**1. Introduction**

The edible bird's nest (EBN) is the nest of the swift. It is constructed with the saliva secreted from the pair of its sublingual glands as the main material. *Aerodramus fuciphagus* (white nests) and *Aerodramus maximus* (black nests) are the two main species of swiftlets that are known to produce valuable EBNs. Malaysia is the second largest exporter in the world, contributing about 20% to the total market of EBN production. Less than half of the EBN produced are consumed by locals, while a larger percentage is exported to other countries, such as Hong Kong, Singapore, and China [1]. Due to its high nutritional and medicinal therapeutic values, EBNs can cost USD 2000–10,000 per kilogram and are regarded as the most expensive animal by-product in the world [2]. White EBNs are regarded as the "Caviar of the East" due to their unique taste and smooth texture, they have also been widely used by people, especially the Chinese community, as traditional

**Citation:** Mohamad Nasir, N.N.; Mohamad Ibrahim, R.; Abu Bakar, M.Z.; Mahmud, R.; Ab Razak, N.A. Characterization and Extraction Influence Protein Profiling of Edible Bird's Nest. *Foods* **2021**, *10*, 2248. https://doi.org/10.3390/ foods10102248

Academic Editor: Antonello Santini

Received: 20 July 2021 Accepted: 18 September 2021 Published: 23 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

medicines. In traditional Chinese medicine, EBNs are prescribed to treat diseases such as tuberculosis, asthma, dry cough, hemoptysis, asthenia, difficulty in breathing, gastric troubles, and general bronchial ailments [3].

Interestingly, EBNs have also been the focus of modern research and technology, which have revealed their nutritional value and pharmacological activities, including anti-aging, anti-cancer, cough-suppressing, anti-tuberculosis, voice-improving, and phlegm-dissolving properties [4]. Meanwhile, in the food industry, EBN extract has been used as one of the ingredients in foods, drinks, and nutraceutical products. For example, ice cream with 0.2% EBN extract was found to be more favorable than the others due to high melting rates and lower sweetness [4]. These positive nutritional and health effects have increased the demand and supply of EBNs and have generated increasing interest among researchers. Investigations of the hidden nutritive and pharmacological properties of EBNs have become the primary focus of this research due to their potential as a therapeutic agent.

EBNs consist of protein (42.0–63.0%), carbohydrate (10.63–27.26%), moisture (7.5–12.9%), ash (2.1–7.3%), and fat (0.14–1.28%) [5]. Bioactive peptides released from EBN hydrolysate can exhibit various biological activities and nutritional properties, such as lowering blood pressure by ACE inhibitory peptides [6]. The main carbohydrate found in EBNs is sialic acid, which is a type of glycoprotein that consists of oligosaccharide chains (glycans) covalently attached to amino acid sidechains. Sialic acid has been proven to ameliorate cardiovascular disease biomarkers [7] and significantly improve the learning and memory function of the preterm infant [8]. To date, EBN containing bioactive peptides were found to improve bone loss and skin aging in post-menopausal women [9], to be effective in the treatment of neurodegenerative diseases [10], to prevent obesity-related inflammation and oxidative stress in rats [11], to lower oxidative stress and inflammatory markers [12], and to improve spatial learning performance in children [13].

Consumption of EBN-based nutritional beverages has been found to promote the improvement of human health. EBN glycopeptides were used in the formulation of readyto-drink products and have been shown to have significantly higher antioxidant activity (*p* < 0.05) compared to non-EBN drinks [14]. In the cosmetic industry, EBN extracts have been incorporated into skincare products and have been used to promote skin cell renewal, radiant complexion, and anti-aging [4]. In some studies, EBN has been shown to improve digestive problems [5], with the prebiotic properties of EBN promoting a healthy human gu<sup>t</sup> environment.

The bird's nest industry in Malaysia suffered a setback in 2011 when China banned EBN exports due to high concentrations of nitrate, lead, and arsenic detected in certain EBN products. As a result, strict regulations regarding EBN standards and specifications were implemented and enforced. The requirement listed in the EBN protocol for China specifies that EBN products must be free from avian influenza and the nitrite content must be less than 30 ppm. The quality of EBNs from Malaysia for export to China is set by [15], a Malaysian Standard that includes (1) MS 2333:2010 Good manufacturing practice (GMP) for processing raw-unclean and raw-clean edible birdnest (EBN); (2) MS 2334:2011 Edible birdnest (EBN)-specification; (3) MS 2612:2015 Raw-unclean edible birdnest (EBN)- house nest specification; and (4) MS 2509:2012 (P) Test method for edible bird nest (EBN)- determination for nitrite (NO2 −) and nitrate (NO3 −) content. The Malaysian Standards also provide requirements for EBN swiftlet farming—MS 2273:2012 and MS 2333:2010—as references for the EBN industry to reduce potential contamination in EBNs. The Standard MS 2273:2012 specifies guidelines for ranching practices of edible nest swiftlets, including ranch design and maintenance, hygiene of the premises, and signs of illness in swiftlets. The Standard MS 2333:2010 provides guidelines for the design of processing facilities in order to avoid cross-contamination and to control procedures that affect operations, building maintenance, personal hygiene, and animal hygiene control systems (contaminant control).

Grading of harvested EBNs depend on factors such as shape (half-cup or stripeshaped), type (white, red, or grass), color (white, yellow, or red), and cleanliness of the nest. In addition, the dry mass, duration of nest building, and protein content of cleaned

house-farmed EBNs also contribute to the EBN grading, reflecting quality [5]. The detailed characteristics and grading of EBNs, including physicochemical analysis (physical [12], morphology [16], elemental composition [17], and microbial content [18]) and chemical analysis (crude protein [2], amino acid [19] and sialic acid [7]) have been reported. However, research on the relationship between the physical and chemical characteristics of EBNs and their nutritional and bioactive properties is lacking.

Several methods have been reported for obtaining EBN extract and bioactive peptides, such as solvent extraction, heat extraction, enzymatic hydrolysis, and microbial fermentation [4]. Solvent extraction may result in low extraction efficiency, low selectivity, solvent residue, and environmental pollution. In contrast, enzymatic hydrolysis is preferable compared to solvent extraction and microbial fermentation due to high recovery, less solvent residue, and being more environmentally friendly, making this method popular among food and pharmaceutical industries [20]. However, data on the impact of characterization and extraction of different shapes of EBNs on the protein profiling and bioactive content is insufficient; this research area needs to be explored for a better understanding of EBN applications. A bioactive compound, when extracted using different extraction methods, may potentially produce a wide range of bioactivity, which may be attributed to the synergistic effects of diverse constituents of the food [21].

Proteomic research into EBNs has resulted in a bottleneck, due to its poor solubility and low extractive rate [22] and the limited number of protein sequences deposited in the database. The effects of simulated gastrointestinal digestion provide insight into the amounts of bioactive peptides that are likely to be derived from hydrolysis of EBNs in real conditions of humans. The optimum conditions implicated for the bioavailability of EBN glycopeptides is thus double boiling followed by enzymatic hydrolysis; this combination of methods uncoils the glycoprotein structure, which in turn may unlock the potential benefit of EBNs [4].

Even though both half-cup and stripe-shape EBNs originate from the same source, the comparison data from physicochemical analysis (physical, morphology, elemental composition, and microbial content) and chemical analysis (crude protein, amino acid, and sialic acid) is important to understand and correlate between protein hydrolysis and bioactive peptide studies. Four types of water extraction combined with heat treatment were selected to simulate the common traditional preparation of EBN, aimed to profile the water-soluble protein. Protein profiling portrayed by SDS-PAGE and LC-MS/MS analysis demonstrates confirmation of the good quality of protein produced by the hydrolysis technique, which is related to the functional characterization of bioactive glycopeptides. This peptide sequence clarified the specific bioactive activity with different characteristics, which is important for food, pharmaceutical, and medical applications.

#### **2. Materials and Methods**

#### *2.1. Edible Bird's Nest (EBN)*

Different types (half-cup and stripe-shaped) of raw-cleaned house Edible Bird's Nest (EBN) were purchased from Blossom View Sdn. Bhd in Terengganu (East Coast of Malaysia). Feathers and impurities identified during the screening procedure were manually removed using tweezers, ground with a mortar to form EBN powder, and stored in an air-tight container at 4 ◦C until further use.
