Preparation and characterization of hen egg proteins-soybean protein isolate composite gels

Food Hydrocolloids 97 (2019) 105191

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Preparation and characterization of hen egg proteins-soybean protein isolate composite gels

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Mengqi Zhanga,b,1, Junhua Lia,b,1, Yujie Sua,b, Cuihua Changa,b, Xin Lia,b, Yanjun Yanga,b,∗, Luping Gua,b,∗∗ a b

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China

ARTICLE INFO

ABSTRACT

Keywords: Hen egg proteins Soybean protein isolate Gel properties Microstructure

The physicochemical properties, rheology, texture, water state and microstructure of gels formed from different ratio of egg proteins and soybean protein isolate were investigated. Results showed that composite solution that contained 75% egg proteins had the highest protein surface hydrophobicity while the biggest solubility was found when the proportion of egg proteins was 25%. Solid content and total sulfhydryl groups content before centrifugation increased as the proportion of egg proteins raised while total sulfhydryl groups content after centrifugation had less fluctuations because of the changes of protein solubility. Egg proteins-soybean protein isolate composite solutions had various growth ratio of G’ during different stages of solution-gel transition process, suggesting the composition of the solutions affected the molecular interactions and then changed the gelling properties. Besides, composite gels that contained the whole egg had higher hardness and lower springiness than those gels that contained egg yolk. The gel hardness after 50% addition of the whole egg and egg yolk was about 561% and 329% of the original gel strength while the gel springiness was 0.91 and 0.94, respectively. From results of microstructure, egg yolk-soybean protein isolate composite gels possessed denser gel network, which may be related with higher solid content of egg yolk. Conversely, the whole egg-soybean protein isolate composite gels showed rigid fiber network with holes, leading to better water mobility, higher hardness and lower springiness.

1. Introduction Gelation property is one of important functional properties of proteins, which provide unique texture, sensory and flavor for food products (Harfmann, 2016). Among these, the whole egg (TWE), a type of animal proteins, is commonly used as food ingredients to improve gelation properties of food products (Alamprese, Casiraghi, & Rossi, 2009; Dawson, Sheldon, & Ball, 1990; Lambrecht, Rombouts, Nivelle, & Delcour, 2017; Shimoyamada et al., 2004). TWE consists of two edible fractions, egg white (EW) and egg yolk (EY), accounting for 28.1–32.6% and 67.4–71.9%, respectively (Silversides & Budgell, 2004). Our previous study showed that EW and EY had different gelling properties, that is, EW gels were harder while EY gels had higher springiness (Zhang et al., 2019). These differences of gelling properties led to their distinct functions in pasta. It was proved that higher values of albumen/ yolk produced stiffer and tougher raw or cooked pasta sheets

(Alamprese et al., 2009). This illustrates that the use of the whole egg at natural or even greater albumen/yolk values in fresh egg pasta production should be taken into account by the producers when deciding pasta recipe. In addition, Dawson et al. (1990) found that the higher addition of spray-dried egg white into mechanically deboned chicken meat increased cook yield and decreased deformability of the gels. Lambrecht et al. (2017) showed that the covalent protein network of egg white noodles was superior to whole egg and egg yolk noodles; however, the rapidly formed protein network in egg white noodles could not cope with starch swelling. Soybean protein isolate (SPI), a type of plant proteins, consists mainly of 7S and 11S (Mujoo, Trinh, & Ng, 2003; Pires Vilela, Cavallieri, & Lopes Da Cunha, 2011). It is commonly used as an additive to improve functional properties of other proteins due to its good gelling properties, abundant resources and low cost (Feng & Xiong, 2002; Luo, Shen, Pan, & Bu, 2008; Wang et al., 2015). Feng et al. (2002) found

Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China. Corresponding author. School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China. E-mail addresses: [email protected] (Y. Yang), [email protected] (L. Gu). 1 These authors contributed equally. ∗

∗∗

https://doi.org/10.1016/j.foodhyd.2019.105191 Received 15 January 2019; Received in revised form 17 June 2019; Accepted 26 June 2019 Available online 27 June 2019 0268-005X/ © 2019 Published by Elsevier Ltd.

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that the addition of preheated SPI can accelerate the disappearance of myosin heavy chain in the gelling process and significantly increased the elasticity and hardness of pork myofibrillar protein isolate (MPI) gels. Wang et al. (2015) further explained the enhancement of gel properties was mainly resulted from hydrophobic interactions and hydrogen bonds, instead of disulfide bonds. Luo et al. (2008) reported that with the addition of 10% SPI, a higher breaking force of silver carp surimi was observed. From above, combination with other proteins may provide a useful approach for protein gels to obtain better functional properties (Comfort & Howell, 2003; Ma, Yiu, & Harwalkar, 1990; Su et al., 2015). However, fewer studies have been reported about the egg proteins and soybean protein isolate composite gels. Our previous study suggested that the soybean protein isolate/egg white proteins blended in ratio of 1:1 showed higher enhancement in springiness and water-holding capacity (Su et al., 2015). Nevertheless, a phase separation in the mixture of egg white and SPI was observed after placed for a while and we think this incompatibility have an adverse effect on the gelling properties. Many schemes have been tried and an interesting phenomenon was found that no phase separation was observed in the mixture of the whole egg/egg yolk and SPI. And thus, the aim of the work was to prepare and investigate the gelling properties, water state and microstructure of composite gels based on the whole egg (TWE)/egg yolk (EY) and soybean protein isolate (SPI). Meanwhile, the characterization of composite protein solutions, including solubility, solid content, surface hydrophobicity, total sulfhydryl content and rheological properties, was also investigated to elaborate gelling behavior of composite proteins. This research may not only provide a theoretical basis for the mixed study of plant proteins and animal proteins but also expand the usage range of egg protein and develop new products.

was assayed by the biuret method (Kaewmanee, Benjakul, & Visessanguan, 2011). Solubility was expressed as the percentage of protein content in supernatant to that in composite solutions. Solid content was determined by drying the samples in a forceddraft oven (DGG-9240, Shanghai Senxin, China) at 105 °C for 5 h to ensure that they had reached a constant weight. Solid content was calculated by dividing weight of dried samples to the initial weight of composite solutions. 2.3.2. Protein surface hydrophobicity Protein surface hydrophobicity was measured as described by Benjakul, Visessanguan, Ishizaki, and Tanaka (2001) with some modification. Firstly, the composite solutions were centrifuged at 10,000×g for 10 min, and then the supernatants were collected and adjusted to pH 7.0. After that, the collected solution was diluted with distilled water to 0.005, 0.01, 0.05, 0.1, 0.2, 0.3 mg/mL, respectively. Subsequently, 20 μL of 8 mM/L ANS was added to 4 mL of each solution. After standing in the dark at 25 °C for 10 min, a fluorescence spectrometer (F7000, Hitachi, Japan) was used to determine fluorescence intensity with an excitation wavelength of 390 nm (5 nm slit) and an emission wavelength of 470 nm (5 nm slit). Protein surface hydrophobicity was calculated by initial slope of curve of fluorescence intensity against protein concentration using linear regression analysis. 2.3.3. Total sulfhydryl content The total sulfhydryl content was measured as described by Zhao et al. (2016) with some modification. Each sample of before and after centrifugation was prepared for measurement. Two milliliters of an 8M urea-Tris-Gly buffer solution (0.1M Tris, 0.1M glycine, 4 mM EDTA, pH 8.0) and 0.02 mL of Ellman's reagent (4 mg/mL DTNB dissolved in 0.1M, pH 8.0 Tris-glycine buffer solution) were added to 0.6 mL of the solution as described above. The solution was placed at 25 °C for 1 h to develop full color (Ellman, 1959; Hansen & Winther, 2009). After centrifuging at 10,000×g for 10 min at 25 °C, the absorbance of supernatant was evaluated at 412 nm (UH5300, Hitachi, Japan) and the sulfhydryl content was calculated according to eq (1). An 8M urea-TrisGly buffer solution was used as control.

2. Materials and methods 2.1. Materials Fresh hen eggs were provided by Anhui Rong Da Egg Industry Co., Ltd., (Guangde, Anhui, China). Fresh eggs were washed and broken to get the whole egg (TWE), and then egg yolk (EY) was obtained by a separator. Soybean protein isolate (SPI) powder was purchased from Shandong Yu Wang group (Shandong, China). The protein content of TWE, EY and SPI was determined using the Kjeldahl method (N × 6.25) and was 13.0%, 17.5% and 90.8% (dry basis), respectively (w/w). All chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

SH(µM) = 73.53 × A 412 × D

(1)

where 73.53 is calculated from unit conversion (106/ 1.36 × 104 M−1 cm−1), A412 is the absorption value at 412 nm and D refers to the dilution factor (4.37 in this study). 2.3.4. Rheological behavior The rheological behavior of composite solutions was determined by DHR-3 dynamic rheometer (Waters, USA) using the method of Maltais, Remondetto, and Subirade (2008) with some modification. Firstly, the samples were loaded onto the rheometer (gap 1000 μm) and a parallel plate (40 mm diameter) was used in the test. Shear rate was set from 0.1 to 100 1/s at 25 °C and equilibration time was 5 s. The initial viscosity value was defined as the viscosity of composite solutions. Then reloaded samples and the temperature sweep of solutions was performed from 25 to 85 °C at the heating rate of 5 °C/min, 1 Hz and 1.0% strain. Time sweep of the sample was then carried out at 85 °C for 10 min. After that, samples were cooled to 25 °C at a descending rate of 10 °C/ min. Storage modulus (G′) and loss modulus (G″) were recorded to analyze the effects of temperature and time on the rheological properties of samples.

2.2. Preparation of composite solutions TWE/EY-SPI composite solutions were prepared according to the following procedure: firstly, SPI solution (10%, w/w) was prepared by dissolving power in distilled water, and stirring for 4 h to ensure complete dispersion and dissolution. TWE/EY solution (10%, w/w) was obtained by diluting original TWE/EY solution with distilled water. Then TWE/EY and SPI solutions were mixed together at the ration of 0:1, 1:3, 1:1, 3:1, 1:0 (w/w), respectively. After stirring slowly for 1 h, the mixture was adjusted to pH 7.0 with 1 M NaOH or HCl, and then used for analysis and the preparation of composite protein gels. 2.3. Characteristics of composite solutions

2.4. Preparation of composite gels

2.3.1. Solubility and solid content The solubility of composite solutions was determined using a modified method previously described (Li et al., 2018a,b). Firstly, the composite solutions were diluted to 10 mg/mL with distilled water and the pH value was adjusted to 7.0. After centrifugation at 10,000×g for 10 min at 25 °C, the supernatants were collected and the protein content

Firstly, 5 g composite solutions were transferred into 10 mL beakers and sealed with plastic wrap. Then they were placed in a pre-heated water bath and heated at 85 °C for 30 min to promote gelation. Finally, the gels were cooled down using ice water immediately and then stored at 4 °C for 12 h to allow the maturation of gels. 2

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2.5. Characteristics of composite gels

Table 1 Solubility and solid content of composite gels based on different mixing ratio of the whole egg (TWE), egg yolk (EY) and soybean protein isolate (SPI).

2.5.1. Texture profile analysis (TPA) A TA-XT Plus texture analyzer (SMS Co. Ltd, England) was used to determine the texture of composite gels and texture profile analysis (TPA) was chosen as the text mode. Compression test was performed at a crosshead speed of 2 mm/s and compression deformation of 50% of the initial sample height. The time pause between two compressions was 5 s. All samples (height of 12 mm and diameter of 22 mm) were placed on the platform of the TA-XT Plus fitted with a 1 kg load cell and a flat plunger 35 mm in diameter (SMS-P/35). Hardness was defined as peak force during the first compression cycle. Springiness was defined as the recovery degree of gels after decompression to their initial shape.

Samples

Solubility (%)

SPI TWE:SPI = 1:3 TWE:SPI = 1:1 TWE:SPI = 3:1 TWE:SPI = 1:0 EY:SPI = 1:3 EY:SPI = 1:1 EY:SPI = 3:1 EY:SPI = 1:0

53.56 64.44 61.34 59.28 54.90 58.30 43.86 34.01 24.48

± ± ± ± ± ± ± ± ±

0.92d 1.12 g 0.87 f 0.90 e 0.84 d 0.63 e 1.08 c 0.86 b 0.99 a

Solid content (%) 11.04 11.86 14.91 18.01 19.58 15.37 20.17 23.49 29.67

± ± ± ± ± ± ± ± ±

1.21 a 0.94 a 2.02 b 1.04 c 0.83 cd 0.79 b 0.97 d 1.06 e 1.01 f

Data with different letters (a-g) in a column are significantly different (p < 0.05).

2.5.2. Low-field NMR Low-field NMR relaxation measurement was performed according to the method of Han, Wang, Xu, and Zhou (2014) with some modification. Approximately 5 g of composite gels were encased in a plastic film and then placed in cylindrical glass tubes (25 mm in diameter). Subsequently, they were inserted into NMR probe of a Niumag pulsed NMR analyzer (NMI20-Analyst, Niumag Electric Corporation, Shanghai, China), operating at 21 MHz. The transverse relaxation time (T2) was measured using the Carr–Purcell–Meiboom–Gill (CPMG) sequence. The Spectral Width (SW) was set to 200 kHz, the Receiver Gain (RG) was 20 dB, and the Number of Scans (NS) was 4, respectively. A total of 4000 echoes were acquired for analysis. The T2 relaxation curve was fitted to a multi-exponential curve with the MultiExp Inv Analysis software (Niumag Electric Corporation, Shanghai, China).

content of the egg white-soybean meal blended adhesives. When the ratio of TWE/SPI or EY/SPI was 1:3, composite solutions exhibited maximum solubility. However, a further increase in the proportion of egg proteins, the solubility of composite solutions sharply decreased, which may be contributed to high viscosity of SPI (Table 2). The solution became dispersed and less viscosity as some egg was added, so it was more difficult for soluble proteins to be carried by insoluble proteins into precipitate when solubility was measured by centrifugation, resulting in higher solubility. However, with the increasing adding amount of egg, the low solubility of egg proteins led to the declining trend of composite solutions, though solutions became dispersed as the proportion of egg increased. The intermolecular bonds of proteins play key roles in their ability to form gel (Clark, 1992). Consequently, the main influences on the characteristics of heat-set gels were determined, including the hydrophobic interaction and disulfide bond. Fig. 1 and Fig. 2 show the changes of surface hydrophobicity in solutions after centrifugation and total sulfhydryl groups in solutions before (bc) and after centrifugation (ac). It is obvious that surface hydrophobicity of composite solutions represented an upward trend firstly then decreased as the proportion of egg proteins increased, reaching the peak at the ratio of 3:1 (egg proteins/SPI). At this point, EY-SPI composite solutions had significantly higher surface hydrophobicity than TWE-SPI composite solutions, which may be due to the facts that EY had higher surface hydrophobicity and the interactions between egg yolk and soybean protein isolate promote exposure of more hydrophobic groups. As shown in Fig. 2, samples before centrifugation had more SH groups than supernatant and compared to TWE-SPI composite solutions, EY-SPI composite solutions had higher amount of SH groups before centrifugation. This suggested that the amount of egg yolk was the main factor on changes of SH groups in the hen egg. After centrifugation, SPI and EY had lowest quantity of total sulfhydryl groups and all composite solutions contained more SH groups, which may be related with the solubility of solutions. And the ratio of total sulfhydryl groups (ac)/total sulfhydryl groups (bc) was positive in correlation with the solubility and the correlation coefficient was 0.884, p < 0.1.

2.5.3. Scanning electron microscopy (SEM) Samples were cut into pieces, fixed with 2.5% glutaraldehyde, dehydrated with ethanol, and dried with a CentriVap Concentrator (Labconco, USA). Dried samples were then sputter-coated with gold (SCD 005, BAL-TEC, Switzerland) and the microstructure was observed using a scanning electron microscopy (Quanta 200, Fei, Holland). 2.6. Statistical analysis At least three replicates were performed for each measurement. SPSS 17.0 package was used for statistical analysis of means and standard deviations. One-way variance analysis was carried out using Duncan's Multiple Range Test (p < 0.05) to detect significant difference between mean values. Figures were edited using OriginPro 9.0 software. 3. Results and discussion 3.1. Characteristics of composite solutions 3.1.1. Solubility, solid content, surface hydrophobicity and total sulfhydryl content Characteristics of composite protein solutions, including solubility, solid content, surface hydrophobicity and total sulfhydryl content, are important factors contributing to their gelation properties. Initially, solubility and solid content of composite solutions containing TWE/SPI and EY/SPI with different ratios were measured. As shown in Table 1, the solubility of SPI, TWE and EY was 53.56%, 54.90% and 24.48%, respectively. And the solid content was 11.04%, 19.58% and 29.67%, respectively. With the addition of egg proteins (TWE or EY), the solubility of composite solutions increased firstly then decreased, while solid content kept an ascendant trend. Meanwhile, samples contained TWE had higher solubility and lower solid content than samples contained EY. This phenomenon can be ascribed to the low solubility of egg yolk proteins and high oil content in EY (Anton & Gandemer, 1997). Luo et al. (2017) also proved that the solid component of the egg was the major contributor to the increase in the solid

3.1.2. Rheological analysis Rheological analysis was carried out to provide more information about the characteristics of composite protein solutions. Typical storage modulus (G′) and loss modulus (G″) vs. temperature curves of composite solutions at different egg proteins/SPI ratios during heating are shown in Fig. 3. It can be seen that both G′ and G″ of composite solutions increased with the rise of temperature. This phenomenon should be resulted from denaturation of proteins by thermal treatment, leading to exposure of hydrophobic groups and hydrophobic interactions (Petruccelli & Anon, 1995). In addition, there were G′-G″ crossover among samples except three samples (the whole egg accounted for 0%, 25% and 50%). And it is also observed that G′ of EY-SPI composite solutions was lower than corresponding TWE-SPI composite solutions at 3

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Table 2 Viscosity of composite solutions made with different egg proteins/SPI. Egg proteins/SPI

Viscosity (Pa.s) 0:1

TWE:SPI EY:SPI

1:3 d

510.04 ± 0.86 510.04 ± 0.86 d

18.94 ± 0.23 18.87 ± 0.15

1:1 c

8.35 ± 0.12 1.67 ± 0.01

c

3:1 b a

0.26 ± 0.08 0.05 ± 0.00

1:0 a a

0.06 ± 0.01 0.01 ± 0.00

a a

Data with different letters (a-d) in the table are significantly different (p < 0.05).

Fig. 1. Changes in the surface hydrophobicity of egg proteins (TWE or EY)-SPI dispersions with different ratios. Data with different letters (a–h) in a figure are significantly different (p < 0.05).

Fig. 3. Effect of egg proteins/SPI ratio on the storage modulus (G′) and loss modulus (G″) of composite solutions during heating process (a: TWE/SPI; b: EY/SPI).

Fig. 2. Changes in the total sulfhydryl groups of egg proteins (TWE or EY)-SPI dispersions with different ratios (bc: before centrifugation; ac: after centrifugation.). Data with different letters (A-H/a-e) in a figure are significantly different (p < 0.05).

consistent with the results from Fig. 1. As for the G′ decrease of SPI, which may be resulted from the decline of SPI's viscosity during heating, which can be observed by eyes. During constant temperature stage, G′ value of gels further ascended, which indicated that thermal process still promoted the molecular interactions between proteins. On the contrary, the G’ growth ratio of samples that contained TWE and SPI was faster than that of samples contained EY and SPI, which suggested that other molecular forces were playing a leader role, rather than hydrophobic interactions. The formation of a three-dimensional network is caused by various molecular forces, including hydrogen bonding, ionic attractions, disulfide bonding, hydrophobic interactions or a combination of these forces (Sun & Arntfield, 2012; Zhao, Sun, Li, Liu, & Kong, 2017). In

the same ratio. The above results seem to be associated with the descending order of viscosity of SPI, TWE and EY (Table 2). High viscosity of SPI could endow the weak gel state of composite dispersions when the proportion of SPI was over 50%, leading to the inexistence of G′-G″ crossover in the whole heating process. Meanwhile, Table 3 also reflects the G′ change using specific data. It was found that the G′ growth ratio of samples increased obviously as the proportion of egg proteins increased at heating stage. Meanwhile the G′ growth ratio of samples that contained EY and SPI was almost higher than that of samples contained TWE and SPI. This suggests that EY-SPI composite solutions had higher surface hydrophobicity, which is 4

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Table 3 Rheological analysis comparing the storage modulus (G’) values for soybean protein isolate (SPI), the whole egg (TWE), egg yolk (EY) and their composite gels at 25 °C (beginning of heating cycle), 85 °C (just reached and preservation ended), and 25 °C (end of cooling cycle). Egg proteins/SPI

G’ (Pa) at 25 °C before heating

G’ (Pa) at 85 °C (Just reached)

G’ (Pa) at 85 °C (heat preservation ended)

G’ (Pa) at 25 °C after cooling

0:1 (SPI) TWE:SPI = 1:3 TWE:SPI = 1:1 TWE:SPI = 3:1 TWE:SPI = 1:0 EY:SPI = 1:3 EY:SPI = 1:1 EY:SPI = 3:1 EY:SPI = 1:0

120.52 ± 7.98 d 12.82 ± 4.22 c 4.64 ± 0.94 ab 0.30 ± 0.05 a 0.26 ± 0.02 a 8.96 ± 1.33 bc 0.90 ± 0.10 a 0.22 ± 0.03 a 0.10 ± 0.01 a

52.04 ± 9.54 a 172.13 ± 11.45 b 467.50 ± 31.22 d 769.05 ± 19.65 g 958.08 ± 33.34 h 278.61 ± 29.67 c 531.84 ± 38.95 e 627.97 ± 43.87 f 982.09 ± 20.11 h

201.36 ± 23.75 a 892.47 ± 17.99 c 2003.87 ± 47.98 g 3517.97 ± 87.43 h 3525.77 ± 38.67 h 772.15 ± 42.11 b 1065.13 ± 34.51 d 1298.83 ± 63.77 e 1701.69 ± 52.82 f

1833.41 ± 34.78 a 3241.97 ± 76.32 e 6157.46 ± 56.97 f 11503.80 ± 110.48 h 10178.23 ± 84.56 g 2291.62 ± 48.87 b 2407.49 ± 36.79 c 2638.93 ± 56.78 d 2636.64 ± 64.72 d

Data with different letters (a-h) in a column are significantly different (p < 0.05).

addition, the denaturation of proteins by heating was a precondition for other forces to occur (Wang et al., 2015) and constant temperature stage provided time for forces to react and contribute to gel stiffness. Finally, G′ of all gels further increased during subsequent cooling process. The appearance of growth was called gel reinforcement, a typical characteristic of protein gels, which was generally attributed to consolidation of attractive forces such as hydrogen bonds and ionic interactions between proteins (Clark & Lee-tuffnell, 1986). It is obvious that G′ value of SPI had the largest increase in the cooling process compared to the heating and constant temperature stages, which was also observed in another work (Zhao et al., 2017). Meanwhile, compared with composite protein gels, the G’ growth ratio of SPI gels during cooling process was higher, suggesting that hydrogen bonding might be more important to SPI than composite proteins and egg proteins (Li, Zhang, et al., 2018; Nagano, Mori, & Nishinari, 1994). 3.2. Gel properties of composite gels

Fig. 5. Hardness and springiness of gels prepared with different egg proteins/ SPI ratios. Data with different letters (A-F/a-f) in a figure are significantly different (p < 0.05).

3.2.1. Macroscopic visual observations As shown in Fig. 4, composite gels exhibited different morphology coefficient for its different source and composition. TWE contributed to a smooth appearance for composite gels, while gels were difficult to be removed from beakers in the presence of EY (Fig. 4a and b). However, an obvious phase separation was observed in the composite gels which consisted of egg white proteins and SPI (Fig. 4c). This phenomenon may be resulted from high viscidity and low denaturation temperature of egg white proteins, leading to egg white proteins aggregation and then separation from the composite solutions during thermal processing. Meanwhile, the yellowness of gels was deeper with increase in the proportion of egg proteins. The yellowness in mainly originated from carotenoids in egg yolk, including lutein, beta-carotene, zeaxanthin, etc.

taste for food products. Hardness and springiness both were common parameters used in the analysis of textural properties. Fig. 5 shows significant differences in hardness and springiness between the composite gels with different ratios of egg proteins/SPI. An increase in the proportion of egg proteins resulted in a steady increase in the hardness until to a stable value. The gel hardness after 50% addition of TWE/EY was about 561%/329% of the original gel strength. The stronger gel could be due to the increasing function of hydrophobic interactions and disulfide bonds (Figs. 1 and 2), increased crosslink between egg proteins and SPI, as well as the formation of hydrogen bond among protein molecules (Li, Zhang, et al., 2018; Zhao et al., 2017). In addition, it is obvious that EY had a weaker effect than TWE to enhance the hardness of gels. The variation trend in gels hardness was in accordance with that of G’ at 25 °C after cooling (Table 3), indicating that there was a strong relationship between rheological property and textural property.

3.2.2. Hardness and springiness Proteins play a major role in the field of food processing, because the textural properties of proteins gels could contribute to a required

Fig. 4. Typical macroscopic aspect of the gels (a: gels that were made of TWE and SPI; b: gels that were made of EY and SPI; c: gels that were made of egg white and SPI). 5

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Table 4 Water state of protein gels made with egg proteins and SPI in different ratios. T21 and T22 represent the distributions of T2 relaxation times; PT21 and PT22 represent the corresponding area fractions. Egg proteins/SPI

T21 (ms)

T22 (ms)

PT21 (%)

PT22 (%)

0:1 (SPI) TWE:SPI = 1:3 TWE:SPI = 1:1 TWE:SPI = 3:1 TWE:SPI = 1:0 EY:SPI = 1:3 EY:SPI = 1:1 EY:SPI = 3:1 EY:SPI = 1:0

114.98 ± 0.85 g 100.00 ± 1.31 f 86.97 ± 1.14 e 75.65 ± 0.79 d 75.65 ± 0.82 d 86.97 ± 1.04 e 65.79 ± 2.39 c 49.77 ± 0.98 b 43.29 ± 1.12 a

– – – – 4328.76 ± 4.73 b – – 533.67 ± 1.87 a 533.67 ± 1.29 a

100 100 100 100 98.71 ± 0.16 100 100 98.81 ± 0.38 98.45 ± 0.77

– – – – 1.29 ± 0.16 – – 1.19 ± 0.38 1.55 ± 0.77

a

a a

a

a a

Data with different letters (a-g) in a column are significantly different (p < 0.05).

Therefore, this result implies that the addition of egg proteins may be a useful approach to improve the hardness of soybean products at neutral pH condition. Springiness represents the degree to which the gel returns to its original shape after it has been decompressed. As shown in Fig. 5, when the adding amount of TWE was no more than 50%, springiness of composite gels stayed higher. And with a further increase in the percentage of TWE, the springiness of composite gels decreased. However, the springiness of EY-SPI composite gels could keep a high trend with the addition of EY. It implied that egg yolk was the main reason to maintain the springiness of the composite gels, which may be resulted from its high solid content (Table 1). Too much egg white proteins would decrease the elastic springiness of composite gels due to incompatibility between egg white proteins and soybean proteins; poor springiness of egg white proteins itself was another reason (Zhang et al., 2019).

motion, which is commonly used to analyze water state in gel system through low-field NMR technique (Kuntz et al., 1974; Yasui, Ishioroshi, Nakano, & Samejima, 1979). Shorter relaxation time indicated that water molecules tend to bound and stable state (Yasui et al., 1979). Table 4 shows the distributions of T2 relaxation times of composite gels. There were two distinct water populations centered at approximately 50–150 ms (T21), 3000–5000 ms (T22), which represented water trapped within the gel structure and free water in the gel system, respectively (Han et al., 2014). On the one hand, with the addition of TWE and EY, the T21 relaxation time decreased from 114.98 ms to 75.65 ms, 43.29 ms, respectively, indicating that water molecules became more stable in composite gels, especially in EY-SPI composite gels. This may be resulted from two reasons: (1) high solubility of composite solutions led to more accessible protein molecules to participate in the formation of water-ion bonds (Li et al., 2018a,b); (2) high solid content of egg proteins reduced water mobility and water evaporation in gels (Luo et al., 2017). On the other hand, peak that represented free water in gels was only detected in TWE gel, EY-SPI (75:25) composite gel and EY gel. The PT22 of them was 1.29% at 4328.76 ms, 1.19% at 533.67 ms and 1.55% at 533.67 ms, respectively.

3.2.3. Low-field nuclear magnetic resonance (low-field NMR) Water molecules were presented in materials as bound and free state (Kuntz & Kauzmann, 1974). T2 relaxation time is sensitive to molecular

Fig. 6. Scanning electron microscopy (SEM) photograph of gels (10% protein, w/w); (a: SPI; b: TWE:SPI = 1:1; c: TWE; d: EY:SPI = 1:1; e: EY. Magnification: 5000 × ; scale bar = 10.0 μm). 6

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These phenomena indicate that molecular interactions between egg proteins and SPI converted free-state water in egg protein gels to bound water.

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3.2.4. Microscopic examination The microstructure of egg proteins-SPI composite gel at the ratio of 1:1 was observed to analyze the effects of protein interactions on the gelling properties. As seen in Fig. 6, gels with different kinds and compositions of proteins exhibited obvious different interior morphology. SPI gel had a loose cross-linked structure with holes, which may result in the lowest hardness of the gel, and TWE gel exhibited granule packing structure (Fig. 6c). TWE-SPI composite gel was compact and had a linear structure padding with granules (Fig. 6b). Because of the differences in the molecular sizes and interactions between TWE and SPI, egg proteins can fill the macromolecular gaps of SPI and produce a dense structure, which was also observed by Luo et al. (2017). Meanwhile, the insoluble solid of composite solutions can act as a non-gelling component and intersperse throughout the gel network, making the structure dense. EY gel also exhibited granule packing structure but the size of particles agglomeration may be much bigger than the space of the cross-linked structure of SPI, leading to the accumulation of EY molecules onto the SPI surface. Results above illustrated that compared with EY-SPI composite gels, the microstructure of TWE-SPI composite gels was more compact, which provided higher hardness for the composite gels. 4. Conclusions In this study the gelling properties of egg proteins and soybean protein isolate composite gels were analyzed. Results indicated that mixing proteins changed the physicochemical properties of solutions. As the proportion of egg proteins increasing, hydrophobic interactions and disulfide bonding, rather than hydrogen bonding, played main roles in the formation of gels. The addition of egg proteins into SPI could increase the hardness and springiness, which may be mainly due to better molecular interactions between proteins and more compact microstructure of composite gels. However, too much egg proteins would decrease the springiness, which may be because of the incompatibility between egg white proteins and soybean proteins, as well as poor springiness of egg white proteins itself. In conclusion, it is possible that the mixture of egg proteins and soybean proteins could provide desired texture and high nutritive value for food products. Conflicts of interest The authors declared that no conflict of interest exists. Acknowledgments The authors would like to thank for the financial supporting from the National Key Research and Development Program of China [No. 2018YFD0400303]; the Natural Science Foundation of Jiangsu Province for the Youth [No. BK20180610]; the National Natural Science Foundation for the Youth of China [No. 31801483] and the project of China Scholarship Council. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.105191. References Alamprese, C., Casiraghi, E., & Rossi, M. (2009). Modeling of fresh egg pasta characteristics for egg content and albumen to yolk ratio. Journal of Food Engineering, 93(3), 302–307.

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