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A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate
A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate Chenyao Tong, Fang Geng, Zhenjiao He, Zhaoxia Cai,1 and Meihu Ma1 National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
Key words: chicken egg yolk immunoglobulin, isolation, polysaccharide, delipidation 2015 Poultry Science 94:104–110 http://dx.doi.org/10.3382/ps/peu005
INTRODUCTION
considerable attention as a means of preventing and controlling such diseases as bovine mastitis, diarrhea in piglets, campylobacteriosis, and shrimp white spot syndrome virus (Wang et al., 2011). In addition, IgY can be used as an immunological supplement in infant formula and other food (Facon et al., 1993). However, the practical use of IgY in research and diagnostics is limited because of necessary complex and time-consuming purification steps. The key to isolating IgY from egg yolk is to remove the water-insoluble components such as lipids and lipoproteins to get water-soluble protein fraction (WSF). Numerous methods are used to isolate IgY, including water dilution (Akita and Nakai, 1993), salt precipitation (Jensenius et al., 1981), high polymer precipitation (Polson et al., 1985), organic solvent extraction (Hamada et al., 1991), ultrafiltration (Kim and Nakai, 1998), and chromatography (Ko and Ahn, 2007). However, most of these methods have drawbacks, such as low IgY yield, complexity of procedures, or compatibility with human uses. Among the various methods, Chang et al. (2000) suggested that anionic polysaccharides could interact with
Chicken egg yolk has been considered an ideal source of immunoglobulin, and IgY is understood to be the predominant antibody in chicken egg yolk (Hamal et al., 2006). It has many significant advantages over mammalian IgG. First, the production of IgY is noninvasive, which makes it suitable for large-scale production (∼40 g IgY/y/hen). Second, because of the phylogenetic distance between chickens and mammals, IgY does not interact with rheumatoid factors, bacterial fragment crystallizable (Fc) receptors, or activate mammalian complement (Hadge and Anbrosuius, 1984). Also, IgY can recognize more epitopes of the highly conserved mammalian proteins than other mammalian IgGs are able to, so IgY can induce an efficient immune response (Edzard et al., 2012). IgY has also attracted C 2014 Poultry Science Association Inc. Received November 12, 2014. Accepted 0, 2014. 1 Corresponding authors: Zhaoxia Cai: E-mail: [email protected], Tel.: +86 27 87283177; Meihu Ma: E-mail: [email protected]; Tel.: +86 27 87283177
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conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energyand time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale.
ABSTRACT Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4◦ C before being centrifuged at 4◦ C. The resulting supernatant was added to 35% (w/v) (NH4 )2 SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to
THE ISOLATION OF EGG YOLK IMMUNOGLOBULIN
MATERIALS AND METHODS Materials Fresh eggs laid within 24 h were purchased from a local hennery (Jiufeng Farm, Wuhan, China). Pectin, λ-carrageenan, dextran sulfate, carboxymethylcellulose, and methylcellulose were purchased from Sigma-Aldrich (St. Louis, MO). The reagents used in SDS-PAGE were purchased from Guge Biotechnology (Wuhan, China), and the prestained protein molecular weight markers were supplied by Fermentas China Co., Ltd. (Shenzhan, China). Horseradish peroxidase (HRP)–conjugated rabbit antichicken IgG was from Bioss (Beijing, China). The standard of IgY was purchased from Abcam (Shanghai, China). All the other chemicals used in this experiment were of analytical grade and obtained from Sinopharm Chemical Reagent (Shanghai, China). The distilled water used for all the experiments was produced by a water purification system.
Separation of IgY Egg yolk was diluted with 6-fold delipidation solution made from distilled water of 5 different concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) and 5 different types of polysaccharides (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate), respectively. The diluted yolk was mixed with a magnetic stirrer for 1 h at 4◦ C. Then the pH of the diluted sample was adjusted to 5.0 by HCl (1 M) and
was kept overnight at 4◦ C before being centrifuged at 10,000 × g for 25 min at 4◦ C. The pellets (e.g., lipids and lipoproteins) were discarded, and the supernatant was collected. Ammonium sulfate was added to the supernatant to a final concentration of 35% (w/v), and the mixture was stirred for 2 h at 4◦ C and then centrifuged at 12 000 × g for 25 min at 4◦ C. Finally, the pellets were dissolved in distilled water, and excess salt was removed through dialyze.
Determination of Protein Concentration IgY concentration was determined using a quartz cuvette at 280 nm and an extinction coefficient of 1.33 (Pauly et al., 2011) (NANO 2000C, Thermo, USA).
HPLC The purity of the optimized products was analyzed by high-performance liquid chromatography (HPLC), and the purity was calculated simultaneously. Reverse phase chromatography was performed on a GraceVydacC4 (214TP, 5 μm, 250 × 4.6 mm) column with a Waters 2695 Separations Module (Waters). A linear gradient of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) was used for elution at a flow rate of 1.0 mL/min. The linear gradient elution increased from 5 to 80% ACN in 15 min, decreased to 5% ACN in 1 min, then remained at a constant concentration for 4 min (Geng et al., 2012). The temperature of the column was kept constant at 25◦ C. Detection was carried out at 280 nm with a Waters 2696 photodiode array detector.
SDS-PAGE SDS-PAGE was performed under both reducing and nonreducing conditions. SDS gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (10% acryl-amide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel. Approx. 10 μg protein sample was loaded on the gel. After migration, gels were stained with Coomassie brilliant blue R-250 composed of 25% ethanol and mixed with 8% acetic acid for 30 min at 4◦ C. Destaining steps were carried out after immersion of the gels in 7% acetic acid. The molecular weights and purity of IgY were estimated by Gel-Pro Analyzer 4.0 (Media Cybernetics).
Western Blotting After nonreducing SDS-PAGE, the samples on gel were transferred to a nitrocellulose membrane under semidry conditions for 40 min at 10 V, kept in blocking buffer containing bovine serum albumin (BSA) for 1 h, and incubated with rabbit antichicken IgG conjugated
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lipoproteins to isolate IgY, but they just focused on the interactions between polysaccharides and lipoproteins; they did not consider the purification of IgY. Souza et al. (2013) further pointed out that polysaccharides and lipoproteins could form complex coacervates, and the changes of pH value in egg yolk solution influenced the extent of the precipitation of polysaccharidelipoprotein complexes. Tan et al. (2012) reported a rapid way to isolate IgY using a combination solution followed by ammonium sulfate. But the yield and purity of IgY isolated in this way were not very satisfying, mainly because many of their factors (e.g., pH, types and concentrations of polysaccharides, and diluted rate) were not in the optimized condition (Luo et al., 2010). In this study, we adopted a simple, safe, and effective extraction method using delipidation solution to remove insoluble lipids and lipoproteins in the first step, followed by ammonium sulfate precipitation. Polysaccharides, as safe and nontoxic materials widely existing in nature, can be used as food additives, so the isolated IgY can be applied to large-scale production in the food industry and medical field. Also, the whole process can be easily implemented through 2-step precipitation to obtain a relatively high yield and purity, and the production of IgY through this method may be scaled up to commercial quantities.
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with horseradish peroxidase for 30 min. Thus, the color reaction could be observed after using diaminobenzidine solution for coloration.
ELISA
Statistical Analysis All samples were observed in triplicate, and data were analyzed using the IBM SPSS statistical software. Oneway ANOVA was calculated, and Duncan test was employed (P < 0.05) to separate means.
RESULTS AND DISCUSSION Optimization of the Isolated IgY Figure 1 shows the effect of concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) and types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) of delipidation solutions on the yield and purity of the isolated IgY. The reason these delipidation solutions could remove lipids and lipoproteins effectively was that polysaccharides could interact with egg yolk lipoproteins to form complex coacervations, whose formation was based on ionic bonds, hydrophobic interactions, and hydrogen bonds (Souza et al., 2013). In addition, the force of each bond of the polysaccharide-lipoprotein complexes varied with the types of the polysaccharides. The charge density, the conformational structures of the polysaccharides, and the dissociation constant of carbonyl and sulfate groups all contributed to the different effects on the separation of IgY.
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The antibody immunoactivity was tested by ELISA (Wen et al., 2012) with some modifications. Standards of IgY and isolated IgY were dissolved in carbonatebicarbonate buffer (0.05 M, pH 9.6) and diluted to the final concentration of 200 ng/L. Each well of the plate (Probe, Shanghai, China) was coated with 50 μL of the IgY samples, respectively, and was incubated overnight at 4◦ C. Plates were washed 5 times with 100 μL phosphate buffer solution containing 0.05% Tween 20 (PBS-Tw), followed by a blocking step using 100 μL/well of 3% BSA for 1 h at 37◦ C. The wells were washed with PBS-Tw 5 times. Then 100 μL/well (HRP)-conjugated rabbit antichicken IgY (1:5000) (Probe, Shanghai, China) was added to each well. After incubated at 37◦ C for 30 min in the precoated and blocked plate for 1 h the bound IgY was detected. Then the plate was washed with PBS-Tw, 50 μL/well of tetramethylbenzidin substrate solution was added onto the plate, and the corresponding reaction continued for 15 min at room temperature. The reaction was stopped by 2 M sulfuric acid, and the developed color was measured at 490 nm.
Pectin, which is obtained from cells and the intercellular walls of plants and fruits, is a kind of anionic commercial polysaccharide with acid D-galacturonic unities. The pKa is around 3.6, and its main monomer is glucoronate (Jafar and Fadia, 2010). As a natural polymer with good gelling and emulsifying stability, pectin has been widely used in food, pharmaceutical, and other industries. Figure 1A shows that the yield of IgY increased with pectin concentration increasing from 0.01 to 0.1%. The yield reached the maximum value (8.36 mg/mL) at 0.1% pectin. When the concentration of pectin increased further, the yield decreased rapidly and then rose slowly. The purity of IgY was relatively stable with the various pectin concentrations. It reached the highest point (83.3%) at 0.1% pectin and then decreased when the concentration of pectin increased further. Thus, 0.1% pectin was found to be the optimal condition to isolate IgY. However, Chang et al. (2000) reported that 0.15% pectin was the optimal condition for the removal of lipoproteins. The difference in optimal pectin concentration between this reference and our work might due to standing time in process. Under the action of hydrogen ions, IgY molecules lose the protection of charge repulsion and hydration film to polymerize into large particles and deposited (Bruno and Andre, 2001). Thus, prolonging standing time could enhance the separation efficiency of IgY. In our experiment, we chose 10 h as the standing time and used a 0.1% concentration of pectin delipidation solution to improve the yield and purity of IgY. Carrageenan obtained from several genera and species of marine algae class Rodophyta is a kind of anionic polysaccharide polymerized by sulfated galactan (Ruiter and Rudolph, 1997), and the main characteristics of carrageenan’s biopolymer formation are its linear/helical structures with pKa around 4.3. Carrageenan falls into three different categories: kappa (κ), iota (ι), and lambda (λ) (Shchipunov, 2003). Figure 1B shows that the yield of IgY reduced during the whole period with an increased concentration of λ-carrageenan. Also, it can be seen that purity increased with the concentration of λ-carrageenan from 0.01% to 0.1% and then decreased. When isolating IgY, the optimal condition for λ-carrageenan is 0.1%. In this best condition, the yield and purity were 7.64 mg/mL and 80.94%, respectively, which was consistent with the results of a previous study (Hatta et al., 1990). Some researchers have also suggested that additional λ-carrageenan can result in effective delipidation of egg yolk solution (Kim and Nakai, 1996). However, comparing the two methods, we found the addition of λcarrageenan was not as effective as the addition of pectin in improving the purity and yield of IgY. Dextran sulfate is a kind of anionic polysaccharide esterified by glucan and chlorosulfonic acid. Figure 1C shows that when isolating IgY, the optimal condition for dextran sulfate is 0.1%, with the yield reaching 9.31 mg/mL and purity 80.1%. Jensenius et al. (1981) were the first to introduce the use of dextran sulfate in the
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extraction of IgY from egg yolk. They added a certain amount of dextran sulfate and calcium chloride to water-soluble protein fraction (WSF) centrifuged by 4-fold Tris-HCl buffer. After centrifuging, the precipitate was removed and the supernatant mixed with sodium sulfate. The purity of IgY reached approx. 80%. Based on these previous studies, our approach simplified the entire isolation procedure from 3 to 2 steps, leading to both saved time and improved purity. Carboxymethylcellulose obtained from plants is also a kind of anionic polysaccharide polymerized by methylated glucose. Its hydroxyl groups from glucose, which is partly replaced by –CH2 –COOH via the Williamson reaction. This process leads to the formation of modified cellulose’s functional properties, such as high solubility and viscosity in cold or hot water. Its pKa is approx.
2.0 (Kita et al., 2007). As can be seen in Figure 1D, the yield of IgY reached its lowest yield of 4.46 mg/mL at a concentration of 0.05%, but at the same concentration of 0.05%, the purity of IgY reached its highest point, 81.23%. It was also found that when isolating IgY, the optimal condition for carboxymethylcellulose is carboxymethylcellulose 0.1%, with yield reaching 8.1 mg/mL and purity 79.94%. These findings consistent with earlier results (Hung, 2000). Methylcellulose is a kind of nonionic polysaccharide polymerized by methylated glucose, and its hydroxyl groups from glucose are partly replaced by –CH3 . Because of the nonionic property of methylcellulose, it can be compatible with other emulsifiers. It can be seen in Figure 1E that with the increase of methylcellulose from 0.01 to 0.15%, the yield and purity of IgY slightly rose
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Figure 1. Effect of concentrations and types of delipidation solutions on the yield and purity of IgY. (A) Pectin, (B) λ -carrageenan, (C) dextran sulfate, (D) carboxymethylcellulose, (E) methylcellulose. There was a difference (P < 0.05) between all the yield data.a,b,c Values with no common letters differ significantly (P < 0.05) (n = 3).
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and then decreased. Thus, methylcellulose 0.15% was the best isolating concentration, with the yield of IgY reaching 8.13 mg/mL and purity 78.17%. Because carboxymethylcellulose and methylcellulose were similar in structure but different in ionic types, they were chosen to indicate the effect of ionic types of polysaccharides on the isolation of IgY. The results of this study revealed that the yield of isolated IgY through delipidation solutions containing carboxymethylcellulose and solutions containing methylcellulose had no significant difference, but the purity of IgY isolated through solutions containing methylcellulose was lower than IgY isolated through carboxymethylcellulose. These results might be due to interactions between polysaccharides and lipoproteins that partly depended on ionic bonds.
Quality Evaluation of IgY It can be concluded that optimal conditions for using pectin, λ-carrageenan, dextran sulfate, or carboxymethylcellulose to isolate IgY were at a concentration of 0.1%, and the optimal methylcellulose concentration was 0.15%. Figure 2 presents the separation effect of IgY with the use of these optimized delipidation solutions. IgY is a big biological molecule with a molecular weight of 180 KDa. IgY consists of two heavy chains weighing approx. 65 KDa and two light chains of approx. 28 KDa. Under reducing condition, the disulfide bond could easily be broken, which resulted in the separation between heavy chain and light chain. As can be seen from Figure 2, all the samples had two major bands, one of which corresponds to the heavy chain and one of which corresponds to the light chain of IgY. Some other bands also could be observed, which might be the β -livetin or phosvitin around 35 to 55 KDa. Although the bands indicated that there was no significant difference among the 5 different delipidation solutions, analysis by Gel-Pro Analyzer confirmed that the purity of IgY isolated by delipidation solutions containing pectin was highest among the types of delipidation solutions,
Figure 3. Identification of IgY by Western blot. Lane 1 = standard IgY; lane 2 = marker; lanes 3 to 7 = purified IgY by delipidation solution containing 0.1% pectin, 0.1% λ -carrageenan, 0.1% dextran sulfate, 0.1% carboxymethylcellulose, and 0.15% methylcellulose, respectively, with 35% ammonium sulfate.
and the purity of IgY isolated by delipidation solutions containing methylcellulose was the lowest. Figure 3 shows the Western blot results of the obtained IgY. We could compare those results with bands of the standard IgY and the samples, and infer there was a specific combination of rabbit antichicken IgG conjugated with horseradish peroxidase and thus the target protein was IgY. The results showed that IgY isolated by delipidation solution containing 0.1% pectin provided the highest purity. IgY isolated in this optimized delipidation solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC chromatogram displayed a single symmetrical peak (Figure 4) with a retention time of 16.07 min. The analysis was carried out in triplicate, and an average purity of 83.0 ± 0.3% was obtained by the percentage of the peak area.
Immunoreactivity of IgY Biological activity of the protein is an important indicator of the process of separation and purification. During the purification process, IgY suffered the loss of antigen binding activity to a certain extent, and the immunoreactivity of the purified IgY was determined by ELISA. The results are displayed in Figure 5. The value of absorbance of IgY standard was 0.504 ± 0.4. Because the 5 kinds of polysaccharides were all relatively mild (they are even used as food additives), the isolated IgY kept a high immunoreactivity level. However, it also could be seen that the immunoreactivity of IgY suffered a loss as the concentration of polysaccharides increased. Delipidation solutions with a lower concentration of polysaccharides (<0.05%) could help keep the immunoreactivity of IgY at a relatively high level, whereas a higher concentration (>0.05%) would result in a relatively high loss. It could also be observed that IgY isolated by
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Figure 2. The SDS-PAGE patterns of IgY obtained from delipidation solutions. Lane 1 = protein marker; lanes 2 to 6 = purified IgY by delipidation solution containing 0.1% λ -carrageenan, 0.1% carboxymethylcellulose, 0.15% methylcellulose, 0.1% dextran sulfate, and 0.1% pectin, respectively, with 35% ammonium sulfate.
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Figure 4. HPLC elution profile of IgY obtained by delipidation solution containing 0.1% pectin with 35% ammonium sulfate at 280 nm
delipidation solutions containing pectin maintained a higher immunoreactivity level than IgY isolated by the other 4 delipidation solutions. In addition, IgY isolated by λ-carrageenan and dextran sulfate maintained the lowest immunoreactivity levels. The findings might be explained by the adverse effect caused by the sulfate groups of polysaccharides. The final protocol is shown in Figure 6.
Figure 6. Schematic diagram of the separation of IgY from egg yolk
such as ultrafiltration or use of chromatography. Moreover, the polysaccharides used in this method are all nontoxic, and they are even widely used as food additives, which ensured the safety of isolated IgY. Thus, it might be applied to large-scale food production.
CONCLUSION Under the optimized condition of 0.1% pectin, the yield and the purity of IgY could reach 8.36 mg/mL and 83.3%, respectively, with a minimal negative effect on the immunoreactivity of IgY. Based on our experimental results, a delipidation solution made from polysaccharides coupled with (NH4 )2 SO4 precipitation provides an effective and simple separation method for IgY, although the purity is not very high. However, considering the focus on developing a process of larger production, the yield is relatively satisfying. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods
ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 31371810). Project 2013PY036 was supported by the Fundamental Research Funds for the Central Universities.
REFERENCES Akita, E. M., and S. Nakai. 1993. Comparison of four purication methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. J. Immunol. Methods. 160:207–214.
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Figure 5. Effect of delipidation solutions on the immunoreactivity of IgY. At each concentration point, data followed by different letters are significantly different (P < 0.01).
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TONG ET AL. Jensenius, J. C., I. Andersen, J. Hau, M. Crone, and C. Koch. 1981. Eggs: Conveniently packaged antibodies. Methods for purification of yolk IgG. J. Immunol. Methods. 46:63–68. Kim, H., and S. Nakai. 1996. Immunoglobulin separation from egg yolk: A serial filtration system. J. Food Sci. 61:510–514. Kim, H., and S. Nakai. 1998. Simple separation of immunoglobulin from egg yolk by ultrafiltration. J. Food Sci. 63:485–490. Kita, K., F. Korlos, and V. Kiosseoglou. 2007. Improvement, by dryheating, of the emulsion-stabilizing properties of a whey protein concentrate obtained through carboxymethylcellulose complexation. Food Chem. 104:1153–1159. Ko, K. Y., and D. U. Ahn. 2007. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. Poult. Sci. 86:400–407. Luo, Y., R. S. Xiong, L. F. Hou, J. D. Li, and J. C. Chen. 2010. Process for coproduction of crude immunoglobulin Y and highdensity lipoproteins from hen egg yolk. J. Agric. Food Chem. 58:11420–11427. Polson, A., T. Coetzer, J. Kruger, E. von Maltzahn, and K. J. van der Merwe. 1985. Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol Invest. 14:323– 327. Pauly, D., P. A. Chacana, E. G. Calzado, B. Brembs, and R. Schade. 2011. IgY technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J. Vis. Exp. 51:3791–3084. Ruiter, G. A. E., and B. Rudolph. 1997. Carrageenan biotechnology. Trends. Food. Sci. Tech. 8:389–395. Shchipunov, Y. A. 2003. Sol-gel-derived biomaterials of silica and carrageenans. J. Colloid. Interf. Sci. 268:68–76. Tan, S. H., A. Mohamedalia, A. Kapur, L. Lukjanenko, and M. S. Baker. 2012. A novel, cost-effective and efficient chicken egg IgY purification procedure. J. Immunol. Methods. 380:73–76. Wang, L. H., X. Y. Li, L. J. Jin, J. S. You, Y. Zhou, S. Y. Li, and Y. P. Xu. 2011. Characterization of chicken egg yolk immunoglobulins (IgYs) specific for the most prevalent capsular serotypes of mastitis-causing Staphylococcus aureus. Vet. Micro. 149:415–421. Wen, J. L., S. Q. Zhao, D. G. He, Y. N. Yang, Y. M. Li, and S. S. Zhu. 2012. Preparation and characterization of egg yolk immunoglobulinY specific to influenza B virus. Antivir. Res. 93:154–159.
Downloaded from http://ps.oxfordjournals.org/ at University of New England on January 17, 2015
Al-Hakkak, J., and J. F. Al-Hakkak. 2010. Functional egg whitepectin conjugates prepared by controlled maillard reaction. J. Food Eng. 100:152–159. Bruno, M., and H. Andre. 2001. Isolation and purification of chicken egg yolk immunoglobulins: A review. Food. Agr. Immunol. 13:275–288. Chang, H. M., T. C. Lu, C. C. Chen, Y. Y. Tu, and J. Y. Hwang. 2000. Isolation of immunoglobulin from egg yolk by anionic polysaccharides. J. Agric. Food Chem. 48:995–999. Clitor, J. F. S., E. Edwin, R. Garcia, R. M. Nath´ alia, A. Gaspar, and J. F. C. Lins. 2013. Complex coacervates obtained from interaction egg yolk lipoprotein and polysaccharides. Food Hydrocolloid. 30:375–381. Edzard, S., B. Ingke, G. Kerstin, S. Henning, B. Simon, and P. Dion. 2012. Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy. Biologicals. 40:313– 322. Facon, M., B. J. Skura, and S. Nakai. 1993. Potential for immunological supplementation of foods. Food Agric. Immunol. 5: 85–91. Geng, F., Q. Huang, X. F. Wu, G. D. Ren, Y. Y. Shan, G. F. Jin, and M. H. Ma. 2012. Co-purification of chicken egg white proteins using polyethylene glycol precipitation and anion-exchange chromatography. Sep. Purif. Technol. 96:75–80. Hadge, D., and H. Anbrosuius. 1984. Evolution of low molecular weight immunoglobulins-IV: IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Mol. Immunol. 21:699–707. Hatta, H., M. Kim, and T. Yamamoto. 1990. A novel isolation method for hen egg yolk antibody, “IgY”. Agric. Biol. Chem. 54:2531–2535. Hamada, S., T. Horikoshi, T. Minami, S. Kawabata, J. Hiraoka, T. Fujiwara, and T. Ooshima. 1991. Oral passive immunization against dental caries in rats by use of egg yolk antibodies specific for cell-associated glucosyltransferase of streptococcus mutants. Infect. Immun. 59:4161–4167. Hamal, K. R., S. C. Burgess, I. Y. Pevzner, and G. F. Erf. 2006. Maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens. Poult. Sci. 85:1364– 1372.
Key words: chicken egg yolk immunoglobulin, isolation, polysaccharide, delipidation 2015 Poultry Science 94:104–110 http://dx.doi.org/10.3382/ps/peu005
INTRODUCTION
considerable attention as a means of preventing and controlling such diseases as bovine mastitis, diarrhea in piglets, campylobacteriosis, and shrimp white spot syndrome virus (Wang et al., 2011). In addition, IgY can be used as an immunological supplement in infant formula and other food (Facon et al., 1993). However, the practical use of IgY in research and diagnostics is limited because of necessary complex and time-consuming purification steps. The key to isolating IgY from egg yolk is to remove the water-insoluble components such as lipids and lipoproteins to get water-soluble protein fraction (WSF). Numerous methods are used to isolate IgY, including water dilution (Akita and Nakai, 1993), salt precipitation (Jensenius et al., 1981), high polymer precipitation (Polson et al., 1985), organic solvent extraction (Hamada et al., 1991), ultrafiltration (Kim and Nakai, 1998), and chromatography (Ko and Ahn, 2007). However, most of these methods have drawbacks, such as low IgY yield, complexity of procedures, or compatibility with human uses. Among the various methods, Chang et al. (2000) suggested that anionic polysaccharides could interact with
Chicken egg yolk has been considered an ideal source of immunoglobulin, and IgY is understood to be the predominant antibody in chicken egg yolk (Hamal et al., 2006). It has many significant advantages over mammalian IgG. First, the production of IgY is noninvasive, which makes it suitable for large-scale production (∼40 g IgY/y/hen). Second, because of the phylogenetic distance between chickens and mammals, IgY does not interact with rheumatoid factors, bacterial fragment crystallizable (Fc) receptors, or activate mammalian complement (Hadge and Anbrosuius, 1984). Also, IgY can recognize more epitopes of the highly conserved mammalian proteins than other mammalian IgGs are able to, so IgY can induce an efficient immune response (Edzard et al., 2012). IgY has also attracted C 2014 Poultry Science Association Inc. Received November 12, 2014. Accepted 0, 2014. 1 Corresponding authors: Zhaoxia Cai: E-mail: [email protected], Tel.: +86 27 87283177; Meihu Ma: E-mail: [email protected]; Tel.: +86 27 87283177
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conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energyand time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale.
ABSTRACT Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4◦ C before being centrifuged at 4◦ C. The resulting supernatant was added to 35% (w/v) (NH4 )2 SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to
THE ISOLATION OF EGG YOLK IMMUNOGLOBULIN
MATERIALS AND METHODS Materials Fresh eggs laid within 24 h were purchased from a local hennery (Jiufeng Farm, Wuhan, China). Pectin, λ-carrageenan, dextran sulfate, carboxymethylcellulose, and methylcellulose were purchased from Sigma-Aldrich (St. Louis, MO). The reagents used in SDS-PAGE were purchased from Guge Biotechnology (Wuhan, China), and the prestained protein molecular weight markers were supplied by Fermentas China Co., Ltd. (Shenzhan, China). Horseradish peroxidase (HRP)–conjugated rabbit antichicken IgG was from Bioss (Beijing, China). The standard of IgY was purchased from Abcam (Shanghai, China). All the other chemicals used in this experiment were of analytical grade and obtained from Sinopharm Chemical Reagent (Shanghai, China). The distilled water used for all the experiments was produced by a water purification system.
Separation of IgY Egg yolk was diluted with 6-fold delipidation solution made from distilled water of 5 different concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) and 5 different types of polysaccharides (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate), respectively. The diluted yolk was mixed with a magnetic stirrer for 1 h at 4◦ C. Then the pH of the diluted sample was adjusted to 5.0 by HCl (1 M) and
was kept overnight at 4◦ C before being centrifuged at 10,000 × g for 25 min at 4◦ C. The pellets (e.g., lipids and lipoproteins) were discarded, and the supernatant was collected. Ammonium sulfate was added to the supernatant to a final concentration of 35% (w/v), and the mixture was stirred for 2 h at 4◦ C and then centrifuged at 12 000 × g for 25 min at 4◦ C. Finally, the pellets were dissolved in distilled water, and excess salt was removed through dialyze.
Determination of Protein Concentration IgY concentration was determined using a quartz cuvette at 280 nm and an extinction coefficient of 1.33 (Pauly et al., 2011) (NANO 2000C, Thermo, USA).
HPLC The purity of the optimized products was analyzed by high-performance liquid chromatography (HPLC), and the purity was calculated simultaneously. Reverse phase chromatography was performed on a GraceVydacC4 (214TP, 5 μm, 250 × 4.6 mm) column with a Waters 2695 Separations Module (Waters). A linear gradient of acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) was used for elution at a flow rate of 1.0 mL/min. The linear gradient elution increased from 5 to 80% ACN in 15 min, decreased to 5% ACN in 1 min, then remained at a constant concentration for 4 min (Geng et al., 2012). The temperature of the column was kept constant at 25◦ C. Detection was carried out at 280 nm with a Waters 2696 photodiode array detector.
SDS-PAGE SDS-PAGE was performed under both reducing and nonreducing conditions. SDS gel electrophoresis was performed in 1.0 M Tris-HCl buffer (pH 6.8) for stacking gel (5% acrylamide) and 1.5 M Tris-HCl buffer (pH 8.8) for separating gel (10% acryl-amide). Migration was performed at 80 V in the stacking gel and at 120 V in the separating gel. Approx. 10 μg protein sample was loaded on the gel. After migration, gels were stained with Coomassie brilliant blue R-250 composed of 25% ethanol and mixed with 8% acetic acid for 30 min at 4◦ C. Destaining steps were carried out after immersion of the gels in 7% acetic acid. The molecular weights and purity of IgY were estimated by Gel-Pro Analyzer 4.0 (Media Cybernetics).
Western Blotting After nonreducing SDS-PAGE, the samples on gel were transferred to a nitrocellulose membrane under semidry conditions for 40 min at 10 V, kept in blocking buffer containing bovine serum albumin (BSA) for 1 h, and incubated with rabbit antichicken IgG conjugated
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lipoproteins to isolate IgY, but they just focused on the interactions between polysaccharides and lipoproteins; they did not consider the purification of IgY. Souza et al. (2013) further pointed out that polysaccharides and lipoproteins could form complex coacervates, and the changes of pH value in egg yolk solution influenced the extent of the precipitation of polysaccharidelipoprotein complexes. Tan et al. (2012) reported a rapid way to isolate IgY using a combination solution followed by ammonium sulfate. But the yield and purity of IgY isolated in this way were not very satisfying, mainly because many of their factors (e.g., pH, types and concentrations of polysaccharides, and diluted rate) were not in the optimized condition (Luo et al., 2010). In this study, we adopted a simple, safe, and effective extraction method using delipidation solution to remove insoluble lipids and lipoproteins in the first step, followed by ammonium sulfate precipitation. Polysaccharides, as safe and nontoxic materials widely existing in nature, can be used as food additives, so the isolated IgY can be applied to large-scale production in the food industry and medical field. Also, the whole process can be easily implemented through 2-step precipitation to obtain a relatively high yield and purity, and the production of IgY through this method may be scaled up to commercial quantities.
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with horseradish peroxidase for 30 min. Thus, the color reaction could be observed after using diaminobenzidine solution for coloration.
ELISA
Statistical Analysis All samples were observed in triplicate, and data were analyzed using the IBM SPSS statistical software. Oneway ANOVA was calculated, and Duncan test was employed (P < 0.05) to separate means.
RESULTS AND DISCUSSION Optimization of the Isolated IgY Figure 1 shows the effect of concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) and types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) of delipidation solutions on the yield and purity of the isolated IgY. The reason these delipidation solutions could remove lipids and lipoproteins effectively was that polysaccharides could interact with egg yolk lipoproteins to form complex coacervations, whose formation was based on ionic bonds, hydrophobic interactions, and hydrogen bonds (Souza et al., 2013). In addition, the force of each bond of the polysaccharide-lipoprotein complexes varied with the types of the polysaccharides. The charge density, the conformational structures of the polysaccharides, and the dissociation constant of carbonyl and sulfate groups all contributed to the different effects on the separation of IgY.
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The antibody immunoactivity was tested by ELISA (Wen et al., 2012) with some modifications. Standards of IgY and isolated IgY were dissolved in carbonatebicarbonate buffer (0.05 M, pH 9.6) and diluted to the final concentration of 200 ng/L. Each well of the plate (Probe, Shanghai, China) was coated with 50 μL of the IgY samples, respectively, and was incubated overnight at 4◦ C. Plates were washed 5 times with 100 μL phosphate buffer solution containing 0.05% Tween 20 (PBS-Tw), followed by a blocking step using 100 μL/well of 3% BSA for 1 h at 37◦ C. The wells were washed with PBS-Tw 5 times. Then 100 μL/well (HRP)-conjugated rabbit antichicken IgY (1:5000) (Probe, Shanghai, China) was added to each well. After incubated at 37◦ C for 30 min in the precoated and blocked plate for 1 h the bound IgY was detected. Then the plate was washed with PBS-Tw, 50 μL/well of tetramethylbenzidin substrate solution was added onto the plate, and the corresponding reaction continued for 15 min at room temperature. The reaction was stopped by 2 M sulfuric acid, and the developed color was measured at 490 nm.
Pectin, which is obtained from cells and the intercellular walls of plants and fruits, is a kind of anionic commercial polysaccharide with acid D-galacturonic unities. The pKa is around 3.6, and its main monomer is glucoronate (Jafar and Fadia, 2010). As a natural polymer with good gelling and emulsifying stability, pectin has been widely used in food, pharmaceutical, and other industries. Figure 1A shows that the yield of IgY increased with pectin concentration increasing from 0.01 to 0.1%. The yield reached the maximum value (8.36 mg/mL) at 0.1% pectin. When the concentration of pectin increased further, the yield decreased rapidly and then rose slowly. The purity of IgY was relatively stable with the various pectin concentrations. It reached the highest point (83.3%) at 0.1% pectin and then decreased when the concentration of pectin increased further. Thus, 0.1% pectin was found to be the optimal condition to isolate IgY. However, Chang et al. (2000) reported that 0.15% pectin was the optimal condition for the removal of lipoproteins. The difference in optimal pectin concentration between this reference and our work might due to standing time in process. Under the action of hydrogen ions, IgY molecules lose the protection of charge repulsion and hydration film to polymerize into large particles and deposited (Bruno and Andre, 2001). Thus, prolonging standing time could enhance the separation efficiency of IgY. In our experiment, we chose 10 h as the standing time and used a 0.1% concentration of pectin delipidation solution to improve the yield and purity of IgY. Carrageenan obtained from several genera and species of marine algae class Rodophyta is a kind of anionic polysaccharide polymerized by sulfated galactan (Ruiter and Rudolph, 1997), and the main characteristics of carrageenan’s biopolymer formation are its linear/helical structures with pKa around 4.3. Carrageenan falls into three different categories: kappa (κ), iota (ι), and lambda (λ) (Shchipunov, 2003). Figure 1B shows that the yield of IgY reduced during the whole period with an increased concentration of λ-carrageenan. Also, it can be seen that purity increased with the concentration of λ-carrageenan from 0.01% to 0.1% and then decreased. When isolating IgY, the optimal condition for λ-carrageenan is 0.1%. In this best condition, the yield and purity were 7.64 mg/mL and 80.94%, respectively, which was consistent with the results of a previous study (Hatta et al., 1990). Some researchers have also suggested that additional λ-carrageenan can result in effective delipidation of egg yolk solution (Kim and Nakai, 1996). However, comparing the two methods, we found the addition of λcarrageenan was not as effective as the addition of pectin in improving the purity and yield of IgY. Dextran sulfate is a kind of anionic polysaccharide esterified by glucan and chlorosulfonic acid. Figure 1C shows that when isolating IgY, the optimal condition for dextran sulfate is 0.1%, with the yield reaching 9.31 mg/mL and purity 80.1%. Jensenius et al. (1981) were the first to introduce the use of dextran sulfate in the
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extraction of IgY from egg yolk. They added a certain amount of dextran sulfate and calcium chloride to water-soluble protein fraction (WSF) centrifuged by 4-fold Tris-HCl buffer. After centrifuging, the precipitate was removed and the supernatant mixed with sodium sulfate. The purity of IgY reached approx. 80%. Based on these previous studies, our approach simplified the entire isolation procedure from 3 to 2 steps, leading to both saved time and improved purity. Carboxymethylcellulose obtained from plants is also a kind of anionic polysaccharide polymerized by methylated glucose. Its hydroxyl groups from glucose, which is partly replaced by –CH2 –COOH via the Williamson reaction. This process leads to the formation of modified cellulose’s functional properties, such as high solubility and viscosity in cold or hot water. Its pKa is approx.
2.0 (Kita et al., 2007). As can be seen in Figure 1D, the yield of IgY reached its lowest yield of 4.46 mg/mL at a concentration of 0.05%, but at the same concentration of 0.05%, the purity of IgY reached its highest point, 81.23%. It was also found that when isolating IgY, the optimal condition for carboxymethylcellulose is carboxymethylcellulose 0.1%, with yield reaching 8.1 mg/mL and purity 79.94%. These findings consistent with earlier results (Hung, 2000). Methylcellulose is a kind of nonionic polysaccharide polymerized by methylated glucose, and its hydroxyl groups from glucose are partly replaced by –CH3 . Because of the nonionic property of methylcellulose, it can be compatible with other emulsifiers. It can be seen in Figure 1E that with the increase of methylcellulose from 0.01 to 0.15%, the yield and purity of IgY slightly rose
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Figure 1. Effect of concentrations and types of delipidation solutions on the yield and purity of IgY. (A) Pectin, (B) λ -carrageenan, (C) dextran sulfate, (D) carboxymethylcellulose, (E) methylcellulose. There was a difference (P < 0.05) between all the yield data.a,b,c Values with no common letters differ significantly (P < 0.05) (n = 3).
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and then decreased. Thus, methylcellulose 0.15% was the best isolating concentration, with the yield of IgY reaching 8.13 mg/mL and purity 78.17%. Because carboxymethylcellulose and methylcellulose were similar in structure but different in ionic types, they were chosen to indicate the effect of ionic types of polysaccharides on the isolation of IgY. The results of this study revealed that the yield of isolated IgY through delipidation solutions containing carboxymethylcellulose and solutions containing methylcellulose had no significant difference, but the purity of IgY isolated through solutions containing methylcellulose was lower than IgY isolated through carboxymethylcellulose. These results might be due to interactions between polysaccharides and lipoproteins that partly depended on ionic bonds.
Quality Evaluation of IgY It can be concluded that optimal conditions for using pectin, λ-carrageenan, dextran sulfate, or carboxymethylcellulose to isolate IgY were at a concentration of 0.1%, and the optimal methylcellulose concentration was 0.15%. Figure 2 presents the separation effect of IgY with the use of these optimized delipidation solutions. IgY is a big biological molecule with a molecular weight of 180 KDa. IgY consists of two heavy chains weighing approx. 65 KDa and two light chains of approx. 28 KDa. Under reducing condition, the disulfide bond could easily be broken, which resulted in the separation between heavy chain and light chain. As can be seen from Figure 2, all the samples had two major bands, one of which corresponds to the heavy chain and one of which corresponds to the light chain of IgY. Some other bands also could be observed, which might be the β -livetin or phosvitin around 35 to 55 KDa. Although the bands indicated that there was no significant difference among the 5 different delipidation solutions, analysis by Gel-Pro Analyzer confirmed that the purity of IgY isolated by delipidation solutions containing pectin was highest among the types of delipidation solutions,
Figure 3. Identification of IgY by Western blot. Lane 1 = standard IgY; lane 2 = marker; lanes 3 to 7 = purified IgY by delipidation solution containing 0.1% pectin, 0.1% λ -carrageenan, 0.1% dextran sulfate, 0.1% carboxymethylcellulose, and 0.15% methylcellulose, respectively, with 35% ammonium sulfate.
and the purity of IgY isolated by delipidation solutions containing methylcellulose was the lowest. Figure 3 shows the Western blot results of the obtained IgY. We could compare those results with bands of the standard IgY and the samples, and infer there was a specific combination of rabbit antichicken IgG conjugated with horseradish peroxidase and thus the target protein was IgY. The results showed that IgY isolated by delipidation solution containing 0.1% pectin provided the highest purity. IgY isolated in this optimized delipidation solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC chromatogram displayed a single symmetrical peak (Figure 4) with a retention time of 16.07 min. The analysis was carried out in triplicate, and an average purity of 83.0 ± 0.3% was obtained by the percentage of the peak area.
Immunoreactivity of IgY Biological activity of the protein is an important indicator of the process of separation and purification. During the purification process, IgY suffered the loss of antigen binding activity to a certain extent, and the immunoreactivity of the purified IgY was determined by ELISA. The results are displayed in Figure 5. The value of absorbance of IgY standard was 0.504 ± 0.4. Because the 5 kinds of polysaccharides were all relatively mild (they are even used as food additives), the isolated IgY kept a high immunoreactivity level. However, it also could be seen that the immunoreactivity of IgY suffered a loss as the concentration of polysaccharides increased. Delipidation solutions with a lower concentration of polysaccharides (<0.05%) could help keep the immunoreactivity of IgY at a relatively high level, whereas a higher concentration (>0.05%) would result in a relatively high loss. It could also be observed that IgY isolated by
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Figure 2. The SDS-PAGE patterns of IgY obtained from delipidation solutions. Lane 1 = protein marker; lanes 2 to 6 = purified IgY by delipidation solution containing 0.1% λ -carrageenan, 0.1% carboxymethylcellulose, 0.15% methylcellulose, 0.1% dextran sulfate, and 0.1% pectin, respectively, with 35% ammonium sulfate.
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Figure 4. HPLC elution profile of IgY obtained by delipidation solution containing 0.1% pectin with 35% ammonium sulfate at 280 nm
delipidation solutions containing pectin maintained a higher immunoreactivity level than IgY isolated by the other 4 delipidation solutions. In addition, IgY isolated by λ-carrageenan and dextran sulfate maintained the lowest immunoreactivity levels. The findings might be explained by the adverse effect caused by the sulfate groups of polysaccharides. The final protocol is shown in Figure 6.
Figure 6. Schematic diagram of the separation of IgY from egg yolk
such as ultrafiltration or use of chromatography. Moreover, the polysaccharides used in this method are all nontoxic, and they are even widely used as food additives, which ensured the safety of isolated IgY. Thus, it might be applied to large-scale food production.
CONCLUSION Under the optimized condition of 0.1% pectin, the yield and the purity of IgY could reach 8.36 mg/mL and 83.3%, respectively, with a minimal negative effect on the immunoreactivity of IgY. Based on our experimental results, a delipidation solution made from polysaccharides coupled with (NH4 )2 SO4 precipitation provides an effective and simple separation method for IgY, although the purity is not very high. However, considering the focus on developing a process of larger production, the yield is relatively satisfying. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods
ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (No. 31371810). Project 2013PY036 was supported by the Fundamental Research Funds for the Central Universities.
REFERENCES Akita, E. M., and S. Nakai. 1993. Comparison of four purication methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. J. Immunol. Methods. 160:207–214.
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Figure 5. Effect of delipidation solutions on the immunoreactivity of IgY. At each concentration point, data followed by different letters are significantly different (P < 0.01).
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TONG ET AL. Jensenius, J. C., I. Andersen, J. Hau, M. Crone, and C. Koch. 1981. Eggs: Conveniently packaged antibodies. Methods for purification of yolk IgG. J. Immunol. Methods. 46:63–68. Kim, H., and S. Nakai. 1996. Immunoglobulin separation from egg yolk: A serial filtration system. J. Food Sci. 61:510–514. Kim, H., and S. Nakai. 1998. Simple separation of immunoglobulin from egg yolk by ultrafiltration. J. Food Sci. 63:485–490. Kita, K., F. Korlos, and V. Kiosseoglou. 2007. Improvement, by dryheating, of the emulsion-stabilizing properties of a whey protein concentrate obtained through carboxymethylcellulose complexation. Food Chem. 104:1153–1159. Ko, K. Y., and D. U. Ahn. 2007. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography. Poult. Sci. 86:400–407. Luo, Y., R. S. Xiong, L. F. Hou, J. D. Li, and J. C. Chen. 2010. Process for coproduction of crude immunoglobulin Y and highdensity lipoproteins from hen egg yolk. J. Agric. Food Chem. 58:11420–11427. Polson, A., T. Coetzer, J. Kruger, E. von Maltzahn, and K. J. van der Merwe. 1985. Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol Invest. 14:323– 327. Pauly, D., P. A. Chacana, E. G. Calzado, B. Brembs, and R. Schade. 2011. IgY technology: Extraction of chicken antibodies from egg yolk by polyethylene glycol (PEG) precipitation. J. Vis. Exp. 51:3791–3084. Ruiter, G. A. E., and B. Rudolph. 1997. Carrageenan biotechnology. Trends. Food. Sci. Tech. 8:389–395. Shchipunov, Y. A. 2003. Sol-gel-derived biomaterials of silica and carrageenans. J. Colloid. Interf. Sci. 268:68–76. Tan, S. H., A. Mohamedalia, A. Kapur, L. Lukjanenko, and M. S. Baker. 2012. A novel, cost-effective and efficient chicken egg IgY purification procedure. J. Immunol. Methods. 380:73–76. Wang, L. H., X. Y. Li, L. J. Jin, J. S. You, Y. Zhou, S. Y. Li, and Y. P. Xu. 2011. Characterization of chicken egg yolk immunoglobulins (IgYs) specific for the most prevalent capsular serotypes of mastitis-causing Staphylococcus aureus. Vet. Micro. 149:415–421. Wen, J. L., S. Q. Zhao, D. G. He, Y. N. Yang, Y. M. Li, and S. S. Zhu. 2012. Preparation and characterization of egg yolk immunoglobulinY specific to influenza B virus. Antivir. Res. 93:154–159.
Downloaded from http://ps.oxfordjournals.org/ at University of New England on January 17, 2015
Al-Hakkak, J., and J. F. Al-Hakkak. 2010. Functional egg whitepectin conjugates prepared by controlled maillard reaction. J. Food Eng. 100:152–159. Bruno, M., and H. Andre. 2001. Isolation and purification of chicken egg yolk immunoglobulins: A review. Food. Agr. Immunol. 13:275–288. Chang, H. M., T. C. Lu, C. C. Chen, Y. Y. Tu, and J. Y. Hwang. 2000. Isolation of immunoglobulin from egg yolk by anionic polysaccharides. J. Agric. Food Chem. 48:995–999. Clitor, J. F. S., E. Edwin, R. Garcia, R. M. Nath´ alia, A. Gaspar, and J. F. C. Lins. 2013. Complex coacervates obtained from interaction egg yolk lipoprotein and polysaccharides. Food Hydrocolloid. 30:375–381. Edzard, S., B. Ingke, G. Kerstin, S. Henning, B. Simon, and P. Dion. 2012. Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy. Biologicals. 40:313– 322. Facon, M., B. J. Skura, and S. Nakai. 1993. Potential for immunological supplementation of foods. Food Agric. Immunol. 5: 85–91. Geng, F., Q. Huang, X. F. Wu, G. D. Ren, Y. Y. Shan, G. F. Jin, and M. H. Ma. 2012. Co-purification of chicken egg white proteins using polyethylene glycol precipitation and anion-exchange chromatography. Sep. Purif. Technol. 96:75–80. Hadge, D., and H. Anbrosuius. 1984. Evolution of low molecular weight immunoglobulins-IV: IgY-like immunoglobulins of birds, reptiles and amphibians, precursors of mammalian IgA. Mol. Immunol. 21:699–707. Hatta, H., M. Kim, and T. Yamamoto. 1990. A novel isolation method for hen egg yolk antibody, “IgY”. Agric. Biol. Chem. 54:2531–2535. Hamada, S., T. Horikoshi, T. Minami, S. Kawabata, J. Hiraoka, T. Fujiwara, and T. Ooshima. 1991. Oral passive immunization against dental caries in rats by use of egg yolk antibodies specific for cell-associated glucosyltransferase of streptococcus mutants. Infect. Immun. 59:4161–4167. Hamal, K. R., S. C. Burgess, I. Y. Pevzner, and G. F. Erf. 2006. Maternal antibody transfer from dams to their egg yolks, egg whites, and chicks in meat lines of chickens. Poult. Sci. 85:1364– 1372.