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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

大米肽的酶法制备工艺及其特性的研究

该文利用蛋白酶水解大米蛋白制备得到大米肽。比较碱性蛋白酶、中性蛋白酶、复合蛋白酶和风味酶水解大米蛋白的进程曲线,结果显示碱性蛋白酶的水解效果最好,其较佳作用条件为：底物浓度10%、pH值9.0、温度45℃、酶与底物比48 AU/kg、时间150 min。在此条件下,大米肽的得率为46.8%,纯度为71.3%。大米肽具有溶解性较好和黏度较低的特性,可以在食品中广泛应用。     

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.     

大米降压肽酶法制备工艺及其性质研究

该文利用碱性蛋白酶水解大米蛋白制备大米降压肽，确定碱性蛋白酶水解终点为水解度18.17%。采用9种大孔吸附树脂吸附大米降压肽，结果显示NKA型树脂对大米降压肽的吸附效果较好，大米降压肽中灰分含量由脱盐前的16.64%减少到脱盐后的2.32%。对大米降压肽的相对分子质量、溶解性进行分析，大米降压肽的相对分子质量在138～1461之间；在pH 3～11的范围内，大米降压肽的溶解度在97.6%左右。     

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.     

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%.     

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.     

Sweetness and enzymatic activity of lysozyme

Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste.     

中性蛋白酶水解鸡骨泥制备短肽工艺优化

为了探究酶法制备鸡骨泥短肽的最佳工艺,该文研究了以中性蛋白酶酶解鸡骨泥时各因素对短肽得率和羟自由基清除率的影响,以及鸡骨泥肽清除超氧阴离子自由基和1,1-二苯基-2-三硝基苯肼自由基(2,2-diphenyl-1-picrylhydrazyl)的能力。试验探究了酶解过程中不同底物浓度、酶浓度、反应温度和反应时间对短肽得率和羟自由基清除率的影响,采用响应曲面优化得到了以短肽得率为目标的中性蛋白酶酶解鸡骨泥的最优工艺,当反应液p H值为7.2,反应温度42℃,反应时间2 h,底物质量分数5%,酶添加量200 mg/g,该条件下制备鸡骨泥肽,测得其短肽得率为56.16%,羟自由基清除率为54.12%。采用该酶解工艺制备鸡骨泥肽,测得其对超氧阴离子自由基最高清除率为56.01%,对DPPH自由基最高清除率为81.57%,说明鸡骨泥肽具有一定的抗氧化活性,研究结果为酶法制备鸡骨泥肽提供参考。     

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.     

Aggregation of peptides during hydrolysis as a cause of reduced enzymatic extractability of soybean meal proteins

With the purpose of analyzing the size and composition of enzyme-unextractable proteins in differently heat-treated soybean meals, a selection of extractants was screened for their ability to extract these proteins from enzyme-unextractable residues. The largest effects were obtained with urea, urea plus beta-mercaptoethanol, and dilute alkali; the latter extracted up to 87% of the enzyme-unextractable protein. Gel permeation chromatography indicated that a large proportion of the extracted material was of high molecular weight. However, the combined results from gel electrophoresis, LC-MS, and MALDI-ToF MS showed that the extracted protein material was composed of aggregated peptides. The largest aggregates were observed in the enzymatic residues originating from meals heat-treated at high humidity. Extracted aggregates were fully degraded upon subsequent proteolytic treatment.