Comparative Studies of Yolkin Preparations Isolated from Egg Yolks of Selected Bird Species.

The results of our research have proven that yolkin preparations isolated from eggs of different bird species show a high similarity in polypeptide composition. Despite the small differences in protein patterns, all of yolkin preparations showed also strong immunomodulatory activity, comparable with yolkin obtained previously from hen egg yolk. It can therefore be deducted that the presence of this polypeptide complex in the egg is not accidental and performs an important biological function for developing embryo.

ß-Amyloid Peptide 1-42-Conjugated Magnetic Nanoparticles for the Isolation and Purification of Glycoproteins in Egg White.

Aß1-42-conjugated magnetic nanoparticles, Aß1-42@MNP, were prepared by covalently coupling Aß1-42 to hyperbranched polyethyleneimine (PEI)-modified magnetic nanoparticles via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). Aß1-42's high binding capacity to glycosyl groups facilitates Aß1-42@MNP composite to be a promising selective adsorbent for glycoproteins in egg whites. In our study, under conditions of pH 4.0, the adsorption efficiency of Aß1-42@MNP composite for ovalbumin (100 µg mL-1) was 98.4% and its maximum adsorption capacity was 344.8 mg g -1; under the condition of pH 4.0 and 200 mmol L-1 NaCl, its adsorption efficiencies for ovalbumin and ovotransferrin were 96.9% and 60.0%, respectively. According to these primary data, in practice, ovalbumin was removed from egg white by Aß1-42@MNP composite at pH 4.0 (step I), and then after adding NaCl until the final salt concentration reached 200 mmol L-1 (pretreated egg white), we utilized the same adsorbent to further isolate/purify glycoproteins (step II). SDS-PAGE results showed that Aß1-42@MNP composite could largely remove ovalbumin in step I and could isolate/purify the remaining ovalbumin and ovotransferrin in step II. LC-MS/MS analysis results showed that the removal of ovalbumin reduced its percentage in egg white samples from 32.93% to 11.05% in step I and the remaining ovalbumin and ovotransferrin were enriched in step II, where the final percentage reached 11.6% and 12.6%, respectively. In summary, 81 protein species were identified after two-step extraction with Aß1-42@MNP on egg white, while only 46 protein species were identified directly from raw egg white without any pretreatment. This work well illustrates the excellent adsorption performance of Aß1-42@MNP composite to glycoproteins and its potential in the application of proteomic studies on low-abundance proteins in egg white.

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

Recovery of Sea Star Egg Cell Surface Proteins Released at Fertilization.

To provide a better understanding of the composition of the egg cell membrane, we describe a method in which proteins and peptides that are either naturally released by the egg or cleaved by sperm proteases can be collected, analyzed, and identified. Such molecules are captured and isolated from the surrounding seawater via biotinylation, before being concentrated by an affinity interaction and subsequently analyzed by western blotting and mass spectrometry.

Simultaneous separation of the four major allergens of hen egg white.

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.

An easy and rapid separation method for five major proteins from egg white: Successive extraction and MALDI-TOF-MS identification.

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.

New methods for purification of Paralichthys olivaceus lipovitellin and immunoassay-based detection of vitellogenin.

Increasing levels of estrogenic pollution in marine environments has made the development of reliable biological detection techniques urgently needed. In this study, Japanese flounder (Paralichthys olivaceus) lipovitellin (Lv) was purified and used to establish three immunological methods for the detection of vitellogenin (Vtg), a biomarker for environmental estrogens. Firstly, five different methods were employed to purify Lv, among which water-precipitation was the fastest and easiest way to purify Lv. Japanese flounder Lv was characterized as a phospholipoglycoprotein with a molecular weight of ∼369 kDa. Using purified Lv and its specific polyclonal antibody, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This assay had a working range from 7.8 to 250 ng/mL and a detection limit of 3.1 ng/mL. Furthermore, we developed an immunohistochemistry (IHC) and an immunofluorescence (IF) assay, both of which allowed visual detection of liver Vtg. Finally, Vtg induction in plasma and liver of juvenile Japanese flounders exposed to 17ß-ethinylestradiol (EE2) was measured using these three methods. Exposure to 10 and 50 ng/L EE2 significantly increased plasma Vtg levels, and obvious positive fluorescence signals were observed near the liver sinusoidal vessels. These results confirmed that the methods developed effectively detected estrogenic activity of exogenous chemicals. Therefore, this study provides reliable methodologies for biomonitoring of estrogenic pollution in marine environments.

Isolation and identification of peptides from simulated gastrointestinal digestion of preserved egg white and their anti-inflammatory activity in TNF-α-induced Caco-2 cells.

Simulated gastrointestinal digestion of preserved egg white (SGD-PEW) exerts anti-inflammatory effects on Caco-2 cells and a mouse model of DSS-induced colitis. Here, we aimed to separate peptides derived from SGD-PEW and evaluate their anti-inflammatory effects using an in vitro inflammatory model. Six peptides were isolated and identified. DEDTQAMPFR (DR-10), DEDTQAMPF (DF-9), MLGATSL (ML-7) and MSYSAGF (MF-7) significantly inhibited IL-8 secretion and markedly decreased gene expression, including TNF-α, IL-8, IL-6, IL-1ß and IL-12 and promoted IL-10 gene expression in Caco-2 cells. DR-10, DF-9, ML-7 and MF-7 significantly inhibited the phosphorylation of JNK. In the meantime, DR-10 and DF-9 significantly reduced the phosphorylation of IκB and p38. These results indicated that ML-7 and MF-7 exerted their anti-inflammatory activity through the MAPK signaling pathway in TNF-α-induced Caco-2 cells. Whereas, DR-10 and DF-9 inhibited the NF-κB and MAPK signaling pathways. The results suggested that DR-10, DF-9, ML-7 and MF-7 derived from SGD-PEW may be a new type of prophylactic food for the treatment of inflammation.

Antibacterial activity of lysozyme-binding proteins from chicken egg white.

The purpose of this study was to establish a method for determining the bacteriolytic activity after separation of lysozyme-binding proteins from egg white. Lysozyme-binding proteins such as ovotransferrin and ovalbumin were separated by non-denaturing two-dimensional electrophoresis (2DE) and transferred to a membrane. The lysozyme activity of the separated and immobilized egg white proteins was assessed directly to produce a non-denaturing 3D map of the egg white proteins by incorporating an axis that combined each spot's lysozyme-activity with the non-denaturing 2DE pattern. Lysozyme-ovotransferrin and lysozyme-ovalbumin complexes could be reconstructed in vitro after the cathode end fraction containing lysozyme was added to purified ovotransferrin and ovalbumin, respectively. These complexes retained lysozyme activity even after separation by non-denaturing 2DE. Furthermore, when the lysozyme-ovotransferrin complex from egg white was extracted after separation by isoelectric focusing by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, the complex possessed bacteriolytic activity against both Bacillus subtilis and Escherichia coli. These methods can be applied to investigate protein complexes possessing bacteriolytic activity against a wide range of both Gram-positive and Gram-negative bacteria.

Egg proteins: fractionation, bioactive peptides and allergenicity.

Eggs are an important source of macro and micronutrients within the diet, comprised of proteins, lipids, vitamins, and minerals. They are constituted by a shell, the white (containing 110 g kg-1 proteins: ovalbumin, ovotransferrin, ovomucoid, lysozyme and ovomucin), and the yolk (containing 150-170 g kg-1 proteins: lipovitellins, phosvitin, livetins, and low-density lipoproteins). Owing to their nutritional value and biological characteristics, both the egg white and yolk proteins are extensively fractionated using different techniques (e.g., liquid chromatography, ultrafiltration, electrophoresis, and chemical precipitation), in which liquid chromatography is the most commonly used technique to obtain individual proteins with high protein recovery and purity to develop novel food products. However, concerns over allergenic responses induced by certain egg proteins (e.g., ovomucoid, ovalbumin, ovotransferrin, lysozyme, α-livetin, and lipoprotein YGP42) limit their widespread use. As such, processing technologies (e.g., thermal processing, enzymatic hydrolysis, and high-pressure treatment) are investigated to reduce the allergenicity by conformational changes. In addition, biological activities (e.g., antioxidant, antimicrobial, antihypertensive, and anticancer activities) associated with egg peptides have received more attention, in which enzyme hydrolysis is demonstrated as a promising way to break polypeptides sequences and produce bioactive peptides to provide nutritional and therapeutic benefits for human health. © 2018 Society of Chemical Industry.

Purification and identification of adipogenic-differentiating peptides from egg white hydrolysate.

Egg proteins are a good source of bioactive peptides. Previous work from our research group has demonstrated the potential of egg white hydrolysate (EWH) for enhancing adipogenic differentiation and insulin signalling in 3T3-F442A pre-adipocytes. EWH was prepared by a combination of thermolysin and pepsin. Here, in this study, we aimed to identify the responsible peptide(s) in EWH. EWH was fractionated stepwise by ultrafiltration, C18 Sep-Pack cartridge, cation-exchange chromatography, and reverse-phase chromatography. The two most active fractions were analyzed by LC-MS/MS and 42 peptides were identified. Eleven peptides were synthesized and WEKAFKDED, QAMPFRVTEQE, ERYPIL, and VFKGL from ovalbumin were validated with peroxisome proliferator-associated receptor gamma stimulatory activity in adipocytes. For the first time, adipogenic differentiating peptides were characterized from egg white proteins. This data is pivotal for future structure-function studies of adipogenic peptides.

Preparation of eggshell membrane protein hydrolysates and culled banana resistant starch-based emulsions and evaluation of their stability and behavior in simulated gastrointestinal fluids.

This study aims to evaluate the effect of resistant starch (RS) derived from culled banana on the stability and characteristics of emulsions stabilized by eggshell membrane (ESM) protein hydrolysates. It was observed that incorporation of resistant starch improved the properties of the emulsions. Furthermore, the most stable emulsions developed (using a combination of RS and pretreated alcalase hydrolysates) were assessed for their behavior in an in-vitro gastrointestinal model system and changes in their particle size, zeta potential and morphology were evaluated. It was seen that these emulsions underwent flocculation and coalescence in the presence of pepsin and at higher concentrations of mucin enzyme and further coalescence and fatty acid release were observed after their passage through the small intestine. These insightful results about emulsion behavior in the gastrointestinal tract can be useful for designing delivery systems for controlled release of bioactive compounds.

The effect of ultrasound on the alkali extraction of proteins from eggshell membranes.

BACKGROUND: Eggshell contains two layers formed by a dense network of fibrous proteins. These proteins are highly insoluble in a broad variety of solvents, but their composition makes them suitable for a broad range of applications. In this study, in order to extract and solubilise these proteins, the eggshell membranes were treated in an alkali solution. A Box-Behnken design was employed to determine the influence of the treatment variables on the amount of protein solubilised. Furthermore, the effect of ultrasound on the protein recovery yield was also evaluated and compared with the unmodified process. RESULTS: A solubilised protein yield close to 100% of the total eggshell membrane protein was obtained. The optimal conditions could be set at 70 °C in a 1.0 mol L-1 NaOH solution for 60 min. However, when ultrasound was applied, it was possible to decrease the time of reaction by half. In the two processes, the temperature was found to be the most important independent variable evaluated. Finally, the antioxidant properties of the proteins obtained in each case were similar. CONCLUSIONS: Ultrasound favours the detachment of big clumps of proteins from the eggshell membrane, facilitating the solubilisation of its compounds. The ultrasound had no effect on the protein properties tested in this study. © 2017 Society of Chemical Industry.

Separation of proteins from complex bio-matrix samples using a double-functionalized polymer monolithic column.

A double-functionalized polymer monolithic column was fabricated within the confines of a stainless-steel column (50 mm × 4.6 mm i.d.) via a facile method using iron porphyrin, ionic liquid (1-allyl-3-methylimidazolium chloride) and 1,10-decanediol dimethacrylate as tri-monomers; ethylene dimethacrylate as a crosslinker; polyethylene glycol 400 and N,N-dimethylformamide as co-porogens; benzoyl peroxide and N,N-dimethyl aniline as the redox initiation system. Results obtained from scanning electron microscopy, nitrogen adsorption-desorption, and mercury intrusion porosimetry confirmed the uniform pore structure and the pore size distribution of macro-pores. The home-made monolith was further characterized by elemental analysis to investigate the elemental composition of Fe supplied by iron porphyrin, confirming the synthetic process. The resulting optimized monolithic column was used as the stationary phase in high performance liquid chromatography for separating proteins, such as mixture of standard proteins, egg white, and human plasma, exhibiting good selectivity and high performance. It is worth noting that the home-made double-functionalized polymer monolithic column shows excellent selectivity for fractionation separation of human plasma proteins, and it is a promising separation tool for complex bio-samples in proteomic research.

Effect of Freezing, Thermal Pasteurization, and Hydrostatic Pressure on Fractionation and Folate Recovery in Egg Yolk.

In this study, the impact of pasteurization and freezing of raw material, as performed at a commercial scale, on egg yolk fractionation and folate recovery was assessed. Freezing induced denaturation of the lipoproteins in egg yolk, which prevented further fractionation of the yolk. Thermal pasteurization of egg yolk at 61.1 °C for 3.5 min as well as high hydrostatic pressure (HHP) treatment (400 MPa for 5 min) did not change (p < 0.05) the composition of egg yolk or yolk fractions after their recovery by centrifugation. Expressed as dry matter, folate in pasteurized yolk was measured to be 599 µg/100 g, while its concentration reached 1969.7 µg/100 g for pasteurized granule and 1902.5 µg/100 g for HHP-treated granule. Folate was not detected in plasma, emphasizing the complete separation of yolk folate into granule. Further, we studied the effect of HHP on different dilutions of egg yolk, which were then fractionated. Egg yolk was diluted with water at different concentrations (0.1, 1.0, 10, 25, and 50%), HHP-treated at 400 MPa for 5 min, and centrifuged. Characterization of the compositions of the separated granule and plasma followed. Folate was stable under the HHP conditions used. However, HHP caused separation of folate from the yolk structure into water-soluble plasma. After HHP processing, the amount of folate detected in the plasma fraction was significantly (p < 0.05) higher (1434.9 µg/100 g) in the 25% diluted samples but was significantly (p < 0.05) lower in HHP-treated granule samples. Native sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that phosvitin, α-livetin, and apovitellenin VIa were the proteins most resistant to HHP. This study confirms that dilution of egg yolk before HHP treatment can significantly (p < 0.05) change the composition of granule and plasma fractions after centrifugal fractionation of egg yolk.

Partial biochemical characterization of Cu,Zn-superoxide dismutase extracted from eggs of hens (Gallus gallus domesticus).

Biochemical characteristics of Cu,Zn-SOD derived from hen egg white and egg yolk were determined, and compared with those of enzymes from erythrocytes of hens and SOD standard. The presence of dimer with a molecular weight of 33.38±0.34kDa, and pI of 6.30±0.15 was confirmed in samples of SOD extracted from egg yolk. Cu,Zn-SOD isolated from egg yolk had an optimum at pH 6. Average SOD activity in egg yolk was 98.5±19.5U·g-1 while in egg white reached 6.1±0.8U·g-1. Changes in SOD activity of the egg yolk during its storage for 200days were also described. FTIR analysis confirmed that the enzymatic protein described in this study was SOD, while MALDI-TOF analysis confirmed only SOD from erythrocytes. Since eggs are a cheap and easily obtainable source of SOD, this enzymatic protein could be used in food, cosmetic or pharmaceutical industries.

Preparation of a novel Zr(4+)-immobilized metal affinity membrane for selective adsorption of phosphoprotein.

In this study, a novel phosphate-Zr(4+) immobilized metal affinity membrane (IMAM) was prepared based on the surface initiated-atom transfer radical polymerization technique for the selective adsorption of phosphoprotein. The adsorption capacity and selectivity of the phosphate-Zr(4+) IMAM were evaluated by using the mixture of standard phosphoproteins (ß-casein, ovalbumin) and nonphosphoproteins (bovine serum albumin and lysozyme) as model samples. The adsorption isotherms and competitive adsorption results demonstrated that the phosphate-Zr(4+) IMAM had higher binding capacity and selectivity for phosphoproteins over nonphosphoproteins. Moreover, the phosphate-Zr(4+) IMAM exhibited good re-usability and re-productivity. Finally, the phosphate-Zr(4+) IMAM was applied to separate phosphoprotein from real samples with high purity. Therefore, the as-prepared phosphate-Zr(4+) IMAM could be a promising affinity material for the efficient enrichment of phosphoprotein from complex bio-samples.

Lipovitellin as an antigen to improve the precision of sandwich ELISA for quantifying zebrafish (Danio rerio) vitellogenin.

Vitellogenin (Vtg) in zebrafish (Danio rerio) is a core biomarker for screening environmental estrogens in test guidelines of the Organization for Economic Cooperation and Development. To accurately quantify zebrafish Vtg, lipovitellin (Lv), the main Vtg-derived yolk protein, was used as the antigen to establish a sandwich enzyme-linked immunosorbent assay (ELISA). The purified Lv was a phospholipoglycoprotein with apparent molecular weight of ~445kDa, and separated into three polypeptides corresponding to ~117, ~102, and ~23.8kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunological analysis confirmed the specificity of the anti-Lv antibody for Vtg and the immunological similarity between Vtg and Lv. Using the purified Lv and anti-Lv antibody, a sandwich ELISA with a detection limit of 4.3ng/mL and a detection range from 7.8 to 250ng/mL was developed. The intra- and inter-assay coefficients of variation were both below 10%. Moreover, the Lv standard curve was nearly identical to the Vtg standard curve, and paralleled serial whole-body homogenate dilutions of male zebrafish exposed to 17ß-estradiol, demonstrating that the Lv-based ELISA could be used for quantification of zebrafish Vtg. Zebrafish Lv showed high stability during purification process, heat treatment, -80°C storage, and repeated freeze/thaw cycles. Additionally, the standard curve of Lv stored at -80°C for 3months exhibited higher robustness than that of Vtg stored under the same conditions. Finally, the usefulness of the ELISA for detecting estrogenic activity was verified by quantifying Vtg inductions in zebrafish exposed to monocrotophos.

Lysozyme fractionation from egg white at pilot scale by means of tangential flow membrane adsorbers: Investigation of the flow conditions.

The application of membrane adsorbers instead of classical packed bed columns for protein fractionation is still a growing field. In the case of egg white protein fractionation, the application of classical chromatography is additionally limited due to its high viscosity that impairs filtration. By using tangential flow membrane adsorbers as stationary phase this limiting factor can be left out, as they can be loaded with particle containing substrates. The flow conditions existing in tangential flow membrane adsorbers are not fully understood yet. Thus, the aim of the present study was to gain a deeper understanding of the transport mechanisms in tangential flow membrane adsorbers. It was found that loading in recirculation mode instead of single pass mode increased the binding capacity (0.39 vs. 0.52 mg cm(-2)). Further, it was shown that either higher flow rates (0.39 mg cm(-2) vs. 0.57 mg cm(-2) at 1 CV min(-1) or 20 CV min(-1), respectively) or higher amounts of the target protein in the feed (0.24 mg cm(-2) vs. 0.85 mg cm(-2) for 2.5 or 39.0 g lysozyme, respectively) led to more protein binding. These results show that, in contrast to radial flow or flat sheet membrane adsorbers, the transport in tangential flow membrane adsorbers is not purely based on convection, but on a mix of convection and diffusion. Additionally, investigations concerning the influence of fouling formation were performed that can lead to transport limitations. It was found that this impact is neglectable. It can be concluded that the usage of tangential flow membrane adsorbers is very recommendable for egg white protein fractionations, although the transport is partly diffusion-limited.

Recent Research in Antihypertensive Activity of Food Protein-derived Hydrolyzates and Peptides.

Year to year obesity prevalence, reduced physical activities, bad habits/or stressful lifestyle, and other environmental and physiological impacts lead to increase in diseases such as coronary heart disease, stroke, cancer, diabetes, and hypertension worldwide. Hypertension is considered as one of the most common serious chronic diseases; however, discovery of medications with high efficacy and without side effects for treatment of patients remains a challenge for scientists. Recent trends in functional foods have evidenced that food bioactive proteins play a major role in the concepts of illness and curing; therefore, nutritionists, biomedical scientists, and food scientists are working together to develop improved systems for the discovery of peptides with increased potency and therapeutic benefits. This review presents a recent research carried out to date for the purpose of isolation and identification of bioactive hydrolyzates and peptides with angiotensin I converting enzyme inhibitory activity and antihypertensive effect from animal, marine, microbial, and plant food proteins. Effects of food processing and hydrolyzation conditions as well as some other impacts on formation, activity, and stability of these hydrolyzates and peptides are also presented.