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Characterization of edible bird’s nest by peptide fingerprinting with principal component analysis

Characterization of edible bird’s nest by peptide fingerprinting with principal component analysis 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. which has been proven to have nutritious and therapeutic values, such Introduction as anti-influenza viruses, antioxidant, skin lightening, bone strength Edible bird’s nest (EBN), or cubilose, is a health food supplement improvement, anti-inflammatory, and epidermal growth enhancement originated from salivary secretion by specific swiftlets, mainly from (Kong et  al., 1987; Kong et  al., 1989; Guo et  al., 2006; Aswir and Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), 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. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. 84 C.-F. Wong et al., 2017, Vol. 1, No. 1 Chan et  al., 2015). Southeast Asian countries, including Indonesia, Besides, most of the publications still retained in elucidating chemi- Malaysia, Vietnam, and Thailand, are the major exporting countries of cal composition as the quality control parameters: since no official EBN. Human consumption and medicinal application of EBN could be method has been established for quality surveillance of EBN (Deng dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty et al., 2006; Wang et al., 2006; Wu et al., 2010; Chan et al., 2013a). (960–1279 A.D.) in China (Koon and Cranbrook, 2002). Here, we attempt to find a key to open these proteome barri- Although EBN has been served as an esteemed food in Chinese ers by high performance liquid chromatography (HPLC) peptide community for over 1000 years, limited research has been conducted fingerprinting. HPLC fingerprinting is one promising tool widely on EBN and its proteins. Protein is a major part of EBN accounting used in the modern standardization of herbal extracts (Department for 50% of EBN dried weight on average (Jiangsu New Medicine of Health, Hong Kong, 2010; Chinese Pharmacopoeia Commission, College, 1977); it is conjectured to be a key factor of its nourishing and/ China, 2015), which could be applied to EBN as a robust technique or medicinal functions. The epidermal growth factor (EGF)-like pep- in qualitative and quantitative controls. Firstly, an over-stewing tide was partially purified with Bio-Gel P-10 columns from aqueous method was developed to extract most of the EBN protein. Secondly, extracts of EBN that stimulated cell division and growth and enhanced simulated gastric fluid (SGF) was used to digest EBN protein fully tissue growth and regeneration (Kong et al., 1987; Kong et al., 1989). into peptides that can be separated by HPLC according to their Two major bands (~106 kDa and ~128 kDa) were identified by sodium polarity. Thirdly, according to the most relevant NCBI protein data- dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as base, the characteristic peaks in chromatograms were identified ‘sialo-glycoprotein’. Nevertheless, no satisfactory result was obtained and quantified. In addition, principal component analysis (PCA) from protein identification studies that include N-terminal sequence and hierarchical cluster analysis (HCA) were adopted to reveal the determination (Edman degradation), matrix-assisted laser desorption relationships of factors within the data, including colour, country of ionization–tandem time of flight (MALDI–TOF/TOF), and liquid origin, and production site of EBN. The results therefore contributed chromatography–tandem mass spectrometry (LC–MS/MS) (Zhang to the authentication and classification of EBN. This study aimed et al., 2012). Acidic mammalian chitinase-like (AMCase-like) protein to provide comprehensive information for the quality evaluation of fragments from Meleagris gallopavo and an allergen homologous EBN protein at the peptide level, which would be a valuable refer- to ovo-inhibitor have been identified by 2-DE assays followed by ence for further study on EBN proteins. MALDI–TOF/TOF/MS analysis in EBN extract (Liu et  al., 2012). In addition, a microbial nitrate reductase, converting nitrate to nitrite and Materials and Methods 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 Chemicals and EBN unclear whether those identified proteins could accurately represent Pepsin, SGF without enzyme (contains 0.07 M hydrochloric acid the majority of EBN protein. The difficulties encountered in research and 0.1 M sodium chloride), and trifluroacetic acid were purchased of EBN proteins are: (i) extracting and purifying proteins; and (ii) lack- from Sigma/Aldrich (St Louis, MO). LC-MS-grade acetonitrile was ing of full Aerodramus genome sequence. obtained from JT Baker (Center Valley, PA). Aprotinin, a monomeric Owing to the limited supply and labour-intensive cleaning process, globular polypeptide with a molecular weight of 6512, was obtained EBN is always expensive with current prices ranging from USD 500 from GE Healthcare (Buckinghamshire, UK). Twenty-five batches of to 15 000/kg. Driven by the lucrative return, various materials, includ- EBN, including different colour, country of origin, and production ing Tremella fungus, fried porcine skin, carrageenan, agar, and gelatin, site, from different commercial bands were randomly purchased in which are almost indistinguishable from the genuine samples by visual Hong Kong market, and all samples were in standard ‘cup’ grade. inspection, were commonly adulterated into EBN in order to increase Samples were labelled and stored at room temperature upon arrival. the net weight (Ma and Liu, 2012). Some businesses have been known The sample information was listed in Table 1. to mix low-quality EBN into high-quality EBN and selling that at a high price. Occasionally, consumers have been counterfeited into pur- Extraction and digestion of EBN chasing lower priced house EBN at a premium price associated with cave EBN. About 40 publications are found in PubMed today, and A cup (a common market size) of dried EBN was accurately nearly one-third of the publications are published in the last 5 years. 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 85 3–15 h (3 h for White EBN; 10 h for Yellow EBN; 15 h for Red parameters were set as follows: (i) pepsin A as the digestion enzyme, EBN), as described previously (Chan et al., 2013b). After discard- (ii) allowing absence of two internal cleavage sites, (iii) oxidation of ing the soaked water to remove contaminants, the EBN was rinsed methionine as a variable modification, (iv) ±1 Da for peptide mass with water three times. The soaked EBN was put into a stewing tolerance, and (v) ±0.8 Da for fragment mass tolerance. The false pot with 30 volumes of water. The soaked EBN was stewed for discovery rate was below 5% by evaluating the number of matched 8–40 h at 98 ± 2°C until it was completely molten (8 h for White proteins in the search of real database and decoy database. The EBN; 16 h for Yellow; 40 h for Red EBN). The sample was dia- peptide sequences were searched in the NCBI library and Chaetura lyzed overnight against water in a dialysis bag with 2000 cut- pelagica (Chimney swift) database. off molecular weight and then filtered, freeze-dried, and stored at 4°C until use. The EBN lyophilized powder was dissolved and Data analysis digested with a SGF (pH 2), consisting of pepsin and SGF without PCA is an unsupervised statistical model that transforms a set of enzyme, for 1 h at 37°C. The digest was neutralized with sodium observations of possibly correlated variables into a smaller group hydroxide and then filtered by 0.45  µm hydrophilic filter. The of linearly uncorrelated variables, thereby avoiding subjective deci- total protein content of EBN was determined by Kjeldahl method sions, making data easy to explore, and allowing for the visualiza- (Codex Alimentarius International Food Standards, 1999), and tion of significant differences in complex data sets with many factors the extracted protein was determined by the Bradford method (Abdi and William, 2010). Only those characteristic peaks having (Bio-Rad, Herculues, CA). higher than their respectively limit of quantification (LOQ) values were quantified and used for subsequent data analysis. Five peaks SDS–PAGE analysis (A, B, C, D, and E) could reach the LOQ requirement, and they were The EBN lyophilized powder was suspended in 1 ml Milli-Q water. selected and quantified with reference to the spiked protein apro- The samples were treated with direct lysis buffer (0.125 M Tris–Cl, tinin. PCA was performed using SIMCA 13 (Umetrics, Sweden) with pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.02% the parameter set to ‘PCA-X’. The relative amount of peaks A–E bromophenol blue) at 95°C for 5  min, and subsequently 10 μg of from the chromatographic fingerprints was used for PCA, and the extracted proteins of EBN were loaded onto a 15% SDS polyacryla- score plot, loading plot, and dendrogram were examined in order mide gel and run at 60 V for electrophoresis. After separation of to reveal possible relationship between peptide mapping and EBN. protein, the gel was stained by Coomassie blue reagent and then Statistical tests were done by using one-way analysis of variance destained by a solution consisting of water, methanol, and acetic (ANOVA) provided in GraphPad Prism 6.0. Statistically significant acid. Gel photos were captured by a scanner. changes were classed as [*] where P < 0.05, [**] where P < 0.01, and [***] where P < 0.001. HPLC conditions The chromatographic separation was performed on an Agilent HPLC 1200 series system (Agilent, Waldbronn, Germany), which Results was equipped with a diode array detector (DAD), a degasser, a Optimization of protein digestion on EBN binary pump, an autosampler, and a thermo-stated column compart- Twenty-five EBN samples collected from a Hong Kong market ment. The EBN samples were separated by a SymmetrySheild™ RP (Table  1) were extracted by the over-stewing method in order to C18 column (5 mm i.d., 250 mm × 4.6 mm). The mobile phase was ensure the complete solubility of EBN protein. To calculate the total composed of 0.1% trifluroacetic acid in water (A) and acetonitrile amount of protein in EBN (soluble or insoluble), nine crude EBN (B) using the following gradient program: 0–10  min, linear gradi- samples, including different colour, country of origin, and production ent 5–15% (B); 10–30 min, linear gradient 15–30% (B); 30–50 min, site, were sent to a food laboratory for protein analysis by Kjeldahl isocratic gradient 30% (B); 50–60 min, linear gradient 30–50% (B); method (Codex Alimentarius International Food Standards, 1999), a a pre-equilibration period of 20  min was used between each run. well-established test to investigate the total protein amount of food. The flow rate was 0.6 ml/min; the column temperature was 25°C; The average protein content of three White EBN was 48.5 ± 5.1% and the injection volume was 10 µl. The DAD wavelength was set (Sample 1, 2, 6); Yellow EBN was 55.7 ± 2.0% (Sample 15, 16, 17); to 214 nm since the peptide bond showed the highest absorbance at Red EBN was 53.1 ± 3.2% (Sample 19, 21, 25) of the dried weight this wavelength. that was very close to the literature value, i.e. 50% (Jiangsu New Medicine College, 1977). Thus, the reported value was adopted as Protein identification by LC–MS/MS the denominator in calculating the extraction rate for simplicity. By The eluent at 9–40 min from the peptide fingerprint was collected determining the extracted protein, 83.7 ± 10.0% (Sample 1–14) and by a Gilson FC203B fraction collector at 1  min per fraction. Five 81.5  ±  10.9% (Sample 15–18) of proteins were extracted among fractions representing peaks A, B, C, D, and E were collected and White and Yellow EBN samples, respectively; whereas 65.6 ± 7.3% subjected to protein identification. The samples were desalted, con- (Sample 19–25) were found in Red EBN samples. The low extrac- centrated, and purified by ZipTip and then analysed by a Thermo tion efficiency revealed in Red EBN was consistent with historical Scientific LTQ Velos Dual-Pressure Ion Trap Mass Spectrometer cou- wisdom of subjecting this EBN to a longer stewing time (Chan pled with a Thermo Accela 600 pump, an Accela autosampler LC, et al., 2013b). and ETD source (spray voltage 1.6 kV, capillary temperature 250°C). The extracted proteins of White EBN (Sample 1) were separated A Thermo Scientific BioBasic-18 column was used (150.0 × 0.1 mm, by SDS polyacrylamide gel, and most of them were stacked at the top 5 μm). Formic acid at 0.1% in MS-grade water and 0.1% formic of gel, which suggested that most of the soluble EBN proteins were acid in MS-grade acetonitrile were used as mobile phase A  and B, over 200  kDa (Figure  1A). After SGF digestion, the size of White respectively; with a flow rate of 0.15 ml/min. Mascot Daemon (ver - EBN proteins was decreased in a time-dependent manner. The pep- sion 2.3.0) was used as a sequence database searching engine. The sin from SGF served as a control at ~35 kDa. The EBN digest was 86 C.-F. Wong et al., 2017, Vol. 1, No. 1 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 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 lane represents EBN extract (indicated) without digestion; SGF lane with pepsin (indicated) served as blank control. The digested EBN protein (10 μl) 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 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 EBN, and Red EBN were revealed, and similar peaks B–E were identical. The common fake products, including agar, Tremella fungus, gelatin, pig 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 skin, and carrageenan, showed very different pattern. (B) Aprotinin was spiked into the EBN digest for HPLC fingerprinting. A  calibration curve of 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 aprotinin protein content was shown in the insert. The peak content in the fingerprint was quantified with reference to aprotinin. n   =  4. EBN, edible time points in peptide fingerprint indicated the digestion was completed after 60  min. n  =  4. EBN, edible bird’s nest; HPLC, high performance liquid bird’s nest; HPLC, high performance liquid chromatography; SGF, simulated gastric fluid. chromatography; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SGF, simulated gastric fluid. Characterization of edible bird’s nest, 2017, Vol. 1, No. 1 87 collected every 15 min and analysed by SDS–PAGE. The protein con- and Supplementary Table S1 show the estimated peptide contents tent of EBN digests, as well as the disappearance of high molecular of all identified fragments of different types of EBN. In general, the weight protein, was decreased to a steady status after 60 min indi- peptide contents of Red EBN were lower than the others: this low cating a full digestion by SGF (Figure 1A and 1B). The EBN digests amount could be an outcome of lower amount of extractable protein (Sample 1, 2, 3, and 4) at different time intervals were injected into derived from Red EBN. HPLC for fingerprinting analysis. Absorption at 214 nm was chosen To verify the authenticating power of peptide fingerprint, the pep- to obtain maximal peak height. Four distinguishable and dominant tide fingerprints of three White EBNs from different sources and two peaks (B–E) were selected as the reference points (Figure  2A). The adulterated EBN (being mixed with fake EBN) were generated. The areas of those peaks were analysed along the time course, and they chromatographic patterns of all samples were revealed, and similar reached maximum peak area after 60 min of digestion (Figure 1C). peaks B–E were observed (Supplementary Figure S2). The relative This digestion profile was revealed also from Yellow EBN (Sample amounts of peaks B–E were determined. As illustrated in Figure 3B, 15, 16, 17, and 18)  in Supplementary Figure S1A, and Red EBN the amounts of peaks B–E for EBN (Indonesia), EBN (Malaysia), (Sample 19, 20, 21, and 25)  in Supplementary Figure S1B. Thus, and EBN (Vietnam) were comparable to that of genuine EBN. The 60 min was decided as the complete digestion time of EBN extract. amounts of peaks of EBN mixed with fake EBN were significantly To identify major EBN proteins, the peaks of A–E were col- lower than genuine EBN (Figure  3B). Muc-5AC-like protein (peak lected and sent to ion-trap LC–MS/MS analysis. Hundreds of pro- D) was therefore being considered as a tentative marker with the teins, including origins from avian, mammals, insects, and bacteria, highest discriminative power in authenticating EBN. were matched; and some of them had been reported in previous studies, including AMCase-like (Liu et  al., 2012; You et  al., 2015) Principal component analysis and transferrin (Hou et  al., 2015). None of the proteins identified PCA was performed on the peptide mapping of 25 EBN to detect from EBN extracts matched with Aerodramus, likely because the anomalies and to study relationships in the data. Each sample num- genome sequence of swiftlet was not completed. We then searched ber was labelled in the model for a better visual illustration and the protein sequences from C. pelagica, a species in the same family interpretation. By using the relative peak amount of HPLC peptide as A. fuciphagus. The common proteins were selected among three fingerprints, the dendrogram, score plot, and loading plot based on trials, and blank SGF was served as a control. Only the fragments different classifications of EBN were generated. of AMCase-like, lysyl oxidase homolog 3, Muc-5AC-like fragment, From the score plot (Figure  4A), Red EBN and White/Yellow and AMCase-like were identified in peak B, C, D, and E, respec- EBN could be unambiguously identified utilizing the first two princi- tively, in all samples, which confirmed the identities of EBN proteins pal components, PC1 (t1) and PC2 (t2), which accounted for 85.8% (Table 2). Although fragments from peaks B and E were identified as of the total variation. The values of fitness of data (R2) and predic- AMCase-like proteins, they represented unique AMCase-like frag- tive ability (Q2) were 85.6% and 92.3%, respectively, in the model. ments matching to accession numbers in the database. Analysis showed no samples being outside the Hotelling T2 95% confidence ellipse that could influence the analyses. Based on the Peptide fingerprint and its application loading plot (Figure 4B), Mucin-5AC-like (peak D) showed the high- The HPLC peptide fingerprints were generated from 25 SGF-digested est positive loading score of 0.70, accounting for the colour discrimi- EBN samples, and typical fingerprints of White, Yellow, and Red nation of samples by PC1. In agreement with the results of PCA, EBN were given in Figure  2A. In contrast, the products commonly application of PCA–HCA (Figure 4C) for the whole data set resulted used to imitate EBN (e.g. agar, Tremella fungus, gelatin, pig skin, and in a distinct classification of the samples according to their colours. carrageenan) did not show any similarity to the EBN fingerprint. The Twenty-five samples were correctly classified according to their col- peptide fingerprints showed a close similarity among White, Yellow, our; nonetheless, White EBN and Yellow EBN were not distinguish- and Red EBN, except that the peak heights were varied (Figure 2A). able according to the score plot and dendrogram. To quantify the characteristic peaks of EBN fingerprints, apro- Since colour of EBN shows significant variation in fingerprint tinin, a small protein of ~6 kDa, was used as an external standard. and PCA, only White EBN samples were used for comparison of Aprotinin was spiked into EBN samples, and it did not interfere with production site of EBN to hold other factors constant. House White most of the characteristic peaks (Figure 2B). By referring to aprotinin EBN and cave White EBN could be distinctly classified into two peak, the peak retention time and peak height could be calibrated, clusters using the first two principal components (t1 and t2), which and a calibration curve from 0.5 to 10 µg of aprotinin was estab- explained a total of 82.7% variation (Supplementary Figure S3). lished by absorption at 214 nm (Figure 2B insert). Using this stand- Analysis showed no samples being outside the Hotelling T2 95% ard curve, the relative peptide content could be estimated. Figure 3A confidence ellipse. Fitness of data (R2) of this model was 82.7%, 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 Score Accession*** A Unknown B AMCase-like LYEGPSDTGDLV 43 XP_010005363.1 C Lysyl oxidase homolog 3 LKGGAKVGEGRVEVLR 71 XP_010006484.1 D Muc-5AC-like MWDKKTSIF 41 XP_009994736 E AMCase-like AIGGWNFGTAKF 39 XP_010006620.1 *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. 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 Figure  4. The differentiation of chromatographic fingerprints of various EBN evaluate the predictive power of this model, the third and fourth colour. Twenty-five EBN peptide fingerprints from different colours were principal components (t3 and t4) were also investigated. Although generated: White (1–14); Yellow (15–18); Red (19–25). (A) Score plot of EBN house White EBN and cave White EBN could be classified into two samples. The ellipse represents the Hotelling T2 with 95% confidence. (B) Loading clusters using the first and third principal components (t1 and t3) plot for five characteristic peaks (A–E as shown in Figure 2A). (C) Dendrogram in both score plot and dendrogram, as indicated in Supplementary showing hierarchical clustering results of EBN samples. EBN, edible bird’s nest. Figure S4, the predictive ability (Q2) of this model dropped to 57.46%. The R2 and Q2 value distinctly increased to 98.0% and (t1 and t4) in the model. Furthermore, house White EBN and cave 88.9%, respectively, by using first and fourth principal components 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 C.-F. Wong et al., 2017, Vol. 1, No. 1 separation of the samples; and all of them were correctly classified water. In order to enhance good extraction efficiency of EBN, an according to their production sites. over-stewing method was developed. Only water was utilized in our In order to eliminate the critical variation from EBN colour in extraction: because this should be the method used for preparation fingerprint and PCA, only White EBN samples were used for com- of EBN for human consumption. About 66~84% of EBN proteins parison of EBN from different countries of origin. As illustrated in were extracted, supporting the hypothesis that over-stewing method the score plot (Figure 6A), White EBN from different countries could could be a suitable way to extract EBN proteins. Using the same not be clearly classified into clusters by using the first two princi- extraction protocol, White and Yellow EBN showed better extrac- pal components (t1 and t2), which explained 82.7% of the total tion efficiency as compared to that of Red EBN. The difference in variation. The values of fitness of data (R2) and predictive ability extraction rate of White EBN (~84%) and Red EBN (~66%) may be (Q2) were 82.7% and 63.0%, respectively, in this model. Analysis due to conformational or structural difference of the major proteins. showed no samples being outside the Hotelling T2 95% confidence Indeed, much longer time was required to extract Red EBN, which ellipse. PCA–HCA (Figure  6C) showed that the peptide fingerprint implied Red EBN possessed higher resistance to protein digestion. could not clearly determine country of origin, which was consistent Therefore, the protein in Red EBN might not be fully extracted by with the results of PCA. Moreover, EBN samples from Indonesia stewing. Moreover, very limited research has been reported on Red and Malaysia clustered on the right of score plot, and EBN samples EBN, especially the protein part. Thus, further studies have to be came from Vietnam and Thailand clustered on the left. From the conducted on the proteomics of Red EBN. loading plot (Figure  6B), Mucin-5AC (peak D) showed the highest The lack of a complete genome sequence of Aerodramus pre- positive loading score at 0.80: this could serve as a tentative maker sents a challenge for protein identification in EBN. The identified in discriminating the samples between Indonesia/Malaysia EBN and peptide sequences were matched with animal species in NCBI data- Vietnam/Thailand EBN. 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- Method validation ica, a member of the Apodidae, the EBN proteins were searched with Sensitivity, linearity, accuracy, and precision of the HPLC peptide fin- this database. Muc-5AC-like, AMCase-like, and lysyl oxidase pro- gerprint technique were evaluated. Sensitivity was evaluated by the tein fragments were identified in the fingerprint. Mucins are a family limit of detection (LOD) and limit of quantification (LOQ). A series of glycoprotein with high molecular weight produced by epithelial of decreasing concentrations of aprotinin was evaluated to deter- tissues in most organisms (Marin et al., 2007). The major character- mine the LOD and LOQ. LOD was determined as the concentra- istic of mucin is that it is able to form a gel, which therefore is a key tion with a signal-to-noise ratio (S/N) of at least 3; while LOQ was component in most gel-like secretions, e.g. saliva. In addition, the the lowest concentration with a S/N of at least 10. Linearity was mucin secreted in airways is Muc-5AC, and as a result it is found evaluated by five calibrators prepared by spiking the aprotinin in in the saliva. Chitinases have a family of 18 glycosyl hydrolases; blank matric at concentrations of 0.5, 1, 2, 5, and 10 µg. Precision however, their physiological functions are still not fully resolved. and accuracy were evaluated on the peak area of the characteristic AMCase belongs to the glycosyl hydrolase family in mammals (Boot peaks. Intra-batch precision was evaluated by six determinations per et  al., 2001; Bussink et  al., 2007). In the present work, AMCase- single concentration in a day. Inter-batch precision was evaluated like protein was reported in EBN for the third time, and it seemed by one determination per single concentration at six different days. strange that ‘mammalian’ chitinase was found in birds. Until now, Precision was expressed as the percent of relative standard deviation chitinase protein sequences have not been found in the databases (RSD) calculated by using six determinations. The value of recov- of C. pelagica and M. gallopavo, and thus only the homologues of ery was expressed as the ratio of determined concentration from AMCase-like protein were matched. Insects are the common food six individual tests to corresponding known concentration of the of Aerodramus, and the existence of chitinase in swiftlets could spiked marker. A  known amount of aprotinin was spiked into the facilitate the digestion of insect. Lysyl oxidase is a copper-dependent EBN digest, and recovery was calculated based on the concentration amine oxidase that plays a critical role in the biogenesis of connec- of obtained from the chromatogram and the spiked amount. The tive tissue matrices by cross-linking the extracellular matrix proteins, results of method validation were listed on Table 3. collagen, and elastin (Li and Kagan, 1998). The presence of lysyl oxidase could explain why a previous study found that EBN could Discussion facilitate the synthesis of collagen (Chua et al., 2013). Traditionally, EBN is stewed with water for 1–2  h before eating, In a 2011 study, nitrite was detected in EBN available in the mar- but most of the EBN proteins still remain insoluble: this stewing ket, especially Red EBN. This incident revealed the insufficient qual- method accounts only ~5% protein extraction rate. Before any ity control of EBN products in the industry. A variety authentication kind of protein analysis, the EBN proteins need to be dissolved in methods based on the appearance (Liu and Zhang, 1995; Wu et al., Table 3. Method validation. EBN, edible bird’s nest; LOD, limit of detection; LOQ, limit of quantification. LOD* LOQ* Linearity** Correlation coefficient** (R) Intra-batch precision*** Inter-batch precision*** Recovery**** 0.2 µg 0.5 µg 0.5–10 µg >0.995 1.5% 3.5% 90.7% *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. Characterization of edible bird’s nest, 2017, Vol. 1, No. 1 91 2007), total proteins (Hu and Lai, 1999; Liu et al., 2012; Zhang et al., clearly demonstrate peptide profile differences between genuine and 2012), N-acetylneuraminic acid (Chan et al., 2013a), sialic acid (Wang adulterated EBN samples, which provide a comprehensive picture et al., 2006), DNA (Wu et al., 2010), and sodium nitrate (Chan et al., in quality assurance of EBN. The protein identification results sug- 2013b) of EBN have been established; but no official method could be gested that Muc-5AC-like protein was the major protein in EBN. To found for quality control of EBN by specific protein analysis, regard- our knowledge, the present work is the first report highlighting the less of its abundance. Here, we utilized SGF digestion and HPLC to use of protein, which is main component of EBN, in HPLC finger - generate the peptide fingerprint, which could authenticate EBN on a printing studies. qualitative and quantitative basis robustly and therefore provide a comprehensive picture in quality control of EBN. The peptide finger - Supplementary Material print was able to clearly demonstrate the differences between genuine and fake EBN samples, and thus this method could serve as an authen- Supplementary material is available at Food Quality and Safety ticating tool for EBN. Muc-5AC-like fragment (peak D) was the major online. peptide in the fingerprint of EBN, accounting of ~20% total content in the fingerprinting. This indicated mucin-like protein could be the most Funding abundant protein in EBN. In addition, this mucin peak could serve as an internal marker for the fingerprinting. The chromatographic pat- This research was supported by the grants of Hong Kong Research tern of peptide fingerprint was highly similar among EBN samples Grants Council TBS (T13-607/12R), GRF (661110, 662911, with different colours, suggesting that total extractable protein com- 660411, 663012, 662713), ITC (UIM/254), TUYF12SC02, position does not vary widely based on colour. TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club From the score plot and dendrogram, significant differences were Charities Trust (HKJCCT12SC01) and Foundation of The recorded between White EBN and Red EBN. Red EBN is known Awareness of Nature (TAON12SC01), SRFDP and RGC ERG Joint to dissolve poorly in water, and indeed the protein extraction from Fund (2013009614001/M-HKUST604/13) to Karl Wah-Keung EBN is much lower than that of White or Yellow EBN, as shown Tsim; and National Natural Science Foundation Item (81173498) in the present work. This low solubility of Red EBN protein could to Xiao-Ping Lai. 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 Acknowledgements et  al., 2013). Herein, Paydar group suggested Red EBN could be Chun-Fai Wong received a HK JEBN scholarship. a result of formation of aryl-C-N and NO side group in aromatic Conflict of interest statement. None declared. 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 References digestion. Moreover, further studies have to be conducted on the Abdi, H., William, L. J. (2010). Principal component analysis. Wiley Interdis- underlying mechanism of how EBN gets red. ciplinary Reviews: Computational Statistics, 2: 433–459. No significant difference was found among the country of ori- Aswir, A. R., Wan Nazaimoon, W. M. (2011). Effect of edible bird’s nest on gin of White EBN, thus implying the protein composition should be cell proliferation and tumor necrosis factor-alpha (TNF-α) release in vitro. more or less the same regardless their production sources. However, International Food Research, 18: 1123–1127. EBN samples from Indonesia and Malaysia clustered on the right of Boot, R. G. et al. (2001). Identification of a novel acidic mammalian chitinase score plot, and EBN samples from Vietnam and Thailand clustered distinct from chitotriosidase. Journal of Biological Chemistry, 276: 6770– on the left. The geographic location may explain the difference, i.e. Indonesia and Malaysia or Vietnam and Thailand are neighbouring Bussink, A. P., Speijer, D., Aerts, J. M. F. G., Boot, R. G. (2007). Evolution of countries. mammalian chitinase (-like) members of family 18 glycosyl hydrolases. Genetics, 177: 959–970. The PCA results suggested that there was a difference between But, P. H., Jiang, R. W., Shaw, P. C. (2013). Edible bird’s nests—how do the red house and cave White EBN in both dendrogram and score plot. The ones get red? Journal of Ethnopharmacology, 145: 378–380. protein composition is probably different due to habitat of the swift- Chan, G. K., Zheng, K. Y., Zhu, K. Y., Dong, T. T., Tsim, K. W. (2013a). Deter- lets. Most of the caves are located at the seafront and on islands, mination of free N-acetylneuraminic acid in edible bird nest: a develop- whereas bird nest houses often are built in suburban areas or forests. ment of chemical marker for quality control. Journal of Ethnobiology and The diet of swiftlets could be different and thus affect the secretion Traditional Medicine, 120: 620–628. of EBN. Besides, the environment of a bird nest house can be main- Chan, G. K., Zhu, K. Y., Chou, J. Y., Guo, J. Y., Dong, T. X., Tsim, W. K. (2013b). tained and regulated by the farmers, which may be different from the Surveillance of nitrite level in cubilose: evaluation of removal method and natural cave environment. These findings complement a recent study proposed origin of contamination. Food Control, 34: 637–644. by Cheng’s group whose differentiated between house and cave EBN Chan, K. L. et al. (2015). Edible bird’s nest, an Asian health food supplement, possesses skin lightening activities: identification of N-acetylneuraminic based on amino acid composition (Seow et al., 2016). acid as active ingredient. Journal of Cosmetics, Dermatological Sciences and Applications, 5: 262–274. Chinese Pharmacopoeia Commission, China. (2015). Pharmacopoeia of the Conclusion People’s Republic of China. China Medical Science Press. PCA suggested that the peptide fingerprint could serve as a promis- Chua, K. H. et  al. (2013). Edible bird’s nest extract as a chondro-protective ing and useful tool to classify EBN samples by colour and production agent for human chondrocytes isolated from osteoarthritic knee: in vitro site. As proposed by the model developed in this study, Muc-5AC- study. BMC Complementary and Alternative Medicine, 13: 19. like and AMCase-like proteins were the markers with the highest Codex Alimentarius International Food Standards. (1999). Recommended discriminative power. In addition, HPLC fingerprints were able to Methods of Analysis and Sampling, CODEX STAN 234. 92 C.-F. Wong et al., 2017, Vol. 1, No. 1 Deng, Y. E., Sun, S. Q., Zhou, Q., Li, A. (2006). Analysis and discrimination of Liu, X., Zhang, J. S. (1995). Research on the identification of edible bird’s Collocalia esculenta L. via FTIR spectroscopy. Spectroscopy and Spectral nest and its products. Chinese Journal of Tour Medical Science, 1: 26– Analysis, 26: 1242–1245. 28. Department of Health, Hong Kong. (2010). 4.3 High-performance liquid Ma, F., Liu, D. (2012). Sketch of the edible bird’s nest and its important bioac- chromatography fingerprinting. Hong Kong Chinese Materia Medica tivities. Food Research International, 48: 559–567. Standards, Vol. 3. pp. 13–20. Marin, F. D.  R., Luquet, G., Marie, B, Medakovic, D. (2007). Molluscan Guo, C.-T. et  al. (2006). Edible bird’s nest inhibits influenza virus infection. shell proteins: primary structure, origin, and evolution. Current Topics in Antiviral Research, 70: 140–146. Developmental Biology, 80: 209. Hou, Z. et al. (2015). Lactoferrin and ovotransferrin contribute toward anti- Matsukawa, N. et al. (2011). Improvement of bone strength and dermal thick- oxidative effects of Edible Bird’s Nest against hydrogen peroxide-induced ness due to dietary edible bird’s nest extract in ovariectomized rats. Biosci- oxidative stress in human SH-SY5Y cells. Bioscience, Biotechnology, and ence, Biotechnology, and Biochemistry, 75: 590–592. Biochemistry, 79: 1570–1578. Paydar M., Wong Y. L., Wong W. F., Hamdi O. A. A., Kadir N. A., Looi C. Y., Hu, S. M., Lai, D. M. (1999). SDS-PAGE identification of edible bird’s nest. (2013). Prevalence of Nitrite and Nitrate Contents and Its Effect on Edible Journal of Chinese Medicine, 24: 331–334. Bird Nest’s Color. Journal of Food Science, 78: 1940–1947. Jiangsu New Medicine College (Former of Nanjing Medical University). Seow, E. K., Ibrahim, B., Muhammad, S. A., Lee, L. H., Cheng, L. H. (2016). (1977). Dictionary of Traditional Chinese Medicine, Vol. 2. Shanghai Peo- Differentiation between house and cave edible bird’s nests by chemometric ple’s Publishing House, China, pp. 2653–2654. analysis of amino acid composition data. LWT - Food Science and Tech- Kang, N., Hails, C. J., Sigurdsson, J. B. (1991). Nest construction and egg- nology, 65: 428–435. laying in edible-nest swiftlets Aerodramus spp. and the implications for Wang, H., Ni, K. Y., Wang, Y. (2006). Determination of sialic acid in edible harvesting. IBIS, 133: 170–177. bird’s nest. Chinese Journal of Pharmaceutical Analysis, 26: 1251–1253. Kong, Y. C., Keung, W. M., Yip, T. T., Ko, K. M., Tsao, S. W., Ng, M. H. (1987). Wu, R. H., Chen, Y., Wu, Y. J., Zhao, J. Y., Ge, Y. Q. (2007). Review of EBN Evidence that epidermal growth factor is present in swiftlet’s (Collocalia) authentication method. Inspection and Quarantine Science, 17: 60–62. nest. Comparative Biochemistry and Physiology, Part B: Biochemistry & Wu, Y. J. et  al. (2010). Application of SYBR green PCR and 2DGE methods Molecular Biology, 87: 221–226. to authenticate edible bird’s nest food. Food Research International, 43: Kong, Y. C., Tsao, S. W., Song, M. E., Ng, M. H., Lin, Z. F. (1989). Potentiation 2020–2026. of mitogenic response by extracts of the swiftlet’s (Apus) nest collected Yew, M. Y., Koh, R. Y., Chye, S. M., Othman, I., Ng, K. Y. (2014). Edible bird’s from Huai-Ji. Acta Zoologica Sinica, 35: 429–435. nest ameliorates oxidative stress-induced apoptosis in SH-SY5Y human Koon, L. C., Cranbrook, E. (2002). Swiftlets of Borneo – Builders of Edible neuroblastoma cells. BMC Complementary and Alternative Medicine, 14: Nests. Natural History Publication (Borneo) SDN, B.H.D., Sabah, Malay- 391. sia, pp. 1–171. You, Y. Y. et  al. (2015). Purification and identification of α 2–3 linked sialo- Li, S. M., Kagan, H. M. (1998). Lysyl oxidase: properties, regulation and mul- glycoprotein. European Food Research and Technology, 240: 389–397. tiple functions in biology. Matrix Biology, 16: 387–398. Zhang, S. W. et al. (2012). Competitive enzyme-linked immunoassay for sialo- Liu, X. Q. et al. (2012). Proteomic profile of edible bird’s nest proteins. Journal glycoprotein of edible bird’s nest in food and cosmetics. Journal of Agri- of Agricultural and Food Chemistry, 60: 12477–12481. cultural and Food Chemistry, 60: 3580–3585. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food Quality and Safety Oxford University Press

Characterization of edible bird’s nest by peptide fingerprinting with principal component analysis

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Oxford University Press
Copyright
© The Author 2017. Published by Oxford University Press on behalf of Zhejiang University Press.
ISSN
2399-1399
eISSN
2399-1402
DOI
10.1093/fqs/fyx002
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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. which has been proven to have nutritious and therapeutic values, such Introduction as anti-influenza viruses, antioxidant, skin lightening, bone strength Edible bird’s nest (EBN), or cubilose, is a health food supplement improvement, anti-inflammatory, and epidermal growth enhancement originated from salivary secretion by specific swiftlets, mainly from (Kong et  al., 1987; Kong et  al., 1989; Guo et  al., 2006; Aswir and Aerodramus fuciphagus and Aerodramus maximus (Kang et al., 1991), 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. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. 84 C.-F. Wong et al., 2017, Vol. 1, No. 1 Chan et  al., 2015). Southeast Asian countries, including Indonesia, Besides, most of the publications still retained in elucidating chemi- Malaysia, Vietnam, and Thailand, are the major exporting countries of cal composition as the quality control parameters: since no official EBN. Human consumption and medicinal application of EBN could be method has been established for quality surveillance of EBN (Deng dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty et al., 2006; Wang et al., 2006; Wu et al., 2010; Chan et al., 2013a). (960–1279 A.D.) in China (Koon and Cranbrook, 2002). Here, we attempt to find a key to open these proteome barri- Although EBN has been served as an esteemed food in Chinese ers by high performance liquid chromatography (HPLC) peptide community for over 1000 years, limited research has been conducted fingerprinting. HPLC fingerprinting is one promising tool widely on EBN and its proteins. Protein is a major part of EBN accounting used in the modern standardization of herbal extracts (Department for 50% of EBN dried weight on average (Jiangsu New Medicine of Health, Hong Kong, 2010; Chinese Pharmacopoeia Commission, College, 1977); it is conjectured to be a key factor of its nourishing and/ China, 2015), which could be applied to EBN as a robust technique or medicinal functions. The epidermal growth factor (EGF)-like pep- in qualitative and quantitative controls. Firstly, an over-stewing tide was partially purified with Bio-Gel P-10 columns from aqueous method was developed to extract most of the EBN protein. Secondly, extracts of EBN that stimulated cell division and growth and enhanced simulated gastric fluid (SGF) was used to digest EBN protein fully tissue growth and regeneration (Kong et al., 1987; Kong et al., 1989). into peptides that can be separated by HPLC according to their Two major bands (~106 kDa and ~128 kDa) were identified by sodium polarity. Thirdly, according to the most relevant NCBI protein data- dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) as base, the characteristic peaks in chromatograms were identified ‘sialo-glycoprotein’. Nevertheless, no satisfactory result was obtained and quantified. In addition, principal component analysis (PCA) from protein identification studies that include N-terminal sequence and hierarchical cluster analysis (HCA) were adopted to reveal the determination (Edman degradation), matrix-assisted laser desorption relationships of factors within the data, including colour, country of ionization–tandem time of flight (MALDI–TOF/TOF), and liquid origin, and production site of EBN. The results therefore contributed chromatography–tandem mass spectrometry (LC–MS/MS) (Zhang to the authentication and classification of EBN. This study aimed et al., 2012). Acidic mammalian chitinase-like (AMCase-like) protein to provide comprehensive information for the quality evaluation of fragments from Meleagris gallopavo and an allergen homologous EBN protein at the peptide level, which would be a valuable refer- to ovo-inhibitor have been identified by 2-DE assays followed by ence for further study on EBN proteins. MALDI–TOF/TOF/MS analysis in EBN extract (Liu et  al., 2012). In addition, a microbial nitrate reductase, converting nitrate to nitrite and Materials and Methods 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 Chemicals and EBN unclear whether those identified proteins could accurately represent Pepsin, SGF without enzyme (contains 0.07 M hydrochloric acid the majority of EBN protein. The difficulties encountered in research and 0.1 M sodium chloride), and trifluroacetic acid were purchased of EBN proteins are: (i) extracting and purifying proteins; and (ii) lack- from Sigma/Aldrich (St Louis, MO). LC-MS-grade acetonitrile was ing of full Aerodramus genome sequence. obtained from JT Baker (Center Valley, PA). Aprotinin, a monomeric Owing to the limited supply and labour-intensive cleaning process, globular polypeptide with a molecular weight of 6512, was obtained EBN is always expensive with current prices ranging from USD 500 from GE Healthcare (Buckinghamshire, UK). Twenty-five batches of to 15 000/kg. Driven by the lucrative return, various materials, includ- EBN, including different colour, country of origin, and production ing Tremella fungus, fried porcine skin, carrageenan, agar, and gelatin, site, from different commercial bands were randomly purchased in which are almost indistinguishable from the genuine samples by visual Hong Kong market, and all samples were in standard ‘cup’ grade. inspection, were commonly adulterated into EBN in order to increase Samples were labelled and stored at room temperature upon arrival. the net weight (Ma and Liu, 2012). Some businesses have been known The sample information was listed in Table 1. to mix low-quality EBN into high-quality EBN and selling that at a high price. Occasionally, consumers have been counterfeited into pur- Extraction and digestion of EBN chasing lower priced house EBN at a premium price associated with cave EBN. About 40 publications are found in PubMed today, and A cup (a common market size) of dried EBN was accurately nearly one-third of the publications are published in the last 5 years. 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 85 3–15 h (3 h for White EBN; 10 h for Yellow EBN; 15 h for Red parameters were set as follows: (i) pepsin A as the digestion enzyme, EBN), as described previously (Chan et al., 2013b). After discard- (ii) allowing absence of two internal cleavage sites, (iii) oxidation of ing the soaked water to remove contaminants, the EBN was rinsed methionine as a variable modification, (iv) ±1 Da for peptide mass with water three times. The soaked EBN was put into a stewing tolerance, and (v) ±0.8 Da for fragment mass tolerance. The false pot with 30 volumes of water. The soaked EBN was stewed for discovery rate was below 5% by evaluating the number of matched 8–40 h at 98 ± 2°C until it was completely molten (8 h for White proteins in the search of real database and decoy database. The EBN; 16 h for Yellow; 40 h for Red EBN). The sample was dia- peptide sequences were searched in the NCBI library and Chaetura lyzed overnight against water in a dialysis bag with 2000 cut- pelagica (Chimney swift) database. off molecular weight and then filtered, freeze-dried, and stored at 4°C until use. The EBN lyophilized powder was dissolved and Data analysis digested with a SGF (pH 2), consisting of pepsin and SGF without PCA is an unsupervised statistical model that transforms a set of enzyme, for 1 h at 37°C. The digest was neutralized with sodium observations of possibly correlated variables into a smaller group hydroxide and then filtered by 0.45  µm hydrophilic filter. The of linearly uncorrelated variables, thereby avoiding subjective deci- total protein content of EBN was determined by Kjeldahl method sions, making data easy to explore, and allowing for the visualiza- (Codex Alimentarius International Food Standards, 1999), and tion of significant differences in complex data sets with many factors the extracted protein was determined by the Bradford method (Abdi and William, 2010). Only those characteristic peaks having (Bio-Rad, Herculues, CA). higher than their respectively limit of quantification (LOQ) values were quantified and used for subsequent data analysis. Five peaks SDS–PAGE analysis (A, B, C, D, and E) could reach the LOQ requirement, and they were The EBN lyophilized powder was suspended in 1 ml Milli-Q water. selected and quantified with reference to the spiked protein apro- The samples were treated with direct lysis buffer (0.125 M Tris–Cl, tinin. PCA was performed using SIMCA 13 (Umetrics, Sweden) with pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.02% the parameter set to ‘PCA-X’. The relative amount of peaks A–E bromophenol blue) at 95°C for 5  min, and subsequently 10 μg of from the chromatographic fingerprints was used for PCA, and the extracted proteins of EBN were loaded onto a 15% SDS polyacryla- score plot, loading plot, and dendrogram were examined in order mide gel and run at 60 V for electrophoresis. After separation of to reveal possible relationship between peptide mapping and EBN. protein, the gel was stained by Coomassie blue reagent and then Statistical tests were done by using one-way analysis of variance destained by a solution consisting of water, methanol, and acetic (ANOVA) provided in GraphPad Prism 6.0. Statistically significant acid. Gel photos were captured by a scanner. changes were classed as [*] where P < 0.05, [**] where P < 0.01, and [***] where P < 0.001. HPLC conditions The chromatographic separation was performed on an Agilent HPLC 1200 series system (Agilent, Waldbronn, Germany), which Results was equipped with a diode array detector (DAD), a degasser, a Optimization of protein digestion on EBN binary pump, an autosampler, and a thermo-stated column compart- Twenty-five EBN samples collected from a Hong Kong market ment. The EBN samples were separated by a SymmetrySheild™ RP (Table  1) were extracted by the over-stewing method in order to C18 column (5 mm i.d., 250 mm × 4.6 mm). The mobile phase was ensure the complete solubility of EBN protein. To calculate the total composed of 0.1% trifluroacetic acid in water (A) and acetonitrile amount of protein in EBN (soluble or insoluble), nine crude EBN (B) using the following gradient program: 0–10  min, linear gradi- samples, including different colour, country of origin, and production ent 5–15% (B); 10–30 min, linear gradient 15–30% (B); 30–50 min, site, were sent to a food laboratory for protein analysis by Kjeldahl isocratic gradient 30% (B); 50–60 min, linear gradient 30–50% (B); method (Codex Alimentarius International Food Standards, 1999), a a pre-equilibration period of 20  min was used between each run. well-established test to investigate the total protein amount of food. The flow rate was 0.6 ml/min; the column temperature was 25°C; The average protein content of three White EBN was 48.5 ± 5.1% and the injection volume was 10 µl. The DAD wavelength was set (Sample 1, 2, 6); Yellow EBN was 55.7 ± 2.0% (Sample 15, 16, 17); to 214 nm since the peptide bond showed the highest absorbance at Red EBN was 53.1 ± 3.2% (Sample 19, 21, 25) of the dried weight this wavelength. that was very close to the literature value, i.e. 50% (Jiangsu New Medicine College, 1977). Thus, the reported value was adopted as Protein identification by LC–MS/MS the denominator in calculating the extraction rate for simplicity. By The eluent at 9–40 min from the peptide fingerprint was collected determining the extracted protein, 83.7 ± 10.0% (Sample 1–14) and by a Gilson FC203B fraction collector at 1  min per fraction. Five 81.5  ±  10.9% (Sample 15–18) of proteins were extracted among fractions representing peaks A, B, C, D, and E were collected and White and Yellow EBN samples, respectively; whereas 65.6 ± 7.3% subjected to protein identification. The samples were desalted, con- (Sample 19–25) were found in Red EBN samples. The low extrac- centrated, and purified by ZipTip and then analysed by a Thermo tion efficiency revealed in Red EBN was consistent with historical Scientific LTQ Velos Dual-Pressure Ion Trap Mass Spectrometer cou- wisdom of subjecting this EBN to a longer stewing time (Chan pled with a Thermo Accela 600 pump, an Accela autosampler LC, et al., 2013b). and ETD source (spray voltage 1.6 kV, capillary temperature 250°C). The extracted proteins of White EBN (Sample 1) were separated A Thermo Scientific BioBasic-18 column was used (150.0 × 0.1 mm, by SDS polyacrylamide gel, and most of them were stacked at the top 5 μm). Formic acid at 0.1% in MS-grade water and 0.1% formic of gel, which suggested that most of the soluble EBN proteins were acid in MS-grade acetonitrile were used as mobile phase A  and B, over 200  kDa (Figure  1A). After SGF digestion, the size of White respectively; with a flow rate of 0.15 ml/min. Mascot Daemon (ver - EBN proteins was decreased in a time-dependent manner. The pep- sion 2.3.0) was used as a sequence database searching engine. The sin from SGF served as a control at ~35 kDa. The EBN digest was 86 C.-F. Wong et al., 2017, Vol. 1, No. 1 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 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 lane represents EBN extract (indicated) without digestion; SGF lane with pepsin (indicated) served as blank control. The digested EBN protein (10 μl) 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 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 EBN, and Red EBN were revealed, and similar peaks B–E were identical. The common fake products, including agar, Tremella fungus, gelatin, pig 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 skin, and carrageenan, showed very different pattern. (B) Aprotinin was spiked into the EBN digest for HPLC fingerprinting. A  calibration curve of 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 aprotinin protein content was shown in the insert. The peak content in the fingerprint was quantified with reference to aprotinin. n   =  4. EBN, edible time points in peptide fingerprint indicated the digestion was completed after 60  min. n  =  4. EBN, edible bird’s nest; HPLC, high performance liquid bird’s nest; HPLC, high performance liquid chromatography; SGF, simulated gastric fluid. chromatography; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SGF, simulated gastric fluid. Characterization of edible bird’s nest, 2017, Vol. 1, No. 1 87 collected every 15 min and analysed by SDS–PAGE. The protein con- and Supplementary Table S1 show the estimated peptide contents tent of EBN digests, as well as the disappearance of high molecular of all identified fragments of different types of EBN. In general, the weight protein, was decreased to a steady status after 60 min indi- peptide contents of Red EBN were lower than the others: this low cating a full digestion by SGF (Figure 1A and 1B). The EBN digests amount could be an outcome of lower amount of extractable protein (Sample 1, 2, 3, and 4) at different time intervals were injected into derived from Red EBN. HPLC for fingerprinting analysis. Absorption at 214 nm was chosen To verify the authenticating power of peptide fingerprint, the pep- to obtain maximal peak height. Four distinguishable and dominant tide fingerprints of three White EBNs from different sources and two peaks (B–E) were selected as the reference points (Figure  2A). The adulterated EBN (being mixed with fake EBN) were generated. The areas of those peaks were analysed along the time course, and they chromatographic patterns of all samples were revealed, and similar reached maximum peak area after 60 min of digestion (Figure 1C). peaks B–E were observed (Supplementary Figure S2). The relative This digestion profile was revealed also from Yellow EBN (Sample amounts of peaks B–E were determined. As illustrated in Figure 3B, 15, 16, 17, and 18)  in Supplementary Figure S1A, and Red EBN the amounts of peaks B–E for EBN (Indonesia), EBN (Malaysia), (Sample 19, 20, 21, and 25)  in Supplementary Figure S1B. Thus, and EBN (Vietnam) were comparable to that of genuine EBN. The 60 min was decided as the complete digestion time of EBN extract. amounts of peaks of EBN mixed with fake EBN were significantly To identify major EBN proteins, the peaks of A–E were col- lower than genuine EBN (Figure  3B). Muc-5AC-like protein (peak lected and sent to ion-trap LC–MS/MS analysis. Hundreds of pro- D) was therefore being considered as a tentative marker with the teins, including origins from avian, mammals, insects, and bacteria, highest discriminative power in authenticating EBN. were matched; and some of them had been reported in previous studies, including AMCase-like (Liu et  al., 2012; You et  al., 2015) Principal component analysis and transferrin (Hou et  al., 2015). None of the proteins identified PCA was performed on the peptide mapping of 25 EBN to detect from EBN extracts matched with Aerodramus, likely because the anomalies and to study relationships in the data. Each sample num- genome sequence of swiftlet was not completed. We then searched ber was labelled in the model for a better visual illustration and the protein sequences from C. pelagica, a species in the same family interpretation. By using the relative peak amount of HPLC peptide as A. fuciphagus. The common proteins were selected among three fingerprints, the dendrogram, score plot, and loading plot based on trials, and blank SGF was served as a control. Only the fragments different classifications of EBN were generated. of AMCase-like, lysyl oxidase homolog 3, Muc-5AC-like fragment, From the score plot (Figure  4A), Red EBN and White/Yellow and AMCase-like were identified in peak B, C, D, and E, respec- EBN could be unambiguously identified utilizing the first two princi- tively, in all samples, which confirmed the identities of EBN proteins pal components, PC1 (t1) and PC2 (t2), which accounted for 85.8% (Table 2). Although fragments from peaks B and E were identified as of the total variation. The values of fitness of data (R2) and predic- AMCase-like proteins, they represented unique AMCase-like frag- tive ability (Q2) were 85.6% and 92.3%, respectively, in the model. ments matching to accession numbers in the database. Analysis showed no samples being outside the Hotelling T2 95% confidence ellipse that could influence the analyses. Based on the Peptide fingerprint and its application loading plot (Figure 4B), Mucin-5AC-like (peak D) showed the high- The HPLC peptide fingerprints were generated from 25 SGF-digested est positive loading score of 0.70, accounting for the colour discrimi- EBN samples, and typical fingerprints of White, Yellow, and Red nation of samples by PC1. In agreement with the results of PCA, EBN were given in Figure  2A. In contrast, the products commonly application of PCA–HCA (Figure 4C) for the whole data set resulted used to imitate EBN (e.g. agar, Tremella fungus, gelatin, pig skin, and in a distinct classification of the samples according to their colours. carrageenan) did not show any similarity to the EBN fingerprint. The Twenty-five samples were correctly classified according to their col- peptide fingerprints showed a close similarity among White, Yellow, our; nonetheless, White EBN and Yellow EBN were not distinguish- and Red EBN, except that the peak heights were varied (Figure 2A). able according to the score plot and dendrogram. To quantify the characteristic peaks of EBN fingerprints, apro- Since colour of EBN shows significant variation in fingerprint tinin, a small protein of ~6 kDa, was used as an external standard. and PCA, only White EBN samples were used for comparison of Aprotinin was spiked into EBN samples, and it did not interfere with production site of EBN to hold other factors constant. House White most of the characteristic peaks (Figure 2B). By referring to aprotinin EBN and cave White EBN could be distinctly classified into two peak, the peak retention time and peak height could be calibrated, clusters using the first two principal components (t1 and t2), which and a calibration curve from 0.5 to 10 µg of aprotinin was estab- explained a total of 82.7% variation (Supplementary Figure S3). lished by absorption at 214 nm (Figure 2B insert). Using this stand- Analysis showed no samples being outside the Hotelling T2 95% ard curve, the relative peptide content could be estimated. Figure 3A confidence ellipse. Fitness of data (R2) of this model was 82.7%, 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 Score Accession*** A Unknown B AMCase-like LYEGPSDTGDLV 43 XP_010005363.1 C Lysyl oxidase homolog 3 LKGGAKVGEGRVEVLR 71 XP_010006484.1 D Muc-5AC-like MWDKKTSIF 41 XP_009994736 E AMCase-like AIGGWNFGTAKF 39 XP_010006620.1 *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. 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 Figure  4. The differentiation of chromatographic fingerprints of various EBN evaluate the predictive power of this model, the third and fourth colour. Twenty-five EBN peptide fingerprints from different colours were principal components (t3 and t4) were also investigated. Although generated: White (1–14); Yellow (15–18); Red (19–25). (A) Score plot of EBN house White EBN and cave White EBN could be classified into two samples. The ellipse represents the Hotelling T2 with 95% confidence. (B) Loading clusters using the first and third principal components (t1 and t3) plot for five characteristic peaks (A–E as shown in Figure 2A). (C) Dendrogram in both score plot and dendrogram, as indicated in Supplementary showing hierarchical clustering results of EBN samples. EBN, edible bird’s nest. Figure S4, the predictive ability (Q2) of this model dropped to 57.46%. The R2 and Q2 value distinctly increased to 98.0% and (t1 and t4) in the model. Furthermore, house White EBN and cave 88.9%, respectively, by using first and fourth principal components 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 C.-F. Wong et al., 2017, Vol. 1, No. 1 separation of the samples; and all of them were correctly classified water. In order to enhance good extraction efficiency of EBN, an according to their production sites. over-stewing method was developed. Only water was utilized in our In order to eliminate the critical variation from EBN colour in extraction: because this should be the method used for preparation fingerprint and PCA, only White EBN samples were used for com- of EBN for human consumption. About 66~84% of EBN proteins parison of EBN from different countries of origin. As illustrated in were extracted, supporting the hypothesis that over-stewing method the score plot (Figure 6A), White EBN from different countries could could be a suitable way to extract EBN proteins. Using the same not be clearly classified into clusters by using the first two princi- extraction protocol, White and Yellow EBN showed better extrac- pal components (t1 and t2), which explained 82.7% of the total tion efficiency as compared to that of Red EBN. The difference in variation. The values of fitness of data (R2) and predictive ability extraction rate of White EBN (~84%) and Red EBN (~66%) may be (Q2) were 82.7% and 63.0%, respectively, in this model. Analysis due to conformational or structural difference of the major proteins. showed no samples being outside the Hotelling T2 95% confidence Indeed, much longer time was required to extract Red EBN, which ellipse. PCA–HCA (Figure  6C) showed that the peptide fingerprint implied Red EBN possessed higher resistance to protein digestion. could not clearly determine country of origin, which was consistent Therefore, the protein in Red EBN might not be fully extracted by with the results of PCA. Moreover, EBN samples from Indonesia stewing. Moreover, very limited research has been reported on Red and Malaysia clustered on the right of score plot, and EBN samples EBN, especially the protein part. Thus, further studies have to be came from Vietnam and Thailand clustered on the left. From the conducted on the proteomics of Red EBN. loading plot (Figure  6B), Mucin-5AC (peak D) showed the highest The lack of a complete genome sequence of Aerodramus pre- positive loading score at 0.80: this could serve as a tentative maker sents a challenge for protein identification in EBN. The identified in discriminating the samples between Indonesia/Malaysia EBN and peptide sequences were matched with animal species in NCBI data- Vietnam/Thailand EBN. 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- Method validation ica, a member of the Apodidae, the EBN proteins were searched with Sensitivity, linearity, accuracy, and precision of the HPLC peptide fin- this database. Muc-5AC-like, AMCase-like, and lysyl oxidase pro- gerprint technique were evaluated. Sensitivity was evaluated by the tein fragments were identified in the fingerprint. Mucins are a family limit of detection (LOD) and limit of quantification (LOQ). A series of glycoprotein with high molecular weight produced by epithelial of decreasing concentrations of aprotinin was evaluated to deter- tissues in most organisms (Marin et al., 2007). The major character- mine the LOD and LOQ. LOD was determined as the concentra- istic of mucin is that it is able to form a gel, which therefore is a key tion with a signal-to-noise ratio (S/N) of at least 3; while LOQ was component in most gel-like secretions, e.g. saliva. In addition, the the lowest concentration with a S/N of at least 10. Linearity was mucin secreted in airways is Muc-5AC, and as a result it is found evaluated by five calibrators prepared by spiking the aprotinin in in the saliva. Chitinases have a family of 18 glycosyl hydrolases; blank matric at concentrations of 0.5, 1, 2, 5, and 10 µg. Precision however, their physiological functions are still not fully resolved. and accuracy were evaluated on the peak area of the characteristic AMCase belongs to the glycosyl hydrolase family in mammals (Boot peaks. Intra-batch precision was evaluated by six determinations per et  al., 2001; Bussink et  al., 2007). In the present work, AMCase- single concentration in a day. Inter-batch precision was evaluated like protein was reported in EBN for the third time, and it seemed by one determination per single concentration at six different days. strange that ‘mammalian’ chitinase was found in birds. Until now, Precision was expressed as the percent of relative standard deviation chitinase protein sequences have not been found in the databases (RSD) calculated by using six determinations. The value of recov- of C. pelagica and M. gallopavo, and thus only the homologues of ery was expressed as the ratio of determined concentration from AMCase-like protein were matched. Insects are the common food six individual tests to corresponding known concentration of the of Aerodramus, and the existence of chitinase in swiftlets could spiked marker. A  known amount of aprotinin was spiked into the facilitate the digestion of insect. Lysyl oxidase is a copper-dependent EBN digest, and recovery was calculated based on the concentration amine oxidase that plays a critical role in the biogenesis of connec- of obtained from the chromatogram and the spiked amount. The tive tissue matrices by cross-linking the extracellular matrix proteins, results of method validation were listed on Table 3. collagen, and elastin (Li and Kagan, 1998). The presence of lysyl oxidase could explain why a previous study found that EBN could Discussion facilitate the synthesis of collagen (Chua et al., 2013). Traditionally, EBN is stewed with water for 1–2  h before eating, In a 2011 study, nitrite was detected in EBN available in the mar- but most of the EBN proteins still remain insoluble: this stewing ket, especially Red EBN. This incident revealed the insufficient qual- method accounts only ~5% protein extraction rate. Before any ity control of EBN products in the industry. A variety authentication kind of protein analysis, the EBN proteins need to be dissolved in methods based on the appearance (Liu and Zhang, 1995; Wu et al., Table 3. Method validation. EBN, edible bird’s nest; LOD, limit of detection; LOQ, limit of quantification. LOD* LOQ* Linearity** Correlation coefficient** (R) Intra-batch precision*** Inter-batch precision*** Recovery**** 0.2 µg 0.5 µg 0.5–10 µg >0.995 1.5% 3.5% 90.7% *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. Characterization of edible bird’s nest, 2017, Vol. 1, No. 1 91 2007), total proteins (Hu and Lai, 1999; Liu et al., 2012; Zhang et al., clearly demonstrate peptide profile differences between genuine and 2012), N-acetylneuraminic acid (Chan et al., 2013a), sialic acid (Wang adulterated EBN samples, which provide a comprehensive picture et al., 2006), DNA (Wu et al., 2010), and sodium nitrate (Chan et al., in quality assurance of EBN. The protein identification results sug- 2013b) of EBN have been established; but no official method could be gested that Muc-5AC-like protein was the major protein in EBN. To found for quality control of EBN by specific protein analysis, regard- our knowledge, the present work is the first report highlighting the less of its abundance. Here, we utilized SGF digestion and HPLC to use of protein, which is main component of EBN, in HPLC finger - generate the peptide fingerprint, which could authenticate EBN on a printing studies. qualitative and quantitative basis robustly and therefore provide a comprehensive picture in quality control of EBN. The peptide finger - Supplementary Material print was able to clearly demonstrate the differences between genuine and fake EBN samples, and thus this method could serve as an authen- Supplementary material is available at Food Quality and Safety ticating tool for EBN. Muc-5AC-like fragment (peak D) was the major online. peptide in the fingerprint of EBN, accounting of ~20% total content in the fingerprinting. This indicated mucin-like protein could be the most Funding abundant protein in EBN. In addition, this mucin peak could serve as an internal marker for the fingerprinting. The chromatographic pat- This research was supported by the grants of Hong Kong Research tern of peptide fingerprint was highly similar among EBN samples Grants Council TBS (T13-607/12R), GRF (661110, 662911, with different colours, suggesting that total extractable protein com- 660411, 663012, 662713), ITC (UIM/254), TUYF12SC02, position does not vary widely based on colour. TUYF12SC03, TUYF15SC01, The Hong Kong Jockey Club From the score plot and dendrogram, significant differences were Charities Trust (HKJCCT12SC01) and Foundation of The recorded between White EBN and Red EBN. Red EBN is known Awareness of Nature (TAON12SC01), SRFDP and RGC ERG Joint to dissolve poorly in water, and indeed the protein extraction from Fund (2013009614001/M-HKUST604/13) to Karl Wah-Keung EBN is much lower than that of White or Yellow EBN, as shown Tsim; and National Natural Science Foundation Item (81173498) in the present work. This low solubility of Red EBN protein could to Xiao-Ping Lai. 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 Acknowledgements et  al., 2013). Herein, Paydar group suggested Red EBN could be Chun-Fai Wong received a HK JEBN scholarship. a result of formation of aryl-C-N and NO side group in aromatic Conflict of interest statement. None declared. 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 References digestion. Moreover, further studies have to be conducted on the Abdi, H., William, L. J. (2010). Principal component analysis. Wiley Interdis- underlying mechanism of how EBN gets red. ciplinary Reviews: Computational Statistics, 2: 433–459. 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