About Bird Nest - Types of Bird Nests

Bird Nests are classified according to the location where the nests are built, edibility, external features, colour, quality and cleanliness and they can basically be classified into two main categories:

• Cave Bird Nest: (including whole cave Bird Nest pieces, cave Hydrochous Gigas and Collacalia Maxima)

Cave Bird Nest refers to the nests built by Bird Nests in natural stone walls or limestone caves. These Bird Nests absorb lots of minerals and they are usually beige or yellow in colour and they are more solid. They contain more fine feathers compared to house Bird Nests and contain more impurities. They expand 2-3 times upon soaking and require longer soaking time. They are delicious after stewing, taste better and can be stewed for longer hours.

• House Bird Nest: (including common white Bird Nest pieces and house Hydrochous Gigas Bird Nest)

House Bird Nest are Bird Nests built in swift houses and used by swifts for nesting. Bird Nests build their nests on the beams and walls in these houses, resulting in a slight taste of sawdust in such Bird Nests. Houses used by Bird Nests to build nests are known as swift houses. Workers check the Bird Nests on a regular basis and will only harvest them when the time is right. As Bird Nests are covered with feathers and other impurities, it is necessary to send them for cleansing at the Bird Nest factory where feathers and other impurities are removed. House Bird Nests are higher in quality, with less feathers and less impurities. They expand better in water and on the average, they expand 5 to 7 times their original size. They taste smoother but they cannot be stewed for long hours. They are normally stewed for 30-45 minutes before they are ready to be served. If stewing exceeds such periods (more than 1-2 hours), the Bird Nest will dissolve in water and disappear.

About Bird Nest - Types of Bird Nests

*Whether it is house Bird Nest or cave Bird Nest, they are nests built by Collocalia ciphaga Bird Nests and their nutritional value is almost at par.

• Edibility

Bird Nests normally use twigs, straws and earth to build their nests and there are no edible contents. There are only three major types of edible Bird Nests and the purity in protein contents and nutritional value of the saliva secreted by Collocalia ciphaga Bird Nests is the highest.

• Collocalia ciphaga Bird Nest {Official Bird Nest}:

Collocalia ciphaga Bird Nest means Bird Nests built with pure saliva of Bird Nests, with minimum impurities, in strips, with high affinity for water, able to expand 7-8 times and is the best quality Bird Nest. They are usually white, yellow, red or grey in colour.

• Collacalia Maximus Bird Nest:

Collacalia Maximus is a type of cave Bird Nest and they have less saliva. They build their nests using feathers mixed with saliva and they are lighter in colour, usually white or yellow.
Collacalia Maximus Bird Nest require more processing. The nests must be broken up and soaked in water before feathers and impurities are removed. They are then made into Bird Nest strips, Bird Nest balls and Bird Nest biscuits.
As it requires more processing, their nutritional value and price are lower than Collocalia ciphaga Bird Nests. Collacalia Maximus Bird Nests also have a lower water affinity compared to Collocalia ciphaga Bird Nests, thus they do not expand so much.

• Hydrochous Gigas Bird Nest:

Hydrochous Gigas Bird Nest may be divided into house Hydrochous Gigas Bird Nest and cave Hydrochous Gigas Bird Nest. They are mostly found along the coastal regions. From their name (in Chinese), we know that Hydrochous Gigas Bird Nests use their saliva and straws to build their nests, and that is why these Bird Nests contain pieces of straws. Hydrochous Gigas Bird Nests come from Bird Nests with specially sticky saliva. These Bird Nests love to use young straw and their saliva to build their nests. Their Bird Nests therefore are high in impurities and their edible Bird Nest content is the lowest among the three types of Bird Nests and so is its price. They are usually sold as broken Bird Nest and Bird Nest strips.

• External features and shape

They are principally divided into whole Bird Nest pieces, narrow Bird Nest pieces, Bird Nest corners, broken Bird Nest, Bird Nest balls, Bird Nest biscuits and Bird Nest strips. Whole Bird Nest pieces refer to Bird Nests which are intact after having undergone cleansing.

• Whole piece Bird Nest:

These are curved or boat-shaped Bird Nest which has been selected from the Bird Nest harvested, with feathers removed, before they are sold. As whole piece Bird Nests are naturally obtained, their shapes and sizes differ but these type of Bird Nests are thicker than other types of Bird Nests. They also possess higher nutritional value, with better flavor and better expandability when soaked. They are the cleanest among all Bird Nests and they have high expandability. If Bird Nests built their nests on the beams in swift houses, the Bird Nests produced will be boat-shaped.

• Narrow Bird Nest pieces:

These are whole piece Bird Nests which have been accidentally broken during harvesting and delivery or whole piece Bird Nests which have been cut into narrow strips or Bird Nests which have been separated after careful selection. Although they are not attractive physically, their quality and method of preparation are similar to whole piece Bird Nests.

• Bird Nest corners:

They are the hardest portions at both ends of whole piece Bird Nests and they are the places where the concentration of saliva from Bird Nests is the highest. They require longer soaking time (six to eight hours) and Bird Nest corners are normally separated from whole piece Bird Nests and soaked separately. The Bird Nest corners are usually torn into smaller pieces prior to soaking so that they expand more easily. They expand more compared to whole piece Bird Nests and are more crispy.

• Broken Bird Nests:

These are as a result of breakage during harvesting and transportation or during the cleansing process and they are made up of small amounts of edible Bird Nest.

• Bird Nest biscuit:

These are made from broken Bird Nest during harvesting which have been processed and compressed into different shapes (such as rhombus, sphere, crescent and rectangular) and their quality is one grade below that of whole piece Bird Nests.

Characterization of edible Bird Nest by peptide fingerprinting with principal component analysis

Bird Nest, or cubilose, is a health food supplement originated from salivary secretion by specific Bird Nests, mainly from Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), which has been proven to have nutritious and therapeutic values, such as anti-influenza viruses, antioxidant, skin lightening, bone strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong et al., 1987; Kong et al., 1989; Guo et al., 2006; Aswir and Wan Nazaimoon, 2011; Matsukawa et al., 2011; Yew et al., 2014; Chan et al., 2015). Southeast Asian countries, including Indonesia, Malaysia, Vietnam, and Thailand, are the major exporting countries of Bird Nest. Human consumption and medicinal application of Bird Nest could be dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty (960–1279 A.D.) in China (Koon and Cranbrook, 2002).

OBJECTIVES

Proteins are the major component and play a key role in nutritious and therapeutic functions of Bird Nest; however, limited studies have been conducted on the protein due to difficulties in extraction, isolation as well as identification. This study aimed to provide comprehensive information for the quality evaluation of Bird Nest peptides, which would be a valuable reference for further study on Bird Nest proteins.

METHODS

Here, we developed a quality control method using high performance liquid chromatography (HPLC) peptide fingerprints deriving from Bird Nest being digested with simulated gastric fluid. The characteristic peptide peaks were collected and identified by LC-MS/MS.

RESULTS

The characteristic peptide peaks, corresponding to the protein fragments of acidic mammalian chitinase-like, lysyl oxidase, and Mucin-5AC-like, were identified and quantified. Interestingly, the principal component analysis indicated that the fingerprints were able to discriminate colour of Bird Nest (white/red) and production sites (cave/house) of White Bird Nest on the same weight basis. As proposed by the model developed in this study, Muc-5AC-like and AMCase-like proteins were the markers with the highest discriminative power.

CONCLUSIONS

The overall findings suggest that HPLC peptide fingerprints were able to clearly demonstrate peptide profile differences between genuine and adulterated Bird Nest samples; and classify Bird Nest samples by its color and production site. In addition, the protein identification results suggested that Muc-5AC-like protein was the major protein in Bird Nest.

Introduction

Bird Nest, or cubilose, is a health food supplement originated from salivary secretion by specific Bird Nests, mainly from Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), which has been proven to have nutritious and therapeutic values, such as anti-influenza viruses, antioxidant, skin lightening, bone strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong et al., 1987; Kong et al., 1989; Guo et al., 2006; Aswir and Wan Nazaimoon, 2011; Matsukawa et al., 2011; Yew et al., 2014; Chan et al., 2015). Southeast Asian countries, including Indonesia, Malaysia, Vietnam, and Thailand, are the major exporting countries of Bird Nest. Human consumption and medicinal application of Bird Nest could be dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty (960–1279 A.D.) in China (Koon and Cranbrook, 2002).
Although Bird Nest has been served as an esteemed food in Chinese community for over 1000 years, limited research has been conducted on Bird Nest and its proteins. Protein is a major part of Bird Nest accounting for 50% of Bird Nest dried weight on average (Jiangsu New Medicine College, 1977); it is conjectured to be a key factor of its nourishing and/or medicinal functions. The epidermal growth factor (EGF)-like peptide was partially purified with Bio-Gel P-10 columns from aqueous extracts of Bird Nest that stimulated cell division and growth and enhanced tissue growth and regeneration (Kong et al., 1987; Kong et al., 1989). Two major bands (~106 kDa and ~128 kDa) were identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as ‘sialo-glycoprotein’. Nevertheless, no satisfactory result was obtained from protein identification studies that include N-terminal sequence determination (Edman degradation), matrix-assisted laser desorption ionization–tandem time of flight (MALDI–TOF/TOF), and liquid chromatography–tandem mass spectrometry (LC–MS/MS) (Zhang et al., 2012). Acidic mammalian chitinase-like (AMCase-like) protein fragments from Meleagris gallopavo and an allergen homologous to ovo-inhibitor have been identified by 2-DE assays followed by MALDI–TOF/TOF/MS analysis in Bird Nest extract (Liu et al., 2012). In addition, a microbial nitrate reductase, converting nitrate to nitrite and playing a role in the colour change of White and Red Bird Nest, was identified by mass spectroscopy (Chan et al., 2013b). Nonetheless, it remains unclear whether those identified proteins could accurately represent the majority of Bird Nest protein. The difficulties encountered in research of Bird Nest proteins are: (i) extracting and purifying proteins; and (ii) lacking of full Aerodramus genome sequence.
Owing to the limited supply and labour-intensive cleaning process, Bird Nest is always expensive with current prices ranging from USD 500 to 15000/kg. Driven by the lucrative return, various materials, including Tremella fungus, fried porcine skin, carrageenan, agar, and gelatin, which are almost indistinguishable from the genuine samples by visual inspection, were commonly adulterated into Bird Nest in order to increase the net weight (Ma and Liu, 2012). Some businesses have been known to mix low-quality Bird Nest into high-quality Bird Nest and selling that at a high price. Occasionally, consumers have been counterfeited into purchasing lower priced house Bird Nest at a premium price associated with cave Bird Nest. About 40 publications are found in PubMed today, and nearly one-third of the publications are published in the last 5 years. Besides, most of the publications still retained in elucidating chemical composition as the quality control parameters: since no official method has been established for quality surveillance of Bird Nest (Deng et al., 2006; Wang et al., 2006; Wu et al., 2010; Chan et al., 2013a).
Here, we attempt to find a key to open these proteome barriers by high performance liquid chromatography (HPLC) peptide fingerprinting. HPLC fingerprinting is one promising tool widely used in the modern standardization of herbal extracts (Department of Health, Hong Kong, 2010; Chinese Pharmacopoeia Commission, China, 2015), which could be applied to Bird Nest as a robust technique in qualitative and quantitative controls. Firstly, an over-stewing method was developed to extract most of the Bird Nest protein. Secondly, simulated gastric fluid (SGF) was used to digest Bird Nest protein fully into peptides that can be separated by HPLC according to their polarity. Thirdly, according to the most relevant NCBI protein database, the characteristic peaks in chromatograms were identified and quantified. In addition, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were adopted to reveal the relationships of factors within the data, including colour, country of origin, and production site of Bird Nest. The results therefore contributed to the authentication and classification of Bird Nest. This study aimed to provide comprehensive information for the quality evaluation of Bird Nest protein at the peptide level, which would be a valuable reference for further study on Bird Nest proteins