Fabrication and characterization of acid-induced gels from thermally-aggregated egg white protein formed at alkaline condition

Food Hydrocolloids 99 (2020) 105337

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Fabrication and characterization of acid-induced gels from thermallyaggregated egg white protein formed at alkaline condition

T

Farhad Alavia, Zahra Emam-Djomeha,b,d,∗, Shima Momena, Elnaz Hosseinic, Ali Akbar Moosavi-Movahedic,d a

Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran Transfer Phenomena Laboratory (TPL), Controlled Release Center, Department of Food Science, Engineering and Technology, College of Agriculture & Natural Resources, University of Tehran, Karaj Campus, Karaj, Iran c Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran d Center of Excellence in Biothermodynamics, University of Tehran, Tehran, Iran b

ABSTRACT

Cold gelation of egg white proteins (EWP) has still remained a challenge because they suffer from heat-induced gelation at low protein concentrations during the preheating step. This study suggested that adjusting pH of EWP solution at relatively high alkaline pH (i.e. 11.3) could inhibit the heat-induced gelation of EWP, allowing to produce thermally aggregated EWP (TA-EWP) at relatively high protein concentrations during the preheating step. Regarding SDS-PAGE, free –SH groups, circular dichroism, and surface hydrophobicity data, the preheating step at alkaline pH probably caused the TA-EWP are assembled from the partially unfolded protein molecules which stabilized by numerous disulfide bonds as well as hydrophobic interactions that develop a network of the β-sheet structures. The TA-EWPs are able to form acid-induced cold-set gels. SEM images showed the TA-EWP acid-induced gels had a honeycomb-like microstructure contained large serum pools. With increasing preheating temperature and protein content, the microstructure of the gels became more compact and all texture parameters including hardness, springiness, chewiness, cohesiveness as well as Young's modulus, true fracture stress (σ) and Hencky strain tend to increase. Furthermore, the water holding capacity (WHC) improved with increasing protein content and preheating temperature. All TA-EWP acid-induced gels exhibited a weak dependence of storage modulus (G′) with frequency and the G′ value of the acid-induced gels increased with increasing preheating temperature and protein content.

1. Introduction Gelation is one of the most important functional properties of globular proteins. Proteins can form gels through physically or chemically cross-linked protein monomers that can entrap lots of water or other biological liquids inside their three-dimensional network (Abaee, Mohammadian, & Jafari, 2017). Protein-based gels can be classed in two groups of heat-set gels and cold-set gels, based on their fabrication process. Heat-set gelation is a one-step process, where globular proteins denature at a temperature above denaturation point, inducing modifications in the conformational structures of the protein. The unfolding process, in turn, triggers the creation of high molecular weight aggregates through disulfide and thiol-disulfide or by hydrogen bonding, hydrophobic and electrostatic interaction. Consequently, the solution viscosity progressively increases as a result of the formation of the aggregates with great hydrodynamic diameters until a gel network is lastly formed (Brodkorb, Croguennec, Bouhallab, & Kehoe, 2016, pp. 155–178). In the other hand, cold gelation is a two-step process: in the first step, the protein solution is denatured and unfolded by preheating

the solution beyond the denaturation temperature and below the critical gelation concentration threshold at a pH well above the isoelectric point, leading to exposure of hydrophobic patches and free sulfhydryl groups (free –SH) and form soluble aggregates. Then, the protein solution is cooled and in the second step the gelation triggers by charging with acidulant agents such as glucose-d-lactone (GDL) (called acid-induced gelation) or adding salts such as NaCl and CaCl2 (called saltinduced gelation) (Alting, Hamer, de Kruif, Paques, & Visschers, 2003; Brodkorb et al., 2016, pp. 155–178). Cold-set gels have been considered as potential carriers for the production of healthy foods, as they let the incorporation of thermalsensitive bioactive ingredients such as enzymes, vitamins, and probiotics as well as volatile compounds in a cold aggregated protein solution before triggering of gelation by adding of acidulant agents or salts (Weijers, van de Velde, Stijnman, van de Pijpekamp, & Visschers, 2006; Yang, Wang, & Chen, 2017). Besides, due to their ability to produce gel structures in a food matrix without the need for heating, the cold-gelable soluble aggregates may be considered as an attractive alternative for carbohydrate-based thickening ingredients (Alting et al., 2003).

∗ Corresponding author. Transfer Phenomena Laboratory (TPL), Department of Food Science, Technology and Engineering Faculty of Agricultural Engineering and Technology, Agricultural Campus of the University of Tehran, 31587-11167, Karadj, Iran. Tel./Fax:+982632248804 E-mail address: [email protected] (Z. Emam-Djomeh).

https://doi.org/10.1016/j.foodhyd.2019.105337 Received 14 March 2019; Received in revised form 5 July 2019; Accepted 24 August 2019 Available online 28 August 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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at least 2 h. To ensure complete hydration of EWP proteins, these solutions were kept at 4 °C overnight. The solutions were then adjusted to pH 11.3 using 8 M NaOH and transferred to a tightly sealed glass vial and heated at a different temperature of 65, 70, 80, and 90 °C by a controlled-temperature heater stirrer for 30 min. Subsequently, heated samples were cooled in an ice bath. After cooling, the pH of all samples was adjusted to 8.0 by adding HCl (3 M). The solutions prepared by the method described above were called thermally-aggregated egg white proteins (TA-EWP). The obtained TA-EWP were coded as a-TA-EWP, bTA-EWP, c-TA-EWP, and d-TA-EWP, corresponding to the TA-EWP samples prepared by heating of EWP solutions with protein content of 45 mg/mL at temperatures 65, 70, 80, and 90 °C, respectively, and eTA-EWP, and f-TA-EWP, corresponding to the TA-EWP samples prepared by heating of EWP solutions with protein content of 60 mg/mL at temperatures 65, and 70 °C, respectively.

Cold-set gels has been successfully fabricated from different types of globular proteins including whey protein isolate (Svanberg, Wassén, Gustinelli, & Öhgren, 2019), soy protein isolate (Chen, Chassenieux, & Nicolai, 2018), pea proteins (Mession, Chihi, Sok, & Saurel, 2015), and oat proteins (Yang et al., 2017). Egg white is considered as an inexpensive and popular source of high-quality protein. Nonetheless, though cold gelation has been used effectively for pure ovalbumin (main protein of egg white) (Alting et al., 2003), this method is not easily used for commercial egg white protein isolates, as they already formed gel network at the preheating part of the process at as low as 3% protein solutions due to their high ion content (Tomczyńska-Mleko, Nishinari, & Handa, 2014; Weijers et al., 2006). At high ionic strength, electrostatic repulsive forces become weak and the intermolecular attractive forces (usually hydrophobic interaction) induces random aggregation and gelation of the already denatured egg white protein molecules. To address the problem and produce cold-set gels of egg white protein (EWP) with higher protein content, some authors suggested a desalting step to reduce the ionic commercial EW to fabricate cold-set gels or removing of ovotransferrin as most heat sensitive of EWP (Tomczyńska-Mleko et al., 2014; Weijers et al., 2006). Tomczyńska-Mleko et al. (2014) fabricated the calciuminduced cold-set gels with a maximum protein concentration of 5.5% through the preheating (at 80 °C for 30 min) of a demineralized egg white isolate powder. Furthermore, a new gelation method based on a radical-induced aggregation of EWP by a redox pair involving ascorbic acid + hydrogen peroxide was suggested by Alavi, Momen, EmamDjomeh, Salami, and Moosavi-Movahedi (2018). This technique eliminates the preheating step for the formation of soluble aggregates and leads to gelation of non-heat-treated EWP. However, cold-set gels obtained by the method had weak mechanical properties as compared to heat-set EWP gels. In addition to decrease ionic strength, adjusting of pH of egg white solution far from the pI may be an alternative method to thermally denature egg white proteins without forming coagula, because in this condition the electrostatic repulsive forces compete with attractive hydrophobic interactions, which may prevent the aggregate formation (Gharbi & Labbafi, 2018). This study hypothesizes that increasing pH of EWP solution to high alkaline pH (above 11.0) may constrain the intermolecular interactions and random aggregation and thereby inhibit the heat-induced protein gelation of EWP during the preheating step. In this extreme alkaline pH, we expected to be able to produce gelable thermally aggregated EWP at higher initial protein concentration than those at neutral or weak alkaline pH. Therefore, the main aim of the present study was to use a high alkaline condition (pH 11.3) as a simple strategy to inhibit the heat-induced gelation of EWP for the fabrication of acid-induced cold-set gels from EWP. Furthermore, the impact of the preheating temperature and protein concentration in the preheating step on microstructure, mechanical properties and water holding capacity of the resulting gels was investigated.

2.3. Molecular characterization of TA-EWP 2.3.1. Free –SH content determination All sample solutions were diluted to 40 mg/mL protein content by distilled water and then 0.2 mL of the diluted protein solution was added to 1.8 mL of Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 0.004 M EDTA, pH 8.0) in a 2 mL microcentrifuge tubes, to reach a final protein concentration of 4 mg/mL. Also, the same buffer having 2.5% SDS was exploited to measure the total free –SH groups. After the addition of 20 μL of Ellman's reagent to the tubes, samples were incubated for 15 min at room temperature. Consequently, the samples were centrifuged at 15,000×g (11,570 rpm) at 25 °C for 10 min to remove insoluble turbid particles and the supernatant fractions were analyzed for free –SH content at 412 nm using a CecilCE2502 UV–Vis spectrophotometer (Cecil Ins., Cambridge, UK). The free –SH content (μmol SH g−1) was calculated as follows:

µmol SH / g = (75.53 × A 412 × D )/ C

(Eq 1)

Where C is the protein content in reaction mixture (4 mg/mL), A412 = the absorbance at 412 nm; D = the dilution factor (2.02 2.00 ), and the factor 73.53 is from 104/(1.36 × 104); 1.36 × 104 = the molar absorptivity constant (M−1 cm−1). 2.3.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoreses (SDSPAGE) The molecular weight distribution of TA-EWP was determined by SDS-PAGE. To do this, TA-EWP solutions diluted to a protein concentration of 4 mg/mL and were directly mixed with 2 × sample buffer under non-reducing and reducing (in presence of β-mercaptoethanol) conditions, and incubated at 90 °C for 4 min. The samples were then injected to 15% polyacrylamide gels at 100 V with the following staining with Coomassie Brilliant Blue R-250. 2.3.3. Circular dichroism (CD) Variations in the secondary structures of TA-EWP as a function of preheating temperature and protein content were checked with a spectropolarimeter (Aviv model 215, New Jersey, USA). The CD spectra of the samples in the far-UV region (190–260 nm) was measured with 1 mm path length quartz cell at 25 °C. The samples were diluted to a protein concentration of 0.4 mg/mL by distilled water with a pH of 8.0.

2. Materials and methods 2.1. Materials EWP powder containing 80% protein (wet base) was obtained from Pulviver Company (EAP-R™, Bastogne, Belgium). Fast Green FCF, 5, 5′dithiobis-(2-nitrobenzoic acid) (DTNB), sodium hydroxide (NaOH), 8Anilino-1-naphthalene sulfonic acid (ANS), and hydrochloric acid (HCl) were purchased from Sigma-Aldrich (Sa. Louis, MO, USA). All of the chemicals that used for the running of gel electrophoresis were also purchased from Merck and Sigma-Aldrich.

2.3.4. Surface hydrophobicity The surface hydrophobicity (H0) of native EWP and TA-EWP samples was measured according to the method of Alavi et al. (2018) using ANS as a fluorescent probe. To do this, the samples diluted to a protein content of 0.02–0.2 mg/mL by distilled water with a pH of 8.0. Then, 20 μL of ANS solution (8 mM) was added to 2 mL of these diluted samples, vortexed, and kept for 10 min in the dark. Afterwards, the fluorescence intensities of the mixtures were determined by a Cary Eclipse spectrofluorimeter (Varian, Cary Eclipse) at excitation and

2.2. Preparation of thermally-aggregated EWP (TA-EWP) EWP powder was dissolved in distilled water to obtain a protein concentration of 45 and 60 mg/mL and stirred at room temperature for 2

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emission wavelengths of 390 nm and 488 nm, respectively. The excitation and emission slits were adjusted at 10 nm. H0 was calculated through the measuring of initial slope of the fluorescence emission intensity plot versus protein concentration.

for scanning electron microscopy, small species of freeze-dried gels were cut from their cross-sectional area and mounted on aluminium stubs and then sputter-coated with gold prior to imaging. A Vega scanning electron microscope (Tescan, USA) was used to capture SEM images of the gels at 200 × and 500 × magnifications.

2.3.5. Turbidity The changes in turbidity of BWP and TA-EWP solutions as a function of preheating temperature and protein content were recorded with a CecilCE2502 UV–Vis spectrophotometer (Cecil Ins., Cambridge, UK). After preheating, all protein solutions were diluted to 10 mg/mL by distilled water with a pH of 8.0. The absorbance of the samples was monitored at 500 nm as an indicator of turbidity. Furthermore, to evaluate impact of the high alkaline pH (i.e. 11.3) during preheating step on inhibition of random aggregation, a control preheated EWP solution also produced by heating a EWP solution with protein concentration of 30 mg/mL at pH 9.0 (as natural pH of egg white) and its turbidity was recorded after its dilutions to 10 mg/mL. It should be noted that at protein concentration above 30 mg/mL, the EWP solutions produced a gel-like structure or a heat-set gel during heating.

2.6.2. Texture analysis For texture analysis, the acid-induced gels were prepared in 10 mL plastic syringe with 15 mm diameter by the method described in section 2.6. The gel samples were removed from syringes and cut as cylindrical pieces with 15 mm diameter and 10 mm length by a blade. A Brookfield CT3 instrument (Brookfield, USA) equipped a 38 mm diameter plate probe (TA4:1000) with compression speed of 0.5 mm s−1 was used to the uniaxial compression experiment until gel fracture (~80% compression). Force (N) and displacement (m) in fracture point were applied to calculate true fracture stress σ (Eq. (3)) and Hencky strain ε (Eq. (4)):

2.4. Flow behaviour of EWPA solutions The flow behaviour of TA-EWP solutions was determined via a rotational LV-DV3T viscometer (Brookfield Engineering Inc, MA, USA) equipped with a small spindle sampler adaptor with spindle SC4-34 at 25 °C at the shear rate range of 1–70 1/s. Before viscosity measurement, a pre-shear rate of 50 1/s was applied on samples to obtain a homogenous suspension. The obtained data were fitted to the Power law model Eq. (2) with Curve Expert Professional software version 2.6.4.

= K

F H A Hi

=

ln

(3)

H Hi

(4)

Where F is a force (N) at the fracture point, A is the cross-sectional area of the gel samples (m2), Hi is the initial height of uncompressed specimen (m), and H is the height which fracture occurred during compression. Fracture of the gel is described as a peak or discontinuity of the stress over strain curve. Texture profile analysis (TPA) was performed through two consecutive penetrations to a predetermined distance (50%) within the gel by a flat-bottomed cylindrical probe (TA4:1000, 38 mm diameter) at a speed of 0.5 mm s−1. Four parameters of TPA was measured including hardness (g), springiness index, chewiness (mJ), and cohesiveness. For each sample, five pieces of gel from the two syringes were used. The Young's modulus was also calculated as the slope of the initial linear region of the first curve of TPA for a true strain from 0 to 0.05.

(2)

n 1

=

n

Where K is the consistency coefficient (mPa. S ), γ is the shear rate (s−1), n is the flow behaviour index (dimensionless), and η is the apparent viscosity (mPa. s). 2.5. Gelation experiments

2.6.3. Small-strain dynamic rheology Physica MCR 300 dynamic rheometer (Anton Paar GmbH, Graz, Austria) was used to measure the rheological properties of TA-EWP solutions during acid-induced gelation. Just after GDL addition (0.19 g/ g protein), approximately 3 mL of TA-EWP solutions were loaded onto the rheometer. A CP50-1 cone/plate measuring system (gap 3 mm) was used in the test. Time sweep of samples was performed at 40 °C for 120 min with an angular frequency of 1 Hz and a strain of 1% that was within the linear viscoelastic range (LVE). Storage and loss moduli (G’, G”) were recorded as functions of time. After that, the sample was cooled to 10 °C at a descending rate of 10 °C/min, and a frequency sweep of the sample was then carried out at an angular frequency between 1 Hz and 100 Hz at a fixed strain of 1% (LVE range).

Acid-induced cold gelation of TA-EWP samples was induced via GDL. TA-EWP solutions were charged with GDL powder and then stirred for 1 min. Based on our preliminary tests, the GDL to protein ratio of 0.19 g/g protein was selected to reach a final pH of 4.5, according to the isoelectric point of ovalbumin as main protein fraction of EWP (Gharbi & Labbafi, 2018). After GDL addition, samples were incubated at an incubator for 2 h at 40 °C and then the formed gels were kept to the refrigerator (4 °C) for 48 h until further analyses. 2.6. Characterization of acid-induced TA-EWP gels 2.6.1. Microstructural morphology 2.6.1.1. Confocal laser scanning microscopy (CLSM). TA-EWP Solutions were prepared as above described (section 2.2). Twenty μl of an aqueous solution of Fast Green FCF (5 mg/ml) was added to 2 mL TAEWP solutions. GDL (0.19 g/g protein) was added to the protein solutions. Aliquots of the mixtures were poured onto a concave slide and covered by a lamel and aluminium foil to prevent exposure to light. The slides were incubated at 40 °C for a 2 h period and then the formed gels were transferred to the refrigerator (4 °C) for 48 h until CLSM test. The apparatus used to examine the microstructure of the formed gels was a Leica TCS SPE confocal laser-scanning microscope equipped with an inverted microscope (Leica DMi8) (Leica Microsystems (CMS) GmbH, Mannheim, Germany). The light source was a helium-neon laser beam and the objective lens used had a magnification of 40 × . The emission and excitation wavelengths were 633 nm and between 655 and 755 nm, respectively.

2.6.4. Water holding capacity TA-EWP gels were fabricated in 5 mL centrifuge tube and were centrifuged at 8000×g (8450 rpm) for 10 min at 4 °C using a refrigerated centrifuge (Model RFS-REFRI, PART AZMA, Iran), according to Li et al. (2018). The water was drained out using a syringe. WHC was calculated as follows:

WHC (%) =

Wt

Wr Wt

× 100

(Eq. 5)

Where Wt = the gram water in gels before centrifugation and Wr = gram of water removed from gels. 2.7. Statistical analysis

2.6.1.2. Scanning electron microscopy (SEM). To prepare the gel samples

Statistical analysis of the data was performed by one-way analyses 3

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exposed free –SH groups have involved in the formation of S-S bonds by SH oxidation or SH/S-S interchange reaction (Mine, Noutomi, & Haga, 1990). Furthermore, the results also indicate that increasing preheating temperature intensifies exposing of free –SH groups as well as their involvement in the formation of S-S bonds. The SDS- PAGE was performed to assess the effect of the thermal treatment at alkaline pH on the possible formation of covalent bonds in EWP (Fig. 1). In lane N corresponded to native EWP, three main

of variance (ANOVA) and Duncan's multiple range method was used to determine significant differences (p < 0.05) between the samples via SPSS software (version 23, IBM software, NY, USA). Each experiment was replicated at least three times. 3. Results and discussion In this study, pH of EWP solution was selected sufficiently far from the pI of all protein fractions of egg white even lysozyme (with pI of 10.7), so that the electrostatic repulsive forces compete with hydrophobic interactions and constrain the random aggregation and gel formation during heating (Gharbi & Labbafi, 2018). In the primary evaluation, we found that in protein concentration above 70 mg/mL, heating at pH 11.3 caused an extensive viscosity increase or in situ gelations even when the solutions were heated in 65 °C. Furthermore, as protein concentration was 70 mg/mL, after cooling and then pH adjusting to 8.0 (before GDL adding), the viscosity of the solutions (even those heated at 65 °C) extremely increased and a paste-like texture with a high portion of air bubble was formed. In this condition, we were not able to form acid-induced gels with homogenous and uniform structure. Thus, we selected the protein concentration of 45 and 60 mg/mL to form acid-induced EWP gel. To study the impact of preheating temperature on resulting acid-induced EWP gels, various temperatures including 65, 70, 80 and 90 °C were investigated. With protein concentration of 60 mg/mL, only temperatures of 65 and 70 °C could be used, because higher temperature (e.g. 80 and 90 °C) caused extensive viscosity increase and in situ gelations upon heating. Also, it should be noted that preheating at pH values below 11.0 also resulted in heat-set gelation at the protein content of 60 mg/mL. Furthermore, as EWP solutions with a protein content of 45 mg/mL were heated at pH values lower 9.0, heat-set gelation was observed during the preheating step. 3.1. Thermal induced-molecular changes in EWP solutions The average contents of total and surface free –SH groups of native EWP were 41.59 and 2.49 μmol/g protein, respectively (Table 1). It indicates that in native (un-heated) EWP the free –SH groups mostly existed in the interior of protein molecules and were not available to react with DNTB, but they were exposed in the presence of 2.5% SDS (a denaturant agent). Table 1 shows the impact of preheating temperature on the –SH groups of the TA-EWPs. With 45 mg/mL protein concentration, the surface –SH groups in egg white protein significantly increased as preheating temperature raised to 90 °C. For 60 mg/mL protein concentration, there was a similar trend where surface –SH groups in EWP significantly increased with temperature increase from 65 to 70 °C while total –SH groups decreased. These results indicate that most of buried free –SH groups in native EWP have become exposed by the heating. Furthermore, the amount of total –SH groups of EWP decreased constantly to 80 °C but with temperature increase to 90 °C, it did not show any significant changes (Table 1). This decrease in total –SH groups of EWP after heating at alkaline pH indicated that some the Table 1 Surface and total free –SH content and hydrophobicity of native and TA-EWP samples. Sample Native EWP a-TA-EWP b-TA-EWP c-TA-EWP d-TA-EWP e-TA-EWP f-TA-EWP

Surface –SH (μmol/g) f

2.49 ± 0.05 11.33 ± 0.32e 12.86 ± 0.20d 15.47 ± 0.03c 16.50 ± 0.25b 15.75 ± 0.20c 17.33 ± 0.38a

Total –SH (μmol/g) 41.59 28.08 24.12 15.66 15.94 34.77 30.03

± ± ± ± ± ± ±

a

2.43 0.81d 0.70e 0.12f 0.27f 0.93b 0.29c

Hydrophobicity 354.5 ± 10.2f 2004 ± 45.4e 1995 ± 60.1e 2243 ± 47.0d 4484 ± 27.5a 2445 ± 31.2c 3026 ± 41.2b

Fig. 1. Non-reducing (A) and reducing (B) SDS-PAGE of native EWP (lane N), aTA-EWP (lane a), b-TA-EWP (lane b), c-TA-EWP (lane c), d-TA-EWP (lane d), eTA-EWP (lane e), f-TA-EWP (lane f), and Marker molecular weight (lane M). Circular dichroism spectrum of native and TA-EWP samples (C).

Different superscripts in each column represent a significant difference (p < 0.05). Data are means ± SD. 4

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fractions of EWP, namely, lysozyme (band 1, 14.3 kDa), ovalbumin (band 2, 45 kDa), and ovotransferrin (band 3, 78 kDa) were obvious at non-reducing SDS-PAGE by comparison of their molecular weight with those from Marker lane (M lane, Fig. 1). For SDS-PAGE profile of TAEWP samples produced by 45 mg/mL protein concentration, the intensity of protein bands corresponding to ovalbumin fraction diminished, indicating the involvement of the protein fraction in thermally induced of aggregates with a high molecular weight that observed on the border of stacking gel and running gel (Fig. 1A). The large aggregates were unable to enter the 15% stacking gel and formed dark bands at the top of SDS-PAGE gels of TA-EWPs. The disappearing degree of ovalbumin band increased by a temperature increase to 90 °C where the band related to ovalbumin completely disappeared in the dTA-EWP sample (heating at 90 °C). The same fading in ovalbumin band intensity was observed in TA-EWP samples with 60 mg/mL protein content heated at 65 and 70 °C (lane e and f, Fig. 1A). Unlike ovalbumin band that some of that remained when EWP solutions heated at 65 and 70 °C (Fig. 1A), however, the ovotransferrin band disappeared almost completely in the electrophoretogram of all TA-EWP samples, regardless to preheating temperature. In egg white, ovotransferrin is recognized as the most heat-labile protein, and aggregation of this protein happens on heating of egg white to a temperature as low as 60 °C (Matsudomi, Oka, & Sonoda, 2002). Given that lower denaturation temperature of ovotransferrin (~60 °C) compared to ovalbumin (~73–79 °C), one can expect that all of the ovotransferrin molecules denature upon heating condition used in this study (even heating temperature of 65 °C) and involve in forming of high molecular weight aggregates. At reducing condition (at the presence of mercaptoethanol, Fig. 1B), the dark points of TA-EWP samples at the top of non-reducing SDSPAGE gel decreased and ovalbumin bond rather completely reappeared. It indicated that the main type of binding in aggregates originating from ovalbumin is likely to be disulfide interactions that forming through sulfhydryl-disulfide interchange reactions during heating (Mine et al., 1990; Liu, Oey, Bremer, Carne, & Silcock, 2017). Most of the studies have shown that protein aggregates in heat-treated egg white solutions remained un-dissociated under non-reducing SDS-PAGE but were dissociated in the presence of β-mercaptoethanol (reducing condition). In our study, however, some large un-dissociated aggregates still remained and were visible at the top of the SDA-PAGE gel at the presence of βmercaptoethanol, predominantly for the d-TA-EWP sample. Also, ovotransferrin band was not reappeared. By reviewing past studies in which the SDS-PAGE profile of egg proteins was investigated after heating, we observed that some authors have reported the reduction of the ovotransferrin band and formation of such high molecular weight aggregates that were not dissociated even in the presence of β-mercaptoethanol, especially as high temperature and alkaline pH were used during heating (Liu et al., 2017a, 2017b; Mine, 1990; Nicorescu et al., 2010). Given that Tris-glycine, SDS and β-mercaptoethanol in the reducing sample buffer of SDS-PAGE dissociate hydrogen bonds, hydrophobic interactions, and disulfide bridges, it highlights that besides of disulfide bonds and other non-covalent interactions, some strong unclear covalent bonds also might involve in protein aggregation of EWP upon heating condition used in our study. Fig. 1C show heat-induced modifications in the far UV-CD spectra of TA-EWP. The estimated values of secondary structure elements (αhelix, β-sheets, β-turns, and random coil) derived from the UV-CD spectra of the samples were also presented in Table 2. For both protein concentrations, upon heating at alkaline pH, there was a loss of α-helix and random coil structures and an increase in the proportion of β-sheet structures as compared native EWP. It has been reported that increasing β-sheet value is usually associated with protein aggregation (Momen, Salami, Alavi, Emam-Djomeh, & Moosavi-Movahedi, 2019). These results are in good accordance with the results of Mine et al. (1990) who observed a marked increase of β-sheet structure in the sacrifice of the helical structure during heat denaturation of EWP.

Table 2 Estimations of secondary structures content for native and TA-EWP samples. Sample

α- helix

β-sheet

β-turn

Random coil

Native EWP a-TA-EWP b-TA-EWP c-TA-EWP d-TA-EWP e-TA-EWP f-TA-EWP

23.6 21.5 21.8 18.9 18.8 18.8 19.9

26.1 32.0 32.6 38.9 40.3 32.9 34.8

15.5 15.3 15.5 14.3 14.9 15.4 14.2

34.6 31.1 30.0 27.9 25.8 30.3 30.9

Heating also significantly influenced on surface hydrophobicity (H0) of EWP (Table 1). While native EWP showed a low H0, heating significantly increased H0 of EWP. It represents the permanent modifications of conformation structure of EWP as a result of unfolding and exposing hydrophobic areas. Furthermore, in both protein concentration, the more substantial increases in H0 were observed in the TA-EWP produced in higher temperature, except for a-TA-EWP and b-TA-EWP samples that did not show any significant difference in H0. Regarding the above data, it could be supposed that the TA-EWPs are assembled from the partially unfolded molecules which stabilized by numerous disulfide bonds as well as hydrophobic interactions that develop a network of the β-sheet structures. The turbidity of a colloidal dispersion can be measured as an indicator of its particle size because larger particles scatter light to a greater extent. Thus, turbidity increase is a direct indication of protein aggregation, and therefore it can be used to measure the extent of aggregation qualitatively (Momen et al., 2019). The native EWP solution (pH 8.0) was not completely transparent and its turbidity was quite high. Due to this fact that SDS-PAGE showed no large aggregates in native egg white protein, the turbidity of native EWP solution at pH 8.0 presented the existence of some large aggregates formed through noncovalent weak interactions which disrupted by SDS buffer. It is well known that in fresh egg white, part of the positively charged lysozyme is bound to negatively charged ovomucin or other egg white network protein fractions through electrostatic attraction and form some microsized aggregates with large light scattering characteristic (Gharbi & Labbafi, 2018; Panozzo et al., 2014). This was in accordance with our visual observations, in which as the pH of native EWP solutions was increased from 8.0 to 11.3, the turbidity of the solutions completely disappeared due to constraining of the electrostatic attractions in this pH After preheating step and adjusted pH of the TA-EWP solutions at 8.0, interestingly we observed a significant decrease in turbidity of the TA-EWP solutions, in particular at TA-EWP solutions with protein content of 45 mg/mL. It seems to be contrasted with the SDS-PAGE result, in which we observed the formation of a large number of aggregates after the heating of the EWP solution at alkaline pH. The origin of this decrease in turbidity after heating could be attributed to forming the small soluble aggregates with low light scattering characteristics and, on the other hand, disrupt the electrostatically-originated primary large aggregates as a result of existent of high electrostatic repulsion between egg white protein molecules during heating at high alkaline pH (11.3). Generally, it could be supposed that upon preheating condition used in our study the egg white protein molecules significantly unfolded and soluble aggregates (as primary aggregation step) with low hydrodynamic size formed by intermolecular disulfide bridges as confirmed by surface hydrophobicity and SDS-PAGE data. However, the random aggregation (as a secondary step of aggregation) does not sufficiently proceed to form large protein aggregates with a high hydrodynamic diameter which are responsible for turbidity increasing of the system due to the presence of high electrostatic repulsive forces during heating at pH 11.3. To confirm the role of high alkaline pH (i.e. pH 11.3) to constrain random aggregation and formation of large protein aggregates with 5

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Fig. 2. Visual images and (B) turbidity values of native EWP (N) and TA-EWP solutions at protein concentration of 10 mg/mL and pH of 8.0.

solutions upon heating are determined by the size of aggregates formed during heating (Brodkorb et al., 2016, pp. 155–178). A two-step mechanism has been suggested for protein aggregation including an initial unfolding step followed by an irreversible aggregation step; Based on this mechanism, in low heating temperature the unfolding step becomes rate-limiting step of protein aggregation, while with high heating temperature the aggregation step has to become rate limiting (Brodkorb et al., 2016, pp. 155–178). The unfolding step of globular proteins mainly depends on the heating temperature where higher temperatures cause increasing of protein unfolding. Protein concentration mainly affects the irreversible aggregation step rather than the unfolding (Wolz, Mersch, & Kulozik, 2016). After unfolding step unfolded proteins aggregates initially into oligomers and subsequently into small size aggregates called soluble aggregates through hydrophobic interactions and disulfide bonds. Upon further heating, the soluble aggregates form larger aggregates with high hydrodynamic size, increasingly augment bulk viscosity of heated protein solutions. With a protein concentration of 45 mg/mL, it could be supposed that due to low protein concentration and presence of high electrostatic repulsive forces between unfolded protein molecules during heating at pH 11.3, the aggregation step does not sufficiently proceed to form large protein aggregates with high hydrodynamic diameter which are responsible for viscosity increasing of the system. It might explain why when the preheating temperature rises to 80–90 °C, the apparent viscosity of the TA-EWP solutions with protein content 45 mg/mL did not increase considerably, even though the data of surface free –SH content, CD, and H0 showed that most of the protein molecules became unfolded in the high preheating temperature (e.g. 80 and 90 °C) and aggregated by hydrophobic interactions as well as disulfide and other covalent bonds, as observed in SDS-PAGE (Fig. 1A).

high hydrodynamic diameter, a control preheated EWP solution also was produced by heating an EWP solution with a protein concentration of 30 mg/mL at pH 9.0 (as natural pH of egg white). As seen in Fig. 2A, the EWP solution heated in this condition, visually got a milky appearance and its turbidity was significantly higher than those of native EWP and TA-EWP solutions. It suggested that the high alkaline pH used in the current study is necessary to produce soluble aggregates and constrain random aggregation and heat-induced gelation during the preheating step. 3.2. Flow behaviour of TA-EWP solutions The apparent viscosity of native EWP and TA-EWP solutions in the range of shear rate from 1 to 70 1/s as a function of preheating temperature are presented in Fig. 3A. Furthermore, Table 3 shows the consistency coefficient (K value) and flow behaviour index (n) obtained by fitting the flow curve data with Power law model. EWP solution obtained by solubilizing of the egg white powder showed low viscosity at both protein concentration of 45 and 60 mg/mL. In our previous study (Alavi et al., 2019), we observed that the egg white powder contained ~4.0% insoluble aggregates, which were responsible for turbidity of the EWP solutions. Because these insoluble aggregates form only a small fraction of the total protein of the EWP, it seems that these turbid insoluble aggregates do not play a dominant role in creating the viscosity in the EWP solution. For TA-EWP solutions prepared with 45 mg/mL protein concentration, the “K” and “n” values only slightly increased with temperature (Fig. 2B). With protein concentration 60 mg/mL, however, increasing temperature form 65 °C–70 °C caused a huge increase in K value and a tremendous decrease in “n” value. The viscosity changes of the protein 6

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Fig. 3. Viscosity as a function of shear rate for TA-EWP solutions.

3.3. Characterization of acid-induced gels of TA-EWP

Table 3 Parameters obtained from Power law model fitting for native EWP (N) and TAEWP solutions. K, n, and R2 are consistency coefficient (mPa sn), flow behavior index (dimensionless) and confidence of fit, respectively. Sample

K (mPa sn)

n

N (45 mg/mL) N (65 mg/mL) a-TA-EWP b-TA-EWP c-TA-EWP d-TA-EWP e-TA-EWP f-TA-EWP

2.95 ± 0.15h 3.51 ± 0.20g 4.23 ± 0.14f 4.90 ± 0.09e 7.08 ± 0.24d 8.56 ± 0.30c 12.28 ± 0.20b 1181.9 ± 104.21a

0.89 0.88 0.91 0.90 0.89 0.84 0.84 0.41

After GDL addition, all TA-EWP solutions yielded self-supporting acid-induced gels at both protein concentration 45 and 60 mg/mL, while native EWP did not form a gel at both 60 and 45 mg/mL. A control native EWP solution that its pH was increased to 11.3 and kept at room temperature for 30 min at this pH and then its pH was adjusted to 8.0 did not develop a gel after GDL addition. It suggested the preheating step is required to develop EWP acid-induced gels after GDL addition. Furthermore, except for f-TA-EWP that tend to post-order during storage and formed a flowable gel, no gelation event occurred when the TA- EWP solutions (pH 8.0) were kept for 24 h in 40 °C without GDL addition and these solutions were completely flowable after this incubation period.

R2 ± ± ± ± ± ± ± ±

0.02a 0.02a 0.02a 0.01a 0.01a 0.02b 0.02b 0.05c

0.97 0.96 0.98 0.98 0.98 0.98 0.98 1.00

Different superscripts in each column represent a significant difference (p < 0.05). Data are means ± SD.

3.3.1. Microstructural morphology To investigate the relationship between the preheating temperature of TA-EWP and their protein content with gel morphology, both SEM and CLSM were applied (Fig. 4). For protein concentration of 45 mg/ mL, CLSM images of all TA-EWP gels prepared by preheating temperature from 65 to 90 °C were homogenous. However, in protein content of 60 mg/mL, CLSM images of the gels became moderately heterogeneous when the preheating temperature of TA-EWP increased from 65 to 70 °C (i.e. f-TA-EWP gel). The heterogeneity of gel microstructure in the f-TA-EWP gel might be a consequence of the very high apparent viscosity of its gel pre-solution limiting movement of the aggregates and protein clusters during gelation. Therefore, after GDL addition the protein clusters in f-TA-EWP solution probably had lower opportunity to completely fuse together to develop absolutely homogenous gel networks. In SEM images, the TA-EWP acid-induced gels presented a honeycomb-like microstructure contained large serum pools. With both protein concentration, increasing preheating temperature has a positive effect on the compactness and uniformity of the gel network structure and led to an ordered matrix with smaller voids (Fig. 4). Furthermore, the TA-EWP gels with 60 mg/mL protein content had more compact microstructures compared to 45 mg/mL protein concentration. It might be attributed to this fact that at higher protein content more protein molecules were devoted to gel network formation (Yang et al., 2017).

With protein concentration of 60 mg/mL, regarding this fact that preheating temperature of 65 °C is far from denaturation temperature of ovalbumin (the main protein fraction of EWP), the degree of protein unfolding might be little in this low preheating temperature (i.e. 65 °C). Thus, in this condition, the unfolding step is rate limiting step and proteins cannot enter the aggregation stage sufficiently and viscosity of e-TA-EWP solution will not increase greatly (Table 3) due to inadequate protein aggregation. However, when the preheating temperature rises to 70 °C (f-TA-EWP), the protein unfolding rate increases and more unfolded proteins can enter the aggregation step. Regarding higher protein concentration of the f-TA-EWP sample compared to TA-EWP sample with 45 mg/mL, the probability of collision between the unfolded protein molecules rises resulting in the formation of a greater number of aggregates with a high hydrodynamic diameter that entangling with each other and resist against the flow (Wolz et al., 2016). It could describe the great apparent viscosity detected for the f-TA-EWP solution and is in line with turbidity data (Fig. 2B) where the f-TA-EWP solution had the highest turbidity (means the highest aggregate particle size) between TA-EWP solutions. However, the aggregates increasingly detangle and orientate toward the shear field direction with the shear rate increase, thus f-TA-EWP showed an intensive shear-thinning character (very low “n” value) as compared to other TA-EWP samples (Table 3) (Alavi et al., 2019).

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and cohesiveness) and Young modulus of the gels were determined (Table 4). For both protein content, with increasing temperature, all parameters of TPA including hardness, springiness, chewiness, cohesiveness as well as Young's modulus constantly increased. On the other hand, with increasing protein content, TPA parameters significantly increase for gels of TA-EWP prepared from the same preheating temperature. True fracture stress (σ) (the stress that is needed to break a gel) and Hencky strain (ε) (the strain at which a gels breaks and specifies how much a gel needs to be deformed until it breaks) showed an increase with increasing preheating temperature for both protein concentration. It signifies that the weakest connections (regarded as breaking point) in the gels of TA-EWP prepared by higher preheating temperatures is stronger than gels of TA-EWP made by lower preheating temperatures. This coincides with the results from SEM, where the gels of TA-EWP prepared from higher preheating temperatures showed a more ordered structure than the lower preheating temperature. Higher TPA parameters at the gels of TA-EWP prepared from higher preheating temperature may be explained by the more intensive formation of hydrophobic and disulfide bonds in the gels during acidifying step because their TA-EWP as building blocks of the gels had higher surface hydrophobic and surface free –SH groups. Same results were reported by Hongsprabhas and Barbut (1996) who observed higher preheating temperature resulted in stiffer cold-set whey protein isolate gels. Furthermore, at the same preheating temperature, as protein concentration increased from 45 to 60 mg/mL, the values of fracture stress and Hencky strain considerably amplified (Table 4). As observed in SEM images, higher protein content caused a denser gel network as a result of greater protein-protein interactions formed between protein molecules during gel network formation leading to higher fracture stress in the resulting gels. It is worth noting that the acid-induced gels of TAEWP produced with 60 mg/mL protein concentration showed comparable or higher fracture stress compared to heat-set egg white gels with 100 mg/mL protein content (14–18 kPa) reported in past studies (Babaei, Khodaiyan, & Mohammadian, 2019; Khemakhem, Attia, & Ayadi, 2019). 3.3.3. Rheological properties 3.3.3.1. Gelation kinetics. The gelation kinetic of TA-EWP solutions after GDL addition was recorded by small strain rheometry (Fig. 5A). Generally, gelation time is defined as the crossover point of the viscoelastic moduli i.e. tanδ = G’/G’’ = 1. However, in the present study, the storage modulus (G′) of all TA-EWP solutions just after GDL addition was higher than loss modulus G”, proposing that the systems became predominately elastic after GDL addition (It should be noted that the pH of all TA-EWP solution just after the GDL addition decreased from 8.0 to 7.4). Hence, G′ was selected as the indicator to illustrate the TA-EWP gelation process. Fig. 5A shows the progress of G′ values of different TA-EWP solutions as a function of time. After GDL addition, G′ showed a sharp increase in the first 2000 s and then gradually levelled off, indicating that the systems experience a quite fast transformation from a fluid to viscoelastic solid. As seen in Fig. 5A, the fast increase of G′ value coincided with the rapid decrease of pH

Fig. 4. The microstructure of TA-EWP gels. Right column (A) – CLSM; Middle and left columns– SEM at ×200 (B, scale bar 200 μm) and ×500 (C, scale bar 50 μm) magnification, respectively.

3.3.2. Textural properties To understand how variations in preheating temperature and protein content affect the mechanical properties of acid-induced TA-EWP gels, four parameters of TPA (hardness, springiness index, chewiness,

Table 4 Parameters of texture profile analysis, Yung's modulus, true axial stress (σ) and Hencky strain (ε) of acid-induced TA-EWP gels. Samples

a-TA-EWP

b-TA-EWP

c-TA-EWP

d-TA-EWP

e-TA-EWP

f-TA-EWP

Hardness (g) Springiness Index Chewiness (mJ) Cohesiveness Young's modulus (kPa) σ (kPa) ε

62.00 ± 4.24e 0.62 ± 0.04e 0.35 ± 0.07e 0.19 ± 0.04d 3.44 ± 0.24e 2.61 ± 0.26f 0.79 ± 0.04d

122.50 ± 0.71d 0.75 ± 0.01d 1.90 ± 0.28d 0.39 ± 0.01c 6.80 ± 0.07d 3.96 ± 0.09e 0.79 ± 0.01d

170.67 ± 9.07c 0.83 ± 0.04c 2.77 ± 0.61c 0.40 ± 0.05c 9.47 ± 0.50c 6.46 ± 0.43d 0.84 ± 0.04c

193.50 ± 1.06b 0.88 ± 0.02b 3.90 ± 0.07b 0.46 ± 0.01ab 10.42 ± 0.28b 8.72 ± 1.06c 0.92 ± 0.07bc

165.00 ± 4.24c 0.87 ± 0.01b 3.45 ± 0.35b 0.49 ± 0.05ab 9.16 ± 0.23c 11.03 ± 1.31b 0.98 ± 0.05b

322.00 ± 18.03a 0.94 ± 0.01a 7.73 ± 0.62a 0.50 ± 0.02a 17.87 ± 1.00a 20.48 ± 0.54a 1.07 ± 0.04a

Different superscripts in each column represent a significant difference (p < 0.05). Data are means ± SD. 8

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Fig. 5. A) Storage modulus (G′) and pH value evolution of TA-EWP solutions after GDL addition as a function of time. (B) Frequency dependence of storage modulus (G′, solid) and loss modulus (G″, open) of TA-EWP gels.

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(with “n” value 0.11) (Yang et al., 2017). Furthermore, the “a” value of the acid-induced gels increased with increasing preheating temperature and protein content. For 45 mg/mL protein content, as preheating temperature increased from 65 to 80 °C the “a” value substantially raised, while with further increasing preheating temperature (from 80 to 90 °C) the increase was no significant (p > 0.05). Furthermore, the highest “a” value was related to the f-TA-EWP gel followed by d-TAEWP gel suggesting these gels are more solid and form the stronger gel networks compared to other gels. It was in line with SEM and texture analysis data, where more compact structure and higher texture parameters were observed for f-TA-EWP gel.

Table 5 Power law parameters fitted from storage modulus G’ of TA-EWP gels. Sample

a

n

a-TA-EWP b-TA-EWP c-TA-EWP d-TA-EWP e-TA-EWP f-TA-EWP

1478.5 ± 45.4d 3810.4 ± 84.1c 6992.6 ± 74.3b 7507.3 ± 64.5b 3690.0 ± 48.1c 15639 ± 111.8a

0.13 0.14 0.14 0.14 0.14 0.11

R2 ± ± ± ± ± ±

0.00a 0.01a 0.01a 0.00a 0.01a 0.01b

0.986 0.994 0.996 0.999 0.999 0.994

Different superscripts in each column represent a significant difference (p < 0.05). Data are means ± SD.

value in the first 2000 s (from 7.4 to ~5.2). The trend of evolution of G′ with time was similar in all the samples regardless of their protein content and preheating temperature. At the end of the test time (7200 s), gels of TA-EWP prepared from higher preheating temperature showed significantly greater the G′ value compared to those of TA-EWP prepared from lower preheating temperature. Regarding protein content, at the same preheating temperature, gels with higher protein concentration showed substantially greater G’ at the end of the test time (Fig. 5A).

3.3.4. Water holding capacity (WHC) WHC is one of the most important functional properties of protein gels and gels with a large WHC are generally desirable for food applications since water loss can cause gel shrinkage, texture changes and unattractive quality attributes (Yang et al., 2017). As showed in Fig. 6, for 45 mg/mL protein content, as preheating temperature of TA-EWP increased from 65 °C to 80 °C, the WHC of the gels increased considerably from 61.87% to 92.67%, and then with more increasing temperature to 90 °C slightly but insignificantly dropped to 92.15% (p > 0.05). At same preheating temperature, TA-EWP gels with a protein concentration of 60 mg/mL tend to have significantly higher WHC as compared those gels with 45 mg/mL protein concentration (aand b-TA-EWP gels compared to e- and f-TA-EWP gels, respectively, Fig. 6). Same with gels with 45 mg/mL protein content, increasing the preheating temperature from 65 to 70 °C significantly improved WHC of gels with 65 mg/mL protein content (p < 0.05). It can be observed that stiffer (with greater fracture stress, Table 4) and more elastic gels (with higher storage module, Fig. 5B) had higher WHC than less stiff and elastic gels. It may be due to this fact that upon centrifugal force the less stiff gels experience a network compression and water remove easier from the gels. In line with our study, Li et al. (2018) observed a positive correlation between gel fracture strength and WHC in heat-set gels of EWP. The WHC of the cold-set gels prepared in our study (except a- and b-TA-EWP gels) was comparable with those of heat-set egg white gels that reported by other authors who measured WHC at centrifugal forces between 1500–10,000 g (WHC 88–96%) (Babaei et al., 2019; Khemakhem et al., 2019; Li et al., 2018).

3.3.3.2. Frequency dependence of TA-EWP acid-induced gels. To do a further rheological analysis of the TA-EWP acid-induced gels, a frequency sweep test was done on formed gels. The frequency sweeps plots of different gels are illustrated in Fig. 5B. As it can be observed from this figures, for all gel samples the G′ values are significantly higher than G″ values at a frequency range of 1–100 Hz suggesting the TA-EWP solutions produced a three-dimensional gel network. The frequency (ω) dependence of G′ can indicate the structural strength of the gels. To quantitative analyse the degree of frequency dependence of G′ of the gels, a Power law model as G = a n was fitted to the frequency sweep data from Fig. 5B. The fitted Power law parameters are shown in Table 5. The coefficients “a” is related to strength (elastic structure) of a gel. The “n” value represents a measure of frequency dependence of the storage module and an “n” value close to zero is characteristic of a more solid-like material (Chaux-Gutiérrez, PérezMonterroza, & Mauro, 2019). All gels exhibited a weak dependence of G’ with ω (low “n” value, Table 5), indicating the formation of the gels with high shear strength by all TA-EWP gels, especially f-TA-EWP gel

Fig. 6. Water holding capacity (WHC) of TA-EWP gels. Different superscripts in each column chart represent a significant difference (p < 0.05).

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4. Conclusion

denaturation, aggregation and gelation of whey proteins. Advanced dairy chemistry. New York, NY: Springer. Chaux-Gutiérrez, A. M., Pérez-Monterroza, E. J., & Mauro, M. A. (2019). Rheological and structural characterization of gels from albumin and low methoxyl amidated pectin mixtures. Food Hydrocolloids, 92, 60–68. Chen, N., Chassenieux, C., & Nicolai, T. (2018). Kinetics of NaCl induced gelation of soy protein aggregates: Effects of temperature, aggregate size, and protein concentration. Food Hydrocolloids, 77, 66–74. Gharbi, N., & Labbafi, M. (2018). Effect of processing on aggregation mechanism of egg white proteins. Food Chemistry, 252, 126–133. Hongsprabhas, P., & Barbut, S. (1996). Ca2+-induced gelation of whey protein isolate: Effects of pre-heating. Food Research International, 29(2), 135–139. Khemakhem, M., Attia, H., & Ayadi, M. A. (2019). The effect of pH, sucrose, salt and hydrocolloid gums on the gelling properties and water holding capacity of egg white gel. Food Hydrocolloids, 87, 11–19. Li, J., Li, X., Wang, C., Zhang, M., Xu, Y., Zhou, B., ... Yang, Y. (2018). Characteristics of gelling and water holding properties of hen egg white/yolk gel with NaCl addition. Food Hydrocolloids, 77, 887–893. Liu, Y. F., Oey, I., Bremer, P., Carne, A., & Silcock, P. (2017a). Effects of pH, temperature and pulsed electric fields on the turbidity and protein aggregation of ovomucin-depleted egg white. Food Research International, 91, 161–170. Liu, Y. F., Oey, I., Bremer, P., Silcock, P., & Carne, A. (2017b). In vitro peptic digestion of ovomucin-depleted egg white affected by pH, temperature and pulsed electric fields. Food Chemistry, 231, 165–174. Matsudomi, N., Oka, H., & Sonoda, M. (2002). Inhibition against heat coagulation of ovotransferrin by ovalbumin in relation to its molecular structure. Food Research International, 35(9), 821–827. Mession, J. L., Chihi, M. L., Sok, N., & Saurel, R. (2015). Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation. Food Hydrocolloids, 46, 233–243. Mine, Y., Noutomi, T., & Haga, N. (1990). Thermally induced changes in egg white proteins. Journal of Agricultural and Food Chemistry, 38(12), 2122–2125. Momen, S., Salami, M., Alavi, F., Emam-Djomeh, Z., & Moosavi-Movahedi, A. A. (2019). The techno-functional properties of camel whey protein compared to bovine whey protein for fabrication a model high protein emulsion. LWT, 101, 543–550. Nicorescu, I., Vial, C., Talansier, E., Lechevalier, V., Loisel, C., Della Valle, D., ... Legrand, J. (2011). Comparative effect of thermal treatment on the physicochemical properties of whey and egg white protein foams. Food Hydrocolloids, 25(4), 797–808. Panozzo, A., Manzocco, L., Calligaris, S., Bartolomeoli, I., Maifreni, M., Lippe, G., et al. (2014). Effect of high pressure homogenisation on microbial inactivation, protein structure and functionality of egg white. Food Research International, 62, 718–725. Svanberg, L., Wassén, S., Gustinelli, G., & Öhgren, C. (2019). Design of microcapsules with bilberry seed oil, cold-set whey protein hydrogels and anthocyanins: Effect of pH and formulation on structure formation kinetics and resulting microstructure during purification processing and storage. Food Chemistry, 280, 146–153. Tomczyńska-Mleko, M., Nishinari, K., & Handa, A. (2014). Ca2+-Induced egg white isolate gels with various microstructure. Food Science and Technology Research, 20(6), 1207–1212. Weijers, M., van de Velde, F., Stijnman, A., van de Pijpekamp, A., & Visschers, R. W. (2006). Structure and rheological properties of acid-induced egg white protein gels. Food Hydrocolloids, 20(2–3), 146–159. Wolz, M., Mersch, E., & Kulozik, U. (2016). Thermal aggregation of whey proteins under shear stress. Food Hydrocolloids, 56, 396–404. Yang, C., Wang, Y., & Chen, L. (2017). Fabrication, characterization and controlled release properties of oat protein gels with percolating structure induced by cold gelation. Food Hydrocolloids, 62, 21–34.

In the current study, EWP was heated at high alkaline pH to produce gelable TA-EWP. With the increased preheating temperature, exposing free –SH groups, the formation of disulfide bonds, and participation of egg white protein fractions to form high molecular weight aggregates were increased, and more hydrophobic residues were exposed in EWP molecules. CD spectra suggested that the β-sheet structures had the predominant role in the arrangement of the TA-EWP. Microstructure compactness, mechanical and rheological properties, and WHC of the gels were improved by increasing preheating temperature and protein content. This technique allows fabricating cold-set EWP gels with different mechanical properties and relatively high protein content. As compared their heat-set counterparts, the cold-set EWP gels could have more potential for application in the development of healthy foods and drug delivery systems, as they let the incorporation of heat sensible ingredients for example probiotics enzymes, and vitamins. Acknowledgement The support of the University of Tehran, UNESCO Chair on Interdisciplinary Research in Diabetes is acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.105337. References Abaee, A., Mohammadian, M., & Jafari, S. M. (2017). Whey and soy protein-based hydrogels and nano-hydrogels as bioactive delivery systems. Trends in Food Science & Technology, 70, 69–81. Alavi, F., Emam-Djomeh, Z., Momen, S., Mohammadian, M., Salami, M., & MoosaviMovahedi, A. A. (2019). Effect of free radical-induced aggregation on physicochemical and interface-related functionality of egg white protein. Food Hydrocolloids, 87, 734–746. Alavi, F., Momen, S., Emam-Djomeh, Z., Salami, M., & Moosavi-Movahedi, A. A. (2018). Tailoring egg white proteins by a GRAS redox pair for production of cold-set gel. LWT, 98, 428–437. Alting, A. C., Hamer, R. J., de Kruif, C. G., Paques, M., & Visschers, R. W. (2003). Number of thiol groups rather than the size of the aggregates determines the hardness of cold set whey protein gels. Food Hydrocolloids, 17(4), 469–479. Babaei, J., Khodaiyan, F., & Mohammadian, M. (2019). Effects of enriching with gellan gum on the structural, functional, and degradation properties of egg white heat-induced hydrogels. International Journal of Biological Macromolecules, 128, 94–100. Brodkorb, A., Croguennec, T., Bouhallab, S., & Kehoe, J. J. (2016). Heat-induced

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