Article pubs.acs.org/JAFC

Emulsifying Properties of Soy Protein Nanoparticles: Influence of the Protein Concentration and/or Emulsification Process Fu Liu† and Chuan-He Tang*,†,‡ †

Department of Food Science and Technology and ‡State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, 510640 Guangdong, People’s Republic of China ABSTRACT: The interfacial and emulsifying properties of soy protein isolate nanoparticles formed by combined treatments of heating and electrostatic screening, as affected by variation of the protein concentration (c) and emulsification process, were investigated. These nanoparticles (with a z-average diameter of 103 nm at c = 0.1%, w/v) tended to aggregate at higher c values, and their internal structure was mainly maintained by hydrophobic interactions and disulfide bondings. In general, increasing c progressively favored diffusion and/or adsorption at the interface and formation of finer emulsions; increasing the energy input level of emulsification improved the emulsification efficiency and extent of droplet flocculation, as well as the emulsion coalescence and creaming stability. The rheological and creaming behavior of these emulsions was predominately determined by the amount of proteins adsorbed at the interface. The results confirmed that these nanoparticles can formulate Pickering emulsions with properties tailored by selecting c and the emulsification process. KEYWORDS: soy protein nanoparticle, Pickering emulsions, emulsification process, emulsifying property

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INTRODUTION There is increasing interest in developing Pickering oil-in-water (O/W) emulsions stabilized by food-grade particles, due to their compatibility with food and some outstanding characteristics, e.g., extraordinary stability against coalescence, sustained release of some encapsulated ingredients, and enhancement of stability against lipid oxidation.1−4 To date, the food-grade particles or nanoparticles that have been confirmed to stabilize Pickering emulsions include chitin nanocrystal particles,5,6 water-insoluble zein,7 microcrystalline cellulose (MMC),3 modified starch,3,8,9 some flavonoids,10 and even solid lipid nanoparticles.11 Recently, we reported that soy protein isolate (SPI) can also be developed into Pickering nanoparticle stabilizers with sizes of ∼100 nm by means of protein aggregation induced by thermal treatment (95 °C, 15 min) coupled with electrostatic screening.12 The emulsions stabilized by the SPI nanoparticles at higher protein concentration (c) values exhibited much better stability against coalescence and creaming.12 Compared with the particles mentioned above, the SPI nanoparticles as Pickering stabilizers have the following advantages: (i) they are nutritional and functional food ingredients themselves and are abundantly and commercially available; (ii) they can perform “double duty” and not only emulsify, but also provide steric stabilization; (iii) they do not need any chemical modification to improve the surface property of the particles; (iv) finally, and importantly, they are compatible with high-pressure emulsification. On the other hand, a variety of homogenizers have been available for preparation of conventional O/W emulsions, including high-shear mixers, high-pressure homogenizers, colloid mills, ultrasonic homogenizers, and membrane homogenizers.13 In contrast, the choice of homogenization mode or homogenizer for formulating particle-stabilized Pickering emulsions should be cautious, since for most classic Pickering stabilizers, e.g., inorganic silica nanoparticles, the high-pressure © 2014 American Chemical Society

mode of emulsification may cause damage to the process device.14 Due to this consideration, most of the Pickering emulsions reported in the literature, including those stabilized by many food-grade particles, are formed using high-shear7−9 or ultrasonic5,6 homogenizers. In general, the energy input level of emulsification usually increases in the order high-shear mixers < ultrasonic homogenizers < high-pressure homogenizers. The energy input level of emulsification not only is related to its efficiency (with emulsion size as an indicator), but also may affect the nature (e.g., surface hydrophobicity, size, and aggregated state) of many Pickering particles. Thus, the properties of the Pickering emulsions produced by different emulsification techniques might vary considerably. To date, however, few studies on particle-stabilized Pickering emulsions have addressed the application of high-pressure emulsification. It has been well recognized that denatured or aggregated proteins in SPI undergo changes in size, surface hydrophobicity (Ho), and even protein solubility when subjected to a highpressure or ultrasonic treatment.15−17 For example, Arzeni and others15 found that high-intensity ultrasound treatment resulted in decreased size but enhanced Ho and solubility of the aggregates in commercial SPI dispersions. Similar effects on Ho and the solubility of the proteins have been observed in native or heated SPI subjected to microfluidization.17 Keerati-U-Rai and Corredig16 even pointed out that, during high-pressure treatment, the proteins in soy protein dispersions might undergo partial disruption and subsequent rearrangement of the supramolecular structure. These observations imply that when soy protein nanoparticles are applied as the emulsifiers for the emulsions, an emulsification process with a higher Received: Revised: Accepted: Published: 2644

November 27, 2013 March 6, 2014 March 6, 2014 March 6, 2014 dx.doi.org/10.1021/jf405348k | J. Agric. Food Chem. 2014, 62, 2644−2654

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energy input level will be more favorable for producing finer emulsions. Furthermore, the application of a high energy level of emulsification may impart some novel characteristics (e.g., enhanced interdroplet interactions and, subsequently, formation of a uniform and gel-like emulsion) to the emulsions stabilized by these nanoparticles. Another noteworthy issue is that, for any particle dispersion, interparticle interactions and particle−particle associations (or aggregation) are highly determined by the particle concentration. The concentration of particles may affect their adsorption at the oil/water interface during emulsification, as well as formation of a multilayer-structured interfacial film, since at high particle concentrations, most of the particles with good surface hydrophobicity (e.g., modified silica nanoparticles) are usually present in the aggregated form.19 Once these aggregated particles are adsorbed at the interface, they could provide supplementary stabilization to the formed emulsions.19 More importantly, the association or aggregation of particles in the continuous phase (which is more favorable at higher particle concentrations) may lead to formation of a gellike network in the emulsions, as in the chitin nanocrystal case.5 The gel-like network formation can thus impart extraordinary stability against creaming to the emulsions. Using steady viscosity and dynamic oscillatory measurements, gel-like network formation has also been confirmed for the emulsions stabilized by preheated SPI or whey proteins at high c values, produced by microfluidization as the emulsification process (e.g., 6% in the continuous phase).19−21 If the oil fraction is high enough, the gel-like behavior can also be observed for the emulsions stabilized by SPI nanoparticles, produced even by a high-shear mixer with a low-energy input.21 In this work we aim to investigate the influence of the emulsification process on the interfacial and emulsifying properties of SPI nanoparticles and the properties of the resultant Pickering emulsions. The SPI nanoparticles were formed by the same process as in our previous work.12 For comparison, three emulsification processes (I−III) with different energy input levels were applied to formulate the emulsions: (I) only high-shear homogenization; (II) combined applications of high-shear homogenization and ultrasonic treatment; (III) combined applications of high-shear homogenization and microfluidization. It is reasonably hypothesized that increasing the energy input level of emulsification may greatly improve the adsorption of the nanoparticles at the interface and, subsequently, can impart some unique characteristics to the resultant Pickering emulsions, e.g., extraordinary stability against coalescence and creaming and a gel-like emulsion structure.

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w/v), diluted with the same buffer to an appropriate concentration (in the range of 0.01−6.0%, w/v) if needed, was applied to the emulsion preparation. SPI Nanoparticle Characterization. The particle size (z-average diameter) of nanoparticles in the preheated SPI dispersions was determined using the dynamic light scattering (DLS) technique, using a Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., Worcestershire, U.K.) equipped with a 4 mW He−Ne laser (633 nm wavelength) at 25 °C. The SPI nanoparticle dispersion (6%, w/v) was diluted with the same background buffer (containing 300 mM NaCl) to different individual c values (0.01−1.5%, w/v), and the diluted dispersions were kept at ambient temperature for 30 min, before being subjected to the determination. All the samples were centrifuged at 10000g for 20 min to remove any insolubles in the dispersions prior to the determination. The z-average diameter was obtained with a single-exponential fitting, with each datum reported as the average over three readings. To unravel the interactive forces involved in the formation and maintenance of the nanoparticle structure, the z-average diameter of the SPI nanoparticle dispersion at c = 0.05% (w/v) in the presence of 6.0 M urea, 0.5% SDS, and 30 mM DTT, alone or in combinations, was also determined according to the same process above. Dynamic Surface Pressure (π) Measurement. The adsorption kinetics of SPI nanoparticles at the oil/water (pH 7.0, 300 mM NaCl) interface, at varying c values of 0.01−1.5% (w/v), were determined using an optical contact angle meter (OGA-20) with an oscillating drop accessory, ODG-20 (Dataphysics Instruments GmbH, Germany), at 25 °C. The interfacial tension (γ) of the oil/water interface, formed with an oil drop submerged into a cuvette filled with the nanoparticle dispersions, was determined by the drop shape analysis.22 The surface pressure (π) was calculated as γs − γp, where γs and γp are the surface tensions of the aqueous background buffer and the corresponding nanoparticle dispersion (at any test time), respectively. All the measurements with a period of up to 3 h were performed in duplicate. For each sample two consecutive measurements were performed (with the average used for statistics analysis), and each sample was replicated at least two times. Emulsion Preparation. The SPI nanoparticle dispersions at varying c values of 0.5−6% (w/v), obtained as mentioned above, were applied to form the emulsions, with types I, II, and III corresponding to the three different emulsification processes. All the emulsions were formed at a fixed oil fraction (ϕ) of 0.2. The type I emulsions were prepared as follows: In brief, 16 mL of any SPI nanoparticle dispersion was added to a glass vial, and 4 mL of soybean oil was slowly added to the dispersion, with mixing via an Ultra Turrax homogenizer (model IKA-ULTRA-TURRAX T25 basic, IKA Works, Inc., Wilmington, NC) with a dispersing head operating at 10000 rpm. After all the oil was added, the sample was mixed for an additional 2 min. The type I emulsions were further processed using ultrasonic equipment with a probe at 250 W for 3 min to produce the type II emulsions or homogenized through a microfluidizer (M110EH model, Microfluidics International Corp., Newton, MA) for one pass at a pressure level of 40 MPa to produce the type III emulsions. These resultant emulsions (fresh) were directly subjected to analysis or stored at room temperature for various periods of time for emulsion stability evaluation, e.g., 7 or 40 days for coalescence stability analysis. Droplet Size Distribution Profiles and Volume-Average Droplet Size (d4,3) of Emulsions. Droplet size distribution profiles of various freshly prepared or stored (7 or 40 days) emulsions were obtained with a Malvern MasterSizer 2000 (Malvern Instruments Ltd.). Deionized water or 1.0% (w/v) SDS solution containing 300 mM NaCl was used as the dispersant. The relative refractive index of the emulsion was taken as 1.095, that is, the ratio of the refractive index of soy oil (1.456) to that of the continuous phase (1.33). Droplet size measurements are reported as the volume-average droplet size, d4,3 (=∑nidi4/∑nidi3), where ni is the number of droplets with diameter di. All determinations were conducted on individual samples at least in duplicate. Flocculation Index (FI, %). The FI (%) of the fresh emulsions was evaluated according to the method described by Castellani and

MATERIALS AND METHODS

Materials. Freeze-dried SPI was the same as that applied in our previous work,12 the protein content of which was about 91% (wet basis). Soy oil was purchased from a local supermarket in Guangzhou (China). Nile Blue A, Nile Red, and dithiothreitol (DTT) were obtained from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin (BSA) was obtained from Fitzgerald Industries International Inc. (Concord, MA). All other chemicals used were of analytical grade. SPI Nanoparticle Preparation. The SPI nanoparticles were formed according to the same process described in our previous work.12 In brief, an SPI solution (6%, w/v) in water, containing 0.02% (w/v) sodium azide, was heated in a bath at 95 °C for 15 min. After being cooled in an ice bath to room temperature, the preheated SPI dispersion was added to the salt powder little by little to a final concentration of 300 mM. Last, the SPI nanoparticle dispersion (6%, 2645

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Figure 1. (A) Protein concentration dependence of the z-average diameter (Dz) for heat-induced SPI nanoparticles. The nanoparticles were formed by heating the SPI dispersion (c = 6%, w/v) at 95 °C for 15 min and, subsequently, electrostatic screening with addition of 300 mM NaCl. The preheated SPI dispersion was diluted with the same solvent (with 300 mM NaCl) to a specific c value in the range of 0.01−1.5% (w/v). (B) Effects of various protein-perturbing solvents on the Dz of nanoparticles in a preheated SPI dispersion at c = 0.05%. Different characters on the tops of the columns represent the significant difference at the p < 0.05 level (n = 3). others.23 The emulsions were diluted in deionized water with and without 1% (w/v) SDS, and d4,3 of the droplets was determined as described above. The FI was calculated as follows:

concentrated oil droplets) at the top of the tube and an aqueous phase of the emulsion at the bottom. The cream layer was carefully removed using a syringe, and the subnatant was filtered through a 0.22 μm filter (Millipore Corp.). The protein concentration of the filtrate (Cf) was determined with the Lowry method using BSA as the standard. The percentage of adsorbed proteins (AP) was calculated as [(Co − Cf) × 100]/Co, where Co is the initial protein concentration in SPI aggregate dispersions. The amount of adsorbed proteins (g/100 mL) was calculated on the basis of the total adsorbed protein content per 100 mL of emulsion. Statistics. An analysis of variance (ANOVA) of the data was performed, and a least significant difference (LSD) with a confidence interval of 95% was applied to compare the means of duplicate or triplicate measurements on separate samples (n = 2 or 3).

FI (%) = [d4,3(in water)/d4,3(in 1% SDS) − 1.0] × 100 Microstructure of Fresh Emulsions. The microstructure of the fresh emulsions (types I−III), formed at varying c values of 0.5−6.0% (w/v), was evaluated by confocal laser scanning microscopy (CLSM) using a Leica TCS SP5 confocal laser scanning head mounted on a Leica DMRE-7 (SDK) upright microscope (Leica Microsystems Inc., Heidelberg, Germany). Each fresh emulsion sample (1 mL) was mixed with about 40 μL of fluorescence dye mixtures of Nile Blue (0.1%, w/ v) and Nile Red (0.1%, w/v). The mixtures were then placed on concave confocal microscope slides (Sail, Sailing Medical-Lab Industries Co. Ltd., Suzhou, China), covered with glycerol-coated coverslips, and examined with a 100× magnification lens and an argon/krypton laser with an excitation line of 488 nm and a He−Ne laser with excitation at 633 nm, respectively. Creaming Stability. The creaming stability of various emulsions was assessed visually upon quiescent storage for up to 30 days. Each emulsion (10 mL) in a glass test tube (1.5 cm internal diameter × 12 cm height) was perpendicularly stored at ambient temperature. After different periods of storage, the height of the serum (Hs) and total height of the emulsion (Ht) were recorded. The creaming index (%) was calculated as (Hs/Ht) × 100. Each datum is reported as the mean ± standard deviation of three replicates performed on separated samples. Rheological Characterization. Steady Shear Viscosities. Steady shear viscosities (η) of various fresh emulsions were characterized using a HAAKE RS600 rheometer (HAAKE Co., Germany) with parallel plates (d = 27.83 mm) at 25 °C. The gap between two plates was set to 1.0 mm. After 10 min of equilibration, η of the emulsions was recorded as the shear rate was increased from 1 to 100 s−1. Dynamic Oscillatory Measurements. The dynamic viscoelastic properties of the gel-like type III emulsions were characterized by the same RS600 rheometer using a small-amplitude oscillatory strain or frequency sweep mode. For the strain sweep experiments, the strain (γ) was varied from 0.002 to 1.0 at a constant frequency of 1.0 Hz, while, for the frequency sweep experiments, the frequency was varied from 0.1 to 100 rad/s at a constant strain of 0.5%. The elastic modulus (G′), loss modulus (G″), and loss tangent (tan δ) were recorded. All the experiments were carried out at 25 °C unless stated otherwise. Amount of Adsorbed or Entrapped Proteins. The amount of adsorbed or entrapped proteins in the fresh emulsions (types I−III) was evaluated by a centrifugation method. In brief, each fresh emulsion (1.0 mL) was centrifuged at 10000g for 30 min at room temperature. After the centrifugation, two phases were observed: a cream layer (or

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RESULTS AND DISCUSSION SPI Nanoparticle Characteristics. Figure 1 A shows the zaverage diameter (Dz) of SPI nanoparticles as affected by the c (in the range 0.01−1.5%, w/v) at which the DLS measurements were performed. Dz (about 103 nm) basically remained constant at c values in the range of 0.01−0.1% (w/v), but it significantly increased to 122 nm as c was increased to 0.5% (w/v) (Figure 1 A). A further increase in c to 1.5% (w/v) led to a slight but insignificant increase in Dz. Dz at c = 0.5% (w/v; 122 nm) is basically similar to that (84−112 nm) observed for the glycinin and β-conglycinin mixtures heated at 100 °C for 30 min at the same c value but without 300 mM NaCl.24 The increases in Dz might be associated with the increased interparticle interactions and subsequent formation of larger particles.12 To unravel the pattern of interactive forces involved in formation and maintenance of the nanoparticle structure, we determined the Dz of nanoparticles at c = 0.05% (w/v) in the presence of various protein perturbants, including 6 M urea, 0.5% SDS, and 30 mM DTT, or their combinations, as displayed in Figure 1 B. The results interestingly showed that the presence of 0.5% SDS or 30 mM DTT led to a significant reduction in Dz, with a higher extent of reduction observed in the presence of 0.5% SDS; the presence of both 0.5% SDS and 30 mM DTT led to a considerable decrease in Dz (77% vs 39% in the presence of 0.5% SDS; Figure 1B). On the other hand, the presence of 6 M urea did not decrease Dz, regardless of the presence or absence of 0.5% SDS and/or 30 mM DTT, and in some cases, Dz on the contrary significantly increased (Figure 1 2646

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Figure 2. (A) Time evolution of the surface pressure (π) for the adsorption of SPI nanoparticles at the oil/water interface (SPI concentration in solution, 0.01−1.5%, w/v; temperature, 25 °C). (B) Typical profile of the molecular penetration and configurational rearrangement steps at the interface for SPI nanoparticles at c = 0.5%. kP and kR represent first-order rate constants of penetration and rearrangement, respectively.

Table 1. Characteristic Parameters, Including the Apparent Diffusion Rate (kdiff°), Constants of Penetration and Structural Rearrangement at the Interface (kP and kR), and Surface Pressure at the End of Adsorption (11800 s; π11800), for the Adsorption of SPI Nanoparticles at the Oil/Water Interface at Varying Protein Concentrations of 0.01−1.5% (w/v) protein concn (%) 0.01 0.05 0.1 0.5 1.5 a

kdiff° a (mN m−1 s−0.5) (LRb) 0.5822 0.2986 0.2117 0.1779 0.1552

kP × 104 (s−1) (LR)

(0.9459) (0.9274) (0.9550) (0.9495) (0.9755)

−2.5609 −2.6183 −2.5456 −2.4088 −2.6482

(0.969) (0.979) (0.982) (0.982) (0.989)

kR × 104 (s−1) (LR) −12.2 −11.4 −11.0 −11.1 −11.7

(0.808) (0.870) (0.861) (0.937) (0.949)

π11800 (mN m−1) 14.13 14.14 14.54 15.99 16.10

π vs t1/2 slope during the initial adsorption. bLR = linear regression coefficient.

(w/v) was the adsorption of the nanoparticles at the interface diffusion-controlled, while, at c values of 0.05% (w/v) or higher, the adsorption was more complex. A similar adsorption behavior has been previously observed for native and heattreated (at 90 or 120 °C for 20 min) SPIs.30 At c values above 0.01% (w/v), the π value at the beginning of adsorption (0 s; π0) was far above zero, suggesting that the initial adsorption at these c values was too fast to be detected. However, it can still be observed that π0 progressively increased from about 0 to 10.2 mN/m as c increased from 0.01 to 1.5% (w/v; Figure 2 A). This observation reflects that increasing the particle concentration progressively accelerated the most initial diffusion and/ or adsorption of the nanoparticles from the bulk onto the interface. The increasing rate of π, or slope for the plot of π against t1/2 during initial adsorption (e.g., less than 100 s), gradually decreased with increasing c (from 0.01% to 1.5%, w/v; Table 1), which might reflect the presence of an energy barrier at the interface.31 On the other hand, π at the end of the adsorption period (11800 s; π11800) was nearly unchanged by variation in c from 0.01% to 0.1%, irrespective of the differences in π changes during initial adsorption; π11800 considerably increased from about 14.5 to 16.0 mN/m when c was increased from 0.1% to 0.5% (w/v), and it did not change upon a further increase in c (to 1.5%, w/v) (Table 1). Interestingly, the changing pattern of π11800 upon an increase in c is basically the same as that observed for the particle size (Figure 1A), implying that the potential for these nanoparticles to be adsorbed at the interface and participate in interfacial film formation was largely determined by their particle size. Following the initial adsorption, penetration and subsequent rearrangement of previously adsorbed nanoparticles at the interface occur. These processes can be monitored using a first-

B). Usually, SDS, urea, and DTT mainly disrupt hydrophobic interactions, hydrogen bonding, and disulfide bonding, respectively.25,26 Thus, the observations suggest that hydrophobic interactions and disulfide bondings are the major intramolecular interactive forces maintaining the internal structure of the nanoparticles, while the hydrogen bonds play a crucial role in their external structure. The interactive force pattern seems to be a bit different from that observed for SPI nanoparticles induced by the calcium ion, where the driving forces for the nanoparticle formation and stabilization were largely attributed to hydrogen bonding and hydrophobic interactions, while disulfide bonding was minor.27 Interfacial Adsorption Behavior: Adsorption Kinetics and Structural Rearrangements at the Interface. The interfacial adsorption behavior of the SPI nanoparticles in relation to c was also investigated, with the aim to elucidate the role of the particle concentration in the formation dynamics of the films at the oil/water interface. The surface pressure (π) at the interface for SPI nanoparticles at any test c value (0.01− 1.5%, w/v) progressively increased with time (Figure 2A), reflecting protein or particle adsorption at the interface.22 Due to the consideration that various kinds of emulsifiers (including solid particles, globular proteins, and surfactants) exhibit high similarities in emulsification and emulsion stability,28 the adsorption kinetics of SPI nanoparticles at the interface can also be described by three main steps: (i) diffusion of particles from the bulk onto the interface, (ii) penetration and structural deformation at the interface, and (iii) rearrangement of adsorbed nanoparticles at the interface and multilayer formation, as has been widely applied to proteins.29 When the adsorption process is controlled by diffusion, a plot of π against t1/2 will be linear and the slope of this plot the diffusion rate constant (kdiff).29 Figure 2 A shows that only at c = 0.01% 2647

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Figure 3. Typical droplet size distribution profiles of various fresh emulsions stabilized by the SPI nanoparticle aggregates at a comparable initial c of 0.5−6.0% (w/v), as determined with water (A−C) or 1% SDS (A′−C′) as the dispersing solvent: (A, A′) emulsification with an Ika T25 (emulsification I); (B, B′) emulsification with an Ika T25 in combination with ultrasonic equipment (emulsification II); (C, C′) emulsification with an Ika T25 in combination with a microfluidizer (emulsification III).

Table 2. Mean z-Average Diameter (d4,3, in Water or 1% SDS) Values and Flocculation Index (FI, %) of Droplets in the Fresh Emulsions Stabilized by the SPI Nanoparticle Aggregates at a Comparable Initial c Value of 0.5−6.0% (w/v), Produced by Three Emulsification Processes (I−III)a emulsification process Ib

emulsification process II

d4,3 (μm) protein concn (%) 0.5 1.0 2.0 4.0 6.0

water 57.3 53.2 49.4 49.7 36.3

± ± ± ± ±

3.5 2.4 1.1 1.3 1.7

d4,3 (μm) SDS

a a b b c

53.6 52.2 52.3 50.3 38.9

emulsification process III

± ± ± ± ±

6.4 1.4 1.6 1.6 1.8

FI (%) a a a b c

6.9 ± 0.6 a 2.0 (± 0.1 b

d4,3 (μm)

water 15.8 6.8 4.4 3.7 3.6

± ± ± ± ±

2.3 0.8 0.5 0.3 0.4

SDS a b c c c

8.6 3.9 3.8 3.8 3.9

± ± ± ± ±

1.9 0.2 0.3 0.2 0.3

FI (%) a b b b b

83.7 ± 2.5 a 74.4 ± 2.8 b 22.2 ± 0.6 c

water 23.2 23.1 27.2 19.7 6.1

± ± ± ± ±

2.1 1.8 1.2 1.3 0.5

SDS b b a c d

1.8 0.89 0.57 0.44 0.42

± ± ± ± ±

0.1 a 0.01 b 0.02 c 0.02 d 0.01 d

FI (%) 1189 2496 4672 4377 1352

± ± ± ± ±

81 d 224 b 137 a 258 a 102 c

The SPI nanoparticles were formed by heating a 6% (w/v) SPI dispersion at 95 °C for 15 min (and then immediately cooling it in an ice bath to room temperature), followed by addition of 300 mM NaCl. The heated SPI dispersion (containing the nanoparticles) was diluted with pH 7.0 water with the same ionic strength to the required initial c value (in the range of 0.5−4.0%, w/v). Emulsifications I−III are the same as in the caption of Figure 3. Each datum is the mean ± standard deviation of triplicate measurements on separate samples. Different online letters (a−d) represent the significant difference at the p < 0.05 level due to the difference in c (within the same column). bThe data are the same as in our previous work.12

a

order phenomenological equation: ln[(π11800 − πt)/(π11800 − π0)] = −kit, where π11800, πt, and π0 are the π values at the final time (11800 s) of each step, at any time (t), and at the initial time (t0), respectively, and ki is the first-order rate constant.32 Figure 2 B shows a typical application of the above equation to the adsorption of the nanoparticles at the interface at c = 0.5% (w/v). In general, the first slope is taken as a first-order rate constant of penetration (kP), while the next slope refers to a first-order rate constant of molecular rearrangement (kR) for the adsorbed nanoparticles. The results showed that kP and kR for the adsorbed nanoparticles were slightly affected by increasing c from 0.01% to 1.5% (w/v) ((−2.41 to −2.65) × 10−4 s−1 and (−11 to −12) × 10−4 s−1 for kP and kR, respectively; Table 1). At any c value, kR for the adsorbed nanoparticles was considerably higher than kP, indicating the

greater importance of the structural rearrangement of adsorbed nanoparticles at the interface to film formation (than their penetration). The observations suggest that, after initial adsorption, penetration at the interface and subsequent structural rearrangement of adsorbed nanoparticles seem to be slightly dependent on the applied c in the range of 0.01− 1.5% (w/v). Emulsifying Properties and Emulsion Characteristics. Emulsifying Ability or Emulsification Efficiency. The emulsifying properties of these nanoparticles, including emulsifying ability, flocculated state of a droplet in fresh emulsions, and emulsion stability against coalescence and creaming, were evaluated at varying c values of 0.5−6.0% (w/v) and at a fixed oil fraction (ϕ) of 0.2. Three emulsification processes (I−III; see section 2.3) with different levels of energy input were 2648

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to fully cover the newly created interfacial area during the emulsification. The progressive but slight decrease in d4,3 upon an increase of c from 1.0% to 4.0% (w/v), in the type III emulsions, reflects that increasing the c of the nanoparticles was favorable for the coalescence stability of newly formed oil droplets during microfluidization. Considering that the initial adsorption of the nanoparticles at the interface was highly related to their concentration, and the adsorption of these nanoparticles was very fast during the initial adsorption, especially at high c values (Figure 2), it can thus be reasonably hypothesized that the droplet size in 1% SDS of the emulsions stabilized by these nanoparticles is mainly determined by the interfacial area created during the emulsification, and increasing c may facilitate the stabilization of newly created oil droplets through an enhanced rate of adsorption during the initial adsorption. In the emulsions stabilized by bare silica, it has been observed that the interfacial area was linearly proportional to the silica content.18 Decreases in droplet size with an increase in particle concentration have been similarly observed in emulsions stabilized by modified silica nanoparticles,18,33 clay particles,34 and chitin nanocrystal particles.5 This observation is expected because more particles or nanoparticles are available to stabilize smaller oil droplets. At low c values (e.g., 0.5%, w/v) bridging flocculation and/or coalescence may take place, while, when the SPI nanoparticles are in excess of that needed to fully cover the oil droplet surface, more particles may be adsorbed to form interfacial multilayers or remain dispersed in the aqueous phase.5,33 Flocculated State of Droplets in Fresh Emulsions. In the protein-stabilized emulsion system, protein-coated oil droplets may be present in the flocculated form, subsequently affecting the coalescence and even creaming stability of the emulsions. At low c values, bridging flocculation may occur, while, at high c values, the flocculation may be induced by interdroplet attraction interactions (e.g., hydrophobic interactions) between adsorbed proteins on individual droplets. The flocculated state of oil droplets in various fresh emulsions (types I−III) stabilized by SPI nanoparticles was evaluated in terms of the flocculation index (FI, %) by comparison of d4,3 between water and 1% SDS (as the dispersant), as included in Table 2. For the type I emulsions, the droplet size distribution profiles without 1% SDS were basically the same as those with 1% SDS (Figure 3 A,A′), indicating a low extent of droplet flocculation in the fresh emulsions. However, it can be still observed that FI progressively decreased from 7.0 to nearly 0 as c increased from 0.5% to 2.0% (w/v; Table 2), suggesting complete inhibition of bridging flocculation at c values above 1.0% (w/v). The decrease in FI with c can be similarly observed in the type II emulsions, where FI progressively decreased from about 84 to near 0 as c increased from 0.5% to 4.0% (w/v; Table 2). This can also be seen from the bimodal droplet size distribution profiles (in water) of this type of emulsion (Figure 3 B), wherein increasing c led to a progressive shift of the larger size distribution peak (toward lower sizes) and, concomitantly, a progressive increase in the volume fraction of the smaller size peak. In contrast, FI at any comparable c value for the type III emulsions was considerably higher than that for the type II counterparts (Table 2), indicating a much higher extent of flocculation. For this type of emulsion, FI on the contrary progressively increased from 1189% to 4672%, as c increased from 0.5% to 2.0% (w/v); when c was above 2.0% (w/v), a contrary trend was observed (Table 2). In this case, the

applied to form the emulsions. Figure 3 shows the typical droplet size distribution profiles of these fresh emulsions, diluted in water or 1% SDS, and the corresponding volumeaverage diameter (d4,3) data are summarized in Table 2. One noteworthy point is that the data for the type I emulsions produced with only a high-shear homogenization have been reported in our previous work,12 but are still presented herein for comparison between the different emulsification processes. In general, the emulsifying ability of a protein can be reflected in terms of the droplet size of the freshly formed emulsion in the presence of 1% SDS, with smaller sizes indicative of higher emulsifying ability, since SDS can keep individual oil droplets separate in the emulsions. As expected, the droplet size distribution profiles and the d4,3 data (in 1% SDS) were highly dependent on the applied emulsification process and c (Figure 3 and Table 2), indicating that the ability of these nanoparticles to help dispersion of the oil phase into an aqueous phase (at a specific ϕ) might be closely related to the interfacial area of droplets created in the emulsification and the concentration of proteins in the continuous phase. As previously indicated, the type I emulsions exhibited a prominent size distribution peak at around 60−70 μm, with d4,3 (in water or 1% SDS) slightly decreasing with an increase in c (from 0.5% to 6.0%) (Figure 3 A,A′ and Table 2). For this type of emulsion, d4,3 (in 1% SDS; 52−53 μm) did not significantly change upon an increase of c to 2.0% (w/v), and it progressively decreased as c further increased above 2.0% (w/v; Table 2). This observation suggests that, at c values >2.0% (w/ v), the total interfacial area nonlinearly increased with c, due to enough nanoparticles being available to aid formation and stabilization of the newly created interface. The progressive increase in total interfacial area with c (above 2.0%, w/v) is very consistent with the fact that at higher c values the initial adsorption of nanoparticles at the interface was much faster (Figure 2 A), and as a consequence, more interfacial area newly created during the emulsification could be coated and stabilized. When the type I emulsions were further processed by ultrasonic equipment (II) or microfluidization (III), it was observed that, at any specific c, the size distribution profiles in the presence of 1% SDS remarkably shifted toward lower sizes, with a much higher extent of the changes observed for the type III emulsions than the type II counterparts (Figure 3 A′−C′). This can also be reflected in the changes in d4,3 data (in 1% SDS; Table 2), confirming that the emulsification efficiency considerably increased with the energy level input order of the emulsification processes: I < II < III. For the type II emulsions, the droplet size distribution profiles (in 1% SDS) were basically bimodal (Figure 3 B′), reflecting that the additional ultrasonic treatment increased the polydispersity of droplets. In this case, increasing c from 0.5% to 1.0% (w/v) resulted in a shift of the larger size peak toward lower sizes and an increased volume of the smaller peak, and concomitantly, d4,3 (in 1% SDS) considerably decreased from 8.6 to 3.9 μm, while a further increase in c to 6.0% (w/v) did not significantly change the size distribution profiles and the d4,3 data (Figure 3 B′ and Table 2). In contrast, in the case of type III emulsions, the monomodal size distribution peak (in 1% SDS) progressively shifted toward lower sizes, and d4,3 (in 1% SDS) accordingly decreased from 1.76 to about 0.4 μm, as c increased from 0.5% to 4.0% (w/v), though the magnitude of the reduction in d4,3 was relatively marginal (Figure 3 C′ and Table 2). The observations suggest that, in both the cases, the amount of nanoparticles at c = 1% (w/v) seemed to be enough 2649

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increasing extent of flocculation upon an increase in c to 2.0% (w/v) might be largely attributed to the enhanced attraction interactions between the adsorbed proteins, while the progressive decrease in FI at c values above 2.0% (w/v) could also be explained to be a result of inhibited flocculation, as in the type I or II emulsions. The flocculated state of oil droplets in these fresh emulsions can be further corroborated by their CLSM observations, as illustrated in Figure 4. For the type I emulsions, only a portion

droplets are basically consistent with the droplet size data (in 1% SDS; Figure 3 C′ and Table 2). Coalescence Stability. Extraordinary coalescence stability is one of the most striking features for Pickering emulsions.33 Table 3 shows the influence of storage after specific periods (7 and 40 days) on d4,3 (in 1% SDS) of the various fresh emulsions. In general, we can observe that the coalescence stability of the emulsions was higher at higher c values, or when a higher energy input level of emulsification was applied (Table 3). For the type I emulsions, for example, a minimal c of approximately 1% was needed to keep them stable against coalescence upon storage for 7 days, while a minimal c of 4% (w/v) was needed when the storage was extended to 40 days (Table 3). The type II emulsions, at c values >0.5% (w/v), were basically stable against coalescence upon storage for up to 40 days, though significant but marginal increases in d4,3 (in 1% SDS) were observed at c = 1.0% or 2.0% (w/v) after more than 7 days of storage (Table 3). In this case, the emulsion at c = 0.5% (w/v) was unstable against coalescence after 40 days of storage (Table 3). In contrast, all the type III emulsions exhibited extraordinary stability against coalescence upon storage for up to 40 days, even at c = 0.5% (w/v; Table 3). For particle-stabilized emulsions, the extraordinary coalescence stability can be largely attributed to the effective and irreversible adsorption of particles at the interface, because several thousand kT (k is the Boltzmann constant and T the temperature) of energy might be needed for one particle to desorb from the interface.35 Besides, the steric hindrance of the particles at the interface,5,36 as well as a network formation of flocculated particles at the interface or in the continuous phase at high solid concentrations,5,37 can also contribute to the stability of droplets against coalescence. In the present study, the particle size analyses confirmed that increasing c above 0.1% (w/v) progressively increased the flocculation or aggregation of SPI nanoparticles (Figure 1A), which suggests that the increase in coalescence stability at higher c values might be largely due to the formation of a network involving associated nanoparticles in the system. On the other hand, it should be worth noting that the SPI nanoparticles share some common features of SPI; e.g., once the nanoparticles adsorb at the interface, unfolding or deformation of their structures and subsequent structural rearrangement may occur. This situation may become more distinct at lower c values, e.g., 0.5% (w/v) in the present work. Thus, the differences in coalescence stability at low c values between the different emulsification processes might be to a certain extent attributed to the differences in viscoelasticity and/or intermolecular interactions between adsorbed proteins of interfacial protein films. This can be indirectly corroborated

Figure 4. Typical CLSM images of emulsions I−III stabilized by the SPI nanoparticle aggregates at a comparable initial c of 0.5−6.0% (w/ v). Emulsions I−III correspond to those produced by the three emulsification processes, as described in the caption of Figure 3.

of the oil droplets at c = 0.5% (w/v) were associated together, while, in the case of the type II emulsions, the microstructural morphology of oil droplets gradually changed from a clustered to a dissociated state as c increased from 0.5% to 4% (w/v; Figure 4, the top two rows). In the latter case, most of the individual oil droplets seemed to be present in the dissociated form, which is well in accordance with the droplet size data (Figure 3 A and Table 2). As compared with the type II emulsions, the extent of droplet flocculation and/or aggregation for the type III emulsions was much higher (Figure 4, middle and bottom rows), indicating a much higher interdroplet interaction of droplets. When c was high enough, e.g., 4% or above, a homogeneous network of the emulsions could be observed, which seems to be in agreement with the decrease in the extent of droplet flocculation (Table 2). On the other hand, the CLSM observations of the contour size of individual oil

Table 3. Changes of d4,3 (in 1% SDS; μm) of SPI-Nanoparticle-Stabilized Emulsions I−III Formed at Varying c Values of 0.5− 6.0% (w/v) upon Storage for up to 40 daysa emulsion I protein concn (%) 0.5 1.0 2.0 4.0 6.0

0 days 53.6 52.2 52.3 50.3 38.9

± ± ± ± ±

6.4 1.4 1.6 1.6 1.8

7 days a a a a a

64.1 54.4 49.2 51.6 38.6

± ± ± ± ±

1.1 2.6 3.3 0.4 2.3

emulsion II 40 days

b a a a a

63.5 61.8 60.8 51.0 39.4

± ± ± ± ±

0.8 4.4 2.4 2.1 3.3

0 days b b b a a

8.6 3.9 3.8 3.8 3.9

± ± ± ± ±

1.9 0.2 0.3 0.2 0.3

a a a a a

emulsion III

7 days

40 days

10.6 ± 4.3 ab 4.3 ± 0.1 b 4.3( ± 0.2 b 3.8 ± 0.3 a 3.9 ± 0.4 a

16.0 ± 3.2 b 4.3 ± 0.1 b 4.4 ± 0.5 b 3.8 ± 0.2 a 4.0 ± 0.4 a

0 days 1.8 ± 0.1 a 0.89 ± 0.01 0.57 ± 0.02 0.44 ± 0.02 0.42 ± 0.01

a a a a

7 days

40 days

2.0 ± 0.2 a 1.0 ± 0.03 ab 0.59 ± 0.01 a 0.45 ± 0.01 a 0.40 ± 0.01 a

2.0 ± 0.2 a 1.2 ± 0.2 b 0.64 ± 0.09 b 0.54 ± 0.07 a 0.44 ± 0.02 a

Emulsification processes I−III correspond to those described in the caption of Figure 3. Each datum is the mean ± standard deviation of at least two replicates on separate samples. Different online letters (a−c) represent the significant difference at the p < 0.05 level between different storage periods (0−40 days) for a specific emulsion at a specific c value. a

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concentration has been observed for the emulsions stabilized by microcrystalline cellulose and modified starch,3 chitin nanocrystal particles,5 and zein colloidal particles.7 The c dependence of creaming behavior is very consistent with the droplet size data in water (Table 2), reflecting that, within each type of emulsion, the creaming behavior of droplets might still satisfy the Stokes rule. Compared with the type II emulsions, the type III emulsions at any test c value showed much larger d4,3 in water but exhibited much higher creaming stability (Table 2 and Figure 5), implying that the creaming behavior in the latter case was predominated by other crucial factors rather than the droplet size in the system. In our previous work, the improved creaming stability for the type I emulsions at higher c or ϕ values has been attributed to the formation of a gel-like network involving server droplet flocculation.12 This explanation is distinctly applicable to the extraordinary creaming stability of the type III emulsions relative to the type II counterparts, since in the former case the droplet flocculation was much higher (Table 2 and Figure 5). The importance of the gel-like network formation to the creaming stability has also been confirmed in the emulsions stabilized by preheated whey protein or SPI19,21 and chitin nanocrystal particles.5 Rheological Properties of Gel-like Emulsions. To ascertain the formation of a gel-like network in the emulsions, we evaluated the flow behavior and dynamic viscoelastic properties of various freshly prepared emulsions. As expected, all the test emulsions (fresh) exhibited a shear-thinning behavior in the shear rate range of 0.2−100 s−1 (Figure 6), mainly reflecting deflocculation of associated droplets in the emulsions.21,38−40 For the type I emulsions, η at a specific shear rate above 1.0 s−1 gradually increased upon an increase in c from 0.5% to 6.0% (w/v) (Figure 6 A), indicating enhanced association or flocculation at higher c values. In this case, all the formed emulsions exhibited a good flow behavior with very low η values, e.g.,