Hen Egg Yolk Phosvitin Research Articles

Bioactivities of hen's egg yolk phosvitin and its functional phosphopeptides in food industry and health.

Phosvitin, one of the most noteworthy bioactive components of hen egg yolk, is an amphiphilic protein that stands out with its unique composition and functionality in the food industry and health. Phosvitin consists of 4% of egg yolk dry matter and 11% of egg yolk proteins. It is considered as the most phosphorylated protein with 10% phosphorus. Besides, some potential novel phosphopeptides containing clusters of phosphoserines can be derived from hen's egg yolk phosvitin. Phosvitin, which has many functional features thanks to its unique structure, is known primarily for its metal bonds binding (iron, calcium, etc.) feature. On the other hand, its phosphopeptides may increase the bioavailability of metals compared to phosvitin. Although this feature of phosvitin may partially decrease the bioavailability of especially iron in the egg, it allows the phosvitin to have many bioactivities in the food industry and health. Lipid oxidation, which is a serious problem in the food industry, can be inhibited by adding phosvitin and its derived phosphopeptides to the food production chain via inhibiting bivalent iron. Because phosvitin is an amphiphilic protein capable of chelating, it also shows potential antibacterial effects against the Gram-negative bacteria. Moreover, the literature has recently been attempting to define the promising relationship between phosvitin and its phosphopeptides and plenty of health-promoting activities such as immune-enhancing, melanogenesis inhibitor, anti-ageing, and anticancer. In this review, current information on the hen's egg yolk phosvitin and its phosphopeptides and their bioactivities in the food industry and health are discussed and some future directions are given.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid.

Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation. © 2017 Society of Chemical Industry.

Phosphopeptides (PPPs) from hen egg yolk phosvitin exert anti-inflammatory activity via modulation of cytokine expression

In order to investigate the anti-inflammatory activity of egg yolk-derived phosphopeptides (PPPs), intestinal epithelial (Caco-2 cells, HT-29 cells) and macrophage RAW 264.7 cell lineages were stimulated with either tumor necrosis factor alpha (TNF-α) or Salmonella enterica lipopolysaccharide (LPS). Upon pre-incubation with different doses of PPPs, the secretion of interleukin 8 (IL-8) and TNF-α in cell supernatants was quantified by enzyme-linked immunosorbent assay (ELISA). The gene expression levels of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines (i.e., IL-8, IL-12, IL-6, monocyte chemoattractant protein-1 (MCP-1), TNF-α, IL-1β) were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results revealed that PPPs significantly inhibit the secretion of IL-8 in TNF-α-stimulated Caco-2 and HT-29 cells in a dose-dependent manner; while no significant difference was observed in LPS-stimulated Caco-2 and HT-29 cells. Interestingly, the expression of IL-8, MCP-1 and IL-12 was inhibited by PPPs in both TNF-α- and LPS-stimulated HT-29 cells, and similarly, the expression of TNF-α, IL-1β, IL-6, and iNOS was downregulated significantly in LPS-stimulated macrophage RAW 264.7 cells pre-incubated with PPPs. We conclude that PPPs possesses anti-inflammatory properties evidenced by the down-regulation of key pro-inflammatory markers. Thus our results suggest that PPPs may serve as nutraceutical compounds with anti-inflammatory properties.

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.

Antioxidant activity of tryptic digests of hen egg yolk phosvitin

Food protein-derived peptides with antioxidant activity have great potential for use as natural antioxidants in food products. However, neither the structure-activity relationship nor the antioxidant mechanism of peptides is fully understood. Egg yolk phosvitin has been acknowledged to have strongly antioxidant protein owing to its highly phosphorylated form. In this study the antioxidant activities of tryptic digests of hen egg yolk phosvitin (PPPs), which were prepared by partial dephosphorylation and subsequent hydrolysis by trypsin, were compared with that of intact phosvitin. Compared with intact phosvitin, PPPs exhibited distinctly stronger capability of inhibiting lipid oxidation in a linoleic acid system and more efficient radical-scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. However, the chelating capability of PPPs on iron(II) was weaker than that of intact phosvitin. The strong antioxidant activity of PPPs can be mainly attributed to the amino acid composition rather than to the content of phosphorylserine ligands. Histidine, methionine and tyrosine are the amino acids most likely to be responsible for the strong antioxidant activity of phosvitin peptides. The present study indicated that PPPs could be used in foods as natural antioxidants with strong antioxidant activity. Copyright © 2007 Society of Chemical Industry.

Oligophosphopeptides Derived from Egg Yolk Phosvitin Up-regulate γ-Glutamylcysteine Synthetase and Antioxidant Enzymes against Oxidative Stress in Caco-2 Cells

Previously, we have found phosphopeptides (PPPs) from hen egg yolk phosvitin possess a potent antioxidative activity against oxidative stress in human intestinal epithelial cells, Caco-2. However, their biological activity at the cellular level has not yet fully understood. The objective of this study is to evaluate the regulation of glutathione (GSH) biosynthesis-associated and antioxidant enzymes against oxidative stress in Caco-2 cells using an in vitro model. Treatment of 1 mM H2O2-induced Caco-2 cells with PPPs increased cellular GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit mRNA. Furthermore, intracellular glutathione reductase, glutathione S-transferase, and catalase activities were elevated by PPPs. In addition, PPPs with high content of phosphorus showed higher induction of these enzyme activities than PPPs without phosphorus. These data indicate that oligophosphopeptides from hen egg yolk phosvitin can up-regulate cellular GSH biosynthesis-associated enzymes activity and antioxidative activities, which play key roles against tissue oxidative stress in the human intestinal epithelial cells.

Antioxidative Stress Activity of Oligophosphopeptides Derived from Hen Egg Yolk Phosvitin in Caco-2 Cells

The protective effects of hen egg yolk phosvitin phosphopeptides (PPPs) against hydrogen peroxide (H2O2)-induced oxidative stress were evaluated in an in vitro assay using human intestinal epithelial cells. Caco-2 cells were stimulated with 1 mM H2O2 for 6 h, and the secretion of IL-8, a proinflammatory mediator, was determined by ELISA as a biomarker of oxidative stress. The inhibition of H2O2-induced IL-8 secretion from Caco-2 cells was observed by pretreatment for 2 h with PPPs, but not with phosvitin. PPPs also suppressed the formation of malondialdehyde in H2O2-treated Caco-2 cells. Furthermore, intracellular glutathione levels and glutathione reductase activity were elevated by the addition of PPPs. The protective effects of PPPs against H2O2-induced oxidative stress were almost the same as that of glutathione, and PPPs with a high content of phosphorus exhibited higher protective activity than PPPs without phosphorus; however, phosphoserine itself did not show any significant antioxidative stress activity. These findings suggest that oligophosphopeptides from hen egg yolk phosvitin possess novel antioxidative activity against oxidative stress in intestinal epithelial cells and that phosphorus and peptide structure seem to have a key role in the activity.

Oil-in-water emulsion properties and interfacial characteristics of hen egg yolk phosvitin

Phosvitin is an egg yolk protein, constituted by 50% of serines, which are all phosphorylated. This singularity makes of phosvitin one of the most safety natural iron binding molecules. When hen egg yolk is used as emulsifying agent, the adsorption of phosvitin could alter this iron binding property. Consequently, this work was performed to better understand emulsifying and interfacial properties of phosvitin in optimal metal binding conditions. We have studied sunflower oil-in-water emulsions of phosvitin at pH 5 and 6, and ionic strengths of 0.05 and 0.15 M. The oil droplet size, the stability against coalescence and flocculation, the composition of the interfacial protein film and the interfacial activity at model interfaces were analysed. Finally, the capacity of phosvitin to bind iron when it is anchored at the emulsion interface was investigated. Phosvitin showed satisfactory emulsifying capacity in conditions favouring iron fixation. In these conditions, emulsifying activity is sensitive to pH whereas flocculation is influenced by ionic strength. Coalescence destabilisation is not extended and the interfacial protein film has better characteristics at pH 5. Phosvitin was not efficient to reduce the oil-in-water interfacial tension, although at 0.1 mg/ml the interfacial tension was reduced from 31 mN/m to 15 mN/m. The high iron binding capacity of phosvitin in solution is kept by interfacial adsorbed phosvitin. Finally, to explain the poor adsorption efficiency coupled with the suitable emulsifying properties of phosvitin and the preservation of the iron binding capacity, it is likely that phosvitin is anchored at the interface only by a tiny terminal portion, presenting the rest of the molecule solvated on the aqueous phase.

Egg yolk phosvitin: preparation of metal-free purified protein by fast protein liquid chromatography using aqueous solvents

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.

Phosphopeptides derived from hen egg yolk phosvitin: effect of molecular size on the calcium-binding properties.

Two different phosphopeptide (PPP) fragments derived from partially dephosphorylated hen egg yolk phosvitin were prepared by tryptic digestion, and their Ca2+ binding property compared with that of commercial casein phosphopeptides (CPP). The smaller fragment of less than 1 kDa and O-phospho-1-serine did not bind Ca2+ to any significant extend, while PPP of 1-3 kDa showed a higher ability than CPP to render soluble calcium. The results show that not only the phosphoserine residues are critical for Ca2+ binding, but also the molecular size of the phosphopeptides.

Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin.

Novel hen egg phosvitin phosphopeptides (PPP) with molecular masses of 1-3 kDa were prepared from tryptic hydrolysis following partial alkaline dephosphorylation. The phosvitin treated with various NaOH concentrations (0.05-0.4 N) resulted in different dephosphorylated proteins ranging from 17.5 to 96.3% of phosphate retention. The protein was digested into 10-20 amino acid peptides with trypsin. Calcium-binding properties of PPP were compared with those of commercial casein phosphopeptides in vitro. The PPP with 35% phosphate retention is shown to be effective for enhancing calcium binding capacity and inhibiting the formation of insoluble calcium phosphate. The results provide potential novel functional oilgophosphopeptides as nutraceuticals.

Effect of Protease Digestion and Dephosphorylation on High Emulsifying Properties of Hen Egg Yolk Phosvitin

The emulsifying properties, particularly the emulsion stability, of phosvitin was found to be higher than those of other food proteins. The emulsifying activity and emulsion stability were greatly decreased by protease and phosphatase treatment. The protease digestion of phosvitin resulted in the peptide cleavage of large fragment (a highly phosphorylated core region, 50 to 210 peptide) and small fragments (N-terminal 1 to 49 and C-terminal 211 to 217 peptides). The large fragment lacking the small fragments did not show the excellent emulsifying properties, suggesting that small fragments of protein moiety play an important role in emulsifying properties. On the other hand, the effect of phosphatase treatment showed that electrostatic repulsive force of phosphate in phosvitin has a significant affect on its emulsifying properties and that the protein moiety with abundant phosphorylated residues is also considered to be essential for the high emulsifying properties.

A Raman spectroscopic study of hen egg yolk phosvitin: structures in solution and in the solid state.

Laser Raman spectroscopy has been employed to study the structure of the hen egg yolk protein phosvitin in H2O and D2O solutions at neutral and acidic pH (pD) and in the solid state. The Raman data indicate an unusual conformation for phosvitin in neutral aqueous solution, which is deficient in both alpha-helix and conventional beta-sheet conformations. This unusual pH 7 structure is, however, largely converted to a beta-sheet conformation in strongly acidic media (pH less than 2). beta-Sheet is also the predominant secondary structure for phosvitin in the solid state, obtained by lyophilization of the protein from aqueous solution at neutral pH. The imidazolium rings of histidyl residues remain significantly protonated near neutrality, which suggests substantial elevation of the pK for imidazolium ring ionizations of phosvitin in aqueous solution. This may result from extensive ion-pair interactions involving positively charged histidines and negatively charged phosphoserines, which are prevalent in the phosvitin sequence. The present results suggest that antiparallel beta-sheets may not be the secondary structure most characteristic of native phosvitin (physiological pH), even though beta-sheet is the predominant conformation for phosvitin in acidic solutions (pH 1.5) and in the lyophilized solid. Phosvitin appears to be the first protein for which the major component to the Raman amide I band is centered near 1685 cm-1, which is 10-40 cm-1 higher than proteins heretofore examined in aqueous solution by Raman spectroscopy.

Characterization of phosvitin and phosphopeptides of rainbow trout eggs.

Rainbow trout egg phosvitin prepared by the method of WALLACE et al. had molecular weight of 20, 000 which was about half of 41, 000 of hen's egg yolk phosvitin. It contained 57 residues of serine per molecule, followed by 15 of arginine and 10 of aspartic acid, and also contained 7 residues of glucosamine, 5 of mannose, and 3 of galactose. The molar ratio of serine and phos-phate was 1:1, and all of the phosphate were released by β-elimination reaction. Other inorganic components such as iron and copper were not detected in the phosvitin.The precipitates obtained from the trichloroacetic acid extracts of rainbow trout eggs by precipitating with ethanol were separated into glycoprotein, phosvitin, and phosphopeptides by gel filtration. The phosphopeptides were composed mainly of components having molecular weights of about 1, 300 and 5, 400. Serine content of phosphopeptides was similar to that of phosvitin, but the composition of several amino acids differed with each other. These results show that phosphopeptides are naturally occurring in the rainbow trout eggs. The amounts of phosvitin and phosphopeptides are 0.49g and 0.47g per 100g of the rainbow trout eggs, respectively, while hen's egg yolks were lacking in phosphopeptides.

Fractionation and Characterization of Hen's Egg Yolk Phosvitin

ABSTRACTTWO methods for phosvitin preparation were evaluated, based on purity of the products. Phosvitin was found to be heterogeneous in the polyacrylamide‐gel electropherogram in which it migrated into two components designated α and β‐phosvitin. They were partially fractionated using gel filtration. The β‐phosvitin contained 9.20% phosphorus, while α had 2.97%. Phosvitin was dissociated into several bands ranging in molecular weights from 18,500—60,000 daltons, in the SDS polyacrylamide‐gel electropherogram. Results indicate that phosvitin is composed of several subcomponents or polypeptides which interact or polymerize in aqueous solution to form larger molecular weight aggregates.