Proteomics analysis of egg white proteins from different egg varieties

The market of specialty eggs, such as omega-3-enriched eggs, organic eggs, and free-range eggs, is continuously growing. The nutritional composition of egg yolk can be manipulated by feed diet; however, it is not known if there is any difference in the composition of egg white proteins among different egg varieties. The purpose of the study was to compare the egg white proteins among six different egg varieties using proteomics analysis. Egg white proteins were analyzed using two-dimensional gel electrophoresis (2-DE), and 89 protein spots were subjected to LC-MS/MS. A total of 23 proteins, belonging to Gallus gallus , were identified from 72 detected protein spots. A quiescence-specific protein precursor in egg white was identified for the first time in this study. Significant differences in the abundant levels of 19 proteins (from 65 protein spots) were observed among six egg varieties. Four proteins, ovalbumin-related protein Y, cystatin, avidin, and albumin precursor, were not different among these six egg varieties. These findings suggest that the abundance, but not the composition, of egg white proteins varied among the egg varieties.     

Comparison of different electrophoretic separations of hen egg white proteins

The hen egg white protein composition has not yet been fully defined. To improve the knowledge of this biological fluid, the most usual and recently developed electrophoretic methods have been used: SDS-PAGE, native-PAGE, isoelectric focusing (IEF), and 2-dimensional electrophoresis (2DE). Seven of the major known proteins were thus identified in at least one electrophoretic system. Isoforms of ovotransferrin, ovalbumin, and ovomucoid were visualized when pI was used for the separation. Two-dimensional electrophoresis allowed separation of a very large number of spots. In each of the four systems, some components were revealed but not identified, and unknown spots were particularly numerous with 2DE. With this technique, many spots corresponding to small acidic proteins were highlighted, among which was the Ch21 protein, whose presence in hen egg white was thus confirmed. This study thus constitutes, to our knowledge, the first proteomic investigation of hen egg white.     

Influence of thiol metabolism of lactobacilli on egg white proteins in wheat sourdoughs

In wheat sourdoughs, the degradation of gluten proteins is favored by acidification and reducing conditions. This study aimed to determine the proteolytic degradation of egg white proteins in wheat sourdoughs acidified with lactobacilli differing in their thiol metabolism. Ovotransferrin was the only major egg white protein that degraded during sourdough fermentations. An extensive degradation of ovotransferrin required a heterofermentative lactobacilli starter, Lactobacillus sanfranciscensis, with glutathione reductase activity. Ovotransferrin was more resistant to breakdown when sourdoughs were acidified with homofermentative lactobacilli or a mutant strain of L. sanfranciscensis lacking the glutathione reductase. Its susceptibility to proteolysis in L. sanfranciscensis sourdoughs is thus attributable to thiol accumulation by L. sanfranciscensis, which apparently altered the structure of ovotransferrin through a reduction of disulfide bonds. Proteolytic degradation of ovotransferrin was attributable to wheat aspartic proteinases. In addition to the susceptibility to proteolysis, other functional properties of egg proteins may be influenced by thiol-exchange reactions.     

Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.     

Heat-induced changes in the susceptibility of egg white proteins to enzymatic hydrolysis: a kinetic study

A kinetic study was conducted on the effect of heating in the temperature range of 50-92 degrees C, on the susceptibility of ovalbumin and albumen solutions to enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Heat treatment resulted in an increase in degree of hydrolysis after 10 min of enzymatic reaction of both ovalbumin and albumen, as measured using the pH-stat method. The time-dependent change in the susceptibility to enzymatic hydrolysis after heat treatment was described by a fractional conversion model (based on an apparent first-order reaction kinetic model). Different end levels of degree of hydrolysis were obtained after heating for a long time at different temperatures, which suggests that the final degree of unfolding of the protein is temperature dependent.     

Comparative studies on antigenicity and allergenicity of native and denatured egg white proteins

The binding activities of IgG and IgE antibodies from egg-allergic patients to physically or chemically treated egg white proteins were examined and compared with those of rabbit anti-egg white IgG antibodies. The sera from eight patients and four rabbit antibodies were used in this study. The binding activities of human IgG antibody to partially denatured ovotransferrin (Tf), ovalbumin (OA), and lysozyme (Lys) forms were increased, whereas carboxymethylation (RCM) and heat treatment caused a dramatic decrease in the antigenicity of Tf and ovomucoid (OVM). Tf and OVM were major immunogenic antigens for the rabbit IgG response. Urea also caused Tf to exhibit greater rabbit IgG binding activity. In contrast, human and rabbit antibodies did not react with ovomucin. Partially denatured Tf and Lys also induced strong IgE binding activities. The allergenicity of Tf, OVM, and Lys was decreased by RCM, whereas OA retained its binding capacity. These results suggested that anti-OA IgE recognizes more sequential epitopes and that anti-OVM and Lys antibodies recognize both conformational and sequential epitopes. Tf and OVM were dominant allergens for the IgE antibodies of anaphylaxis patients, whereas IgE from atopic patients bound more strongly with OA and OVM.     

Inhibiting effects of egg white dry-heated at 120 degrees C on heat aggregation and coagulation of egg white and characteristics of dry-heated egg white.

Dialyzed and freeze-dried egg white (FDEW) was dry-heated at 120 degrees C for up to 6 h. The inhibiting effects of the dry-heated egg white (DHEW) on the heat aggregation and coagulation of egg white (as 10% FDEW solution) and characteristics of the DHEW were examined. From the changes in turbidities and soluble protein contents of supernatant in various mixtures of 10% FDEW and DHEW solutions induced by heating (60 degrees C, 5 min), it was found that the inhibiting capacity increased with increases in the dry-heating time (DHT). The FDEW proteins were denatured with a mild conformational change (not secondary but tertiary structure) with the increase in DHT and aggregated partially. However, the more transparent solutions of DHEW containing soluble aggregates according to DHT were also obtained after heating. The transparency according to DHT came to be scarcely affected by the NaCl concentration and the dilution with diluents containing SDS, urea, and 2-mercaptoethanol. These findings suggest that the heat aggregations and coagulations of ovotransferrin and lysozyme in the FDEW were inhibited by their bindings with the soluble aggregates in DHEW.     

Kinetic study on the changes in the susceptibility of egg white proteins to enzymatic hydrolysis induced by heat and high hydrostatic pressure pretreatment

A kinetic study was conducted on the effect of heat pretreatment in the temperature range of 50-85 degrees C at atmospheric pressure and of high hydrostatic pressure pretreatment (100-700 MPa) at four temperatures (10, 25, 40, and 60 degrees C) on the susceptibility of egg white solutions (10% v/v, pH 7.6) to subsequent enzymatic hydrolysis by a mixture of trypsin and alpha-chymotrypsin at 37 degrees C and pH 8.0. Both heat pretreatment at atmospheric pressure and high-pressure pretreatment resulted in an increase in degree of hydrolysis (DH) after 10 min of enzymatic reaction (DH10) of egg white solutions, as measured using the pH-stat method, which could be described by a fractional conversion model (based on an apparent first-order reaction kinetic model). The temperature dependence of the corresponding rate constants could be described by the Arrhenius equation. At elevated pressure, a negative apparent activation energy was obtained, implying an antagonistic effect of pressure and temperature. The pressure dependence of the rate constants could be described by the Eyring equation, and negative activation volumes were observed, which demonstrates the positive effect of pressure on the susceptibility of egg white solutions to subsequent enzymatic hydrolysis.     

Proteomic analysis of hen egg white

Hen egg white is an original biological fluid in which major proteins have been widely studied, unlike the minor components. In this study, two-dimensional electrophoresis associated with mass spectrometry enabled the separation of 69 protein spots and their matching with major proteins, which were already known, and with minor proteins. Sixteen proteins were identified, and among them, two had never been previously detected in hen egg white, i.e., Tenp, a protein with strong homology with a bacterial permeability-increasing protein family (BPI), and VMO-1, an outer layer vitelline membrane protein. Thirteen proteins present a very wide polymorphism (ovotransferrin, ovomucoid, clusterin, etc.), some of them up to nine isoforms (ovoinhibitor). Eleven functional protein families were identified (serpin, transferrin, protease inhibitors Kazal, glycosyl hydrolases, lipocalin, bactericidal permeability-increasing protein, clusterin, UPAR/CD59/Ly6/ snake neurotoxin, cysteine protease inhibitor, VMO-1, and folate receptor families). These various biological functions could be interesting for further valorizations. In addition, three spots remain unidentified, probably because these proteins are not yet indexed in the international protein databanks.     

Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme

This work was aimed at the isolation, purification, and characterization of novel antimicrobial peptides from chicken egg white lysozyme hydrolysate, obtained by peptic digestion and subsequent tryptic digestion. The hydrolysate was composed of over 20 small peptides of less than 1000 Da, and had no enzymatic activity. The water-soluble peptide mixture showed bacteriostatic activity against Gram-positive bacteria (Staphylococcus aureus 23-394) and Gram-negative bacteria (Escherichia coli K-12). Two bacteriostatic peptides were purified and sequenced. One peptide, with the sequence Ile-Val-Ser-Asp-Gly-Asp-Gly-Met-Asn-Ala-Trp, inhibited Gram-negative bacteria E. coli K-12 and corresponded to amino acid residues 98-108, which are located in the middle part of the helix-loop-helix. Another novel antimicrobial peptide inhibited S. aureus 23-394 and was determined to have the sequence His-Gly-Leu-Asp-Asn-Tyr-Arg, corresponding to amino acid residues 15-21 of lysozyme. These peptides broadened the antimicrobial activity of lysozyme to include Gram-negative bacteria. The results obtained in this study indicate that lysozyme possesses nonenzymatic bacteriostatic domains in its primary sequence and they are released by proteolytic hydrolysis.     

alpha-Casein improves the gel properties of dried egg white

The effects of addition of alpha-casein (alpha-CN) to dried egg white (DEW) were investigated by measuring transparency, hardness, and water-holding capacity (WHC) of the heat-induced gels. A DEW concentration of 8% (w/w) was required for formation of a self-supporting gel following heating at 80 degrees C for 20 min at pH 7. Solutions of alpha-CN, even up to a protein concentration of 12% (w/w), did not gel under the same conditions. The addition of alpha-CN (0.5-4%) to 8% DEW caused the increase in gel hardness gels, as compared with DEW gels alone at a total amount of protein concentrations, and the mixed gels became transparent with the increase of added alpha-CN concentrations. The 10% mixed protein solutions of alpha-CN (3-6%) and DEW (4-7%) formed transparent gels, although each protein did not gel individually at their protein concentrations. Mixture with 2:8 mixing ratio of alpha-CN to DEW at a total protein concentration of 10% showed synergistic effects in improving DEW gel properties above pH 7 and below 25 mM NaCl. The improvements (hardness, transparency, and WHC) of DEW gel by alpha-CN seem to be caused mainly by the inhibition of alpha-CN against heat coagulation of DEW protein.     

Improvement of gel properties of dried egg white by modification with galactomannan through the Maillard reaction

The effects of Maillard reaction on gel properties of dried egg white (DEW) with galactomannan (GM) were investigated. Maillard-reacted DEW (MDEW) was prepared by dry-heating a mixture with a weight ratio of 1:4 of GM to DEW at 60 degrees C and 65% relative humidity. The modification of amino groups and polymerization of DEW proteins dry-heated with GM proceeded with increasing the dry-heating time. The covalent attachment of GM to DEW was confirmed from SDS-PAGE analysis. Gel strength and water-holding capacity of MDEW gels were higher than those of DEW dry-heated without GM (control DEW) and reached maximum after 3 days of dry-heating. The appearance of MDEW gels became transparent with increasing the dry-heating time, but control DEW gels were still turbid. MDEW dry-heated for 3 days was almost soluble even after heating of its solution at 90 degrees C, whereas control DEW proteins precipitated. The modification of DEW with GM through the Maillard reaction was an effective method to make a firm and transparent gel from DEW at broader range of pH and NaCl concentration of the medium.     

Immunochemical and structural analysis of pepsin-digested egg white ovomucoid

Ovomucoid, an egg protein comprising approximately 10% egg white, was digested using the enzyme pepsin, and fragments were isolated by anion-exchange and reverse phase HPLC. Four distinct fragments were identified by analysis with SDS-PAGE, including three large fragments with molecular weights of around 24, 18, and 14 kDa. N- and C-terminal and amino acid sequencing analyses identified the fragments as V134-C186 (domain 3), V21-A133, and A1-A133 (domain 1+2). Further separation and sequencing of the fraction composed of small peptides, to determine their exact makeup and location in the protein, remained to be carried out and identified a peptide G51-Y73. All four fragments showed IgE-binding activity, as measured by ELISA, using human sera from egg-allergic individuals. Little change in the digestibility of ovomucoid by trypsin and chymotrypsin was observed following digestion with pepsin, indicating that pepsin-digested ovomucoid retains its trypsin (protease) inhibitor activities. Reduced carboxymethylated ovomucoid was prepared, and digestion with pepsin produced significantly more peptides than did the digestion of the native ovomucoid, indicating that the disulfide bonds play a significant role in the digestive resistance of ovomucoid. The reduction of ovomucoid enhanced its digestibility and lower allergenicity of the protein.     

Immunomodulatory effects of heated ovomucoid-depleted egg white in a BALB/c mouse model of egg allergy

Oral immunotherapy (OIT) is a promising therapeutic approach for treating food allergy. The treatment with heated ovomucoid-depleted egg white (HOMEW) in egg-allergic patients is noteworthy; however, OIT protocols are still experimental, and a better knowledge of the underlying mechanism is required. The objective of this work was to investigate the immunomodulatory effects of HOMEW and characterize the underlying mechanism in a BALB/c mouse model of egg allergy. Mice were sensitized with EW and treated with HOMEW. Post treatment, mice were challenged with EW and euthanized for collecting blood and spleen. Markers of allergic clinical outcomes were measured as histamine concentration, serum antibody activity, and cytokine production from cultured splenocytes. Digestibility of HOMEW was assessed mimicking physiological conditions in vitro. The HOMEW demonstrated high digestibility. The treatment induced a marked increase of the Th1/Th2 ratio in the high-dose treatment group. Treated mice had significantly less histamine, EW-specific IgE, and IL-4 and more IFN-γ and IL-10. This study confirms the mechanisms involved in successful tolerance induction with OIT using HOMEW and allows understanding of the vital role of surrogate allergy markers involved in immune modulation.     

Bacterial precipitation of calcium carbonate in presence of phosphate

The formation of calcium carbonate (CaCO₃) crystals under different concentrations of K₂HPO₄ have been determined in 90 bacterial strains isolated from soil. Crystal formation decreased when PO^3?₄ ion concentration increased (in culture media) and precipitation was completely inhibited at 1.4 g I^?1 of K₂HPO₄.     

Identification and characterization of ovalbumin gene Y in hen egg white

Ovalbumin gene Y has been known as a member of the ovalbumin gene family since 1982, when its encoding gene was sequenced. In the present study, ovalbumin gene Y has been demonstrated as a new minor protein of hen egg white. This protein has been isolated by isoelectrofocalization and two-dimensional polyacrylamide gel electrophoresis and has been characterized using peptide mass fingerprinting. The concentration ratio of ovalbumin gene Y:ovalbumin is about 13:100. Unlike ovalbumin, ovalbumin gene Y is not phosphorylated, but like ovalbumin, this protein is glycosylated. Ovalbumin gene Y exists as a mixture of three molecular species, which differ in their isoelectric points. The polymorphism of this protein cannot be explained by various glycosylation levels.     

Bacterial growth on proteins in the presence of clay minerals

The growth of microorganisms on gelatin, bovine serum albumin and lysozyme in the presence of the clay minerals, montmorillonite and kaolinite was studied. The growth of microorganisms was diauxic, protein hydrolysis taking place in the second phase of growth. Depending on the protein-to-clay ratio, the second phase of growth could be divided into one of three types. At high protein-to-clay ratios, growth was not affected by the clay; at intermediate protein-to-clay ratios, growth rate but not final yield was reduced; and at low protein-to-clay ratios, the protein was unavailable for hydrolysis. The protein-to-clay ratios differentiating the three growth types were not well correlated with change in the affinity of adsorption of the protein to the clay and were different for different organisms.A model that explains the results is proposed. The main assumption of this model is that protein can bind strongly to clay and is either available or unavailable to proteases. The quantities of protein bound in these two modes are defined by the clay mineral, the protein and the protease.     

Metabolites of oxytetracycline, tetracycline, and chlortetracycline and their distribution in egg white, egg yolk, and hen plasma

4-epioxytetracycline and N-demethyloxytetracycline, as metabolites of oxytetracycline (OTC), 4-epitetracycline and N-demethyltetracycline, as metabolites of tetracycline (TC), and 4-epichlortetracycline, isochlortetracycline (ICTC), 4-epi-ICTC, and N-demethyl-ICTC, as metabolites of chlortetracycline (CTC), were detected in egg yolk and plasma obtained from feeding studies with either OTC, TC, or CTC. In egg white, only OTC, TC with its 4-epimer, and ICTC with its 4-epimer were detected in substantial concentrations. The ratios of epimerization and N-demethylation in the eggs did not change during the medication period. The samples were analyzed by an automated HPLC system (ASTED) with UV, fluorescence, or MS-MS detection.     

Effects of pulsed electric fields on ovalbumin solutions and dialyzed egg white

Ovalbumin solutions (2%, pH 7.0, 200 ohm.cm) and dialyzed fresh egg white (pH 9.2, 200-250 ohm.cm) were subjected to 50-400 exponential decay pulses with an electric field strength of 27-33 kV/cm. The pulse width was ca. 0.3 micros (at a capacitance of 20 nF) or 0.9 micros (at 80 nF), and the corresponding dissipated energy was 0.7 or 2.3 J/(pulse.mL) of solution. The sample temperature was maintained below 29 degrees C. While the four sulfhydryl groups of native ovalbumin did not react with DTNB, they became reactive immediately after pulse processing, indicating either partial protein unfolding or enhanced SH ionization. The extent of SH reactivity increased with dissipated energy, 3.7 SH groups becoming reactive after 100 or 200 pulses at 31.5 kV/cm and 80 nF. However, SH reactivity was reversible, since only 0.79 or 0.2 SH group was found to remain reactive 30 min or 8 h after pulse processing. The fourth derivatives of UV spectra of ovalbumin were determined, before and 15-30 min after pulse processing, to assess possible polarity and conformation changes in the environment of tyrosine and tryptophan. No differences were observed. Thermal gels prepared from fresh or dialyzed egg white had markedly different mechanical and water retention characteristics. Pulse processing of dialyzed egg white (200 pulses, 30 kV/cm, 80 nF) only slightly reduced its gelling properties. Thus electric pulses known to induce significant microbial inactivation did not cause notable changes in the proteins investigated.     

Glycation and phosphorylation of beta-lactoglobulin by dry-heating: effect on protein structure and some properties

Beta-lactoglobulin (beta-Lg) was glycated with maltopentaose and subsequently phosphorylated by dry-heating in the presence of pyrophosphate to investigate the structural and functional properties of phosphorylated beta-Lg. The circular dichroism spectra showed that the change of the secondary structure in the beta-Lg molecule by glycation and subsequent phosphorylation was small. The differential scanning calorimetry thermograms of beta-Lg showed that the denaturation temperature of the most stable domain was only slightly affected, whereas the retinol-binding activity of beta-Lg was somewhat reduced by glycation and subsequent phosphorylation. These results indicated that the conformational changes of the beta-Lg molecule by glycation and subsequent phosphorylation were mild. The anti-beta-Lg antibody response was somewhat reduced by glycation, but significant changes were not observed by phosphorylation. Although the stability of beta-Lg against heat-induced insolubility was improved by glycation alone, it was further enhanced by phosphorylation. The calcium phosphate solubilizing ability of beta-Lg was enhanced by phosphorylation following glycation.