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Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin
Accepted Manuscript Title: Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin Author: Tao Huang Zong-cai Tu Prof. Hui Wang Prof. Xinchen Shangguan Lu Zhang Peipei Niu Xiao-mei Sha PII: DOI: Reference:
S0927-7757(16)30867-6 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.10.013 COLSUA 21080
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
27-8-2016 25-9-2016 11-10-2016
Please cite this article as: Tao Huang, Zong-cai Tu, Hui Wang, Xinchen Shangguan, Lu Zhang, Peipei Niu, Xiao-mei Sha, Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.10.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin
Tao Huanga, Zong-cai Tua,b*[email protected], Hui Wanga, **
Xinchen-Shangguana,c,d [email protected], Lu Zhangb, Peipei Niua, Xiao-mei Shab
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China
b
Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China
c
College of Food Science, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China
d
Food and Drug Administration of Jiangxi Province, Nanchang, Jiangxi 330029, China
*
Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang
University, Nanchang , China Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science, Jiangxi Normal University, Nanchang, China
**
Prof. Xinchen-Shangguan, Food and Drug Administration of Jiangxi Province, Nanchang, China,
Graphical abstract
Highlights • • •
SW could improve the foam-ability, but decrease the foam-stability of EWP obviously. Addition of FSG could enhance the foam-ability and foam-stability of SWEWP. SWEWP-FSG formed stronger mechanical film and showed higher foam properties.
Abstract The aim of this work was to study the effect of fish scales gelatin (FSG) on the foam and interfacial properties of subcritical water (SW) treated egg white protein (EWP) systems. The foam ability of the SW treated EWP (SWEWP) system is obviously better than that of untreated EWP system, although the former had poorer foam stability. The foam ability of the SWEWP system was further enhanced by the addition of FSG by reducing the surface tension. FSG seemed to build an interfacial viscoelastic network at the air – water interface with the increased surface dilational rheological behavior, causing the low drainage of liquid and inhibiting the bubbles coalescence of complex systems. Moreover, variations of surface elasticity matched the foam stability as the FSG concentration increased. This study described the effect of the biopolymer mixing ratio on the foam properties of SWEWP and FSG. This study also offered the possibility to design the production of protein powder with an outstanding capacity for foams formation and stabilization.
Keywords: egg white protein; subcritical water; fish scales gelatin; foam properties; interfacial properties
1. Introduction
Egg white protein (EWP) is frequently used as a foaming agent in the food industry to improve
and maintain the quality (texture and volume) of aerated food, such as meringues, beverages, fermentation, cakes, whipped creams and chocolate mousses [1-3]. However, thermodynamic instability is a basic property of foams, thereby leading to their disintegration over time and their large surface area, this instability could significantly influence the final quality of the products [1, 4].
Several techniques have been employed to improve the foaming properties of EWP, such as heat [5, 6], high pressure [7, 8], pulsed electric fields followed by heat treatment [9], irradiation [10-12] and acylation [13]. Nevertheless, high pressure treatment has several limitations during mass production and may increase the production cost [12]. Irradiation produces unattractive odors [14], whereas chemical modifications may generate hazardous substances. Therefore, a novel method should be developed to improve the foaming properties of EWP. Subcritical water (SW) is defined as hot water at high temperatures ranging from 100 to 374 oC under high pressure to maintain water in the liquid state. SW is an extraordinary medium or solvent for various chemical reactions and the extraction of different compounds [15]. SW is also an alternative to the traditional methods of protein hydrolysis because water is an environment - friendly reaction medium does not need acids or enzymes [16, 17]. SW has been used to produce products with excellent foam stability from soy meal [18]. To the best of our knowledge, information is available on the foaming mechanism SW - treated protein. Moreover, the synergistic effect of blending proteins and hydrocolloids generally produces a more significant improvement of the functional properties of several foods than those of proteins and hydrocolloids alone [19]. Gelatin, low in calories, is normally applied in foodstuffs to enhance protein levels, and is especially useful in stabilizing foamed food products because of its excellent surface-active and gelling properties upon cooling [20, 21]. Nevertheless, concerns over bovine
spongiform encephalopathy, vegetarianism and religious beliefs have driven the concerted efforts to find alternative protein sources that can provide similar functions for food systems [22]. Fish gelatin has been regarded as a promising alternative in the food industry, because of its similar properties with mammalian gelatin [23]. The foaming [24] and interfacial properties [25] of mammalian gelatin or casein glycomacropeptide (CMP) – mammalian gelatin [21] have been studied. However, the influence of fish gelatin on the foaming properties of other food proteins has been rarely reported. In our previous work (Fig S1), FSG enhanced the foaming properties of untreated EWP and SW - treated EWP systems, the latter presented better foaming capacity and foaming stability. At present, many studies on surface dilational rheology of various surfactant at the air – water interface have been published to better understand the interfacial behavior of a film [26, 27]. Therefore, the present work was performed to study the foaming and interfacial properties of EWP as influenced by pre-treatment with SW and the addition of FSG. The foaming mechanism of protein systems was explored, to develop a process that can produce protein powder product with high foaming properties, which has a potential in future expand the application of FSG and EWP through an industrially applicable process.
2. Materials and methods
2.1.Materials
Fresh eggs (Lao nan gou) were bought from local market in Nanchang, China. Liquid EWP with protein content of 10.21 ± 1.12% [28] was prepared by separating egg white and yolk manually. FSG
was extracted from bighead carp scales as described in our previous study [23]. The crude protein content of lyophilized gelatin was 90.21 ± 1.23 % with molecular weight of 30 – 200 kDa [23]. Double-distilled water (with conductivity of 0 µs / cm) was used throughout all the experiments.
2.2 .Methods
2.2.1.Preparation of SW treated EWP Fresh liquid EWP was mixed with distilled water at a ratio of 1:4 (w/w) for 0.5 h at room temperature to obtain a homogenous mixture. The SW treatment was performed at 140 °C for 1 h. After the reaction, the treated EWP solution was rapidly cooled by submerging the reactor in an ice bath for 5 min. The treated and untreated EWP solutions were collected and sprayed for further analysis.
2.2.2 Preparation of single and complex protein systems Single system: The EWP and SW treated EWP (SWEWP) powders were dissolved with deionized water in a water bath (55 oC, 1 h, ～400 rpm) to prepare the protein solutions (10 mg mL-1, pH 6.5). The gelation of gelatin was hindered because the critical concentration for gelation is 20 mg mL-1 [21]. To discriminate the foaming properties of gelatin from its gelling properties, the max FSG concentration was 6 mg mL-1. The single system was named as EWP, SWEWP and FSG, respectively Complex systems: The SWEWP- FSG systems were prepared by adding FSG to the prepared SWEWP solution. The concentrations of FSG were 2, 4 and 6 mg mL-1, the mixtures were incubated in a water bath (40 oC, ～400 rpm) until complete dissolutions, and were named as SWEWP-FSG1, SWEWP-FSG2, and SWEWP-FSG3, respectively
2.2.3. Measurements of foam properties The foam properties of single and complex systems were characterized based on their foam formation and stability on a Foamscan instrument (IT Concept, Teclis Co., France) according to the method of Zhang et al. [29] with some modifications. The foam ability, foam stability and drainage from the foams were determined by conductivity measurement of the foam column. The change of the bubbles was observed by a CCD camera which photographed every 5 s. The size and distribution of the bubbles were analyzed from the foam pictures with a CAS software. Briefly, the foams were generated by blowing air at a constant flow rate of 200 mL min-1 through a porous glass filter at the bottom of a glass tube, where 60 mL of prepared protein solution was located. The blowing of air was immediately stopped when the foam volume reached 100 mL. The blowing time “t” was defined as the foamability. The time of foam volume decays “t1/2” was recorded as the foam volume decayed from 100 mL to 50 mL. The quantity of the liquid that remained after foaming and that was drained out from the foam was measured by a pair of electrodes at the bottom of the glass column. The volume of the liquid in the foam was measured by conductimetry with three pairs of electrodes along the glass column. Each sample was performed in triplicate at room temperature.
2.2.4. Measurements of surface tension The surface tension of various solution systems were carried out on the interface expansion rheometer (Tracker, TECUS-IT Concept, France) using pendant drop method. The average values of surface tension were obtained in triplicate at room temperature.
2.2.5. Measurements of surface dilational viscoelasticity The parameters of surface dilational viscoelasticity of the systems with various components were measured using the oscillating bubble rheometer (Tracker, TECUS-IT Concept, France) as reported by He et al. [30] with few modifications. The dilational viscoelasticity measurements were started after a pre-equilibrium period of 2 h. The prepared bubble was expanded and compressed sinusoidally with a small amplitude (3%) and oscillational frequency (0.1 Hz). The ratio of small changes in the surface tension (γ) and surface area (A) are defined as the surface dilational modulus ε:
where εd is the dilational elasticity, ηd is dilational viscosity and ωηd is the dilational viscosity component, which can be calculated by: where |ε| is the absolute modulus, and θ is phase angle. All the measurements were performed in at least triplicate at 25±0.1 oC .
2.2.6. Statistical analysis All the experiments were performed in three times. Data were subjected to analysis of variance (ANOVA) (P < 0.05) using Statistic program (Statsoft Inc.). Analysis was performed using SPSS 17.0 (SPSS Inc, Chicago, IL, USA).
3. Results and discussion
3.1. Foam properties
3.1.1. Foam morphology The variation of bubbles generated from single and complex systems was measured and analyzed after stopping nitrogen blowing when foam volumes reached 100 mL. Obviously, the diameters of bubbles generated from all systems gradually increased as the bubbles changed in shape from sphere to polyhedrons with increasing time (Fig. 1). These results were similar with those of Martinez et al.[21], who reported that the diameter of bubbles generated from a CMP / gelatin system and the CMP – gelatin complex systems gradually increased with time. According to the Laplace – Young law, gas diffusion occurred through liquid film from smaller bubbles to larger bubbles because of the higher pressure in smaller bubbles, thereby decreasing the number of smaller bubbles while increasing the size and collapse of the larger bubbles. Zhang et al. [29] also reported that the liquid between the bubbles drained out from the liquid channels and plateau borders under gravity, making adjacent bubbles coalesce as the liquid film became thin enough . As shown in Fig. S2, FSG also possesses smaller bubbles than pig skin gelatin (PSG), whereas SWEWP-FSG has smaller bubbles than SWEWP-PSG. Therefore, FSG displays better steric protection and elastic properties to interfaces and could replace PSG in some aerated foods. Compared with EWP, treated EWP took less time to reach 100 mL (Table 1) and possessed smaller bubbles at 0 s (Fig 1). However, these bubbles easily enlarged within a short of period of time,
namely, 300 s in this study. SW could improve the foaming capacity of EWP, but reduced its foaming stability (Fig. S1). The images of complex systems (t = 0) presented smaller bubbles sizes as the FSG concentration increased, revealing that FSG could strengthen the bubbles stability and improve the stability of complex systems. The foam of the complex systems was more stable than the single system, but was more heterogeneous, implying that FSG could influence the structure of bubbles and delay foam collapse by decreasing the initial bubbles size and delaying the increase of bubbles. The evolution of bubbles in foam could be described by observing the size distribution of bubbles in the foamy network with time [31]. In this study, images of the bubbles size distribution (at least 200) and the mean radii in different systems at 100 s were analyzed by the CSA software, the corresponding data are displayed in Fig. 2 and Table 1, respectively. Obviously, the bubble size of EWP was approximately below 0.075 mm (frequency of 134), which was larger than that of the SWEWP bubbles (below 0.066 mm, frequency of 239). In the complex systems, the distributions of the bubble radius were significantly dependent upon the FSG concentration. The SWEWP-FSG3 system had the smallest bubbles diameters, which were approximately below 0.036 mm (frequency of 202) and at 0.036 - 0.059 mm (frequency of 182). Furthermore, the invariant mean value of each group was practically obtained by our analysis. A similar observation was previously reported by Barik and Roy [29].
3.1.2. Foam ability The foaming power or foam ability is related to the level of air phase volume upon the introduction of a gas into the protein solution [2]. The time “t” could be described as the time needed to make the foam volume reach 100 mL, which reflects the foaming capacity[29]. Table 1 shows that single EWP system has higher foam time (95.63 ± 1.07) than single A system (41.53 ± 1.11) (p < 0.05),
implying that SW could improve the foam ability of EWP. EWP could be hydrolyzed by SW under high temperature and pressure, producing numerous substances with low molecular weights, and lower adsorption energies, these substances could rapidly diffuse to the surface within a shorter time than the original EWP. FSG is a linear polymer with a typical molecular weight of 30 - 200 kDa [23]. SWEWP has substances with lower molecule weights, which could rapidly diffuse to the surface within a shorter period of time. Thus SWEWP keeps the constant molecule exchange between the surface and bulk phase, as well as lower the surface tension (Table 2), leading to improve foaming ability of complex systems with the increasing of FSG concentration.
3.1.3. Foam stability and drainage Foam stability is important to prolong the shelf-life and improve the product appearance of food foams, this property must be maintained even when the foam is subjected to various processes, such as heating, mixing and cutting [2]. Given the effects of gravity, the liquid and gas in the foam tend to separate and leak liquid out of the foam, thereby drying the foam as time increases [32]. The half life (t1/2) of foams is an important parameter that is used to evaluate foam stability. The stability and kinematic viscosity of SWEWP are lower than those of EWP (Table 1), descending the stability of the film and speeding up the breaking up of bubbles. On the other hand, FSG had significant effect on the t1/2 values, as SWEWP-FSG3 had the highest t1/2 value (11233.33 ± 57.73) (p < 0.05). The higher t1/2, the higher foam stability. FSG could affect the viscosity of the continuous phase and the interface, whereas EWP mainly affects the interface alone. Thus a positive behavior of t1/2 raised from the ratio of SWEWP and FSG in the mixture systems. SWEWP could be diffused to the interface where it interacts with FSG, forming a mixture with highly viscoelastic characteristics, and films with considerably high
rigidity at the interface. This behavior was also attributed to the interactions between these biopolymers at the air - water interface and in the aqueous solutions. These interactions created thick visco-elastic networks that could delay foam destabilization [33] [21]. Foam collapse is analyzed from the time evolution of the foam volume after stopping gas bubbling (Fig 4A). The slope of this plot is taken as the collapse rate (kcollapse) of the system (Fig 4B). SWEWP showed a rapid collapse,which is also consistent with the results in Fig 1. The foam behavior of complex systems was dominated by the increasing FSG concentration because the kcollapse value of the foams declined. This trend was probably caused by the increased hydrophilic properties of adsorbed gelatin and the strong absorption or retention of water in the foams lamella, thereby inhibiting drainage and foam collapse [21]. The liquid content in the foams is related to the foam stability, and is usually denoted by the liquid volume [29]. The variation of liquid volume in the foams generated from protein systems is exhibited in Fig. 4C. The liquid volume in the foams first increased to the maximum values, and then decreased gradually with time. The maximum liquid volume of the complex systems increased as the FSG increased. The drainage rate is mainly affected by the packing pattern of the foam films, the composition of the interface and aqueous phase, the random network, and other properties of the foam films [29, 34]. The drainage process reduces the liquid in foam films and makes the smaller bubbles become closer to each other, eliminating the smaller foams and forming larger bubbles, this drainage process continues until equilibrium is reached between gravity and capillarity pressure [29]. Therefore, we speculated that FSG predominantly contributes to the foaming properties with the increasing FSG content in complex systems. Moreover, FSG is a good candidate for the stabilization of foams in aerated food consumed by Muslims, Jews and Hindus et al [20].
3.2 . Mechanism of foam stabilization
3.2.1. Surface tension Surface tension is used to explain the foaming mechanism [29]. Protein can spontaneously adsorb at various surfaces and form mechanically stable layers with low surface tension [35] [36]. As observed in Table 2, the surface tension of untreated EWP (48.3 mN m-1) is lower than that of SWEWP (45.16 mN m-1) (p < 0.05), because of the hydrolysis effect of SW. FSG system showed the lowest surface tension among the single protein system, indicating its best foaming ability. Moreover, the surface tension of complex systems was lower than those of individual protein system, and this surface tension decreased from 43.54 to 40.57 as the FSG concentration increased from 2 to 6 mg mL-1 ( p < 0.05). This was mainly attributed to that hydrolyzed EWP molecules at the interface were gradually displaced by competing FSG molecules in the adsorbed layer via weak electrostatic and hydrophobic interactions. Lu et al. [37] showed that concentration and configuration change of proteins on the drop surface might influence upon their adsorption rate and the film stability at interface. Schmidt et al. [38] reported that the re-organization of proteins at the interface or presence of multicouches would decrease the surface tension with increasing napin concentration. Therefore, FSG could rapidly reduce the surface tension of the protein system as a good foaming agent.
3.2.2. Surface dilational viscoelasticity Dilational rheology is a very effective indicator to characterize film properties [26, 39]. The effect of the FSG concentration on the surface dilational visco-elasticity of protein systems at a constant
frequency (0.1 Hz) is shown in Table 2. SW could sharply decrease the absolute modulus |ε|, producing a weaker foam stability as depicted in Fig. 1. The |ε| increased with the increasing FSG concentration, due to the increased concentration in the bulk. The absolute modulus |ε| is influenced by several factors, such as the frequency, temperature, protein concentration. Zhang et al. [29] reported that |ε| increased with increasing oscillational frequencies by relaxation processes at the interface, including the exchange and conformational changes of molecules in the surface layer. Gelatin imparts high viscosity because of its hydrodynamic volume, flexibility and expanded coil structure [24]. Therefore, the complex systems showed an increasing surface dilational viscoelasticity with the increasing FSG concentration at a constant frequency (0.1 Hz) (Table 2). FSG and SWEWP were supposed to form a viscoelastic layer in the films between bubbles and in the plateau, producing a gel-like network formed by hydrogen bonds, Van der Waals' force et al. The viscosity affected the immobile liquid film by reducing the drainage rate. Therefore, the higher viscosity and mechanical strength of the adsorbed layer, the higher foam stability will be given. In addition, the εd values of all systems were much higher than their ωηd, revealing that the dominance of the elastic characteristic in the single component and mixed layers at the air – water surface., as suggested by Zhang et al. [29]. This phenomenon was attributed to two reasons: (1) Conformational rearrangement following adsorption that causes the formation of an interconnected surface protein network; (2) The intra-protein structural rigidity at the surface that resists surface compression and/or expansion [37, 40]. Therefore, the addition of FSG could form a gel-like network structure at the surface of the complex systems. What’s more, the formation of a surface network along with the intrinsic structural stability of individual protein units within the network strengthened the ability of the film to withstand
a disturbance, improving foam stability [40].
4.Conclusions The single EWP system had weaker foam ability and a lower drainage rate, whereas the single SWEWP had better foam ability but faster drainage and weaker stable foams. This difference as caused by SW, could decrease the surface tension and surface dilational viscoelasticity. The addition of FSG could decrease the surface tension, but increased the kinematic viscosity, absolute modulus |ε| and surface dilational elasticity εd, thereby promoting the foaming properties of complex systems. Moreover, all the films of protein surface were elastic. The variation of foam stability agreed well with the kinematic viscosity and surface dilational elasticity of the corresponding systems as the FSG concentration increased. The higher stability of the foams was mirrored in the higher |ε| and εd values. In comparison to PSG, FSG contributed more to the foaming properties of the protein system. Therefore, FSG can act as an effective stabilizer of foam systems instead of mammalian gelatin. The SWEWP-FSG powder is also a promising alternative for aerated products processing, because of its outstanding performance.
Acknowledgement We gratefully acknowledge financial support from the National High Technology Research and Development Program of China (863 Program) (No, 2013AA102205), the Freedom Explore Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-ZZB-201310), National Natural Science of Foundation of China (No, 31660487), Collaborative Innovation Center for Major Ecological Security Issue of Jiangxi Province and Monitoring
Implementation (JXS-EW-00) and Earmarked fund for Jiangxi Agriculture Research System (JXARS-03).
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Figure caption Fig. 1. The variation of the foams generated from protein solution beginning with the stop of air flow (their is a 100 seconds interval between adjacent images respectively). EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 2. The bubble radius distribution of different protein systems at time of 100 s. EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 3. Time evolution of foam volume for different foam systems generated from various protein system. EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 4. Foam volume (A), rate of collapse (kcollapse) (B) and liquid volume (C) of different systems generated from protein systems as a function of time. EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; 1:0.2、1:0.4、1:0.6: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Table 1 Foam time (t), decay time (t1/2) and mean radius of different protein systems System
t (s)
t1/2 (s)
Mean radius
EWP
95.63±1.07e
8586.67±15.28e
0.092±0.085a
SWEWP
41.53±1.11d
353.67±4.73a
0.066±0.062a
FSG
38.17±0.67c
6780±10d
0.085±0.077a
SWEWP-FSG1
38.06±0.41c
1470±10b
0.086±0.033a
SWEWP-FSG2
36.8±1.15b
3540±10c
0.045±0.025a
SWEWP-FSG3
27.87±0.93a
11233.33±57.73f
0.041±0.021a
Values in the same column with different letters are significantly different (p < 0.05); EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: fish scales gelatin; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 (w/v) SWEWP solution was 2 mg mL-1, 4 mg mL-1 and 6 mg mL-1, respectively. Table 2 Surface tension (γ), absolute modulus |ε|, surface dilational elasticity (εd) and surface dilational viscous component (ωηd) of different protein systems System
γ(mN/m)
|ε|
εd
ωηd
EWP
48.3±0.02f
44.11±0.33e
43.18±0.32e
9.05±0.16e
SWEWP
45.16±0.06e
22.05±0.35a
20.98±0.22a
6.07±0.03b
FSG
44.08±0.17d
27.49±0.08c
27.29±0.08c
3.34±0.05a
SWEWP-FSG1
43.54±0.08c
25.51±0.16b
25.44±0.11b
6.41±0.25c
SWEWP-FSG2
42.6±0.02b
33.24±0.07d
32.77±0.11d
7.17±0.06d
SWEWP-FSG3
40.57±0.02a
47.53±0.06f
45.88±0.04f
9.28±0.04f
Values in the same column with different letters are significantly different (p < 0.05); EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: fish scales gelatin; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 (w/v) SWEWP solution was 2 mg mL-1, 4 mg mL-1 and 6 mg mL-1, respectively.
S0927-7757(16)30867-6 http://dx.doi.org/doi:10.1016/j.colsurfa.2016.10.013 COLSUA 21080
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
27-8-2016 25-9-2016 11-10-2016
Please cite this article as: Tao Huang, Zong-cai Tu, Hui Wang, Xinchen Shangguan, Lu Zhang, Peipei Niu, Xiao-mei Sha, Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2016.10.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Promotion of foam properties of egg white protein by subcritical water pre-treatment and fish scales gelatin
Tao Huanga, Zong-cai Tua,b*[email protected], Hui Wanga, **
Xinchen-Shangguana,c,d [email protected], Lu Zhangb, Peipei Niua, Xiao-mei Shab
a
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China
b
Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China
c
College of Food Science, Jiangxi Agricultural University, Nanchang, Jiangxi 330045, China
d
Food and Drug Administration of Jiangxi Province, Nanchang, Jiangxi 330029, China
*
Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang
University, Nanchang , China Key Laboratory of Functional Small Organic Molecule, Ministry of Education and College of Life Science, Jiangxi Normal University, Nanchang, China
**
Prof. Xinchen-Shangguan, Food and Drug Administration of Jiangxi Province, Nanchang, China,
Graphical abstract
Highlights • • •
SW could improve the foam-ability, but decrease the foam-stability of EWP obviously. Addition of FSG could enhance the foam-ability and foam-stability of SWEWP. SWEWP-FSG formed stronger mechanical film and showed higher foam properties.
Abstract The aim of this work was to study the effect of fish scales gelatin (FSG) on the foam and interfacial properties of subcritical water (SW) treated egg white protein (EWP) systems. The foam ability of the SW treated EWP (SWEWP) system is obviously better than that of untreated EWP system, although the former had poorer foam stability. The foam ability of the SWEWP system was further enhanced by the addition of FSG by reducing the surface tension. FSG seemed to build an interfacial viscoelastic network at the air – water interface with the increased surface dilational rheological behavior, causing the low drainage of liquid and inhibiting the bubbles coalescence of complex systems. Moreover, variations of surface elasticity matched the foam stability as the FSG concentration increased. This study described the effect of the biopolymer mixing ratio on the foam properties of SWEWP and FSG. This study also offered the possibility to design the production of protein powder with an outstanding capacity for foams formation and stabilization.
Keywords: egg white protein; subcritical water; fish scales gelatin; foam properties; interfacial properties
1. Introduction
Egg white protein (EWP) is frequently used as a foaming agent in the food industry to improve
and maintain the quality (texture and volume) of aerated food, such as meringues, beverages, fermentation, cakes, whipped creams and chocolate mousses [1-3]. However, thermodynamic instability is a basic property of foams, thereby leading to their disintegration over time and their large surface area, this instability could significantly influence the final quality of the products [1, 4].
Several techniques have been employed to improve the foaming properties of EWP, such as heat [5, 6], high pressure [7, 8], pulsed electric fields followed by heat treatment [9], irradiation [10-12] and acylation [13]. Nevertheless, high pressure treatment has several limitations during mass production and may increase the production cost [12]. Irradiation produces unattractive odors [14], whereas chemical modifications may generate hazardous substances. Therefore, a novel method should be developed to improve the foaming properties of EWP. Subcritical water (SW) is defined as hot water at high temperatures ranging from 100 to 374 oC under high pressure to maintain water in the liquid state. SW is an extraordinary medium or solvent for various chemical reactions and the extraction of different compounds [15]. SW is also an alternative to the traditional methods of protein hydrolysis because water is an environment - friendly reaction medium does not need acids or enzymes [16, 17]. SW has been used to produce products with excellent foam stability from soy meal [18]. To the best of our knowledge, information is available on the foaming mechanism SW - treated protein. Moreover, the synergistic effect of blending proteins and hydrocolloids generally produces a more significant improvement of the functional properties of several foods than those of proteins and hydrocolloids alone [19]. Gelatin, low in calories, is normally applied in foodstuffs to enhance protein levels, and is especially useful in stabilizing foamed food products because of its excellent surface-active and gelling properties upon cooling [20, 21]. Nevertheless, concerns over bovine
spongiform encephalopathy, vegetarianism and religious beliefs have driven the concerted efforts to find alternative protein sources that can provide similar functions for food systems [22]. Fish gelatin has been regarded as a promising alternative in the food industry, because of its similar properties with mammalian gelatin [23]. The foaming [24] and interfacial properties [25] of mammalian gelatin or casein glycomacropeptide (CMP) – mammalian gelatin [21] have been studied. However, the influence of fish gelatin on the foaming properties of other food proteins has been rarely reported. In our previous work (Fig S1), FSG enhanced the foaming properties of untreated EWP and SW - treated EWP systems, the latter presented better foaming capacity and foaming stability. At present, many studies on surface dilational rheology of various surfactant at the air – water interface have been published to better understand the interfacial behavior of a film [26, 27]. Therefore, the present work was performed to study the foaming and interfacial properties of EWP as influenced by pre-treatment with SW and the addition of FSG. The foaming mechanism of protein systems was explored, to develop a process that can produce protein powder product with high foaming properties, which has a potential in future expand the application of FSG and EWP through an industrially applicable process.
2. Materials and methods
2.1.Materials
Fresh eggs (Lao nan gou) were bought from local market in Nanchang, China. Liquid EWP with protein content of 10.21 ± 1.12% [28] was prepared by separating egg white and yolk manually. FSG
was extracted from bighead carp scales as described in our previous study [23]. The crude protein content of lyophilized gelatin was 90.21 ± 1.23 % with molecular weight of 30 – 200 kDa [23]. Double-distilled water (with conductivity of 0 µs / cm) was used throughout all the experiments.
2.2 .Methods
2.2.1.Preparation of SW treated EWP Fresh liquid EWP was mixed with distilled water at a ratio of 1:4 (w/w) for 0.5 h at room temperature to obtain a homogenous mixture. The SW treatment was performed at 140 °C for 1 h. After the reaction, the treated EWP solution was rapidly cooled by submerging the reactor in an ice bath for 5 min. The treated and untreated EWP solutions were collected and sprayed for further analysis.
2.2.2 Preparation of single and complex protein systems Single system: The EWP and SW treated EWP (SWEWP) powders were dissolved with deionized water in a water bath (55 oC, 1 h, ～400 rpm) to prepare the protein solutions (10 mg mL-1, pH 6.5). The gelation of gelatin was hindered because the critical concentration for gelation is 20 mg mL-1 [21]. To discriminate the foaming properties of gelatin from its gelling properties, the max FSG concentration was 6 mg mL-1. The single system was named as EWP, SWEWP and FSG, respectively Complex systems: The SWEWP- FSG systems were prepared by adding FSG to the prepared SWEWP solution. The concentrations of FSG were 2, 4 and 6 mg mL-1, the mixtures were incubated in a water bath (40 oC, ～400 rpm) until complete dissolutions, and were named as SWEWP-FSG1, SWEWP-FSG2, and SWEWP-FSG3, respectively
2.2.3. Measurements of foam properties The foam properties of single and complex systems were characterized based on their foam formation and stability on a Foamscan instrument (IT Concept, Teclis Co., France) according to the method of Zhang et al. [29] with some modifications. The foam ability, foam stability and drainage from the foams were determined by conductivity measurement of the foam column. The change of the bubbles was observed by a CCD camera which photographed every 5 s. The size and distribution of the bubbles were analyzed from the foam pictures with a CAS software. Briefly, the foams were generated by blowing air at a constant flow rate of 200 mL min-1 through a porous glass filter at the bottom of a glass tube, where 60 mL of prepared protein solution was located. The blowing of air was immediately stopped when the foam volume reached 100 mL. The blowing time “t” was defined as the foamability. The time of foam volume decays “t1/2” was recorded as the foam volume decayed from 100 mL to 50 mL. The quantity of the liquid that remained after foaming and that was drained out from the foam was measured by a pair of electrodes at the bottom of the glass column. The volume of the liquid in the foam was measured by conductimetry with three pairs of electrodes along the glass column. Each sample was performed in triplicate at room temperature.
2.2.4. Measurements of surface tension The surface tension of various solution systems were carried out on the interface expansion rheometer (Tracker, TECUS-IT Concept, France) using pendant drop method. The average values of surface tension were obtained in triplicate at room temperature.
2.2.5. Measurements of surface dilational viscoelasticity The parameters of surface dilational viscoelasticity of the systems with various components were measured using the oscillating bubble rheometer (Tracker, TECUS-IT Concept, France) as reported by He et al. [30] with few modifications. The dilational viscoelasticity measurements were started after a pre-equilibrium period of 2 h. The prepared bubble was expanded and compressed sinusoidally with a small amplitude (3%) and oscillational frequency (0.1 Hz). The ratio of small changes in the surface tension (γ) and surface area (A) are defined as the surface dilational modulus ε:
where εd is the dilational elasticity, ηd is dilational viscosity and ωηd is the dilational viscosity component, which can be calculated by:
2.2.6. Statistical analysis All the experiments were performed in three times. Data were subjected to analysis of variance (ANOVA) (P < 0.05) using Statistic program (Statsoft Inc.). Analysis was performed using SPSS 17.0 (SPSS Inc, Chicago, IL, USA).
3. Results and discussion
3.1. Foam properties
3.1.1. Foam morphology The variation of bubbles generated from single and complex systems was measured and analyzed after stopping nitrogen blowing when foam volumes reached 100 mL. Obviously, the diameters of bubbles generated from all systems gradually increased as the bubbles changed in shape from sphere to polyhedrons with increasing time (Fig. 1). These results were similar with those of Martinez et al.[21], who reported that the diameter of bubbles generated from a CMP / gelatin system and the CMP – gelatin complex systems gradually increased with time. According to the Laplace – Young law, gas diffusion occurred through liquid film from smaller bubbles to larger bubbles because of the higher pressure in smaller bubbles, thereby decreasing the number of smaller bubbles while increasing the size and collapse of the larger bubbles. Zhang et al. [29] also reported that the liquid between the bubbles drained out from the liquid channels and plateau borders under gravity, making adjacent bubbles coalesce as the liquid film became thin enough . As shown in Fig. S2, FSG also possesses smaller bubbles than pig skin gelatin (PSG), whereas SWEWP-FSG has smaller bubbles than SWEWP-PSG. Therefore, FSG displays better steric protection and elastic properties to interfaces and could replace PSG in some aerated foods. Compared with EWP, treated EWP took less time to reach 100 mL (Table 1) and possessed smaller bubbles at 0 s (Fig 1). However, these bubbles easily enlarged within a short of period of time,
namely, 300 s in this study. SW could improve the foaming capacity of EWP, but reduced its foaming stability (Fig. S1). The images of complex systems (t = 0) presented smaller bubbles sizes as the FSG concentration increased, revealing that FSG could strengthen the bubbles stability and improve the stability of complex systems. The foam of the complex systems was more stable than the single system, but was more heterogeneous, implying that FSG could influence the structure of bubbles and delay foam collapse by decreasing the initial bubbles size and delaying the increase of bubbles. The evolution of bubbles in foam could be described by observing the size distribution of bubbles in the foamy network with time [31]. In this study, images of the bubbles size distribution (at least 200) and the mean radii in different systems at 100 s were analyzed by the CSA software, the corresponding data are displayed in Fig. 2 and Table 1, respectively. Obviously, the bubble size of EWP was approximately below 0.075 mm (frequency of 134), which was larger than that of the SWEWP bubbles (below 0.066 mm, frequency of 239). In the complex systems, the distributions of the bubble radius were significantly dependent upon the FSG concentration. The SWEWP-FSG3 system had the smallest bubbles diameters, which were approximately below 0.036 mm (frequency of 202) and at 0.036 - 0.059 mm (frequency of 182). Furthermore, the invariant mean value of each group was practically obtained by our analysis. A similar observation was previously reported by Barik and Roy [29].
3.1.2. Foam ability The foaming power or foam ability is related to the level of air phase volume upon the introduction of a gas into the protein solution [2]. The time “t” could be described as the time needed to make the foam volume reach 100 mL, which reflects the foaming capacity[29]. Table 1 shows that single EWP system has higher foam time (95.63 ± 1.07) than single A system (41.53 ± 1.11) (p < 0.05),
implying that SW could improve the foam ability of EWP. EWP could be hydrolyzed by SW under high temperature and pressure, producing numerous substances with low molecular weights, and lower adsorption energies, these substances could rapidly diffuse to the surface within a shorter time than the original EWP. FSG is a linear polymer with a typical molecular weight of 30 - 200 kDa [23]. SWEWP has substances with lower molecule weights, which could rapidly diffuse to the surface within a shorter period of time. Thus SWEWP keeps the constant molecule exchange between the surface and bulk phase, as well as lower the surface tension (Table 2), leading to improve foaming ability of complex systems with the increasing of FSG concentration.
3.1.3. Foam stability and drainage Foam stability is important to prolong the shelf-life and improve the product appearance of food foams, this property must be maintained even when the foam is subjected to various processes, such as heating, mixing and cutting [2]. Given the effects of gravity, the liquid and gas in the foam tend to separate and leak liquid out of the foam, thereby drying the foam as time increases [32]. The half life (t1/2) of foams is an important parameter that is used to evaluate foam stability. The stability and kinematic viscosity of SWEWP are lower than those of EWP (Table 1), descending the stability of the film and speeding up the breaking up of bubbles. On the other hand, FSG had significant effect on the t1/2 values, as SWEWP-FSG3 had the highest t1/2 value (11233.33 ± 57.73) (p < 0.05). The higher t1/2, the higher foam stability. FSG could affect the viscosity of the continuous phase and the interface, whereas EWP mainly affects the interface alone. Thus a positive behavior of t1/2 raised from the ratio of SWEWP and FSG in the mixture systems. SWEWP could be diffused to the interface where it interacts with FSG, forming a mixture with highly viscoelastic characteristics, and films with considerably high
rigidity at the interface. This behavior was also attributed to the interactions between these biopolymers at the air - water interface and in the aqueous solutions. These interactions created thick visco-elastic networks that could delay foam destabilization [33] [21]. Foam collapse is analyzed from the time evolution of the foam volume after stopping gas bubbling (Fig 4A). The slope of this plot is taken as the collapse rate (kcollapse) of the system (Fig 4B). SWEWP showed a rapid collapse,which is also consistent with the results in Fig 1. The foam behavior of complex systems was dominated by the increasing FSG concentration because the kcollapse value of the foams declined. This trend was probably caused by the increased hydrophilic properties of adsorbed gelatin and the strong absorption or retention of water in the foams lamella, thereby inhibiting drainage and foam collapse [21]. The liquid content in the foams is related to the foam stability, and is usually denoted by the liquid volume [29]. The variation of liquid volume in the foams generated from protein systems is exhibited in Fig. 4C. The liquid volume in the foams first increased to the maximum values, and then decreased gradually with time. The maximum liquid volume of the complex systems increased as the FSG increased. The drainage rate is mainly affected by the packing pattern of the foam films, the composition of the interface and aqueous phase, the random network, and other properties of the foam films [29, 34]. The drainage process reduces the liquid in foam films and makes the smaller bubbles become closer to each other, eliminating the smaller foams and forming larger bubbles, this drainage process continues until equilibrium is reached between gravity and capillarity pressure [29]. Therefore, we speculated that FSG predominantly contributes to the foaming properties with the increasing FSG content in complex systems. Moreover, FSG is a good candidate for the stabilization of foams in aerated food consumed by Muslims, Jews and Hindus et al [20].
3.2 . Mechanism of foam stabilization
3.2.1. Surface tension Surface tension is used to explain the foaming mechanism [29]. Protein can spontaneously adsorb at various surfaces and form mechanically stable layers with low surface tension [35] [36]. As observed in Table 2, the surface tension of untreated EWP (48.3 mN m-1) is lower than that of SWEWP (45.16 mN m-1) (p < 0.05), because of the hydrolysis effect of SW. FSG system showed the lowest surface tension among the single protein system, indicating its best foaming ability. Moreover, the surface tension of complex systems was lower than those of individual protein system, and this surface tension decreased from 43.54 to 40.57 as the FSG concentration increased from 2 to 6 mg mL-1 ( p < 0.05). This was mainly attributed to that hydrolyzed EWP molecules at the interface were gradually displaced by competing FSG molecules in the adsorbed layer via weak electrostatic and hydrophobic interactions. Lu et al. [37] showed that concentration and configuration change of proteins on the drop surface might influence upon their adsorption rate and the film stability at interface. Schmidt et al. [38] reported that the re-organization of proteins at the interface or presence of multicouches would decrease the surface tension with increasing napin concentration. Therefore, FSG could rapidly reduce the surface tension of the protein system as a good foaming agent.
3.2.2. Surface dilational viscoelasticity Dilational rheology is a very effective indicator to characterize film properties [26, 39]. The effect of the FSG concentration on the surface dilational visco-elasticity of protein systems at a constant
frequency (0.1 Hz) is shown in Table 2. SW could sharply decrease the absolute modulus |ε|, producing a weaker foam stability as depicted in Fig. 1. The |ε| increased with the increasing FSG concentration, due to the increased concentration in the bulk. The absolute modulus |ε| is influenced by several factors, such as the frequency, temperature, protein concentration. Zhang et al. [29] reported that |ε| increased with increasing oscillational frequencies by relaxation processes at the interface, including the exchange and conformational changes of molecules in the surface layer. Gelatin imparts high viscosity because of its hydrodynamic volume, flexibility and expanded coil structure [24]. Therefore, the complex systems showed an increasing surface dilational viscoelasticity with the increasing FSG concentration at a constant frequency (0.1 Hz) (Table 2). FSG and SWEWP were supposed to form a viscoelastic layer in the films between bubbles and in the plateau, producing a gel-like network formed by hydrogen bonds, Van der Waals' force et al. The viscosity affected the immobile liquid film by reducing the drainage rate. Therefore, the higher viscosity and mechanical strength of the adsorbed layer, the higher foam stability will be given. In addition, the εd values of all systems were much higher than their ωηd, revealing that the dominance of the elastic characteristic in the single component and mixed layers at the air – water surface., as suggested by Zhang et al. [29]. This phenomenon was attributed to two reasons: (1) Conformational rearrangement following adsorption that causes the formation of an interconnected surface protein network; (2) The intra-protein structural rigidity at the surface that resists surface compression and/or expansion [37, 40]. Therefore, the addition of FSG could form a gel-like network structure at the surface of the complex systems. What’s more, the formation of a surface network along with the intrinsic structural stability of individual protein units within the network strengthened the ability of the film to withstand
a disturbance, improving foam stability [40].
4.Conclusions The single EWP system had weaker foam ability and a lower drainage rate, whereas the single SWEWP had better foam ability but faster drainage and weaker stable foams. This difference as caused by SW, could decrease the surface tension and surface dilational viscoelasticity. The addition of FSG could decrease the surface tension, but increased the kinematic viscosity, absolute modulus |ε| and surface dilational elasticity εd, thereby promoting the foaming properties of complex systems. Moreover, all the films of protein surface were elastic. The variation of foam stability agreed well with the kinematic viscosity and surface dilational elasticity of the corresponding systems as the FSG concentration increased. The higher stability of the foams was mirrored in the higher |ε| and εd values. In comparison to PSG, FSG contributed more to the foaming properties of the protein system. Therefore, FSG can act as an effective stabilizer of foam systems instead of mammalian gelatin. The SWEWP-FSG powder is also a promising alternative for aerated products processing, because of its outstanding performance.
Acknowledgement We gratefully acknowledge financial support from the National High Technology Research and Development Program of China (863 Program) (No, 2013AA102205), the Freedom Explore Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-ZZB-201310), National Natural Science of Foundation of China (No, 31660487), Collaborative Innovation Center for Major Ecological Security Issue of Jiangxi Province and Monitoring
Implementation (JXS-EW-00) and Earmarked fund for Jiangxi Agriculture Research System (JXARS-03).
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Figure caption Fig. 1. The variation of the foams generated from protein solution beginning with the stop of air flow (their is a 100 seconds interval between adjacent images respectively). EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 2. The bubble radius distribution of different protein systems at time of 100 s. EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 3. Time evolution of foam volume for different foam systems generated from various protein system. EWP: original egg white protein; SWEWP: EWP treated by SW at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Fig. 4. Foam volume (A), rate of collapse (kcollapse) (B) and liquid volume (C) of different systems generated from protein systems as a function of time. EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: Fish scales gelatin solution; 1:0.2、1:0.4、1:0.6: the addition of FSG in the 10 mg mL-1 SWEWP solution was 2, 4 and 6 mg mL-1 respectively. Table 1 Foam time (t), decay time (t1/2) and mean radius of different protein systems System
t (s)
t1/2 (s)
Mean radius
EWP
95.63±1.07e
8586.67±15.28e
0.092±0.085a
SWEWP
41.53±1.11d
353.67±4.73a
0.066±0.062a
FSG
38.17±0.67c
6780±10d
0.085±0.077a
SWEWP-FSG1
38.06±0.41c
1470±10b
0.086±0.033a
SWEWP-FSG2
36.8±1.15b
3540±10c
0.045±0.025a
SWEWP-FSG3
27.87±0.93a
11233.33±57.73f
0.041±0.021a
Values in the same column with different letters are significantly different (p < 0.05); EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: fish scales gelatin; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 (w/v) SWEWP solution was 2 mg mL-1, 4 mg mL-1 and 6 mg mL-1, respectively. Table 2 Surface tension (γ), absolute modulus |ε|, surface dilational elasticity (εd) and surface dilational viscous component (ωηd) of different protein systems System
γ(mN/m)
|ε|
εd
ωηd
EWP
48.3±0.02f
44.11±0.33e
43.18±0.32e
9.05±0.16e
SWEWP
45.16±0.06e
22.05±0.35a
20.98±0.22a
6.07±0.03b
FSG
44.08±0.17d
27.49±0.08c
27.29±0.08c
3.34±0.05a
SWEWP-FSG1
43.54±0.08c
25.51±0.16b
25.44±0.11b
6.41±0.25c
SWEWP-FSG2
42.6±0.02b
33.24±0.07d
32.77±0.11d
7.17±0.06d
SWEWP-FSG3
40.57±0.02a
47.53±0.06f
45.88±0.04f
9.28±0.04f
Values in the same column with different letters are significantly different (p < 0.05); EWP: original egg white protein; SWEWP: EWP treated by subcritical water at temperature of 140 °C for 1h; FSG: fish scales gelatin; SWEWP-FSG1、SWEWP-FSG2、SWEWP-FSG3: the addition of FSG in the 10 mg mL-1 (w/v) SWEWP solution was 2 mg mL-1, 4 mg mL-1 and 6 mg mL-1, respectively.