Anti-biofilm effect of enzymatic hydrolysates of ovomucin in Listeria monocytogenes and Staphylococcus aureus.

Despite modern advances in food hygiene, food poisoning due to microbial contamination remains a global problem, and poses a great threat to human health. Especially, Listeria monocytogenes and Staphylococcus aureus are gram-positive bacteria found on food-contact surfaces with biofilms. These foodborne pathogens cause a considerable number of food poisoning and infections annually. Ovomucin (OM) is a water-insoluble gel-type glycoprotein in egg whites. Enzymatic hydrolysis can be used to improve the bioactive properties of OM. This study aimed to investigate whether ovomucin hydrolysates (OMHs) produced using five commercial enzymes (Alcalase®, Bromelain, α-Chymotrypsin, Papain, and Pancreatin) can inhibit the biofilm formation of L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7. Particularly, OMH prepared with papain (OMPP; 500 µg/mL) significantly inhibited biofilm formation in L. monocytogenes ATCC 15313, L. monocytogenes H7962, S. aureus KCCM 11593, and S. aureus 7 by 85.56 %, 80.28 %, 91.70 %, and 79.00 %, respectively. In addition, OMPP reduced the metabolic activity, exopolysaccharide production (EPS), adhesion ability, and gene expression associated with the biofilm formation of these bacterial strains. These results suggest that OMH, especially OMPP, exerts anti-biofilm effects against L. monocytogenes and S. aureus. Therefore, OMPP can be used as a natural anti-biofilm agent to control food poisoning in the food industry.

Dynamic changes in the aggregation-depolymerization behavior of Ovomucin-Complex and its binding to urease during in vitro simulated gastric digestion.

Ovomucin-Complex extracted from egg white is expected to have a barrier function similar to gastric mucin. In this study, the dynamic changes in structure, rheological properties and binding ability of Ovomucin-Complex during in vitro simulated gastric digestion were investigated. The results from HPLC and CLSM showed that extremely acidic pH (pH = 2.0) promoted Ovomucin-Complex to form aggregation. Acid-induced aggregation may hinder its binding to pepsin, thus rendering Ovomucin-Complex resistant to pepsin. Consequently, most of the polymer structure and weak gel properties of Ovomucin-Complex retained after simulated gastric digestion as verified by HPLC, CLSM and rheological measurement, although there was a small breakdown of the glycosidic bond as confirmed by the increased content of reducing sugar. The significantly reduced hydrophobic interactions of Ovomucin-Complex were observed under extremely acidic conditions and simulated gastric digestion compared with the native. Noticeably, the undigested Ovomucin-Complex after simulated gastric digestion showed a higher affinity (KD = 5.0 ± 3.2 nm) for urease - the key surface antigen of Helicobacter pylori. The interaction mechanism between Ovomucin-Complex and urease during gastric digestion deserves further studies. This finding provides a new insight to develop an artificial physical mucus barrier to reduce Helicobacter pylori infection.

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%.

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.

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.

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.

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.

Effect of proteolysis on the sialic acid content and bifidogenic activity of ovomucin hydrolysates.

Ovomucin, accounting for ∼3.5% of egg white proteins, contains 2.6-7.4% of sialic acid; sialic acid is suggested to play important roles in host-recognition, cognition and memory development. However, ovomucin's limited water solubility might restrict its future applications. The objective of the study was to examine the effect of proteolysis of ovomucin on the sialic acid content and bifidogenic activity of ovomucin hydrolysates. Ovomucin extract was hydrolyzed by 14 proteases with yields and DHs ranging from 42.6% (flavourzyme) to 97.4% (protease N), and 2.4% (flavourzyme) to 46.3% (pronase), respectively. Ovomucin hydrolyzed by pronase and protex 26L showed molecular weight (Mw) distributions less than 40kDa while others larger than 200kDa. Allergenicity of ovomucin hydrolysates was significantly reduced (P<0.05) in comparison to ovomucin extract. The content of sialic acid in hydrolysates ranged from 0.1% (protex 26L) to 3.7% (pronase). Ovomucin hydrolysates did not generally support growth of Bifidobacterium spp. in vitro.

Searching the Evolutionary Origin of Epithelial Mucus Protein Components-Mucins and FCGBP.

The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying mucins based on profile hidden Markov models, we have found a large number of such proteins in Metazoa, aiding in their classification and allowing evolutionary studies. Most vertebrates have 5-6 gel-forming mucin genes and the genomic arrangement of these genes is well conserved throughout vertebrates. An exception is the frog Xenopus tropicalis with an expanded repertoire of at least 26 mucins of this type. Furthermore, we found that the ovomucin protein, originally identified in chicken, is characteristic of reptiles, birds, and amphibians. Muc6 is absent in teleost fish, but we now show that it is present in animals such as ghost sharks, demonstrating an early origin in vertebrate evolution. Public RNA-Seq data were analyzed with respect to mucins in zebrafish, frog, and chicken, thus allowing comparison in regard of tissue and developmental specificity. Analyses of invertebrate proteins reveal that gel-forming-mucin type of proteins is widely distributed also in this group. Their presence in Cnidaria, Porifera, and in Ctenophora (comb jellies) shows that these proteins were present early in metazoan evolution. Finally, we examined the evolution of the FCGBP protein, abundant in mucus and related to gel-forming mucins in terms of structure and localization. We demonstrate that FCGBP, ubiquitous in vertebrates, has a conserved N-terminal domain. Interestingly, this domain is also present as an N-terminal sequence in a number of bacterial proteins.

Evolutionary formation of gene clusters by reorganization: the meleagrin/roquefortine paradigm in different fungi.

The biosynthesis of secondary metabolites in fungi is catalyzed by enzymes encoded by genes linked in clusters that are frequently co-regulated at the transcriptional level. Formation of gene clusters may take place by de novo assembly of genes recruited from other cellular functions, but also novel gene clusters are formed by reorganization of progenitor clusters and are distributed by horizontal gene transfer. This article reviews (i) the published information on the roquefortine/meleagrin/neoxaline gene clusters of Penicillium chrysogenum (Penicillium rubens) and the short roquefortine cluster of Penicillium roqueforti, and (ii) the correlation of the genes present in those clusters with the enzymes and metabolites derived from these pathways. The P. chrysogenum roq/mel cluster consists of seven genes and includes a gene (roqT) encoding a 12-TMS transporter protein of the MFS family. Interestingly, the orthologous P. roquefortine gene cluster has only four genes and the roqT gene is present as a residual pseudogene that encodes only small peptides. Two of the genes present in the central region of the P. chrysogenum roq/mel cluster have been lost during the evolutionary formation of the short cluster and the order of the structural genes in the cluster has been rearranged. The two lost genes encode a N1 atom hydroxylase (nox) and a roquefortine scaffold-reorganizing oxygenase (sro). As a consequence P. roqueforti has lost the ability to convert the roquefortine-type carbon skeleton to the glandicoline/meleagrin-type scaffold and is unable to produce glandicoline B, meleagrin and neoxaline. The loss of this genetic information is not recent and occurred probably millions of years ago when a progenitor Penicillium strain got adapted to life in a few rich habitats such as cheese, fermented cereal grains or silage. P. roqueforti may be considered as a "domesticated" variant of a progenitor common to contemporary P. chrysogenum and related Penicillia.

Enzymatic hydrolysis of ovomucoid and the functional properties of its hydrolysates.

Ovomucoid is well known as a "trypsin inhibitor" and is considered to be the main food allergen in egg. However, the negative functions of ovomucoid can be eliminated if the protein is cut into small peptides. The objectives of this study were to hydrolyze ovomucoid using various enzyme combinations, and compare the functional properties of the hydrolysates. Purified ovomucoid was dissolved in distilled water (20 mg/mL) and treated with 1% of pepsin, α-chymotrypsin, papain, and alcalase, singly or in combinations. Sodium sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) results of the hydrolysates indicated that pepsin (OMP), alcalase (OMAl), alcalase+trypsin (OMAlTr), and alcalase+papain (OMAlPa) treatments best hydrolyzed the ovomucoid, and the 4 treatments were selected to determine their functional characteristics. Among the 4 enzyme treatments, hydrolysate from OMAlTr showed the highest iron-chelating and antioxidant activities, while OMP showed higher ACE-inhibitory activity, but lower Fe-chelating activity than the other treatments. However, no difference in the copper-chelating activity among the treatments was found. MS/MS analysis identified numerous peptides from the hydrolysates of OMAlPa and OMAlTr, and majority of the peptides produced were <2 kDa. Pepsin treatment (OMP), however, hydrolyzed ovomucoid almost completely and produced only amino acid monomers, di- and tri-peptides. The ACE-inhibitory, antioxidant and iron-chelating activities of the enzyme hydrolysates were not consistent with the number and size of peptides in the hydrolysates, but we do not have information about the quantity of each peptide present in the hydrolysates at this point.

Mg2+ binding affects the structure and activity of ovomucin.

Metal-protein interaction for regulating the function and structure of proteins is of major interest. We demonstrate here that the addition of 9 µmol Mg2+ per mg ovomucin resulted in a 2.0-fold increase in the adhesion capacity of ovomucin to New Disease Virus (NDV) using ELISA method. The hemagglutinin inhibition rate of 100 µg/mL ovomucin-Mg2+ mixture (9 µmol Mg2+ per mg ovomucin) to NDV increased to 55.61%, which is significantly higher than that of the pure ovomucin (P<0.01). Binding characteristics of ovomucin to Mg2+ were then evident by fluorescence and FT-IR spectroscopy as well as dynamic light scattering (DLS) technology. Fluorescence and FT-IR spectroscopy results suggest that Mg2+ prefers to interact with the carbohydrate moiety of ovomucin without significant changes in the secondary structure when Mg2+ concentration is less than 9 µmol/mg ovomucin. When Mg2+ increases to 16 µmol/mg ovomucin, it gives rise to changes in secondary structure, and forms a more compact configuration. This is supported by the hydrodynamic radius of ovomucin with and without Mg2+ binding as examined by DLS. The Mg2+ bound in regulating the structure and activity of ovomucin provided important insight into the effect of metal ions on ovomucin binding to pathogens.

Rab1a regulates sorting of early endocytic vesicles.

We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered.

Concentration-dependent viscosity of binary and ternary mixtures of nonassociating proteins: measurement and analysis.

Using a recently developed automated viscometer (Grupi, A.; Minton, A. P. Anal. Chem. 2012, 84, 10732-10736), the dependence of solution viscosity upon the concentrations of bovine serum albumin, hen egg ovomucoid, and human fibrinogen have been measured individually and in binary and ternary mixtures over a wide range of compositions and at total concentrations of up to 300 g/L. The concentration dependence of viscosity of all solutions is quantitatively described over the entire range of concentrations and compositions by a semiempirical equation requiring specification of only two composition-independent global parameters per protein.

pH-selective mutagenesis of protein-protein interfaces: in silico design of therapeutic antibodies with prolonged half-life.

Understanding the effects of mutation on pH-dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein-protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH-dependent energy profiles demonstrate excellent agreement with experimentally measured pH-dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH-dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH-selective binding. The method can be used to select mutations that change the pH-dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH-dependent binding behavior of IgG to FcRn with the aim of improving the half-life of therapeutic antibodies in the target organism.

Simple pH treatment as an effective tool to improve the functional properties of ovomucin.

UNLABELLED: Ovomucin has been considered to contribute a lot to the excellent functional properties of egg white. This work focused on investigating the effects of pH and protein concentration on foaming and emulsifying properties of ovomucin to evaluate the proper use of this egg protein as a functional food ingredient, and to clarify its contribution to the functional properties of egg white under different pH conditions. Protein solubility and surface hydrophobicity were measured through the pH ranged from 2.3 to 11.0. Alkali treatment gave ovomucin improved emulsification properties, which were correlated well with the surface hydrophobicity (r ≥ 0.89, P < 0.01). Although ovomucin showed lower foaming capacity in acid and neutral solution, enhanced foaming stability was observed with weak acid-treated ovomucin (pH 5 to 6) compared to native ovomucin. These results demonstrated that acid and alkali treatment, which leads to partial unfolding of ovomucin can improve functional properties of ovomucin, with the greatest improvement for emulsification properties being from the alkali treatment and for foaming stability being from weak acid treatment. These results are helpful to produce unfolding ovomucin suitable for wide range of applications in food industry, and to provide useful information on the proper use of egg white in different food systems. PRACTICAL APPLICATION: Ovomucin plays a critical functional role in egg white products. However, it is typically insoluble in distilled water or common salt solutions, which has thus limited its commercial applications. Alkaline treatment resulted in gradual increase in solubility, which markedly enhanced the emulsifying properties, on the other hand foaming stability of ovomucin can be promoted by weak acid treatment. The results of this work help to produce unfolding ovomucin suitable for wide range of applications in food industry, and to provide useful information on the proper use of egg white in different food systems.

Role of magnesium chloride on the purity and activity of ovomucin during the isolation process.

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl(2) on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl(2) is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl(2) against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05 mol/L CaCl(2) followed by precipitation with 0.05 mol/L MgCl(2) was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl(2) was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity.

Yeast thioredoxin-enriched extracts for mitigating the allergenicity of foods.

Thioredoxin (TRX) catalyzes the reduction of disulfide bonds in proteins via the NADPH-dependent thioredoxin reductase system. Reducing the disulfide bonds of allergenic proteins in food by TRX lowers the allergenicity. We established in this study a method to prepare TRX-enriched extracts from the edible yeast, Saccharomyces cerevisiae, on a large and practical scale, with the objective of developing TRX-containing functional foods to mitigate food allergy. Treating with the yeast TRX-enriched extracts together with NADPH and yeast thioredoxin reductase enhanced the pepsin cleavage of ß-lactoglobulin and ovomucoid (OM). We also examined whether yeast TRX can mitigate the allergenicity of OM by conducting immediate allergy tests on guinea pigs. The treatment with TRX reduced the anaphylactic symptoms induced by OM in these tests. These results indicate that yeast TRX was beneficial against food allergy, raising the possibility that yeast TRX-enriched extracts can be applied to food materials for mitigating food allergy.

pH replica-exchange method based on discrete protonation states.

We propose a new algorithm for obtaining proton titration curves of ionizable residues. The algorithm is a pH replica-exchange method (PHREM), which is based on the constant pH algorithm of Mongan et al. (J Comput Chem 2004;25:2038-2048). In the original replica-exchange method, simulations of different replicas are performed at different temperatures, and the temperatures are exchanged between the replicas. In our PHREM, simulations of different replicas are performed at different pH values, and the pHs are exchanged between the replicas. The PHREM was applied to a blocked amino acid and to two protein systems (snake cardiotoxin and turkey ovomucoid third domain), in conjunction with a generalized Born implicit solvent. The performance and accuracy of this algorithm and the original constant pH method (PHMD) were compared. For a single set of simulations at different pHs, the use of PHREM yields more accurate Hill coefficients of titratable residues. By performing multiple sets of constant pH simulations started with different initial states, the accuracy of predicted pK(a) values and Hill coefficients obtained with PHREM and PHMD methods becomes comparable. However, the PHREM algorithm exhibits better samplings of the protonation states of titratable residues and less scatter of the titration points and thus better precision of measured pK(a) values and Hill coefficients. In addition, PHREM exhibits faster convergence of individual simulations than the original constant pH algorithm.

Recognition and uptake of free and nanoparticle-bound betalactoglobulin--a food allergen--by human monocytes.

SCOPE: To improve our understanding of the interaction of food allergens with cells of the immune system, the endocytosis by human monocytes of bovine ß-lactoglobulin (BLG) and ovomucoid (OM)--two major food allergens--and human serum albumin (HSA) was studied. METHODS AND RESULTS: BLG was covalently conjugated to dextran-coated magnetic nanoparticles (MNPs) without affecting its structure and immunoreactivity. BLG-conjugated MNPs were taken up by human monocytes much more efficiently than non-conjugated MNPs, allowing easy magnetic separation of cells that had adsorbed the allergen. BLG, OM, and HSA were conjugated to MNPs also labeled with a fluorescent probe. The uptake of these materials by human monocytes was monitored through flow cytometry, and compared with fluorescent MNPs and the free fluorescently labeled proteins, confirming higher uptake of the BLG-conjugated MNPs versus non-conjugated MNPs. OM but not HSA conjugation to particles enhanced uptake of the MNPs. Confocal microscopy provided direct evidence of the actual internalization of BLG-MNP conjugates into the cytoplasm. CONCLUSIONS: These results contribute to the current understanding of the interaction between food allergens and antigen-presenting cells, and demonstrate that the BLG is readily endocytosed by monocytes both as the single protein and as a conjugate.