Enzyme immunoassay for hen egg white lysozyme used as a food additive

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.     

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.     

Dry-heating makes hen egg white lysozyme an efficient foaming agent and enables its bulk aggregation

Dry-heating is considered to be one of the most promising approaches to improving the functionality of food proteins. It has been shown that even if only minor structural modifications occur during dry-heating, the foaming properties of proteins are highly improved. With the recent results obtained in the field of foam stabilization by nanoparticles or protein aggregates in mind, a study was undertaken on the impact of dry-heating of lysozyme, used as a model protein, on its foaming properties. This work highlighted the fact that dry-heated hen egg white lysozyme simultaneously exhibited enhanced foaming properties and aggregation capacity. Although the conditions that favored bulk aggregation (high ionic strength, pH, treatment duration, and protein concentration) also favored foaming properties, the large bulk aggregates were not essential to obtain the best functionality. It is envisaged that heat-treated lysozyme may self-associate at the air/water interface, stabilizing air bubbles.     

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.     

Isolation and characterization of immunoglobulin in yolk (IgY) specific against hen egg white lysozyme by immunoaffinity chromatography

Six hens were intramuscularly (im) immunized once a week for 3 weeks using chicken egg white lysozyme (LS) as antigen. Antibody (immunoglobulin in yolk, IgY) ELISA values of 10(3)-fold diluted yolk were almost as high as 1.879 in the sixth week and maintained a value of 0.756 in the eighth week after the initial immunization treatment. The purification efficiency (specific activity of purified IgY against LS/specific activity of antibody in yolk against LS) of IgY specific against LS isolated by laboratory-prepared LS-bound (IgY-) Sepharose 4 Fast Flow immunoaffinity column was approximately 3380. By applying various amounts (0-22 mg) of the thusly obtained IgY specific against LS to the immunoaffinity column, the binding capacity (q(m)) and dissociation constant (K(d), M(-1)) of such immunoaffinity gel for IgY against LS were found to be 0.68 mg of IgY/mL of wet gel (0.54 mg of IgY/mg of LS) and 7.13 x 10(-6) M, respectively, as determined by Langmuir-type adsorption isotherms.     

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.     

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.     

Cross-linking of hen egg white lysozyme by microbial transglutaminase under high hydrostatic pressure: localization of reactive amino acid side chains

After incubation of hen egg white lysozyme (HEWL) with microbial transglutaminase (mTG) under high pressure (400-600 MPa for 30 min at 40 °C), the formation of HEWL oligomers was observed via SDS electrophoresis. At atmospheric pressure, HEWL represents no substrate for mTG. Likewise, enzymatic treatment following a pretreatment with high pressure did not lead to oligomerization. Reactive amino acid side chains were identified by peptide mapping after tryptic digestion using RP-HPLC with ESI-TOF-MS. Isopeptide-containing peptide fragments were found only in HEWL samples simultaneously treated with enzyme and pressure. It was found that mTG exclusively cross-links HEWL under high pressure by formation of an isopeptide between lysine at position 1 and glutamine at position 121 in the peptide chain. Therefore, a pressure-induced partial and reversible unfolding of the protein with exposure of lysine and glutamine side chains has to occur, resulting in a site-directed oligomerization of HEWL by mTG. The enzymatic modification of HEWL by mTG under high pressure offers interesting perspectives for further functionalization reactions.     

Toward better understanding of salt-induced hen egg white protein aggregation using field-flow fractionation

Field-flow fractionation techniques including sedimentation field-flow fractionation (SdFFF) and flow field-flow fractionation (FlFFF) were applied to investigate hen egg white protein aggregation. The thermally induced aggregation of hen egg white protein was observed at temperatures of 60 degrees C and higher. Particle size and size distribution of hen egg white protein aggregates were characterized by SdFFF to investigate parameters affecting ZnCl 2-induced aggregation of hen egg white protein. At a fixed concentration of 1.0 M ZnCl 2 and an incubation time of 15 min, the mean particle diameters of the aggregates were determined to be 0.43, 0.67, and 0.80 mum for hen egg white protein contents of 5, 6.25, and 7.5% (w/v), respectively. With the incubation time of 15 min, increasing the concentration of ZnCl 2 from 0.5 to 1.0 and to 1.5 M caused the mean particle diameter of the aggregates to grow from 0.37 to 0.42 and to 0.68 mum, respectively at 5% (w/v) hen egg white protein. Upon prolonged contact time, larger aggregates were formed. Furthermore, FlFFF was employed as a novel approach to determine the efficiency of protein utilization for aggregation. The pH values as well as ZnCl 2 and protein concentrations influenced the efficiency of protein utilization for aggregation. With the optimum condition, that is, a protein concentration higher than 2% (w/v) and a pH greater than 5, the efficiency of protein utilization was approximately 65%.     

Rapid separation of lysozyme from chicken egg white by reductants and thermal treatment

Reductants (0.1-2.0% ascorbic acid, cysteine, or cystine and 0.04-1. 0% beta-mercaptoethanol) were added to 5-fold diluted, salted duck egg whites (commercially and laboratory prepared) and fresh egg whites (chicken and duck), and subsequently the mixtures were heated at 70 degrees C for 1-10 min. The maximal recovery and purification fold of lysozyme obtained from fresh chicken egg whites added with 1. 0% ascorbic acid were 78% and 2.4, respectively. Storage tests showed that the obtained lyophilized lysozyme powder after dialysis was stable when refrigerated at 4 degrees C for 3 months.     

Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies

To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.     

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.     

Effect of cholecalciferol-enriched hen feed on egg quality

Eggs are one of the most important sources of vitamin D in the human diet, and their vitamin D content can be further increased by adding more vitamin D to hen feed. To investigate this issue more closely, we performed two feeding experiments. In both, zero egg samples were collected while the hens were fed regular feeds with a vitamin D content of 1720 or 4280 IU/kg. In experiment 1, egg samples were collected 2, 4, 7, 9, 11, 13, 16, 23, and 30 days after beginning the high-cholecalciferol (11 200 IU/kg) feeding period. In experiment 2, samples were collected 2, 4, 6, 8, 13, 28, 56, 84, 112, 140, and 168 days after beginning the high-cholecalciferol (12 000 IU/kg) diet. The egg samples were then assayed for their cholecalciferol content, and some samples, also for the presence of 25-hydroxycholecalciferol by an HPLC method. Further, the vitamin D-fortified eggs were compared with the controls by a sensory evaluation, by conducting fatty acid and functional analyses (emulsion capacity, gel forming capacity, foaming properties) and by measuring eggshell strength. Because vitamin D can be toxic in high doses, we also performed histopathological tests on the hens at the end of experiment 2. The top cholecalciferol contents in egg yolk (ca. 30 microg/100 g) were reached 8-13 days from starting the high-cholecalciferol diet. After 112 days feeding the cholecalciferol content gradually decreased to ca. 22 microg/100 g. When added to eggs as described above, vitamin D did not affect their sensory or functional properties or their fatty acid composition. Moreover, the cholecalciferol levels used in this study appeared not to affect eggshell strength or to be harmful for hens.     

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.     

Identification of an IgE reactive peptide in hen egg riboflavin binding protein subjected to simulated gastrointestinal digestion

Riboflavin binding protein (RfBP) is a minor protein in hen egg; its potential involvement in egg allergy has seldom been studied. The aim of this work was to investigate the IgE binding capacity of RfBP before and after simulated gastrointestinal digestion. It was shown that digestion of RfBP mainly occurred during the gastric phase. The protein fragments resulting from the subsequent duodenal phase remained linked through disulfide bonds. Both the intact protein and its digests were subjected to inhibition ELISA with sera obtained from patients allergic to egg. The results revealed significant IgE binding to intact RfBP, whereas the digests showed reduced but substantial IgE binding levels, with serum-to-serum variability. The RfBP digests were then subjected to immunoblot with allergic patients' sera, and the IgE-reactive peptides were further analyzed by MALDI-TOF/TOF mass spectrometry for sequence determination. The RfBP sequence 41-84 was identified as a novel IgE binding peptide in patients allergic to egg.     

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.     

Preparation of novel functional oligophosphopeptides from hen egg yolk phosvitin

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

Protein components of low-density lipoproteins purified from hen egg yolk

To identify apoproteins present in purified low-density lipoproteins from hen egg yolk in relation with their emulsifying properties, they have been separated by SDS-PAGE. We identified two different proteins by liquid chromatography-tandem mass spectrometry analysis of the peptides obtained by the trypsin digestion of protein gel bands. Apovitellenin I was identified as a monomer and a dimer. Its amino acid sequence was totally confirmed, and molecular mass determination by liquid chromatography-mass spectrometry showed that it did not present post-translational modifications but only a slight heterogeneity by the loss of one or two amino acids at the C-terminal part of the protein. Apolipoprotein B was identified into seven bands corresponding to fragments resulting of a processing of the hen blood apo-B protein. The identity of the fragments was determined by the observation of the sequence coverage by trypsin peptides and the sequence alignment with homologous human blood apolipoprotein B-100.     

Competitive adsorption of proteins from total hen egg yolk during emulsification

In this article, the competitive adsorption of egg yolk proteins at oil/water interfaces during emulsification is studied. By using two-dimensional polyacrylamide electrophoresis and mass spectrometry, it was possible to characterize and identify adsorbing and non-adsorbing protein species. The egg yolk contains proteins with a wide range of molecular weights and pI. Lipoproteins adsorbed selectively throughout the pH range investigated. It is suggested that selectivity is determined by the average hydrophobic and hydrophilic domain lengths in the protein sequences where long average hydrophobic domain lengths result in high affinity for the interface and thus strong preferential adsorption.