Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.     

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.     

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.     

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.     

Characterization of anti-irradiation-denatured ovalbumin monoclonal antibodies. Immunochemical and structural analysis of irradiation-denatured ovalbumin

Five monoclonal antibodies (OVA-01, -02, -03, -04, -06) produced against irradiated ovalbumin were investigated in relation to the conformational change in the ovalbumin molecule induced by irradiation with Cobalt-60 gamma-rays. Four antibodies (OVA-01, -02, -04, -06) recognized both native and irradiated ovalbumin, but OVA-03 reacted only with irradiated ovalbumin. These antibodies were classified by modified competitive ELISA, and their K(d) values were determined by the Klotz equation. Epitope analyses were also performed on OVA-03 using CNBr-cleaved peptide fragments from ovalbumin, and it was confirmed that OVA-03 bound to the fragment corresponding to residues Val173-Met196 of the ovalbumin molecule that consists of internal beta-sheet strand 3A and helix F1 containing one open turn. These results demonstrate that dramatic conformational changes in proteins can be induced or that some tertiary or secondary structures can be broken down by gamma-ray irradiation, producing new antigenic sites.     

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.     

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.     

Emulsifying and foaming properties of ultraviolet-irradiated egg white protein and sodium caseinate

The physicochemical and functional properties of ultraviolet (UV)-treated egg white protein (EW) and sodium caseinate (SC) were investigated. UV irradiation of the proteins was carried out for 30, 60, 90, and 120 min. However, the SC samples were subjected to extended UV irradiation for 4 and 6 h as no difference was found on the initial UV exposure time. Formol titration, SDS-PAGE, and FTIR analyses indicated that UV irradiation could induce cross-linking on proteins and led to improved emulsifying and foaming properties (P < 0.05). These results indicated that the UV-irradiated EW and SC could be used as novel emulsifier and foaming agents in broad food systems for stabilizing and foaming purposes.     

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.     

Stability of acidic egg white protein emulsions containing xanthan gum

The influence of xanthan gum concentration on the physicochemical stability of model oil-in-water emulsions prepared with egg white protein at pH 3.8 and containing 150 mM NaCl was investigated by following droplet aggregate formation, rheological changes, and serum separation with storage time. Egg white emulsions were more strongly flocculated and exhibited higher stability against creaming than those of yolk, irrespective of the presence or absence of xanthan. Depletion effects, originating from the presence in the continuous phase of the emulsions of nonadsorbing xanthan molecules, intensified droplet-droplet flocculation effects and resulted in large droplet flocs. At relatively low xanthan contents, the emulsions exhibited higher stability against creaming compared to the respective control emulsions probably due to the formation of a continuous droplet aggregate network structure. At higher xanthan contents, less extensive droplet interactions, due to slowly evolving microstructure of phase-separated xanthan-rich and xanthan-depleted regions, resulted in emulsions exhibiting increased stability against creaming. The role of interactions between protein molecules adsorbed on neighboring droplets in these changes and their effect on emulsion aging are discussed.     

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.     

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.     

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.     

辐照杀菌对鸡蛋蛋白液特性的影响

为了探明液态蛋蛋白液经辐照处理后有关特性的变化情况,为液态蛋的辐照杀菌技术应用提供试验依据。试验研究了不同辐照条件下鸡蛋蛋白液的pH值、色度、黏度、热变性、起泡性和乳化性的变化。试验表明,在辐照剂量0～3.0 kGy范围内随辐照剂量增大,蛋白液的pH值有所下降,但变化相对不大;蛋白液的黏度在辐照剂量0～0.4 kGy范围内随辐照剂量增大有较大下降,但剂量大于0.4 kGy 以后蛋白黏度随辐照剂量增大变化较小;蛋白液的色度随剂量增大无变化,但蛋液经加热凝固后,2.0 kGy以上剂量辐照组蛋白胶体颜色出现褐色,且随辐照剂量增大而加深;随辐照剂量增大,蛋白液起泡性能增强,但泡沫稳定性下降;随辐照剂量增大辐照后蛋白液的乳化性、乳化稳定性均下降。     

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.     

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.     

Development of an ELISA for quantifying lysozyme in hen egg white

An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.     

Correlation of the protein structure and gelling properties in dried egg white products

The relationship between protein structure and aggregation, as well as heat-induced gelling properties, of seven dried egg white (DEW) products was investigated. Strong correlations were found between average molecular weight and hydrophobicity plus surface SH groups of DEW-soluble protein aggregate (SPA). This suggests that hydrophobic interactions and disulfide bond formation between protein molecules were involved in the aggregation. The average molecular weight of DEW products with alkaline pHs was relatively higher than those with neutral pHs and the same degree of protein unfolding, probably because of more disulfide bond formation between protein molecules. In addition, strong correlations were found between hydrophobicity, surface SH groups plus average molecular weight of DEW-SPA, and physical properties of the gels from DEW products. These data indicated that controlling the aggregation of DEW proteins in the dry state is crucial to controlling the gelling properties of DEW.