Ultrasonic pretreatment enhanced the glycation of ovotransferrin and improved its antibacterial activity.

This study aims to evaluate the effect of ultrasonic pretreatment combined with glycation on the structural characteristics and antibacterial activity of ovotransferrin (OVT). Firstly, OVT (purity >90%) was isolated from egg white with a simple and efficient method. After the treatment of ultrasound and glycation, the browning degree of OVT increased with the rising power of ultrasound, while the number of free amino groups obviously decreased to 25.4%. Various spectrum detection showed that the structures of OVT have changed significantly, indicating the tertiary structure became more flexible and looser. The minimal inhibitory concentration of ultrasound glycated OVT were 25.0 and 32.1 µmol/L for E. coli and S. aureus, respectively. In summary, ultrasound-assisted glycation is an effective technique to improve the biological activity of OVT.

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.

Isotope-coded protein label based quantitative proteomic analysis reveals significant up-regulation of apolipoprotein A1 and ovotransferrin in the myopic chick vitreous.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching -10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing -10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

Optimization of Extraction Parameters for Enhanced Production of Ovotransferrin from Egg White for Antimicrobial Applications.

Ovotransferrin is the second most abundant protein (~12-13% of the total egg protein) in egg white after ovalbumin. Ovotransferrin is a potent natural antimicrobial agent as it possesses antibacterial, antifungal, and antiviral properties and is also the major metal binding protein found in egg, which makes it an industrially important protein. Ovotransferrin was extracted from egg white using its metal (iron) binding properties. In the present study, eggs from two different sources were used (fresh local eggs from domestic household source and poultry eggs from shops) to compare the results and Response Surface Methodology was used for the experiment design and data analysis. The following extraction conditions were optimized so as to maximize the yield of ovotransferrin from egg white: ethanol % (v/v) and pH and volume (mL) of 25 mM FeCl3/50 mL of egg white. A maximum yield of ~85 ± 2.5% was obtained near the optimum extraction conditions. The yield was calculated based on the theoretical value (934 mg) of ovotransferrin in 100 mL of 1.5x diluted egg white solution. Our results suggest that efficient downstream processing may reduce the cost of overall production process of this promising enzyme, making it a natural and cost-effective alternative to the existing chemically synthesized antimicrobial agents.

Lactoferrin and ovotransferrin contribute toward antioxidative effects of Edible Bird's Nest against hydrogen peroxide-induced oxidative stress in human SH-SY5Y cells.

There are reports of improved redox outcomes due to consumption of Edible Bird's Nest (EBN). Many of the functional effects of EBN can be linked to its high amounts of antioxidants. Interestingly, dietary components with high antioxidants have shown promise in the prevention of aging and its related diseases like Alzheimer's disease. In this study, the antioxidative potentials of EBN and its constituents, lactoferrin (LF) and ovotransferrin (OVF), were determined and protective effects against hydrogen peroxide (H2O2)- induced toxicity on SH-SY5Y cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and acridine orange and propidium iodide (AO/PI) staining with microscopy were examined. Results showed that EBN and its constituents attenuated H2O2-induced cytotoxicity, and decreased radical oxygen species (ROS) through increased scavenging activity. Furthermore, LF, OVF, and EBN produced transcriptional changes in antioxidant related genes that tended towards neuroprotection as compared to H2O2-treated group. Overall, the results suggest that LF and OVF may produce synergistic or all-or-none antioxidative effects in EBN.

Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white.

Ovalbumin, ovotransferrin, ovomucin, and lysozyme are a few of the egg white proteins that can be used as functional components. The objective of this study was to develop a simple, sequential separation method for multiple proteins from egg white. Separated proteins are targeted for human use, and thus any toxic compounds were excluded. The methods for individual components and the sequential separation were practiced in laboratory scale first, and then tested for scale-up. Lysozyme was separated first using FPC3500 cation exchange resin and then ovomucin using isoelectric precipitation. Ovalbumin and ovotransferrin were separated from the lysozyme- and ovomucin-free egg white by precipitating ovotransferrin first using 5.0% (wt/vol) (NH4)2SO4 and 2.5% (wt/vol) citric acid combination. After centrifugation, the supernatant (S1) was used for ovalbumin separation and the precipitant was dissolved in water, and reprecipitated using 2.0% ammonium sulfate (wt/vol) and 1.5% citric acid (wt/vol) combination. The precipitant was used as ovotransferrin fraction, and the supernatant (S2) was pooled with the first supernatant (S1), desalted using ultrafiltration, and then heat-treated to remove impurities. The yield of ovomucin and ovalbumen was >98% and that of ovotransferrin and lysozyme was >82% for both laboratory and scale-up preparations. The SDS-PAGE and western blotting of the separated proteins, except for ovomucin, showed >90% purity. The ELISA results indicated that the activities of separated ovalbumin, ovotransferrin, and lysozyme were >96%. The protocol separated 4 major proteins in sequence, and the method was simple and easily scaled up.

Separation of ovotransferrin and ovomucoid from chicken egg white.

Ovotransferrin and ovomucoid were separated using 2 methods after extracting the ovotransferrin- and ovomucoid-containing fraction from egg white. Diluted egg white (2×) was added to Fe(3+) and treated with 43% ethanol (final concentration). After centrifugation, the supernatant was collected and treated with either a high-level ethanol (61% final concentration) or an acidic salt combination (2.5% ammonium sulfate and 2.5% citric acid) to separate ovotransferrin and ovomucoid. For the high-level of ethanol method, ovotransferrin was precipitated using 61% ethanol. After centrifugation, the precipitant was dissolved in 9 vol. of distilled water and the residual ethanol in the solution was removed using ultrafiltration. The supernatant, mainly containing ovomucoid, was diluted with 4 vol. of water, had ethanol removed, and was then concentrated and used as the ovomucoid fraction. For the acidic salt precipitation method, the ethanol in the supernatant was removed first. The ethanol-free solution was then concentrated and treated with a 2.5% ammonium sulfate and 2.5% citric acid combination. After centrifugation, the precipitant was used as the ovotransferrin and the supernatant as the ovomucoid fraction. The ovomucoid fraction from both of the protocols was further purified by heating at 65°C for 20 min and the impurities were removed by centrifugation. The yields of ovomucoid and ovotransferrin were >96 and >92%, respectively. The purity of ovomucoid was >89% and that of the ovotransferrin was >88%. The ELISA results confirmed that the activity of the separated ovotransferrin was >95%. Both of the protocols separated ovotransferrin and ovomucoid effectively and the methods were simple, fast, and easy to scale up.

Photopolymerized sol-gel monoliths for separations of glycosylated proteins and peptides in microfluidic chips.

Photopolymerized silica sol-gel monoliths, functionalized with boronic acid ligands, have been developed for protein and peptide separations in polydimethylsiloxane microfluidic devices. Pore size characterization of the monoliths was carried out with SEM, image analysis, and differential scanning calorimetry to evaluate both the micron-sized macropores and the nanometer-sized mesopores. Monoliths were functionalized with boronic acid using three different immobilization techniques. Batch experiments were conducted to determine the capacity of the monoliths and selectivity toward cis-diol-containing compounds. Conalbumin was used as a model glycoprotein, and a tryptic digest of the glycoprotein horseradish peroxidase was used as a peptide mixture to demonstrate proof-of-concept extraction of glycoproteins and glycopeptides by the monoliths formulated in polydimethylsiloxane microfluidic chips. For proteins, fluorescence detection was used, whereas the peptide separations employed off-line analysis using MALDI-MS.

Separation of ovotransferrin from chicken egg white without using organic solvents.

Ovotransferrin is one of the major egg white proteins that have antimicrobial activity as well as iron binding capability. The objective of this study was to develop a simple and easy method to separate ovotransferrin without using organic solvents. Egg white was separated from yolk, added in a 1:1 ratio to distilled water (DW), and then homogenized. The ovomucin in the diluted egg white was removed by centrifugation, adjusting the pH to 4.5 to 5.0. The resulting supernatant was added to different ratios of ammonium sulfate and citric acid, and then centrifuged after holding overnight at 4°C. The precipitant, which contains ovotransferrin, was dissolved in DW, and ovotransferrin was precipitated using different ratios of ammonium sulfate and citric acid. The precipitant collected after centrifugation was dissolved with DW and subjected to ultrafiltration to remove salts and concentrate the solution. The purity of the ovotransferrin was determined using SDS-PAGE, the protein identified using Western blot, and the estimated yield calculated by weighing the ovotransferrin after freeze drying. Over 85% purity and over 83% yield were obtained from the combinations of 5.0% (wt/vol) ammonium sulfate and 2.5% (wt/vol) citric acid followed by 2.0% (wt/vol) ammonium sulfate and 1.5% (wt/vol) citric acid. Activity of the ovotransferrin showed similar activity with previously separated ovotransferrin. However, this method is simpler and more cost effective than the previous method. The isolated ovotransferrin can be used as is or after modifications for various applications such as antimicrobial treatments, anticancer treatments, and iron-supplementing agents for humans.

Purification and characterization of ovotransferrin from Crocodylus siamensis.

Ovotransferrin (OTf) is the major glycoprotein in reptile egg whites. However, knowledge concerning its functional and biological properties remains limited. In this study, OTf from Crocodylus siamensis was purified and characterized. The proteins were precipitated with 80 % ammonium sulfate and then purified by anion exchange chromatography followed by hydrophobic interaction chromatography. The purified crocodile ovotransferrin (cOTf) had a molecular weight of 79 kDa. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) indicated multiple isoforms of cOTf, which had isoelectric points ranging from 6.0 to 6.8. cOTf was N-linked glycosylated protein identified by using PNGase F deglycosylation technique. Optimal autoproteolysis of cOTf occurred under acidic conditions and pH values more than 5, which differs from that of OTf.

Microfluidic free-flow electrophoresis chips with an integrated fluorescent sensor layer for real time pH imaging in isoelectric focusing.

Functional microfluidic free-flow electrophoresis chips with integrated fluorescent pH sensors are presented. Polyethylene glycol based structures were fabricated that allowed for integration of both functions on a single microchip. Microchips were applied in free-flow isoelectric focusing of model compounds and proteins with on-line monitoring of pH during microscale electrophoretic separation.

Zirconia nanoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins.

A ZrO(2) nanoparticles (ZrO(2)NPs)-coated column was prepared through a sol-gel process using zirconium(iv) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO(2)NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5-6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO(2)NPs. The α- and ß-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.

Affinity purification of egg-white allergens for improved component-resolved diagnostics.

BACKGROUND: Egg is a common cause of food-allergic reactions, especially among young children. Some egg-allergic patients do, however, tolerate heated egg products and component-resolved diagnostics (CRD) may facilitate prediction of different disease manifestations. Commercially available preparations of the egg-white allergens, ovomucoid, ovalbumin, conalbumin and lysozyme, have been reported to contain impurities which interfere with accurate CRD. METHODS: Commercial preparations of the 4 egg-white allergens were characterized using allergen-specific monoclonal chimeric human/mouse IgE antibodies in experimental ImmunoCAP® tests. Further purification of commercial ovomucoid, ovalbumin and conalbumin preparations was performed by chromatography based on affinity to monoclonal antibodies. Purity was monitored by size exclusion chromatography, SDS-PAGE, Western blotting and experimental ImmunoCAP tests using allergen-specific chimeric IgE antibodies. IgE reactivity to the highly purified egg components was analyzed in 83 samples from egg white-sensitized individuals. RESULTS: Preparations of commercially available ovomucoid, ovalbumin and conalbumin were found to contain other egg allergens which were removed by chromatographic purification. No impurities were detected in the commercial lysozyme preparation. Previously unknown complexes between the target allergens and contaminating allergens were detected and removed by affinity chromatography. IgE reactivity to ovalbumin was most common in the analyzed samples (87%), followed by ovomucoid (72%), conalbumin (69%) and lysozyme (58%). CONCLUSIONS: In this study we demonstrate the advantage of using monoclonal antibodies for purification, and monoclonal chimeric IgE antibodies for characterization, of egg allergens intended for CRD. Our study also established that ovalbumin, ovomucoid, conalbumin and lysozyme are all major allergens.

Comparative study of strong anion exchangers: structure-related chromatographic performances.

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.

Thermodynamic studies of partitioning behavior of lysozyme and conalbumin in aqueous two-phase systems.

The objective of this study was to determine the thermodynamic parameters (Delta(tr)G, Delta(tr)H and Delta(tr)S) associated with lysozyme and conalbumin partitioning in aqueous two-phases systems (ATPS). Influence of salt type and polyethylene glycol (PEG) concentrations on the partition coefficient of lysozyme and conalbumin from egg white was studied. The evaluated ATPS were composed of PEG 1500 and inorganic salts (sodium citrate and sodium sulfate) at a temperature of 25 degrees C and pH 7.0, with PEG 1500 g mol(-1) concentrations of 14%, 16% and 18% (mass basis). Partitioning of lysozyme in PEG-citrate ATPS was enthalpically driven, however the PEG-sulfate ATPS was entropically driven. The tested systems can be employed for the separation of these two proteins in egg white, due to the fact that lysozyme migrates toward the polymeric phase and conalbumin to the saline phase in both ATPS. A high recovery of conalbumin in the saline phase of the PEG-sulfate ATPS was determined to be enthalpically driven.

The panel of egg allergens, Gal d 1-Gal d 5: Their improved purification and characterization.

Egg proteins represent one of the most important sources evoking food allergic reactions. In order to improve allergy diagnosis, purified and well-characterized proteins are needed. Although the egg white allergens Gal d 1, 2, 3 and 4 (ovomucoid, ovalbumin, ovotransferrin, and lysozyme) are commercially available, these preparations contain impurities, which affect exact in vitro diagnosis. The aim of the present study was to set up further purification protocols and to extend the characterization of the physicochemical and immunological properties of the final batches. The egg white allergens Gal d 1-4 were purified from commercial preparations, whereas Gal d 5 (alpha-livetin) was purified from egg yolk. The final batches of Gal d 1-5 consisted of a range of isoforms with defined tertiary structure. In addition, the IgE binding capacity of the purified egg allergens was tested using allergic patients' sera. The allergen batches will be further used to set up allergen specific diagnostic assays and to screen a larger collection of patients' sera.

The Biflow: an instrument for transfer-loop mediated, continuous, preparative-scale isoelectric trapping separations.

The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4

Isolation and characterisation of crocodile and python ovotransferrins.

Transferrins play a major role in iron homeostasis and metabolism. In vertebrates, these proteins are synthesised in the liver and dispersed within the organism by the bloodstream. In oviparous vertebrates additional expression is observed in the oviduct and the synthesised protein is deposited in egg white as ovotransferrin. Most research on ovotransferrin has been performed on the chicken protein. There is a limited amount of information on other bird transferrins, and until our previous paper on red-eared turtle protein there was no data on the isolation, sequencing and biochemical properties of reptilian ovotransferrins. Recently our laboratory deposited ten new sequences of reptilian transferrins in the EMBL database. A comparative analysis of these sequences indicates a possibility of different mechanisms of iron release among crocodile and snake transferrin. In the present paper we follow with the purification and analysis of the basic biochemical properties of two crocodile (Crocodilus niloticus, C. rhombifer) and one snake (Python molurus bivittatus) ovotransferrins. The proteins were purified by anion exchange and hydrophobic chromatography, and their N-terminal amino-acid sequences, molecular mass and isoelectric points were determined. All three proteins are glycosylated and their N-glycan chromatographic profiles show the largest contribution of neutral oligosaccharides in crocodile and disialylated glycans in python ovotransferrin. The absorption spectra of iron-saturated transferrins were analysed. Iron release from these proteins is pH-dependent, showing a biphasic character in crocodile ovotransferrins and a monophasic type in the python protein. The reason for the different types of iron release is discussed.

Evaluation of radial chromatography versus axial chromatography, practical approach.

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).

High-capacity purification of hen egg-white proteins by ion-exchange electrochromatography with an oscillatory transverse electric field.

The ion-exchange electrochromatography with an oscillatory electric field perpendicular to the mobile-phase flow driven by pressure (pIEEC) was used to separate hen egg-white (HEW) proteins. The results were compared with those of normal ion-exchange chromatography (IEC). The column was designed as three-compartment rectangular column of 2-mL with dimensions (length x width x depth) of 40 x 10 x 5 mm(3) and the electric field was applied across the direction of column width. Q Sepharose FF was packed into the central compartment as the chromatographic bed. It was confirmed that the dynamic binding capacity (DBC) of different proteins (ovotransferrin and ovalbumin) in the HEW solution increased 2.3 times when an oscillatory electric current of 30 mA at 1/20 Hz was applied in the transverse column direction. Then, the HEW proteins were separated by the pIEEC at loading amounts 2.3-fold higher than those by the IEC. When the feedstock of about one-third of the DBC was applied to the columns (i.e., 7 mL for the pIEEC and 3 mL for the IEC), similar separation efficiencies of the two chromatographic modes were achieved. Both the recovery yield and purity reached 73% to over 90%. The results indicate that the pIEEC is promising for high-capacity purification of proteins.