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. 2026 Sep 26;91(9):e71491. doi: 10.1111/1750-3841.71491

Developing an Environmentally Friendly Sequential Separation and Purification Process of Six Functional Components From Egg Yolk

Yao Yao 1,2, Zihong Ma 1,3, Yonggang Tu 2, Dong Uk Ahn 1,✉
PMCID: PMC13615658  PMID: 42799634

ABSTRACT

Egg yolk contains multiple high‐value bioactive components, yet most existing technologies only achieve single/dual‐component extraction and suffer from low comprehensive utilization efficiency. This study established an integrated sequential separation and purification process of six functional components, which was rationally designed based on the inherent differences in solubility, isoelectric precipitation pH, and salt tolerance of yolk fractions. Egg yolk was diluted with nine volumes of water, adjusted to pH 5.2 and centrifuged to separate supernatant (SUP) and precipitate (PPT). Livetins‐rich fraction was harvested from SUP by ultrafiltration, while 1% NaCl and centrifugation produced low‐density lipoprotein (LDL)‐rich SUP and granules. Note that 20% (NH4)2SO4 + 10% NaCl was selected to separated immunoglobulin Y (IgY) and IgY‐free livetins (IgY‐free). Phospholipids (PLs) were extracted from granules with ethanol. The post‐extraction residues were homogenized in 10% NaCl (pH 10.5), adjusted pH to 4.0 and centrifuged to obtain phosvitin (PV) and phosvitin‐free fraction (PV‐free). Experiments were conducted in three replicates, employing 60 eggs per replicate. The yields of IgY, IgY‐free livetins, LDL, PLs, PV, and PV‐free fraction were 0.73%, 1.41%, 31.82%, 1.16%, 0.96%, and 6.75%, respectively. Total yield of the six components reached (86.06 ± 1.34%), with the two key components, IgY and PV, exhibiting purity of 81.86% and 97.23%.

Practical Applications

This process realizes full‐value graded utilization of egg yolk by fully recovering six distinct functional fractions, avoiding resource waste caused by partial component extraction. Benefiting from accessible and scalable separation techniques, it delivers reliable technical backing for the scaled‐up production and practical commercialization of egg yolk‐derived bioactive substances within the food and pharmaceutical sectors.

Keywords: egg yolk, immunoglobulin Y, phosvitin, sequential separation

1. Introduction

Over the past 15 years, the consumption of eggs in the United States has exhibited a steady upward trend, increasing from 242.4 per capita in 2011 to 275.9 eggs in 2025 (Ozbun 2025). However, the price and demand for egg yolk products are much lower than those of egg white products because of the negative consumer perception of egg yolk as a high‐fat and high‐cholesterol food (Huang and Ahn 2019). In 2015, the avian influenza outbreak caused significant volatility in egg and egg product prices, with egg white powder ranging from $9.13 to $18.75 per pound, being approximately two to five times the price of the yolk powder (Sterk 2015). Although the whole liquid egg dominated the liquid egg market with 55.54% of the revenue in 2024, liquid egg whites emerged as the fastest‐growing segment by product type, projected to grow at a compound annual growth rate of 6.78% (Mordor Intelligence 2024). Therefore, developing novel processing strategies to facilitate the utilization of egg yolk and, consequently, to balance the utilization of the whole egg is extremely important for both consumers and the egg industry.

As a crucial component of avian eggs, egg yolk is rich in various functional lipids and proteins with excellent physiological properties, which demonstrate its high application value in nutritional fortification and pharmaceutical active ingredient preparation. Among these components, yolk immunoglobulin Y (IgY), a counterpart to immunoglobulin G (IgG) of mammals, has garnered widespread attention due to its distinct advantages over IgG, including a more robust immune response to conserved mammalian proteins, low cross‐reactivity, high stability, and the absence of reported toxic side effects in humans and animals (Xia et al. 2023). As a protein synthesized by the avian immune system and transferred from serum to egg yolk to confer immune protection on offspring, IgY exhibited a series of diagnostic and therapeutic effects against bacterial and viral infections, such as Helicobacter pylori (Horie et al. 2004), diarrheagenic E. coli (Karthikeyan et al. 2022), rotavirus (Thu et al. 2017), or even COVID‐19 (Somasundaram et al. 2020; Wei et al. 2021). The oral intake of IgY was considered as a non‐antibiotic alternative for the prophylaxis and treatment of a wide variety of infectious diseases, including dental caries, periodontitis and gingivitis, gastritis and gastric ulcer, oral thrush, and diarrhea (Rahman et al. 2013). The prospective analysis of the commercialization and marketing potential of IgY‐based commercial products revealed that the number of IgY patent applications increased steeply after 2010, with a total of 819 patent applications recorded in the Patentscope database from 2010 to 2022, while approximately 3729 and 846 products are used as the primary and secondary antibodies, respectively (Yakhkeshi et al. 2022).

PV, which is recognized as the most highly phosphorylated protein in nature, distinguishes itself through its excellent metal‐chelating, antioxidant, emulsifying, and antibacterial properties (S. Marcet et al. 2022). When subjected to high‐temperature medium‐pressure pretreatment combined with enzymatic hydrolysis, it can be hydrolyzed into hundreds of peptides that exhibit better functional activities than the parent protein, including higher bioavailability, greater solubility, metal‐carrying effects, and more efficient intestinal absorption (Huang et al. 2019). Both PV itself and its hydrolyzed peptides demonstrated tremendous application potential in the food and pharmaceutical industries (Liu et al. 2022). PLs are not only key components of cell membranes but also play a critical role in regulating blood lipids and enhancing memory, and they act as essential components of special medicinal emulsions with great potential for new drug development (Zhao et al. 2023; Xiao et al. 2020). Thus, employing fractionation and modification technologies to produce distinctive egg yolk fractions with unique compositional, nutritional, and functional characteristics represents a promising strategy to enhance the utilization of egg yolk.

However, previous studies on egg yolk separation have predominantly focused on the individual extraction of single or dual high‐value components, such as independent purification of IgY, PV, or PLs (Wang et al. 2021; Zhang et al. 2024; Bernardo et al. 2019). Most conventional protocols rely on organic solvent extraction, repeated salting‐out, or long‐time centrifugation procedures, which are tailored for the maximum recovery of a single target component while the coexisting functional ingredients are often discarded as residues due to the lack of effective separation and recovery methods. (Abeyrathne et al. 2022; Xia et al. 2023; Chalamaiah et al. 2018). This resulted in the waste of egg yolk resources and substantially diminished the comprehensive economic benefits of egg yolk, and imposed environmental burdens during waste disposal. This situation was clearly contrary to the current concept of green and low‐carbon development, making it challenging to meet the actual needs of large‐scale and value‐enhanced egg industry development.

To address this critical gap, the present work focused on developing an environmentally friendly sequential separation and purification process for six functional components from egg yolk, with the aim of fully utilizing these egg yolk components in areas such as food processing, functional food, nutraceuticals, pharmaceuticals, and cosmeceuticals.

2. Materials and Methods

2.1. Materials

Large‐sized chicken eggs were purchased from a local market. Ammonium sulfate, sodium chloride, sodium hydroxide, and hydrochloric acid were purchased from Fisher Scientific (Waltham, MA). Standards of chicken IgY and PV were from (Santa Ana, CA) and Sigma‐Aldrich (St. Louis, MO), respectively.

2.2. Sequential Separation of Proteins and Phospholipids From Yolk

A total of 180 white‐shelled large chicken eggs were equally divided into three replications (60 eggs each) to separate six fractions from yolk. The process begins by separating the egg yolk from the egg white using an egg splitter, then diluting it with nine volumes of distilled water (DW, v/w) and homogenizing for 1 min using a hand blender (KitchenAid) at high speed. To investigate the effect of pH on the extraction efficiency of livetins, the pH of the yolk diluent was either maintained at its natural value of 6.2 or adjusted to 5.2 using 1 mol/L hydrochloric acid. After standing at 4°C for 12 h, the mixture was centrifuged at 3500 × g for 30 min at 4°C to separate the SUP and PPT. The resulting SUP was concentrated using an ultrafiltration unit (cartridge cut‐off size 10,000 MW, Flexstand, GE Healthcare, Piscataway, NJ) to obtain the livetin‐rich fraction. To determine the effects of pH and ionic strength on the separation of LDL and granules from the PPT, 10 g PPT was blended with two or three volumes (v/w) of 0.5% or 1% NaCl solution and centrifuged (3500 × g for 30 min at 4°C) to separate the LDL‐containing SUP and granules. After optimizing the LDL separation conditions by these small‐scale tests, the SUP obtained from large‐scale experiments (using 60 eggs) was freeze‐dried for yield calculation. PLs were extracted from the crude granules using four volumes (v/w) of 100% ethanol, followed by centrifugation (3500 × g for 30 min at 4°C). The ethanol in the SUP was removed using a vacuum evaporator (Buchi Rotavapro 200), and the collected fraction was used as PLs. The resulting PPT after PLs extraction was mixed with 4 volumes (v/w) of DW, homogenized for 1 min using a hand blender (KitchenAid) at high speed, and centrifuged (3500 × g for 30 min at 4°C). The resulting SUP was discarded, and the PPT was collected and used to extract PV.

For IgY separation, three methods (M1: 35% (NH4)2SO4; M2: 11.4% (NH4)2SO4 + 15% NaCl; M3: 20% (NH4)2SO4 + 10% NaCl) were tested to establish the best salting‐out conditions from the concentrated livetins‐rich fraction. Each method utilized 120 mL of livetins‐rich concentrate, which was divided into three aliquots, with each aliquot serving as a replicate. The salting out process was conducted at 20°C for 12 h, followed by centrifugation to collect the IgY PPT and the IgY‐free SUP. After redissolving the IgY PPT in DW, IgY, and IgY‐free SUP were separately desalted via dialysis and freeze‐drying.

For PV separation, the PV‐rich PPT was homogenized with four volumes (v/w) of 10% NaCl in 0.05 N NaOH solution (pH 10.5) using a Polytron at 20,000 r/min for 5 min. After adjusting the pH to 4.0, the mixture was then centrifuged (3500 × g for 30 min at 4°C) to obtain SUP 1 and crude PV‐free PPT. The crude PV‐free PPT was re‐extracted with four volumes (v/w) of DW under the same homogenization conditions and then centrifuged to separate the SUP 2 from the PV‐free fraction. The PV‐free fraction was then directly collected and freeze‐dried. These two SUPs were pooled, desalted, and concentrated through ultrafiltration to obtain a crude PV. The crude PV was stored overnight in a refrigerator at 4°C, followed by centrifugation to remove precipitated impurities. A schematic diagram (Figure 1) shows the sequential separation process as well as the three optimization steps.

FIGURE 1.

FIGURE 1

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Schematic diagram of the sequential separation process of and proteins and PLs from egg yolk. DW = distilled water.

2.3. Determination of Lipid Class With Thin‐Layer Chromatography

Lipid class was measured as reported previously by Ahn et al. (2006). PLs were dissolved in Folch solution (chloroform:methanol = 2:1) to make the lipid content approximately 100 mg/mL. Two drops of ethanol were added to help solubilize PLs. PLs sample and standard mixture containing triglyceride (TG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingomyelin (SM), free fatty acid, and cholesterol (10 µL each) were applied onto a pre‐coated silica gel‐G plate (20 cm × 20 cm, Merck, Billerica, MA, USA) that had been previously activated by heating at 120°C for 2 h. The plates were developed in a chloroform:methanol:water (98:38:6) solution to 10 cm from the origin line (about 50 min). Then it was air‐dried for 30 min and developed in a hexane and diethyl ether (4:1) solution for about 40 min. After air‐drying for another 30 min, the plate was sprayed with 0.1% (w/v) 2′,7′‐dichlorofluororescein in ethanol. The spots corresponding to PE, PC, cholesterol, and TG were identified under UV light.

2.4. Yield and Purity Calculation

The yield was calculated as the weight of the lyophilized fractions to the weight of egg yolk, and the results were expressed as grams per egg yolk (g/egg yolk) and grams per 100 g of egg yolk (g/100 g egg yolk), respectively. The average weight of whole eggs and egg yolk was 60.55 ± 1.52 and 17.27 ± 0.22 g, both conforming to the relevant weight standards for large eggs in the United States Department of Agriculture (USDA), with a common egg yolk weight of 17–18 g.

To assess the separation efficiency, SDS‐PAGE (10 cm wide × 8 cm tall × 1.0 mm thick) was performed using a 10% separating gel and a 4% stacking gel using a Mini‐Protein II cell (Bio‐Rad, Hercules, CA, USA). Each sample was mixed with 2 × non‐reducing buffer (62.5 mM Tris‐HCl, pH 6.8, 2.00% w/v SDS, 25.00% v/v glycerol, and 0.02% w/v bromophenol blue), and 5 µL of the mixture was loaded for electrophoresis. A broad‐range Precision Plus Protein Dual Xtra Standard (10–250 kDa; Bio‐Rad Laboratories Inc.) was used as a marker. Electrophoresis was run at 120 V for 1 h. The proteins in the gel were stained using the Coomassie Blue method with 0.1 M aluminum nitrate. The purity was estimated by converting the density of protein bands in the gel image to the percentage of total protein in the sample using ImageJ software (NIH, Bethesda, MD).

2.5. Statistical Analysis

The results were calculated as means ± standard deviation (SD). Analysis of variance (ANOVA) was performed using SPSS 26.0 statistical analysis software (SPSS Inc., Chicago, IL, USA). p < 0.05 indicated a significant difference. Images were plotted using the OriginPro 2021 software (OriginLab Co., MS, USA).

3. Results and Discussion

3.1. Effect of pH on the Separation of Water‐Soluble Proteins and Water‐Insoluble Fractions, and the Components Therein

The 6‐fold water dilution method proposed by Akita and Nakai (1992) was a classic technique that achieved isolation of livetins by leveraging the polarity and solubility differences between lipids and proteins in water. Subsequent studies demonstrated that increasing the dilution ratio to 9‐ to 10‐fold was more conducive to the extraction of livetins and the removal of lipids (Nilsson et al. 2008; Xia et al. 2023; Ko et al. 2011), thus 9‐fold dilution was adopted in this study. As the pH of diluted egg yolk was also an important factor for the separation of livetins and insoluble fractions, two different pH conditions were tested on a small scale using 4 g of egg yolk (Figure 2A,B). The initial pH of the egg yolk dilution was approximately 6.2. After standing overnight at 4°C, the egg yolk granules and lipids aggregated to form a compact PPT accounting for approximately 1/8 of the total volume. The corresponding livetins could be easily separated by pouring or siphoning. Notably, the livetins appeared slightly yellowish, indicating the presence of a small amount of lipids in the system. In contrast, when the pH of the egg yolk dilution was adjusted to 5.2, aggregates became loose, and the sedimented volume increased to approximately 7/8 of the total volume. The color of diluted egg yolk became milky white, and the aggregated parts could only be obtained by centrifugation. The protein composition in the electrophoretogram of the SUP and PPT under different pH conditions is shown in Figure 3 (Lanes 2–5). As reported by Navidghasemizad et al. (2014), the bands with molecular weights of approximately 221, 203, 122, 93, 85, and 68 kDa corresponded to lipoprotein components that belong to LDL. These bands were more intense in the SUP separated at pH 6.2 (shown in the red box on Lane 2) than those at pH 5.2. At the same time, their intensity was significantly reduced in the SUP separated at pH 5.2, indicating that LDL, the primary carrier of lipids, was more likely to precipitate at this pH.

FIGURE 2.

FIGURE 2

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Effect of pH on the separation and degreasing of livetins. (A) and (B) Separation of livetins and PPT by overnight standing and centrifugation at two different pH values; (C) degreasing effect after freeze–thaw and centrifugation of concentrated livetins obtained at pH 6.2; (D) impurity removal of concentrated livetins obtained at pH 5.2 after overnight standing and centrifugation.

FIGURE 3.

FIGURE 3

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SDS‐PAGE profile of egg yolk components. (lane M: marker protein; Lane 1: Egg yolk; Lane 2: Egg yolk SUP obtained at pH 6.2; Lane 3: Egg yolk PPT obtained at pH 6.2; Lane 4: Egg yolk SUP obtained at pH 5.2; Lane 5: Egg yolk PPT obtained at pH 5.2; Lane 6: PV standard; Lane 7: Crude PV; Lane 8: Purified PV after overnight standing and impurity removal; Lane 9: PV‐free fraction).

To verify the reliability of this phenomenon, a large‐scale experiment involving 60 eggs was further conducted, and the results were consistent with those of the small‐scale experiment. The livetins prepared at pH 5.2 exhibited lower lipid levels, which not only ensured a smooth ultrafiltration and concentration process but also allowed the residual lipids in the system to be removed through the simple procedure of overnight standing plus centrifugation (Figure 2D). The lipid separation behavior of livetin concentrate was dependent on lipid content and emulsion stability, since livetins acted as a natural emulsifier to form oil‐in‐water emulsions with lipids (Zhang et al. 2024). At low lipid levels, the large spacing between oil droplets reduced the emulsification burden of livetins, which further destabilized the entire emulsion system. Conversely, livetins isolated at pH 6.2 contained abundant lipids, which tend to block the membrane pores and markedly retard the ultrafiltration process. The resulting concentrate thus retained considerable residual lipids, in which oil droplets were densely encapsulated by livetins to form a high‐viscosity, thermodynamically stable emulsion that resisted gravitational separation even after prolonged standing. Consequently, lipid removal could only be achieved via freezing‐thawing cycles, which disrupted the protein interfacial film and promoted oil droplet coalescence for efficient lipid separation (Figure 2C). This was consistent with the findings of Wang et al. (2021), who reported that boundary water crystallization during freezing induced LDL aggregation and greatly facilitated subsequent lipid removal. This confirmed that the pH of the egg yolk dilution not only significantly affected the precipitation state of lipids but also directly correlated with the separation efficiency of livetins. Previous research also suggested that IgY was more easily dissociated from lipoprotein particles around pH 5.2 (Chalamaiah et al. 2017), so this pH was employed in the subsequent separation of livetins and water‐insoluble fractions.

3.2. Optimization of Salting‐Out Conditions for IgY

Salting‐out was one of the most commonly used methods for separating IgY from livetins, which relied on the differences in the structural and surface properties between IgY (γ‐livetin) and α‐/β‐livetins. IgY contained more surface‐exposed hydrophobic residues and adopted a looser conformation than α‐ and β‐livetins, rendering its hydration layer more susceptible to salt disruption, whereas α‐/β‐livetins buried hydrophobic residues internally with compact structures and superior hydration stability (Pashkuleva and Teixeira 2025). In mixed salt systems, two or more types of ions follow the principle of the Hofmeister effect, which exerts additive or complementary regulatory effects, acting independently through synergistic cooperation (Egelhaaf et al. 2009). Although various salt combinations have been employed in previous studies, the conclusions differ among researchers. Tong et al. (2015) employed 35% (NH4)2SO4 (w/w), while Lee et al. (2014) used a combination of 25% saturation (NH4)2SO4 (i.e., 11.4% w/w) + 15% NaCl, and Xia et al. (2023) proposed a mixture of 20% (NH4)2SO4 + 10% NaCl (w/w). To verify the optimal conditions for IgY separation, comparative experiments were conducted using a 40 mL aliquot of the livetins concentrate. Our results revealed significant differences in the yield and purity of IgY among the three methods. Method 1, which used 35% (NH4)2SO4, yielded the highest IgY (0.58 ± 0.02 g), but with the lowest purity (50.35 ± 1.57%); Method 2, which used 25% saturation (NH4)2SO4 (i.e.,), 11.4% w/w) + 15% NaCl, exhibited the lowest yield (0.22 ± 0.02 g) yet the highest purity (82.33 ± 1.55%); Method 3, which used 20% (NH4)2SO4 + 10% NaCl, achieved a moderate yield and a high purity, which were 0.29 ± 0.01 g and 81.68 ± 2.03%, respectively (Figure 4). The net yield of IgY, calculated as the product of purity multiplied by yield, was 0.29, 0.18, and 0.23 g for Methods 1, 2, and 3, respectively. Complementary evidence from the IgY‐free fractions further supported these findings. The weight of the IgY‐free livetins from Method 1 (0.29 ± 0.03 g) was significantly (p < 0.05) lower than those from Method 2 (0.61±0.03 g) and Method 3 (0.56±0.02 g), indicating that part of the α‐ and β‐livetins in the SUP co‐precipitated with IgY. No significant difference in IgY purity was observed between Methods 2 and 3 (p < 0.05), but IgY was still detectable in the IgY‐free fraction prepared by Method 2 (Figure 4B, Lane 3), indicating that the 11.4% (NH4)2SO4 + 15% NaCl combination was insufficient to achieve complete precipitation of IgY. Method 3 achieved both high purity and net yield of IgY among the three tested methods, thus was identified as the optimal separation process in this study.

FIGURE 4.

FIGURE 4

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Optimization of salting out conditions for IgY. (A) Yield and purity of IgY and IgY‐free fraction, different lowercase letters (a–c) indicate significant differences at p < 0.05. (B): SDS‐PAGE profile of IgY and IgY‐free livetins fraction (Lane M: marker protein; Lane 1: Concentrated livetins; Lanes 2–4: IgY‐free livetins fraction obtained by Method 1–3; Lane 5–7: IgY obtained by Method 1–3; Lane 8: IgY standard.)

The ability of Method 3 to achieve a balance between moderate yield and high purity was probably due to the precise regulation of the mixed salt system. Note that 20% (NH4)2SO4 was within the critical precipitation range of IgY, which could only trigger the selective precipitation of the hydrophobic IgY. However, this concentration of (NH4)2SO4 was not enough to reach the precipitation threshold of α‐ and β‐livetins, and thus avoided their co‐precipitation with IgY (Schade and Csutak 2019). Meanwhile, 10% sodium chloride played dual roles. It increased the exposure of hydrophobic regions of the IgY surface to ensure a moderate precipitation yield and simultaneously weakened the electrostatic adsorption between IgY and α‐/β‐livetins through the charge shielding effect (Laber et al. 2017), which reduced contaminant adsorption by miscellaneous proteins.

In the large‐scale extraction employing 60 egg yolks, the yields of IgY and IgY‐free livetin fractions were 0.13 ± 0.01 and 0.24 ± 0.02 g per yolk, respectively (or 0.73 ± 0.03 g/100 g yolk and 1.41 ± 0.07 g/100 g yolk). By comparing the results, the yields of the IgY‐free livetin fraction from the small‐ and larger‐scale trials were 1.98‐ and 1.92‐fold that of IgY, respectively, showing consistency between the small‐ and large‐scale extraction processes. The yield of IgY reported by Tong et al. (2015) was 8.36 mg/mL egg yolk (Method 1), which was equivalent to approximately 0.84 g/100 g egg yolk after conversion. Lee et al. (2014) calculated the IgY extraction yield as 98.7% based on the standard amount of 0.6% IgY in egg yolk, corresponding to 0.59 g/100 g of egg yolk. In this study, the yield of IgY reached 0.73 g/100 g egg yolk, which was comparable to the above‐mentioned studies in terms of extraction efficiency. Using rabbit anti‐chicken IgY polyclonal antibodies as the detection reagent, Carlander et al. (2001) dynamically monitored the IgY concentration in egg yolks from 10 randomly selected Single Comb White Leghorn for four consecutive weeks. The results showed that significant differences existed in the IgY concentration among individual egg yolks, with the fluctuation ranging from 2.8 ± 0.3 to 7.0 ± 0.5 mg/mL (0.28–0.70 g/100 g egg yolk), confirming that the extraction process adopted in this experiment efficiently recovered natural IgY from egg yolks. The significant individual differences in the amount of IgY in the egg yolk indicated that genetic factors were the primary determinants influencing the content of IgY in the egg yolk (Carlander et al. 2001). This provides important guidance for the large‐scale preparation of IgY, specifically that the industrial production efficiency of IgY could be further improved by breeding chicken strains with high IgY concentration.

Currently, research on the utilization of the IgY‐free livetins is scarce. However, existing studies have confirmed that the intact livetins possesses diverse application potentials. First, this fraction is rich in essential amino acids and exhibits high bioavailability, making it a promising ingredient for the development of functional products, such as high‐protein nutritional powders and sports nutrition foods (Gu et al. 2023). Second, livetins and their derived peptides not only possess distinct anti‐inflammatory activities but also demonstrated significant potential in the prevention and intervention of oxidative stress‐related diseases, thereby holding substantial application value in the fields of nutraceuticals and pharmaceuticals (Chalamaiah and Wu 2017; Ibrahim et al. 2023). Based on these characteristics, the IgY‐free livetins isolated in this study show broad prospects for the development and application in various areas.

3.3. Effects of pH and Ionic Strength on the Separation of LDL and Egg Granules

The PPT obtained after extracting livetins was a mixture of LDL and yolk granules, with the yolk granules primarily consisting of high‐density lipoprotein (HDL) and PV, while PLs as a high‐value ingredient were predominantly present in HDL (S. Marcet et al. 2022). To achieve efficient separation, this study investigated the effects of pH, NaCl concentration, and dilution on the separation efficiency of LDL from the yolk granules.

As shown in Figure 5, after adding two volumes (w/w) of DW to the loose lipoprotein PPT obtained at pH 5.2, the mixture was homogenized, and the pH was adjusted to 6.2. The mixture exhibited a water absorption phenomenon and was difficult to precipitate by centrifugation at 3500 × g for 30 min. This was attributed to the fact that the pH range of 6.2–6.3 is the natural pH of egg yolk, under which lipoproteins restore their intact hydrophilic structure and reassemble into stable colloidal particles. Water molecules bound to the charged groups of the colloidal particles to form a protective hydration layer, while electrostatic repulsion between particles with the same charge prevented aggregation. Eventually, the lipoproteins were uniformly dispersed in the aqueous phase as tiny colloidal particles.

FIGURE 5.

FIGURE 5

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Effects of pH and ionic strength on the separation of LDL and granules.

When two or three volumes of a 0.5% NaCl solution were added to the PPT obtained at pH 5.2, the SUP appeared pale yellow, and the PPT was orange and viscous. This was due to the solubility difference between the LDL and HDL under low ionic strength, and the consequent changes in the dissolution and dispersion of lipophilic pigments. Carotenoids represent less than 1% of total lipids and were responsible for the yellow/orange color of the yolk (Li et al. 2021). The low ionic strength of the added NaCl (0.5%) could only slightly shield part of the surface charge of the lipoprotein particles, which was insufficient to break the stable structure of LDL aggregates. Consequently, the solubility of LDL was marginally improved, and only a small amount of LDL (along with encapsulated carotenoids) was dissolved into the SUP, resulting in a pale‐yellow color. In contrast, HDL had even lower solubility under low ionic strength, and the complex formed by HDL and PV via calcium‐phosphate bridges remained intact and was retained mainly in the PPT. Meanwhile, most of the undissolved LDL was tightly bound to the yolk granules containing HDL, and carotenoids were highly concentrated in the PPT along with the LDL, giving the PPT an intense orange color.

When the pH of the 0.5% NaCl system was adjusted to 6.2, the color intensity of the SUP changed to orange, and the volume of the PPT decreased. This phenomenon was due to the synergistic effects of pH and low ionic strength. At pH 6.2, the hydrophilic structure of LDL was further restored, and its solubility was significantly higher than that at pH 5.2. More LDL and encapsulated pigments were dissolved and moved from the PPT to the SUP, leading to the orange color of the SUP. However, HDL remained in the PPT due to the complex structure under low ionic strength, and its solubility was not significantly improved. With the dissolution of a large amount of LDL, the total amount of residual lipoproteins in the PPT decreased, and the volume was correspondingly reduced. This indicated that pH 6.2 could improve the separation efficiency of LDL in a low ionic strength system by increasing the solubility of LDL. However, a complete separation of LDL from the PPT was not achieved.

When two volumes of 1% NaCl were added, regardless of whether the PPT was obtained from the homogenate at pH 5.2 or 6.2, the PPT appeared pale‐yellow and the SUP was orange. This could be due to the high ionic strength of 1% NaCl, which efficiently screens the electrostatic repulsion on the surface of the lipoprotein particles, thereby completely restoring the stability of the LDL aggregates. As a result, a vast majority of the LDL was dissolved into the SUP, and carotenoids were enriched in the SUP along with the LDL, resulting in an orange color. In contrast, the PPT was primarily composed of undissolved HDL, PLs, and PV, which appeared pale yellow due to its extremely low pigment content. When three volumes of 1% NaCl were added, the ionic strength was further increased, disrupting the calcium‐phosphate bridge complex between the HDL and PV, which led to more HDL being dissolved into the SUP and resulted in a reduction in the amount of PPT. Although this seemingly improved the separation efficiency of LDL, the reduced PPT yield diminished the subsequent recovery of PV as the primary separation target, thereby lowering the overall extraction efficiency.

Based on the above analysis, it was clear that the solubility of lipoproteins was jointly regulated by ionic strength and pH, which synergistically affected the dissolution behaviors of LDL and HDL, thereby determining the separation effect. Considering the cost of large‐scale production and the requirements for subsequent component recovery, two volumes of 1% NaCl were determined as the optimal separation condition, offering balanced separation efficiency, comprehensive recovery of target components, and production economy.

LDL accounted for 85% of yolk plasma and yolk plasma makes up 78% of the total yolk dry matter (S. Marcet et al. 2022), the theoretical content of LDL was approximately 33.15% of the egg yolk. The yield of LDL in this study was 5.49 ± 0.15 g/yolk or 31.82 ± 0.89 g/100 g yolk, equivalent to 31.82%, and in good agreement with the theoretical value. With outstanding pH/ionic strength‐tolerant emulsifying properties, distinct heat‐induced gelation capacity, and effective cryoprotective activity (Anton 2013; Moussa et al. 2002; Lv et al. 2022), the obtained LDL represents a promising natural functional ingredient with substantial potential in the food processing industry.

3.4. Phospholipids Extraction From the Egg Granules

The HDLs present in yolk granules are protein–PL complexes, whose particulate structure is maintained by five disulfide bridges, and comprise a hydrophobic cavity that can hold up to 40 PL molecules per HDL molecule (Li et al. 2021). Latest studies have demonstrated that PLs, which can enhance the bioavailability of drugs by facilitating their transport, are confirmed to inhibit the proliferation of tumor cells and play a crucial role in brain development and neural network formation (Xiao et al. 2020). Compared with soybean PLs, egg yolk PLs had a PC content as high as 73%, which was more than three times that of soybean PLs. Due to this compositional advantage, their lipid profile was similar to that of human cell membranes, and they exhibited superior emulsifying stability and hydrophilicity (Zhao et al. 2023). In an experiment on rats fed a high‐fat and high‐fructose diet, the levels of hepatic TG, malondialdehyde (MDA), and other related indicators were significantly reduced by both egg yolk PL and soybean PL (p < 0.05). At the same time, egg yolk PLs exerted a more favorable effect on improving lipid metabolism and protecting the liver (Yin et al. 2021).

PLs can be easily extracted using solvent extraction or supercritical extraction methods, and solvent extraction can employ single solvents or mixed solvent systems, including ethanol, n‐hexane, acetone, petroleum ether, and isopropanol (Abeyrathne et al. 2022). Ethanol, a polar extractant, was one of the most widely used solvents for PLs extraction, as it exhibited good solubility and compatibility, along with low toxicity, operational safety, and high compliance with food/pharmaceutical‐grade standards. Using four volumes of ethanol for PL extraction was preferred because it yielded high‐purity PL (> 85%). Although increasing the volume of ethanol (e.g., nine volumes) reduced impurities, it also increased the solvent volume and cost (Ahn et al. 2006).

Ahn et al. (2006) and Zhao et al. (2023) reported a PLs content of 9.4 g per 100 g liquid egg yolk; Anton and Gandemer (1997) and Xiao et al. (2020) documented 17.7 g and 20.6 g/100 g dry weight. When converted based on the 50% moisture content of whole egg yolk, the approximate range of PL content on a fresh egg yolk basis is 8.35–10.3 g/100 g yolk. However, in the present study, only 1.16 ± 0.08 g of PL was extracted from 100 g of yolk, which was attributed to the fact that the PLs were exclusively extracted from the egg yolk granule fraction, which comprises 22% of egg yolk. The yolk granules are composed of 70% HDL, 16% PV, and 12% LDL (Xiao et al. 2020). The total granule‐derived components obtained from 100 g of egg yolk was 8.87 g, which was composed of 0.96 ± 0.04 g of PV, 6.75 ± 0.31 g of PV‐free fraction, and 1.16 ± 0.08 g of PL, where PL accounted for 13.1%. This value was slightly higher than the previously reported PL proportion of 11.1% in the granule fraction (Anton and Gandemer 1997). This difference was most likely attributed to the trace amount of impurities in the extract. Previous studies have confirmed that in the phospholipid–ethanol system, PL can act as cosolvents to self‐assemble into reverse micelles or organogels, and their outward‐facing hydrophobic tails can encapsulate nonpolar lipid molecules in the polar ethanol medium, thereby enhancing the solubility of neutral lipids (Shchipunov 2001; Saini et al. 2021). The PLs extracted in this study were dominated by PC, followed by PE, with trace amounts of SM (Figure 6). In addition, minor quantities of cholesterol and free fatty acids (FFAs) were detected. As the intrinsic content of FFAs in fresh eggs was extremely low, the FFAs identified were probably derived from the degradation of PLs and TGs during rotary evaporation and subsequent storage (Luo et al. 2023).

FIGURE 6.

FIGURE 6

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Thin layer chromatogram for component distribution of egg yolk extracts. TG: triglyceride; FFAs: free fatty acids; Chol: cholesterol; PE: phosphatidylethanolamine; PC: phosphatidylcholine; SM: sphingomyelin.

3.5. Separation of PV From the Lipid‐Free Egg Granules

Methods such as ion exchange chromatography and gel filtration were used to separate PV previously, but they were complex to operate, required high‐cost materials, and had a processing capacity confined to only tens of milligrams of product per batch (Ko et al. 2011; Lei and Wu 2012). For a large‐scale production of PV, a salt extraction method was proposed by Ko et al. (2011), and the extraction efficiencies of 9‐fold volume of (NH4)2SO4 at different concentrations (5%, 10%, 15%, 20%, 25%) and 10% NaCl were compared. Results demonstrated that the yield of the 10% NaCl extraction (97%) was higher than that of the 10% (NH4)2SO4 (72%), but the purity of PV obtained by these two methods was both at approximately 85%. Further investigations by Lee et al. (2014) revealed that the addition of 0.05 mol/L NaOH to the 10% NaCl could not only improve the purity and yield of PV but also reduce the extraction volume to 4‐fold. So, this study employed this modified alkaline–NaCl extraction system for PV isolation.

The crude PV obtained by ultrafiltration contained trace amounts of impurities (Figure 3; Lane 7). However, these impurities could be effectively removed by overnight standing followed by centrifugation, which significantly improved the purity of PV to 97.23% (Figure 3; Lane 8). The PV yield was 0.17 g/yolk or 0.96 g/100 g of yolk, equivalent to 1.92% of yolk dry matter (Table 1). Variations in the reported PV contents of egg yolk have been documented across different studies. A content of 5.3% of yolk dry matter was reported by Lei and Wu (2012), who used high‐performance liquid chromatography (HPLC). Yilmaz and Ağagündüz (2020) reported that PV accounted for 4% of yolk dry matter or 11% of total yolk protein based on nitrogen and phosphorus contents. S. Marcet et al. (2022) indicated that PV constituted 18% of egg yolk granules, which was estimated to be 3.96% of the yolk dry matter, equivalent to about 2% of yolk. The extraction yields of 97% and 98.7% reported by Ko et al. (2011) and Lee et al. (2014), respectively, were calculated on the basis that 1 g of egg yolk contained 1.2% and 1.25% PV, which corresponded to 2.4%–2.5% of yolk dry matter. Following the calculation methods of Ko et al. (2011) and Lee et al. (2014), the yield of the current research reached 76.8%–80%, which was approximately 20% lower than that of the aforementioned two groups. Unlike single‐component extraction, where parameters could be specifically optimized to maximize the recovery of an individual fraction, the integrated extraction strategy required balancing the recovery performance of multiple functional components. Therefore, partial dissolution and loss of yolk granules occur during LDL separation from the loose precipitate, which constituted an unavoidable limitation of this integrated co‐extraction route.

TABLE 1.

Weight and percentage of extracted egg components.

Fractions In one yolk* g/100 g yolk
① IgY 0.13 ± 0.01 0.73 ± 0.03
② IgY‐free Livetin 0.24 ± 0.02 1.41 ± 0.07
③ LDL 5.49 ± 0.15 31.82 ± 0.89
④ Phospholipids 0.20 ± 0.02 1.16 ± 0.09
⑤ Phosvitin 0.17 ± 0.01 0.96 ± 0.04
⑥ PV‐free 1.17 ± 0.05 6.75 ± 0.31
Fractions total 7.38 ± 0.19 43.73 ± 1.11
Extraction rate (86.06 ± 1.34)%
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Note: The weight of one yolk was based on an average of 17.27 g.

*

The values in Table 1 only list the yields of each separated component; therefore, no statistical comparison is performed.

Recent studies have reported novel approaches for PV separation, including high hydrostatic pressure (HHP) treatment of egg yolk at 400 MPa for 5 min (Giarratano et al. 2020) and ultrasonic treatment at 600 W for 10 min (Jiang et al. 2019). These emerging processing methods can serve as valuable references for the scaled‐up separation of PV in future industrial applications. It is worth noting that egg yolk granules were also identified as a favorable source of 5‐methyltetrahydrofolate (5‐MTHF), a folate derivative with extremely high bioavailability (Patanwala et al. 2014). Studies have demonstrated that HHP treatment at 200–600 MPa can disrupt the compact protein structure of the granules, thereby improving the release and extraction efficiency of 5‐MTHF (Naderi et al. 2017). Therefore, for future processes of PV extraction, HHP treatment can be employed synchronously to facilitate the co‐extraction of 5‐MTHF, thereby further enhancing the comprehensive utilization efficiency of egg yolk resources.

Studies have demonstrated that the phosphorylation sites of PV are masked by its compact spatial structure, which limits its metal‐chelating activity (Yilmaz and Ağagündüz 2020). In contrast, phosvitin phosphopeptides (PPPs) prepared by enzymatic hydrolysis can break this structural constraint, allowing the exposed phosphoserine residue clusters to bind efficiently with metal ions, thereby expanding their biological activities to multiple dimensions, including antioxidant, antibiosis, osteogenesis promotion, and α‐amylase‐inhibiting and amylase‐alleviating insulin resistance (Hu et al. 2023; Liu et al. 2023; Xue, Gong, et al. 2025; Xue, Zhao, et al. 2025). Therefore, converting extracted PV into PPPs could significantly improve its utilization.

3.6. Yield and Potential Utilization of PV‐Free Fraction

The yield of the leftover of PV extraction, or PV‐free fraction, was 1.17 ± 0.05 g per egg yolk, or 6.75 ± 0.31 g/100 g yolk. This accounts for 13.5% of dried egg yolk weight, which is similar to the value of 12.6% reported by Chalamaiah et al. (2018). The yield ratio of PV, PL, and PV‐free fraction was 1:1.21:7.02, and these three components accounted for 10.84%, 13.12%, and 76.04% of the total mass of dry egg yolk granules, respectively. Previous studies were mainly focused on egg yolk or defatted egg yolk granules, whereas minimal research had been conducted on egg yolk granules with PV removal. To the best of our knowledge, this was the first report on the specific yield ratio of the three components isolated from egg yolk granules. Chalamaiah et al. (2018) demonstrated that the removal of PV induced the structural disruption of egg yolk granules, and the resulting granules exhibited higher apparent viscosity than the intact granules, along with superior foaming and emulsifying properties. Therefore, these leftover granules can be used as a food ingredient and applied in the production of various food products such as mayonnaise, salad dressings, cream‐style sauces, and bakery items.

The existing research findings on egg yolk could also provide an important reference for the development and utilization of the PV‐free fraction. One promising approach is to fabricate edible films by the casting method (S. Marcet et al. 2022). In addition, the incorporation of substances such as nisin and thymol into the system can provide the resultant films with antimicrobial properties, thereby expanding their application in food packaging (I. Marcet et al. 2017). Another effective approach is the use of enzyme hydrolysis to obtain functional peptides. Guo et al. (2025) established a simple, efficient, and scalable separation and enrichment technology for egg yolk peptides. By using DA201‐C resin as an adsorbent, adjusting the pH of the loading solution to 7.5, and combining the gradient ethanol elution strategy, they successfully achieved the simultaneous enrichment and separation of three types of functional peptide fractions with osteogenic and metal‐chelating, antioxidant, and ACE‐inhibitory activities from a defatted egg yolk hydrolysate, with the total recovery rate of active peptides reaching 87.65%. Overall, the insights gained from existing research on egg yolk can be effectively translated into practical strategies for utilizing the PV‐free fraction.

4. Conclusion

This study optimized separation conditions and established an integrated continuous separation and purification strategy for six functional components from egg yolk, and the general pathway was as follows: (1) Diluting egg yolk with distilled water, adjusting pH, and centrifuging to separate the SUP and PPT; (2) Performing salting‐out to isolate IgY from other livetins; (3) Treating the PPT with a salt solution to remove LDL; (4) Isolating PL with ethanol; (5) Extracting PV with a high‐salt alkaline solution and simultaneously obtaining the PV‐free fraction. The total recovery of six target fractions was 86.06%, with IgY, IgY‐free livetins, LDL, PLs, PV, and PV‐free fraction made up 1.76%, 3.25%, 74.39%, 2.71%, 2.30%, and 15.85% of all recovered materials. Compared with single‐component extraction protocols, the present strategy obtained IgY with satisfactory yield and purity, PV, and PLs with high purity yet moderate yields, alongside substantial co‐production of LDL, IgY‐free livetins, and PV‐free fraction. Only five conventional reagents, namely ethanol, HCl, NaOH, (NH4)2SO4, and NaCl were employed in the process. Featuring simple operation and environmental friendliness, this method provides technical support for the large‐scale application of these value‐added egg yolk components in food ingredients and functional food development.

Author Contributions

Yao Yao: formal analysis, writing – original draft, investigation, data curation, methodology, conceptualization. Zihong Ma: formal analysis, investigation, data curation. Yonggang Tu: supervision, writing – review and editing. Dong Uk Ahn: conceptualization, writing – review and editing, supervision, funding acquisition, resources, methodology.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

This study is supported by the National Institute of Food and Agriculture/USDA (No. 2024‐67017‐42821). Sincere gratitude is extended to Byungrok Min for his valuable contribution to the manuscript review and editing.

References

  1. Abeyrathne, E. D. N. S. , Nam K. C., Huang X., and Ahn D. U.. 2022. “Egg Yolk Lipids: Separation, Characterization, and Utilization.” Food Science and Biotechnology 31: 1243–1256. 10.1007/s10068-022-01138-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ahn, D. U. , Lee S. H., Singam H., Lee E. J., and Kim J. C.. 2006. “Sequential Separation of Main Components From Chicken Egg Yolk.” Food Science and Biotechnology 15: 189–195. [Google Scholar]
  3. Akita, E. M. , and Nakai S.. 1992. “Immunoglobulins From Egg Yolk: Isolation and Purification.” Journal of Food Science 57: 629–634. 10.1111/j.1365-2621.1992.tb08058.x. [DOI] [Google Scholar]
  4. Anton, M. 2013. “Egg Yolk: Structures, Functionalities and Processes.” Journal of the Science of Food and Agriculture 93, no. 12: 2871–2880. 10.1002/jsfa.6247. [DOI] [PubMed] [Google Scholar]
  5. Anton, M. , and Gandemer G.. 1997. “Composition, Solubility and Emulsifying Properties of Granules and Plasma of Egg Yolk.” Journal of Food Science 62: 484–487. 10.1111/j.1365-2621.1997.tb04411.x. [DOI] [Google Scholar]
  6. Bernardo, J. , Videira R. A., Valentão P., Veiga F., and Andrade P. B.. 2019. “Extraction of Phospholipid‐Rich Fractions From Egg Yolk and Development of Liposomes Entrapping a Dietary Polyphenol With Neuroactive Potential.” Food and Chemical Toxicology 133: 110749. 10.1016/j.fct.2019.110749. [DOI] [PubMed] [Google Scholar]
  7. Carlander, D. , Wilhelmson M., and Larsson A.. 2001. “Limited Day to Day Variation of IgY Levels in Eggs From Individual Laying Hens.” Food and Agricultural Immunology 13, no. 2: 87–92. 10.1080/09540100120055657. [DOI] [Google Scholar]
  8. Chalamaiah, M. , Esparza Y., Hong H., Temelli F., and Wu J.. 2018. “Physicochemical and Functional Properties of Leftover Egg Yolk Granules After Phosvitin Extraction.” Food Chemistry 268: 369–377. 10.1016/j.foodchem.2018.06.033. [DOI] [PubMed] [Google Scholar]
  9. Chalamaiah, M. , Esparza Y., Temelli F., and Wu J.. 2017. “Physicochemical and Functional Properties of Livetins Fraction From Hen Egg Yolk.” Food Bioscience 18: 38–45. 10.1016/j.fbio.2017.04.002. [DOI] [Google Scholar]
  10. Chalamaiah, M. , and Wu J.. 2017. “Anti‐Inflammatory Effects of Egg Yolk Livetins (α, β, and γ‐Livetin) Fraction and Its Enzymatic Hydrolysates in Lipopolysaccharide‐Induced RAW 264.7 Macrophages.” Food Research International 100: 449–459. 10.1016/j.foodres.2017.07.032. [DOI] [PubMed] [Google Scholar]
  11. Egelhaaf, S. U. , Platten F., and Hansen J.. 2009. “Analysis of Hofmeister Effects on the Density Profile of Protein Adsorbates: A Neutron Reflectivity Study.” Journal of Physical Chemistry B 113, no. 45: 14952–14958. 10.1021/jp904065w. [DOI] [PubMed] [Google Scholar]
  12. Giarratano, M. , Duffuler P., Chamberland J., et al. 2020. “Combination of High Hydrostatic Pressure and Ultrafiltration to Generate a New Emulsifying Ingredient From Egg Yolk.” Molecules 25: 1184. 10.3390/molecules25051184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gu, L. , Liu Y., Zhang W., et al. 2023. “Novel Extraction Technologies and Potential Applications of Egg Yolk Proteins.” Food Science and Biotechnology 32: 121–133. 10.1007/s10068-022-01209-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guo, X. , Liu H., and Li B.. 2025. “The Simultaneous Enrichment and Separation of Various Bioactive Peptides in Defatted Egg Yolk Hydrolysates by Macroporous Resins.” Food Bioscience 74: 107841. 10.1016/j.fbio.2025.107841. [DOI] [Google Scholar]
  15. Horie, K. , Horie N., Abdou A. M., et al. 2004. “Suppressive Effect of Functional Drinking Yogurt Containing Specific Egg Yolk Immunoglobulin on Helicobacter pylori in Humans.” Journal of Dairy Science 87, no. 12: 4073–4079. 10.3168/jds.S0022-0302(04)73549-3. [DOI] [PubMed] [Google Scholar]
  16. Hu, L. , Sun C., Wang S., Su F., and Zhang S.. 2023. “Lipopolysaccharide Neutralization by a Novel Peptide Derived From Phosvitin.” International Journal of Biochemistry & Cell Biology 45, no. 11: 2622–2631. 10.1016/j.biocel.2013.09.002. [DOI] [PubMed] [Google Scholar]
  17. Huang, X. , and Ahn D. U.. 2019. “How Can the Value and Use of Egg Yolk Be Increased?” Journal of Food Science 84, no. 2: 205–212. 10.1111/1750-3841.14430. [DOI] [PubMed] [Google Scholar]
  18. Huang, X. , Moon S. H., Lee J., et al. 2019. “Effective Preparation Method of Phosphopeptides From Phosvitin and the Analysis of Peptide Profiles Using Tandem Mass Spectrometry.” Journal of Agricultural and Food Chemistry 67, no. 51: 14086–14101. 10.1021/acs.jafc.9b05973. [DOI] [PubMed] [Google Scholar]
  19. Ibrahim, H. R. , Hitotsumatsu A., and Miyata T.. 2023. “Identification of Potent Antioxidant Bioactive Peptides From the Soluble Proteins of Chicken Egg Yolk.” Functional Food Science 3, no. 6: 63–78. 10.31989/ffs.v3i6.1110. [DOI] [Google Scholar]
  20. Jiang, B. , Wang L., Wang X., et al. 2019. “Ultrasonic Thermal‐Assisted Extraction of Phosvitin From Egg Yolk and Evaluation of Its Properties.” Polymers 11: 1353. 10.3390/polym11081353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Karthikeyan, M. , Indhuprakash S. T., Gopal G., Ambi S. V., Krishnan U. M., and Diraviyam T.. 2022. “Passive Immunotherapy Using Chicken Egg Yolk Antibody (IgY) Against Diarrheagenic E. coli: A Systematic Review and Meta‐Analysis.” International Immunopharmacology 102: 108381. 10.1016/j.intimp.2021.108381. [DOI] [PubMed] [Google Scholar]
  22. Ko, K. Y. , Nam K. C., Jo C., Lee E. J., and Ahn D. U.. 2011. “A Simple and Efficient Method for Preparing Partially Purified Phosvitin From Egg Yolk Using Ethanol and Salts.” Poultry Science 90: 1096–1104. 10.3382/ps.2010-01138. [DOI] [PubMed] [Google Scholar]
  23. Laber, J. R. , Dear B. J., Martins M. L., et al. 2017. “Charge Shielding Prevents Aggregation of Supercharged GFP Variants at High Protein Concentration.” Molecular Pharmaceutics 14, no. 10: 3269–3280. 10.1021/acs.molpharmaceut.7b00322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee, H. Y. , Abeyrathne E. D., Choi I., Suh J. W., and Ahn D. U.. 2014. “Sequential Separation of Immunoglobulin Y and Phosvitin From Chicken Egg Yolk Without Using Organic Solvents.” Poultry Science 93, no. 10: 2668–2677. 10.3382/ps.2014-04093. [DOI] [PubMed] [Google Scholar]
  25. Lei, B. , and Wu J.. 2012. “Purification of Egg Yolk Phosvitin by Anion Exchange Chromatography.” Journal of Chromatography A 1223: 41–46. 10.1016/j.chroma.2011.12.023. [DOI] [PubMed] [Google Scholar]
  26. Li, J. , Zhai J., Gu L., et al. 2021. “Hen Egg Yolk in Food Industry—A Review of Emerging Functional Modifications and Applications.” Trends in Food Science & Technology 115: 12–21. 10.1016/j.tifs.2021.06.031. [DOI] [Google Scholar]
  27. Liu, W. , Tang C., Cai Z., Jin Y., Ahn D. U., and Huang X.. 2023. “The Effectiveness of Polypeptides From Phosvitin and Eggshell Membrane in Enhancing the Bioavailability of Eggshell Powder Calcium and Its Accumulation in Bones.” Food Bioscience 51: 102257. 10.1016/j.fbio.2022.102257. [DOI] [Google Scholar]
  28. Liu, W. , Zhao M., Li S., Ahn D. U., Chen N., and Huang X.. 2022. “Advances in Preparation and Bioactivity of Phosvitin Phosphopeptides.” Journal of Future Foods 2, no. 3: 213–222. 10.1016/j.jfutfo.2022.06.003. [DOI] [Google Scholar]
  29. Luo, W. , Liu X., Wang B., Wu D., Wang J., and Geng F.. 2023. “Quantitative Lipidomics Analysis of Changes in Egg Yolk Lipids During Spray‐Drying and Subsequent Accelerated Storage.” Current Research in Food Science 6: 100503. 10.1016/j.crfs.2023.100503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lv, X. , Huang X., Ma B., et al. 2022. “Modification Methods and Applications of Egg Protein Gel Properties: A Review.” Comprehensive Reviews in Food Science and Food Safety 21, no. 3: 2233–2252. 10.1111/1541-4337.12907. [DOI] [PubMed] [Google Scholar]
  31. Marcet, I. , Sáez S., Rendueles M., and Díaz M.. 2017. “Edible Films From Residual Delipidated Egg Yolk Proteins.” Journal of Food Science and Technology 54, no. 12: 3969–3978. 10.1007/s13197-017-2861-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Marcet, S. , Sáez‐Orviz M., Rendueles M., and Díaz M.. 2022. “Egg Yolk Granules and Phosvitin. Recent Advances in Food Technology and Applications.” LWT 153: 112442. 10.1016/j.lwt.2021.112442. [DOI] [Google Scholar]
  33. Mordor Intelligence . 2024. “Liquid Egg Market Size, Share & 2030 Growth Trends Report.” https://www.mordorintelligence.com/industry‐reports/liquid‐egg‐market.
  34. Moussa, M. , Martinet V., Trimeche A., Tainturier D., and Anton M.. 2002. “Low Density Lipoproteins Extracted From Hen Egg Yolk by an Easy Method: Cryoprotective Effect on Frozen–Thawed Bull Semen.” Theriogenology 57, no. 6: 1695–1706. 10.1016/S0093-691X(02)00682-9. [DOI] [PubMed] [Google Scholar]
  35. Naderi, N. , Pouliot Y., House J. D., and Doyen A. J.. 2017. “High Hydrostatic Pressure Effect in Extraction of 5‐Methyltetrahydrofolate (5‐MTHF) From Egg Yolk and Granule Fractions.” Innovative Food Science & Emerging Technologies 43: 191–200. 10.1016/j.ifset.2017.08.009. [DOI] [Google Scholar]
  36. Navidghasemizad, S. , Temelli F., and Wu J.. 2014. “Physicochemical Properties of Leftover Egg Yolk After Livetins Removal.” LWT—Food Science and Technology 55: 170–175. 10.1016/j.lwt.2013.08.002. [DOI] [Google Scholar]
  37. Nilsson, E. , Hanrieder J., Bergquist J., and Larsson A.. 2008. “Proteomic Characterization of IgY Preparations Purified With a Water Dilution Method.” Journal of Agricultural and Food Chemistry 56, no. 24: 11638–11642. 10.1021/jf802626t. [DOI] [PubMed] [Google Scholar]
  38. Ozbun, T. 2025. “Per Capita Consumption of Eggs in the United States From 2000–2026.” https://www.statista.com/statistics/183678/per‐capita‐consumption‐of‐eggs‐in‐the‐us‐since‐2000/?srsltid=AfmBOoqoZEgbJUZKAnF0m2P9KEWFbOIhBeOBbnnkWWn9NDsXZ86aCkPk.
  39. Pashkuleva, I. , and Teixeira B.. 2025. “Latest Findings in Immunoglobulin Y Technologies and Applications.” International Journal of Molecular Sciences 26, no. 13: 6380. 10.3390/ijms26136380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Patanwala, I. , King M. J., Barrett D. A., et al. 2014. “Folic Acid Handling by the Human Gut: Implications for Food Fortification and Supplementation.” American Journal of Clinical Nutrition 100, no. 2: 593–599. 10.3945/ajcn.113.080507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rahman, S. , Van Nguyen S. A., Icatlo F. C. Jr., Umeda K., and Kodama Y.. 2013. “Oral Passive IgY‐Based Immunotherapeutics: A Novel Solution for Prevention and Treatment of Alimentary Tract Diseases.” Human Vaccines & Immunotherapeutics 9, no. 5: 1039–1048. 10.4161/hv.23383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saini, R. K. , Prasad P., Shang X., and Keum Y. S.. 2021. “Advances in Lipid Extraction Methods—A Review.” International Journal of Molecular Sciences 22: 13643. 10.3390/ijms222413643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schade, R. , and Csutak A.. 2019. “Egg Yolk Antibodies (IgY) and Their Applications in Human and Veterinary Health: A Review.” Veterinary Medicine and Science 5, no. 3: 221–232. 10.1002/vms3.245. [DOI] [Google Scholar]
  44. Shchipunov, Y. U. A. 2001. “Lecithin Organogel: A Micellar System With Unique Properties.” Colloids and Surfaces A: Physicochemical and Engineering Aspects 183–185: 541–554. 10.1016/S0927-7757(01)00511-8. [DOI] [Google Scholar]
  45. Somasundaram, R. , Choraria A., and Antonysamy M.. 2020. “An Approach Towards Development of Monoclonal IgY Antibodies Against SARS CoV‐2 Spike Protein (S) Using Phage Display Method: A Review.” International Immunopharmacology 85: 106654. 10.1016/j.intimp.2020.106654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sterk, R. 2015. “Egg Product Prices Retreat.” Food Business News, November 3. https://www.foodbusinessnews.net/articles/5305‐egg‐product‐prices‐retreat.
  47. Thu, H. M. , Myat T. W., Win M. M., et al. 2017. “Chicken Egg Yolk Antibodies (IgY) for Prophylaxis and Treatment of Rotavirus Diarrhea in Human and Animal Neonates: A Concise Review.” Korean Journal for Food Science of Animal Resources 37, no. 1: 1–9. 10.5851/kosfa.2017.37.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tong, C. , Geng F., He Z., Cai Z., and Ma M.. 2015. “A Simple Method for Isolating Chicken Egg Yolk Immunoglobulin Using Effective Delipidation Solution and Ammonium Sulfate.” Poultry Science 94: 104–110. 10.3382/ps/peu005. [DOI] [PubMed] [Google Scholar]
  49. Wang, X. , Li J., Su Y., Chang C., and Yang Y.. 2021. “Freeze‐Thaw Treatment Assists Isolation of IgY From Chicken Egg Yolk.” Food Chemistry 364: 130225. 10.1016/j.foodchem.2021.130225. [DOI] [PubMed] [Google Scholar]
  50. Wei, S. , Duan S., Liu X., et al. 2021. “Chicken Egg Yolk Antibodies (IgYs) Block the Binding of Multiple SARS‐CoV‐2 Spike Protein Variants to Human ACE2.” International Immunopharmacology 90: 107172. 10.1016/j.intimp.2020.107172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Xia, M. , Liu C., Ahn D. U., Huang X., Jin Y., and Cai Z.. 2023. “Large‐Scale Isolation and Purification of Yolk Immunoglobulin With Different Purity Levels via a Combination Technique Based on High‐Speed‐Shear Crossflow Membrane Separation.” Food Hydrocolloids 140: 108618. 10.1016/j.foodhyd.2023.108618. [DOI] [Google Scholar]
  52. Xiao, N. , Zhao Y., Yao Y., et al. 2020. “Biological Activities of Egg Yolk Lipids: A Review.” Journal of Agricultural and Food Chemistry 68, no. 7: 1948–1957. 10.1021/acs.jafc.9b06616. [DOI] [PubMed] [Google Scholar]
  53. Xue, T. , Gong T., Zhao W., et al. 2025. “A Novel Phosvitin Phosphopeptide (Glu‐Asp‐Asp‐pSer‐pSer) as a Potential Food Ingredient to Intervene in Osteoporosis: A Potential Mechanism Study on Promoting the Osteogenic Differentiation of MC3T3‐E1 Cells.” Journal of Functional Foods 128: 106821. 10.1016/j.jff.2025.106821. [DOI] [Google Scholar]
  54. Xue, T. , Zhao W., Hao C., Zhi H., Xu Q., and Zhang X.. 2025. “Two Novel Phosvitin Peptides FGTEPDAK and IWGR: the Dual Effects and Associated Mechanisms for Inhibiting α‐Amylase and Alleviating Insulin Resistance in IR‐HepG2 Cells.” Food Chemistry 475: 143314. 10.1016/j.foodchem.2025.143314. [DOI] [PubMed] [Google Scholar]
  55. Yakhkeshi, S. , Wu R., Chelliappan B., and Zhang X.. 2022. “Trends in Industrialization and Commercialization of IgY Technology.” Frontiers in Immunology 13: 991931. 10.3389/fimmu.2022.991931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yilmaz, B. , and Ağagündüz D.. 2020. “Bioactivities of Hen's Egg Yolk Phosvitin and Its Functional Phosphopeptides in Food Industry and Health.” Journal of Food Science 85: 2969–2976. 10.1111/1750-3841.15447. [DOI] [PubMed] [Google Scholar]
  57. Yin, M. , Matsuoka R., Xi Y., and Wang X.. 2021. “Comparison of Egg Yolk and Soybean Phospholipids on Hepatic Fatty Acid Profile and Liver Protection in Rats Fed a High‐Fructose Diet.” Foods 10: 1569. 10.3390/foods10071569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhang, F. , Ding Y., Zhang Z., et al. 2024. “Developing a Simple and Feasible Process for the Crude Extraction of Livetins and Phosvitin From Egg Yolk.” Foods 13: 3990. 10.3390/foods13243990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhao, F. , Li R., Liu Y., and Chen H.. 2023. “Perspectives on Lecithin From Egg Yolk: Extraction, Physicochemical Properties, Modification, and Applications.” Frontiers in Nutrition 9: 1082671. 10.3389/fnut.2022.1082671. [DOI] [PMC free article] [PubMed] [Google Scholar]

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