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.

LC-MS characterization of N/O-glycans of α- and ß-subunits of chicken ovomucin separated by SDS-PAGE.

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and ß-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and ß-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and ß-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the ß-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and ß-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

Crucial effect of ovomucin on alkali-induced egg white gel formation: Properties, structure and facilitation mechanism.

Alkali-induced preserved egg gel formation is a dynamic process that involves complex protein changes. Ovomucin (OVM) is closely associated with the gel properties of egg white. In this study, the effect of OVM in alkali-induced egg white gel (AEWG) formation was investigated. The results suggested that OVM reduced the gel formation time by 15 %. The mechanical properties of the fully formed gel were also improved by OVM. Specifically, OVM increased the storage modulus (G') of the gel by 1.5-fold, while the hardness significantly increased from 78.90 ± 4.24 g to 99.80 ± 9.23 g. Low-field nuclear magnetic resonance (LF-NMR) demonstrated that OVM significantly shortened T23 relaxation time and reduced the water mobility, thus increasing the water holding capacity (WHC). Meanwhile, the presence of OVM resulted in a more homogeneous and denser microscopic morphology of the gel. Selective solubility experiments revealed that disulfide bonds are the primary force in gel formation. OVM promoted the formation of more disulfide bonds, which increased the strength and stability of the gel network. Overall, this research proved OVM plays a critical role in the performance improvement of AEWG, which provides a new insight into the quality control of preserved egg and protein gel foods.

Preparation and characterization of ovomucin self-assembly nanoparticles under glycerol compression for astaxanthin delivery: Sustained release and antioxidant activity.

Astaxanthin (AST) is a natural hydrophobic nutrient with various biological activities, but its low solubility limits its application. In this study, self-assembly nanoparticles were prepared by ovomucin (OVM) and Ca2+ with the enhancement of glycerol to deliver AST. Glycerol compressed the particle size of nanoparticles from 175.7 ± 1.8 to 142.9 ± 0.6 nm, and the nanoparticles had a strong negative charge (-28.9 ± 0.6 mV). Ultraviolet-visible spectroscopy and X-ray diffraction (XRD) confirmed the successful encapsulation of AST in an amorphous form with a high encapsulation efficiency (82.9% ± 2.1%). Fourier transform infrared and circular dichroism analyses demonstrated that nanoparticles formation mainly involved electrostatic interactions and hydrophobic interactions. AST in nanoparticles presented excellent gastric juice resistance and sustained release ability, whereas free radical scavenging efficiency reached up to 75%. In addition, the nanoparticles had no apparent toxicity to cell viability. This study is expected to provide a new insight into the safe and efficient delivery of AST, while demonstrating the potential of OVM as a delivery carrier in the food and health industries.

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.

Immunostimulatory Effect of Ovomucin Hydrolysates by Pancreatin in RAW 264.7 Macrophages via Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway.

Ovomucin (OM), which has insoluble fractions is a viscous glycoprotein, found in egg albumin. Enzymatic hydrolysates of OM have water solubility and bioactive properties. This study investigated that the immunostimulatory effects of OM hydrolysates (OMHs) obtained by using various proteolytic enzymes (Alcalase®, bromelain, α-chymotrypsin, Neutrase®, pancreatin, papain, Protamax®, and trypsin) in RAW 264.7 cells. The results showed that OMH prepared with pancreatin (OMPA) produced the highest levels of nitrite oxide in RAW 264.7 cells, through upregulation of inducible nitric oxide synthase mRNA expression. The production of pro-inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 were increased with the cytokines mRNA expression. The effect of OMPA on mitogen-activated protein kinase signaling pathway was increased the phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase in a concentration-dependent manner. Therefore, OMPA could be used as a potential immune-stimulating agent in the functional food industry.

Chain reactions of temperature-induced egg white protein amorphous aggregates: Formation, structure and material composition of thermal gels.

Egg white protein is widely used in food, chemical, medical and other fields due to its excellent thermal gel properties. However, the regularity of egg white thermal gel (EWTG) by temperature influence is still unknown. In this study, we investigated the potential mechanism of temperature (75-95 °C, 15 min) gradient changes inducing thermal aggregation and gel formation of EWTG. The results showed that changes in textural characteristics and water holding capacity (WHC) of EWTGs depended on switching in protein aggregation morphology (spherical shape - chain shape - regiment shape) and gel network structure differences ("irregular bead-like" - "regular lamellar structure"). In addition, proteomics indicated that the generation of amorphous protein aggregates at 95 °C might be related to Mucin 5B as the aggregation core. The research revealed the EWTG formation from "whole egg white protein" to "single molecules", aiming to provide a reference for quality control in gel food processing.

Ovomucin Hydrolysates Reduce Bacterial Adhesion and Inflammation in Enterotoxigenic Escherichia coli (ETEC) K88-Challenged Intestinal Epithelial Cells.

Enterotoxigenic Escherichia coli (ETEC) K88 is the most common cause of diarrhea in neonatal and postweaning pigs. After adhering to small intestinal epithelial cells via glycoprotein receptor recognition, the pathogen can produce enterotoxins, impair intestinal integrity, trigger watery diarrhea, and induce inflammation via nuclear factor κB (NF-κB) and mitogen-activated protein kinase phosphatase (MAPK) pathways. Inhibiting ETEC K88 adhesion to cell surfaces by interfering with the receptor-fimbriae recognition provides a promising strategy to prevent the initiation and progression of infection. Ovomucin is a highly glycosylated protein in chicken egg white with diverse bioactivities. Ovomucin hydrolysates prepared by the enzymes Protex 26L (OP) and pepsin/pancreatin (OPP) were previously revealed to prevent adhesion of ETEC K88 to IPEC-J2 cells. Herein, we investigated the protective effects of ovomucin hydrolysates on ETEC K88-induced barrier integrity damage and inflammation in IPEC-J2 and Caco-2 cells. Both hydrolysates inhibited ETEC K88 adhesion to cells and protected epithelial cell integrity by restoring transepithelial electronic resistance (TEER) values. Removing sialic acids in the hydrolysates reduced their antiadhesive capacities. Ovomucin hydrolysates suppressed ETEC-induced activation of NF-κB and MAPK signaling pathways in both cell lines. The ability of ETEC K88 in activating calcium/calmodulin-dependent protein kinase 2 (CaMK II), elevating intracellular Ca2+ concentration, and inducing oxidative stress was attenuated by both hydrolysates. In conclusion, this study demonstrated the potential of ovomucin hydrolysates to prevent ETEC K88 adhesion and alleviate inflammation and oxidative stress in intestinal epithelial cells.

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.

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.

Physicochemical Index Analyses of the Egg White in Blue-Shelled Eggs and Commercial Brown-Shelled Eggs during Storage.

To compare the physical and chemical changes in egg whites during storage, assisting in the evaluation of differences in egg freshness between various chicken breeds, we chose 240 blue-shelled eggs (Blue group) and 240 commercial brown-shelled eggs (Brown group) that 28-week-old hens had laid. In this study, all eggs were stored at 25 °C. The egg weight, egg components' weight and proportion, Haugh Unit value and the contents of S-ovalbumin, ovomucin and lysozyme in the thick albumen (KA) and thin albumen (NA) were measured at eight time points every 3 days until the 21st day of storage. The eggshell, yolk and KA proportions in the Brown group were significantly lower, whereas the NA proportion was significantly higher than that in the Blue group (p < 0.001). The Haugh Unit value and S-ovalbumin in the Brown group were significantly higher, whereas KA ovomucin and NA lysozyme were significantly lower than those in the Blue group (p < 0.001). There existed significant negative correlations between the KA and NA, irrespective of weight or proportion. The Haugh Unit value was significantly positively correlated with lysozyme and ovomucin, but significantly negatively correlated with S-ovalbumin. During storage, the KA weight (proportion), Haugh Unit value, lysozyme and ovomucin decreased, whereas the NA weight (proportion) and S-ovalbumin increased. At each time point, the NA lysozyme in the Brown group was lower than that in the Blue group (p < 0.05). After storage for 6 days, the KA ovomucin in the Brown group began to be lower than that in the Blue group (p < 0.05). The study showed that the weight (proportion) differences in egg components between blue-shelled eggs and commercial brown-shelled eggs are mainly due to the NA. The Haugh Unit value and albumin protein indexes of blue-shelled eggs were better than those of brown-shelled eggs, and showed mild changes during storage, indicating the better storage performance of blue-shelled eggs.

Ovomucin may be the key protein involved in the early formation of egg-white thermal gel.

Clarification of the mechanism of heat-induced gel formation by proteins under natural food systems could provide important references for the regulation of food texture. In the present study, the proteins involved in the early stage (heating at 72 °C for 8 min) of egg-white thermal gel (EWG) formation were studied quantitatively through comparative proteomic analysis. We discovered that the abundance of ovalbumin and ovomucoid increased significantly (p < 0.01), whereas that of ovotransferrin, lysozyme, ovomucin (mucin 5B and mucin 6) decreased significantly (p < 0.01), in the supernatant of EWG. If the initial interaction of egg white proteins was altered by ultrasonic pretreatment, the abundance of ovomucin and lysozyme in the supernatant of EWG increased, and was accompanied by the change from a solid gel to a fluid gel. Based on these results, we hypothesize that ovomucin has a key role in the formation and regulation of EWG properties.

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.

Ovomucin Ameliorates Intestinal Barrier and Intestinal Bacteria to Attenuate DSS-Induced Colitis in Mice.

Egg white ovomucin (OVM) is homologically related to MUC2, the key component of colonic mucous layer. This study investigated the effects of orally administered OVM from egg white on the colonic mucosal barrier and the development of colitis using a colitis C57BL/6J mice model. The results showed that daily supplementation of 125 and 250 mg/kg BW of OVM partially relieved the villous destruction and loss of intestinal barrier integrity, and hence decreased the epithelial barrier permeability. The supplementation also reduced the secretion of proinflammatory cytokines TNF-α and IL-6. Besides, OVM administration significantly increased the relative abundance of intestinal beneficial bacteria including Lactobacilli, Faecalibaculum, Ruminococcus, etc. and further upregulated the production of bacterial metabolites such as short-chain fatty acids (SCFAs), which is a direct source of energy for the proliferation of epithelia and goblet cells. In conclusion, OVM from egg white ameliorates colitis by enhancing the intestinal barrier function and abundance of intestinal bacteria, thereby increasing the number of SCFAs.

Soybean ß-conglycinin and catfish cutaneous mucous p22 glycoproteins deteriorate sporangial cell walls of Pseudoperonospora cubensis and suppress cucumber downy mildew.

BACKGROUND: Cucumber plants suffer from a serious threatening disease, downy mildew, throughout the growing seasons irrespective of the weather temperature. The causal agent, Pseudoperonospora cubensis, tends to evolve rapidly upon sequential applications of chemical fungicides and generate new progeny possessing tolerance to such fungicides. Glycoproteins represent an environmentally safe alternative for chemically synthetized fungicides and do not trigger fungicide resistance. We studied the antifungal activity of four glycoproteins namely soybean ß-conglycinin, chickpea vicilin, duck egg ovomucin and catfish p22 against P. cubensis. Ten commercial fungicides of different chemical groups were used as positive controls of glycoprotein treatments. RESULTS: The results revealed that soybean ß-conglycinin and catfish p22 glycoproteins possess significant antifungal activity against P. cubensis. The amount of disease suppression caused by ß-conglycinin and p22 was comparable to the highly efficient chemical fungicides containing copper oxychloride, cymoxanil and fosetyl Al as active ingredients. Vicilin and ovomucin were less efficient biocides as they gave moderate suppression of disease severity. However, all tested glycoproteins provided full protection for the newly emerged cucumber leaves. Microscopic examination of glycoprotein-treated leaves inferred the ability of catfish p22 and soybean ß-conglycinin to disrupt the integrity of sporangial cell walls of P. cubensis rendering them non-viable compared to untreated ones. Expression levels of total phenolic compounds and the antioxidant enzymes catalase, superoxide dismutase and peroxidase were elevated upon glycoproteins application, which infers their involvement in disease suppression. CONCLUSION: This report emphasizes the direct and indirect roles of glycoproteins in safe management of cucumber downy mildew disease. © 2021 Society of Chemical Industry.

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.

Sialylated O -Glycans from Hen Egg White Ovomucin are Decomposed by Mucin-degrading Gut Microbes.

Ovomucin, a hen egg white protein, is characterized by its hydrogel-forming properties, high molecular weight, and extensive O -glycosylation with a high degree of sialylation. As a commonly used food ingredient, we explored whether ovomucin has an effect on the gut microbiota. O- Glycan analysis revealed that ovomucin contained core-1 and 2 structures with heavy modification by N -acetylneuraminic acid and/or sulfate groups. Of the two mucin-degrading gut microbes we tested, Akkermansia muciniphila grew in medium containing ovomucin as a sole carbon source during a 24 h culture period, whereas Bifidobacterium bifidum did not. Both gut microbes, however, degraded ovomucin O -glycans and released monosaccharides into the culture supernatants in a species-dependent manner, as revealed by semi-quantified mass spectrometric analysis and anion exchange chromatography analysis. Our data suggest that ovomucin potentially affects the gut microbiota through O -glycan decomposition by gut microbes and degradant sugar sharing within the community.

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.

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.