Effects of ascorbic acid and sugars on solubility, thermal, and mechanical properties of egg white protein gels

International Journal of Biological Macromolecules 62 (2013) 397–404

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Effects of ascorbic acid and sugars on solubility, thermal, and mechanical properties of egg white protein gels Abdorreza Mohammadi Nafchi a,∗ , Ramin H. Tabatabaei b , Bita Pashania a , Hadiseh Z. Rajabi b , A.A. Karim c a

Food Biopolymer Research Group, Department of Food Science and Technology, Islamic Azad University, Damghan Branch, Damghan, Semanan, Iran Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Beheshti Avenue, Gorgan 49138-15739, Iran c Food Biopolymer Research Group, Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia b

a r t i c l e

i n f o

Article history: Received 16 August 2013 Received in revised form 20 September 2013 Accepted 27 September 2013 Available online 4 October 2013 Keywords: Egg white protein Maillard reaction Crosslinking

a b s t r a c t The effects of reducing sugars (fructose, glucose, ribose, and arabinose), sucrose, and ascorbic acid were studied on thermo-mechanical properties and crosslinking of egg white proteins (EWP) through Maillard reaction. Sugars (0%, 1%, 5%, and 10%) and ascorbic acid (0%, 0.25%, 0.5%, and 2.5%) were added to EWP solutions. Thermal denaturation and crosslinking of EWP were characterized by differential scanning calorimetry (DSC). Mechanical properties (failure strength, failure strain and Young’s modulus) of modified and unmodified EWP gels were evaluated by texture analyzer. Ascorbic acid decreased thermal denaturation temperature of EWP, but the reducing sugars increased the denaturation temperature. DSC thermograms of EWP showed that ascorbic acid exhibited an exothermic transition (≈110 ◦ C) which was attributed to Maillard crosslinking of the protein. The reduction in pH (from 7.21 to ≈6) and protein solubility of egg white protein gel (from ≈70% to ≈10%) provides further evidence of the formation of Maillard cross-linking. Reactive sugars (ribose and arabinose) increased the mechanical properties of EWP gels, whereas ascorbic acid decreased the mechanical properties. Generally, the effect of ascorbic acid was more pronounced than that of various reducing sugars on the thermal and mechanical properties of egg white proteins. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Protein gelation can be defined as a protein aggregation phenomenon in which polymer–polymer and polymer–solvent interactions are so balanced that a tertiary or three-dimensional network is formed [1]. This network can immobilize or entrap a large amount of water in addition to other food components. In addition to effects of the size, shape and arrangement of the primary protein strands comprising the gel network, the characteristics of protein gels are affected by intra- and inter-strand cross-linking [2]. The covalent bond formation between polypeptide chains or proteins, leads to the intra- or inter-molecular crosslinking in protein gels. Hydrophobic interactions, hydrogen bonds, and disulfide bonds also play an important role in crosslinking and stabilizing the structure of those proteins gels [3,4]. In addition to these types of bonds, the occurrence of the Maillard cross-link (within a protein gel in the presence of reducing

∗ Corresponding author. Tel.: +98 232 522 5045; fax: +98 232 522 5039. E-mail addresses: [email protected], [email protected] (A. Mohammadi Nafchi). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.09.050

sugars could be partly responsible for enhanced protein gelation [5,6]. It is known that the incubation of proteins with sugars often results in a loss of solubility, and it has been suggested that this is due to the polymerization of protein molecules by covalent crosslinks being formed as a result of the Maillard reaction [7]. On the other hand sugars can act to stabilize proteins by increasing the onset temperature of heat denaturation and altering bond formation during gelation [8], consequently effects on gel strength [9]. The Maillard reaction can also cause a pH reduction due to the production of acidic side products [10]. Other than reducing sugars, l-ascorbic acid has also been reported to undergo a Maillard-type reaction. Ascorbic acid is commonly used in food products and is notable for its potential to be involved in both Maillard reactions and free radical cycles [11]. Farahnaky et al. [11] also observed that storing gelatin powders containing ascorbic acid at 80 ◦ C for 6 h at 50% relative humidity resulted in decreased protein solubility and increased molecular weight. The changes were attributed to Maillard cross-linking. There are about 40 different proteins in egg white, which are a dietary source of various amino acids and nitrogen that are required for growth, maintenance, and the general well-being of humans. Egg proteins have a high nutritional value, and they also have

398

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

valuable functional properties that make them as useful ingredient in food manufacturing such as noodles, mayonnaise, cakes, and candies [12,13]. For example, the irreversible, heat-induced coagulation or gelation of egg proteins often controls the success of certain cooked food products. By understanding the mechanisms of EWP gelation and stabilization of the EWP, greater utilization of egg white protein ingredients in foods can be obtained. To our knowledge, thermomechanical properties of cross-linked EWP through Maillard reaction and impact of sugars and ascorbic acid has not been studied. Therefore, the objectives of the present study were to examine the effects of selected sugars and ascorbic acid on Maillard cross-linking in egg white protein with respect to the thermal and mechanical properties of egg white protein gels. Also the indirect evidences of protein cross-linking, (pH changes and protein solubility) were evaluated.

Compression measurements were performed with the TA.XT2i Texture Analyzer (Stable Micro Systems, Godalming, Surrey, UK) with a 5.0 kg load cell according to the method of Medina-Torres et al. [15]. The samples were placed between two rigid parallel plates. The upper plate moved downwards at a crosshead speed of 0.5 mm s−1 and the axial force was recorded as a function of time. To obtain fracture stress and Young’s modulus, displacement and axial force data were transformed to stress and Hencky strain [16,17]. The gels were compressed up to a deformation of 70%. For each test, eight gels were tested. The engineering stress ( eng = force/original cross-sectional area) and engineering strain (εeng = reduction of length/original length) curves were obtained. Young’s modulus was calculated as the slope line drawn at the initial linear zone in the stress–strain curve [15]. 2.5. pH measurements

2. Materials and methods 2.1. Materials Sucrose, d-glucose, d-fructose, d-arabinose, d-ribose, and lascorbic acid were purchased from R&M Chemicals (Essex, UK). All other chemicals were of analytical grade. 2.2. EWP preparation Egg white separated from fresh hen eggs and then dialyzed against deionized water (18 ␮) for 2 days at 4 ◦ C. The dialyzed egg white was freeze-dried, and stored at −18 ◦ C before use [14]. 2.3. Thermal properties Egg white protein solutions were prepared in phosphate buffer (pH 7.0) at 10% (w/v). 0% (control), 1%, 5%, and 10% (w/v) concentration of sugars and ascorbic acid were used. All samples were stirred thoroughly at room temperature for 30 min to obtain homogenous mixtures. The differential scanning calorimetry (DSC) analysis was performed using a modulated differential scanning calorimeter (DSC-Q100, TA Instruments, Newcastle, DE, USA) with refrigerator cooler. Calibration of heat flow, temperature, and heat capacity was done using pure indium and sapphire. An empty aluminum pan was used as a reference to balance the heat capacity of the sample pan. The samples (10–20 mg each) were analyzed in modulation mode (MDSC). After equilibration at 40 ◦ C, samples were heated at 2 ◦ C min−1 , (modulated at 60 s and amplitude 0.318 ◦ C) up to 140 ◦ C. All samples were analyzed in triplicate. Onset (To ) and peak (TP ) temperatures and overall enthalpy (Hd , J/g) associated with denaturation and cross-linking were analyzed and determined with Universal Analysis 2000 software (TA Instruments, New Castle, USA). 2.4. Mechanical properties of EWP gels Egg white protein gels were prepared by mixing 5.0% (w/v) egg white proteins with sugars and/or ascorbic acid at different concentrations. The concentration of egg white was chosen based on preliminary experiments for a convenient handling of the gel. The sugar concentration used were 0% (control), 1%, and 5% (w/v) and the concentration of ascorbic acid used were 0% (control), 0.25%, 0.5%, and 2.5% (w/v). The mixtures were poured into universal bottles and heated in a water bath at 80 ◦ C for 5 min to ensure uniform gel formation, then heated at 120 ◦ C in the oven for 1 h. After the gels were cooled to room temperature, a 15-mm-diameter cork borer was used to remove the gels from the bottles. The gels then were cut into 10-mm height cylinders.

EWP gels were freeze-dried using a Labconco freeze dryer (Missouri, USA). The dried samples (0.5%, w/v) dissolved in deionized water (triplicated). The pH of the solution was determined using a Cyberscan 2500 pH meter (Eutech Instruments, Singapore). 2.6. Protein solubility Freeze-dried EWP samples (0.1 g) were dispersed in 10 ml of sodium dodecyl sulphate plus 1% ␤-mercaptoethanol solvent. The dispersions were shaken in an orbital shaker at 37 ◦ C for 14 h. The dispersions were then centrifuged at 2330 × g for 15 min and filtered through a Whatman No. 4 filter paper to remove undissolved materials. Lowry’s method [18] based on folin phenol reagent was used for estimation of soluble protein content in the supernatant after 2-fold dilutions were made. The protein solubility was done on triplicate samples. 2.7. Statistical analysis ANOVA and Tukey’s Post Hoc tests were used to compare the effect of sugars and ascorbic acid concentrations on mechanical properties, thermal properties, protein solubility, and pH, of EWP gels at the 5% significance level. Statistical analysis was conducted using IBM SPSS21.0 for windows (SPSS Inc. Chicago, IL) or GraphPad Prism 5 (GraphPad Software Inc., La Jolla, USA). 3. Results and discussions 3.1. pH of egg white protein gels Table 1 shows the pH of EWP gels containing different concentrations of sugars and ascorbic acid. By increasing the concentration of reducing sugars or ascorbic acid, the pH of the EWP gels slightly (but significantly) decreased. These results were similar to those reported by other researchers for other protein systems [10]. The reduction in pH of the system could be attributed to the formation of acidic by-products (such as organic acid) and the loss of protons from the ␧-amino groups during the Maillard reaction [19]. The Maillard reaction is expected to occur when egg white proteins are heated with chemical agents containing carbonyl groups. Watanabe et al. [20] also reported that the Maillard reaction would cause a pH decline in the gelling system via the breakdown of sugars to organic acid. On the other hand, the addition of sucrose into the egg white proteins did not influence the pH value of the system. As is generally known, sucrose is not a reducing sugar and the Maillard reaction may not occur in the presence of this non-reducing sugar. The observed decrease in pH of egg white proteins in the presence of ascorbic acid (Table 1) might be partly due to the acidification of ascorbic acid. According to Liao and Seib [21], the

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

399

Table 1 Effect of sugars and ascorbic acid at different concentrations on the pHa of egg white protein gels. Concentration of sugar (w/v) 0% (control) Sucrose Ribose Arabinose Fructose Glucose

7.21a 7.21a 7.21a 7.21a 7.21a

± ± ± ± ±

0.5%

0.06 0.06 0.06 0.06 0.06

7.19a 6.80b 6.85b 6.99b 7.07b

2.5% ± ± ± ± ±

0.06 0.06 0.03 0.01 0.04

7.19a 6.28c 6.48c 6.71c 6.85c

5.0% ± ± ± ± ±

0.01 0.03 0.01 0.01 0.04

7.16a 5.95d 6.35d 6.54d 6.68d

± ± ± ± ±

0.00 0.07 0.01 0.04 0.02

Concentration of ascorbic acid (w/v)

Ascorbic acid a

0% (control)

0.25%

0.5%

2.5%

7.21a ± 0.06

6.87b ± 0.00

6.52c ± 0.01

4.71d ± 0.01

Values are mean ± standard deviation (n = 3). Means related to a particular compound with different letters are significantly different at p ≤ 0.05.

l-ascorbic acid radical (first oxidation product formed from lascorbic acid) is a strong acid with a pKa of 0.45. Therefore, the addition of ascorbic acid might also influence the pH value of egg white proteins. 3.2. Protein solubility

Fig. 1. The effect of various sugars at different concentrations on the solubility of egg white protein gels in the mixed 1% (v/v) ␤-mercaptoethanol and 1% (w/v) sodium dodecyl sulphate solvent.

3.2.1. Effects of sugars Fig. 1 shows the effect of various sugars on the solubility of egg white protein gels in the 1% (v/v) ␤-mercaptoethanol + 1% (w/v) sodium dodecyl sulphate solvent. According to Mitchell and Hill [19], the Maillard gels had a low solubility in solvents that disrupted disulfide bridges, hydrophobic interactions and hydrogen bonds. It is evident that a significant decrease in solubility occurred in all gels (Fig. 1). These data indicate that non-disulfide covalent cross-links were present in the gels because ␤-mercaptoethanol reduces disulfide linkages and sodium dodecyl sulphate disrupts non-covalent interactions. Thus, the formation of covalent links (Maillard cross-linkages) likely occurred during

Table 2 Effects of sugars and ascorbic acid on onset (To ) and peak temperatures (TP ) as well as enthalpy (Hd ) associated with denaturation of ovalbumin and ovotransferrin in egg white during programmed heating in the DSC.a Compound

Ovalbumin ◦

Ovotransferrin

To ( C)

TP ( C)

Hd (J/g)

To (◦ C)

TP (◦ C)

Hd (J/g)

Sucrose 0% (control) 1% 5% 10%

76.94a 77.08a 77.47b 78.65c

82.29a 82.48a 83.09b 83.77c

0.58a 0.64a 0.62a 0.65a

63.76a 64.14b 64.74c 65.13d

67.66a 67.46a 68.11b 68.76c

0.19a 0.14a 0.09a 0.16a

Ribose 0% (control) 1% 5% 10%

76.94a 77.50b 77.60bc 77.80c

82.29a 82.67b 82.77bc 82.98c

0.58a 0.61a 0.60a 0.61a

63.76a 64.23b 64.34b 64.37b

67.66ab 67.66ab 67.56a 67.92b

0.19a 0.09b 0.09ab 0.12ab

Fructose 0% (control) 1% 5% 10%

76.94a 77.15a 78.19b 78.68c

82.29a 82.59b 83.35c 84.04d

0.58a 0.63a 0.63a 0.60a

63.76a 63.86a 64.90b 65.72c

67.66a 67.56a 68.14b 68.88c

0.19a 0.13a 0.10a 0.08a

Glucose 0% (control) 1% 5% 10%

76.94a 77.69b 78.45c 79.34d

82.29a 82.61a 83.59b 84.44c

0.58a 0.69a 0.66a 0.61a

63.76a 63.94a 64.94b 65.56c

67.66a 67.92b 68.50c 68.97d

0.19a 0.15a 0.12a 0.11a

Ascorbic acid 0% (control) 1% 5% 10%

76.94a 75.61b 64.46c 60.55d

82.29a 80.95a 70.75b 67.80c

0.58a 0.53ab 0.49ab 0.41b

63.76a 62.20b nd nd

67.66a 66.61b nd nd

0.19a 0.08a nd nd

a

◦

Values are mean ± SD (n = 3). Means within a column related to a particular chemical agent with different letters are significantly different at p ≤ 0.05.

400

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

Fig. 2. The effect of ascorbic acid concentrations on the solubility of egg white protein gels in the mixed 1% (v/v) ␤-mercaptoethanol and 1% (w/v) sodium dodecyl sulphate solvent.

the heating process used to make the gels. The results of this study in agreement with those reported previously by other researchers [10,20,22]. According to Kato et al. [23], the protein–sugar complex produced in the early Maillard reaction exhibits specific physicochemical properties, such as high solubility and heat stability. However, such protein properties result in poor solubility, browning, and a reduction in nutritional availability in the advanced stage of the reaction. The egg white protein gels containing 0.5% (w/v) arabinose, or ribose exhibited lower solubility than those of other sugars (Fig. 1). This suggests that these sugars were much more reactive in the Maillard reaction compared to the others. Rizzi [24] reported that pentoses (ribose, xylose, arabinose) are more reactive than hexoses (glucose, galactose, fructose) in the Maillard reaction. Such a substantial difference in the reaction rate among sugars might be explained by the stereochemistry of each sugar, and this has been discussed by Kato et al. [23]. The addition of sucrose (a non-reducing sugar) into the egg white protein gels unexpectedly decreased the solubility of the gel in the mixed solvent, although to a lesser extent compared to the reducing sugars (Fig. 1). These results indicate that the high temperature (120 ◦ C) used during the preparation of the gel might have hydrolyzed the glycosidic bonds of sucrose and released its constituent monosaccharides to facilitate the Maillard reaction. According to Mottram [25], the role of sucrose in the Maillard reaction is less clear-cut compared to reducing sugars. At temperatures below 37 ◦ C, food systems containing sucrose appear to be stable for long periods of storage; however, at higher temperatures the glycosidic bonds of sucrose may be hydrolyzed, thereby releasing the constituent monosaccharide and allowing the Maillard reaction to proceed in the normal way [25]. Thus, a higher temperature is needed with sucrose to obtain the same degree of reaction as can be obtained under lower temperatures with glucose. On the other hand, the EWP gels containing sucrose did not turn into brown (data not shown); this result suggests that in these gels only the early stage of the Maillard reaction occurred and the reaction products were mainly present as the colorless, non-volatile Amadori compound. Browning occurs and flavor compounds are formed mainly from reducing sugars and proteins as a result of the advanced Maillard reaction [24,25]. 3.2.2. Effects of ascorbic acid Fig. 2 shows the effect of ascorbic acid on the solubility of EWP gels in the mixed 1% (v/v) ␤-mercaptoethanol + 1% (w/v) sodium dodecyl sulphate solvent. The solubility of the gels exhibited a significant decreasing trend when the concentration of ascorbic acid

Fig. 3. The effect of ascorbic acid on the transition temperatures associated with the denaturation and cross-linking of protein in egg white during heating in the differential scanning calorimeter. Two major endothermic transitions were due to the denaturation of ovotransferrin (a) and the denaturation of ovalbumin (b). An exothermic transition is shown at 5% and 10% ascorbic acid (c, d).

increased from 0.25% to 2.5% (w/v). Only ∼14.8% of the EWP in the gels became soluble in the solvent even at a very low concentration of ascorbic acid (2.5%, w/v). The large insoluble fraction must represent a matrix held together by non-disulfide covalent bonds presumably formed as a consequence of the Maillard reaction. The amine groups of EWP gels are expected to react with the carbonyl groups of ascorbic acid in this reaction to form additional Maillard cross-linkages within the EWP gel network. According to Fayle et al. [26], ascorbic acid can cross-link proteins at 37 ◦ C by mechanisms that do not involve disulfide bonding. The mechanism is thought to involve the Maillard reaction.

3.3. Thermal properties of EWP 3.3.1. Effects of sugars Ma and Harwalkar [27] reported that thermal denaturation of proteins involves conformational changes from the native structure and the disruption of chemical forces that maintain the structural integrity of the protein molecules. The ruptures of hydrogen bonds during the denaturation of proteins are endothermic, whereas the breakup of hydrophobic interactions and aggregations are exothermic. Fig. 3 shows the DSC curve of the EWP (control) during programmed heating in the DSC. For EWP containing sugars, the DSC curves for different sugars at different concentrations were qualitatively very similar to the control, and therefore they are not shown because they almost overlapped. Data in Table 2 show the effect of sugars on the transition temperatures and enthalpy (Hd ) associated with denaturation of protein in egg white during programmed heating in the DSC. At a heating rate of 5 ◦ C min−1 from 30 to 120 ◦ C, EWPs at pH 7 exhibited two major endothermic transitions (shown by arrows in Fig. 3): The first was due to the denaturation of ovotransferrin at ∼67.7 ◦ C and the second was due to the denaturation of ovalbumin at ∼82.3 ◦ C. These results are similar to those reported by other researchers [28,29]. In general, a slight but significant increase in onset (To ) and peak (TP ) transition temperatures was associated to an increase in the protein stability of ovalbumin and ovotransferrin when the sugar concentration was increased (Table 2). These results are consistent with those reported earlier [30].

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

Both reducing sugars (glucose, ribose) and the non-reducing sugar (sucrose) exhibited the same effect on the thermal denaturation temperatures of EWPs. The increase of thermal denaturation temperature of EWPs containing sugars suggests that sugars may stabilize proteins against thermal denaturation. Back et al. [31] suggested that sugars form specific cooperative bonding with groups on the outside of the protein molecule, thereby displacing water and reducing the amount of water molecules that compete for hydrogen bonds in the interior of the protein molecules; therefore, the protein structures are stabilized. Hydrophobic interactions generally are considered to be the major factor responsible for stabilizing the three-dimensional structure of proteins. The effects of sugars on hydrophobic interactions, and consequently on the thermal stability of proteins, depend on how they affect the three-dimensional hydrogen-bonded structure of water. Levy and Onuchic [32] noted that sugars are able to influence the structure of water, decrease the surface hydrophobicity of protein molecules, and indirectly strengthen the internal hydrophobic interaction in protein. Back et al. [31] also showed that hydrophobic interactions between pairs of hydrophobic groups are stronger in sucrose or glycerol solutions than in pure water. Hence, thermal stabilization of protein denaturation is greatly due to the effects of sugars on hydrophobic interactions. Wright [33] suggested that the enthalpy of denaturation is largely attributable to the heat of rupture of intra-chain hydrogen bonds. The results in Table 2 show no significant differences in enthalpy of denaturation (Hd ) when various sugars were heated with EWPs in the DSC. Donovan et al. [28] reported similar findings. These results suggest that there were no major changes in protein conformation as a result of the stabilization of sugars. 3.3.2. Effects of ascorbic acid 3.3.2.1. Protein denaturation. In contrast to sugar, when EWPs were heated with ascorbic acid in the DSC, a significant decrease in thermal denaturation temperature occurred. Table 2 shows that the onset (To ) and peak (TP ) temperatures of ovalbumin and ovotransferrin decreased significantly as the concentration of ascorbic acid increased. A similar decreasing trend was observed for the Hd of ovalbumin when the concentration of ascorbic acid was increased. On the other hand, ovotransferrin did not exhibit any significant difference in the Hd value with increasing concentration of ascorbic acid. In fact, the thermal denaturation transitions of ovotransferrin were not detectable when the concentration of ascorbic acid exceeded 5% (w/v). These results suggest that ascorbic acid might promote the thermal denaturation of EWPs. Lapanje [34] noted that both low and high pH values far removed from the isoelectric point favor protein denaturation. In this experiment, the inclusion of ascorbic acid at high concentration (exceeding 5%, w/v) might have decreased the pH value of protein so that it was far from its isoelectric point, thereby resulting in partial or complete protein unfolding. Nishimura et al. [35,36] also proposed that free radicals are produced during the oxidation of ascorbic acid. These free radicals, especially • O2 , cleave peptide bonds, change the conformation, and increase the surface hydrophobicity of protein molecules. Similar studies by Howell [37] have shown that ascorbic acid increases the surface and exposed hydrophobicity of proteins by enhancing protein unfolding. Thus, in this study ascorbic acid likely enhanced the unfolding of the protein molecules and subsequently decreased the thermal denaturation transition temperature of proteins. 3.3.2.2. Protein crosslinking. Fig. 3 shows the DSC thermogram of EWPs containing ascorbic acid at different concentrations during heating. No exothermic transition appeared when <1.0% (w/v) ascorbic acid was added into EWPs, but an exothermic transition was evident (shown by arrows – c and d – in Fig. 3) over a broad

401

range of temperature (90–140 ◦ C) when EWPs were heated with 5% and 10% (w/v) ascorbic acid. The transition was quite large and it may represent the heat released as EWPs were cross-linked in the presence of ascorbic acid. It is well known that the cross-linking reaction is exothermic. The cross-linking in proteins is similar to the curing reaction in synthetic polymers; as the polymer is cured, energy is released and there is an exothermic transition in the thermal profile. As reported by Back et al. [31], the browning reaction of protein amino groups results in an exothermic heat change. This reaction occurs above the denaturation transition of proteins (possibly being governed by the exposure of amino groups on denaturation). The exothermic transition observed in Fig. 3 is not likely to be due to the aggregation of protein (disulphide bonds and hydrophobic interactions), even though the aggregation of protein also is an exothermic reaction. This is because the aggregation of proteins always occurs in the temperature range of the denaturation transition; the kinetic of this reaction is low relative to denaturation, and it always is concealed by the denaturation transition. According to Wright [33], the initial unfolding of proteins is accompanied by a significant uptake of heat, seen as an endothermic peak in the DSC thermogram. The subsequent aggregation reactions are exothermic in nature, and if they occur in the temperature range of the denaturation transition it can complicate the determination of the denaturation enthalpy. In contrast, an exothermic transition was not observed when EWPs were heated with sugars, probably because reducing sugars are not as reactive as ascorbic acid. Therefore, the kinetic of the reaction might be slow and undetectable by DSC. At high temperature, ascorbic acid can undergo a Maillard type reaction with protein. As proposed by Feather [38], ascorbic acid serves as a very active participant in Maillard reactions. At pH 7.0 and 37 ◦ C in the presence of oxygen, ascorbic acid is very unstable and may well decompose to yield carbonyl-containing compounds such as threose. Threose can interact with amine groups of protein to produce Maillard reaction products. These additional covalent cross-linkages would increase with increasing reaction time and temperature [39]. 3.4. Mechanical properties of EWP gels 3.4.1. Influences of sugars Fig. 4 shows the influence of different sugars on the (a) failure stress, (b) failure strain, and (c) Young’s modulus of EWP gels when heated at 120 ◦ C for 1 h. Failure stress of EWP gels significantly increased (p < 0.05) by increasing the concentration of reducing sugars. Failure stress increased markedly at lower concentration of sugars and there is no significant changes observed when the concentration of sugars reached ∼2.5% (w/v). The reducing sugars (glucose, fructose, arabinose, and ribose) clearly promoted the gelation of EWP gels, as indicated by the increasing value of failure stress. It would be expected that the formation of additional Maillard cross-links in EWP gel networks would affect the rupture and viscoelastic properties of the gel. Thus, the inclusion of reducing sugars into EWP gels might strengthen the gel via the formation of new covalent bonds in addition to hydrogen and disulphide bonds. These results are similar to those reported previously for other protein gels [10,40]. EWP gels contained ribose and arabinose were showed the higher failure stress than other sugars; suggesting that these sugars were much more reactive in the Maillard reaction compared to the other sugars. Mitchell and Hill [19] reported that more reactive the reducing sugars, the stronger the gel. In contrast, the inclusion of sucrose into the EWP gels did not cause any significant difference (p > 0.05) on failure stress. The fact that these gels did not appear brown, in contrast to the reducing sugar containing gels, suggests the absence of advanced Maillard reactions that promote the formation of Maillard cross-linking.

402

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

of syneresis when the concentrations of these two reactive sugars were increased. At lower concentrations of these sugars, the EWP gels showed a superior water holding capability. This may be due to the reduction in the number of positively charged amino groups (Maillard reaction occur by modifying the ␧-amino group of lysine) present, resulting in a lowering of the isoelectric point of the EWPs. Armstrong et al. [10] reported that gels have reduced water-holding capability as the isoelectric pH is approached. However, as the concentration of ribose and arabinose were increased in this study, more Maillard cross-linking occurred and the acidic by-products produced during this reaction were expected to decrease the pH value of the gels. Therefore, when the pH value of the gels was close to its isoelectric point, syneresis was expected to occur. The loss of water in these Maillard cross-linked gels might decrease the failure strain because the presence of water molecules is important in the formation of a deformable gel. A similar study by Armstrong et al. [10] revealed that bovine serum albumin gels without xylose and glucono-␦-lactone showed no syneresis, but the glucono-␦-lactone gels lost a lot of water during the reaction. The inclusion of two types of reactive sugars (ribose and arabinose) resulted in a significantly increasing trend of Young’s modulus (Fig. 4c). Gunasekaran and Mehmet [41] noted that the firmer the gels, the higher the force or stress needed to cause a given deformation or strain. In this study, a large Young’s modulus value likely was due to the increasing degree of covalent bonding within the gel network as a result of the formation of additional Maillard-induced cross-links, and hence resulted in a stiffer gel. The addition of other reducing sugars (glucose, fructose) and the non-reducing sugar (sucrose) did not have a significant effect on Young’s modulus of EWP gels. These results suggest that these reducing sugars were less reactive in the Maillard reaction in comparison to arabinose and ribose. For sucrose, Maillard cross-linking was not expected to occur and therefore no changes in Young’s modulus were recorded.

Fig. 4. The influence of different sugars at different concentrations on the (a) failure stress, (b) failure strain, and (c) Young’s modulus of egg white protein gels when heated at 120 ◦ C for 1 h.

The failure strain (Fig. 4b) of the gels increased significantly for reducing sugars, except sucrose (the non-reducing sugar). The inclusion of different sugars in EWP gels was expected to yield a more deformable gel. The increase in failure strain suggests that Maillard cross-links formed in the EWP gel network. The EWP gels with 0.5% (w/v) of ribose and arabinose had a higher failure strain than other sugars at the same concentration. However, when the concentration of these sugars was increased to 5.0% (w/v), the failure strain of the EWP gels began to decrease (i.e., they became less deformable), possibly because the gels exhibited a greater level

3.4.2. Influences of ascorbic acid Fig. 5 shows the effect of ascorbic acid on the (a) failure stress, (b) failure strain, and (c) Young’s modulus of EWP gels when heated at 120 ◦ C for 1 h. Low concentrations of ascorbic acid (0.25%, 0.50% (w/v)) had no significant effects on failure stress of EWP gels, but when the concentration increased to 2.5% (w/v) a drastic increase in failure stress (up to 4.7 kPa) occurred. This observation suggests that the Maillard cross-linking that occurs between ascorbic acid and EWPs increased the degree of covalent bonding within the gel network and strengthened the texture of the gel. The failure strain of EWP gels increased significantly as the concentration of ascorbic acid increased. This result suggests that more deformable EWP gels were formed due to the formation of additional Maillard cross-links (in addition to disulphide bonds) within the gel networks. Thus, higher failure strain values for Maillard gels might represent additional cross-linking of the protein network. In contrast to gels containing reducing sugars, a significant decrease in Young’s modulus occurred when the concentration of ascorbic acid was increased from 0 to 2.5% (w/v), especially to 0.5% (w/v). The reduction in the firmness or rigidity of the EWP gels as the concentration of ascorbic acid increased occurred presumably because of syneresis. Kitabatake et al. [42] reported that the reduction in density of the gel network would result less elastic of heat-induced ovalbumin gels. Therefore, the loss of water in EWP gels as the concentration of ascorbic acid increases might reduce the density of the gel network and consequently decrease Young’s modulus values. The presence of ascorbic acid presumably decreases the pH of EWPs. When the pH of EWP approaches the isoelectric point, the proteins tend to aggregate and the protein–protein interaction is more pronounced than the

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

403

4. Conclusion The results of this study show that reducing sugars and ascorbic acid influence the thermal and mechanical properties of EWPs. The sugars increased the thermal denaturation temperature of EWPs through their ability to stabilize the native EWP structures. Conversely, ascorbic acid decreased the thermal denaturation temperature of EWPs, probably by altering their conformational structures or pH value. The mechanical properties of EWP gels containing reducing sugars and ascorbic acid changed to a varying degree. In the presence of reactive reducing sugars (arabinose and ribose), more rigid gels were formed upon heating, possibly due to the formation of Maillard cross-linkages in addition to disulphide bonds within the EWP gel network. DSC thermograms of EWPs containing ascorbic acid exhibited an exothermic transition, which might be attributable to the crosslinking of EWPs via the Maillard reaction. Reduced pH and solubility indicated the formation of Maillard cross-linkages in these systems. References

Fig. 5. The influence of ascorbic acid at different concentrations on the (a) failure stress, (b) failure strain, and (c) Young’s modulus of egg white protein gels when heated at 120 ◦ C for 1 h.

protein–solvent interaction [40,43]. Therefore, the EWP gels that form under these conditions might have a lower water-holding capacity and show a tendency for syneresis. Young and Baldwin [44] reported that changes in the structure of EWP molecules from the fluid (sol) to the solid or semisolid (gel) state might be brought about by the addition of acid. According to Van Kleef [40], gels formed near the protein isoelectric point generally have highly aggregated structures. These highly aggregated structures are opaque, less homogenous, and generally show a tendency for syneresis.

[1] S. Raccosta, M. Manno, D. Bulone, D. Giacomazza, V. Militello, V. Martorana, P. Biagio, European Biophysics Journal 39 (2010) 1007–1017. [2] R.H. Schmidt, in: J.P. Cherry (Ed.), Protein Functionality in Foods, American Chemical Society, 1981, pp. 131–147. [3] J.A. Gerrard, Trends in Food Science & Technology 13 (2002) 391–399. [4] C.M. Bryant, D.J. McClements, Trends in Food Science & Technology 9 (1998) 143–151. [5] G.R. Ziegler, E.A. Foegeding, in: E.K. John (Ed.), Advances in Food and Nutrition Research, Academic Press, 1990, pp. 203–298. [6] A.M. Easa, H.J. Armstrong, J.R. Mitchell, S.E. Hill, S.E. Harding, A.J. Taylor, International Journal of Biological Macromolecules 18 (1996) 297–301. [7] J. Buchert, D. Ercili Cura, H. Ma, C. Gasparetti, E. Monogioudi, G. Faccio, M. Mattinen, H. Boer, R. Partanen, E. Selinheimo, R. Lantto, K. Kruus, Annual Review of Food Science and Technology 1 (2010) 113–138. [8] J.M. Garrett, R.A. Stairs, R.G. Annett, Journal of Dairy Science 71 (1988) 10–16. [9] T. Arakawa, S.N. Timasheff, Biochemistry 21 (1982) 6536–6544. [10] H.J. Armstrong, S.E. Hill, P. Schrooyen, J.R. Mitchell, Journal of Texture Studies 25 (1994) 285–298. [11] A. Farahnaky, D.A. Gray, J.R. Mitchell, S.E. Hill, Food Hydrocolloids 17 (2003) 321–326. [12] M. Yoshinori, R. Prithy, in: Y.i. Mine, F. Shahidi (Eds.), Nutraceutical Proteins and Peptides in Health and Disease, CRC Press, 2005, pp. 445–459. [13] Y. Mine, Trends in Food Science & Technology 6 (1995) 225–232. [14] L.-T. Lim, Y. Mine, M.A. Tung, Journal of Agricultural and Food Chemistry 46 (1998) 4022–4029. [15] L. Medina-Torres, F. Calderas, J. Gallegos-Infante, R.-F. Gonzalez-Laredo, N. Rocha-Guzman, F. Harte, Food and Bioprocess Technology 3 (2010) 150–154. [16] L. Medina-Torres, E. Brito-De La Fuente, B. Torrestiana-Sanchez, S. Alonso, Carbohydrate Polymers 52 (2003) 143–150. [17] J. Tang, M.A. Tung, Y. Zeng, Carbohydrate Polymers 29 (1996) 11–16. [18] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Journal of Biological Chemistry 193 (1951) 265–275. [19] J.R. Mitchell, S.E. Hill, Trends in Food Science & Technology 6 (1995) 219–224. [20] K. Watanabe, Y. Sato, Y. Kato, Journal of Food Processing and Preservation 3 (1980) 263–274. [21] M.-L. Liao, P.A. Seib, Food Chemistry 30 (1988) 289–312. [22] S. Benjakul, W. Visessanguan, Food Research International 36 (2003) 253–266. [23] Y. Kato, T. Matsuda, N. Kato, K. Watanabe, R. Nakamura, Journal of Agricultural and Food Chemistry 34 (1986) 351–355. [24] P.R. George, Controlling Maillard Pathways To Generate Flavors, American Chemical Society, 2010, pp. 121–128. [25] S. Mottram Donald, Thermally Generated Flavors, American Chemical Society, 1993, pp. 104–126. [26] S.E. Fayle, J.A. Gerrard, L. Simmons, S.J. Meade, E.A. Reid, A.C. Johnston, Food Chemistry 70 (2000) 193–198. [27] C.Y. Ma, V.R. Harwalkar, Journal of Food Science 53 (1988) 531–534. [28] J.W. Donovan, C.J. Mapes, J.G. Davis, J.A. Garibaldi, Journal of the Science of Food and Agriculture 26 (1975) 73–83. [29] P.-O. Hegg, H. Martens, B. Löfqvist, Journal of the Science of Food and Agriculture 30 (1979) 981–993. [30] A. Kulmyrzaev, C. Bryant, D.J. McClements, Journal of Agricultural and Food Chemistry 48 (2000) 1593–1597. [31] J.F. Back, D. Oakenfull, M.B. Smith, Biochemistry 18 (1979) 5191–5196.

404

A. Mohammadi Nafchi et al. / International Journal of Biological Macromolecules 62 (2013) 397–404

[32] Y. Levy, J.N. Onuchic, Annual Review of Biophysics and Biomolecular Structure 35 (2006) 389–415. [33] D.J. Wright, in: H.W.S. Chan (Ed.), Biophysical Methods in Food Research, Blackwell Scientific Publications, London, 1984, pp. 1–36. [34] S. Lapanje, in: S. Lapanje (Ed.), Physicochemical Aspects of Protein Denaturation, John Wiley & Sons, Inc., Mississauga, Canada, 1978. [35] K. Nishimura, M. Ohtsuru, K. Nigota, Journal of Agricultural and Food Chemistry 37 (1989) 1539–1543. [36] K. Nishimura, M. Ohtsuru, K. Nigota, Journal of Agricultural and Food Chemistry 37 (1989) 1544–1547. [37] N.K. Howell, in: A.G. Gaonkar (Ed.), Ingredient Interactions. Effects on Food Quality, Marcel Dekker, Inc., New York, 1995.

[38] M.S. Feather, in: T.H. Parliment, M.J. Morello, R.J. McGorrin (Eds.), Thermally Generated Flavours. Maillard, Microwave, and Extrusion Process, American Chemical Society, Washington, D.C., 1994, pp. 127–141. [39] J.A. Gerrard, P.K. Brown, International Congress Series 1245 (2002) 211–215. [40] F.S.M. Van Kleef, Biopolymers 25 (1986) 31–59. [41] M. Mehmet, S. Gunasekaran, Cheese Rheology and Texture, CRC Press, 2002. [42] N. Kitabatake, Y. Tani, E. Doi, Journal of Food Science 54 (1989) 1632–1638. [43] M.A. Blanco, E. Sahin, Y. Li, C.J. Roberts, Journal of Chemical Physics 134 (2011) 225103. [44] S.C. Young, R.E. Baldwin, in: W.J. Stadelman, O.J. Cotterill (Eds.), Egg Science and Technology, 4th ed., The Haworth Press, Binghamton, NY, 1995, pp. 405–464.