Interface Adsorption Behavior of Heated Soy Proteins - American

Article pubs.acs.org/JAFC

Emulsifying Properties and Oil/Water (O/W) Interface Adsorption Behavior of Heated Soy Proteins: Effects of Heating Concentration, Homogenizer Rotating Speed, and Salt Addition Level Zhumei Cui,†,§ Yeming Chen,† Xiangzhen Kong,† Caimeng Zhang,† and Yufei Hua*,† †

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, People’s Republic of China § Department of Biology and Food Engineering, Changshu Institute of Technology, Suzhou, Jiangsu Province 215500, People’s Republic of China ABSTRACT: The adsorption of heat-denatured soy proteins at the oil/water (O/W) interface during emulsification was studied. Protein samples were prepared by heating protein solutions at concentrations of 1−5% (w/v) and were then diluted to 0.3% (w/v). The results showed that soy proteins that had been heated at higher concentrations generated smaller droplet size of emulsion. Increase in homogenizer rotating speed resulted in higher protein adsorption percentages and lower surface loads at the O/W interface. Surface loads for both unheated and heated soy proteins were linearly correlated with the unadsorbed proteins’ equilibrium concentration at various rotating speeds. With the rise in NaCl addition level, protein adsorption percentage and surface loads of emulsions increased, whereas lower droplet sizes were obtained at the ionic strength of 0.1 M. The aggregates and non-aggregates displayed different adsorption behaviors when rotating speed or NaCl concentration was varied. KEYWORDS: soybean proteins, emulsions, aggregates, adsorption, oil−water interface

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INTRODUCTION Whereas soybean contains hundreds of different proteins, soy proteins are usually referred to those storage proteins in soybean seeds, mainly composed of glycinin and β-conglycinin. Glycinin, commonly referred to as 11S, is a hexamer with a molecular mass of 300−380 kDa. β-Conglycinin, also known as 7S, is a trimeric glycoprotein with a molecular mass of 150−200 kDa.1 Soy protein isolate (SPI) is a commercial soy protein product with a protein content of >90%, which is widely used as a proteinaceous ingredient in food processing. Functionality is very crucial to SPI, and many protein modification methods have been proposed. Proteins in a commercial SPI product can rarely remain in the native state, because heat sterilization is a necessary step in SPI processing. Heated SPI has been efficiently used for the stabilization of emulsions, which can improve emulsion stability against creaming and freeze−thawing in the presence of salt.2 Soy protein aggregates, which are prepared by combined treatments of heating and electrostatic screening, were reported to exhibit potential as Pickering-type stabilizers.3 Heat treatment at 95 °C of SPI leads to an increase of surface hydrophobicity,4 resulting in smaller average oil droplet size of emulsions, higher adsorbed protein percentage, and lower protein surface load at the oil/ water (O/W) interface.5 If soy proteins were denatured under more severe conditions, such as at 120 °C, a higher surface activity and more rapid development of intermolecular interactions in the adsorbed layer than those at 90 °C could be observed.6 However, it was also reported that β-conglycinin and glycinin had the same strong adsorbing ability at the O/W interface whether SPI was heated at 95 °C or not before emulsification.1,7,8 Structural transitions associated with the © 2014 American Chemical Society

thermal treatment of soy proteins depended highly on protein concentration, temperature, and processing time.9 At low concentration, both protein subunit dissociation and protein aggregation occur rapidly upon heating of soy proteins. With the increase in protein concentration, more proteins are involved in the formation of larger aggregates, and eventually a protein gel is obtained.10,11 The adsorption behavior of protein aggregates at the O/W interface is far from been fully understood. From a kinetic point of view, the diffusion of protein to the interfacial region decreases as a result of the increase in particle size.12 However, Keerati-u-rai and Corredig1,7,8 reported that heating of soy protein solution before emulsification resulted in higher protein load at the interface. Tcholakova et al.13 considered that the excess adsorption of protein aggregates was responsible for the higher protein surface load than monolayer coverage in whey protein stabilized emulsions. They observed a sharp onset of the multilayer formation at a protein concentration of about 0.13% (w/v) and proposed that the first layer served as a substrate for the adsorption of the aggregates.13 This study was focused on the adsorption behavior of heatdenatured soy protein at the O/W interface. Soy protein solutions with different protein concentrations were heated to obtain samples with different degrees of aggregation. The heated soy proteins were characterized in terms of aggregate content, particle size, surface hydrophobicity, and interfacial Received: Revised: Accepted: Published: 1634

October 9, 2013 January 21, 2014 January 27, 2014 January 27, 2014 dx.doi.org/10.1021/jf404464z | J. Agric. Food Chem. 2014, 62, 1634−1642

Journal of Agricultural and Food Chemistry

Article

filtered through a 0.45 μm filter. The appropriate viscosity and refractive index parameters for each solution were set. DLS analysis was carried out at a fixed angle of 173° using a Zetasizer Nano-ZS instrument (Malvern Instruments, British) at 25 °C. All measurements were performed in at least duplicate at room temperature, and the results reported are averaged over three readings. Surface Hydrophobicity. Surface hydrophobicity of protein samples was determined according to the method of Wang et al.6 The aliquots (1.0 mL) of protein solutions (0.2 mg/mL) were placed in the cell of an F7000 fluorescence spectrophotometer (Hitachi Co., Japan), and aliquots (10 μL) of 1-anilino-8-naphthalenesulfonate (ANS; 5 mM in 10 mM phosphate buffer, pH 7.0) were titrated to reach a final concentration of 50 μM. The molar coefficient (5000 M−1 cm−1 at 350 nm) was used to calculate ANS concentration. The relative fluorescence intensity (F) was measured at 390 nm (excitation; slit width, 5 nm) and 470 nm (emission; slit width, 5 nm). Data were elaborated using the Lineweaver−Burk equation (eq 3)

tension. After adjustment to the same protein concentration, the heated SPI was used to prepare emulsions using a highshear homogenizer. We examined the effects of homogenizer rotating speed and ionic strength on soy protein aggregates’ adsorption. Results of the study could provide more information as to the effects of heating conditions on the emulsifying properties of soy proteins.

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MATERIALS AND METHODS

Materials. Low-denatured, defatted soy flakes were kindly provided by Shandong Gushen Industrial & Commercial Co., Ltd. (Dongying, Shandong, China). For the preparation of the emulsions, soybean oil was obtained from a local supermarket and purified with Florisil (60− 100 mesh, Sigma-Aldrich) to remove surface-active impurities. All other reagents and chemicals were of analytical grade. Preparation of Soy Proteins. The defatted soy flakes were dispersed in 15-fold distilled water and adjusted to pH 7.0 with 2 M NaOH. After stirring for 1 h, the suspension was centrifuged at 15500g for 30 min at 4 °C, and then the supernatant was subjected to isoelectric precipitation by adjusting the pH to 4.5 with 2 M HCl and centrifuged at 4 °C for 1 h. The protein precipitate was washed twice with distilled water and then adjusted to pH 7.0 by 2 M NaOH. This protein suspension was centrifuged at 15500g for 30 min at 4 °C (CR21G, Hitachi, Japan), and the supernatant was then freeze-dried and ground to yield soy proteins. Protein content of the prepared soy protein was 90.6% (w/w) as determined by using the micro-Kjeldahl method with a nitrogen conversion factor of 6.25. Native soy protein (NSP) solution with concentrations of 1, 3, and 5% (w/v) were prepared by dissolving soy proteins prepared as above in 10 mM sodium phosphate buffer and stored at 4 °C overnight. The final pH was adjusted to 7.0 by adding 1 M NaOH or 1 M HCl. The protein dispersions were filtered to remove the insoluble particles through 0.45 μm filters (Φ50 mm × 0.45 μm, low protein binding, Sinopharm Chemical Reagent Co., Ltd.), using a sand core filter device by vacuum extraction filtering method. The filtrates were heated in a 95 °C water bath for 30 min and cooled to room temperature in an ice bath to obtain H1, H3, and H5, which was heated at protein concentrations 1, 3, and 5%, respectively. The protein concentration of the final solutions was checked by using the Lowry method.14 In our experiment, high-purity native soy protein was used as a protein standard. The high-purity native soy protein was prepared in this laboratory and has a protein content of 99.0% as determined by the micro-Kjeldahl method. High-Performance Size Exclusion Chromatography (HPSEC). The molecular weight distribution was determined according to the method of Wu et al.15 Native and heated soy protein solutions were filtered through a cellulose acetate membrane with a pore size of 0.45 μm (Sartorius Co., Ltd., Gottingen, Germany). A Waters 2690 liquid chromatograph system (Waters Chromatography Division, Milford, MA, USA) equipped with a Shodex protein KW-804 column (Shodex Separation and HPLC Group, Tokyo, Japan) was used. The elution was performed with 50 mM phosphate buffer (pH 7.0, including 0.3 M NaCl) at a flow rate of 1.0 mL/min. About 20 μL of protein solutions was injected into the column, and the fluent was monitored at 280 nm. The relative amount of the aggregates was expressed as the percentage of its peak areas (PA) as related to the total area (TA) (eq 1). The content of non-aggregated fraction was calculated by taking the difference in relative protein content reading in the whole solution and the aggregates (eq 2).

Caggregates =

PA aggregates TA

× Csolution

Cnon‐aggregates = Csolution − Caggregates

1/F = 1/Fmax + (Kd /L0)(1/Fmax )

(3)

where L0 is the fluorescent probe concentration (μM), Fmax is the maximum fluorescence intensity of the ANS probe (at saturating probe concentration), and Kd is the apparent dissociation constant of a supposedly monomolecular protein/ANS complex. Fmax can be calculated by standard linear regression fitting procedures. Interfacial Tension. The interfacial tensions between soybean oil and 10 mM phosphate buffer or the protein solutions were measured according to the Wilhelmy plate method,16 using a dynamic contact angle meter and tensiometer (DCAT21, Dataphysics, Germany), at 25 °C. The aqueous phase was water or SPI solution (0.3%, w/v) in water, pH adjusted to 7.0. Emulsion Preparation. Two different series of emulsions were prepared. In the first series, the pH (7.0) and the salt addition level (0 M) were kept constant, whereas the homogenizer rotating speed was varied between 10000 and 22000 rpm. In the second series, the homogenizer rotating speed (16000 rpm) and the pH (7.0) were kept constant, whereas the salt addition level was adjusted to 0, 0.1, 0.2, 0.3, and 0.4 M, respectively. All protein samples were diluted to 0.3% (w/ v) using 10 mM sodium phosphate buffer, pH 7.0, containing 0.02% sodium azide as an antimicrobial agent. Emulsions were prepared by mixing 10% soybean oil with 90% SPI solution and emulsified using a high-shear homogenizer (FA25 model, Fluko Equipment Co., Ltd., Shanghai, China) for 2 min at room temperature. Each emulsion was prepared at least three times to check for processing repeatability and stored at 4 °C for further analysis (within 2 h). Mean Particle Size of Emulsion. The particle size distribution of the emulsion was determined using a Mastersizer 2000 laser particlesize analyzer (Malvern Instruments, Malvern, U.K.). The refractive indices of soybean oil and phosphate buffer were taken as 1.472 and 1.330, respectively; absorption index, 0.001; obscuration value, 15%. Half a milliliter of fresh emulsion was taken and diluted in 10 mL of 1% SDS solution (1% SDS in 10 mM phosphate buffer). The particle sizes measured are reported as the area-weighted mean diameter, d32. Determination of Protein Concentration at the O/W Interface. The concentration of protein adsorbed at the O/W interface was determined according to the method described by Keerati-u-rai et al.1 with some modifications. Emulsions were centrifuged twice at 15500g at 4 °C for 45 min. The serum phase (unadsorbed protein) was withdrawn carefully using a syringe, and the protein concentration was determined according to the method of Lowry et al.14 The aggregates (A) or non-aggregates (NA) remaining in serum phase were determined using high-performance size exclusion chromatography (HPSEC).15 The serum phase was concentrated by ultrafiltration tubes to about 3 mg/mL before the HPSEC experiment. The surface load (Γ) was obtained by relating the adsorbed amount to the specific surface area of the emulsion.13,17 Equation 4 was used to calculate the surface load (Γ) in this study:13

(1) (2)

Dynamic Light Scattering. Apparent hydrodynamic radius (Rh) was determined according to method of Wang et al.6 To avoid multiple particle effects, protein samples were diluted to 1.0 mg/mL with 10 mM phosphate buffer (pH 7.0), and the native proteins were

Γ (mg/m 2) = 1635

d32(C INI − CSER )(1 − Φ) 6Φ

(4)

dx.doi.org/10.1021/jf404464z | J. Agric. Food Chem. 2014, 62, 1634−1642


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CINI and CSER are the protein concentration in the initial solution (prior to emulsification) and the concentration in the aqueous phase after the centrifugation procedure, respectively. Φ is the volume fraction of oil phase. Statistical Analysis. Experiment data were analyzed by analysis of variance (ANOVA) using the SAS 9.1 package (SAS 2005). A least significant difference (LSD) test with a confidence interval of 95% was used to compare the means. All treatments were run in triplicate. Data were expressed as the mean ± SD (n = 3).

heated soy proteins. The surface hydrophobicity of all heated proteins was significantly (p < 0.05) higher than that of NSP. Several previous studies had also reported that heat-treated soy protein possessed higher surface hydrophobicity than the native protein.20,21 These changes might be induced by a modification of the native structure of soy proteins during heat treatment: more hydrophobic group clusters in the interior of molecule transferred to protein’s surface as a consequence of heatinduced protein unfolding.22 Furthermore, the heated proteins that were heated at higher concentration possessed more surface hydrophobicity. This may seem surprising because aggregation of heat-denatured proteins was thought to take place through hydrophobic interactions between nonpolar groups; thus, more aggregates mean fewer exposed hydrophobic groups. Semisotnov et al.23 pointed out that protein− ANS binding was not only affected by accessibility of hydrophobic area but also influenced by the protein structure. Most probably, soy proteins heated at higher concentrations were more “molten globule” like and had higher β-structure, both of which displayed a higher affinity to ANS. The interfacial tension of the soybean oil−phosphate buffer (10 mM) was 18.1 mN/m. When soy protein (0.3%) was added to the aqueous phase, the interfacial tension of the O/W interface substantially decreased. However, heat treatment induced only a slight decrease in interfacial tension of soy proteins (Table 1). Possibly in addition to hydrophobicity, the interfacial tension is also related to properties such as molecular size and flexibility.24,25 Adsorption of Soy Protein at the O/W Interface As Affected by Homogenizer Rotating Speed. Droplet Size Distribution of the Emulsions. As shown in Figure 2,

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RESULTS AND DISCUSSION Characterization of Soy Proteins. Heat treatment above the denaturation temperature caused protein unfolding and subsequent protein aggregation. As shown in Figure 1, native

Figure 1. HPSEC profiles of native and heat-denatured soy proteins.

soy proteins (NSP) resulted in two peaks at 9−10 and 11−12 min, corresponding to molecular weights of 150 kDa (20−630 kDa) and 14.1 kDa (2−20 kDa). Elution profiles for heated soy proteins revealed that protein aggregates’ peak emerged at about 6 min, and heating at higher protein concentration resulted in more aggregates. Protein aggregates’ content increased from 0 to 1.92 mg/mL for H5, whereas the nonaggregated protein decreased from 2.78 to 0.857 mg/mL. Ratios of non-aggregated proteins to protein aggregates were approximately 1:2, 1:1, and 2:1 for H1, H3, and H5, respectively. At the same time, Rh values also increased with the heating concentration of proteins (Table1). These findings are well in agreement with previous studies.8,18,19 Surface hydrophobicity of protein has long been used to characterize the structural changes and recognized as a related factor controlling surface activity of protein. Table 1 summarizes the surface hydrophobicity data of native and

Figure 2. Mean particle size of emulsion (μm) at various homogenizer rotating speeds and zero salt addition. Letters (a−o) following each point indicate significant differences at the 0.05 level (n = 3).

Table 1. Surface Hydrophobicity (Fmax), Interfacial Tension (σ), and Hydrodynamic Radius (Rh) of NSP or HeatDenatured Soy Proteinsa sample

Fmax

σ (mN m−1)

Rh (nm)

NSP H1 H3 H5

299.2c 680.1b 714.3b 759.2a

12.1a 11.1b 10.9b 11.7a

7.45d 14.0c 18.1b 23.0a

homogenizer rotating speed was a major factor determining the droplet size of emulsions. According to Walstra,26 the size of an emulsion’s drops depended mainly on the average power density in the emulsification chamber. This could explain why droplet sizes of emulsions stabilized by different proteins decreased in almost the same pattern. Heat treatment of soy proteins also affected droplet size of emulsions. For every rotating speed, NSP-stabilized emulsions possessed larger droplet sizes than emulsions stabilized by heated proteins. Li et al.5 reported that heated soy proteins yielded smaller droplet

a

Letters (a−d) following each value indicate significant differences at the 0.05 level (n = 3). 1636



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sizes, whereas Keerati-u-rai et al.1 found that heated soy proteins resulted in larger sized droplets. Li-Chan et al.27 considered that emulsifying and fat-binding properties of heated proteins were improved under the conditions favoring combinations of high solubility and hydrophobicity. Because surface hydrophobicity of heated soy proteins increased substantially and protein solubility remained unchanged in this experiment, it was found that heated proteins displayed better emulsifying property than NSP. This was in agreement with previous studies, which have reported that increasing the heat-induced aggregate concentration resulted in the formation of emulsions with smaller droplet size.3 The marked difference in emulsifying behavior among H1, H3, and H5 could not be solely explained by the surface hydrophobicity. Instead, a difference in the content of protein aggregates was found to parallel the changes in droplet size of emulsions (Figure 2). Different adsorption behaviors for aggregates and nonaggregates are considered in the following parts. Adsorption of Total Proteins at the O/W Interface. Protein surface loads obtained in this study (Figure 3) were much

adsorption at the O/W interface would be expected to increase at higher homogenizer rotating speed if there were enough proteins in the system. In the present systems, however, increase of rotating speed resulted in emulsions with small droplet size (larger interface area), at the expense of surface protein load. Good linear relationships were found when protein surface loads (Γ) were plotted against concentrations of unadsorbed proteins in the aqueous phase (CSER) under the respective homogenizer rotating speed (Figure 4). Thus, adsorption of soy

Figure 4. Linear relationships of protein surface loads (Γ) and unadsorbed proteins in the aqueous phase (CSER) at various homogenizer rotating speeds.

proteins at the O/W interface could be described by a simple linear equation. Tcholakova et al.13 also used an empirical equation to describe protein adsorption behavior at the O/W interface after an unsuccessful attempt of fitting data with those well-known adsorption isotherms. Unfortunately, there were no data describing adsorption behavior when the protein concentration approached 0. To simplify discussion, a linear adsorption isotherm was also assumed in this range (as represented by dashed lines in Figure 4). In the linear Henry isotherm equation (eq 5)

Figure 3. Total protein surface load (mg/m2) of emulsion at various homogenizer rotating speeds and zero salt addition. Letters (a−n) following each point indicate significant differences at the 0.05 level (n = 3).

Γ = kc

higher than the typical globular protein monolayer (about 1−3 mg/m2).16 The values of all heated samples were >9.16 mg/m2. High protein surface load values were also reported by Puppo et al.28 (>9.0 mg/m2) and Waninge et al.29 (4−5 mg/m2) on emulsifying properties of soy protein and milk protein, respectively. As shown in Figure 3, heat treatment enhanced the surface load of soy protein; the protein surface load of H5 (22.2 mg/m2) was almost doubled compared to that of H1 (13.3 mg/m2) at the homogenizer rotating speed of 10000 rpm. Nilsson and Bergenståhl30 considered that diffusional transport of polymers to the interface became less important; instead, convective transport would dominate during homogenization conditions. In the homogenization shear field, transportation to the O/W interface of large molecules was favored over small ones, leading to higher surface loads by causing jamming at the interface. Tcholakova et al.13 attributed the much higher protein surface load at the O/W interface to the presence of aggregates in their study. From Figure 3, it could also be noted that the increasing rotating speed decreased the surface protein loads of all emulsions to various degrees. The protein

(5)

The amount of surface adsorbate was proportional to the unadsorbed protein concentrations in the aqueous phase. Therefore, a two-stage Henry isotherm was proposed to describe the adsorption of soy proteins at the O/W interface. The first stage, starting from the origin and ending at the point obtained at a homogenizer rotating speed of 22000 rpm, was assumed to be the adsorption of insufficient proteins relative to large interface area. The second stage, represented by the original fitted line, was the adsorption of excess proteins relative to small interface area. The Henry isotherm constant (k) was the adsorbed protein quantity corresponding to unit concentration of unadsorbed proteins and revealed the affinity of the adsorbate to the adsorbent. For both adsorption stages, k values changed according to the order H5 > H3 > H1 > NSP, again suggesting that proteins heated at higher concentration were prone to be adsorbed at the O/W interface. If k values of different adsorption stages were compared, we could find that for heated proteins, k values of the first stage were higher than those of the second stage. For NSP, however, k values for the 1637



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Table 2. Adsorption Percentages of Aggregates (A) or Non-aggregates (NA) at the O/W Interface at Different Homogenizer Rotating Speedsa NSP

a

H1

H3

H5

homogenizer rotating speed (rpm)

NA (%)

NA (%)

A (%)

NA (%)

A (%)

NA (%)

A (%)

10000 13000 16000 19000 22000

12.1u 14.7s 19.7q 24.4o 27.4n

27.0n 29.5m 34.4k 38.02j 40.6i

17.4r 28.1m 48.3h 62.9f 72.5d

23.7o 28.0n 32.4l 37.3j 40.8i

35.5k 45.8h 68.8e 85.3b 90.4b

10.7v 13.2t 19.70 22.9p 24.4o

58.5g 67.1e 84.2c 96.4a 99.8a

Letters (a−u) following each value indicate significant differences at the 0.05 level (n = 3).

mass components in the polymer were selectively adsorbed in dynamic homogenization condition, which favored transport to the interface of the high molar mass polymers in the sample. Next, surface protein loads for aggregated and nonaggregated soy proteins were studied (Figure 5) as a function of concentration of the corresponding unadsorbed protein species in the aqueous phases (Figure 6). Intriguingly, the behavior for aggregates in both H3 and H5 resulted in downward convex curves, whereas that for non-aggregates in both H1 and H3 showed upward concave curves. The adsorption energy of the convex type adsorption isotherm decreases as surface load increases, whereas that of concave type increases as surface load increases. Thus, the adsorbed aggregates expelled the adsorbing aggregates, possibly due to the steric effects, whereas the adsorbed non-aggregates attracted the adsorbing non-aggregates, probably because of the hydrophobic interactions between unfolded polypeptides. The adsorption behavior of H1 aggregates and H5 non-aggregates was abnormal because the ΓA for H1 in the high CSER region decreased, whereas ΓN for H5 was exceptionally low. This phenomenon could be a demonstration of the interaction between two protein fractions: for H1, the adsorption of aggregates was suppressed by non-aggregates, but for H5 the reverse may be the case. Adsorption of Soy Proteins at the O/W Interface As Affected by Ionic Strength. Particle Size and Total Protein Surface Load of the Emulsions. Soy protein stabilized emulsions were prepared with different levels of NaCl addition. Variation in oil droplet sizes and protein surface loads for heated and unheated soy protein stabilized emulsions followed the same pattern with the increase of NaCl concentration, although the effects differed quantitatively. At low salt concentration ( H3 > H1). However, non-aggregates formed by heating in lower concentrations were more extensively adsorbed by the O/W interface (H1 > H3 > H5). Higher adsorption percentage for aggregates than for nonaggregates could be related to their difference in structure. As revealed in previous studies, heat-induced aggregation of soy proteins was a consequence of several steps, including dissociation, unfolding, reassociation, and aggregation.10 Unfolding of heat-dissociated polypeptides from glycinin and conglycinin resulted in the loss of α-helical structure, whereas much of the β-sheet structure remained. Because the transition of structure and the exposure extent of hydrophobic sites for the protein molecules were closely related, the surface hydrophobicity increased.10 Specific associations between basic polypeptides or between basic polypeptides and βsubunits through disulfide bonds or noncovalent bonds have been reported.9 When soy proteins were heated at gradually increased concentrations, aggregation of unfolded polypeptides and reassociated oligomers would have more chances to occur and, as a result, those proteins with more hydrophilic nature would be left as non-aggregates. In addition to hydrophobicity, electric charge frequency and molecular flexibility31 were important for protein function at an interface. With the increase of homogenizer rotating speed, adsorption percentages for both aggregated and non-aggregated proteins rose. For aggregated proteins of H5, adsorption percentage increased from 58.5% at 10000 rpm to nearly 100% at 22000 rpm. However, under no circumstance did adsorption percentages of non-aggregated proteins exceed 40.8%. The low adsorption percentages suggested the low affinity of nonaggregated proteins toward the oil phase. On the other hand, the adsorption of larger sized protein aggregates was limited by the diffusion rate at low rotating speed, but effectively enhanced at higher speed. Nilsson et al.32 reported that the high molar 1638



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Figure 6. Relationships of surface load for non-aggregates (ΓN) or aggregates (ΓA) of heated proteins and the respective unadsorbed protein concentrations in aqueous phases (CSER): (A) non-aggregates; (B) aggregates.

concentration of NaCl and facilitated the adsorption of soy proteins at the interface. Adsorption of Aggregated and Non-aggregated Proteins at the O/W Interface at Different Ionic Strengths. Addition of salt was very effective in enhancing the adsorption of aggregated proteins. In fact, >95% of aggregated proteins were found to transfer to the O/W interface at a salt concentration of ≥0.2 M. Adsorptions of non-aggregated proteins as well as the unheated soy proteins displayed similar increasing trends, but the maximum adsorption percentage was much lower than that of aggregates. In contrast to the effect of homogenizer rotating speed, salt enhanced adsorption percentages of non-aggregates of H5 more effectively than other proteins (Table 3). It seems that adsorption of non-aggregates of H5 was more severely hindered by electrical repulsion, and the screening of electric charges on proteins resulted in a more remarkable increase in adsorption percentage. It was clear that aggregate/non-aggregate profiles on the O/W interfaces did not change substantially as a result of increasing salt concentration (Figure 8). With the elevation of salt concentration, aggregates in H5 remained as predominantly adsorbed components but both protein fractions contributed to the adsorbed layer for H1- and H3-stabilized emulsions. On the other hand, increase in NaCl concentration effected a marked decrease of ΓA/ΓN for H5 emulsions, from 8.21 to 3.66, whereas ΓA/ΓN was observed to increase from 0.76 to 1.17 for H1-stabilized emulsion.

Figure 5. Protein surface load of aggregates and non-aggregates in emulsions at various homogenizer rotating speeds. Letters (a−i) above each column indicate significant differences at the 0.05 level (n = 3).

encouraged unfolding of protein molecules and subsequent increase in protein solubility. Lawal et al.39 considered that such an increase in protein solubility enhanced a rapid migration to the O/W interface and improved the emulsifying activity of the protein. With further increase in ionic strength (>0.1 M), screening of the surface charges increased and this encouraged protein−protein interaction but reduced protein−oil interaction. Protein surface loads were also influenced markedly by increasing ionic strength (Figure 7B). More proteins, whether heated or unheated, were found to be adsorbed at the O/W interface with increasing of NaCl concentration. Tcholakova et al.35 also found that the protein surface loads of emulsions stabilized by β-lactoglobulin (0.02 or 0.1%, w/v) increased with increasing NaCl concentration (5−1000 mM, pH 6.2.) In this study, the enhanced Γ was probably due to the electrostatic screening, which reduced electrostatic repulsion with increasing 1639



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Figure 7. Mean particle size of emulsion (μm) at various ionic strengths and constant homogenizer rotating speed of 16000 rpm (A); total protein surface load (mg/m2) of emulsion at various ionic strengths and constant homogenizer rotating speed of 16000 rpm (B). Letters (a−j) following each point indicate significant differences at the 0.05 level (n = 3).

In summary, the present study showed that not only the heat treatment but also the heating concentration affected the emulsifying properties of soy proteins. Better emulsifying properties, as judged from droplet size, adsorption percentage, and surface load, could be obtained by heating soy proteins at higher concentrations. Levels of protein aggregates in heated samples were more important than other variables in affecting adsorption properties of proteins at the O/W interface. Adsorption properties of total proteins at the O/W interface as a function of homogenizer rotating speed or salt concentration seem to follow a similar trend, regardless of the use or not of heating and the heating concentrations. However, if aggregated and non-aggregated proteins were

Figure 8. Protein surface load of aggregates and non-aggregates in emulsions at various ionic strengths and constant homogenizer rotating speed of 16000 rpm. Letters (a−j) above each column indicate significant differences at the 0.05 level (n = 3).

Table 3. Adsorption Percentages of Aggregates (A) or Non-aggregates (NA) at the O/W Interface at Different Ionic Strengthsa NSP

a

H1

H3

H5

ionic strength (M)

NA (%)

NA (%)

A (%)

NA (%)

A (%)

NA (%)

A (%)

0 0.1 0.2 0.3 0.4

19.7l 37.5i 46.1g 47.2g 47.4 g

33.7j 43.2h 50.9f 51.9f 52.5f

48.3g 87.0c 96.3a 96.2a 96.8a

34.7j 53.3f 66.6d 66.6d 65.9d

69.1d 91.8b 95.0a 96.1a 96.8a

23.9k 39.0i 61.1e 63.6e 64.0e

86.7c 99.6a 99.8a 99.9a 99.9a

Letters (a−l) following each value indicate significant differences at the 0.05 level (n = 3). 1640



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considered separately, different adsorption behaviors that depended on heating concentrations could be observed. Thus, the adsorption of soy proteins at the O/W interface could not be considered as the sum of the independent adsorption of aggregates and non-aggregates.

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AUTHOR INFORMATION

Corresponding Author

*Postal address: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu Province, PR China. Phone/fax: 051085329091. E-mail: [email protected]. Funding

We gratefully acknowledge financial support received from the National High Technology Research and Development Program of China (2013AA102204-3) and the Key Project in the National Science and Technology Pillar Program during the Eleventh Five-year Plan Period (2012BAD34B04-1). Notes

The authors declare no competing financial interest.

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Interface Adsorption Behavior of Heated Soy Proteins - American

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