Effect of oxidation on the emulsifying properties of soy protein isolate

Food Research International 52 (2013) 26–32

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Effect of oxidation on the emulsifying properties of soy protein isolate Nannan Chen a, Mouming Zhao a, b, Weizheng Sun a,⁎, Jiaoyan Ren a, Chun Cui a a b

College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 19 November 2012 Accepted 18 February 2013 Keywords: Soy protein isolate Protein oxidation Emulsifying properties Structural properties AAPH

a b s t r a c t Soy protein isolate (SPI) was oxidized by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and the structural and emulsifying properties of oxidized SPI were evaluated. Increasing extent of oxidation resulted in gradual carbonyl group generation, free sulfhydryl group degradation and dityrosine formation. Moderate oxidization could generate soluble protein aggregates with more flexible structure while over-oxidization would induce the formation of insoluble aggregates. Compared with the control, emulsions stabilized by moderately oxidized SPI had smaller droplet size and better thermal stability. Results from creaming index and microstructure measurement after 15 days indicated that emulsions stabilized by SPI of over-oxidation underwent severe droplet aggregation during storage while moderate oxidation had a positive effect on the emulsion stability. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Soy protein is an important food ingredient used in a lot of protein-based food formulations, since it possesses high nutrition and good ability to improve the food quality (Tang, Wang, Yang, & Li, 2009). Soy protein isolate (SPI) is the most refined commercially available soy protein powder, containing 90% protein on a moisture-free basis, and possesses some desirable functionalities (Morr, 1990). One of the most important applications of SPI is being used as an emulsifier in the manufacture of food, such as meat products (Chen, Chen, Ren, & Zhao, 2011). As an emulsifier, SPI can form and stabilize small droplets. When proteins adsorb to oil–water interface in emulsion, they reduce the interfacial tension and therefore promote droplet generation and form an interfacial layer which stabilizes droplets against flocculation or coalescence via electrostatic repulsion (Walstra, 1993). The emulsifying capability of proteins depends on their molecular structure and physicochemical characteristics (Keerati-u-rai, Miriani, Iametti, Bonomi, & Corredig, 2012). Under quiescent conditions, the most obvious manifestation of instability is creaming (visible separation of bulk dispersed phase), and the rate of creaming is very sensitive to droplet-size distribution. Stability against creaming is usually correlated with coalescence and flocculation of the droplets (Dickinson, 2001). Environment conditions like pH and temperature also affect the emulsion stability (McClements, 2004). Soy protein, similar to other components such as lipids and pigments, is vulnerable to oxidative attack during processing and storage (Harel & Kanner, 1985). Protein oxidation is the structural modification induced ⁎ Corresponding author. Tel./fax: +86 20 22236089. E-mail address: [email protected] (W. Sun). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.02.028

directly by reactive oxygen species or indirectly by reaction with the by-products of lipid peroxidation (Shacter, 2000). Oxidative modification can trigger a number of changes in amino acid residue side-chains and protein polypeptide backbone, resulting in protein fragmentation, cross-linking, unfolding, and conformational changes (Davies, 2005; Stadtman & Berlett, 1998). Radical oxidant will attack almost all kinds of amino acid side chains while aromatic and sulfur-containing amino acid side chains are particularly vulnerable to oxidation (Davies, 2005). Currently, oxidation that happens in animal meat during storage have been extensively investigated. And it turns out that oxidation has both positive and negative influences on the meat quality (Rowe, Maddock, Lonergan, & Huff-Lonergan, 2004; Santé-Lhoutellier, Engel, Aubry, & Gatellier, 2008). Some industry processes will also induce protein oxidation. Researchers have found that heat-induced oxidation would reduce SPI's nutritional qualities (Tang, Wu, Le, & Shi, 2012) and protein oxidation accelerated during the first stage production of Parma Ham (Koutina, Jongberg, & Skibsted, 2012). As SPI is widely used in many lipid-enriched food systems, the effect of lipid on the SPI has aroused scientific attention. Lipid is sensitive to oxidation and will produce lipid peroxidation-derived free radicals as well as lipid hydroperoxides and reactive aldehydes which will interact with protein, leading to protein oxidation (Refsgaard, Tsai, & Stadtman, 2000). Besides, the remaining lipoxygenase in soy protein would catalyze the lipid peroxidation (Wu, Wu, & Hua, 2010). Recently, some researchers had mimicked various kinds of lipid peroxidation-derived by-products and other free radical-generating system such as FeCl3/H2O2/ascorbate to trigger protein oxidation and found that oxidation had either positive or negative effects on the gelling properties of soy protein depending on different oxidants (Liu, Xiong, & Butterfield, 2000; Wu, Hua, Lin, & Xiao, 2011). Other investigators have confirmed that lipid peroxidation happened during the storage of emulsion and its by-products may cause

N. Chen et al. / Food Research International 52 (2013) 26–32

the protein oxidation (Estévez, Kylli, Puolanne, Kivikari, & Heinonen, 2008; Hu, Mcclements, & Decker, 2003). Despite these prior studies, the relationship between emulsifying properties and the structural characteristics of oxidized SPI is rarely investigated. This relationship is expected to give guidance to better use of SPI in industry process and its storage. Thermal decomposition of 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) can generate peroxyl radicals at a known and constant rate under the stable temperature of 37 °C. The amount of peroxyl radicals from thermal decomposition of AAPH is proportional to its concentration (Gieseg, Duggan, & Gebicki, 2000). Therefore, AAPH-derived peroxyl radicals were selected as a representative by-product of lipid peroxidation in this study to evaluate the effect of oxidation on the emulsifying properties of SPI. 2. Materials and methods 2.1. Samples and materials Defatted soy flakes were purchased from Yuwang Group (Shangdong, China). 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical reagent grade. 2.2. SPI preparation Defatted soy flakes powder (200 g) was mixed with 15-fold deionized water, and the mixture (pH 6.7) was adjusted to pH 7.5 with 2.0 M NaOH. After stirring for 2 h, the resulting suspension was centrifuged at 8000 g for 20 min at 4 °C to remove the insoluble material. Then the pH of the supernatant was adjusted to pH 4.5 with 2.0 M HCl, and the precipitate was collected by centrifugation at 8000 g, for 10 min at 4 °C. The precipitate was then redissolved in 5-fold deionized water and the pH was adjusted to 7.0 with 2.0 M NaOH. The neutral SPI solution was frozen at −18 °C, and then freeze-dried using a freeze drier (Marin Christ, Germany). The freeze-dried samples were then sealed in polyethylene bags and stored at 4 °C and 40% RH until use. 2.3. SPI oxidation Oxidized SPI was prepared according to the method described by Wu, Zhang, Kong, and Hua (2009). SPI dispersion (40 mg/mL containing 0.5 mg/mL sodium azide, suspended in 10 mM sodium phosphate buffer, pH 7.4) was mixed with a serial concentration of AAPH and then incubated by continuous shaking under air at 37 °C in dark for 24 h. The final concentration of AAPH was 0, 0.05, 0.2, 0.5, 1, 3, and 5 mM. The reaction was stopped by immediately cooling the solution to 4 °C by ice-bathing and then centrifuged at 8000 g for 15 min at 4 °C. The supernatant was dialyzed against deionized water at 4 °C for 72 h to remove residual AAPH and salt. The dialysis membrane has a molecular cut off of 14,000 Da (Juyuang Biota Technology Co., Ltd., Shanghai, China). The dialyzed supernatant was frozen at −18 °C, and then freeze-dried using a freeze drier (Marin Christ, Germany). The freeze-dried samples were sealed in polyethylene bags and stored at 4 °C and 40% RH until use. 2.4. Emulsion preparation and heat treatment The SPI dispersion (20 mg/mL) was prepared in sodium phosphate buffer (10 mM, pH 7.0). Sodium azide was added to the dispersion as an antimicrobial agent with a final concentration of 0.2 mg/mL. Oil-in-water emulsions containing 20% (v/v) corn oil was then prepared at ambient temperature using an APV-1000 homogenizer (APV Gaulin, Abvertslund, Denmark) operating at 30 MPa. Heat treatment involved incubating the emulsion in a water bath set to the desired temperature ranging from 30 °C to 90 °C for 20 min. Heat treatment at 120 °C was

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placing the emulsion in an autoclave (Zhejiang, China) for 20 min. This approach is widely used when studying the thermal stability of emulsion because it mimics the thermal history that such emulsions undergo during processing.

2.5. Protein carbonyl group measurement Protein carbonyl groups in native and oxidized SPI were quantified according to the method described by Huang, Hua, and Qiu (2006) using SP-721 UV spectrophotometer (Shanghai, China). SPI samples were suspended in deionized water with a concentration of 5 mg/mL, stirring for 30 min at room temperature (25 ± 2 °C). In 15-ml capped polyethylene centrifuge tubes, 1 mL SPI dispersion was mixed with 3 mL 10 mM 2,4-dinitrophenylhydrazine(DNPH) dissolved in 2 M HCl and incubated at room temperature for 2 h. A matching aliquot was mixed in 3 mL 2 M HCl as an absorbance blank. Then 4 mL 20% trichloroacetic acid was added to each tube and blended. The mixture was centrifuged at 10,000 g for 10 min at 4 °C, after standing for 20 min. The supernatant was discarded, and the pellet was washed three times with 5 mL ethanol/ethyl acetate solution (1:1, v/v). The protein, free of DNPH, was then dissolved in 3 mL 6 M guanidine hydrochloride in 0.1 M sodium phosphate buffer (pH 7.0). The absorbance at 367 nm was corrected by the absorbance in the HCl blank. Soluble protein concentration in guanidine hydrochloride solution was evaluated by the Biuret method (Chang, 2010) with bovine serum albumin (BSA) (Dingguo Biota Technology Co., Ltd., Beijing, China) as the standard. The results were expressed as nmole of carbonyl groups per milligram of soluble protein with molar extinction coefficient of 22,000 M −1 cm −1.

2.6. Free sulfhydryl group measurement Contents of free sulfhydryl (free SH) groups in native and oxidized SPI were determined using Beveridge's procedure (Beveridge, Toma, & Nakai, 1974). SPI samples were dissolved with 8 M urea in Trs-Gly buffer (pH 8.0) with a concentration of 5 mg/mL and the mixture was stirred at room temperature for 30 min. The suspension was then centrifuged under 10,000 g for 15 min at 4 °C and soluble protein concentration in the supernatant was evaluated by the Biuret method (Chang, 2010) with bovine serum albumin(BSA) (Dingguo Biota Technology Co., Ltd., Beijing, China) as the standard. Then supernatant (3 mL) reacted with 0.02 ml DTNB reagent (4 mg/mL) dissolved in Tris-Gly buffer. After standing at room temperature for 1 h, the absorbance was measured at 412 nm using SP-721 UV spectrophotometer (Shanghai, China). The results were expressed as nmole of SH per milligram of soluble protein with molar extinction coefficient of 13,600 M−1 cm−1.

2.7. Dityrosine measurement Dityrosine formation of native and oxidize SPI was estimated using the method of Morzel, Gatellier, Sayd, Renerre, and Laville (2006) with slight modifications. SPI dispersion (2 mg/mL) was prepared in 10 mM sodium phosphate buffer (pH 7.0). The mixture was then centrifuged at 10,000 g for 10 min at 4 °C. Fluorescent intensity of the supernatant was estimated at 420 nm using F7000 fluorescence spectrophotometer (Hitachi Co., Japan), with an excitation wavelength of 325 nm and constant slit of 5 nm for both excitation and emission. Soluble protein concentration in the supernatant was evaluated by the Biuret method (Chang, 2010) with bovine serum albumin (BSA) (Dingguo Biota Technology Co., Ltd., Beijing, China) as the standard. Corrected fluorescence was obtained by dividing measured fluorescence by protein concentration (mg/mL). The results were expressed in arbitrary units (A.U.).

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2.8. Particle size distribution of SPI Particle size distribution was measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcestershire, United Kingdom). SPI was suspended in 10 mM sodium phosphate buffer (pH 7.0) at a concentration of 0.2 mg/mL and magnetically stirred thoroughly. The suspension was centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant was filtered through cellulose acetate membranes with pore size of 0.45 μm to remove any other insoluble particles. Then 0.8 mL protein solution was transferred to a square cuvette for DLS measurement. The particle size distribution was analyzed by Dispersion Technology Software (DTS) version 4.20 supplied by the manufacturer (Malvern Instruments Ltd.). Results were expressed as volume percentage (%) versus particle size diameter (nm).

characterized by the creaming index (CI, %): CI = (HS / HE) × 100%, where HS is the height of serum layer, and HE is the total height of the emulsion. 2.14. Microstructure of emulsions Images of the emulsions were observed on the 15th day after emulsion preparation using a CX31-12C04 microscope (Olympus Co., Tokyo, Japan) according to the method described by Sun, Zhou, Sun, and Zhao (2012). Samples of the emulsion were taken from the emulsion as a whole after carefully blending. A drop of the non-diluted emulsion was placed on a microscope slide which was then covered with a coverslip, and visualized at a magnification of 400×. The images were captured by a charge coupled device camera (Olympus Co., Tokyo, Japan) connected to the microscope, and the photographs of the emulsions were recorded for analysis.

2.9. Turbidity measurement 2.15. Statistical analysis Turbidity was determined according to the method of Huang et al. (2006). SPI was dissolved in 10 mM sodium phosphate buffer at pH 7.0 at a concentration of 1% (w/v) and stirred magnetically at room temperature for 60 min. Absorbance of the dispersions was determined at 600 nm. 2.10. Intrinsic fluorescence emission spectra Intrinsic fluorescence emission spectra of protein samples were determined in the F7000 fluorescence spectrophotometer (Hitachi Co., Japan). Protein solutions (0.2 mg/mL) were prepared in 10 mM phosphate buffer (pH 7.0). To minimize the contribution of tyrosyl residues to the emission spectra, protein solutions were excited at 290 nm, and emission spectra were recorded from 300 to 400 nm at a constant slit of 5 nm for both excitation and emission. The maximum fluorescence emission wavelength (λmax) of the spectra was recorded for analysis. 2.11. Determination of mean droplet size of emulsion Droplet size distribution (individual droplets or droplet aggregates) of emulsion samples was determined within 12 h after preparation using a Malvern Mastersizer 2000 (Malvern Instruments, Worcestershire, United Kingdom). The mean droplet size was characterized in terms of the volume mean diameter (d4,3) and surface area mean diameter (d3,2). The d4,3 and d3,2 calculations were done by using the Malvern Mastersizer 2000 software version 5.60 supplied by the manufacturer (Malvern Instruments Ltd.). 2.12. Emulsion ζ-potential measurements The ζ-potential in the emulsion was measured within 12 h after preparation using the Zetasizer Nano-ZS instrument. The emulsion was diluted (50 μL in 5 mL of 10 mM sodium phosphate buffer, pH 7.0) and injected into a folded capillary cell with electrodes at either end to which a potential was applied. The cell was placed in a cell chamber controlled at 25 °C, and the velocity of the charged droplet movement toward the oppositely charged electrode was measured. The ζ-potential was then calculated from the droplet velocity using Dispersion Technology Software (DTS) version 4.20. 2.13. Visual assessment of creaming Stability against creaming was determined according to the method of Chen et al. (2011). Emulsion samples were poured into 10 mL glass tubes immediately after preparation. Subsequently, the tubes were sealed to prevent evaporation. The emulsion samples were stored quiescently at ambient temperature for 15 days. The extent of creaming was

Statistical calculations were performed using the statistical package SPSS 11.5 (SPSS Inc., Chicago, IL, USA) for one-way ANOVA. Leastsquares difference was used for comparison of mean values among treatments and to identify significant differences (p b 0.05) among treatments. All the data were expressed as means ± standard deviations of triplicate determinations. 3. Results and discussion 3.1. Characterization of oxidized SPI Carbonylation is an irreversible and non-enzymatic modification of proteins that involves the formation of carbonyl moieties induced under oxidative stress (Stadtman & Berlett, 1998). Total protein carbonyl group content is a routine indicator used to evaluate the protein oxidation. Effect of AAPH on total carbonyl group content of SPI is given in Table 1. The carbonyl group level of SPI (control) was 5.78 nmol/mg soluble protein. This value was close to those reported by Liu et al. (2000), but higher than that observed by Wu et al. (2009). No significant difference (p > 0.05) of carbonyl group content was observed until AAPH concentration reached 1 mM. Enhancing oxidation extent at 3 and 5 mM AAPH leads to further significant increase (p b 0.05) of carbonyl group content. Carbonylation was promoted by the reaction of peroxyl radicals with protein backbone and vulnerable amino acid side chain, and yielded proteinperoxyl radicals under aerobic conditions (Davies, 2005). Some of the proteinperoxyl radicals then evolved into carbonyl derivatives in the subsequent reactions (Headlam & Davies, 2004). Oxidation can alter the secondary and tertiary structures of proteins, leading to changes in physicochemical and functional properties of proteins (Wu et al., 2009). Protein oxidation is also accompanied by a decrease in free sulfhydryl groups, which can be oxidized to reversible form (protein disulphide

Table 1 Protein carbonyl groups, free sulfhydryl groups, and dityrosine content in soluble protein of SPI incubated with increasing concentration of AAPH for 24 h at 37 °C. AAPH (mM)

Carbonyl (nmol/mg)

Free SH (nmol/mg)

Dityrosine (A.U.)

0 0.05 0.2 0.5 1 3 5

5.78 5.75 5.73 5.75 5.89 6.02 6.04

3.08 2.84 2.87 2.70 2.34 1.87 1.57

100.40 98.045 99.44 103.65 104.55 117.20 122.95

± ± ± ± ± ± ±

0.04a 0.06a 0.02a 0.02a 0.02b 0.04c 0.06c

± ± ± ± ± ± ±

0.07a 0.02b 0.01b 0.02c 0.03d 0.01e 0.02f

± ± ± ± ± ± ±

0.42a 0.02a 0.06a 0.07b 0.49b 0.57c 0.21d

Least-squares difference was used for comparison of mean values among treatments, and to identify significant differences. Values in the same column followed by different letters (a–f) are significant difference (p b 0.05).

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and sulfenic acid) or irreversible form (sulfinic and sulfonic acids) in different oxidative environments (Eaton, 2006). Increasing concentration of AAPH resulted in a significant decline (p b 0.05) in free sulfhydryl groups of oxidized SPI (Table 1). It was noteworthy that incubation with 5 mM AAPH resulted in 49% loss of free sulfhydryl groups which may be that sulfur-containing amino acid side chains of Cys and Met were particularly sensitive to oxidation (Davies, 2005). Sulfhydryl degradation is obvious in many oxidation systems (Morzel et al., 2006; Wu et al., 2009). Cysteine residues and disulphide bonds had important influence on the structure of proteins (Visschers & De Jongh, 2005). Therefore, decline in free sulfhydryl groups could induce structural changes of oxidized SPI. Dityrosine is formed from the oxidative C\C coupling of two L-tyrosine molecules. It is a fluorescent bulky amino acid, and can be used as a useful marker for protein oxidation (Kim, Lee, Lee, & Ahn, 2012). Morzel et al. (2006) reported a sudden increase of dityrosine formation when chicken myofibrils were exposed to 1 mM oxidizing agent. In this work, no noticeable change of dityrosine was observed at first (Table 1). Significant increase (p b 0.05) of dityrosine formation occurred when SPI was exposed to 0.5, 1, 3 and 5 mM AAPH, reaching the maximum of 122.95 A.U. Dityrosine could be produced by the reaction of two tyrosyl radicals belonging to either two different or the same protein chain and lead to the cross-linking of proteins intra- or inter molecularly (Saeed, Gillies, Wagner, & Howell, 2006). Therefore, it could modify the protein structure and influence its functionality.

3.2. Oxidation aggregation of SPI Dynamic light scattering (DLS) as a quantitative technique has been proven to be a sensitive and powerful method in monitoring the formation of aggregates. Particle size distributions of native and oxidized SPI are shown in Fig. 1A. SPI gradually shifted to larger molecule within AAPH ≤ 1 mM while at the concentration between 3 and 5 mM, the particle size became smaller than the control (0 mM). In this method, only soluble component of sample was investigated. Results indicated that SPI incubated with low concentration of oxidant reagent (AAPH ≤ 1 mM) induced the formation of soluble aggregates. Further increasing extend of oxidation could promote insoluble component formation and cleavage of peptide bonds by peroxyl radicals (Davies, 2005). Therefore, at 3 and 5 mM AAPH, some larger soluble aggregates broke into smaller soluble peptides while some shifted to insoluble components through covalent and non-covalent interaction that were removed by centrifugation. This result was in agreement with the previous studies (Huang et al., 2006; Wu et al., 2011). The difference in soluble particle size might contribute to different functionalities, and some larger protein soluble aggregates might be more suitable for stabilization at fluid interfaces such as water/oil than the smaller one (Schmitt, Bovay, Rouvet, Shojaei-Rami, & Kolodziejczyk, 2007). Turbidity of the native and oxidized SPI dispersions was evaluated to further elaborate the oxidation aggregation of protein. Turbidity arises mainly from the change in mass and size of aggregates in the solution (Cromwell, Hilario, & Jacobson, 2006). As shown in Fig. 1B, turbidity first remarkably decreased with slight increase between 0.2 and 0.5 mM AAPH, and then increased at 3 and 5 mM AAPH. The initial decreasing turbidity is due to the soluble protein aggregates formation from some insoluble aggregates in the control. The slight increase at 0.2 and 0.5 mM AAPH might arise from the larger soluble aggregates formed. At 3 and 5 mM some soluble protein aggregates evolve into insoluble aggregates leading to the increase of turbidity due to their larger mass and size. At 5 mM, the turbidity was coincidently similar to the control, which may be due to the combining effects of soluble and insoluble aggregates proportion in both suspensions. This confirmed the results of DLS and was in agreement with previous research (Huang et al., 2006).

Fig. 1. Particle size distribution (A) and turbidity (B) change of SPI incubated with increasing concentration of AAPH for 24 h at 37 °C. Least-squares difference was used for comparison of mean values among treatments in panel B, and to identify significant differences. Columns with different letters (a–d) are significant difference (p b 0.05).

3.3. Intrinsic fluorescence emission spectra Tryptophan residues are able to emit fluorescence in the range 300–400 nm when excited at 290 nm. Tryptophan maximum fluorescence emission wavelength (λmax) is an indicator of changes in protein tertiary structure because it denotes the relative position of the tryptophan residues within proteins (Keerati-u-rai et al., 2012). As shown in Fig. 2, λmax increased gradually from 332.0 to 333.4 nm between 0 and 1 mM AAPH, and then decreased to 332.6 and 331.7 nm respectively at 3 and 5 mM AAPH. Shifts of fluorescence emission to longer wavelengths (red shift) indicate exposure of tryptophan residues to a hydrophilic

Fig. 2. Maximum fluorescence emission wavelength (λmax) of SPI incubated with increasing concentration of AAPH for 24 h at 37 °C. Least-squares difference was used for comparison of mean values among treatments, and to identify significant differences. Columns with different letters (a–d) are significant difference (p b 0.05).

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Table 2 Effect of temperature on the mean droplet size of emulsion stabilized by SPI incubated with increasing concentration of AAPH for 24 h at 37 °C. AAPH (mM)

d4,3 (μm)

d3,2 (μm)

30 °C 0 0.05 0.2 0.5 1 3 5

4.73 3.49 1.96 1.63 1.53 5.46 4.15

± ± ± ± ± ± ±

60 °C 0.07 0.03 0.03 0.00 0.00 0.04 0.06

a,w b,w c,w d,w e,w f,w g,w

4.87 3.63 2.21 1.60 1.52 5.96 6.57

± ± ± ± ± ± ±

90 °C 0.09 0.04 0.02 0.01 0.02 0.10 0.13

a,x b,w c,x d,x e,w f,w g,x

5.92 4.54 2.53 1.66 1.45 9.96 6.23

± ± ± ± ± ± ±

120 °C 0.07 0.03 0.01 0.01 0.02 0.92 1.74

a,y b,wx c,y d,y e,w f,x g,x

4.79 5.04 2.07 1.52 1.84 16.90 4.87

± ± ± ± ± ± ±

30 °C 0.05 1.12 0.12 0.01 0.25 1.33 0.03

a,wx b,wx c,w d,z e,x f,y g,wx

1.79 1.51 0.62 0.49 0.49 1.91 1.50

± ± ± ± ± ± ±

60 °C 0.01 a,w 0.01 b,w 0.02 c,w 0.01 d,w 0.01 d,w 0.01 e,w 0.06 b,w

1.82 1.49 1.04 0.49 0.48 2.02 1.99

± ± ± ± ± ± ±

90 °C 0.01 0.00 0.03 0.00 0.00 0.01 0.01

a,x b,w c,xy d,x d,x e,x f,x

1.96 1.73 1.18 0.48 0.47 2.01 1.54

± ± ± ± ± ± ±

120 °C 0.01 0.00 0.00 0.00 0.00 0.01 0.01

a,y b,x c,y d,y d,y e,x f,y

1.79 1.70 0.86 0.46 0.45 1.93 1.69

± ± ± ± ± ± ±

0.01 0.02 0.22 0.00 0.01 0.04 0.00

a,w a,x b,x c,z c,z e,w a,z

Least-squares difference was used for comparison of mean values among treatments, and to identify significant differences. Values in the same column followed by different letters (a–g) and in the same row followed by different letters (w–z) are significant difference (p b 0.05).

environment while shorter wavelength (blue shift) to a hydrophobic environment (Keerati-u-rai et al., 2012). Both red shift and blue shift of λmax in tryptophan fluorescent emission spectrum have been observed on oxidized protein in previous researches (Viljanen, Kivikari, & Marina, 2004; Wu et al., 2009). In this work, under 1 mM AAPH, the protein gradually exposed the tryptophan residues to the protein surface indicated by increasing λmax. As a result of oxidation and protein denaturation, there was an increase in structural flexibility and some initially protected backbone amide groups were exposed (Saeed et al., 2006). Therefore, with further increased oxidation extent, some other hydrophobic groups might also expose to the hydrophilic environment, which might finally contribute to formation of protein aggregates due to the hydrophobic interaction. And the tryptophan residues were buried into the inner hydrophobic environment of the oxidized SPI again, resulting in decreased λmax.

have effects on their structure rearrangement on the oil–water interface, resulting in their different emulsifying capabilities. ζ-Potential as an indicator of the surface charge property of particle in solution is another important index of the emulsion properties. Slight increase of surface charge was observed at 0.5 mM AAPH pretreated sample (Fig. 3A). Between 3.0 and 5.0 mM AAPH, surface charge showed a significant decline (p b 0.05). Higher surface charge would enhance the electrostatic repulsive force between emulsion droplets to overcome the attractive interactions like Van der Waals and hydrophobic attraction (Kim, Decker, & McClements, 2002). Surface charge was greatly affected by the amino acid residue (Spector et al., 2000). The exposure of previously buried amino acid and the disturbance of acid /basic amino acid proportion caused by selective oxidation on vulnerable amino acid might be the reason of the change of protein surface charge.

3.4. Emulsifying capabilities Emulsifying capability refers to the ability of an emulsifier to form and stabilize small droplets. Generally, emulsifying capability is related to the mean droplet size d4,3 and d3,2. Low d4,3 and d3,2 values signify high emulsifying activity (Chen et al., 2011). The emulsifying activity of SPI and emulsion's stability against heating was therefore assessed by measuring the mean droplet size in response to different temperatures ranging from 30 to 120 °C. As shown in Table 2, at 30 °C, with the increase of oxidizing agent from 0 to 1 mM, the d4,3, and d3,2 values of emulsion showed a significant decrease. The mean droplet size increased in the emulsion stabilized by SPI in over-oxidized condition of 3 and 5 mM AAPH. Compared with the 3 mM treatment, the mean droplet size of emulsion at 5 mM AAPH decreased, which was similar to a previous research that droplet size decreased in the emulsion stabilized by over-oxidized protein (Sun et al., 2012). No noticeable increase of mean droplet size was observed as the temperature raised from 30 to 120 °C in the emulsion prepared with moderately oxidized SPI. On the contrary, mean droplet size of emulsion stabilized by over-oxidized SPI at 3 and 5 mM AAPH showed a more noticeable increase as temperature raised. Significantly, d4,3 value of the emulsion stabilized by SPI oxidized by 3 mM AAPH increased from 5.46 to 16.90 μm in the observed temperature range. Therefore, during the thermal processing, stability of emulsion stabilized by moderately oxidized SPI was better than that stabilized by highly oxidized SPI. Moderate oxidation caused the unfolding of protein structure exposing more hydrophobic groups and formation of soluble aggregates. High-pressure homogenization during emulsion preparation could further induce the soluble aggregates and attribute to the diversity of structure flexibility of the aggregates which could easily attach to the interface and form a thicker adsorbed layer, resulting in better emulsifying properties (Yuan, Ren, Zhao, Luo, & Gu, 2012). Homogenization would have less effect on highly oxidized SPI because of their non-flexible structure. When protein adsorbed to oil–water interface, it would undergo structure rearrangement, which had great influence on the emulsion properties (Keerati-u-rai et al., 2012).Different molecular structures of native and oxidized SPI would

Fig. 3. ζ-Potential (A) and creaming index (B) of emulsion stabilized by SPI incubated with increasing concentration of AAPH for 24 h at 37 °C. Least-squares difference was used for comparison of mean values among treatments, and to identify significant differences. Columns with different letters (a–g) are significantly different (p b 0.05).

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3.5. Emulsion stability As shown in Fig. 3B, the creaming index declined gradually from 9.43% at 0 mM to the minimum of 5.74% at 1 mM AAPH. Rapid creaming was observed at 3 and 5 mM AAPH. Results from visual creaming measurement after 15 days were in close agreement with the mean droplet size measurement. During the storage, emulsion droplets would undergo aggregation (coalescence or flocculation) and then the particle size increases, usually resulting in creaming. Creaming is also observed as a result of poor emulsion forming like the presence of large emulsion droplets after homogenizing (Van der Ven, Gruppen, De Bont, & Voragen, 2001). In this work, the larger droplet with lower surface charge in the emulsion stabilized by

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over-oxidized SPI combined with the droplet aggregation, promoted the considerable creaming during storage. The micrographs of the different emulsions stabilized by native and oxidized SPI after 15 days are shown in Fig. 4. The emulsions showed significant structural difference as a function of AAPH concentration. Between 0 and 1 mM AAPH, the degree of flocculation and droplet size gradually decreased. Compared with the sample of 0 mM, emulsion droplet evolved gradually from large, coarse, and flocculated image to small and fine appearance at AAPH ≤ 1 mM. Droplet coalescence and flocculation appeared in the emulsions stabilized by over-oxidized SPI. These observations were well in accordance with the results of emulsion mean droplet size, ζ-potential and creaming index measurements as described above.

Fig. 4. Microstructure of the emulsions stabilized by SPI incubated with increasing concentration of AAPH for 24 h at 37 °C.

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