Highly efficient extraction and purification of low-density lipoprotein from hen egg yolk

Highly efficient extraction and purification of low-density lipoprotein from hen egg yolk ABSTRACT Low-density lipoprotein (LDL) from hen egg yolk has high nutritional value and plays an important role in the fields of biology, medicine, and materials. To develop fundamental research about LDL, a highly efficient extraction method is necessary. We found that 30% saturated ammonium sulfate can extract more crude LDL than 40% saturation. We selected polyethylene glycol (PEG; nonionic type) to obtain crude LDL. Three factors were employed, namely, degree of polymerization, concentration of PEG, and pH of egg yolk plasma. The optimized condition was 5% PEG 4,000 and plasma pH 6.0, and the best extraction efficiency was 68.1 ± 0.5 g lipid /100 g DM and 69.9 ± 2.0% protein. The crude LDL oil of PEG precipitation was very significantly higher (P < 0.01) than ammonium sulfate precipitation (ASP), while there was no significant difference in protein, which indicates that PEG can extract more crude LDL. When ascorbic acid was added, hydrosulfuryl (SH) groups and lipids oxidation degree of crude LDL extracted by PEG (PEG-LDL) was very significantly lower than ASP (P < 0.01). We also obtained both purified LDL and yolk immunoglobulin (IgY) with an appropriate purification column. This paper proposes a highly efficient method to extract LDL with high activity using PEG and ensures co-purification of LDL and IgY. INTRODUCTION Low-density lipoprotein (LDL) is a spherical nanoscale lipid-protein complex with an average diameter of 35 to 40 nm (Anton et al., 2003). Accounting for about two-thirds wet weight of egg yolk (Anton, 2007), LDL mainly exists in egg yolk plasma but also appears in small amounts in egg yolk granules (Mann and Mann, 2008). LDL possesses important functional properties and nutritional values. On one hand, this protein-lipid compound has excellent emulsifying (Dauphas et al., 2007a; Dauphas et al., 2007b; Le Denmat et al., 2000; Mizutani and Nakamura, 1985), gelation (Blume et al., 2015), and cryoprotective (Amirat-Briand et al., 2010; Bencharif et al., 2008; Moussa et al., 2002) properties. For example, this compound can be used to prepare nanogels to control the release of doxorubicin (He et al., 2015) based on amphipathic property. LDL plays an important role in lipid-mediated antimicrobial activity (Brady et al., 2002). On the other hand, it is a major nutritional source. LDL consists of 12% protein and 87% lipids, which are composed of about 71% triacylglycerol, 25% phospholipids, and 4% cholesterol (Anton et al., 2003). LDL also provides essential fatty acids, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid (Gao and Charter, 2000; Wang et al., 2014). However, little research about the metabolic path and possible effect of this extraneous protein-lipid compound on the human body has been done. Numerous polyunsaturated fatty acids exist in LDL, which may lead to lipid peroxidation when in contact with the environment and result in ox-LDL, which is atherogenic. To explore its functional mechanism, purified LDL with high activity (low oxidation degree) must be obtained from hen egg yolk. LDL is extracted by a combination of salting out and ultracentrifugation, followed by purification via molecular screen gel filtration chromatography of Ultrogel AcA 34 (Bee and Cotterill, 1979; Jolivet et al., 2008; Martin et al., 1964; Moussa et al., 2002). However, the salting-out procedure requires further dialysis, which is time consuming and may lead to peroxidation of PUFA of phospholipids in the surface of lipid-rich LDL. Ammonium sulfate precipitation (ASP) is an appropriate method to fractionate proteins prior to purification (Cohn et al., 1940; Isenberg, 1995; Mirica et al., 2012). In the present LDL extraction method, 40% ammonium sulfate saturation (ASS) was employed (Anton et al., 2001; He et al., 2015; Neves et al., 2014). In this experiment, we selected different ASS to obtain an optimized extraction effect. Polyethylene glycol (PEG) is an inexpensive, neutral, water-soluble, biocompatible, FDA-approved polymer, so it is probably the most widely applied synthetic polymer in biotechnology and medicine (Duncan et al., 2006; Lutz and Hoth, 2006). Moreover, it is commonly applied to precipitate and crystallize proteins (Geng et al., 2012; Zhang et al., 2011). On the basis of the molecular space exclusion effect, PEG can precipitate proteins by water adsorption and squeezing protein molecules together (Kumar et al., 2009). This method is mild and can stabilize the protein conformation, which is in favor of extracting bioactive proteins. As a bioactive molecule and major component of egg yolk plasma, yolk immunoglobulin (IgY) is separated from other proteins by PEG precipitation (Pauly et al., 2011). LDL have a larger molecular weight than IgY and may be easily “squeezed out” by PEG. In this experiment, PEG was employed for the first time, to the best of our knowledge, to extract LDL, which may simplify the operating steps and reduce the oxidation degree of samples. Both ammonium sulfate and PEG were used to separate LDL, and the yield and oxidation degree of crude LDL extracted by the two methods were compared to determine the optimum separation method. High-performance HiPrep 26/600 Sephacryl S-300 HR was applied to increase the purity of LDL. Finally, the purified LDL could be used for subsequent research and application. MATERIALS AND METHODS Chemicals and Reagents Fresh hen eggs laid within 24 h from Hy-Line Brown were bought from a local hennery (Jiufeng Farm, Wuhan, China), stored at 4 °C, and used for experiments within 30 days. NaCl, PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, 2,4-dinitrophenyl hydrazine, 5,5'-dithio-bis-(2-nitrobenzoic acid), ascorbic acid, trichloromethane, and methanol were obtained from SINOPHARM Chemical Reagent Co., Ltd. (Shanghai, China). The reagents used for SDS-PAGE were purchased from Guge Biotechnology Co., Ltd. (Wuhan, China). Lowry reagent was procured from Biosharp (Hefei, China). HiPrep 26/600 Sephacryl S-300 HR was purchased from GE Healthcare Bio-Science AB (Uppsala, Sweden). Plasma Extraction Hen egg yolk plasma was separated according to McBee's method (Bee and Cotterill, 1979). Fresh hen eggs were manually broken, and most albumen was eliminated. Yolks were carefully rolled on a filter paper to remove albumen and chalazion adhering to the vitelline membrane. This membrane was then perforated to collect unspoiled egg yolk in a beaker cooled in iced water. Yolk was diluted with an equal volume of 0.17 M NaCl solution and stirred with a magnetic stirrer for 1 h at 4°C. This solution was centrifuged at 15,000 × g for 15 min at 4°C, and the supernatant (plasma) was separated from the sediment (granules). Plasma was then centrifuged under the same conditions for complete removal of granules. Ammonium Sulfate Precipitation Ammonium sulfate of suitable saturation (20 to 45%) was added to 25 g of plasma to precipitate LDL, and the mixture was stirred for 1 h at 4°C and centrifuged at 15,000 × g for 15 min at 4°C. The precipitate was discarded, and the supernatant was dialyzed against deionized water for at least 6 h (the bath was changed every 2 h). The supernatant was then centrifuged at 10,000 × g for 30 min at 4°C. The resulting floating material containing LDL was pooled. PEG Precipitation Plasma (pH from 5.00 to 7.00) mixed with PEG of different polymerization degrees (PD; ranging from 4,000 to 20,000) and different concentrations (2, 4, 6, 8, and 10%) was stirred for 1 h at 4°C and centrifuged at 22,000 × g for 15 min at 4°C. The upper floating material was pooled, and the subnatant was discarded. Molecular Sieve Chromatography The extracted crude LDL was dissolved in 0.05 M Tris-HCl buffer (pH 8.5) containing 0.15 M NaCl and filtered through a 0.22 μm membrane. The sample was applied to a pre-packed column (26/600) of Sephacryl S-300 (High Resolution, Uppsala, Sweden) and eluted with 0.05 M Tris-HCl buffer (pH 8.5) containing 0.15 M NaCl at a flow rate of 1 mL/min. Each peak was collected through a fraction collector (F9-R, Uppsala, Sweden), and the purity of LDL and components of these was identified by SDS-PAGE analysis. SDS-PAGE Analysis Electrophoreses were run on polyacrylamide gels (stacking: 5% and resolving: 12%) with a migration buffer consisting of 0.025 M Tris (pH 8.3), 0.2 M Gly, and 0.1% SDS solution (DYY-12, Beijing, China). PageRuler Prestained Protein Ladder (26616, MBI, Thermo Scientific, Shanghai, China) with a molecular weight ranging from 10 kDa to 170 kDa was loaded to compare the molecular weight of LDL proteins. The protein bands were stained with Coomassie Brilliant Blue R-250 (0.1% in the staining solution containing 25% ethanol and 8% acetic acid) for 30 min at 45°C and de-stained by diffusion in de-staining solution (25% ethanol and 8% acetic acid). Protein Determination Protein content was determined by the modified procedure of Markwell (Markwell et al., 1978). The protein content (between 10 and 100 μg/mL) was calculated using a linear regression equation, with the absorbency of bovine serum albumin solution as a function of their concentration. Results were expressed as grams of protein per 100 g DM. Lipid Determination Lipid content was determined by the modified procedure of Folch (Folch et al., 1957). About 10 mL of chloroform–methanol (v/v, 2:1) were added to 1 g of sample, and the mixture was homogenized with a vortex mixer. After standing for 15 min, the mixture was centrifuged (5,000 × g for 10 min) and filtered. Subsequently, 5 mL of 0.88% NaCl were added to the filtrate to promote stratification. The organic phase was collected, and solvents were evaporated with a rotary evaporator (R201D, Gongyi, China). Lipid content was estimated by weighing lipid extract after solvent evaporation. Results were expressed as grams of lipid per 100 g DM. Sulfhydryl Determination Sulfhydryl content was measured by the reaction with Ellman's reagent [5,5'-dithio-bis-(2-nitrobenzoic acid)]. Approximately 0.5 mL of crude LDL solution (10 mg/mL) was mixed with 2.5 mL of 8 M urea in Tris-glycine buffer and 0.02 mL of 4 mg/mL Ellman's reagent. After reaction for 20 min, the absorbance at 412 nm was read. A molar extinction coefficient of 1.36 × 104 M−1cm−1 was used to convert absorbance to the sulfhydryl concentration (Beveridge et al., 1974; Zhao et al., 2013). Carbonyl Determination Carbonyl content was measured by the reaction with 2,4-dinitrophenyl hydrazine according to Levine (Levine et al., 1990). Results were expressed as micromoles of carbonyl groups per gram of LDL calculated with a molar extinction coefficient of 2.2 × 104 M−1cm−1. Malondialdehyde Determination Malondialdehyde (MDA) content was measured by the reaction with 2-thiobarbituric acid according to Tatum (Tatum et al., 1990). A standard solution of 1,1,3,3-tetraethoxypropane (4 to 32 μmol/L) was employed to make a calibration curve. The TBA–MDA adduct was quantitated with a fluorescence detector set at 515 nm excitation and 550 nm emission. The MDA content of samples was calculated according to the calibration curve. Statistical Analysis Except for the experimental design, 3 replicates were prepared for all the measurements. The results of separation and chemical analysis were subjected to Duncan analysis using SPSS. Confidence intervals were set at 95% (P < 0.05). RESULTS AND DISCUSSION Results of ASP The results of ASP are shown in Table 1. ASS significantly influenced the protein content and DM weight of crude LDL (P < 0.01). The results showed that 30% ASS could extract 57.72 g/100 g DM lipids and 69.41% protein, whereas 40% ASS could extract 56.80 g/100 g DM lipids and 73.78% protein. Table 1. Extraction results of ammonium sulfate precipitation. ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e Duncan analysis at a 95% confidence interval (P < 0.05). a-eIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Table 1. Extraction results of ammonium sulfate precipitation. ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e Duncan analysis at a 95% confidence interval (P < 0.05). a-eIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Compared with 40% ASS (Anton et al., 2003; Moussa et al., 2002), 30% ASS could extract more lipids (1.62% higher) and less protein (6.30% lower), but the difference was not significant. Fewer proteins were extracted, and subsequent purification was simplified. Thus, we considered 30% ASS as the optimized experimental condition of ASP. Results of PEG Precipitation Single-factor Experiment of PEG Precipitation In the single-factor experiment, three factors, namely, PEG PD (4% PEG and plasma pH 6.00), PEG concentration (PEG 6,000 and plasma pH 6.00), and plasma pH (4% PEG 6,000), were employed to develop unidirectional optimization experiments. The crude LDL extraction results (Figure 1A, C, and E) showed that the extraction rate of crude LDL increased significantly with increasing PD and PEG concentration (P < 0.05), and the most suitable plasma pH was 6.00 (similar to natural plasma pH 6.02). Some previous research has demonstrated that the PD and concentration of PEG can significantly affect protein extraction efficiency, which improved with the increase of PEG polymerization degree and concentration (Hammerschmidt et al., 2016; Li and Zydney, 2017). SDS-PAGE analysis results (Figure 1B, D, and F) showed that the molecular weight of most proteins in the subnatant of the PEG PD and plasma pH experiment were 100, 70, 60, and 35 to 40 kDa, whereas the major proteins in the subnatant of the PEG experiment were high and light chains of IgY. Figure 1. View largeDownload slide Crude LDL extraction results of single-factor experiment and corresponding SDS-PAGE analysis of the subnatant. A and B correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG PD, respectively; for SDS-PAGE analysis, lanes refer to PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, plasma, and PageRuler Prestained Protein Ladder 10–170 KD (26616) (marker). C and D correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG concentration, respectively; for SDS-PAGE analysis, lanes refer to 10, 8, 6, 4, and 2% PEG concentration, plasma, and marker. E and F correspond to crude LDL extraction rate and SDS-PAGE analysis result of plasma pH, respectively; for SDS-PAGE analysis, lanes refer to pH 7.00, pH 6.50, pH 6.00, pH 5.50, pH 5.00, plasma, and marker. All the analyses were carried out as Duncan analysis at a 95% confidence interval (P < 0.05). In each BAR, different letters mean significant variation, whereas the same letter means no significant variation. Figure 1. View largeDownload slide Crude LDL extraction results of single-factor experiment and corresponding SDS-PAGE analysis of the subnatant. A and B correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG PD, respectively; for SDS-PAGE analysis, lanes refer to PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, plasma, and PageRuler Prestained Protein Ladder 10–170 KD (26616) (marker). C and D correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG concentration, respectively; for SDS-PAGE analysis, lanes refer to 10, 8, 6, 4, and 2% PEG concentration, plasma, and marker. E and F correspond to crude LDL extraction rate and SDS-PAGE analysis result of plasma pH, respectively; for SDS-PAGE analysis, lanes refer to pH 7.00, pH 6.50, pH 6.00, pH 5.50, pH 5.00, plasma, and marker. All the analyses were carried out as Duncan analysis at a 95% confidence interval (P < 0.05). In each BAR, different letters mean significant variation, whereas the same letter means no significant variation. High PEG PD may keep more proteins in crude LDL, but low PD enables easy decondensation (Ramos et al., 2005). Thus, PEG 4,000, PEG 6,000, and PEG 8,000 were selected for the following orthogonal experiment. For the PEG experiment, more LDL were resolved in the subnatant even with a high crude LDL extraction rate. We selected 4 to 8% PEG concentration for the orthogonal experiments. Proteins in the subnatant of plasma pH optimization were almost similar to one another. The most crude LDL were extracted in pH 6.00, which was eventually selected. Orthogonal Experiment of PEG Precipitation On the basis of single-factor experiment results, we selected appropriate factor levels for orthogonal experiments (see Supporting Information Table S1). The range analysis results (Table 2) showed that PD of PEG exerted the highest influence on the lipid content and protein concentration of the subnatant, PEG concentration had the highest effect on crude LDL extract efficiency, and plasma pH had the largest effect on the protein content. Table 2. Orthogonal experimental result. Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 A: PEG PD, B: PEG concentration, C: plasma pH, D: Lipid content (g/100 g DM), E: protein content (%), F: crude LDL extract efficiency (%), G: protein concentration of subnatant (μg/mL). View Large Table 2. Orthogonal experimental result. Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 A: PEG PD, B: PEG concentration, C: plasma pH, D: Lipid content (g/100 g DM), E: protein content (%), F: crude LDL extract efficiency (%), G: protein concentration of subnatant (μg/mL). View Large ANOVA (see Supporting Information Table S2) demonstrated that PD of PEG significantly affected the protein content (P < 0.05) and very significantly influenced the protein concentration of the subnatant (P < 0.01); PEG concentration significantly affected crude LDL extract efficiency (P < 0.05). Research showed that PD and concentration of PEG can significantly affect the extraction efficiency and purity of protein, while pH has no significant influence on either (Polson et al., 1964; Sommer et al., 2014). On the basis of range analysis and ANOVA results, we selected A1B2C2, with an oil extraction rate of 68.14 g/100 g DM, protein extraction rate of 69.91%, crude LDL extraction rate of 90.10%, and subnatant protein concentration of 134.89 μg/mL for LDL extraction and compared these values with the ASP method. Correlation analysis was conducted among the 4 experimental indices (see Supporting Information Table S3). The results showed a very significant negative correlation between the oil extraction rate and subnatant protein concentration, as well as a significant positive correlation between the crude LDL extraction rate and subnatant protein concentration. The oil extraction rate and protein extraction rate were negatively related. Comparison of 2 Separate Methods Comparison of Extract Results ASP extracted 56.80 g/100 g DM lipid and 73.78% protein in 40% ASS. In 30% ASS, the extract efficiency of lipid and protein was 57.72 g/100 g DM and 69.41%, respectively. The optimized PEG precipitation extracted 68.14 g/100 g DM lipid and 69.91% protein. To compare the extraction efficiency of the 2 extraction methods, the ANOVA results showed that the oil extraction rate of crude LDL extracted by PEG precipitation is very significantly higher than the ASP-LDL (P < 0.01), and the protein extraction rate of the 2 samples has no significant difference, which indicates that PEG precipitation could obtain more crude LDL than ASP and allowed a simple purification procedure. Comparison of Crude LDL Oxidation Degree In this section, we selected Vc (0.02% additive amount) as an exogenous antioxidant to inhibit LDL oxidation (Pantelidis et al., 2007; Rice-Evans et al., 1997). The sulfhydryl, carbonyl, and MDA contents were selected to compare the oxidation degree of crude LDL extracted by the 2 methods. The results are shown in Table 3. Table 3. Comparison of crude LDL oxidation degree. Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Duncan analysis at a 95% confidence interval (P < 0.05). ND = no detection. **= P < 0.01. NS = no significance. a,bIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Table 3. Comparison of crude LDL oxidation degree. Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Duncan analysis at a 95% confidence interval (P < 0.05). ND = no detection. **= P < 0.01. NS = no significance. a,bIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large The results showed that no carbonyl was detected in all the crude LDL samples. Compared with the no Vc addition group, sulfhydryl contents are significantly high when antioxidant is added in both LDL samples (P < 0.05). In other ways, the sulfhydryl content of PEG-LDL was very significantly higher than that in ASP-LDL with or without Vc addition (P < 0.01). For the PEG-LDL sample, Vc addition can significantly decrease the MDA content (P < 0.05). With antioxidant added, the MDA content of PEG-LDL was very significantly lower than that in ASP-LDL (P < 0.01). However, no significant difference was detected between the 2 LDL samples with no Vc addition. The results indicated that both LDL samples developed no protein oxidation (Zhang et al., 2018), and crude LDL extracted by PEG precipitation has a lower oxidation degree of hydrosulfuryl (SH) groups (Jiang et al., 2017) and lipids (Smialowska et al., 2017) than that in ASP-LDL. We also found that ASP was a diluting procedure that may cause further loss of target compounds during dialysis, centrifugation, and purification (Papala et al., 2017). By contrast, PEG could condense the target compounds during extraction and ensured a credible calculation of purified LDL after quantitative dilution and purification based on the water content of crude LDL. Purification of LDL LDL are the largest compounds in hen egg yolk plasma (Anton, 2013; Martin et al., 1964). LDL can be effectively purified via molecular sieve chromatography. We employed Sephacryl S-200 HR (16/600) and Sephacryl S-300 HR (26/600) to purify LDL. Both LDL (Jolivet et al., 2008) and IgY (Tan et al., 2012) were detected in crude LDL. Sephacryl S-200 HR (separation range of 5 to 250 kDa) could not completely separate LDL from IgY (data not shown). Three eluting peaks were obtained when crude LDL were applied to the Sephacryl S-300 HR column (Figure 2A). Figure 2. View largeDownload slide Sephacryl S-300 HR (26/600) chromatography of crude LDL (A) and SDS-PAGE analysis of collected fractions (B). Three main peaks were obtained. Fractions 1 to 6 were collected with a volume of 5 mL, fractions 8 to 12 were collected with a volume of 10 mL, and the remaining fractions were collected with a volume of 20 mL. Fractions collected by Sephacryl S-300 HR (26/600) both at 1 and 1.5 mL/min were applied to SDS-PAGE analysis. Lanes 1 to 5 were fractions of 1.0 mL/min. Lane 1 = fraction 1, lane 2 = fractions 3 to 5, lane 3 = fractions 6 to 8, lane 4 = fractions 18 to 21, and lane 5 = fractions 30 to 32. Lanes 6 to 10 were fractions of 1.5 mL/min. Lane 6 = fraction 1, lane 7 = fractions 3 to 5, lane 8 = fractions 6 to 9, lane 9 = fractions 15 to 18, lane 10 = fractions 22 and 23, and lane 11 = marker. Figure 2. View largeDownload slide Sephacryl S-300 HR (26/600) chromatography of crude LDL (A) and SDS-PAGE analysis of collected fractions (B). Three main peaks were obtained. Fractions 1 to 6 were collected with a volume of 5 mL, fractions 8 to 12 were collected with a volume of 10 mL, and the remaining fractions were collected with a volume of 20 mL. Fractions collected by Sephacryl S-300 HR (26/600) both at 1 and 1.5 mL/min were applied to SDS-PAGE analysis. Lanes 1 to 5 were fractions of 1.0 mL/min. Lane 1 = fraction 1, lane 2 = fractions 3 to 5, lane 3 = fractions 6 to 8, lane 4 = fractions 18 to 21, and lane 5 = fractions 30 to 32. Lanes 6 to 10 were fractions of 1.5 mL/min. Lane 6 = fraction 1, lane 7 = fractions 3 to 5, lane 8 = fractions 6 to 9, lane 9 = fractions 15 to 18, lane 10 = fractions 22 and 23, and lane 11 = marker. Subsequent SDS-PAGE analysis revealed that LDL and IgY corresponded with the first and second peaks, respectively (Figure 2B), and the 2 constituents reached electrophoretic purity. With a separation range of 10 to 1,500 kDa, Sephacryl S-300 HR could obtain purified LDL and IgY at flow rates of 1.0 and 1.5 mL/min. Compared with previous experiments (Jolivet et al., 2006; Jolivet et al., 2008), LDL obtained in our experiment had high purity with no other protein between 20 and 50 kDa. Co-purification of IgY The main proteins in egg plasma are LDL and livetin (Hatta et al., 2008). The 3 types of livetin are α, β, and γ livetin. γ livetin results in immunity, and its major fraction is called IgY. Several studies have investigated the purification of IgY because of its unique biophysical function and high requirements for purity and bioactivity (Dong et al., 2008; Gee et al., 2003; Hodek et al., 2013). The previous experiments have proved that the molecule weight of LDL is much larger than IgY (Juneja, 2008), and PEG can be used for antibody extraction without denaturation (Chen et al., 2017; Kuczewski et al., 2011); thus; in our experiment, the remaining IgY can be easily purified following the purified LDL. The SDS-PAGE analysis results of the orthogonal experiment (Figure 3) showed that a variation in the 3 experiment factors could alter the IgY content in the subnatant. Figure 3. View largeDownload slide SDS-PAGE analysis results of orthogonal experiment. A) SDS-PAGE analysis of the subnatant. Lanes M to 9 are the marker and subnatant of experiments 1 to 9. B) SDS-PAGE analysis of crude LDL. Lanes 1 to M are 1 to 9 crude LDL and marker. Figure 3. View largeDownload slide SDS-PAGE analysis results of orthogonal experiment. A) SDS-PAGE analysis of the subnatant. Lanes M to 9 are the marker and subnatant of experiments 1 to 9. B) SDS-PAGE analysis of crude LDL. Lanes 1 to M are 1 to 9 crude LDL and marker. CONCLUSIONS In this study, we constructed an extraction method of high efficiency and low oxidative degree for hen egg yolk LDL using PEG, which demonstrated higher LDL yield and purity than ASP. The optimized experimental conditions of 5% PEG 4,000 and pH 6.00 with 0.02% Vc addition were obtained. In PEG-LDL, the oil concentration was very significantly higher than ASP-LDL(P < 0.01), while the protein concentration showed no significant difference between both crude LDL samples. The oxidation degree of SH groups and lipids in PEG-LDL was very significantly lower than ASP-LDL (P < 0.01), and no LDL protein oxidation was detected in either sample. Thus, PEG precipitation may allow a large amount of extraction of hen egg yolk LDL and benefit other fundamental research on LDL. We employed HiPrep 26/600 Sephacryl S-300 HR molecular sieve chromatography to purify LDL, and at the same time obtained purified IgY. If a further experiment was developed, a more effective extraction method of LDL and IgY could be constructed. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Appendix A. Supplementary Material Supplementary data associated with this article can be found in the online version. ACKNOWLEDGMENTS This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-41-K23) and Chinese Ministry of Agriculture nonprofit industry special research project (No.201303084). REFERENCES Amirat-Briand L. , Bencharif D. , Vera-Munoz O. , Pineau S. , Thorin C. , Destrumelle S. , Desherces S. , Anton M. , Jouan M. , Shmitt E. . 2010 . In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: Preliminary results of artificial inseminations . Anim. Reprod. Sci. 122 : 282 – 287 . Google Scholar CrossRef Search ADS PubMed Anton M. 2007 . 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Changes in structural characteristics of antioxidative soy protein hydrolysates resulting from scavenging of hydroxyl radicals . J. Food Sci. 78 : C152 – C159 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Highly efficient extraction and purification of low-density lipoprotein from hen egg yolk

Poultry Science , Volume Advance Article (6) – Mar 8, 2018

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey059
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Abstract

ABSTRACT Low-density lipoprotein (LDL) from hen egg yolk has high nutritional value and plays an important role in the fields of biology, medicine, and materials. To develop fundamental research about LDL, a highly efficient extraction method is necessary. We found that 30% saturated ammonium sulfate can extract more crude LDL than 40% saturation. We selected polyethylene glycol (PEG; nonionic type) to obtain crude LDL. Three factors were employed, namely, degree of polymerization, concentration of PEG, and pH of egg yolk plasma. The optimized condition was 5% PEG 4,000 and plasma pH 6.0, and the best extraction efficiency was 68.1 ± 0.5 g lipid /100 g DM and 69.9 ± 2.0% protein. The crude LDL oil of PEG precipitation was very significantly higher (P < 0.01) than ammonium sulfate precipitation (ASP), while there was no significant difference in protein, which indicates that PEG can extract more crude LDL. When ascorbic acid was added, hydrosulfuryl (SH) groups and lipids oxidation degree of crude LDL extracted by PEG (PEG-LDL) was very significantly lower than ASP (P < 0.01). We also obtained both purified LDL and yolk immunoglobulin (IgY) with an appropriate purification column. This paper proposes a highly efficient method to extract LDL with high activity using PEG and ensures co-purification of LDL and IgY. INTRODUCTION Low-density lipoprotein (LDL) is a spherical nanoscale lipid-protein complex with an average diameter of 35 to 40 nm (Anton et al., 2003). Accounting for about two-thirds wet weight of egg yolk (Anton, 2007), LDL mainly exists in egg yolk plasma but also appears in small amounts in egg yolk granules (Mann and Mann, 2008). LDL possesses important functional properties and nutritional values. On one hand, this protein-lipid compound has excellent emulsifying (Dauphas et al., 2007a; Dauphas et al., 2007b; Le Denmat et al., 2000; Mizutani and Nakamura, 1985), gelation (Blume et al., 2015), and cryoprotective (Amirat-Briand et al., 2010; Bencharif et al., 2008; Moussa et al., 2002) properties. For example, this compound can be used to prepare nanogels to control the release of doxorubicin (He et al., 2015) based on amphipathic property. LDL plays an important role in lipid-mediated antimicrobial activity (Brady et al., 2002). On the other hand, it is a major nutritional source. LDL consists of 12% protein and 87% lipids, which are composed of about 71% triacylglycerol, 25% phospholipids, and 4% cholesterol (Anton et al., 2003). LDL also provides essential fatty acids, such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid (Gao and Charter, 2000; Wang et al., 2014). However, little research about the metabolic path and possible effect of this extraneous protein-lipid compound on the human body has been done. Numerous polyunsaturated fatty acids exist in LDL, which may lead to lipid peroxidation when in contact with the environment and result in ox-LDL, which is atherogenic. To explore its functional mechanism, purified LDL with high activity (low oxidation degree) must be obtained from hen egg yolk. LDL is extracted by a combination of salting out and ultracentrifugation, followed by purification via molecular screen gel filtration chromatography of Ultrogel AcA 34 (Bee and Cotterill, 1979; Jolivet et al., 2008; Martin et al., 1964; Moussa et al., 2002). However, the salting-out procedure requires further dialysis, which is time consuming and may lead to peroxidation of PUFA of phospholipids in the surface of lipid-rich LDL. Ammonium sulfate precipitation (ASP) is an appropriate method to fractionate proteins prior to purification (Cohn et al., 1940; Isenberg, 1995; Mirica et al., 2012). In the present LDL extraction method, 40% ammonium sulfate saturation (ASS) was employed (Anton et al., 2001; He et al., 2015; Neves et al., 2014). In this experiment, we selected different ASS to obtain an optimized extraction effect. Polyethylene glycol (PEG) is an inexpensive, neutral, water-soluble, biocompatible, FDA-approved polymer, so it is probably the most widely applied synthetic polymer in biotechnology and medicine (Duncan et al., 2006; Lutz and Hoth, 2006). Moreover, it is commonly applied to precipitate and crystallize proteins (Geng et al., 2012; Zhang et al., 2011). On the basis of the molecular space exclusion effect, PEG can precipitate proteins by water adsorption and squeezing protein molecules together (Kumar et al., 2009). This method is mild and can stabilize the protein conformation, which is in favor of extracting bioactive proteins. As a bioactive molecule and major component of egg yolk plasma, yolk immunoglobulin (IgY) is separated from other proteins by PEG precipitation (Pauly et al., 2011). LDL have a larger molecular weight than IgY and may be easily “squeezed out” by PEG. In this experiment, PEG was employed for the first time, to the best of our knowledge, to extract LDL, which may simplify the operating steps and reduce the oxidation degree of samples. Both ammonium sulfate and PEG were used to separate LDL, and the yield and oxidation degree of crude LDL extracted by the two methods were compared to determine the optimum separation method. High-performance HiPrep 26/600 Sephacryl S-300 HR was applied to increase the purity of LDL. Finally, the purified LDL could be used for subsequent research and application. MATERIALS AND METHODS Chemicals and Reagents Fresh hen eggs laid within 24 h from Hy-Line Brown were bought from a local hennery (Jiufeng Farm, Wuhan, China), stored at 4 °C, and used for experiments within 30 days. NaCl, PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, 2,4-dinitrophenyl hydrazine, 5,5'-dithio-bis-(2-nitrobenzoic acid), ascorbic acid, trichloromethane, and methanol were obtained from SINOPHARM Chemical Reagent Co., Ltd. (Shanghai, China). The reagents used for SDS-PAGE were purchased from Guge Biotechnology Co., Ltd. (Wuhan, China). Lowry reagent was procured from Biosharp (Hefei, China). HiPrep 26/600 Sephacryl S-300 HR was purchased from GE Healthcare Bio-Science AB (Uppsala, Sweden). Plasma Extraction Hen egg yolk plasma was separated according to McBee's method (Bee and Cotterill, 1979). Fresh hen eggs were manually broken, and most albumen was eliminated. Yolks were carefully rolled on a filter paper to remove albumen and chalazion adhering to the vitelline membrane. This membrane was then perforated to collect unspoiled egg yolk in a beaker cooled in iced water. Yolk was diluted with an equal volume of 0.17 M NaCl solution and stirred with a magnetic stirrer for 1 h at 4°C. This solution was centrifuged at 15,000 × g for 15 min at 4°C, and the supernatant (plasma) was separated from the sediment (granules). Plasma was then centrifuged under the same conditions for complete removal of granules. Ammonium Sulfate Precipitation Ammonium sulfate of suitable saturation (20 to 45%) was added to 25 g of plasma to precipitate LDL, and the mixture was stirred for 1 h at 4°C and centrifuged at 15,000 × g for 15 min at 4°C. The precipitate was discarded, and the supernatant was dialyzed against deionized water for at least 6 h (the bath was changed every 2 h). The supernatant was then centrifuged at 10,000 × g for 30 min at 4°C. The resulting floating material containing LDL was pooled. PEG Precipitation Plasma (pH from 5.00 to 7.00) mixed with PEG of different polymerization degrees (PD; ranging from 4,000 to 20,000) and different concentrations (2, 4, 6, 8, and 10%) was stirred for 1 h at 4°C and centrifuged at 22,000 × g for 15 min at 4°C. The upper floating material was pooled, and the subnatant was discarded. Molecular Sieve Chromatography The extracted crude LDL was dissolved in 0.05 M Tris-HCl buffer (pH 8.5) containing 0.15 M NaCl and filtered through a 0.22 μm membrane. The sample was applied to a pre-packed column (26/600) of Sephacryl S-300 (High Resolution, Uppsala, Sweden) and eluted with 0.05 M Tris-HCl buffer (pH 8.5) containing 0.15 M NaCl at a flow rate of 1 mL/min. Each peak was collected through a fraction collector (F9-R, Uppsala, Sweden), and the purity of LDL and components of these was identified by SDS-PAGE analysis. SDS-PAGE Analysis Electrophoreses were run on polyacrylamide gels (stacking: 5% and resolving: 12%) with a migration buffer consisting of 0.025 M Tris (pH 8.3), 0.2 M Gly, and 0.1% SDS solution (DYY-12, Beijing, China). PageRuler Prestained Protein Ladder (26616, MBI, Thermo Scientific, Shanghai, China) with a molecular weight ranging from 10 kDa to 170 kDa was loaded to compare the molecular weight of LDL proteins. The protein bands were stained with Coomassie Brilliant Blue R-250 (0.1% in the staining solution containing 25% ethanol and 8% acetic acid) for 30 min at 45°C and de-stained by diffusion in de-staining solution (25% ethanol and 8% acetic acid). Protein Determination Protein content was determined by the modified procedure of Markwell (Markwell et al., 1978). The protein content (between 10 and 100 μg/mL) was calculated using a linear regression equation, with the absorbency of bovine serum albumin solution as a function of their concentration. Results were expressed as grams of protein per 100 g DM. Lipid Determination Lipid content was determined by the modified procedure of Folch (Folch et al., 1957). About 10 mL of chloroform–methanol (v/v, 2:1) were added to 1 g of sample, and the mixture was homogenized with a vortex mixer. After standing for 15 min, the mixture was centrifuged (5,000 × g for 10 min) and filtered. Subsequently, 5 mL of 0.88% NaCl were added to the filtrate to promote stratification. The organic phase was collected, and solvents were evaporated with a rotary evaporator (R201D, Gongyi, China). Lipid content was estimated by weighing lipid extract after solvent evaporation. Results were expressed as grams of lipid per 100 g DM. Sulfhydryl Determination Sulfhydryl content was measured by the reaction with Ellman's reagent [5,5'-dithio-bis-(2-nitrobenzoic acid)]. Approximately 0.5 mL of crude LDL solution (10 mg/mL) was mixed with 2.5 mL of 8 M urea in Tris-glycine buffer and 0.02 mL of 4 mg/mL Ellman's reagent. After reaction for 20 min, the absorbance at 412 nm was read. A molar extinction coefficient of 1.36 × 104 M−1cm−1 was used to convert absorbance to the sulfhydryl concentration (Beveridge et al., 1974; Zhao et al., 2013). Carbonyl Determination Carbonyl content was measured by the reaction with 2,4-dinitrophenyl hydrazine according to Levine (Levine et al., 1990). Results were expressed as micromoles of carbonyl groups per gram of LDL calculated with a molar extinction coefficient of 2.2 × 104 M−1cm−1. Malondialdehyde Determination Malondialdehyde (MDA) content was measured by the reaction with 2-thiobarbituric acid according to Tatum (Tatum et al., 1990). A standard solution of 1,1,3,3-tetraethoxypropane (4 to 32 μmol/L) was employed to make a calibration curve. The TBA–MDA adduct was quantitated with a fluorescence detector set at 515 nm excitation and 550 nm emission. The MDA content of samples was calculated according to the calibration curve. Statistical Analysis Except for the experimental design, 3 replicates were prepared for all the measurements. The results of separation and chemical analysis were subjected to Duncan analysis using SPSS. Confidence intervals were set at 95% (P < 0.05). RESULTS AND DISCUSSION Results of ASP The results of ASP are shown in Table 1. ASS significantly influenced the protein content and DM weight of crude LDL (P < 0.01). The results showed that 30% ASS could extract 57.72 g/100 g DM lipids and 69.41% protein, whereas 40% ASS could extract 56.80 g/100 g DM lipids and 73.78% protein. Table 1. Extraction results of ammonium sulfate precipitation. ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e Duncan analysis at a 95% confidence interval (P < 0.05). a-eIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Table 1. Extraction results of ammonium sulfate precipitation. ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e ASS 20% 25% 30% 35% 40% 45% Lipid content (g/100 g DM) 54.29 ± 1.3a,b 55.56 ± 1.17a,b 57.72 ± 2.9a 50.6 ± 4.44b 56.8 ± 3.16a,b 54.29 ± 3.78a,b Protein content (%) 85.19 ± 1.41a 63.72 ± 1.63d 69.41 ± 1.66c 66.17 ± 1.27d 73.78 ± 1.17b 48.65 ± 1.68e Duncan analysis at a 95% confidence interval (P < 0.05). a-eIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Compared with 40% ASS (Anton et al., 2003; Moussa et al., 2002), 30% ASS could extract more lipids (1.62% higher) and less protein (6.30% lower), but the difference was not significant. Fewer proteins were extracted, and subsequent purification was simplified. Thus, we considered 30% ASS as the optimized experimental condition of ASP. Results of PEG Precipitation Single-factor Experiment of PEG Precipitation In the single-factor experiment, three factors, namely, PEG PD (4% PEG and plasma pH 6.00), PEG concentration (PEG 6,000 and plasma pH 6.00), and plasma pH (4% PEG 6,000), were employed to develop unidirectional optimization experiments. The crude LDL extraction results (Figure 1A, C, and E) showed that the extraction rate of crude LDL increased significantly with increasing PD and PEG concentration (P < 0.05), and the most suitable plasma pH was 6.00 (similar to natural plasma pH 6.02). Some previous research has demonstrated that the PD and concentration of PEG can significantly affect protein extraction efficiency, which improved with the increase of PEG polymerization degree and concentration (Hammerschmidt et al., 2016; Li and Zydney, 2017). SDS-PAGE analysis results (Figure 1B, D, and F) showed that the molecular weight of most proteins in the subnatant of the PEG PD and plasma pH experiment were 100, 70, 60, and 35 to 40 kDa, whereas the major proteins in the subnatant of the PEG experiment were high and light chains of IgY. Figure 1. View largeDownload slide Crude LDL extraction results of single-factor experiment and corresponding SDS-PAGE analysis of the subnatant. A and B correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG PD, respectively; for SDS-PAGE analysis, lanes refer to PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, plasma, and PageRuler Prestained Protein Ladder 10–170 KD (26616) (marker). C and D correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG concentration, respectively; for SDS-PAGE analysis, lanes refer to 10, 8, 6, 4, and 2% PEG concentration, plasma, and marker. E and F correspond to crude LDL extraction rate and SDS-PAGE analysis result of plasma pH, respectively; for SDS-PAGE analysis, lanes refer to pH 7.00, pH 6.50, pH 6.00, pH 5.50, pH 5.00, plasma, and marker. All the analyses were carried out as Duncan analysis at a 95% confidence interval (P < 0.05). In each BAR, different letters mean significant variation, whereas the same letter means no significant variation. Figure 1. View largeDownload slide Crude LDL extraction results of single-factor experiment and corresponding SDS-PAGE analysis of the subnatant. A and B correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG PD, respectively; for SDS-PAGE analysis, lanes refer to PEG 4,000, PEG 6,000, PEG 8,000, PEG 20,000, plasma, and PageRuler Prestained Protein Ladder 10–170 KD (26616) (marker). C and D correspond to the crude LDL extraction rate and SDS-PAGE analysis result of PEG concentration, respectively; for SDS-PAGE analysis, lanes refer to 10, 8, 6, 4, and 2% PEG concentration, plasma, and marker. E and F correspond to crude LDL extraction rate and SDS-PAGE analysis result of plasma pH, respectively; for SDS-PAGE analysis, lanes refer to pH 7.00, pH 6.50, pH 6.00, pH 5.50, pH 5.00, plasma, and marker. All the analyses were carried out as Duncan analysis at a 95% confidence interval (P < 0.05). In each BAR, different letters mean significant variation, whereas the same letter means no significant variation. High PEG PD may keep more proteins in crude LDL, but low PD enables easy decondensation (Ramos et al., 2005). Thus, PEG 4,000, PEG 6,000, and PEG 8,000 were selected for the following orthogonal experiment. For the PEG experiment, more LDL were resolved in the subnatant even with a high crude LDL extraction rate. We selected 4 to 8% PEG concentration for the orthogonal experiments. Proteins in the subnatant of plasma pH optimization were almost similar to one another. The most crude LDL were extracted in pH 6.00, which was eventually selected. Orthogonal Experiment of PEG Precipitation On the basis of single-factor experiment results, we selected appropriate factor levels for orthogonal experiments (see Supporting Information Table S1). The range analysis results (Table 2) showed that PD of PEG exerted the highest influence on the lipid content and protein concentration of the subnatant, PEG concentration had the highest effect on crude LDL extract efficiency, and plasma pH had the largest effect on the protein content. Table 2. Orthogonal experimental result. Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 A: PEG PD, B: PEG concentration, C: plasma pH, D: Lipid content (g/100 g DM), E: protein content (%), F: crude LDL extract efficiency (%), G: protein concentration of subnatant (μg/mL). View Large Table 2. Orthogonal experimental result. Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 Experimental results Factor levels A B C Blank D E F G 1 1 1 1 1 65.03 82.89 90.51 149.78 2 1 2 2 2 68.14 69.91 90.10 134.89 3 1 3 3 3 65.21 94.11 91.86 134.22 4 2 1 2 3 66.00 82.83 90.13 165.56 5 2 2 3 1 60.68 89.46 90.59 175.11 6 2 3 1 2 64.57 84.56 91.00 165.33 7 3 1 3 2 60.58 88.97 90.88 178.89 8 3 2 1 3 63.29 78.64 91.65 202.22 9 3 3 2 1 60.16 82.09 92.82 212.44 R1 4.78 0.72 2.61 2.88 R2 3.31 7.58 12.57 4.05 R3 1.21 1.39 0.093 0.65 R4 58.22 6.00 9.70 19.41 A: PEG PD, B: PEG concentration, C: plasma pH, D: Lipid content (g/100 g DM), E: protein content (%), F: crude LDL extract efficiency (%), G: protein concentration of subnatant (μg/mL). View Large ANOVA (see Supporting Information Table S2) demonstrated that PD of PEG significantly affected the protein content (P < 0.05) and very significantly influenced the protein concentration of the subnatant (P < 0.01); PEG concentration significantly affected crude LDL extract efficiency (P < 0.05). Research showed that PD and concentration of PEG can significantly affect the extraction efficiency and purity of protein, while pH has no significant influence on either (Polson et al., 1964; Sommer et al., 2014). On the basis of range analysis and ANOVA results, we selected A1B2C2, with an oil extraction rate of 68.14 g/100 g DM, protein extraction rate of 69.91%, crude LDL extraction rate of 90.10%, and subnatant protein concentration of 134.89 μg/mL for LDL extraction and compared these values with the ASP method. Correlation analysis was conducted among the 4 experimental indices (see Supporting Information Table S3). The results showed a very significant negative correlation between the oil extraction rate and subnatant protein concentration, as well as a significant positive correlation between the crude LDL extraction rate and subnatant protein concentration. The oil extraction rate and protein extraction rate were negatively related. Comparison of 2 Separate Methods Comparison of Extract Results ASP extracted 56.80 g/100 g DM lipid and 73.78% protein in 40% ASS. In 30% ASS, the extract efficiency of lipid and protein was 57.72 g/100 g DM and 69.41%, respectively. The optimized PEG precipitation extracted 68.14 g/100 g DM lipid and 69.91% protein. To compare the extraction efficiency of the 2 extraction methods, the ANOVA results showed that the oil extraction rate of crude LDL extracted by PEG precipitation is very significantly higher than the ASP-LDL (P < 0.01), and the protein extraction rate of the 2 samples has no significant difference, which indicates that PEG precipitation could obtain more crude LDL than ASP and allowed a simple purification procedure. Comparison of Crude LDL Oxidation Degree In this section, we selected Vc (0.02% additive amount) as an exogenous antioxidant to inhibit LDL oxidation (Pantelidis et al., 2007; Rice-Evans et al., 1997). The sulfhydryl, carbonyl, and MDA contents were selected to compare the oxidation degree of crude LDL extracted by the 2 methods. The results are shown in Table 3. Table 3. Comparison of crude LDL oxidation degree. Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Duncan analysis at a 95% confidence interval (P < 0.05). ND = no detection. **= P < 0.01. NS = no significance. a,bIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large Table 3. Comparison of crude LDL oxidation degree. Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Oxidation index Sample Vc No Vc Sulfhydryl content (μM/g) PEG-LDL 23.24 ± 0.7a 20.13 ± 0.67b ASP-LDL 18.49 ± 0.79a 16.74 ± 0.23b P ** ** Carbonyl content PEG-LDL ND ND ASP-LDL ND ND MDA content (nM/g) PEG-LDL 0.024±0.0004b 0.046±0.0025a ASP-LDL 0.042±0.0014a 0.048±0.0029a P ** NS Duncan analysis at a 95% confidence interval (P < 0.05). ND = no detection. **= P < 0.01. NS = no significance. a,bIn the top-right of data, different letters mean significant variation, whereas the same letter means no significant variation. View Large The results showed that no carbonyl was detected in all the crude LDL samples. Compared with the no Vc addition group, sulfhydryl contents are significantly high when antioxidant is added in both LDL samples (P < 0.05). In other ways, the sulfhydryl content of PEG-LDL was very significantly higher than that in ASP-LDL with or without Vc addition (P < 0.01). For the PEG-LDL sample, Vc addition can significantly decrease the MDA content (P < 0.05). With antioxidant added, the MDA content of PEG-LDL was very significantly lower than that in ASP-LDL (P < 0.01). However, no significant difference was detected between the 2 LDL samples with no Vc addition. The results indicated that both LDL samples developed no protein oxidation (Zhang et al., 2018), and crude LDL extracted by PEG precipitation has a lower oxidation degree of hydrosulfuryl (SH) groups (Jiang et al., 2017) and lipids (Smialowska et al., 2017) than that in ASP-LDL. We also found that ASP was a diluting procedure that may cause further loss of target compounds during dialysis, centrifugation, and purification (Papala et al., 2017). By contrast, PEG could condense the target compounds during extraction and ensured a credible calculation of purified LDL after quantitative dilution and purification based on the water content of crude LDL. Purification of LDL LDL are the largest compounds in hen egg yolk plasma (Anton, 2013; Martin et al., 1964). LDL can be effectively purified via molecular sieve chromatography. We employed Sephacryl S-200 HR (16/600) and Sephacryl S-300 HR (26/600) to purify LDL. Both LDL (Jolivet et al., 2008) and IgY (Tan et al., 2012) were detected in crude LDL. Sephacryl S-200 HR (separation range of 5 to 250 kDa) could not completely separate LDL from IgY (data not shown). Three eluting peaks were obtained when crude LDL were applied to the Sephacryl S-300 HR column (Figure 2A). Figure 2. View largeDownload slide Sephacryl S-300 HR (26/600) chromatography of crude LDL (A) and SDS-PAGE analysis of collected fractions (B). Three main peaks were obtained. Fractions 1 to 6 were collected with a volume of 5 mL, fractions 8 to 12 were collected with a volume of 10 mL, and the remaining fractions were collected with a volume of 20 mL. Fractions collected by Sephacryl S-300 HR (26/600) both at 1 and 1.5 mL/min were applied to SDS-PAGE analysis. Lanes 1 to 5 were fractions of 1.0 mL/min. Lane 1 = fraction 1, lane 2 = fractions 3 to 5, lane 3 = fractions 6 to 8, lane 4 = fractions 18 to 21, and lane 5 = fractions 30 to 32. Lanes 6 to 10 were fractions of 1.5 mL/min. Lane 6 = fraction 1, lane 7 = fractions 3 to 5, lane 8 = fractions 6 to 9, lane 9 = fractions 15 to 18, lane 10 = fractions 22 and 23, and lane 11 = marker. Figure 2. View largeDownload slide Sephacryl S-300 HR (26/600) chromatography of crude LDL (A) and SDS-PAGE analysis of collected fractions (B). Three main peaks were obtained. Fractions 1 to 6 were collected with a volume of 5 mL, fractions 8 to 12 were collected with a volume of 10 mL, and the remaining fractions were collected with a volume of 20 mL. Fractions collected by Sephacryl S-300 HR (26/600) both at 1 and 1.5 mL/min were applied to SDS-PAGE analysis. Lanes 1 to 5 were fractions of 1.0 mL/min. Lane 1 = fraction 1, lane 2 = fractions 3 to 5, lane 3 = fractions 6 to 8, lane 4 = fractions 18 to 21, and lane 5 = fractions 30 to 32. Lanes 6 to 10 were fractions of 1.5 mL/min. Lane 6 = fraction 1, lane 7 = fractions 3 to 5, lane 8 = fractions 6 to 9, lane 9 = fractions 15 to 18, lane 10 = fractions 22 and 23, and lane 11 = marker. Subsequent SDS-PAGE analysis revealed that LDL and IgY corresponded with the first and second peaks, respectively (Figure 2B), and the 2 constituents reached electrophoretic purity. With a separation range of 10 to 1,500 kDa, Sephacryl S-300 HR could obtain purified LDL and IgY at flow rates of 1.0 and 1.5 mL/min. Compared with previous experiments (Jolivet et al., 2006; Jolivet et al., 2008), LDL obtained in our experiment had high purity with no other protein between 20 and 50 kDa. Co-purification of IgY The main proteins in egg plasma are LDL and livetin (Hatta et al., 2008). The 3 types of livetin are α, β, and γ livetin. γ livetin results in immunity, and its major fraction is called IgY. Several studies have investigated the purification of IgY because of its unique biophysical function and high requirements for purity and bioactivity (Dong et al., 2008; Gee et al., 2003; Hodek et al., 2013). The previous experiments have proved that the molecule weight of LDL is much larger than IgY (Juneja, 2008), and PEG can be used for antibody extraction without denaturation (Chen et al., 2017; Kuczewski et al., 2011); thus; in our experiment, the remaining IgY can be easily purified following the purified LDL. The SDS-PAGE analysis results of the orthogonal experiment (Figure 3) showed that a variation in the 3 experiment factors could alter the IgY content in the subnatant. Figure 3. View largeDownload slide SDS-PAGE analysis results of orthogonal experiment. A) SDS-PAGE analysis of the subnatant. Lanes M to 9 are the marker and subnatant of experiments 1 to 9. B) SDS-PAGE analysis of crude LDL. Lanes 1 to M are 1 to 9 crude LDL and marker. Figure 3. View largeDownload slide SDS-PAGE analysis results of orthogonal experiment. A) SDS-PAGE analysis of the subnatant. Lanes M to 9 are the marker and subnatant of experiments 1 to 9. B) SDS-PAGE analysis of crude LDL. Lanes 1 to M are 1 to 9 crude LDL and marker. CONCLUSIONS In this study, we constructed an extraction method of high efficiency and low oxidative degree for hen egg yolk LDL using PEG, which demonstrated higher LDL yield and purity than ASP. The optimized experimental conditions of 5% PEG 4,000 and pH 6.00 with 0.02% Vc addition were obtained. In PEG-LDL, the oil concentration was very significantly higher than ASP-LDL(P < 0.01), while the protein concentration showed no significant difference between both crude LDL samples. The oxidation degree of SH groups and lipids in PEG-LDL was very significantly lower than ASP-LDL (P < 0.01), and no LDL protein oxidation was detected in either sample. Thus, PEG precipitation may allow a large amount of extraction of hen egg yolk LDL and benefit other fundamental research on LDL. We employed HiPrep 26/600 Sephacryl S-300 HR molecular sieve chromatography to purify LDL, and at the same time obtained purified IgY. If a further experiment was developed, a more effective extraction method of LDL and IgY could be constructed. SUPPLEMENTARY DATA Supplementary data are available at Poultry Science online. Appendix A. Supplementary Material Supplementary data associated with this article can be found in the online version. ACKNOWLEDGMENTS This work was supported by the Earmarked Fund for Modern Agro-industry Technology Research System (No. CARS-41-K23) and Chinese Ministry of Agriculture nonprofit industry special research project (No.201303084). REFERENCES Amirat-Briand L. , Bencharif D. , Vera-Munoz O. , Pineau S. , Thorin C. , Destrumelle S. , Desherces S. , Anton M. , Jouan M. , Shmitt E. . 2010 . In vivo fertility of bull semen following cryopreservation with an LDL (low density lipoprotein) extender: Preliminary results of artificial inseminations . Anim. Reprod. Sci. 122 : 282 – 287 . Google Scholar CrossRef Search ADS PubMed Anton M. 2007 . 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Poultry ScienceOxford University Press

Published: Mar 8, 2018

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