Food Quality and Safety, 2017, 1, 83–92

doi:10.1093/fqs/fyx002
Research Paper

Research Paper

Characterization of edible bird’s nest by peptide

fingerprinting with principal component
analysis
Chun-Fai Wong*, Gallant Kar-Lun Chan*, Ming-Lu Zhang*, Ping Yao*,
Huang-Quan Lin*, Tina Ting-Xia Dong*,**, Geng Li***, Xiao-Ping Lai****
and Karl Wah-Keung Tsim*,**
*Division of Life Science and Center for Chinese Medicine R&D, The Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, **HKUST Shenzhen Research Institute, Hi-Tech Park, Nanshan, ***School of Chinese
Herbal Medicine, Guangzhou University of Chinese Medicine, and ****Dongguan Mathematical Engineering
Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, China

Correspondence to: Karl Wah-Keung Tsim, Division of Life Science, The Hong Kong University of Science and Technology,
Clear Water Bay Road, Kowloon, Hong Kong SAR, China. E-mail: botsim@ust.hk
Received 8 November 2016; Revised 27 November 2016; Editorial Decision 30 December 2016.

Abstract
OBJECTIVES: Proteins are the major component and play a key role in nutritious and therapeutic
functions of edible bird’s nest (EBN); 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 EBN peptides, which would be a valuable
reference for further study on EBN proteins.
METHODS: Here, we developed a quality control method using high performance liquid
chromatography (HPLC) peptide fingerprints deriving from EBN 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 EBN (white/red) and production sites (cave/house) of White EBN 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 EBN samples;
and classify EBN samples by its color and production site. In addition, the protein identification
results suggested that Muc-5AC-like protein was the major protein in EBN.

Key words: Edible bird’s nest; Peptide fingerprint; Mucin-5AC-like; Acidic mammalian chitinase-like; Principal component analysis.

Introduction
Edible bird’s nest (EBN), or cubilose, is a health food supplement
originated from salivary secretion by specific swiftlets, 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;

© The Author 2017. Published by Oxford University Press on behalf of Zhejiang University Press.
83
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84 C.-F. Wong et al., 2017, Vol. 1, No. 1

Chan et  al., 2015). Southeast Asian countries, including Indonesia,
Malaysia, Vietnam, and Thailand, are the major exporting countries of
EBN. Human consumption and medicinal application of EBN 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 EBN has been served as an esteemed food in Chinese
community for over 1000 years, limited research has been conducted
on EBN and its proteins. Protein is a major part of EBN accounting
for 50% of EBN 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 pep-
tide was partially purified with Bio-Gel P-10 columns from aqueous
extracts of EBN 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 EBN 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 EBN, was identi-
fied by mass spectroscopy (Chan et al., 2013b). Nonetheless, it remains
unclear whether those identified proteins could accurately represent
the majority of EBN protein. The difficulties encountered in research
of EBN proteins are: (i) extracting and purifying proteins; and (ii) lack-
ing of full Aerodramus genome sequence.
Owing to the limited supply and labour-intensive cleaning process,
EBN is always expensive with current prices ranging from USD 500
to 15 000/kg. Driven by the lucrative return, various materials, includ-
ing Tremella fungus, fried porcine skin, carrageenan, agar, and gelatin,
which are almost indistinguishable from the genuine samples by visual
inspection, were commonly adulterated into EBN in order to increase
the net weight (Ma and Liu, 2012). Some businesses have been known
to mix low-quality EBN into high-quality EBN and selling that at a
high price. Occasionally, consumers have been counterfeited into pur-
chasing lower priced house EBN at a premium price associated with
cave EBN. 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 chemi-
cal composition as the quality control parameters: since no official
method has been established for quality surveillance of EBN (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 barri-
ers 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 EBN as a robust technique
in qualitative and quantitative controls. Firstly, an over-stewing
method was developed to extract most of the EBN protein. Secondly,
simulated gastric fluid (SGF) was used to digest EBN protein fully
into peptides that can be separated by HPLC according to their
polarity. Thirdly, according to the most relevant NCBI protein data-
base, 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 EBN. The results therefore contributed
to the authentication and classification of EBN. This study aimed
to provide comprehensive information for the quality evaluation of
EBN protein at the peptide level, which would be a valuable refer-
ence for further study on EBN proteins.
Materials and Methods
Chemicals and EBN
Pepsin, SGF without enzyme (contains 0.07 M hydrochloric acid
and 0.1 M sodium chloride), and trifluroacetic acid were purchased
from Sigma/Aldrich (St Louis, MO). LC-MS-grade acetonitrile was
obtained from JT Baker (Center Valley, PA). Aprotinin, a monomeric
globular polypeptide with a molecular weight of 6512, was obtained
from GE Healthcare (Buckinghamshire, UK). Twenty-five batches of
EBN, including different colour, country of origin, and production
site, from different commercial bands were randomly purchased in
Hong Kong market, and all samples were in standard ‘cup’ grade.
Samples were labelled and stored at room temperature upon arrival.
The sample information was listed in Table 1.
Extraction and digestion of EBN
A cup (a common market size) of dried EBN was accurately
weighed and soaked in a 100-fold volume of water (w/v) for

Table 1.  Information of edible bird’s nest (EBN) samples.

Sample code Colour Country of origin* Production site* Sample code Colour Country of origin* Production site*

1 White Indonesia House 14 White Thailand Cave

2 White Vietnam Cave 15 Yellow Indonesia House
3 White Thailand Cave 16 Yellow Indonesia House
4 White Indonesia House 17 Yellow Indonesia House
5 White Indonesia House 18 Yellow Indonesia House
6 White Malaysia House 19 Red Indonesia House
7 White Thailand Cave 20 Red Indonesia House
8 White Vietnam Cave 21 Red Indonesia House
9 White Thailand Cave 22 Red Indonesia House
10 White Malaysia House 23 Red Indonesia House
11 White Indonesia House 24 Red Indonesia House
12 White Vietnam Cave 25 Red Malaysia Cave
13 White Malaysia House

*The country of origin and production site of EBN were provided by the merchants.
Characterization of edible bird’s nest, 2017, Vol. 1, No. 1
3–15 h (3 h for White EBN; 10 h for Yellow EBN; 15 h for Red
EBN), as described previously (Chan et al., 2013b). After discard-
ing the soaked water to remove contaminants, the EBN was rinsed
with water three times. The soaked EBN was put into a stewing
pot with 30 volumes of water. The soaked EBN was stewed for
8–40 h at 98 ± 2°C until it was completely molten (8 h for White
EBN; 16 h for Yellow; 40 h for Red EBN). The sample was dia-
lyzed overnight against water in a dialysis bag with 2000 cut-
off molecular weight and then filtered, freeze-dried, and stored
at 4°C until use. The EBN lyophilized powder was dissolved and
digested with a SGF (pH 2), consisting of pepsin and SGF without
enzyme, for 1 h at 37°C. The digest was neutralized with sodium
hydroxide and then filtered by 0.45  µm hydrophilic filter. The
total protein content of EBN was determined by Kjeldahl method
(Codex Alimentarius International Food Standards, 1999), and
the extracted protein was determined by the Bradford method
(Bio-Rad, Herculues, CA).
SDS–PAGE analysis
The EBN lyophilized powder was suspended in 1 ml Milli-Q water.
The samples were treated with direct lysis buffer (0.125 M Tris–Cl,
pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.02%
bromophenol blue) at 95°C for 5  min, and subsequently 10 μg of
extracted proteins of EBN were loaded onto a 15% SDS polyacryla-
mide gel and run at 60 V for electrophoresis. After separation of
protein, the gel was stained by Coomassie blue reagent and then
destained by a solution consisting of water, methanol, and acetic
acid. Gel photos were captured by a scanner.
HPLC conditions
The chromatographic separation was performed on an Agilent
HPLC 1200 series system (Agilent, Waldbronn, Germany), which
was equipped with a diode array detector (DAD), a degasser, a
binary pump, an autosampler, and a thermo-stated column compart-
ment. The EBN samples were separated by a SymmetrySheild™ RP
C18 column (5 mm i.d., 250 mm × 4.6 mm). The mobile phase was
composed of 0.1% trifluroacetic acid in water (A) and acetonitrile
(B) using the following gradient program: 0–10  min, linear gradi-
ent 5–15% (B); 10–30 min, linear gradient 15–30% (B); 30–50 min,
isocratic gradient 30% (B); 50–60 min, linear gradient 30–50% (B);
a pre-equilibration period of 20  min was used between each run.
The flow rate was 0.6 ml/min; the column temperature was 25°C;
and the injection volume was 10 µl. The DAD wavelength was set
to 214 nm since the peptide bond showed the highest absorbance at
this wavelength.
Protein identification by LC–MS/MS
The eluent at 9–40 min from the peptide fingerprint was collected
by a Gilson FC203B fraction collector at 1  min per fraction. Five
fractions representing peaks A, B, C, D, and E were collected and
subjected to protein identification. The samples were desalted, con-
centrated, and purified by ZipTip and then analysed by a Thermo
Scientific LTQ Velos Dual-Pressure Ion Trap Mass Spectrometer cou-
pled with a Thermo Accela 600 pump, an Accela autosampler LC,
and ETD source (spray voltage 1.6 kV, capillary temperature 250°C).
A Thermo Scientific BioBasic-18 column was used (150.0 × 0.1 mm,
5 μm). Formic acid at 0.1% in MS-grade water and 0.1% formic
acid in MS-grade acetonitrile were used as mobile phase A and B,
respectively; with a flow rate of 0.15 ml/min. Mascot Daemon (ver-
sion 2.3.0) was used as a sequence database searching engine. The
85
parameters were set as follows: (i) pepsin A as the digestion enzyme,
(ii) allowing absence of two internal cleavage sites, (iii) oxidation of
methionine as a variable modification, (iv) ±1 Da for peptide mass
tolerance, and (v) ±0.8 Da for fragment mass tolerance. The false
discovery rate was below 5% by evaluating the number of matched
proteins in the search of real database and decoy database. The
peptide sequences were searched in the NCBI library and Chaetura
pelagica (Chimney swift) database.
Data analysis
PCA is an unsupervised statistical model that transforms a set of
observations of possibly correlated variables into a smaller group
of linearly uncorrelated variables, thereby avoiding subjective deci-
sions, making data easy to explore, and allowing for the visualiza-
tion of significant differences in complex data sets with many factors
(Abdi and William, 2010). Only those characteristic peaks having
higher than their respectively limit of quantification (LOQ) values
were quantified and used for subsequent data analysis. Five peaks
(A, B, C, D, and E) could reach the LOQ requirement, and they were
selected and quantified with reference to the spiked protein apro-
tinin. PCA was performed using SIMCA 13 (Umetrics, Sweden) with
the parameter set to ‘PCA-X’. The relative amount of peaks A–E
from the chromatographic fingerprints was used for PCA, and the
score plot, loading plot, and dendrogram were examined in order
to reveal possible relationship between peptide mapping and EBN.
Statistical tests were done by using one-way analysis of variance
(ANOVA) provided in GraphPad Prism 6.0. Statistically significant
changes were classed as [*] where P < 0.05, [**] where P < 0.01, and
[***] where P < 0.001.
Results
Optimization of protein digestion on EBN
Twenty-five EBN samples collected from a Hong Kong market
(Table  1) were extracted by the over-stewing method in order to
ensure the complete solubility of EBN protein. To calculate the total
amount of protein in EBN (soluble or insoluble), nine crude EBN
samples, including different colour, country of origin, and production
site, were sent to a food laboratory for protein analysis by Kjeldahl
method (Codex Alimentarius International Food Standards, 1999), a
well-established test to investigate the total protein amount of food.
The average protein content of three White EBN was 48.5 ± 5.1%
(Sample 1, 2, 6); Yellow EBN was 55.7 ± 2.0% (Sample 15, 16, 17);
Red EBN was 53.1 ± 3.2% (Sample 19, 21, 25) of the dried weight
that was very close to the literature value, i.e. 50% (Jiangsu New
Medicine College, 1977). Thus, the reported value was adopted as
the denominator in calculating the extraction rate for simplicity. By
determining the extracted protein, 83.7 ± 10.0% (Sample 1–14) and
81.5  ±  10.9% (Sample 15–18) of proteins were extracted among
White and Yellow EBN samples, respectively; whereas 65.6 ± 7.3%
(Sample 19–25) were found in Red EBN samples. The low extrac-
tion efficiency revealed in Red EBN was consistent with historical
wisdom of subjecting this EBN to a longer stewing time (Chan
et al., 2013b).
The extracted proteins of White EBN (Sample 1) were separated
by SDS polyacrylamide gel, and most of them were stacked at the top
of gel, which suggested that most of the soluble EBN proteins were
over 200  kDa (Figure  1A). After SGF digestion, the size of White
EBN proteins was decreased in a time-dependent manner. The pep-
sin from SGF served as a control at ~35 kDa. The EBN digest was
86
Figure  1.  Digestion of White EBN with SGF. (A) Fifty milligrams White EBN
extract (Sample 1) was digested in 5 ml SGF for 2 h. The digest was collected
every 15 min and then neutralized immediately by sodium hydroxide. EBN
lane represents EBN extract (indicated) without digestion; SGF lane with
pepsin (indicated) served as blank control. The digested EBN protein (10 μl)
was separated and analysed by 15% SDS–PAGE followed by Coomassie blue
staining. (B) The protein contents of above different time of SGF digests were
analysed by Bradford protein assay. Values are expressed in mg/ml, mean
± SEM, n  =  4. Statistically significant is as [***] P  <  0.005. (C) EBN digest
was collected at different time points and analysed by HPLC, as shown in
Figure 2A. The area of peaks B, C, D, E and the total peaks area at different
time points in peptide fingerprint indicated the digestion was completed
after 60 min. n = 4. EBN, edible bird’s nest; HPLC, high performance liquid
chromatography; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel
electrophoresis; SGF, simulated gastric fluid.
C.-F. Wong et al., 2017, Vol. 1, No. 1
Figure  2.  HPLC chromatogram of EBN digests and its fake products. Fifty
milligrams of EBN, or same amount of fake products extract, was digested in
5 ml SGF for 1 h and then neutralized.Ten microliters of the digest was injected
into HPLC system. (A) The chromatographic patterns of White EBN, Yellow
EBN, and Red EBN were revealed, and similar peaks B–E were identical.
The common fake products, including agar, Tremella fungus, gelatin, pig
skin, and carrageenan, showed very different pattern. (B) Aprotinin was
spiked into the EBN digest for HPLC fingerprinting. A  calibration curve of
aprotinin protein content was shown in the insert. The peak content in the
fingerprint was quantified with reference to aprotinin. n  =  4. EBN, edible
bird’s nest; HPLC, high performance liquid chromatography; SGF, simulated
gastric fluid.
Characterization of edible bird’s nest, 2017, Vol. 1, No. 1
collected every 15 min and analysed by SDS–PAGE. The protein con-
tent of EBN digests, as well as the disappearance of high molecular
weight protein, was decreased to a steady status after 60 min indi-
cating a full digestion by SGF (Figure 1A and 1B). The EBN digests
(Sample 1, 2, 3, and 4) at different time intervals were injected into
HPLC for fingerprinting analysis. Absorption at 214 nm was chosen
to obtain maximal peak height. Four distinguishable and dominant
peaks (B–E) were selected as the reference points (Figure 2A). The
areas of those peaks were analysed along the time course, and they
reached maximum peak area after 60 min of digestion (Figure 1C).
This digestion profile was revealed also from Yellow EBN (Sample
15, 16, 17, and 18)  in Supplementary Figure S1A, and Red EBN
(Sample 19, 20, 21, and 25)  in Supplementary Figure S1B. Thus,
60 min was decided as the complete digestion time of EBN extract.
To identify major EBN proteins, the peaks of A–E were col-
lected and sent to ion-trap LC–MS/MS analysis. Hundreds of pro-
teins, including origins from avian, mammals, insects, and bacteria,
were matched; and some of them had been reported in previous
studies, including AMCase-like (Liu et al., 2012; You et al., 2015)
and transferrin (Hou et  al., 2015). None of the proteins identified
from EBN extracts matched with Aerodramus, likely because the
genome sequence of swiftlet was not completed. We then searched
the protein sequences from C. pelagica, a species in the same family
as A. fuciphagus. The common proteins were selected among three
trials, and blank SGF was served as a control. Only the fragments
of AMCase-like, lysyl oxidase homolog 3, Muc-5AC-like fragment,
and AMCase-like were identified in peak B, C, D, and E, respec-
tively, in all samples, which confirmed the identities of EBN proteins
(Table 2). Although fragments from peaks B and E were identified as
AMCase-like proteins, they represented unique AMCase-like frag-
ments matching to accession numbers in the database.
Peptide fingerprint and its application
The HPLC peptide fingerprints were generated from 25 SGF-digested
EBN samples, and typical fingerprints of White, Yellow, and Red
EBN were given in Figure 2A. In contrast, the products commonly
used to imitate EBN (e.g. agar, Tremella fungus, gelatin, pig skin, and
carrageenan) did not show any similarity to the EBN fingerprint. The
peptide fingerprints showed a close similarity among White, Yellow,
and Red EBN, except that the peak heights were varied (Figure 2A).
To quantify the characteristic peaks of EBN fingerprints, apro-
tinin, a small protein of ~6 kDa, was used as an external standard.
Aprotinin was spiked into EBN samples, and it did not interfere with
most of the characteristic peaks (Figure 2B). By referring to aprotinin
peak, the peak retention time and peak height could be calibrated,
and a calibration curve from 0.5 to 10 µg of aprotinin was estab-
lished by absorption at 214 nm (Figure 2B insert). Using this stand-
ard curve, the relative peptide content could be estimated. Figure 3A
Table  2.  Protein identification by liquid chromatography–tandem mass spectrometry (LC–MS/MS). AMCase-like, acidic mammalian chi-
tinase-like.
Peak* Protein** Major matched fragment
A Unknown
B AMCase-like LYEGPSDTGDLV
C Lysyl oxidase homolog 3 LKGGAKVGEGRVEVLR
D Muc-5AC-like MWDKKTSIF
E AMCase-like AIGGWNFGTAKF
*Peaks A–E in peptide fingerprint of EBN were collected (Figure 2A) and subjected to LC–MS/MS for protein identification. n = 3.
**The peptide sequences were matched with Chaetura pelagica (Chimney swift) protein database in NCBI.
***Accession number was reference to C. pelagica in NCBI database.
87
and Supplementary Table S1 show the estimated peptide contents
of all identified fragments of different types of EBN. In general, the
peptide contents of Red EBN were lower than the others: this low
amount could be an outcome of lower amount of extractable protein
derived from Red EBN.
To verify the authenticating power of peptide fingerprint, the pep-
tide fingerprints of three White EBNs from different sources and two
adulterated EBN (being mixed with fake EBN) were generated. The
chromatographic patterns of all samples were revealed, and similar
peaks B–E were observed (Supplementary Figure S2). The relative
amounts of peaks B–E were determined. As illustrated in Figure 3B,
the amounts of peaks B–E for EBN (Indonesia), EBN (Malaysia),
and EBN (Vietnam) were comparable to that of genuine EBN. The
amounts of peaks of EBN mixed with fake EBN were significantly
lower than genuine EBN (Figure 3B). Muc-5AC-like protein (peak
D) was therefore being considered as a tentative marker with the
highest discriminative power in authenticating EBN.
Principal component analysis
PCA was performed on the peptide mapping of 25 EBN to detect
anomalies and to study relationships in the data. Each sample num-
ber was labelled in the model for a better visual illustration and
interpretation. By using the relative peak amount of HPLC peptide
fingerprints, the dendrogram, score plot, and loading plot based on
different classifications of EBN were generated.
From the score plot (Figure  4A), Red EBN and White/Yellow
EBN could be unambiguously identified utilizing the first two princi-
pal components, PC1 (t1) and PC2 (t2), which accounted for 85.8%
of the total variation. The values of fitness of data (R2) and predic-
tive ability (Q2) were 85.6% and 92.3%, respectively, in the model.
Analysis showed no samples being outside the Hotelling T2 95%
confidence ellipse that could influence the analyses. Based on the
loading plot (Figure 4B), Mucin-5AC-like (peak D) showed the high-
est positive loading score of 0.70, accounting for the colour discrimi-
nation of samples by PC1. In agreement with the results of PCA,
application of PCA–HCA (Figure 4C) for the whole data set resulted
in a distinct classification of the samples according to their colours.
Twenty-five samples were correctly classified according to their col-
our; nonetheless, White EBN and Yellow EBN were not distinguish-
able according to the score plot and dendrogram.
Since colour of EBN shows significant variation in fingerprint
and PCA, only White EBN samples were used for comparison of
production site of EBN to hold other factors constant. House White
EBN and cave White EBN could be distinctly classified into two
clusters using the first two principal components (t1 and t2), which
explained a total of 82.7% variation (Supplementary Figure S3).
Analysis showed no samples being outside the Hotelling T2 95%
confidence ellipse. Fitness of data (R2) of this model was 82.7%,
Score Accession***
43 XP_010005363.1
71 XP_010006484.1
41 XP_009994736
39 XP_010006620.1
88 C.-F. Wong et al., 2017, Vol. 1, No. 1

Figure  3.  The relative amounts of peaks A–E in peptide fingerprint of

EBN. The peptide contents of peaks A–E, as indicated in Figure  2A, were
calibrated according to the spike aprotinin, as shown in Figure 2B. (A) White
EBN contained the highest amount of the five distinguishable peaks, while
Red EBN contained the least. Minimum and maximum peptide content of
different groups are depicted by black caps, the box signifies the upper and
lower quartiles, and the median is represented by a short black line within
the box for each types of EBN. (B) The peptide fingerprints of three White
EBNs from different sources (Indonesia, Malaysia, and Vietnam) and two
adulterated EBN being mixed with fake EBN [Sample 3 + Tremella fungus (1:1
by weight); Sample 4 + fried porcine skin (1:1)] were generated as indicated
in Supplementary Figure S2. The amounts of peaks B–E for EBN (Indonesia),
EBN (Malaysia), and EBN (Vietnam) were comparable to that of genuine
EBNs. The amounts of peaks of EBN mixed with fake EBN were significantly
lower than genuine EBN. Values are presented in relative amount as
compared to that of aprotinin. Mean ± SEM, n = 4. EBN, edible bird’s nest.

and the model was of predictive ability (Q2) of 63.0%. In order to

evaluate the predictive power of this model, the third and fourth
principal components (t3 and t4) were also investigated. Although
house White EBN and cave White EBN could be classified into two
clusters using the first and third principal components (t1 and t3)
in both score plot and dendrogram, as indicated in Supplementary
Figure S4, the predictive ability (Q2) of this model dropped to
57.46%. The R2 and Q2 value distinctly increased to 98.0% and
88.9%, respectively, by using first and fourth principal components
Figure  4.  The differentiation of chromatographic fingerprints of various EBN
colour. Twenty-five EBN peptide fingerprints from different colours were
generated: White (1–14); Yellow (15–18); Red (19–25). (A) Score plot of EBN
samples.The ellipse represents the HotellingT2 with 95% confidence. (B) Loading
plot for five characteristic peaks (A–E as shown in Figure 2A). (C) Dendrogram
showing hierarchical clustering results of EBN samples. EBN, edible bird’s nest.
(t1 and t4) in the model. Furthermore, house White EBN and cave
White EBN could be clearly classified into two clusters using the
Characterization of edible bird’s nest, 2017, Vol. 1, No. 1 89

Figure  6.  The differentiation of chromatographic fingerprints of EBN from

Figure  5.  The differentiation of chromatographic fingerprints of EBN from
country of origin. Fourteen White EBN peptide fingerprints from different
production sites. Fourteen White EBN peptide fingerprints from different
country of origin were generated according to the procedures: Indonesia (1,
production sites were generated according to the procedures: House (1, 4,
4, 5, and 11); Malaysia (6, 10, and 13); Vietnam (2, 8, and 12); Thailand (3,
5, 6, 10, 11, and 13); cave (2, 3, 7, 8, 9, 12, and 14). The relative peak area
7, 9, and 14). The relative peak area from the chromatographic fingerprints
from the chromatographic fingerprints was calculated and used for PCA. (A)
was calculated and used for PCA. (A) Score plot of EBN samples. The ellipse
Score plot of EBN samples. The ellipse represents the Hotelling T2 with 95%
represents the Hotelling T2 with 95% confidence. (B) Loading plot for five
confidence. (B) Loading plot for five characteristic peaks (A–E as shown in
characteristic peaks (A–E as shown in Figure 2A). (C) Dendrogram showing
Figure  2A). (C) Dendrogram showing hierarchical clustering results of EBN
hierarchical clustering results of EBN samples. EBN, edible bird’s nest; PCA,
samples. EBN, edible bird’s nest; PCA, principal component analysis.
principal component analysis.

first and fourth principal components (t1 and t4) in score plot two clusters based on their production site, as illustrated in loading
(Figure 5A). AMCase-like (peak E) showed the highest positive load- plot (Figure  5B). In agreement with results of PCA-X, application
ing score at 0.60 accounting for the discrimination of samples into of PCA–HCA (Figure  5C) for the whole data set resulted to good
90
separation of the samples; and all of them were correctly classified
according to their production sites.
In order to eliminate the critical variation from EBN colour in
fingerprint and PCA, only White EBN samples were used for com-
parison of EBN from different countries of origin. As illustrated in
the score plot (Figure 6A), White EBN from different countries could
not be clearly classified into clusters by using the first two princi-
pal components (t1 and t2), which explained 82.7% of the total
variation. The values of fitness of data (R2) and predictive ability
(Q2) were 82.7% and 63.0%, respectively, in this model. Analysis
showed no samples being outside the Hotelling T2 95% confidence
ellipse. PCA–HCA (Figure 6C) showed that the peptide fingerprint
could not clearly determine country of origin, which was consistent
with the results of PCA. Moreover, EBN samples from Indonesia
and Malaysia clustered on the right of score plot, and EBN samples
came from Vietnam and Thailand clustered on the left. From the
loading plot (Figure 6B), Mucin-5AC (peak D) showed the highest
positive loading score at 0.80: this could serve as a tentative maker
in discriminating the samples between Indonesia/Malaysia EBN and
Vietnam/Thailand EBN.
Method validation
Sensitivity, linearity, accuracy, and precision of the HPLC peptide fin-
gerprint technique were evaluated. Sensitivity was evaluated by the
limit of detection (LOD) and limit of quantification (LOQ). A series
of decreasing concentrations of aprotinin was evaluated to deter-
mine the LOD and LOQ. LOD was determined as the concentra-
tion with a signal-to-noise ratio (S/N) of at least 3; while LOQ was
the lowest concentration with a S/N of at least 10. Linearity was
evaluated by five calibrators prepared by spiking the aprotinin in
blank matric at concentrations of 0.5, 1, 2, 5, and 10 µg. Precision
and accuracy were evaluated on the peak area of the characteristic
peaks. Intra-batch precision was evaluated by six determinations per
single concentration in a day. Inter-batch precision was evaluated
by one determination per single concentration at six different days.
Precision was expressed as the percent of relative standard deviation
(RSD) calculated by using six determinations. The value of recov-
ery was expressed as the ratio of determined concentration from
six individual tests to corresponding known concentration of the
spiked marker. A  known amount of aprotinin was spiked into the
EBN digest, and recovery was calculated based on the concentration
of obtained from the chromatogram and the spiked amount. The
results of method validation were listed on Table 3.
Discussion
Traditionally, EBN is stewed with water for 1–2  h before eating,
but most of the EBN proteins still remain insoluble: this stewing
method accounts only ~5% protein extraction rate. Before any
kind of protein analysis, the EBN proteins need to be dissolved in
Table 3.  Method validation. EBN, edible bird’s nest; LOD, limit of detection; LOQ, limit of quantification.
LOD* LOQ* Linearity** Correlation coefficient** (R)
0.2 µg 0.5 µg 0.5–10 µg >0.995
*LOD and LOQ were calculated by aprotinin. n = 6.
**Linearity and correlation coefficient (R) were calculated by calibration curve of aprotinin. n = 6.
***Intra-batch precision and inter-batch precision were reference to peak D in EBN peptide fingerprint. n = 6.
****A known amount of aprotinin was spiked into the EBN digest; and recovery was calculated on the concentration of obtained from the chromatogram
and the spiked amount. n = 6.
C.-F. Wong et al., 2017, Vol. 1, No. 1
water. In order to enhance good extraction efficiency of EBN, an
over-stewing method was developed. Only water was utilized in our
extraction: because this should be the method used for preparation
of EBN for human consumption. About 66~84% of EBN proteins
were extracted, supporting the hypothesis that over-stewing method
could be a suitable way to extract EBN proteins. Using the same
extraction protocol, White and Yellow EBN showed better extrac-
tion efficiency as compared to that of Red EBN. The difference in
extraction rate of White EBN (~84%) and Red EBN (~66%) may be
due to conformational or structural difference of the major proteins.
Indeed, much longer time was required to extract Red EBN, which
implied Red EBN possessed higher resistance to protein digestion.
Therefore, the protein in Red EBN might not be fully extracted by
stewing. Moreover, very limited research has been reported on Red
EBN, especially the protein part. Thus, further studies have to be
conducted on the proteomics of Red EBN.
The lack of a complete genome sequence of Aerodramus pre-
sents a challenge for protein identification in EBN. The identified
peptide sequences were matched with animal species in NCBI data-
base; however, no useful result was achieved. The present identified
protein is based on C. pelagica that belongs to the same family as
Aerodramus. Thanks to the complete genome sequence of C. pelag-
ica, a member of the Apodidae, the EBN proteins were searched with
this database. Muc-5AC-like, AMCase-like, and lysyl oxidase pro-
tein fragments were identified in the fingerprint. Mucins are a family
of glycoprotein with high molecular weight produced by epithelial
tissues in most organisms (Marin et al., 2007). The major character-
istic of mucin is that it is able to form a gel, which therefore is a key
component in most gel-like secretions, e.g. saliva. In addition, the
mucin secreted in airways is Muc-5AC, and as a result it is found
in the saliva. Chitinases have a family of 18 glycosyl hydrolases;
however, their physiological functions are still not fully resolved.
AMCase belongs to the glycosyl hydrolase family in mammals (Boot
et  al., 2001; Bussink et  al., 2007). In the present work, AMCase-
like protein was reported in EBN for the third time, and it seemed
strange that ‘mammalian’ chitinase was found in birds. Until now,
chitinase protein sequences have not been found in the databases
of C. pelagica and M. gallopavo, and thus only the homologues of
AMCase-like protein were matched. Insects are the common food
of Aerodramus, and the existence of chitinase in swiftlets could
facilitate the digestion of insect. Lysyl oxidase is a copper-dependent
amine oxidase that plays a critical role in the biogenesis of connec-
tive tissue matrices by cross-linking the extracellular matrix proteins,
collagen, and elastin (Li and Kagan, 1998). The presence of lysyl
oxidase could explain why a previous study found that EBN could
facilitate the synthesis of collagen (Chua et al., 2013).
In a 2011 study, nitrite was detected in EBN available in the mar-
ket, especially Red EBN. This incident revealed the insufficient qual-
ity control of EBN products in the industry. A variety authentication
methods based on the appearance (Liu and Zhang, 1995; Wu et al.,
Intra-batch precision*** Inter-batch precision*** Recovery****
1.5% 3.5% 90.7%
Characterization of edible bird’s nest, 2017, Vol. 1, No. 1
2007), total proteins (Hu and Lai, 1999; Liu et al., 2012; Zhang et al.,
2012), N-acetylneuraminic acid (Chan et al., 2013a), sialic acid (Wang
et al., 2006), DNA (Wu et al., 2010), and sodium nitrate (Chan et al.,
2013b) of EBN have been established; but no official method could be
found for quality control of EBN by specific protein analysis, regard-
less of its abundance. Here, we utilized SGF digestion and HPLC to
generate the peptide fingerprint, which could authenticate EBN on a
qualitative and quantitative basis robustly and therefore provide a
comprehensive picture in quality control of EBN. The peptide finger-
print was able to clearly demonstrate the differences between genuine
and fake EBN samples, and thus this method could serve as an authen-
ticating tool for EBN. Muc-5AC-like fragment (peak D) was the major
peptide in the fingerprint of EBN, accounting of ~20% total content in
the fingerprinting. This indicated mucin-like protein could be the most
abundant protein in EBN. In addition, this mucin peak could serve as
an internal marker for the fingerprinting. The chromatographic pat-
tern of peptide fingerprint was highly similar among EBN samples
with different colours, suggesting that total extractable protein com-
position does not vary widely based on colour.
From the score plot and dendrogram, significant differences were
recorded between White EBN and Red EBN. Red EBN is known
to dissolve poorly in water, and indeed the protein extraction from
EBN is much lower than that of White or Yellow EBN, as shown
in the present work. This low solubility of Red EBN protein could
account for such a difference, as observed in our PCA. White EBN
could change to Red EBN by treating with sodium nitrite in acidic
medium, as reported (But et al., 2013; Chan et al., 2013b; Paydar
et  al., 2013). Herein, Paydar group suggested Red EBN could be
a result of formation of aryl-C-N and NO2 side group in aromatic
amino acids of White EBN by sodium nitrite (Paydar et al., 2013).
Thus, we proposed that this structural change in Red EBN could be
the reason of low water solubility and high resistance to enzymatic
digestion. Moreover, further studies have to be conducted on the
underlying mechanism of how EBN gets red.
No significant difference was found among the country of ori-
gin of White EBN, thus implying the protein composition should be
more or less the same regardless their production sources. However,
EBN samples from Indonesia and Malaysia clustered on the right of
score plot, and EBN samples from Vietnam and Thailand clustered
on the left. The geographic location may explain the difference, i.e.
Indonesia and Malaysia or Vietnam and Thailand are neighbouring
countries.
The PCA results suggested that there was a difference between
house and cave White EBN in both dendrogram and score plot. The
protein composition is probably different due to habitat of the swift-
lets. Most of the caves are located at the seafront and on islands,
whereas bird nest houses often are built in suburban areas or forests.
The diet of swiftlets could be different and thus affect the secretion
of EBN. Besides, the environment of a bird nest house can be main-
tained and regulated by the farmers, which may be different from the
natural cave environment. These findings complement a recent study
by Cheng’s group whose differentiated between house and cave EBN
based on amino acid composition (Seow et al., 2016).
Conclusion
PCA suggested that the peptide fingerprint could serve as a promis-
ing and useful tool to classify EBN samples by colour and production
site. As proposed by the model developed in this study, Muc-5AC-
like and AMCase-like proteins were the markers with the highest
discriminative power. In addition, HPLC fingerprints were able to
91
clearly demonstrate peptide profile differences between genuine and
adulterated EBN samples, which provide a comprehensive picture
in quality assurance of EBN. The protein identification results sug-
gested that Muc-5AC-like protein was the major protein in EBN. To
our knowledge, the present work is the first report highlighting the
use of protein, which is main component of EBN, in HPLC finger-
printing studies.
Supplementary Material
Supplementary material is available at Food Quality and Safety
online.
Funding
This research was supported by the grants of Hong Kong Research
Grants Council TBS (T13-607/12R), GRF (661110, 662911,
660411, 663012, 662713), ITC (UIM/254), TUYF12SC02,
TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club
Charities Trust (HKJCCT12SC01) and Foundation of The
Awareness of Nature (TAON12SC01), SRFDP and RGC ERG Joint
Fund (2013009614001/M-HKUST604/13) to Karl Wah-Keung
Tsim; and National Natural Science Foundation Item (81173498)
to Xiao-Ping Lai.
Acknowledgements
Chun-Fai Wong received a HK JEBN scholarship.
Conflict of interest statement. None declared.
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