without heating

Food Hydrocolloids 103 (2020) 105654

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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd

Enhancement of gel characteristics of NaOH-induced duck egg white gel by adding Ca(OH)2 with/without heating Minmin Ai a, b, 1, Quan Zhou a, b, 1, Nan Xiao a, b, 1, Shanguang Guo a, b, c, Yuanyuan Cao a, b, Hong Fan a, b, Ziting Ling a, b, Ledan Zhou a, b, Shuchang Li a, b, Jiaoli Long a, b, Aimin Jiang a, b, c, * a

College of Food Science, South China Agricultural University, Guangzhou, 510642, China The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, South China Agricultural University, Guangzhou, 510642, China c The Guangdong Province Livestock and Poultry Products Processing Technology Engineering Research Center, South China Agricultural University, Guangzhou, 510642, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Egg white gel Heating process Rheology Microstructure Texture

Egg white protein (EWP) can form a gel under alkali condition, but there have been few reports on the gelling of EWP with the addition of Ca(OH)2. In this paper, the effects of Ca(OH)2 and the heating process on the rheo­ logical, mechanical and microstructural properties, and intermolecular interactions of egg white gel (EWG) were investigated. Results suggested that the pH of EWG increased markedly upon addition of Ca(OH)2 but declined after heating, and the surface hydrophobicity of EWP decreased significantly due to the embedding and destruction of hydrophobic groups. And sulfhydryl groups and disulfide bonds contents decreased after heating. Secondary structural analysis showed that β-sheet content increased, and β-turn content decreased after the addition of Ca(OH)2, while heating decreased random structures and α-helical content and increased β-turn content. The results of electrophoresis revealed that the addition of Ca(OH)2 degraded protein patterns gradu­ ally, while heating destroyed EWP. Textural test results demonstrated the hardness value increased remarkably upon addition of Ca(OH)2 and heating, but springiness declined gradually. The addition of Ca(OH)2 improved storage (G0 ) and loss (G00 ) modulus of EWG, which might relate to the formation of calcium bridges and ion interactions. The surface morphology of EWG transformed from yellow to brown or red after heating. Rough and irregular microstructure formed when the amount of Ca(OH)2 increased, while the microstructure became more compact and regular after heating. These results suggested that addition of Ca(OH)2 significantly affected the represented characteristics of EWG, and heating changed the intermolecular interactions and physicochemical properties.

1. Introduction More than 100 types of proteins are contained in egg white (EW), the most abundant being ovalbumin, followed by ovotransferrin, ovomu­ coid and lysozyme (Mine, 2008). EW protein (EWP) can form a gel under various conditions, and the gelling properties are mainly influenced by the environment and treatment conditions. Duck eggs are often used to prepare salted eggs or preserved eggs because of their poor smell and unpopularity. The preserved egg is a traditional egg product in China, which is prepared from soaking fresh duck egg in alkaline solution by the ‘immersion method’ (Wang & Fung, 1996). The main functional

ingredients in the alkaline solution are sodium hydroxide (NaOH) and metal ions such as Cu2þ or Zn2þ, which are effective alternatives to the use of Pb2þ with alkaline solution, since Pb2þ can control the amount of alkali that gets into eggs (Ganesan & Benjakul, 2010). Alkali denatur­ ation of the EWP occurs under the strong alkaline conditions, with the denatured EWP interacting to form a loose, linear network gel (Zhao et al., 2016). Hence, the preparation of gels using alkali, and always NaOH, has attracted a lot of attention. The addition of NaOH into EW dispersion, ovalbumin or preserved egg to explore the mechanism of EW gel (EWG) formation has been extensively studied (Zhao et al., 2016, 2016; Chen et al., 2015; Li et al.,

* Corresponding author. College of Food Science, South China Agricultural University, Guangzhou, 510642, China. E-mail address: [email protected] (A. Jiang). 1 Co-first author. https://doi.org/10.1016/j.foodhyd.2020.105654 Received 22 September 2019; Received in revised form 5 January 2020; Accepted 7 January 2020 Available online 9 January 2020 0268-005X/© 2020 Elsevier Ltd. All rights reserved.

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2018). It has been found that the intermolecular forces to support the gel are different for these different egg products, resulting in different physicochemical characteristics of the resulting gel, but that excessive alkali always causes the formed gel to be hydrated (Chen et al., 2015). There have been few reports on the effects of Ca(OH)2 on the formation of EWG. The alkalinity of Ca(OH)2 is weaker than that of NaOH due to its lower solubility, and Ca(OH)2 is often used in the production and pro­ cessing of food. For example, konjac gel is prepared by adding Ca(OH)2 into konjac dispersion, and Ca(OH)2 is used in the sugar industry to neutralize excess acids (Huang, Chu, Huang, Wu, & Tsai, 2015; Nakano, Ugwu, & Tokiwa, 2012). Ca(OH)2 is also used in the production of rubber to improve its elasticity, sealability and durability (Nascimento et al., 2014). In the production of preserved eggs, quicklime is often added to water to generate Ca(OH)2, and which further generates NaOH to denature duck EW and preserve the eggs (Ma, 2007). In previous studies, we found that the addition of tea polyphenols and Ca(OH)2 into EWG had a significant effect on the gelling characteristics of EWG (Ai et al., 2019). However, the preparation of EWG was at room tempera­ ture; the properties of EWG after heating have not been studied. Heat treatment is a common method for food processing and prep­ aration. It can denature proteins and facilitate their digestion and ab­ sorption by the human body. Preserved eggs are often processed at high temperatures to prepare porridge in China. Based on a large number of previous studies (Ai et al., 2019; Huang et al., 2019; Li et al., 2018), we found that the addition of a small amount of Ca(OH)2 on the basis of 0.57% NaOH and heating had a great impact on the characteristics of EWG after lots of preliminary experiments, such as its mechanical and microstructural properties. Therefore, the objective of this study was to investigate the gelling properties of NaOH-induced EWG by adding Ca (OH)2 with/without thermal modification.

equilibrium before detection. The unheated group and heated group of EWG were defined as UEWG and HEWG respectively. 2.3. pH and zeta potential The pH of EWG was determined according to the methods described by a previous study (Zhang, Jiang, Chen, Ockerman, & Chen, 2015). Five grams of each sample was weighed and homogenized at 10,000 rpm for 2 min (T25 easy clean digital, IKA, Germany) with 50 mL 0.1 mol/L KCl solution. Then, the samples were filtered and the filtrate was collected to determine its pH using a pH meter (PB-10, Sartorius, Berlin, Germany). Another five grams of EWG was dispersed at 10,000 rpm for 2 min with 95 mL distilled water, and the mixture was placed at room temperature for 4 h before absorbing 4 mL of the mixture into a test tube with 6 mL distilled water. Then, the zeta potential of the diluted mixture was analyzed using particle electrophoresis (Zetasizer Nano ZS-90, Malvern Instruments, Worcestershire, UK). All experiments were conducted in triplicate. 2.4. Surface hydrophobicity Determination of surface hydrophobicity was performed according to the method described by Ai et al. (2019) using a fluorescence spec­ trophotometer (RF-5301PC, Shimadzu, Tokyo, Japan). All analysis was conducted three times under the same experimental conditions. 2.5. Total and free sulfhydryl content The total and free sulfhydryl contents were performed by the method described by previous study (Beveridge & Arntfield, 1979; Yarnpakdee; Benjakul; Visessanguan, & Kijroongrojana, 2009) with some modifica­ tions. Three grams of EWG was weighed and dispersed into 27 mL so­ dium phosphate buffer (0.1 mol/L pH 8.0), homogenized at 10,000 rpm for 2 min and centrifuged at 10,000�g for 15 min, and the supernatant was collected. The protein content in the supernatant was measured by Bradford’s method. For the determination of free sulfhydryl content (SHF) content, 0.2 mL of the supernatant with 2.8 mL Tris-Gly buffers solution (pH 8.0 containing 0.089 mol/L Tris, 0.09 mol/L glycine, 0.004 mol/L EDTA and 8 mol/L urea) and 0.02 mL Ellman reagent (4 mg/mL DTNB was dissolved in Tris-Gly buffer) reacted in a 40 � C water bath for 15 min before measuring the absorbance at 412 nm (UV2600, Shi­ madzu, Tykyo, Japan). For the determination of total sulfhydryl content (SHT), another 0.2 mL of the supernatant with 2.8 mL of Tris-Gly buffer (pH 8.0 contains 1.5 mg/mL of β-Me, 0.5% SDS and 8 mol/L urea) reacted in a 40 � C water bath for 60 min. The proteins were then precipitated with 12% (w/v) TCA for 1 h and centrifuged at 4000�g for 10 min. The pellets were collected and rinsed with 12% (w/v) TCA for 3 times. Then, the pellets were dissolved in 10 mL Tris-glycine buffer (pH 8.0 containing 8 mol/L urea), and the absorbance of 4 mL of the solution with the addition of 0.02 mL Ellman reagent was measured at a wave­ length of 412 nm after staining for 15 min at 40 � C. The SH content was calculated using an extinction coefficient of 13,600 M 1cm 1. A blank was analyzed using the treatment buffer for determining SHT and SHF. All analyses were conducted in triplicate. The content of SHF, SHT and disulfide bonds (SS) were calculated as following:

2. Materials and methods 2.1. Materials Fresh duck eggs were bought from a supermarket in Guangzhou, China. Potassium bromide (KBr), methanol, trichloroacetic acid (TCA), acetic acid, Coomassie brilliant blue G-250, glutaraldehyde, Ca(OH)2, NaOH, glycine (Gly) and ethylenediaminetetraacetic acid (EDTA) were bought from the Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). 1-nilino-8-naphthalenesulfonic acid (ANS), 5,50 -dithiobis-(2-nitro­ benzoic acid) (DTNB), trimethylaminomethane (Tris), β-mercaptoetha­ nol (β-Me), sodium dodecyl sulfate (SDS) were obtained from Sigma (St. Louis, MO, USA). 2.2. Preparation of EWG One- or two-days old duck eggs were cleaned with tap water, and those were hand-broken, EW and yolk were separated carefully from the eggshell. The EW was mixed homogeneously under magnetic stirrer (841A6, Shanghai Sile Instrument Co., Ltd, Shanghai, China) at 2000 rpm for 15 min. According to our previous study (Ai et al., 2019), EWP could form a gel as influenced by adding 0.57% NaOH, of which was also used in this study. Two groups of samples were prepared. 1 mL of a mixed solution with NaOH and Ca(OH)2 was added into 20 g EW dispersion (protein concentration almost 11.0%) in order to achieve the NaOH concentration of 0.57% (w/w) and the Ca(OH)2 concentration of 0.005%, 0.01%, 0.015% and 0.03% (w/w) respectively. Then, all EW was stirred well with a glass rod immediately. One group of samples was sealed with a plastic wrap and placed at 25 � C for 3 days to keep equi­ librium before chemical testing and instrumental analysis. Another group of samples was heated 30 min at 90 � C in a thermally controlled water bath (HH-4, Changzhou Aohua Instrument Co., Ltd, Changzhou, China) according to the previous study (Cordobes, Partal, & Guerrero, 2004). The heated samples were cooled down to normal temperature by tap water and stored at the same condition (25 � C for 3 days) to keep

SHðμmol = gproteinÞ ¼ A412 � 73:53 � D = C

(1)

SSðμmol = gproteinÞ ¼ ðSHT

(2)

SHF Þ = 2

where A412 is the absorbance at 412 nm, D is the dilution factor, C is the concentration of protein in mg/mL and 73.53 is derived from 106/ 13,600 (13,600 is Ellman’s reagent molar absorptivity).

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Fig. 1. Changes in pH and zeta potential (A) of egg white gel, surface hydrophobicity (B) of egg white protein dispersions as affected by Ca(OH)2. Un, unheated; He, heated. Different letters in the same index denote the significance difference (P < 0.05).

2.6. Secondary structure analysis

adjusted to 1 mg/mL 100 μL of supernatant mixed with 100 μL of sample diluent. Then, a 10 μL of sample and 10 μL of standard proteins were loaded onto the stacking gel. Electrophoresis was conducted using a vertical gel electrophoresis unit (Mini-protein II; Bio-Rad Laboratories, Richmond, CA, USA) at the constant voltage of 120 V/plate. The gels were stained with Coomassie brilliant blue G-250, 25% methanol, and 10% acetic acid. Destaining was performed using 40% methanol and 10% acetic acid.

The infrared spectrum of EWG was determined by using a FTIR spectrometer (Bruker vertex70, Coventry, Germany). 1.2 mg of vacuum freeze-dried sample powder with 100 mg of KBr were ground in an agate mortar and determined in the range of 4000–400 cm 1 for 16 scans at 4 cm 1 resolution. Secondary structure components derived from the amide I band (1700–1600 cm 1) were analyzed using PeakFit software 4.12 (SeaSolve, Framigham, MA, USA). Pure KBr was used as a blank. All analyses were conducted in triplicate.

2.8. Textural profile analysis (TPA) TPA was conducted according to a previous study (Ai, Guo, Zhou, Wu, & Jiang, 2018) with some modifications using a TA-XT Plus texture analyzer (Stable Microsystems, Surrey, England). The samples were prepared in the size of 10 mm � 10 mm � 15 mm with a knife and compressed 50% of its original height with 1 mm/s pretest and test speed fitted with a probe (P/36R). The texture parameters of hardness and springiness were calculated. Hardness values were obtained for the

2.7. Electrophoresis Protein patterns of EWG were performed by SDS-PAGE. Stacking and separating gel concentration were 5% and 12%, respectively. 3 g sample was dispersed into 27 mL 0.1 mol/L sodium phosphate buffer, and then the mixture was centrifuged at 10,000�g for 15 min to obtain the su­ pernatant. The protein content was measured by Bradford’s method and 3

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peak value at the time of the first compression. Springiness values were the ratio of the time from the start of secondary compression to the peak value for the first compression. All samples were conducted in 8–10 times. 2.9. Rheological properties Rheological properties of EWG were performed using a rheometer (MCR301, Anton Parr, Austria) fitted with a 50 mm steel parallel plate (PP50). A sequence of the following sweep was conducted: a, time sweep was conducted and lasted for 3 h, the mixture was adsorbed and dropped on the platform immediately when the alkali solution was added into EW, the strain was set to 2% and the temperature was set 25 � C; b, frequency sweep was determined under 1–100 rad/s at 25 � C with 0.2% strain. The obtained time sweep results were simulated as following (Kuan, Nafchi, Huda, Ariffin, & Karim, 2016): G0 t ¼ kgelln(tgel) þ C, where G0 t is the value of G0 at time t, C is a constant, tgel is gelation time, and kgel is the gelation rate constant. EWG without adding Ca(OH)2 was used as the model to obtain the target storage modulus (G0 C) after 3 h gelling at 25 � C. The time (tmodel) required for a gelling system to reach 0 G0 C was calculated using the following equation: tmodel ¼ eðG C CÞ=kgel . The 0 G -value of frequency sweep was simulated by a power-law model 0 equation described in the previous study ðG’ ¼ K0 ωn Þ (Razi, Mota­ medzadegan, Shahidi, & Rashidinejad, 2018). 2.10. Color and surface morphology The samples were tiled on a color dish and the chromatic value of L*, a*, and b* were measured using a portable spectrophotometer (SP62, XRite corporation, USA) with using blackboard and whiteboard as blanks. Five points of the samples were analyzed, and the average value was adopted. The total color change △E was calculated as follows (Santi­ panichwong & Suphantharika, 2007): qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ΔE ¼ ðΔL*Þ2 þ ðΔa*Þ2 þ ðΔb*Þ2 (3) where L* stands for brightness, a* stands for red (þ) or green ( ), b* stands for yellow (þ) or blue ( ). A 12-megapixel digital camera was used to capture the surface features of EWG. 2.11. Scanning electron microscopy (SEM) SEM was performed according to a previously described method (Chen et al., 2015) with some modifications using an SEM (SEM, Quanta-200F, FEI, Netherlands). An approximately 1.5 g sample was fixed in 2.5% (v/v) glutaraldehyde (0.1 mol/L sodium phosphate buffer, pH 7.2) for approximately 0.5 h at room temperature. Subsequently, the sample was rinsed with 0.1 mol/L sodium phosphate buffer (pH 7.2) thrice for approximately 15 min. Afterward, the samples were dehy­ drated in a graded ethanol series (30%, 50%, 70%, 90%, and 100%) for 30 min for each step. At last, the fixed samples were freeze-dried in a lyophilizer (Alpha1-2, Martin Christ, Germany) and observed by a SEM at an acceleration voltage of 5 kV in low vacuum mode with 3000 magnification.

Fig. 2. Changes in free and total sulfhydryl content and disulfide bonds of egg white protein as affected by Ca(OH)2. Un, unheated; He, heated. Different let­ ters in the same index denote the significance difference (P < 0.05). SHF, free sulfhydryl; SHT, total sulfhydryl; SS, disulfide bonds.

3. Results and discussion 3.1. pH, zeta potential, and surface hydrophobicity The pH of EWG, zeta potential of EWG dispersion and surface hy­ drophobicity of EWP are shown in Fig. 1. The pH increased significantly after the addition of Ca(OH)2 (P < 0.05; Fig. 1A). Specifically, for UEWG the pH increased from 10.84 to 10.97, and for HEWG the pH increased from 9.59 to 10.15. There was a significant decrease in pH after heating. The absolute value of zeta potential decreased significantly (P < 0.05; Fig. 1A) upon addition of Ca(OH)2, and was lower for HEWG than for UEWG. The surface hydrophobicity of EWG protein decreased signifi­ cantly (P < 0.05; Fig. 1B), from 735.89 S0–451.36 S0 for UEWG and from

2.12. Data analysis The results were reported as the mean � standard deviation. The data was analyzed using SPSS V.20 software (SPSS Inc., Chicago, IL). Significant differences between means (P < 0.05) were studied through Duncan’s multiple-range test by one way ANOVA using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). 4

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Fig. 3. Secondary structure changes of egg white protein as affected by Ca(OH)2. Different letters in the same index denote the significance difference (P < 0.05).

617.41 S0–102.15 S0 for HEWG. Non-significant changes occurred upon addition of 0.015% and 0.03% Ca(OH)2 for UEWG (P > 0.05). The addition of Ca(OH)2 released OH to increase the pH of EWG. The dissociation of Ca2þ neutralized the negative charges on the surface of EWP, resulting in the decreased of absolute zeta potential. It indicated that the addition of Ca(OH)2 increased the possibility of protein aggre­ gation to affect the physicochemical properties of EWG formed, such as rheology, texture and microstructure. Comparison of the pH and zeta potentials of UEWG and HEWG showed that heating accelerated the Maillard reaction to produce acidic compounds, causing the decrease in pH, as well as promoting the embedding of Ca2þ into the gel structure, decreasing the amount of positive charge on the surface. Meanwhile, the surface charge changing also affected the protein hydrophobic group distribution, thus affecting the surface hydrophobicity value of EWP. Our previous study, which used a combination of tea polyphenols and Ca (OH)2, revealed that the pH increased with increasing Ca(OH)2 con­ centration (Ai et al., 2019), but to a lesser extent than in the present study due to the acidity of tea polyphenols. This change may affect the physicochemical properties of EWG, for instance, the hardness of the gel with tea polyphenols was less than that of Ca(OH)2. Most hydrophobic groups at the surface of EWP are embedded inside the molecular structure in an alkaline environment (Croguennec, Renault, Beaufils, Dubois, & Pezennec, 2007). Since Ca(OH)2 is an al­ kali, the addition of Ca(OH)2 increases the alkalinity of the gel system and the surface hydrophobicity declines. Additionally, heating can destroy the forces that maintain the three-dimensional protein structure, such as hydrogen bonds, van der Waals forces and electrostatic in­ teractions (Davis & Williams, 2010); therefore, some hydrophobic groups supported by intermolecular forces were destroyed. Further­ more, protein aggregates closely after heating, causing some hydro­ phobic groups to be fixed inside the gel structure formed. Finally, salt ions compete with water molecules in binding to proteins, influencing the surface hydrophobicity (Arakawa, Bhat, & Timasheff, 1990); in addition, another possibility is that heating induced the high-speed vi­ bration of water molecules and Ca2þ, causing Ca2þ to compete with water molecules for binding to hydrophobic amino acid residues, and resulting in decreased surface hydrophobicity.

3.2. Total and free sulfhydryl contents Changes in SHT, SHF and SS contents of EWG due to Ca(OH)2 and heating are shown in Fig. 2. For UEWG, the SHT decreased significantly (P < 0.05) with increasing Ca(OH)2, and the SHF increased significantly (P < 0.05) in the range of 0.01–0.03% Ca(OH)2. For HEWG, the SHT, SHF contents and SS bonds increased obviously (P < 0.05); SHT, SHF and SS contents decreased significantly (P < 0.05) compared with UEWG after heating, indicating that SH oxidation was enhanced by heating. EWP has many SH and SS sources. For example, EW contains a large amount of cysteine, and ovalbumin is the only protein containing SHF content. Ovotransferrin, lysozyme and ovomucoid contain many SS bridges in their interiors (Mine, 1995). EWG rapidly underwent SH-SS transformation in a very short period of time under strong alkaline conditions with adding NaOH (Mine, 1996; Chen et al., 2015), and some proteins (such as ovotransferrin and lysozyme) are denatured under strongly alkaline. This rapid denaturation caused the molecular struc­ ture of the protein to expose and enhanced SS-SH exchanging rapidly, thus increasing the content of SHF. However, the SHT groups decreased obviously, which might be related to the protein oxidation of EWP. Cysteine is easily oxidized under alkaline conditions to form cystine via forming SS bonds (Dische & Zil, 1951). The conversion rate of SS-SH and the formation rate of SS are unbalanced, and as the proportion of Ca (OH)2 increased, the oxidation rate of cysteine decreased, and the rate of formation of SS bonds is lower than that of SS-SH conversion, resulting in a decrease in SS content. At the same time, the EWP was denatured under alkaline conditions to expose the SHF groups, leading to the reduction of SHF groups. The previous analysis indicated (Fig. 1A) that the addition of Ca(OH)2 to the EWG system promoted aggregation be­ tween proteins, indicating the increase of SHF groups improved the attractive interaction between EWP. However, heating seemed to change this pattern of changing. The changing tends in SHF groups and SS bonds after heating were completely opposite to those in the case of the UEWG, and the contents of SHT groups and SS bonds were much smaller than those in the UEWG. Based on this change, we speculated that the main reasons were as follows: Sulfur-containing amino acids are a class of amino acids that have a great influence on food flavor during heat treatment. During heating, sulfurized amino acids (such as methi­ onine, cysteine, cysteine), in addition to formation hydrogen sulfide, ammonia, acetaldehyde, cysteamine, etc., also produce thiazoles, 5

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Fig. 4. SDS-PAGE profile of egg white gel as affected by Ca(OH)2. M, marker; C, control group with adding 0.57% NaOH; F, fresh egg white protein.

thiophenes and many sulfur compounds, most of which are volatile and strong olfactory substances (Cavallini, Ricci, Federici, Costa, & Achilli, 1982; Sekhon, Schilling, Phillips, Aikins, & Mikel, 2010). It is the reason why a pungent smell exited for the HEWG after heated at 90 � C for 30 min. Heating improved the vibration of water and ion molecules, and protein aggregation enhanced, resulting in the increase of retained SHT, SHF groups and SS bonds. Matsudomi, Takahashi, and Miyata (2001) reported that dry heating exposed the SH groups inside a protein molecule on the surface. Also, Lee (1992) found that new SS bridge formed when protein samples were heated to 90–100 � C. These results were consistent with the present experimental results. The formation of covalent bonds between protein molecules significantly affected the gelling properties of protein gels (Totosaus, Montejano, Salazar, & Guerrero, 2002). Therefore, SS bonds might be the main supporting intermolecular forces for HEWG, affecting gelling properties such as texture and rheology.

structures results in the formation of a three-dimensional network (Berhe, Lawaetz, Engelsen, Hviid, & Lametsch, 2015). Therefore, EWG formed a tighter network structure after heating. Protein secondary structure was maintained by hydrogen bonds between the hydrogen atoms of amino acid residue amino groups (–NH–) and the carbonyl group oxygen atoms in peptide chains (Kabsch & Sander, 2010). Addi­ tionally, β-turns are common secondary protein structures that exist on the surface of globular proteins, with their specific conformation depending in part on the constituent amino acids (Hutchinson & Thornton, 2010). The addition of Ca(OH)2 changed the surface distri­ bution β-turns, while heating increased the vibration of the various protein functional groups, bringing the proteins closer together and increasing the amount of β-turn structure (Hutchinson & Thornton, 2010). All of these results suggested that the addition of Ca(OH)2 into EWG mainly affected the β-sheet and β-turn content, and heating influenced the random structure, α-helices and β-turns. However, the complex factors existed in secondary structure changes for EWP during alkali-denauration by adding NaOH and Ca(OH)2, thus more in­ vestigations were needed to explore the changing mechanism.

3.3. Secondary structure analysis Secondary protein structure is usually classified according to protein absorption in the amide I region (1700–1600 cm 1) with using FTIR. Peaks are assigned as follows: 1600–1640 cm 1 represents β-sheets; 1640-1650 cm 1 is random structure; 1650–1660 cm 1 is ɑ-helices; and, 1660-1700 cm 1 represents β-turns (Ulrichs, Drotleff, & Ternes, 2015). The secondary structural changes of EWG protein are shown in Fig. 3. For UEWG, β-sheets increased significantly (P < 0.05) and β-turns decreased significantly (P < 0.05); there was no significant change in random structure or α-helices (P > 0.05). For HEWG, β-sheets decreased significantly (P < 0.05), β-turns increased significantly (P < 0.05), and the random structure and α-helices changed slightly (P > 0.05). Com­ parison of UEWG and HEWG revealed that heating caused the propor­ tion of random structure and α-helices to decrease and β-turn content to increase. β-Sheet structures indicate strong hydrogen-bonding interactions, which are the main force that maintains the protein secondary structure (Levy-Moonshine, Amir, & Keasar, 2009). Therefore, the addition of Ca (OH)2 increased the hydrogen bonding between EWP for UEWG. How­ ever, the hydrogen bonds were destroyed after heating. Comparison with previous studies revealed that the addition of NaOH (0.1–0.4%) decreased the β-sheet content, but that β-sheets still formed more than 20% of all secondary structure, with a lower percentage of α-helical and random structures present (Li et al., 2018). This meant that the high concentration of alkali (0.57%) caused more serious damage to the secondary structure of EWP. It is known that a decrease in ɑ-helical

3.4. Electrophoresis The protein electrophoresis patterns (SDS-PAGE) of EWP under different concentrations of Ca(OH)2 are shown in Fig. 4. The molecular weight of ovomucin, ovotransferrin, ovalbumin, ovomucoid and lyso­ zyme is 110, 76, 44.5, 28 and 14.3 KDa, respectively (Kaewmanee, Benjakul, & Visessanguan, 2011; Mine, 1995). It has been reported that ovotransferrin is sensitive to extreme environments, especially under alkaline conditions, and that other proteins such as ovomucin and lysozyme are also degraded due to the decreased proportion of these proteins in EW following treatment (Chen et al., 2015; Su & Lin, 1998). In the present study, only ovalbumin and ovomucoid bands appeared for UEWG, with most protein bands showing degradation compared with fresh EWP. This result was also consistent with protein changes in traditional preserved EWG (Zhao et al., 2016). And the EWP, mainly ovalbumin and ovomucoid, became degration when the Ca(OH)2 con­ centration increased. For HEWG, all protein bands disappeared, indi­ cating that the proteins that were not denatured by alkali were thermally denatured at 90 � C. The changes to protein components at different Ca (OH)2 concentrations and due to heating might affect the properties of the resulting EWG.

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Fig. 5. Changes in textural properties, including hardness and springiness, of egg white gel as affected by Ca(OH)2. Un, unheated; He, heated. Different letters in the same index denote the significance difference (P < 0.05).

3.5. Textural properties

formation of inter-protein calcium bridges that increased the rigidity of the formed gel. The formed inter-protein calcium bridges immobilized the polypeptide segments and enhanced resistance to a certain amount of applied external force, showing greater hardness; but, when the external force exceeded a certain limit, as promoted by high concen­ trations of Ca2þ, the energy between polypeptide segments could not be transmitted through the calcium bridge sufficiently quickly, resulting in weaker resistance to larger strain. The HEWG possessed better hardness, which was attributed to the expansion of part of the protein conforma­ tion or the decomposition of part of the protein and the resulting enhancement of thermal aggregation of EWP (Chen et al., 2015; Li et al., 2018). Springiness reflects the fragmentation of gel blocks under the action of external forces. When the gel is broken into several large blocks under

For gels prepared from EW, textural quality is an important factor for selling and consumption. EWG textural properties of hardness and springiness are shown in Fig. 5. The hardness values of UEWG and HEWG increased significantly (P < 0.05) upon addition of Ca(OH)2, while the springiness values of UEWG and HEWG were significantly decreased (P < 0.05). The hardness value for HEWG was larger than that of UEWG, but the springiness value for HEWG was lower than for UEWG. Therefore, the alkali-induced gels formed in this study had excellent textural characteristics, especially HEWG, similar to the findings of previous studies (Handa, Takahashi, Kuroda, & Froning, 2010; Ai et al., 2019). Interestedly, the hardness values of EWG with the addition of a small amount of Ca(OH)2 increased markedly. This might be due to the

Fig. 6. Time sweep results of egg white gel with adding Ca(OH)2. 0.005–0.3, the concentration of Ca(OH)2. 7

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Table 1 Parameters for the logarithmic model used to fit the gelation profiles of egg white gelling system with adding Ca(OH)2. Rheological parameters

Gelling system

G0 c (Pa)

kgel

G -value

Control 0.005% 0.01% 0.015% 0.03%

634 634 634 634 634

214.27 362.23 659.93 872.26 995.47

0

C 1469.90 2461.90 4374.90 5683.90 6239.40

R2

tmodel (s)

Final G0 (Pa)

0.8467 0.8787 0.9060 0.9428 0.968

18378.19 5150.14 1978.41 1719.00 996.93

634 1060 2000 2590 3080

G0 c: Egg white gel with adding 0.57% NaOH without Ca(OH)2.

Fig. 7. Changes in frequency sweep and power-law model fitting results of egg white gel as affected by Ca(OH)2. A. frequency sweep of egg white gel; B, K0 and n of power-law fitting; Un, unheated; He, heated. Different letters in the same index denote the significance difference (P < 0.05).

the action of external forces, the springiness value is relatively large; in contrast, when the sample is broken into many small blocks, the springiness value is relatively small (Lau, Tang, & Paulson, 2000). The springiness decreased gradually with increasing Ca(OH)2 concentration, indicating that EWG was more easily broken into small pieces. This could be due to calcium ions immobilizing the protein, resulting in decreased EW ductility and lower springiness. Thermal modification changed the protein aggregation mode of EWP, increasing the ability to

resist external pressure, showing the short of the second compression time and the lower springiness values. 3.6. Rheological behavior 3.6.1. Gelation kinetics To study the influence of Ca(OH)2 on the gelation process of EW, the changes in the storage modulus (G0 ) and viscous modulus (G00 ) during 8

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Food Hydrocolloids 103 (2020) 105654

(OH)2 and tea polyphenols into alkali-induced EWG decreased the n0 value (Ai et al., 2019). That might be attributed to the limited effects of tea polyphenols (0.01–0.1%) on Ca(OH)2. These results suggested the covalent bonds became markedly weaker after adding Ca(OH)2, prob­ ably due to the strong ionic concentration and hydrogen-bonding formed. For HEWG, the K0 value increased significantly (P < 0.05) and n0 value, except in the range of 0.015–0.03% added Ca(OH)2, decreased significantly (P < 0.05). The alteration in the K0 value was consistent with changes in the hardness value. Li et al. (2018) reported that K0 and n0 values decreased gradually for formed EWG by the addition of NaOH with heat modification, indicating that a highly crosslinked covalent elastic gel network was formed. In contrast, the gel induced by Ca(OH)2 without heat modification tended to be a highly rigid gel owing to ovotransferrin unfolding and the increase in hydrogen bonds. The pre­ pared EWG after heating became a covalent, highly elastic gel, which agreed with the results reported by Li et al. (2018).

Table 2 Changes in color parameters of egg white gel with adding Ca(OH)2 and heating process. Process methods

Ca(OH)2 concentration

L*

a*

b*

△E*

Unheated

0.005

49.05 � 0.79a 48.59 � 0.82b 47.54 � 0.90c 47.19 � 0.86c 32.56 � 0.73d 32.51 � 0.42d 31.94 � 0.81e 31.06 � 0.37f 31.05 � 0.55f

0.29 � 0.02d 0.53 � 0.02d 0.75 � 0.03cd 0.90 � 0.04c 10.69 � 0.25b 11.07 � 0.22b 11.08 � 0.18b 12.09 � 0.32a 12.21 � 0.10a

7.60 � 0.14e 7.09 � 0.05f 6.70 � 0.08f 6.10 � 0.13g 15.47 � 0.31d 15.78 � 0.42d 18.48 � 0.78c 20.04 � 0.38b 21.04 � 0.59a

49.64 1.52a 49.12 1.06b 48.02 1.32c 47.59 1.40d 37.59 0.46g 37.79 0.86g 38.53 0.79f 38.89 0.97f 39.44 1.58e

0.01 0.015 0.03 Heated

Control 0.005 0.01 0.015 0.03

� � � � � � � �

3.7. Gel color and appearance

�

Color parameters, including L* (brightness), a* (redness) and b* (yellowness), of the EWG are shown in Table 2. For UEWG, brightness, redness, yellowness and △E were all significantly decreased (P < 0.05) as the amount of Ca(OH)2 content increased. For HEWG, brightness decreased significantly (P < 0.05) at concentrations of lower than 0.015% Ca(OH)2, but redness, yellowness and △E increased signifi­ cantly (P < 0.05). The HEWG showed lower brightness but more redness and yellowness than UEWG, and the △E for HEWG was lower than for UEWG. The EWG after the addition of calcium hydroxide forms a cal­ cium bridge due to the action of calcium ions, and the degree of protein aggregation is enhanced, resulting in a lower light trichrome effect and a decrease in the L* value. The degree of protein aggregation after heating is increased, and a protein molecule aggregate having a smaller surface area is formed, the light scattering effect is weakened, and the L* value is smaller. EW contains free reducing sugar, the aldehyde group of which can react with protein amino acid –NH2 groups to undergo the Maillard reaction, which is accelerated by heat. The first glycosylation product rearranges into a more stable ketoamine or Amadori product (Ganesan & Benjakul, 2010 and 2011), and the Amadori product can form crosslinks between adjacent proteins or other amino groups, resulting in a polymer known as the advanced glycosylation end product (Friedman, 1996). The brown polymer produced by these Maillard reactions showed a more enrichment effect during heating than at room temper­ ature, which also caused the HEWG to exhibit a brown color, showing the increase of a* and b* values. Furthermore, the addition of Ca(OH)2 increased the alkalinity of the gel system so that the a* and b* values of HEWG increased with increasing of Ca(OH)2 concentration by acceler­ ating the Maillard reaction. The UEWG showed a yellowish color, the a* showed a negative value, and the b* showed a positive value. However, the effect of Ca2þ enhanced the partial opacity of the gel color, and the Maillard reaction progressed slowly, so that the a* and b* values decreased as the Ca(OH)2 concentrations increased. Digital photographs of the EWG formed with different Ca(OH)2 ra­ tios are shown in Fig. 8A and B. Previous research revealed that, when the aggregation rate of the EWP or polypeptide was less than the protein denaturation rate, the gel formed was relatively regular and translucent (Totosaus et al., 2002). The prepared gel in this study was translucent or transparent, indicating the rapid protein denaturation of EWG as affected by Ca(OH)2 and 0.57% NaOH. The HEWG appeared more red or brown than UEWG, which was more yellow; these findings were consistent with changes in color parameters.

*Different letters indicate significant difference (p < 0.05).

the gelation process were explored by time sweep to evaluate the ki­ netics and mechanical stability of gelation. The results are shown in Fig. 6. All gel systems showed typical curves of gel modulus change, and G0 was larger than G00 , indicating that the gel was forming. The gelation rate (Kgel) has been widely used to explore the modulus change during gelation. As shown in Table 1, Kgel was proportional to the addition amount of Ca(OH)2. This might be related to the bonding properties of Ca2þ and alkalinity of Ca(OH)2, resulting in the formation of calcium bridges and acceleration of the accumulation of protein ag­ gregates. The final formed EWG showed an increase in hardness (Fig. 5), which was also related to the formation of the gel network structure and was consistent with the changes in G0 and G00 . All gel systems had little time to reach the Tmodel, indicating that the addition of Ca(OH)2 pro­ moted the formation of the gel network. Compared with the G0 C (Table 1), the addition of Ca(OH)2 improved the final G0 -value (Table 1) of EWG, demonstrating that the formed EWG had superior mechanical properties, such as the hardness value (Fig. 5). Addition of calcium ions to other raw materials, such as sodium alginate, pectin or k-carrageenan can also promote the formation of gel (Funami et al., 2009; Han et al., 2017; Liu & Li, 2016), but the mechanisms are different in terms of the physical and chemical interactions. 3.6.2. Frequency sweep The storage modulus (G0 ) reflects the ability to store energy under a certain strain or stress and represents the elastic part of the gel (Mon­ tesinos-Herrero, Cottell, O’Riordan, & O’Sullivan, 2006). The G0 changes of EWG as affected by different concentrations of Ca(OH)2 are shown in Fig. 7A. The gel exhibited a gradual increase in G0 -vaule when higher ratios of Ca(OH)2 were added. The G0 -vaule for HEWG appeared to be larger than for the UEWG, which was closely related to the inter­ action between the EWP upon heating. There was a high-frequency correlation between G0 -vaule and the angular frequency, indicating that the gel structure was enhanced after the addition of Ca(OH)2; this was consistent with the changes in textural properties (Fig. 5). The results of frequency sweep were simulated using a power-law model, resulting in high-coefficient degree of fitting (R2 > 0.978). The K0 value was used to reflect the rigidity of the formed gel, and the n0 value represented the covalent bonds with lower values of n0 repre­ senting a higher proportion of covalent bonds in the formed gel (Razi et al., 2018; Wang et al., 2015). The K0 and n0 values are shown in Fig. 7B. For UEWG, there was a non-significant increase in K0 upon addition of 0.01–0.15% Ca(OH)2, and the n0 value increased signifi­ cantly (P < 0.05). Our previous report revealed that the addition of Ca

3.8. SEM and schematic model The microstructural changes of the EWG observed using SEM are shown in Fig. 8C and D. It has been reported that proteins at high ionic concentrations aggregate to form granular chain-like microstructures 9

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Food Hydrocolloids 103 (2020) 105654

Fig. 8. Surface morphology features, microstructural properties (3000 � f) and proposed scheme of egg white gel as influenced by Ca(OH)2. A and B, egg white gel surface morphology; C, egg white microstructure with heating modification; D, egg white microstructure without heating modification. E, proposed a scheme for duck egg white gelation as influenced by Ca(OH)2 and heating.

(Handa et al., 2010). Preserved EWG exhibited a looser network struc­ ture when Ca2þ was added into the pickling solution (Ganesen & Ben­ jakul, 2011). Interestingly, in this study, a partially granular and irregular structure formed under the influence of Ca(OH)2. This was likely due to the formation of salt bridges between Ca2þ and the protein, resulting in a coarser and more hierarchical microstructure. The microstructure of UEWG tended to be rough and irregular, and more cavity structure existed, whereas the gel microstructure for HEWG was closer and more compact. These results agreed with those reported by Duan, Zhao, and Chi (2018). A proposed explanation for these microstructures is as follows. At alkaline pH, repulsive electrostatic forces between protein molecules and aggregation of linear molecules dominate, leading to the formation of a uniform network (Uquillas & Akkus, 2012). Therefore, the addition of Ca(OH)2 enhanced the electrostatic interactions between protein molecules. Upon heating, the protein molecules vibrated and further aggregated into a dense structure; therefore, heat modification improved the formation of a uniform network. It was these changes in protein microstructure that led to the changes in physical characteristics (hardness, springiness, G0 and G00 ) of EWG; a compact and regular

microstructure was responsible for the improved physical characteristics of the protein gel. The proposed formation mechanism of EWG upon the addition of Ca (OH)2 is reflected in Fig. 8E. After adding the mixed solution of Ca(OH)2 and NaOH to EWP, the pH increased, resulting in the embedding and destruction of hydrophobic groups and decrease in surface hydropho­ bicity. With the increase of positive ions on the protein surface, elec­ trostatic repulsion decreased and hydrogen bond interactions improved. Simultaneously, the alkali-denatured EWP began to unfold and rear­ rangement of cross-linking occurred. The ionic effects of Ca2þ and the formation of calcium bridges not only promoted the cross-linking of proteins but also caused the formation of gel with a rough and irregular microstructure and enhanced textural characteristics (hardness). Heat­ ing promoted the vibration of water molecules in the gel and accelerated the physicochemical interactions, damaging the formed hydrogen bonds and changing the distribution of hydrophobic groups on the protein surface. Concurrently, heating accelerated the conversion of SH groups to SS bonds, thus promoting the further aggregation between EWP and resulting in the compact microstructure and greater hardness of HEWG than UEWG. 10

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4. Conclusion

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This paper reports the gelling properties of alkali-induced EWG formation with the addition of Ca(OH)2. Surprisingly, the addition of Ca (OH)2 significantly improved the mechanical and rheological properties of the EWG, including the gradual increase of hardness, G0 and G00 . Heating further enhanced the represented physical properties. Addition of Ca(OH)2 accelerated the formation of the gel network structure, resulting in the embedding of hydrophobic groups to decrease surface hydrophobicity, as well as the formation of more inter-protein hydrogen bonds, increase in SHF and decline in SHT. However, the microstructure was rough and non-regular. Heating decreased the surface hydropho­ bicity and SHT/SHF content compared with unheated group, leading to a more compact and regular microstructure. Further studies are needed to investigate the EWG formation mechanism in greater detail. Declaration of competing interest There are no conflicts of interest to declare. CRediT authorship contribution statement Minmin Ai: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Quan Zhou: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Nan Xiao: Conceptualization, Methodology, Writing - original draft, Writing - review & editing. Shanguang Guo: Software. Yuanyuan Cao: Visu­ alization, Investigation. Hong Fan: Visualization, Investigation. Ziting Ling: Visualization, Investigation. Ledan Zhou: Visualization, Investi­ gation. Shuchang Li: Visualization, Investigation. Jiaoli Long: Visual­ ization, Investigation. Aimin Jiang: Supervision. Acknowledgement The author thanked all those involved in the design and operation of the experiment and thanked the laboratory for the financial support: Guangdong province livestock and poultry products processing tech­ nology engineering research center construction (2014B090904075); International training plan for excellent young scientific research talents in Guangdong Province (2019YQGP_BS008); The National Center for Precision Machining and Safety of Livestock and Poultry Products Joint Engineering Research Center, China. And thanked for grammar and spell-checking help from my best friend Wenkai Zhou. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2020.105654. References Ai, M., Guo, S., Zhou, Q., Wu, W., & Jiang, A. (2018). The investigation of the changes in physicochemical, texture and rheological characteristics of salted duck egg yolk during salting. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 88, 119–125. Ai, M., Tang, T., Zhou, L., Ling, Z., Guo, S., & Jiang, A. (2019). Effects of different proteases on the emulsifying capacity, rheological and structure characteristics of preserved egg white hydrolysates. Food Hydrocolloids, 87, 933–942. Ai, M., Zhou, Q., Guo, S., Ling, Z., Zhou, L., Fan, H., et al. (2019). Effects of tea polyphenol and Ca(OH)2 on the intermolecular forces and mechanical, rheological, and microstructural characteristics of duck egg white gel. Food Hydrocolloids, 94, 11–19. Arakawa, T., Bhat, R., & Timasheff, S. N. (1990). Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry, 29(7), 1924–1931. Berhe, D. T., Lawaetz, A. J., Engelsen, S. B., Hviid, M. S., & Lametsch, R. (2015). Accurate determination of endpoint temperature of cooked meat after storage by Raman spectroscopy and chemometrics. Food Control, 52, 119–125. Beveridge, T., & Arntfield, S. (1979). Heat induced changes in sulfhydryl levels in egg white. Canadian Institute of Food Science and Technology Journal, 12(4), 173–176.

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