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Effects of single- and dual-frequency ultrasound on the functionality of egg white protein
Journal of Food Engineering 277 (2020) 109902
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Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng
Effects of single- and dual-frequency ultrasound on the functionality of egg white protein Sun Jun, Mu Yaoyao, Jing Hui, Mohammed Obadi, Chen Zhongwei, Xu Bin * School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Egg white protein Ultrasound frequency Physicochemical properties Secondary structure
The effects of high-power ultrasound (HP-US) treatment with different frequencies, temperatures, and durations on the physicochemical and structural properties of untreated and HP-US-treated egg white protein (EWP) were evaluated. Results indicated that the solubility of EWP was significantly improved after HP-US treatment, especially under the continuous dual frequency of 20/40 kHz. The foaming capacity of the EWP also depended on the treatment frequency, temperature, and duration, and foaming stability increased considerably in the HPUS-treated EWP compared with the untreated one. The particle size of the HP-US-treated EWP greatly decreased compared with that of untreated EWP. Results of low-field nuclear magnetic resonance analysis indicated that more water molecules were trapped in the EWP gel structure as free water after HP-US treatment. Therefore, the green technology of HP-US treatment can improve the physicochemical properties of EWP because of its sono chemical effect on the molecular conformation of EWP.
1. Introduction Egg white protein (EWP), which contains nearly 90% of proteins as functional ingredients in food processing, is attracting considerable attention because of its excellent nutritional and functional properties, such as foaming and gelation properties. The EWP contains 40 different proteins, with its main protein fractions as ovalbumin (~55%), ovo transferrin (~12%), ovomucoid (~11%), and lysozyme (~3.5%) (Powrie and Nakai, 1986; Andrea et al., 2017). The functional properties of the EWP powder cannot meet the current multiple needs of customers. Thus, the effective modification methods for EWP processing are being developed due to the insufficient supply for the new demand of EWP powder. The functional properties of proteins can be modified by physical, chemical, or enzymatic treatments. The application of chemical methods to modify protein in the food industry is still limited, because the chemical reagents used are harmful to human health (Mirmoghtadaie et al., 2016). The popularity and application of enzymatic modification is also limited because of the high cost of the enzymes (Beilen and Li, 2002). Therefore, the physical modification of protein has attracted wide attention. Among the physical methods for protein modification, ultrasound has been paid special interest. Ultrasound, with frequency above the threshold of human hearing (�16 kHz) is generally considered
as a safe, non-toxic, and environment-friendly technique (Knorr et al., 2004). Ultrasound can be classified into high-frequency low-energy diagnostic ultrasound (100 kHz–1 MHz) and low-frequency high power ultrasound (HP-US, 16 kHz–100 kHz) (Soria et al., 201 0). HP-US is primarily based on the high shear force and turbulence generated by the cavitation to break covalent bonds in protein (Chemat et al., 2017; Mason et al., 2005). Thus, HP-US is a rapid, efficient, and reliable technique to improve the functional quality of proteins. At present, the impact of ultrasound on the functional properties of animal and vegetable proteins, such as whey protein (Gordon and Pilosof, 2010; Jambrak et al., 2008), soy protein isolate/concentrate (Hu et al., 2013), millet protein concentrate (Nazari et al., 2018), rice protein (Yang et al., 2017), and EWP (Arzeni et al., 2012a, 2012b; Knorr et al., 2004), are increasingly being investigated. Jambrak et al. (2008) stud ied the effect of ultrasound on the solubility and foaming properties of whey protein and observed that ultrasound treatment at 20 kHz can affect these properties of whey proteins. Meanwhile, ultrasound treat ment at 40 kHz shows less effect on the properties of whey protein, but ultrasound treatment at 500 kHz displays no effect on the foaming properties (Chemat et al., 2017). Arzeni et al. (2012a, 2012b) studied the impact of US (20 kHz, 4.27 � 0.71 W) on the functionality of whey protein concentrate, soy protein isolate, and EWP. The results show that ultrasound promotes a decrease in the consistency index of all protein
* Corresponding author. E-mail addresses: [email protected] (S. Jun), [email protected] (X. Bin). https://doi.org/10.1016/j.jfoodeng.2020.109902 Received 13 September 2019; Received in revised form 19 October 2019; Accepted 2 January 2020 Available online 11 January 2020 0260-8774/© 2020 Elsevier Ltd. All rights reserved.
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Journal of Food Engineering 277 (2020) 109902
solutions, primarily on the soy protein isolate, but the gelation of EWP is not modified by the ultrasound. Nazari et al. (2018) determined the effects of ultrasound treatment at 20 kHz (18.4, 29.58, and 73.95 W/cm2) on the functional properties of millet protein concentrate. The results show that the solubility of millet protein concentrate is sub stantially improved after ultrasound treatment, and the foaming ca pacity of the concentrate declines and increases at low and high ultrasound intensities, respectively. Recent studies have focused on the modification of proteins by using an ultrasonic equipment, primarily an ultrasonic bath and an ultrasonic probe. To the best of our knowledge, little is known about the effects of single- and dual-frequency ultrasound on the functional and structural properties of EWP. Two type of ultrasound radiation modes are available. Although ul trasound baths are more widely used, this mode have two main disad vantages, namely, the lack of uniformity in the distribution of ultrasound and decline in power with time, that substantially decrease experimental repeatability and reproducibility. Hence, the most com mon ultrasound baths with ultrasound probes are less applied. The countercurrent ultrasound equipment manufactured by Meibo Biotech nology Co., Ltd. (Zhenjiang, Jiangsu, China) was used in this study to elucidate the effects of HP-US on the physicochemical properties of EWP. This study was performed to explore the effect of HP-US fre quencies, temperatures, and durations on the EWP to improve the physicochemical properties of the protein. Moreover, the structural properties of EWP upon HP-US treatment were analyzed to evaluate the relationship between the physicochemical and structural properties of the untreated and HP-US-treated EWP.
untreated EWP solutions were considered as the control group. All EWP solutions after HP-US treatment were lyophilized in a freeze dryer (FD1A-50, Bo Yi Kang Experimental Instrument Co., Ltd., Beijing, China) and then stored in air- and water-tight containers at room temperature until analyzed. 2.4. Characterization of the HP-US treated and untreated EWPs 2.4.1. Solubility The protein content of the supernatant was determined according to the method by Salvador et al. (2009) after calibration with bovine serum albumin (BSA) as a standard protein. After HP-US treatment, the freeze-dried EWP powders (1 g) were dispersed in 100 mL of deionized water (1% w/w) and centrifuged at 5000 rpm for 30 min at 4 � C. Protein solubility was expressed as grams of soluble protein per 100 g of initial EWP powder. 2.4.2. Foaming properties The foaming properties were determined according to the method described by Jambrak et al. (2008) with slight modification. Then, 100 mL (V0) of the untreated or HP-US treated EWP solution was dispersed with a homogenizer (Griffin & George, Co Ltd., UK) at 15 000 rpm for 5 min at 25 � C. The expanded EWP solution, which was treated by high-speed dispersion, was transferred into a 250 mL graduated flask to measure the foam volume V1. After resting for 30 min, the residual foam volume (V2) of the EWP solution was measured again. The foaming capacity (FC) and foaming stability (FS) of the EWP solution were calculated as follows:
2. Materials and methods
FCð%Þ ¼
2.1. Materials The protein content (total basis) of the EWP powder was 88.93% � 1.18% (N � 6.25) (AOAC, 1980), and the EWP powder was supplied by Jiangsu Kangde Eggs Co., Ltd. 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB) and 8-anilinonaphthalene-sulfonic acid (ANS) were purchased from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), NaH2PO4, Na2HPO4, other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
FSð%Þ ¼
V1
V2 V1
V0
� 100
(1)
V0 � 100 V0
(2)
V0
where V0 is the initial volume of the EWP solution (mL), V1 is the volume of EWP solution after dispersion, and V2 is the volume of the EWP so lution after resting for 30 min. 2.4.3. Free sulfhydryl groups The concentration of the free sulfhydryl groups of the untreated and HP-US treated EWP solutions were determined using Ellman’s reagent (DTNB solution). DTNB (50 mg) was solvated with 10 mL of Tris-glycine buffer solution (pH 8.0, 0.1 M) according to the method by Lagrain et al. (2008) with slight modification. Then, 1% freeze-dried EWP solution, which was dissolved in 0.1 M Tris-glycine buffer solutions (pH 8.0) were shaken in a water bath for 1 h at room temperature and then centrifuged at 5000 rpm for 15 min. After centrifugation, the supernatants were used to determine the free sulfhydryl groups. DTNB solutions (125 μL) were added into 3 mL of the supernatants of the EWP solution and allowed to stand for 30 min at room temperature. Then, the absorbance of the so lution was measured at 412 nm on a UV–vis spectrophotometer (Varian Inc., Palo Alto, CA, USA). The free sulfhydryl groups was calculated as follows:
2.2. Preparation of the EWP solution The EWP powders were dissolved in distilled water to obtain 10% (w/w) solution. Then, the EWP solutions were stirred in an ice-water bath for 1 h and centrifuged at 6000 rpm for 20 min. Afterward, the supernatant was collected, and the pH of the EWP solutions was adjusted to 7.0 � 0.2 by using 0.1 M HCl. 2.3. Experimental design of ultrasound treatment The effects of HP-US treatment at different frequencies, tempera tures, and durations on the physicochemical and structural properties of EWP were investigated using single- and continuous dual-frequency ultrasound equipment which equipped with probes operating at five different frequencies, including 20 (the fundamental one), 28, 35, 40 and 50 kHz. (Meibo Biotechnology Co., Ltd. Zhenjiang, Jiangsu, China). The continuous dual frequency of 20/40 kHz and 20/28 KHz refers to that two kinds of ultrasound frequency, which is generated in a suc cessive mode without interval, that is, one is pulsed on-time while the other is pulsed off-time. In addition, the treatment temperature is controlled by a thermostatic water bath, the mode of HP-US action (action 5 s, Intermittent 3 s). The sonochemical effects of HP-US treatment on the EWP solution were determined at different frequencies (20, 28, 35, 40, 50, 20/28, and 20/40 KHz), temperatures (25, 35, 45, and 55 � C), and durations (10, 20, 30, 40, 50, and 60 min). The power density was kept at 375 W/L. The
SH ðμm = gÞ EWP powder ¼ 75:53 � A412 � D = C
(3)
where C is the concentration of the EWP solution (g/mL), and D is the dilution factor (EWP is 30.2). 2.4.4. Surface hydrophobicity (H0) The H0 of the untreated and HP-US treated EWP solutions were determined with the fluorescence probe 1-anilino-8-naphathalene-sulfo nate (ANS) according to the method of Kato and Nakai (Kato and Nakai, 1980). The freeze-dried EWP powders were dissolved with phosphate buffer solution (PBS, 0.01 M, pH 7.0) to different concentrations (0.1–0.5%). ANS solution (20 μL, 0.05 M PBS, pH 7.0) was mixed with 4 2
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Journal of Food Engineering 277 (2020) 109902
mL of the EWP solution, and the fluorescence intensity (FI) was deter mined by a fluorophotometer (Card-F98, Varian Inc., Palo Alto, CA, USA) at room temperature under excitation wavelength of 393 nm and emission wavelength of 481 nm. The protein concentration was plotted by FI, and the initial slope was calculated as H0. The H0 of the control EWP was defined as 100%. 2.4.5. UV–visible (UV–Vis) spectroscopy The concentration of the untreated and HP-US treated EWP solution were diluted to 1 mg/mL using 0.01 M PBS (pH 7.0). The UV–Vis spectra of the sample solutions were recorded in the wavelength range of 240–400 nm using a UV–Vis spectrophotometer (Varian Inc., Palo Alto, CA, USA) with a path length of 1.0 cm for the quartz cell at room tem perature. The spectrum of 0.01 M PBS (pH 7.0) was used as blank for the EWP solution. 2.4.6. Fourier transform infrared (FTIR) spectroscopy The secondary structure of the untreated and HP-US treated EWPs were determined by an FTIR spectrometer (Nicolet is50 spectrometer, Thermo Electron Corporation, MA, USA) in the range of 2000–1000 cm 1. For each spectrum, 32 scans at a resolution of 4 cm 1 were per formed. The conformational changes of the EWP were analyzed in the range of 1600–1700 cm 1 using PeakFit v4.12 (SeaSolve, Framigham, MA, USA). 2.4.7. Scanning electron microscopy (SEM) The effects of ultrasound treatments on the microstructure and par ticle size of the EWP were observed with a scanning electron microscopy system (S–4800II SEM, Hitachi Limited, Tokyo, Japan) at an accelera tion voltage of 15 kV. Prior to the SEM analysis, the powder samples were coated with gold in an argon atmosphere using a spatter coater. 2.4.8. Low-field nuclear magnetic resonance (LF-NMR) The relaxation time (T2) of the EWP solution and gels were deter mined using a low-field NMR analyzer (NMI20-030V–I, Niumag Co., Ltd., Suzhou, China). The sample (3 g) was placed in a 25-mm diameter NMR glass tubes and inserted in the NMR probe. The T2 relaxation times were measured using Call-Purcell-Meiboom-Gill (CPMG) sequence. The echo time, wait time, and the number of scans were set to 0.05 ms, 5000 ms, and 8, respectively. A total of 17 000 echoes were recorded for analysis. T21, T22, and T23 were designed as the relaxation components, and PT21, PT22, and PT23 were designed as the corresponding area fractions. 2.5. Statistical analysis All experimental data were presented as the means from two parallel experimental data. One-way ANOVA) was used to identify significant differences (P < 0.05). 3. Results and discussion 3.1. Solubility Solubility is the most important physicochemical property of protein that can reflect the denaturation and aggregation of protein in the so lution. Thus, this property affects other functional properties of proteins, such as foaming and gelation. As indicated in Fig. 1, the solubility of the EWP solution after HP-US treatment under the different frequencies, temperatures, and treatment times increased significantly compared with that of the untreated EWP. With increased frequency, the solubility of EWP solution initially increased and then decreased before peaking at the continuous dual-frequency mode of 20/40 kHz at 25 � C for 30 min. This phenomenon is probably due to the fact that a certain frequency of ultrasound can loosen the molecular structure of the EWP, thereby increasing the solubility. However, when the ultrasound frequency
Fig. 1. Effects of HP-US treatment on the solubility of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C.
3
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exceeded a certain intensity, EWP was denatured and aggregated, resulting in reduced solubility. At the same time, as shown in Fig. 1B, the solubility of EWP solution decreased with increased treatment temper ature from 25 � C to 55 � C probably because a small amount of aggre gated protein can be formed during sonication at higher temperature (Arzeni et al., 2012a, 2012b). Similarly, as shown by Beck et al. (2017) who reported that an increase in shear treatment resulted in an increase in protein solubility, because the application of shear could lead to a disruption of the newly formed aggregates, thus increase unfolding protein could subsequently interact with the water and increase the solubility. As indicated in Fig. 1C, the solubility of EWP solution initially increased and then decreased with prolonged treatment time from 10 min to 60 min. This phenomenon may be due to the conformation and structural changes of the EWP induced by US cavitation effects (Fig. 4). Such changes caused hydrophilic parts of amino acids to move from the interior to the surface toward water. This process increased the pro tein–water interactions and then affected the solubility of protein (Hu et al., 2013; Morel et al., 2000). Jambrak et al. (2008) obtained similar results for the solubility of whey proteins and hydrolyzed whey protein at 10% (w/w) under the 20–40 kHz treatment for 15–30 min. 3.2. Foaming properties The application of EWPs in the food industry is highly dependent on the foaming properties. Thus, foaming capacity and foaming stability, as the most important functional properties of protein, are important in the baking industry. As shown in Fig. 2A, the acoustic effect of HP-US considerably increased the foaming capacity and foaming stability of the EWP solution under 40, 20/28, and 20/40 kHz, especially under the continuous dual frequency of 20/40 kHz. Xiong et al. (2016) obtained similar results in the foaming capacity and foaming stability of oval bumin under 20 kHz treatment. Moreover, the foaming capacity of the EWP was dependent on the frequency, temperature, and duration during HP-US treatment (Fig. 2), whereas foaming stability, which was not significantly dependent on the frequency, temperature, and duration of HP-US, increased considerably, compared with that of the untreated EWP. The temperature is harmful to the improvement of foaming capacity of the EWP solution probably because of the aggregation of protein as the temperature increased. Moreover, the foaming capacity of the HP-US treated EWP solution initially increased and then decreased with prolonged treatment time from 10 min to 60 min. The maximum value of foaming capacity was reached at 30 min of treatment. The enhanced foaming properties of EWP might be attributed to the higher solubility after HP-US treatment (Fig. 1). Partial protein unfolding, which reflected the exposure of hy drophobic and free sulfhydryl groups, allowed the EWP molecule to be effectively adsorbed onto the air–water interface (Mirmoghtadaie et al., 2016), thereby promoting the foam properties of proteins. However, Xiong et al. (2016) obtained a contrasting result in their study on the effect of ultrasound on the foaming properties of ovalbumin. The discrepancy may be due to the fact that the EWP is a mixture of over 40 other proteins, so that changes in the EWP are associated with all protein fractions in the EWP rather than a single component like ovalbumin. Van der Plancken et al. (2017) pointed out that the improvement of foaming properties is primarily achieved by enhancing the hydrophobic protein–protein interactions at the interface. Thus, the exposure of hy drophobic protein and the conformational change of EWP during HP-US treatment can lead to the breakage of EWP in the internal interactions. However, in the current study, the variation in the foaming capacity and surface hydrophobicity (Fig. 4) of the EWP was just the opposite under the HP-US temperature from 25 � C to 55 � C. Hence, HP-US is considered as an effective technique to improve the foaming capacity and foaming stability of the EWP solution.
Fig. 2. Effects of ultrasonic treatment on the foaming properties of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C.
3.3. Free sulfhydryl groups As the main protein fractions of the EWP, the ovalbumin is the only component that contained 4 free thiol groups buried within the core of the protein (Sheng et al., 2018). The increased free sulfhydryl groups content can generally be ascribed to the exposure of the inner sulfhydryl, resulting in dissociation of the subunit and breaking of the disulfide bond (Sheng et al., 2018). Thus, the free sulfhydryl groups content of the untreated and HP-US treated EWP might influence the physicochemical 4
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Journal of Food Engineering 277 (2020) 109902
properties, such as solubility and foaming properties. As shown in Fig. 3, the free sulfhydryl groups content initially increased and then decreased with increased ultrasound temperature and treatment time. However, the changes were not significant (P < 0.05), which agree well with the
Fig. 4. Effects of ultrasonic treatment on the surface hydrophobicity (H0) of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HPUS durations researched at 40 kHz and 25 � C. Fig. 3. Effects of ultrasonic treatment on the free SH groups of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B⋅HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C. 5
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results of Chandrapala et al. (2010) and Arzeni et al. (2012a, 2012b). Jin et al. (2015) studied the ultrasound treatment on corn gluten meal. The results indicated that the free sulfhydryl groups content in creases significantly after ultrasound treatment probably because the ultrasound changed the protein conformation, and more sulfhydryl groups have been exposed to the surface of proteins. However, in 2007, Gulseren et al. (2007) pointed out that the ultrasound cavitation effect can cause the oxidization of the sulfhydryl groups, resulting in decreased free sulfhydryl groups in the protein. Comprehensive analysis of existing reports show that HP-US has no significant effect on the free sulfhydryl groups of EWP. This phenomenon is due to the fact that the HP-US treatment can cause the exposure of sulfhydryl groups to the surface, but the simultaneous occurrence of the reduction of S–S (disulfide bond) to the sulfhydryl groups and the oxidization of the sulfhydryl groups caused by the heat of HP-US may lead to no significant changes in the free sulfhydryl groups content, or any transient changes that occurred was reversible. 3.4. Surface hydrophobicity The surface hydrophobicity of proteins, which is an index of the number of hydrophobic groups on the surface of a protein molecule in contact with the polar aqueous environment, can be used to evaluate the change in the protein conformation. This property impacts the physi cochemical and structural properties of protein. As shown in Fig. 4, surface hydrophobicity increased significantly after HP-US treatment (P < 0.05). The ultrasonic frequency mode with continuous dual frequency of 20/28 kHz and 20/40 kHz especially showed higher surface hydro phobicity. This result is probably due to the fact that the EWP solution subjected to ultrasonic wave and the produced bubble size is not a single value, such that a range of values exists. Therefore, the ultrasonic fre quency should be applied in a range of values. Moreover, the increase in surface hydrophobicity after HP-US treatment indicated that HP-US with frequencies of 20/28 kHz and 20/40 kHz exposed some of the hydrophobic groups, which were initially buried in the interior of the EWP molecule. This process increases the surface hydrophobicity of EWP. This phenomenon also shows that the EWP molecules might be broken under the ultrasonic cavitation, which is proved in the “sec ondary structure” section (Fig. 5). Moreover, as shown in Fig. 4B and C, surface hydrophobicity increased with increased ultrasound temperature and treatment time but decreased significantly after 60 min of HP-US treatment (P < 0.05). This result suggested that the temperature was advantageous to expose the hydrophobic groups and regions inside the molecules toward the aqueous environment. This finding is consistent with the study of Gul seren et al. (2007) on the ultrasound treatment of bovine serum albumin. Furthermore, negative correlation was found between the surface hydrophobicity and solubility which represented the hydrophilic effect of EWP in the different HP-US temperatures, which is consistent with the study of Jin et al. (2016). However, Wagner et al. (2000) showed in their study on soy protein isolates a positive correlation between surface hydrophobicity and the solubility of proteins. The main reason might be that different protein molecules have distinct molecular structures and the concentration of hydrophobic groups in and on the surface of the proteins.
Fig. 5. Effects of ultrasonic treatment on the secondary structure of the egg white protein (EWP) solution. A. UV–Vis and B. FTIR spectra.
UV–Vis spectrum can be used as one of the indices to determine the conformational change in protein, and its principle is the absorption of UV light by the side chain groups of tryptophan (279 nm), tyrosine (275 nm), and phenylalanine (257 nm) (Jin et al., 2016). Therefore, the conformational change in protein can be deduced by analyzing the po sition and intensity of the maximum absorption wavelength of protein in the UV–Vis spectra. The UV–Vis spectra of the EWP samples before and after HP-US treatment are shown in Fig. 5A. The characteristic absorption at 278 nm may be due to the combined effect of tyrosine and tryptophan. The UV absorption intensity of EWP increased after HP-US treatment, especially under the continuous dual frequency of 20/40 kHz at 25 � C for 30 min. The cavitation effect of HP-US might have changed the molecules of EWP, resulting in the exposure of more protein with UV absorption so that the UV absorption intensity of EWP increased. Moreover, FTIR can be used to quantitatively determine the sec ondary structural changes of EWP upon HP-US treatment. In the IR spectra, the bands at 1600–1700 cm 1 correspond to the amide I vi bration and N–H bending, which can be used to analyze the secondary structure of protein. Given that the α-helix, β-sheet, T-turn, and γ-random coil are related to the overall protein structures, their relative assigned percentages were analyzed. The content of α-helix, β-sheet, Tturn, and γ-random coil were quantitatively estimated using Gaussian peaks and curve fitting models according to the description of Byler and Susi (Susi and Michael Byler, 1983). The contents of the different secondary structures of the controlled
3.5. Secondary structure According to the results of solubility, foaming properties, free sulf hydryl groups, and surface hydrophobicity, the ultrasonic frequency mode with single-fixed frequency of 40 kHz and continuous dual fre quency of 20/28, 20/40 kHz at 25 � C for 30 min were chosen as the optimized ultrasonic conditions. Thus, the UV–Vis and FTIR spectra of the untreated and HP-US treated EWP were evaluated under the opti mized ultrasonic conditions. 6
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and HP-US treated EWP are shown in Fig. 5B. The secondary structure of EWP changed after HP-US treatment, and the β-sheets increased slightly after HP-US treatment, except under the continuous dual frequency of 20/40 kHz. Moreover, the HP-US-treated EWP exhibited an increase in the α-helix structure upon HP-US treatment under the continuous dual frequency of 20/28 kHz and 20/40 kHz. The results suggested that the single frequency of 40 kHz caused the unfolding of the α-helix, followed by the formation of β-sheet and T-turn. However, the continuous dual frequency of 20/40 kHz caused the unfolding of the β-sheet, followed by the formation of α-helix and γ-random coil. The differences in the sec ondary structure contents might be related to the distinct sonication effects and the shear forces of the ultrasound mechanical action, which disrupted the interactions between the protein molecules and influenced the internal structure of the protein molecule. 3.6. SEM The SEM can be used to characterize the shape and size of the protein with a higher resolution. Fig. 6 shows the microstructure and particle size of the control and HP-US-treated EWP under continuous dualfrequency mode of 20/40 kHz. The results showed that, compared with the untreated EWP, the particle sizes of the EWP powder were greatly reduced (Fig. 6B) under the HP-US treatment. This means that HP-US increased the EWP molecular surface area, thus increased the contact chance between EWP and water, this finding can also be a good explanation for the improved solubility and FC after HP-US treatment. In addition, because of the hydrophobic interaction, native EWP particles existed in sheet structure and accumulated together, and after sonication treatments, different degrees of deformation occurred with small cavities. The microstructure of the EWP by continuous dual fre quency of 20/40 kHz treatment presented a hole structure. These phe nomena are primarily based on the high shear force and turbulence generated by the cavitation and mechanical effects (Chemat et al., 2017).
Fig. 7. Effects of HP-US treatment on the water type and water content of egg white protein (EWP) gel. Table 1 Effects of HP-US treatment on T2 relaxation parameters of egg white protein (EWP) gel. sample
T21
T22
control
\
151.99 0.2a 151.99 0.2a 151.99 0.2a 151.99 0.2a
40 20/28
\
20/40
\
� � � �
T23
PT21
PT22
PT23
5722.37 � 14.50c 2848.04 � 17.68b 2848.04 � 13.42b 2477.08 � 16.81a
\
99.65 � 0.49b 96.33 � 1.47ab 96.66 � 1.43ab 95.15 � 1.10a
0.35 � 0.49a 3.67 � 1.47ab 3.34 � 1.43ab 4.85 � 1.10b
\ \ \
Mean � SD values denoted by different letters differ statistically significantly (p < 0.05). \: indicates that no signal was detected.
3.7. LF-NMR LF- NMR can be used to study the water type, water distribution, and the moisture content in food systems. Fig. 7 and Table 1 show the T2 relaxation spectra and T2 relaxation parameters of the control and HPUS-treated EWP gels. The T22 peak in the range of 10–100 ms can be attributed to the immobilized water, while the slowest T23 peak of >1000 ms represented the free/bound water. The position of T22 of the EWP before and after HP-US treatment showed no changes for the HPUS-treated EWP gels, while the T23 of the HP-US-treated EWP exhibi ted a shift to low relaxation times compared with the control EWP. These results indicated that the HP-US-treated EWP gel exhibited low water mobility compared with the control EWP gel. The HP-US treatments probably increased the hydrophobic interaction among the gel mole cules (Fig. 7). Thus, the mobility of the free water in the EWP gels
decreased and promoted the transition of the high mobility T23 component to low mobility component. Furthermore, PT22 and PT23 represent the areas of relaxation times T22 and T23, respectively. As shown in Table 1, the PT23 component of the control EWP gel increased from 0.35% to 3.67% (40 kHz), 3.35% (20/28 kHz), and 4.85% (20/40 kHz), while the PT22 component of native EWP gel decreased from 99.65% to 95.15% (20/40 kHz). These results indicated that more water molecules were trapped in the EWP gel structure as free water after HPUS treatment.
Fig. 6. Microstructure and particle sizes of the untreated and ultrasound treated (dual-fixed-frequency combination of 20/40 kHz) egg white protein (EWP). 7
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4. Conclusion
Chemat, F., Rombaut, N., Sicaire, A.G., et al., 2017. Ultrasound assisted extraction of food and natural products. Mechanisms, techniques, combinations, protocols and applications. A review. Ultrason. Sonochem. 34, 540–560. Gordon, L., Pilosof, A.M.R., 2010. Application of high intensity ultrasounds to control the size of whey proteins particles. Food Biophys. 5, 203–210. Gulseren, I., Guzey, D., Bruce, B.D., et al., 2007. Structural and functional changes in ultrasonicated bovine serum albumin solutions. Ultrason. Sonochem. 14, 173–183. Hu, H., Wu, J., Li-Chan, E.C.Y., et al., 2013. Effects of ultrasound on structural and physical properties of soy protein isolate (SPI) dispersions. Food Hydrocolloids 30, 647–655. Jin, J., Ma, H., Wang, B., et al., 2016. Effects and mechanism of dual-frequency power ultrasound on the molecular weight distribution of corn gluten meal hydrolysates. Ultrason. Sonochem. 30, 44–51. Jin, J., Ma, H., Wang, K., et al., 2015. Effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten meal. Ultrason. Sonochem. 24, 55–64. Jambrak, A.R., Mason, T.J., Lelas, V., et al., 2008. Effect of ultrasound treatment on solubility and foaming properties of whey protein suspensions. J. Food Eng. 86, 281–287. Kato, A., Nakai, S., 1980. Hydrophobicity determined by a fluorescence probe method and its correlation with surface properties of proteins. Biochim. Biophys. Acta 624, 13–20. Knorr, D., Zenker, M., Heinz, V., et al., 2004. Applications and potential of ultrasonics in food processing. Trends Food Sci. Technol. 15, 261–266. Lagrain, B., Brijs, K., Delcour, J.A., 2008. Reaction kinetics of gliadineglutenin crosslinking in model systems and in bread making. J. Agric. Food Chem. 56, 10660–10666. Mason, T., Riera, E., Vercet, A., et al., 2005. Application of ultrasound. In: Sun, D.W. (Ed.), Emerging Technologies for Food Processing. Elsevier Academic Press, California, pp. 323–350. Mirmoghtadaie, L., Aliabadi, S.S., Hosseini, et al., 2016. Recent approaches in physical modification of protein functionality. Food Chem. 199, 619–627. Morel, M.H., Dehlon, P., Autran, J.C., et al., 2000. Effects of temperature, sonication time, and power settings on size distribution and extractability of total wheat flour proteins as determined by size-exclusion high-performance liquid chromatography. Cereal Chem. 77, 685–691. Nazari, B., Mohammadifar, M.A., Shojaee-Aliabadi, S., et al., 2018. Effect of ultrasound treatments on functional properties and structure of millet protein concentrate. Ultrason. Sonochem. 41, 382–388. Powrie, W.D., Nakai, S., 1986. The chemistry of eggs and egg products. In: Cotterill, O.J. (Ed.), Egg Science and Technology, pp. 97–139. Salvador, P., Toldra, M., Saguer, E., et al., 2009. Microstructure-function relationships of heat-induced gels of porcine haemoglobin. Food Hydrocolloids 23, 1654–1659. Sheng, L., Huang, M., Wang, J., et al., 2018. A study of storage impact on ovalbumin structure of chicken egg. J. Food Eng. 219, 1–7. Susi, H., Michael Byler, D., 1983. Protein structure by Fourier transform infrared spectroscopy: second derivative spectra. Biochem. Biophys. Res. Commun. 115, 391–397. Wagner, J.R., Sorgentini, D.A., Anon, M.C., 2000. Relation between solubility and surface hydrophobicity as an indicator of modifications during preparation processes of commercial and laboratory-prepared soy protein isolates. J. Agric. Food Chem. 48, 3159–3165. Xiong, W., Wang, Y., Zhang, C., et al., 2016. High intensity ultrasound modified ovalbumin: structure, interface and gelation properties. Ultrason. Sonochem. 31, 302–309. Yang, X., Li, Y., Li, S., et al., 2017. Effects of ultrasound pretreatment with different frequencies and working modes on the enzymolysis and the structure characterization of rice protein. Ultrason. Sonochem. 38, 19–28.
The solubility, foaming capacity and foaming stability of HP-UStreated EWP samples were significantly higher than those of the un treated EWP (P < 0.05), especially under the continuous dual frequency of 20/40 kHz. Moreover, compared with the untreated EWP, HP-US treatment did not increase significantly the free sulfhydryl groups in the EWP (P > 0.05), but surface hydrophobicity greatly increased after HP-US treatment. HP-US treatment changed the secondary structure of the EWP, and the structure of the treated EWP was less stable because of the increase in γ-random coil, especially under the continuous dual frequency of 20/40 kHz. Finally, the particle size of the HP-US-treated EWP was greatly reduced compared with that of the untreated EWP. More water molecules would be trapped in the EWP gel structure as free water after HP-US treatment. Hence, the HP-US, which is a green technology, can improve the physicochemical properties of EWP because of its sonochemical effect on the molecular conformation of protein. Acknowledgements This work was supported by the National Key Research and Devel opment Program of China (2018YFD0400303), Program of National Natural Science Foundation of China (31701530). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jfoodeng.2020.109902. References Andrea, B.S., Jelena, R.J., Marina, B.D., 2017. Effect of the controlled high-intensity ultrasound on improving functionality and structural changes of egg white proteins. Food Bioprocess Technol. 10, 1224–1239. Arzeni, C., Martínez, K., Zema, P., et al., 2012a. Comparative study of high intensity ultrasound effects on food proteins functionality. J. Food Eng. 108, 463–472. Arzeni, C., P� erez, O.E., Pilosof, A.M.R., 2012b. Functionality of egg white proteins as affected by high intensity ultrasound. Food Hydrocolloids 29, 308–316. Beck, S.M., Knoerzer, K., Sellahewa, J., et al., 2017. Effect of different heat-treatment times and applied shear on secondary structure, molecular weight distribution, solubility and rheological properties of pea protein isolate as investigated by capillary rheometry. J. Food Eng. 208, 66–76. Beilen, J.B.V., Li, Z., 2002. Enzyme technology: an overview. Curr. Opin. Biotechnol. 13, 338–344. Chandrapala, J., Zisu, B., Palmer, M., et al., 2010. Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrason. Sonochem. 8, 951–957.
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Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng
Effects of single- and dual-frequency ultrasound on the functionality of egg white protein Sun Jun, Mu Yaoyao, Jing Hui, Mohammed Obadi, Chen Zhongwei, Xu Bin * School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Egg white protein Ultrasound frequency Physicochemical properties Secondary structure
The effects of high-power ultrasound (HP-US) treatment with different frequencies, temperatures, and durations on the physicochemical and structural properties of untreated and HP-US-treated egg white protein (EWP) were evaluated. Results indicated that the solubility of EWP was significantly improved after HP-US treatment, especially under the continuous dual frequency of 20/40 kHz. The foaming capacity of the EWP also depended on the treatment frequency, temperature, and duration, and foaming stability increased considerably in the HPUS-treated EWP compared with the untreated one. The particle size of the HP-US-treated EWP greatly decreased compared with that of untreated EWP. Results of low-field nuclear magnetic resonance analysis indicated that more water molecules were trapped in the EWP gel structure as free water after HP-US treatment. Therefore, the green technology of HP-US treatment can improve the physicochemical properties of EWP because of its sono chemical effect on the molecular conformation of EWP.
1. Introduction Egg white protein (EWP), which contains nearly 90% of proteins as functional ingredients in food processing, is attracting considerable attention because of its excellent nutritional and functional properties, such as foaming and gelation properties. The EWP contains 40 different proteins, with its main protein fractions as ovalbumin (~55%), ovo transferrin (~12%), ovomucoid (~11%), and lysozyme (~3.5%) (Powrie and Nakai, 1986; Andrea et al., 2017). The functional properties of the EWP powder cannot meet the current multiple needs of customers. Thus, the effective modification methods for EWP processing are being developed due to the insufficient supply for the new demand of EWP powder. The functional properties of proteins can be modified by physical, chemical, or enzymatic treatments. The application of chemical methods to modify protein in the food industry is still limited, because the chemical reagents used are harmful to human health (Mirmoghtadaie et al., 2016). The popularity and application of enzymatic modification is also limited because of the high cost of the enzymes (Beilen and Li, 2002). Therefore, the physical modification of protein has attracted wide attention. Among the physical methods for protein modification, ultrasound has been paid special interest. Ultrasound, with frequency above the threshold of human hearing (�16 kHz) is generally considered
as a safe, non-toxic, and environment-friendly technique (Knorr et al., 2004). Ultrasound can be classified into high-frequency low-energy diagnostic ultrasound (100 kHz–1 MHz) and low-frequency high power ultrasound (HP-US, 16 kHz–100 kHz) (Soria et al., 201 0). HP-US is primarily based on the high shear force and turbulence generated by the cavitation to break covalent bonds in protein (Chemat et al., 2017; Mason et al., 2005). Thus, HP-US is a rapid, efficient, and reliable technique to improve the functional quality of proteins. At present, the impact of ultrasound on the functional properties of animal and vegetable proteins, such as whey protein (Gordon and Pilosof, 2010; Jambrak et al., 2008), soy protein isolate/concentrate (Hu et al., 2013), millet protein concentrate (Nazari et al., 2018), rice protein (Yang et al., 2017), and EWP (Arzeni et al., 2012a, 2012b; Knorr et al., 2004), are increasingly being investigated. Jambrak et al. (2008) stud ied the effect of ultrasound on the solubility and foaming properties of whey protein and observed that ultrasound treatment at 20 kHz can affect these properties of whey proteins. Meanwhile, ultrasound treat ment at 40 kHz shows less effect on the properties of whey protein, but ultrasound treatment at 500 kHz displays no effect on the foaming properties (Chemat et al., 2017). Arzeni et al. (2012a, 2012b) studied the impact of US (20 kHz, 4.27 � 0.71 W) on the functionality of whey protein concentrate, soy protein isolate, and EWP. The results show that ultrasound promotes a decrease in the consistency index of all protein
* Corresponding author. E-mail addresses: [email protected] (S. Jun), [email protected] (X. Bin). https://doi.org/10.1016/j.jfoodeng.2020.109902 Received 13 September 2019; Received in revised form 19 October 2019; Accepted 2 January 2020 Available online 11 January 2020 0260-8774/© 2020 Elsevier Ltd. All rights reserved.
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solutions, primarily on the soy protein isolate, but the gelation of EWP is not modified by the ultrasound. Nazari et al. (2018) determined the effects of ultrasound treatment at 20 kHz (18.4, 29.58, and 73.95 W/cm2) on the functional properties of millet protein concentrate. The results show that the solubility of millet protein concentrate is sub stantially improved after ultrasound treatment, and the foaming ca pacity of the concentrate declines and increases at low and high ultrasound intensities, respectively. Recent studies have focused on the modification of proteins by using an ultrasonic equipment, primarily an ultrasonic bath and an ultrasonic probe. To the best of our knowledge, little is known about the effects of single- and dual-frequency ultrasound on the functional and structural properties of EWP. Two type of ultrasound radiation modes are available. Although ul trasound baths are more widely used, this mode have two main disad vantages, namely, the lack of uniformity in the distribution of ultrasound and decline in power with time, that substantially decrease experimental repeatability and reproducibility. Hence, the most com mon ultrasound baths with ultrasound probes are less applied. The countercurrent ultrasound equipment manufactured by Meibo Biotech nology Co., Ltd. (Zhenjiang, Jiangsu, China) was used in this study to elucidate the effects of HP-US on the physicochemical properties of EWP. This study was performed to explore the effect of HP-US fre quencies, temperatures, and durations on the EWP to improve the physicochemical properties of the protein. Moreover, the structural properties of EWP upon HP-US treatment were analyzed to evaluate the relationship between the physicochemical and structural properties of the untreated and HP-US-treated EWP.
untreated EWP solutions were considered as the control group. All EWP solutions after HP-US treatment were lyophilized in a freeze dryer (FD1A-50, Bo Yi Kang Experimental Instrument Co., Ltd., Beijing, China) and then stored in air- and water-tight containers at room temperature until analyzed. 2.4. Characterization of the HP-US treated and untreated EWPs 2.4.1. Solubility The protein content of the supernatant was determined according to the method by Salvador et al. (2009) after calibration with bovine serum albumin (BSA) as a standard protein. After HP-US treatment, the freeze-dried EWP powders (1 g) were dispersed in 100 mL of deionized water (1% w/w) and centrifuged at 5000 rpm for 30 min at 4 � C. Protein solubility was expressed as grams of soluble protein per 100 g of initial EWP powder. 2.4.2. Foaming properties The foaming properties were determined according to the method described by Jambrak et al. (2008) with slight modification. Then, 100 mL (V0) of the untreated or HP-US treated EWP solution was dispersed with a homogenizer (Griffin & George, Co Ltd., UK) at 15 000 rpm for 5 min at 25 � C. The expanded EWP solution, which was treated by high-speed dispersion, was transferred into a 250 mL graduated flask to measure the foam volume V1. After resting for 30 min, the residual foam volume (V2) of the EWP solution was measured again. The foaming capacity (FC) and foaming stability (FS) of the EWP solution were calculated as follows:
2. Materials and methods
FCð%Þ ¼
2.1. Materials The protein content (total basis) of the EWP powder was 88.93% � 1.18% (N � 6.25) (AOAC, 1980), and the EWP powder was supplied by Jiangsu Kangde Eggs Co., Ltd. 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB) and 8-anilinonaphthalene-sulfonic acid (ANS) were purchased from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), NaH2PO4, Na2HPO4, other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
FSð%Þ ¼
V1
V2 V1
V0
� 100
(1)
V0 � 100 V0
(2)
V0
where V0 is the initial volume of the EWP solution (mL), V1 is the volume of EWP solution after dispersion, and V2 is the volume of the EWP so lution after resting for 30 min. 2.4.3. Free sulfhydryl groups The concentration of the free sulfhydryl groups of the untreated and HP-US treated EWP solutions were determined using Ellman’s reagent (DTNB solution). DTNB (50 mg) was solvated with 10 mL of Tris-glycine buffer solution (pH 8.0, 0.1 M) according to the method by Lagrain et al. (2008) with slight modification. Then, 1% freeze-dried EWP solution, which was dissolved in 0.1 M Tris-glycine buffer solutions (pH 8.0) were shaken in a water bath for 1 h at room temperature and then centrifuged at 5000 rpm for 15 min. After centrifugation, the supernatants were used to determine the free sulfhydryl groups. DTNB solutions (125 μL) were added into 3 mL of the supernatants of the EWP solution and allowed to stand for 30 min at room temperature. Then, the absorbance of the so lution was measured at 412 nm on a UV–vis spectrophotometer (Varian Inc., Palo Alto, CA, USA). The free sulfhydryl groups was calculated as follows:
2.2. Preparation of the EWP solution The EWP powders were dissolved in distilled water to obtain 10% (w/w) solution. Then, the EWP solutions were stirred in an ice-water bath for 1 h and centrifuged at 6000 rpm for 20 min. Afterward, the supernatant was collected, and the pH of the EWP solutions was adjusted to 7.0 � 0.2 by using 0.1 M HCl. 2.3. Experimental design of ultrasound treatment The effects of HP-US treatment at different frequencies, tempera tures, and durations on the physicochemical and structural properties of EWP were investigated using single- and continuous dual-frequency ultrasound equipment which equipped with probes operating at five different frequencies, including 20 (the fundamental one), 28, 35, 40 and 50 kHz. (Meibo Biotechnology Co., Ltd. Zhenjiang, Jiangsu, China). The continuous dual frequency of 20/40 kHz and 20/28 KHz refers to that two kinds of ultrasound frequency, which is generated in a suc cessive mode without interval, that is, one is pulsed on-time while the other is pulsed off-time. In addition, the treatment temperature is controlled by a thermostatic water bath, the mode of HP-US action (action 5 s, Intermittent 3 s). The sonochemical effects of HP-US treatment on the EWP solution were determined at different frequencies (20, 28, 35, 40, 50, 20/28, and 20/40 KHz), temperatures (25, 35, 45, and 55 � C), and durations (10, 20, 30, 40, 50, and 60 min). The power density was kept at 375 W/L. The
SH ðμm = gÞ EWP powder ¼ 75:53 � A412 � D = C
(3)
where C is the concentration of the EWP solution (g/mL), and D is the dilution factor (EWP is 30.2). 2.4.4. Surface hydrophobicity (H0) The H0 of the untreated and HP-US treated EWP solutions were determined with the fluorescence probe 1-anilino-8-naphathalene-sulfo nate (ANS) according to the method of Kato and Nakai (Kato and Nakai, 1980). The freeze-dried EWP powders were dissolved with phosphate buffer solution (PBS, 0.01 M, pH 7.0) to different concentrations (0.1–0.5%). ANS solution (20 μL, 0.05 M PBS, pH 7.0) was mixed with 4 2
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mL of the EWP solution, and the fluorescence intensity (FI) was deter mined by a fluorophotometer (Card-F98, Varian Inc., Palo Alto, CA, USA) at room temperature under excitation wavelength of 393 nm and emission wavelength of 481 nm. The protein concentration was plotted by FI, and the initial slope was calculated as H0. The H0 of the control EWP was defined as 100%. 2.4.5. UV–visible (UV–Vis) spectroscopy The concentration of the untreated and HP-US treated EWP solution were diluted to 1 mg/mL using 0.01 M PBS (pH 7.0). The UV–Vis spectra of the sample solutions were recorded in the wavelength range of 240–400 nm using a UV–Vis spectrophotometer (Varian Inc., Palo Alto, CA, USA) with a path length of 1.0 cm for the quartz cell at room tem perature. The spectrum of 0.01 M PBS (pH 7.0) was used as blank for the EWP solution. 2.4.6. Fourier transform infrared (FTIR) spectroscopy The secondary structure of the untreated and HP-US treated EWPs were determined by an FTIR spectrometer (Nicolet is50 spectrometer, Thermo Electron Corporation, MA, USA) in the range of 2000–1000 cm 1. For each spectrum, 32 scans at a resolution of 4 cm 1 were per formed. The conformational changes of the EWP were analyzed in the range of 1600–1700 cm 1 using PeakFit v4.12 (SeaSolve, Framigham, MA, USA). 2.4.7. Scanning electron microscopy (SEM) The effects of ultrasound treatments on the microstructure and par ticle size of the EWP were observed with a scanning electron microscopy system (S–4800II SEM, Hitachi Limited, Tokyo, Japan) at an accelera tion voltage of 15 kV. Prior to the SEM analysis, the powder samples were coated with gold in an argon atmosphere using a spatter coater. 2.4.8. Low-field nuclear magnetic resonance (LF-NMR) The relaxation time (T2) of the EWP solution and gels were deter mined using a low-field NMR analyzer (NMI20-030V–I, Niumag Co., Ltd., Suzhou, China). The sample (3 g) was placed in a 25-mm diameter NMR glass tubes and inserted in the NMR probe. The T2 relaxation times were measured using Call-Purcell-Meiboom-Gill (CPMG) sequence. The echo time, wait time, and the number of scans were set to 0.05 ms, 5000 ms, and 8, respectively. A total of 17 000 echoes were recorded for analysis. T21, T22, and T23 were designed as the relaxation components, and PT21, PT22, and PT23 were designed as the corresponding area fractions. 2.5. Statistical analysis All experimental data were presented as the means from two parallel experimental data. One-way ANOVA) was used to identify significant differences (P < 0.05). 3. Results and discussion 3.1. Solubility Solubility is the most important physicochemical property of protein that can reflect the denaturation and aggregation of protein in the so lution. Thus, this property affects other functional properties of proteins, such as foaming and gelation. As indicated in Fig. 1, the solubility of the EWP solution after HP-US treatment under the different frequencies, temperatures, and treatment times increased significantly compared with that of the untreated EWP. With increased frequency, the solubility of EWP solution initially increased and then decreased before peaking at the continuous dual-frequency mode of 20/40 kHz at 25 � C for 30 min. This phenomenon is probably due to the fact that a certain frequency of ultrasound can loosen the molecular structure of the EWP, thereby increasing the solubility. However, when the ultrasound frequency
Fig. 1. Effects of HP-US treatment on the solubility of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C.
3
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exceeded a certain intensity, EWP was denatured and aggregated, resulting in reduced solubility. At the same time, as shown in Fig. 1B, the solubility of EWP solution decreased with increased treatment temper ature from 25 � C to 55 � C probably because a small amount of aggre gated protein can be formed during sonication at higher temperature (Arzeni et al., 2012a, 2012b). Similarly, as shown by Beck et al. (2017) who reported that an increase in shear treatment resulted in an increase in protein solubility, because the application of shear could lead to a disruption of the newly formed aggregates, thus increase unfolding protein could subsequently interact with the water and increase the solubility. As indicated in Fig. 1C, the solubility of EWP solution initially increased and then decreased with prolonged treatment time from 10 min to 60 min. This phenomenon may be due to the conformation and structural changes of the EWP induced by US cavitation effects (Fig. 4). Such changes caused hydrophilic parts of amino acids to move from the interior to the surface toward water. This process increased the pro tein–water interactions and then affected the solubility of protein (Hu et al., 2013; Morel et al., 2000). Jambrak et al. (2008) obtained similar results for the solubility of whey proteins and hydrolyzed whey protein at 10% (w/w) under the 20–40 kHz treatment for 15–30 min. 3.2. Foaming properties The application of EWPs in the food industry is highly dependent on the foaming properties. Thus, foaming capacity and foaming stability, as the most important functional properties of protein, are important in the baking industry. As shown in Fig. 2A, the acoustic effect of HP-US considerably increased the foaming capacity and foaming stability of the EWP solution under 40, 20/28, and 20/40 kHz, especially under the continuous dual frequency of 20/40 kHz. Xiong et al. (2016) obtained similar results in the foaming capacity and foaming stability of oval bumin under 20 kHz treatment. Moreover, the foaming capacity of the EWP was dependent on the frequency, temperature, and duration during HP-US treatment (Fig. 2), whereas foaming stability, which was not significantly dependent on the frequency, temperature, and duration of HP-US, increased considerably, compared with that of the untreated EWP. The temperature is harmful to the improvement of foaming capacity of the EWP solution probably because of the aggregation of protein as the temperature increased. Moreover, the foaming capacity of the HP-US treated EWP solution initially increased and then decreased with prolonged treatment time from 10 min to 60 min. The maximum value of foaming capacity was reached at 30 min of treatment. The enhanced foaming properties of EWP might be attributed to the higher solubility after HP-US treatment (Fig. 1). Partial protein unfolding, which reflected the exposure of hy drophobic and free sulfhydryl groups, allowed the EWP molecule to be effectively adsorbed onto the air–water interface (Mirmoghtadaie et al., 2016), thereby promoting the foam properties of proteins. However, Xiong et al. (2016) obtained a contrasting result in their study on the effect of ultrasound on the foaming properties of ovalbumin. The discrepancy may be due to the fact that the EWP is a mixture of over 40 other proteins, so that changes in the EWP are associated with all protein fractions in the EWP rather than a single component like ovalbumin. Van der Plancken et al. (2017) pointed out that the improvement of foaming properties is primarily achieved by enhancing the hydrophobic protein–protein interactions at the interface. Thus, the exposure of hy drophobic protein and the conformational change of EWP during HP-US treatment can lead to the breakage of EWP in the internal interactions. However, in the current study, the variation in the foaming capacity and surface hydrophobicity (Fig. 4) of the EWP was just the opposite under the HP-US temperature from 25 � C to 55 � C. Hence, HP-US is considered as an effective technique to improve the foaming capacity and foaming stability of the EWP solution.
Fig. 2. Effects of ultrasonic treatment on the foaming properties of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C.
3.3. Free sulfhydryl groups As the main protein fractions of the EWP, the ovalbumin is the only component that contained 4 free thiol groups buried within the core of the protein (Sheng et al., 2018). The increased free sulfhydryl groups content can generally be ascribed to the exposure of the inner sulfhydryl, resulting in dissociation of the subunit and breaking of the disulfide bond (Sheng et al., 2018). Thus, the free sulfhydryl groups content of the untreated and HP-US treated EWP might influence the physicochemical 4
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properties, such as solubility and foaming properties. As shown in Fig. 3, the free sulfhydryl groups content initially increased and then decreased with increased ultrasound temperature and treatment time. However, the changes were not significant (P < 0.05), which agree well with the
Fig. 4. Effects of ultrasonic treatment on the surface hydrophobicity (H0) of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B. HP-US temperature researched at 40 kHz and 30 min; and C. HPUS durations researched at 40 kHz and 25 � C. Fig. 3. Effects of ultrasonic treatment on the free SH groups of the egg white protein (EWP) solution. A. HP-US frequency researched at 30 min and 25 � C; B⋅HP-US temperature researched at 40 kHz and 30 min; and C. HP-US durations researched at 40 kHz and 25 � C. 5
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results of Chandrapala et al. (2010) and Arzeni et al. (2012a, 2012b). Jin et al. (2015) studied the ultrasound treatment on corn gluten meal. The results indicated that the free sulfhydryl groups content in creases significantly after ultrasound treatment probably because the ultrasound changed the protein conformation, and more sulfhydryl groups have been exposed to the surface of proteins. However, in 2007, Gulseren et al. (2007) pointed out that the ultrasound cavitation effect can cause the oxidization of the sulfhydryl groups, resulting in decreased free sulfhydryl groups in the protein. Comprehensive analysis of existing reports show that HP-US has no significant effect on the free sulfhydryl groups of EWP. This phenomenon is due to the fact that the HP-US treatment can cause the exposure of sulfhydryl groups to the surface, but the simultaneous occurrence of the reduction of S–S (disulfide bond) to the sulfhydryl groups and the oxidization of the sulfhydryl groups caused by the heat of HP-US may lead to no significant changes in the free sulfhydryl groups content, or any transient changes that occurred was reversible. 3.4. Surface hydrophobicity The surface hydrophobicity of proteins, which is an index of the number of hydrophobic groups on the surface of a protein molecule in contact with the polar aqueous environment, can be used to evaluate the change in the protein conformation. This property impacts the physi cochemical and structural properties of protein. As shown in Fig. 4, surface hydrophobicity increased significantly after HP-US treatment (P < 0.05). The ultrasonic frequency mode with continuous dual frequency of 20/28 kHz and 20/40 kHz especially showed higher surface hydro phobicity. This result is probably due to the fact that the EWP solution subjected to ultrasonic wave and the produced bubble size is not a single value, such that a range of values exists. Therefore, the ultrasonic fre quency should be applied in a range of values. Moreover, the increase in surface hydrophobicity after HP-US treatment indicated that HP-US with frequencies of 20/28 kHz and 20/40 kHz exposed some of the hydrophobic groups, which were initially buried in the interior of the EWP molecule. This process increases the surface hydrophobicity of EWP. This phenomenon also shows that the EWP molecules might be broken under the ultrasonic cavitation, which is proved in the “sec ondary structure” section (Fig. 5). Moreover, as shown in Fig. 4B and C, surface hydrophobicity increased with increased ultrasound temperature and treatment time but decreased significantly after 60 min of HP-US treatment (P < 0.05). This result suggested that the temperature was advantageous to expose the hydrophobic groups and regions inside the molecules toward the aqueous environment. This finding is consistent with the study of Gul seren et al. (2007) on the ultrasound treatment of bovine serum albumin. Furthermore, negative correlation was found between the surface hydrophobicity and solubility which represented the hydrophilic effect of EWP in the different HP-US temperatures, which is consistent with the study of Jin et al. (2016). However, Wagner et al. (2000) showed in their study on soy protein isolates a positive correlation between surface hydrophobicity and the solubility of proteins. The main reason might be that different protein molecules have distinct molecular structures and the concentration of hydrophobic groups in and on the surface of the proteins.
Fig. 5. Effects of ultrasonic treatment on the secondary structure of the egg white protein (EWP) solution. A. UV–Vis and B. FTIR spectra.
UV–Vis spectrum can be used as one of the indices to determine the conformational change in protein, and its principle is the absorption of UV light by the side chain groups of tryptophan (279 nm), tyrosine (275 nm), and phenylalanine (257 nm) (Jin et al., 2016). Therefore, the conformational change in protein can be deduced by analyzing the po sition and intensity of the maximum absorption wavelength of protein in the UV–Vis spectra. The UV–Vis spectra of the EWP samples before and after HP-US treatment are shown in Fig. 5A. The characteristic absorption at 278 nm may be due to the combined effect of tyrosine and tryptophan. The UV absorption intensity of EWP increased after HP-US treatment, especially under the continuous dual frequency of 20/40 kHz at 25 � C for 30 min. The cavitation effect of HP-US might have changed the molecules of EWP, resulting in the exposure of more protein with UV absorption so that the UV absorption intensity of EWP increased. Moreover, FTIR can be used to quantitatively determine the sec ondary structural changes of EWP upon HP-US treatment. In the IR spectra, the bands at 1600–1700 cm 1 correspond to the amide I vi bration and N–H bending, which can be used to analyze the secondary structure of protein. Given that the α-helix, β-sheet, T-turn, and γ-random coil are related to the overall protein structures, their relative assigned percentages were analyzed. The content of α-helix, β-sheet, Tturn, and γ-random coil were quantitatively estimated using Gaussian peaks and curve fitting models according to the description of Byler and Susi (Susi and Michael Byler, 1983). The contents of the different secondary structures of the controlled
3.5. Secondary structure According to the results of solubility, foaming properties, free sulf hydryl groups, and surface hydrophobicity, the ultrasonic frequency mode with single-fixed frequency of 40 kHz and continuous dual fre quency of 20/28, 20/40 kHz at 25 � C for 30 min were chosen as the optimized ultrasonic conditions. Thus, the UV–Vis and FTIR spectra of the untreated and HP-US treated EWP were evaluated under the opti mized ultrasonic conditions. 6
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and HP-US treated EWP are shown in Fig. 5B. The secondary structure of EWP changed after HP-US treatment, and the β-sheets increased slightly after HP-US treatment, except under the continuous dual frequency of 20/40 kHz. Moreover, the HP-US-treated EWP exhibited an increase in the α-helix structure upon HP-US treatment under the continuous dual frequency of 20/28 kHz and 20/40 kHz. The results suggested that the single frequency of 40 kHz caused the unfolding of the α-helix, followed by the formation of β-sheet and T-turn. However, the continuous dual frequency of 20/40 kHz caused the unfolding of the β-sheet, followed by the formation of α-helix and γ-random coil. The differences in the sec ondary structure contents might be related to the distinct sonication effects and the shear forces of the ultrasound mechanical action, which disrupted the interactions between the protein molecules and influenced the internal structure of the protein molecule. 3.6. SEM The SEM can be used to characterize the shape and size of the protein with a higher resolution. Fig. 6 shows the microstructure and particle size of the control and HP-US-treated EWP under continuous dualfrequency mode of 20/40 kHz. The results showed that, compared with the untreated EWP, the particle sizes of the EWP powder were greatly reduced (Fig. 6B) under the HP-US treatment. This means that HP-US increased the EWP molecular surface area, thus increased the contact chance between EWP and water, this finding can also be a good explanation for the improved solubility and FC after HP-US treatment. In addition, because of the hydrophobic interaction, native EWP particles existed in sheet structure and accumulated together, and after sonication treatments, different degrees of deformation occurred with small cavities. The microstructure of the EWP by continuous dual fre quency of 20/40 kHz treatment presented a hole structure. These phe nomena are primarily based on the high shear force and turbulence generated by the cavitation and mechanical effects (Chemat et al., 2017).
Fig. 7. Effects of HP-US treatment on the water type and water content of egg white protein (EWP) gel. Table 1 Effects of HP-US treatment on T2 relaxation parameters of egg white protein (EWP) gel. sample
T21
T22
control
\
151.99 0.2a 151.99 0.2a 151.99 0.2a 151.99 0.2a
40 20/28
\
20/40
\
� � � �
T23
PT21
PT22
PT23
5722.37 � 14.50c 2848.04 � 17.68b 2848.04 � 13.42b 2477.08 � 16.81a
\
99.65 � 0.49b 96.33 � 1.47ab 96.66 � 1.43ab 95.15 � 1.10a
0.35 � 0.49a 3.67 � 1.47ab 3.34 � 1.43ab 4.85 � 1.10b
\ \ \
Mean � SD values denoted by different letters differ statistically significantly (p < 0.05). \: indicates that no signal was detected.
3.7. LF-NMR LF- NMR can be used to study the water type, water distribution, and the moisture content in food systems. Fig. 7 and Table 1 show the T2 relaxation spectra and T2 relaxation parameters of the control and HPUS-treated EWP gels. The T22 peak in the range of 10–100 ms can be attributed to the immobilized water, while the slowest T23 peak of >1000 ms represented the free/bound water. The position of T22 of the EWP before and after HP-US treatment showed no changes for the HPUS-treated EWP gels, while the T23 of the HP-US-treated EWP exhibi ted a shift to low relaxation times compared with the control EWP. These results indicated that the HP-US-treated EWP gel exhibited low water mobility compared with the control EWP gel. The HP-US treatments probably increased the hydrophobic interaction among the gel mole cules (Fig. 7). Thus, the mobility of the free water in the EWP gels
decreased and promoted the transition of the high mobility T23 component to low mobility component. Furthermore, PT22 and PT23 represent the areas of relaxation times T22 and T23, respectively. As shown in Table 1, the PT23 component of the control EWP gel increased from 0.35% to 3.67% (40 kHz), 3.35% (20/28 kHz), and 4.85% (20/40 kHz), while the PT22 component of native EWP gel decreased from 99.65% to 95.15% (20/40 kHz). These results indicated that more water molecules were trapped in the EWP gel structure as free water after HPUS treatment.
Fig. 6. Microstructure and particle sizes of the untreated and ultrasound treated (dual-fixed-frequency combination of 20/40 kHz) egg white protein (EWP). 7
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4. Conclusion
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The solubility, foaming capacity and foaming stability of HP-UStreated EWP samples were significantly higher than those of the un treated EWP (P < 0.05), especially under the continuous dual frequency of 20/40 kHz. Moreover, compared with the untreated EWP, HP-US treatment did not increase significantly the free sulfhydryl groups in the EWP (P > 0.05), but surface hydrophobicity greatly increased after HP-US treatment. HP-US treatment changed the secondary structure of the EWP, and the structure of the treated EWP was less stable because of the increase in γ-random coil, especially under the continuous dual frequency of 20/40 kHz. Finally, the particle size of the HP-US-treated EWP was greatly reduced compared with that of the untreated EWP. More water molecules would be trapped in the EWP gel structure as free water after HP-US treatment. Hence, the HP-US, which is a green technology, can improve the physicochemical properties of EWP because of its sonochemical effect on the molecular conformation of protein. Acknowledgements This work was supported by the National Key Research and Devel opment Program of China (2018YFD0400303), Program of National Natural Science Foundation of China (31701530). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jfoodeng.2020.109902. References Andrea, B.S., Jelena, R.J., Marina, B.D., 2017. Effect of the controlled high-intensity ultrasound on improving functionality and structural changes of egg white proteins. Food Bioprocess Technol. 10, 1224–1239. Arzeni, C., Martínez, K., Zema, P., et al., 2012a. Comparative study of high intensity ultrasound effects on food proteins functionality. J. Food Eng. 108, 463–472. Arzeni, C., P� erez, O.E., Pilosof, A.M.R., 2012b. Functionality of egg white proteins as affected by high intensity ultrasound. Food Hydrocolloids 29, 308–316. Beck, S.M., Knoerzer, K., Sellahewa, J., et al., 2017. Effect of different heat-treatment times and applied shear on secondary structure, molecular weight distribution, solubility and rheological properties of pea protein isolate as investigated by capillary rheometry. J. Food Eng. 208, 66–76. Beilen, J.B.V., Li, Z., 2002. Enzyme technology: an overview. Curr. Opin. Biotechnol. 13, 338–344. Chandrapala, J., Zisu, B., Palmer, M., et al., 2010. Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate. Ultrason. Sonochem. 8, 951–957.
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