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Impact of microwave assisted phosphorylation on the physicochemistry and rehydration behaviour of egg white powder
Food Hydrocolloids 100 (2020) 105380
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Impact of microwave assisted phosphorylation on the physicochemistry and rehydration behaviour of egg white powder
T
Peishan Li, Yongguo Jin∗∗, Long Sheng∗ National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Albumen Powder Structure Rehydration
Three egg white protein powders under different treatments (untreated, microwave-alone treated and microwave-assisted phosphorylation modification) were prepared and their physicochemistry and rehydration behavior were investigated in this study. Scanning electron microscopy (SEM) and physical property measurements showed that the particle size and specific surface area decreased and increased, respectively, in phosphorylated egg white protein and that the particles became looser and more porous. Fourier Transform infrared spectroscopy (FTIR) showed that phosphate groups were successfully introduced into the egg white protein side chain. After phosphorylation modification via microwave treatment of the egg white protein, the egg white protein had increased electronegativity and reduced surface hydrophobicity. Treatment with microwave heating had a negative impact on the rehydration of egg white powder, and phosphorylation modification inhibited this effect to some extent. Compared with microwave-alone treated egg white protein powder, phosphorylated egg white powder showed increased wettability, dispersibility and solubility under alkaline conditions and at 20–40 °C. The maximum solubility of phosphorylated egg white powder (95.52 g/100 g) was higher than that of native egg white powder (93.16 g/100 g) and microwave-alone treated egg white powder (92.76 g/100 g) at pH 10.0.
1. Introduction Egg white is an important resource for food processing applications, and its excellent foaming and gelling properties are widely used in the food industry (Duan et al., 2018). Additionally, egg white contains various proteins, such as ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme (Sheng et al., 2018a); thus, it can be developed as a nutrient enrichment product. In the food industry, egg white powder is more common than fresh or liquid eggs, has a lower packaging cost and is easier to store and transport. The main methods of drying egg white to powder are freeze drying and spray drying. Compared with freeze-drying technology, spray-drying technology is more frequently used in egg powder processing because it consumes less power and shorter drying time (Hammami & René, 1997). Similarly, it is easier to control some of the quality indicators of egg white powder, such as the particle size, humidity and sensory indicators. The mechanism of spray drying is as follows: the material is initially dispersed into fine liquid droplets that increase the evaporation area of water and accelerate the drying process. Then, the fine liquid droplets that contact hot air and most moisture are instantly removed. Finally, the solid in the material is
∗
dried into a powder (Vehring, Foss, & Lechuga-Ballesteros, 2007). Some scholars have studied the rehydration characteristics of various food powders, such as whey protein, skim milk, 73% high fat milk, milk protein isolate (MPI), sodium caseinate powder, sugar, and corn flour (Fitzpatrick et al., 2016). It was not possible to quantify the entire rehydration process according to traditional standard methods. The process of powder rehydration is dynamic and continuous; thus, it is difficult to fully define. To characterize the entire rehydration process, powder rehydration was identified as occurring in three sequential stages: wetting, dispersing and solubilization. Wetting is the first step, in which powder is detached into primary particles; dispersing is the stage, in which materials are released from particles into the aqueous phase; and solubilization is the critical phase, in which primary particles start to release materials from the particle surface into the liquid (Ji, Fitzpatrick, Cronin, Maguire, et al., 2016; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). It is difficult for many milk protein powders to be wet. Powder particles float on the surface of water and cannot completely sink into water, even after 20 min, because milk powder has a high protein content (> 80%), is usually hydrophobic (Carter, Patel, Barbano, & Drake, 2018; Havea, 2006;
Corresponding author. College of Food Science and Technology of Huazhong Agricultural University, Wuhan, Hubei Province, PR China. Corresponding author. E-mail addresses: [email protected] (Y. Jin), [email protected] (L. Sheng).
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https://doi.org/10.1016/j.foodhyd.2019.105380 Received 14 May 2019; Received in revised form 8 September 2019; Accepted 10 September 2019 Available online 12 September 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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respectively. The degree of phosphorylation was determined by the method of Li, Sheng, and Jin (2019). The egg white powders were digested in nitric acid, sulfuric acid, and perchloric acid and the amount of phosphorus in the digest was regarded as the total phosphorus of the protein. For the determination of inorganic phosphorus (Pi), 5 mL of 10 mg/mL sample solution was added with the same volume of 15% trichloroacetic acid and then centrifuged at 3,000 g for 20 min. The amount of phosphorus bound to proteins was evaluated as the difference between the total phosphorus and the Pi content.
Hussain, Gaiani, & Scher, 2012) and has relatively poor wettability and dispersibility (Gaiani, Scher, Schuck, Desobry, & Banon, 2009; Richard et al., 2013). However, the rehydration characteristics of egg white powder, which has a high protein content, have rarely been studied. When spray-dried egg white powder is completely rehydrated, its functional properties can be fully utilized. Therefore, its solubility affects other functional properties, such as emulsifying properties, thermo stability and gel properties (Gouda, Zu, Ma, Sheng, & Ma, 2018; Sheng et al., 2018b). Some researchers have shown that the phosphorylated protein has higher solubility than the nonphosphorylated protein, likely attributed to its higher electronegativity (Nayak, Arora, Sindhu, & Sangwan, 2006). There is a strong electrostatic repulsion between phosphorylated protein molecules, promoting their dissolution in the dispersant. Furthermore, the surface charge of protein molecules enhances the interaction between the protein molecules and water molecules, forming a hydration layer and soluble aggregates (Pelegrine & Gasparetto, 2005). Phosphorylation modification is usually carried out by dry heating or water bath heating. In previous studies (Li, Zhuo, Ma, Jin, & Long, 2018), it was shown that microwave-assisted phosphorylation could dramatically accelerate the reaction extremely and that phosphorylation of egg white protein led to better foaming properties. However, the effect of microwave-assisted phosphorylation on the powder properties remains unknown. In this study, microwave-assisted phosphorylated egg white protein powder, natural egg white powder and microwave-alone treated egg white powder were selected as the research materials. The effect of the different treatments on the rehydration properties of protein powders and the corresponding relationship among the physical properties, protein structure and rehydration characteristics were explored, laying the foundation for subsequent development of egg white protein powder.
2.4. Powder characterization 2.4.1. Particle size The particle size distribution (PSD) of the protein powders was measured by laser light scattering using a Malvern Mastersizer 2000 equipped with a Scirocco 2000 dry dispersing unit (Malvern Instruments, Worcestershire, UK). Moderate powders were added to dry the dispersing device to achieve the required opacity of the instrument, and the PSD was determined at least three times (Beck, Knoerzer, & Arcot, 2017). 2.4.2. Scanning electron microscopy (SEM) The morphologies of the egg white protein powders were imaged by a Tungsten-filament SEM (JSM-6390LV, JEOL, Japan). Before SEM, the samples were coated with approximately 20 nm gold-palladium under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The sputtered time was approximately 30 s, and the accelerating voltage was 15 kV (Schmidmeier et al., 2019). 2.4.3. Surface tension and porosity The surface area of the egg white powders was calculated by the Brunauer-Emmett-Teller (BET) method using N2 sorption experiments on a Micromeritics ASAP 2460 BET system. The samples were degassed for 8 h at 150 °C prior to the N2 sorption measurement. All specific surface areas and N2 adsorption–desorption isotherms were determined using a micropore surface area analysis system (BET, Micromeritics ASAP 2020) at 77 K and relative pressures (P/Po) of 0–0.99 (Liu, 2019).
2. Materials and methods 2.1. Materials Fresh eggs were purchased from a local farm. Tris, citric acid monohydrate, 1-anilino-8-naphthalenesulfonate (ANS), sodium hydroxide, hydrochloric acid, disodium phosphate, potassium dihydrogen phosphate and sodium chloride were of analytical grade. Sodium tripolyphosphate (STP) was purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). All other reagents were of analytical grade.
2.5. Protein characterization 2.5.1. Fourier infrared spectroscopy (FITR) Each sample was analyzed over the wave number range of 4000400 cm−1 at 25 °C using an FTIR spectrometer (Nicolet Nexus 470). The resolution of the instrument was 4 cm−1, and air was scanned as the background for each sample. Each measurement was a superposition of 32 scans. The samples were mixed with KBr and pressed to pellets before measurement.
2.2. Preparation of the egg white protein solution The egg white protein solution was prepared as follows. Egg white protein was separated from infertile eggs and equilibrated using a magnetic stirrer for 12 h at 4 °C. Next, the egg white protein was added together with twice its volume of deionized water and equilibrated with a magnetic stirrer for 6 h at 4 °C, and then, insoluble protein was removed. This solution was used to prepare egg white protein powder.
2.5.2. Hydrophobicity (Ho) The 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL egg white dispersions were dissolved in 0.1 mol/L phosphate buffer (pH 7.2). Next, 4 mL of each dilution was added to 20 μL of 8 mmol/L ANS solution (0.1 mol/L phosphate buffer solution, pH 7.2). The fluorescence intensity was then determined using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The ANS solution lacking protein was used to correct for background fluorescence. Ho was determined according to the slope method used in a previous paper (Sheng et al., 2017a).
2.3. Preparation of phosphorylated egg white protein powder STP was dissolved in the egg white protein solution to form a 22.28 g/L mixed solution at pH 8 by adjusting the pH with citric acid monohydrate (pH 2). Next, the solutions were placed into a microwave reactor (PLC microwave reactor; Guangzhou Kewei Microwave Energy Co., Ltd) at 300 W for 150 s. When the reaction was finished, the solutions were immediately placed in ice water to prevent further reaction. The solutions were dialyzed for 24 h at 4 °C against deionized water to remove free phosphate. Finally, powders were obtained using a spray dryer, in which the outlet temperature was 170 °C and the speed of fluid flow was 1200 mL/h. The phosphorylated samples are referred to as PP-EWP. The blank control group and the microwave-alone treated group were called native egg protein (N-EWP) and MW-EWP,
2.5.3. Zeta potential Each sample (0.1 mg/mL) was dissolved in 0.1 mol/L phosphate buffer solution (pH 7.2). After filtration through a 0.45 mm microporous membrane, the diluted samples were directly injected into the chamber of a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcester-shire, UK) prior to zeta potential analysis at 25 °C (Gouda 2
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degree of phosphorylation of the proteins. Scanning electron micrographs of spray-dried egg white powder particles under different treatments are shown in Fig. 1. The spray-dried egg white protein particles exhibited loose and porous microspheres and had uneven particles. After microwave-alone treatment, the microstructure of the egg white protein particles showed no significant change. Fig. 1 and Table 1 show that, compared with the N-EWP (5.79 μm) samples, the particle size of the MW-EWP powder (5.32 μm) was smaller, the number of small particles adhering to the large particles was reduced, and the particle distribution became more uniform. The PP-EWP powder particle size (4.74 μm) and particle distribution were more reduced and uniform and there was less particle aggregation. The specific surface area refers to the entire surface area of the powder per unit mass or volume. Small particles had a larger specific surface area than large particles (Lv & Wang, 2012)—that is, PP-EWP had a large specific surface area (6.04 m2/g). The results of the microscopic observations were consistent with the data presented in Table 1. Particles with a larger specific surface area were more difficult to wet and were prone to undergo particle agglomeration (Yang & Hu, 2000). The pore volume is the total pore volume of the unit mass of the porous material. Table 1 shows that, compared with the N-EWP sample, the total volume of the MW-EWP powder particles was higher and that the total volume of PP-EWP was even higher than that of the MW-EWP powder. However, no significant difference in pore size was found among the three samples. The results suggested that microwave-alone treatment and phosphorylation modification might have little effect on the pore size of spray-dried egg white powder. The pore size and pore volume results indicated that PP-EWP powder became more porous, which might be attributed to a more uniform particle distribution. In summary, the particle size of PP-EWP decreased and its structure became looser and more porous.
et al., 2018). 2.6. Powder rehydration 2.6.1. Contact angles by spreading wetting The contact angle was the tangent angle at the point of contact of three phases (liquid, solid and air) and is a widely used parameter to determine the wettability of powders. Because wetting behavior is considered a dynamic process, it was necessary to monitor the change rate of the contact angle to quantify the wetting process (Yuan & Lee, 2013). Approximately 20 mg of N-EWP, PP-EWP and MW-EW powders was loaded into the testing platform and smoothed surface, which was necessary to determine the correct contact point and calculate the angle. The syringe filled with ultrapure water was mounted on the OCA15EC's auto-injector control system. The setting parameters were a 4 μL water droplet volume and medium titration rate. The contact angles at 0 s, 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s and 90 s were recorded. The measurement of each sample was repeated at least 5 times (Ji et al., 2016b). 2.6.2. Particle size measurement by laser light scattering method The laser light scattering method based on the Malvern Mastersizer 2000 system (Malvern Instruments Ltd, Worcestershire, UK) was used to quantify the powder dispersion process in terms of the decreasing rate of the particle size. The detailed measurement principle of the machine is described in the study of Ji, Cronin, Fitzpatrick, Fenelon, and Song (2015). To reach the ideal obscuration range of the machine, approximately 1,000 mg was added to the dispersion unit with 1,000 mL distilled water at a temperature of 25 °C ± 2 °C, and then, it was agitated at 2000 rpm. Triplicate measurements were carried out for each powder. The particle size distributions were recorded every 2 min in 30 min. 2.6.3. Solubility of powders at different pH and temperature Protein suspensions were prepared at 10 mg/mL and were then adjusted to pH 3.0–10.0 by 0.1 mol/L NaOH or 0.1 mol/L HCl at 20 °C or were incubated at 20–80 °C for 30 min at pH 7.0. The solutions were mixed for 0.5 h and then centrifuged at 3000 g for 15 min at 20 °C. The concentration of supernatant proteins and total proteins was determined by Coomassie brilliant blue staining and according to the total nitrogen content in the sample, separately. The solubility (%) was calculated using the following equation:
3.2. Protein structure 3.2.1. Fourier infrared spectroscopy The FTIR results of the N-EWP, MW-EWP and PP-EWP samples are shown in Fig. 2. Compared with the MW-EWP and N-EWP samples, PPEWP showed new absorption peaks at wave numbers of 976 cm−1 and 894 cm−1, which belonged to P–O stretching and P]O stretching, respectively. This result was consistent with that of Tang, Yu, Lu, Fu, and Cai (2019), indicating that the egg white protein side chain was successfully introduced to phosphate groups. The infrared region of protein has several characteristic absorption bands that can be used to study secondary structures, such as the amide I band (1600-1700 cm−1) and amide II band (1480-1575 cm−1) (Sheng et al., 2017b). Compared with the N-EWP sample, PP-EWP and MW-EWP had a slightly lower wave number for the amide I and amide II bands, indicating that microwavealone treatment and phosphorylation modification had an impact on the secondary structure. The wave numbers at 3298 cm−1 and 1397 cm−1 were assigned to –NH and –CN stretching, respectively. Compared with the N-EWP sample, PP-EWP had an approximately a 2 cm−1 red shift at –NH stretching and a 2 cm−1 blue shift at –CN stretching, while MW-EWP only had a 1 cm−1 blue shift at –CN stretching. In general, phosphorylation modification might cause further changes in the protein structure.
Solubility (%) = the content of protein in the supernatant / total content of protein in the sample × 100%
2.7. Statistical analysis The results are expressed as the means ± standard deviation and were analyzed using SPSS 19.0 software (SPSS Statistical Software, Inc., IL, USA). One-way analysis of variance (ANOVA) was used to the analyze data, and Duncan's multiple-range tests were conducted to determine differences between means. The results were considered statistically significant at P < 0.05. 3. Results and discussion 3.1. Physical properties
3.2.2. Hydrophobicity (Ho) Surface hydrophobicity is typically measured to assess protein conformational changes (Cui, Li, Lu, Liu, & Duan, 2019). As shown in Fig. 3, the MW-EWP sample behaved more hydrophobically than the NEWP sample, which might be attributed to the microwave-alone treatment causing the hydrophobic group hidden inside to be exposed (Jiang et al., 2014). The Ho of PP-EWP was significantly reduced, in agreement with the results of Xiong, Zhang, and Ma (2016). The Ho of
It could been seen in Table 1 that degree of phosphorylation of untreated, microwave-alone and phosphorylated samples had been determined, which were 0.60 mg/g, 0.57 mg/g and 1.79 mg/g. In our previous study (Li et al., 2019), we found that moderate microwavealone treatment could promote the egg white proteins phosphorylation levels, but excessive microwave treatment had a negative effect on the 3
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Table 1 Degree of phosphorylation, BET specific surface area, pore volume, pore size and particle size of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave-assisted (PP-EWP). Sample
Degree of phosphorylation(mg/g)
BET specific surface area(m2/g)
Pore volume(cm3/g)
Pore size(nm)
D(4,3) (μm)
N-EWP MW-EWP PP-EWP
0.60 ± 0.02a 0.57 ± 0.03a 1.79 ± 0.04b
4.68 ± 0.05c 5.60 ± 0.05b 6.04 ± 0.07a
0.0067 ± 0.0003c 0.0081 ± 0.0001b 0.0094 ± 0.0001a
3.70 ± 0.04b 3.94 ± 0.05a 3.75 ± 0.03b
5.79 ± 0.13a 5.32 ± 0.07b 4.74 ± 0.18c
Mean ± standard deviation values (n = 3) in the same row denoted with different letters indicate significant differences between groups (P < 0.05).
Fig. 1. Scanning electron micrography (SEM) of spray-dried native egg white protein powder (A1, A2, A3), egg white protein powder with microwave treatment (B1,B2,B3) and phosphorylated protein powder with microwave treatment (C1, C2, C3).
glutinous rice had increased electronegativity and an increased absolute value of the zeta potential due to the connection of negatively charged phosphate groups with the protein (Wang, Zhang, Fan, Yang, & Chen, 2019). No significant difference was found in the zeta potential between N-EWP and MW-EWP (P < 0.05). This result indicated that short-term microwave radiation might have no significant effect on the distribution of the surface charge.
phosphorylated ovalbumin at pH 7.0 and 9.0 was lower than that of native ovalbumin. Phosphorylation modification could restrain further exposure of the hydrophobic groups, instead rearranging them inside the protein molecules (Stănciuc, Banu, Turturică, & Aprodu, 2016). 3.2.3. Zeta potential The zeta potential is a scientific term for the electromotive force in colloidal systems and is commonly used to indicate the stability of protein suspension (Sheng et al., 2019). The zeta potentials of the NEWP, PP-EWP and MW-EWP samples were measured. As shown in Fig. 4, the absolute zeta potential value of the PP-EWP sample (−20.70 mV) was higher than those of MW-EWP and N-EWP (P < 0.05), suggesting the phosphorylated protein solution was more stable than the other samples. This result was in agreement with the previous finding that phosphorylated rice glutelin isolated from
3.3. Powder rehydration 3.3.1. Wetting The contact angle (θ) is a common indicator that is used to evaluate the wettability of powders (Gao & Mccarthy, 2006). The change in the θ value as a function of time quantifies the wetting dynamics (Mittal & Mittal, 2009). When particles contact water, a film is formed on the 4
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Fig. 2. FTIR spectra of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
Fig. 3. Surface hydrophobicity (Ho) of native egg white protein powder (NEWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP). Values are expressed as the means ± standard deviation (n = 3). Different letters indicate significant differences between groups (P < 0.05).
Fig. 4. Zeta potential of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP). Values are expressed as the means ± standard deviation (n = 3). Different letters indicate significant differences between groups (P < 0.05).
interface between the powder and water to prevent water from further penetrating the powder matrix. Thus, it would take a long time for water droplets to penetrate into the powder (Ji, Fitzpatrick, Cronin, Maguire, et al., 2016). As seen in Fig. 5, the contact angles of N-EWP, PP-EWP and MW-EWP were 135.57°, 137.67° and 142.85° at 0 s, respectively. This result showed that all the samples not only had a large initial contact angle but were also all above 130°. Additionally, the contact angle was only reduced by approximately 21° over 90 s among all the samples. The MW-EWP sample was the most difficult to wet, followed by PP-EWP. Particle size and surface tension affected the wetting behavior, and the smaller the particles, the less likely they were to be wetted. Because low porosity or small-sized particles might have capillary effects (Depalo & Santomaso, 2013), loose and porous structures or large particles had more advantages and water can flow freely inside the powder particles (Forny, Marabi, & Palzer, 2011). According to the SEM observations and the physical properties of the particles, the PP-EWP sample had a looser and more porous structure but the smallest
particle size and largest specific surface area. Thus, at 0 s, the PP-EWP powder was less hydrophilic than the N-EWP powder. At 90 s, the contact angle of all the samples continuously decreased due to some water entering the interior of the powder. The contact angle of N-EWP and PP-EWP deceased to 129.78° and 133.92° at 1 s, respectively, and both rates of deduction were approximately 4°/s. However, the rate of deduction of the MW-EWP sample deceased from 142.85° to 140.60°, only approximately 2°/s. The rapid decrease in the contact angle mainly occurred over the first 10 s, consistent with the result of milk protein wettability (Ji et al., 2016b). Additionally, the trends of the three samples were similar at 10–90 s. It was speculated that when water partially enters the interior of the powder, microwave-alone treatment and phosphorylation modification had no significant effect on the wettability. The strategies for improving the wettability were to overcome the film formed at the interface between the particles and water 5
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Fig. 7. Solubility of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP) at pH 3–10.
Fig. 5. Dynamic contact angle of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
other samples. The methods for increasing the rate of dispersion are usually the addition of mineral salts, phosphate or citrate solutions (Schuck et al., 2002) or physical methods, such as ultrasound (Mccarthy, Kelly, Maher, & Fenelon, 2014). Loose porous structures and phosphates groups introduced into PP-EWP were responsible for its increased dispersibility. Additionally, the final particle size (91.66 μm) of PP-EWP showed no significant difference compared with that of NEWP (94.48 μm). However, the final particle size of MW-EWP (174.96 μm) was larger than that of the others. This result might be attributed to microwave-alone treatment leading to denaturation of the protein and forming a large particle size-denatured protein aggregate. Protein phosphorylation modification could inhibit the formation of denatured protein aggregates to some extent and even reduce the formation of aggregates.
or to accelerate the capillary flow, which is a primary limiting rate factor of the rehydration process of spray-dried egg white protein powder.
3.3.2. Dispersion Generally, as primary particles are released into the dispersant, the corresponding particle size decreases (Fang et al., 2011). Therefore, observing changes in the particle size distribution (PSD) is a suitable method to monitor the dispersion of protein powder (Mimouni et al., 2009). As shown in Fig. 6, at 0 s, the particle size (D50) of the N-EWP (758.82 μm) sample was approximately 1.3 times larger than that of PPEWP (542.66 μm) and MW-EWP (597.04 μm), suggesting the powder granules of N-EWP easily aggregated to form larger granules. During the first 2 min, the particle size (D50) of N-EWP decreased the most, followed by PP-EWP and MW-EWP. This phenomenon might be due to the adhesion of large particles to small particles of N-EWP powder. When the aggregate began to contact water, small particles quickly fell off the surface of large particles, causing a rapid drop in particle size. As the powder further rehydrated, the PP-EWP dispersed faster than the
3.3.3. Solubilization The solubilities of N-EWP, MW-EWP and PP-EWP at different pH values and temperatures are shown in Fig. 7 and Fig. 8. Fig. 7 shows that all the samples under alkaline conditions were more soluble than
Fig. 6. Dynamic particle size change of native egg white protein powder (NEWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
Fig. 8. Solubility of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP) at 20–80 °C. 6
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for the Central Universities (Program No. 2662018JC022) and Modern Agro-Industry Technology Research System (Project No. CARS-40-K24).
those under acidic conditions. N-EWP, MW-EWP and PP-EWP achieved maximum solubility at pH 10.0: 93.16 g/100 g, 92.76 g/100 g, and 95.52 g/100 g, respectively. These results were consistent with the previous finding of Kakalis and Regenstein (2010). Spray-dried egg white powder had better solubility under higher pH conditions. The proteins had minimal solubility at the isoelectric point. The lowest solubility of N-EWP and MW-EWP was at pH 4.0, while that of PP-EWP was at pH 3.0. The isoelectric point of the PP-EWP sample might shift to a lower pH. Combined with the zeta potential and hydrophobicity data, PP-EWP had the greatest electronegativity of all samples at pH 7.4. Thus, there was greater electrostatic repulsion between phosphorylated protein molecules, which could promote dissolution in the dispersant. Additionally, the surface charge of the protein molecule enhanced the interaction between the protein molecules and water molecules, forming a hydration layer and soluble aggregates (Pelegrine & Gasparetto, 2005). Additionally, the PP-EWP sample had fewer hydrophobic groups on the surface and thus higher solubility in water than the other two groups. Fig. 8 shows that as the temperature increased, the solubility of all the samples first increased and then decreased. The initial improvement in the temperature did not cause thermal denaturation of the protein and promoted protein solubility because large molecules of protein could be dispersed into many soluble small-molecule aggregates when heated to some extent (Sorgentini, Wagner, & Anon, 1995). However, too-high temperatures might cause thermal denaturation of proteins, resulting in decreased solubility (Guo et al., 2012). When the temperature was 20–40 °C, the solubility of PP-EWP was better than that of the other two samples. The egg white protein globular molecule with exposed hydrophobic groups was more likely to form insoluble aggregates. N-EWP and MW-EWP had maximum solubilities at 50 °C (90.30 g/100 g and 88.10 g/100 g, respectively), while PP-EWP had a maximum solubility of 91.47 g/100 g at 40 °C. When the temperature was higher than 50 °C, the solubility of all samples showed a downward trend. At 80 °C, the solubility of all the samples was the lowest, and that of the PP-EWP sample was lower than that of the other two samples. These results indicated that the heat resistance of PP-EWP at 80 °C was inferior to that of N-EWP and MW-EWP.
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(2019). Enhancement of dewaterability and heavy metals solubilization of waste activated sludge conditioned by natural vanadium-titanium magnetite-activated peroxymonosulfate oxidation with rice husk. Chemical Engineering Journal, 359, 217–224. Li, P., Zhuo, S., Ma, M., Jin, Y., & Long, S. (2018). Effect of microwave-assisted phosphorylation modification on the structural and foaming properties of egg white powder. LWT- Food Science and Technology, 97, 151–156. Lv, J., & Wang, Z. (2012). Review of urban atmospheric dust fall research methods. Northern Environment, 23(7), 97–99. Mccarthy, N. A., Kelly, P. M., Maher, P. G., & Fenelon, M. A. (2014). Dissolution of milk protein concentrate (MPC) powders by ultrasonication. Journal of Food Engineering, 126(126), 142–148. Mimouni, A., Deeth, H. C., Whittaker, A. K., Gidley, M. J., & Bhandari, B. R. (2009). Rehydration process of milk protein concentrate powder monitored by static light scattering. Food Hydrocolloids, 23(7), 1958–1965. Mittal, K. L., & Mittal, K. L. (2009). Contact angle, wettability and adhesion. Advances in Chemistry, 43(4 Pt 1), 888–892. Nayak, S. K., Arora, S., Sindhu, J. S., & Sangwan, R. B. (2006). Effect of chemical phosphorylation on solubility of buffalo milk proteins. International Dairy Journal, 16(3), 268–273. Pelegrine, D. H. G., & Gasparetto, C. A. (2005). Whey proteins solubility as function of temperature and pH. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 38(1), 77–80. Richard, B., Page, J. F. L., Schuck, P., Andre, C., Jeantet, R., & Delaplace, G. (2013).
4. Conclusion In this work, the rehydration properties of spray-dried egg protein powders under different treatments (microwave-alone and phosphorylation modification) and their agglomerates were investigated. The introduced phosphate groups and microwave-alone treatment caused changes in the physical properties and protein structure of spray-dried protein powder particles, affecting the rehydration properties. The smaller particle size of the modified phosphorylated powder caused a capillary effect and hindered the flow of water. The dispersibility of phosphorylated protein was improved due to the addition of hydrophilic groups to the side chain. However, microwave assistance had a certain effect on the powder properties, and the final particle size was not significantly different from that of the natural egg white powder. Phosphorylated egg white powder had better solubility than the other two samples at alkaline pH and 20–40 °C. Conflicts of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgement This research was supported by the National Natural Science Foundation of China (No. 31701622), the Fundamental Research Funds 7
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phosphorylation on the structural and foaming properties of ovalbumin under wetheating conditions. Food Hydrocolloids, 91, 166–173. Stănciuc, N., Banu, I., Turturică, M., & Aprodu, I. (2016). pH and heat induced structural changes of chicken ovalbumin in relation with antigenic properties. International Journal of Biological Macromolecules, 93, 572–581. Tang, S., Yu, J., Lu, L., Fu, X., & Cai, Z. (2019). Interfacial and enhanced emulsifying behavior of phosphorylated ovalbumin. International Journal of Biological Macromolecules, 131, 293–300. Vehring, R., Foss, W. R., & Lechuga-Ballesteros, D. (2007). Particle formation in spray drying. Journal of Aerosol Science, 38(7), 728–746. Wang, Y.-R., Zhang, B., Fan, J.-L., Yang, Q., & Chen, H.-Q. (2019). Effects of sodium tripolyphosphate modification on the structural, functional, and rheological properties of rice glutelin. Food Chemistry, 281, 18–27. Xiong, Z., Zhang, M., & Ma, M. (2016). Emulsifying properties of ovalbumin: Improvement and mechanism by phosphorylation in the presence of sodium tripolyphosphate. Food Hydrocolloids, 60, 29–37. Yang, S. B., & Hu, H. Q. (2000). Development of surface area and pore structure of carbon adsorbent with large surface area. Journal of Fuel Chemistry and Technology, 28(5), 476–477. Yuan, Y., & Lee, T. R. (2013). Contact angle and wetting properties. Surface Science Techniques, 3–34.
Towards a better control of dairy powder rehydration processes. International Dairy Journal, 31(1), 18–28. Schmidmeier, C., Wen, Y., Drapala, K. P., Dennehy, T., McGuirke, A., Cronin, K., et al. (2019). The effect of agglomerate integrity and blending formulation on the mechanical properties of whey protein concentrate powder tablets. Journal of Food Engineering, 247, 160–167. Schuck, P., Davenel, A., Mariette, F., Briard, V., Méjean, S., & Piot, M. (2002). Rehydration of casein powders: Effects of added mineral salts and salt addition methods on water transfer. International Dairy Journal, 12(1), 51–57. Sheng, L., He, Z., Chen, J., Liu, Y., Ma, M., & Cai, Z. (2017a). The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk. International Journal of Biological Macromolecules, 96, 129–136. Sheng, L., Huang, M., Wang, J., Xu, Q., Hammad, H. H. M., & Ma, M. (2018a). A study of storage impact on ovalbumin structure of chicken egg. Journal of Food Engineering, 219, 1–7. Sheng, L., Su, P., Han, K., Chen, J., Cao, A., Zhang, Z., et al. (2017b). Synthesis and structural characterization of lysozyme–pullulan conjugates obtained by the maillard reaction. Food Hydrocolloids, 71, 1–7. Sheng, L., Wang, Y., Chen, J., Zou, J., Wang, Q., & Ma, M. (2018b). Influence of highintensity ultrasound on foaming and structural properties of egg white. Food Research International, 108, 604–610. Sheng, L., Ye, S., Han, K., Zhu, G., Ma, M., & Cai, Z. (2019). Consequences of
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Impact of microwave assisted phosphorylation on the physicochemistry and rehydration behaviour of egg white powder
T
Peishan Li, Yongguo Jin∗∗, Long Sheng∗ National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Albumen Powder Structure Rehydration
Three egg white protein powders under different treatments (untreated, microwave-alone treated and microwave-assisted phosphorylation modification) were prepared and their physicochemistry and rehydration behavior were investigated in this study. Scanning electron microscopy (SEM) and physical property measurements showed that the particle size and specific surface area decreased and increased, respectively, in phosphorylated egg white protein and that the particles became looser and more porous. Fourier Transform infrared spectroscopy (FTIR) showed that phosphate groups were successfully introduced into the egg white protein side chain. After phosphorylation modification via microwave treatment of the egg white protein, the egg white protein had increased electronegativity and reduced surface hydrophobicity. Treatment with microwave heating had a negative impact on the rehydration of egg white powder, and phosphorylation modification inhibited this effect to some extent. Compared with microwave-alone treated egg white protein powder, phosphorylated egg white powder showed increased wettability, dispersibility and solubility under alkaline conditions and at 20–40 °C. The maximum solubility of phosphorylated egg white powder (95.52 g/100 g) was higher than that of native egg white powder (93.16 g/100 g) and microwave-alone treated egg white powder (92.76 g/100 g) at pH 10.0.
1. Introduction Egg white is an important resource for food processing applications, and its excellent foaming and gelling properties are widely used in the food industry (Duan et al., 2018). Additionally, egg white contains various proteins, such as ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme (Sheng et al., 2018a); thus, it can be developed as a nutrient enrichment product. In the food industry, egg white powder is more common than fresh or liquid eggs, has a lower packaging cost and is easier to store and transport. The main methods of drying egg white to powder are freeze drying and spray drying. Compared with freeze-drying technology, spray-drying technology is more frequently used in egg powder processing because it consumes less power and shorter drying time (Hammami & René, 1997). Similarly, it is easier to control some of the quality indicators of egg white powder, such as the particle size, humidity and sensory indicators. The mechanism of spray drying is as follows: the material is initially dispersed into fine liquid droplets that increase the evaporation area of water and accelerate the drying process. Then, the fine liquid droplets that contact hot air and most moisture are instantly removed. Finally, the solid in the material is
∗
dried into a powder (Vehring, Foss, & Lechuga-Ballesteros, 2007). Some scholars have studied the rehydration characteristics of various food powders, such as whey protein, skim milk, 73% high fat milk, milk protein isolate (MPI), sodium caseinate powder, sugar, and corn flour (Fitzpatrick et al., 2016). It was not possible to quantify the entire rehydration process according to traditional standard methods. The process of powder rehydration is dynamic and continuous; thus, it is difficult to fully define. To characterize the entire rehydration process, powder rehydration was identified as occurring in three sequential stages: wetting, dispersing and solubilization. Wetting is the first step, in which powder is detached into primary particles; dispersing is the stage, in which materials are released from particles into the aqueous phase; and solubilization is the critical phase, in which primary particles start to release materials from the particle surface into the liquid (Ji, Fitzpatrick, Cronin, Maguire, et al., 2016; Mimouni, Deeth, Whittaker, Gidley, & Bhandari, 2009). It is difficult for many milk protein powders to be wet. Powder particles float on the surface of water and cannot completely sink into water, even after 20 min, because milk powder has a high protein content (> 80%), is usually hydrophobic (Carter, Patel, Barbano, & Drake, 2018; Havea, 2006;
Corresponding author. College of Food Science and Technology of Huazhong Agricultural University, Wuhan, Hubei Province, PR China. Corresponding author. E-mail addresses: [email protected] (Y. Jin), [email protected] (L. Sheng).
∗∗
https://doi.org/10.1016/j.foodhyd.2019.105380 Received 14 May 2019; Received in revised form 8 September 2019; Accepted 10 September 2019 Available online 12 September 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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respectively. The degree of phosphorylation was determined by the method of Li, Sheng, and Jin (2019). The egg white powders were digested in nitric acid, sulfuric acid, and perchloric acid and the amount of phosphorus in the digest was regarded as the total phosphorus of the protein. For the determination of inorganic phosphorus (Pi), 5 mL of 10 mg/mL sample solution was added with the same volume of 15% trichloroacetic acid and then centrifuged at 3,000 g for 20 min. The amount of phosphorus bound to proteins was evaluated as the difference between the total phosphorus and the Pi content.
Hussain, Gaiani, & Scher, 2012) and has relatively poor wettability and dispersibility (Gaiani, Scher, Schuck, Desobry, & Banon, 2009; Richard et al., 2013). However, the rehydration characteristics of egg white powder, which has a high protein content, have rarely been studied. When spray-dried egg white powder is completely rehydrated, its functional properties can be fully utilized. Therefore, its solubility affects other functional properties, such as emulsifying properties, thermo stability and gel properties (Gouda, Zu, Ma, Sheng, & Ma, 2018; Sheng et al., 2018b). Some researchers have shown that the phosphorylated protein has higher solubility than the nonphosphorylated protein, likely attributed to its higher electronegativity (Nayak, Arora, Sindhu, & Sangwan, 2006). There is a strong electrostatic repulsion between phosphorylated protein molecules, promoting their dissolution in the dispersant. Furthermore, the surface charge of protein molecules enhances the interaction between the protein molecules and water molecules, forming a hydration layer and soluble aggregates (Pelegrine & Gasparetto, 2005). Phosphorylation modification is usually carried out by dry heating or water bath heating. In previous studies (Li, Zhuo, Ma, Jin, & Long, 2018), it was shown that microwave-assisted phosphorylation could dramatically accelerate the reaction extremely and that phosphorylation of egg white protein led to better foaming properties. However, the effect of microwave-assisted phosphorylation on the powder properties remains unknown. In this study, microwave-assisted phosphorylated egg white protein powder, natural egg white powder and microwave-alone treated egg white powder were selected as the research materials. The effect of the different treatments on the rehydration properties of protein powders and the corresponding relationship among the physical properties, protein structure and rehydration characteristics were explored, laying the foundation for subsequent development of egg white protein powder.
2.4. Powder characterization 2.4.1. Particle size The particle size distribution (PSD) of the protein powders was measured by laser light scattering using a Malvern Mastersizer 2000 equipped with a Scirocco 2000 dry dispersing unit (Malvern Instruments, Worcestershire, UK). Moderate powders were added to dry the dispersing device to achieve the required opacity of the instrument, and the PSD was determined at least three times (Beck, Knoerzer, & Arcot, 2017). 2.4.2. Scanning electron microscopy (SEM) The morphologies of the egg white protein powders were imaged by a Tungsten-filament SEM (JSM-6390LV, JEOL, Japan). Before SEM, the samples were coated with approximately 20 nm gold-palladium under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The sputtered time was approximately 30 s, and the accelerating voltage was 15 kV (Schmidmeier et al., 2019). 2.4.3. Surface tension and porosity The surface area of the egg white powders was calculated by the Brunauer-Emmett-Teller (BET) method using N2 sorption experiments on a Micromeritics ASAP 2460 BET system. The samples were degassed for 8 h at 150 °C prior to the N2 sorption measurement. All specific surface areas and N2 adsorption–desorption isotherms were determined using a micropore surface area analysis system (BET, Micromeritics ASAP 2020) at 77 K and relative pressures (P/Po) of 0–0.99 (Liu, 2019).
2. Materials and methods 2.1. Materials Fresh eggs were purchased from a local farm. Tris, citric acid monohydrate, 1-anilino-8-naphthalenesulfonate (ANS), sodium hydroxide, hydrochloric acid, disodium phosphate, potassium dihydrogen phosphate and sodium chloride were of analytical grade. Sodium tripolyphosphate (STP) was purchased from Aladdin Industrial Co. Ltd. (Shanghai, China). All other reagents were of analytical grade.
2.5. Protein characterization 2.5.1. Fourier infrared spectroscopy (FITR) Each sample was analyzed over the wave number range of 4000400 cm−1 at 25 °C using an FTIR spectrometer (Nicolet Nexus 470). The resolution of the instrument was 4 cm−1, and air was scanned as the background for each sample. Each measurement was a superposition of 32 scans. The samples were mixed with KBr and pressed to pellets before measurement.
2.2. Preparation of the egg white protein solution The egg white protein solution was prepared as follows. Egg white protein was separated from infertile eggs and equilibrated using a magnetic stirrer for 12 h at 4 °C. Next, the egg white protein was added together with twice its volume of deionized water and equilibrated with a magnetic stirrer for 6 h at 4 °C, and then, insoluble protein was removed. This solution was used to prepare egg white protein powder.
2.5.2. Hydrophobicity (Ho) The 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL egg white dispersions were dissolved in 0.1 mol/L phosphate buffer (pH 7.2). Next, 4 mL of each dilution was added to 20 μL of 8 mmol/L ANS solution (0.1 mol/L phosphate buffer solution, pH 7.2). The fluorescence intensity was then determined using a fluorescence spectrophotometer at an excitation wavelength of 390 nm and an emission wavelength of 470 nm. The ANS solution lacking protein was used to correct for background fluorescence. Ho was determined according to the slope method used in a previous paper (Sheng et al., 2017a).
2.3. Preparation of phosphorylated egg white protein powder STP was dissolved in the egg white protein solution to form a 22.28 g/L mixed solution at pH 8 by adjusting the pH with citric acid monohydrate (pH 2). Next, the solutions were placed into a microwave reactor (PLC microwave reactor; Guangzhou Kewei Microwave Energy Co., Ltd) at 300 W for 150 s. When the reaction was finished, the solutions were immediately placed in ice water to prevent further reaction. The solutions were dialyzed for 24 h at 4 °C against deionized water to remove free phosphate. Finally, powders were obtained using a spray dryer, in which the outlet temperature was 170 °C and the speed of fluid flow was 1200 mL/h. The phosphorylated samples are referred to as PP-EWP. The blank control group and the microwave-alone treated group were called native egg protein (N-EWP) and MW-EWP,
2.5.3. Zeta potential Each sample (0.1 mg/mL) was dissolved in 0.1 mol/L phosphate buffer solution (pH 7.2). After filtration through a 0.45 mm microporous membrane, the diluted samples were directly injected into the chamber of a Zetasizer Nano-ZS instrument (Malvern Instruments, Worcester-shire, UK) prior to zeta potential analysis at 25 °C (Gouda 2
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degree of phosphorylation of the proteins. Scanning electron micrographs of spray-dried egg white powder particles under different treatments are shown in Fig. 1. The spray-dried egg white protein particles exhibited loose and porous microspheres and had uneven particles. After microwave-alone treatment, the microstructure of the egg white protein particles showed no significant change. Fig. 1 and Table 1 show that, compared with the N-EWP (5.79 μm) samples, the particle size of the MW-EWP powder (5.32 μm) was smaller, the number of small particles adhering to the large particles was reduced, and the particle distribution became more uniform. The PP-EWP powder particle size (4.74 μm) and particle distribution were more reduced and uniform and there was less particle aggregation. The specific surface area refers to the entire surface area of the powder per unit mass or volume. Small particles had a larger specific surface area than large particles (Lv & Wang, 2012)—that is, PP-EWP had a large specific surface area (6.04 m2/g). The results of the microscopic observations were consistent with the data presented in Table 1. Particles with a larger specific surface area were more difficult to wet and were prone to undergo particle agglomeration (Yang & Hu, 2000). The pore volume is the total pore volume of the unit mass of the porous material. Table 1 shows that, compared with the N-EWP sample, the total volume of the MW-EWP powder particles was higher and that the total volume of PP-EWP was even higher than that of the MW-EWP powder. However, no significant difference in pore size was found among the three samples. The results suggested that microwave-alone treatment and phosphorylation modification might have little effect on the pore size of spray-dried egg white powder. The pore size and pore volume results indicated that PP-EWP powder became more porous, which might be attributed to a more uniform particle distribution. In summary, the particle size of PP-EWP decreased and its structure became looser and more porous.
et al., 2018). 2.6. Powder rehydration 2.6.1. Contact angles by spreading wetting The contact angle was the tangent angle at the point of contact of three phases (liquid, solid and air) and is a widely used parameter to determine the wettability of powders. Because wetting behavior is considered a dynamic process, it was necessary to monitor the change rate of the contact angle to quantify the wetting process (Yuan & Lee, 2013). Approximately 20 mg of N-EWP, PP-EWP and MW-EW powders was loaded into the testing platform and smoothed surface, which was necessary to determine the correct contact point and calculate the angle. The syringe filled with ultrapure water was mounted on the OCA15EC's auto-injector control system. The setting parameters were a 4 μL water droplet volume and medium titration rate. The contact angles at 0 s, 1 s, 2 s, 3 s, 4 s, 5 s, 10 s, 30 s, 60 s and 90 s were recorded. The measurement of each sample was repeated at least 5 times (Ji et al., 2016b). 2.6.2. Particle size measurement by laser light scattering method The laser light scattering method based on the Malvern Mastersizer 2000 system (Malvern Instruments Ltd, Worcestershire, UK) was used to quantify the powder dispersion process in terms of the decreasing rate of the particle size. The detailed measurement principle of the machine is described in the study of Ji, Cronin, Fitzpatrick, Fenelon, and Song (2015). To reach the ideal obscuration range of the machine, approximately 1,000 mg was added to the dispersion unit with 1,000 mL distilled water at a temperature of 25 °C ± 2 °C, and then, it was agitated at 2000 rpm. Triplicate measurements were carried out for each powder. The particle size distributions were recorded every 2 min in 30 min. 2.6.3. Solubility of powders at different pH and temperature Protein suspensions were prepared at 10 mg/mL and were then adjusted to pH 3.0–10.0 by 0.1 mol/L NaOH or 0.1 mol/L HCl at 20 °C or were incubated at 20–80 °C for 30 min at pH 7.0. The solutions were mixed for 0.5 h and then centrifuged at 3000 g for 15 min at 20 °C. The concentration of supernatant proteins and total proteins was determined by Coomassie brilliant blue staining and according to the total nitrogen content in the sample, separately. The solubility (%) was calculated using the following equation:
3.2. Protein structure 3.2.1. Fourier infrared spectroscopy The FTIR results of the N-EWP, MW-EWP and PP-EWP samples are shown in Fig. 2. Compared with the MW-EWP and N-EWP samples, PPEWP showed new absorption peaks at wave numbers of 976 cm−1 and 894 cm−1, which belonged to P–O stretching and P]O stretching, respectively. This result was consistent with that of Tang, Yu, Lu, Fu, and Cai (2019), indicating that the egg white protein side chain was successfully introduced to phosphate groups. The infrared region of protein has several characteristic absorption bands that can be used to study secondary structures, such as the amide I band (1600-1700 cm−1) and amide II band (1480-1575 cm−1) (Sheng et al., 2017b). Compared with the N-EWP sample, PP-EWP and MW-EWP had a slightly lower wave number for the amide I and amide II bands, indicating that microwavealone treatment and phosphorylation modification had an impact on the secondary structure. The wave numbers at 3298 cm−1 and 1397 cm−1 were assigned to –NH and –CN stretching, respectively. Compared with the N-EWP sample, PP-EWP had an approximately a 2 cm−1 red shift at –NH stretching and a 2 cm−1 blue shift at –CN stretching, while MW-EWP only had a 1 cm−1 blue shift at –CN stretching. In general, phosphorylation modification might cause further changes in the protein structure.
Solubility (%) = the content of protein in the supernatant / total content of protein in the sample × 100%
2.7. Statistical analysis The results are expressed as the means ± standard deviation and were analyzed using SPSS 19.0 software (SPSS Statistical Software, Inc., IL, USA). One-way analysis of variance (ANOVA) was used to the analyze data, and Duncan's multiple-range tests were conducted to determine differences between means. The results were considered statistically significant at P < 0.05. 3. Results and discussion 3.1. Physical properties
3.2.2. Hydrophobicity (Ho) Surface hydrophobicity is typically measured to assess protein conformational changes (Cui, Li, Lu, Liu, & Duan, 2019). As shown in Fig. 3, the MW-EWP sample behaved more hydrophobically than the NEWP sample, which might be attributed to the microwave-alone treatment causing the hydrophobic group hidden inside to be exposed (Jiang et al., 2014). The Ho of PP-EWP was significantly reduced, in agreement with the results of Xiong, Zhang, and Ma (2016). The Ho of
It could been seen in Table 1 that degree of phosphorylation of untreated, microwave-alone and phosphorylated samples had been determined, which were 0.60 mg/g, 0.57 mg/g and 1.79 mg/g. In our previous study (Li et al., 2019), we found that moderate microwavealone treatment could promote the egg white proteins phosphorylation levels, but excessive microwave treatment had a negative effect on the 3
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Table 1 Degree of phosphorylation, BET specific surface area, pore volume, pore size and particle size of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave-assisted (PP-EWP). Sample
Degree of phosphorylation(mg/g)
BET specific surface area(m2/g)
Pore volume(cm3/g)
Pore size(nm)
D(4,3) (μm)
N-EWP MW-EWP PP-EWP
0.60 ± 0.02a 0.57 ± 0.03a 1.79 ± 0.04b
4.68 ± 0.05c 5.60 ± 0.05b 6.04 ± 0.07a
0.0067 ± 0.0003c 0.0081 ± 0.0001b 0.0094 ± 0.0001a
3.70 ± 0.04b 3.94 ± 0.05a 3.75 ± 0.03b
5.79 ± 0.13a 5.32 ± 0.07b 4.74 ± 0.18c
Mean ± standard deviation values (n = 3) in the same row denoted with different letters indicate significant differences between groups (P < 0.05).
Fig. 1. Scanning electron micrography (SEM) of spray-dried native egg white protein powder (A1, A2, A3), egg white protein powder with microwave treatment (B1,B2,B3) and phosphorylated protein powder with microwave treatment (C1, C2, C3).
glutinous rice had increased electronegativity and an increased absolute value of the zeta potential due to the connection of negatively charged phosphate groups with the protein (Wang, Zhang, Fan, Yang, & Chen, 2019). No significant difference was found in the zeta potential between N-EWP and MW-EWP (P < 0.05). This result indicated that short-term microwave radiation might have no significant effect on the distribution of the surface charge.
phosphorylated ovalbumin at pH 7.0 and 9.0 was lower than that of native ovalbumin. Phosphorylation modification could restrain further exposure of the hydrophobic groups, instead rearranging them inside the protein molecules (Stănciuc, Banu, Turturică, & Aprodu, 2016). 3.2.3. Zeta potential The zeta potential is a scientific term for the electromotive force in colloidal systems and is commonly used to indicate the stability of protein suspension (Sheng et al., 2019). The zeta potentials of the NEWP, PP-EWP and MW-EWP samples were measured. As shown in Fig. 4, the absolute zeta potential value of the PP-EWP sample (−20.70 mV) was higher than those of MW-EWP and N-EWP (P < 0.05), suggesting the phosphorylated protein solution was more stable than the other samples. This result was in agreement with the previous finding that phosphorylated rice glutelin isolated from
3.3. Powder rehydration 3.3.1. Wetting The contact angle (θ) is a common indicator that is used to evaluate the wettability of powders (Gao & Mccarthy, 2006). The change in the θ value as a function of time quantifies the wetting dynamics (Mittal & Mittal, 2009). When particles contact water, a film is formed on the 4
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Fig. 2. FTIR spectra of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
Fig. 3. Surface hydrophobicity (Ho) of native egg white protein powder (NEWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP). Values are expressed as the means ± standard deviation (n = 3). Different letters indicate significant differences between groups (P < 0.05).
Fig. 4. Zeta potential of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP). Values are expressed as the means ± standard deviation (n = 3). Different letters indicate significant differences between groups (P < 0.05).
interface between the powder and water to prevent water from further penetrating the powder matrix. Thus, it would take a long time for water droplets to penetrate into the powder (Ji, Fitzpatrick, Cronin, Maguire, et al., 2016). As seen in Fig. 5, the contact angles of N-EWP, PP-EWP and MW-EWP were 135.57°, 137.67° and 142.85° at 0 s, respectively. This result showed that all the samples not only had a large initial contact angle but were also all above 130°. Additionally, the contact angle was only reduced by approximately 21° over 90 s among all the samples. The MW-EWP sample was the most difficult to wet, followed by PP-EWP. Particle size and surface tension affected the wetting behavior, and the smaller the particles, the less likely they were to be wetted. Because low porosity or small-sized particles might have capillary effects (Depalo & Santomaso, 2013), loose and porous structures or large particles had more advantages and water can flow freely inside the powder particles (Forny, Marabi, & Palzer, 2011). According to the SEM observations and the physical properties of the particles, the PP-EWP sample had a looser and more porous structure but the smallest
particle size and largest specific surface area. Thus, at 0 s, the PP-EWP powder was less hydrophilic than the N-EWP powder. At 90 s, the contact angle of all the samples continuously decreased due to some water entering the interior of the powder. The contact angle of N-EWP and PP-EWP deceased to 129.78° and 133.92° at 1 s, respectively, and both rates of deduction were approximately 4°/s. However, the rate of deduction of the MW-EWP sample deceased from 142.85° to 140.60°, only approximately 2°/s. The rapid decrease in the contact angle mainly occurred over the first 10 s, consistent with the result of milk protein wettability (Ji et al., 2016b). Additionally, the trends of the three samples were similar at 10–90 s. It was speculated that when water partially enters the interior of the powder, microwave-alone treatment and phosphorylation modification had no significant effect on the wettability. The strategies for improving the wettability were to overcome the film formed at the interface between the particles and water 5
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Fig. 7. Solubility of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP) at pH 3–10.
Fig. 5. Dynamic contact angle of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
other samples. The methods for increasing the rate of dispersion are usually the addition of mineral salts, phosphate or citrate solutions (Schuck et al., 2002) or physical methods, such as ultrasound (Mccarthy, Kelly, Maher, & Fenelon, 2014). Loose porous structures and phosphates groups introduced into PP-EWP were responsible for its increased dispersibility. Additionally, the final particle size (91.66 μm) of PP-EWP showed no significant difference compared with that of NEWP (94.48 μm). However, the final particle size of MW-EWP (174.96 μm) was larger than that of the others. This result might be attributed to microwave-alone treatment leading to denaturation of the protein and forming a large particle size-denatured protein aggregate. Protein phosphorylation modification could inhibit the formation of denatured protein aggregates to some extent and even reduce the formation of aggregates.
or to accelerate the capillary flow, which is a primary limiting rate factor of the rehydration process of spray-dried egg white protein powder.
3.3.2. Dispersion Generally, as primary particles are released into the dispersant, the corresponding particle size decreases (Fang et al., 2011). Therefore, observing changes in the particle size distribution (PSD) is a suitable method to monitor the dispersion of protein powder (Mimouni et al., 2009). As shown in Fig. 6, at 0 s, the particle size (D50) of the N-EWP (758.82 μm) sample was approximately 1.3 times larger than that of PPEWP (542.66 μm) and MW-EWP (597.04 μm), suggesting the powder granules of N-EWP easily aggregated to form larger granules. During the first 2 min, the particle size (D50) of N-EWP decreased the most, followed by PP-EWP and MW-EWP. This phenomenon might be due to the adhesion of large particles to small particles of N-EWP powder. When the aggregate began to contact water, small particles quickly fell off the surface of large particles, causing a rapid drop in particle size. As the powder further rehydrated, the PP-EWP dispersed faster than the
3.3.3. Solubilization The solubilities of N-EWP, MW-EWP and PP-EWP at different pH values and temperatures are shown in Fig. 7 and Fig. 8. Fig. 7 shows that all the samples under alkaline conditions were more soluble than
Fig. 6. Dynamic particle size change of native egg white protein powder (NEWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP).
Fig. 8. Solubility of native egg white protein powder (N-EWP), egg white protein powder with microwave treatment (MW-EWP) and phosphorylated protein powder with microwave treatment (PP-EWP) at 20–80 °C. 6
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for the Central Universities (Program No. 2662018JC022) and Modern Agro-Industry Technology Research System (Project No. CARS-40-K24).
those under acidic conditions. N-EWP, MW-EWP and PP-EWP achieved maximum solubility at pH 10.0: 93.16 g/100 g, 92.76 g/100 g, and 95.52 g/100 g, respectively. These results were consistent with the previous finding of Kakalis and Regenstein (2010). Spray-dried egg white powder had better solubility under higher pH conditions. The proteins had minimal solubility at the isoelectric point. The lowest solubility of N-EWP and MW-EWP was at pH 4.0, while that of PP-EWP was at pH 3.0. The isoelectric point of the PP-EWP sample might shift to a lower pH. Combined with the zeta potential and hydrophobicity data, PP-EWP had the greatest electronegativity of all samples at pH 7.4. Thus, there was greater electrostatic repulsion between phosphorylated protein molecules, which could promote dissolution in the dispersant. Additionally, the surface charge of the protein molecule enhanced the interaction between the protein molecules and water molecules, forming a hydration layer and soluble aggregates (Pelegrine & Gasparetto, 2005). Additionally, the PP-EWP sample had fewer hydrophobic groups on the surface and thus higher solubility in water than the other two groups. Fig. 8 shows that as the temperature increased, the solubility of all the samples first increased and then decreased. The initial improvement in the temperature did not cause thermal denaturation of the protein and promoted protein solubility because large molecules of protein could be dispersed into many soluble small-molecule aggregates when heated to some extent (Sorgentini, Wagner, & Anon, 1995). However, too-high temperatures might cause thermal denaturation of proteins, resulting in decreased solubility (Guo et al., 2012). When the temperature was 20–40 °C, the solubility of PP-EWP was better than that of the other two samples. The egg white protein globular molecule with exposed hydrophobic groups was more likely to form insoluble aggregates. N-EWP and MW-EWP had maximum solubilities at 50 °C (90.30 g/100 g and 88.10 g/100 g, respectively), while PP-EWP had a maximum solubility of 91.47 g/100 g at 40 °C. When the temperature was higher than 50 °C, the solubility of all samples showed a downward trend. At 80 °C, the solubility of all the samples was the lowest, and that of the PP-EWP sample was lower than that of the other two samples. These results indicated that the heat resistance of PP-EWP at 80 °C was inferior to that of N-EWP and MW-EWP.
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