage (AP%) and the surface protein concentration of emulsions were largely affected by the casein to whey protein ratios and the applied sono-energy density (table 1). The AP% and surface load increased (p < 0.05) with increasing energy density while the highest raise in AP% was observed at low energy densities with the presence of whey proteins in the emulsions. For instance, the highest raise in AP% was observed in WPI solutions under 58.5 J/mL energy density while C:W ratios of 80:20 and 60:40 showed the highest raise in AP% under 117. J/mL energy density. Moreover, emulsions stabilized with C:W ratio of 50:50 exhibited the highest AP% under all energy densities and the highest surface load was also observed in the same system under all energy densities except 11.7 J/mL. However, under the 117.0 J/mL energy density, ~57% caseins and 43% whey proteins were presented in the interfacial layer of C:W ratio of 50:50 (figure 2A). Furthermore, the lowest AP% and surface load resulted in emulsions prepared by C:W ratio of 80:20 under all sonication times with the maximum adsorption of ~14% and 2.70 mg/m2 of surface load (table 1) under 117.0 J/mL energy density. Moreover, the interfacial layer in the same system primarily consisted of casein proteins denoting ~62% fraction (figure 2A). 3.3.

Size of emulsions

Upon sonication, the width of the bimodal particle size distribution decreased significantly, creating a unimodal distribution with smaller emulsion droplets in all milk protein ratios depending on the composition of milk proteins in the aqueous phase of emulsion (figure 3A). Emulsions having a higher whey protein fraction obtained unimodal size distribution within shorter sonication times. For instance, emulsions stabilized by WPI and C:W ratio of 40:60 attained unimodal distribution below 58.5 J/mL energy density whereas 81.9 J/mL was

required for C:W ratio of 60:40 and 50:50. However, caseins-rich systems (MPI and 80:20) attained unimodal distribution up on 117.0 J/mL energy density. Droplet size decreased (p < 0.05) with increase in the applied energy density from 11.7 J/mL to 117.0 J/mL regardless of the composition of the proteins in the system (figure 4A). Emulsions prepared with C:W ratio of 40:60 showed the highest particle size (3.13 µm) whereas smallest particle size (1.58 µm) was observed in WPI emulsions under 1 min sonication. However, all the emulsions acquired significantly similar droplet size (p>0.05) at higher applied energy density (117.0 J/mL). The highest percentage of reduction in particle size was observed at 35.1 J/mL energy density followed by a gradual decrease in size thereafter at higher sono-energy. Moreover, the decrease in droplets size as the applied energy density increases resulted in adsorption of more proteins at the oil-water interface (figure 3B). 3.4.

Emulsion activity and stability indexes

The ability of proteins to form an emulsion is described by the emulsion activity index (EAI) (Pearce & Kinsella, 1978) and the stability of the emulsions over a defined time period is described by the emulsion stability index (ESI). The EAI and ESI depend on the milk protein ratio in the aqueous phase and applied energy density (table 1). Emulsions stabilized with WPI showed the highest initial EAI of ~30 m2/g while the lowest EAI (~1 m2/g) was observed in C:W ratio of 50:50 at 11.7 J/mL energy density. However, all the emulsions exhibited significantly higher EAI (>30 m2/g) values at higher applied energy density (117.0 J/mL). Moreover, a C:W ratio of 40:60 obtained their highest EAI at 35.1 J/mL while C:W ratio of 60:40 and WPI obtained highest EAI under 58.5 J/mL and remained constant (p > 0.05) upon increasing energy density. However, continuous increase in EAI was observed in

C:W ratio of 80:20 with increasing sono-energy while EAI of MPI-emulsions increased up to 81.9 J/mL energy density followed by a decrease at 117.0 J/mL. In terms of ESI, emulsions stabilized with WPI showed the highest ESI under all sonication times while lowest ESI observed in C:W ratio of 50:50. Interestingly, ESI of C:W ratio of 60:40, 50:50, prepared with MPI and WPI increased gradually up to 81.9 J/mL followed by a reduction at 117.0 J/mL energy density. 3.5.

Surface charge of emulsion

Figure 4B shows the zeta potential values of GSO-M emulsions at different processing times. Emulsions stabilized with WPI showed the highest negative zeta potential at all energy densities (p < 0.05) and the values were not affected with an increase in the applied energy. Similarly, the zeta potential of C:W ratio of 80:20 also remained unchanged during sonication. In the system of C:W ratio of 40:60, increases in zeta potential were observed up to 35.1 J/mL energy density followed by constant values at higher applied energy. Moreover, the zeta potential of C:W ratio of 60:40, 50:50 and MPI increased up to 35.1 J/mL then reduced at 117.0 J/mL applied energy.

4. Discussion Sono-emulsification comprises a two-step mechanism. The first step involves the eruption of dispersed phase droplets into the continuous phase leading to smaller droplet formation which is driven by the mechanical vibrations generated from turbulence. The second step involves breaking of the droplets through the shear forces generated by acoustic cavitation near the interface (Canselier, Delmas, Wilhelm, & Abismail, 2002). Laplace pressure (p) which denote the energy (in the form of shear) required by the continuous phase to deform droplets of the dispersed phase, depends on the radius of emulsion droplets (R) and interfacial tension

(IT). Laplace pressure can be calculated using p = (IT/2R) equation and the emulsions can be formed when the applied shear stress is greater than the particular Laplace pressure (Canselier, Delmas, Wilhelm, & Abismail, 2002; Walstra, 1993). Therefore, interfacial tension and the droplet size of the emulsion provide valuable information regarding the feasibility of emulsion formation. Moreover, the composition and the physico-chemical characteristics in the interfacial layer are also important in determining the emulsion stability. Furthermore, zeta potential relationship between EAI and ESI are key indicators for shortterm stability of emulsions. 4.1.

Milk systems with C:W ratio of 80:20 and MPI

Sono-emulsification of GSO and milk protein solutions with C:W ratio of 80:20 and MPI produced stable emulsions at energy densities of 35.1 J/mL, 58.5 J/mL, 81.9 J/mL and 117.0 J/mL. The MPI solution and C:W ratio of 80:20 showed the equilibrium interfacial tension value around 14.5 mN/m before the application of ultrasound (figure 1). DIFT of MPI decreased with increase in sono-energy while increased in DIFT was observed in C:W ratio of 80:20 and this may be attributed to the sonication-induced structural changes of milk proteins. As observed in our previous work (Silva, Zisu, & Chandrapala, 2018), milk solutions with C:W ratio of 80:20 produced more hydrophobically mediated aggregates during sonication which hinders the adsorption of proteins at oil-water interface, resulting a higher DIFT. However, the application of ultrasound reduce the particle size of MPI solution due to the disruption of hydrophobic bonds exposing more hydrophobic sites to bind at the oil-water interface and reducing the interfacial tension easily. Reduced interfacial tension resulted in a higher rate of oil droplet breakup during the emulsification leading to the formation of smaller droplets (O'Sullivan, Arellano, Pichot, & Norton, 2014). As a consequence, emulsions stabilized with MPI produced smaller droplets compared to the C:W

ratio of 80:20 (figure 4A). An increase in applied energy density further decreased the droplet size and create a unimodal size distribution (figure 3A). This may be attributed to the higher rate of collision provided by the physical effects generated in the liquid system such as acoustic cavitation, acoustic streaming, mechanical vibrations, microsteaming, shear and turbulence at higher energy densities (Abbas, Hayat, Karangwa, Bashari, & Zhang, 2013; Ashokkumar, Bhaskaracharya, Kentish, Lee, Palmer, & Zisu, 2010). Correspondingly, the percentage of adsorbed proteins and the surface load increased as the sonication time increases resulting in more stable emulsions at higher applied energy densities (table 1). Moreover, the adsorbed protein layer of these two systems were primarily consisted with the caseins indicating 69% and 62% for MPI and C:W ratio of 80:20 respectively at 117.0 J/mL applied energy density (figure 2A). Generally, a monolayer surface coverage can be observed in the interfacial layer of caseins stabilized emulsions and 1-2 mg/m2 of surface coverage of caseins is sufficient to produce fine stable emulsions (Singh, 2005). Moreover, the minimum casein surface coverage was found to be 1 mg/m2 with a 5 nm of interfacial layer thickness and the maximum coverage reported as 3 mg/m2 with 10 nm thickness (Fang & Dalgleish, 1993). Higher surface load values (>3 mg/m2) observed in the present study at higher energy densities may be attributed with the formation of the secondary protein layer around the emulsion droplet (Hunt & Dalgleish, 1994) or the formation of casein aggregates (Srinivasan, Singh, & Munro, 1996). The presence of 31% and 37% whey proteins (figure 2A) at the adsorbed layer of MPI and 80:20 respectively at 117.0 J/mL energy density denotes the formation of aggregates with un-adsorbed whey protein rather than the formation of a secondary layer. The decrease in EAI and ESI values and increase in zeta potential values in MPI emulsions at 117.0 J/mL energy density may be attributed with the depletion flocculation phenomenon (table 1). Unfolding and surface denaturation of β-Lactoglobulin may lead to the depletion flocculation of emulsion droplets

(Dickinson, 2010) and it was insensitive with particle size measurements as emulsion droplets maintain their individual integrity during the depletion flocculation. 4.2.

Milk systems with C:W ratio of 60:40 and 50:50

Stable emulsions were produced using milk protein solutions with C:W ratio of 60:40 at 58.5 J/mL, 81.9 J/mL and 117.0 J/mL sono-energy density whereas a C:W ratio of 50:50 produced stable emulsions at 81.9 J/mL and 117.0 J/mL sono-energy density. However, there were no differences of DIFT between untreated and sonicated (up to 117.0 J/mL) milk solutions with C:W ratio of 60:40 and 50:50 (figure 1). This suggested that the rate of the protein adsorbed at the oil-water interface was approximately similar for the untreated and ultrasound treated solutions (O'Sullivan, Arellano, Pichot, & Norton, 2014). Upon increasing applied energy density, an increase in the percentage of adsorbed proteins was observed although interfacial properties were not improved (table 1). Therefore, it can be suggested that besides the interfacial lowering effect of milk proteins, mechanical and shear forces generated from sonication also greatly improve the emulsifying ability and emulsion properties such as the formation of smaller droplets (Shanmugam & Ashokkumar, 2014) which thereby leads to the higher adsorption of proteins at the interface. Nevertheless, the interfacial layer of these two systems primarily consisted of casein proteins (~60% at 117.0 J/mL energy density – figure 2A) and a considerable fraction of whey proteins remained in the continuous phase. Presence of more un-adsorbed proteins in the aqueous phase during sono-emulsification induced the unfolding and denaturation of whey proteins leading to the formation of aggregates via hydrophobic and thiol-disulphide interactions. Moreover, denatured whey proteins show a higher affinity for κ-caseins than the whey proteins resulting in the formation of bonds between denatured whey proteins and κ-caseins than between the whey proteins themselves (Donato, Guyomarc’h, Amiot, & Dalgleish, 2007). These thiol-disulphide interchange

reactions lead to strong crosslinking between denatured globular proteins. Furthermore, there was a possibility to form hydrophobic interactions between non-adsorbed globular proteins and interfacial proteins. As a result of these interactions, flocculates and/or aggregates were formed leading to the instability of the emulsion (S. Euston, Finnigan, & Hirst, 2000; S. R. Euston, Al-Bakkush, & Campbell, 2009; Liang, Patel, Matia-Merino, Ye, & Golding, 2013; McSweeney, Mulvihill, & O'Callaghan, 2004). Therefore, emulsions prepared by C:W ratio of 50:50 at shorter sonication times showed layer separation (figure 2B) and lowest EAI and ESI values (table 1) although it had the highest AP% at 1 min sonication. Upon sonication, higher energy densities helps in the mixing of oil and aqueous phases and to overcome the Laplace pressure to form stable emulsions (Shanmugam & Ashokkumar, 2014) with the higher surface load. However, increasing applied energy density may lead to the instability of emulsion due to the depletion flocculation as showed in reduced ESI (table 1) and increased zeta potential (figure 4B) at 117.0 J/mL applied energy density. 4.3.

Milk systems with C:W ratio of 40:60

Emulsions containing milk protein solution with C:W ratio of 40:60 formed stable emulsions at 35.1 J/mL -117.0 J/mL sono-energy density. The composition of interfacial layer (~59% whey proteins under 117.0 J/mL energy density – figure 2A) suggests that prolong sonication induced the aggregation of whey proteins leading to the adsorption of more whey protein aggregates at the interface. Lower amounts of caseins compared to the whey proteins in the continuous phase may form a thick and heterogeneous interface with more protruding fractal whey proteins due to the denaturation and aggregation of whey proteins. It reduces the chance of aggregation between caseins and whey proteins (Surel, et al., 2014). Presence of more aggregated structures at the droplet interface led to the highest particle size at 11.7 J/mL energy density compared to the other ratios (figure 4A). Moreover, aggregated whey proteins

in the adsorbed layer hinder the further adsorption of proteins upon sonication due to the low availability of thio-disulphide interchanges or hydrophobic sites for further aggregation resulting significantly smaller AP% at 117.0 J/mL energy density compared to the other emulsions (table 1). This behaviour was further justified by the EAI and zeta potential values as they remained unchanged after the 35.1 J/mL applied energy density (table 1 & figure 4B). 4.4.

Milk systems with WPI

Emulsions prepared by WPI alone produced stable emulsions at very low energy density (11.7 J/mL) showing higher emulsifying ability. Mechanical and shear properties generated through ultrasound and reduction of interfacial tension upon sonication enhance the emulsifying ability of WPI system. However, equilibration time was long for WPI compared to the other casein containing milk protein solutions (figure 1). Globular proteins such as whey proteins take a long time to unfold and subsequently reduce the interfacial tension due to the nature of tertiary and secondary structures compared to the flexible random-coiled structures of the caseins (Wilde, 2000). Generally, two distinct phases can be observed in the DIFT curves of globular proteins before reaching to the equilibrium; first phase – a sharp decrease of DIFT due to the diffusion and adsorption of proteins to the surface, second phase – slow decrease in DIFT due to the conformational arrangement of adsorbed protein at the interface. Flexible proteins such as caseins are more effective in reducing interfacial tension due to the presence of more non-polar groups than rigid proteins or proteins with fewer nonpolar groups (Nakai & Li-Chan, 1988). Application of ultrasound induces the dissociation, denaturation and unfolding of β-Lactoglobulin, which thereby leads to aggregation themselves (Silva, Zisu, & Chandrapala, 2018). Therefore, at the moment of emulsion formation, WPI solution contained native, unfolded and aggregated whey proteins. As native whey proteins are better emulsifiers than the aggregated proteins (Surel, et al., 2014), native

whey proteins may preferentially adsorb to the interface as their dimer structures (Mackie, Mingins, & Dann, 1991). Therefore, WPI emulsions showed lower AP% (3.8%) (table 1) and the smallest globule size at 11.7 J/mL energy density (figure 4A). Upon sonication adsorbed proteins may also undergo confirmation reorganization allowing further exposure of hydrophobic sites (Zhai, Miles, Pattenden, Lee, Augustin, Wallace, et al., 2010). At the same time, prolong sonication may lead to the unfolding of unabsorbed whey proteins which thereby improves the surface activity of un-adsorbed β-lactoglobulin (Das & Kinsella, 1990) leading to the formation of aggregation via thiol-disulphide exchanges between adsorbed and un-adsorbed whey proteins. Therefore, a higher percentage of whey protein adsorption could be observed as the applied energy density increases resulting in 26.2% of AP% at 117.0 J/mL (figure 2A). Correspondingly, emulsions stabilized with WPI showed the highest negative zeta potential at all the sonication times and did not changed upon ultrasonic treatment (figure 4B). Higher magnitude in charged showed the higher stability of an emulsion due to the enhanced electrostatic repulsions between droplets (Guzey & McClements, 2007). Similarly, highest EAI and ESI observed even at 11.7 J/mL energy density compared to the other emulsions (table 1). Sono-emulsification properties of GSO primary emulsions were influenced by the composition of the milk protein present in the aqueous phase and the applied energy density. As shown in table 2, EAI was negatively correlated with particle size and positively correlated with the percentage of adsorbed proteins. Similar to the EAI, ESI values were also found to be negatively correlated with particle size and positively correlated with the percentage of adsorbed proteins. Furthermore, there was a positive correlation between EAI and ESI values. For instance, increasing applied energy density during the production of WPI emulsions from 11.7 J/mL to 117.0 J/mL decreased the particle size from 1.580 µm to 0.637 µm along with the increasing EAI values ranging from 30.68 m2/g to 34.59 m2/g. Moreover,

more than a threefold increase in ESI was observed at 81.9 J/mL sonication compared to the 11.7 J/mL. These relationships among emulsion properties can provide valuable insight into the prediction of emulsion stability in future studies. 5. Conclusion The application of low-frequency ultrasound at 20 kHz produced stable emulsions of 10% (w/w) grape seed oil and 3.3% (w/w) milk protein solutions. The composition of the milk proteins and the applied energy density influence on the interfacial and emulsification properties of milk protein stabilized emulsions during the sono-emulsification. The increase in energy density resulted in smaller droplet size with narrow size distribution, increasing percentage of adsorbed proteins with higher surface load, increasing emulsion activity index and emulsion stability index. If the milk protein solution contained equal or more caseins than the whey proteins, the interface was mainly constituted by the caseins and casein-whey protein aggregates due to the ultrasound-induced denaturation of whey proteins during the sonication leading to the aggregation via hydrophobic and thiol-disulphide interactions. However, when the concentration of casein was less than the whey protein, the interface is primarily covered by the whey-whey and casein-whey aggregates. Moreover, adsorption of aggregated protein structures led to the larger droplet size. If the aqueous phase consisted with only whey proteins, the interface was mainly covered by the native whey proteins and whey-whey aggregates resulting smaller particle size. Therefore, it should finally be stated that under different energy densities, the amount of adsorbed milk proteins at the surface could be mainly attributed to the different molecular structure and bonding nature of caseins and/or whey proteins in the continues phase. This may provide valuable insight to future researches in designing milk protein-stabilized emulsions with varying milk composition Moreover, sonication can be used as an effective emulsification method with the careful selection of process parameters. Pilot scale experiments are recommended under similar

energy densities and compositions as possible in order to understand the gaps between the laboratory and scaling-up applications. Acknowledgements The authors gratefully acknowledge the financial and technical support from Royal Melbourne Institute of Technology, Australia and international postgraduate research scholarship (RMIT IPRS).

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Figure legends

Figure 1: Dynamic interfacial tension (DIFT) behaviour at oil/water interface between purified grape seed oil and milk protein solutions (casein :whey - 80:20, 60:40, 50:50, 40:60, MPI and WPI) sonicated at different energy densities (A-untreated, B-11.7 J/mL and C-117.0 J/mL). Figure 2: A - Relative proportion of adsorbed caseins and whey proteins at the droplet surface (cream phase) of GSO-M emulsions, B - photographs of emulsions; 80:20, 60:40, 50:50 (top – left to right), 40:60, MPI and WPI (bottom – left to right) at different applied energy densities;11.7 J/mL (1), 35.1 J/mL (2), 58.5 J/mL (3), 81.9 J/mL (4), 117.0 J/mL (5) and protein solutions (P). Layer separation can be observed in following emulsions; 80:20 – (1), 60:40 – (1 & 2), 50:50 - (1, 2 & 3), 40:60 – (1) and MPI – (1). Figure 3: A - Volume size distribution, B - relationship between particle size and percentage of adsorbed proteins in GSO-M emulsions prepared with varying casein to whey ratios of 80:20, 60:40, 50:50, 40:60 and WPI and MPI at different energy densities of 11.7 J/mL, 35.1 J/mL, 58.5 J/mL, 81.9 J/mL and 117.0 J/mL. Capital and simple superscripts denote the significant difference between sonication times and milk protein ratios respectively. Figure 04: A - Volume mean diameter D(4,3), B - surface charge of GSO-M emulsions prepared using different casein to whey protein ratios of 80:20, 60:40, 50:50, 40:60, MPI and WPI at different energy densities of 11.7 J/mL, 35.1 J/mL, 58.5 J/mL, 81.9 J/mL and 117.0 J/mL. Capital and simple superscripts denote the significant difference between sonication times and milk protein ratios respectively.

Figure 01

Figure 02

A

B

Figure 03

A

B

Figure 04

Table 1: The percentage of adsorbed protein, surface load, emulsion activity index and emulsion stability index of GSO-M emulsions prepared using different casein to whey protein ratios of 80:20, 60:40, 50:50, 40:60, MPI and WPI under different energy densities of 11.7 J/mL, 35.1 J/mL, 58.5 J/mL, 81.9 J/mL and 117.0 J/mL. Energy density (J/mL)

Adsorbed protein (%) 80:20

60:40

50:50

40:60

MPI

Surface load (mg/m2) WPI

80:20

60:40

50:50

40:60

MPI

WPI

11.7

2.48a

3.70a

8.62a

4.44a

2.42a

3.77a

0.32a

1.88a

0.80a

2.84a

0.43a

1.56a

35.1

5.49b

6.67b

10.17a

6.20b

5.72b

6.60b

1.55b

2.65b

4.25b

2.76a

1.66b

2.28b

58.5

6.63c

9.90c

14.55b

8.96c

8.48c

16.36c

2.31c

3.45c

5.04c

2.86a

2.46c

4.90c

81.9

8.05d

12.12d

20.81c

12.59d

9.56d

16.77c

2.67c

3.29c

6.41d

3.68b

2.76d

4.64c

117.0

13.64e

22.36e

26.06d

13.67e

14.13e

26.20d

2.70c

5.74d

7.68e

3.66b

2.88d

6.56d

EAI (m2/g)

ESI (h)

11.7

15.85a

17.94a

1.17a

18.04a

12.54a

30.68a

88.16a

45.18a

-

38.42a

26.57a

170.93a

35.1

20.48b

29.55b

9.10b

33.60b

26.72b

32.91b

103.44b

221.05b

128.49a

209.28b

73.63b

429.95b

58.5

34.85c

34.23c

29.56c

34.47b

33.80c

34.84c

754.29c

833.13c

171.98b

380.99c

727.55c

1609.96c

81.9

35.69d

34.08c

33.80d

34.89b

34.69d

34.25c

805.41d

1716.83e

843.34d

817.12d

948.52d

2104.77e

117.0

36.32e

35.20c

34.98e

34.00b

31.55d

34.59c

1295.32e

1211.27d

529.55c

1356.85e

729.56c

1760.40d

1

1

Values in the same column and row with different capital and simple superscripts

respectively are significantly different (p < 0.05).

Table 2: Pearson correlation coefficients (r) for emulsifying properties of GSO-M emulsions (EAI: Emulsion activity index, ESI: Emulsion stability index, PS: particle size, AP%: adsorbed protein %).

Correlation variables

80:20

60:40

Casein : whey protein ratio 50:50 40:60

MPI

WPI

EAI & PS ESI & PS

-0.893* -0.801*

-0.969** -0.832*

-0.888* -0.784*

-0.978** -0.697*

-0.945* -0.741*

-0.964** -0.893*

EAI & AP ESI & AP

0.720* 0.917*

0.726* 0.693*

0.890* 0.742*

0.690* 0.949*

0.820* 0.822*

0.839* 0.862*

EAI & ESI

0.918*

0.772*

0.686*

0.559*

0.846*

0.886*

PS & AP

-0.720*

-0.811*

-0.753*

-0.810*

-0.907*

-0.909*

2

* Correlation is significant at the 0.05 level **Correlation is significant at the 0.01 level. 2

Highlights 

Stable GSO-M emulsions were produced using low frequency ultrasound (20 kHz).



Emulsification properties improved with increase in the applied energy density for all milk protein ratios.



Casein:whey protein ratio determined the nature and the composition of the interfacial layer.

Highlights 

Stable GSO-M emulsions were produced using low frequency ultrasound (20 kHz).



Emulsification properties improved with increase in the applied energy density for all milk protein ratios.



Casein:whey protein ratio determined the nature and the composition of the interfacial layer.

 Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

