Dry-heat-induced phosphoserine-specific fragmentation of ovalbumin.

When subjected to dry-heating, egg white ovalbumin, a phosphoglycoprotein, undergoes fragmentation and forms soluble aggregates. We investigated the mechanisms of dry-heat-induced fragmentation of ovalbumin. SDS-PAGE analysis showed that ovalbumin fragmented into five polypeptides, and their amount increased over 6 h of dry-heat treatment at 120 °C. The fragments contained fewer or no phosphoserine, compared with that in crude ovalbumin. Liquid chromatography-tandem mass spectrometry analysis of tryptic digests revealed that the fragmentation sites were located on phosphoserine residues, S68 and S344. During fragmentation, the phosphoserine residues underwent conversion into dehydroalanine residues, which were subsequently hydrolyzed. The nitrogen from the dehydroalanine became a newly formed terminal amide group on the N-terminal fragment, while the remaining molecule predominantly formed a new terminal pyruvoyl group. Furthermore, the fragments were incorporated into monomers or soluble aggregates of ovalbumin via covalent and non-covalent bonds. This study demonstrated a novel mechanism for dry-heat-induced fragmentation of phosphoproteins.

Transcription activator-like effector nuclease-mediated deletion safely eliminates the major egg allergen ovomucoid in chickens.

Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.

Lysozyme impacts gel properties of egg white protein via electrostatic interactions, polarity differences, local pH regulation, or as a filler.

The effects of lysozyme on egg white gel properties and their underlying causes were investigated under comparison between lysozyme removed with ion exchange resin and three levels of commercial lysozyme powder (1/2, 2/2, 3/2 the natural concentration in egg white) re-added in the lysozyme-removed system. Results showed that a lysozyme-removed gel obtained the best water holding capacity (61.61 %), lowest cooking loss (11.85 %), and enhanced textural properties (hardness, 638.04 g; resilience, 0.57; and gumminess), which was attributed to lysozyme promoting protein aggregation and weakening electrostatic repulsion by charge neutralization and competition for water, and this could be eliminated by removing lysozyme. Besides, the stronger intermolecular interactions (enhanced ionic bonds, hydrogen bonds and inhibited hydrophobic interactions), the shorter transverse relaxation time (T21 and T22), as well as more uniform microstructure formed in the lysozyme-removed gel, allowing the gels to bind more water molecules. With return of lysozyme, the gel properties were weakened to varying degrees, which was also ascribed to the filling of lysozyme in gel matrix narrowed interspace for binding and storage of water. In sum, adjustment on the content of lysozyme can regulate the gel properties of egg white, so as to obtain gels with regulable gel quality and processing characteristics.

A comparative study of unpasteurized and pasteurized frozen whole hen eggs using size-exclusion chromatography and small-angle X-ray scattering.

Hen eggs are rich in proteins and are an important source of protein for humans. Pasteurized frozen whole hen eggs are widely used in cooking and confectionery and can be stored for long periods. However, processed eggs differ from raw eggs in properties such as viscosity, foaming ability, and thermal aggregation. To develop pasteurized frozen whole egg products with properties similar to those of unpasteurized whole eggs, it is necessary to establish a method that can differentiate between the two egg types with respect to the structures of their proteins. In this study, size-exclusion chromatography (SEC) and SEC coupled with small-angle X-ray scattering (SEC-SAXS) were successfully used to differentiate between the proteins in unpasteurized and pasteurized frozen whole eggs. We found that proteins in the plasma fraction of egg yolk, especially apovitellenins I and II, formed large aggregates in the pasteurized eggs, indicating that their structures are sensitive to temperature changes during pasteurization, freezing, and thawing. The results suggest that SEC and SEC-SAXS can be used to differentiate between unpasteurized and pasteurized frozen whole eggs. Additionally, they may be useful in determining molecular sizes and shapes of multiple components in various complex biological systems such as whole eggs.

Thermal Processing of Liquid Egg Yolks Modulates Physio-Chemical Properties of Mayonnaise.

In this study, the effect of various heating temperatures (61−70 °C) and times (1−10 min) on physical and chemical properties of liquid egg yolk (LEY) and mayonnaise were investigated. Initially, we found that the increase of LEY protein denaturation was highly correlated with the increase of temperature and time, without causing either protein degradation or aggregation. In addition, the viscosity and particle size of LEY were significantly increased with greater heating temperature and time. Furthermore, the emulsification stability of mayonnaise prepared from thermally processed LEY were significantly better than that of the unheated control group, in particular, the emulsion stability of mayonnaise was higher at a temperature ranging from 62 °C to 68 °C, whereas the emulsion stability decreased above 69 °C. A rheological analysis showed that mayonnaise prepared from thermally processed LEY has higher shear stress when compared with the control group. Indeed, a sharp increase in the shear stress was observed when LEY was heated above 67 °C. Results from storage behavior analysis suggest that mayonnaise prepared from thermally processed LEY failed to affect the chemical qualities of mayonnaise, as evidenced by the fact that acid values and TBA values were not statistically significant with the unheated control group. Microscopic observation indicates that the number of complete oil droplets were significantly reduced at higher heating (70 °C/5 and 10 min) conditions. Finally, the sensory evaluation results suggest that mayonnaise prepared from thermally processed LEY does not influence the appearance, aroma, taste, greasy feeling, and overall acceptance of mayonnaise, as indicated by there being no significant differences between the experimental group and the control group (p > 0.05). We conclude from our study that a combination of heating conditions over 67 °C/5 min can allow the mayonnaise to retain better quality in terms of stability.

Characterization of the effect of dry-heat treatment on the gelation of alkaline dried egg whites using dynamic viscoelastic measurement and ultrasound spectroscopy.

In the present study, the mechanism for gelation of dried egg whites (DEWs) samples, which were differentially pretreated with dry-heat treatment and alkaline pH, were investigated using dynamic viscoelastic measurements and ultrasound spectroscopy for gels formed with at high protein concentrations. Rheological measurements showed that DEW gels with dry-heat treatments have a higher dynamic complex modulus than DEW gels without dry-heat treatments. Furthermore, ultrasonic attenuation analyses showed that subjecting DEWs to dry-heat treatment and alkaline pH induced the formation of an increased number of DEW protein "soluble aggregates" compared with unheated and neutral DEWs. Our data suggests that pretreatment of DEW samples led to partially unfolded DEW proteins with more "soluble aggregates", which were strongly correlated with the formation of gels with a more homogeneous and rigid texture. Understanding their structural properties at a molecular detail would enable desirable textural modifications for the development of new food products.

Effect of pH and Heat Treatment on the Antioxidant Activity of Egg White Protein-Derived Peptides after Simulated In-Vitro Gastrointestinal Digestion.

The study aimed to analyze pH and heat treatment's effect in modulating the release of peptides with antioxidant activity after simulated gastrointestinal (GI) digestion of Egg white powder (EWP). EWP samples with neutral (EWPN) and alkaline (EWPA) pH were heat-treated at 20, 60, and 90 °C and analyzed for protein aggregation, solubility, and GI digestibility. Heat treatment decreased solubility and induced protein aggregation, which was higher for EWPN as compared to EWPA. The unfolding of EWPA proteins at 60 °C exhibited a higher GI digestibility and antioxidant activity via Oxygen Radical Absorbance Capacity (ORAC) assay as compared to EWPN. Interestingly, a reverse trend was observed in the cellular antioxidant assay, and the GI-digest of EWPN exhibited a higher antioxidant activity. The LC-MS/MS analysis are in concordance with cellular antioxidant activity assay and showed a higher intensity for peptides with potential antioxidant activity in the GI-digest of EWPN. The results indicate that heat treatment but not the pH is a critical factor in improving the protein digestibility and releasing peptides with antioxidant activity after GI digestion.

Egg White Hydrolysate Retains the Nutritional Value of Proteins and Is Quickly Absorbed in Rats.

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.

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide.

Ovalbumin (OVA) is the most abundant protein in egg whites that is unnecessary in the egg yolk-based food industry. The development of OVA-based functional materials is of great interest. Collagen is a major component of the extracellular matrix. In this study, an OVA fragment, the OVA secretory signal peptide (OVA SP), was loaded in collagen gels, which were used as a cell scaffold for various types of cells. We examined the effect of OVA SP loaded in collagen gels to cell properties. The peptide was initially bound to the collagen fibers and then released from the gel. Our results indicate that the released OVA SP suppressed the integrin-mediated cell adhesion and focal adhesion formation. However, the adhesion of NIH3T3 cells was not suppressed by treatment with a chelating agent and an anti-ß1 antibody. Our results suggest that OVA SP nonspecifically interacts with cell surface proteins. The adhesion of various cell types on collagen gels were changed by the addition of OVA SP, depending on their integrin expression pattern. Additionally, the differentiation of MC3T3-E1 osteoblastic cells was promoted on the OVA SP-loaded collagen gels. This suggests that OVA SP may modulate both the differentiation and the adhesion of cells cultured on the collagen gels.

Coacervates and coaggregates: Liquid-liquid and liquid-solid phase transitions by native and unfolded protein complexes.

Coacervates are self-assemblies formed by oppositely charged macromolecules in aqueous solution. Although coacervates usually take a homogeneous spherical shape with flowability, they have the potential to adopt unexpected macroscopic structures. In this study, we investigated the influence of the interaction mode and morphology on unfolded proteins constituting coacervates and coaggregates using ovalbumin (OVA) and lysozyme (LYZ) as the model systems. The unfolded proteins were prepared via heating at 80 °C and then incubated at ambient temperature. OVA and LYZ formed complexes at pH values between their respective isoelectric points in both their native and unfolded states. The unfolded proteins were more prone to form complexes than the native proteins due to hydrophobic interactions rather than electrostatic attraction. Interestingly, native OVA and LYZ formed liquid-like coacervates with spherical shapes, whereas unfolded OVA and LYZ formed solid-like coaggregates with amorphous structures. Understanding the difference between coacervates and coaggregates will provide fundamental information regarding differences between the amorphous aggregation and liquid-liquid phase separation of proteins.

Observations using Phosphorus-31 nuclear magnetic resonance (31P-NMR) of structural changes in freeze-thawed hen egg yolk.

Hen egg yolk (EY) has a complicated structure consisting of lipids and proteins, and its structure is deeply related with its functional properties. 31P-NMR is an efficient technique to non-destructively detect the dynamic behaviour of phospholipids, the main component of bio-membranes. We determined conditions for measuring the 31P NMR spectra of EY and identified the components. 31P-NMR was used to detect phosvitin, inorganic phosphate, and lipoprotein as well as structural changes such as granule collapse and freeze-thaw denaturation as signal changes. Freeze-thaw denaturation generated a new denaturation peak. We separated aggregates of LDL from freeze-thawed plasma using centrifugation. TEM and 31P-NMR observations revealed that the denaturation peak corresponded to LDL aggregates. The 31P-NMR spectra suggested the formation of multiple forms of LDL aggregates in which the head groups of phospholipid molecules adopt a face-to-face orientation, similar to that observed following the flocculation of lipoproteins or in the lamellar-like structures of phospholipids.

Arginine prevents thermal aggregation of hen egg white proteins.

The control of aggregation and solubilization of hen egg white protein (HEWP) is an important issue for industrial applications of one of the most familiar food protein sources. Here, we investigated the effects of edible amino acids on heat-induced aggregation of HEWP. The addition of 0.6M arginine (Arg) completely suppressed the formation of insoluble aggregates of 1mgmL-1 HEWP following heat treatment, even at 90°C for 20min. In contrast, lysine (Lys), glycine (Gly), and sodium chloride (NaCl) did little to suppress the aggregation of HEWP under the same conditions. SDS-PAGE indicated that Arg suppresses the thermal aggregation of almost all types of HEWP at 1mgmL-1. However, Arg did not suppress the thermal aggregation of HEWP at concentrations ≥10mgmL-1 and prompted the formation of aggregates. Transmission electron micrographs revealed a high-density structure of unfolded proteins in the presence of Arg. These results indicate that Arg exerts a greater suppressive effect on a protein mixture, such as HEWP, than on a single model protein. These observations may propose Arg as a safe and reasonable additive to HEWP for the elimination of microorganisms by allowing an increase in sterilization temperature.

Influence of processing factors on the stability of model mayonnaise with whole egg during long-term storage.

Mayonnaise-like oil-in-water emulsions with different stabilities-evaluated from the degree of macroscopic defects, e.g., syneresis-were prepared by different formulations and processing conditions (egg yolk weight, homogenizer speed, and vegetable oil temperature). Emulsions prepared with lower egg yolk content were destabilized for shorter periods. The long-term stability of emulsions was weakly related to initial properties, e.g., oil droplet distribution and protein coverage at the interface. Protein aggregation between oil droplets was observed and would be responsible for the instability of emulsions exhibited by the appearance defects. SDS-PAGE results for adsorbed and unadsorbed proteins at the O/W interface suggested that predominant constituents adsorbed onto the interface were egg white proteins as compared with egg yolk components when the amount of added egg yolk was low. In present condition, egg white proteins adsorbed at the O/W interface could be a bridge of neighboring oil droplets thereby causing flocculation in emulsions.

Anodic Oxidative Modification of Egg White for Heat Treatment.

A new functionalization of egg white was achieved by an electrochemical reaction. The method involves electron transfer from thiol groups of egg white protein to form disulfide bonds. The oxidized egg white produced less hydrogen sulfide during heat treatment; with sufficient application of electricity, almost no hydrogen sulfide was produced. In addition, gels formed by heating electrochemically oxidized egg white exhibited unique properties, such as a lower gelation temperature and a softened texture, presumably due to protein aggregation and electrochemically mediated intramolecular disulfide bond formation.

Ovalbumin Delivery by Guanidine-Terminated Dendrimers Bearing an Amyloid-Promoting Peptide via Nanoparticle Formulation.

Development of protein delivery systems is important for biomedical applications such as immunotherapy. Ovalbumin (OVA) is a major component of egg whites, and is a possible cause of egg allergy. In this study, OVA was used as a model protein to develop a delivery system using guanidine-terminated dendrimers (Gdn-den) bearing an amyloid-promoting peptide derived from the helix B (hB) region of OVA (hB-Gdn-den). OVA nanoparticles (NPs) were prepared by heat treatment of OVA/hB-Gdn-den mixtures. The NP size and the surface charge were controlled by adjusting the ratio of hB-Gdn-den to OVA. The NPs were around 200 nm in diameter and stably dispersed, and their encapsulation efficiency for OVA was more than 80%. Although OVA NPs were also prepared using Gdn-den, the NPs aggregated readily. Complexation with hB-Gdn-den induced conformational changes in the OVA, and the hB peptide promoted digestion of OVA. These suggest that the hB peptide of the Gdn-den works as a possible anchor to OVA. The positively charged OVA NPs effectively associated with RAW264 cells. Thus, the amyloid-promoting Gdn-den, when mixed with OVA at a suitable molar ratio to form NPs, could act as a carrier for delivery of antigen proteins to immune cells.

Thermal aggregation of hen egg white proteins in the presence of salts.

Hen egg white contains more than 40 kinds of proteins with concentrations reaching 100 mg/mL. Highly concentrated protein mixtures are common in the food industry, but the effects of a crowded environment containing salts on protein stability and aggregation have only been investigated using pure protein solutions. Here, we investigated the thermal aggregation of hen egg white protein (EWP) at various concentrations in the presence of inorganic salts by solubility measurements and SDS-PAGE. EWP at 1 mg/mL formed aggregates with increasing temperature above 55 °C; the aggregation temperatures increased in the presence of inorganic salt with the Hofmeister series. Namely, the chaotrope 0.5 M NaSCN completely suppressed the thermal aggregation of 1 mg/mL EWP. As the protein concentration increased, NaSCN unexpectedly enhanced the protein aggregation; the aggregation temperature of 10 and 100 mg/mL EWP solutions were dramatically decreased at 62 and 47 °C, respectively. This decrease in aggregation temperatures due to the chaotrope was described by the excluded volume effect, based on a comparative experiment using Ficoll 70 as a neutral crowder. By contrast, the kosmotrope Na2SO4 did not affect the aggregation temperature at concentrations from 1 to 100 mg/mL EWPs. The unexpected fact that a chaotrope rather enhanced the protein aggregation at high concentration provides new insight into the aggregation phenomena with the Hofmeister effect as well as the crude state of highly concentrated proteins.

Role of the N-terminal amphiphilic region of ovalbumin during heat-induced aggregation and gelation.

Ovalbumin (OVA), a major globular protein in egg white, forms semiflexible fibrillar aggregates during heat-induced gelation. The N-terminal amphiphilic region (pN1-22) of OVA is removed after treatment with pepsin at pH 4 to leave a large OVA fragment (pOVA). The conformation and thermal stability of pOVA and OVA are similar, but the rheological strength of the heat-induced gel of pOVA is much lower compared to that of OVA. The aggregation rate of pOVA, which forms spherical aggregates, was lower than that of OVA. These results suggest that the N-terminal amphiphilic region of OVA facilitates the α-to-ß conformational transition, which triggers OVA fibril formation. Heat treatment of OVA in the presence of pN1-22 consistently resulted in the formation of straight fibrils. The strength of OVA and collagen gels was increased when prepared in the presence of pN1-22, suggesting that pN1-22 may be used to control the properties of protein gels.

Effect of various types of egg white on characteristics and gelation of fish myofibrillar proteins.

Three types of egg white protein (regular dried egg white [REW], special dried egg white [SEW], and liquid egg white [LEW]) were compared for their effect in surimi (CON), containing no egg white (EW). They were characterized for enzyme inhibition and time of EW addition, either with cryoprotectants prior to freezing or during gel preparation, using Pacific whiting surimi. In addition, the setting (suwari) effect and fish protein-EW protein interactions (dynamic rheological properties, total sulfhydryl groups, and fracture gel analysis) were evaluated using Alaska pollock surimi. After 12 mo frozen storage, adding 2% and 3% SEW to Pacific whiting surimi during chopping significantly (P < 0.05) increased the force and deformation values compared to adding the respective EW before freezing. The highest (P < 0.05) force (175.2 g) and deformation (9.0 mm) values after 12 mo were obtained when 3% SEW was added during chopping. Enzyme autolysis showed that addition of 2% EW protein was effective at inhibiting enzyme activity. During setting, adding 2% SEW maintained force (1047.4 g) and deformation (17.9 mm) values similar (P > 0.05) to CON (1055.1 g; 19.3 mm) and significantly (P < 0.05) better than 2% REW (666.1 g; 15.6 mm). Texture results corresponded well to other data where 2% SEW showed the lowest total sulfhydryl groups (48.3 mole/10(5) g), and higher elastic modulus (G'), which suggests improved protein interaction for gel formation. Overall, the addition of 2% to 3% SEW improved gel textural properties of Pacific whiting and Alaska pollock fish protein.