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Formation mechanism of low-density lipoprotein gel induced by NaCl
Formation mechanism of low-density lipoprotein gel induced by NaCl Lilan Xu,∗ Yan Zhao,†,‡,1 Mingsheng Xu,∗ Xuliang Nie,∗ Na Wu,∗ and Yonggang Tu∗,1 ∗
Jiangxi Key Laboratory of Natural Products and Functional Food, Jiangxi Agricultural University, Nanchang 330045, China; † Engineering Research Center of Biomass Conversion, Ministry of Education, Nanchang University, Nanchang 330047, China; and ‡ Jiangxi Shenzhu Tianyuan Food Co., Ltd., Nanchang University, Nanchang 330219, China
ABSTRACT Salted eggs, which are a traditional Chinese egg product, are favored by Chinese consumers and have become very popular in other Asian countries due to their unique features such as “fresh, fine, tender, loose, gritty and oily texture.” In order to illuminate the forming process of salted egg, the gelation behavior and mechanism of low-density lipoprotein (LDL) induced by NaCl were investigated using marinated model outside the eggshell. Results showed that as the salting proceeded, the moisture content exhibited a decreasing trend. The NaCl content, oil exudation, hardness, and surface hydrophobicity showed constantly increasing trends. In the early stages of salting, the size of the LDL particles, soluble protein content, and T21 increased, whereas T21 (with D2 O), T22 , and the free sulfhydryl content declined. In the later stages
of salting, LDL formed a multiple composite aggregate gel structure with filamentous apoproteins and nonspherical lipid particles intertwined with each other. The soluble protein content and T23 (without D2 O) decreased, whereas T21 (with D2 O), T22 and the free sulfhydryl content increased. Fourier transform infrared spectroscopy revealed that the fresh LDL mainly consisted of α-helix and β -sheet structures. After the gel becomes hardened, the LDL secondary structure was changed remarkably, characterized by the decrease of α-helix elements and increase of β -sheet elements. The results suggested that the oil exudation of salted LDL gels was mainly due to LDL destruction and the release of components (apoproteins, phospholipids, and neutral lipids) facilitated by increased interactions between apoproteins and lipids.
Key words: LDL gel, NaCl-induced, microstructure, intermolecular forces 2019 Poultry Science 98:5166–5176 http://dx.doi.org/10.3382/ps/pez232
INTRODUCTION Salted eggs, which are a famous traditional preserved egg product originally created in China, are mainly pickled with high concentration of salt at room temperature for 4 to 5 wk (Lai et al., 1999). The production of outstanding features (loose, gritty, and oily) of salted egg yolks requires the treatment of fresh duck eggs with a high salt concentration. However, the salt contents in the maturated salted egg whites reach 7 to 10% after salting (Xu et al., 2017). Recently, several studies have emphasized that egg yolks are separated from egg whites before bringing to reduce the salt in the egg whites (Woodward and Cotterill, 2010; Singh and Ramaswamy, 2013). Despite the benefits of a shorter salting cycle and retaining protein integrity, these salted egg yolks do not achieve good quality. A major reason is that the processes and mechanisms involved in the mat-
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2019 Poultry Science Association Inc. Received October 19, 2018. Accepted April 22, 2019. 1 Corresponding authors: [email protected] (TY); zhaoyan@ ncu.edu.cn (ZY)
uration of salted eggs during salting are unclear due to the complexity of the egg yolk structures. Low-density lipoprotein (LDL), which consists of approximately 83 to 89% lipids (74% neutral lipids and 26% phospholipids) and 11 to 17% protein, is the most abundant protein in egg yolk (Martin et al., 1963). LDL is considered to be a spherical particle of approximately 35 to 40 nm in diameter, consisting of a core of neutral lipids (triglycerides and cholesterol) surrounded by a monolayer of apoproteins and phospholipids (Evans et al., 1973; Anton et al., 2003). Furthermore, Dauphas et al. (2007) reported that LDL should exhibit protein– phospholipid and protein–triglyceride interactions in the film at the interface. It is well known that LDL plays a main role in the functional properties of egg yolk (Burley and Cook, 1961). One of the important functional properties of LDL is its ability to undergo aggregation based upon external factors (i.e., pH, storage conditions, freezing, and heat treatments), which plays an important role in the textural properties of final food products (Castellani et al., 2005; Marcet et al., 2016; Valverde et al., 2016; Liu et al., 2018). During the pickling of salted eggs, high salt induces the sol-gel transformation of egg
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yolks. The egg yolk granules are partially destroyed and gradually decreased by treatment with high salt (Xu et al., 2018). Moreover, the study of egg yolks, plasma, and granules induced by NaCl proposed that LDL plays a dominant role in egg yolk gel formation and lipid exudation under high salt conditions (Xu et al., 2019). Egg yolk has a complex system of multiscale supramolecular assemblies of proteins and lipids (Anton, 2013). It is mainly composed of 2 main fractions including plasma and granules. Although the plasma mainly consists of 85% LDL, it also contains 15% globular glycoproteins (α-, β -, and γ -livetins) (Anton, 2013). Thus, the molecular mechanism of gel formation under high salt conditions in the complex system of the egg yolk of salted eggs is difficult to explain in detail. Therefore, the objective of this study was to investigate the gelation behavior, microstructure, protein structures, and intermolecular force of LDL gels during salting. The intrinsic molecular mechanism of LDL coagulation under high salt was analyzed. The gained knowledge can be used to improve low salinization process for the salted egg.
MATERIALS AND METHODS Chemicals NaCl, potassium ferrocyanide, potassium chromate, silver nitrate, sodium dihydrogen phosphate, n-hexane, iso-propyl alcohol, and ethanol were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The 5 ,5-dithiobis-(2-nitrobenzoic acid) (DTNB), ammonium sulfate (40%), and D2 O were purchased from Sigma-Aldrich, Co., Ltd (St Louis, MO). Other main analytical grade chemicals were obtained from the Solarbio Science & Technology Co., Ltd. (Beijing, China).
Preparation of LDL Gels Fresh duck eggs, less than 3 days old after laying, with a weight range of 65 to 75 g, were purchased from a farm in Nanchang County, Jiangxi Province, China. The eggs were manually broken, and the yolk was collected. Yolks were carefully rolled on a filter paper to remove the albumen attached to the vitellin membrane. This membrane was then disrupted, and unspoiled egg yolk was collected in a beaker cooled in ice water at 4◦ C. Plasma was prepared from the yolks according to the method of Bee and Cotterill (1979). The yolks were diluted with an equal volume of a 0.17 M NaCl solution and stirred with a magnetic stirrer for 1 h at 4◦ C. The sample solution was then centrifuged at 10,000 g for 30 min at 4◦ C, and the supernatant (plasma) was separated from the sediment. Under the same conditions, plasma was centrifuged again for the complete removal of granules. Plasma was then preserved at 4◦ C for further LDL extraction. LDL was extracted from the plasma according to a method previously described (97% purity) (Moussa et al., 2002). Ammonium sul-
fate (40%) was slowly added to the stirring plasma solution and then stirred at 4◦ C for 1 h to precipitate the livetins. The pH of the plasma solution was controlled to be 8.7. Centrifugation at 10,000 g for 30 min at 4◦ C separated the supernatant from the sediment (livetins). The supernatant was then dialyzed against deionized water for at least 12 h (the bath was changed every 4 h) to eliminate ammonium sulfate. After complete ammonium sulfate elimination, the sample solution was then centrifuged at 10,000 g for 30 min at 4◦ C. The resulting floating residue, rich in LDL, was pooled. LDL was mixed with finely ground NaCl (1% concentration) and then manually stirred in the beaker until the NaCl was completely dissolved. The sample (6 g) was then poured into round small aluminum boxes (29 × 15 mm) and salted at 25◦ C, with samples taken every day during salting for up to 4 D. During salting, 3 salted LDL samples were chosen every day. For each treatment, 3 salted LDL gels pooled as composite samples. During salting, the samples were taken out at 1, 2, 3, and 4 D for determination and analysis. Before salting, the moisture of the LDL was adjusted to the same moisture content as that of fresh yolk (46.65 ± 0.42%) using distilled water.
Determination of the Moisture and NaCl Contents Moisture and NaCl contents of LDL gels were analyzed according to the methods of AOAC (2000).
Determination of Oil Exudation Oil exudation of LDL gel was determined using the method of Lai et al. (2010) with a slight modification. The samples (about 5 g) were homogenized with 25 mL of distilled water at 10,000 rpm, using an IK homogenizer (IKA T18 digital, IKA Works Guangzhou Co., Ltd., China) for 30 s. The mixture was centrifuged at 7,500 g (Anke, Model TGL-20B, Shanghai, China) for 30 min. Twenty-five milliliters of organic solution (n-hexane: isopropanol = 3:2, v/v) was added to the supernatant to dissolve the float. The top solvent-lipid layer obtained was separated using a separatory funnel and transferred into a water bath (55◦ C) to evaporate the majority of the organic solution, and then dried at 105◦ C to a constant weight. The obtained residue was weighed and taken as free lipid. Approximately 3 g of LDL gel sample was homogenized with 35 mL of n-hexane/2-propanol (3:2 v/v) at 10,000 rpm for 1 min. The homogenate was filtered via a filter paper and placed in a water bath (55◦ C), followed by heating the residue at 105◦ C to obtain a constant weight. The obtained residue was weighed and taken as the total lipids. Oil exudation was calculated based on the following formula: Oil exudation =
Free lipid content × 100% Total lipid content
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Texture Profile Analysis Texture profile analysis (TPA) of LDL gel was performed as described by Kaewmanee et al. (2009) with a TPA texture analyzer (Stable Micro System, Surrey, England). The LDL gels were taken directly from the round small aluminum boxes and subjected to TPA analysis. The LDL gel samples compression ratio were 50% of their original height with a compression cylindrical P/36R probe. A force–distance deformation curve was recorded at a pre-recording speed of 5 mm/s and the recording speed was 1 mm/s. All tests were repeated 6 times in parallel.
Determination of the Low-Field Nuclear Magnetic Resonance (LF-NMR) Spin–Spin Relaxation Time (T2 ) LF-NMR spin–spin relaxation (T2 ) measurements can provide direct information about interactions between exchange protons in proteins. The T2 measurements of LDL gel samples with and without deuterium oxide (D2 O) substitution were measured using a low-field pulsed NMR analyzer (Niumag Co., Ltd., Shanghai, China) according to the methods of Shao et al. (2016) with slight modifications. Deuterium oxide (D2 O) experiments were performed in order to eliminate exchanging protons (water) using the method of Li et al. (2015). Samples were soaked in excess D2 O (Sigma-Aldrich) for 12 h in 25 mm diameter NMR glass tubes and subjected to vacuum freeze-drying (Labconco Co., Ltd., Kansas City, MO). Both the cycle operations were repeated 4 times. The sample without D2 O substitution was placed in a 25 mm diameter NMR glass tube. The T2 relaxation times with and without deuterium oxide (D2 O) substitution were measured using the Carr–Purcell–Meiboom–Gill sequence. The T2 measurements were performed at 38◦ C with a τ -value (time between 90◦ and 180◦ pulses) of 100 μs. Data were acquired as 16 scan repetitions. Distributed fitting of CPMG decay curves were performed in analysis software (Niumag Co., Ltd., Shanghai, China). Each LDL gel sample was determined within less than 2 min. These steps were repeated 4 times, and the T2 was determined as an average.
Determination of the Microstructure by Transmission Electron Microscopy The microstructures of LDL gels were determined using transmission electron microscopy (TEM) (JEOL JEM 2010, Tokyo, Japan) at 160 kV. LDL gels (approximately 0.5 g) were fixed with 2.5% glutaraldehyde for 12 h at 4◦ C, and then gradient dehydrated using ethanol solutions. LDL gels were embedded in EPON resin and polymerized for 24 h at 70◦ C. Thin sections were cut with a diamond knife in an LKB Ultramicrotome (LKB Ultrascan XL, Bromma, Sweden). The
80 nm thick sections were deposited on copper grids, stained with 1% uranyl acetate, and photographed.
Determination of the Soluble Protein and the Free Sulfhydryl Content Bradford method was applied to determine the soluble protein content in LDL gels (Goren and Li, 1986). The free sulfhydryl group content of LDL gel was measured according to the method of Ellman (1959). Approximately 1 g of LDL gel sample was mixed with 10 mL of phosphate buffer (0.1 mol/L, pH = 8.0) and homogenized at 12,000 rpm for 2 min. The mixture was centrifuged at 10,000 g at 4◦ C for 20 min. TrisGly buffer treatment solutions (3.7 mL) and 40 μL of Ellman’s reagent (4 mg/mL DTNB in a Tris-glycine buffer) were added to 0.3 mL of the supernatant. Then, the mixed solution was incubated at 40◦ C for 15 min. After cooling, the absorbance of the supernatant was determined using a T6 spectrophotometer (Persee Co., Ltd., Beijing, China) at 412 nm. A blank was conducted by replacing the sample with treatment buffer. The free SH content was calculated as follows: μM SH/g protein = 73.53 × A412 × D/C, where A412 is the absorbance at 412 nm, C is the sample concentration in mg/mL, and D is the dilution factor of 15.01.
Determination of the Surface Hydrophobicity The surface hydrophobicity of LDL gel was analyzed according to the method of Benjakul et al. (2010). LDL gel samples were mixed with phosphate buffer (0.01 mol/L, pH = 7.0) and homogenized at 12,000 rpm for 2 min. The homogenate mixture was centrifuged at 10,000 g at 4◦ C for 20 min. LDL gel sample solutions were diluted to obtain concentrations between 0.005 and 0.30 mg/mL (5 gradients), using a 0.01 M sodium phosphate buffer (pH = 7). Four milliliters of solutions were mixed with 20 μL of 8 mM ANS. A spectrofluorometer RF-1501 (Shimadzu, Kyoto, Japan) was used to measure the fluorescence intensity at excitation and emission wavelengths of 395 and 475 nm, respectively. The initial slope of the fluorescence intensity vs. protein concentration was refined to the surface hydrophobicity.
Fourier Transform Infrared Spectroscopy (FTIR) Analysis Freeze-dried samples of LDL gel were milled into powder and ground with KBr (spectroscopic grade), followed by tablet preparation. The FTIR spectrum (Thermo Scientific Nicolet iS5) was recorded in the region of 400 to 4,000 cm−1 for 16 scans. For each sample, 3 replicates were automatically taken, averaged from 16 scans at 4 cm−1 resolution. OMNIC6.0 data collection software program (Thermo Scientific Nicolet iS5) was
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used for spectra evaluation. The second derivative was used to ascertain the positions of the absorbance peaks located in the amide I region (1,600 to 1,700 cm−1 ).
Differential Scanning Calorimeter (DSC) Analysis The thermally induced transition of the LDL gel was determined using DSC (NETZSCH Scientific Instruments Trading Ltd, DSC 214 Polyma, Germany). The LDL gels were conditioned in hermetic aluminum pans, weighed (6 to 7 mg) using a precision balance (Analytical Plus, Mettler Toledo), and heated at a rate of 2◦ C/min between 20 and 130◦ C under an inert atmosphere (50 mL min−1 of dry N2 ). The reference was an empty aluminum pan. The denaturation temperature (Tmax ) was determined by measuring the top endothermic peak.
Sodium Dodecyl Sulfate –Polyacrylamide Gel Electrophoresis Protein patterns of the LDL gel were determined according to the previously described method of Laemmli (1970), using 5% stacking gel (1.75 mL deionized water, 0.42 mL 30% acrylamide, 0.32 mL 1 M Tris-Hcl, 25 μL 10% SDS, 25 μL 10% persulfamide 50 μL, 4 μL TEMED) and 12% separating gel (1.6 mL deionized water, 2 mL 30% acrylamide, 1.3 mL 1.5 M TrisHcl, 50 μL 10% SDS, 50 μL 10% persulfamide 50 μL, 6 μL TEMED). About 3 g of LDL sample was added to 27 mL deionized water using a homogenizer (Ultra Turrax homogenizer, IKA T18 digital, IKAWorks Guangzhou Co., Ltd., China) at a speed of 12,000 rpm for 2 min, followed by centrifugation at 10,000 g for 20 min at room temperature using a centrifuge (Anke, Model TGL-20B, Shanghai, China). Then the centrifuged supernatant samples were diluted (1:1 v/v) in a sample buffer (0.125 M Tris–HCl (pH 6.8), 20% glycerol, 10% β -mercaptoethanol, and 4% SDS solution) to bring sample concentrations to approximately 1 μg protein/μL. After dilutions, samples were heated in boiling water bath for 5 min. The prepared sample (10 μL protein) was loaded onto the gel. Electrophoresis was performed using a vertical gel electrophoresis unit (Bio-Rad, Richmond, CA) at a constant voltage of 120 V/plate. After electrophoresis was completed, the gels were stained with Coomassie Brilliant Blue R125 (0.125%) in 25% methanol and 10% acetic acid. Destaining was performed using 25% ethanol and 8% acetic acid.
Statistical Analysis Except for TPA and TEM, the experimental design was a completely random design with 3 replications. Data were presented as mean values with standard deviations. Statistical analyses were per-
Figure 1. Changes in the moisture and NaCl contents of LDL gels induced by NaCl (a: moisture content; b: NaCl content).
formed using the SPSS statistics ver. 17.0 software (SPSS Inc., Chicago, IL). One-way analysis of variance was carried out, and the comparison of means was performed using Duncan’s multiple range tests (P < 0.05).
RESULTS AND DISCUSSION Changes in the Moisture and NaCl Contents of LDL Gels Induced by NaCl As shown in Figures 1a and 1b, the moisture and NaCl contents were 46.65 and 0.56% in the fresh LDL, respectively. As the salting proceeded, the moisture content of salted LDL gels exhibited a significant reduction (P < 0.05). After 4 D of salting, the moisture content decreased to a minimal level of 6.05% in the salted LDL gels. The NaCl content was at 0.56% initially and then significantly (P < 0.05) increased to 2.42% at the end of salting. During salting, the reduction in the moisture contents of salted LDL gels was in agreement with the increase in NaCl content. This may be attribute to the loss of water from the salted LDL to the outside caused by the osmosis process.
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Figure 3. Texture profile analysis (TPA) of LDL gels induced by NaCl.
Figure 2. Changes in the oil exudation of LDL gels induced by NaCl.
Changes in the Oil Exudation of LDL Gels Induced by NaCl As shown in Figure 2, increasing the salting time led to a significant increase in the oil exudation in the salted LDL gels (P < 0.05). The increase in oil exudation during salting might be associated with the destruction of LDLs, whereas the dehydration effect of salt can further facilitate the release of LDL constitutes (Lai et al., 1999). After 4 D of salting, the oil exudation of the salted LDL gels was higher compared to our previous research on the model of salted egg yolk, plasma, and granules (P < 0.05). This further indicated that the salting effect of NaCl increased the release of free lipids, mainly due to the structural changes in the LDLs. A portion of the LDLs lose their emulsification properties, which enhances moisture diffusion and increases the opportunities for the aggregation of lipophilic groups and the subsequent formation of visible oil liquid or oil droplets (Lai et al. 2010).
TPA of LDL Gels Induced by NaCl The LDL gels began to harden, and their viscosities began to increase until they became easy to detect by TPA on day 1 of salting. Thus, the LDL gels were processed after 1 D of salting, and the TPA properties were measured to obtain the 2 primary parameters (hardness and springiness). The hardness and springiness of LDL gels during salting are depicted in Figure 3. The hardness of LDL gels significantly increased with the increase in the salting time (P < 0.05). The springiness of the LDL gels showed an overall trend in which it first increased and then decreased (P < 0.05) during salting. The hardness of LDL gels was in agreement with the oil exudation increase during salting. After 4 D of salting, LDL gels having relatively high oil exudation produced a harder gel structure, suggesting that the free lipid molecules may somehow be involved in gel
structure formation. After salting, salted egg yolk underwent gelation and hardening due to the structural changes in LDL induced by dehydration (Ai et al., 2018). In addition, the denaturation of proteins and/or the coalescence of LDLs caused the leakage of phospholipids, which aided interactions between aggregates (Wakamatsu, Handa and Chiba, 2018) Springiness represents the recuperation rate from a deformation. When the gel was broken down into fewer large pieces during the first compression, the springiness showed higher values. The springiness of LDL gels increased remarkably (P < 0.05) during the early phase of salting, demonstrating that LDL gels were broken down into fewer large pieces in the process of TPA compression. However, after day 4 of salting, the springiness of LDL gels decreased. This result might be attributed to the destruction of LDL particles into small pieces and the exudation of free lipids.
LF-NMR Spin–Spin Relaxation Time (T2 ) Analysis of LDL Gels Induced by NaCl In this study, LF-NMR was used to determine the changes in the mobility of proton signals involved with water and lipids during the NaCl-induced LDL gelforming process. D2 O substitution and freeze-drying were used to eliminate the water proton signals (Yang et al., 2015). Thus, only the lipid proton signals of the gel system were represented in the LF-NMR study. Figure 4 shows the variation of the relaxation time T2 of the gels with and without replacement of water by deuterated water. For the salted LDL gels with D2 O substitution during salting, 3 different fractions of protons were detected: T21 , T22 , and T23 . As the salting proceeded, the T21 and T22 of salted LDL gels with D2 O substitution showed an overall trend in which they first decreased and then increased (P < 0.05). This indicates that the tightness of the binding of lipid protein shows an increase followed by a decrease. In fact, it was observed that the LDL gels were completely hardened on the second day of salting, which corresponds to the lowest T21 and T22 values. Therefore, it can be inferred
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2016). The relaxation time T22 showed an overall trend in which they first decreased and then increased (P < 0.05) during salting. The relaxation time T23 , however, showed an opposite trend, for which the increase in salting times led to an increase and then decrease (P < 0.05). Such behavior can be attributed to the effects of salting-in and oil exudation, thus enhancing the mobility of water and lipids in the early stages of salting. Another factor to be considered is the protein aggregation in the latter stages of salting, resulting in the restriction of the migration of protons related to water and lipid molecules.
Changes in the Microstructure of LDL Gels Induced by NaCl Figure 4. Changes in the T2 of LDL gels induced by NaCl. a, b, and c indicate significant differences between T21 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). d, e, and f indicate significant differences between T22 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). h, I, and j indicate significant differences between T23 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). A and B indicate significant differences between T21 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). C, D, and E indicate significant differences between T22 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). F, G, and H indicate significant differences between T23 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05).
that the decreases in T21 and T22 may be related to protein aggregation. The T23 of salted LDL gels showed a significant increase, which was consistent with the trend of oil exudation, indicating that the increase in T23 may be related to the free lipids. Three proton fractions were obtained in the case of no D2 O substitution of fresh LDL, the relaxation times for which were designated as T21 , T22 , and T23 , respectively. The T21 of salted LDL gels significantly decreased on the first day of salting (P < 0.05). However, after 1 D of salting, the T21 of salted LDL gels was not detected. This may be due to the mutual interference of proton signals from water and lipids (Au et al.,
In Figure 5, TEM microphotographs of LDL gels during salting are shown. TEM images of fresh LDL show spherical particles of 15 to 90 nm in diameter. Similar findings have been made by Anton et al. (2003). After salting for 0 to 2 D, the size of the LDL spherical particles becomes larger. These TEM images (larger LDL spherical particles) corresponded well with the lipid proton signal (T21 and T22 ) results and clearly indicated that proteins and lipids are tightly bound. Actually, it was observed that the LDL on the second day of salting has basically formed a gel. After 4 D of salting, the salted LDL gels showed various fine fiber filamentary structures as well as nonspherical protein and lipid particle structures. Such complex structures associated with each other to generate huge aggregates. These fine fiber filamentous structures may be some of the smaller LDL apoproteins that are released through the destruction of the LDL(Chang et al., 1977). This indicates that NaCl induced LDL destruction and releases its components (LDL apoproteins, phospholipids, and neutral lipids), facilitated by increased interactions between apoproteins-phospholipids and apoproteinsneutral lipids. When proteins are mostly hydrophobic, they insert into a phospholipid monolayer by interaction with neutral lipids to form mixed structures, and the phospholipid monolayer membrane structure is destroyed (Phillips et al., 1975). Most hydrophilic proteins
Figure 5. Changes in the microstructure of LDL gels induced by NaCl.
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Figure 6. Changes in the soluble protein of LDL gels induced by NaCl.
Figure 7. Changes in the free sulfhydryl content of LDL gels induced by NaCl.
would rather form complexes via electrostatic interactions with the polar headgroups of phospholipids.
Changes in the Soluble Protein of LDL Gels Induced by NaCl The effect of NaCl on the soluble protein of LDL gels during salting is shown in Figure 6. As the salting proceeded, the soluble protein contents of LDL gels showed an overall trend in which they first increased and then decreased (P < 0.05) during salting. In the case of salting for 2 D, the soluble protein of LDL gels increases to a maximum, which coincided with larger LDL spherical particles. After 3 D of salting, these results of soluble protein contents corresponded well with the TEM images, which indicated that the lower contents of soluble protein in LDL are due to the formation of insoluble aggregates. The addition of a larger amount of NaCl induced a salting-out effect. Moreover, at higher NaCl concentrations, the interactions of negatively charged chloride ions with positively charged protein molecules lead to a decrease in the electrostatic repulsion, thus enhancing hydrophobic interactions (Rdecs et al., 2007).
Changes in the Free Sulfhydryl Content of LDL Gels Induced by NaCl In Figure 7, the free sulfhydryl contents of LDL gels during salting are shown. The free sulfhydryl contents of LDL gels showed a significant decreasing trend, followed by a dramatic increase during salting (P < 0.05). It was found that a hardened gel appeared on the outer surface of the LDL samples on the first day of salting, which indicated that the LDL gels had started to form. Therefore, we can assume that the reduction of free sulfhydryl groups to the minimum may be attributed to intermolecular aggregation of LDLs. Maintaining these LDL aggregates may be related to the formation of new
Figure 8. Changes in the surface hydrophobicity of LDL gels induced by NaCl.
disulfide bonds, which are obtained via the sulfhydryl– disulfide (SH–SS) exchange reaction. However, these new disulfide bonds are likely to be more easily destroyed, resulting in an increase in the free sulfhydryl content after salting for 1 D. In the presence of atmospheric O2 and free lipids, disulfide bond formation could be impeded (Kaewmanee et al., 2011), resulting in an increased free sulfhydryl content in the later stages of salting.
Changes in the Surface Hydrophobicity of LDL Gels Induced by NaCl The surface hydrophobicity values of LDL gels during salting are shown in Figure 8. With increasing salting times, the surface hydrophobicity of LDL gels remarkably increased (P < 0.05). This increase might occur in steps and include the exposure of hydrophobic proteins, which were originally buried in the molecule, to the unfolding of LDL in the
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Figure 10. DSC curves of LDL gels induced by NaCl.
Figure 9. Secondary structural analysis of LDL gels induced by NaCl (a: FTIR spectrum; b: second-derivative FTIR spectrum).
later stages of salting. Although some aggregates were formed in the later stages of salting, a large number of intramolecular hydrophobic groups are still exposed simultaneously. The surface hydrophobicity of LDL gels increased during salting, which coincided with higher hardness. These results indicated that NaCl induced LDL unfolding and the formation of LDL gels, which was facilitated by increased hydrophobic interactions during salting.
Secondary Structural Analysis of LDL Gels Induced by NaCl The FTIR spectra of LDL gels during salting are depicted in Figure 9a. No shift in the peak shape and peak reformation of the LDL gels was observed during salting. Two stretching bands at 2925.48 and 2854.18 cm−1 were found in salted LDL gels. Such behavior may be attributed to the free lipids in salted LDL gels. Moreover, the absorbance at 1747.19 cm−1 may correspond to the stretching vibration of C=O in phospholipids and triglycerides in LDL (Liu et al., 2002). Figure 9b shows a typical second-derivative FTIR spectrum of the amide I absorption band (1,700 to 1,600 cm−1 ) of LDL gels during salting. The peak ranges from 1,615 to 1,625 cm−1 was assigned to intermolecular
β -sheets (Lilienthal et al., 2015). Meanwhile, the peak at 1,650 to 1,652 cm−1 was found to correspond to αhelices (Li et al., 2018). The intensity of the main peak of fresh LDL was centered at 1,629 and 1,656 cm−1 . Even though the second-derivative FTIR spectrum reinforced the possibility of resolving the overlapping components, it was difficult to clearly assign the peak at 1,626 cm−1 to a single secondary structural element. Thus, it may originate from a mixture of intermolecular and intramolecular β -sheets, whereas the peak at 1,656 cm−1 was assignable to α-helices (Lilienthal, Drotleff and Ternes, 2015). This indicated that the fresh LDL has more α-helix and β -sheet structures, which was in good agreement with the results of Blume et al. (2015). With increasing salting time, the β -sheet structural elements increased. However, the α-helix structural elements exhibited a decrease in the later stages of salting. According to Matheus et al. (2006), the intramolecular β -sheet is called a protein gel band. Additionally, our results indicated that the higher β -sheet structural elements of salted LDL gels during salting were in agreement with the strong hardness. These results suggested that great change occurred in the salted LDL gels during salting. It is possible that the α-helix structure is sacrificed and converted to a β -sheet structure for participation in LDL aggregation.
DSC Curves of LDL Gels Induced by NaCl Figure 10 shows the DSC thermograms of LDL gels during salting. A pronounced maximum endothermic peak, located at 75.2◦ C, was found in the fresh LDL. The irreversible endothermic peak of fresh LDL was related to the denaturation temperature. The denaturation temperatures were found at 108.2 and 112.2◦ C for LDLs on days 1 and 2 of salting, respectively. Wang et al. (2018) reported that the Tmax values of salted egg yolk plasma was located 98.3◦ C. After 2 D of salting, no endothermic peaks of salted LDL gels were detected. Such behavior can be attributed to the unfolding
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indicated that NaCl did not affect the structure of the LDL peptide chain.
Proposed Scheme for the LDL Gel-Forming Mechanism Induced by NaCl
Figure 11. SDS–polyacrylamide gel electrophoresis (SDS-PAGE).
temperature of the LDL structure to those found in previous studies (Mohr and Simon, 1992). The unfolding of LDL begins at above 70 to 75◦ C and complete denaturation temperature of the LDL structures at 76◦ C (Le et al., 1999). Thus, it can be inferred that no endothermic peaks were detectable due to the higher degree of structural unfolding of salted LDL gels. The higher degree of structural unfolding of salted LDL gels coincided with a higher surface hydrophobicity during salting.
Sodium Dodecyl Sulfate –Polyacrylamide Gel Electrophoresis The SDS-PAGE profiles of LDL gels during salting are depicted in Figure 11. The SDS-PAGE results indicated that salted LDL consists of 6 major apoproteins with molecular weights of about 185,130, 85, 65, 60, and 15 kDa. The results of the fresh LDL bands were similar to the to those found in previous studies (Moussa et al., 2002). The molecular weights of salted LDL gel proteins did not change after salting. A comparison of the electrophoretograms of the molecular weights between the salted LDL gel proteins and fresh LDL proteins showed that similar changes were observed, which
A possible mechanism for LDL gelation is proposed in Figure 12. First, the formation of LDL aggregates corresponded to the initial formation of the LDL gel. The formation of the gel mainly depends on intermolecular disulfide bonds and hydrophobic interactions. Second, the LDLs were disrupted and released fine fiber filamentary structures (LDL apoproteins) and non-spherical lipid particle structures (phospholipids and neutral lipids). Such released components are bound together by hydrophobic interactions to generate huge aggregates, facilitated by increased interactions between apoproteins-lipids (phospholipids and neutral lipids). The aggregation of LDL caused a disruption of the phospholipid monolayers by interaction between apoproteins (inserting into phospholipid monolayers) and neutral lipids. The disruption of phospholipid monolayers was consistent with the higher mobility of lipid proton signals, aiding the release of phospholipids and neutral lipids. The release of phospholipids and neutral lipids formed lipid particles between aggregated LDLs, aiding the exudation of oil.
CONCLUSIONS NaCl induced the aggregation of LDL, facilitated by increased oil exudation and gel strength texture. Additionally, the treatment of NaCl increased the surface hydrophobicity and β -sheet structural elements of salted LDL gels. Although NaCl can change the spatial structure of egg yolk proteins, it did not affect the structure of the LDL peptide chain. The formation of salted LDL gels during salting mainly depends on disulfide bonds and hydrophobic interactions. Therefore, the next studies should focus on the formation mechanism and role of disulfide bonds and hydrophobic interactions in LDL gels by a non-destructive detection method.
Figure 12. Proposed scheme for the LDL gel-forming mechanism induced by NaCl.
LOW-DENSITY LIPOPROTEIN GEL INDUCED BY NaCl
ACKNOWLEDGMENTS We gratefully acknowledge the financial support provided by the Program of the National Natural Science Foundation of China (Grant Nos. 31760467), Key Research and Development Project of Jiangxi Province (Grant No. 20171BBF60038), the Young Scientist Training Objects Program of Jiangxi Province (Grant No. 20153BCB23028), and Jiangxi Province Outstanding Youth Talent Funded Projects (Grant No. 20162BCB23031).
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Jiangxi Key Laboratory of Natural Products and Functional Food, Jiangxi Agricultural University, Nanchang 330045, China; † Engineering Research Center of Biomass Conversion, Ministry of Education, Nanchang University, Nanchang 330047, China; and ‡ Jiangxi Shenzhu Tianyuan Food Co., Ltd., Nanchang University, Nanchang 330219, China
ABSTRACT Salted eggs, which are a traditional Chinese egg product, are favored by Chinese consumers and have become very popular in other Asian countries due to their unique features such as “fresh, fine, tender, loose, gritty and oily texture.” In order to illuminate the forming process of salted egg, the gelation behavior and mechanism of low-density lipoprotein (LDL) induced by NaCl were investigated using marinated model outside the eggshell. Results showed that as the salting proceeded, the moisture content exhibited a decreasing trend. The NaCl content, oil exudation, hardness, and surface hydrophobicity showed constantly increasing trends. In the early stages of salting, the size of the LDL particles, soluble protein content, and T21 increased, whereas T21 (with D2 O), T22 , and the free sulfhydryl content declined. In the later stages
of salting, LDL formed a multiple composite aggregate gel structure with filamentous apoproteins and nonspherical lipid particles intertwined with each other. The soluble protein content and T23 (without D2 O) decreased, whereas T21 (with D2 O), T22 and the free sulfhydryl content increased. Fourier transform infrared spectroscopy revealed that the fresh LDL mainly consisted of α-helix and β -sheet structures. After the gel becomes hardened, the LDL secondary structure was changed remarkably, characterized by the decrease of α-helix elements and increase of β -sheet elements. The results suggested that the oil exudation of salted LDL gels was mainly due to LDL destruction and the release of components (apoproteins, phospholipids, and neutral lipids) facilitated by increased interactions between apoproteins and lipids.
Key words: LDL gel, NaCl-induced, microstructure, intermolecular forces 2019 Poultry Science 98:5166–5176 http://dx.doi.org/10.3382/ps/pez232
INTRODUCTION Salted eggs, which are a famous traditional preserved egg product originally created in China, are mainly pickled with high concentration of salt at room temperature for 4 to 5 wk (Lai et al., 1999). The production of outstanding features (loose, gritty, and oily) of salted egg yolks requires the treatment of fresh duck eggs with a high salt concentration. However, the salt contents in the maturated salted egg whites reach 7 to 10% after salting (Xu et al., 2017). Recently, several studies have emphasized that egg yolks are separated from egg whites before bringing to reduce the salt in the egg whites (Woodward and Cotterill, 2010; Singh and Ramaswamy, 2013). Despite the benefits of a shorter salting cycle and retaining protein integrity, these salted egg yolks do not achieve good quality. A major reason is that the processes and mechanisms involved in the mat-
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2019 Poultry Science Association Inc. Received October 19, 2018. Accepted April 22, 2019. 1 Corresponding authors: [email protected] (TY); zhaoyan@ ncu.edu.cn (ZY)
uration of salted eggs during salting are unclear due to the complexity of the egg yolk structures. Low-density lipoprotein (LDL), which consists of approximately 83 to 89% lipids (74% neutral lipids and 26% phospholipids) and 11 to 17% protein, is the most abundant protein in egg yolk (Martin et al., 1963). LDL is considered to be a spherical particle of approximately 35 to 40 nm in diameter, consisting of a core of neutral lipids (triglycerides and cholesterol) surrounded by a monolayer of apoproteins and phospholipids (Evans et al., 1973; Anton et al., 2003). Furthermore, Dauphas et al. (2007) reported that LDL should exhibit protein– phospholipid and protein–triglyceride interactions in the film at the interface. It is well known that LDL plays a main role in the functional properties of egg yolk (Burley and Cook, 1961). One of the important functional properties of LDL is its ability to undergo aggregation based upon external factors (i.e., pH, storage conditions, freezing, and heat treatments), which plays an important role in the textural properties of final food products (Castellani et al., 2005; Marcet et al., 2016; Valverde et al., 2016; Liu et al., 2018). During the pickling of salted eggs, high salt induces the sol-gel transformation of egg
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yolks. The egg yolk granules are partially destroyed and gradually decreased by treatment with high salt (Xu et al., 2018). Moreover, the study of egg yolks, plasma, and granules induced by NaCl proposed that LDL plays a dominant role in egg yolk gel formation and lipid exudation under high salt conditions (Xu et al., 2019). Egg yolk has a complex system of multiscale supramolecular assemblies of proteins and lipids (Anton, 2013). It is mainly composed of 2 main fractions including plasma and granules. Although the plasma mainly consists of 85% LDL, it also contains 15% globular glycoproteins (α-, β -, and γ -livetins) (Anton, 2013). Thus, the molecular mechanism of gel formation under high salt conditions in the complex system of the egg yolk of salted eggs is difficult to explain in detail. Therefore, the objective of this study was to investigate the gelation behavior, microstructure, protein structures, and intermolecular force of LDL gels during salting. The intrinsic molecular mechanism of LDL coagulation under high salt was analyzed. The gained knowledge can be used to improve low salinization process for the salted egg.
MATERIALS AND METHODS Chemicals NaCl, potassium ferrocyanide, potassium chromate, silver nitrate, sodium dihydrogen phosphate, n-hexane, iso-propyl alcohol, and ethanol were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The 5 ,5-dithiobis-(2-nitrobenzoic acid) (DTNB), ammonium sulfate (40%), and D2 O were purchased from Sigma-Aldrich, Co., Ltd (St Louis, MO). Other main analytical grade chemicals were obtained from the Solarbio Science & Technology Co., Ltd. (Beijing, China).
Preparation of LDL Gels Fresh duck eggs, less than 3 days old after laying, with a weight range of 65 to 75 g, were purchased from a farm in Nanchang County, Jiangxi Province, China. The eggs were manually broken, and the yolk was collected. Yolks were carefully rolled on a filter paper to remove the albumen attached to the vitellin membrane. This membrane was then disrupted, and unspoiled egg yolk was collected in a beaker cooled in ice water at 4◦ C. Plasma was prepared from the yolks according to the method of Bee and Cotterill (1979). The yolks were diluted with an equal volume of a 0.17 M NaCl solution and stirred with a magnetic stirrer for 1 h at 4◦ C. The sample solution was then centrifuged at 10,000 g for 30 min at 4◦ C, and the supernatant (plasma) was separated from the sediment. Under the same conditions, plasma was centrifuged again for the complete removal of granules. Plasma was then preserved at 4◦ C for further LDL extraction. LDL was extracted from the plasma according to a method previously described (97% purity) (Moussa et al., 2002). Ammonium sul-
fate (40%) was slowly added to the stirring plasma solution and then stirred at 4◦ C for 1 h to precipitate the livetins. The pH of the plasma solution was controlled to be 8.7. Centrifugation at 10,000 g for 30 min at 4◦ C separated the supernatant from the sediment (livetins). The supernatant was then dialyzed against deionized water for at least 12 h (the bath was changed every 4 h) to eliminate ammonium sulfate. After complete ammonium sulfate elimination, the sample solution was then centrifuged at 10,000 g for 30 min at 4◦ C. The resulting floating residue, rich in LDL, was pooled. LDL was mixed with finely ground NaCl (1% concentration) and then manually stirred in the beaker until the NaCl was completely dissolved. The sample (6 g) was then poured into round small aluminum boxes (29 × 15 mm) and salted at 25◦ C, with samples taken every day during salting for up to 4 D. During salting, 3 salted LDL samples were chosen every day. For each treatment, 3 salted LDL gels pooled as composite samples. During salting, the samples were taken out at 1, 2, 3, and 4 D for determination and analysis. Before salting, the moisture of the LDL was adjusted to the same moisture content as that of fresh yolk (46.65 ± 0.42%) using distilled water.
Determination of the Moisture and NaCl Contents Moisture and NaCl contents of LDL gels were analyzed according to the methods of AOAC (2000).
Determination of Oil Exudation Oil exudation of LDL gel was determined using the method of Lai et al. (2010) with a slight modification. The samples (about 5 g) were homogenized with 25 mL of distilled water at 10,000 rpm, using an IK homogenizer (IKA T18 digital, IKA Works Guangzhou Co., Ltd., China) for 30 s. The mixture was centrifuged at 7,500 g (Anke, Model TGL-20B, Shanghai, China) for 30 min. Twenty-five milliliters of organic solution (n-hexane: isopropanol = 3:2, v/v) was added to the supernatant to dissolve the float. The top solvent-lipid layer obtained was separated using a separatory funnel and transferred into a water bath (55◦ C) to evaporate the majority of the organic solution, and then dried at 105◦ C to a constant weight. The obtained residue was weighed and taken as free lipid. Approximately 3 g of LDL gel sample was homogenized with 35 mL of n-hexane/2-propanol (3:2 v/v) at 10,000 rpm for 1 min. The homogenate was filtered via a filter paper and placed in a water bath (55◦ C), followed by heating the residue at 105◦ C to obtain a constant weight. The obtained residue was weighed and taken as the total lipids. Oil exudation was calculated based on the following formula: Oil exudation =
Free lipid content × 100% Total lipid content
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Texture Profile Analysis Texture profile analysis (TPA) of LDL gel was performed as described by Kaewmanee et al. (2009) with a TPA texture analyzer (Stable Micro System, Surrey, England). The LDL gels were taken directly from the round small aluminum boxes and subjected to TPA analysis. The LDL gel samples compression ratio were 50% of their original height with a compression cylindrical P/36R probe. A force–distance deformation curve was recorded at a pre-recording speed of 5 mm/s and the recording speed was 1 mm/s. All tests were repeated 6 times in parallel.
Determination of the Low-Field Nuclear Magnetic Resonance (LF-NMR) Spin–Spin Relaxation Time (T2 ) LF-NMR spin–spin relaxation (T2 ) measurements can provide direct information about interactions between exchange protons in proteins. The T2 measurements of LDL gel samples with and without deuterium oxide (D2 O) substitution were measured using a low-field pulsed NMR analyzer (Niumag Co., Ltd., Shanghai, China) according to the methods of Shao et al. (2016) with slight modifications. Deuterium oxide (D2 O) experiments were performed in order to eliminate exchanging protons (water) using the method of Li et al. (2015). Samples were soaked in excess D2 O (Sigma-Aldrich) for 12 h in 25 mm diameter NMR glass tubes and subjected to vacuum freeze-drying (Labconco Co., Ltd., Kansas City, MO). Both the cycle operations were repeated 4 times. The sample without D2 O substitution was placed in a 25 mm diameter NMR glass tube. The T2 relaxation times with and without deuterium oxide (D2 O) substitution were measured using the Carr–Purcell–Meiboom–Gill sequence. The T2 measurements were performed at 38◦ C with a τ -value (time between 90◦ and 180◦ pulses) of 100 μs. Data were acquired as 16 scan repetitions. Distributed fitting of CPMG decay curves were performed in analysis software (Niumag Co., Ltd., Shanghai, China). Each LDL gel sample was determined within less than 2 min. These steps were repeated 4 times, and the T2 was determined as an average.
Determination of the Microstructure by Transmission Electron Microscopy The microstructures of LDL gels were determined using transmission electron microscopy (TEM) (JEOL JEM 2010, Tokyo, Japan) at 160 kV. LDL gels (approximately 0.5 g) were fixed with 2.5% glutaraldehyde for 12 h at 4◦ C, and then gradient dehydrated using ethanol solutions. LDL gels were embedded in EPON resin and polymerized for 24 h at 70◦ C. Thin sections were cut with a diamond knife in an LKB Ultramicrotome (LKB Ultrascan XL, Bromma, Sweden). The
80 nm thick sections were deposited on copper grids, stained with 1% uranyl acetate, and photographed.
Determination of the Soluble Protein and the Free Sulfhydryl Content Bradford method was applied to determine the soluble protein content in LDL gels (Goren and Li, 1986). The free sulfhydryl group content of LDL gel was measured according to the method of Ellman (1959). Approximately 1 g of LDL gel sample was mixed with 10 mL of phosphate buffer (0.1 mol/L, pH = 8.0) and homogenized at 12,000 rpm for 2 min. The mixture was centrifuged at 10,000 g at 4◦ C for 20 min. TrisGly buffer treatment solutions (3.7 mL) and 40 μL of Ellman’s reagent (4 mg/mL DTNB in a Tris-glycine buffer) were added to 0.3 mL of the supernatant. Then, the mixed solution was incubated at 40◦ C for 15 min. After cooling, the absorbance of the supernatant was determined using a T6 spectrophotometer (Persee Co., Ltd., Beijing, China) at 412 nm. A blank was conducted by replacing the sample with treatment buffer. The free SH content was calculated as follows: μM SH/g protein = 73.53 × A412 × D/C, where A412 is the absorbance at 412 nm, C is the sample concentration in mg/mL, and D is the dilution factor of 15.01.
Determination of the Surface Hydrophobicity The surface hydrophobicity of LDL gel was analyzed according to the method of Benjakul et al. (2010). LDL gel samples were mixed with phosphate buffer (0.01 mol/L, pH = 7.0) and homogenized at 12,000 rpm for 2 min. The homogenate mixture was centrifuged at 10,000 g at 4◦ C for 20 min. LDL gel sample solutions were diluted to obtain concentrations between 0.005 and 0.30 mg/mL (5 gradients), using a 0.01 M sodium phosphate buffer (pH = 7). Four milliliters of solutions were mixed with 20 μL of 8 mM ANS. A spectrofluorometer RF-1501 (Shimadzu, Kyoto, Japan) was used to measure the fluorescence intensity at excitation and emission wavelengths of 395 and 475 nm, respectively. The initial slope of the fluorescence intensity vs. protein concentration was refined to the surface hydrophobicity.
Fourier Transform Infrared Spectroscopy (FTIR) Analysis Freeze-dried samples of LDL gel were milled into powder and ground with KBr (spectroscopic grade), followed by tablet preparation. The FTIR spectrum (Thermo Scientific Nicolet iS5) was recorded in the region of 400 to 4,000 cm−1 for 16 scans. For each sample, 3 replicates were automatically taken, averaged from 16 scans at 4 cm−1 resolution. OMNIC6.0 data collection software program (Thermo Scientific Nicolet iS5) was
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used for spectra evaluation. The second derivative was used to ascertain the positions of the absorbance peaks located in the amide I region (1,600 to 1,700 cm−1 ).
Differential Scanning Calorimeter (DSC) Analysis The thermally induced transition of the LDL gel was determined using DSC (NETZSCH Scientific Instruments Trading Ltd, DSC 214 Polyma, Germany). The LDL gels were conditioned in hermetic aluminum pans, weighed (6 to 7 mg) using a precision balance (Analytical Plus, Mettler Toledo), and heated at a rate of 2◦ C/min between 20 and 130◦ C under an inert atmosphere (50 mL min−1 of dry N2 ). The reference was an empty aluminum pan. The denaturation temperature (Tmax ) was determined by measuring the top endothermic peak.
Sodium Dodecyl Sulfate –Polyacrylamide Gel Electrophoresis Protein patterns of the LDL gel were determined according to the previously described method of Laemmli (1970), using 5% stacking gel (1.75 mL deionized water, 0.42 mL 30% acrylamide, 0.32 mL 1 M Tris-Hcl, 25 μL 10% SDS, 25 μL 10% persulfamide 50 μL, 4 μL TEMED) and 12% separating gel (1.6 mL deionized water, 2 mL 30% acrylamide, 1.3 mL 1.5 M TrisHcl, 50 μL 10% SDS, 50 μL 10% persulfamide 50 μL, 6 μL TEMED). About 3 g of LDL sample was added to 27 mL deionized water using a homogenizer (Ultra Turrax homogenizer, IKA T18 digital, IKAWorks Guangzhou Co., Ltd., China) at a speed of 12,000 rpm for 2 min, followed by centrifugation at 10,000 g for 20 min at room temperature using a centrifuge (Anke, Model TGL-20B, Shanghai, China). Then the centrifuged supernatant samples were diluted (1:1 v/v) in a sample buffer (0.125 M Tris–HCl (pH 6.8), 20% glycerol, 10% β -mercaptoethanol, and 4% SDS solution) to bring sample concentrations to approximately 1 μg protein/μL. After dilutions, samples were heated in boiling water bath for 5 min. The prepared sample (10 μL protein) was loaded onto the gel. Electrophoresis was performed using a vertical gel electrophoresis unit (Bio-Rad, Richmond, CA) at a constant voltage of 120 V/plate. After electrophoresis was completed, the gels were stained with Coomassie Brilliant Blue R125 (0.125%) in 25% methanol and 10% acetic acid. Destaining was performed using 25% ethanol and 8% acetic acid.
Statistical Analysis Except for TPA and TEM, the experimental design was a completely random design with 3 replications. Data were presented as mean values with standard deviations. Statistical analyses were per-
Figure 1. Changes in the moisture and NaCl contents of LDL gels induced by NaCl (a: moisture content; b: NaCl content).
formed using the SPSS statistics ver. 17.0 software (SPSS Inc., Chicago, IL). One-way analysis of variance was carried out, and the comparison of means was performed using Duncan’s multiple range tests (P < 0.05).
RESULTS AND DISCUSSION Changes in the Moisture and NaCl Contents of LDL Gels Induced by NaCl As shown in Figures 1a and 1b, the moisture and NaCl contents were 46.65 and 0.56% in the fresh LDL, respectively. As the salting proceeded, the moisture content of salted LDL gels exhibited a significant reduction (P < 0.05). After 4 D of salting, the moisture content decreased to a minimal level of 6.05% in the salted LDL gels. The NaCl content was at 0.56% initially and then significantly (P < 0.05) increased to 2.42% at the end of salting. During salting, the reduction in the moisture contents of salted LDL gels was in agreement with the increase in NaCl content. This may be attribute to the loss of water from the salted LDL to the outside caused by the osmosis process.
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Figure 3. Texture profile analysis (TPA) of LDL gels induced by NaCl.
Figure 2. Changes in the oil exudation of LDL gels induced by NaCl.
Changes in the Oil Exudation of LDL Gels Induced by NaCl As shown in Figure 2, increasing the salting time led to a significant increase in the oil exudation in the salted LDL gels (P < 0.05). The increase in oil exudation during salting might be associated with the destruction of LDLs, whereas the dehydration effect of salt can further facilitate the release of LDL constitutes (Lai et al., 1999). After 4 D of salting, the oil exudation of the salted LDL gels was higher compared to our previous research on the model of salted egg yolk, plasma, and granules (P < 0.05). This further indicated that the salting effect of NaCl increased the release of free lipids, mainly due to the structural changes in the LDLs. A portion of the LDLs lose their emulsification properties, which enhances moisture diffusion and increases the opportunities for the aggregation of lipophilic groups and the subsequent formation of visible oil liquid or oil droplets (Lai et al. 2010).
TPA of LDL Gels Induced by NaCl The LDL gels began to harden, and their viscosities began to increase until they became easy to detect by TPA on day 1 of salting. Thus, the LDL gels were processed after 1 D of salting, and the TPA properties were measured to obtain the 2 primary parameters (hardness and springiness). The hardness and springiness of LDL gels during salting are depicted in Figure 3. The hardness of LDL gels significantly increased with the increase in the salting time (P < 0.05). The springiness of the LDL gels showed an overall trend in which it first increased and then decreased (P < 0.05) during salting. The hardness of LDL gels was in agreement with the oil exudation increase during salting. After 4 D of salting, LDL gels having relatively high oil exudation produced a harder gel structure, suggesting that the free lipid molecules may somehow be involved in gel
structure formation. After salting, salted egg yolk underwent gelation and hardening due to the structural changes in LDL induced by dehydration (Ai et al., 2018). In addition, the denaturation of proteins and/or the coalescence of LDLs caused the leakage of phospholipids, which aided interactions between aggregates (Wakamatsu, Handa and Chiba, 2018) Springiness represents the recuperation rate from a deformation. When the gel was broken down into fewer large pieces during the first compression, the springiness showed higher values. The springiness of LDL gels increased remarkably (P < 0.05) during the early phase of salting, demonstrating that LDL gels were broken down into fewer large pieces in the process of TPA compression. However, after day 4 of salting, the springiness of LDL gels decreased. This result might be attributed to the destruction of LDL particles into small pieces and the exudation of free lipids.
LF-NMR Spin–Spin Relaxation Time (T2 ) Analysis of LDL Gels Induced by NaCl In this study, LF-NMR was used to determine the changes in the mobility of proton signals involved with water and lipids during the NaCl-induced LDL gelforming process. D2 O substitution and freeze-drying were used to eliminate the water proton signals (Yang et al., 2015). Thus, only the lipid proton signals of the gel system were represented in the LF-NMR study. Figure 4 shows the variation of the relaxation time T2 of the gels with and without replacement of water by deuterated water. For the salted LDL gels with D2 O substitution during salting, 3 different fractions of protons were detected: T21 , T22 , and T23 . As the salting proceeded, the T21 and T22 of salted LDL gels with D2 O substitution showed an overall trend in which they first decreased and then increased (P < 0.05). This indicates that the tightness of the binding of lipid protein shows an increase followed by a decrease. In fact, it was observed that the LDL gels were completely hardened on the second day of salting, which corresponds to the lowest T21 and T22 values. Therefore, it can be inferred
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2016). The relaxation time T22 showed an overall trend in which they first decreased and then increased (P < 0.05) during salting. The relaxation time T23 , however, showed an opposite trend, for which the increase in salting times led to an increase and then decrease (P < 0.05). Such behavior can be attributed to the effects of salting-in and oil exudation, thus enhancing the mobility of water and lipids in the early stages of salting. Another factor to be considered is the protein aggregation in the latter stages of salting, resulting in the restriction of the migration of protons related to water and lipid molecules.
Changes in the Microstructure of LDL Gels Induced by NaCl Figure 4. Changes in the T2 of LDL gels induced by NaCl. a, b, and c indicate significant differences between T21 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). d, e, and f indicate significant differences between T22 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). h, I, and j indicate significant differences between T23 of salted LDL gels with D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). A and B indicate significant differences between T21 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). C, D, and E indicate significant differences between T22 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05). F, G, and H indicate significant differences between T23 of salted LDL gels without D2 O substitution obtained from different salting times and from fresh LDL (P < 0.05).
that the decreases in T21 and T22 may be related to protein aggregation. The T23 of salted LDL gels showed a significant increase, which was consistent with the trend of oil exudation, indicating that the increase in T23 may be related to the free lipids. Three proton fractions were obtained in the case of no D2 O substitution of fresh LDL, the relaxation times for which were designated as T21 , T22 , and T23 , respectively. The T21 of salted LDL gels significantly decreased on the first day of salting (P < 0.05). However, after 1 D of salting, the T21 of salted LDL gels was not detected. This may be due to the mutual interference of proton signals from water and lipids (Au et al.,
In Figure 5, TEM microphotographs of LDL gels during salting are shown. TEM images of fresh LDL show spherical particles of 15 to 90 nm in diameter. Similar findings have been made by Anton et al. (2003). After salting for 0 to 2 D, the size of the LDL spherical particles becomes larger. These TEM images (larger LDL spherical particles) corresponded well with the lipid proton signal (T21 and T22 ) results and clearly indicated that proteins and lipids are tightly bound. Actually, it was observed that the LDL on the second day of salting has basically formed a gel. After 4 D of salting, the salted LDL gels showed various fine fiber filamentary structures as well as nonspherical protein and lipid particle structures. Such complex structures associated with each other to generate huge aggregates. These fine fiber filamentous structures may be some of the smaller LDL apoproteins that are released through the destruction of the LDL(Chang et al., 1977). This indicates that NaCl induced LDL destruction and releases its components (LDL apoproteins, phospholipids, and neutral lipids), facilitated by increased interactions between apoproteins-phospholipids and apoproteinsneutral lipids. When proteins are mostly hydrophobic, they insert into a phospholipid monolayer by interaction with neutral lipids to form mixed structures, and the phospholipid monolayer membrane structure is destroyed (Phillips et al., 1975). Most hydrophilic proteins
Figure 5. Changes in the microstructure of LDL gels induced by NaCl.
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Figure 6. Changes in the soluble protein of LDL gels induced by NaCl.
Figure 7. Changes in the free sulfhydryl content of LDL gels induced by NaCl.
would rather form complexes via electrostatic interactions with the polar headgroups of phospholipids.
Changes in the Soluble Protein of LDL Gels Induced by NaCl The effect of NaCl on the soluble protein of LDL gels during salting is shown in Figure 6. As the salting proceeded, the soluble protein contents of LDL gels showed an overall trend in which they first increased and then decreased (P < 0.05) during salting. In the case of salting for 2 D, the soluble protein of LDL gels increases to a maximum, which coincided with larger LDL spherical particles. After 3 D of salting, these results of soluble protein contents corresponded well with the TEM images, which indicated that the lower contents of soluble protein in LDL are due to the formation of insoluble aggregates. The addition of a larger amount of NaCl induced a salting-out effect. Moreover, at higher NaCl concentrations, the interactions of negatively charged chloride ions with positively charged protein molecules lead to a decrease in the electrostatic repulsion, thus enhancing hydrophobic interactions (Rdecs et al., 2007).
Changes in the Free Sulfhydryl Content of LDL Gels Induced by NaCl In Figure 7, the free sulfhydryl contents of LDL gels during salting are shown. The free sulfhydryl contents of LDL gels showed a significant decreasing trend, followed by a dramatic increase during salting (P < 0.05). It was found that a hardened gel appeared on the outer surface of the LDL samples on the first day of salting, which indicated that the LDL gels had started to form. Therefore, we can assume that the reduction of free sulfhydryl groups to the minimum may be attributed to intermolecular aggregation of LDLs. Maintaining these LDL aggregates may be related to the formation of new
Figure 8. Changes in the surface hydrophobicity of LDL gels induced by NaCl.
disulfide bonds, which are obtained via the sulfhydryl– disulfide (SH–SS) exchange reaction. However, these new disulfide bonds are likely to be more easily destroyed, resulting in an increase in the free sulfhydryl content after salting for 1 D. In the presence of atmospheric O2 and free lipids, disulfide bond formation could be impeded (Kaewmanee et al., 2011), resulting in an increased free sulfhydryl content in the later stages of salting.
Changes in the Surface Hydrophobicity of LDL Gels Induced by NaCl The surface hydrophobicity values of LDL gels during salting are shown in Figure 8. With increasing salting times, the surface hydrophobicity of LDL gels remarkably increased (P < 0.05). This increase might occur in steps and include the exposure of hydrophobic proteins, which were originally buried in the molecule, to the unfolding of LDL in the
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Figure 10. DSC curves of LDL gels induced by NaCl.
Figure 9. Secondary structural analysis of LDL gels induced by NaCl (a: FTIR spectrum; b: second-derivative FTIR spectrum).
later stages of salting. Although some aggregates were formed in the later stages of salting, a large number of intramolecular hydrophobic groups are still exposed simultaneously. The surface hydrophobicity of LDL gels increased during salting, which coincided with higher hardness. These results indicated that NaCl induced LDL unfolding and the formation of LDL gels, which was facilitated by increased hydrophobic interactions during salting.
Secondary Structural Analysis of LDL Gels Induced by NaCl The FTIR spectra of LDL gels during salting are depicted in Figure 9a. No shift in the peak shape and peak reformation of the LDL gels was observed during salting. Two stretching bands at 2925.48 and 2854.18 cm−1 were found in salted LDL gels. Such behavior may be attributed to the free lipids in salted LDL gels. Moreover, the absorbance at 1747.19 cm−1 may correspond to the stretching vibration of C=O in phospholipids and triglycerides in LDL (Liu et al., 2002). Figure 9b shows a typical second-derivative FTIR spectrum of the amide I absorption band (1,700 to 1,600 cm−1 ) of LDL gels during salting. The peak ranges from 1,615 to 1,625 cm−1 was assigned to intermolecular
β -sheets (Lilienthal et al., 2015). Meanwhile, the peak at 1,650 to 1,652 cm−1 was found to correspond to αhelices (Li et al., 2018). The intensity of the main peak of fresh LDL was centered at 1,629 and 1,656 cm−1 . Even though the second-derivative FTIR spectrum reinforced the possibility of resolving the overlapping components, it was difficult to clearly assign the peak at 1,626 cm−1 to a single secondary structural element. Thus, it may originate from a mixture of intermolecular and intramolecular β -sheets, whereas the peak at 1,656 cm−1 was assignable to α-helices (Lilienthal, Drotleff and Ternes, 2015). This indicated that the fresh LDL has more α-helix and β -sheet structures, which was in good agreement with the results of Blume et al. (2015). With increasing salting time, the β -sheet structural elements increased. However, the α-helix structural elements exhibited a decrease in the later stages of salting. According to Matheus et al. (2006), the intramolecular β -sheet is called a protein gel band. Additionally, our results indicated that the higher β -sheet structural elements of salted LDL gels during salting were in agreement with the strong hardness. These results suggested that great change occurred in the salted LDL gels during salting. It is possible that the α-helix structure is sacrificed and converted to a β -sheet structure for participation in LDL aggregation.
DSC Curves of LDL Gels Induced by NaCl Figure 10 shows the DSC thermograms of LDL gels during salting. A pronounced maximum endothermic peak, located at 75.2◦ C, was found in the fresh LDL. The irreversible endothermic peak of fresh LDL was related to the denaturation temperature. The denaturation temperatures were found at 108.2 and 112.2◦ C for LDLs on days 1 and 2 of salting, respectively. Wang et al. (2018) reported that the Tmax values of salted egg yolk plasma was located 98.3◦ C. After 2 D of salting, no endothermic peaks of salted LDL gels were detected. Such behavior can be attributed to the unfolding
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indicated that NaCl did not affect the structure of the LDL peptide chain.
Proposed Scheme for the LDL Gel-Forming Mechanism Induced by NaCl
Figure 11. SDS–polyacrylamide gel electrophoresis (SDS-PAGE).
temperature of the LDL structure to those found in previous studies (Mohr and Simon, 1992). The unfolding of LDL begins at above 70 to 75◦ C and complete denaturation temperature of the LDL structures at 76◦ C (Le et al., 1999). Thus, it can be inferred that no endothermic peaks were detectable due to the higher degree of structural unfolding of salted LDL gels. The higher degree of structural unfolding of salted LDL gels coincided with a higher surface hydrophobicity during salting.
Sodium Dodecyl Sulfate –Polyacrylamide Gel Electrophoresis The SDS-PAGE profiles of LDL gels during salting are depicted in Figure 11. The SDS-PAGE results indicated that salted LDL consists of 6 major apoproteins with molecular weights of about 185,130, 85, 65, 60, and 15 kDa. The results of the fresh LDL bands were similar to the to those found in previous studies (Moussa et al., 2002). The molecular weights of salted LDL gel proteins did not change after salting. A comparison of the electrophoretograms of the molecular weights between the salted LDL gel proteins and fresh LDL proteins showed that similar changes were observed, which
A possible mechanism for LDL gelation is proposed in Figure 12. First, the formation of LDL aggregates corresponded to the initial formation of the LDL gel. The formation of the gel mainly depends on intermolecular disulfide bonds and hydrophobic interactions. Second, the LDLs were disrupted and released fine fiber filamentary structures (LDL apoproteins) and non-spherical lipid particle structures (phospholipids and neutral lipids). Such released components are bound together by hydrophobic interactions to generate huge aggregates, facilitated by increased interactions between apoproteins-lipids (phospholipids and neutral lipids). The aggregation of LDL caused a disruption of the phospholipid monolayers by interaction between apoproteins (inserting into phospholipid monolayers) and neutral lipids. The disruption of phospholipid monolayers was consistent with the higher mobility of lipid proton signals, aiding the release of phospholipids and neutral lipids. The release of phospholipids and neutral lipids formed lipid particles between aggregated LDLs, aiding the exudation of oil.
CONCLUSIONS NaCl induced the aggregation of LDL, facilitated by increased oil exudation and gel strength texture. Additionally, the treatment of NaCl increased the surface hydrophobicity and β -sheet structural elements of salted LDL gels. Although NaCl can change the spatial structure of egg yolk proteins, it did not affect the structure of the LDL peptide chain. The formation of salted LDL gels during salting mainly depends on disulfide bonds and hydrophobic interactions. Therefore, the next studies should focus on the formation mechanism and role of disulfide bonds and hydrophobic interactions in LDL gels by a non-destructive detection method.
Figure 12. Proposed scheme for the LDL gel-forming mechanism induced by NaCl.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support provided by the Program of the National Natural Science Foundation of China (Grant Nos. 31760467), Key Research and Development Project of Jiangxi Province (Grant No. 20171BBF60038), the Young Scientist Training Objects Program of Jiangxi Province (Grant No. 20153BCB23028), and Jiangxi Province Outstanding Youth Talent Funded Projects (Grant No. 20162BCB23031).
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