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Studies on foaming and physicochemical properties of egg white during cold storage
Colloids and Surfaces A 582 (2019) 123916
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Studies on foaming and physicochemical properties of egg white during cold storage
T
Yinxia Chena, Long Shenga, Mostafa Goudaa,b, Meihu Maa,
⁎
a b
National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, PR China Department of human nutrition & food science, National Research Centre, Dokki, Cairo, Egypt
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Chemical compounds studied in this article: ANS (8-Anilino-1-naphthalenesulfonic acid) (PubChem CID: 1369) DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) (PubChem CID: 6254) SDS (Sodium dodecyl sulfate) (PubChem CID: 3423265) Tris (Tris(hydroxymethyl)aminomethane) (PubChem CID: 6503) EDTA (Ethylenediaminetetraacetic acid) (PubChem CID: 6049) Glycine (PubChem CID: 750) HCl (PubChem CID: 313) NaCl (PubChem CID: 5234) KBr (Potassium bromide) (PubChem CID: 253877)
The effects of shell egg cold storage (4 °C) on the foaming properties of its egg white were investigated by evaluating its physicochemical changes. The foaming ability decreased significantly (p < 0.05) while the foaming stability decreased slightly during cold storage. Specifically, the foam ability reached the minimum (25.17%) at 40 d of storage, which was decreased by 44.49% versus that at 0 d (45.33%). In the first 40 d, the decrease in foaming ability was mainly ascribed to the decreased surface hydrophobicity and the increased surface tension. Moreover, the exposed sulfhydryl groups tended to cause protein cross-linking probably by sulfhydryl-disulfide exchange reaction, leading to an increase of particle size and viscosity. However, during the 60–80 d storage, the decrease of sulfhydryl content and viscosity tended to inhibit foam formation. Additionally, more larger protein aggregates were observed with prolonging of storage period under a scanning electron microscope. This investigation reveals the change mechanisms of egg white properties during different storage periods and provides guidance for the processing of egg products.
Abbreviations: ANS, 8-anilino-1-naphthalenesulfonic acid; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); SDS, sodium dodecyl sulfate; Tris, Tris(hydroxymethyl)aminomethane; FA, foaming ability; FS, foaming stability; SH, sulfhydryl groups (μmol/g protein); H0, surface hydrophobicity (a.u.); FI, fluorescence intensity; d3,2, volume-surface mean diameter (μm); d4,3, volume-mean diameter (μm); FT-IR, Fourier transform infrared spectroscopy; SEM, scanning electron microscope; ANOVA, analysis of variance; G′, storage modulus (Pa); G″, loss modulus (Pa); tan δ, G″/G′; k, consistency coefficient; n, flow behavior index ⁎ Corresponding author. E-mail address: [email protected] (M. Ma). https://doi.org/10.1016/j.colsurfa.2019.123916 Received 23 June 2019; Received in revised form 9 August 2019; Accepted 1 September 2019 Available online 03 September 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces A 582 (2019) 123916
Y. Chen, et al. Keywords: Egg white Cold storage Foaming properties Physicochemical properties
1. Introduction
2. Material and methods
Egg white not only contains a broad range of nutritional components, but also possesses multiple functional characteristics such as foaming, emulsifying, gelling and binding adhesion [1]. Among the large amounts of proteins present in egg white, the four main proteins are ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%) and lysozyme (3.5%) [2]. These proteins have a combined effect on the functional properties of egg white and display different roles in the processing of many products, suggesting their important functions in food industry [3]. Due to its prominent foaming properties, egg white has been widely applied in many food products, including dessert shells, cakes, bread and ice cream [4]. In the processing of these products, egg white foams can provide desirable and unique textures with a larger product volume [5]. Additionally, it plays an important role in surface-active ingredients due to the interaction of protein components in the media of products. Furthermore, globulin contributes to foam formation, lysozyme improves film strength and foam stability by interacting with other proteins, and ovomucoid prevents foam drainage [6]. Many factors have been reported to affect the foaming properties of egg white, including pH [7], protein concentration [8], ionic strength [9], intermolecular interaction [10], protein composition [11], and temperature [12]. In addition, storage conditions have a huge influence on the quality of egg freshness, such as the content of s-ovalbumin, Haugh units, albumen height, yolk index, egg weight and air cell size, all of which are correlated with egg freshness [13–15]. Based on these factors, different modifications methods have been developed to improve the foaming properties of egg white, which are mainly divided into physical, chemical and enzymatic methods [16,17]. Cold storage contributes to control microorganisms, especially Salmonella in the egg products. As the most convenient and effective storage method, cold storage displays a certain degree of limitation on the quality deterioration of egg white [12]. With the extension of storage time, the functional properties of egg white were subjected to destruction, especially at a high temperature [18]. Many previous studies have shown that the occurrence of egg white thinning with the increment of storage duration at room or high temperature due to the sulfhydryl-disulfide exchange reaction of ovalbumin [19], the loss of lysozyme activity [20] and the dissociation of ovomucin-lysozyme [21]. Although, cold storage was reported to moderately spoil the foaming capacity of egg white, little further research has been undertaken on the potential mechanism for the change in foaming properties so far, which is essential to seek suitable modification methods for improving foaming properties. In this study, shell eggs stored at ten different time periods (at an interval of 10 d) were used to explore the potential explanation mechanisms for the cold storage effect on the foaming properties of egg white, by measuring its free sulfhydryl content, surface hydrophobicity, surface tension, particle size, zeta potential, rheological properties, and secondary protein structure. This research has shed light on the changes in egg white foaming properties under cold storage and contributes to the development of a feasible and effective method for egg storage.
2.1. Materials Hy-Line Brown eggs were collected from animal husbandry of Hubei Academy of Agricultural Sciences (Wuhan, China). 1Anilinonaphthalene-8-sulfonic acid (ANS) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents used in this study were all of analytical grade. 2.2. Sample preparation Fresh shell eggs (laid within 3 d) were stored at 4 ± 1 °C and total storage period was 90 d. Eggs from different storage periods (0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 d) were washed cleanly and broken manually to separate egg white and egg yolk carefully. Finally, a uniform sample system was obtained by stirring the original egg white for 3 h using a magnetic stirrer (IKA, IKA Works Inc., Wilmington, NC, USA). 2.3. Foams preparation and properties Foams were prepared as previously reported with slight modifications [22]. Briefly, an aliquot volume of egg white sample (20 mL, 10% v/v) diluted with phosphate buffer (10 mM, pH 7.0) was added into a 50 mL glass cylinder (internal diameter of 22 mm, height of 150 mm). Then, foam was produced by a high-speed homogenizer (XHF-DY, Scientz, Zhejiang, China) equipped with a probe (internal diameter of 7.5 mm, height of 145 mm) at 8000 rpm for 1 min at 25 °C. The whipping foam volumes were recorded at 2, 30, 60, 180 and 300 min to evaluate the foam decay, and the corresponding visual appearance of the foam was captured to observe the bubble change with time. Besides, the foam microscopy images at 2, 10, 30 and 60 min were obtained by an optical microscope (Sunny Optical Technology Co., LTD, Zhejiang, China). Finally, the foaming ability (FA) and the foaming stability (FS) were calculated according to the following equations:
FA(%) = V2/20 × 100
(1)
FS(%) = V30/ V2 × 100
(2)
where V2 and V30 are the foam volume at 2 and 30 min after whipping, respectively. 2.4. Physicochemical changes in egg white 2.4.1. Free sulfhydryl (SH) content The free sulfhydryl group content of egg white was measured according to the method of Duan et al. [17]. Briefly, Ellman’s reagent (4 mg/mL) was prepared by dissolving 4 mg of DTNB in 1 mL of TrisGlycine buffer (10.4 g Tris, 6.9 g Glycine and 1.2 g EDTA dissolved in 1 L distilled water, pH 8.0). Meanwhile, 1.5 g egg white was diluted to 10 mL with 1% (m/v) NaCl solution in Tris-Glycine buffer. Then, 2.9 mL 0.5% (m/v) SDS solution in Tris-Glycine buffer and 0.02 mL Ellman’s reagent were mixed with 0.1 mL of the above diluent sample solution. Subsequently, the mixture was shaken fully and incubated in dark for 15 min. The absorbance of the prepared sample was measured at 412 nm on a UV/VIS spectrophotometer (Nanodrop-2000C, Thermo Scientific, USA) against the blank (without Ellman’s reagent and protein 2
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sample). The protein content in all samples was determined by the Bradford method, and the free SH group content was calculated by the following equation:
SH (µ mol/g protein) = 73.53 × A 412 × D/C
defined as the initial slope of the plot of fluorescence intensity (FI) as a function of protein concentration (C).
FI = FI1
(3)
FI0 = H0 × C + B
(4)
where FI1 and FI0 are respectively the fluorescence intensity with and without ANS and C is protein concentration of each diluted sample.
where A412 is the absorbance of the sample at 412 nm, D is the dilution factor, and C is the concentration of egg white protein solution.
2.4.3. Surface tension The surface tension of egg white protein solution was determined as previously described by Sheng et al. [23]. Briefly, liquid egg white was diluted to 10% (v/v) using phosphate buffer (10 mM, pH 7.0), followed by measuring the sample surface tension with a tensiometer (k100, Krüss, Germany) using the Wilhelmy’s plate method at 25 °C. For each measurement, an aliquot volume of diluted egg white sample (30 mL) was used and 40 data points were recorded for 3600 s. Finally, the surface tension plot versus time was used for analysis.
2.4.2. Surface hydrophobicity (H0) The surface hydrophobicity of egg white protein solution was determined as reported by Duan et al. [5] with some modifications. Specifically, the egg white sample was diluted to five concentrations (0.01%, 0.02%, 0.05%, 0.1% and 0.2%, v/v) using phosphate buffer (10 mM, pH 7.0), followed by filtration through 0.45 μm membrane. Then, 4 mL of each diluted protein solution was supplemented with 20 μL of 8 mM 1-anilinonaphthalene-8-sulphonic acid in the same buffer, followed by incubation in dark for 3 min and measuring the fluorescence intensity (with ANS and without ANS) with excitation at 390 nm, emission at 470 nm and slit width 5 nm using a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). The spectrum of the buffer subtracted to correct the background fluorescence. H0 was
2.4.4. Particle size and zeta potential Before measurement, egg white was diluted to 1% (v/v) in phosphate buffer (10 mM, pH 7.0) and filtered through 0.45 μm membrane. The particle size of freshly prepared egg white protein solution was
Fig. 1. The foam collapse curve (A), foam ability (B), foam stability (C), foam visual appearance (D) and foam microscopy image (E) of egg white from shell eggs stored (4℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 3
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determined by refractive index (1.450) and absorption index (0.001) using a laser light scattering analyzer (APA2000, Malvern Instrument, Ltd., UK). The zeta potential of egg white solution was determined using a ZS Zetasizer Nano (Malvern Instrument, Ltd., UK). All measurements were carried out at 25 ℃ and repeated three times.
storage time prolonging, coupled with an increase of bubble size, due to bubble disproportion and liquid drainage (Fig. 1A, D and E) [27]. In Fig. 1B, it was shown that the foaming ability decreased from 45.33 to 25.17% at 40 d versus that at 0 d (p < 0.05), indicating the great effect of cold storage on foam formation. However, the foaming ability increased from 40 to 60 d (25.17 to 31.33%, p < 0.05), followed by an decrease from 60 to 80 d (31.33 to 28.50%, p < 0.05). These dynamic variations may be related to multiple protein structures and interaction change. The changes in the ovomucin characteristic during storage had an effect on its interaction with lysozyme between the thick and thin white, leading to an increase in the interaction of lysozyme with soluble ovomucin as the storage proceeded [28]. Therefore, the participation of soluble ovomucin in foam formation contributed to the increase of foaming ability from 40 to 60 d. Sheng et al. (2018b) [29] reported that the foam properties of ovalbumin decreased significantly after storage of 50 d at 22 °C, which may be ascribed to less exposure of hydrophobic amino acid caused by the aggregation of ovalbumin. In Fig. 1C, the foaming stability was shown to decrease slightly after storage, which was attributed to the thinning of egg white [13]. Robinson [30] reported that the thick albumen gradually became thin with the extension of egg storage time, thus increasing the content of the thin albumen. Additionally, it was reported that, when the pH increased during storage, n-ovalbumin was transformed into s-ovalbumin [31], and the less hydrophobic s-ovalbumin interfered with the formation of a cohesive film on the air-water interface, causing a reduction in foam stability. On the other hand, a gradual larger bubble size was observed with storage time prolonging throughout the experiment (Fig. 1E), implying foam drainage, foam coarsening and bubble size distribution [28].
2.4.5. Rheological behaviors Dynamic rheological measurements of egg white were conducted using a DHR2 rheometer (Waters, USA) as described by Wang et al. [24]. All the procedures were performed at 25 °C using a parallel plate (60 mm) with the gap being set to 1 mm. Specifically, the first step was to determine the region of the linear viscoelasticity at the mode of oscillation amplitude in the strain range of 0.01 to 100% at a constant frequency (1 Hz). Then, the elasticity modulus G′ (storage modulus) and viscosity modulus G″ (loss modulus) were obtained in the frequency range of 0.1–10 Hz within the linear area of viscoelasticity. Finally, under the mode of flow sweep, the apparent viscosity was obtained at a varying shear rate from 1 to 100 1/s. 2.4.6. Fourier transform infrared spectroscopy (FT-IR) Quantitative analysis of the changes in the secondary structures of lyophilized egg white samples was performed in amide Ⅰ band (17001600 cm−1) using a Fourier transform infrared spectrophotometer (Nexus 470, Metier, USA). The lyophilized egg white samples (2 mg) were mixed with KBr at the ratio of 1:200 and ground to power in a mortar with a pestle. The FT-IR spectra were recorded in the range of 4000-400 cm−1 with air as background. The baselines were subtracted automatically and the initial values of the peak positions were determined by Fourier deconvolution. Additionally, the obtained spectra were smoothed by correcting the background noise with air data using the Omnic software (Version 9.2. 86, Thermo Fisher Scientific Inc). Finally, the second derivative calculation and Gaussian curve-fitting analysis were performed to estimate the position and percentage of the component bands using PeakFit v4.04 (Aisn Software Inc) [25,26].
3.2. Analysis of physicochemical properties 3.2.1. Free sulfhydryl content The alteration in free sulfhydryl (SH) content is an indicator for changes in disulfide bonds, which are a contributor to protein aggregation. It has been reported that film formation in the bubble can be improved by the variation in the molecular structure induced by reduction of disulfide bonds among protein molecules [32]. Fig. 2 shows the free SH content of egg white from shell eggs stored at 4 ℃ for different time periods. The free SH content showed an increase of 11.97 to 19.57 μmol/g protein from 0 to 20 d (p < 0.05), which was attributed to the exposure of inner sulfhydryl group and subunit dissociation. A similar trend was observed during 30 to 40 d (11.97 to 16.32 μmol/g protein, p < 0.05). In the first 40 d of storage,
2.5. Morphology observation The morphology of egg white samples was observed by a SEM. Briefly, samples were lyophilized by Alpha 2–4 LD plus freeze dryer (Christ, Germany), followed by sputter-coated a small amount of each dried sample with gold using a gold injection instrument (ACE200, Leica, Germany). Finally, the prepared samples were examined under a scanning electron microscope (Quanta 250, FEI, USA) at 10 kV under 200 and 2000 x magnification. During SEM operation, several different parts of each sample were observed, and images with a similar morphology were selected to represent the whole sample. 2.6. Statistical analysis All experiments were carried out in triplicate and each sample was measured in duplicate for analytical assays. All data were presented as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) with a 95% confidence interval to estimate the significance of the results. Means values between treatments were compared by Duncan's multiple range tests using the SPSS software program (Version 19). Graphs were plotted using Origin software (Version 9.0.0). 3. Results and discussion 3.1. Foam analysis Foam formation was determined by the migration, unfolding and rearrangement of protein molecules at the air-water interface. Fig. 1A shows the variation of foam volume in the time range from 2 to 300 min. Generally, the foam volume showed a downward trend with
Fig. 2. Free sulfhydryl (SH) group content for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 4
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the increase in free SH content meant that protein molecule was exposed to a more polar environment, which was adverse to foam formation, thus decreasing the foam ability. At the same time, the exposure of SH group increased the interaction among protein molecules and protein cross-linking induced by sulfhydryl-disulfide exchange reaction. Additionally, Zhao et al. [33] reported that an increment in SH content was correlating with formation of new cross-links. However, after the storage of 40 d, the free SH content showed a similar tendency to that of foaming ability, with an increase first and then a decrease from 40 to 80 d. This increase may be attributed to breakage of interchain disulfide bonds and exposure of sulfhydryl group induced by the dissociation of lysozyme-ovomucin, resulting in improved foam formation, while this decrease may be ascribed to protein oxidation during storage. The dissociation of lysozyme with ovomucin was observed after 40 d of storage, causing a different protein structure state.
Normally, the foaming ability was largely dependent on the surface tension at the air-water interface and lower surface tension was beneficial for the foam formation. The samples from all tested storage time periods showed higher surface tension than the control, confirming that the foaming ability was also reduced during all storage periods. Additionally, the surface tension increased during 50 to 80 d (47.23 to 50.45, p < 0.05), suggesting that protein molecules became more rigid and compact, leading to less ability to adsorb and unfold at the airwater interface. Xiong et al. [38] reported that lower surface tension was related to smaller particle size and less surface charge, which could decrease the electrostatic barrier and thus have a more rapid adsorption rate at the air-water interface. 3.2.4. Particle size and zeta potential Particle size is usually presented as volume-surface mean diameter (d3,2) and volume-mean diameter (d4,3), with an effect on the interfacial properties, including foaming and emulsification [39]. Fig. 5A shows the tendency of d3,2 and d4,3, with an increase for both of them increased after storage versus 0 d. Specifically, after 20 d of storage, d3,2 and d4,3 increased significantly from 21.24 to 42.74 μm and from 67.27 to 91.97 μm (p < 0.05), respectively, and a similar increase was observed between 30 and 40 d (30.17 to 36.69 μm, 69.40 to 78.45 μm, respectively, p < 0.05). In the first 40 d, the increased particle size significantly restrained the foaming ability, implicating that the exposure of SH group increased the interaction (probably by disulfide or hydrogen bond) among protein molecules. Meanwhile, the increased particle size might also cause a higher surface tension, leading to a lower adsorption rate at the air-water interface. The higher value of d4,3 was still observed after 40 d storage, indicating the continuous aggregation behavior of protein molecules. In Fig. 5B, it was shown that a high degree of heterogeneity (high span value) occurred at 0 d, probably due to the formation of noncovalent cross-linked protein aggregates in its native state [40]. However, the span value was decreased from 3.92 to 2.75 (p < 0.05) after 10 d storage, suggesting that the particle size distribution was narrowed by the formation of the soluble aggregates through covalent and noncovalent interactions [41]. In the first 40 d, the increase of particle size coupled with the decrease of span value indicated the presence of large but uniform protein aggregates. Moreover, the same tendency between the span value and foaming ability in the first 40 d demonstrated that higher heterogeneity facilitated protein adsorption at the air-water interface during this storage period. Nevertheless, during 50 to 80 d storage, the span value showed a gradual increase from 2.41 to 3.94
3.2.2. Surface hydrophobicity (H0) Surface hydrophobicity (H0), a common structural characteristic for evaluating protein conformation change, is correlated with foaming properties [34]. The H0 values of egg white samples from shell eggs stored at 4 ℃ for different time periods are shown in Fig. 3A. The H0 values exhibited a decrease from 798.4 to 607.9 (p < 0.05) at 20 d compared with 0 d, probably due to the potential aggregation behavior of egg white proteins as indicated by the increased particle size. The decline in H0 could decrease the number of amphipathic molecules and affect the adsorption behavior at the air-water interface, thus reducing foam formation. Additionally, in the first 40 d of storage, the H0 values exhibited the same tendency as that of foaming ability, implying that foam formation was determined by the exposure degree of hydrophobic group and unfolding of protein molecules. Furthermore, the polar environment induced by the exposure of SH group explained the reduced H0 value, thus decreasing the foaming ability in the first 40 d of storage. Kuan et al. [35] reported that the foaming ability of proteins could be enhanced by conformational changes with increasing surface hydrophobicity. Meanwhile, a dramatic increase of H0 was observed at 30 d (1062.7) compared with 20 d (607.9) (p < 0.05) (Fig. 1B), which was due to the exposure of hydrophobic groups buried in protein molecules. Combined with other parameters, the 30 d of storage was a turning point due to great change was obtained. Meanwhile, this significant increase in H0 value coincided with reduced surface tension and increased foaming ability. A possible explanation for this is that the increasing exposure of hydrophobicity groups reduce energy absorption barrier at the air-water interface, thus enhancing the adsorption kinetics [36]. 3.2.3. Surface tension Surface tension at the air-water interface is referred to as Van de Waals interactions between amphiphilic molecules. These amphiphilic protein molecules absorb at the interface, leading to the reduction of surface tension [37]. Fig. 4A shows the time evolution of surface tension for egg white protein at the air-water interface over different shell egg storage time periods. In the inset, the rapid reduction of surface tension at the first 1200s, indicated that protein molecules were rapidly diffused and adsorbed at the air-water interface in all the samples, suggesting that egg white proteins were adsorbed from the bulk onto the interface. However, with adsorption time prolonging, the surface tension showed a gradual decrease in all the samples, implying that fewer proteins were involved in the saturated adsorption interface, contributing to energy barrier formation during the adsorption and rearrangement process. Fig. 4B displays the specific surface tension value of egg white solution at 550 s. The direct increase of surface tension was ascribed to the (from 46.47 to 49.20, p < 0.05) led to an obvious decrease of foaming ability at 10 d, and this increment of surface tension was due to less absorption of egg white protein at the air-water interfaces.
Fig. 3. Changes in surface hydrophobicity (H0) for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 5
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Fig. 4. Time evolution of surface tension for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d) (A). Data were recorded at the adsorption time of 550 s (B). Different small letters indicate significant differences between groups. Error bars represent mean values ± standard deviations (n = 3).
Fig. 5. The particle size (A), span value (B) and zeta potential (C) for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between storage groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3).
(p < 0.05), while the viscosity exhibited a decrease, probably due to the formation of different particle size induced by loose structure. Zeta potential can be used to evaluate the stability of colloidal dispersion systems and elaborate the degree of electrostatic repulsion and interaction between protein molecules [42]. As shown in Fig. 5C, the absolute zeta potential was higher in egg white at 10 d (-12.47 mV) than in the control (-10.80 mV). This increment in the surface net charge increased the electrostatic barrier and thus decreased the adsorption rate [43], which was proved by the decrease of foaming ability and increase of surface tension at 10 d. However, during 10 to 40 d storage, the absolute zeta potential decreased from -12.47 to -9.58 mV
(p < 0.05), which was probably due to the reduction in the energy barrier to adsorption at the air-water interface induced by the interaction of protein molecules [44]. Additionally, a similar trend was observed between 50 and 90 d (-11.47 to -9.32 mV, p < 0.05), which was harmful to the system stability. The decreased foaming stability was largely related to the lower absolute zeta potential and it also indicated that the aggregation behavior caused by oxidation reaction during storage. 3.2.5. Rheological properties Identification of rheological 6
characteristics
facilitates
the
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Fig. 6. Changes in rheological properties for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d), including G′ (A), G″ (B), viscosity (C) and shear stress as a function of shear rate (D). Table 1 The frequency sweep data (G′, G″ and tan δ) were obtained at a frequency of 1 Hz, and the flow model parameters (k and n) were calculated from the Power-law for liquid egg white of shell egg stored (4 ℃) for different time periods (0 to 90 d). d
Frequency sweep G'/Pa
0 10 20 30 40 50 60 70 80 90
0.08 0.15 0.23 0.05 0.16 0.41 0.26 0.15 0.17 0.21
± ± ± ± ± ± ± ± ± ±
Flow sweep G”/Pa
0.02e 0.00d 0.02bc 0.00e 0.01d 0.05a 0.02b 0.01d 0.02d 0.03c
0.10 0.15 0.16 0.13 0.13 0.20 0.16 0.13 0.21 0.18
± ± ± ± ± ± ± ± ± ±
tan δ 0.01e 0.01bcd 0.03b 0.00de 0.02de 0.01a 0.00bc 0.00cde 0.04a 0.01ab
1.35 1.02 0.72 2.43 0.81 0.50 0.61 0.88 1.20 0.87
k ± ± ± ± ± ± ± ± ± ±
0.23b 0.06c 0.06de 0.03a 0.10d 0.07f 0.05ef 0.08cd 0.09b 0.09cd
0.06 0.07 0.30 0.03 0.14 0.38 0.30 0.22 0.11 0.12
R2
n ± ± ± ± ± ± ± ± ± ±
0.01fg 0.02efg 0.04b 0.01g 0.06d 0.03a 0.05b 0.01c 0.04def 0.03de
0.68 0.83 0.49 0.93 0.54 0.38 0.38 0.40 0.66 0.54
± ± ± ± ± ± ± ± ± ±
0.04c 0.04b 0.07de 0.06a 0.02d 0.10e 0.07e 0.02e 0.04c 0.07d
0.9975 0.9724 0.9747 0.9847 0.9843 0.9789 0.9733 0.9446 0.9069 0.9840
Different small letters in the same column indicate significant differences between groups (p < 0.05). Value was presented as mean ± standard deviation (n = 3).
understanding of the molecular changes during storage [45]. Fig. 6 shows the changes in the rheological properties of egg white samples from shell eggs stored for different time periods. In the first 40 d, the increased G′ meant the increasing interaction among protein molecules [24], which had a negative effect on the foam formation of egg white (Fig. 6A). Meanwhile, a higher particle size was observed in accompany with an increased G′, suggesting that the protein structure became more rigid proved by the decreased surface hydrophobicity and increased SH content, leading to lower foam formation. Compared with G′, G″ showed no dramatic differences in all the egg white samples (Fig. 6B). Liquid egg white was proved to be shear-thinning fluids [46], and Fig. 6C shows that the apparent viscosities of egg white decreased with
increasing shear rate. The shear-thinning behavior could be resulted from the breakdown of weak linkages between protein molecules and their rearrangement towards the direction of shear and decreasing flow resistance. An obvious increase in the viscosity was observed after the storage of 20 d, and a similar result was obtained during 30 to 40 d storage, which might result from small aggregates and cross-linking interaction of protein molecules proved by increasing particle size and decreasing surface hydrophobicity. In the first 40 d, the increased viscosity showed an adverse effect on the foaming ability because of the formation of rigid structure. Moreover, the increase in viscosity during storage could probably contribute to the insolubilization and aggregation of egg white proteins via intermolecular bonds. As reported by Herald et al. [47], the 7
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behavior index (n) remained below 1 and exhibiter the same tendency with other parameters.
Table 2 FTIR spectral analysis of changes in the secondary structure of egg white protein during shell eggs storage. d
α-helix 0 10 20 30 40 50 60 70 80 90
3.2.6. FT-IR spectroscopy Changes in the secondary structures of protein were investigated by Fourier transform infrared spectroscopy (FTIR). Amide I band (16001700 cm−1) in the FTIR spectra of protein was the characteristic band for the secondary structure and can be characterized by α-helix (16501660 cm−1), β-sheet (1610-1640 cm−1), β-turn (1660-1700 cm−1) and the random coil (1640-1650 cm−1) [49]. Table 2 summarizes the percentages changes in α-helix, β-sheet, βturn, and random coil. It can be seen that the content of α-helix and random coil remained relatively stable, in contrast to a significant change in the content of β-structure. The content of β-turn decreased from 10 to 30 d (28.00 to 26.33%, p < 0.05), but increased from 50 to 90 d (25.50 to 28.90%, p < 0.05) at the expense of β-sheet content (30.39 to 26.03%, p < 0.05). This change indicated that the protein structure became relatively loose with prolonging storage time due to protein dissociation, aggregation and partial denaturation, which could be well supported by the results of SEM, the increase of particle size, and the reduction of surface hydrophobicity.
Secondary structures (%)
13.86 13.91 13.80 13.47 13.88 13.29 13.29 13.77 13.67 14.21
± ± ± ± ± ± ± ± ± ±
β-sheet 0.28b 0.05b 0.02b 0.17cd 0.07b 0.11d 0.18d 0.16b 0.06bc 0.02a
28.29 27.40 28.24 29.61 28.02 30.39 30.32 28.49 28.60 26.03
± ± ± ± ± ± ± ± ± ±
β-turn 0.81c 0.02d 0.16c 0.60b 0.29cd 0.25a 0.41ab 0.75c 0.15c 0.09e
27.07 28.00 27.04 26.33 27.33 25.50 25.61 26.97 26.98 28.90
random coil ± ± ± ± ± ± ± ± ± ±
0.61c 0.06b 0.13cd 0.53d 0.27c 0.13e 0.42e 0.73cd 0.17cd 0.06a
30.77 30.69 30.92 30.60 30.78 30.82 30.79 30.77 30.75 30.86
± ± ± ± ± ± ± ± ± ±
0.08abc 0.05bc 0.10a 0.10c 0.06abc 0.06ab 0.20abc 0.14abc 0.08abc 0.03ab
Different small letters in the same structure indicate significant differences between groups (p < 0.05). Value was presented as mean ± standard deviation (n = 3).
viscosity of liquid whole egg increased during frozen storage, probably due to freeze-induced denaturation of the protein. A gradual decrease in the viscosity during 50 to 80 d storage meant the formation of loose structure. To illustrate the functionality loss of egg white during cold storage, the data of G′, G″ and tan δwere recorded at 1 Hz and the rheological flow models (Power-law) were also used to fit the experimental data (Fig. 6D, Table 1). G′ and G″ showed an increase from 0 to 20 d (0.08 to 0.23 Pa, 0.10 to 0.16 Pa, respectively) and from 30 to 50 d (0.05 to 0.41 Pa, 0.13 to 0.20 Pa, respectively, p < 0.05). Generally, tan δis considered as an indicator of protein quality [48], and a decrease in tan δindicated that the samples tend to become more elastic. In Table 1, the tan δwas shown to decrease from 0 to 20 d (1.35 to 0.72) and from 30 to 40 d (2.43 to 0.81) (p < 0.05). In the first 40 d, the decreased tan δindicated the protein structure was more elastic and solid-like, which was adverse to foam formation. However, the increase from 0.5 to 1.2 during 50 to 80 d suggested the transition of the protein structure to a more viscous one. Furthermore, the consistency coefficient (k value) also showed an increase from 0 to 20 d (0.06 to 0.30) and from 30 to 40 d (0.03 to 0.14), and a decrease from 50 to 80 d (0.38 to 0.11) (p < 0.05). The increase in the k value illustrated that cold storage increased both the system viscosity and flow resistance. Additionally, the pseudoplastic behavior of egg white appeared to be unchanged since the flow
3.3. Morphology change Scanning electron microscopy (SEM) was carried out to obtain tangible evidence for changes in egg white protein microstructure under different storage time periods. Fig. 7 displays a series of common SEM images of egg white samples at 200 x and 2000 x magnification. It could be seen that the 0 d sample exhibited a characteristic morphology with a smooth surface and sheet-like structures. However, after 30 d of storage, small aggregates were observed on the surface, and them became larger with prolonging storage time and transformed into larger aggregates at 50 and 70 d. This alteration may be attributed to protein cross-linking through non-covalent bond induced by oxidation, leading to enhanced interaction between proteins and more complex network composed of randomly aggregated protein molecules. 4. Conclusion In this study, we investigated the change mechanisms underlying the physicochemical and foaming properties of egg white samples from
Fig. 7. The SEM images for egg white from shell eggs stored (4 ℃) for different time periods (0, 30, 50 and 70 d). 8
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shell eggs stored at 4 ℃ for different time periods. In the first 40 d storage, the exposure of SH group increased the interaction among molecules via hydrogen and disulfide bond coupled with the increasing polarity of protein molecules, leading to the decrease of surface hydrophobicity. Meanwhile, the increased SH content caused the crosslinking between protein molecules as proved by the increased G′, resulting in the increased particle size and viscosity. The surface tension and the interface properties were generally affected by the exposure of SH group and the changes in surface hydrophobicity, particle size and viscosity. Additionally, the changes in the protein secondary structure influenced the surface and foaming properties, and the existence of oxidation during storage could be derived from the increase of particle size and the emergence of protein aggregation. Overall, the decrease in the foaming properties during cold storage was ascribed to the exposure of SH group, the decrease of surface hydrophobicity and the increase of surface tension caused by the increase of particle size and viscosity, giving rise to a lower adsorption rate of protein at the air-water interface. The changes and correlation of physicochemical properties of egg white from shell eggs storage were presented in the work, providing theoretical basis for food production.
[18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
Conflict of interest statement
[28]
All the authors declare that they have no conflict of interest.
[29] [30]
Acknowledgments
[31]
This work was supported by the National Natural Science Foundation of China (grant numbers 31571784 and < GN1 > 31701622); the H < /GN1 > ubei Provincial Natural Science Foundation of China (grant number 2018CFB606).
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Studies on foaming and physicochemical properties of egg white during cold storage
T
Yinxia Chena, Long Shenga, Mostafa Goudaa,b, Meihu Maa,
⁎
a b
National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, PR China Department of human nutrition & food science, National Research Centre, Dokki, Cairo, Egypt
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Chemical compounds studied in this article: ANS (8-Anilino-1-naphthalenesulfonic acid) (PubChem CID: 1369) DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) (PubChem CID: 6254) SDS (Sodium dodecyl sulfate) (PubChem CID: 3423265) Tris (Tris(hydroxymethyl)aminomethane) (PubChem CID: 6503) EDTA (Ethylenediaminetetraacetic acid) (PubChem CID: 6049) Glycine (PubChem CID: 750) HCl (PubChem CID: 313) NaCl (PubChem CID: 5234) KBr (Potassium bromide) (PubChem CID: 253877)
The effects of shell egg cold storage (4 °C) on the foaming properties of its egg white were investigated by evaluating its physicochemical changes. The foaming ability decreased significantly (p < 0.05) while the foaming stability decreased slightly during cold storage. Specifically, the foam ability reached the minimum (25.17%) at 40 d of storage, which was decreased by 44.49% versus that at 0 d (45.33%). In the first 40 d, the decrease in foaming ability was mainly ascribed to the decreased surface hydrophobicity and the increased surface tension. Moreover, the exposed sulfhydryl groups tended to cause protein cross-linking probably by sulfhydryl-disulfide exchange reaction, leading to an increase of particle size and viscosity. However, during the 60–80 d storage, the decrease of sulfhydryl content and viscosity tended to inhibit foam formation. Additionally, more larger protein aggregates were observed with prolonging of storage period under a scanning electron microscope. This investigation reveals the change mechanisms of egg white properties during different storage periods and provides guidance for the processing of egg products.
Abbreviations: ANS, 8-anilino-1-naphthalenesulfonic acid; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); SDS, sodium dodecyl sulfate; Tris, Tris(hydroxymethyl)aminomethane; FA, foaming ability; FS, foaming stability; SH, sulfhydryl groups (μmol/g protein); H0, surface hydrophobicity (a.u.); FI, fluorescence intensity; d3,2, volume-surface mean diameter (μm); d4,3, volume-mean diameter (μm); FT-IR, Fourier transform infrared spectroscopy; SEM, scanning electron microscope; ANOVA, analysis of variance; G′, storage modulus (Pa); G″, loss modulus (Pa); tan δ, G″/G′; k, consistency coefficient; n, flow behavior index ⁎ Corresponding author. E-mail address: [email protected] (M. Ma). https://doi.org/10.1016/j.colsurfa.2019.123916 Received 23 June 2019; Received in revised form 9 August 2019; Accepted 1 September 2019 Available online 03 September 2019 0927-7757/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces A 582 (2019) 123916
Y. Chen, et al. Keywords: Egg white Cold storage Foaming properties Physicochemical properties
1. Introduction
2. Material and methods
Egg white not only contains a broad range of nutritional components, but also possesses multiple functional characteristics such as foaming, emulsifying, gelling and binding adhesion [1]. Among the large amounts of proteins present in egg white, the four main proteins are ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%) and lysozyme (3.5%) [2]. These proteins have a combined effect on the functional properties of egg white and display different roles in the processing of many products, suggesting their important functions in food industry [3]. Due to its prominent foaming properties, egg white has been widely applied in many food products, including dessert shells, cakes, bread and ice cream [4]. In the processing of these products, egg white foams can provide desirable and unique textures with a larger product volume [5]. Additionally, it plays an important role in surface-active ingredients due to the interaction of protein components in the media of products. Furthermore, globulin contributes to foam formation, lysozyme improves film strength and foam stability by interacting with other proteins, and ovomucoid prevents foam drainage [6]. Many factors have been reported to affect the foaming properties of egg white, including pH [7], protein concentration [8], ionic strength [9], intermolecular interaction [10], protein composition [11], and temperature [12]. In addition, storage conditions have a huge influence on the quality of egg freshness, such as the content of s-ovalbumin, Haugh units, albumen height, yolk index, egg weight and air cell size, all of which are correlated with egg freshness [13–15]. Based on these factors, different modifications methods have been developed to improve the foaming properties of egg white, which are mainly divided into physical, chemical and enzymatic methods [16,17]. Cold storage contributes to control microorganisms, especially Salmonella in the egg products. As the most convenient and effective storage method, cold storage displays a certain degree of limitation on the quality deterioration of egg white [12]. With the extension of storage time, the functional properties of egg white were subjected to destruction, especially at a high temperature [18]. Many previous studies have shown that the occurrence of egg white thinning with the increment of storage duration at room or high temperature due to the sulfhydryl-disulfide exchange reaction of ovalbumin [19], the loss of lysozyme activity [20] and the dissociation of ovomucin-lysozyme [21]. Although, cold storage was reported to moderately spoil the foaming capacity of egg white, little further research has been undertaken on the potential mechanism for the change in foaming properties so far, which is essential to seek suitable modification methods for improving foaming properties. In this study, shell eggs stored at ten different time periods (at an interval of 10 d) were used to explore the potential explanation mechanisms for the cold storage effect on the foaming properties of egg white, by measuring its free sulfhydryl content, surface hydrophobicity, surface tension, particle size, zeta potential, rheological properties, and secondary protein structure. This research has shed light on the changes in egg white foaming properties under cold storage and contributes to the development of a feasible and effective method for egg storage.
2.1. Materials Hy-Line Brown eggs were collected from animal husbandry of Hubei Academy of Agricultural Sciences (Wuhan, China). 1Anilinonaphthalene-8-sulfonic acid (ANS) and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other reagents used in this study were all of analytical grade. 2.2. Sample preparation Fresh shell eggs (laid within 3 d) were stored at 4 ± 1 °C and total storage period was 90 d. Eggs from different storage periods (0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 d) were washed cleanly and broken manually to separate egg white and egg yolk carefully. Finally, a uniform sample system was obtained by stirring the original egg white for 3 h using a magnetic stirrer (IKA, IKA Works Inc., Wilmington, NC, USA). 2.3. Foams preparation and properties Foams were prepared as previously reported with slight modifications [22]. Briefly, an aliquot volume of egg white sample (20 mL, 10% v/v) diluted with phosphate buffer (10 mM, pH 7.0) was added into a 50 mL glass cylinder (internal diameter of 22 mm, height of 150 mm). Then, foam was produced by a high-speed homogenizer (XHF-DY, Scientz, Zhejiang, China) equipped with a probe (internal diameter of 7.5 mm, height of 145 mm) at 8000 rpm for 1 min at 25 °C. The whipping foam volumes were recorded at 2, 30, 60, 180 and 300 min to evaluate the foam decay, and the corresponding visual appearance of the foam was captured to observe the bubble change with time. Besides, the foam microscopy images at 2, 10, 30 and 60 min were obtained by an optical microscope (Sunny Optical Technology Co., LTD, Zhejiang, China). Finally, the foaming ability (FA) and the foaming stability (FS) were calculated according to the following equations:
FA(%) = V2/20 × 100
(1)
FS(%) = V30/ V2 × 100
(2)
where V2 and V30 are the foam volume at 2 and 30 min after whipping, respectively. 2.4. Physicochemical changes in egg white 2.4.1. Free sulfhydryl (SH) content The free sulfhydryl group content of egg white was measured according to the method of Duan et al. [17]. Briefly, Ellman’s reagent (4 mg/mL) was prepared by dissolving 4 mg of DTNB in 1 mL of TrisGlycine buffer (10.4 g Tris, 6.9 g Glycine and 1.2 g EDTA dissolved in 1 L distilled water, pH 8.0). Meanwhile, 1.5 g egg white was diluted to 10 mL with 1% (m/v) NaCl solution in Tris-Glycine buffer. Then, 2.9 mL 0.5% (m/v) SDS solution in Tris-Glycine buffer and 0.02 mL Ellman’s reagent were mixed with 0.1 mL of the above diluent sample solution. Subsequently, the mixture was shaken fully and incubated in dark for 15 min. The absorbance of the prepared sample was measured at 412 nm on a UV/VIS spectrophotometer (Nanodrop-2000C, Thermo Scientific, USA) against the blank (without Ellman’s reagent and protein 2
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sample). The protein content in all samples was determined by the Bradford method, and the free SH group content was calculated by the following equation:
SH (µ mol/g protein) = 73.53 × A 412 × D/C
defined as the initial slope of the plot of fluorescence intensity (FI) as a function of protein concentration (C).
FI = FI1
(3)
FI0 = H0 × C + B
(4)
where FI1 and FI0 are respectively the fluorescence intensity with and without ANS and C is protein concentration of each diluted sample.
where A412 is the absorbance of the sample at 412 nm, D is the dilution factor, and C is the concentration of egg white protein solution.
2.4.3. Surface tension The surface tension of egg white protein solution was determined as previously described by Sheng et al. [23]. Briefly, liquid egg white was diluted to 10% (v/v) using phosphate buffer (10 mM, pH 7.0), followed by measuring the sample surface tension with a tensiometer (k100, Krüss, Germany) using the Wilhelmy’s plate method at 25 °C. For each measurement, an aliquot volume of diluted egg white sample (30 mL) was used and 40 data points were recorded for 3600 s. Finally, the surface tension plot versus time was used for analysis.
2.4.2. Surface hydrophobicity (H0) The surface hydrophobicity of egg white protein solution was determined as reported by Duan et al. [5] with some modifications. Specifically, the egg white sample was diluted to five concentrations (0.01%, 0.02%, 0.05%, 0.1% and 0.2%, v/v) using phosphate buffer (10 mM, pH 7.0), followed by filtration through 0.45 μm membrane. Then, 4 mL of each diluted protein solution was supplemented with 20 μL of 8 mM 1-anilinonaphthalene-8-sulphonic acid in the same buffer, followed by incubation in dark for 3 min and measuring the fluorescence intensity (with ANS and without ANS) with excitation at 390 nm, emission at 470 nm and slit width 5 nm using a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). The spectrum of the buffer subtracted to correct the background fluorescence. H0 was
2.4.4. Particle size and zeta potential Before measurement, egg white was diluted to 1% (v/v) in phosphate buffer (10 mM, pH 7.0) and filtered through 0.45 μm membrane. The particle size of freshly prepared egg white protein solution was
Fig. 1. The foam collapse curve (A), foam ability (B), foam stability (C), foam visual appearance (D) and foam microscopy image (E) of egg white from shell eggs stored (4℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 3
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determined by refractive index (1.450) and absorption index (0.001) using a laser light scattering analyzer (APA2000, Malvern Instrument, Ltd., UK). The zeta potential of egg white solution was determined using a ZS Zetasizer Nano (Malvern Instrument, Ltd., UK). All measurements were carried out at 25 ℃ and repeated three times.
storage time prolonging, coupled with an increase of bubble size, due to bubble disproportion and liquid drainage (Fig. 1A, D and E) [27]. In Fig. 1B, it was shown that the foaming ability decreased from 45.33 to 25.17% at 40 d versus that at 0 d (p < 0.05), indicating the great effect of cold storage on foam formation. However, the foaming ability increased from 40 to 60 d (25.17 to 31.33%, p < 0.05), followed by an decrease from 60 to 80 d (31.33 to 28.50%, p < 0.05). These dynamic variations may be related to multiple protein structures and interaction change. The changes in the ovomucin characteristic during storage had an effect on its interaction with lysozyme between the thick and thin white, leading to an increase in the interaction of lysozyme with soluble ovomucin as the storage proceeded [28]. Therefore, the participation of soluble ovomucin in foam formation contributed to the increase of foaming ability from 40 to 60 d. Sheng et al. (2018b) [29] reported that the foam properties of ovalbumin decreased significantly after storage of 50 d at 22 °C, which may be ascribed to less exposure of hydrophobic amino acid caused by the aggregation of ovalbumin. In Fig. 1C, the foaming stability was shown to decrease slightly after storage, which was attributed to the thinning of egg white [13]. Robinson [30] reported that the thick albumen gradually became thin with the extension of egg storage time, thus increasing the content of the thin albumen. Additionally, it was reported that, when the pH increased during storage, n-ovalbumin was transformed into s-ovalbumin [31], and the less hydrophobic s-ovalbumin interfered with the formation of a cohesive film on the air-water interface, causing a reduction in foam stability. On the other hand, a gradual larger bubble size was observed with storage time prolonging throughout the experiment (Fig. 1E), implying foam drainage, foam coarsening and bubble size distribution [28].
2.4.5. Rheological behaviors Dynamic rheological measurements of egg white were conducted using a DHR2 rheometer (Waters, USA) as described by Wang et al. [24]. All the procedures were performed at 25 °C using a parallel plate (60 mm) with the gap being set to 1 mm. Specifically, the first step was to determine the region of the linear viscoelasticity at the mode of oscillation amplitude in the strain range of 0.01 to 100% at a constant frequency (1 Hz). Then, the elasticity modulus G′ (storage modulus) and viscosity modulus G″ (loss modulus) were obtained in the frequency range of 0.1–10 Hz within the linear area of viscoelasticity. Finally, under the mode of flow sweep, the apparent viscosity was obtained at a varying shear rate from 1 to 100 1/s. 2.4.6. Fourier transform infrared spectroscopy (FT-IR) Quantitative analysis of the changes in the secondary structures of lyophilized egg white samples was performed in amide Ⅰ band (17001600 cm−1) using a Fourier transform infrared spectrophotometer (Nexus 470, Metier, USA). The lyophilized egg white samples (2 mg) were mixed with KBr at the ratio of 1:200 and ground to power in a mortar with a pestle. The FT-IR spectra were recorded in the range of 4000-400 cm−1 with air as background. The baselines were subtracted automatically and the initial values of the peak positions were determined by Fourier deconvolution. Additionally, the obtained spectra were smoothed by correcting the background noise with air data using the Omnic software (Version 9.2. 86, Thermo Fisher Scientific Inc). Finally, the second derivative calculation and Gaussian curve-fitting analysis were performed to estimate the position and percentage of the component bands using PeakFit v4.04 (Aisn Software Inc) [25,26].
3.2. Analysis of physicochemical properties 3.2.1. Free sulfhydryl content The alteration in free sulfhydryl (SH) content is an indicator for changes in disulfide bonds, which are a contributor to protein aggregation. It has been reported that film formation in the bubble can be improved by the variation in the molecular structure induced by reduction of disulfide bonds among protein molecules [32]. Fig. 2 shows the free SH content of egg white from shell eggs stored at 4 ℃ for different time periods. The free SH content showed an increase of 11.97 to 19.57 μmol/g protein from 0 to 20 d (p < 0.05), which was attributed to the exposure of inner sulfhydryl group and subunit dissociation. A similar trend was observed during 30 to 40 d (11.97 to 16.32 μmol/g protein, p < 0.05). In the first 40 d of storage,
2.5. Morphology observation The morphology of egg white samples was observed by a SEM. Briefly, samples were lyophilized by Alpha 2–4 LD plus freeze dryer (Christ, Germany), followed by sputter-coated a small amount of each dried sample with gold using a gold injection instrument (ACE200, Leica, Germany). Finally, the prepared samples were examined under a scanning electron microscope (Quanta 250, FEI, USA) at 10 kV under 200 and 2000 x magnification. During SEM operation, several different parts of each sample were observed, and images with a similar morphology were selected to represent the whole sample. 2.6. Statistical analysis All experiments were carried out in triplicate and each sample was measured in duplicate for analytical assays. All data were presented as mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) with a 95% confidence interval to estimate the significance of the results. Means values between treatments were compared by Duncan's multiple range tests using the SPSS software program (Version 19). Graphs were plotted using Origin software (Version 9.0.0). 3. Results and discussion 3.1. Foam analysis Foam formation was determined by the migration, unfolding and rearrangement of protein molecules at the air-water interface. Fig. 1A shows the variation of foam volume in the time range from 2 to 300 min. Generally, the foam volume showed a downward trend with
Fig. 2. Free sulfhydryl (SH) group content for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 4
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the increase in free SH content meant that protein molecule was exposed to a more polar environment, which was adverse to foam formation, thus decreasing the foam ability. At the same time, the exposure of SH group increased the interaction among protein molecules and protein cross-linking induced by sulfhydryl-disulfide exchange reaction. Additionally, Zhao et al. [33] reported that an increment in SH content was correlating with formation of new cross-links. However, after the storage of 40 d, the free SH content showed a similar tendency to that of foaming ability, with an increase first and then a decrease from 40 to 80 d. This increase may be attributed to breakage of interchain disulfide bonds and exposure of sulfhydryl group induced by the dissociation of lysozyme-ovomucin, resulting in improved foam formation, while this decrease may be ascribed to protein oxidation during storage. The dissociation of lysozyme with ovomucin was observed after 40 d of storage, causing a different protein structure state.
Normally, the foaming ability was largely dependent on the surface tension at the air-water interface and lower surface tension was beneficial for the foam formation. The samples from all tested storage time periods showed higher surface tension than the control, confirming that the foaming ability was also reduced during all storage periods. Additionally, the surface tension increased during 50 to 80 d (47.23 to 50.45, p < 0.05), suggesting that protein molecules became more rigid and compact, leading to less ability to adsorb and unfold at the airwater interface. Xiong et al. [38] reported that lower surface tension was related to smaller particle size and less surface charge, which could decrease the electrostatic barrier and thus have a more rapid adsorption rate at the air-water interface. 3.2.4. Particle size and zeta potential Particle size is usually presented as volume-surface mean diameter (d3,2) and volume-mean diameter (d4,3), with an effect on the interfacial properties, including foaming and emulsification [39]. Fig. 5A shows the tendency of d3,2 and d4,3, with an increase for both of them increased after storage versus 0 d. Specifically, after 20 d of storage, d3,2 and d4,3 increased significantly from 21.24 to 42.74 μm and from 67.27 to 91.97 μm (p < 0.05), respectively, and a similar increase was observed between 30 and 40 d (30.17 to 36.69 μm, 69.40 to 78.45 μm, respectively, p < 0.05). In the first 40 d, the increased particle size significantly restrained the foaming ability, implicating that the exposure of SH group increased the interaction (probably by disulfide or hydrogen bond) among protein molecules. Meanwhile, the increased particle size might also cause a higher surface tension, leading to a lower adsorption rate at the air-water interface. The higher value of d4,3 was still observed after 40 d storage, indicating the continuous aggregation behavior of protein molecules. In Fig. 5B, it was shown that a high degree of heterogeneity (high span value) occurred at 0 d, probably due to the formation of noncovalent cross-linked protein aggregates in its native state [40]. However, the span value was decreased from 3.92 to 2.75 (p < 0.05) after 10 d storage, suggesting that the particle size distribution was narrowed by the formation of the soluble aggregates through covalent and noncovalent interactions [41]. In the first 40 d, the increase of particle size coupled with the decrease of span value indicated the presence of large but uniform protein aggregates. Moreover, the same tendency between the span value and foaming ability in the first 40 d demonstrated that higher heterogeneity facilitated protein adsorption at the air-water interface during this storage period. Nevertheless, during 50 to 80 d storage, the span value showed a gradual increase from 2.41 to 3.94
3.2.2. Surface hydrophobicity (H0) Surface hydrophobicity (H0), a common structural characteristic for evaluating protein conformation change, is correlated with foaming properties [34]. The H0 values of egg white samples from shell eggs stored at 4 ℃ for different time periods are shown in Fig. 3A. The H0 values exhibited a decrease from 798.4 to 607.9 (p < 0.05) at 20 d compared with 0 d, probably due to the potential aggregation behavior of egg white proteins as indicated by the increased particle size. The decline in H0 could decrease the number of amphipathic molecules and affect the adsorption behavior at the air-water interface, thus reducing foam formation. Additionally, in the first 40 d of storage, the H0 values exhibited the same tendency as that of foaming ability, implying that foam formation was determined by the exposure degree of hydrophobic group and unfolding of protein molecules. Furthermore, the polar environment induced by the exposure of SH group explained the reduced H0 value, thus decreasing the foaming ability in the first 40 d of storage. Kuan et al. [35] reported that the foaming ability of proteins could be enhanced by conformational changes with increasing surface hydrophobicity. Meanwhile, a dramatic increase of H0 was observed at 30 d (1062.7) compared with 20 d (607.9) (p < 0.05) (Fig. 1B), which was due to the exposure of hydrophobic groups buried in protein molecules. Combined with other parameters, the 30 d of storage was a turning point due to great change was obtained. Meanwhile, this significant increase in H0 value coincided with reduced surface tension and increased foaming ability. A possible explanation for this is that the increasing exposure of hydrophobicity groups reduce energy absorption barrier at the air-water interface, thus enhancing the adsorption kinetics [36]. 3.2.3. Surface tension Surface tension at the air-water interface is referred to as Van de Waals interactions between amphiphilic molecules. These amphiphilic protein molecules absorb at the interface, leading to the reduction of surface tension [37]. Fig. 4A shows the time evolution of surface tension for egg white protein at the air-water interface over different shell egg storage time periods. In the inset, the rapid reduction of surface tension at the first 1200s, indicated that protein molecules were rapidly diffused and adsorbed at the air-water interface in all the samples, suggesting that egg white proteins were adsorbed from the bulk onto the interface. However, with adsorption time prolonging, the surface tension showed a gradual decrease in all the samples, implying that fewer proteins were involved in the saturated adsorption interface, contributing to energy barrier formation during the adsorption and rearrangement process. Fig. 4B displays the specific surface tension value of egg white solution at 550 s. The direct increase of surface tension was ascribed to the (from 46.47 to 49.20, p < 0.05) led to an obvious decrease of foaming ability at 10 d, and this increment of surface tension was due to less absorption of egg white protein at the air-water interfaces.
Fig. 3. Changes in surface hydrophobicity (H0) for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3). 5
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Fig. 4. Time evolution of surface tension for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d) (A). Data were recorded at the adsorption time of 550 s (B). Different small letters indicate significant differences between groups. Error bars represent mean values ± standard deviations (n = 3).
Fig. 5. The particle size (A), span value (B) and zeta potential (C) for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d). Different small letters indicate significant differences between storage groups (p < 0.05). Error bars represent mean values ± standard deviations (n = 3).
(p < 0.05), while the viscosity exhibited a decrease, probably due to the formation of different particle size induced by loose structure. Zeta potential can be used to evaluate the stability of colloidal dispersion systems and elaborate the degree of electrostatic repulsion and interaction between protein molecules [42]. As shown in Fig. 5C, the absolute zeta potential was higher in egg white at 10 d (-12.47 mV) than in the control (-10.80 mV). This increment in the surface net charge increased the electrostatic barrier and thus decreased the adsorption rate [43], which was proved by the decrease of foaming ability and increase of surface tension at 10 d. However, during 10 to 40 d storage, the absolute zeta potential decreased from -12.47 to -9.58 mV
(p < 0.05), which was probably due to the reduction in the energy barrier to adsorption at the air-water interface induced by the interaction of protein molecules [44]. Additionally, a similar trend was observed between 50 and 90 d (-11.47 to -9.32 mV, p < 0.05), which was harmful to the system stability. The decreased foaming stability was largely related to the lower absolute zeta potential and it also indicated that the aggregation behavior caused by oxidation reaction during storage. 3.2.5. Rheological properties Identification of rheological 6
characteristics
facilitates
the
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Fig. 6. Changes in rheological properties for egg white from shell eggs stored (4 ℃) for different time periods (0 to 90 d), including G′ (A), G″ (B), viscosity (C) and shear stress as a function of shear rate (D). Table 1 The frequency sweep data (G′, G″ and tan δ) were obtained at a frequency of 1 Hz, and the flow model parameters (k and n) were calculated from the Power-law for liquid egg white of shell egg stored (4 ℃) for different time periods (0 to 90 d). d
Frequency sweep G'/Pa
0 10 20 30 40 50 60 70 80 90
0.08 0.15 0.23 0.05 0.16 0.41 0.26 0.15 0.17 0.21
± ± ± ± ± ± ± ± ± ±
Flow sweep G”/Pa
0.02e 0.00d 0.02bc 0.00e 0.01d 0.05a 0.02b 0.01d 0.02d 0.03c
0.10 0.15 0.16 0.13 0.13 0.20 0.16 0.13 0.21 0.18
± ± ± ± ± ± ± ± ± ±
tan δ 0.01e 0.01bcd 0.03b 0.00de 0.02de 0.01a 0.00bc 0.00cde 0.04a 0.01ab
1.35 1.02 0.72 2.43 0.81 0.50 0.61 0.88 1.20 0.87
k ± ± ± ± ± ± ± ± ± ±
0.23b 0.06c 0.06de 0.03a 0.10d 0.07f 0.05ef 0.08cd 0.09b 0.09cd
0.06 0.07 0.30 0.03 0.14 0.38 0.30 0.22 0.11 0.12
R2
n ± ± ± ± ± ± ± ± ± ±
0.01fg 0.02efg 0.04b 0.01g 0.06d 0.03a 0.05b 0.01c 0.04def 0.03de
0.68 0.83 0.49 0.93 0.54 0.38 0.38 0.40 0.66 0.54
± ± ± ± ± ± ± ± ± ±
0.04c 0.04b 0.07de 0.06a 0.02d 0.10e 0.07e 0.02e 0.04c 0.07d
0.9975 0.9724 0.9747 0.9847 0.9843 0.9789 0.9733 0.9446 0.9069 0.9840
Different small letters in the same column indicate significant differences between groups (p < 0.05). Value was presented as mean ± standard deviation (n = 3).
understanding of the molecular changes during storage [45]. Fig. 6 shows the changes in the rheological properties of egg white samples from shell eggs stored for different time periods. In the first 40 d, the increased G′ meant the increasing interaction among protein molecules [24], which had a negative effect on the foam formation of egg white (Fig. 6A). Meanwhile, a higher particle size was observed in accompany with an increased G′, suggesting that the protein structure became more rigid proved by the decreased surface hydrophobicity and increased SH content, leading to lower foam formation. Compared with G′, G″ showed no dramatic differences in all the egg white samples (Fig. 6B). Liquid egg white was proved to be shear-thinning fluids [46], and Fig. 6C shows that the apparent viscosities of egg white decreased with
increasing shear rate. The shear-thinning behavior could be resulted from the breakdown of weak linkages between protein molecules and their rearrangement towards the direction of shear and decreasing flow resistance. An obvious increase in the viscosity was observed after the storage of 20 d, and a similar result was obtained during 30 to 40 d storage, which might result from small aggregates and cross-linking interaction of protein molecules proved by increasing particle size and decreasing surface hydrophobicity. In the first 40 d, the increased viscosity showed an adverse effect on the foaming ability because of the formation of rigid structure. Moreover, the increase in viscosity during storage could probably contribute to the insolubilization and aggregation of egg white proteins via intermolecular bonds. As reported by Herald et al. [47], the 7
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behavior index (n) remained below 1 and exhibiter the same tendency with other parameters.
Table 2 FTIR spectral analysis of changes in the secondary structure of egg white protein during shell eggs storage. d
α-helix 0 10 20 30 40 50 60 70 80 90
3.2.6. FT-IR spectroscopy Changes in the secondary structures of protein were investigated by Fourier transform infrared spectroscopy (FTIR). Amide I band (16001700 cm−1) in the FTIR spectra of protein was the characteristic band for the secondary structure and can be characterized by α-helix (16501660 cm−1), β-sheet (1610-1640 cm−1), β-turn (1660-1700 cm−1) and the random coil (1640-1650 cm−1) [49]. Table 2 summarizes the percentages changes in α-helix, β-sheet, βturn, and random coil. It can be seen that the content of α-helix and random coil remained relatively stable, in contrast to a significant change in the content of β-structure. The content of β-turn decreased from 10 to 30 d (28.00 to 26.33%, p < 0.05), but increased from 50 to 90 d (25.50 to 28.90%, p < 0.05) at the expense of β-sheet content (30.39 to 26.03%, p < 0.05). This change indicated that the protein structure became relatively loose with prolonging storage time due to protein dissociation, aggregation and partial denaturation, which could be well supported by the results of SEM, the increase of particle size, and the reduction of surface hydrophobicity.
Secondary structures (%)
13.86 13.91 13.80 13.47 13.88 13.29 13.29 13.77 13.67 14.21
± ± ± ± ± ± ± ± ± ±
β-sheet 0.28b 0.05b 0.02b 0.17cd 0.07b 0.11d 0.18d 0.16b 0.06bc 0.02a
28.29 27.40 28.24 29.61 28.02 30.39 30.32 28.49 28.60 26.03
± ± ± ± ± ± ± ± ± ±
β-turn 0.81c 0.02d 0.16c 0.60b 0.29cd 0.25a 0.41ab 0.75c 0.15c 0.09e
27.07 28.00 27.04 26.33 27.33 25.50 25.61 26.97 26.98 28.90
random coil ± ± ± ± ± ± ± ± ± ±
0.61c 0.06b 0.13cd 0.53d 0.27c 0.13e 0.42e 0.73cd 0.17cd 0.06a
30.77 30.69 30.92 30.60 30.78 30.82 30.79 30.77 30.75 30.86
± ± ± ± ± ± ± ± ± ±
0.08abc 0.05bc 0.10a 0.10c 0.06abc 0.06ab 0.20abc 0.14abc 0.08abc 0.03ab
Different small letters in the same structure indicate significant differences between groups (p < 0.05). Value was presented as mean ± standard deviation (n = 3).
viscosity of liquid whole egg increased during frozen storage, probably due to freeze-induced denaturation of the protein. A gradual decrease in the viscosity during 50 to 80 d storage meant the formation of loose structure. To illustrate the functionality loss of egg white during cold storage, the data of G′, G″ and tan δwere recorded at 1 Hz and the rheological flow models (Power-law) were also used to fit the experimental data (Fig. 6D, Table 1). G′ and G″ showed an increase from 0 to 20 d (0.08 to 0.23 Pa, 0.10 to 0.16 Pa, respectively) and from 30 to 50 d (0.05 to 0.41 Pa, 0.13 to 0.20 Pa, respectively, p < 0.05). Generally, tan δis considered as an indicator of protein quality [48], and a decrease in tan δindicated that the samples tend to become more elastic. In Table 1, the tan δwas shown to decrease from 0 to 20 d (1.35 to 0.72) and from 30 to 40 d (2.43 to 0.81) (p < 0.05). In the first 40 d, the decreased tan δindicated the protein structure was more elastic and solid-like, which was adverse to foam formation. However, the increase from 0.5 to 1.2 during 50 to 80 d suggested the transition of the protein structure to a more viscous one. Furthermore, the consistency coefficient (k value) also showed an increase from 0 to 20 d (0.06 to 0.30) and from 30 to 40 d (0.03 to 0.14), and a decrease from 50 to 80 d (0.38 to 0.11) (p < 0.05). The increase in the k value illustrated that cold storage increased both the system viscosity and flow resistance. Additionally, the pseudoplastic behavior of egg white appeared to be unchanged since the flow
3.3. Morphology change Scanning electron microscopy (SEM) was carried out to obtain tangible evidence for changes in egg white protein microstructure under different storage time periods. Fig. 7 displays a series of common SEM images of egg white samples at 200 x and 2000 x magnification. It could be seen that the 0 d sample exhibited a characteristic morphology with a smooth surface and sheet-like structures. However, after 30 d of storage, small aggregates were observed on the surface, and them became larger with prolonging storage time and transformed into larger aggregates at 50 and 70 d. This alteration may be attributed to protein cross-linking through non-covalent bond induced by oxidation, leading to enhanced interaction between proteins and more complex network composed of randomly aggregated protein molecules. 4. Conclusion In this study, we investigated the change mechanisms underlying the physicochemical and foaming properties of egg white samples from
Fig. 7. The SEM images for egg white from shell eggs stored (4 ℃) for different time periods (0, 30, 50 and 70 d). 8
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shell eggs stored at 4 ℃ for different time periods. In the first 40 d storage, the exposure of SH group increased the interaction among molecules via hydrogen and disulfide bond coupled with the increasing polarity of protein molecules, leading to the decrease of surface hydrophobicity. Meanwhile, the increased SH content caused the crosslinking between protein molecules as proved by the increased G′, resulting in the increased particle size and viscosity. The surface tension and the interface properties were generally affected by the exposure of SH group and the changes in surface hydrophobicity, particle size and viscosity. Additionally, the changes in the protein secondary structure influenced the surface and foaming properties, and the existence of oxidation during storage could be derived from the increase of particle size and the emergence of protein aggregation. Overall, the decrease in the foaming properties during cold storage was ascribed to the exposure of SH group, the decrease of surface hydrophobicity and the increase of surface tension caused by the increase of particle size and viscosity, giving rise to a lower adsorption rate of protein at the air-water interface. The changes and correlation of physicochemical properties of egg white from shell eggs storage were presented in the work, providing theoretical basis for food production.
[18] [19] [20] [21] [22] [23] [24] [25] [26] [27]
Conflict of interest statement
[28]
All the authors declare that they have no conflict of interest.
[29] [30]
Acknowledgments
[31]
This work was supported by the National Natural Science Foundation of China (grant numbers 31571784 and < GN1 > 31701622); the H < /GN1 > ubei Provincial Natural Science Foundation of China (grant number 2018CFB606).
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