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In this study, a proteome analysis of Tibetan chicken egg yolk as well as the comparison to that of lowland chicken were performed by label-free quantitative proteomics. A total of 135 proteins were identified and abundances of 19 of these proteins were significantly different between these two groups. These differential proteins were mainly associated with oxidative stress, defense, energy metabolism and tissue development through bioinformatics analysis. To further verify the species-specific diversity of the antioxidant capacity, the antioxidative activities of egg yolk proteins were further invested in vitro and in Caco-2 cells. It was observed that both Tibetan and lowland chicken egg yolk proteins showed antioxidant activities, but the former exerted a much stronger effect. PIT54 and glutathione peroxidase 3 were considered to be antioxidant-related candidate proteins. These results provide substantial evidence for the molecular mechanisms of enhancing physical activity levels of egg yolk proteins, especially with regard to the cross-species protective mechanisms against oxidative stress., Competing Interests: Declaration of Competing Interest The authors declare that ‘NO Conflict of interest’ exist in this research article., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Posttranslational N-glycosylation of food proteins plays a critical role in their structure and function. However, the N-glycoproteome of chicken egg yolk (CEY) has not been studied yet. Glycopeptides hydrolyzed from CEY proteins were enriched, with deglycosylation occurring using PNGase F, and then were identified using a shotgun glycoproteomics strategy. A total of 217 N-glycosylation sites and 86 glycoproteins were identified in CEY, and these glycoproteins are mainly involved in the binding, biological regulation, catalytic activity, and metabolic processes. Among the identified CEY glycoproteins, 22 were recognized as proteases and protease inhibitors, suggesting that a proteinase/inhibitor regulation system exists in CEY; further, 15 were members of the complement and immune systems, which provide protection against potential threats during hatching. The study provides important structural information about CEY glycoproteins and aids in the understanding of the underlying mechanism of embryo development as well as changes in CEY functional characteristics during storage and processing.

Chicken egg white (CEW) is a perfect source of natural proteins that possesses outstanding functional properties and various bioactivities. The glycosylation structure of CEW proteins plays important roles in their functions, bioactivities, and allergies. The present work attempted to identify N-glycosites of CEW proteins using an omics strategy. CEW proteins were digested with trypsin and chymotrypsin; glycopeptides were enriched and deglycosylated using PNGase F in H 2 18 O water, followed by analysis using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). A total of 71 N-glycosites in 26 CEW glycoproteins were identified. Web-Logo analysis showed that most of the N-glycosites were at N-X-T (55%) and N-X-S (32%). Furthermore, two-dimensional electrophoresis of CEW clusterin demonstrated a series of spots horizontally distributed at 35-37 kDa with an extremely wide isoelectric point range of 4.54-6.68, indicating the heterogeneity of glycosylation of CEW clusterin. These results provided important information for the understanding of the structures, functions, and bioactivities of CEW glycoproteins.

Background: Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C., Results: Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α 2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β 2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40)., Conclusion: It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α 2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Egg white proteins provide essential nutrients and antimicrobial protection during embryonic development. Although various biological functions of major egg white proteins have been investigated via embryogenesis, understanding of global changes in low-abundance proteins has been limited. In the current study, a proteomic analysis of low-abundance egg white proteins was conducted using combinatorial peptide ligand libraries (CPLL), two-dimensional gel electrophoresis (2-DE), and matrix-assisted laser desorption/ionization-time-of-flight with two mass analyzers for tandem mass spectrometry (MALDI-TOF MS/MS) during the rapid embryonic growth period. Significant increases in the relative abundance of 88 protein spots (P ≤ 0.05), of which 47 spots were found to correspond to 10 proteins from 8 protein families were identified over 16 d incubation. During this developmental process, the protein concentration increased and the amount of albumin solid material decreased in the residual egg white. Clusterin precursors were observed over a wide range of pH values and the tenp protein increased continuously during embryonic development. Low-abundance proteins were identified in a comparison of optimal incubation conditions to the altered conditions of 2 control groups to better understand the function of these proteins in egg whites. Collectively, these findings provide insight into the supportive role of the egg white during embryonic development, enabling a broader understanding of chick embryogenesis., (© 2015 Poultry Science Association Inc.)

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin. However, the practical application of IgY in research, diagnostics, and functional food is limited due to complex or time-consuming purification procedures. The objective of this study was to develop a simple, safe, large-scale separation method for IgY from egg yolk. Egg yolk was diluted with 6-fold delipidation solutions made of different types (pectin, λ-carrageenan, carboxymethylcellulose, methylcellulose, and dextran sulfate) and concentrations (0.01, 0.05, 0.1, 0.15, and 0.2%) of polysaccharides, respectively. The yolk solution was adjusted to pH 5.0, and then kept overnight at 4°C before being centrifuged at 4°C. The resulting supernatant was added to 35% (w/v) (NH4)2SO4 and then centrifuged. The precipitant, which contained IgY, was dissolved in distilled water and then dialyzed. SDS-PAGE and Western blotting were utilized to conduct qualitative analysis of IgY; high-performance liquid chromatography (HPLC) was used for quantitative analysis. The immunoreactivity of IgY was measured by ELISA. The results showed that yield, purity, and immunoreactivity varied with types and concentrations of polysaccharides. The optimal isolation of IgY for pectin, λ-carrageenan, dextran sulfate, and carboxymethylcellulose was at the concentration of 0.1%; for methylcellulose, optimal isolation was at 0.15%. The best results were obtained in the presence of 0.1% pectin. In this condition, yield and purity can reach 8.36 mg/mL egg yolk and 83.3%, respectively, and the negative effect of IgY on immunoreactivity can be minimized. The procedure of isolation was simplified to 2 steps with a higher yield of IgY, avoiding energy- and time-consuming methods. Therefore, the isolation condition under study has a great potential for food industry production of IgY on a large scale., (© 2014 Poultry Science Association Inc.)

Background: Some hydrolyzed peptides derived from food proteins possess antioxidant and anti-fatigue activities. In this study, egg white protein powder (EWPP) was hydrolyzed with pepsin for various times, and four peptide fractions were separated from the hydrolysates by ultrafiltration. The antioxidant activity of the four peptide fractions was determined. The peptide fraction with the strongest antioxidant activity was used to evaluate its anti-fatigue effect and probable mechanisms., Results: The egg white peptides (EWPs) fraction with molecular weight 2-5 kDa (named EWPs2) showed stronger antioxidant activity than the other peptide fractions (P < 0.05). The swimming time to exhaustion of mice administered EWPs2 was longer (P < 0.05) than that of the control group. EWPs2 increased the levels of blood glucose (by 28.4-42.2%), muscle glycogen (by 6.4-10.6%) and liver glycogen (by 10.7-23.8%) and significantly decreased the levels of lactic acid in muscle and urea nitrogen in blood (P < 0.05)., Conclusion: Among the four peptide fractions, EWPs2 possessed the strongest antioxidant activity and exhibited an anti-fatigue effect. The experimental data could clarify partially the anti-fatigue mechanisms of EWPs and provide an important basis for developing EWPs as safe and natural antioxidants and anti-fatigue agents for wide use., (© 2014 Society of Chemical Industry.)

Little is known regarding the diversification of relative low-abundance proteins in hen egg white that underlie the process of chicken embryonic development. The purpose of this study was to identify changes that may be involved in the development of chick embryos. Comparative proteomic analysis was conducive to revealing the potential biological function of low-abundance proteins and embryonic development demand. In addition, those proteins that are increased in abundance seem to have advantages to be targeted as new biomarkers for animal disease or animal production. However, the huge disparity in abundance in chicken egg white and poor performance of affinity depletion methods were specific challenges. This study applied combinatorial peptide ligand library for expanding the deep proteomics. Two-dimensional gel electrophoresis (2-DE) was performed with the hen embryo egg white, which was obtained from embryos at d 0 and 7. The protein spots showing significant increases were selected for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry/mass spectrometry (2 mass analyzers for tandem mass spectrometry). Many precursors were identified as strongly increased after 7 d of incubation. They were ovoinhibitor precursor, clusterin precursor, apolipoprotein D precursor, and extracellular fatty acid-binding protein precursor. Moreover, clusterin precursor were observed concentrated in 2 regions: one is the range of molecular weight increase and the other is the range of high pH. In addition, ovalbumin and ovotransferrin were observed in high pH area in a 2-DE map. These findings will give insight into the changes in fertilized egg white proteins during hatching and provide a better understanding of the biochemical changes in the egg development process.

The egg white protein alterations during the early phase of chicken embryonic development were recently reported by our laboratory. Nevertheless, the original albumen differences between fresh unfertilized and fertilized chicken eggs have not been investigated. By using 2-dimensional gel electrophoresis (2-DE), coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS) method, 1 ovalbumin protein spot as well as 6 ovalbumin-related protein Y spots were identified showing more than 10-fold differences (P < 0.01) in abundance between fresh unfertilized and fertilized chicken egg whites. Six of these protein spots represented higher intensity in fertilized eggs through 2-DE analysis. It was thus concluded that ovalbumin protein family, especially ovalbumin-related protein Y, may play an important role in embryonic development, which still needs to be validated. This finding will provide insight into embryogenesis to improve our understanding of the functions of ovalbumin family proteins in regulating or supporting embryonic development.

Although the effect of storage temperature was suggested to be a more important factor than that of storage time on changes in unfertilized egg white proteins, no comprehensive analysis of the thermally induced egg white protein changes was carried out. This study presents a proteomic analysis of the changes in unfertilized egg white proteins after 15 days of storage at 4, 20, and 37 °C. Using two-dimensional electrophoresis followed by MALDI-TOF MS/MS, 32 protein spots representing 8 proteins were identified with significant differences in abundance when stored at different temperatures. An accelerated degradation of ovalbumin, possibly resulting from the reduction of antiprotease, was observed after the storage at higher temperature. In addition, an increase in the formation of ovalbumin complexes and a decrease in lipocalin family proteins were detected with increasing storage temperature, which may indicate a thermally promoted change in chicken eggs. The decrease of clusterin during the high-temperature storage was suggested to be an effective biomarker for egg quality evaluation. These findings will give insight into the effects of storage temperature on changes in unfertilized egg white proteins during storage and provide a better understanding of the thermally induced biochemical changes that may affect the egg deteriorative process.

Unlabelled: Ovomucin has been considered to contribute a lot to the excellent functional properties of egg white. This work focused on investigating the effects of pH and protein concentration on foaming and emulsifying properties of ovomucin to evaluate the proper use of this egg protein as a functional food ingredient, and to clarify its contribution to the functional properties of egg white under different pH conditions. Protein solubility and surface hydrophobicity were measured through the pH ranged from 2.3 to 11.0. Alkali treatment gave ovomucin improved emulsification properties, which were correlated well with the surface hydrophobicity (r ≥ 0.89, P < 0.01). Although ovomucin showed lower foaming capacity in acid and neutral solution, enhanced foaming stability was observed with weak acid-treated ovomucin (pH 5 to 6) compared to native ovomucin. These results demonstrated that acid and alkali treatment, which leads to partial unfolding of ovomucin can improve functional properties of ovomucin, with the greatest improvement for emulsification properties being from the alkali treatment and for foaming stability being from weak acid treatment. These results are helpful to produce unfolding ovomucin suitable for wide range of applications in food industry, and to provide useful information on the proper use of egg white in different food systems., Practical Application: Ovomucin plays a critical functional role in egg white products. However, it is typically insoluble in distilled water or common salt solutions, which has thus limited its commercial applications. Alkaline treatment resulted in gradual increase in solubility, which markedly enhanced the emulsifying properties, on the other hand foaming stability of ovomucin can be promoted by weak acid treatment. The results of this work help to produce unfolding ovomucin suitable for wide range of applications in food industry, and to provide useful information on the proper use of egg white in different food systems., (© 2012 Institute of Food Technologists®)

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A new kind of soluble eggshell membrane protein (SEP) was prepared from eggshell membrane (ESM). The extraction rate of SEP could rise to 90% by two times, basically accomplishing the complete utilization of the whole ESM. Five proteases were employed as hydrolytic enzyme for the preparation of antioxidative peptides from SEP, and the antioxidative activities of the hydrolysates were investigated using a chemiluminescence method. Among the hydrolysates, alcalase hydrolysates with the highest free radical scavenging activity were further separated into three fractions using Sephadex G-25 gel filtration chromatography of the 4 h hydrolysate (SP1, SP2, and SP3). Among these three fractions, SP2 with an average molecular weight of 618.86 Da possessed the strongest fraction of scavenging activity. The IC50 values of the superoxide radicals, hydroxyl scavenging activities, and protective effect on DNA damage caused by hydroxyl radicals generated were 0.10, 0.18, and 0.95 mg/mL, respectively. These results demonstrate that inexpensive ESM waste could be a new alternative in the production of antioxidative peptides.