Roua Lajnaf
email: roua_lajnaf@yahoo.fr

Ines Trigui

Olfa Samet-Bali

Hamadi Attia

M A Ayadi

Comparative study on emulsifying and physico-chemical properties of 3 bovine and camel acid and sweet wheys 4 5

Keywords: Milk protein, techno-functional properties, aggregation, heat treatment, pH

Main goal of this research was to examine the emulsifying properties of acid and sweet wheys extracted from bovine and camel fresh milks after heating at 70 and 90 °C for 30 min at laboratory scale. Specifically, emulsifying properties (emulsification stability (ESI) and activity (EAI) indexes) and the physico-chemical characteristics (surface hydrophobicity, ζpotential, interfacial tension and denaturation rate) of wheys were assessed. Maximum EAI and ESI were found for sweet wheys (EAI~ 2 m²/g; ESI~ 65%), with higher EAI values for the camel whey. This behavior was explained by the strongest electrostatic-repulsive forces between oil droplets under conditions away from the isoelectric-point of proteins in agreement with the ζ-potential measurements.

Findings indicate that heating affected the physico-chemical properties of camel and bovine whey proteins in acidic conditions by increasing surface hydrophobicity and the ability to reduce the interfacial tension. These results confirmed the protein-protein aggregation of heated acid wheys as proved by electrophoreses.

Introduction

Whey is the main by-product of caseins or cheese manufacture. It is of great importance in the dairy industry. Its demand is increasing for whey proteins preparation due to the high functional and nutritional values with application in food ingredients industry [START_REF] Baldasso | Concentration and purification of whey proteins by ultrafiltration[END_REF]. Indeed, whey proteins have become the most employed proteins in food formulations due to their excellent functional characteristics such as emulsification [START_REF] Nishanthi | Properties of whey protein concentrate powders obtained by spray drying of sweet, salty and acid whey under varying storage conditions[END_REF][START_REF] Tosi | Foaming properties of sweet whey solutions as modified by thermal treatment[END_REF].

Techno-functional properties such as emulsifying properties of bovine whey proteins have been intensively studied and reported with special interest in the effect of the physicochemical factors such as the applied heat treatment, the chemical environment and the pH value (Lam andNickerson, 2015a, 2015b;[START_REF] Mellema | Effect of acidification and heating on the rheological properties of oil-water interfaces with adsorbed milk proteins[END_REF][START_REF] Slack | Foaming and emulsifying characteristics of fractionated whey protein[END_REF].

Indeed, the β-lactoglobulin (β-lg), which is the main protein of soluble cow milk fraction, was found to have a lower emulsion stability at acid pH values when compared to that at pH 6.2, in spite of the greater adsorption rate near the isoelectric-point (pI) of this protein [START_REF] Tcholakova | Coalescence stability of emulsions containing globular milk proteins[END_REF]. Furthermore, the α-lactalbumin (α-la) protein was found to coat the emulsion droplets better at neutral pH than that in acid conditions (Lam and Nickerson, 2015b).

After a heat treatment, denatured milk proteins may polymerize to create aggregates, depending on both pH level and the heating temperature value (de la [START_REF] De La Fuente | Recent advances in the characterization of heat-induced aggregates and intermediates of whey proteins[END_REF].

Thus, the resulting emulsifying and foaming properties of proteins will be a competitive adsorption phenomenon between aggregates and non-aggregated denatured milk proteins [START_REF] Schmitt | Whey protein soluble aggregates from heating with NaCl: physicochemical, interfacial, and foaming properties[END_REF][START_REF] Lajnaf | The foaming properties of camel and bovine whey: The impact of pH and heat treatment[END_REF].

Camels are well-known producers of milk which is used as main food resource for populations in the arid countries and hot regions of the world. Camel milk is a potential source of all the essential nutrients already found in cow milk. Besides, it is rich in iron, lactoferrin and vitamin C [START_REF] Kappeler | Sequence analysis of camel (Camelus dromedarius) lactoferrin[END_REF]. Compared to the bovine whey, camel whey is devoid of the β-lg which has been considered as one of the most dominant bovine milk allergen limiting the use of this milk for the preparation of infant formulae [START_REF] Uchida | Process for preparing a fraction having a high content of α-lactalbumin from whey and nutritional compositions containing such fractions[END_REF]. Thus, the α-la is the major protein of camel soluble protein fraction, with a concentration of 2.2 g/L [START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF]. As bovine α-la, camel α-la is a calciummetalloprotein composed of 123 amino acid residues, with a molecular weight of 14.43 kDa [START_REF] Beg | The primary structure of alpha-lactalbumin from camel milk[END_REF]. However, when compared to bovine α-la, camel one presents a different structure justified by a difference in the nature of 39 aminoacids between these two different proteins. Thus, the percentage sequence similarity and identity between the sequences are 82.9 % and 69.1 %, respectively. Camel α-la has a considerably more hydrophobic core than its bovine counterpart at positions 25-35. The secondary structure of this protein is more preserved during heat treatment, so, the camel α-la is more thermostable than its bovine equivalent. However, its conformation is more sensitive to calcium loss [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF].

The α-la can be denatured in several ways, such as pH and heating temperature. For instance, at pH < 5, the α-la has been found to lose its bound calcium ions and assumes the molten globular state [START_REF] Matsumura | Competitive adsorption of αlactalbumin in the molten globule state[END_REF]. Furthermore, the α-la denatures at relatively low temperatures (~64 °C) but does not aggregate at this temperature value because of its lack of free thiol groups. However, when held at temperatures > 85 °C, the α-la evolves free -SH groups that form intermolecular disulfide-bonded aggregates [START_REF] Doi | Heat-induced complex formation between κ-casein and α-lactalbumin[END_REF][START_REF] Mcguffey | Denaturation and aggregation of three α-lactalbumin preparations at neutral pH[END_REF].

Few studies describe the emulsifying behavior and interfacial properties of camel whey proteins [START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF] even if for cow proteins, these properties were extensively studied and reported [START_REF] Fachin | Effect of pH and heat treatment of cheese whey on solubility and emulsifying properties of whey protein concentrate produced by ultrafiltration[END_REF]Lam and Nickerson, 2015a;[START_REF] Jiang | Effect of heat treatment on physicochemical and emulsifying properties of polymerized whey protein concentrate and polymerized whey protein isolate[END_REF].

Thus, the mechanism of emulsion creation of camel milk proteins has not been widely studied in the literature and the absence of the β-lg is suggested to have a significant impact on the resulting emulsifying properties at different pH values.

Therefore, the goal of this work is to examine the effect of different heat treatments (70 and 90 °C for 30 min) on the emulsifying properties of the extracted camel and bovine sweet and acid wheys at laboratory scale. The temperature values of the heat treatment (70 and 90 °C) were chosen according to previous works as [START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF] and [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF]. These authors demonstrated that at 70 °C, the β-lg molecules are reduced from dimers to monomers and begin to unfold. Besides, the denaturation temperature values of both bovine and camel α-la are near 70 °C. [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF] reported also that 90 °C is the temperature of the total denaturation and aggregation of whey proteins. This work would allow the valorization of the camel and bovine whey by-product of the cheese industry, as an ingredient in food industry.

Materials and methods

Whey separation

Fresh raw camel milk (Camelus dromedarius) was obtained from a cattle belonging to a farm which is located in the region of Medenine in Tunisia. Fresh bovine milk was derived from a local breed in the region of Sfax (Tunisia).

When arriving to the laboratory (at 4 °C), the physico-chemical composition of camel and bovine milk was systematically determined according to AOAC Official Method (AOAC, 1984). Both milk samples were skimmed by centrifugation at 3000g for 20 min at 4 °C using a Thermo Scientific Heraeus Megafuge Centrifuge (Germany) and pH values of milk were measured using the pH meter "Metrohm" [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF].

Sweet wheys of bovine and camel milks were extracted from skimmed milks after an enzymatic coagulation at 37 °C for 1-2 h in the presence of microbial rennet enzyme (0.35 and 1.4 mL per liter of skimmed bovine and camel milks, respectively) (Parachimic, Laboratories Arrazi, Sfax, Tunisia, strength = 1:10,000). Whereas, acid wheys were isolated after the acidification of skimmed milks using a solution of HCl (1 M) until pH = 4.3 and 4.6 for camel and bovine milks, respectively [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF].

Afterwards, wheys were separated from casein fraction by a centrifugation at 3000g for 20 min at 20 °C (Thermo Scientific Heraeus Megafuge Centrifuge, Germany).

Whey solution preparation

The protein content of the isolated wheys was determined using the Kjeldahl method (AOAC, 1984).

Camel and bovine heated wheys were obtained after a heat treatment using water-bath at 70 and 90 °C for 30 min followed by an ice incubation to stop the proteins denaturation. The control whey sample was at 20 °C. It corresponds to the native conditions. The selected parameters of the thermal treatment temperatures are chosen according to previous studies [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF][START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF].

Emulsifying properties

Oil-in-water emulsions were prepared by mixing 15% (v/v) corn oil (3.75 mL) and 85% (v/v) (21.25 mL) whey protein solutions at a concentration of 5 g/L using the Ultra-Turrax T25 high-speed mixer (IKA Labortechnik, Staufen, Germany) at a speed of 21,500 rpm for 30 seconds.

A 100 µL aliquot of freshly created emulsion was taken from the bottom of the beaker and dispersed into 900 µL of 0.1% sodium dodecyl-sulfate (SDS) solution (w/v). The mixture was vortexed for 10 seconds and the absorbance was measured at λ = 500 nm using a UV mini-1240 PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

The created emulsions were kept undisturbed for 10 min and then 100 mL aliquots were taken and dispersed into 900 µL of 0.1% SDS solution. The absorbance of the emulsion-SDS mixture was also measured at 500 nm as described above. Emulsifying activity index (EAI, m²/g) and emulsion stability index (ESI, %) of whey solutions were calculated using Eq. (1) and Eq. (2) suggested by [START_REF] Pearce | Emulsifying properties of proteins: evaluation of a turbidimetric technique[END_REF]:

EAI (m²/g) = [2×2.303×A500×dilution]/[C×(1-Φ)×10 4 ]
(1)

ESI (%) = [A10 /A0] ×100 (2)
where A500 represents the absorbance of analysis whey sample at 500 nm, C is the protein concentration (g/mL), Φ is the volume of the oil fraction (v/v) of the created emulsion (Φ = 0.15), 100 is the dilution, A0 and A10 represent the absorbance at 500 nm at time zero and after 10 min, respectively.

Emulsifying activity index (EAI) and emulsion stability index (ESI) of Whey Protein Isolate (WPI, Lactalis Ingredient, Laval, France) and Sodium caseinates (C8654, Lot# BCBC3986V, Product of New Zealand) were also determined (at a protein concentration of 5 g/L) in order to compare emulsifying properties of camel and bovine wheys-based emulsifier agents to commercial emulsifiers.

SDS-PAGE electrophoresis

SDS-PAGE electrophoresis was carried out on gels (12% acrylamide gel) using the technique described by [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF] and [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Electrophoresis was performed at a constant current 120V for 1.5-2 h (Mini Protean Tetra Cell, BioRad laboratories, USA).

Quantitative estimation of each whey protein amount was carried out using an appropriate software (Gel-Quant.NET; biochemlabsolutions.com).

Thiol groups content and denaturation rate

Free thiol groups were quantified as described by [START_REF] Ellman | Tissue sulfhydryl groups[END_REF]. 300 µL of the extracted wheys at a concentration of 1 g/L were mixed with 50 µL of DTNB (5,5′-dithio-bis(2nitrobenzoic acid) solution (2 mM DTNB, 50 mM sodium acetate (NaAc) in H2O), 100 µL of Tris solution (1 M Tris, pH 8.0) and 550 μL of distilled water.

Mixture was incubated for 5 min at 37 °C using water bath. Then, the optical density (DO) was measured at λ = 412nm.

The free SH groups concentration (CSH) was calculated by Eq. ( 3):

CSH (M) = (DO412nm/ ε412) × (1000/300) ( 3
)
where DO412nm is the absorbance at λ = 412 nm; ε412 is the DTNB extinction coefficient (13,600 M -1 /cm) at λ = 412nm. 1000 µL is the cuvette volume and 300 µL is the protein sample volume.

In this assay, the whey proteins' denaturation rate (DR) was calculated by the Eq. ( 4):

Denaturation rate (%) = ( ) ( ) ( ) ⨯100 (4)

ζ-potential measurements

The ζ-potential values of the extracted whey proteins were determined at 25 ± 1 °C using the Zetasizer Nano-ZS90 appartus (Malvern Instruments, Westborough, MA) at a protein concentration of 0.5 g/L as suggested by Lam and Nickerson (2015b) .

The ζ-potential value (mV) was measured using Henry's equation (Eq. ( 5)):

UE= ( ) ɳ (5)
where: UE is the electrophoretic mobility, ε the permittivity (Farad/m); k, the Debye length; f(kα) the function of the radius of particle (α, nm); ɳ the viscosity of the dispersion (mPa s).

2.7.Determination of hydrophobicity

The surface hydrophobicity of the whey proteins was measured using the method described by [START_REF] Chelh | A simplified procedure for myofibril hydrophobicity determination[END_REF]. Briefly, 1 mL of whey sample (at a protein concentration of 1 mg/mL) and 200 µL of 1 mg/mL bromophenol blue (BPB) were added and mixed well.

A control was prepared using Tris-HCl buffer (20 mM, pH 8.0) instead of sample.

Whey samples were kept under agitation for 10 min at room temperature and then centrifuged at 2,000g for 15 min at 25 °C.

Supernatants were diluted 1:10 and the DO was then measured at λ = 595 nm against a blank Tris-HCl buffer.

In this assay, a higher amount of bound-BPB indicates a higher proteins' hydrophobicity which was calculated by Eq. ( 6):

Bound-BPB (µg) = !" # !$ #! "!% - !" # !$ '(% !" # !$ #! "!% ⨯200 µg (6)

Interfacial tension

Interfacial tension for each whey protein solution was measured using a TSD-971

Tensiometry System Digital (Gibertini Elettronica, Italia) via the "Du Noüy methodology" as described by Lam and Nickerson (2015b).

Thus, for the determination of the surface tension, the whey solution was placed in an oil-water system. Indeed, within a 40 mm diameter glass sample beaker, 20 mL of whey solution at a protein concentration of 1 g/L were added, followed by the immersion of the Du Nüoy ring (20 mm diameter) and then the addition of upper corn oil layer (20 mL).

Finally, the ring was pulled upwards to stretch the oil-water interface to determine the maximum force and then to calculate the interfacial tension value (mN/m).

The interfacial tension was calculated by the Eq. ( 7)

γ = )' * +,-. ( 7
)
where γ is the interfacial tension (mN/m), Fmax is the maximum force (mN), R is the radius of the used ring (20 mm), β is a correction factor which depends on two factors : the density of the liquid and the dimensions of the ring. All interfacial tension analyses were realized at 25 °C.

Statistics

All experimental analyses and measurements in this work were performed in triplicate and mentioned as the mean value ± standard deviation.

The ANOVA test was used to test for significance in the main effects of the extracted whey samples and heat treatment conditions, along with their associated interactions on the hydrophobicity, surface tension, thiol groups' content, ζ-potential measurements and emulsifying properties indexes of proteins.

Statistical analyses were determined using SPSS-statistics (Version 19, IBM, USA).

Results and discussion

Chemical composition of camel and bovine milk and whey proteins content

The physico-chemical composition of camel and bovine milk was determined in this study as shown in the Table 1. Table 1 shows that the main values of protein concentration in camel milk (22.06 ± 0.73 g/L) were significantly lower than that in bovine milk (28.36 ± 0.27 g/L) (p<0.05). Moreover, the main components of camel milk were relatively close to that of bovine milk including fat, lactose, ash and total solids.

Analysis of the protein content of the different extracted wheys indicated that the protein concentration in acid and sweet bovine whey samples were 10.79 ± 0.10 and 11.55 ± 0.12 g/L of whey, respectively. The protein content in sweet bovine whey was significantly higher (p<0.05) than that in its acid counterpart probably due to the presence of caseinomacropeptide in the sweet whey after rennet coagulation.

Whereas, no significant differences in the proteins concentration were observed between acid and sweet camel wheys (6.44±0.18; 6.52±0.01 g/L for acid and sweet camel wheys, espectively). This behavior could be explained by the lack of the caseinomacropeptide in camel whey which is mainly related to the reduced κ-casein content in camel milk [START_REF] Ekstrand | Casein micelle size and composition related to the enzymatic coagulation process[END_REF][START_REF] Al Haj | Compositional, technological and nutritional aspects of dromedary camel milk[END_REF].

3.2.Emulsifying properties

The EAI measures how well proteins can coat an oil droplet surface within a dilute emulsion, while the ESI gives an estimate of the emulsion's stability as function of time [START_REF] Pearce | Emulsifying properties of proteins: evaluation of a turbidimetric technique[END_REF]. EAI values of camel and bovine wheys' solutions at a protein concentration of 5 g/L as function of the temperature of the thermal treatment (70 and 90 °C) are shown in Figure 1A.

Thus, it was found that sweet wheys, regardless of milk origin and heating temperature (70 and 90 °C for 30 min) coated the oil droplets better than acid wheys with higher EAI values of the sweet camel whey (2.2 and 1.9 m²/g for sweet camel and bovine wheys, respectively under native conditions). Whereas, in acidic conditions, a better emulsification activity was achieved with the bovine whey when compared to its camel counterpart (1.7 and 1.3 m²/g for both acid bovine and camel wheys, respectively). Statistical analyses showed that heating reduced significantly the EAI values (p<0.05) for acid wheys probably due to the extensive aggregation and denaturation of preheated whey proteins at acid pH values (Lam and Nickerson, 2015a).

The emulsification ability significantly increased for both sweet wheys after heating at 90 °C for 30 min (p < 0.05) and achieved maximum EAI values in these conditions (EAI ~ 2.5 min). These results are in agreement with those of [START_REF] Dissanayake | Functional properties of whey proteins affected by heat treatment and hydrodynamic high-pressure shearing[END_REF] who reported that the EAI values of whey proteins increased with thermal treatment and proteins denaturation. On the other hand, [START_REF] Fachin | Effect of pH and heat treatment of cheese whey on solubility and emulsifying properties of whey protein concentrate produced by ultrafiltration[END_REF] found that the emulsifying properties were considerably improved after heating at pH 7. This improvement in emulsifying properties was justified by the greater denaturation of whey proteins in these conditions. [START_REF] Mellema | Effect of acidification and heating on the rheological properties of oil-water interfaces with adsorbed milk proteins[END_REF] reported that the heat treatment (85 °C for 20 min) of whey protein solutions at pH 6.7 improved the ability of proteins to adsorb at oil-water interface resulting in lower interfacial tension values, and clearly elevated values for the viscoelastic modulus as compared to the native whey solutions (without heating). On the other hand, [START_REF] Lajnaf | The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk[END_REF] found that, the foamability and the interfacial properties of the purified camel α-lactalbumin solution at neutral pH are significantly improved by heat treatment (i.e. 70 and 90 °C for 30 min).

Difference between camel and bovine wheys can be attributed to the difference in protein composition of both wheys and their conformational change at neutral and acidic pH values. [START_REF] Suttiprasit | The surface activity of α-lactalbumin, βlactoglobulin, and bovine serum albumin[END_REF] noted that at neutral pH, the α-la is more efficient to reduce the interfacial tension than the β-lg, as it is more flexible and smaller. Whereas, [START_REF] Laleye | Comparative study on heat stability and functionality of camel and bovine milk whey proteins[END_REF] reported that the lower emulsifying properties of pre-acidified camel whey are believed to be due to the pronounced aggregation of camel whey protein molecules. These authors noted that the aggregation behavior of camel milk proteins at lower pH levels is explained by the high content of the α-la. Thus, the high proportion of the α-la can explain the low EAI values of acid camel whey compared to acid bovine whey.

The ESI values of camel and bovine whey solutions as a function of temperature pretreatment are shown in Figure 1B. ESI values of sweet wheys were higher than those of acid wheys regardless of the heating temperature value in agreement with the findings of Lam andNickerson (2015a, 2015b). [START_REF] Yamauchi | Emulsifying properties of whey protein[END_REF] and [START_REF] Leman | Creaming stability of fluid emulsions containing different milk protein preparations[END_REF] reported that the stability of whey protein emulsions increases when pH level is increased from 5 to 7, which is probably due to an increase in repulsion by the electrostatic charge of the proteins Indeed, the magnitude of the electrical charge on the oil droplets surface decreased at pH values close to the pI of proteins. Consequently, the negative charge of acid wheys proteins is insufficient to generate electrostatic-repulsive forces between the created oil droplets leading to various attractive droplet-droplet interactions as Van der Waals and the hydrophobic forces and then, droplets aggregate forming large flocculates (McClements, 2004).

For sweet bovine whey, ESI data were similar under native conditions and at 70 °C (~65%), and then increased to 80% min at 90 °C (Figure 2B). For sweet camel whey, ESI values increased significantly from 54% at 70 °C, to 75% at 90 °C (p<0.05).

ESI of acid wheys rose significantly (p<0.05) as function of temperature. Thus, ESI reached their maximum at 90 °C with values of 54% and 60% for acid bovine and camel wheys, respectively.

This behavior could be attributed to the increase in the adsorption velocity and the diffusion of heated milk proteins at the oil-water interface as reported by [START_REF] Dickinson | Hydrocolloids at interfaces and the influence on the properties of dispersed systems[END_REF]. [START_REF] Jiang | Effect of heat treatment on physicochemical and emulsifying properties of polymerized whey protein concentrate and polymerized whey protein isolate[END_REF] have found that heated whey proteins (at 85 °C for 30 min) give more stable emulsions than unheated proteins due to the creation of large protein aggregates and the further negative charge after heating. For the camel whey, [START_REF] Lajnaf | The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk[END_REF] noted that the open structure of the camel α-la molecule and the reduced electrostatic repulsion of this protein near its pI are all factors that could promote the creation of large aggregates resulting in a greater ability to stabilize foam and emulsions.

A comparison between camel and bovine wheys-based emulsifier agents and commercial dairy emulsifiers which are whey protein isolate (WPI) and sodium caseinates (Na-cas) reveled that, for both dairy emulsifiers, EAI values were higher than those of camel and bovine wheys-based emulsifiers' agents (EAI~ 4.33 and 4.87 m²/g for WPI and Na-cas, respectively). On the other hand, emulsions created by camel and bovine wheys were more stable than those of WPI solutions (ESI~ 38.65%). Na-cas had the best stabilizing properties (ESI~ 88.8%). Furthermore, the heat treated WPI and Na-cas solutions (i.e. 70 and 90 °C for 30 min) were found to coat the emulsion droplets better than both heated camel and bovine wheys reaching EAI values ∼ 5.57 and 4.91 m²/g for heated WPI and Na-cas, respectively.

No significant difference was observed between ESI values of heated camel and bovine wheys and WPI solutions (ESI~ 66.66%). Heat treated Na-cas exhibited the highest emulsion stabilizing abilities when compared to the extracted wheys (ESI> 90%). These results are in agreement with the findings of [START_REF] Casper | Functional properties of whey protein concentrates from caprine and ovine specialty cheese wheys[END_REF]. Indeed, these authors reported that Bovine cheese whey showed lower emulsifying capability at pH 8 when compared to WPI.

Therefore, this behavior depended on protein composition and physico-chemical conditions. As expected, the free thiol groups' concentration of both wheys raised significantly after heating as function of temperature. Consequently, the DR values reached their maximum at 90 °C with values of 1220±211% and 183±50% for acid and sweet bovine wheys, respectively and 1143±150% and 419±100% for acid and sweet camel wheys, respectively.

Protein denaturation and aggregation

Denaturation rate

It could be explained by the milk proteins denaturation which happened during 30 min of heating at 90 °C regardless of the milk proteins origin as reported by [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF].

Acid wheys carried higher DR values compared to their sweet counterparts regardless of heating temperature value. This result confirmed that acid wheys are characterized by a higher thermal sensitivity than the sweet wheys. Lam and Nickerson (2015a) reported that the size of whey proteins' aggregates is the greatest in acidic conditions due to the minimized electrostatic repulsion between neighboring proteins molecules leading them to interact and aggregate. Whereas, the high electronegative charge of sweet whey proteins may electrostatically keep them from aggregation after heating.

Electrophoresis patterns

We characterized the aggregation and the denaturation behavior of camel and bovine wheys after heating (at 70 and 90 °C for 30 min) by the gel electrophoretic patterns (Figure 3).

The densitometry analysis of the gel corresponding to native acid and sweet bovine wheys (Figure 3A L1 and Figure 3B L1, respectively) showed that five major protein bands (150 kDa, 70 kDa, 30 kDa, 18 kDa and 14 kDa) were identified as immunoglobulins (~3%), BSA (~8%), protein fraction F (~4%), β-lg (~65%) and α-la (~20%).

For native acid and sweet camel wheys (Figure 3C L1 and Figure 3D L1, respectively), five major protein bands with molecular weight of 80 kDa, 66 kDa, 22 kDa, 19 kDa and 14 kDa were identified, corresponding to lactoferrin (~2%), camel serum albumin (CSA) (~21%), protein fractions F1 (~10%) and F2 (~15%) and α-la (~52%), respectively. As expected, no band corresponding to β-lg was detected as already reported by previous authors [START_REF] Ereifej | Comparison and characterisation of fat and protein composition for camel milk from eight Jordanian locations[END_REF][START_REF] Omar | Quantification of major camel milk proteins by capillary electrophoresis[END_REF].

Figure 3A shows that when acid bovine whey was heated, the serum albumin and the αla appeared to decrease in intensity with temperature value (L2 and L3); whereas, for sweet bovine whey (Figure 3B), no effect was recorded on β-lg and α-la protein bands heated at 70 and 90 °C for 30 min. Heated bovine whey samples showed the appearance of a protein band (42 kDa) which is probably formed during heating.

When acid camel whey was heated at (70 or 90 °C), α-la and protein fractions (F1 and F2) bands appeared to increase in intensity (Figure 3C). For raw sweet camel whey (Figure 3D), the thermal treatment at 70 °C (L2) and 90 °C (L3) for 30 min induced an immediate disappearance of the α-la and the appearance of several faint distinct bands, which were probably intermediate protein species (e.g., α-la dimers, trimers) that were formed during heating.

Similar results were obtained by [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF]. These authors noticed that the disappearance of the CSA and α-la electrophoretic bands could be the consequence of camel proteins denaturation and/or aggregation. For bovine wheys, the denaturation is maintained by the presence of the β-lg as reported by de la Fuente et al. (2002). Thus, these authors noted that the β-lg dominates the behavior of the milk protein aggregation under different thermal treatments due to its particular molecular characteristics. Thus, this protein reacts more easily with itself than with the α-la leading to the creation of heat induced complexes rich in β-lg.

However, the rate of disappearance of the α-la was greater when compared to the β-lg because of the higher number of intramolecular disulphide bonds and lower thermal transition temperature of the α-la [START_REF] Schokker | Heat-induced aggregation of β-lactoglobulin A and B with α-lactalbumin[END_REF].

In order to understand the aggregation phenomena in camel whey, [START_REF] Felfoul | A laboratory investigation of cow and camel whey proteins deposition under different heat treatments[END_REF] showed that the α-la in acid camel whey has a lower denaturation temperature (60.5 °C) than that in sweet camel whey (73.8 °C). Besides, [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF] found that the camel α-la apo state is more sensitive to heat treatment than the holo state.

Surface characteristics

Surface hydrophobicity

Hydrophobicity of bovine and camel isolated wheys at a protein concentration of 1g/L as a function of heating temperature value (70 and 90 °C for 30 min) is shown in Figure 4.

The BPB-bound amounts were 58.4±7.1 µg/mL and 5.5±1.1 µg/mL for acid and sweet bovine wheys, respectively, and 98.9±7 µg/mL and 11.5±4 µg/mL for acid and sweet camel wheys, respectively.

Acid wheys carried higher BPB-bound amounts compared to sweet wheys and the maximum hydrophobicity values were achieved with the acid wheys regardless of the temperature of the thermal treatment. Indeed, after heating at 90 °C for 30 min, BPB-bound amounts of acid camel and bovine wheys reached 174 and 147 µg/mL of whey, respectively.

For both sweet wheys, statistical analysis showed that heating has no significant effect on the protein's hydrophobicity with the lowest BPB-bound amounts (~15 µg/mL) after heating at 90 °C for 30 min.

In support of these results, [START_REF] Voutsinas | Relationships of hydrophobicity to emulsifying properties of heat denatured proteins[END_REF] found that the surface hydrophobicity of whey proteins increases with heating (80 °C for 4 min). Lam and Nickerson (2015a) have found that the heat-treatment (85 °C for 30 min) of a pre-acidified whey protein isolate (WPI) solution (pH 5) resulted in high hydrophobicity rate values. These authors reported that the high hydrophobicity is mainly attributed to the combined effect of reduced net charge and protein denaturation of whey. Indeed, the combination of acidification and heating of whey proteins led to the exposure of the buried hydrophobic moieties to the surface of the unfolded protein. But, the reduced hydrophobicity of whey proteins at neutral pH after heating may reflect the greater contribution of surface negative charge which could restrict complete whey protein unfolding [START_REF] Kato | Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins[END_REF]Lam and Nickerson, 2015a).

The different behavior of both acid wheys could be explained by their protein composition. For the bovine whey, [START_REF] Zhang | Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins[END_REF] reported that the β-lg is thermodynamically stable in acidic conditions and more rigid than the α-la.

The highest surface hydrophobicity of acid camel whey proteins can be attributed to the lack of the β-lg in this serum and also to the greatest hydrophobicity of camel α-la. Thus, fluorescence results obtained by [START_REF] Atri | Comparative study on heat stability of camel and bovine apo and holo αlactalbumin[END_REF] reported that camel α-la shows a greater surface hydrophobicity than its bovine counterpart. Indeed, primary structure of camel α-la contains more hydrophobic residues such as Tryptophan (Trp). Thus, after calcium removal, the camel α-la showed a greater surface hydrophobicity due to the greater hydrophobicity of the N-terminal part of its α-helical domain.

Determination of ζ-potential

Surface charge values (or ζ-potential) for bovine and camel wheys (acid and sweet) at a protein concentration of 0.5g/L after heating at 70 and 90 °C for 30 min were measured and given in Figure 5.

Overall, the ζ-potential values of acid wheys were significantly lower than that of their sweet counterparts (p<0.05). Under native conditions, the ζ-potential values were ~-2.5±0.4

and ~-18.12±0.6 mV for acid and sweet camel wheys, respectively and ~-3.85±0.5 and ~-20.79±1.2 mV for acid and sweet bovine wheys, respectively. These findings are in agreement with the ζ-potential results of [START_REF] Momen | Effect of dry heating on physico-chemical, functional properties and digestibility of camel whey protein[END_REF]. These authors confirmed that whey proteins of camel milk carried lower negative charge than cow whey proteins. They suggested that this difference can be explained by the variance in the protein composition, the pI of both α-la (4.87 and 4.65 for camel and bovine α-la, respectively) and the presence of the lactoferrin in camel whey which is a highly basic protein (pI = 8.8).

Figure 5 shows also that, the electronegative charge of both sweet wheys was not significantly modified after heating, while after a heat-treatment at 70 °C, it significantly decreased to ~-1.16 and ~-1.04 mV for acid bovine and camel wheys, respectively.

These results are in agreement and with the findings of Lam and Nickerson (2015a) and with the highest surface hydrophobicity observed with the acid wheys (Figure 4). Indeed, at neutral pH, whey proteins molecules were strongly negatively charged which may electrostatically repel BPB from binding to the protein. In contrast, near whey protein's pI, the reduction of the electrostatic repulsion and the open structure could promote protein-BPB interactions.

Interfacial properties

The interfacial tension between corn oil and whey proteins' solutions (at a protein concentration of 1 g/L) in response to temperature pre-treatments (70 and 90 °C for 30 min)

and pH values (acid and sweet wheys) are shown in Figure 6. Overall, both camel and bovine wheys have significantly reduced the interfacial tension at oil-water interface from 29.1mN/m (p<0.05).

Thus, in native conditions, the order of effectiveness to reduce the surface tension at the oil-water interface was: acid camel whey (γ = 19.6±1.9 mN/m) > acid bovine whey (γ = 23.4±0.8 mN/m) > sweet camel whey (γ = 25.1±0.7 mN/m) > sweet bovine whey (γ = 26.9±0.5 mN/m).

The heat-treatment improved the tensioactivity of whey proteins and their ability to reduce the interfacial tension at oil-water interface depending on the pH value.

Indeed, when heating sweet wheys at 70 °C for 30 min, the surface tension declined significantly (p<0.05) from 26.9 to 24.3 mN/m and from 25.1 to 22.7 mN/m for sweet bovine and camel wheys, respectively. No significant change was found on the evolution of surface tension values of sweet wheys between 70 and 90 °C.

After a heat-treatment of 90 °C for 30 min, acid wheys have retained the best interfacial properties when compared to the sweet wheys. Thus, the order of effectiveness at 90 °C was: acid camel whey (γ = 9.35±1.34 mN/m) > acid bovine whey (γ = 17.5±1.0 mN/m) > sweet camel whey (γ = 21.45±1.5 mN/m) and sweet bovine whey (γ = 23.05±0.8 mN/m).

These results are in agreement with those of Lam and Nickerson (2015a) who noted that pH plays a key role in the adsorption of whey proteins at the oil-water interface. Indeed, electrostatic repulsion between whey proteins molecules leads to greater difficulties in aligning at the interface to create a viscoelastic film. Whereas, in acidic conditions, whey proteins carried less negative charge near their pI, allowing for higher proteins interactions and better adsorption at the interface.

Conclusion

Whey emulsifying properties depended on the pH level, the protein composition and the degree of denaturation of these proteins after a thermal treatment. A Higher oil droplets surface coverage (EAI) was obtained for sweet wheys especially the sweet camel whey.

Furthermore, stability of emulsions seemed greatest for sweet wheys due to the presence of electrostatic repulsive forces between proteins as confirmed by the ζ-potential measurements.

A heat-treatment at 70 and 90 °C of the acid wheys for 30 min resulted in a significant increase in ESI values due to the denaturation and aggregaton of proteins. Finally, these results confirmed the strong potential of camel and bovine wheys as emulsifier agent for potential applications in industrial emulsion production. Total solids (%) 13.01±0.12 12.95±0.17

Figure 2

 2 Figure 2 compares the DR values of wheys at a protein concentration of 1g/L as a

FiguresFigure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Effect of temperature (70 (∎) and 90 °C (⧠) for 30 min) on camel and bovine whey

Figure 3 .

 3 Figure 3. SDS-PAGE electrophoresis patterns of acid bovine whey (A), sweet bovine whey

Figure 4 .

 4 Figure 4. Surface hydrophobicity (µg of BPB-bound) of camel and bovine whey protein

Figure 5 .

 5 Figure 5. ζ-potential measurements (mV) of camel and bovine whey protein solutions, at a

Figure 6 .

 6 Figure 6. The interfacial tension (mN/m) of camel and bovine whey protein solutions, at a

Table 1 .

 1 Chemical composition of camel and bovine milk(AOAC, 1984)

	Tables
		Bovine milk	Camel milk
	Protein (g/L)	28.36±0.27	22.06±0.73
	Fat (g/L)	34.1±0.5	35.4 ± 0.6
	Lactose (g/L)	46.1±2.2	43.5±1.1
	Ash (%)	0.89±0.01	0.69±0.04
	NPN (non-protein
		0.69±0.03	0.43±0.03
	nitrogen) (g/L)

a-d Samples represented with different letters are significantly different from each other (p<0.05). Error bars show the standard deviations of mean values of DR.

Acknowledgements This work was funded by the Ministry of Scientific Research and

Technology of Tunisia. We are greatly indebted to Professor A. Ben Ali for editing the manuscript.

Previous studies [START_REF] Shimizu | Emulsifying and structural properties of βlactoglobulin at different pHs[END_REF][START_REF] Hunt | Effect of pH on the stability and surface composition of emulsions made with whey protein isolate[END_REF]) noted that at low pH values the α-la is more dominant at the interface than the β-lg, which could explain the lower surface tension values of the acid wheys. [START_REF] Mellema | Effect of acidification and heating on the rheological properties of oil-water interfaces with adsorbed milk proteins[END_REF] have shown by surface tension measurements that the heat-treatment of pre-acidified bovine whey proteins (85 °C for 20 min) are not stable in solution and will have a high tendency to aggregate or adsorb leading to surface active aggregates and giving high viscoelastic modulus values. These authors reported that, for whey proteins isolates, pre-acidification dominates the interfacial behavior at the oil-water interface over pre-heating. Thus, the combination of pre-acidification and pre-heating of these proteins was found to give results similar to pre-acidification without heating. [START_REF] Lajnaf | The effect of pH and heat treatments on the foaming properties of purified α-lactalbumin from camel milk[END_REF] observed that the α-la isolated from camel milk was more flexible at acid pH, regardless of heating temperature, due to the reduced negative charge of this proteins and its molten globular state at low pH values.

Thus, it can be suggested that the interfacial behavior of camel whey, at the oil-water interface, is maintained by the major protein which is the camel α-la regardless of the applied heat-treatment. Thus, a greater tensioactivity of the acid camel whey can be attributed to the highest hydrophobicity rate of camel α-la (section 3.3.1).

Despite the highest tensioactivity of acid wheys, greater droplet coverage and higher emulsion stability were found for sweet wheys regardless of the milk origin. These findings suggest that better oil droplet coverage could not be maintained by neutrally charged proteins even if they exhibited the highest effectiveness to reduce the surface tension at the oil-water interface in agreement with of Lam and Nickerson (2015a). Whereas, for sweet wheys, electrostatic repulsion may help proteins to spread out at the oil droplet surface leading to droplet coverage by the negatively charged proteins.


Ashkan Madadlou

Marie-Hélène Famelart

Stéphane Pezennec

Florence Rousseau

Juliane Floury

Didier Dupont
email: didier.dupont@inra.fr

Marie-Helene Famelart

Interfacial and (emulsion) gel rheology of hydrophobised whey proteins

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Different types of whey proteins, such as β-lactoglobulin (β-lg) and bovine serum albumin, as well as whey protein products such as whey protein isolate (WPI) and whey protein concentrate (WPC), are widely used in various fields of food technology, including foam and emulsion stabilisation (Jaishankar, Sharma, & McKinley, 2011), fat replacement, and gel formation [START_REF] Zhang | Properties of partially denatured whey protein products: Viscoelastic properties[END_REF]. Each application may require modification of one or several functional properties of whey proteins. Enzymatic hydrolysis, heat-induced polymerisation [START_REF] Foegeding | Advances in modifying and understanding whey protein functionality[END_REF], high pressure processing [START_REF] Liu | Modification of whey protein concentrate hydrophobicity by high hydrostatic pressure[END_REF], and chemical functionalisation [START_REF] Zhao | Study of succinylated food proteins by Raman Spectroscopy[END_REF]) are examples of whey protein modifications.

Recently, WPI was hydrophobised through a process consisting of two consecutive steps including a pre-acetylation stage followed by a combined acetylation/heat treatment period. The process resulted in acetylation of ≥90% of available amino groups of whey proteins. Details about the structural characterisation of the hydrophobised WPI have been provided elsewhere [START_REF] Madadlou | Structural assessment and catalytic oxidation activity of hydrophobized whey proteins[END_REF]. The modified product was then used to form a water-in-water (W/W) emulsion of alginate and WPI, which was attributed to the adsorption of protein particles to the liquid-liquid interface. The interfacial tension between the two incompatible biopolymer phases was further modulated by addition of erythritol, a low-calorie sugar alcohol [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF].

Adsorption of macromolecules and particles to the liquid/liquid interface during emulsification forms a viscoelastic layer, which can be assessed by shear rheology components (elasticity and viscosity). Shear rheology characterises the response of the (particle-laden) interface to a shape deformation caused by an external mechanical perturbation, in a similar way done for bulk rheology [START_REF] Mendoza | Particle laden fluid interfaces: Dynamics and interfacial rheology[END_REF]. Inter-particle attractive and repulsive forces such as electrostatic and hydrophobic interactions govern the interface rheology. Indeed, interfacial rheology is very sensitive to the structure and composition of a protein layer formed at the interface, as well as to the nature of interactions within the layer [START_REF] Dickinson | Time-dependent polymerization of β-lactoglobulin through disulphide bonds at the oil-water interface in emulsions[END_REF]. Therefore, it was presumed that probing interfacial rheology is advantageous for assessing the emulsification behaviour of whey proteins as influenced by hydrophobisation.

Rheological analysis is also helpful to study the gelation properties of whey proteins.

Cold-set gelation of whey proteins is of great value in forming particles that can modify viscosity [START_REF] Zhang | Properties of partially denatured whey protein products: Viscoelastic properties[END_REF], and encapsulate heat-labile cargos [START_REF] Farjami | Modulating the textural characteristics of whey protein nanofibril gels with different concentrations of calcium chloride[END_REF][START_REF] Hashemi | Functional and in vitro gastric digestibility of the whey protein hydrogel loaded with nanostructured lipid carriers and gelled via citric acid-mediated crosslinking[END_REF]. This property can also be used to form whey protein-stabilised emulsion gels for fortification and technological purposes. For example, such emulsions can be incorporated into cultured milk products like yoghurt to improve texture [START_REF] Boutin | Characterisation and acid-induced gelation of butter oil emulsions produced from heated whey protein dispersions[END_REF]. The cold-set gelation process takes place by a two-step consecutive procedure. Initially, the proteins are heat-denatured at non-gelling conditions (i.e., at low ionic strength, low protein content and at pH values far from the proteins' isoelectric point, pI), which causes formation of whey protein soluble aggregates. Then the protein dispersion is gelled at ambient temperature by adding salts and/or adjusting pH toward the pI [START_REF] Ju | Gelation of pH-aggregated whey protein isolate solution induced by heat, protease, calcium salt, and acidulant[END_REF][START_REF] Meydani | Influence of the Maillard reaction on the properties of cold-set whey protein and maltodextrin binary gels[END_REF]. The rheology of such gels is governed by the interaction between the aggregating protein particles, which is highly dependent on the aggregate chemistry [START_REF] Alting | Cold-set globular protein gels: Interactions, structure and rheology as a function of protein concentration[END_REF]. Therefore, the modification of whey proteins via hydrophobisation is expected to alter the cold-set gelation behaviour and the gel rheology of WPI.

The first objective of the present study was to analyse the interfacial rheology of WPI at the air-liquid interface as influenced by heat denaturation and hydrophobisation treatments, as well as, erythritol (E) addition into the hydrophobised WPI. Subsequently, bulk rheological experiments were carried out to assess the acid-induced cold-set gelation process and the gel properties of the hydrophobised WPI (with and without E) and the alginate-supplemented Eadded hydrophobised WPI (i.e., W/W emulsion).

Materials and methods

Materials

WPI was received as a gift from Lactalis ingredients (Bourgbarré, France). It contained, according to the information provided by the manufacturer, 90% protein, 5.1% water, 3.0% lactose, and 1.97% ash. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis indicated that the α-lactalbumin and β-lactoglobulin content of the WPI was 16% and 56% of total protein, respectively. Erythritol was purchased from Vita-World GmbH (Taunusstein, Germany). Alginic acid sodium salt from brown algae was procured from Sigma-Aldrich (SKU W201502, Sigma-Aldrich Chemie S.a.r.l., Saint-Quentin Fallavier, France). Sodium hydroxide and hydrochloric acid were bought from Sigma-Aldrich. Ultrapure, 15 mΩ-resistivity water prepared by reverse osmosis was used throughout the study and all chemicals were analytical grade.

Protein modification, emulsion preparation and gelation

WPI was hydrophobised following the method described in our previous report [START_REF] Madadlou | Structural assessment and catalytic oxidation activity of hydrophobized whey proteins[END_REF]. Briefly, WPI powder was hydrated (65 mg mL -1 ) for 18 h and supplemented with acetic anhydride at a ratio of 0.18 g g -1 protein. After 30 min acetylation while the pH was kept over 8.0 through successive additions of 1 M NaOH, the WPI dispersion was heated at 85 °C for 20 min. A non-acetylated but heat-denatured WPI sample was also prepared by sequentially adding 1 M HCl (200 μL), and 1 M NaOH (300 μL) to a native WPI dispersion (9.5 mL, pH 6.7) to set pH to 8.25 ± 0.05, and subsequent heat treatment at 85 °C for 20 min. The sequential addition of the acid and alkali was carried out to mimic the fall and rise of pH during the hydrophobisation process. After heat treatment, the pH value of the hydrophobised and heat-denatured WPI samples was adjusted to 7.50 ± 0.05 using approximately 20 μL of either 1 M HCl or 1 M NaOH.

The alginate-in-WPI W/W emulsions were prepared according to the method described elsewhere [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF] with minor changes. At first, erythritol was added (100 mg mL -1 ) into the hydrophobised WPI dispersion and stirred, followed by addition of sodium alginate solution (30 mg mL -1 ) into WPI-erythritol mixture at a volume ratio of 1:9.

The mixture was vortexed at a rotating frequency of 40 Hz (Bioblock Top Mix 11118, Fisher Scientific, Waltham, MA, USA) for 90 s to obtain the W/W emulsion.

The hydrophobised (with and without added erythritol) and heat-denatured WPI samples, as well as the alginate-in-WPI mixed (i.e., W/W emulsion) system (pH 7.50 ± 0.1)

were gelled at 37 °C by acidification. Glucono-δ-lactone (GDL) was added (5, 10 and 20 mg mL -1 ) into the samples to gradually hydrolyse into gluconic acid, causing acid gel formation.

The samples were stored at 37 °C for 24 h before analyses. The pH value of the samples during gelation was monitored using a digital pH meter (CyberScan Series pH 110, Eutech

Instrument, Singapore). The accuracy of the pH measurement was ±0.02 pH unit.

Interfacial shear measurements

A stress-controlled rheometer (Discovery Series Hybrid Rheometer (DHR) HR-2, New Castle, DE, USA) using the double wall ring (DWR) geometry was used to perform the interfacial shear analyses including strain amplitude sweep and creep tests. The WPI sample was poured into a round trough and an annular ring made of a platinum-iridium alloy was positioned at the liquid-air interface. The DWR geometry has been thoroughly described [START_REF] Vandebril | A double wallring geometry for interfacial shear rheometry[END_REF]) and provides certainty that high values of the Boussinesq number are attained. Therefore, the contribution of the induced subphase flow to torque is negligible and the interfacial torque dominates [START_REF] Jaishankar | Power-law rheology in the bulk and at the interface: quasi-properties and fractional constitutive equations[END_REF]. Based on time sweep tests at a strain amplitude of 0.1% and an angular frequency of 10 rad s -1 and on the literature review [START_REF] Jaishankar | Interfacial viscoelasticity, yielding and creep ringing of globular protein-surfactant mixtures[END_REF], all steady state experiments were carried out after waiting for 3 h at 20 °C. WPI dispersions (56 mg mL -1 )

were loaded into the trough, the annular ring was positioned at the interface and after the rest period, the experiments were carried out.

It is noteworthy that in the current study, the interfacial shear rheology of WPI dispersions was analysed at the same concentration as emulsion preparation was done.

Commonly, the interfacial rheology tests are performed at much lower concentrations of amphiphilic compounds to make sure that a monolayer of the compounds is present at the interface. However, as noted earlier [START_REF] Lexis | Relating foam and interfacial rheological properties of β-lactoglobulin solutions[END_REF], the structure of the interfacial layers of proteins depends largely on concentration; therefore, a correlation between properties such as foaming and emulsification, and interfacial rheology is achieved only when the interfacial layers at both conditions (i.e., emulsion formation and interfacial rheology test) are comparable. Nonetheless, as the thickness of the interfacial layer may not be negligible anymore, the measured moduli are treated as apparent values. Similar concentrations have already been used for the interfacial rheological analysis of bovine serum albumin [START_REF] Jaishankar | Power-law rheology in the bulk and at the interface: quasi-properties and fractional constitutive equations[END_REF][START_REF] Jaishankar | Interfacial viscoelasticity, yielding and creep ringing of globular protein-surfactant mixtures[END_REF][START_REF] Sharma | Rheology of globular proteins: Apparent yield stress, high shear rate viscosity and interfacial viscoelasticity of bovine serum albumin solutions[END_REF].

After amplitude sweep tests, creep tests were performed on WPI interfacial layers over short durations. In stress-controlled rheometry, for sufficiently viscoelastic fluids, even if they are simple Newtonian fluids, a ringing event (i.e., damped oscillations) is observed at the start of a creep test [START_REF] Ewoldt | Creep ringing in rheometry or how to deal with oft-discarded data in step stress tests![END_REF]. The coupling of fluid elasticity and instrument inertia causes periodic oscillations which decay exponentially due to viscous dissipation [START_REF] Jaishankar | Power-law rheology in the bulk and at the interface: quasi-properties and fractional constitutive equations[END_REF]. The creep ringing logarithmic decrement, ∆ and ringing frequency, * [START_REF] Nagarkar | Some mechanistic insights into the gelation of regenerated silk fibroin sol[END_REF] of WPI interfacial layers were calculated (where applicable) using the following equations:

∆= 2 [Eq. 1] * = [Eq. 2]
where , , and are the compliance values of the 1 st peak, 1 st valley and 2 nd peak of the creep ringing data, respectively [START_REF] Nagarkar | Some mechanistic insights into the gelation of regenerated silk fibroin sol[END_REF] and and are the times of the 2 nd peak and 1 st peak, respectively.

Particle size and ζ-potential of WPI dispersions during gelation

The hydrodynamic diameter and ζ-potential of protein particles during gelation were approximated by dynamic light scattering (DLS) following the methods of Ju and Kilara (1998) and de Jong, Klok, and van de Velde (2009a) for acid-induced gelation of whey proteins. A Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) was used for this purpose. Protein samples (at 37 °C) were supplemented with GDL (20 mg mL -1 for hydrophobised WPI and 5 mg mL -1 for heat-treated WPI), and stored at 37 °C (de Jong, Klok, & van de Velde, 2009b) for different durations, i.e., 130 min for the hydrophobised WPI samples and 35 min for the heat-denatured WPI. At various time intervals, the samples were diluted 100 fold with deionised water and subjected to DLS analysis. Dilution did not have a remarkable influence (less than 0.2 unit) on the pH value of the samples. The refractive index of dispersant (water) was 1.33, and the dispersant viscosity was set to 0.69 mPa s. A laserbackscattering angle of 173° and a wavelength of 633 nm were applied in size measurements.

Rheological analyses of WPI and alginate-WPI gel samples

The gelation time (Tig) and rheological properties of WPI and emulsion samples were determined using an Anton Paar Physica MCR 301 rheometer (Anton Paar GmbH, Graz, Austria) equipped with a cone-plate geometry (diameter 49.96 mm and cone angle 1.99°).

Samples supplemented with GDL (20 mg mL -1 for the hydrophobised WPI and the emulsion and 5, 10, and 20 mg mL -1 for the heat-denatured WPI) were poured onto the plate, followed by introducing the cone. Temperature was kept at 37 °C. Low-density paraffin was added around the samples to prevent evaporation. Changes in storage modulus (Gʹ), loss modulus (Gʺ), and damping factor (tan δ) during gelation of the samples were measured at an angular frequency of 1 rad s -1 and 0.1% strain. Gelation time (Tig) was arbitrarily defined as Gʹ = 1 Pa [START_REF] Andoyo | Acid gelation of whey protein microbeads of different sizes[END_REF]. Gels were oscillated in situ for 24 h to obtain a fully developed network. Then, a frequency sweep test was performed to determine the effect of time scale of deformation on the rheological properties when the samples were formed [START_REF] Lee | Rheological properties, whey separation, and microstructure in set-style yogurt: Effects of heating temperature and incubation temperature[END_REF]. Frequency was increased from 0.0628 to 628 rad s -1 at 0.1% strain and 37 °C.

Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) observation of gel microstructure was performed using the ZEISS LSM 880 inverted confocal microscope (Carl Zeiss AG, Oberkochen, Germany) set at magnification 63× (Plan Apochromat objective, oil immersion, NA 1.4) using the Airyscan detection unit. The dispersion of WPI and emulsion containing GDL were mixed with a 1% (w/v) solution of Fast Green at a ratio of 9:1. Each sample (1 mL) was injected into the chamber of a gel cassette® system [START_REF] Brocklehurst | Detection of microbial growth[END_REF] and vertically incubated at 37 °C for 24 h. After gelification, Airyscan images were acquired using a He/Ne laser with a wavelength of 633 nm, using with a main beam splitter MBS488/561/633 and no additional emission filter. The zoom was set at 4 for gel imaging and 1.8 for W/W emulsion sample. For further image analysis of W/W emulsion, ten micrographs of 1336×1336 pixels (1 µm = 18.17 pixels) were taken on different regions in a constant z-position. Zen Black 2.1 (Version 13.0.0.0) software was used to process the acquired datasets using the 2D mode at default settings of the Airyscan processing function, including a Wiener filter deconvolution.

Airyscan confocal micrographs of the W/W emulsions were converted to binary images using the "Default" thresholding algorithm of Fiji-win64 software. The segmentation procedure was validated by visual comparison of the resulted binary image with its original image. The particle areas were measured using the Analyze Particles measurements tool (in µm²). Corresponding diameters were calculated, based on the hypothesis that all particles were spherical.

Statistical analysis

Samples were prepared in triplicate and the experiments were done at least two times.

The results were analysed by one-way ANOVA with SPSS software version 16 (IBM software, New York, NY, USA) using Duncan's test at a significance level of P <0.05.

Results and discussion

Interfacial rheology

Amplitude sweep

The dynamic moduli of air-WPI interfaces were first measured over time at a fixed strain amplitude and oscillation frequency. The interfacial shear storage ( ) and loss moduli reached equilibrium 2-3 h after sample loading, which indicates that the structure of interface reached steady state [START_REF] Jaishankar | Power-law rheology in the bulk and at the interface: quasi-properties and fractional constitutive equations[END_REF]. Then, small amplitude oscillatory strain sweeps were carried out to find out the onset of non-linearity of the samples viscoelastic response. Typical curves of the results of strain sweep tests are illustrated in Fig.

1.

In agreement with the literature on native β-lactoglobulin [START_REF] Ridout | Rheology of mixed β-casein/βlactoglobulin films at the air-water interface[END_REF], the of the air-liquid interfacial layer of native WPI had a value of about 13 mN m -1 (Table 1). Heat denaturation and hydrophobisation decreased the value of the air-liquid interfacial layer of WPI. Native β-lactoglobulin, as the major whey protein, when adsorbed at interface polymerises over time, concomitant with lack of any appreciable exchange between adsorbed polymers and non-adsorbed monomers. The polymerisation takes place by the gradual exposure of the free -SH groups embedded in the interior of the molecule, triggering progressive formation of disulphide crosslinks. It develops a remarkable extent of βlactoglobulin oligomers at interface 2 h after interfacial adsorption [START_REF] Dickinson | Time-dependent polymerization of β-lactoglobulin through disulphide bonds at the oil-water interface in emulsions[END_REF].

In contrast to native proteins, extensive conformational changes and disulphide bond formation take place before interfacial adsorption in heat-processed proteins [START_REF] Meydani | Influence of the Maillard reaction on the properties of cold-set whey protein and maltodextrin binary gels[END_REF], i.e., the heat-denatured and hydrophobised WPI samples. This reduces the extent of permanent crosslinks formation after interfacial adsorption. Furthermore, the greater absolute values of negatively signed ζ-potentials of heat-denatured and hydrophobised WPIs in comparison with native WPI at comparable pH values [START_REF] Madadlou | Structural assessment and catalytic oxidation activity of hydrophobized whey proteins[END_REF] suggest a stronger electrostatic repulsion between protein particles at the interfacial layer, reducing the stiffness and hence the of the layer. Likewise, a smaller extent of crosslinking between protein molecules was argued to explain the lower interfacial viscosity of β-lactoglobulin at the air-water interface when compared with oil-water interface [START_REF] Roth | Interfacial shear rheology of aged and heattreated β-lactoglobulin films: Displacement by nonionic surfactant[END_REF].

The point at which deviated by more than 10% from a constant value was considered as the onset of non-linearity (TA Instruments, New Castle, DE, USA: http://www.tainstruments.com/pdf/literature/RS23.pdf), which took place at a strain amplitude of 1.8%, 1.6%, and 1.2% for the air-liquid interfacial layers of native, heatdenatured and hydrophobised WPI samples, respectively. For these three samples, a shorter linear region coincided a lower value (Table 1).

Although erythritol addition slightly decreased the and of the hydrophobised WPI interfacial layer, the reduction was not statistically significant (Fig. 1; Table 1).

Likewise, the phase-shift angle, δ of the layer was not influenced by erythritol addition (Table 1). Furthermore, the δ values of the air-liquid interfacial layers of the hydrophobised WPI samples (either E-free or E-added) were not different from that of the heat-denatured sample.

In contrast, native WPI had a remarkably smaller δ in the linear region. Within the linear region, was larger than for all samples, indicating that the interfacial layers were predominately elastic [START_REF] Langevin | Influence of interfacial rheology on foam and emulsion properties[END_REF][START_REF] Sharma | Rheology of globular proteins: Apparent yield stress, high shear rate viscosity and interfacial viscoelasticity of bovine serum albumin solutions[END_REF].

All interfacial layers underwent a yielding process at high strain amplitudes. The process is shown by a steep collapse in the magnitude of and the domination of beyond a certain amplitude [START_REF] Jaishankar | Interfacial viscoelasticity, yielding and creep ringing of globular protein-surfactant mixtures[END_REF]. The values of oscillation flow stress ( ) and flow strain ( of the WPI interfacial layers were affected by heat denaturation and hydrophobisation. The crossovers of and , which indicate the flow points of the interfacial layers, are specified by the circles in Fig. 1, and the corresponding and values are reported in Table 1. In accordance with the influence on , heat-denaturation and hydrophobisation decreased the values of and of the interfacial layer, and erythritol addition into the hydrophobised WPI did not influence the flow-point criteria (i.e., and )

of the air-liquid interfacial layer. Again, hydrophobisation caused a greater reduction of the flow-point criteria than heat denaturation.

Creep ringing

The creep compliance of the interfacial layers as a function of time at different magnitudes of interfacial stress, over a few seconds is illustrated in Fig. 1. A typical creep ringing behaviour, i.e., damping free oscillation, is observed for the interfacial film of native WPI. In the linear regime, strain is a linear function of stress; hence, the compliance curves should overlap for an ideal linear viscoelastic material. This happened at times shorter than 2 s for the interfacial layer of native WPI and indicates that the elastic component of the sample controls the first instant response, causing comparable ringing behaviours irrespective of the value of applied [START_REF] Goudoulas | Viscoelastic properties of polyacrylamide solutions from creep ringing data[END_REF]. Likewise, the creep compliance curves of polyacrylamide solutions [START_REF] Goudoulas | Viscoelastic properties of polyacrylamide solutions from creep ringing data[END_REF] and bovine serum albumin interfacial films [START_REF] Jaishankar | Interfacial viscoelasticity, yielding and creep ringing of globular protein-surfactant mixtures[END_REF] deviated at periods longer than 2 s and 5 s, respectively, indicating the onset of non-linear behaviour and the domination of viscous component.

Creep ringing was weakly observed for the air-liquid interfacial film of the heatdenatured WPI. In addition, the compliance curves of this sample (heat-denatured WPI) commenced to deviate only after 0.8 s. The interfacial films of the hydrophobised WPI and the E-added hydrophobised WPI did not show any creep ringing property. Indeed, to observe creep ringing, the sample must have sufficient elasticity, so that > !" , where is the modulus of spring in the Jeffreys model [START_REF] Ewoldt | Creep ringing in rheometry or how to deal with oft-discarded data in step stress tests![END_REF]. Therefore, the absence of the ringing property in the air-water interfacial layers of the hydrophobised WPIs (with and without added erythritol) agrees with the results of the strain amplitude oscillatory test.

∆ and * values of the interfacial layer of native WPI-air were 0.125 ± 0.010 and 1.17 ± 0.06 Hz, respectively. The short time creep response is quadratic in time and according to Struick's formula, the interfacial elastic modulus is proportional to the square of * [START_REF] Jaishankar | Interfacial viscoelasticity, yielding and creep ringing of globular protein-surfactant mixtures[END_REF]:

≈ % * &1 + ) ∆ * + , [Eq. 3]
The parameter % = -. is an instrumentation parameter and remains unchanged for the samples because the total moment of inertia (/) of the system and the factor 0 which is determined by the geometry are identical. The ratio of the square values of * of native WPIair layer was 2.25, which is very close to the ratio of the samples values in the linear region, i.e., 2.35. This indicates that the linear elastic modulus can be directly determined using the data from the creep ringing test.

Buffering capacity and particle size of WPI during acid gelation

Interfacial rheology analyses indicated that hydrophobisation decreased the dynamic moduli of air-liquid interfacial layers of WPI. Next, we assessed the influence of hydrophobisation on the acid-induced cold-set gelation properties of WPI using bulk rheology. The WPI samples and the W/W emulsion produced were gelled with GDL. The pH value of the heat-denatured WPI supplemented with GDL at a level of 20 mg mL -1 decreased to ≈3.8, after acidification for 130 min, whereas its value for hydrophobised WPI supplemented with a comparable GDL concentration (20 mg mL -1 ) decreased only to 5.07 after an identical period of time (results not shown). On the other hand, the acidification profiles (pH versus time) of the heat-denatured and hydrophobised WPI samples supplemented with GDL at the respective concentration of 5 mg mL -1 and 20 mg mL -1 were very close. Therefore, the buffering capacity (i.e., resistance to pH changes) of whey proteins increased due to hydrophobisation.

The carboxylic acid group of amino acids has a pKa value of approximately 2.3, which is significantly lower than the pKa value (4.8) of acetic acid due to negative inductive (electron withdrawing) effect on the carboxylic acid group by the -NH 3 group. Accordingly, the carboxyl acid group of α-amino acids is about 300 times stronger an acid than acetic acid.

In proteins and peptides, the pKa of terminal α-carboxyl groups is 3.1-3.8 (intermediate between the carboxylic acid groups of acetic acid and glycine) because of the lower negative inductive effect of proximal peptide bond rather than the -NH 3 group in an α-amino acid. It is noteworthy that the β and γ carboxylic acid groups of aspartic (pKa = 3.9) and glutamic (pKa = 4.3) acids are somewhat insulated from these electronic effects by intervening saturated carbons [START_REF] Barrett | Amino acids and peptides[END_REF]. Hydrophobisation, which occurred through the acetylation of a great majority (>90%) of free amino groups of whey proteins [START_REF] Madadlou | Structural assessment and catalytic oxidation activity of hydrophobized whey proteins[END_REF], decreased the inductive effect and increased the pKa values of terminal carboxylic acid groups. A large amount of acid or base is needed to change pH near the pKa, so that buffer solutions are stronger at pH levels near their pKa values (Shimadzu Corporation, 2015). The hydrophobised WPI that had a higher pKa value was a stronger buffer and required a 4-fold higher amount of GDL to undergo an acidification profile comparable with that of the heatdenatured WPI.

During formation of acid-induced protein gels, reduction of pH towards the pI decreases the net negative charge on protein particles. Then, the particles begin to crosslink, forming clusters of different but finite size. At a certain threshold of interconnection, the final gel network is formed [START_REF] Madadlou | Acid-induced gelation behavior of sonicated casein solutions[END_REF].

The particle size of WPI dispersions was measured during acidification immediately after GDL addition. The hydrophobised protein particles had a mean hydrodynamic diameter of approximately 82 nm, 3 min after GDL addition. Fig. 2A indicates that during the acidification, the particle size of hydrophobised WPI initially decreased, followed by a progressive increase over the rest of acidification. Plotting the mean hydrodynamic diameter of protein particles versus mean pH during acidification until gel formation time (Fig. 2B) indicated that the size of hydrophobised protein particles decreased with decreasing pH to approximately 5.5 (identified by an arrow in Fig. 2B). This pH value corresponded to 30 min after GDL addition. Likewise, the particle diameter of heat-denatured WPI showed a small reduction in the beginning of acidification, followed by a remarkable increase (Fig. 2A). As soon as GDL was added, the pH value of WPI dispersions sharply decreased (results not shown), and then reached a plateau after gel formation over long durations. The initial reduction and the subsequent increase of protein particle size during acid gelation, which were observed for both heat-denatured and hydrophobised WPI dispersions are attributed respectively to protein particles shrinkage due to the decrease of intra-particle electrostatic repulsion and succeeding aggregation of the particles.

Erythritol addition did not influence the initial particle size of hydrophobised WPI dispersion, which suggests that erythritol did not interact electrostatically with WPI particles [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF]. It did not also affect the aggregate size as a function of pH reduction (Fig. 2B). However, erythritol addition postponed the onset of aggregation of protein particles to a lower pH value (approximately 5.4) and caused formation of comparatively smaller aggregates just before the gelation point (Fig. 2A).

Gelation time

Different WPI samples supplemented with GDL (20 mg mL -1 for the hydrophobised WPI samples, and 5, 10, and 20 mg mL -1 for the heat-denatured WPI) were gelled at 37 °C on a rheometer and the corresponding gelation times are illustrated in Fig. 3A. The heatdenatured WPI gelled approximately 5, 13, and 42 min after adding GDL at respective concentrations of 20, 10, and 5 mg mL -1 . The values of the heat-denatured WPI gels prepared using GDL at 20 and 10 mg mL -1 levels decreased after a peak value (Fig. 3B). The reduction of was substantial for the heat-denatured WPI supplemented with 20 mg mL -1 GDL, and is attributed to gel disintegration due to over-acidification, which reduced pH to values well below the proteins pI.

Gelation time ( 4 ) of whey proteins was influenced by hydrophobisation, as well as by erythritol and alginate addition (Fig. 3A). Hydrophobised WPI required about 125 min to gel (pH 5.07 ± 0.01) after adding GDL at 20 mg mL -1 . Erythritol addition, which did not alter the pH value of the hydrophobised WPI, postponed the 4 (Fig. 3A). The E-added hydrophobised WPI gelled approximately 134 min (pH 5.06 ±0.02) after GDL supplementation (20 mg mL - 1 ), which is 9 min longer compared with the counterpart without erythritol. On the contrary, alginate addition into the E-added hydrophobised WPI resulted in a shorter 4 (94 ± 2 min) in comparison with both E-added and E-free hydrophobised WPIs.

Dynamic rheology of the WPI gels

The gels developed in situ on the rheometer were subjected to frequency sweep test to gain an insight into their time-dependent behaviour. This rheological analysis is useful to gather information on the internal structure of samples (polymer solutions, gels, emulsions, etc.) and the stability of dispersions during storage. The results of the frequency sweep test are illustrated in Fig. 4. The values of and ′ of all samples increased with increasing frequency and was greater than ′ at any frequency for all samples, which suggests significant protein-protein interactions at the gels [START_REF] Zhang | Properties of partially denatured whey protein products: Viscoelastic properties[END_REF] and domination of the elastic component to the viscous one (Madadlou, Khosroshahi, & Mousavi, 2005). However, the difference between the and ′ of all gels was smaller than 1 log cycle, indicating that the gels were rather weak [START_REF] Kamal | Effects of heating and calcium and phosphate mineral supplementation on the physical properties of rennet-induced coagulation of camel and cow milk gels[END_REF].

In a manner similar to that of the interfacial rheology results, the hydrophobised WPI gel had lower values than the heat-denatured WPI gel throughout the whole frequency range. Moreover, erythritol addition decreased the maximum value of the hydrophobised WPI gel in the frequency sweep analysis from approximately 5 kPa to 4 kPa. The ′ of the WPI gel decreased at a comparable extent (≈1.27 fold) due to erythritol addition (Fig. 4). Both E-free and E-added hydrophobised gels had a final (i.e., after 24 h) pH value of 4.75 ± 0.1.

Erythritol might interrupt gel network development [START_REF] Cai | Confectionery gels: Effects of low calorie sweeteners on the rheological properties and microstructure of fish gelatin[END_REF][START_REF] Torres | Effect of sucrose, stevia and xylitol on rheological properties of gels from blends of chestnut and rice flours[END_REF]. We hypothesise that erythritol increased the hydration of protein particles, reducing the formation of non-covalent bonds [START_REF] He | Effects of sugars on the crosslinking formation and phase separation of high-pressure induced gel of whey protein from bovine milk[END_REF] between the particles, impairing gel development. This may explain why the Eadded hydrophobised WPI had relatively smaller aggregates than E-free sample just before the gelation point. Although erythritol did not interact with whey proteins [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF], it could increase protein hydration because of excluded volume effect, a phenomenon that weakly depends on the native or denatured state of proteins [START_REF] Ebel | Probing protein-sugar interactions[END_REF]. Addition of carbohydrates such as sugar alcohols to native protein solutions is known to delay the heat-induced aggregation and gel formation [START_REF] Wijayanti | Stability of whey proteins during thermal processing: A review[END_REF].

In accordance with the influence on gelation time, alginate addition increased the and ′ values of the E-added hydrophobised WPI gel. One may assume that alginate gelation along with whey proteins accelerated the gelation and increased gel firmness. Alginate can gel without the aid of calcium (and other multivalent cations) when pH is gradually lowered (for example by GDL) because the fraction of unionised carboxyl groups on the uronic acid residues increases and water is squeezed out [START_REF] Yotsuyanagi | Acid-induced and calciuminduced gelation of alginic acid: bead formation and pH-dependent swelling[END_REF].

Gradual pH reduction is requisite as an abrupt acidification leads to precipitation of alginic acid rather than gel formation. It has been estimated that a minimum alginate concentration of 3.5 mg mL -1 is needed to obtain a GDL-induced gel [START_REF] Draget | Alginic acid gels: the effect of alginate chemical composition and molecular weight[END_REF], which is remarkably lower than the alginate concentration employed in the current study (30 mg mL -1 ), although alginate would partition between the protein-rich and alginate-rich phases of the emulsion [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF].

To assess alginate gelation, we added different concentrations of GDL to the alginate solution (30 mg mL -1 ) and found that a GDL concentration of 1.1 mg mL -1 decreased pH after about 90 min (similar to the emulsion 4 ) to 5.15 ± 0.04, which is comparable with the emulsion pH at gelation point. Nevertheless, the alginate solution did not gel. The alginateadded WPI gel had a final (after 24 h) pH of approximately 4.75, similar to that of heatdenatured and hydrophobised WPI gels, associated with the protein buffering capacity [START_REF] Léonard | Aqueous two-phase system cold-set gelation using natural and recombinant probiotic lactic acid bacteria as a gelling agent[END_REF]. Also, de Jong et al. (2009a) observed a constant pH, independent of polysaccharide type and amount for the acid-induced gels of WPI/polysaccharide mixtures. It was presumed that the continuous and disperse phases of the W/W emulsion had equal pH values [START_REF] Singh | On the encapsulation and viability of probiotic bacteria in edible carboxymethyl cellulose-gelatin water-in-water emulsions[END_REF]. Adding higher concentrations of GDL, namely 1.4 and 1.7 mg mL -1 , decreased pH (after 90 min) to approximately 4.85 and 4.75, respectively; however, gelation did not happen. Indeed, acid gelation of alginate happens at pH values below the 67 ! of the uronic acid residues which is approximately 3.7 [START_REF] Bu | Effects of pH on dynamics and rheology during association and gelation via the Ugi reaction of aqueous alginate[END_REF].

Although the WPI powder used in the current study contained a small amount of minerals, which could cause alginate hardening/gelation when pH was reduced, the shorter tg and higher dynamic moduli of alginate-added gel compared with the alginate-free WPI systems is mainly attributed to the microstructural organisation of the composite gel.

Likewise, a higher of an alginate/caseinate aqueous two-phase system gel than individual gels of either alginate or caseinate was ascribed to the development of a more organised and interconnected structure in the mixed gel [START_REF] Léonard | Aqueous two-phase system cold-set gelation using natural and recombinant probiotic lactic acid bacteria as a gelling agent[END_REF]. Hence, the microstructural features of the samples were assessed to explain the difference between the dynamic moduli of the alginate-added gel and the alginate-free WPI gels.

Microstructure of the WPI gels

The microstructures of well developed (i.e., stored for 24 h) gel samples were imaged by CLSM and the typical images are depicted in Fig. 5. The polysaccharide-free WPI gels had homogenous microstructures, composed of a protein-rich phase (green) and a serum phase.

The latter is observed as black micro-channels interconnected throughout the gel matrix.

Microscopic images (Fig. 5) showed that alginate was dispersed as droplets in a continuous matrix of proteins. Micro-phase separation of protein-polysaccharide pairs is usual and known to increase the firmness of the subsequently formed protein gels [START_REF] Zand-Rajabi | Caffeine-loaded whey protein hydrogels reinforced with gellan and enriched with calcium chloride[END_REF]. Micro-phase separation concentrates the incompatible polymers in separated domains, causing higher effective concentrations than nominal [START_REF] Beaulieu | Rheology, texture and microstructure of whey proteins/low methoxyl pectins mixed gels with added calcium[END_REF].

The diameter of approximately 1700 droplets were measured and the result is illustrated in Fig. 5. The emulsion gel droplet size was highly inhomogeneous and droplets as large as 20 μm were present. It is also observed in Fig. 5D that some adjacent alginate droplets were in contact and partially coalesced. Modification of the gelation method might be helpful to confine the droplet disproportionation kinetics.

Conclusions

Hydrophobisation of whey proteins through acetylation decreased the liquid-air interfacial viscoelasticity of WPI. Air is a very hydrophobic medium. Hence, concurrent reduction of the interfacial elasticity (observed in the current study) and interfacial tension (observed in a previous study; [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF] of hydrophobised WPI at the liquid-air interface indicates that more hydrophobic protein particles which accumulated at the interface were less elastic. An opposite process took place in the alginate-protein emulsions, i.e. more elastic but less hydrophobic protein particles stabilised the all-aqueous interfaces. This conclusion relies on the assumption that at least two populations of particles with different hydrophobicities were present at the protein phase. It is noteworthy that macroscopic phase separation of non-gelled W/W emulsions is conventionally slowed down by accumulation of third component particles at the water-water interface. However, as communicated in an earlier report [START_REF] Madadlou | Development of an aqueous two-phase emulsion using hydrophobized whey proteins and erythritol[END_REF], stable W/W emulsions can be made using a pair consisting of only hydrophobised protein and a co-charged polysaccharide without a third component. The fabrication of the two-component stable W/W emulsions is thrilling. Should the above-mentioned conclusion be correct, one may design a wide range of two-component (protein and polysaccharide only) stable and fluid (not gelled) W/W emulsions by masterful manipulation of the shape, size, and surface hydrophobicity of protein particles. Acidification of the emulsion produced soft gels which might be used for encapsulation of drugs, nutraceuticals and enzymes as two-phase oil-free systems. In addition, the emulsion could be utilised to perform reactions at the interface of two discrete, but interfacially touching, aqueous phases. Addition of erythritol to the hydrophobised WPI dispersion, which facilitated emulsification, resulted in an inferior interfacial and gel viscoelasticity. Therefore, it is possible to tune the interfacial and gel viscoelasticity of hydrophobised WPI and the W/W emulsions using a non-caloric sweetener.

Fig. 1 .

 1 Fig. 1. Interfacial strain sweep (A, C, E, G) and creep (B, D, F, H) tests of whey protein

Fig. 2 .

 2 Fig. 2. The particle diameter, as a function of (A) time, and (B) pH, and ζ-potential (C) of

Fig. 3 .

 3 Fig. 3. Evolution of the storage modulus (G') of whey protein isolate (WPI) solutions during

Fig. 4 .

 4 Fig. 4. Mean dynamic moduli of different whey protein isolate (WPI) samples: and ,

Fig. 5 .

 5 Fig. 5. CLSM images of acid-induced WPI gels and alginate-in-WPI emulsion gel: A, heat-

  Figure1

Table 1

 1 Interfacial properties of whey protein isolate (WPI) samples. a Abbreviations are: , storage modulus in the linear viscoelastic region; , phase-shift angle in the linear viscoelastic region; , flow stress; , flow strain of the interfacial layer. The and values were acquired from Fig.1, which shows typical curves of the strain sweep test at the liquid-air interface of different WPI samples; means with different superscript letters within a column differ significantly (P < 0.05).

	Interface
		(mN m -1 )	(°)	(μN m -1 ) (%)
	Native WPI-air	12.95 a 0.36	10.9 b 0.6	3.8	8.0
	Heat-denatured WPI-air	4.40 b 1.3	30.3 a 2.8	1.2	3.1
	Hydrophobised WPI-air	0.86 c 0.13	30.9 a 2.7	0.2	2.6
	Erythritol-added hydrophobised WPI-air	0.63 c 0.11	30.5 a 2.1	0.2	2.6

a

Acknowledgements

The authors are thankful to the support of the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an AgreenSkills+ fellowship (grant agreement n°609398). The ZEISS LSM880 confocal microscope equipped with the Fast Airyscan detector was funded by the European Union (FEDER), the French Ministry of Education, Research and Innovation, the Regional Council of Brittany and INRA.