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The insect eggshell is a multifunctional structure with several important roles, including generating an entry point for sperm via the micropyle before oviposition, serving as an oviposition substrate attachment surface, and functioning as a protective layer during embryo development. Eggshell proteins play major roles in eggshell tanning and hardening following oviposition and provide molecular cues that define dorsal-ventral axis formation. Precise eggshell formation during ovarian follicle maturation is critical for normal embryo development and the synthesis of a defective eggshell often gives rise to inviable embryos. Therefore, simple and accurate methods for identifying eggshell proteins will facilitate our understanding of the molecular pathways regulating eggshell formation and the mechanisms underlying normal embryo development. This protocol describes how to isolate and enrich eggshells from mature oocytes of Aedes aegypti mosquitoes and how to extract their eggshell proteins for liquid chromatography with tandem mass spectrometry (LC-MS/MS) proteomic analysis. Although this methodology was developed for studying mosquito eggshells, it may be applicable to eggs from a variety of insects. Mosquitoes are ideal model organisms for this study as their ovarian follicle development and eggshell formation are meticulously regulated by blood feeding and their follicles develop synchronously throughout oogenesis in a time-dependent manner., (© 2024 Cold Spring Harbor Laboratory Press.)

The effect of the diet modification (soybean and lupine addition) on the content of protein and amino acids (AA) in eggs was studied. Both the sampling day and the diet influenced the total protein content. In albumen, the lowest protein content (10.6%) was noted after administering a diet containing 25% lupine; in the same egg the yolk contained the most proteins (16.7%). In the content of nonessential AA (NAA) in egg yolks, differences were noted only for cysteine, with its the highest content in the yolks of the control group. The stable content of essential yolk amino acids (EAA) was observed only for isoleucine, leucine, tryptophan and phenylalanine. The highest contents of EAA and NAA were recorded in the yolks of the control group (~47 and ~53 g/100 g of protein, respectively) and in the group with 25% additions of lupine (~42 and ~51 g/100 g of protein, respectively). AA with constant content in the tested albumens were methionine, tryptophan and alanine. The highest content of EAA (>~42 g/100 g of protein) and NAA (>~62 g/100 g of protein) were determined in albumen of eggs determined in the group with at least 20% additions of lupine. The highest content of EAA for humans delivered eggs from groups 4-6 (with the addition of soy into the diet ≤5%). The protein sources used in the hen diet significantly influenced the content of protein and individual AA in the produced eggs.

Background: Sturgeon species are living fossils that exhibit unique reproductive characteristics, and elucidation of the molecular processes governing the formation and quality of sturgeon eggs is crucial. However, comprehensive data on the protein composition of sturgeon ovarian fluid (OF) and eggs and their functional significance are lacking. To address this knowledge gap, the aim of the present study was to conduct a comprehensive comparative proteomic analysis of Siberian sturgeon OF and eggs using liquid chromatography-mass spectrometry (LC-MS/MS)., Results: A total of 617 proteins were identified in OF, and 565 proteins were identified in eggs. A total of 772 proteins showed differential abundance. Among the differentially abundant proteins, 365 were more abundant in OFs, while 407 were more abundant in eggs. We identified 339 proteins unique to OFs and 287 proteins specific to eggs, and further investigated the top 10 most abundant proteins in each. The functional annotation of the OF proteins highlighted their predominant association with immune system processes, including the complement and coagulation cascade, neutrophil and leukocyte-mediated immunity, cholesterol metabolism, and regulation of the actin cytoskeleton. Analysis of egg proteins revealed enrichment in metabolic pathways, such as oxidative phosphorylation and fatty acid metabolism, and protein ubiquitination and translation. OF-specific proteins included extracellular matrix and secretory vesicles, and eggs were enriched in proteins localized to mitochondria and ribosome components., Conclusions: This study presents the first comprehensive characterization of the protein composition of sturgeon OF and eggs and elucidates their distinct functional roles. These findings advance our understanding of sturgeon reproduction, OF-egg signaling and the origin of OF proteins. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD044168 to ensure accessibility for further research., (© 2024. The Author(s).)

In this study, delipidated egg yolk proteins were used for the first time to prepare nanoparticles by the self-assembling method at pH 8.0, then treated with UV-C as a crosslinking agent, and their stability tested at pH 7.0, which is a more convenient pH for food applications. According to the results obtained, non-irradiated nanoparticles had a size of 431.8 ± 75.7 nm at pH 7.0, but the 10 min UV-C irradiated nanoparticles had an average size of 139.7 ± 5.9 nm. These nanoparticles also showed a high resistance to destabilization by SDS, urea or DTT and noticeable antioxidant and ferrous chelating activities. Pickering emulsions prepared at the nanoparticle concentration of 1 % (w/w) showed the smallest average droplet size and the lowest Turbiscan stability index value after 80 days of storage. All in all, these results have important implications for the utilisation of these proteins as a conventional Pickering emulsifying agent., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The dry separate curing of duck egg yolks was carried out by ultrasonic synergize NaCl (sodium chloride) and NaCl alone. The mechanism of the amelioration of salted egg yolk quality by ultrasonic synergistic NaCl dry-curing was studied. The quality variations of the salted egg yolks were analyzed for the same curing time and NaCl content achieved by ultrasonic synergistic NaCl curing and NaCl curing alone. The results showed that under the same salting time, the NaCl content, oil exudation and chewiness of U48-SEY (ultrasonic for 48 h-salted egg yolk) were higher than those in SEY (salted egg yolk). At the same NaCl content, the oil exudation and chewiness of U44-SEY (ultrasonic for 44 h-salted egg yolk) were still significantly increased. Compared to SEY, the soluble protein content and H 0 of U44-SEY and U48-SEY were augmented. Scanning electron microscopy (SEM) indicated that the polyhedral particles in the salted egg yolks prepared by ultrasonic synergistic NaCl dry-curing were closely aligned and evenly distributed, and the salted egg yolks were sandier. Structural analysis revealed that the secondary and tertiary structures of egg yolk protein were changed although the ultrasonic synergistic NaCl dry-curing did not cause the fragmentation or aggregation of the peptide chain structure. The above results suggested that ultrasonic not only perfected the quality of salted egg yolk by promoting NaCl penetration, but also modified the structures of egg yolk protein by the action of ultrasonic itself, which prominently improved the quality of salted egg yolks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the formation mechanism of salted egg yolk (SEY) mudding during storage. Results showed that the soluble protein, hardness, and intrinsic fluorescence intensity of SEY decreased significantly during storage, while total volatile basic nitrogen, sulfhydryl group, dityrosine, adhesiveness, and surface hydrophobicity increased significantly, and the intrinsic fluorescence peak position red-shifted at first and then blue-shifted. In addition, from the results of infrared and microstructure analyses, there was an obvious oxidation reaction between protein and lipid in the late storage stage; the structure of SEY was destroyed, many random coils were formed, and the degree of protein-lipid binding and the crystallinity of SEY protein decreased during storage. Finally, the heatmap analysis revealed that the protein and lipid oxidation and conformational changes might be the main reasons for SEY mudding. This study can provide theoretical guidance for the control of SEY mudding., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Amaranth (Amaranthus L.), like other pseudocereals as quinoa (Chenopodium quinoa Willd.), chia (Salvia hispanica L.) and buckwheat (Fagopyrum sp.), is a promising source of dietary protein. Depending on the subspecies and breeds of amaranth, the protein content in its grain is estimated from 13.1 to 21.5%, and its amino acid score varies over a significant range and can be limited. The aim of this study was to obtain a protein concentrate from amaranth (Amaranthus L.) grain of the Voronezh breed, enrich it with chicken egg protein, determine the amino acid score of the obtained protein module, and experimentally evaluate in vivo its true digestibility and biological value. Material and methods . The amaranth protein concentrate was obtained from grain according to the technological scheme, including its enzymatic treatment, alkaline extraction, acid precipitation of proteins, microfiltration and lyophilization. The amino acid composition and amino acid score of the concentrate were determined. The protein module was obtained by mixing amaranth protein concentrate and chicken egg protein in a weight ratio of 58:42. The true digestibility and biological value of the protein module has been determined in vivo. The experiment was carried out on 32 Wistar male rats divided into 2 groups (n=16 rats): control group 1 with a body weight of 118.7±3.1 g and experimental group 2 with a body weight of 119.5±3.0 g. Animals of groups 1 and 2 received diets in which egg protein and a protein module were used as a protein source, respectively. Within 15 days of the experiment, individual indicators of food intake and body weight gain of each animal were determined. From the 14th to the 15th day food intake was determined and feces were collected. The amount of nitrogen in the food and feces was determined for each rat using the Kjeldahl method. The true digestibility of the protein was determined according to obtained data. Results . The resulting amaranth protein concentrate contained 70.4±0.6% of protein, 17.0±1.0% fat, 9.8±0.8% carbohydrates, 1.8±0.2% ash, its moisture content was 1.4±0.1%. There were no significant differences in food intake and body weight gain between animals of both groups. The calculated value of the true digestibility of chicken egg protein was 98.8±0.1% for the control group 1, of the protein module was 99.0±0.1% for the experimental group 2, the differences between the groups were not significant. Conclusion . The results of amino acid analysis and the in vivo study of the true digestibility of the protein module (composition amaranth protein/chicken egg protein) indicate the absence of limitation relative to the amino acid scale of the "ideal" protein (FAO/WHO, 2007) and high true digestibility. The biological value of the protein module, calculated according to PDCAAS, is 99.0±0.1%, which confirms the prospects for its inclusion in specialized foods., Competing Interests: Authors declare no conflict of interest., (Copyright© GEOTAR-Media Publishing Group.)

Preparation of egg yolk powder (EYP) with excellent hydration properties and dissolution stability is important for the efficient utilization of its functional properties and nutritional properties in food. In this work, a new method utilizing ultrasound-assisted enzymolysis (UP-EM) was investigated to obtain EYP. Compared to enzymolysis-alone treated (EM), ultrasonic pre-treatment (UP) significantly increased the enzymatic hydrolysis rate by 106.28%. In particular, the UP 60 W 20 min-EM group obtained the desired solubility and coefficient of stability. The observed microstructure of EYP showed that the egg yolk particles obtained by UP-EM were more uniformly distributed and smaller in particle size. The protein structure was confirmed by Fourier transform infrared spectrum, which showed that UP enhanced the hydrogen bonding force between egg yolk proteins. Therefore, it can be believed that UP-EM can significantly improve the hydration properties and dispersion stability of EYP, laying the foundation for its wide applications in food industry., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The rapid evolution of fertilization proteins has generated remarkable diversity in molecular structure and function. Glycoproteins of vertebrate egg coats contain multiple zona pellucida (ZP)-N domains (1-6 copies) that facilitate multiple reproductive functions, including species-specific sperm recognition. In this report, we integrate phylogenetics and machine learning to investigate how ZP-N domains diversify in structure and function. The most C-terminal ZP-N domain of each paralog is associated with another domain type (ZP-C), which together form a "ZP module." All modular ZP-N domains are phylogenetically distinct from nonmodular or free ZP-N domains. Machine learning-based classification identifies eight residues that form a stabilizing network in modular ZP-N domains that is absent in free domains. Positive selection is identified in some free ZP-N domains. Our findings support that strong purifying selection has conserved an essential structural core in modular ZP-N domains, with the relaxation of this structural constraint allowing free N-terminal domains to functionally diversify., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Dietary supplementation of green tea powder (GTP) changes egg quality of hens, however, whether these changes affect incubation is still unknown. This study was to compare the proteomic difference of incubated eggs from hens with GTP supplemented or not. Huainan partridge chickens (1,080) at 35 wk of age were allocated into 2 groups, one group fed basal diet (CG) and one group fed basal diet plus 1% GTP (EG). After 4 wk feeding, artificially fertilized eggs were collected for yolk cholesterol determination and incubation. During incubation, 6 embryos from each group were randomly selected in each day for yolk protein extraction and quantification. Yolk cholesterol content was significantly lower, while the hatchability was significantly higher in EG than that of the CG group (P < 0.05). Yolk protein concentration at embryonic days (ED) of 0, 2, 6, and 13 showed significant changes and were selected for proteomic analysis by 2-dimensional gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Fifty-one differentially expressed (DE) protein spots were identified among different incubation stages between CG and EG group which were mainly classified into vitellogenin, immunoglobulin, and ovoinhibitor, and occupied 45.1, 23.5, and 15.7%, respectively, to the total DE proteins. Ovotransferrin, participated in extracellular sequestering of iron ion process, was significantly lower in EG group than that of the CG group (P < 0.05). Ig light chain precursor (Immunoglobulin) exhibited higher expression at ED6 in EG group as compared with that of the CG group, and was participated in immune response related processes. Ovoinhibitor, mainly involved in protease binding activity, showed lower abundance at ED13 in EG group as compared with that of the CG group. Vitellogenin-3, showed lower expression in EG group as compared with that of the CG group, was mainly participated in lipid transportation and localization according to GO enrichment. Chickens fed diet with GTP provided eggs more antioxidant ability that increased hatchability, indicated that GTP could be considered as additive in breeding layer., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Egg white protein (EWP) is susceptible to denaturation and coagulation when exposed to high temperatures, adversely affecting its flavour, thereby influencing consumers' decisions. Here, we employ high-voltage cold plasma (HVCP) as a novel nonthermal technique to investigate its influence on the EWP's flavour attributes using E-nose, E-tongue, and headspace gas-chromatography-ion-mobilisation spectrometry (HS-GC-IMS) due to their rapidness and high sensitivity in identifying flavour fingerprints in foods. The EWP was investigated at 0, 60, 120, 180, 240, and 300 s of HVCP treatment time. The results revealed that HVCP significantly influences the odour and taste attributes of the EWP across all treatments, with a more significant influence at 60 and 120 s of HVCP treatment. Principal component analyses of the E-nose and E-tongue clearly distinguish the odour and taste sensors' responses. The HS-GC-IMS analysis identified 65 volatile compounds across the treatments. The volatile compounds' concentrations increased as the HVCP treatment time was increased from 0 to 300 s. The significant compounds contributing to EWP characterisation include heptanal, ethylbenzene, ethanol, acetic acid, nonanal, heptacosane, 5-octadecanal, decanal, p-xylene, and octanal. Thus, this study shows that HVCP could be utilised to modify and improve the EWP flavour attributes.

Whey and hen egg white proteins are characterized by high nutritional value, but possess antigenic properties, which limit their use in the production of dietary products. Enzymatic hydrolysis decreases significantly the allergenicity of proteins. The efficiency of hydrolysis depends on the specificity of the proteases used. The aim of this work was to determine the effectiveness of EP-96 enzyme preparation obtained from Bacillus subtilis-96 culture liquid in the hydrolysis of whey and egg white proteins in comparison with commercial bacterial proteases preparations - Alcalase, Neutrase, and Protosubtilin. Material and methods . Whey and egg white protein concentrates were used as substrates. Commercial enzyme preparations Alcalase, Neutrase, and Protosubtilin, and an experimental sample of EP-96 preparation obtained from Bacillus subtilis-96 culture liquid were used for hydrolysis. Hydrolysis was carried out at a substrate concentration of 100 g/L for 3 h at 55 °C or for 24 h at 50 °C. After hydrolysis, the reaction mixture was incubated at 90 °C for 15 min to inactivate the enzymes. The content of peptides with a molecular weight of less than 10 kDa was determined in the obtained hydrolysates. The hydrolysis of the main allergenic proteins was assessed by the disappearance of the corresponding protein bands on the hydrolysate supernatants electrophoregrams. Results and discussion . All the studied preparations showed high efficiency in the hydrolysis of whey proteins and provided the yield of low molecular weight peptides at the level of 18.8-22.8% after 3 h of hydrolysis and 39.4-41.6% after 24 h. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a residual amount of protein with a molecular weight of about 14 kDa, corresponding to α-lactoalbumin, after 3 h of hydrolysis when using Neutrase. The preparations containing serine protease, including EP-96, provided more intensive hydrolysis of whey proteins. In the hydrolysis of egg white protein, Neutrase showed the greatest efficiency. The efficiency of EP-96 was comparable to Neutrase both in the yield of low molecular weight peptides and in the intensity of cleavage of the main allergenic proteins. The effectiveness of preparations with predominant content of serine proteases - Alcalase and Protosubtilin was significantly lower. Conclusion . The optimal ratio of neutral and serine proteases in the EP-96, obtained on the basis of the B. subtilis-96 strain, provided the high efficiency and its versatility in the hydrolysis of the main allergenic proteins of whey and egg white. The parameters of the hydrolysis technology using EP-96 are recommended, which provide intensive conversion of the main immunogenic proteins of whey and egg white to soluble and low molecular weight fractions (duration 3 h at a temperature of 55 °C and the proteolytic activity of the preparation is not less than 2 units per g of substrate) and an increase of subsequent ultrafiltration efficiency in the production of protein hydrolysates for foods for special dietary uses., Competing Interests: The authors declare no overt and potential conflict of interest related to the publication of this article., (Copyright© GEOTAR-Media Publishing Group.)

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We evaluated by oral food challenge the reactivity to a brand of PAL-labelled milk- and egg-free biscuits of children with severe milk and egg allergy. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in such biscuits. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed using immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were undetectable with a limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Milk/egg content of the biscuits is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergens in foods, and can be used to help avoiding unnecessary PAL.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as "entolecithal" eggs. However, only Neoophora, in the phylum Platyhelminthes, produce "ectolecithal" eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy- energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17β-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R 2 ) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200-2400 kg h -1 , the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000-3000 kg (for long pasta) and 3000-4000 kg (for short pasta).

Lactic acid bacteria fermentation is a safe and green technology that can modify the function of food ingredients (including proteins). In this article, egg yolks were subjected to fermentation with commercial lactic acid bacteria for 0, 3, 6 and 9 h, respectively. After fermentation treatment, the microbial composition has changed obviously (Streptococcus thermophilus increased significantly). The free sulfhydryl group (SH) contents and surface hydrophobicity of egg yolk proteins were significantly reduced. The rheological results indicated that the treated egg yolks possessed a decreased apparent viscosity. Correspondingly, the emulsifying activity of egg yolk was enhanced from 9.07 to 19.55, 23.40 and 24.61 m 2 /g for 3, 6 and 9 h of fermentation, respectively. And the emulsifying stability reached the maximum after 3 h of fermentation. This study investigated the relationship between structure and properties of yolk proteins, and showed that lactic acid fermentation endued egg yolk with better emulsifying properties., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The chicken egg vitelline membrane (CEVM) is an important structure for the transmembrane transport of egg yolk components, protection of the blastodisc, and separation of egg white and egg yolk. In this study, the N-glycoproteome of the CEVM was mapped and analyzed in depth. Total protein of the CEVM was digested, and the glycopeptides were enriched by a hydrophilic interaction liquid chromatography microcolumn and identified by nano liquid chromatography/tandem mass spectrometry. A total of 435 N-glycosylation sites on 208 N-glycoproteins were identified in CEVM. Gene Ontology enrichment analysis showed that CEVM N-glycoproteins are mainly involved in the regulation of proteinases/inhibitors and transmembrane transport of lipids. Mucin-5B is the primary N-glycoprotein in the CEVM. Comparison of the main N-glycoproteins between the CEVM and other egg parts revealed the tissue specificity of N-glycosylation of egg proteins. The results provide insights into protein N-glycosylation in the chicken egg, CEVM functions and underlying mechanisms., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020. Published by Elsevier B.V.)

Hen egg is a worldwide top-consumed food that has attracted public health concerns because it can induce allergic reactions in sensitized individuals. Food allergy investigations need highly purified egg allergens. However, a limited number of purification methods have been described for the combined separation of more than two egg allergens and only few of them have evaluated the immunological activity of these purified proteins. The aim of this work was to develop a chromatographic method for the separation of the four major egg allergens (ovomucoid, ovalbumin, ovotranferrin, and lysozyme) with a demonstrated immunological activity. After a pre-processing step for ovomucin precipitation and pH adjustment, remaining egg white proteins were loaded onto CM-Sepharose column and major egg allergens were separated using cation-exchange chromatography. Yield of ovomucoid, ovalbumin, ovotranferrin, and lysozyme was 60.0%, 52.1%, 29.6%, and 90.2%, respectively. Purified allergens were compared with their commercial standards, showing a high purity as well as a maintained antigenicity. The protocol described in this work is simple, quick, low-cost, and suitable for the study of the immunological properties of these allergens. For higher ovalbumin demand in the lab, 2.1 g ovalbumin can be produced in a single process with high purity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Yolk is the most important temporary biological compartment of the early life stages of fish embryos. The sorption strength of a chemical to yolk components may significantly influence the distribution of that chemical in the fish embryo. We determined yolk-water partition coefficients (K yolk/water , in liters of water per kilogram of yolk, normalized to dry wt) for 70 neutral organic chemicals. The log K yolk/water values range from 0.76 to 6.56. On the basis of these values, we developed polyparameter linear free energy relationship models to predict yolk-water partitioning for a broad range of neutral organic chemicals with a root mean squared error of 0.37 and r 2 of 0.919. These models can be applied for the prediction of internal concentrations at equilibrium (neglecting biotransformation and active transport) in the zebrafish embryo test system. Environ Toxicol Chem 2020;39:1506-1516. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Five major proteins from egg white were separated using a successive extraction/precipitation protocol. The yield and purity of the separated proteins were measured. The separated proteins were confirmed by MALDI-TOF-MS, and their structures were characterized by CD spectrum. Lysozyme was first separated using FPC 3500 resin and then ovomucin from the lysozyme-free egg white. Ammonium sulfate and citric acid were added to the resulting lysozyme- and ovomucin-free egg white solution to precipitate ovotransferrin. Ovomucoid and ovalbumin were separated from the resulting supernatant using ethanol. The separated proteins were further purified and the optimal conditions for the further purifications were suggested. The purity and yield of lysozyme, ovotransferrin, ovalbumin, and ovomucoid were higher than 90% and 77%, while those of ovomucin were about 72% and 75%, respectively. This study separated five major proteins in egg white successively using resin adsorption, pH adjustment, salt/ethanol precipitation, and ultrafiltration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate energetic source used by juveniles of a terrestrial oviparous invertebrate during the earliest periods of their life. Growth, behavioural activities and energy contents of Pardosa saltans spiderlings' residual vitellus were monitored during 8 days after their emergence from their egg-sac until they disperse autonomously. The life-cycle of juvenile after emergence can be divided into three periods: a gregarious while juveniles are aggregated on their mother, dismounting off their mother's back and dispersion. We present the first biochemical study of residual vitellus and energy expenditure during these three periods. At emergence, the mean weight of juveniles was 0.59 mg and energy stock from residual vitellus averaged 51 cal/g wet mass. During gregarious period, the weight of the juveniles aggregated on their mother did not vary significantly and juveniles utilized only 1 cal/day from their residual vitellus. During the period from dismounting until their first exogenous feed, juveniles lost weight and used 30% of their residual vitellus stock. Proteins from the residual vitellus contributed principally to their energy expenditure during this period: 1.5 µg protein/day. Juveniles' first exogenous feeding was observed 7-8 days after emergence, when 70% of residual vitellus energy had been utilized. Juveniles dispersed after eating, reconstituting an energy stock comparable to that observed at emergence from egg-sac (50 cal/g wet mass). This new energy stock contains mainly lipids unlike the energy stock from the residual vitellus.

Rationale: Proteins from the egg perivitelline fluid (PVF) are assumed to play critical roles in embryonic development, but for many groups of animals their identities remain unknown. Identifying egg PVF proteins is a critical step towards understanding their functions including their roles in evolutionary transition in habitats., Methods: We applied proteomic and transcriptomic analysis to investigate the PVF proteome of the eggs of Pomacea diffusa, an aerial ovipositing freshwater snail in the family Ampullariidae. The PVF proteins were separated with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and proteomic analysis was conducted using an LTQ Velos ion trap mass spectrometer coupled with liquid chromatography. Comparison of PVF proteomes and evolution analyses was performed between P. diffusa and other ampullariids., Results: In total, 32 egg PVF proteins were identified from P. diffusa. They were categorized as PV1-like subunits, immune-responsive proteins, protein degradation, signaling and binding, transcription and translation, metabolism, oxidation-reduction and proteins with unknown function. Interestingly, the proteome includes a calcium-binding protein important in forming the hard eggshell that enabled the terrestrial transition. However, it does not include PV2, a neurotoxic protein that was assumed to be present in all Pomacea species., Conclusions: The PVF proteome data from P. diffusa can help us better understand the roles that reproductive proteins played during the transition from underwater to terrestrial egg deposition. Moreover, they could be useful in comparative studies of the terrestrialization in several groups of animals that occurred independently during their evolution., (© 2019 John Wiley & Sons, Ltd.)

A follow-up study was conducted to further evaluate the marking efficiency of broadcast spray applications of egg albumin (from chicken egg whites) on Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae) in alfalfa. A previous study recorded exceptional marking efficiency (e.g., >95% of the population) on H. convergens when using relatively high concentrations (10 to 50%) of chicken egg whites. The present study examines marking efficiency of egg whites using lower concentrations of 2.5, 5.0, and 10.0%. We used cadaver and free-roaming beetles to measure protein mark acquisition (and retention) of each protein concentration by direct contact with the spray application and incidental contact with protein residue on the plant tissue, respectively. The vertical distribution of the protein mark was also determined by sampling the upper and lower portions of the alfalfa canopy. The data indicate, regardless of the egg white treatment, that the backpack sprayer provided uniform coverage of egg albumin on the alfalfa plants and cadaver beetles. Also, almost every free-roaming beetle acquired a mark within 24 h after contact exposure to protein marked plants. This study shows that a very low concentration of egg albumin is sufficient for marking arthropods directly in the field., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Underestimation of egg allergen from processed foods prompted the evaluation of critical Enzyme-Linked Immunosorbent Assay (ELISA) parameters: (1) extraction of egg proteins from a processed matrix; (2) use of anti-heat processed egg antibodies (Abs) on detectability of modified proteins, and (3) utilization of incurred material as standards. The relative affinity of two combinations of raw (R), boiled (B) and fried (F) Abs to unprocessed/processed egg proteins with or without matrix was determined from antibody (Ab) binding curves. In ELISAs using RBF-Abs and BF-Abs, denaturing buffer, and incurred standards, the Limit of Detection (LOD) and Limit of Quantitation (LOQ) were 0.47 and 0.25; and 1.58 and 0.85, respectively, and the linear range was 0-24 μg g -1 egg protein. The recoveries of egg protein from cookies, cereal bar, and muffin (incurred levels 4.8-48 μg g -1 ) with the developed ELISAs were in an acceptable range (50-130%). These ELISAs consistently detected more declared/undeclared egg proteins in market samples compared to assays using PBS for extraction. Overall, better assay performance was observed using BF-Abs. An ELISA combining anti-processed egg Abs, denaturing buffer, and incurred standards promises improved quantitation of egg proteins in processed foods., (Published by Elsevier B.V.)

A modified CUPRAC (cupric reducing antioxidant capacity) method was developed for the simultaneous estimation of protein oxidation and counteracting antioxidant defense, and the results were compared with those of a modified 2,4-dinitrophenylhydrazine (DNPH) carbonyl assay. The alkaline carbonyl method was cleared off interferences by solvent extraction using a cationic surfactant. Both solution and Nafion membrane sensor CUPRAC methods were used to measure the oxidative hazard in protein solutions. Bovine serum albumin, fetal bovine serum and egg white were used as protein probes, exposed to oxidation by Fe(II)-induced Fenton reaction in the absence and presence of selected antioxidants (ascorbic acid, cysteine, gallic acid, glutathione, and N-acetyl cysteine). Protein probes were initially unreactive toward the CUPRAC and DNPH reagents, but produced colored products upon Fenton oxidation which were bleached by antioxidants, enabling an indirect measurement of antioxidant activity (AOA) by difference. Spearman's rank test for antioxidants demonstrated that there was a strong correlation (+0.7 to +0.9) between the modified CUPRAC and carbonyl assays. There was also a strong correlation between the results of the solution phase and optical sensing CUPRAC methods (R 2  > 0.95). As opposed to conventional antioxidant assays not using biologically relevant probes, this work utilizes protein probes for AOA assessment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Guinea fowl eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. Both eggshells are composed of 95% calcite and 3.5% organic matrix, which control its structural organization. Chicken eggshell is made of columnar calcite crystals arranged vertically. In the Guinea fowl, the same structure is observed in its inner half, followed by a dramatic change in crystal size and orientation in the outer region. Guinea fowl eggshell is thicker than chicken eggshell. Both structure and shell thickness confer a superior resistance to breakage compared to eggshells of other bird species. To understand the underlying mechanisms controlling the structural organization of this highly resistant material, we used quantitative proteomics to analyze the protein composition of the Guinea fowl eggshell organic matrix at key stages of the biomineralization process. We identified 149 proteins, which were compared to other bird eggshell proteomes and analyzed their potential functions. Among the 149 proteins, 9 are unique to Guinea fowl, some are involved in the control of the calcite precipitation (Lysozyme, Ovocleidin-17-like, Ovocleidin-116 and Ovalbumin), 61 are only found in the zone of microstructure shift and 17 are more abundant in this zone. SIGNIFICANCE: The avian eggshell is a critical physical barrier to protect the contents of this autonomous reproductive enclosure from physical and microbial assault. The Guinea fowl (Numida meleagris) eggshell exhibits a unique microstructure (texture), which confers exceptional mechanical properties compared to eggshells of other species. In order to understand the mechanisms that regulate formation of this texture in the Guinea fowl eggshell, we performed comparative quantitative proteomics at key stages of shell mineralization and particularly during the dramatic shift in shell microstructure. We demonstrate that the Guinea fowl eggshell proteome comprises 149 proteins, of which 61 were specifically associated with the change in size and orientation of calcite crystals. Comparative proteomics analysis with eggshell of other bird species leads to new insights into the biomineralization process. Moreover, our data represents a list of organic compounds as potential additives to regulate material design for industrial fabrication of ceramics. This information also provides molecular markers for efficient genomic selection of chicken strains to lay eggs with improved shell mechanical properties for enhanced food safety., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ovalbumin-related protein X (OVAX) is a 50 kDa egg-white protein which has heparin-binding affinity. In this study, migration of OVAX and its heparin-binding performance during embryogenesis of fertilized egg were investigated to explore a possible involvement in chick embryo development. Western blotting of egg yolks at different stages using an anti-OVAX antibody showed that OVAX accumulates in yolk during incubation of fertilized egg. Immunohistochemical analysis of embryo resided in 10-day-incubated eggs showed that OVAX existed in almost all tissues of the embryo. These suggest that OVAX is incorporated from egg white into the embryo through yolk sac. Heparin-sepharose chromatography, isothermal titrating calorimetry using fondaparinux as a ligand, and zeta potential measurement indicated that OVAX retained the heparin-binding affinity (Kd = 0.185 ± 0.037) even after 10 D incubation of fertilized egg, although the affinity was slightly decreased during egg incubation because of acidification of molecular surface charge. In conclusion, although heparin-binding ability of egg-white OVAX slightly decreases during embryogenesis, OVAX incorporated into embryo can retain heparin-binding affinity. Our findings provide a new insight that OVAX participates in the functions of heparin during embryogenesis., (© 2019 Poultry Science Association Inc.)

Egg white protein has a high net protein utilisation, with a score of 100 in the amino acid rating system. Although the enzymatic breakdown of egg white yields hydrolysates that are rapidly absorbed and various physiological activities can be expected from them, flavouring egg white to meet taste requirements as a food has been a difficult challenge. Herein, we developed a high-molecular-weight egg white hydrolysate and compared the absorption rate and nutritional value of the hydrolysate with those of egg white proteins obtained from raw materials, whey proteins, and hydrolysates, also known as high-quality proteins. The absorption rate of egg white hydrolysates was faster than that of egg white and whey proteins in portal vein cannulated rats, and their bioavailability values were higher than those of whey proteins and hydrolysates. According to the protein digestibility-corrected amino acid score and digestible indispensable amino acid score, the scores for egg white hydrolysates were equivalent to those of egg white and whey proteins but higher than those of whey hydrolysates. Our results show that egg white hydrolysates maintain the nutritional value of egg whites and are rapidly absorbed by the body., Competing Interests: The authors declare that they have no conflicts of interest.

New protein ingredients are used to support pet food market growth and the development of new products while maintaining animal dietary needs. However, novel protein sources (e.g., spray-dried chicken, and (or) rice, pea, and potato protein concentrates) have limited data available regarding their protein quality. The objective of this study was to evaluate protein ingredients used in the pet food industry by laboratory analysis and a chick growth assay as a model. Following analysis for proximate and amino acid composition, chicks (six birds per pen with four pens per treatment) were fed experimental diets for 10 d. Diets contained 10% crude protein from each of the experimental protein sources (spray-dried egg-SDEG; spray-dried egg white-SDEW, spray-dried inedible whole egg-SDIE, chicken by-product meal-CBPM, chicken meal-CKML, low-temperature fluid bed air-dried chicken-LTCK, low-temperature and pressure fluid bed dried chicken-LTPC, spray-dried chicken-SDCK, whey protein concentrate-WPCT, corn gluten meal-CGML, corn protein concentrate-CPCT, potato protein isolate-PPIS, rice protein concentrate-RPCT, pea protein isolate-PEPI, soy protein isolate-SPIS, and soybean meal-SBML) along with an N-free diet (negative control). Chicks fed SDEG, SDIE, and LTPC had the highest protein efficiency ratio (PER; P < 0.0001; 5.18, 5.37, and 5.33, respectively), LTCK and SDCK were intermediate (4.54 and 4.79), and the CBPM and CKML were the lowest among the poultry proteins for EAA:NEAA, PER, and Lys availability. Among the vegetable proteins, PPIS and SBML had the highest PER values (3.60 and 3.48, P < 0.0001). In general, the chick PER method ranked the quality of animal protein sources higher than vegetable proteins, and these results were consistent with the EAA:NEAA ratio and Lys availability., (© The Author 2018. Published by Oxford University Press on behalf of Poultry Science Association.)

We present an implementation of the absolute quantification (AQUA) method for monitoring of peptide abundance in complex mixtures of processed proteins. Specific peptide markers from meats (chicken, duck, goose, pork and beef) and common protein allergenic additives (soy, milk and egg white preparations) were chosen and synthesised with stable isotopes ( 13 C and 15 N) for use as internal standards. A wide range of food samples, from cooked or raw meat to sterilised pâté, was analysed by a triggered multiple reaction monitoring mode experiment and triple quadrupole mass spectrometry for the direct measure of tryptic peptides representing the amounts of specific proteins. Considerable differences among the abundances of meat and non-meat proteins were observed, and illegal addition and replacement of ingredients were discovered, i.e. undeclared addition of pork and egg white proteins, and illegal substitution of veal, goose and duck meat with cheaper pork., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Food allergy is a growing health problem worldwide; thus, there is an urgent need for robust, specific, and sensitive analytical methods for detecting allergens. Mass spectrometry is an alternative to the existing methods, and it can overcome their limitations. One of the first steps in the development of any analytical method is the identification of the analytes to be further studied. In the case of allergen detection by mass spectrometry, the analytes are peptides. In this study, a strategy was developed for identifying potential peptide biomarkers in processed food products. This strategy was applied to processed egg matrices, and 16 potential peptide biomarkers were identified for the further detection and quantification of egg by means of mass spectrometry. With an empirical approach based on dedicated sample preparation, including tandem Lys-C/trypsin enzymatic digestion and high-resolution mass spectrometry analysis, hundreds of peptides from egg proteins were identified. This list of peptides was further refined with a series of criteria, obtained from empirical evidence, to identify the ideal biomarkers for the development of a quantitative method. These criteria include the resistance to food processing and the specificity of the peptides for eggs but also the effects of amino acid modifications and enzymatic digestion efficiency., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The glycan part of glycoproteins is known to be involved in the structure and modulatory functions of glycoproteins, serving as ligands for cell-to-cell interactions, and as specific ligands for cell-to-microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation exist. As an approach to better understand glycan diversity, egg whites (EW) from four different quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and analysis method. N-Glycans were classified and the profiles were established for quail egg white samples which showed 21 relevant glycan peaks; 18 peaks were expressed significantly, and 10 glycan peaks are found to be abundant in certain species. The result establishes glycan profiles for Blue Scaled, Bobwhite, Japanese, and Mountain Quail egg whites and shows a unique difference among glycan expressions, particularly, high mannose in Japanese Quail and tetra-antennary glycan structure for other quail species.

Maternal investment directly shapes early developmental conditions and therefore has long-term fitness consequences for the offspring. In oviparous species prenatal maternal investment is fixed at the time of laying. To ensure the best survival chances for most of their offspring, females must equip their eggs with the resources required to perform well under various circumstances, yet the actual mechanisms remain unknown. Here we describe the blue tit egg albumen and yolk proteomes and evaluate their potential to mediate maternal effects. We show that variation in egg composition (proteins, lipids, carotenoids) primarily depends on laying order and female age. Egg proteomic profiles are mainly driven by laying order, and investment in the egg proteome is functionally biased among eggs. Our results suggest that maternal effects on egg composition result from both passive and active (partly compensatory) mechanisms, and that variation in egg composition creates diverse biochemical environments for embryonic development., Competing Interests: During the study, one of the authors (R.M.S.) left the University of Hohenheim to take up a position at a manufacturer of carotenoid supplements (DSM Nutritional Products, Kaiseraugst, Switzerland). The remaining authors declare no competing interests.

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.

Chemical hydrolysis assisted by microwave irradiation has been proposed as an alternative method for the analysis of proteins in highly insoluble matrices. In this work, chemical hydrolysis was applied for the first time to detect degraded proteins from paintings and polychromies. To evaluate the performance of this approach, the number of identified peptides, protein sequence coverage (%), and PSMs were compared with those obtained using two trypsin-based proteomics procedures used for the analysis of samples from cultural heritage objects. It was found that chemical hydrolysis allowed the successful identification of all proteinaceous materials in all paint samples analyzed except for egg proteins in one extremely degraded sample. Moreover, in one sample, casein was only identified by chemical digestion. In general, chemical hydrolysis identified more peptides, more PSM's, and greater sequence coverage in the samples containing caseins, and often also in animal glue, highlighting the great potential of this approach for the rapid digestion and identification of insoluble and degraded proteins from the field of the cultural heritage.

Current analytical methods used for composition analysis of egg products are time consuming and laborious. We developed a near-infrared reflectance spectroscopy (NIRS)-based method to determine the fat, moisture, and protein contents in homogenized egg yolk and the moisture and protein contents in homogenized egg albumen to substitute for conventional methods. The coefficients of determination in the external validation set (R2P) were over 0.8 for all chemical compositions. The ratios of performance to standard deviation (RPD) were 4.38, 2.25, 2.28, 2.31, and 3.03 for fat, moisture, protein and moisture in the egg yolk, and protein in the egg albumen, respectively. Thus, NIR spectroscopy could be an efficient tool for quantitative analysis of the nutrients in chicken eggs.

Food allergy is a considerable heath problem, as undesirable contaminations by allergens during food production are still widespread and may be dangerous for human health. To protect the population, laboratories need to develop reliable analytical methods in order to detect allergens in various food products. Currently, a large majority of allergen-related food recalls concern bakery products. It is therefore essential to detect allergens in unprocessed and processed foodstuffs. In this study, we developed a method for detecting ten allergens in complex (chocolate, ice cream) and processed (cookie, sauce) foodstuffs, based on ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Using a single protocol and considering a signal-to-noise ratio higher than 10 for the most abundant multiple reaction monitoring (MRM) transition, we were able to detect target allergens at 0.5mg/kg for milk proteins, 2.5mg/kg for peanut, hazelnut, pistachio, and cashew proteins, 3mg/kg for egg proteins, and 5mg/kg for soy, almond, walnut, and pecan proteins. The ability of the method to detect 10 allergens with a single protocol in complex and incurred food products makes it an attractive alternative to the ELISA method for routine laboratories., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Polymeric monoliths fabricated in tips with embedded materials of choice are important in separation science. Polymeric backbone however interferes in the enrichment and thus affects efficiency. This work focuses on the in-tip fabrication of lanthanum oxide porous monolith and its application in the enrichment of phosphorylated peptides and lipids. Polycondensation reaction uses an aqueous solution of LaCl 3 ·7H 2 O with N-methyl formamide as porogen and propylene oxide as initiator. The aging time of monolith and temperature condition for the reaction are optimized to attain porous monolithic tip. A comparison of (i) solid phase batch extraction using La 2 O 3 , (ii) La 2 O 3 embedded in poly(glycidyl methacrylate (GMA)/divinylbenzene (DVB)) tip, and (iii) pure La 2 O 3 monolithic tip shows improved enrichment efficiency in the case of pure La 2 O 3 monolithic tip. The monolithic tip achieves selectivity of 1:4500 as compared to solid phase extraction (SPE)(1:3500) and limit of detection down to 0.25 fmol. The in-tip La 2 O 3 monolith strategy has better batch to batch reproducibility, reduced time of enrichment, and ease of operation in comparison to solid phase batch extraction. The developed strategy enriches phospho- content from biological samples like phosvitin and lipovitellin from egg yolk and phospholipids/phosphopeptides from human serum. The enriched phospho- moieties are analyzed by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) except the phospholipids where laser desorption ionization (LDI)-MS is employed.

1. The aim of this experiment was to compare the effects of dietary supplementation of hesperidin, naringin and quercetin on laying hen performance, egg quality and egg yolk lipid and protein profiles. 2. A total of 96 Lohmann White laying hens weighing an average of 1500 g at 28 weeks of age were randomly assigned to a basal diet and the basal diet supplemented (0.5 g/kg) with either hesperidin, naringin or quercetin. Each treatment was replicated in 6 cages in an 8-week experimental period. Data were analysed using one-way analysis of variance. 3. None of the dietary flavonoids affected laying performance and eggshell quality. Hesperidin and quercetin supplementations decreased albumen and yolk indexes. 4. As compared to the control group, egg yolk cholesterol content decreased and egg yolk protein content increased in response to dietary hesperidin and quercetin supplementation. The mean egg yolk cholesterol (mg/g) and protein (g/100 g) contents were 10.08/14.28, 16.12/14.08, 14.75/15.04 and 15.15/14.85 for the control group and groups supplemented with naringin, hesperidin and quercetin, respectively. 5. Egg yolk lipid and protein profiles were variable. 6. In conclusion, dietary supplementation of hesperidin or quercetin could be used in the diets during the early laying period to reduce egg yolk cholesterol and increase egg yolk protein, which may be attractive to consumers.

Guppy (Poecilia reticulata) is an ideal model for studying environmental estrogens, and its large caudal fin has a high capacity to regenerate. This study analyzed the feasibility of caudal fin for detecting vitellogenin (Vtg), the most commonly used biomarker of environmental estrogens. Firstly, a sandwich ELISA for guppy Vtg was developed using purified lipovitellin and its antibody and it had a working range of 7.8-1000 ng/mL and detection limit of 3.1 ng/mL. The ELISA was used to detect tissue distribution of Vtg. In male guppy exposed to 50 and 100 ng/L 17β-estradiol (E 2 ), Vtg concentration in caudal fin was higher than that in whole fish, brain, eyes, gonad, and skin, and was close to that in the liver. Furthermore, male guppies were exposed to environmental concentrations of 17a-ethinylestradiol (EE 2 ) and bisphenol S (BPS) to validate the utility of caudal fin Vtg for detecting estrogenic activities. The lowest observed effect concentration of EE 2 and BPS were lower than 2 ng/L and 1 μg/L, which were below or equal to the values reported for other species, demonstrating that caudal fin Vtg was highly sensitive to estrogenic chemicals. Therefore, caudal fins of guppies are suggested as alternative samples for Vtg biomarker detection.

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.)

In this research, partridge eggs were stored during zero to 7, 8 to 14, 15 to 21, 22 to 28, 29 to 35 and 36 to 42 d, respectively. The effect of different egg storage periods on egg protein and moisture rates, mineral contents, and some egg quality and hatching characteristics were investigated. The extension of egg storage times didn't affect the external quality features like shell weight, shell thickness, and albumen rate, but it affected the yolk weight, yolk index, albumen index, and Haugh unit (P < 0.05). Yolk weight was increased and yolk index, albumen index, and Haugh unit values were decreased with the extension of storage time. Different storage times of partridge eggs did not have important effects on the protein rates. Protein and humidity rate of the eggs were 14.21 and 67.64%, respectively. There were some elements such as S, P, Na, K, and Ca, but Al, Cd, Co, Mo, and Pb could not be found in the partridge eggs. There is no effect of the storage time on the mineral content of the egg. Storage time had negative effect on fertility and hatchability after 21 d of storage d, but no effect on hatchability of fertile eggs. As this study showed, partridge eggs are resistant to long storage times, as partridge egg proteins can resist degradation in the optimum storage times. To determine egg degradation, the study should be focused on partridge eggshell features, which are responsible for degradation. As a result of this research, partridge eggs can be stored for 21 d under optimum storage conditions without any negative hatchability results., (© 2017 Poultry Science Association Inc.)

The aim of this study was to analyse the effect of heat treatment parameters on liquid whole egg (LWE) proteins by using ultraviolet-visible (UV-VIS) spectroscopy and capillary electrophoresis (CE). Heat treatment (at 60-68°C for 1-5min) was applied to LWE. Treated LWE was centrifuged and supernatant was taken for measurement of UV-VIS spectroscopy and CE. The change in UV absorbance showed loss of protein solubility depending on heat treatments parameters. Electropherograms of samples demonstrated the effect of treatment parameters on composition of LWE proteins. It was found that conalbumin and lysozyme were influenced by the treatment, while ovalbumin and ovomucoid were not affected. CE combined with principal component analysis (PCA) was used for classification of samples untreated or treated and treated at different treatment parameters. The results of the study revealed that the extent of heat treatment in LWE samples could be determined with PCA of the CE measurements., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Egg white proteins play an important role in avian reproductive systems and are an ideal resource for bioreactor construction. In this study, 1D electrophoresis and MALDI-TOF-MS were performed to analyze egg white proteins in four species. In total, 18, 11, 28, and 13 proteins were identified in the egg whites of the chicken, duck, goose, and pigeon, respectively. Egg white proteins in chickens have been studied previously; therefore, we focused on the proteins in goose and duck egg whites. Based on the amino acid sequence analysis and a comparison of the unique peptides, high similarity was observed between the goose and duck egg whites. Diversity in the regulatory region of the ovalbumin gene explained the higher ovalbumin expression in the duck and goose than in the chicken. These data clarify the evolutionary processes underlying to the unique peptides contributing to the differential expression of ovalbumin in avians., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Unlabelled: In environmental science, omics-based approaches are widely used for the identification of gene products related to stress response. However, when dealing with non-model species, functional prediction of genes is challenging. Indeed, functional predictions are often obtained by sequence similarity searches and functional data from phylogenetically distant organisms, which can lead to inaccurate predictions due to quite different evolutionary scenarios. In oviparous females, vitellogenin production is vital for embryonic development, ensuring population viability. Its abnormal presence in fish male organisms is commonly employed as a biomarker of exposure to xenoestrogens, named endocrine disruptors. Here, in the freshwater amphipod Gammarus fossarum, we identified vitellogenin proteins by means of a proteome temporal dynamics analysis during oogenesis and embryogenesis. This exhaustive approach allows several functional molecular hypotheses in the oogenesis process to be drawn. Moreover, we revealed an unsuspected diversity of molecular players involved in yolk formation as eight proteins originating from different families of the large lipid transfer protein superfamily were identified as "true vitellogenins"., Biological Significance: In non-model species, next generation sequencing technologies development enables quickly deciphering gene and protein sequences but accuracy of associated functional prediction remains to be established. Here, in the crustacean Gammarus fossarum, a key sentinel species in freshwater biomonitoring, we identified key molecular players involved in the female reproduction by studying the proteome dynamics of ovaries and embryos. An unsuspected diversity of vitellogenin proteins was evidenced. These proteins being vital for offspring development, their high diversity may be advantageous for the organism's reproduction. Phylogenetic analysis showed that some forms are true vitellogenin orthologs while others are included in the apolipoprotein family, a paralogous group from the vitellogenin family. Among crustaceans, Gammarus fossarum is the first documented case where diverse protein families are involved in the yolk formation process., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Oviparous animals store yolk proteins within the developing oocyte. These proteins are used in gametogenesis and as a nutritional source for embryogenesis. Vitellogenin and the major yolk protein are two of the most important yolk proteins among diverse species of invertebrates and vertebrates. Among the echinoderms, members of the subphyla Echinozoa (sea urchins and sea cucumbers) express the major yolk protein (MYP) but not vitellogenin (Vtg), while an initial report has documented that two Asterozoa (sea stars) express a vitellogenin. Our results show that sea stars contain two vitellogenins, Vtg 1 and Vtg 2, and MYP. In Patiria miniata, these genes are differentially expressed in the somatic and germ cells of the ovary: Vtg 1 is enriched in the somatic cells of the ovary but not in the oocytes, and Vtg 2 accumulates in both oocytes and somatic cells; MYP is not robustly present in either. Remarkably, Vtg 2 and MYP mRNA reappear in larvae; Vtg 2 is detected within cells of the ectoderm, and MYP accumulates in the coelomic pouches, the intestine, and the posterior enterocoel (PE), the site of germ line formation in this animal. Additionally, the Vtg 2 protein is present in oocytes, follicle cells, and developing embryos, but becomes undetectable following gastrulation. These results help elucidate the mechanisms involved in yolk dynamics, and provide molecular information that allows for greater understanding of the evolution of these important gene products., (© 2016 Marine Biological Laboratory.)

Low-abundance samples and salt interference are always of great challenges for the practical protein profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of α-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and evaluated as matrices for MALDI-MS analysis of protein. Among them, (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) was found to exhibit excellent assay performance for intact proteins by improving the detection sensitivity 10 folds compared with the traditional matrices [i.e., super2,5-dihydroxybenzoic acid (superDHB), sinapic acid (SA), and CHCA]. In addition, CHCA-C3 was shown to have high tolerance to salts, the ion signal of myoglobin was readily detected even in the presence of urea (8 M), NH4HCO3 (2 M), and KH2PO4 (500 mM), meanwhile sample washability was robust. These achievements were mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in comparison with hydrophilic matrixes, leading to more efficient ionization of analyte. Furthermore, direct analysis of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low-abundance proteins in complex biological samples. These outstanding performances indicate the tremendous potential use of CHCA-C3 in protein profiling by MALDI-MS. Graphical Abstract ᅟ.

A deglycosylation step using Peptide-N-Glycosidase F (PNGaseF) has been introduced in a standard proteomic protocol to more confidently identify egg based binders. The ingenuity of introducing a PNGaseF digestion was aimed at removing the molecular hindrance, made up by the heavily glycosylated egg proteins, before the protease(s) hydrolysis. This novelty in the protocol resulted in obtaining a significant increase of proteolytic egg peptides thus improving the quality and reliability of egg identification in artwork samples. The protocol has been set up on paint replicas and successfully tested on two historical samples of different origin.