Dextran sulfate acting as a chaperone-like component on inhibition of amorphous aggregation and enhancing thermal stability of ovotransferrin.

The tendency of ovotransferrin (OVT) to unfold and aggregate under 60 °C severely restricted sterilization temperature during egg processing. Searching for efficient strategies to improve OVT thermal stability is essential for improving egg product quality and processing suitability. Here, we investigated the effect of sulfate polysaccharide (dextran sulfate, DS) on heat-induced aggregation of OVT. We found that DS can effectively suppress amorphous aggregation of OVT at pH 7.0 after heating. Strikingly, the addition of 5 µM DS fully suppressed insoluble aggregates formation of 0.5 mg/mL OVT. Structure analysis confirmed that DS preserves nearly the entire secondary and tertiary structure of OVT during heating. The steric hindrance effect arising from strong electrostatic interactions between OVT and DS, coupled with reduced OVT hydrophobicity, is the underlying mechanism in suppressing protein-protein interactions, thus enhancing thermal stability. These findings suggest DS could act as protein stabilizers and chaperones, enhancing the thermostability of heat-sensitive proteins.

Recent development of egg protein fractions and individual proteins as encapsulant materials for delivery of bioactives.

Egg proteins, as one of the most abundant animal protein sources, have received considerable attention for developing delivery systems. Among all egg proteins, egg white (ovalbumin) is the most promising encapsulant due to its excellent properties such as gelling, digestibility, self-assembly, amphiphilic nature. In this review paper, we focused particularly on egg protein-based delivery systems with superior encapsulation and delivery functions, including polymeric nanoparticles, emulsions, hydrogels and aerogels. Egg protein-based delivery systems across a wide range of geometry and dimensions have been applied to protect or control-release bioactive small molecules and macromolecules, probiotics and metal nanostructures. However, there are challenges that must be carefully addressed for advancing the practical applications of egg protein-based delivery system in foods, including allergenicity from ovalbumin and ovotransferrin, intolerance to environmental conditions, limited processing technologies. More efforts are warranted to fill knowledge gaps related to fabrication, utilization and digestive mechanisms of egg protein-derived delivery systems.

Effects of ultrasound-assisted glycosylation on the interface and foaming characteristics of ovotransferrin.

Ovotransferrin (OVT) is one of the major functional proteins in egg white protein. Most of the industry only paid attention the biological activity of OVT in iron supplement, antibacterial and other aspects, few reports were carried out on its processing characteristics such as foaming, interfacial behavior such as emulsification and foaming, which was an important processing functional attribute affecting its application scenario. In this study, the effects of ultrasound-assisted glycosylation on the interface and foaming characteristics of OVT were investigated. The results showed that proper ultrasonic treatment had a significant effect on the structure and physicochemical properties of OVT glycosylation products. When ultrasonic treatment lasted for 20 min, the grafting degree of OVT was 20.98%, the particle size decreased and the absolute value of potential increased. The foaming ability of OVT increased first and then decreased after ultrasonic-assisted glycosylation treatment. The foaming ability of OVT increased from 43.54% to 96.73% and the foaming stability increased from 68.92% to 89.19% after ultrasonic-assisted glycosylation treatment for 20 min. The experimental study effectively discovered the effect of ultrasound-assisted glycosylation on the foaming property of OVT, and would provide important technical references for expanding its application in food, biology, medicine and other fields.

Different fluorescence emitting copper nanoclusters protected by egg white and double-emission fluorescent probe for fast detection of ethanol.

Green emitting copper nanoclusters (G-Cu NCs), yellow emitting Cu NCs (Y-Cu NCs), orange emitting Cu NCs (O-Cu NCs) and red emitting Cu NCs (R-Cu NCs) were prepared using chicken egg white as the stabilizer by changing the reaction conditions. This is a green, facile and cheap method to explore different color emitting CuNCs by the same precursor and stabilizers. The G-Cu NCs were employed for the detection of ethanol due to their aggregation induced emission enhancement (AIEE) effect. The fluorescence emission of Cu NCs at 526 nm under the excitation of 444 nm can be effectively enhanced in the presence of ethanol due to AIEE effect, thus realizing the quantitative determination of ethanol content in the range 5-60%. In addition, a visual dual-emission fluorescence probe with the combination of G-Cu NCs and silicon nanoparticles (Si NPs/G-Cu NCs) was designed to evaluate ethanol content conveniently and rapidly. Desirable linear relationship is observed between ratio of fluorescence intensity (I525/I441) and ethanol content under the excitation of 383 nm. Visible color transformation of this probe is observed in the ethanol content range 2-20%. Moreover, the ethanol sensing platforms were applied to the detection and evaluation of the alcohol content of liquor, and the recoveries in liquor were in the range 99.7% to 113%, broadening the applications of Cu NCs and providing a sensitive detection method for ethanol.

Expanding the Cross-Link Coverage of a Carboxyl-Group Specific Chemical Cross-Linking Strategy for Structural Proteomics Applications.

Carboxyl-group specific chemical cross-linking is gaining an increased interest as a structural mass spectrometry/structural proteomics technique that is complementary to the more commonly used amine-specific chemistry using succinimide esters. One of these protocols uses a combination of dihydrazide linkers and the coupling reagent DMTMM [4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium] chloride, which allows performing the reaction at neutral pH. The reaction yields two types of products, carboxyl-carboxyl cross-links that incorporate the dihydrazide linker and zero-length carboxyl-amine cross-links induced by DMTMM alone. Until now, it has not been systematically investigated how the balance between the two products is affected by experimental conditions. Here, we studied the role of the ratios of the two reagents (using pimelic dihydrazide and DMTMM) and demonstrate that the concentration of the two reagents can be systematically adjusted to favor one reaction product over the other. Using a set of five model proteins, we observed that the number of identified cross-linked peptides could be more than doubled by a combination of three different reaction conditions. We also applied this strategy to the bovine 20S proteasome and the Escherichia coli 70S ribosome, again demonstrating complementarity and increased cross-link coverage.

Effect of ultrasound-irradiation combined pretreatment on the foamability of liquid egg white.

This study aims to optimize the ultrasound-irradiation combined pretreatment conditions to enhance the liquid egg white (LEW) foamability and investigate the changing mechanism about the physical and structural properties of LEW during the processing. Results indicated that the foamability of the LEW was increased by ultrasound-irradiation combined pretreatment to the highest value of 92.6% (irradiation dose = 33 kGy, ultrasound time = 6 min, and ultrasound power = 300 W). Three significant proteins in LEW (ovalbumin, ovotransferrin, and lysozyme) were chosen to explore the change law on their physical and structural properties. Results indicated that ultrasound-irradiation combined pretreatment increased the solubility and reduced the pH and particle size of ovalbumin and lysozyme, thus enhancing the foamability of LEW. Furthermore, the fluorescence spectra changes implied the un-folding and destruction of ovalbumin, ovotransferrin, and lysozyme during the ultrasound-irradiation combined pretreatment. Moreover, circular dichroism spectroscopy analysis revealed that the pretreatment decreased α-helix and ß-sheet of the ovalbumin, ovotransferrin, and lysozyme, which bring out the improvement of LEW foamability. The present study indicated that ultrasound-irradiation combined pretreatment had the potential to be implemented to enhance the foamability of LEW.

Hydrogels assembled from ovotransferrin fibrils and xanthan gum as dihydromyricetin delivery vehicles.

The present study aimed to assemble protein fibril-polysaccharide hydrogels as nutraceutical delivery vehicles. Turbidity titrations confirmed that complexations between ovotransferrin (OVT) fibrils and xanthan gum (XG) indeed occurred, and electrostatic interaction was the major driving force of OVT fibril-XG complexation. After optimization of the pH and acidifier, stable OVT fibril-XG hydrogels could be fabricated by adjusting the pH to 4.0 with glucono delta-lactone. To better understand the physicochemical properties of OVT fibril-XG gel, characterization of XG gel was also conducted. Scanning electron microscopy indicated that OVT fibril-XG gel had a denser network than XG gel. Rheological measurements revealed that OVT fibril-XG gel had higher gel strength and viscosity than XG gel. OVT fibril-XG gel and XG gel could be used as dihydromyricetin (DMY) delivery vehicles with a higher DMY loading (2 mg mL-1). DMY release was investigated using an in vitro gastrointestinal digestion model. All DMY was released from OVT fibril-XG gel after gastrointestinal digestion, and only 41.7% of DMY was released from XG gel after gastrointestinal digestion, indicating that OVT fibril-XG gel was more efficient in DMY delivery. DMY was released via a non-Fickian transport mechanism in both OVT fibril-XG gel and XG gel. The results of this study could provide new insight into the assembly of protein fibril-polysaccharide hydrogels and rational design of hydrogels as nutraceutical delivery vehicles.

Ovotransferrin nanofibril formation in the presence of glycerol or sorbitol.

Impact of glycerol and sorbitol on assembly of iron-bound ovotransferrin (OVT) into nanofibrils was investigated. Thioflavin T fluorescence result indicated that the presence of glycerol or sorbitol could reduce the rate of OVT fibrillation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that 60% sorbitol could retard hydrolysis of OVT completely for a period of time during thermal treatment, and decoupling hydrolysis from fibril self-assembly helped to identify the building blocks of OVT nanofibrils. OVT nanofibrils were composed of both intact OVT monomers and OVT-derived peptides. Influence of glycerol and sorbitol on morphology of OVT nanofibrils was studied using atomic force microscopy. The presence of glycerol or sorbitol shortened OVT nanofibrils, and the presence of 60% glycerol or sorbitol could increase thickness of OVT nanofibrils. Hopefully, this work may provide new insight about building blocks of protein nanofibrils and impact of polyols on protein fibrillation.

Iron Transport Tocopheryl Polyethylene Glycol Succinate in Animal Health and Diseases.

Gut health is the starting place for maintaining the overall health of an animal. Strategies to maintain gut health are, thus, an important part in achieving the goal of improving animal health. A new strategy to do this involves two molecules: the iron transport protein ovotransferrin (IT) and α-tocopheryl polyethylene glycol succinate (TPGS), which result in the novel formulation of ITPGS. These molecules help reduce gut pathogens, while enhancing the absorption and bioavailability of therapeutic drugs, phytomedicines, and nanomedicines. This, in turn, helps to maintain normal health in animals. Maintaining the gastrointestinal tract (GIT) in its normal condition is key for successful absorption and efficacy of any nutrient. A compromised GIT, due to an imbalance (dysbiosis) in the GIT microbiome, can lead to an impaired GI barrier system with impaired absorption and overall health of the animal. The molecules in ITPGS may address the issue of poor absorption by keeping the GI system healthy by maintaining the normal microbiome and improving the absorption of nutrients through multiple mechanisms involving antioxidative, anti-inflammatory, immunomodulatory, and antimicrobial activities. The ITPGS technology can allow the dose of active pharmaceutical or herbal medicine to be significantly reduced in order to attain equal or better efficacy. With complimentary actions between IT and TPGS, ITPGS presents a novel approach to increase the bioavailability of drugs, phytoconstituents, nutrients, and nanomedicines by enhanced transport to the tissues at the site of action, while reducing gut pathogen load. The ITPGS approach appears to be a novel strategy for maintaining the health of animals by manipulation of microbiota.

Novel ACE inhibitory tripeptides from ovotransferrin using bioinformatics and peptidomics approaches.

Food-derived ACE inhibitory peptides have recently attracted increased attention. This work focused on a more efficient in silico method to find ACE inhibitory peptides from ovotransferrin. In this work, ovotransferrin was digested into peptides by virtual enzymolysis. Subsequently, in vitro ACE inhibitory activity of potential tripeptides was conducted following the peptide score, toxicity, and water solubility prediction. Both pharmacophore study and flexible docking were applied to analyze ACE inhibition mechanism of tripeptides. Our results demonstrated that EWL was a potent ACE inhibitory tripeptide with IC50 value of 380 ± 10 µM. Besides, pharmacophore and flexible docking showed that the pi interaction and hydrogen bond were the key interactions in ACE-EWL complex. It appears that the in vitro ACE inhibitory activity of tripeptide EWL was consistent with its molecular modeling.

Ovotransferrin fibril-stabilized Pickering emulsions improve protection and bioaccessibility of curcumin.

The present study aimed to investigate protection and bioaccessibility of curcumin in ovotransferrin (OVT) fibril-stabilized Pickering emulsions. Curcumin protection of OVT fibril-stabilized emulsions against ultraviolet light exposure was studied. OVT fibril-stabilized Pickering emulsion at an ionic strength of 1000 mM provided the best curcumin protection. OVT fibril-stabilized Pickering emulsion at pH 6 provided better curcumin protection than those at pH 2 and 4. Afterwards, digestion of OVT fibril-stabilized curcumin emulsion was investigated in both TNO dynamic digestion model (TIM-1) and pH-stat static digestion model. In terms of TIM-1 result, curcumin bioaccessibility in OVT fibril-stabilized emulsion increased by 129% when compared with that in bulk oil. In pH-stat digestion model, curcumin bioaccessibility increased by 114% after formulated into OVT fibril-stabilized droplets, which was due to higher extent of lipolysis. Interestingly, both TIM-1 and pH-stat digestion models gave almost consistent measurements of improved percentage in curcumin bioaccessibility. Curcumin bioaccessibility of the emulsion in TIM-1 and pH-stat model was 15.3% and 33.8% respectively, indicating bioaccessibility overestimation in pH-stat model. The novel findings in this work could facilitate designing food-grade Pickerinng emulsion with excellent nutraceutical protection and enhanced nutraceutical bioaccessibility.

Modulation of Formation, Physicochemical Properties, and Digestion of Ovotransferrin Nanofibrils with Covalent or Non-Covalent Bound Gallic Acid.

The impact of covalent or non-covalent bound gallic acid (GA) on the formation, physicochemical properties, and digestion of ovotransferrin (OTF) nanofibrils was comprehensively studied. Thioflavin T fluorescence results revealed that bound GA could inhibit OTF nanofibrillation and that the fibril-inhibitory activity of bound GA was dose dependent. Covalent bound GA exerted stronger inhibition on OTF nanofibrillation than an equal amount of non-covalent bound GA. Atomic force microscopy revealed that covalent bound GA shortened OTF nanofibrils significantly, while non-covalent bound GA did not change the contour length of OTF fibrils remarkably. Bound GA altered diameter of OTF nanofibrils. Both covalent and non-covalent bound GA could alter the zeta potential, surface hydrophobicity, and rheological properties of OTF nanofibrils. Bound GA endowed OTF nanofibrils with a strong antioxidant activity. In vitro gastrointestinal digestion results showed that covalent bound GA elevated the fibril digestion rate better than non-covalent bound GA. Polyphenol binding provided a new approach to modulating the physicochemical properties of protein nanofibrils.

Curcumin-loaded Pickering emulsion stabilized by insoluble complexes involving ovotransferrin-gallic acid conjugates and carboxymethyldextran.

The present work aimed to fabricate antioxidant particle-stabilized Pickering emulsions with outstanding protection of encapsulated nutraceuticals. Antioxidant ovotransferrin-gallic acid conjugates (OTGCONJ) were prepared using the alkaline method, and the electrostatic assembly technique was utilized to construct OTGCONJ-CMD particles with OTGCONJ and carboxymethyldextran (CMD) as the building blocks. After the investigation of the particle size, insoluble nature and intermediate wettability of the OTGCONJ-CMD particles, the OTGCONJ-CMD particles were verified as eligible Pickering stabilizers. Visual observation showed that the stable OTGCONJ-CMD particle-stabilized Pickering emulsion consisted of the emulsified phase alone. Rheological analysis revealed that the Pickering emulsion had a high viscosity and a gel-like structure. In terms of the protective effect, the OTGCONJ-CMD particle-stabilized Pickering emulsion could significantly retard curcumin degradation under UV light. An in vitro digestion study revealed that the OTGCONJ-CMD particle-stabilized Pickering emulsion improved both the extent of lipolysis and curcumin bioaccessibility remarkably, suggesting that the OTGCONJ-CMD particle-stabilized Pickering emulsion was an excellent nutraceutical delivery vehicle. The novel findings in this work could have important implications for the design of nutraceutical-loaded Pickering emulsions with an excellent protective effect and nutraceutical delivery efficiency.

Noncovalently Associated Peptides Observed during Liquid Chromatography-Mass Spectrometry and Their Effect on Cross-Link Analyses.

Cross-linking mass spectrometry draws structural information from covalently linked peptide pairs. When these links do not match to previous structural models, they may indicate changes in protein conformation. Unfortunately, such links can also be the result of experimental error or artifacts. Here, we describe the observation of noncovalently associated peptides during liquid chromatography-mass spectrometry analysis, which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure. Noncovalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing coelution of the corresponding linear peptides in MS1 spectra, as well as the presence of the individual (intact) peptide fragments in MS2 spectra. To suppress noncovalent peptide formations, increasingly disruptive ionization settings can be used, such as in-source fragmentation.

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.

Formation and characterization of peptides in egg white during storage at ambient temperature.

Egg white thinning during ambient storage is a well-known phenomenon. The objective of the study was to characterize the formation of peptides <10 kDa in egg white during storage at room temperature. The results indicated that the content of peptides in the egg white fraction of <10 kDa increased gradually. Similar but a faster trend was observed for the fraction of <3 kDa. Gallin, also called ovodefensin (∼7 kDa), was the main component in 10-3 kDa egg white fraction, which rapidly degraded and disappeared at 28 d of storage. Mass spectrometry analysis of <3 kDa fraction identified 6 peptide fragments from ovotransferrin and 11 peptides from ovomucin. Ovodefensin, ovotransferrin and ovomucin are the major innate components of egg defense; thus the degradation of these proteins during storage contributes to egg white thinning and increased susceptibility to bacterial contamination. This study provides the insights on the molecular mechanism of egg deterioration during prolonged ambient storage.

Proteolytic pattern, protein breakdown and peptide production of ovomucin-depleted egg white processed with heat or pulsed electric fields at different pH.

Protein susceptibility to in vitro gastrointestinal digestion of ovomucin-depleted egg white (OdEW) adjusted to pH 4, 5, 7 and 9 and processed by heat (60 and 80 °C for 10 min) or pulsed electric fields (PEF) (1.4-1.8 kV/cm, 259-695 kJ/kg) was studied by assessing peptide production, proteolytic pattern, and the final peptide profile. Ovotransferrin was more susceptible to pepsin hydrolysis than lysozyme, with ovalbumin showing the highest proteolytic resistance. Ovalbumin was, however, hydrolyzed by pancreatin to produce a stable fragment. Heat treatment of OdEW solutions at 60 °C had little impact on protein susceptibility with the ovalbumin dimers formed having a comparable resistance to pepsinolysis as ovalbumin. Heating at 80 °C significantly enhanced protein susceptibility, as ovalbumin and protein aggregates formed were completely hydrolyzed within 30 min of pepsinolysis. Adjusting OdEW solution to pH 4 and treating with PEF at 695 kJ/kg enhanced protein susceptibility, similar to heat treatment at 80 °C, mainly owing to the enhanced enzymatic hydrolysis of ovalbumin. PEF processing can, therefore, increase protein digestion while minimizing protein aggregation, which will enhance protein functionality in egg whites.

Microscale isolation of native forms of lysozyme from chicken egg white by gel isoelectric focusing.

To separate and extract the native states of lysozyme from chicken egg white, a hybrid method for the mobilization of proteins after non-denaturing gel isoelectric focusing (IEF) combined with detection of lysozyme activity was developed. When the proteins in the chicken egg white were first separated using non-denaturing gel IEF, a lysozyme was obtained at the top of the gel column at the cathode end of the IEF. And, when the IEF-separated proteins of the egg white were mobilized by replacing the cathodic sodium hydroxide solution with phosphoric acid solution, an additional active state of the lysozyme that could be bound to proteins, such as ovotransferrin, was extracted from the solution. Furthermore, it was shown that the addition of lysozyme, obtained via IEF, to pure ovotransferrin generated a complex manifesting lysozyme activity, clearly indicating a successful reconstruction of the lysozyme-ovotransferrin complex in vitro. Therefore, the obtained results demonstrated that the native states of lysozymes, such as lysozyme and the lysozyme-ovotransferrin complex, can be effectively separated and extracted using non-denaturing gel IEF. Thus, this method can be applied to separate and extract different charge states of native proteins that retain their biological activities.

Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry.

In proteomics, dodecyl sulfate (DS-) as sodium salt is commonly used in protein solubilization prior to tryptic digestion, but the presence of the DS- hampers the electrospray ionization mass spectrometric (ESI-MS) analysis. The development of DS- depletion techniques is therefore important especially when dealing with small samples where there could be poor sensitivity due to sample loss or dilution during sample preparation. Here, we present a simple and fast electrokinetic removal method of DS- from small volumes of peptide and digested protein samples prior to ESI-MS. The selective removal was accomplished using an acidic extraction solution (ES) containing acetonitrile (ACN) inside a fused-silica capillary that was dipped into the sample. The use of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic movement of DS- monomers and micelles into the capillary. The high amount of ACN present at the tip of the capillary served to collapse the micelles migrating into the capillary, thereby releasing the peptides that were bound to these micelles, facilitating peptide retention in the sample and efficient DS- removal. Increased % MS signal intensity (SI) restoration of the peptide was observed, while DS- removal was unaffected when the amount of ACN in the ES was increased. This is because of the micelle to solvent stacking mechanism (effective electrophoretic mobility reversal) working at high concentration of ACN for the improved recovery of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin peptides were 75-83% while % MS SI reduction of DS- was up to 99% under optimal conditions, that is, 40% ACN in the ES. Higher % peptide recoveries from digested protein samples were obtained using the proposed method compared to the conventional cold acetone precipitation method.

Adsorption of Proteins on Colloidal Lignin Particles for Advanced Biomaterials.

Coating of colloidal lignin particles (CLPs), or lignin nanoparticles (LNPs), with proteins was evaluated in order to establish a safe, self-assembly mediated modification technique to tune their surface chemistry. Gelatin and poly- l-lysine formed the most pronounced protein corona on the CLP surface, as determined by dynamic light scattering (DLS) and zeta potential measurements. Spherical morphology of individual protein coated CLPs was confirmed by transmission electron (TEM) and atomic force (AFM) microscopy. A mechanistic adsorption study with several random coiled and globular model proteins was carried out using quartz crystal microbalance with dissipation monitoring (QCM-D). The three-dimensional (3D) protein fold structure and certain amino acid interactions were decisive for the protein adsorption on the lignin surface. The main driving forces for protein adsorption were electrostatic, hydrophobic, and van der Waals interactions, and hydrogen bonding. The relative contributions of these interactions were highly dependent on the ionic strength of the surrounding medium. Capillary electrophoresis (CE) and Fourier transform infrared spectroscopy (FTIR) provided further evidence of the adsorption-enhancing role of specific amino acid residues such as serine and proline. These results have high impact on the utilization of lignin as colloidal particles in biomedicine and biodegradable materials, as the protein corona enables tailoring of the CLP surface chemistry for intended applications.