2.3.2. Sonication

SO extraction was performed by sonicating soaked EBN in a beaker for 30 min at room temperature (25 ± 1 ◦C) using an ultrasonic cleaner (42 kHz, 135 W; Branson Ultrasonic Corporation, Fairfield County, CT, USA). The water level in the beaker was kept at the same level of water as the ultrasonic bath, maintained at a constant room temperature (25 ± 1 ◦C). Next, the aliquot was filtered using a muslin cloth to obtain the extract.

## 2.3.3. Hot Water

Direct heat from boiling water was used to obtained the HW extract. The soaked EBN was placed in a beaker and heated for 30 min at 100 ◦C in a water bath. It was then cooled to room temperature and subsequently filtered through a muslin cloth to obtain the extract.

#### *2.4. Determination of Sialic Acid by HPLC*

The sialic acid content was evaluated based on [13] using high-performance liquid chromatography (HPLC) (Agilent1200) with a reversed-phase Agilent HC-C18 column (4.5 × 250 mm, 5 μm). The methanol, acetonitrile, and water solution (7:8:85) were used as a mobile phase, with a flow rate of 0.9 mL/min. A fluorescent detector was used, with the excitation wavelength at 373 nm and emission wavelength at 448 nm.

#### *2.5. Enzymatic Hydrolysis of EBN*

Enzymatic hydrolysis, following simulated gastrointestinal digestion, was performed to produce edible bird's nest (EBN) protein hydrolysate as previously described by [3], with slight modifications. The pH of EBN extract (5 mg/mL) was adjusted to 2.0 using HCl (20 mL) and digested with pepsin (1% (*w/w*) in 0.1 M potassium chloride). The pH-adjusted sample was put in a shaking incubator for 2 h at 37 ◦C and boiled for 20 min to stop the pepsin activity. The pH of the mixture was increased to 8 with 1 M NaOH and further digested with pancreatin (1% (*w/w*) in 0.1 M potassium phosphate buffer) for another 2 h at 37 ◦C to simulate small intestine conditions. The reaction was stopped by boiling the sample for 30 min and immediately cooling at pH 8.9. The hydrolyzed solution was centrifuged at 10,000× *g* at 4 ◦C for 30 min and desalted using snakeskin pleated dialysis tubing at 7.0 K MWCO (Thermo Fisher Scientific Inc., Watham, MA, USA). The hydrolysate was subsequently frozen at −80 ◦C, lyophilized, and stored at −20 ◦C until further use.

#### *2.6. Estimation of Soluble Protein*

The soluble protein content of EBN samples was determined using the Bradford protein assay kit following the manufacturer's instructions (Catalog Number: P010; Gene Copeia, Rockville, MD, USA) [29]. About 100 μL of the sample and bovine serum albumin (BSA) standard were added into 96-well plate, followed by the addition of 100 μL of Bradford reagen<sup>t</sup> to each well. The mixture was mixed with a plate shaker for 30 s and incubated for 5 min at room temperature. Protein concentration was calculated according to the standard protein curve of BSA, and absorbance was read at 595 nm.

#### *2.7. Protein Separation and Molecular Weight Determination Using SDS-PAGE*

The samples for purification and determining the molecular weight of the protein were prepared using optimized half-cup EBN extracts (FS and SE) and hydrolysates (FSh and SEh). The sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) was prepared according to [30], with some modifications. The 5% (*w/v*) of EBN was loaded in 12% resolving gel and 4% stacking gel in a 1:1 (*v/v*) ratio containing Tris buffer. The solution was heated in a 90 ◦C water bath for 20 min and then cooled immediately. Then, 20 μL of the sample and 20 μL of the protein standard were loaded into individual wells and run under the constant current setting of 30 mA and 150 V for 15 min before being increased to 200 V for another 45 min. Proteins were stained with 0.1% (*w/v*) Coomassie blue, with protein markers in the range of 11 to 245 kDa.

#### *2.8. Protein Identification by LC-MS/MS Q-TOF*

#### 2.8.1. EBN Protein Digestion

In-solution digestion of the EBN sample was carried out according to the manufacturer's instructions [31]. RapiGest solution (0.2%, *w/v*) was prepared by resuspending 1 mg RapiGestTM (Waters) in 500 μL of 50 mM ammonium bicarbonate. EBN extract (100 μg) was then dissolved in 50 μL of 0.2% RapiGest solution and vortexed. DTT was added to the mixture to a final concentration of 5 mM for the reduction step before boiling at 60 ◦C for 30 min. The mixture was cooled to room temperature, before being alkylated with iodoacetamide to a final concentration of 15 mM for 30 min in the dark environment. The proteolytic digestion step was performed by adding mass spectrometry grade Trypsin Gold (Promega, Madison, WI, USA) at a ratio of 1:50 (trypsin/protein), followed by incubation overnight at 37 ◦C. At the end of the digestion step, 1 μL of formic acid was added to stop trypsin activity. The digested protein samples were stored at −20 ◦C before protein and peptide identification.

2.8.2. Liquid Chromatography-Tandem Mass Spectrometry Coupled Quadrupole-Time of Flight (LC-MS/MS Q-TOF) Analysis

LC-MS/MS analysis was performed using 6550 iFunnel Q-TOF LC/MS from Agilent Technologies (Santa Clara, CA, USA). A total of 5 μL digested EBN was loaded into an Agilent Large Capacity Chip consisting of a 75 μm × 150 mm analytical column and a 160 mL enrichment column, which was packed by 5 mM of Zorbax 300SB-C18 for chromatographic separation. The mobile phase consisted of solvent A (0.1% formic acid in MilliQ water) and Solvent B (9:1 ratio of 0.1% formic acid in acetonitrile: MilliQ water); a flow rate of 1.0 mL/min was used to elute the peptides. The mobile phase gradient was programmed as 3–50% of solvent B for 30 min; 50–95% of solvent B for 2 min; 95% of solvent B for 7 min; and 95–3% of solvent B for 47 min. The polarity of Q-TOF was set at positive, the voltages for capillary (2050 V) and fragment (300 V) were set accordingly, and the gas flow was set at 5 L/min and 325 ◦C. The peptide spectra were acquired using Agilent MassHunter Workstation Data Acquisition software (Agilent Technologies, Santa Clara, CA, USA) by monitored positive ion acquisition in the range of 110 to 3000 m/z for the MS scan, and 50 to 3000 m/z for the MS/MS scan. The chromatograms obtained were analyzed using the Agilent MassHunter Qualitative Analysis B.05.00 software (Agilent Technologies, Santa Clara, CA, USA).
