key: cord-0861610-119psbio
authors: Aobai, Tu; Xue, Zhao; Yuanyuan, Shan; Xin, Lü
title: Potential role of ovomucin and its peptides in modulation of intestinal health: A review
date: 2020-06-20
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2020.06.148
sha: 24fa23f780c8e052e2cbbd8c99e54793b6723c2f
doc_id: 861610
cord_uid: 119psbio

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.

. The effect of dietary structure on the intestinal mucosal barrier [4, 5, 9, 10] In the past few years, increasing interest has been directed to food-derived bioactive proteins and their peptides, for their excellent potential for maintaining immune homeostasis [11] . For example, milk casein, rice albumin and egg white protein have been proved to have immunomodulatory activity [12] . Biopeptides with anti-inflammatory and anti-oxidant activities can help prevent chronic diseases [13, J o u r n a l P r e -p r o o f 6 other biological activities. As an important structural protein in poultry eggs, ovomucin is an important linear high molecular sulfated glycoprotein, which belongs to the mucin family [16] . It is mainly found in the thick and thin proteins of egg whites, accounting for 2%-4% of the total protein in egg whites, as well as in the frenulum and the outer layer of the yolk membrane. The functions of ovomucin, such as anti-virus [17] [18] [19] [20] [21] [22] [23] , anti-tumor [24] [25] [26] [27] , anti-inflammation [28] , anti-oxidation [29, 30] and immune regulation [31] , have been confirmed one after another in the past decades. Based on the existing studies, this paper summarizes the structure, biological activity of ovomucin and its peptides in the prevention and treatment of chronic metabolic diseases, and looks forward to its further research. It is expected to provide a new strategy for the protection of intestinal mucosal barrier. The potential application of ovomucin and its peptides as functional food ingredients for preventing and auxiliary pretreating intestinal health problems are also highlighted.

J o u r n a l P r e -p r o o f 8 while the β-subunit mainly contains hydroxyl amino acids such as threonine and serine [34] .

Linear structure of ovomucin with multiple glycosylation sites [34, 38, 39] Our previous findings had demonstrated that, ovomucin consists of 60% of carbohydrate cotent [40] . Kato, et al. [41] had elucidated the composition of three carbohydrates side chains of ovomucin, one chain is composed of galactose, galactosamine, sialic acid and sulfate in an equal molar ratio, another is composed of galactose and galactosamine in equal molar ratio, and the last is composed of mannose and galactosamine in equal molar ratio. Sialic acid, also known as neuraminic acid, is a 9-carbon pyranose which was first extracted and isolated from salivary gland mucin. Sialic acid is widely found in various biological tissues and is an important component of glycoproteins, oligosaccharides and glycolipids, which usually located at the ends of glycoproteins and glycolipids [42] . Neu5Ac (N-acetylneuraminic acid) and Neu5Gc (N-Glycolylneuraminic acid) are two types of sialic acid and ovomucin only contains Neu5Ac. The sialic acid group on the glycoside glycoprotein terminal in ovomucin is thought to play an important role in its functional properties because its strong negative electrical charge may affect the J o u r n a l P r e -p r o o f 9 diffusion of molecules and the interaction with other molecules [43] . Sialic acid promotes the structural diversity of complex carbohydrates and can directly participate in various recognition processes [44] . It is the most common ligand for pathogenic and non-pathogenic viruses, bacteria and protozoa. Sialic acid can participate in various recognition processes in the body, and has anti-infective activity against a variety of viruses or bacteria, such as influenza virus, cholera, coronavirus, Escherichia coli, and Helicobacter pylori [45, 46] . In addition, sialic acid can also remove toxic hydrogen peroxide in vitro and inhibit H 2 O 2 -induced cell death [47] .

Therefore, the existence of sialic acid groups may be one of the important reasons why ovomucin has shown many biological activities in various studies. As a kind of high molecular glycoprotein with multiple activities, ovomucin has profound research value in revealing the relationship between carbohydrate side chains and protein functional properties.

J o u r n a l P r e -p r o o f 10 the serum cholesterol level [48] . These important bioactivities reported from ovomucin and its derived components using cell experiments and animal models are given in Table 1 .

Here insert Table1

In the 1940s, Gottschalk and Lind [23] have demonstrated the anti-hemagglutination activity of ovomucin against influenza virus, and found that the interaction between viral enzymes and the viral hemagglutinin inhibitory components of ovomucin results in carbohydrate peptide complex release. Through hemagglutination inhibition and ELISA test, Tsuge et al. [17] [18] [19] found that ovomucin has a high affinity for bovine rotavirus (RV), Newcastle disease virus (NDV) and human influenza virus (IV). N-acetylneuraminic acid (NeuNAc) residues in the β-subunit are the key to the binding of ovomucin to NDV, while the disulfide bond between and within the subunit promoted the binding of ovomucin to antibodies. The J o u r n a l P r e -p r o o f 11 sialic acid groups of pronase-treated ovomucin have the ability of binding to Escherichia coli O157: H7, and this activity is lost after the ovomucin glycopeptide is treated with sialidase. Ovomucin can also inhibit the adhesion of urease produced by Helicobacter pylori to gastric mucosa. Urease is located on the surface of Helicobacter pylori cells, and ovomucin inhibits the adhesion of Helicobacter pylori to gastric mucosa receptors by predominantly binding urease [51] . Sialic acid at the end of the carbohydrate chain can not only be used as sites in "molecule-cell" and "cell-cell" recognition, but also can mask recognition sites. Glycosides linked to sialic acid at the end of glycoconjugates can effectively prevent some important antigen sites and recognition markers on the cell surface, thus protecting cells from enzymatic hydrolysis and immune attacks. [45, 52] . Therefore, the anti-adhesion activity of ovomucin and its derivatives may be caused by the sialic acid group, for they can occupy the recognition site of pathogenic microorganisms. Watanabe, et al. [24] also found pronase treated ovomucin (220 and 120 kDa, highly glycosylated peptides) completely cured the proximal tumor, directly and indirectly inhibit the growth of tumor surrounding tissue. Asialization experiments showed that sialic acid residues in the 120kDa fragment had an indirect effect on distant tumors. Oguro, et al. [33] later demonstrated that massive neutrophils, macrophages and lymphocytes were found to accumulate, and angiogenesis (the formation of new capillaries) is inhibited in tumor tissues of mice injected with highly glycosylated fragment OVMa70F in the α-subunit of streptavidin-treated ovomucin.

Yokota, et al. [27] found that highly glycosylated fragment of β-ovomucin can bind with basic fibroblast growth factor receptor (bFGFR) to competitively block the interaction between basic fibroblast growth factor (bFGF) and bFGFR, which 

shown to induce cytotoxicity and inflammation, disorders of cholesterol metabolism and inflammation have been involved in the pathophysiology of many chronic diseases, and disorders of metabolic and neurological tissues [55] . Ovomucin also showed a cholesterol-lowering effect compared to casein: ovomucin inhibited cholesterol uptake in Caco-2 cells which could significantly reduce serum cholesterol J o u r n a l P r e -p r o o f 14 in rats. Most peptides with high bile acid binding ability can inhibit the reabsorption of bile acid in the ileum and reduce the level of blood cholesterol (ileal effect), which may also be the mechanism of ovomucin [46] . The decrease of solubility of cholesterol micelles may inhibit cholesterol absorption through the direct interaction between cholesterol mixed micelles and ovomucin in jejunal epithelium, which is also part of the mechanism of cholesterol lowering induced by ovomucin (jejunal effect).

Therefore, the cholesterol-lowering effect of ovomucin may involve both jejunal and ileal effects. Many previous studies have revealed the relationship between the sialic acid group of ovomucin and its physiological activity, but whether the cholesterol-lowering effect induced by ovomucin in vivo is related to N-acetylneuraminic acid is currently being studied [48] .

Existing reports have verified the in vitro bacteriostatic effect of the ovomucin derivatives on common spoilage bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella, and it has a certain degree of thermal stability and acid-base stability [56] . The enzymatic hydrolysates (2.0kDa-70.0kDa average molecular weight) obtained by the reaction of ovomucin and serine protease, papain, metalloprotease, trypsin, and pepsin, etc. Fu, et al. [57] analyzed the antibacterial mechanism of ovomucin by flow cytometry and scanning electron microscopy. The J o u r n a l P r e -p r o o f 15 result showed a strong inactivation effect on food poisoning bacteria such as enterotoxigenic Escherichia coli, enteroinvasive Escherichia coli, pathogenic Vibrio bacterium, Bacillus dysentericus, and Pseudomonas aeruginosa [58] . Ovomucin can cause cell membrane damage or apoptosis in Staphylococcus aureus, Staphylococcus saprophyticus and Streptococcus mutans, revealing that the bacteriostatic mechanism of ovomucin derivatives is by affecting the permeability of the cell membrane, leading to the cell wall and membrane eventually ruptured.

Sun, et al. [59] found that ovomucin hydrolysates prepared by acid protease II, rather than intact ovomucin, can interfere with the adhesion of K88 (Ac) enterotoxigenic Escherichia coli to porcine red cells and have the potential to be used as an anti-adhesion agent for infectious diseases. The glycopeptides of α-ovomucin consists of the pentosaccharide core of GlcNAc2man3 and a bisecting GlcNAc, which may contain terminal β-linked galactose, are involved in this anti-agglutinating activity. In a further study, ovomucin-Protex 26L hydrolysates can act as the decoy J o u r n a l P r e -p r o o f 16

Hydrolysates and mock digests of ovomucin have shown varying degrees of antioxidant activity. Chang, et al. [29] evaluated the free radical scavenging activities of oval transferrin, ovalbumin, lysozyme and ovomucin, and found that under the same conditions, ovomucin had the strongest scavenging efficiency. The antioxidant activity of ovomucin hydrolyzed peptides was 6 times higher than that of untreated ovomucin. Two peptide sequences, LDEPDPL and NIQTDDFRT, which mainly exert antioxidant activity, were isolated from α-ovomucin. It is suggested that the antioxidant activity of ovomucin may depend on their amino acid composition.

Abeyrathne, et al. [30] found that the ovomucin hydrolyzed in alkaline condition showed the best metal ion binding activity and antioxidant activity, and hydrolysates treated by papain and alkaline protease showed good ACE inhibitory activity. The LC-MS result also proposed that the number and size of peptides are closely related to the biological activity of ovomucin.

Sun, et al. [28] tested the anti-inflammatory activities of different ovomucin hydrolysates and their components in human skin fibroblasts and found that desalted alkaline protease hydrolysates could significantly reduce the expression of ICAM-1 (intercellular cell adhesion molecule-1) induced by TNF. The desalted alkaline protease hydrolysate is rich in low molecular weight peptides, while the free part only J o u r n a l P r e -p r o o f 17 contains high molecular weight peptides. Only desalted components attenuated the activation of NF-κB mediated by TNF, which further proved that the specific anti-inflammatory potential of ovomucin peptides may be mediated by its low molecular weight peptides. The anti-inflammatory activity of these small molecular peptides is regulated by inhibiting the activity of B cells activated by tumor necrosis factor-mediated nuclear factor-κ light chain enhancer. This result further demonstrates the key role of desalting and reducing the molecular weight of peptides in enhancing potentially beneficial activities such as reducing tissue inflammation.

Based on the above studies, ovomucin and its derivatives may act on intestinal physical, immune and biological barriers, inhibit the incidence of intestinal mucosal disorders and chronic inflammation by the following ways. Adhesion of many infectious bacteria is mediated by surface lectins in the form of submicroscopic multisubunit organelles designated as pili or fimbriae, with distinct carbohydrate specificities. The glycosyl groups on the target cells will be recognized by lectins，further lead to adhesion and infection. Anti-adhesion agents do not directly kill bacteria, so they can effectively reduce the occurrence of drug-resistant strains [63] . Ovomucin or its hydrolysate in vitro has a significant inhibitory effect on the growth of environment-dependent pathogens such as Escherichia coli [50, 56] . And ovomucin has good adhesion activity to Escherichia coli, Salmonella, and J o u r n a l P r e -p r o o f suppress the colonization of these bacteria on intestinal epithelial cells [59] . Adhered microbial antigens such as Escherichia coli are considered to be the activating factor for intestinal mucosal damage [5] . By activating the TLR4 / NF-κB pathway, the secretion of inflammatory cytokines is triggered and exacerbates the inflammatory injury response [64, 65] . Sun, et al. [66] also proved the potential of ovomucin hydrolyzed peptides as prebiotics, suggesting that they may be degraded in the infant gastrointestinal tract by different Bifidobacterium or other members of the intestinal microbiota and form SCFAs. SCFAs can regulate immunity, resist pathogenic microorganisms and anti-inflammatory, regulate intestinal microbiota balance, and protect intestinal mucosal function [67] . SCFAs can reduce the expression of leukocyte adhesion molecules and IL-6 and play an anti-inflammatory effect [68] . Therefore, ovomucin or its metabolites are likely to prevent the intestinal mucus barrier damage by interfering with the structure of the intestinal microbiota, affecting the colonization of intestinal Escherichia coli, then affecting the NF-κB signaling pathway and secretion of inflammatory factors.

The medical significance of oxidative stress has become increasingly recognized.

It is now considered to be a component of virtually every disease process. Intestinal Ovomucin-derived peptides have different acid-base amino acid composition and hydrophobicity from the peptide molecules in other egg protein active substances, which may be the main reason for their stronger antioxidant activity [74] . However, whether ovomucin can still retain its antioxidant and anti-inflammatory activity in the intestine after dietary intake and maintain the intestinal mucosal homeostasis has not been reported yet , which is worthy of further study.

The intestine is one of the tissues in which the animal's body is in close contact Besides, Ovomucin contains a large number of sialic acid residues that have been shown to have immunomodulatory activity. In mouse models of respiratory and intestinal tract inflammation, sialic acid significantly reduces the level of IL-17 secreted by Th cells and supports regulatory T cells differentiation [75] . Th17 cells can secrete IL-17A, IL-17F, IL-6 and TNF-a. These cytokines can collectively mobilize, recruit and activate neutrophils, thus effectively mediating tissue inflammation [76] . Therefore, we believe that the presence of sialic acids make ovomucin hydrolysates take part in signal-recognizing process in the immune system, 

Intestinal injury always accompanied by the disturbance of intestinal physical barrier function, such as the increase of permeability [77] , the decrease of goblet cells, the decrease of mucus layer thickness and the changes of mucin, phosphatidylcholine and other components. Goblet cells are secretory cells in the intestinal epithelium. The gel secreted by goblet cells forms mucin MUC2, to prevent large granular matter and bacteria from invading the epithelial cell layer [78] . Mucin is critical to the integrity of the intestinal mucosa [4] . Studies have shown that MUC2-knockout mice will spontaneously form colitis [79] . Egg white ovomucin is an important member of the mucin family. It has a high degree of genetic homology with MUC2 in human intestinal mucus, it is interesting that β-ovomucin was reported to be a homologous protein of human MUC6; while α-ovomucin is highly homologous to MUC2 protein, lacks only a PTS domain [16] . We have reason to believe that ovomucin and intestinal mucin have theoretical similarities in function. Therefore, we are very interested in whether ovomucin can enter the intestinal tissue in the form of exogenous protein in the case of intestinal mucus damage, so that can replace the damaged intestinal mucin to play the corresponding function. Some experiments have confirmed that ovomucin can resist the effects of digestive enzymes such as pepsin and trypsin to a certain extent [30, 80] . This resistance may be related to its highly glycosylated molecular structure. Therefore, it can be speculated that ovomucin has the potential to enter the gastrointestinal tract after dietary intake. When various stimuli cause intestinal J o u r n a l P r e -p r o o f 24 inflammation, one of the most common phenomena is that the degradation of mucus increases and the thickness of the mucus layer decreases [81] . Ovomucin is highly homologous with biological mucin MUC2, and has great similarity in glycosylation structure (O-link glycosylation, N-link glycosylation and terminal sialic acid group).

The latest study of Takada, et al. [82] have found that ovomucin oligosaccharides are also a high-quality source of carbohydrates for some bacteria, including certain intestinal microbes. These intestinal microorganisms that degrade mucin in humans include Akkermansia maciniopha [83, 84] which is associated with the reduction of obesity and type 2 diabetes, and probiotic Bifidobacterium bifidum. Although sialic acid has been confirmed to be the main active group in ovomucin, there are still many activities that have not been confirmed to be related to sialic acid groups. Besides, on the existing basis, it is of great significance to isolate or identify more specific anti-tumor, antioxidant or immune-activating peptides by desalination, enzymolysis and purification to reveal the structure-activity relationship of these protein peptide groups. Because of the special similarity between ovomucin and human mucin, the possible substitution or synergistic effect between ovomucin and endogenous mucin in the process of maintaining intestinal mucosal barrier is a new direction in research of ovomucin properties. To explore the bioactivities of ovomucin and its peptides in different stages of digestion will be the basis of this study. By using the methods of modern nutrition, immunology and molecular biology, the effects of 

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The authors gratefully acknowledge financial support provided by China Postdoctoral 

The authors have declared that there is no conflict of interest.Journal Pre-proof J o u r n a l P r e -p r o o f