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Egg yolk phosvitin is of particular interest due to its functional and biological properties. Recently, it was demonstrated that high hydrostatic pressure (HHP) (400 MPa for 5 min) induced the transfer of folic acid and phosvitin from the egg yolk granule to the plasma fraction. A granule fraction (G in ) produced by egg yolk centrifugation was pressure-treated at 400 and 600 MPa for 5 and 10 min, and centrifuged to generate granule fractions (G P1 to G P4 ) and plasmas (P P1 to P P4 ). Iron and phosphorus contents were also increased in P P1 to P P4 fractions, confirming the transfer of phosvitins from pressure-treated granule to plasma. Pressurization drastically improved phosvitin recovery in P P fractions, specifically at 600 MPa for 10 min, which had the highest value of phosvitin/100 mg of dry plasma at 33.3 ± 4.39 mg. Consequently, HHP represents an alternative approach for phosvitin transfer and recovery in the egg yolk soluble fraction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this study, the impact of pasteurization and freezing of raw material, as performed at a commercial scale, on egg yolk fractionation and folate recovery was assessed. Freezing induced denaturation of the lipoproteins in egg yolk, which prevented further fractionation of the yolk. Thermal pasteurization of egg yolk at 61.1 °C for 3.5 min as well as high hydrostatic pressure (HHP) treatment (400 MPa for 5 min) did not change (p < 0.05) the composition of egg yolk or yolk fractions after their recovery by centrifugation. Expressed as dry matter, folate in pasteurized yolk was measured to be 599 μg/100 g, while its concentration reached 1969.7 μg/100 g for pasteurized granule and 1902.5 μg/100 g for HHP-treated granule. Folate was not detected in plasma, emphasizing the complete separation of yolk folate into granule. Further, we studied the effect of HHP on different dilutions of egg yolk, which were then fractionated. Egg yolk was diluted with water at different concentrations (0.1, 1.0, 10, 25, and 50%), HHP-treated at 400 MPa for 5 min, and centrifuged. Characterization of the compositions of the separated granule and plasma followed. Folate was stable under the HHP conditions used. However, HHP caused separation of folate from the yolk structure into water-soluble plasma. After HHP processing, the amount of folate detected in the plasma fraction was significantly (p < 0.05) higher (1434.9 μg/100 g) in the 25% diluted samples but was significantly (p < 0.05) lower in HHP-treated granule samples. Native sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that phosvitin, α-livetin, and apovitellenin VIa were the proteins most resistant to HHP. This study confirms that dilution of egg yolk before HHP treatment can significantly (p < 0.05) change the composition of granule and plasma fractions after centrifugal fractionation of egg yolk.

Background: Phosvitin is the principal phosphoprotein in egg yolk and has great potential for use as a functional food ingredient in improving bone health. This study reports a thermal-aided extraction method without using organic solvents or non-food-compatible chemicals., Results: Egg yolk was two times diluted with water and then extracted by 100 g L(-1) NaCl. Effects of pH and heating temperature on the extract were examined. The phosvitin purity increased from 75.7% at pH 8.0 to 80.1% at pH 5.0 and then started to decrease, but the yield decreased at decreasing pHs. The phosvitin purity increased at increasing temperature up to 90 °C and then started to decrease at 95 °C, while the yield increased from 70 to 80 °C and then started to decline at 85 °C., Conclusion: A purity of 88.0% and a yield of 23.5 g kg(-1) yolk dry matter were obtained at 90 °C. The purity and yield were comparable to or higher than those of previously methods. The method developed in this study is simple, including mainly two steps, i.e. water dilution of egg yolk and NaCl extraction with heating, and can be scaled up for industrial production., (© 2015 Society of Chemical Industry.)

A study was conducted to develop a simple sequential separation protocol to separate phosvitin and IgY from egg yolk without using organic solvents. Egg yolk was diluted with 2 volumes of distilled water (DW), homogenized, and centrifuged. The precipitant was collected and homogenized with 4 volumes of 10% NaCl (wt/vol) in 0.05 N NaOH solution to extract phosvitin. The pH of the homogenate was adjusted to 4.0 and the precipitate was removed by centrifugation. The supernatant was collected and then heat-treated at 70°C for 30 min and centrifuged to remove impurities. The supernatant containing phosvitin was collected, had salts removed, and was concentrated and then freeze-dried. The supernatant from the centrifugation of diluted egg yolk was diluted again with 3 volumes of DW, and the precipitate was removed by centrifugation. The resulting supernatant was concentrated using ultrafiltration and then IgY was precipitated using 20% saturated (NH₄)₂SO₄+ 15% NaCl (wt/vol). The precipitant was collected after centrifugation at 3,400 × g for 30 min at 4°C and dissolved with DW, had salts removed, and then was freeze-dried. The purity of separated phosvitin and IgY was checked using SDS-PAGE and the proteins were verified using Western blotting. The purity of phosvitin and IgY was 97.2 and 98.7%, and the yield was 98.7 and 80.9%, respectively. The ELISA results indicated that the activities of separated IgY and phosvitin were 96.3 and 98.3%, respectively. This study proved that both phosvitin and IgY can be separated in sequence from egg yolk without using an organic solvent. Also, the method is very simple and has a high potential for scale-up processing., (©2014 Poultry Science Association Inc.)

A novel TiO2/diatomite composite (TD) was prepared and then characterized by scanning electron microscope (SEM) and Fourier Transform Infrared (FTIR). The results of SEM showed that after modification, the porous surface of diatomite was covered with TiO2. Both diatomite and TD had clear disc-shaped structures with average grain diameters of around 25 μm. Then TD and pure TiO2 were applied in the purification of phosvitin phosphopeptides (PPPs) from the digest of egg yolk protein, and a comparative study of adsorption properties of PPPs on TD and TiO2 was performed. In the study of adsorption kinetics, the adsorption equilibrium of PPPs on TD and TiO2 fitted well with the Langmuir model, and the time needed to reach adsorption equilibrium were both around 10 min. The maximum dynamic adsorption capacity of TD (8.15 mg/g) was higher than that of TiO2 (4.96 mg/g). The results of repeated use showed that TD and TiO2 were very stable after being subjected to ten repeated adsorption-elution cycles, and TD could easily be separated from aqueous solution by filtration. On the other hand, the present synthetic technology of TD was very simple, cost-effective, organic solvent-free and available for large-scale preparation. Thus, this separation method not only brings great advantages in the purification of PPPs from egg yolk protein but also provides a promising purification material for the enrichment of phosphopeptides in proteomic researches., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Functional immobilized metal affinity magnetic carboxymethyl chitosan nanoparticles (abbreviated as Fe(3)O(4) (PEG+CM-CTS) @ Fe (III)) were conveniently applied for phosvitin phosphopeptides (PPPs) enrichment for the first time. The morphology of magnetic nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) was about 20 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. In the PPPs enrichment study, the results obtained emphasized the role of pH, temperature and the initial concentration of the peptides solution in governing the efficiency and mechanism of affinity interactions. Due to the large specific surface area, the enrichment of PPPs onto the Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) nanoparticles was promising. The adsorption equilibrium of PPPs onto the obtained magnetic nanoparticles fitted well with the Langmuir model, and the nitrogen/phosphorus molar ratio (N/P) which at the maximum enrichment capacity for PPPs was 4.83. Due to the small diameter, the decrease of the N/P is particularly rapid in the early enrichment stages (0-30 min) to reach a plateau after 60 min. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and PPPs of higher purity were obtained. Since the preparation, surface modification and affinity separation processes of the magnetic nanoparticles are cost-effective, convenient and efficient, this type of Fe(3)O(4) (PEG+CM-CTS) @ Fe (III) nanoparticles would bring advantages compared to conventional separation techniques of PPPs from chicken egg yolk, as well as for phosphopeptides enrichment in proteomics research., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as IMAN @ Fe (III)) were prepared and employed in extracting of Phosvitin Phosphopeptides (short as PPPs) from egg yolk. It was found that the diameter of the magnetic CS nanoparticles was about 20 nm, and they could easily be aggregated by a magnet when suspending in the aqueous solution. The adsorption equilibrium of PPPs onto the obtained nanocarriers fitted well with the Langmuir model. The adsorption capacity of PPPs onto the superparamagnetic nanoparticles was influenced by pH and the initial concentration of the peptides solution. The final nitrogen/phosphorus molar ratios (short as N/P) of PPPs from crude egg yolk peptides and phosvitin peptides were low to 5.78 and 5.23, respectively. Compared with traditional methods, the need for preparation of phosvitin before purification is obviated and the higher purity of PPPs were obtained. In conclusion, this type of IMAN @ Fe (III) would bring advantages to the conventional separation techniques of PPPs from chicken egg yolk., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The objective of this study was to develop a simple method of phosvitin purification from hen egg yolk without using organic solvents. Egg yolk was diluted with equal volume of water and stirred for one hour at room temperature, followed by centrifugation to remove soluble proteins along with most of the yolk lipids in the supernatant. The granules were collected as the precipitate containing minimum amount of lipids (dry granules). The dry granules were dissolved in 0.05 M carbonate-bicarbonate buffer at pH 9.6, which yields a light yellowish solution used for anion exchange chromatography. Phosvitin fraction was collected from anion exchange chromatography as the last eluting peak with a purity of 92.6% and a yield of 35.4% of total phosvitin in the yolk or a recovery of 1.9% of total yolk dry matter, which are comparable to current methods employing organic solvents or chromatography after salt fractionation and dialysis. This method developed is simple and fast without using organic solvents., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An attempt was made to develop a new protocol for preparing phosvitin that could be easy scaled up using polyethylene glycol (PEG6000). Influence of PEG6000 concentration and pH of sample solution on phosvitin isolation was investigated. Phosvitin of high purity (99%) was obtained in good yield (47%) with the optimal condition of pH 4.0 and 3% PEG6000 precipitation. In addition, through evaluating different anion-exchange chromatography methods, the DEAE procedure at pH 7.5 was finally selected as the best procedure to obtain metal-free phosvitin that lost the least protein. Furthermore, it is observed that the purity and characterizations of prepared phosvitin were similar to those of the standard phosvitin from Sigma, and the random coil of phosvitin converted into more compact structure after removing metal ion. In conclusion, the developed method was simple and suitable for scaled up preparation of phosvitin., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phosvitin, an egg yolk protein constituted by 50% of phosphorylated serines, presents good emulsifying properties whereas its interfacial properties are not yet clearly elucidated and remain object of discussion. Phosvitin has a high charge density and naturally forms aggregates through phosphocalcic bridges in egg yolk. This high charge density, doubled by this capacity to aggregate, limits the adsorption of the protein at the air-water interface. In this work, we investigated the aggregation impact by calcium ions on the organization of the phosvitin interfacial film using the atomic force microscopy. Phosvitin interfacial films without calcium ions are compared to phosvitin interfacial films formed in the presence of calcium ions in the subphase. We demonstrated that phosvitin is able to anchor at air-water interfaces in spite of its numerous negative charges. In the compression isotherm a transition was observed just before 28 mN/m signifying a possible modification of the interfacial film structure or organization. Calcium ions induce a reorganization towards a greater compaction of the phosvitin interfacial film even at low surface pressure. In conclusion we suggest that, in diluted regime, phosvitin molecules could adsorb by their two hydrophobic extremities exhibiting loops in the aqueous phase, whereas in concentred regime (high interfacial concentration) it would be adsorbed at the interface by only one extremity (brush model).

An immobilized metal affinity electrophoresis (IMAEP) method is described here. In this method, metal ions are immobilized in a native polyacrylamide gel to capture phosphoproteins. The capture of phosphoproteins by IMAEP is demonstrated with immobilized metals like iron, aluminum, manganese, or titanium. In the case studies, phosphoproteins alpha-casein, beta-casein, and phosvitin are successfully extracted from a protein mixture by IMAEP.

Two chromatographic methods for hen egg yolk phosvitin purification avoiding organic solvents were evaluated. Hydrophobic interaction and ion-exchange chromatographies were applied to isolated phosvitin. Hydrophobic interaction chromatography has better capacity than ion-exchange chromatography to fractionate phosvitin in their different polypeptides, but its protein yield was lower (0.7 vs. 1.7% of egg yolk dry matter). Finally, ion-exchange chromatography was selected and allowed to fractionate phosvitin polypeptides, including the recovering of phosphoproteins with high electrophoretic mobility: phosvettes. Highly purified (>98%) and free metal protein was obtained in reduced time. Phosvitin polypeptide heterogeneity was evidenced.

Vitellogenin (Vg) and its three egg yolk protein products, lipovitellin (Lv), phosvitin (Pv) and beta'-component, were isolated from mature female Sakhalin taimen (Hucho perryi). Vg had an apparent molecular weight of 540 kDa and appeared as a major 240 kDa band in SDS-PAGE, which resolved into two major bands (165 and 125 kDa) after reduction. The estimated molecular weights of purified Lv, Pv, and beta'-component were 330, 23, and 30 kDa, respectively. Lv appeared as a main band of 150 kDa in SDS-PAGE which resolved into two smaller bands (92 and 29 kDa) after reduction. beta'-component appeared as a 34 kDa band before and as a 17kDa band after reduction. Except for Pv, the purified proteins all reacted with an antiserum to Vg. In SDS-PAGE, Pv appeared as a 23 kDa band and a second < 6.5 kDa diffuse band. An antiserum to Pv dephosphorylated by alkaline phosphatase (Ap) was prepared. In Western blots, the antiserum reacted with dephosphorylated Pv and Vg, but not with Lv and beta'-component. This is the first immunological proof that three egg yolk proteins (Lv, Pv, and beta'-component) are derived from Vg in fish.

1. A method developed for the isolation of phosvitin from chicken egg yolk was successfully applied to the isolation of phosvitin from salmon eggs. 2. Salmon roe phosvitin is smaller in molecular size than chicken egg phosvitin. 3. Circular dichroism spectra of all phosvitins investigated displayed good similarities with spectra showing characteristics of unordered and beta-sheet secondary structure. 4. The main component in the Fourier transform infrared spectra of chicken egg phosvitin is indicative of unordered conformation, whereas the Fourier infrared data of the salmon egg phosvitin are consistent with more of beta-sheet structure compared to the chicken egg phosvitin.

Phosvitin polymorphism in egg yolk of the Japanese quail was found by horizontal polyacrylamide gradient gel electrophoresis. Six phenotypes of yolk phosvitin designated A, B, C, AB, AC, and BC were observed in a population of 281 birds. Analysis of family data revealed that the phenotypic variation of quail yolk phosvitins was controlled by an autosomal Pv locus with three codominant alleles, Pva, Pvb and Pvc. The gene frequencies of Pva, Pvb and Pvc were 0.064, 0.824 and 0.112, respectively.

A family of hitherto undescribed sialoglycoproteins has been isolated from the eggs of Pacific herring in a yield about 10 times as much as phosvitin known as a major protein component of the eggs. The sialoglycoproteins are composed of 60-50% of carbohydrates and 40-50% of protein. The carbohydrate composition is similar to that of glycoproteins known to have complex types of heterosaccharide units whereas the amino acid composition is unique. The molecular weights ranging over 8800-13000 were determined for the sialoglycoprotein fractions by sedimentation equilibrium. The low mobility of the sialoglycoproteins during sodium dodecyl sulfate gel electrophoresis is discussed in terms of low values of hydrophobicity.

Modifications of the method of Wallace et al. [Can. J. Biochem. 44, 1647-1655 (1966)] for phosvitin isolation from vertebrate eggs were devised to enhance the method's general effectiveness. Phosvitins which do not precipitate on dilution from solutions of their lipovitellin complexes may be selectively adsorbed onto, and desorbed from, DEAE-cellulose. Phosvitins which are too small for dialysis or ultrafiltration may be concentrated or desalted by precipitation with stoichiometric amounts of ferric ions, followed by iron removal with EDTA on gel filtration. Since phosvitin distribution among ammonium sulfate fractions depends on initial protein and salt concentrations in a species-specific manner, pilot experiments are needed to establish conditions for optimal fractionation.

The polypeptide and lipid components of the crystallinelipoprotein-phosphoprotein from the yolk system of Xenopus laevis are described. This lipoprotein complex contains 17% lipid of which 75% is phospholipid. The phospholipid fraction consists of mostly phosphatidylcholine and phosphatidylethanolamine. The neutral lipid fraction contains mainly triglyceride. The phosphoprotein, phosvitin, has been separated from the lipoprotein, lipovitellin, by classical methods. Three polypeptide chains can be observed in the lipoprotein and their molecular weights as determined by sodium dodecyl sulfate gel electrophoresis are 105,500, 35,500, and 32,000. Phosvitin behaves abnormally on these gels, but analysis of the results suggests Mr = approximately 16,000 to 19,000/polypeptide chain. The lipovitellin component of the yolk complex contains approximately 100 bound lipid molecules/dimer. The stoichiometry of the components of this crystalline lipoprotein system is discussed in terms of the results obtained in this study and those of other workers.

The effects of estrogen on plasma vitellogenin have been studied in the cockerel by immunoprecipitation techniques using an antiserum prepared against the egg yolk phosphoprotein, phosvitin. The antiserum gave precipitin lines of complete identity to phosvitin and to vitellogenin which was isolated from hen plasma by DEAE-cellulose chromatography and by affinity chromatography using anti-phosvitin coupled to Sepharose 4B. The cross-reactivity of vitellogenin and phosvitin adds support to the concept that plasma vitellogenin is the precursor phosphoprotein of egg yolk phosvitin. In the three-week old cockerel, anti-phosvitin produced no detectable immunoprecipitate in the plasma. However, after a single sc injection of diethylstilbestrol (2.5 mg), plasma vitellogenin levels began to increase at 4 h and reached a maximum 20-30 h after hormone administration. The increase in plasma levels of triglyceride paralleled those of vitellogenin. These studies suggest that there is no significant time lag in the estrogenic induction of plasma vitellogenesis in the cockerel, the longer lag periods observed by other investigators may be a function of the sensitivity of the assays used for detecting vitellogenin.

Chicken phosvitin was prepared from egg yolk by a variety of published methods, including a modification of our own original procedure. Yolk granules and all phosvitin preparations have been previously found to contain major phosphoproteins at Mr 40,000 and 33,000 and minor satellite components when electrophoresed on polyacrylamide gradient gels and stained with Stains-all. However, only our current preparation contained three additional phosphoproteins (Mr 18,000, 15,000 and 13,000) that are also present in yolk granules. Our current phosvitin preparation also appeared to have additional components when compared with other preparations by size-exclusion and anion-exchange chromatography. Particularly complex but entirely reproducible patterns were obtained by hydrophobic-interaction chromatography. However, a cross-referencing of fractions eluted by size-exclusion chromatography to the other procedures employed, including gel electrophoresis, reinforced the notion that unfractionated chicken phosvitin contains at least five major components, designated B, C, E1, E2 and F for the Mr 40,000, 33,000, 15,000, 18,000, and 13,000 phosphoproteins, respectively. Stoichiometric considerations lead us to suggest that vitellogenin I gives rise to phosvitins C and F, vitellogenin II gives rise to phosvitin B, and vitellogenin III gives rise to either phosvitin E1 or E2, but not both. Thus, a fourth, as yet undetected, vitellogenin may exist for the chicken.

Vitellogenin serves as the plasma precursor for the yolk proteins, lipovitellin and phosvitin, in nonmammalian vertebrates. 32P-Vitellogenin was isolated from the plasma of the teleost, Fundulus heteroclitus, and was used both to label phosvitin in the ovary and to indicate the phosvitin region in preparative chromatographs of ovarian extracts on DEAE-cellulose. Crude [32P]phosvitin could be resolved further into two labeled components with shallow gradients on DEAE-cellulose and into eight labeled components by electrophoresis on 12% polyacrylamide gels. Only the two largest electrophoretically resolved components could be correlated with Coomassie Blue staining bands, but several of the smaller components could be indicated with the cationic carbocyanine dye, Stains-all. Stains-all-dyed components were also generally indicated as multiple bands. The ovary of a reproductively active female contains vitellogenic oocytes, postvitellogenic oocytes undergoing maturation prior to ovulation, and ovulated eggs. Examination of various types of follicles and eggs on polyacrylamide gels revealed that during maturation, the largest phosvitin components formed during vitellogenesis either disappear or diminish, while smaller phosvitin components appear. The transformation of phosvitin components can also be achieved in vitro by incubating prematurational follicles in a saline medium containing deoxycorticosterone. These preliminary results demonstrate that a complex array of phosvitin-like components are present within a single ovary of F. heteroclitus. We also postulate that one reason for the anomalous yolk proteins generally found thus far in teleost eggs is that some of the proteins derived from vitellogenin during vitellogenesis undergo further proteolysis during oocyte maturation.

The Ca-binding proteins of hen serum were resolved by gel filtration in 45Ca buffer and were named CaBP(1) and CaBP(2). The major calcium-binding protein, CaBP(1), had a molecular size of 6.0 X 10(5) daltons and appears to be vitellogenin. Deeley et al. (1975) described vitellogenin as the precursor of lipovitellin and phosvitin, the Ca-binding proteins of egg yolk. The present paper demonstrates that "native" vitellogenin is a calcium-binding protein in the serum of laying chickens. The CaBP(2) co-eluted with and was immunologically identical to chicken serum albumin which also bound 45Ca. Data from elution profiles and phosphorus assays indicated that CaBP(1) and CaBP(2) were different from phosvitin and the Ca-binding proteins of the duodenum and uterus.

Phosvitin was prepared from chicken yolk by a variety of published methods, including a new procedure modified from our own original method. Yolk granules and all phosvitin preparations were found to contain a cluster of phosphoproteins ranging in size from Mr 28,000 to 43,000 when electrophoresed on polyacrylamide gradient gels and stained with Stains-all. However, only the current preparation contained three additional phosphoproteins (Mr 13,000, 15,000, and 18,000) that are also present in yolk granules. None of these components could be demonstrated with Coomassie blue.

The catalytic activity of phosvitin in Fe(II) oxidation and the addition of iron to transferrin were studied under various conditions. It was concluded that the Fe(II) oxidized by phosvitin would bind to apotransferrin, although an appreciable fraction of Fe(III) remained bound to phosvitin. Fe(III) also migrated from phosvitin to apotransferrin. This reaction was first-order with respect to Fe(III)-phosvitin concentration with a half-time (t1/2) of 10 min, and a first-order rate constant, k=0.069min-1, in 700 muM-phosphate buffer, pH 7.2, at 30 degrees C. The catalysis of the oxidation of Fe(III) by phosvitin was proportional to O2 concentration, and is quite different from the relative O2 independence of Fe(II) oxidation as catalysed by ferroxidase. A scheme for the mobilization and transfer of iron in the chicken, including the role of ferroxidase, phosyitin and transferrin, is presented.