Germination and Simulated Gastrointestinal Digestion of Chickpea (Cicer arietinum L.) in Exhibiting In Vitro Antioxidant Activity in Gastrointestinal Epithelial Cells.

Plant-based proteins, in particular pulse proteins, have grown in popularity worldwide. Germination, or sprouting, is an effective method to release peptides and other dietary compounds. However, the combination of germination and gastrointestinal digestion in enhancing the release of dietary compounds with potential health-beneficial biological activity has yet to be entirely elucidated. The present study illustrates the impact of germination and gastrointestinal digestion on the release of dietary compounds with antioxidant activity from chickpeas (Cicer arietinum L.). Germination up to 3 days (D0 to D3) increased the peptide content by denaturing chickpea storage proteins and increased the degree of hydrolysis (DH) in the gastric phase. The antioxidant activity was measured at three different dosages (10, 50, and 100 µg/mL) and compared between D0 and D3 on human colorectal adenocarcinoma cells (HT-29). A significant increase in antioxidant activity was observed in the D3 germinated samples in all three tested dosages. Further analysis identified 10 peptides and 7 phytochemicals differentially expressed between the D0 and D3 germinated samples. Among the differentially expressed compounds, 3 phytochemicals (2',4'-dihydroxy-3,4-dimethoxychalcone, isoliquiritigenin 4-methyl ether, and 3-methoxy-4,2',5'-trihydroxychalcone) and 1 peptide (His-Ala-Lys) were identified only in the D3 samples, indicating their potential contribution towards the observed antioxidant activity.

Effect of radiofrequency processing on the structural and bio-functional properties of egg white proteins.

Radiofrequency (RF) assisted thermal processing can significantly enhance the gel firmness of egg white powder compared to the traditional hot room (HR) processing. Thus, the present study aims to delineate the impact of RF processing on the proteins' structure and bio-functional properties of egg white protein gels. The secondary protein conformations of egg white proteins exhibited no significant alteration upon RF-assisted thermal processing over traditional HR processing. In-vitro gastrointestinal (GI) digestion of egg white gels demonstrated that the RF processing did not compromise the accessibility of digestive proteases despite a more robust gel network. Peptides from the GI digest of egg white gel showed that Ovalbumin and Ovotransferrin were the parent proteins of most of the unique peptides generated, and minor structural differences accounted for these peptides. The bioavailability of the egg protein-derived peptides remains unaffected after RF processing without compromising the viability and integrity of the GI epithelial cells.

Comprehensive Review of γ-Glutamyl Peptides (γ-GPs) and Their Effect on Inflammation Concerning Cardiovascular Health.

γ-Glutamyl peptides (γ-GPs) are a group of peptides naturally found in various food sources. The unique γ-bond potentially enables them to resist gastrointestinal digestion and offers high stability in vivo with a longer half-life. In recent years, these peptides have caught researchers' attention due to their ability to impart kokumi taste and elicit various physiological functions via the allosteric activation of the calcium-sensing receptor (CaSR). This review discusses the various food sources of γ-glutamyl peptides, different synthesis modes, allosteric activation of CaSR for taste perception, and associated multiple biological functions they can exhibit, with a special emphasis on their role in modulating chronic inflammation concerning cardiovascular health.

Nonsupercharged Retrosternal Roux-en-Y Esophagojejunostomy for Distal Esophageal Reconstruction.

BACKGROUND: Delayed distal esophageal reconstruction with nonsupercharged jejunum is an option when gastric conduit is not available. This study aimed to describe a single-center experience with distal esophageal reconstruction with retrosternal Roux-en-Y esophagojejunostomy (RYEJ) and compare perioperative outcomes with retrosternal gastric pull-up (GP). METHODS: An Institutional Review Board-exempt retrospective chart review was conducted of patients who underwent esophagostomy closure by the retrosternal route at the University of Minnesota Medical Center (Minneapolis, MN) from January 2009 to July 2019. Patients with colonic conduits were excluded. The study compared patients with RYEJ with a contemporary cohort of patients who underwent GP. The anatomic criteria for RYEJ were the absence of a gastric conduit and an esophageal remnant that reached the sternomanubrial joint. Patient characteristics, anastomotic leak and stricture rate, postoperative complications, hospital length of stay, 30-day readmission, and 90-day mortality were recorded. Statistical analysis was performed using the Fisher exact test and the Wilcoxon rank-sum test with a significance level at P ≤.05. RESULTS: A total of 9 patients underwent RYEJ, and 10 patients had GP. Previous esophageal adenocarcinoma was more common in the RYEJ group (n = 5) compared with the GP group (n = 0) (P = .01). Patient demographics and comorbidities were comparable between the groups. No differences were found in all end points, including operating time, estimated blood loss, anastomotic leak or stricture rate, Clavien-Dindo class III to IV complications, hospital length of stay, or mortality. CONCLUSIONS: Retrosternal RYEJ without microvascular augmentation is a safe alternative for esophagostomy closure in patients with adequate esophageal length when the stomach is not available. The nonsupercharged jejunum can safely reach the level of the sternomanubrial joint.

Anti-hypertensive Peptide Predictor: A Machine Learning-Empowered Web Server for Prediction of Food-Derived Peptides with Potential Angiotensin-Converting Enzyme-I Inhibitory Activity.

Angiotensin converting enzyme-I (ACE-I) is a key therapeutic target of the renin-angiotensin-aldosterone system (RAAS), the central pathway of blood pressure regulation. Food-derived peptides with ACE-I inhibitory activities are receiving significant research attention. However, identification of ACE-I inhibitory peptides from different food proteins is a labor-intensive, lengthy, and expensive process. For successful identification of potential ACE-I inhibitory peptides from food sources, a machine learning and structural bioinformatics-based web server has been developed and reported in this study. The web server can take input in the FASTA format or through UniProt ID to perform the in silico gastrointestinal digestion and then screen the resulting peptides for ACE-I inhibitory activity. This unique platform provides elaborated structural and functional features of the active peptides and their interaction with ACE-I. Thus, it can potentially enhance the efficacy and reduce the time and cost in identifying and characterizing novel ACE-I inhibitory peptides from food proteins. URL: http://hazralab.iitr.ac.in/ahpp/index.php.

Bioactivity of Cooked Standard and Enriched Whole Eggs from White Leghorn and Rhode Island Red in Exhibiting In-Vitro Antioxidant and ACE-Inhibitory Effects.

Hen breed, diet enrichment, cooking methods, and gastrointestinal (GI) digestion modulates the bioaccessibility of the bioactive compounds in eggs, but their synergistic role in modulating bioactivity is still unclear. The present study evaluates the effect of hen breed, diet enrichment, and GI digestion on the cooked whole egg-derived peptides in-vitro antioxidant and antihypertensive activities. Standard and enriched whole eggs from White Leghorn (WLH) and Rhode Island Red (RIR) hens were boiled or fried and subjected to GI digestion. Antioxidant activity was measured through oxygen radical absorbance capacity (ORAC) and gastrointestinal epithelial cell-based assays, and the antihypertensive capacity by in-vitro Angiotensin-I Converting Enzyme (ACE) inhibition assay. WLH fried standard egg hydrolysate showed a high ORAC antioxidant activity but failed to show any significant antioxidant effect in the cell-based assay. No significant differences were observed in the antihypertensive activity, although enriched samples tended to have a higher ACE-inhibitory capacity. The peptide profile explained the antioxidant capacities based on antioxidant structural requirements from different peptide fractions, while previously reported antihypertensive peptides were found in all samples. The study validates the importance of physiologically relevant models and requires future studies to confirm mechanisms that yield bioactive compounds in whole egg hydrolysates.

Evaluating the effect of high-pressure processing in contrast to boiling on the antioxidant activity from alcalase hydrolysate of Great Northern Beans (Phaseolus vulgaris).

Food processing can alter protein structure, modulate enzyme accessibility, and therefore the release of bioactive peptides. Thus, processing techniques, boiling, high-pressure processing (HPP), and a combination of both, were compared for their efficiency to release antioxidant peptides after alcalase hydrolysis of Great Northern Beans (GNBs). The oxygen radical absorbance capacity (ORAC) indicated that boiled hydrolysates had the highest antioxidant activity (370.9 ± 43.8 µmol TEAC/g). Mass spectrometry-based analysis suggested that di- and tri-peptide expression were significantly altered among the three treatments, and either Ile, Leu, Phe, and Arg containing peptides potentially contributed toward the enhanced antioxidant activity. Furthermore, the total phenolic content of the HPP-treated hydrolysate was higher than the other two treatments, with ferulic acid being the most prominent phenolic compound present in the bean hydrolysates. This study indicates that thermal processing such as boiling is more effective in modulating the release of antioxidant peptides. PRACTICAL APPLICATIONS: Common beans (Phaseolus vulgaris), such as Great Northern Beans (GNBs) are one of the major pulse crops in the United States. Storage proteins in beans can release peptides with biological activities after enzymatic hydrolysis. However, processing conditions can modulate the release of peptides. The present study is primarily focused on comparing the two processing methods, boiling and HPP, and their combination for the generation of peptides with potential antioxidant activity in alcalase-digested GNBs. Data from the study suggest that thermal treatment such as boiling is more effective in modulating the release of peptides from alcalase hydrolysate of GNBs with antioxidant activity. This is particularly important because over different cultures around the world, boiling is the most widely used processing method for the cooking of beans, and hence, these data also ensure that boiling is the most effective method in getting the most beneficial effects from the consumption of beans.

Transport of Dietary Anti-Inflammatory Peptide, γ-Glutamyl Valine (γ-EV), across the Intestinal Caco-2 Monolayer.

The present study analyzed the transepithelial transport of the dietary anti-inflammatory peptide, γ-glutamyl valine (γ-EV). γ-EV is naturally found in dry edible beans. Our previous study demonstrated the anti-inflammatory potency of γ-EV against vascular inflammation at a concentration of 1mM, and that it can transport with the apparent permeability coefficient (Papp) of 1.56 × 10-6 ± 0.7 × 10-6 cm/s across the intestinal Caco-2 cells. The purpose of the current study was to explore whether the permeability of the peptide could be enhanced and to elucidate the mechanism of transport of γ-EV across Caco-2 cells. The initial results indicated that γ-EV was nontoxic to the Caco-2 cells up to 5 mM concentration and could be transported across the intestinal cells intact. During apical-to-basolateral transport, a higher peptide dose (5 mM) significantly (p < 0.01) enhanced the transport rate to 2.5 × 10-6 ± 0.6 × 10-6 cm/s. Cytochalasin-D disintegrated the tight-junction proteins of the Caco-2 monolayer and increased the Papp of γ-EV to 4.36 × 10-6 ± 0.16 × 10-6 cm/s (p < 0.001), while theaflavin 3'-gallate and Gly-Sar significantly decreased the Papp (p < 0.05), with wortmannin having no effects on the peptide transport, indicating that the transport route of γ-EV could be via both PepT1-mediated and paracellular.

Evaluating the effect of cooking and gastrointestinal digestion in modulating the bio-accessibility of different bioactive compounds of eggs.

Eggs' nutritional value has been enhanced by enriching hen's diet with bioactive compounds, but factors influencing bio-accessibility are unspecified. This study investigated the effect of hen breed, diet enrichment, and cooking methods in modulating the egg compounds' bio-accessibility after gastrointestinal (GI) digestion. White Leghorn (WLH) and Rhode Island Red (RIR) hens were fed a corn-soybean-based diet enriched with flaxseed and carotenoids; eggs were collected, cooked, and subjected to simulated GI digestion. The results showed that egg proteins were equally digestible with no change in the degree of hydrolysis (DH). The linolenic fatty acid in enriched-cooked samples remained bio-accessible after GI digestion. The lutein bio-accessibility in enriched eggs decreased after GI digestion except in RIR fried sample. Eggs from WLH and RIR achieved similar peptide content after GI digestion. These results elucidate the bio-accessibility of different bioactive compounds in cooked eggs and the use of eggs as potential functional foods.

Effect of pH and Heat Treatment on the Antioxidant Activity of Egg White Protein-Derived Peptides after Simulated In-Vitro Gastrointestinal Digestion.

The study aimed to analyze pH and heat treatment's effect in modulating the release of peptides with antioxidant activity after simulated gastrointestinal (GI) digestion of Egg white powder (EWP). EWP samples with neutral (EWPN) and alkaline (EWPA) pH were heat-treated at 20, 60, and 90 °C and analyzed for protein aggregation, solubility, and GI digestibility. Heat treatment decreased solubility and induced protein aggregation, which was higher for EWPN as compared to EWPA. The unfolding of EWPA proteins at 60 °C exhibited a higher GI digestibility and antioxidant activity via Oxygen Radical Absorbance Capacity (ORAC) assay as compared to EWPN. Interestingly, a reverse trend was observed in the cellular antioxidant assay, and the GI-digest of EWPN exhibited a higher antioxidant activity. The LC-MS/MS analysis are in concordance with cellular antioxidant activity assay and showed a higher intensity for peptides with potential antioxidant activity in the GI-digest of EWPN. The results indicate that heat treatment but not the pH is a critical factor in improving the protein digestibility and releasing peptides with antioxidant activity after GI digestion.

Dietary γ-Glutamyl Valine Ameliorates TNF-α-Induced Vascular Inflammation via Endothelial Calcium-Sensing Receptors.

γ-Glutamyl valine (γ-EV), commonly found in edible beans, was shown to reduce gastrointestinal inflammation via activation of calcium-sensing receptors (CaSRs). The present study aimed to evaluate the efficacy of γ-EV in modulating the tumor necrosis factor-α-induced inflammatory responses in endothelial cells (ECs) via CaSR-mediated pathways. Human aortic ECs (HAoECs) were pretreated (2 h) with γ-EV (0.01, 0.1, and 1 mM). 1 mM pretreatment of γ-EV significantly reduced the upregulation of inflammatory adhesion molecules, VCAM-1 and E-selectin, by 44.56 and 57.41%, respectively. The production of cytokines IL-8 and IL-6 was significantly reduced by 40 and 51%, respectively, with 1 mM pretreatment of γ-EV. Similarly, there was a significant reduction in chemokine MCP-1 from a positive control of 9.70 ± 0.52 to 6.6 ± 0.43 ng/mL, after γ-EV treatment. The anti-inflammatory effect of γ-EV was attenuated by the treatment of the CaSR-specific inhibitor, NPS-2143, suggesting the involvement of CaSR-mediated pathways. Further studies identified the critical role of key modulators, such as ß-arrestin2 and cyclic adenosine monophosphate response element-binding protein, in mediating the CaSR-dependent anti-inflammatory effect of γ-EV. Finally, the transport efficiency of γ-EV was evaluated through a monolayer of intestinal epithelial cells (Caco-2), and the apparent permeability (Papp) of the peptide was found to be 1.56 × 10-6 cm/s.

Immuno-informatics approach for multi-epitope vaccine designing against SARS-CoV-2.

The novel Corona Virus Disease 2019 (COVID-19) pandemic has set the fatality rates ablaze across the world. So, to combat this disease, we have designed a multi-epitope vaccine from various proteins of Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) with an immuno-informatics approach, validated in silico to be stable, non-allergic and antigenic. Cytotoxic T-cell, helper T-cell, and B-cell epitopes were computationally predicted from six conserved protein sequences among four viral strains isolated across the world. The T-cell epitopes, overlapping with the B-cell epitopes, were included in the vaccine construct to assure the humoral and cell-mediated immune response. The beta-subunit of cholera toxin was added as an adjuvant at the N-terminal of the construct to increase immunogenicity. Interferon-gamma inducing epitopes were even predicted in the vaccine. Molecular docking and binding energetics studies revealed strong interactions of the vaccine with immune-stimulatory toll-like receptors (TLR) -2, 3, 4. Molecular dynamics simulation of the vaccine ensured in vivo stability in the biological system. The immune simulation of vaccine evinced elevated immune response. The efficient translation of the vaccine in an expression vector was assured utilizing in silico cloning approach. Certainly, such a vaccine construct could reliably be effective against COVID-19.

Oral intervention of Lactobacillus pentosus S-PT84 attenuates the allergenic responses in a BALB/C mouse model of egg allergy.

Food protein-induced allergies are primarily aggravated due to imbalance immune responses. Earlier studies by different research groups have reported that the intervention of Lactobacillus pentosus (L. pentosus) S-PT84 can modulate T-helper (Th)1/Th2 balance through regulatory T cells and can effectively promote type 1 immunity by activating dendritic cells and natural killer cells, such biological activity makes L. pentosus S-PT84 a potential mediation in controlling food allergy. Thus, this study aimed to evaluate the effects of L. pentosus S-PT84 against egg ovalbumin (OVA)-induced allergic response in mice. BALB/c mice (n = 12/group) were sensitized with OVA (50 µg/mice) via intraperitoneal injection (IP) for four weeks and subsequently administered with three different doses of L. pentosus S-PT84 via pelleted diet. The allergenic status was assessed by clinical signs, serum histamine, mouse mast cell protease (MMCP) level, and antibody activity, cytokines level in splenocytes, and expression of T regulatory cells (T-regs) in blood. The intervention of L. pentosus S-PT84, precisely at the high dose (0.6 % L. pentosus S-PT84 in pelleted diet) group, significantly reduced the clinical allergenic symptoms and reduced the histamine and MMCP levels in serum. However, the intervention of L. pentosus S-PT84 did not affect the OVA-specific IgE, IgG concentration, but led to lower the total IgE and IgG titers, suggesting that the therapeutic effect of L. pentosus S-PT84 may be due to development of immune tolerance. Moreover, differences in the immune response were observed after L. pentosus S-PT84 intervention, as it significantly reduced the production of IL-4, IL-17, and increased the population of CD25+Foxp3+ cells. Thus, it can be concluded that the intervention of L. pentosus S-PT84 provides a potential therapeutic strategy to reduce the chicken egg OVA-induced allergic symptoms.

Prophylactic effects of isomaltodextrin in a Balb/c mouse model of egg allergy.

The aim of this study was to evaluate the potential effects of isomaltodextrin (IMD), a dietary saccharide polymer derived from enzymatically produced from starch, on the ability to alter immune response (IR) bias to hen egg ovalbumin (Ova) induced allergic inflammation in mice. Groups of Balb/c mice were pre-treated with various doses of IMD in drinking water (1.0, 2.5, and 5.0% w/v) for 6 weeks and subsequently sensitized to the Ova together with continuous administration of IMD. To evaluate changes in immune response bias, immunoglobulin isotype-associated antibody activity, concentrations of type 1 and 2 cytokines and the percentage of T-regulatory cells (T-regs) in blood were measured. Clinical signs of allergy were assessed after oral challenge with Ova. Treatment with IMD did not significantly alter the frequency of clinical signs, however there was a trend in the overall reduction of clinical signs. Effect on IR bias was observed in the treatment groups as reflected by reduction in a type 1-biased phenotype as evident by decrease in isotype-specific IgE, IgG and increase in IL-12 cytokine production and a high proportion of T-regs. This study revealed that IMD could be a useful prophylactic candidate for alteration of allergic IR bias in mice and an immune-stimulator for reducing egg induced allergic reactions.

γ-Glutamylvaline Prevents Low-Grade Chronic Inflammation via Activation of a Calcium-Sensing Receptor Pathway in 3T3-L1Mouse Adipocytes.

The calcium-sensing receptor (CaSR), a G-protein receptor, is well recognized for its role in the regulation of adipocyte proliferation, in modulating adipose tissue dysfunction, and as a potential target for therapeutic intervention. In the present study, we investigate the anti-inflammatory effect of γ-glutamylvaline (γ-EV) on mouse adipocytes and explore the role of γ-EV-activated CaSR in the regulation of cellular homeostasis using the mouse 3T3-L1 cell line in vitro model. Our results indicate that the 3T3-L1 adipocyte-like cells accumulated lipids and expressed CaSR after 2 days of differentiation and 7 days of maturation period. The pretreatment with γ-EV (10 µM) suppressed the production of TNF-α-induced pro-inflammatory cytokines, i.e., IL-6 (23.92 ± 5.45 ng/mL, p < 0.05)) and MCP-1 (101.17 ± 39.93 ng/mL, p < 0.05), while enhancing the expression of PPARγ (1.249 ± 0.109, p < 0.001) and adiponectin (7.37 ± 0.59 ng/mL, p < 0.05). Elevated expression of Wnt5a was detected in γ-EV-treated cells (115.90 ± 45.50, p < 0.001), suggesting the involvement of the Wnt/ß-catenin pathway. Also, phosphorylation of ß-catenin was shown to be significantly inhibited (0.442 ± 0.034) by TNF-α but restored when cells were pretreated with γ-EV (0.765 ± 0.048, p < 0.05). These findings suggest that γ-EV-induced CaSR activation not only prevents TNF-α-induced inflammation in adipocytes but also modulates the cross-talk between Wnt and PPARγ pathways. Concentrations of serine phosphorylated IRS-1 were shown to be lower in γ-EV-treated cells, indicating γ-EV may also prevent inflammation in the context of insulin resistance. Thus, γ-EV-activated CaSR plays a significant role in the cross-talk between adipocyte inflammatory and metabolic pathways through the regulation of extracellular sensing.

Structural-features of food-derived bioactive peptides with anti-inflammatory activity: A brief review.

Chronic inflammatory conditions such as obesity, type-2 diabetes, and cardiovascular diseases are the leading causes of mortality worldwide. Hence, much research interest in bioactive peptides has been stimulated due to lack of potent pharmacological interventions. Although many such peptides have been identified from food proteins, insufficient information is available on their structure-function relationship. Presence of hydrophobic and positively charged amino acids is a common occurrence for the peptides with anti-inflammatory properties. However, inconsistent findings have also been reported. Most of the food-derived peptides exhibited their anti-inflammatory activities primarily by inhibiting signaling components of either NF-κB or MAPK pathway, which are the two major pathways involved in chronic inflammation following uncontrolled signal activation. This review highlighted the structural requirements of the peptides to exhibit anti-inflammatory activity based on the current knowledge about food-derived anti-inflammatory peptides and their underlying molecular mechanisms of action. PRACTICAL APPLICATIONS: While research in the food-derived bioactive peptide is gaining momentum, but the ability to translate these new findings into the commercial product such as nutraceuticals and functional foods, remains delayed. The most prominent reasons for this delay are the lack of detailed research on, (i) the structure-function relationship of the peptide and the underlying molecular mechanisms of these bioactive peptides, and (ii) the interaction of these peptides with different cellular elements in the disease pathophysiology. This review gives an insight into the structure-activity relationship of bioactive peptides involved in anti-inflammatory responses. The information provided here would be highly beneficial to describe the possible anti-inflammatory activity of any newly identified peptides from different food sources.

Food-derived bioactive peptides and their role in ameliorating hypertension and associated cardiovascular diseases.

Non-communicable diseases including cardiovascular diseases (CVDs) and associated metabolic disorders are responsible for nearly 40 million deaths globally per year. Hypertension or high blood pressure (BP) is one of the primary reasons for the development of CVDs. A healthy nutritional strategy complementing with physical activity can substantially reduce high BP and prevent the occurrence of CVD-associated morbidity and mortality. Bioactive peptides currently are the next wave of the promising bench to clinic options for potential targeting chronic and acute health issues including hypertension. Peptides demonstrating anti-inflammatory, anti-oxidant, and angiotensin-converting enzyme-I inhibitory activity are widely studied for the amelioration of hypertension and associated CVDs. Isolating these potent bioactive peptides from different food sources is a promising endeavor toward nutraceutical based dietary management and prevention of hypertension. Understanding the pathophysiology of hypertension and the action mechanisms of the bioactive peptides would complement in designing and characterizing more potent peptides and suitable comprehensive dietary plans for the prevention of hypertension and associated CVDs.

Food-Derived Bioactive Peptides in Human Health: Challenges and Opportunities.

Recent scientific evidence suggests that food proteins not only serve as nutrients, but can also modulate the body's physiological functions. These physiological functions are primarily regulated by some peptides that are encrypted in the native protein sequences. These bioactive peptides can exert health beneficial properties and thus are considered as a lead compound for the development of nutraceuticals or functional foods. In the past few decades, a wide range of food-derived bioactive peptide sequences have been identified, with multiple health beneficial activities. However, the commercial application of these bioactive peptides has been delayed because of the absence of appropriate and scalable production methods, proper exploration of the mechanisms of action, high gastro-intestinal digestibility, variable absorption rate, and the lack of well-designed clinical trials to provide the substantial evidence for potential health claims. This review article discusses the current techniques, challenges of the current bioactive peptide production techniques, the oral use and gastrointestinal bioavailability of these food-derived bioactive peptides, and the overall regulatory environment.

N-Glycoproteomic Analysis of Chicken Egg Yolk.

Posttranslational N-glycosylation of food proteins plays a critical role in their structure and function. However, the N-glycoproteome of chicken egg yolk (CEY) has not been studied yet. Glycopeptides hydrolyzed from CEY proteins were enriched, with deglycosylation occurring using PNGase F, and then were identified using a shotgun glycoproteomics strategy. A total of 217 N-glycosylation sites and 86 glycoproteins were identified in CEY, and these glycoproteins are mainly involved in the binding, biological regulation, catalytic activity, and metabolic processes. Among the identified CEY glycoproteins, 22 were recognized as proteases and protease inhibitors, suggesting that a proteinase/inhibitor regulation system exists in CEY; further, 15 were members of the complement and immune systems, which provide protection against potential threats during hatching. The study provides important structural information about CEY glycoproteins and aids in the understanding of the underlying mechanism of embryo development as well as changes in CEY functional characteristics during storage and processing.