Hydrolysis Time Differentially Affects Antioxidant and Anti-Inflammatory Activities of Goose Egg (Anser anser) Ovomucoid Peptides

Eggs are an important source of animal protein, rich in vitamins, minerals, essential fatty acids, phospholipids, sphingomyelin, lutein, and zeaxanthin. Furthermore, they contain various bioactive components, such as antioxidants, anti-inflammatory agents, and choline (Sanlier and Ustun., 2021). A goose egg typically weighs about 162 grams, but in Asia, especially in Indonesia, ranges from approximately 120-130 g and it contains albumen (57.2%), yolk (30.6%), and eggshell (12.2%) (Kokoszyński., 2017; Karabulut., 2021). The bulk composition of fresh raw eggs includes water (76.1%), protein (12.6%), fat (9.5%), ash (1.1%), and carbohydrates (0.7%) (Sarantidi et al., 2023). Currently, very limited literature exists regarding the specific protein composition of goose egg white. However, as a general reference, chicken egg white proteins are composed of ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), lysozyme (3.5%), and ovomucin (3.5%), all of which are closely associated with albumen viscosity. Minor proteins include avidin (0.05%), cystatin (0.05%), ovomacroglobulin (ovostatin) (0.5%), ovoflavoprotein (0.8%), ovoglycoprotein (1.0%), and ovoinhibitor (1.5%) (Abeyrathne et al., 2013; Abeyrathne et al., 2018). Ovomucoid is the most heavily glycosylated protein in egg whites, with a molecular weight of 20.1 kDa, typically visualized in SDS-PAGE gels in the range of 30-40 kDa (Abeyrathne et al., 2013; da Silva et al., 2019). As a glycoprotein, ovomucoid contains a high proportion of carbohydrates (approximately 25-30% of the overall mass), making it heat-stable and resistant to clumping together (Razi et al., 2023).

As a major component of egg white, chicken egg white ovomucoid has been found to exhibit functional properties, including antioxidant and anti-inflammatory activities (Abeyrathne et al., 2015; Xia et al., 2024; Yubolphan et al., 2025). To maximize the utilization of these functional properties, enzymatic hydrolysis is a well-known method. Protein hydrolysates are defined as a mixture of polypeptides, oligopeptides, and amino acids resulting from the hydrolysis of various protein sources (McCarthy et al., 2013). Specifically, enzymatic hydrolysis is employed to break down protein bonds into smaller peptides that are more readily absorbed by the body (Palupi et al., 2010). Enzymes, as biological catalysts, function to accelerate specific biochemical reactions (Chew et al., 2019). Alcalase is known as one of the most efficient enzymes for releasing bioactive peptides from diverse protein sources (Tacias-Pascacio et al., 2020). Alcalase exhibits high cutting specificity, particularly targeting aromatic (Phe, Trp, Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu, Ala), hydroxyl (Ser), and basic (Lys) residues (Cui et al., 2021).

Peptides are protein-derived biomolecules typically composed of 2 to 20 amino acids and have a molecular weight of less than 6000 Da. They possess diverse bioactivities that contribute to human health (Chalamaiah et al., 2019; He et al., 2019; Cruz-Casas et al., 2021). Bioactive peptides are molecules exhibiting significant health-beneficial effects, including potential antimicrobial, antihypertensive, antioxidant, anticancer, immunomodulatory, and anti-inflammatory activities (Fernandez-Tome et al., 2019; Liu et al., 2022). Hydrolyzed proteins are classified based on peptide size into partially hydrolyzed (peptides with a molecular weight of 3-10 kDa) and extensively hydrolyzed (peptides with a molecular weight of < 3 kDa) (Nutten., 2016; Vandenplas et al., 2022). Currently, many studies have investigated the functional properties of ovomucoid and its hydrolysates regarding antioxidant and anti-inflammatory activities in chickens. However, there is still a lack of comprehensive studies specifically examining the effective hydrolysis time of goose egg white ovomucoid hydrolysates to produce optimal antioxidant and anti-inflammatory activities. (Abeyrathne et al., 2015; Yubolphan et al., 2025).

Goose eggs collected 1 to 5 days after laying, were sourced from local suppliers at the Pasty Market in the Special Region of Yogyakarta (Daerah Istimewa Yogyakarta). The eggs used had an average weight of approximately 150 grams. Materials used in this study are Ethanol absolute, Aquades, FeCl3, Bio-Rad electrophoresis gel, SDS-PAGE buffer, Loading dye reagent, Staining gel solution, Destaining gel solution, Smobio protein marker PM 5100, Bradford solution, Ninhydrin solution, Methanol, DPPH reagent (Sigma), Potassium persulfate, ABTS reagent (Sigma), Sodium chloride, Potassium chloride, Sodium phosphate dibasic, potassium phosphate monobasic, Sulfuric acid (H2SO4), Sodium phosphate (Na3PO4), Ammonium molybdate, Phosphomolybdate reagent solution, Potassium ferricyanide (1%). Ovomucoid protein was extracted from the goose egg white. The purified ovomucoid was subsequently subjected to enzymatic hydrolysis using Alcalase. The hydrolysis was performed at various time intervals (0, 15, 30, and 60 minutes) and all treatments were conducted in triplicate.

Ovomucoid was extracted and purified based on the method described by Abeyrathne et al. (2014) with slight modifications. Initially, the egg white was separated from the yolk and diluted with one volume of distilled water (1:1, v/v). Subsequently, Fe3+ was added at a concentration of 0.8 mL/L of 500 mM FeCl3 to separate ovomucoid from ovotransferrin through specific binding to ovotransferrin. The mixture was homogenized using a hand mixer for approximately 2 minutes. Precipitation was induced by the slow addition of cold 100% absolute ethanol to achieve a final concentration of 43%. The solution was centrifuged at 3,400 x g for 20 minutes at 4oC. The resulting supernatant, containing the ovomucoid, was collected, re-extracted with two volumes of 43% ethanol, and subsequently stored overnight at 4oC. The mixture was centrifuged again under the same conditions 3,400 x g for 20 minutes at 4oC. The supernatant was collected, and the residual ethanol was subsequently removed via a rotary evaporator. The extract was then heated in a water bath at 80oC for 30 minutes before undergoing the final drying stage using a freeze dryer (lyophilization).

The hydrolysis was performed based on the method described by (Abeyrathne et al., 2015) with minor modifications. The freeze-dried ovomucoid was weighed using an analytical balance and subsequently dissolved in distilled water to a concentration of 0.23 mg/mL (total volume 1 mL), because the total yield produced during the extraction process was very low, approximately 6.77 g from the total egg weight. The Alcalase enzyme was subsequently added, and the mixture was incubated at 50oC for various durations: 0, 15, 30, and 60 min. To terminate the enzymatic reaction, the samples were immediately heated at 90oC for 10 minutes.

The purity, protein amount, and success of ovomucoid hydrolysis were assessed using the SDS-PAGE method, adapted from the procedures of (Abeyrathne et al., 2014) and (Prihanto et al., 2019). SDS-PAGE analysis was performed on the ovomucoid isolate (after extraction) and on the final hydrolysate samples. Samples 3 μL were prepared in a microtube by adding 7 μL of distilled water and 3 μL of loading dye reagent. The prepared samples were loaded into a 12-well SDS polyacrylamide gel. Each well was filled with a total volume of 13 μL of the prepared sample mixture. Electrophoresis was run at a constant voltage of 80-100 Volts. Following electrophoresis, the gel underwent staining and destaining until the background was sufficiently clear. The protein bands were then visualized and documented using a Gel Doc system.

The yield of ovomucoid obtained after the isolation process was determined based on the AOAC method (2005). The yield was calculated by weighing the initial mass of goose egg white and the final mass of the ovomucoid powder obtained after extraction and freeze-drying. The yield (%) was calculated using the following formula:

Protein concentration was determined using the Bradford assay, following the method described by (Kielkopf et al., 2020). Briefly, 10 μL of the prepared protein extract sample was placed into a microtube, followed by the addition of 180 μL of Bradford solution. The samples were incubated until the color reaction stabilized (i.e., turned blue), and then the absorbance was measured using a spectrophotometer at a wavelength of 595 nm.

The concentration of free amino groups in the ovomucoid hydrolysates was determined using the Ninhydrin assay, based on the method by (McGrath., 1972). Ovomucoid hydrolysate samples (0.131 mg/mL) were analyzed. For the assay, the samples were diluted with distilled water to a total volume of 5 mL. The absorbance of the resulting solutions was measured using a spectrophotometer at an absorbance of 570 nm. The concentration of free amino groups was calculated using the standard linear regression equation of the amino acid glycine (Y = 0.009888 X + 0.06462) (Figure 1).

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was determined following the procedure outlined by (Xue et al., 2019). Briefly, 300 μL of the ovomucoid hydrolysate sample (from the 0, 15, 30, and 60 minute hydrolysis times) was mixed with 300 μL of 0.1 mM DPPH solution in a microtube (1:1 ratio, v/v). The mixture was incubated at room temperature (approx. 25oC) in the dark for 30 minutes. The absorbance of the reaction mixture was measured using a spectrophotometer at 517 nm. The scavenging activity was calculated using the following formula:

The ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity was assessed following the method of (da Silva et al., 2019). with modifications. The ABTS working solution, prepared and incubated for 24 hours, was used for the assay. (Note: The 1:19 ratio mentioned conflicts with the volumes below, so this sentence is simplified). A 300 μL aliquot of the ovomucoid hydrolysate sample (from the 0, 15, 30, and 60 minute hydrolysis times) was mixed with 950 μL of the ABTS working solution. The mixture was then incubated at room temperature in the dark for 30 minutes. The absorbance of the reaction mixture was measured using a spectrophotometer at 734 nm. The scavenging activity was calculated using the following formula:

The reducing power of the ovomucoid hydrolysates was determined using the method described by (Annadurai et al., 2021). The ovomucoid hydrolysate samples (from 0, 15, 30, and 60 minutes hydrolysis) were dissolved in 200 μL of 0.2 M Phosphate Buffered Saline (PBS,pH 6.6). Subsequently, 200 μL of 1% Potassium Ferricyanide was added, and the mixture was incubated in a water bath at 50oC for 20 minutes. The reaction was terminated by adding 200 μL of 10% Trichloroacetic Acid (TCA) solution and centrifuged at 10,000 rpm for 10 minutes. Then, 300 μL of the resulting supernatant was collected, mixed with 300 μL of distilled water and 60 μL of 0.1% Ferric Chloride (FeCl3) solution. The absorbance was measured using a spectrophotometer at 700 nm. The reducing power was calculated using the standard linear regression equation derived from ascorbic acid: (Y = 0.08230 X + 0.09029) (Figure 2).

The Total Antioxidant Capacity (TAC) of the ovomucoid hydrolysates was determined using the phosphomolybdenum assay, based on the methods of (Annadurai et al., 2021) and (Kpemesi et al., 2023). For the TAC assay, 70 μL of the ovomucoid hydrolysate (from 0, 15, 30, and 60 minute hydrolysis) was mixed with 700 μL of the phosphomolybdate reagent (1:10, v/v). The mixture was then incubated at 95oC for 90 minutes. Following incubation, the reaction mixture was centrifuged at 3,000 rpm for 5 minutes, and the supernatant was collected. The absorbance was measured using a spectrophotometer at 765 nm. The total antioxidant capacity was quantified using the standard linear regression equation derived from ascorbic acid: (Y = 0.08230 X + 0.09029) (Figure 3).

Anti-inflammatory activity was assessed using the Egg Albumen Denaturation Inhibition Assay, based on the method reported by Kpemissi et al. (2023). The reaction mixture consisted of 200 μL of fresh chicken egg albumen, 2,800 μL of Phosphate Buffered Saline (PBS, pH 6.4), and 2,000 μL of the ovomucoid hydrolysate (from 0, 15, 30, and 60 minute hydrolysis). The mixture was initially incubated at 37oC for 15 minutes, then subjected to heating at 70oC for 5 minutes (to induce denaturation of the target protein (albumin) as a model of inflammation), followed by immediate cooling to 37oC. The absorbance of the mixture was measured using a spectrophotometer at 660 nm. The percentage inhibition of albumen denaturation was calculated using the following formula:

The data obtained were analyzed using Analysis of Variance (ANOVA). Significant differences between the treatment means were subsequently analyzed using Duncan’s Multiple Range Test (DMRT). All statistical procedures were performed using the Statistical Product and Service Solution (SPSS) software, Version 26.0.

The ovomucoid isolated from goose egg white via ethanol extraction exhibited an average yield (%) of 13.00±0,82 of the total egg white mass (Table 1). The resulting purified protein, obtained through the ethanol extraction and heat treatment steps, is visually represented in Figure 4.

Table 1: Yield of ovomucoid from goose eggs.

SDS-PAGE analysis of the isolated ovomucoid revealed protein bands primarily in the range of 30 kDa and 40 kDa (Figure 4). This observation confirms the successful purification of ovomucoid, as its molecular weight typically falls within this region.

The initial ovomucoid protein concentration, determined using the Bradford assay, was found to be 0.23 mg/mL (corresponding to 1.15 mg of total protein in the 0.2 initial solution). The concentration of free amino acid groups of ovomucoid using ninhydrin protein assay increased over time: (210 μg/mL) at 15 minutes, (340 μg/mL) at 30 minutes, and reached the highest concentration of (510 μg/mL) at 60 minutes (Figure 5). This result suggests rapid and near-complete hydrolysis by Alcalase.

The ovomucoid protein hydrolysate exhibited the highest DPPH radical scavenging activity at the 60 minute hydrolysis time, reaching 75.83%. This optimal activity was statistically significant (P < 0.05) when compared to other hydrolysis times (Figure 6).

The ovomucoid protein hydrolysate demonstrated the highest ABTS radical scavenging activity at the 60 minute hydrolysis time, achieving an average inhibition percentage of 95.21%. This maximum activity was found to be statistically significant (P < 0.05) (Figure 7).

The reducing power of the ovomucoid hydrolysate showed a significant dependence on the hydrolysis time (P < 0.05$, Figure 8). The maximum reducing power was observed at the longest hydrolysis time, reaching an average value of 2.74 mg/g (as Ascorbic Acid Equivalents).

The Total Antioxidant Capacity (TAC) exhibited a significant dependence on the hydrolysis time (P < 0.05) Figure 9. The maximum TAC value was recorded at the 60 minute hydrolysis time, reaching 58.30 mg/g (as Ascorbic Acid Equivalents).

The anti-inflammatory activity, assessed via the egg albumen denaturation inhibition assay, showed a significant effect (P < 0.05) Figure 10. on inhibiting protein denaturation. The highest inhibition of protein denaturation 17.78% was exhibited by the native ovomucoid (unhydrolyzed protein), indicating the intrinsic anti-inflammatory potential of the whole molecule. Among the hydrolysates, the 15 minute sample displayed the greatest inhibitory effect, reaching 13.32 inhibition.

The results reported by Nurliyani et al. (2023). indicate that ovomucoid is a protein with a theoretical molecular weight of 28 kDa. However, the SDS-PAGE bands showed the apparent molecular weight of ovomucoid to be in the range of 30-40 kDa. The high purity is mainly because of the unique chemical properties of ovomucoid. The combination of ethanol extraction and heat treatment effectively denatures and precipitates most egg white proteins, such as ovalbumin, leaving only the more stable ovomucoid and ovotransferrin in solution (Abeyrathne et al., 2014). Ovomucoid’s exceptional stability to heat (up to 90–100°C) and organic solvents like ethanol is linked to its tertiary structure, which is reinforced by nine disulfide bonds (Ji et al., 2020). The initial ovomucoid solution 0.2% was determined to have a protein concentration of 0.23 mg/mL (or 1.15 mg total protein), measured using the Bradford assay. This assay relies on the binding of Coomassie Brilliant Blue G-250 dye to basic amino acid residues, particularly arginine, histidine, phenylalanine, tryptophan, and tyrosine, via hydrophobic interactions, resulting in a color shift to blue (Kielkopf et al., 2020). Variations in protein concentration and composition within the egg white, which subsequently affect the final isolated yield, can be influenced by several factors. These factors include genetics, nutrition, the poultry rearing system, the physiological status of the hen, egg storage conditions, heat treatment applied, and inherent variability among domestic poultry species (Ji et al., 2020; Nurliyani et al., 2023).

The hydrolysis process converts the large ovomucoid protein into simpler protein molecules, or peptides (Abeyrathne et al., 2015). Previous studies suggest that ovomucoid can be hydrolyzed maximally over a prolonged period, typically around 3 hours (Abeyrathne et al., 2015). The efficacy of the hydrolysis in this study is attributed to the protease used. Alcalase exhibits high specificity toward peptide bonds containing aromatic (e.g., Phe, Trp, and Tyr), acidic (Glu), sulfur-containing (Met), aliphatic (Leu and Ala), hydroxyl (Ser), and basic (Lys) amino acid residues (Cui et al., 2021). The SDS-PAGE results confirm the extensive degradation of the ovomucoid; the disappearance or significant fading of the ovomucoid band, combined with the emergence of high soluble protein concentration at 60 minutes, suggests that the protein was nearly or completely hydrolyzed into smaller fragments (Abeyrathne et al., 2015). This band fading occurs because larger proteins migrate more slowly than smaller protein molecules during electrophoresis, while very small peptides may exit the gel entirely (Roy and Kumar., 2014). The resulting small fragments are likely bioactive peptides, which are defined as biomolecules derived from proteins containing typically 2 to 20 amino acid residues and possessing a molecular weight of less than 6000 Da (Cruz-Casas et al., 2021). The presence of these small, soluble peptides are crucial for the observed increase in antioxidant activity.

The DPPH assay is a common method to evaluate the antioxidant capacity of a substance. The mechanism involves the reduction of the stable DPPH free radical upon receiving electrons or hydrogen radicals from the antioxidant, which is visually quantified by a correlated decrease in absorbance (color change). The high antioxidant activity observed in the ovomucoid hydrolysates is primarily influenced by the molecular weight and structure of the resulting peptides (Liu et al., 2021; da Silva et al., 2019). As established, these are typically small fragments containing 2 to 20 amino acid residues (Sukarno et al., 2023). Bioactive peptides from egg hydrolysates work in two main ways to neutralize free radicals that allow them to effectively neutralize free radicals (Sukarno et al., 2023). Firstly, the hydrolysis process is crucial as it reduces the size of the protein, significantly increasing the accessibility of internal functional side chains (R-groups) to reactive oxygen or nitrogen species. Secondly, the fragments function as radical terminators through the Hydrogen Atom Transfer (HAT) mechanism. The hydroxyl group within the aromatic structure of Tyrosine acts as a highly effective hydrogen donor, neutralizing the free radical and effectively breaking the radical chain.

Ovomucoid hydrolysate is what you get when ovomucoid protein is broken down into smaller fragments. These fragments possess bioactive properties and are referred to as bioactive peptides that can prevent oxidative reactions and scavenge free radicals, specifically Reactive Oxygen Species (ROS). Peptides present in egg white protein hydrolysates can inactivate ROS through electron exchange reduction (Jhony et al., 2021; Sureh., 2022).

Egg white hydrolysate exhibits hydrogen/electron donor activity, enabling it to interact with free radicals, thereby halting chain reactions or preventing radical formation (Chen et al., 2012). The antioxidant activity of goose egg ovomucoid hydrolysate, with an average value of 2.74 mg/g (as measured by RPA) and a steep increase between the 15th and 30th minutes, demonstrates a superior electron donor capacity compared to duck egg hydrolysates, which averaged 1.105 mg/g (Sureh., 2022). The bioactive peptides in the ovomucoid hydrolysate exert reducing power by facilitating electron transfer, which reduces the Fe3+ species to the Fe2+ form (Jhony et al., 2021; Sureh., 2022).

The bioactive peptides within ovomucoid hydrolysates act as electron donors, enabling them to break the radical chain and convert reactive free radicals into more stable, non-reactive products (Arabshahi-Delouee and Urooj., 2007). Bioactive peptides resulting from ovomucoid hydrolysis demonstrate the ability to scavenge free radicals, partly through the transfer of electrons to species like the phosphomolybdate complex (Hamez et al., 2018). This mechanism, particularly in the TAC assay, is based on the reduction of phosphomolybdate ions in the presence of antioxidants, resulting in the formation of a green-blue phosphate/MoV complex (Jan et al., 2013). The resulting antioxidant activity is directly proportional to the concentration of bioactive peptides in the hydrolysate. Specifically, the bioactive peptides interact with the yellow MoVI species, reducing it to the greenish-blue MoV form (MoVI + AOH MoV + AO), which confirms the formation of the phosphomolybdate (V) complex (Kpemissi et al., 2023; Sajjad et al., 2023).

Stopping protein denaturation is a major sign in evaluating anti-inflammatory potential in in vitro assays (Kpemissi et al., 2023; Hasan et al., 2024). Compounds capable of inhibiting protein denaturation are considered potential sources of anti-inflammatory agents (Das et al., 2022). The decrease in inhibition observed in the hydrolysates suggests that the smaller ovomucoid peptides may be less resistant to high temperatures during the incubation process. Hydrolysis breaks the larger protein into small peptides and amino acids, which can alter the overall structure, potentially rendering the fragments more susceptible to thermal denaturation. Thermally- or heat-induced protein denaturation, which can lead to auto-antigen formation, causes structural changes by disrupting disulfide, hydrogen, and electrostatic hydrophobic bonds (Anokwah et al., 2022; Kpemissi et al., 2023; Das et al., 2022). In biological systems, effective anti-inflammatory activity often requires the protection of the lysosomal membrane to mitigate the inflammatory response (Kpemissi et al., 2023). Anti-inflammatory substances stabilize liposomal membranes, thereby preventing the release of lysosomal components (such as hydrolytic enzymes) that otherwise increase inflammatory responses and cause tissue damage (Parameswari et al., 2019).

Alcalase effectively broke down goose egg ovomucoid, as indicated by the progressive increase in free amino groups from 210 to 510 μg/mL and the disappearance of ovomucoid protein bands during hydrolysis. Extending the hydrolysis time significantly enhanced antioxidant activity, with the 60 min hydrolysate exhibiting the highest DPPH scavenging activity (75.83%), ABTS scavenging activity (95.21%), reducing power (2.74 mg AAE/g), and total antioxidant capacity (58.30 mg AAE/g). These results demonstrate that prolonged hydrolysis promotes the release of antioxidant bioactive peptides from goose egg ovomucoid. Conversely, anti-inflammatory activity decreased following hydrolysis, with native ovomucoid showing the greatest inhibition of protein denaturation (17.78%), while the highest activity among hydrolysates was observed after 15 min of hydrolysis (13.32%). Therefore, a 60 min hydrolysis is optimal for maximizing antioxidant potential, whereas shorter hydrolysis times better preserve anti-inflammatory activity. However, it should be noted that with a larger sample size, the observed trend might become statistically significant. These findings highlight goose egg ovomucoid as a promising source of functional peptides for antioxidant applications. However, these results are from in vitro assays only, and further studies in living systems are needed.

The authors gratefully acknowledge the Center for Tropical Animal Studies, Faculty of Animal Science, Universitas Gadjah Mada, for providing facilities and research support. This study received no external funding.

The novelty of this study lies in comparing the effect of hydrolysis time on the antioxidant and anti-inflammatory potential of ovomucoid from goose (Anser anser) eggs, thereby identifying the hydrolysis duration that optimizes each bioactivity.

Ekie Maulana Al Buruuj: Methodology, Formal analysis, Investigation, Resources, Data curation, Visualization, Writing-original draft; Muhammad Fathin Hanif: Writing-review and editing; Winda Fatma Sari: Investigation, Writing-review and editing; Mohammad Zainal Abidin: Methodology, Supervision, Conceptualizing; Writing-review and editing; Nurliyani: Methodology, Supervision, Conceptualizing; Writing-review and editing

The authors declare that no generative AI and AI assisted

technology was used in the creation of this manuscript.

The authors declare that there is no conflicts of interest.