Food Bioprocess Technol (2013) 6:2406–2418 DOI 10.1007/s11947-012-0901-y

ORIGINAL PAPER

Effect of Aqueous Phase Composition on Stability of Sodium Caseinate/Sunflower oil Emulsions Cristián Huck-Iriart & Víctor M. Pizones Ruiz-Henestrosa & Roberto J. Candal & María L. Herrera

Received: 2 February 2012 / Accepted: 22 May 2012 / Published online: 1 June 2012 # Springer Science+Business Media, LLC 2012

Abstract The aim of the present work was to investigate the effect of aqueous phase composition on the stability of emulsions formulated with 10 wt% sunflower oil as fat phase. Aqueous phase was formulated with 0.5, 2, or 5 wt% sodium caseinate, or sodium caseinate with the addition of two different hydrocolloids, xanthan gum or locust bean gum, both at 0.3 or 0.5 wt% level or sodium caseinate or with addition of 20 wt% sucrose. Emulsions were processed by Ultra-Turrax and then further homogenized by ultrasound. Creaming and flocculation kinetics were quantified by analyzing the samples with a Turbiscan MA 2000. Emulsions were also analyzed for particle size distribution, microstructure, viscosity, and dynamic surface properties. The most stable systems of all selected in the C. Huck-Iriart : R. J. Candal Instituto de Química Inorgánica, Medio Ambiente y Energía (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Universitaria, Pabellón 2, Piso 3, C1428EHA, Buenos Aires, Argentina C. Huck-Iriart : V. M. Pizones Ruiz-Henestrosa : M. L. Herrera Facultad de Ciencias Exactas y Naturales (FCEN), Universidad de Buenos Aires (UBA), Ciudad Universitaria, Avda. Intendente Güiraldes, 1428 Buenos Aires, Argentina R. J. Candal Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín (UNSAM), Campus Miguelete, 25 de Mayo y Francia, CP 1650 San Martín, Provincia de Buenos Aires, Argentina M. L. Herrera (*) Ciudad Universitaria, Pabellón de Industrias, Intendente Güiraldes S/N, 1428 Buenos Aires, Argentina e-mail: [email protected]

present work were the 0.3 or 0.5 wt% XG or 0.5 wt% LBG/ 0.5 wt% NaCas coarse emulsion and the 20 wt% sucrose/ 5 wt% NaCas fine emulsion. Surprisingly, coarse emulsions with the lower concentration of NaCas, which had greater D4,3, were more stable than fine emulsions when the aqueous phase contained XG or LBG. In these conditions, the overall effect was less negative bulk interactions between hydrocolloids and sodium caseinate, which led to stability. Sugar interacted in a positive way, both in bulk and at the interface sites, producing more stable systems for smalldroplet high-protein-concentration emulsions. This study shows the relevance of components interactions in microstructure and stability of caseinate emulsions. Keywords Emulsions . Stability . Sodium caseinate . Sunflower . Xanthan and locust bean gums . Sucrose

Introduction Emulsions are commonly encountered in food systems. Many traditional food products such as ice cream, low-fat spreads, yoghurt, mayonnaise, cake batters, whipped toppings, dairy creamers, and cream liqueurs are some examples of emulsion-based foods. The physicochemical properties of emulsions play an important role in food systems as they directly contribute to texture, sensory, and nutritional properties of food. One of the most significant aspects of any food emulsion is its stability, which refers to the ability of an emulsion to resist changes in its properties over time. Creaming, flocculation, coalescence, partial coalescence, phase inversion, and Ostwald ripening are examples of physical instability. The length of time that an emulsion must remain stable depends on the nature of the food product. Some food emulsions are formed as

Food Bioprocess Technol (2013) 6:2406–2418

intermediate steps during a manufacturing process and, therefore, only need to remain stable for a few seconds, minutes, or hours (e.g., cake batter, ice cream mix, and margarine premix), whereas others must remain stable for days, months, or even years prior to consumption (e.g., mayonnaise, salad dressings, and cream liqueurs). On the other hand, the production of some foods involves a controlled destabilization of an emulsion during the manufacturing process, for example, margarine, butter, whipped cream, and ice cream. The development of an effective strategy to prevent undesirable changes in the properties of a particular food emulsion depends on the dominant physicochemical mechanism(s) responsible for the changes. In practice, two or more of these mechanisms may operate in concert. It is therefore important for food scientists to identify the relative importance of each mechanism, the relationship between them, and the factors that influence them, so that effective means of controlling the stability and physicochemical properties of emulsions can be established (McClements 2005). Nowadays, the food industry has a growing interest in the replacement of synthetic emulsifiers by natural ones, such as polysaccharides and proteins. The caseins, a group of unique milk-specific proteins, are widely used as an ingredient in the food industry. Casein is of particular importance as an emulsifier because of its ability for rapidly conferring a low interfacial tension during emulsification and because of the strong amphiphilic characteristics of the major individual caseins. In aqueous solution at neutral pH or in foods such as milk, casein is a mixture of monomers, small aggregates, or polydisperse protein particles called “casein micelles” (Dickinson 2006). The micelles may be dispersed by adding a calcium chelator or also by urea, sodium dodecyl sulfate, high pH, or ethanol, indicating that hydrogen bonds, hydrophobic, and electrostatic interactions are also involved in micelle integrity (Fox and Brodkorb 2008). Removing the calcium salts from milk casein and replacing them by sodium salts leads to the production of “sodium caseinate.” Commercial sodium caseinate is a variable multicomponent mixture containing four major constituents, αs1-, αs2-, β-, and κ-casein. In the casein system, the micelle state may be the lowest free energy state of the system. Of particular interest is the micelle structure and the mechanisms, which operate in determining micelle size. Proteins and polysaccharides are commonly used together in many food products. In food emulsions, polysaccharides are usually added to increase the viscosity or to obtain a gel-like product. Xanthan gum (XG), an anionic bacterial polysaccharide, has been extensively studied and widely used in food products due to its specific physical (viscosity, pseudoplasticity) and chemical (water solubility, pH stability) properties (Kobori et al. 2009). Locust bean gum (LBG) is a nondigestible polysaccharide extensively used as a

2407

thickener in food formulations. This polysaccharide has the ability to form very viscous solutions that are almost unaffected by pH or heat processing, mainly due to the neutral character of this gum (Perrechil and Cunha 2010). Emulsions have been studied by numerous techniques, such as dynamic light scattering, microscopy, steady-state viscometry, among others, to characterize their physical properties. Most of these techniques involve some form of dilution. This dilution disrupts emulsion structures modifying the actual system. Therefore, the ability to study the stability of food emulsions in their undiluted forms may reveal subtle nuances about their stability. A relatively recently developed technique, the Turbiscan method, allows scanning the turbidity profile of an emulsion along the height of a glass tube filled with the emulsion. The analysis of the turbidity profiles with time leads to quantitative data on the stability of the studied emulsions and allows making objective comparisons between different emulsions (Mengual et al. 1999). Most of the studies about emulsion stability reported in the literature were done using fat concentrations that led to phase separation or obvious creaming and therefore may be evaluated by visual observation. Few reports showing quantitative description of destabilization mechanisms in turbid or “visual-stable” emulsions may be found in the literature (Chauvierre et al. 2004; Palazolo et al. 2005; Cerdeira et al. 2007; Alvarez Cerimedo et al. 2010), and in none of them, the effect of sucrose or hydrocolloids on stability of emulsions formulated with sunflower oil and sodium caseinate was quantified. There is a general agreement among authors that the most stable systems are obtained for conditions that produce size reduction of the droplets, an increase in viscosity of the continuous phase, and structural changes in emulsions such as gelation. All these conditions decrease the molecular mobility and slow down phase separation. However, this is a simplified picture of factors that determine stability. Bulk and interface interactions among components may be more relevant to emulsion stability than viscosity or particle size and should not be disregarded in stability studies. The aim of the present work was to investigate the effect of aqueous phase composition, and particularly the interactions among aqueous phase components and sodium caseinate, on the stability of emulsions formulated with sunflower oil as fat phase. Creaming and flocculation kinetics were quantified by analyzing the samples with a Turbiscan MA 2000.

Materials and Methods Starting Materials Sodium caseinate was obtained from ICN (ICN Biomedical, Inc., Aurora, OH, USA) and used without any further purification. Fat phase was commercial sunflower oil, which

2408

main fatty acids were identified as C16:0, C18:0, C18:1, and C18:2 with percentages of 6.7, 3.6, 21.9, and 66.3 %, respectively. Sucrose, XG and LBG were analytical grade (Sigma-Aldrich, St Louis, MO, USA).

Food Bioprocess Technol (2013) 6:2406–2418

al. 2006). For D4,3 parameter, values of standard deviations were <0.2 μm. Experiments were done in duplicate, and results were averaged. Emulsion Stability

Emulsion Preparation Aqueous phase was a 0 or 20 wt% solution of sucrose or a 0.3 or 0.5 wt% solution of xanthan or locust bean gums. All emulsions were formulated with a 10 wt% sunflower oil. Sodium caseinate was used as emulsifier at 0.5, 2, and 5 wt%, giving oil/protein ratios of 20:2. Emulsions were kept at 60 °C during preparation. Then, fat and aqueous phases were mixed using an Ultra-Turrax T18 high-speed blender (S 18N-5G dispersing tool, IKA Labortechnik, Janke & Kunkel, GmbH & Co., Staufen, Germany), operated at 20,000 rpm for 1 min. The resulting 18 pre-emulsions (coarse emulsions) were further homogenized for 20 min using an ultrasonic liquid processing Vibra Cell, VCX 750 model (Sonics & Materials, Inc., Newtown, CT, USA), giving 18 fine emulsions. The temperature of the sample cell was controlled by means of a water bath set at 15 °C. By doing this, sample temperature was below 40±1 °C during ultrasound treatment. Then, they were cooled quiescently to ambient temperature (22.5 °C). Subsequently, they were analyzed for droplet size distribution, stability in quiescent conditions, microstructure, viscosity, and dynamic surface properties. The pHs of the sunflower oil emulsions was around 6.66±0.05. No buffer was added to emulsions. Droplet Size Analysis The droplet size distribution of emulsions was determined immediately after emulsion preparation by light scattering using a Mastersizer 2000 with a Hydro 2000MU as dispersion unit (Malvern Instruments Ltd., UK). The pump speed was set at 1,800 rpm. Refraction index for the oil phase was 1.4694. Calculation from 0.1 to 10 μm was expressed as differential volume. Distribution width (W) was expressed as: W ¼ ½dðv; 0:9Þ  dðv; 0:1Þ where d(v, 0.9) and d(v, 0s.1) are the 90 and 10 % volume percentiles of the size distribution. The v in the expression refers to the volume distribution. D 4,3 parameter, the volume-weighted mean diameter of initial emulsions, obtained from droplet size distribution expressed as differential volume, is more sensitive to fat droplet aggregation (coalescence and/or flocculation) than Sauter mean diameter (D3,2) (Relkin and Sourdet 2005). Moreover, the droplet size data were also reported as the volume percentage of particles exceeding 1 μm in diameter (%Vd>1) (Thanasukarn et

The emulsion stability was analyzed using a vertical scan analyzer Turbiscan MA 2000 (Formulaction, Toulouse, France), which was described elsewhere (Pan et al. 2002). This equipment allows for the optical characterization of any type of dispersion (Mengual et al. 1999). The reading head is composed of a pulsed near-IR light source (λ0 850 nm) and two synchronous detectors. The transmission detector receives the light that goes through the sample (0°), while the backscattering detector receives the light backscattered by the sample (135°). The samples were placed in a flat-bottomed cylindrical glass measurement cell and scanned from the bottom to the top in order to monitor the optical properties of the dispersion along the height of the sample placed in the cell. The backscattering (BS) and transmission (T) profiles as a function of the sample height (total height060 mm) were studied in quiescent conditions at 22.5 °C. In this way, the physical evolution of the destabilization process is followed without disturbing the original system and with good accuracy and reproducibility (Mengual et al. 1999). Thus, by repeating the scan of a sample at different time intervals, the stability or the instability of dispersions can be studied in detail. The profiles allow calculation of either creaming, sedimentation, or phase separation rates, as well as flocculation, and the mechanism making the dispersion unstable can be deduced from the transmission or the backscattering data. Measurements were performed immediately after preparation of emulsions and at different times for a week. The curves obtained by subtracting the BS profile at t00 from the profile at t(ΔBS0BSt −BS0, usually called “reference mode”) display a typical shape that allows a better quantification of creaming, flocculation and other destabilization processes. As previously reported, BS level measurements were very reproducible between duplicates (Bordes et al. 2001). In all cases, values differed <0.2 %. Creaming was detected using the Turbiscan as it induced a variation of the concentration between the top and the bottom of the cell. The droplets moved upward because they had a lower density than the surrounding liquid. When creaming takes place in an emulsion, the ΔBS curves show a peak at heights between 0 and 20 mm. The variation of the peak wide, at a fixed height, during the studied time, can be related to the kinetics of migration of small particles (Mengual et al. 1999). The creaming destabilization kinetics was evaluated by measuring the peak thickness at 50 % of the height at different times (bottom zone). The slope of the linear part of a plot of peak thickness vs. time gives an indication of the migration rate.

Food Bioprocess Technol (2013) 6:2406–2418

BS mean values (BSav) change with the increase in particle size. Flocculation was followed by measuring the BSav as a function of storage time in the middle zone of the tube. As was theoretically demonstrated by Mengual et al. (1999), the BS intensity decreased as the particle size increased [when particle size is higher than the wavelength (λ) of the incident light]. It should be mentioned that if the particle size is lower than λ of the incident light, BS values increase with particle size. This phenomenon was used to determine flocculation kinetics (Chauvierre et al. 2004; Palazolo et al. 2005). The optimum zone was the one not affected by creaming (bottom and top of the tube), that is, the 20–50 mm zone. Microstructure The Olympus FV300 (Olympus Ltd, London, UK) confocal laser scanning microscope (CLSM) with an Ar gas laser (k0 488 nm) was used to collect the images. A 10× ocular was used, together with a 60× objective for a visual magnification of 600×. The laser intensity used was 20 %. Images were recorded using confocal assistant Olympus Fluoview version 3.3 software provided with the FV300 CLSM. Nile red was used to color the fat phase. Viscosity Viscosity (η) was measured using a pycnometer and an Ostwald viscometer (IVA, Buenos Aires, Argentina). Immediately after preparation, emulsions were placed in a water bath for 15 min to keep their temperature constant at 22 °C. The pycnometer volume was calibrated with distilled water. Then, an equal volume of emulsion was weight and its density (ρ) was calculated from its weight and volume. After that, emulsion was placed in the viscometer, and the time to flow between two marks was measured. This was called sample time. Viscosity was calculated with the following equation: ηemulsion ρemulsion  temulsion ¼ ηH 2 O ρH2 O  tH2 O Experiments were done in triplicate. The error of the results for ηemulsion was <5 %. Measurements of the Dynamic Surface Properties The NaCas, NaCas/sucrose, or NaCas/XG mixtures adsorption at the oil/water interface was determined by monitoring the evolution of surface pressure (π) and surface dilatational parameters with time. These interfacial parameters were analyzed with a pendant drop tensiometer PAT-1 (Sinterface Technologies, Berlin, Germany) together with the pendant drop method (Liu et al. 2011). A drop was formed at the tip

2409

of a polytetrafluoroethylene capillary immersed into a cuvette filled with sunflower oil, and its volume was kept constant at 12 mm3. Measurements were performed until adsorption equilibrium was reached (around 180 min, 11,100 s). The surface dilatational modulus (E) derived from the change in interfacial tension (σ), resulting from a small change in surface area is a measure of the total material dilatational resistance to deformation. E is a complex quantity and composed of real and imaginary parts. The real part of the dilatational modulus or storage component is the dilatational elasticity, Ed 0∣E∣cosδ. For a perfect elastic material, δ00°, and in case of a perfect viscous material, δ090°. E was measured as a function of adsorption time, at 3 % of deformation amplitude of the drop volume and at an angular frequency (ω) of 0.05 Hz. Measurements were made at least twice. The average standard accuracy of the surface pressure was roughly 0.1 mN/m. The error of the results was <0.5 and 5 % for surface pressure and surface dilatational properties, respectively. The reproducibility of the results was better than 0.5 and 5 % for surface pressure and surface dilatational properties, respectively. Statistical Analysis Significant differences between means were determined by the Student’s t test. An α level of 0.05 was used for significance.

Results and Discussion Droplet Size Analysis Figure 1 shows typical droplet size distributions for the initial emulsions (t00) formulated with 0.5 wt% NaCas and different aqueous phase compositions immediately after homogenization by Ultra-Turrax and with no further homogenization by ultrasound. D4,3, %Vd >1, and W for all emulsions prepared under these processing conditions are reported in Table 1. Emulsions showed almost monomodal distributions with a main peak varying from 9.95 to 28.02 μm. In all cases, D4,3 significantly decreased as NaCas concentration increased (p<0.05), indicating that protein concentration limited the fat globule size in this concentration range. However, Ultra-Turrax homogenization led in all cases to big-droplet emulsions usually called coarse emulsions. In agreement with D4,3 behavior, %Vd>1 was >91.6 % for all emulsions. W was very high in some cases as for example emulsions with LBG in the aqueous phase stabilized with 0.5 wt% NaCas. It was reported that smaller droplet size emulsions or fine emulsions are more stable systems than coarse emulsions (Perrechil and Cunha

2410

Food Bioprocess Technol (2013) 6:2406–2418 Table 1 Volume-weighted mean diameter (D4,3, μm), volume percentage of particles exceeding 1 μm in diameter (%Vd> 1), and width of the distribution (W, μm) of emulsions formulated with sunflower oil (SFO) as fat phase and different concentrations of sodium caseinate (NaCas) immediately after Ultra-Turrax homogenization Sample

Fig. 1 Particle size distribution of emulsions homogenized by UltraTurrax, with 10 wt% sunflower oil (SFO) as fat phase, 0.5 wt% of sodium caseinate (NaCas) as stabilizer, and different aqueous phases: water, black 65; 20 wt% sucrose, black 85; 0.3 wt% xanthan gum, black; 0.5 wt% xanthan gum, dotted black; 0.3 wt% locust bean gum, black 50; 0.5 wt% locust bean gum, dotted black 50

2010). It was expected that the distributions shown in Fig. 1 corresponded to the most unstable systems of all emulsions analyzed in this study. Figure 2 shows droplet size distributions for initial emulsions stabilized by 0.5 wt% NaCas but further homogenized by ultrasound. The emulsion with no sugar and no hydrocolloids added to the aqueous phase was the only one that showed a bimodal distribution. All the others were almost monomodal systems. D4,3, %Vd>1, and W parameters for ultrasound emulsions’ distributions (fine emulsions) are reported in Table 2. The second homogenization with ultrasound produced a noticeable reduction of D4,3. Values varied from 0.24 to 7.40 μm. %Vd>1 was lower than 65.5 % and in the case of emulsions stabilized by 5 wt% NaCas lower than 39.7 %. W values were very small in most cases showing that distributions were narrower when emulsions were further homogenized by ultrasound waves than when prepared using only Ultra-Turrax. There is a general agreement among authors that the smaller the droplet size the higher the stability of an emulsion. Thus, it would be expected that ultrasound emulsions formulated with 5 wt% NaCas would be the most stable of all. Stability by Turbiscan Initial Emulsions Figure 3 shows the BS profiles in reference mode (ΔBS0 BSt −BS0) as a function of the sample height (total height0 65 mm) for the emulsions in Fig. 1. The initial mean value of backscattering along the entire tube (BSav 0, from BS profile at t00) for a, b, c, d, e, and f were 48.17, 36.15,

No additives NaCas 0.5 wt% 2 wt% 5 wt% Sucrose 20 wt% NaCas 0.5 wt% 2 wt% 5 wt% XG 0.3 wt% NaCas 0.5 wt% 2 wt% 5 wt% XG 0.5 wt% NaCas 0.5 wt% 2 wt% 5 wt% LBG 0.3 wt% NaCas 0.5 wt% 2 wt% 5 wt% LBG 0.5 wt% NaCas 0.5 wt% 2 wt% 5 wt%

D4.3a

%Vd>1

W

19.10±0.15 18.21±0.11 16.35±0.20

99.0±0.4 98.1±0.5 95.7±0.2

27.8±0.4 27.1±0.3 26.1±0.3

18.24±0.19 15.29±0.18 9.95±0.09

99.2±0.5 97.6±0.2 91.6±0.3

26.7±0.5 24.8 ±0.1 19.3 ±0.1

15.69±0.17 14.01±0.12 12.10±0.10

100.0±0.0 98.2±0.2 99.9±0.1

14.6±0.1 15.0±0.1 13.9±0.1

23.19±0.20 17.91±0.20 10.64±0.11

100.0±0.0 99.9±0.0 100.0±0.0

26.4±0.1 19.2±0.0 11.7±0.1

26.03±0.19 16.12±0.15 13.05±0.13

100.0±0.0 97.1±0.4 99.4±0.2

42.0±0.0 18.2±0.3 12.9±0.3

28.02±0.20

99.7±0.1

69.0±0.0

17.01±0.15 15.24±0.08

96.2±0.1 97.6±0.5

14.0±0.1 16.0±0.3

Values are the average of duplicates NaCas sodium caseinate, XG xanthan gum, LBG locust bean gum Two values of D4.3 differing more than 0.2 μm are significantly different a

54.05, 45.67, 53.88, and 61.91 %, respectively. BS is a parameter directly dependent on the particle’s mean diameter and on the particle volume fraction (ϕ), i.e., BS0f(D,ϕ) (Mengual et al. 1999). At t00, the distribution of particles is homogeneous along the entire tube for stable systems. In that condition, all emulsions have the same volume fraction, and therefore, BSav at t00 depends predominantly on mean particle diameter. As may be noticed in Fig. 3, some UltraTurrax emulsions are unstable systems (a, b, and e) and showed significant destabilization after 2 min. Thus, BS

Food Bioprocess Technol (2013) 6:2406–2418

2411 Table 2 Volume-weighted mean diameter (D4,3, μm), volume percentage of particles exceeding 1 μm in diameter (%Vd> 1), and width of the distribution (W, μm) of emulsions formulated with sunflower oil (SFO) as fat phase and different concentrations of sodium caseinate (NaCas) immediately after ultrasound homogenization Sample

Fig. 2 Particle size distribution of emulsions homogenized by ultrasound, with 10 wt% sunflower oil (SFO) as fat phase, 0.5 wt% of sodium caseinate (NaCas) as stabilizer, and different aqueous phases: water, black 65; 20 wt% sucrose, black 85; 0.3 wt% xanthan gum, black; 0.5 wt% xanthan gum, dotted black; 0.3 wt% locust bean gum, black 50; 0.5 wt% locust bean gum, dotted black 50

depends on both mean diameter and particle volume fraction. For this reason, BSav at t00 did not correlate with D4,3 values reported in Table 1. Figure 4 shows the BS profiles in reference mode (ΔBS0 BSt −BS0) as a function of the sample height (total height0 65 mm) for emulsions formulated with 5 wt% NaCas as stabilizer and different aqueous phases, further homogenized by ultrasound. BSav 0, from BS profile at t00, for a, b, c, d, e, and f were 75.70, 55.60, 63.86, 60.32, 73.30, and 72.84. BSav at t00 had the lowest value for the emulsion with 20 wt% sucrose in the aqueous phase in agreement with a lower D4,3 value for this emulsion (Table 2). Palazolo et al. (2004) reported a close relationship between BSav at t00 and D4,3 values studying stability of emulsions formulated with whey proteins. The 20 wt% sucrose emulsion is a stable system and it is likely that BSav at t00 depends predominantly on mean particle diameter. It is clear that there is no correlation between BSav at t00 and D4,3 values for emulsions with LBG (Table 2). An inhomogeneous structure might be expected for these emulsions.

No additives NaCas 0.5 wt% 2 wt% 5 wt% Sucrose 20 wt% NaCas 0.5 wt% 2 wt% 5 wt%

D4.3a

%Vd >1

W

1.52±0.01 1.68±0.01 0.75±0.01

61.8±0.1 65.5±0.1 39.7±0.1

2.26±0.08 2.83±0.01 1.90±0.01

0.70±0.10 0.42±0.01 0.24±0.01

19.4±0.1 6.3±0.1 0.0±0.0

1.30±0.10 0.73±0.01 0.24±0.01

38.6±0.1 12.5±0.0 10.0±0.0

1.81±0.01 1.10±0.01 0.96±0.01

53.9±0.1 28.1±0.1 4.6±0.0

16.90±0.20 11.7±0.20 0.63±0.01

46.7±0.1 15.4±0.1 9.9±0.0

2.09±0.01 1.00±0.01 1.00±0.10

60.1±0.1

3.60±0.20

13.3±0.0 14.5±0.0

1.04±0.01 1.12±0.01

Xanthan 0.3 wt% NaCas 0.5 wt% 1.04±0.01 2 wt% 0.97±0.01 5 wt% 0.77±0.01 Xanthan 0.5 wt% NaCas 0.5 wt% 4.47±0.09 2 wt% 4.08±0.05 5 wt% 0.41±0.01 Locust bean 0.3 wt% NaCas 0.5 wt% 1.18±0.01 2 wt% 0.53±0.01 5 wt% 0.55±0.01 Locust bean 0.5 wt% NaCas 0.5 wt% 7.40±0.20 2 wt% 5 wt%

1.45±0.08 2.10±0.10

Values are the average of duplicates NaCas sodium caseinate, XG xanthan gum, LBG locust bean gum Two values of D4.3 differing more than 0.2 μm are significantly different a

Stability with Time 0.5 wt% NaCas Emulsions Processed by Ultra-Turrax To study the global stability of emulsions, the BS profiles in reference mode were analyzed at different storage times. These profiles constitute the macroscopic fingerprint of the emulsion sample at a given time (Mengual et al. 1999). For emulsion in Fig. 3a, the main mechanism of destabilization was creaming of small particles. This may be noticed by the typical mode the profiles change with time when stability is studied by Turbiscan (Mengual et al. 1999). That is, there is a decrease in BS t at the bottom of the tube and a

concomitant increase of BSt in the upper zone attributed to the formation of a cream layer. In addition, the top of the profile formed a peak with a shape indicative of the migration of individual particles. Migration rate was calculated as indicated in Fig. 5, and values of samples stabilized with 0.5 wt% NaCas were summarized in Table 3. The sample without additives in the aqueous phase and homogenized by Ultra-Turrax (control sample) had a great D4,3. Regarding the droplet size distribution, it might be expected that it was unstable. In agreement with the usual correlation observed

2412

Food Bioprocess Technol (2013) 6:2406–2418 30

30

a

10 0

- 10

0

10

20

30

40

50

60

70

80

- 20

ΔBack Scattering (%)

ΔBack Scattering (%)

20

- 30 - 40

20 10 0 30

40

50

60

70

80

-20 -30

30

40

50

60

70

80

-20 Tube Length (mm)

d

20 10 0 -10 0

10

20

30

40

50

60

70

80

-20

10 0 10

20

30

40

50

60

70

80

-20

f

30 ΔBack Scattering (%)

20

Tube length (mm)

40

e

30 ΔBack Scattering (%)

20

-40

Tube length (mm)

40

20 10 0 -10 0

10

20

30

40

50

60

70

80

-20 -30

-30

-40

10

-30

-40

-10 0

0 -10

30 ΔBack Scattering (%)

ΔBack Scattering (%)

30

20

0

40

c

10

10

-30

Tube Length (mm)

40

-10 0

b

20

Tube length (mm)

-40

Tube length (mm)

Fig. 3 Changes in backscattering profiles in reference mode (ΔBS) as a function of the tube length with storage time [samples were stored for 30 min; the first profile (t00) is a straight line at zero; arrow denotes time) in quiescent conditions for emulsions with sunflower oil (SFO) as fat phase, 0.5 wt% sodium caseinate (NaCas) as stabilizer, and

different aqueous phases: a water, b 20 wt% sucrose, c 0.3 wt% xanthan gum (XG), d 0.5 wt% xanthan gum (XG), e 0.3 wt% locust bean gum (LBG), and f 0.5 wt% locust bean gum (LBG). Tube length, 65 mm

between particle size and stability, it had the highest migration rate of all samples shown in Fig. 3, indicating that this formulation is the most unstable of all. Emulsion in Fig. 3b had sucrose added to the aqueous phase. It showed the same behavior as emulsion in Fig. 3a in the way that the main mechanism of destabilization was creaming of small particles but addition of sucrose slow destabilization. For emulsion in Fig. 3b, migration rate was 42.89 mm/h. Emulsions in Fig. 3c–e were coarse emulsions, that is, D4,3 was >10 μm (Table 1), and it would have been expected that

they were very unstable. These emulsions were formulated with hydrocolloids in the aqueous phase. Surprisingly, they were stable for 30 min and after further storage for a week (c and d) or for 3 days (f). There are several reports in literature of food emulsions with these droplet size distributions, which destabilized in minutes. Addition of hydrocolloids led to an unexpected stability. As may be noticed from Fig. 3, there were no significant changes in the profile with time. The ΔBS remained as a straight line during those storage times. Emulsions in Fig. 3c and d contained 0.3

Food Bioprocess Technol (2013) 6:2406–2418

2413

40

10 0

-10 0

10

20

30

40

50

60

70

80

-20

-30 -40

ΔBack Scattering (%)

20

30

20 10

0 20

30

40

50

60

70

80

-20 -30

-50

10

20

30

40

50

60

70

80

-20

-30 Tube length (mm)

d

60 40 20 0

-20

0

10

20

30

40

50

60

70

80

20 10 0

0

10

20

30

40

50

60

70

80

-20 -30

Tube Length (mm)

50

e

f

40

ΔBack Scattering (%)

ΔBack Scatterimg (%)

-10 0

-60

Tube Length (mm)

30

-40

0

-40

-40

-10

10

80

c

10

20

-40

Tube length (mm)

-10 0

b

30

ΔBack Scattering (%)

ΔBack Scattering (%)

30

ΔBack Scattering (%)

40

a

30

20 10 0 -10 0

10

20

30

40

50

60

70

80

-20

-30 -40

Tube Length (mm)

-50

Tube Length (mm)

Fig. 4 Changes in backscattering profiles in reference mode (ΔBS) as a function of the tube length with storage time (samples were stored for a week) in quiescent conditions for emulsions after ultrasonic processing treatment, with sunflower oil (SFO) as fat phase, 5 wt% sodium

caseinate (NaCas) as stabilizer, and different aqueous phases: a water, b 20 wt% sucrose, c 0.3 wt% xanthan gum, d 0.5 wt% xanthan gum, e 0.3 wt% locust bean gum, and f 0.5 wt% locust bean gum. Tube length, 65 mm

and 0.5 wt% XG, respectively. XG is an anionic polysaccharide that was reported to cause gelation of NaCas in aqueous solutions (Braga and Cunha 2004). HadjSadok et al. (2010) studying the effects of XG and NaCas concentrations on the rheological properties of their mixture in an aqueous medium at neutral pH found that the repulsive interactions between XG and NaCas can be inhibited at NaCas concentrations <1.27 wt% with XG concentration ranging from 0 to 0.5 wt%. Our results for coarse emulsions with 0.5 wt% NaCas and 0.3 or 0.5 wt% XG were in

agreement with these reports. LBG is a neutral polysaccharide that has the ability to give very viscous solutions at relatively low concentrations. The one selected for Fig. 3f was enough for stabilizing the emulsion. Perrechil and Cunha (2010) studied the stability of coarse emulsions formulated with 1 wt% NaCas, a soybean oil content of 30 % w/v, and LBG concentrations varying from 0 to 0.8 wt% by visual observation. It might have been expected that emulsions containing 30 % w/v oil were very unstable. However, in their systems, creaming process was slower as polysaccharide concentration

Food Bioprocess Technol (2013) 6:2406–2418

18

Peak Thickness (mm)

16 14 12 10

Δ Back Scattering (%)

2414

stability. Therefore, as was reported for 30 % w/v oil, in our systems, the increase in stability produced by LBG was only due to an increase in viscosity.

0

-10

-20

-30 0

10

20

30

40

Tube Length (mm)

8 6 4 2 -0,2

-0,1

0,0

0,1

0,2

0,3

0,4

0,5

time (min) Fig. 5 Example of the migration rate calculation. The peaks thicknesses (widths at 50 % height) on the bottom of the tube in the reference mode backscattering profile (inset) are plotted against time. The slope in the linear regime was use as a qualitative parameter of the migration rate

increased. The emulsion containing 0.5 wt% LBG was very stable, showing <5 wt% of creaming index, and addition of 0.8 wt% polysaccharide led to a nonphase separated emulsion. In our systems, formulated with 10 wt% sunflower oil, 0.3 wt% LBG was able to improve stability. Although emulsion in Fig. 3e destabilized mainly by creaming of small particles, migration rate was significantly slower than for emulsions in Fig. 3a and b. In agreement with Perrechil and Cunha (2010), stability increased with increase in LBG concentration. Addition of 0.5 wt% LBG to the coarse emulsion stabilized by 0.5 wt% NaCas was enough to prevent creaming (Fig. 3f). Moreover, as LBG does not form gels by itself, there was no additional effect on emulsion structure that improved Table 3 Slope of the linear zone (mm/h) and correlation coefficients (R2) evaluated from peak wide at 50 % height (peak thickness) in the bottom part of the tube from Turbiscan profiles expressed in reference mode at different times showing migration of emulsion samples, stabilized by 0.5 wt% sodium caseinate (NaCas), as represented in Fig. 5 Aqueous Phase

Water 20 wt% sucrose 0.3 wt% XG 0.5 wt% XG 0.3 wt% LBG 0.5 wt% LBG

Ultra-Turrax

Ultrasound

Slope

R2

Slope

R2

66.29 42.89

0.997 0.999

0.063 0.029

0.930 0.940

Stable Stable 12.05 Stable 3 days

– – 0.999 –

0.032 Phase separation Flocculation Flocculation

0.979 – – –

Slopes differing in more than 0.010 are significantly different XG xanthan gum, LBG locust bean gum

0.5 wt% NaCas Emulsions Processed by Ultrasound Table 3 also summarized the main mechanisms of destabilization when emulsions in Fig. 3 were further homogenized by ultrasound. In ultrasound-treated emulsions, droplet size was significantly lower than in the case of processing by Ultra-Turrax (Table 2). Thus, as was expected, further homogenization of samples in Fig. 3a and b led to emulsions that mainly destabilized by creaming but with a significantly slower migration rate. However, the clarification degree was still low after 190 h since the serum phase was still optically opaque and no light reached the transmission detector. All emulsions remained fully turbid along the tube during a week of storage at 22.5 °C. Therefore, the transmission profiles were not reported in this study. Emulsions in Fig. 3a and b further treated with ultrasound would have been considered stable by visual observation. However, the backscattering detector sensed destabilization. Although ultrasound treatment produced fine emulsions (Table 2), which are likely to be stable, further homogenization of samples in Fig. 3c, d, and f led to more unstable emulsions that destabilized by creaming (c), phase separation (d), or flocculation (f). In the case of emulsion in Fig. 3e, application of ultrasound treatment modified the main destabilization mechanism changing from creaming of small particles to flocculation. The BS profile of emulsion in Fig. 3e further homogenized by ultrasound showed a change in BSav (in the range of 20–50 mm height) and had a flat-top wide peak that appeared at the top of the tube (data not shown). The change in BSav value with time and the shape of the peak at the top was indicative of creaming caused for migration of flocculates. In emulsion in Fig. 3e, creaming that took place in coarse- or fine-particle emulsion occurred as a result of two different phenomena: migration of individual particles or migration of flocculates. Both emulsions were formulated in the same way; that is, they have the same oil/protein ratio, but were prepared using different processing conditions, giving very different particle size and therefore showing different creaming behavior. Coarse emulsion had more protein in the bulk phase and lower interfacial area, while fine emulsion is a flocculating emulsion with more interfacial protein. These results suggest that there should be a minimum interfacial area for flocculation. 5 wt% NaCas Emulsions Processed by Ultrasound The emulsion stabilized with 5 wt% NaCas, with no other additives in the aqueous phase, and processed by Ultra-Turrax showed a similar behavior than the one reported in Fig. 3a. The use of ultrasound allowed obtaining a fine emulsion that behaved as shown in Fig. 4a. In the first 23 h, the main

Food Bioprocess Technol (2013) 6:2406–2418

2415

Fig. 6 Confocal laser scanning microscopy (CLSM) images of emulsions with 10 wt% sunflower oil (SFO) as fat phase and 5 wt% sodium caseinate (NaCas) as stabilizer, homogenized by ultrasound waves, kept at 22.5 °C for 24 h: a water, b 20 wt% sucrose, c 0.3 wt% xanthan gum (XG), d 0.5 wt% xanthan gum (XG), e 0.3 wt% locust bean gum (LBG), and f 0.5 wt% locust bean gum (LBG)

mechanism of destabilization was creaming of individual particles, and after that, emulsion destabilized mainly by flocculation as may be noticed by the decrease in BSav in the 20–50 mm zone. Thus, flocculation was favored by the increase in surface area, which occurred when emulsions had smaller droplet sizes, that is, using ultrasound or for the same processing conditions when increasing NaCas concentration (Table 2). Figure 4b shows the behavior for the emulsion that contained 20 wt% of sucrose in the aqueous phase. As may be noticed from the profiles with time, this emulsion is very stable. No destabilization was noticed for at least a week. Figure 6 shows the confocal laser scanning microscopy (CLSM) images of emulsions in Fig. 4. Microstructure of emulsion without additives (Fig. 6a) shows the

presence of flocs after 24 h at 22.5 °C. In agreement with the stability behavior as studied by Turbiscan, emulsion with 20 wt% sucrose in the aqueous phase had a very different microstructure: The image shows small droplets evenly distributed (Fig. 6b). It may be expected from Table 2 that emulsions in Fig. 4 were the most stables of all since D4,3 values were very small and significantly smaller than the ones for Ultra-Turrax homogenization. However, when hydrocolloids were added to the aqueous phase, the use of ultrasound waves for further homogenizing emulsions led to more unstable systems comparing to emulsions with the same formulation but processed with Ultra-Turrax only (Fig. 4c–f). Systems with hydrocolloids showed phase separation to some extent after a few hours of storage at 22.5 °

2416

Food Bioprocess Technol (2013) 6:2406–2418

C. Microstructures of emulsions with XG or LBG show that these systems were heterogeneous and were formed by flocs. No individual droplets were observed in CLSM images (Fig. 6c–f). Rheological Properties Viscosity Figure 7 shows viscosity values for coarse emulsions formulated with 5 wt% NaCas and with or without sucrose or hydrocolloids in the aqueous phase. Addition of sucrose increased the viscosity twofold. However, it was not enough to prevent creaming (Fig. 3b). The high stability found for the 5 wt% NaCas fine emulsion with 20 wt% sucrose (Fig. 4b) may not be explained by an increase in viscosity. It was reported that sugars can alter protein functionality (Kulmyrzaev et al. 2000; Semenova et al. 2002; Belyakova et al. 2003; Guzey et al. 2003; Pizones Ruiz-Henestrosa et al. 2008) and emulsion stability (McClements 2004; Brent 2007). In agreement with those reports, our results suggest that stability effect caused by sugars was more than producing an increase in bulk viscosity of emulsions. In the case of hydrocolloids, the increase in viscosity measured in the emulsion with addition of 0.3 wt% LBG was in agreement with the fact that LBG slow down creaming, and in the case of addition of 0.5 wt% LBG, a stable emulsion was obtained (Fig. 3f) even after 3 days (Table 3). As xanthan gum forms a gel in aqueous solution, the values of viscosity for coarse XG emulsions formulated with 0.5 wt% Na Cas were very high. These emulsions were stable for at least a week (Fig. 3c, d). However, when NaCas concentration increased, emulsions became unstable, which was unexpected since viscosity of emulsions increased with NaCas concentration. The coarse 5 wt% NaCas emulsion with 0.3 wt% XG was an unstable system that mainly destabilized by flocculation (data

Fig. 7 Viscosity of coarse emulsions formulated with 5 wt% sodium caseinate (NaCas), 10 wt% sunflower oil (SFO) and with or without an additive in the aqueous phase. Suc sucrose, LBG locust bean gum, XG xanthan gum

Fig. 8 Effect of the presence of 0.3 wt% xanthan gum (XG) or 20 wt% sucrose on the surface pressure at long-term adsorption (π180) for 5 wt% sodium caseinate (NaCas) solutions at oil/water interfaces. Temperature, 20 °C. Standard deviations were <0.5 %

not shown, profiles were similar to the ones in Fig. 4c–f). Both biopolymers, NaCas and XG, had a negative charge at pH 6.66, and therefore, forces of repulsion were involved in emulsion destabilization. It may be likely that as a result of electrostatic repulsion high concentrations of caseinate disrupt the XG gel, indicating that interactions between protein and hydrocolloids in bulk were also very important since stability seems to depend more on emulsion structure than on emulsion viscosity. In agreement with these results, Liu et al. (2012) proved that viscosity of NaCas/XG mixed solutions was strongly affected by pH, which was indicative of strong interactions between these two biopolymers. Surface Pressure and Surface Dilatational Modulus Protein adsorption at the oil/water interface is the most important step in emulsion formation. During the adsorption

Fig. 9 Effect of the presence of 0.3 wt% xanthan gum (XG) or 20 wt% sucrose on the surface dilatational modulus at long-term adsorption (E180) for 5 wt% sodium caseinate (NaCas) solutions at oil/water interfaces. Temperature, 20 °C

Food Bioprocess Technol (2013) 6:2406–2418

process, protein molecules should undergo conformational changes due to the interaction with the surface or overcome energy barrier to adsorption (Dickinson 2011). Due to the fact that most food dispersions are foams and/or emulsions, protein–polysaccharide interactions, both in the aqueous phase and at fluid interfaces, could have an influence on protein interfacial adsorption and consequently on formation and stability of dispersed colloidal systems. To further investigate this hypothesis, the dynamic characteristics of the interface were studied by measuring the evolution of π with time. Figure 8, where the values of the surface pressure are presented, shows that addition of 0.3 wt% XG to the fine emulsion stabilized by 5 wt% NaCas did not have a significant effect on π180. It was reported that the protein–polysaccharide interactions induced at the interface are sufficiently strong to influence the viscoelastic properties of the adsorbed protein layer (Dickinson 2008). However, in our systems, in agreement with having no significant effects on π180, XG produced only a slight decrease in surface dilatational modulus at longterm adsorption (E180) (Fig. 9). The decrease in E180 values indicated that NaCas/XG mixtures were slightly less elastic compared to NaCas solutions. It was reported that the presence of 0.05 wt% XG in the aqueous phase of sodium caseinate solutions had a significant effect on the caseinate interfacial characteristics and its ability to occupy the oil/water interface sites especially at 0.001 wt% NaCas (Liu et al. 2011). According to these authors, it can be speculated that interactions between protein and polysaccharide reduce the availability of the hydrophobic binding sites on the protein, leading to a lower surface activity. Our results did not support this hypothesis at the concentrations range used in this study. The effect on π and E seemed to indicate that NaCas/XG interactions were weak. On the contrary, addition of 20 wt% sucrose modified the long-term adsorption of NaCas at oil/water interface and significantly diminished dilatational elasticity, indicating changes in surface tension (Fig. 9). The decrease in π and E caused by sucrose indicated that because of sucrose addition, NaCas became a better surfactant. The interactions sucrose/NaCas modified protein functionality, which led to a dramatic change in emulsion microstructure (Fig. 6b) and stability (Fig. 4b). In a previous work (Huck-Iriart et al. 2011), we reported the effect of sugar addition (trehalose or sucrose) on the maximum reciprocal lattice spacing, qmax, determined by small angle X-ray scattering. The studied emulsions contained different NaCas concentration and 10 wt% of a concentrated from fish oils or two vegetable oils, SFO or olive oil, as fat phase. The slightly increase in qmax values with trehalose or sucrose addition suggested that sugar had an effect further than viscosity changes since the aggregation state of the protein changed with the aqueous phase formulation. Therefore, sucrose interacts with NaCas at the interface and may also work as coadjutant in micelle

2417

formation or may change the quality of water as a solvent, improving the solvent–protein interaction, leading to smaller particles sizes and more stable emulsions. Belyakova et al. (2003) reported that there was a pronounced dissociation of NaCas submicelles in the presence of sucrose at a pH above the protein’s isoelectric point due, most likely, to direct hydrogen bonding between NaCas and sucrose. The dissociation of NaCas submicelles was in excellent agreement with the more homogeneous microstructure and the formation of smaller compact protein structures as detected by confocal laser scanning microscopy.

Conclusions The following conclusions can be drawn from the studies reported above. (1) The interactions between sucrose/NaCas at the interface modified protein functionality and led to a dramatic change in emulsion microstructure and stability. Sucrose may also work as coadjuvant in micelle formation or may change the quality of water as a solvent increasing stability of emulsions. (2) The slightly or no effect on surface properties of the hydrocolloids selected for this study showed that for the systems investigated hydrocolloids mainly stabilized emulsions by an increase in viscosity. However, the increase in instability found with increase concentrations of NaCas or with the use of ultrasound strongly suggested that interactions caseinate/hydrocolloid in bulk, which changed with the selected processing method, were very relevant to emulsion stability. In the case of XG, the formation of a gel, which also was affected by NaCas concentration and ultrasound processing, gave emulsions a great stability since this phenomenon strongly modifies emulsion structure. (3) The more stable systems of all selected for this study were the 0.3 or 0.5 wt% XG or 0.5 wt% LBG/0.5 wt% NaCas coarse emulsion and the 20 wt% sucrose/5 wt% NaCas fine emulsion. Acknowledgments María L. Herrera and Roberto J. Candal are researchers of the National Research Council of Argentina (CONICET). This work was supported by CONICET through Project PIP 11220080101504, by the National Agency for the Promotion of Science and Technology (ANPCyT) through Project PICT 0060, and by the University of Buenos Aires through project number 20020100100467.

References Alvarez Cerimedo, M.-S., Huck-Iriart, C., Candal, R.-J., & Herrera, M.-L. (2010). Stability of emulsions formulated with high concentrations of sodium caseinate and trehalose. Food Research International, 43(5), 1482–1493. Belyakova, L.-E., Antipova, A.-S., Semenova, M.-G., Dickinson, E., Matia Merino, L., & Tsapkina, E.-N. (2003). Effect of sucrose on

2418 molecular and interaction parameters of sodium caseinate in aqueous solution: Relationship to protein gelation. Colloids and Surfaces. B, Biointerfaces, 31(1), 31–46. Bordes, C., García, F., Frances, C., Biscans, B., & Snabre, P. (2001). On line optical investigation of concentrated dispersions in precipitation and grinding processes. Kona, 19, 94–108. Braga, A.-L.-M., & Cunha, R.-L. (2004). The effects of xanthan conformation and sucrose concentration on the rheological properties of acidified sodium caseinate xanthan gels. Food Hydrocolloids, 18(6), 977–986. Brent, S.-M. (2007). Stabilization of bubbles and foams. Current Opinion in Colloid & Interface Science, 12(4–5), 232–241. Cerdeira, M., Palazolo, G.-G., Candal, R.-J., & Herrera, M.-L. (2007). Factors affecting initial retention of a microencapsulated sunflower seed oil/milk fat fraction blend. Journal of the American Oil Chemists’ Society, 84(6), 523–531. Chauvierre, C., Labarre, D., Couvreur, P., & Vauthier, C. (2004). A new approach for the characterization of insoluble amphiphilic copolymers based on their emulsifying properties. Colloid & Polymer Science, 282(10), 1097–1104. Dickinson, E. (2006). Structure formation in casein-based gels, foams, and emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 288(1–3), 3–11. Dickinson, E. (2008). Interfacial structure and stability of food emulsions as affected by protein–polysaccharide interactions. Soft Matter, 4(5), 932–942. Dickinson, E. (2011). Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures. Food Hydrocolloids, 25(8), 1966–1983. Fox, P. F., & Brodkorb, A. (2008). The casein micelle: Historical aspects, current concepts and significance. International Dairy Journal, 18(7), 677–684. Guzey, D., McClements, D.-J., & Weiss, J. (2003). Adsorption kinetics of BSA at air–sugar solution interfaces as affected by sugar type and concentration. Food Research International, 36(7), 649–660. HadjSadok, A., Moulai-Mostefa, N., & Rebiha, M. (2010). Rheological properties and phase separation of xanthan–sodium caseinate mixtures analyzed by a response surface method. International Journal of Food Properties, 13(2), 369–380. Huck-Iriart, C., Álvarez Cerimedo, M.-S., Candal, R.-J., & Herrera, M.-L. (2011). Structures and stability of lipid emulsions formulated with sodium caseinate. Current Opinion in Colloid & Interface Science, 16(5), 412–420. Kobori, T., Matsumoto, A., & Sugiyama, S. (2009). pH-dependent interaction between sodium caseinate and xanthan gum. Carbohydrate Polymers, 75(4), 719–723. Kulmyrzaev, A., Bryant, C., & McClements, D.-J. (2000). Influence of sucrose on the thermal denaturation, gelation, and emulsion

Food Bioprocess Technol (2013) 6:2406–2418 stabilization of whey proteins. Journal of Agricultural and Food Chemistry, 48(5), 1593–1597. Liu, L., Zhao, Q., Liu, T., & Zhao, M. (2011). Dynamic surface pressure and dilatational viscoelasticity of sodium caseinate/xanthan gum mixtures at the oil–water interface. Food Hydrocolloids, 25(5), 921–927. Liu, L., Zhao, Q., Liu, T., Long, Z., Kong, J., & Zhao, M. (2012). Sodium caseinate/xanthan gum interactions in aqueous solution: Effect on protein adsorption at the oil–water interface. Food Hydrocolloids, 27(2), 339–346. McClements, D.-J. (2004). Protein-stabilized emulsions. Current Opinion in Colloid & Interface Science, 9(5), 305–313. McClements, D. J. (2005). Emulsion stability. In Food emulsions, principles, practices, and techniques (2nd ed., pp. 269–339). New York: CRC. Mengual, O., Meunier, G., Cayré, I., Puech, K., & Snabre, P. (1999). Turbiscan MA 2000: Multiple light scattering measurement for concentrated emulsion and suspension instability analysis. Talanta, 50(2), 445–456. Palazolo, G.-G., Sorgentini, D.-A., & Wagner, J.-R. (2004). Emulsifying properties and surface behavior of native and denatured whey soy proteins in comparison with other proteins. Creaming stability of oil-in-water emulsions. Journal of American Oil Chemists’ Society, 81(7), 625–632. Palazolo, G.-G., Sorgentini, D.-A., & Wagner, J.-R. (2005). Coalescence and flocculation in o/w emulsions of native and denatured whey soy proteins in comparison with soy protein isolates. Food Hydrocolloids, 19(3), 595–604. Pan, L.-G., Tomás, M.-C., & Añón, M.-C. (2002). Effect of sunflower lecithins on the stability of water-in-oil and oil-in-water emulsions. Journal of Surfactants and Detergents, 5(2), 135–143. Perrechil, F.-A., & Cunha, R.-L. (2010). Oil-in-water stabilized by sodium caseinate: Influence of pH, high-pressure homogenization and locust bean gum addition. Journal of Food Engineering, 97(4), 441–448. Pizones Ruiz-Henestrosa, V.-M., Carrera Sánchez, C., & Rodríguez Patino, J.-M. (2008). Effect of sucrose on functional properties of soy globulins: Adsorption and foam characteristics. Journal of Agricultural and Food Chemistry, 56(7), 2512–2521. Relkin, P., & Sourdet, S. (2005). Factors affecting fat droplet aggregation in whipped frozen protein-stabilized emulsions. Food Hydrocolloids, 19(3), 503–511. Semenova, M.-G., Antipova, A.-S., & Belyakova, L.-E. (2002). Food protein interactions in sugar solutions. Current Opinion in Colloid & Interface Science, 7(5–6), 438–444. Thanasukarn, P., Pongsawatmanit, R., & McClements, D.-J. (2006). Utilization of layer-by-layer interfacial deposition technique to improve freeze-thaw stability of oil-in-water emulsions. Food Research International, 39(6), 721–729.