*2.9. Statistical Analysis*

Statistical analysis was performed using SPSS version 24.0. All results were expressed as mean ± standard deviation (SD). The data were statistically treated by paired-sample t-test and analysis of variance (ANOVA), with *p* < 0.05 considered to be statistically significant, and the mean was compared using Duncan's multiple range tests.

#### **3. Results and Discussion**

#### *3.1. Physicochemical Analysis (Physical, Morphology, Elemental Composition, and Microbial Content)*

The physicochemical analysis included physical measurement, chemical composition, and microbial content of half-cup and stripe-shaped EBNs. The nest cups used were half-cup and stripe-shaped, as shown in Figure 1. The half-cup EBN is U-shaped, and the stripe-shaped EBN is a hard, incomplete cup shape, with some broken parts. Table 1 shows the physical measurement of half-cup and stripe-shaped EBNs. The height, length, and weight were 4.08 ± 0.71 cm, 7.06 ± 1.33 cm, and 5.19 ± 0.18 g (half-cup) and 1.28 ± 0.15 cm, 3.26 ± 0.48 cm, and 2.26 ± 0.18 g (stripe-shaped), respectively; the weight of stripe-shaped EBN was half of that of the half-cup EBN, and the height, length, and weight between half-cup and stripe-shaped EBNs were statistically significantly different (*p* < 0.05). Similar findings of average height, length, and weight of EBNs have also been reported [16], ranging from 3.5 to 5.0 cm, 7.0 to 13.0 cm, and 5.6 ± 1.3 g, respectively. The shape of an EBN is crucial for grading purposes, whereby the price is determined. The shape of the half-cup EBN is Grade A, and the stripe-shaped EBN derived from the broken part of the half-cup EBN is classified as Grade B.

(**a**) (**b**)

**Figure 1.** EBN image: (**a**) half-cup and (**b**) stripe-shaped.



Data shown are means ± standard deviation. a b Different superscript letters in the same row indicate a statistically significant difference between half-cup and striped-shaped EBNs (*p* < 0.05). ND: Not detected; <: Less than the minimum detection limit reported; CFU: Colony-forming unit; MPN: Most probable number.

> Figure 2 shows the surface morphology of half-cup and stripe-shaped EBNs at 50× and 500× magnification by SEM. These structures are seen to be coated with a layer of a partially clear or transparent substance. Substances appearing rough and hard covered some of the surfaces of the EBNs. Micrograph images at 500× magnification showed that the EBN was unevenly structured. The surface formation of the half-cup EBN was uniformly shaped, while a rough surface can be seen in the stripe-shaped EBN. Visualization under 50× magnification for both half-cup and stripe-shaped EBNs revealed ground EBN as an irregular prism with a crystalline structure. Irregular prisms with lustrous translucent to opaque silver crystalline structures were also reported by [16]. The washing and drying process is a common step in processing EBNs, to increase the shelf-life, lower the water activity, and facilitate storage before manufacturing and packaging. Thus, no foreign or unwanted particles, such as mites, fungal structures, and feather strands, were observed.

However, drying involving heat can cause a crystalline appearance, which was observed in both half-cup and stripe-shaped EBNs using SEM.

**Figure 2.** Scanning electron micrograph of EBNs at magnification of 50× for (**a**) half-cup and (**b**) stripe-shaped, and magnification of 500× *g* for (**c**) half-cup and (**d**) stripe-shaped.

SEM with energy-dispersive X-ray (EDX) for elemental analysis was done to characterize the composition and chemical characteristics of both EBNs, as shown in Table 1. Carbon and oxygen elements in stripe-shaped EBNs were five-fold higher than half-cup EBNs. Magnesium (0.22%) was only found in the stripe-shaped EBN sample. Oxalic acid secretion, calcium availability, hydration state, and other environmental factors may influence crystal production and morphology [17]. The high calcium content in the half-cup EBN can be assumed from the translucent crystalline structure (Figure 2a), which is glassier than the stripe-shaped EBN (Figure 2b) under the same resolution at 50×; this structure is supported by calcium, which in the half-cup EBN is two-fold higher (72.94%) than calcium element in striped-shaped EBN (44.16%).

Microorganisms such as coliforms, *Escherichia coli* (*E. coli*), *Salmonella*, *Staphylococcus aureus* (*S*. *aureus*), yeast, and mold were also quantified (Table 1). The results showed that both EBNs had a similar number of aerobic plate counts (1.8 × 10<sup>8</sup> CFU/g), coliforms (1.4 × 10<sup>4</sup> CFU/g), *E. Coli* (not detected at less than 10 CFU/g), *Salmonella* (absent), and *S. aureus* (not detected at less than 3 CFU/g). However, 67 CFU/g of yeas<sup>t</sup> and mold were detected in the half-cup compared to stripe-shaped EBN. Bacteria present in EBNs may lead to food-borne disease if ingested by a human. *Salmonella sp.* and *E. coli* are the most common food-borne pathogens that can affect health conditions; they can cause severe diarrhea or meningitis, which can be fatal if untreated [32]. Based on the latest standards set by the Ministry of Health Malaysia, both types of EBN used in this study were in accordance with the specified standard that raw-clean EBNs should be free from *E. coli*, *Salmonella* sp., and *S. aureus* before being exported to another country [18]. The Standard and Industrial Research Institute of Malaysia (SIRIM) provides guidelines for Edible Bird Nest (EBN) (MS 2334:2010), including the permitted levels of microbial content: total plate count (<2.5 × 106 CFU/g), coliform (<1100 most probably number (MPN)/g), *E. coli* (<100 (MPN)/g), *S. aureus* (<100 (MPN)/g), yeas<sup>t</sup> and mold (<10 CFU), and no presence of *Salmonella sp.* [33].

Yeast, mold, and other contaminants such as mites and feathers can be located within the strands of EBNs. This was shown in the quantification analysis, after the EBN was ground into small pieces. Thus, it is important to educate consumers on the need for additional washing and boiling, which can assist in removing contaminants [33] and significantly reduce the number of bacteria before consumption [18].

#### *3.2. Chemical Analysis (Crude Protein and Amino Acid)*

Table 2 shows the crude protein content and amino acid profile of half-cup and stripe-shaped EBNs. A higher crude protein content in the half-cup (56.96 ± 0.09%) than stripe-shaped (54.70 ± 0.16%) EBN was obtained, which was significantly different for both EBNs (*p* < 0.05). These results were comparable with EBN samples from East Coast Peninsular Malaysia, ECM (Pahang: 55.48 ± 3.60%) [2], Northern Peninsular Malaysia, NM (Perak, Penang, Kedah: 53.8 ± 0.18%) and East Malaysia, EM (Sabah, Sarawak: 52.8 ± 1.04%) [18]. The major amino acids (AAs) found in both half-cup and stripe-shaped EBNs were glutamic acid (Glu), followed by aspartic acid (Asp), serine (Ser), and valine (V), which were also reported in EBN samples from ECM [2]. The similarity in the protein and amino acid profiles of the EBN samples studied by [2] and those of present study was due to the EBN samples being collected from the same region (ECM). However, the major amino acids in EBN from EM [18] were different due to different collection locations (NM and EM). Overall, both half-cup and stripe-shaped EBNs possess a high percentage of amino acids and nonessential amino acid composition. These variances could be due to different locations of EBN harvesting, the effects of processing methods, etc. Breeding sites, climate, and swiftlet diet may also affect the EBN nutrient composition [18].


**Table 2.** Crude protein content and amino acid profile of EBNs.


**Table 2.** *Cont.*

Values are expressed as mean (%) ± S.D. Means with different superscript letters in a row indicate a significant difference between half-cup and striped-shaped EBNs (*p* < 0.05).

> The nutritional value of essential amino acids (EAAs) composition contained in protein may contribute to the functional biopeptides. The readily digestible dietary EAAs are needed at an optimum level for human body requirements, e.g., EAA Gly supplementation has been reported to promote anti-inflammatory effects during endothelial inflammation, while Arg has been found to improve endothelial function in cardiovascular or overweight patients [34]. The presence of nEAA Glu in the body is very important, especially for transamination reactions in amino acid metabolism. It is involved in the synthesis of key molecules such as glutathione, which is important in the alleviation of oxidative stress and modulation of the immune response [35]. Asp plays a role as a regulator in hormone secretion and acts as a precursor for methionine (Met), threonine (Thr), isoleucine (Ile), and lysine (Lys). Similarly, Ser is the precursor of glycine (Gly), cysteine (Cys), and tryptophan (Trp) and is involved in cell signaling.

#### *3.3. EBN Extracts and Soluble Protein Concentration*

EBN extraction is affected by its solubility behavior towards water. Since protein is the major compound in EBNs, preliminary experiments found temperature affected the extracted protein of the EBN. The extraction yield of half-cup and stripe-shaped EBNs is shown in Figure 3a. The FS method showed the highest extraction yield (half-cup = 92.29 ± 2.45%; stripe-shaped = 79.35 ± 0.91%) as compared to the SE method (half-cup = 12.50 ± 0.89%; stripe-shaped = 10.99 ± 0.11%), with significant difference (*p* < 0.05). Meanwhile, less than 5% extraction yield was obtained using the SO and HW methods for both EBNs. The temperature recorded for the double-boiling technique applied for FS and SE was 73 ◦C to 80 ◦C; SO, 25 ◦C; and HW, 100 ◦C.

The double-boiled technique with indirect heating contributes to the maximal yield of the FS and SE extraction methods. There was a significant difference in extraction yield between SE and FS methods for both EBNs (*p* < 0.05), while no significant difference was observed in both EBNs using the SO and HW methods (*p* > 0.05). Thus, the different temperatures and the characteristics of the different EBNs affected the extraction yield, with the optimum temperature between 73 ◦C and 80 ◦C. Meanwhile, low temperature (below 60 ◦C) or high temperature (more than 80 ◦C) resulted in a low extraction yield, as seen with the SO and HW extraction methods, respectively. These results agree with findings reported by [16], emphasizing that the protein concentration of the EBN extract drastically increases under extraction temperatures between 60 ◦C and 80 ◦C, but gradually reduces above 80 ◦C. Thus, temperature influences the extraction and solubility of proteins in EBNs.

**Figure 3.** The (**a**) extraction yield and (**b**) protein concentration of half-cup and stripe-shaped EBNs using different extraction methods. Data shown are means ± standard deviation of triplicates. a,b,c,d,e,f: means with different letters indicate significant differences among the extraction methods and types of EBN (*p* < 0.05).

Figure 3b shows the soluble protein concentration of EBN extracts obtained from four different extraction methods. The highest protein concentration was in the half-cup EBN (SE = 435.6 mg/mL; FS = 375.6 mg/mL; SO = 113.6 mg/mL) compared to the stripe-shaped EBN (SE = 211.6 mg/mL; FS = 115.6 mg/mL; SO = 21.6 mg/mL), while the lowest soluble protein concentration was obtained in the HW extract (half-cup = 15.6 mg/mL). There were significant differences in soluble protein concentration between the two EBNs for the extraction methods applied (*p* < 0.05), but protein concentration of the half-cup FS and SE extracts were not significantly different (*p* > 0.05).

The extraction yield and soluble protein concentration in the SE method were inversely proportional with temperature. The high yield of the FS extract was a combination of soluble and insoluble fractions of the half-cup EBN, while the SE extract was mainly in soluble form. According to [36], water extraction produced the highest protein content as compared to other extraction methods, indicating that EBNs have an abundance of water-soluble proteins. Thus, the concentrated soluble protein fraction in the SE extract was contributed by its protein concentration. The protein in the insoluble fraction of the FS extract may not fully break down during the double-boiling process, resulting in the incomplete release of soluble protein. The high temperature of the HW methods (100 ◦C) may cause denaturation of the protein, and the mild heat of the SO methods (25 ± 1 ◦C) produced an incomplete breakdown of complex protein, resulting in low yield and low soluble protein concentration in both extraction methods.

The optimum extraction condition needs to be chosen to obtain the highest protein concentration and bioactivity of EBNs for further application. The FS and SE extraction methods produced a high yield of crude extract and protein concentration in the half-cup EBN compared to the stripe-shaped EBN. Thus, FS and SE extraction methods for the half-cup EBN were selected to be used for sialic determination and enzymatic hydrolysis.

#### *3.4. Sialic Acid Content of EBN Extracts*

The SE extract of the EBNs contains higher sialic acid than the FS extract, with 8.47% (*w/w*) and 7.91% (*w/w*), respectively, compared to other findings of 8.6% [4] and 11% [11]. In addition, the sialic acid content in EBNs from Malaysia are varied, with 0.70% to 1.50% from North, South, and East Peninsular Malaysia [37] and 1.17% to 3.15%, collected from North, South, and Borneo Sabah [8]. The types of habitats, environment surroundings, breeding site, nest harvesting season, and diet of the swiftlets are the factors that influence the sialic acid content of EBNs from different locations [8].

These findings agree with the protein content of half-cup EBNs in the present study, suggesting that high protein may contributed to high sialic acid content in EBNs. This is because sialic acid is a component of the glycoprotein located in the carbohydrate chains attached to soluble proteins. This results in a high sialic acid content in the SE extract as compared to FS extracts. As mentioned earlier, the soluble and an insoluble fractions of the FS extract resulted from the incomplete breakdown of protein and may reduce the water-soluble bound sialic acid contents.

Both proteins and sialic acid are major nutraceutical ingredients in EBNs. In a study conducted using EBN constituents producing synergistic antioxidative effects, sialic acid was found to ameliorate the progression of atherosclerosis and other cardiovascular disease biomarkers [7]. Sialic acid in EBNs has also shown potential antiviral properties by inhibiting viral genes, strengthening the lungs, improving skin health, and showing anti-aging properties [4]. Taken together, the findings of crude protein, amino acids, and sialic acid analyzed in the half-cup and stripe-shaped EBNs show that EBNs are a good source of protein and glycoprotein, potentially exhibiting nutritional and medicinal properties.

#### *3.5. Soluble Protein Concentration of EBN Hydrolysates*

The high soluble protein found in the half-cup EBN as reported in Section 3.3 indicates a protein-rich sample that yielded a high amount of hydrolysate. The yield of the half-cup FSh (47.5%) was comparable to half-cup SEh (51.5%), as shown in Figure 4a. Enzymatic hydrolysis potentially breaks down complex protein and helps in releasing bioactive peptides from the inactive parent protein, which enables them to exhibit biological properties for therapeutic purposes [6]. Figure 4b shows an uptrend pattern of soluble protein concentration in EBN hydrolysates. Solubility is primarily dependent on the distribution of hydrophobic and hydrophilic amino acids on the surface of the protein and is affected by the protein–water interaction.

A similar concentration of FSh and SEh in the half-cup EBN showed a significant difference in soluble protein concentration (*p* < 0.05), except for at 10,000 ppm, where the soluble protein concentration of SEh (189.80 mg/mL) was comparable to the protein concentration of FSh (179.80 mg/mL). This may be due to the presence of a strong peptide bond in the protein solution of EBNs, whereby any further increase in the concentration of EBN hydrolysate (10,000 ppm) was not able to increase the solubility of the protein due to the saturated state.

**Figure 4.** The (**a**) hydrolysis yield and (**b**) protein concentration of half-cup EBN hydrolysate. Data shown are means ± standard deviation of triplicates. FSh: full stew hydrolysate; SEh: stew hydrolysate. a,b,c Different letters indicate a significant difference between FS and SE hydrolysates at different concentrations (*p* < 0.05).

Enzymatic hydrolysis is often used to improve the solubility of the protein, and the protein degradation results in the formation of amino acids and peptides with smaller molecular masses easily absorbed by the digestive system. However, strong peptide– peptide interactions and the presence of glycoprotein that cannot be hydrolyzed result in less than 100% protein solubility [38]. Pepsin is a proteolytic enzyme secreted by the stomach, while pancreatin is produced by the pancreas, which contains other enzymes such as elastase, trypsin, and chymotrypsin proteases. Optimized extraction followed by simulated gastrointestinal digestion (pepsin and pancreatin hydrolysis) mimics the natural surroundings of the human digestive tract. This process could potentially increase the bioavailability of EBN bioactive peptides and subsequently absorb them through the gu<sup>t</sup> to exert their functional effects.

The EBN hydrolysis process involves cleaving the peptide bonds within EBN proteins into bioactive glycopeptides using enzymes, thus improving the solubility of protein [4]. FS and SE extraction methods, in combination with enzymatic hydrolysis, increased the solubility of EBN extracts and hydrolysate in the respective samples. It is important for high protein food to be able to be well digested, metabolized, and absorbed by the human body, especially in athletes or patients with injuries or burns. Therefore, EBNs are a grea<sup>t</sup> alternative for substituting high protein sources into normal dietary intake.

#### *3.6. Molecular Weight Distribution of Protein in Extracts and Hydrolysates of EBNs*

Characterization of the protein in EBN extracts (FS and SE) and hydrolysates (FSh and SEh) were done by SDS-PAGE with Coomassie blue staining, as shown in Figure 5. The FS and SE extracts depicted a dominant band between 70 kDa and 180 kDa at the top of the gel and a smeared electrophoresis pattern down to 20 kDa, suggesting larger protein sizes had difficulty moving through the gel matrix. This is supported by [21], where the EBN protein band was clearly shown at the top of the SDS-PAGE gel matrix. These band patterns show that EBN extract contains large and complex molecular weight proteins. The protein molecular weight ranges from 100 to 135 kDa, indicating the presence of sialic acid, which has been reported as bound to protein with molecular weights of 106 and 128 kDa in EBN samples, known as 'sialo-glycoprotein' [18].

**Figure 5.** The molecular weight of protein for half-cup EBN extracts and hydrolysates. PM: Protein Marker; FS: Full stew; SE: Stew extract; FSh: Full stew hydrolysate; SEh: Stew hydrolysate.

The FSh and SEh band shows that almost all protein is present in smaller peptides with a molecular weight of less than 11 kDa. Enzymatic hydrolysis breaks down the extracted protein of EBNs by uncoiling the protein and peptide bond, thus producing small molecules with lower molecular weight, as observe using SDS-PAGE. This is also due to the 7.0 KDa cut-off molecular weight of the dialysis tubing used to separate the mixture. The protein size of EBN extract was 140.8, 64.8, and 21.1 kDa, whereas the molecular weight of EBN glycoprotein was 140.8 and 64.8 kDa [39]. Raw EBNs depict more and distinct bands as compared to processed EBNs, where the EBN processing may have reduced the original amount of intact protein, thus resulting in different protein profiles [4]. The different types of EBN depict different protein profiles in SDS-PAGE, with 37–52 kDa as the most abundant protein size found in EBNs from Malaysia [5,36].

#### *3.7. Protein and Peptide Profile of EBN*

The protein and peptide sequences identified in FS and SE extracts from half-cup EBNs using LC-MS/MS are shown in Table 3. There were seven parent proteins identified in EBNs, namely 78 kDa glucose-regulated protein, lysyl oxidase-3, mucin-5AC-like, acidic mammalian chitinase-like (AMCase-like), 45 kDa calcium-binding protein, nucleobindin-2, and ovoinhibitor-like, with a molecular weight ranging from 39.15 to 181.68 kDa.


**Table 3.** Protein and peptide sequences identified by LC-MS/MS in the full stew (FS) and stew extracts (SE) of half-cup EBNs.

MW: Molecular weight; a Accession number is a reference to *Charadrius vociferus* in SwissProt database; b Protein score contributed by each peptide matched in SwissProt database; c Peptide sequences were matched with *C. vociferous* protein databases in SwissProt; d Position of the peptide inside the parent protein; Peptide sequences with **BOLD** text only identified in respective extract.

> About 29 peptide sequences were found matched to the seven identified parent proteins, which had between 8 and 34 amino acid sequences in length and a molecular weight between 318.87 and 1054.19 Da. In this study, 78 kDa glucose-regulated protein was identified in half-cup EBN extract by LC-MS/MS, which has not been reported elsewhere. Meanwhile, six similar parent proteins were reported by [21], with the best-scored proteins

being AMCase-like, mucin-5AC-like, and ovoinhibitor-like proteins. These similar findings may be attributed to the same double-boiled method of water extraction used in the present study.

The most abundant proteins identified in both FS and SE extract were Lysyl oxidase-3 and Mucin-5AC-like protein, which were also frequently detected in EBN samples [18,37]. The authenticity of EBNs from Malaysia, Indonesia, and Vietnam was achieved and was comparable with genuine EBNs, while the outcome of adulterated EBNs (being mixed with faked EBNs) was the opposite [40]. This authenticity assessment concluded that Muc-5AC protein data could serve as an internal marker of EBN fingerprinting with the highest discriminative power, due to this protein abundance in EBNs. However, there were peptide sequences that were only present in either FS or SE half-cup EBNs, corresponding to their parent protein. This might contribute to the different extraction methods used in digestion, resulting in different bioactive peptides.

The 78 kDa glucose-regulated protein or heat shock protein was found only in this study. By referring to their chaperone role, 78 kDa glucose-regulated protein was found to be responsible for regulating cell survival and apoptosis by degradation of misfolded protein and regulation of apoptotic activity, which significantly increases the neuroprotective effects in 6-OHDA-treated neuroblastoma cell model SH-SY5Y [10]. Lysyl oxidase-3 is an enzyme that is essential for the stabilization of collagen fibrils and elasticity of elastin, supporting the evidence on skin complexion improvement in EBN-supplemented ovariectomized rats [9].

Mucin-5Ac-like protein, with a molecular weight of 181.68 kDa, is a gel-forming glycoprotein produced by epithelial tissues and is a key component in most gel-like secretion, like saliva. Muc5Ac is secreted in the gastric and respiratory tract and functions to protect the mucosa from infection and chemical damage [21]. The AMCase-like protein could be highly expressed in the salivary gland of swiftlets and secreted into the saliva for digestion of chitin-containing insects or fungi [10]. The expression of chitinase in the human body could be triggered in response to allergies, possibly accounting for the anti-inflammatory properties of EBNs [11].

The 45 kDa calcium-binding protein consists of 361 amino acids and is associated with cellular and extracellular functions, from calcium homeostasis to calcium signaling pathways [41]. This protein is present in the brain regions and is highly expressed in astrocytes, neurons, oligodendrocyte progenitor cells, myelinating oligodendrocyte, microglia/macrophage, and endothelial cells; it might have fundamental functions in a cell's calcium homeostasis [42].

Nucleobindin-2 is a calcium-binding protein with a suggested role in calcium level maintenance, eating regulation in the hypothalamus, and release of tumor necrosis factor from vascular endothelial cells. In another study on nueclobindinn-2, it was found able to control insulin sensitivity in the condition of caloric excess in the body; loss of this protein increased inflammation and insulin resistance upon high-fat feeding [43]. The protein ovoinhibitor is a serine protease inhibitor that can be found in egg whites. It plays a significant role in the antibacterial defense against *Bacillus subtilis* and *S. aureus* [44].

Proteomic analysis was influenced by the extraction method in projecting the biodiversity of the peptide profile. The application of different extraction methods for similar types of EBN resulted in a homogenous protein profile with respective matched peptide sequences. This input could be used as a future reference by researchers to explore the bioactive peptide and functional activities of EBNs. It is believed that there are still many unknown proteins, as limited databases are available for EBN proteins. Through the protein identification results, hidden potential bioactive peptides can be explored for nutritional and medicinal usage.
