Effects of unfolding treatment assisted glycation on the IgE/IgG binding capacity and antioxidant activity of ovomucoid.

Ovomucoid is the immune-dominant allergen in the egg white of hens. Due to its structure based on nine disulfide bonds as well as its resistance to heat and enzymatic hydrolysis, the allergenicity of this food protein is difficult to decrease by technological processes. We sought to reduce its allergenicity through the Maillard reaction. The unfolding of ovomucoid with L-cysteine-mediated reduction was used to increase accessibility to conformational and linear epitopes by modifying the secondary and tertiary structures of the allergen. Glycation with different saccharides revealed the beneficial effect of maltose glycation on the IgG-binding capacity reduction. By determining the better glycation conditions of unfolded ovomucoid, we produced ovomucoid with reduced IgE binding capacity due to the glycation sites (K17, K112, K129, and K164) on epitopes. Moreover, after simulated infant and adult gastrointestinal digestion, the unfolded plus glycated ovomucoid showed higher ABTS˙+ scavenging activity, O2˙- scavenging activity, ˙OH scavenging activity, Fe2+ chelating activity, and a FRAP value; in particular, for ˙OH scavenging activity, there was a sharp increase of more than 100%.

Quantitative proteomics provides new insights into the mechanism of improving rehydration of egg white powder by ultrasonic pretreatment.

Poor rehydration is one of the key factors affecting the functional properties of egg white powder (EWP). Reducing rehydrated precipitates is important for the processing and application of EWP. In this study, effects of ultrasonic pretreatment on the physicochemical and functional properties of EWP rehydration solutions were studied with the aim of revealing the mechanism of ultrasonic pretreatment to improve rehydration. Compared with freeze-dried EWP (FD) and spray-dried EWP (SD), the percentage of ultrasonic pretreated FD (UFD) and ultrasonic pretreated SD (USD) rehydrated precipitates decreased by 13.0 % and 5.6 %, respectively, after ultrasonic pretreatment (0.25 W/mL for 10 min); and the average particle sizes of UFD and USD solutions decreased by 22.5 % and 15.5 %, respectively. Fourier transform infrared spectroscopy showed that ultrasonic pretreatment caused higher ß-sheet content in the protein secondary structure of UFD rehydrated precipitates (49.2 %). Quantitative proteomic analysis revealed a decrease in the abundance of major egg white proteins (ovalbumin, ovotransferrin, ovomucoid and ovomucin) in the rehydrated precipitates of UFD, except for lysozyme. It was also shown that lysozyme-centered aggregates were disrupted in the rehydrated precipitates of UFD. Our research suggests that ultrasonic pretreatment improves EWP rehydration by reducing the interactions between high abundance proteins as well as improving the solubility of high abundance proteins.

Exploring the binding effect and mechanism of glycyrrhizin to ovomucin by combining spectroscopic analysis and molecular docking.

Ovomucin (OVM) is an ideal natural macromolecular glycoprotein extracted from eggs with good adhesion. Based on the defect that glycyrrhizin (GL) has good antiviral activity but fast metabolism, this study aimed to explore the binding effect and mechanism of GL to OVM, using multi-spectroscopic techniques, isothermal titration calorimetry (ITC), and molecular docking. The adhesion ability of OVM to the hydrophilic interface and GL was first demonstrated by dual polarization interferometry (DPI) analysis and binding capacity assay, and the OVM-GL complex exhibited a similar affinity for the spike protein of COVID-19. The spectroscopic results show that GL can quench the inherent fluorescence and change the glycosidic bond and secondary structure of OVM. The ITC measurements suggested that the binding was exothermic, the hydrogen bond was the dominant binding force for forming OVM-GL. Finally, molecular docking results indicated that GL has hydrogen bond interaction with several amino acid residues located in α-OVM and ß-OVM while embedding into the hydrophobic pocket of OVM via hydrophobic interactions. In conclusion, OVM can adhere to the hydrophilic interface and bind to GL through hydrogen bonding and hydrophobic interactions to form a stable complex, that is expected to be helpful in virus prophylaxis.

Ultrasound improves the thermal stability and binding capacity of ovomucin by promoting the dissociation of insoluble ovomucin aggregates.

Ovomucin (OVM) is a natural glycoprotein with various biological activities but poor solubility. This study aimed to enhance the solubility of OVM by using an ultrasonic-assisted method. The effect of ultrasound (US) on the structure, thermal stability and biological functions of OVM aggregates was evaluated. It was found that insoluble OVM aggregates were dissociated and the solubility increased significantly to 90.0 % after US under 400 W for 45 min. US also improved the onset temperature (To) and denaturation temperature (Td) of OVM. More importantly, the cholesterol binding capacity of both OVM and its digestion products were significantly improved after US (p < 0.05). The gastrointestinal digestion products of US-OVM also showed higher α-amylase and α-glucosidase inhibition than native OVM aggregates. US-induced dissociation of OVM aggregates and the conversion of ß-sheet and ß-turn to random coil, resulting in the exposure of hydrophobic binding sites may be an important reason for the enhanced stability and adsorption capacity. These findings suggested that US was an effective method for preparing soluble OVM and improved its adsorption capacity, which can further facilitate the application of OVM in the food industry.

Effects of ultrasonic treatment on ovomucin: Structure, functional properties and bioactivity.

The effects of ultrasonic treatment on the structure, functional properties and bioactivity of Ovomucin (OVM) were investigated in this study. Ultrasonic treatment could significantly enhance OVM solubility without destroying protein molecules. The secondary structure changes, including ß-sheet reduction and random coil increase, indicate more disorder in OVM structure. After ultrasonic treatment, the OVM molecule was unfolded partially, resulting in the exposure of hydrophobic regions. The changes in OVM molecules led to an increase in intrinsic fluorescence and surface hydrophobicity. By detecting the particle size of protein solution, it was confirmed that ultrasonic treatment disassembled the OVM aggregations causing a smaller particle size. Field emission scanning electron microscopy (FE-SEM) images showed that ultrasonic cavitation significantly reduced the tendency of OVM to form stacked lamellar structure. Those changes in structure resulted in the improvement of foaming, emulsification and antioxidant capacity of OVM. Meanwhile, the detection results of ELISA showed that ultrasonic treatment did not change the biological activity of OVM. These results suggested that the relatively gentle ultrasound treatment could be utilized as a potential approach to modify OVM for property improvement.

Pasteurization induced protein interaction decreased the potential allergenicity of ovalbumin and ovomucoid in egg white.

BACKGROUND: Approximately 9.9% of young children in China suffer from egg allergies. Ovalbumin (OVA) and ovomucoid (OVM) are both the main allergens with higher allergenicity in egg white. The previous studies mainly focused on the effects of pasteurization on the structure and allergenicity of the isolated protein itself. The effects of the interaction between OVA and OVM on their spatial structure and allergenicity under pasteurization are still unclear. Therefore, in this study, the spectroscopic, immunological, and cytological methods were used to investigate the effects on OVA and OVM by their interactions which were induced by the following pasteurization, heating for 10 min at 60, 65, and 70 °C, respectively. RESULTS: Results indicated that OVA and OVM could form macromolecular aggregates by their interaction at 70 °C, and their solubility was decreased while turbidity was increased. The spatial structures of OVA and OVM were both changed by their interaction, when pasteurization temperature was at 70 °C the exposure of their hydrophobic groups and α-helix content were decreased while their ß-sheet was increased. The potential allergenicity of OVA and OVM was also changed, which showed that the immunoglobulin G (IgG)-binding ability of OVA and OVM could be increased, and their IgE-binding ability was decreased a bit. The releases of interleukin 4 (IL-4), IL-6, ß-HEX, histamine and tumor necrosis factor alpha (TNF-α) from OVA-OVM-induced KU812 cells were all decreased at 70 °C. CONCLUSION: Therefore, according to the results, if the liquid egg products were pasteurized for 10 min, the temperature of 70 °C should be carefully considered. © 2022 Society of Chemical Industry.

Effects of deglycosylation and the Maillard reaction on conformation and allergenicity of the egg ovomucoid.

Ovomucoid (OVM), known as the major allergen in egg white, has gained increasing concerns in industrialized countries. Here, we found the deglycosylation and Maillard reaction with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) can induce conformational transformation of OVM from other structures (ß-turn, strang, and random coils) to α-helix. We also introduced an approach to reduce the allergenicity of Gallus domesticus OVM by Maillard reaction with GOS and FOS. However, the OVM glycated by mannosan (MOS) and deglycosylated OVM exhibited higher allergenicity than native OVM. Therefore, GOS and FOS, especially GOS, could be applied in the reduction of the potential allergenicity of OVM through glycation. Furthermore, these findings may provide new insights into the development of hypoallergenic egg products. PRACTICAL APPLICATION: In this study, the allergenicity and conformation of OVM treated with deglycosylation and glycation (GOS, FOS, and MOS) were investigated. The results would provide a better understanding of the effects of deglycosylation and Maillard reaction with different reducing sugars on the molecular characteristics of OVM and further provide new insights into the development of hypoallergenic egg products.

An Enzyme-Mediated Aza-Michael Addition Is Involved in the Biosynthesis of an Imidazoyl Hybrid Product of Conidiogenone B.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,ß-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.

Meleagrin from marine fungus Emericella dentata Nq45: crystal structure and diverse biological activity studies.

The crystal structure and unambiguous absolute configuration of meleagrin (1) isolated from fungus Emericella dentata Nq45 is reported herein to first time on the bases of single crystal X-ray diffraction. Together with 1, haenamindole (2), isorugulosuvine (3), secalonic acid D (4), ergosterol (5) and cerebroside A (6) were obtained and their structures were determined by ESI MS and NMR data analysis. Diverse biological activity of meleagrin (1) was investigated. Compound 1 pronounced potent cytotoxicity against the human cervix carcinoma cell line KB-3-1 and its multidrug resistant sub-clone KB-V1 of IC50 3.07 and 6.07 µM, respectively, in comparison with the reference (+) - griseofulvin (IC50 19, 19.5 µM). Based on the antibiofilm activity, compound 1 displayed as well potent activity against Staphylococcus aureus with an MIC of 0.25 mg/mL. Isolation of the producing fungus and taxonomical characterization is stated as well.

Potential role of ovomucin and its peptides in modulation of intestinal health: A review.

Intestinal dysfunction, which may cause a series of metabolic diseases, has become a worldwide health problem. In the past few years, studies have shown that consumption of poultry eggs has the potential to prevent a variety of metabolic diseases, and increasing attention has been directed to the bioactive proteins and their peptides in poultry eggs. This review mainly focused on the biological activities of an important egg-derived protein named ovomucin. Ovomucin and its derivatives have good anti-inflammatory, antioxidant, immunity-regulating and other biological functions. These activities may affect the physical, biological and immune barriers associated with intestinal health. This paper reviewed the structure and the structure-activity relationship of ovomucin，the potential role of ovomucin and its derivatives in modulation of intestinal health are also summarized. Finally, the potential applications of ovomucin and its peptides as functional food components to prevent and assist in the pretreatment of intestinal health problems are prospected.

Analytical quality by design development of an ecologically acceptable enantioselective HPLC method for timolol maleate enantiomeric purity testing on ovomucoid chiral stationary phase.

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high performance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted as quite expensive with high consumption of organic solvents which leads to chronic exposure of analysts to toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of this study was to develop new method with better eco-friendly features. This was enabled by using protein type Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organic solvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of (S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid column were set using Analytical Quality by Design (AQbD) approach in method development. Optimization step was performed following the Box-Behnken experimental plan and the influence of three critical method parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was examined. CMPs included variation of acetonitrile content in the mobile phase (5-10 %, v/v), pH value of the aqueous phase (6.0-7.0) and ammonium chloride concentration in the aqueous part of the mobile phase (10-30 mmol L-1). The most relevant critical method attributes (CMAs) in this case were the separation criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol. Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance of method's qualitative robustness (π = 95 %). The selected working point situated in the middle of the DS was characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v), pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmol L-1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. The validation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Analytical Eco-Scale metric tool was applied to confirm that developed method represents excellent green analytical method compared to the official one.

Removal of Copper in Microdroplets by Ovomucoid Hydrolysates Bound to Reverse-Phase Chromatography Media Within Pipette Tips.

Ovomucoid (OVM) is a protein found in chicken egg white. When it is hydrolyzed by a protease, subtilisin A from Bacillus licheniformis, it possesses Cu2+-chelating activity. In the present work, we demonstrate that the resulting OVM hydrolysates bind to reverse-phase chromatography media in pipette tips and can be applied to remove Cu2+ within microdroplets. 1.4 nmol of purified OVM was digested in the presence of 17 pmol of subtilisin A at 55 °C for 3 h. The OVM hydrolysates efficiently removed 2.1 and 2.4 nmol of Cu2+ in the droplets by binding to the C4 and C18 chromatography media, respectively. Conversely, 0.6 and 1.0 nmol of Cu2+ were removed by the non-digested OVM bound to the C4 and C18 media, respectively. The removal ratio of Cu2+ increased as more OVM was digested by subtilisin A. The digested OVM polypeptides were stained with Cu2+ after they were separated by non-denaturing electrophoresis. These results indicate that OVM hydrolysates bound to chromatography media in a pipette tip can be applied to remove Cu2+ within microdroplets of biological samples.

Protein-Resistant Property of Egg White Ovomucin under Different pHs and Ionic Strengths.

Ovomucin is a mucin-type glycoprotein accounting for 3.5% (w/w) of total egg white proteins. The purpose of the study was to explore the potential of ovomucin as a protein-resistant material. Using bovine serum albumin (BSA) as a model protein, ovomucin decreased the fluorescence intensities of the adsorbed BSA from 10.90 ± 2.18 to 0.67 ± 0.75, indicating its protein-resistant property. To understand the underlying mechanism, pure repulsive forces between ovomucin and model proteins (e.g., BSA and ovomucin) at various pHs (2.0, 6.0, and 7.2) and ionic strengths (0.1, 10, and 150 mM NaCl) were measured using a surface forces apparatus. Further studies by using atomic force microscope imaging, zeta potential, and dynamic light scattering suggested that the protein-resistant property of ovomucin was mainly attributed to strong electrostatic and steric repulsions between protein layers. This work has demonstrated that ovomucin has antifouling potential with broad applications in the areas of food processing industry and biomedical implants.

Sialic acid involves in the interaction between ovomucin and hemagglutinin and influences the antiviral activity of ovomucin.

Ovomucin (OVM) plays an important role in inhibiting infection of various pathogens. However, this bioactivity mechanism is not much known. Here, the role of sialic acid in OVM anti-virus activity has been studied by ELISA with lectin or ligand. Structural changes of OVM after removing sialic acid were analyzed by circular dichroism and fluorescence spectroscopy. OVM could be binding to the hemagglutinin (HA) of avian influenza viruses H5N1 and H1N1, this binding was specific and required the involvement of sialic acid. When sialic acid was removed, the binding was significantly reduced 71.5% and 64.35%, respectively. Therefore, sialic acid was proved as a recognition site which avian influenza virus bound to. Meanwhile, the endogenous fluorescence and surface hydrophobicity of OVM removing sialic acid were increased and the secondary structure tended to shift to random coil. This indicated that OVM molecules were in an unfolded state and spatial conformation disorder raising weakly. Remarkably, free sialic acid strongly promoted OVM binding to HA and thereby enhanced the interaction. It may contribute to the inhibition of host cell infection, agglutinate viruses. This study can be extended to the deepening of passive immunization field.

Effectively preparing soluble ovomucin with high antiviral activity from egg white.

Ovomucin has a great potential because of its numerous bioactivities, which makes it an attractive molecule for industrials. However, to isolate soluble ovomucin without degradation and contamination remains a challenge. In this study, ovomucin of high purity (99.13%) was obtained in good yield (3.02 g kg-1 fresh egg white) via an improved two-step precipitation followed by gel filtration chromatography. The IC50 of the preparation for three representative new disease virus strains named LaSota, Mukteswar and V-4 is 1.99, 4.95 and 5.78 × 10-3 g L-1, respectively. Produced ovomucin showed limited reduction in the hemagglutination inhibition activity against all of the virus strains during the purification. Infrared spectroscopy of the ovomucin preparation indicated extensive glycosylation and sulfation. Amino acid analysis found that it was rich in alanine, glutamic acid, threonine and valine residues, which is typical in mucins. The improved process developed in this study is an alternative approach to obtain pure ovomucin with elevated antiviral activity and purity, which may significantly push forward the further research on bioactivities of ovomucin.

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.

Advances on the impact of thermal processing on structure and antigenicity of chicken ovomucoid.

BACKGROUND: Ovomucoid (OVM) is the dominant allergen found in egg white. The heat-induced changes on chicken OVM structure and antigenic properties were assessed at acidic, neutral and alkaline pH values. RESULTS: The fluorescence spectroscopy measurements indicated changes in the conformation of OVM caused by both pH and thermal treatment. The OVM molecule exhibited higher exposure of hydrophobic residues at 7.0, as indicated by the synchronous spectra, intrinsic fluorescence and quenching experiments. When heating the protein at pH 9.5, the molecular structure appeared more compact. The antigenic properties of OVM, estimated through the enzyme-linked immunosorbent assay, appeared not to be sensitive to heat at pH 7.0 and 4.5. Single molecule level investigations indicated that the secondary and tertiary structure of OVM was affected by the thermal treatment. CONCLUSIONS: Experimental results indicated over 90% reduction of the antigenicity at pH 9.5 and temperature of 100 °C. Significant changes of the linear epitopes exposure and location of the conformational epitopes were highlighted after performing heating molecular dynamics simulations of OVM from 25 °C to 100 °C. © 2017 Society of Chemical Industry.

Ovomucin nanoparticles: promising carriers for mucosal delivery of drugs and bioactive compounds.

Ovomucin, with a similar structure to mucin and gelation ability at room temperature, is a potential mucosal carrier. To evaluate this potential, the mucoadhesive property of ovomucin was studied by the tensile strength and rheology methods, and compared with three known mucoadhesive polymers, chitosan, polyacrylic acid (PAA), and alginate. Ovomucin particles were basically spherical in shape with an average size of ~572, 235 and 54 nm prepared at pH 5, 6.5, and 9, respectively. The absolute value of the zeta potential increased from ~ -51 mV at pH 5 to ~ -59 and -91 mV at pH 6.5 and 9, respectively. Drug loading efficiency of ovomucin particles for brilliant blue (negatively charged), riboflavin (non-ionic), and ciprofloxacin (positively charged) were ~87.7, 25.4, and 89.1%, respectively. The release of encapsulated ciprofloxacin was evaluated in phosphate-buffered saline (PBS), simulated intestinal fluid (SIF), and simulated gastric fluid (SGF). The mechanism of the releases was studied using different mathematical models: zero-order, first-order, Higuchi, Hixson-Crowell, and Korsmeyer-Peppas models. Approximately 61 and 67% of loaded ciprofloxacin were released from the particles over 7 h of incubation in PBS and SIF, respectively. The kinetics of drug releases in PBS and SIF followed the Fickian diffusion mechanism (Korsmeyer-Peppas model). Therefore, ovomucin could function as a mucoadhesive carrier to efficiently encapsulate drugs and sustainably release them in the non-digestive and intestinal mucosal tissues.

Effects of pH, temperature and pulsed electric fields on the turbidity and protein aggregation of ovomucin-depleted egg white.

The effect of either pulsed electric fields (PEF) or thermal processing on protein aggregation of ovomucin-depleted egg white (OdEW) solutions at different pH was assessed by solution turbidity and SDS-PAGE. Heating to 60°C for 10min caused marked protein aggregation of OdEW at pH5, 7, and 9. At constant electric field strength (E=1.4-1.8kV/cm), PEF processing under high specific energy input (Wspec=260-700kJ/kg) induced some protein aggregation at pH5 and 7, but not at either pH4 or 9. Similar effects of pH on protein aggregation were observed upon PEF processing at varied E (from 0.7 to 1.7kV/cm) but with constant Wspec (713kJ/kg). Analysis by SDS-PAGE revealed that proteins in the OdEW solution at pH5 were most susceptible to both PEF- and heat-induced protein aggregation and lysozyme was only involved in the formation of insoluble aggregates under PEF. The present study shows that PEF treatment has considerable potential for minimizing protein aggregation in the processing of heat-labile egg white proteins. Retaining the OdEW proteins in solution during processing has potential industry application, for example, protein fortification of drinks with OdEW, where minimizing solution turbidity would be advantageous.

Identification and Characterization of Glycopeptides from Egg Protein Ovomucin with Anti-Agglutinating Activity against Porcine K88 Enterotoxigenic Escherichia coli Strains.

Ovomucin is a glycoprotein from egg white with potential to act as an anti-adhesive agent against infectious diseases. This study aimed to determine whether ovomucin or ovomucin hydrolysates could prevent adhesion of two porcine K88 enterotoxigenic Escherichia coli (ETEC) strains. Adhesion was assessed in vitro using a hemagglutination assay. Ovomucin hydrolysates, but not intact ovomucin, prevented adhesion of ETEC to porcine erythrocytes. The ovomucin hydrolysate prepared by acid protease II exhibited the best anti-agglutinating activity against both strains; this hydrolysate was fractionated by cation exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC). The most active fractions, F3(9) and F7(1), with minimal inhibitory concentrations of 0.03 and 0.25 g/L against strains ECL13795 and ECL13998, respectively, were subjected to structural characterization. Six identified glycopeptides were all derived from α-ovomucin, composed of a pentasaccharide core of two N-acetylglucosamine and three mannose residues (GlcNAc2Man3) and a bisecting N-acetylglucosamine (GlcNAc). The terminal ß-linked galactose from these glycopeptides could be one of the binding sites for K88ac fimbriae.