Effect of high-hydrostatic pressure on the digestibility of egg yolk and granule.

The impact of high hydrostatic pressure (HHP) on protein digestibility of egg yolk and egg yolk granule was evaluated by static in vitro digestion using the standardized INFOGEST 2.0 method. The degree of hydrolysis (DH) and the phospholipid content were determined during digestion, and the protein and peptide profiles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse phase-high pressure liquid chromatography (RP-HPLC). The results showed that HHP induced protein aggregation in egg yolk and granule, mainly by disulfide bridges, which were not disrupted in the oral phase. Proteolysis during the gastric phase improved egg yolk and granule protein solubility, regardless of whether HHP was applied. However, the extent of the samples' digestibility was not affected, with DH values ranging from 15% to 20%. During the intestinal phase, the DH of egg yolk protein (∼40%) was higher than that of the granule (∼25%), probably due to the denser structure of the granule reducing the accessibility of intestinal enzymes. The DH, peptide, and protein profiles of control and HHP-treated egg yolk showed similar protein digestion behaviors for both gastric and intestinal phases. Among the different proteins, only the digestibility of ß-phosvitin in HHP-treated granule was enhanced. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin with the potential to generate bioactive phosvitin-derived phosphopeptides. PRACTICAL APPLICATION: High hydrostatic pressure, mainly used as a preservation process, did not impair the nutritional quality of the egg yolk and granule proteins but improved the susceptibility of phosvitin (protein contained in egg yolk) proteolysis to produce bioactive phosphopeptides. Consequently, applying HHP to granules represents an interesting process that improves the digestibility of phosvitin.

Binding mechanism and bioavailability of a novel phosvitin phosphopeptide (Glu-Asp-Asp-pSer-pSer) calcium complex.

Phosvitin has excellent calcium binding capacity, related to its phosphopeptides. The phosphopeptides may be used as functional ingredients for improving calcium bioavailability, but the calcium-binding mechanism is unclear. In this study, a novel phosvitin phosphorylated pentapeptide (Glu-Asp-Asp-pSer-pSer, EDDpSpS) was selected to prepare an EDDpSpS calcium complex (EDDpSpS-Ca), and the calcium-binding mechanism and bioavailability investigated. The calcium-binding capacity of EDDpSpS was up to 468 ± 152.80 mg/g. Calcium ions prompted the folding of the EDDpSpS structure to form spherical nanoparticles. The calcium binding sites of EDDpSpS involved peptide bonds, carboxyl, amino, and phosphate groups. Molecular forces involved in these interactions were electrostatic in nature. Moreover, EDDpSpS-Ca had excellent bioavailability when compared to CaCO3, calcium lactate, and d-calcium gluconate. This study revealed the calcium-binding mechanism of phosvitin phosphopeptide, and suggested that EDDpSpS-Ca has the potential to be a novel, efficient, and promising calcium supplement.

Covalent and Noncovalent Complexation of Phosvitin and Gallic Acid: Effects on Protein Functionality and In Vitro Digestion Properties.

To investigate the effects of different binding modes on the structure, function, and digestive properties of the phosvitin (Pv) and gallic acid (GA) complex, Pv was covalently and noncovalently combined with different concentrations of GA (0.5, 1.5, and 2.5 mM). The structural characterization of the two Pv-GA complexes was performed by Fourier transform infrared, circular dichroism, and LC-MS/MS to investigate the covalent and noncovalent binding of Pv and GA. In addition, the microstructure of the two Pv-GA complexes was investigated by super-resolution microscopy and transmission electron microscopy. The particle size and zeta potential results showed that the addition of GA increased the particle size and the absolute potential of Pv. The determination of protein digestibility, polyphenol content, SH and S-S group levels, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antioxidant capacity of the digests indicated that noncovalent complexes had greater antioxidant and protective effects on polyphenols. Molecular docking revealed that GA was conjugated with Pv through hydrogen bond interactions.

Interaction research of resveratrol and phosvitin based on fluorescence spectroscopy and molecular docking analysis.

Phosvitin (PV) is the main phosphoprotein in egg yolk, with the highest degree of phosphorylation known in nature. The PV and resveratrol (Res) can form a complex, thus effectively improve the solubility of Res. In this work, the interaction between Res and PV was investigated by the fluorescence spectroscopy and molecular docking. The fluorescence emission intensity of PV became weak along with a red shift when it interacted with Res and the antioxidant activity was enhanced. The quenching constants of the interaction systems were 1.12×104  M-1 and 9.40×103  M-1 at 25°C and 35°C, respectively, which indicated the presence of static quenching phenomena between them. The binding constant was 1.80×104  M-1 , and the number of corresponding binding sites was approximately equal to one. The thermodynamic results revealed the combination was spontaneous, and the change of enthalpy and entropy was ∆H = 53.50 kJ/mol, ∆S = 261.00 J/mol·K, respectively. It indicated that the interaction forces between Res and PV were mainly hydrophobic interaction and hydrogen bonding. Molecular docking showed the binding mode, which was consistent with the experiment results. The research on the interaction between Res and PV provided theoretical guidance for the application of Res in food. PRACTICAL APPLICATION: PV is the most highly phosphorylated protein in nature and has pro-calcium absorption effects. Res is a polyphenol with strong antioxidant and anti-inflammatory activity, but its poor solubility limits its application. In this study, the solubility of Res was considerably enhanced by compounding Res and PV, and the antioxidant activity of Res was well retained. It increases the value of Res in food and other applications and opens up new possibilities for processing and utilization of PV.

Phosvitin-wheat gluten complex catalyzed by transglutaminase in the presence of Na2SO3: Formation, cross-link behavior and emulsifying properties.

Phosvitin (PSV) is considered as a good emulsifier, although it has a low proportion of hydrophobic regions and steric hindrance. Wheat gluten (WG) possesses excellent hydrophobicity and macromolecular network structure. In this work, WG was subjected to a series of Na2SO3 solution, followed by cross-linking with PSV under transglutaminase (TGase) catalyzation. The results showed that Na2SO3 could break disulfide bonds of WG and increase its solubility from 7.33% to 42.82% with 1200 mg/L of Na2SO3. Correspondingly, the cross-linking degree was significantly enhanced. Compared to PSV, the cross-linked PSV-WG exhibited a higher surface hydrophobicity and thermal stability, with a lower zeta potential and apparent viscosity. The emulsifying activity of PSV-WG reached 17.42, 20.63 and 20.28 m2/g with Na2SO3 concentration of 300, 600 and 900 mg/L, which were all higher than that of PSV (15.19 m2/g). This work provided a novel strategy to elevate emulsifying properties of PSV by cross-link reaction.

Phosvitin Derived Phospho-Peptides Show Better Osteogenic Potential than Intact Phosvitin in MC3T3-E1 Osteoblastic Cells.

Phosphorylated proteins from food sources have been investigated as regulators of bone formation with potential benefits in treating osteoporosis. Egg, a cheap and nutritious food, is also the source of various proteins and bioactive peptides with applications in human health. Egg yolk is rich in phosvitin, the most phosphorylated protein in nature. Phosvitin has been shown to improve bone health in experimental animals, although the molecular mechanisms and its specific effects on bone-forming osteoblastic cells are incompletely understood. Previous work in our group has identified pancreatin-generated phosvitin phospho-peptides (PPP) as a potential source for bioactive peptides. Given this background, we examined the roles of both phosvitin and PPP in the function of osteoblastic cells. Our results demonstrated their potential to improve bone health by promoting osteoblast differentiation and proliferation, suppressing osteoclast recruitment and the deposition of extracellular matrix, although PPP appeared to demonstrate superior osteogenic functions compared to phosvitin alone.

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.

High hydrostatic pressure induced extraction and selective transfer of ß-phosvitin from the egg yolk granule to plasma fractions.

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 (Gin) produced by egg yolk centrifugation was pressure-treated at 400 and 600 MPa for 5 and 10 min, and centrifuged to generate granule fractions (GP1 to GP4) and plasmas (PP1 to PP4). Iron and phosphorus contents were also increased in PP1 to PP4 fractions, confirming the transfer of phosvitins from pressure-treated granule to plasma. Pressurization drastically improved phosvitin recovery in PP 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.

Fabrication and Characterization of a Microemulsion Stabilized by Integrated Phosvitin and Gallic Acid.

The purpose of this work was to conjugate phosvitin (Pv) with gallic acid (GA) to explore a new emulsifier that had both good emulsifying properties and antioxidant activity. The Pv-GA complex was prepared at a GA concentration of 1.5 mg/mL with pH 9.0. The Pv-GA complex obtained was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and characterized with infrared, ultraviolet, and fluorescence spectra. The emulsifying activity and stability of the Pv-GA complex were slightly improved, and antioxidant activities was significantly enhanced. Furthermore, the Pv-GA complex was used to load conjugated linoleic acid (CLA) for microemulsion preparation. Results showed that the Pv-GA complex could increase the viscosity and lipid antioxidant capacity of Pv-GA/CLA microemulsion. The Pv-GA/CLA microemulsion had remarkable emulsifying activity, emulsifying stability, pH, and thermal stability and poor salt stability.

Effective Preparation Method of Phosphopeptides from Phosvitin and the Analysis of Peptide Profiles Using Tandem Mass Spectrometry.

The effect of high-temperature and mild-pressure (HTMP) pretreatment on the enzymatic hydrolysis of phosvitin and the structural characteristics of the phosphopeptides produced were analyzed using tandem mass spectrometry. The HTMP pretreatment hydrolyzed phosvitin at random sites and helped the subsequent enzyme hydrolysis of the peptides produced. With the HTMP pretreatment alone, 154 peptides were produced, while the use of trypsin, Protex 6L, and Multifect 14L in combination with the pretreatment produced 252, 280, and 164 peptides, respectively. The use of two enzyme combinations (trypsin + Protex 6L and trypsin + Multifect 14L) helped the hydrolysis further. The number of phosphopeptides produced increased when the modifications within the same amino acid sequences were considered. This study indicated that HTMP pretreatment was a breakthrough method to improve the enzymatic hydrolysis of phosvitin that enabled an easy production of phosvitin phosphopeptides for their subsequent functional characterizations.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Role of polysaccharide conjugation in physicochemical and emulsifying properties of egg phosvitin and the calcium binding capacity of its phosphopeptides.

Phosvitin phosphopeptides (PPP) effectively enhanced calcium bioavailability via inhibiting calcium-phosphate deposition. It is difficult to hydrolyze native phosvitin (PSV) to release PPP due to its compact structure. Polysaccharide conjugation could improve the biofunctional properties of proteins via altering their structures. In this research, PSV was subjected to conjugation with pectin, and changes in physicochemical characteristics and functionalities were determined. The results showed that PSV underwent an unfolding process when conjugated with pectin at a mass ratio of 1 : 2, exposing more hydrophobic groups. Excessive glycosylation induced a refolded structure with a lower surface hydrophobicity and a higher thermal stability. These secondary and tertiary structural changes improved the emulsifying properties of PSV and allowed the production of emulsions with smaller oil droplets. Simultaneously, due to redistribution of phosphate groups, the PPP derived from copolymers exhibited a stronger calcium binding capacity, especially at a mass ratio of 1 : 6, possessing a potential to be utilized in functional foods.

Tracking Conformational Changes in Phosvitin throughout a Crowding-Agent-Based Titration.

The sensitivity of Raman optical activity (ROA) towards small conformational changes is explored by tracking the structural changes in an intrinsically disordered protein-phosvitin-induced by different concentrations of crowding agent. It is shown that ROA is capable of tracking small conformational changes involving ß-sheet and α-helical secondary structural properties of the protein. Furthermore, it is indicated that the influences of the crowding agents employed, Ficoll 70 and dextran 70, on the structural properties of phosvitin differ significantly, with the structural changes induced by the presence of Ficoll 70 being more pronounced and already being visible at a lower concentration. The data also suggest that some spectral changes do not arise from a change in the secondary structure of the protein, but are related to differences in interaction between the phosphorylated residues of the protein and the sugar-based crowding agent.

Effects of multiple freeze-thaw treatments on physicochemical and biological activities of egg phosvitin and its phosphopeptides.

Multiple freeze-thaw (F-T) treatments could modify a protein structure and affect its physicochemical and biological activities. In this work, egg phosvitin (PSV) was subjected to multiple F-T treatments, and the changes in physicochemical and functional properties were investigated. The F-T treatments modified the molecular characteristics of PSV involving a decrease in surface hydrophobicity. Differential scanning calorimetry and scanning electron microscopy showed that PSV underwent denaturation, dissociation and possibly aggregation. Correspondingly, the emulsifying ability of PSV dramatically improved from 1.87 m2 g-1 to 3.70 m2 g-1, 3.25 m2 g-1 and 3.15 m2 g-1 after 3, 6, and 9 F-T cycles, respectively. In parallel, the PSV phosphopeptides (PPP) derived from the F-T treated PSV showed a higher calcium binding capacity and protecting activity against H2O2-induced apoptotic cell death of HepG2 cells, when compared with PPP from native PSV. These results indicated that the F-T treatments have potential to be implemented as a strategy to improve the emulsifying and biological activities of PSV.

Physicochemical and functional properties of leftover egg yolk granules after phosvitin extraction.

In this study, leftover egg yolk granules, a by-product after phosvitin extraction, were evaluated for the physicochemical and functional properties and the results were compared with those of the egg yolk and whole granule with phosvitin. Leftover granule after phosvitin removal accounted for 12.6% of the dried egg yolk and contained 84.5% protein and 7.98% fat. Scanning electron microscopy (SEM) images of leftover granules indicated the dissociation of aggregates of high density lipoprotein-phosvitin complexes. Protein solubility of leftover granules was markedly influenced by pH and sodium chloride (0.5 and 1 M). The apparent viscosity of leftover granule was higher than egg yolk and whole granule. Compared to whole granule, leftover granule had significantly (P < 0.05) superior foaming and emulsifying properties, but, lower than those of egg yolk. These findings are useful for the food industry for utilization of leftover egg yolk granules for the preparation of various food products.

Phosphorylation of phosvitin plays a crucial effects on the protein-induced differentiation and mineralization of osteoblastic MC3T3-E1 cells.

This study investigated the effects of phosvitin (PV), one of the major proteins from egg yolk, with different degree of phosphorylation on the physiology of an osteoblast (MC3T3-E1) cell line. The proliferation and differentiation of MC3T3-E1 were analyzed using the CCK-8 and the alkaline phosphatase (ALP) assay, respectively. The effect of PV on the mineralization of MC3T3-E1 was monitored using the Alizarin-red staining. PV at 100 µg/mL increased the ALP activity by 145% of the control after 7 days of incubation. PV also stimulated the proliferation and differentiation of MC3T3-E1 in a phosphorylation level-dependent manner. The RT-PCR reactions indicated that PV stimulated the expression of BMP-2 and OPG mRNA in a phosphorylation-dependent manner, but inhibited RANKL mRNA expression in MC3T3-E1. This result suggested that the phosphate groups in PV not only stimulated the proliferation and differentiation of MC3T3-E1, but also controlled the mineralization by regulating the expression of BMP-2, RANKL and OPG mRNA in the osteoblast cell.

Phosphorylated serine clusters of phosvitin plays a crucial role in the regulatory mineralization.

Phosphorylation of phosvitin has been proved to play an important role in the mineralization, but the active region of phosvitin is still not known yet. Four phosvitin phosphopeptides (PPPs) were obtained after separating from ion exchange column and desalting from gel filtration, and named as PPP0, PPP1, PPP3 and PPP4, respectively. The effect of PPP on the mineralization was investigated by pH-stat system, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscope. SDS-PAGE and circular dichroism were applied to study the composition and the structure of PPP. Results showed that the order of promoting mineralization was as follows: PPP3 > phosvitin > PPP4 > PPP1 > control > PPP0. PPP0 and PPP1 was not involved in the mineralization, while the structure of PPP4 was too compact to promote mineralization because of its high ß-sheet conformation. PPP3, which contained a 10 kDa peptide, is the most effective promoter for mineralization. LTQ-MS/MS results indicated that the phosphorylated serine clusters of phosvitin was the active region for promoting mineralization.

Augmentation of the antibacterial activities of Pt5-derived antimicrobial peptides (AMPs) by amino acid substitutions: Design of novel AMPs against MDR bacteria.

The ever-growing concerns on multi-drug resistant (MDR) bacteria lead to urgent demands for novel antibiotics including antimicrobial peptides (AMPs). Pt5, a peptide consisting of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. Here we used Pt5 as a template to design new AMPs by shortening the sequence and substituting with tryptophan (W) and lysine (K) at selected positions. Among the resultant Pt5-derived peptides, Pt5-1c showed the strongest antimicrobial activity against both Gram-negative and Gram-positive bacteria, including MDR bacteia, with the minimum inhibitory concentrations (MICs) ranging from 1.2 µM to 4.8 µM. Electron microscopic examination showed that Pt5-1c was able to kill the bacteria directly. ELISA revealed that Pt5-1c possessed high affinity to lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Importantly, Pt5-1c was able to disrupt the bacterial membrane by a combined action of membrane depolarization and permeabilization, with little cytotoxicity to mammalian cells. Taken together, these findings suggest that Pt5-1c has considerable potential for future development as novel peptide antibiotics against MDR bacteria.

Observations using Phosphorus-31 nuclear magnetic resonance (31P-NMR) of structural changes in freeze-thawed hen egg yolk.

Hen egg yolk (EY) has a complicated structure consisting of lipids and proteins, and its structure is deeply related with its functional properties. 31P-NMR is an efficient technique to non-destructively detect the dynamic behaviour of phospholipids, the main component of bio-membranes. We determined conditions for measuring the 31P NMR spectra of EY and identified the components. 31P-NMR was used to detect phosvitin, inorganic phosphate, and lipoprotein as well as structural changes such as granule collapse and freeze-thaw denaturation as signal changes. Freeze-thaw denaturation generated a new denaturation peak. We separated aggregates of LDL from freeze-thawed plasma using centrifugation. TEM and 31P-NMR observations revealed that the denaturation peak corresponded to LDL aggregates. The 31P-NMR spectra suggested the formation of multiple forms of LDL aggregates in which the head groups of phospholipid molecules adopt a face-to-face orientation, similar to that observed following the flocculation of lipoproteins or in the lamellar-like structures of phospholipids.

Effect of Freezing, Thermal Pasteurization, and Hydrostatic Pressure on Fractionation and Folate Recovery in Egg Yolk.

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.