Effect of high intensity ultrasound on physicochemical and functional properties of soybean glycinin at different ionic strengths

Innovative Food Science and Emerging Technologies 34 (2016) 205–213

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Effect of high intensity ultrasound on physicochemical and functional properties of soybean glycinin at different ionic strengths Moxi Zhou a, Jian Liu a, Yuanyuan Zhou a, Xingjian Huang a, Fengxia Liu a,b, Siyi Pan a,b, Hao Hu a,b,⁎ a b

College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, PR China

a r t i c l e

i n f o

Article history: Received 17 December 2015 Accepted 16 February 2016 Available online 2 March 2016 Keywords: Ultrasound Soy glycinin Different ionic strengths Conformational structures Emulsifying properties Particle size

a b s t r a c t In this work, soybean glycinin was treated by high intensity ultrasound (HIU; 20 kHz at 80 W cm−2 from 0 to 40 min) in three ionic strengths (I = 0.06, 0.2 and 0.6) at pH 7.0. At all three ionic strengths, HIU of glycinin increased emulsion stability and decreased the turbidity. However, the effects of HIU on the particle size, particle distribution, solubility, emulsifying activity index, and surface hydrophobicity showed different characteristics in three ionic strengths. For example, after HIU, surface hydrophobicity of glycinin increased at I = 0.06 and 0.2, but remained unchanged at I = 0.6. The effects of HIU on glycinin were more pronounced at I = 0.2 than the other two ionic strengths. Furthermore, HIU influenced the glycinin aggregates, but remained the secondary and tertiary structures almost unchanged, which could be demonstrated by circular dichroism and the intrinsic fluorescence spectra. Industrial relevance: Soy protein is a plant protein which is widely employed in food products due to its high nutritional value, low price as well as its good functional properties. However, soy protein, especially glycinin, is easy to form aggregates and therefore limits soy proteins'application in some aspects. High intensity ultrasound (HIU) waves are generally considered as safe, non-toxic, and environmentally friendly. The results of this study suggested that HIU could dissociate soy glycinin and improve some functional properties of soybean glycinin, indicating that HIU can be considered as a potential tool to change soy glycinin's functional property. Moreover, this work found that the effects of HIU on physicochemical and functional properties of soybean glycinin were different in three ionic strengths, which could provide some fundamental information on how HIU influences the soy glycinin structures in different ionic strength and increase the application of HIU in the soy bean industry. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The potential for the application of ultrasound in food industry has been recognized since the 1970s (Mizrach, 2008). Ultrasound is an acoustic wave with frequency greater than 20 kHz (O'Sullivan, Murray, Flynn, & Norton, 2015). A major advantage of ultrasound is that sound waves are generally considered as safe, non-toxic, and environmentally friendly (Kentish & Ashokkumar, 2011). Ultrasound can be classified into two frequency ranges, that is low frequency (20–100 kHz) and high frequency (N 100 kHz). Low frequency and high intensity ultrasound (HIU) also known as “power ultrasound” (20–100 kHz, power in the range 10–1000 W cm−2) can generate strong shear and mechanical forces due to a cavitation phenomenon (Chandrapala, Oliver, Kentish, & Ashokkumar, 2013; Soria & Villamiel, 2010). Therefore, HIU can be used to physically or chemically alter food properties (Ozuna, Paniagua-Martínez, Castaño-Tostado, Ozimek,

⁎ Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China. E-mail address: [email protected] (H. Hu).

http://dx.doi.org/10.1016/j.ifset.2016.02.007 1466-8564/© 2016 Elsevier Ltd. All rights reserved.

& Amaya-Llano, 2015; Shanmugam, Chandrapala, & Ashokkumar, 2012; Tao & Sun, 2015). Among the works of application of HIU to food industry, several researchers used HIU to modify the structures and properties of food proteins (Barukčić, Jakopović, Herceg, Karlović, & Božanić, 2015; Kadam, Tiwari, Álvarez, & O'Donnell, 2015; Marcuzzo, Peressini, Debeaufort, & Sensidoni, 2010; Ozuna et al., 2015). For instance, O'Sullivan et al. (2015) found that HIU reduced the particle size of bovine gelatin, fish gelatin, egg white protein, and pea protein. Yanjun et al. (2014) reported that the HIU of reconstituted milk protein concentrate reduced the particle size, but increased the solubility, emulsifying activity and emulsion stability. Soy protein is a plant protein which is widely employed in food products due to its high nutritional value, low price (Chen, Remondetto, Rouabhia, & Subirade, 2008) as well as its good functional properties such as emulsifying ability, gelation ability, water holding capacity and oil-holding capacity (Kinsella, 1979; Nishinari, Fang, Guo, & Phillips, 2014). Soy protein is consisted of four major water-extractable fractions (2S, 7S, 11S and 15S) which are classified by their sedimentation coefficients (Añón, de Lamballerie, & Speroni, 2012; Riblett, Herald, Schmidt, & Tilley, 2001). Several studies (Arzeni et al., 2012; Chen, Chen,

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Ren, & Zhao, 2011; Jambrak, Lelas, Mason, Krešić, & Badanjak, 2009; O'Sullivan et al., 2015) and our previous works (Hu et al., 2013a) found that the HIU of soy protein isolate increased surface hydrophobicity, emulsifying activity, emulsion stability and protein solubility, changed the rheological properties, but reduced the particle size. Moreover, we noticed that HIU pretreatments improved either the heatinduced gelation property (Hu, Li-Chan, Wan, Tian, & Pan, 2013c; Hu et al., 2013b) or the cold-set gelation property (Zhang et al., 2016) of SPI. The HIU treated SPI cold-set gel was then used as a riboflavin carrier (Hu et al., 2015b). Interestingly, our recent findings showed that the effects of HIU on the physicochemical and functional properties of soybean βconglycinin and glycinin in 0.05 M pH 7.0 Tris–HCl buffer were quite different. The HIU of soybean β-conglycinin decreased particle size, increased, solubility, emulsion stability, emulsifying activity and surface hydrophobicity, while the HIU of glycinin had little effect on its particle size, minimal effects on emulsifying activity, but negative effect on emulsion stability and surface hydrophobicity (Hu, Cheung, Pan, & Li-Chan, 2015a). In other words, in pH 7.0 0.05 M Tris–HCl buffer, the changes of soybean β-conglycinin induced by HIU were consistent with many other food proteins, while those of glycinin were not. The change of protein induced by HIU depends on the nature of the protein, conformational structure as well as its degree of denaturation and aggregation (Ozuna et al., 2015). Thus, it was inferred that the extensive aggregation of soybean glycinin in 0.05 M pH 7.0 Tris–HCl buffer may be the reason for the above phenomenon and the effect of HIU on soybean glycinin may be changed under different degree of aggregation. Therefore, this research focuses on glycinin, a major storage protein in soybeans (around 30%), which is consisted of polypeptides A and B (Hu et al., 2015a). The functional properties and molecular structure of soybean glycinin depends strongly on ionic strength. For instance, the solubility, emulsifying properties and surface hydrophobicity of glycinin is influenced by ionic strength (Lakemond, de Jongh, Hessing, Gruppen, & Voragen, 2000b; Wagner, Sorgentini, & Añón, 2000). At pH 7.6 and at an ionic strength of 0.5 glycinin is mainly present in a hexameric form. Lowering the ionic strength to 0.01 at pH 7.6 causes glycinin to dissociate from the 11S form mainly into the 7S form (Lakemond et al., 2000b). Moreover, when the ionic strength is lowered from 0.5 to 0.03, the basic polypeptides shift more to the exterior of the glycinin complex. This altered the arrangement of acidic and basic polypeptides may influence the functional property of glycinin (Lakemond, de Jongh, Hessing, Gruppen, & Voragen, 2000a). The ionic strength of food products varies from 0.02 to 0.2 (Ruíz-Henestrosa et al., 2007). However, to the best of our knowledge, little is known is about the effects of HIU on the structural and physicochemical properties of soy glycinin in different ionic strength, even though previous works showed that HIU had minor or negative effects on soy glycinin in pH 7.0 0.05 M Tris– HCl buffer. Therefore, in this work, we studied the physicochemical and structural changes of soy glycinin due to HIU treatments in different ionic strength (low ionic strength 0.06, medium ionic strength 0.2 and high ionic strength 0.6) at pH 7.0, so as to provide some basic data on how HIU influences the soy glycinin structures in different ionic strength, which may improve the application of HIU in the soy bean industry. 2. Material and methods 2.1. Materials Defatted 7B soy flour (minimally heat processed) was donated by the Archer Daniels Midland (ADM) company (Decatur, IL, USA). Pure corn oil was purchased from ACROS ORGANICS (Geel, Belgium). Tris base and 1-anilino-8-naphthalene-sulfonate (ANS) were purchased from Sigma-Aldrich. Other chemicals were of analytical grade.

2.2. Isolation of glycinin The glycinin (11S) protein fraction was isolated referring to the method of Nagano, Hirotsuka, Mori, Kohyama, and Nishinari (1992) and Ruíz-Henestrosa et al. (2007). Briefly, the defatted soy flour was extracted with 15-fold volumes of distilled water adjusted to pH 7.5 with 2 M NaOH at room temperature (∼25 °C) for 1 h with constant magnetic stirring. This slurry was filtered through a mesh (200 size), and the filtrate was collected and then centrifuged (Eppendorf AG 5805, Hanburg, Germany) at 9000g, for 30 min. Dry sodium bisulfite (SBS) was then added to the supernatant (0.98 g SBS/L), the pH was then adjusted to 6.4 with 2 M HCl, and the mixture was kept at 4 °C overnight. The following preparation procedure was performed at 4 °C. Centrifugation at 6500g for 20 min. The precipitate was washed three times with distilled water, and the insoluble fraction was glycinin globulin. The glycinin solution was adjusted to pH 7.5 with 2 M NaOH and dialyzed 60 h (change water eight times), then freeze-dried. 2.3. High intensity ultrasound treatment of glycinin 1% (w/v) soy glycinin solutions were prepared by adding different buffers (pH 7.0, ionic strength 0.06, 0.2 and 0.6 buffers) into freezedried soy glycinin and were stirred for 40 min at room temperature. An ultrasound processor (JY92-IIDN, 20 kHz, 0.636 cm diameter titanium probe, NingBo Scientz Biotechnology Co. Ltd., Ningbo, Zhejiang, China) was used to sonicate 20 mL of glycinin solutions in 25 mL flat bottom conical flasks which were immersed in an ice-water bath. Glycinin in different ionic strength buffers were treated at 60% power for 0, 5, 20 or 40 min (pulse duration of on-time 5 s and off-time 1 s). In this work, the ultrasonic intensity that was calculated based on the method of Jambrak et al. (2009) was 80 W cm−2. The I ≈ 0.06 buffer consisted of 2.4 mM NaH2PO4, 2.5 mM Na2HPO4 and 4.9 mM NaCl. The I ≈ 0.2 buffer consisted of 1.8 mM NaH2PO4, 3.1 mM Na2HPO4 and 0.089 M NaCl. The I ≈ 0.6 buffer consisted of 1.6 mM NaH2PO4, 3.3 mM Na2HPO4 and 0.289 M NaCl. Na2HPO4 solutions were used to adjust the pH to 7.0. 2.4. Sodium (SDS-PAGE)

dodecyl

sulfate-polyacrylamide

gel

electrophoresis

Reducing and non-reducing SDS-PAGE was carried out according to the procedure of Liu and Xiong (2000). Under reducing condition, 200 mL of the sample buffer was mixed with 40 mL of 10% SDS, 100 mL water, 10 mL of β-mercaptoethanol and 5 mL of 2% (w/v) bromophenol blue, 25 ml 0.5M Tris–HCl buffer (pH = 6.8) and 20 mL glycerin. 925 uL sample buffer and 75 uL protein (10 mg/mL) were mixed uniformity. The mixtures were boiled for 4 min. 20 uL of the mixtures was added each time after preparing 12% separating gel and 5% stacking gel. Under non-reducing condition, β-mercaptoethanol was not added. Electrophoresis was performed at a constant current of 15 mA for stacking and 30 mA for separation. 2.5. Particle size determination The particle size information of glycinin was determined by a ZS Zetasizer Nano (Malvern Instruments, Malvern, Worcestershire, UK). Zetasizer provides 0.3 nm to 10.0 μm diameter measurement range. Stokes-Einstein equation was used to determine the average hydrodynamic size of glycinin solutions. All determinations were conducted in triplicate. 2.6. Protein solubility The protein solubility of soy glycinin solutions was determined according to the method of Shimada and Cheftel (1988). HIU treated and non-HIU glycinin samples in different buffers were centrifuged at

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2.11. Statistical analysis

20,000g for 20 min at 25 °C. The protein concentration of the supernatants after centrifugation was determined by the Lowry assay kit (Labaide Company, Shanghai, China), using glycinin as the standard. Protein solubility (%) of glycinin = 100 × (protein content of the supernatant) / (total protein content). All determinations were conducted in triplicate.

SPSS (17.0) software was used for statistical analysis. In the figures, the values are the means of triplicates, and the error bars means the standard deviation. Statistical significance of differences among means was evaluated by Duncan's test at p b 0.05.

2.7. Circular dichroism spectra measurement

3. Results and discussion

Circular dichroism (CD) spectroscopy plays an important role in the study of protein structure as it allows the characterization of secondary and tertiary structures of proteins (van Mierlo & Steensma, 2000). Far-UV CD measurements (CD J 1500, Jasco Corp, Japan) were record from 195 to 260 nm in 0.1 cm quartz cuvette (Clara Sze, Kshirsagar, Venkatachalam, & Sathe, 2007), and near-UV CD measurements were record from 350 to 250 nm in 1 cm quartz cuvette. The response, scan rate, and bandwidth were 4 s, 50 nm/min and 1.0 nm, separately. Every spectrum was obtained by the average of the three scans. The proportion of α-helix, β-turn, β-sheet and random coil was calculated according to the Yang's reference software provided by Jasco Corp (Hu et al., 2015a).

The SDS-PAGE patterns of non-HIU treated and HIU treated glycinin under reducing and non-reducing conditions are shown in Supplementary data. Under reducing conditions, bands with molecular-weights of ~ 20 kDa and 30 kDa were respectively assigned to the basic (B) and acidic (A) polypeptide chains. HIU did not significantly change the SDS-PAGE patterns of glycinin either under reducing condition or non-reducing condition.

2.8. Fluorescence emission spectroscopy Protein (0.2 mg/mL) fluorescence was measured with Fl spectorophotometer (F-4600 4.0 5j2-0004) at a 290 nm excitation wavelength and a 300–400 nm emission wavelength. The phosphate buffer used to dissolve soy protein was used as blank solutions (Jiang, Chen, & Xiong, 2009). 2.9. Surface hydrophobicity Surface hydrophobicity (H 0 ) of soy glycinin was determined using ANS as fluorescence probe according to the method of Hayakawa and Nakai (1985). The soy glycinin samples were dissolved in different buffers to get different protein concentrations from 0.0005 to 0.2 mg/mL (Hu et al., 2015a). Relative fluorescence intensity was measured by a Fl spectorophotometer (F-4600 4.0 5j2-0004, Shimadzu Corp., Kyoto, Japan), at wavelengths of 365 nm (excitation) and 484 nm (emission). All determinations were conducted in triplicate.

3.1. Particle size, size distribution, turbidity and protein solubility Particle size is a major factor which can influence the functional properties of soy glycinin (Kinsella, 1979). Several studies found that HIU can reduce the particle size of food proteins. However, the change of protein induced by HIU depends on the nature of the protein and its degree of denaturation and aggregation (Ozuna et al., 2015). Therefore, the effect of HIU on the particle size of the same protein may be diverse, if the protein is in different conformational structure and degree of aggregation. For example, O'Sullivan et al. (2015) pointed out that HIU reduced the particle size of egg white protein but Arzeni et al. (2012) noticed that HIU increased the particle size of egg white protein. The effects of HIU on the particle size of glycinin fractions in different ionic strengths are shown in Fig. 1. The particle size of glycinin was the largest at I = 0.2, followed by I = 0.06 and 0.6. From Fig. 1, it was observed that the particle size of glycinin decreased after HIU treatments in all three ionic strengths. At ionic strength of 0.06, 40 min of HIU of glycinin decreased the particle size from 224.3 nm to 65.2 nm gradually. At ionic strength of 0.6, 5 min of HIU decreased the particle size from 160.9 nm to 45.0 nm, then the particle size increased little (p N 0.05) to 50.0 and 54.0 nm with HIU for 20 and 40 min, respectively. At ionic strength of 0.2, HIU of glycinin decreased the particle size dramatically from 780.1 nm to 105.0 nm. Several researchers studied the effect of HIU on the particle size of food proteins. The above phenomenon could be due to the dissociation of 11S aggregates caused by high

2.10. Emulsifying tests Emulsifying activity index (EAI) and emulsion stability index (ESI) were determined according to the method of Cameron, Weber, Idziak, Neufeld, and Cooper (1991). 1 mL corn oil and 3 mL 1% glycinin solution were added into a 50 mL centrifuge tube, then homogenized (POLYTRON® PT 2100, Littau-Lucerne, Switzerland) for 1 min at 19000 turns/min. From the bottom of the centrifuge tube, 50 uL of the emulsion was taken immediately (time 0) and at subsequent time intervals. The emulsion was diluted into 9.95 mL 0.3% SDS solution. The absorbance of the diluted solutions was measured at 500 nm. All determinations were conducted in triplicate. EAI was calculated as follows:
A0  dilutionfactor EAI m2 =g ¼ 2T c  φ  ð1−θÞ  1000

where T = 2.303, A0 = absorbance at time 0, dilution factor = 200, c = the weight of protein per unit volume (g/mL ), φ (optical path) = 1 cm, and θ = 0.25. ESI was represented by the time (min) when the absorbance was half that of absorbance at time 0 (A0). All determinations were conducted in triplicate.

Fig. 1. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) on particle size of glycinin at different ionic strengths. The different upper case letters (A, B, C, …) or lower case letters (a, b, c, …), (a', b', c', …) indicate significant difference for glycinin samples in I = 0.06, I = 0.2 and I = 0.6, respectively, at p b 0.05 using Duncan's test.

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shear energy waves, local extreme pressures and turbulence from HIU cavitation phenomenon. Interestingly, our previous study found that the particle size of 11S fraction in pH 7.0 0.05 M Tris–HCl buffer did not change after HIU (Hu et al., 2015a). In that work, 11S fraction did not dialyze, therefore, the ionic strength could not be known accurately. Moreover, from the particle size data (around 200 μm), it was obvious that the 11S was highly aggregated in our previous work. This could be the reason for the divergent findings. The effects of HIU on the particle size distribution of glycinin in three ionic strengths are shown in Fig. 2. At ionic strength of 0.06 (Fig. 2A), the non-HIU glycinin had a dominant peak around 370 nm, with a shoulder peak around 60 nm and a small peak around 5400 nm. 5 and 20 min of HIU reduced the fraction of glycinin around 5400 nm. Moreover, 40 min of HIU reduced the dominant peak to 67 nm. At ionic strength of 0.2, ZS Nano Zetasizer was not appropriate to provide the particle size distribution information of non-HIU and 5 min HIU glycinin, which was probably because some of the glycinin aggregates were bigger than the range of ZS Nano Zetasizer. Therefore, only the distributions of 20 min and 40 min HIU treated glycinin were provided in (Fig. 2B). As shown in Fig. 2B, 40 min of HIU showed more particle size in bigger size range than the 20 min HIU sample, suggesting that longer HIU time (40 min) led to the reaggregation of glycinin. At I = 0.6 (Fig. 2C), the untreated sample showed a bimodal distribution of particles, while the HIU treatments reduced the two peaks to the smaller size. Interestingly, HIU increased the intensity of the peak around 5500 nm, indicating that big aggregates were formed (I = 0.6). Fig. 3 shows the appearance of non-HIU and HIU treated glycinin solutions at three ionic strengths. It could be observed that HIU of glycinin solutions at three ionic strengths reduced their turbidity. Our previous work reported similarly that HIU reduced the absorbance of β-conglycinin and glycinin solutions at 600 nm. Madadlou, Mousavi, Emam-Djomeh, Ehsani, and Sheehan (2009) also observed that 35 and 135 kHz sonication treatment led a reduction of the turbidity of case in solutions. HIU reduced the particle size of glycinin, which could be considered as the reason for the decrease in glycinin solution turbidity after the HIU treatments. Fig. 4 shows the effects of HIU on the protein solubility of glycinin in three ionic strengths. The protein solubility of glycinin at I = 0.2 was lower than that of glycinin at I = 0.06 and I = 0.6. At I = 0.06 and I = 0.2, 40 min of HIU treatment increased the protein solubility of glycinin from 88% and 53% to 94% and 77%, respectively. At I = 0.6, 5 min of HIU increased the solubility of glycinin from 87% to 99%. However, longer HIU time (40 min) at I = 0.6 reduced the protein solubility to 88%. The changes of protein solubility at I = 0.6 were consistent with the changes of particle size. At I = 0.6, 5 min of HIU decreased the particle size from 160.9 nm to 45.0 nm, then the particle size increased to 50.0 and 54.0 nm with HIU for 20 and 40 min. Protein solubility of glycinin is determined as the glycinin content in the supernatant after

Fig. 3. Pictures of non-HIU treated glycinin and high intensity ultrasound (HIU; 20 kHz at 80 W cm−2) treated glycinin at three ionic strengths. A): Non-HIU treated, 5 min HIU treated, 20 min HIU treated and 40 min HIU treated glycinin at I = 0.06. B): Non-HIU treated, 5 min HIU treated, 20 min HIU treated and 40 min HIU treated glycinin at I = 0.2. C): Non-HIU treated, 5 min HIU treated, 20 min HIU treated and 40 min HIU treated glycinin at I = 0.6.

20,000g of centrifugation. Smaller particle sizes would be expected to lead to more glycinin in the supernatant after centrifugation and therefore higher protein solubility. From the information of particle size, particle distribution and protein solubility, it could be inferred that the effects of HIU on glycinin in different ionic strengths were different. At I = 0.06 and 0.2, 40 min of HIU dissociated glycinin aggregation and increased the smaller particle size fractions, leading to an improvement of protein solubility. However, at I = 0.6, the HIU induced dissociation and HIU induced aggregation of glycinin aggregates happened simultaneously. At the first 5 min (I = 0.6), the dissociation effect played a dominant effect, while at longer HIU time the dissociated aggregates were re-aggregated, resulting in the constant of particle size at longer HIU time (p N 0.05).

Fig. 2. Particle size distribution of high intensity ultrasound (HIU; 20 kHz at 80 W cm−2) and Non-HIU glycinin at three ionic strengths. Non-HIU samples (■), 5 min HIU sample ( ), 20 min HIU sample ( ) and 40 min HIU sample ( ). A): I = 0.06, B): I = 0.2, C): I = 0.6.

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Fig. 4. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) on protein solubility of glycinin at different ionic strengths. The different upper case letters (A, B, C, …) or lower case letters (a, b, c, …), (a', b', c', …) indicate significant difference for glycinin samples in I = 0.06, I = 0.2 and I = 0.6, respectively, at p b 0.05 using Duncan's test.

3.2. Conformational structures Far-UV circular dichroism (CD) spectra were used to determine the secondary structure of glycinin. The proportions of α-helix, β-sheet,

209

β-turn and random coil, which were derived from Yang's equation are shown in Fig. 5. Regardless of the three ionic strengths, 40 min HIU of glycinin, slightly increased β-sheet, but slightly reduced β-turn (Fig. 5). Similarly, Jiang et al. (2014) found that HIU increased the βsheet of black bean protein isolates and Gülseren, Güzey, Bruce, and Weiss (2007) noticed that HIU reduced the β-turn of bovine serum albumin. Several studies have investigated the effect of sonication on secondary structure of different proteins but divergent results were reported. For instance, our previous study of commercial soy protein isolate demonstrated that lower power HIU treatment (20 kHz, 200 W) showed a decrease in the α-helix and the random coil, and an increase in the β-sheet. In contrast, higher power treatment (20 kHz, 600 W) showed an increase in the α-helix and the random coil, and a decrease in the β-sheet (Hu et al., 2013a). Similarly, Chandrapala, Zisu, Palmer, Kentish, and Ashokkumar (2011) pointed out that 60 min HIU of β-lactoglobulin led to an increase of the α-helix and a decrease of the β-turn and the β-sheet. However, Stathopulos et al. (2004) pointed out that HIU of protein increased the β-structure but decreased the αhelix structure in the protein aggregates. Jin et al. (2015) compared the effects of sweeping frequency and pulsed ultrasound (SFPU) and sequential dual frequency ultrasound (SDFU) on zein and glutelin. They found that SFPU pretreatment had little impact on the secondary structure of zein, while the SDFU decreased the β-sheet and increased the α-helix. Both SFUP and SDFU increased β-sheet and decreased α-helix of glutelin. Interestingly, our previous study of soy β-conglycinin showed that HIU did not change the secondary structure of 7S fractions in Tris–HCl buffer (Hu et al., 2015a). Therefore, many factors, such as the dominant secondary structure type of protein (Marchioni et al., 2009),

Fig. 5. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) on the secondary structure of glycinin at different ionic strengths. I = 0.06 samples (■), I = 0.2 samples ( ), and I = 0.6 samples ( ). A) α-helix, B) β-sheet, C) random coil, and D) β-turn.

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the nature of the protein, the degree of denaturation and aggregation, as well as HIU types and HIU conditions, influenced the effects of HIU on the secondary structures of proteins. Near-UV CD spectra provide an indication of protein tertiary structure (van Koningsveld et al., 2002). All near-UV CD spectra showed two positive peaks (one peak around 285 nm and another one around 291 nm (Fig. 6). The intensity of glycinin near-UV CD spectra at I = 0.2 was lower than that at I = 0.06 and 0.6. The peak at 291 nm points to the presence of tryptophan residues, whereas the peak at 285 indicates the presence of tyrosyl residues (Pouvreau et al., 2005). Similar near-UV CD patterns of glycinin were observed by Lakemond et al. (2000b). At I = 0.06 and 0.6, the HIU treatments slightly reduced the intensity of the two peaks, indicating that HIU unfolded the glycinin tertiary structure (van Koningsveld et al., 2002). However, at I = 0.2, HIU slightly increased the intensity of the two peaks. From the particle size data, glycinin were highly aggregated at I = 0.2, the HIU treatments dissociated the glycinin aggregates and exposed more glycinin molecules to the solution, therefore it increased the soluble tertiary structure. The intrinsic (or Trp) fluorescence spectra are determined by the polarity of the environment of the Trp residues, thus, it is widely used to study the tertiary structure of protein (Jiang et al., 2014; Tang, Sun, & Foegeding, 2011). Usually, if the λmax is N 330 nm, then the Trp is assigned as a “polar” environment; and if the λmax is b330 nm, then the Trp is assigned as a “non-polar” environment (Jiang et al., 2014; Vivian & Callis, 2001). In this work, the Trp of all samples were determined to be present in a “polar” environment (Fig. 7). In all three ionic strengths, HIU did not change the λmax of glycinin. Moreover, minor changes of the fluorescence intensity of glycinin at I = 0.06 and I = 0.6 were observed after the HIU treatments. However, at I = 0.2, HIU increased the fluorescence intensity obviously. Jin et al. (2015) found similarly that HIU resulted in an increase in amounts of the fluorescence intensity of glutelin and zein. They attributed the reason to the fact that multi-frequency power ultrasound pretreatment destroyed hydrophobic interactions of protein molecules, induced molecular unfolding, caused more hydrophobic groups and regions inside the molecules to be exposed to the outside (Jin et al., 2015). Protein surface hydrophobicity (H0) is an index of the amount of hydrophobic groups on the surface of protein molecules contacting with the polar aqueous environment (Chandrapala et al., 2011), therefore, H0 is closely related to the functional properties of a protein and can be considered as an important factor of the conformation of proteins. Before HIU, glycinin at I = 0.6 had the highest H0, followed by I = 0.2 and I = 0.06. The effects of HIU on the H0 of glycinin in different ionic strengths are shown in Fig. 8. It was observed from Fig. 8 that 40 min of HIU increased the H0 of glycinin continuously at I = 0.06 and I = 0.2. The above results indicate that at I = 0.06 and I = 0.2, the cavitation phenomenon induced by 40 min of HIU could disrupt the intermolecular aggregation of glycinin and expose some of the hydrophobic regions of glycinin to the surface. Several previous studies showed that HIU can

increase H0 of food proteins such as soy protein isolate (Arzeni et al., 2012; Chen et al., 2011; Jambrak et al., 2009), egg white protein (Arzeni et al., 2012), peanut protein (Zhang et al., 2014), black bean protein (Jiang et al., 2014), zein protein, glutelin (Jin et al., 2015) and bovine serum albumin (Gülseren et al., 2007). At I = 0.6, HIU of glycinin did not change the H0 (p N 0.05). In Section 3.1, it was inferred that at I = 0.6, the HIU induced dissociation and the HIU induced aggregation of glycinin happened simultaneously. During HIU, the amount of hydrophobic groups exposed due to dissociation of aggregates and the amount of hydrophobic groups buried due to re-aggregation of aggregates may be consistent, leading to the changeless of H0 during the 40 min of HIU. Moreover, from Fig. 8, it can be observed that the influence of HIU on H0 is stronger at I = 0.2 than the other two ionic strengths. This suggested that the effect of HIU on the conformational structure (such as, extent of aggregation and quaternary structure) of glycinin is more obvious at I = 0.2. Similarly, the particle size changes in the different ionic strengths showed a consistent finding (Fig. 1) that the influence of HIU on glycinin is stronger at I = 0.2 than the other two ionic strengths. From the conformational structural changes information, it could be inferred that the effects of HIU on glycinin is more remarkable at I = 0.2 than the other two ionic strengths (I = 0.06 and I = 0.6). Furthermore, based on the data of the far-UV CD spectra, the near-UV CD spectra and the intrinsic fluorescence spectra, it could be found that the secondary and tertiary structure changes induced by HIU were minor. However, HIU can obviously influence glycinin aggregation at all three ionic strengths, as demonstrated by the H0 data, which was consistent with the particle size, solubility as well as the turbidity properties. In other words, HIU influenced the glycinin aggregates, but remained most of the secondary and tertiary structures unchanged. At I = 0.06 and 0.2, HIU of glycinin dissociated the aggregates with increasing HIU time. However, at I = 0.6, the HIU induced dissociation and HIU induced aggregation of glycinin happened simultaneously. 3.3. Emulsifying properties The emulsifying ability of food proteins is very important for many applications such as cake, frozen desserts, mayonnaise, salad dressings, milks, batters and coffee whiteners (Kinsella, 1979). Food proteins aid in the formation of emulsions, mainly due to the decrease interfacial tension between oil and water, and also due to the formation of a physical barrier at the interface (Molina, Papadopoulou, & Ledward, 2001). The emulsifying properties of food proteins depend upon the solubility, molecular flexibility, surface hydrophobicity and stability of the protein structure (Kinsella, 1979; Molina et al., 2001; Wagner & Gueguen, 1999). Emulsifying activity index (EAI) of glycinin at I = 0.2 and I = 0.6 increased from 19.3 and 21.1 m2/g to 24.1 and 24.7 m2/g, respectively, with increasing HIU time to 40 min (Fig. 9). Similarly, Jambrak et al.

Fig. 6. Near-UV circular dichroism spectra of non-HIU treated glycinin and high intensity ultrasound (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) treated glycinin at three ionic strengths. A): I = 0.06, B): I = 0.2, and C): I = 0.6.

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Fig. 7. Fluorescence spectra of non-HIU treated glycinin and high intensity ultrasound (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) treated glycinin at three ionic strengths. A): I = 0.06, B): I = 0.2, and C): I = 0.6.

(2009) and Chen et al. (2011) reported that HIU increased EAI of soy protein isolate and our previous works found that HIU increased EAI of soy β-conglycinin and glycinin in pH 7.0 Tris buffer (Hu et al., 2015a). Several authors reported a positive correlation between solubility, surface hydrophobicity and EAI (Molina et al., 2001; Wagner & Guéguen, 1999). HIU of glycinin increased the protein solubility, surface hydrophobicity and changed the conformational structure, which could make the glycinin have a better potential to adsorb at the interface of oil droplets. However, the EAI of glycinin at I = 0.06 decreased from 24.6 to 20.1 m2/g after 40 min of HIU treatment. This finding was consistent with Chittapalo and Noomhorm (2009) who also found that sonication treatment reduced the EAI of rice proteins. Even though HIU increased the solubility and surface hydrophobicity of glycinin at I = 0.06, the molecular flexibility of protein also changed, which might lead to the reduction of EAI at I = 0.06. Emulsion stability index (ESI) of glycinin at I = 0.06 and 0.6 increased from 4.8 and 6.7 min to 7.4 and 10.0 min, respectively, after 40 min of HIU (Fig. 10). At I = 0.2, ESI increased from 1.0 to 3.0 min after 20 min of HIU and then reduced to 1.7 at 40 min of HIU (Fig. 10). The stabilization of emulsion is usually achieved by the formation of a film around the oil droplets by glycinin (Kinsella, 1979). The HIU of glycinin increased the surface hydrophobicity. This means that the hydrophobic residues may be exposed from the inside of hexameric structure of glycinin and improve its lipophilicity (Molina et al., 2001; Wagner & Guéguen, 1999). Therefore, more stable films may be formed at the oil-water interface. Furthermore, protein solubility was closely

High intensity (20 kHz, 80 W cm−2) ultrasound (HIU) changed the physicochemical and functional properties of glycinin in three different ionic strengths (I = 0.06, 0.2 and 0.6) at pH = 7. The HIU of glycinin reduced the particle size, changed the particle size distributions, decreased the turbidity, increased the surface hydrophobicity, improved the emulsion stability index and changed the emulsifying activity index. However, the effects of HIU were different in three diverse ionic strengths. The effects of HIU on particle size, turbidity, solubility, surface hydrophobicity were more pronounced for glycinin at I = 0.2 than the other two ionic strengths (I = 0.06 and I = 0.6). Moreover, at I = 0.06 and 0.2, The HIU of glycinin dissociated the aggregates with increasing HIU time. However, at I = 0.6, the HIU induced dissociation and HIU induced aggregation of glycinin aggregates happened simultaneously. It was inferred that HIU influenced the glycinin aggregates, but remained the secondary and tertiary structures almost unchanged. In conclusion, the degree of aggregation and conformational structures of glycinin were different in the three ionic strengths, leading to different functional properties of the HIU treated glycinin. Further researches will be carried out to study the HIU effect on soy β-conglycinin at different ionic strengths and pH.

Fig. 8. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) on surface hydrohpobicity of glycinin at different ionic strengths. The different upper case letters (A, B, C, …) or lower case letters (a, b, c, …), (a', b', c', …) indicate significant difference for glycinin samples in I = 0.06, I = 0.2 and I = 0.6, respectively, at p b 0.05 using Duncan's test.

Fig. 9. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W cm−2 for 5, 20 or 40 min) on emulsifying activity index of glycinin at different ionic strengths. Different upper case letters (A, B, C, …) or lower case letters (a, b, c, …), (a', b', c', …) indicate significant difference for glycinin samples in I = 0.06, I = 0.2 and I = 0.6, respectively, at p b 0.05 using Duncan's test.

correlated with ESI. HIU of glycinin could influence the glycinin migration to, adsorption at and rearrangement at the oil–water interface, resulting in the increase of ESI. 4. Conclusions

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Fig. 10. Effect of high intensity ultrasound treatments (HIU; 20 kHz at 80 W/cm−2 for 5, 20 or 40 min) on emulsifying stability index of glycinin at different ionic strengths. The different upper case letters (A, B, C, …) or lower case letters (a, b, c, …), (a', b', c', …) indicate significant difference for glycinin samples in I = 0.06, I = 0.2 and I = 0.6, respectively, at p b 0.05 using Duncan's test.

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