Intrinsically disordered proteins and intrinsically disordered protein regions.

Intrinsically disordered proteins (IDPs) and IDP regions fail to form a stable structure, yet they exhibit biological activities. Their mobile flexibility and structural instability are encoded by their amino acid sequences. They recognize proteins, nucleic acids, and other types of partners; they accelerate interactions and chemical reactions between bound partners; and they help accommodate posttranslational modifications, alternative splicing, protein fusions, and insertions or deletions. Overall, IDP-associated biological activities complement those of structured proteins. Recently, there has been an explosion of studies on IDP regions and their functions, yet the discovery and investigation of these proteins have a long, mostly ignored history. Along with recent discoveries, we present several early examples and the mechanisms by which IDPs contribute to function, which we hope will encourage comprehensive discussion of IDPs and IDP regions in biochemistry textbooks. Finally, we propose future directions for IDP research.

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.

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.

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.

Changes in the protein secondary structure of hen's egg yolk determined by Fourier transform infrared spectroscopy during the first eight days of incubation.

In this study, incubation-induced alterations in the protein secondary structures of egg yolk and its major fractions (granules, plasma, and low-density lipoproteins [LDL]) were monitored during the first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing (IEF). Two factors potentially connected with egg yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and phosvitin, an important egg yolk protein assumed to play an important role in hematopoiesis as the iron carrier during early embryogenesis. However, neither the significant increase in pH value (6.07 to 6.92) of egg yolk during incubation of fertilized eggs, nor the release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that the protein conformation in whole egg yolk, granules, and LDL was stable during incubation, but separate evaluation of the plasma fraction revealed considerable changes in secondary structure. However, it is unlikely that these changes were provoked by structure changes of the proteins originally present in plasma; instead, the physiological influx of albumen into the yolk sac was expected to play an important role in the protein modifications of egg yolk, as was shown both by FTIR and IEF of the water-soluble egg yolk proteins. Moreover, FTIR was used to determine the naturally occurring proportions (%) of the secondary structure elements in egg yolk and its 3 fractions on d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular ß-sheets (57.04%±0.39%). The plasma fraction was found to consist mainly of α-helices (43.23%±0.27%), whereas LDL was composed almost exclusively of intramolecular ß-sheets (67.36%±0.56%) or ß-turns, or both. On the other hand, whole egg yolk was mainly composed of intermolecular ß-sheets (39.77%±0.48%), potentially indicating molecular interchanges between the individual fractions.

Thermal-aided phosvitin extraction from 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.

Sequential separation of immunoglobulin Y and phosvitin from chicken egg yolk without using organic solvents.

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.

Cytotoxic and antigenotoxic activities of phosvitin from egg yolk.

Egg yolk phosvitin is one of the most phosphorylated proteins in nature, and thus has a strong metal-binding ability. The objective of this study was to evaluate the cytotoxic and antigenotoxic activities of phosvitin in vitro. Using the 3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of phosvitin was evaluated in human cancer cell lines of various tissue origins, including the cervix (HeLa), breast (MCF-7), stomach (AGS), lung (A549 and SK-MES-1), liver (HepG2), and larynx (Hep-2). The growth of all cancer cell lines was inhibited in a dose-dependent manner by phosvitin. Among the cancer cell lines tested, MCF-7 and SK-MES-1 were the least sensitive and HeLa, AGS, and HepG2 were the most sensitive to phosvitin. The 50% inhibition of cell viability values of phosvitin were 5.38, 11.57, 4.78, 6.98, 11.82, 3.93, and 9.97 mg/mL for HeLa, MCF-7, AGS, A549, SK-MES-1, HepG2, and Hep-2, respectively. The protective effects of phosvitin against DNA damage in human leukocytes indicated that phosvitin showed protective effects against the oxidative stress-induced DNA damages in human leukocytes. These results suggested that phosvitin has a high potential to be used as an anticancer agent for humans.

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.

Characterization of vitellogenin and its derived yolk proteins in cloudy catshark (Scyliorhinus torazame).

Elasmobranchs (sharks and rays) exhibit unique reproductive characteristics and, in contrast to the situation in teleosts, very little is known about the identity, structure and physical characteristics of their egg yolk proteins. The aims of this study were to (1) detect and purify the vitellogenin (Vtg; egg yolk precursor) and yolk proteins (YPs) of the cloudy catshark (Scyliorhinus torazame), (2) examine the relationships between Vtg and YPs and (3) characterize and classify the deduced primary structure of the Vtg transcript (vtg). The apparent molecular weights of purified Vtg and putative Vtg-related YPs (lipovitellin: Lv, phosvitin: Pv) were determined by gel filtration and were ~560, >669 and ~58 kDa, respectively. Following SDS-PAGE, these purified products (i.e., Vtg, Lv and Pv) appeared as bands of ~210, ~110 and ~22 kDa, respectively. On Western blots, antisera against purified Vtg, Lv and Pv recognized the ~210 kDa Vtg band. Catshark Pv, in contrast to teleost Pvs, had a very low serine content. The catshark Vtg cDNA sequence (vtg) appeared to contain an open-reading frame consisting of domains encoding Lv, Pv and ß'-component (ß'-c). A phylogenetic analysis, with a consideration of genome duplication events, placed catshark vtg into the 'vtgAB type.' It is concluded that at least a single major type of Vtg protein, which is transcribed and translated from catshark vtgAB gene, is the precursor of three egg yolk proteins (Lv, Pv and ß'-c) in catshark.

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.

Phosvitin plays a critical role in the immunity of zebrafish embryos via acting as a pattern recognition receptor and an antimicrobial effector.

How fish embryos that develop externally survive microbial attacks is poorly understood. Here, we clearly demonstrated that the embryo extract of zebrafish and its early embryo both displayed antimicrobial activity against microbes, including pathogenic Aeromonas hydrophila, and phosvitin (Pv), a nutritional protein abundant in eggs, was related to this antimicrobial activity. We also showed that recombinant Pv (rPv) acted as a pattern recognition receptor capable of recognizing the microbial signature molecules LPS, lipoteichoic acid, and peptidoglycan, as well as binding the Gram-negative and -positive microbes Escherichia coli, A. hydrophila, and Staphylococcus aureus and functioned as an antimicrobial agent capable of killing the microbes. Furthermore, we revealed that its C-terminal 55 residues (Pt5) with the functional sites Arg(242) and Ala(201)/Ile(203) were indispensable for Pv antimicrobial activity. Importantly, microinjection of rPv or Pt5 into early embryos significantly enhanced their resistance to A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-Pv antibody plus rPv (or Pt5) but not by injection of anti-actin antibody plus rPv. Moreover, the generated mutants with in vitro antimicrobial activity, when injected into the embryos, could also promote their resistance to A. hydrophila, but those without in vitro antimicrobial activity could not. It is thus proposed that Pv participates in the protection of early embryos against pathogenic attacks via binding and disrupting potential pathogens. This work also opens a new way for the study of the immunological roles of yolk proteins in oviparous animals that rely on yolk proteins for embryonic development.

Effects of high-pressure processing and enzymatic dephosphorylation on phosvitin properties.

BACKGROUND: Egg phosvitin could be a good source of functional peptides. Enzymatic dephosphorylation and high-pressure processing combined with thermal treatment applied before proteolysis could produce phosvitin hydrolysates with different properties compared to its native form. RESULTS: Phosvitin structure was maintained overall during high-pressure treatment of 600 MPa applied at an initial temperature of 65 °C regardless of the pH and duration of treatment, confirming the high structural stability of this phosphoprotein. Treatment of phosvitin with phosphatase increased the degree of dephosphorylation from 24% to 63%, after 2 and 18 h, respectively. Moderate dephosphorylation of phosvitin prior to proteolytic digestion improved its hydrolysis, allowing formation of peptides with a molecular weight lower than 17,000 kDa as determined by size exclusion chromatography. Angiotensin-converting enzyme (ACE) inhibition and antioxidant activity of dephosphorylated and protease-treated phosvitin was increased by 52% and 39%, respectively, as compared to protease-digested native phosvitin. CONCLUSION: Enzymatic dephosphorylation before proteolysis mimicking in vivo gut conditions improved ACE inhibition and antioxidant activity of phosvitin hydrolysates.

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.

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.

Rapid analysis of phosphoamino acids from phosvitin by near-infrared cyanine 1-(ε-succinimydyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium derivatization and polyacrylamide-coated CE with LIF detection.

A polyacrylamide-coated CE method with LIF detection was developed for analyzing three phosphoamino acids including phosphotyrosine (p-Tyr), phosphothreonine (p-Thr), and phosphoserine (p-Ser). A near-infrared dye, 1-(ε-succinimydyl-hexanoate)-1'-methyl-3,3,3',3'-tetramethyl-indocarbocyanine-5,5'-disulfonate potassium (MeCy5-OSu) was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 6.1 min, using 10 mmol/L phosphate buffer (pH 4.0) containing 60 mmol/L SDS as running buffer. The highest derivatization efficiency was achieved in 0.2 mol/L borate buffer (pH 8.8) for 30 min at 30 °C. Linearity of response was found in the range of 0.05-1 µmol/L. The correlation coefficients for these phosphoamino acids were from 0.9940 to 0.9976. The LODs for phosphotyrosine, phosphothreonine, and phosphoserine were about 6, 8, and 8 nmol/L, respectively. The proposed method has been successfully applied to the determination of phosphoamino acids in the hydrolysis sample of a phosphorylated phosvitin. Average recoveries for phosvitin sample were in the range of 94.0-98.0% and coefficients of variation ranged from 2.7 to 4.8%.

Protein-triggered instant disassembly of biomimetic Layer-by-Layer films.

Layer-by-Layer (LbL) coatings are promising tools for the biofunctionalization of biomaterials, as they allow stress-free immobilization of proteins. Here, we explore the possibility to immobilize phosvitin, a highly phosphorylated protein viewed as a model of bone phosphoproteins and, as such, a potential promotive agent of surface-directed biomineralization, into biomimetic LbL architectures. Two immobilization protocols are attempted, first, using phosvitin as the polyanionic component of phosvitin/poly-(L-lysine) films and, second, adsorbing it onto preformed chondroitin sulfate/poly-(L-lysine) films. Surprisingly, it is neither possible to embed phosvitin as the constitutive polyanion of the LbL architectures nor to adsorb it atop preformed films. Instead, phosvitin triggers instant massive film disassembly. This unexpected, incidentally detected behavior constitutes the first example of destructive interactions between LbL films and a third polyelectrolyte, a fortiori a protein, which might open a route toward new stimuli-responsive films for biosensing or drug delivery applications. Interestingly, additional preliminary results still indicate a promotive effect of phosvitin-containing remnant films on calcium phosphate deposition.

Simply and effectively preparing high-purity phosvitin using polyethylene glycol and anion-exchange chromatography.

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.

A simple and efficient method for preparing partially purified phosvitin from egg yolk using ethanol and salts.

The objective of this study was to develop a new protocol that could be used for large-scale separation of phosvitin from egg yolk using ethanol and salts. Yolk granules, which contain phosvitin, were precipitated after diluting egg yolk with 9 volumes of distilled water. The pH of the yolk solution was adjusted to pH 4.0 to 8.0 using 6 N HCl or NaOH, and then yolk granules containing phosvitin was separated by centrifugation at 3,220 × g for 30 min. Lipids and phospholipids were removed from the insoluble yolk granules using 85% ethanol. The optimal volumes and concentration of ethanol in removing lipids from the precipitants were determined. After centrifugation, the lipid-free precipitants were homogenized with 9 volumes of ammonium sulfate [(NH(4))(2)SO(4)] or NaCl to extract phosvitin. The optimal pH and concentration of (NH(4))(2)SO(4) or NaCl for the highest recovery rate and purity for phosvitin in final solution were determined. At pH 6.0, all the phosvitin in diluted egg yolk solution was precipitated. Among the (NH(4))(2)SO(4) and NaCl conditions tested, 10% (NH(4))(2)SO(4) or 10% NaCl at pH 4.0 yielded the greatest phosvitin extraction from the lipid-free precipitants. The recovery rates of phosvitin using (NH(4))(2)SO(4) and NaCl were 72 and 97%, respectively, and their purity was approximately 85%. Salt was removed from the extract using ultrafiltration. The salt-free phosvitin solution was concentrated using ultrafiltration, the impurities were removed by centrifugation, and the resulting solution was freeze-dried. The partially purified phosvitin was suitable for human use because ethanol was the only solvent used to remove lipids, (NH(4))(2)SO(4) or NaCl was used to extract phosvitin, and ultrafiltration was used to remove salt and concentrate the extract. The developed method was simple and suitable for a large-scale preparation of partially purified phosvitin.

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.