Original papers
Effects of Lanthionine and Lysinoalanine on Heat-induced Gelation of Egg White
2020 年 26 巻 6 号 p. 789-795
詳細
2020 年 26 巻 6 号 p. 789-795
Egg white gelation is caused by protein unfolding and aggregation. Intermolecular disulfide bonds have been reported to contribute significantly to the formation and strengthening of gel networks. The covalent crosslinks lanthionine and lysinoalanine, which are collectively known as isopeptide bonds, are also generated in heated egg white proteins. However, little is known regarding the relationship between the isopeptide bonds and the gel properties. Here, we investigated the effect of isopeptide bonds produced during the heating of egg white on the protein aggregation and gel texture. Egg white proteins formed isopeptide bonds, and the number of the bonds increased with increasing temperature. These were mainly composed of lanthionine, which were easily formed in ovotransferrin and lysozyme. A significant correlation coefficient of 0.99 was found between the gel hardness and lanthionine content. These results suggested that lanthionine hardens the egg white gel through strengthening intermolecular crosslinks, resulting in network fortification.
Egg white is widely used around the world due to its various properties, including foaming, emulsification, and heat-induced gelation (Mine, 1995). Gelation is applied to consumer products such as meat and surimi products (Park, 1994; Reppond et al., 1995). The egg white, mainly composed of water and approximately 10.5% (w/w) of protein, has a pH of 7.8–8.0 when it is laid, and a pH of 9.3–9.5 after 10 days (Jin et al., 2011; Silversides and Budgell, 2004). Heat-induced gelation allows the protein to form a 3-dimensional network structure retaining large amounts of water. Above a certain protein concentration, a network is formed by protein denaturation and subsequent intermolecular interactions (Ma and Holme, 1982; Nakamura et al., 1984).
The main proteins in egg white are ovalbumin (OA; 54% of dry matter), ovotransferrin (OT; 12–13%), ovomucoid (11%), lysozyme (LY; 3.4–3.5%), G2 and G3 ovoglobulins (OG; 2%), and ovomucin (1.5–3%) (Mine, 2015). Various studies have reported the protein aggregation temperature in low-concentration solutions. When a dilute egg white solution (protein concentration below 2% (w/w)) at pH 9.0–9.5 is heated, OA coagulates at approximately 75 °C while other proteins aggregate at 55–65 °C (Iwashita et al., 2019; Matsuda et al., 1981; Mine et al., 1990). Few studies have focused on the thermal behavior of the proteins during gelation, because applicable analytical techniques are limited.
Generally, the intermolecular interactions involved in egg white gelation include hydrophobic bonds, ionic bonds, hydrogen bonds, and covalent bonds, such as disulfide (SS) bonds (Alleoni, 2006). Several studies have estimated that the SS bond is essential in gelation and gel hardening based on the thermal changes in free sulfhydryl (SH) content and the band intensity in electropherograms (Mine et al., 1990; Plancken et al., 2005). In addition, the protein solubility in 8 M urea with 5 M 2-mercaptoethanol solution is markedly reduced by heating at 93 °C for 40 min (Beveridge et al., 1980). It is presumed that some covalent bonds other than SS may also contribute to gel formation.
Lanthionine (LAN) and lysinoalanine (LAL) bonds, the covalent bonds called isopeptide (IP) bonds, were found in heat-treated egg white (Germs, 1973; Beveridge et al., 1974; Hasegawa et al., 1987; Zhao et al., 2016). LAN and LAL bonds can be formed via dehydroalanine, which is converted from free SH, SS bonds or serine due to β-elimination, followed by Michael addition with cysteine or lysine, respectively, especially at an alkaline pH and high temperature (Friedman, 1999). Many researchers have reported that IP bonds are also detected in protein-rich ingredients, such as soybeans, wheat, and milk with heating or alkaline treatment (Beveridge et al., 1974; Rombouts et al., 2011; Klostermeyer and Reimerdes, 1977).
Given the above, it is estimated that IP bonds also affect the gel-forming process and the texture of egg white. However, not much is known regarding the effect of IP bonds on heat-induced gelation. Hence, in this study, we attempted to assess the relationship between IP bonds and the physical properties of heat-induced egg white gel.
Reagents Urea, disodium hydrogen phosphate, and acetonitrile (LC/MS grade) were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Cysteic acid and LAN were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). LAL was purchased from Bachem Holding AG (Bubendorf, Switzerland). The other reagents were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan).
Sample preparation Hen eggs were purchased from a local market and used within 3 days of laying. The egg white was separated and blended for 30 s by a high-power homogenizer (Hirosawa Tekkojo, Tokyo, Japan). The pH of the egg white was adjusted to 9.0 with 0.1 M HCl using a pH meter (D-72 LAQUA; HORIBA, Ltd., Kyoto, Japan). The protein content was adjusted to 105 mg/mL (w/v), measured for its absorbance at 280 nm and centrifuged at 5 000 × g for 15 min to remove insoluble matter. The egg white solution was heated at 50–100 °C at intervals of 5 °C for 30 min and immediately cooled to 20 °C using a thermal cycler (Verti; Thermo Fisher Scientific, Waltham, MA, USA).
Texture measurement The hardness of the heated samples was analyzed by a Tensipresser My Boy II system (Taketomo Denki, Osaka, Japan) equipped with a cylindrical plunger (diameter, 2 mm) at a test speed of 3 mm/s. In this analysis, the hardness of the gel was calculated as the compressive stress for 15% deformation.
Scanning electron microscopy (SEM) The microstructure of the gel was observed using SEM. Gel samples were cut from the center of each gel and fixed overnight at room temperature in 2.5% glutaraldehyde. The sample was rinsed thrice in 0.1 M sodium phosphate buffer (pH 7.0). Samples were dehydrated successively in 50–99.5% ethanol and then replaced by t-butanol (Inoue and Osatake, 1988). The samples were then freeze-dried, osmium-coated with HPC-1SW (Vaccume Device Co., Ibaraki, Japan), and observed by SEM (S-4800; Hitachi, Ltd., Tokyo, Japan) at 5 kV.
Soluble protein measurement The heated samples were homogenized by a multibeads shocker (MB1001C; Yasui Kikai Co., Osaka, Japan) with two zirconia beads (diameter 5.0 mm, Yasui Kikai Co.). Then, the samples were suspended in 50 mM Tris-HCl buffer (pH 8.0), and in 50 mM Tris-HCl buffer (pH 8.0) containing 2% (w/v) sodium dodecyl sulfate (SDS), 3 M urea, and 1 mM ethylenediaminetetraacetic acid (EDTA) in the presence or absence of 50 mM dithiothreitol (DTT). The suspensions were diluted 100 times and centrifuged at 16 000 × g for 15 min at room temperature. Thereafter, the absorbance at 280 nm of the supernatant was measured.
Quantitation of free SH groups Free SH groups were measured using the method of Ellman (1958) with slight modifications. Briefly, the heated samples were freeze-dried and shaken for 60 min in 50 mM sodium phosphate buffer (pH 6.5) containing 2% (w/v) SDS, 3 M urea, and 1 mM EDTA. Then, 1 mg/mL 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reagent was added and the samples were shaken for 15 min. After centrifugation at 11 000 × g for 5 min, the absorbance was measured at 412 nm. L-cysteine was used as a standard.
Quantitation of total SH groups and IP bonds The total SH groups (sum of free and SS-bonds forming SH) and IP bonds were liberated from freeze dried proteins in a vacuumed condition for 24 h at 110 °C in 6 M HCl. The samples were dried with a centrifugal concentrator (CVE-3100; EYELA, Tokyo, Japan), dissolved in 0.1 M HCl, filtered with a 0.22-µm nylon filter (TORAST Disc; Shimadzu GLC, Tokyo, Japan) and then analyzed by the liquid chromatography/quadrupole-time of flight measurements (LC/Q-TOF; Infinity 1260 and 6530 Accurate-Mass, Agilent Technologies, Santa Clara, CA, USA). The separation was performed using an Intrada Amino Acid column (100 × 2 mm, 2.7 µm, Imtakt Co., Kyoto, Japan). The mobile phase of acetonitrile with 0.1% formic acid (A) and 100 mM aqueous ammonium formate (B) were employed. Initial mobile phase conditions were 86% A and 14% B, held for 3 min; then B was increased to 100% over 7 min, and maintained for 5 min; B was reduced to 14% over 0.1 min and maintained for 2.5 min. The flow rate was 0.4 mL/min and the column temperature was maintained at 35 °C. Q-TOF was performed using the following conditions: positive ion mode, nebulizer gas 50 psig, drying gas 6 L/min, gas temperature 300 °C, fragmentor 175 V, scan range m/z 74–350. For total cysteine quantitation, cysteine and cystine residues were oxidized to one and two cysteic acid residues, respectively, prior to acid hydrolysis. Performic acid was added to the freeze-dried samples. The mixture was stirred and then left at 4 °C for 16 h. After that, the performic acid was removed with a centrifugal concentrator and the samples were subjected to acid hydrolysis LC/Q-TOF with the negative ion mode. The total SH was calculated as the cysteic acid content.
SDS-polyacrylamide gel electrophoresis (PAGE) SDS-PAGE of heated egg white was performed following the Laemmli method (1970) with minor modifications. Sample suspensions in 50 mM Tris-HCl buffer (pH 8.0), 2% (w/v) SDS, 3 M urea, and 1 mM EDTA in the presence or absence of 50 mM DTT were prepared with the same method described for the soluble protein measurement. The suspensions were mixed with a half volume of 60.6 mM Tris-HCl (pH 6.8) buffer containing 60% glycerol, 3% SDS, and 0.01% bromophenol blue with or without 50 mM DTT and subjected to SDS-PAGE using a 5–20% gradient gel (e-pagel; ATTO Co., Tokyo, Japan) with a molecular weight marker (XL-ladder broad; AproScience Co., Tokushima, Japan). The band detection was performed by One Step CBB (Bio Craft Co., Tokyo, Japan).
Data analysis All results are expressed as an average of three replicates of individual experiments and the data were analyzed using Statcel 4 (OMS Publishing Inc., Tokyo, Japan). Error bars are expressed as the standard deviation of the mean (n = 3).
Heat-induced gelation of egg white Egg white solutions heated at 50–100 °C were observed (Fig. 1). The clear solutions became white and turbid when heated above 60 °C. Sol-like structures formed at 60–70 °C and gelation was observed when heated above 75 °C. Compressive stress measurements of the egg white gel were performed by Tensipressor My Boy II (Fig. 2). The gel showed minimal hardness at 75 °C, and the hardness doubled at 100 °C. The gel hardness increased as a function of temperature; this trend was in agreement with previous reports (Beveridge et al., 1980; Gossett et al., 1984). The gel microstructure was observed by SEM to analyze the relationship between gel hardness and the network structure (Fig. 3). Larger units of the gel were observed when heated at 75 °C, while smaller units were commonly observed above 80 °C. According to previous results (Doi, 1993; Handa et al., 1998), the gel is considered to be harder when the units are smaller and uniformly distributed. Thus, the differences in the texture when heated at 75 °C and at higher temperatures are in agreement with this rule. It is presumed that the increase in gel hardness when heated over 80 °C is caused by reasons other than gel network structures.
Heat induced aggregation and gelation of egg white. Figures show the heating temperature of each sample.
The firmness of heat-induced egg white gel.
The hardness of the gel was measured as a stress with 15% deformation. Data are shown as mean ± SD (n = 15). The different letters indicate significant difference (p < 0.05).
Microstructure of gel network when heated at (A) 75 °C, (B), 80 °C, (C) 90 °C, and (D) 100 °C, observed by scanning electron microscopy S-4800. Scale bar, 1 µm.
Soluble protein content The soluble protein content of the heated samples was measured (Fig. 4). The protein content in Tris-HCl buffers (pH 8.0) with DTT was higher than that in other solutions at 20 °C, which was possibly due to the reduction and solubilization of ovomucin (Omana and Wu, 2009). In the reducing condition, the absorbance by oxidized DTT may also affect the soluble content measurement. The soluble protein content was greatly decreased when heated at 55–85 °C in Tris-HCl buffers (pH 8.0) without DTT. In contrast, the protein content in Tris-HCl buffers (pH 8.0) with DTT was slightly decreased when heated at 70–85 °C. These results indicated that aggregations by non-covalent crosslinks and SS bond formation occurred at 55–85 °C, that more SS bonds contributed to the aggregation as the temperature rose, and that covalent crosslinks other than SS bonds were also formed when heated over 70 °C.
Soluble protein content of heated egg white
Soluble protein content in 0.1 M Tris-HCl buffer (square), 0.1 M Tris-HCl buffer containing 3 M Urea, 2% SDS and 1 mM EDTA with (circle) or without (triangle) 50 mM DTT were determined.
Amino acid content The thermal behavior of free SH groups was determined by Ellman's method (Table 1). The free SH level showed a decreasing tendency when heated over 60 °C; the free SH content decreased by 68.2% upon incubation at 100 °C. This result was in agreement with previous studies performed using various protein concentrations (Mine et al., 1990; Plancken et al., 2005). Total SH and serine content measurements were performed by LC/Q-TOF. The total SH content tended to decrease when heated above 75 °C. The serine content was slightly attenuated when heated over 85 °C. When heated at 100 °C, the total SH and serine residues decreased by 58 and 12 µmol/g, respectively, when compared to those in the unheated samples. These results were similar to the trend observed with bovine serum albumin and gliadin heated to 120 °C (Rombouts et al., 2016).
| Temp. [°C] | Free SH | Total SH | Serine | LAN | LAL |
|---|---|---|---|---|---|
| 20 | 50.7±0.8 | 204±3 | 544±4 | 0±0 | 0±0 |
| 50 | 49.7±0.5 | 202±2 | 536±5 | 0±0 | 0±0 |
| 55 | 49.9±0.7 | 207±5 | 547±6 | 0±0 | 0±0 |
| 60 | 48.2±0.9 | 204±3 | 546±5 | 0±0 | 0±0 |
| 65 | 45.8±1.1 | 204±5 | 550±5 | 0.5±0.2 | 0±0 |
| 70 | 38.1±0.5 | 207±3 | 542±2 | 4.8±1.0 | 0±0 |
| 75 | 25.4±0.7 | 200±3 | 550±6 | 7.7±1.6 | 0±0 |
| 80 | 24.0±1.0 | 199±3 | 538±7 | 12.8±1.6 | 0±0 |
| 85 | 18.9±0.1 | 191±2 | 545±6 | 18.2±2.6 | 0.2±0.1 |
| 90 | 16.7±0.2 | 175±1 | 548±5 | 26.6±4.7 | 0.4±0.2 |
| 95 | 16.1±0.4 | 173±1 | 527±9 | 35.4±4.1 | 1.2±0.6 |
| 100 | 16.1±0.4 | 146±5 | 532±3 | 41.8±5.1 | 3.2±0.4 |
Total SH, serine, and isopeptide bonds liberated from egg white proteins were analyzed. Each content is shown as µmol/g protein.
Quantitation of IP bonds IP content was analyzed by LC/Q-TOF after hydrolysis of the samples (Table 1). LAN and LAL bonds were detected when heated at 65 °C and 85 °C, respectively, and they increased with increasing heating temperature. The LAN bond content of the heated egg white was 13 times higher than the LAL bond content when heated to 100 °C, which corresponded to 40.3% of the total SH in the unheated samples. LAN bonds were the major IP in the heated egg white proteins at all temperatures tested. Germs (1973) reported the detection of 10 µmol/g of LAN bonds by thin layer chromatography in egg white boiled for 20 min. It has also been reported that 5.14–6.00 µmol/g of LAL bonds were observed in egg white boiled for 30 min by HPLC or GC/MS (Hasegawa et al., 1987). The ratio of LAN and LAL bonds in the present study was equivalent to that in the bovine serum albumin and gliadin described in recent research (Rombouts et al., 2016). Positive correlation coefficients of 0.99 and 0.82 were found between the gel hardness and the LAN and LAL bond contents, respectively (Table 2).
| Free SH | SS bond | LAN | LAL | |
|---|---|---|---|---|
| Gel hardness | −0.924 | −0.782 | 0.990 | 0.823 |
SDS-PAGE SDS-PAGE was performed under reducing and non-reducing conditions to assess the heat-induced aggregation of egg white proteins (Fig. 4). The intensity ratio of protein bands was calculated from the electropherogram obtained by SDS-PAGE under the non-reducing condition (Fig. 5). Under this condition, a decrease in the intensity suggested aggregation via covalent crosslinks, such as SS or IP bonds. OT and OG proteins were aggregated at 65 °C, and LY at 60 °C. The main protein, OA, was greatly aggregated at 75–80 °C. For all proteins, the monomers aggregated and the bands disappeared completely at higher temperatures. The protein aggregation temperature was slightly lower in this study than in previous studies (Matsuda et al., 1981; Mine et al., 1990), likely due to differences in the heat treatment condition.
SDS-PAGE patterns under reducing or non-reducing conditions. Protein aggregation was analyzed by SDS-PAGE under (A) non-reducing and (B) reducing conditions. OT, ovotransferrin; OG, ovoglobulins; OA, ovalbumin; LY, lysozyme.
Under the reducing condition, the band did not decay as much as it did under the non-reducing condition. The band intensity of OA was least affected by heating, and that of OT was highly affected when compared to the other proteins. The OT band intensity was greatly attenuated at 60 °C, and disappeared at 95 °C. The band intensities of LY, OG, and OA were attenuated to 77%, 44%, and 26%, respectively, by the heat treatment at 100 °C. It was reported that dehydroalanine is more easily formed from SS bonds than from free SH as a result of using oxidized and reduced glutathione, respectively (Finley et al., 1982). OA contains four free SH groups and one SS bond, and OT and LY have 15 and four SS bonds, respectively, and no SH groups (Alleoni, 2006). These reports suggested that not only the amount of total SH groups, but also the ratio of free SH groups and SS bonds contributes to the differences in LAN bond formation for each protein species.
Changes in relative intensity of the bands under (A) non-reducing and (B) reducing conditions shown in Fig. 3. The figure shows the bands of OT (open circle), OG (open triangle), OA (closed circle), and LY (open square). OT, ovotransferrin; OG, ovoglobulins; OA, ovalbumin; LY, lysozyme.
Relationship between IP bonds and gel texture In the present study, we assessed the relationship between IP and heat-induced egg white gel properties. Egg white gelation occurred when heated at 75 °C, and the egg white became harder with increasing temperature. Both the LAN and LAL contents showed a positive correlation with the gel hardness. It was reported that ε-(γ-glutamyl)-lysine (GL) bonds, which are covalent bonds, are formed between glutamine and lysine residues in the amino acid sequence of proteins by transglutaminase activity (Kornguth and Waelsch, 1963). Several studies have reported that intermolecular GL bonds fortify the hardness of processed protein ingredients made from meat, milk, and soy (Domagała et al., 2016; Li et al., 2018; Wang et al., 2019). In addition, it was reported that egg white gel becomes harder through the formation of 0.2 µmol/g GL crosslinks due to the transglutaminase reaction (Sakamoto et al., 1994). Furthermore, several lines of evidence indicate that LAN bonds contribute to heat stability and irreversible structural changes (Suda et al., 2010). Therefore, it is presumed that IP bonds contribute to stronger intermolecular crosslinks in egg white proteins, and consequently to a harder gel, and that OT is mainly involved in the IP bonds.
In this study, we assessed the relationship between IP and heat-induced egg white gel properties. The main conclusions of this study are as follows:
(1) The hardness of the egg white gel increased as the temperature rose. There were no differences in the gel network when heated above 80 °C.
(2) IP bonds were generated above 65 °C, and they increased with increasing heating temperature. Among them, LAN bonds were predominantly generated, and they showed a significant correlation coefficient with gel hardness.
(3) IP bonds were most easily formed in OT.
These results suggest that the gel hardness increases with increasing heating temperature, because IP bonds, mainly LAN bonds, fortify the intermolecular crosslinks. Conventionally, it has been theorized that intermolecular interactions and SS bonds contribute to the formation of egg white gel. In the present study, we propose the importance of LAN bonds between protein molecules in the heat-induced gel formation of egg white.
Acknowledgements This work was supported by the doctoral program research support system of Tokyo University of Agriculture. The authors thank Ms. S. Shindoh from Tokyo University of Agriculture for her help in the experiments.
