Origin of Red Color in Edible Bird's Nests Directed by the Binding of

Subscriber access provided by UCL Library Services

Food and Beverage Chemistry/Biochemistry

Binding of Fe ion to acidic mammalian chitinase-like protein directs the origin of red color in edible bird’s nest Zack C.F. Wong, Gallant K. L. Chan, Tina T.X. Dong, and Karl W.K. Tsim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01500 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

Journal of Agricultural and Food Chemistry

1

Binding of Fe ion to acidic mammalian chitinase-like protein directs the

2

origin of red color in edible bird’s nest

3 4

Zack C.F. Wong†,§, Gallant K.L. Chan†,§, Tina T.X. Dong†,§, Karl W.K. Tsim†,§*

5 6

†

7

Shenzhen 518057, China;

8

§

9

of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR, China.

Shenzhen Key Laboratory of Edible & Medicinal Bioresources, Hi-Tech Park, Nanshan,

Division of Life Science and Center for Chinese Medicine R&D, The Hong Kong University

10 11

*Correspondence should be addressed to Prof. Karl Tsim, Division of Life Science, The

12

Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong,

13

China.

14 15

Tel: (852) 2358 7924; Fax: (852) 2358 7323; E-mail: [email protected]

16

1 ACS Paragon Plus Environment


17

Abstract

18

The red color of Edible bird’s nest (EBN) remains a mystery over hundreds of years.

19

Here, different analytical methods were employed to identify the color origin of EBN. The

20

treatment of White EBN with NaNO2/HCl turned that into red color. In simulated gastric fluid

21

(SGF)-digested EBN, the HPLC chromatogram, NMR spectrum, circular dichroism spectrum

22

and Raman spectrum of a NaNO2-treated White EBN closely resembled with that of

23

authentic Red EBN. From the HPLC chromatogram of SGF-digested EBN, the peptides

24

associated with red color were identified in Red EBN and NaNO2-treated White EBN.

25

Several lines of evidence indicated that the color-containing peptide could be derived from

26

acidic mammalian chitinase-like (AMCase-like) protein of EBN. Besides, there was a

27

noticeable increase in Fe-O bonding intensity after the color change. Based on the findings,

28

we proposed the oxidation of Fe ion in AMCase-like protein contributed significantly to the

29

color change of EBN.

30 31

Keywords: edible bird’s nest, color, nitrite, acidic mammalian chitinase-like, iron

32


Page 2 of 42

Page 3 of 42

33


■ INTRODUCTION

34

Edible bird’s nest (EBN; cubilose) is a well-known traditional food, used by Chinese

35

popularly, due to its nutritious and/or medicinal properties. Presently, EBN has been

36

reported to exhibit interesting functions, such as anti-inflammatory, anti-influenza virus,

37

anti-oxidant, skin lightening, bone strengthening and epidermal growth enhancement.1–8

38

EBN is the salivary secretion from a unique species of swiftlets, i.e. Aerodramus fuciphagus

39

and Aerodramus maximus.9 Because of its high protein content (up to 60% of dried weight),

40

EBN is being used as a health food supplement in Asia.10 In the food market, EBN has a

41

high price with a range from US$ 1,000 – 15,000 per kilogram due to restricted supply and

42

labor-dependent cleaning process.

43

EBNs can be divided into white, yellow and red based on their colors. White EBNs are

44

softer and smoother; while Yellow and Red (blood) EBNs are crispier in texture. White EBN

45

accounts for over 90% of total EBN supply. Yellow and Red EBN are believed to be an

46

outcome after oxidation of White EBN due to environmental change of the swiftlets’ habitats,

47

e.g. time and humidity. Red EBN appears to have a color of orange-red to brownish red, and

48

which are infrequently found in caves or swiftlet houses. According to literature in traditional

49

Chinese medicine (TCM) in Qing dynasty (~AD 1700), e.g. “Ben Cao Feng Yuan” and “Ben

50

Cao Gang Mu Shi Yi”, Red EBN possesses additional function of replenishing blood, as

51

compared to that of White EBN. Owing to very limited supply and higher health benefits, Red



52

EBN is always being sold at a higher price in the market. However, the origin of red color in

53

EBN has been an age-long mystery.

54

An old folklore asserted swiftlets were hurrying to finish their nests before laying eggs,

55

and the red color came from the blood in their saliva. Other fabrications stated the color

56

came from the seaweed or the food containing rich minerals. In addition, the slow

57

accumulation of minerals through contact surface between the nest and the rock wall in cliffs

58

has also been proposed to be the cause of red color. Various explanations have been given,

59

but there is still lacking scientific evidence to support the claim. The color of EBN has been

60

proposed to be correlated with Fe content.10 In contrast, the vapors from sodium nitrite

61

(NaNO2) dissolved in 2% acid hydrochloric (HCl), or bird’s soil, has been shown to turn

62

White EBNs into red color, and thus the nitro-fixation on aromatic acids in EBN protein

63

backbone could be one of the causes of the red color.11,12 A microbial nitrate reductase,

64

converting nitrate to nitrite and playing a role in the color change of White and Red EBN,

65

was identified by mass spectroscopy.13 Although different lines of evidence have been

66

proposed for origin of EBN color, the chemical nature of red color in Red EBN are still not

67

well understood. Here, we attempted to reveal the color change of EBNs, which could

68

provide a scientific explanation on the color of EBN. By applying numerous analytical

69

methods, including Fourier transform infrared (FTIR), liquid chromatography-mass

70

spectrometry (LC-MS/MS), inductively coupled plasma optical emission spectrometry

71

(ICP-OES), nuclear magnetic resonance (NMR), circular dichroism (CD) and Raman 4 ACS Paragon Plus Environment

Page 4 of 42

Page 5 of 42


72

micro-spectroscopy, the chemical nature was examined in different colors of EBNs.

73

Furthermore, the protein responsible for reddening effect in EBN was identified.

74 75

■ MATERIALS AND METHODS

76

Chemicals and EBN. Copper (II) sulphate, deuterium oxide, formic acid, hydrochloric

77

acid, hydrogen peroxide, Fe (III) chloride, ammonium Fe (II) sulfate, pepsin, simulated

78

gastric fluid without enzyme (contains 0.07 M hydrochloric acid and 0.1 M sodium chloride),

79

sodium hydroxide, sodium nitrite and trifluoroacetic acid (TFA) were purchased from

80

Sigma/Aldrich (St. Louis, MO). 3,3',5,5-Tetramethylbenzidine (TMB) and peroxidase

81

(HRP)-conjugated anti-mouse secondary antibody were purchased from Thermo Fisher

82

Scientific (Waltham, MA). LC-MS grade acetonitrile and water were obtained from Baker

83

(Center Valley, PA). Ten batches of EBN from Indonesia, Malaysia, Thailand and Vietnam

84

were randomly bought in Hong Kong market. EBN samples were labeled and stored at room

85

temperature upon arrival.

86 87

Extraction and digestion of EBN. A cup (a common market size) of dried EBN was

88

accurately weighed and soaked in a 100-fold volume of water (w/v) for 15 hours, as reported

89

before.13 After discarding the soaking water to remove contaminants, the EBN was rinsed

90

with water three times. The soaked EBN was put into a stewing pot with 30 volumes of water.

91

The soaked EBN was stewed for 40 hours at 98 ± 2 oC until it was completely molten. The 5 ACS Paragon Plus Environment


92

sample was filtered, freeze-dried and stored at 4 oC. Fifty mg EBN lyophilized powder was

93

dissolved and digested with a 5 mL SGF (pH 2), consisting of pepsin, 0.07M HCl and 0.1M

94

NaCl for 1 hour at 37 oC. The digest was neutralized with sodium hydroxide and then filtered

95

by 0.45 µm hydrophilic filter. As described previously, the total protein content of EBN was

96

determined by Kjeldahl method,14 and the extracted protein was determined by Bradford

97

method.15

98 99

Color change of EBN extract induced by chemicals. Ten mg/mL White EBN extract

100

was firstly treated with 0.15M NaNO2/ 2%HCl at 25 oC for 2 hours, followed by the addition

101

of 5% H2O2 at 80 oC for 2 hours. Red EBN extract was preliminarily treated with H2O2 at 80

102

o

103

tube without EBN was served as blank control. The color change of EBN extract, induced by

104

chemicals, was monitored by a spectrophotometer at 405 nm in a time-dependent manner.

C for 2 hours followed by addition of 0.15 M NaNO2/ 2%HCl at 25 oC for 2 hours, and the

105 106

Inductively coupled plasma-optical emission spectrometer. Ten mg EBN extract

107

was dissolved in 1 mL concentrated HNO3 and stand for overnight. The samples were

108

heated for 1 hour at 70 oC and diluted with water to a final concentration of 1 mg/mL. The Fe

109

content of samples was analyzed by inductively coupled plasma-optical emission

110

spectrometer (Perkin Elmer, Waltham, MA; Model Optima 7000 DV). The wavelength of Fe

111

was set at 238.204 nm and nebulizers connected to a peristaltic pump were employed to 6 ACS Paragon Plus Environment

Page 6 of 42

Page 7 of 42


112

introduce samples into a nebulization chamber. The samples were then introduced into the

113

plasma formed in the quartz torch by an alumina injector (1.2-mm internal diameter (id)). All

114

measurements were performed in duplicate, and the intensity peak areas were integrated.

115

The standard curve was constructed with 0.08 - 2.00 mg/L FeCl3.

116 117

Fourier transform infrared spectroscopy. EBN sample (0.5 g) was soaked with 50

118

mL water for overnight, then EBN was rinsed with water for three times to remove the

119

impurities. Ten mL 0.15M NaNO2/ 2% HCl was added to White EBN and stood for 25 oC for

120

3 days whereas 10 mL 5% H2O2 was added to Red EBN and stood for 25 oC for 3 days. The

121

solution was discarded, and EBN was rinsed with water for three times to remove the

122

remaining solution. The EBN was lyophilized and ready for FT-IR analysis. The samples

123

were analyzed with Bruker Vertex 70 Hyperion 1000 (Buker, Billerica, MA) under attenuated

124

total reflection (ATR) mode. The C-N stretching (873-874cm-1) and N-O stretching

125

(1406-1408 cm-1) among EBN samples duringthe color change were analyzed.

126 127

Nuclear magnetic resonance. Briefly, dried EBN extract (10 mg) was re-dissolved in

128

750 µL of a D2O solution. All particulates were discarded by centrifugation at 13,000 X g for

129

1 min, and the supernatant was transferred to a standard 5-mm NMR tube. NMR spectra

130

were acquired using a Varian Mercury VX 300 MHz NMR spectrometer (Agilent, Santa Clara,

131

CA). Gradient shimming was employed to improve the magnetic field homogeneity prior to 7 ACS Paragon Plus Environment


132

all acquisition. The 1H NMR spectra of the samples were acquired under standard 1D proton

133

mode. The spinning rate was set to 20 Hz, and regulated speed was chosen. Each sample

134

was performed around 10 min at 25 oC.

135 136

Circular dichroism. The sample vial was firstly cleaned with water and 70% ethanol,

137

which subsequently was dried by compressed air. Four hundred µL EBN extract with the

138

concentration of 0.2 mg/mL was carefully transferred to sample vial and placed inside a

139

Jasco J-815 CD machine (Jasco, Easton, MD). The parameters were set as follows: I)

140

wavelength set: 190-300 nm; II) data pitch: 0.5 nm; III) scanning mode: continuous; IV)

141

scanning speed: 20 nm/min; V) response: 1 sec; VI) bandwidth: 2 nm; and VII) accumulation:

142

1. The CD spectrum and HT value of samples were recorded simultaneously.

143 144

Protein identification by LC-MS/MS. The eluent at 0 - 35 min from the HPLC

145

fingerprint was collected by a Gilson FC203B fraction collector at 1 min per fraction. Five

146

fractions corresponding to peaks 1-5 were collected and then concentrated, desalted and

147

purified by ZipTip. The samples were examined by a Thermo Scientific (Waltham, MA) LTQ

148

Velos Dual-Pressure Ion Trap Mass Spectrometer with a Thermo Accela 600 pump, an

149

Accela autosampler LC and ETD source (spray voltage 1.6 kV, capillary temperature

150

250 °C). A Thermo Scientific BioBasic-18 column was used (150.0 × 0.1 mm, 5 µm). Formic

151

acid at 0.1% in MS-grade water and 0.1% formic acid in MS-grade acetonitrile were used as 8 ACS Paragon Plus Environment

Page 8 of 42

Page 9 of 42


152

mobile phase A and B, respectively, with a flow rate of 0.15 mL/ min. Mascot Daemon

153

(version 2.3.0) was used as a sequence database searching engine. The parameters were

154

selected as follows: (i) pepsin A as the digestion enzyme, (ii) allowing absence of two

155

internal cleavage sites, (iii) oxidation of methionine as a variable modification, (iv) ±1 Da for

156

peptide mass tolerance, and (v) ± 0.8 Da for fragment mass tolerance. The false discovery

157

rate was below 5% by calculating the number of matched proteins in the search of the real

158

database and decoy database. The peptide sequences were searched with Chaetura

159

pelagic (Chimney swift) database in the NCBI library.

160 161

HPLC sizing separation. EBN extract was digested with simulated gastric fluid as

162

described above, and the digested products were separated in a reversed phase column by

163

HPLC.15 Upon the collection of peak 1 in Red EBN digest, the fraction was concentrated and

164

re-suspended in water. The fraction was separated by Superdex 75 10/300 (GE Healthcare

165

Life Science, Chicago, IL). The mobile phase was composed of PBS containing 0.14 M

166

NaCl, 2.68 mM KCl, 0.01 M Na2HPO4 and 1.75 mM K2HPO4. The flow rate was 0.5 mL/min;

167

the column temperature was 25 oC; and the injection volume was 10 µL. The DAD

168

wavelength was set to 214 nm for detection.

169 170

Verification of anti-AMCase-like protein by ELISA. Coating buffer was prepared by

171

dissolving sodium bicarbonate in water and tuned to pH 8.0. EBN fractions collected from 9 ACS Paragon Plus Environment


172

HPLC were dried and solubilized in coating buffer. It was transferred to the 96-well microtiter

173

plates and incubated at 4 °C overnight. Each sample well was washed with 300 µL PBS

174

followed by incubation in 200 µL of blocking solution (5% fat-free dry milk in PBS) for one

175

hour. Wells were then washed three times with 300 µL PBS and incubated in a wet chamber

176

at 4 °C overnight with 100 µL monoclonal antibodies (1:1,000 v/v). After washing, 100 µL of

177

horseradish peroxidase (HRP)-labeled anti-mouse immunoglobulin G (IgG) solution

178

(1:5,000 v/v) was added and subsequently incubated at room temperature for 2 hours. The

179

wells were washed four times and shaken for 15 min with 100 µL of the color substrate, TMB.

180

A hundred µL of 2M sulphuric acid was added to stop the reaction, and the absorbance was

181

monitored at 450 nm.

182 183

Determination of Fe(II) and Fe (III) by post-column derivatization/HPLC.

184

The liquid chromatograph was done with an Agilent HPLC 1100 series system (Agilent,

185

Waldbronn, Germany), which was equipped with a diode array detector (DAD), a degasser,

186

a binary pump, an auto-sampler and a thermo-stated column compartment, and an ionpac

187

CS5A analytical and CG5A guard Column. The mobile phase was composed of 7.0 mM

188

pyridine-2-6-dicarboxylic acid/66 mM potassium hydroxide/5.6 mM potassium sulfate/74

189

mM formic acid in 1 L de-ionized water (pH: 4.2 ± 0.1). The flow rate was 1.2 mL/min; the

190

column temperature was 25 oC; 100 mM Na2SO3 was passed through the column for 1 hour

191

at 1.0 mL/min to eliminate any oxygen build-up on the column at beginning of experiment. 10 ACS Paragon Plus Environment

Page 10 of 42

Page 11 of 42


192

The post-column reagent was pumped with a flow rate of 0.6 mL/min towards the mixing tee.

193

A 100 µL sample, or standard, was injected into the system. The separation of Fe (II) and Fe

194

(III) were realized, and the components reached the mixing tee. They reacted with the

195

post-column derivatization reagent in the knitted reaction coil to give a colored complex, and

196

which were determined with an UV-VIS detector at 521 nm.

197 198

Raman micro-spectroscopy. Raman spectra were collected using Renishaw InVia

199

Micro Raman (Wotton-under-Edge, Gloucestershire, UK) with a CCD detector, with an

200

excitation laser wavelength of 785 nm. The spectral grating was set at 1,200 gr/mm, and an

201

excitation power was set at 25 mW for all samples. EBN samples were prepared on a silicon

202

substrate glass slide, and Raman spectrum was collected in the range 100–2,000 cm-1. The

203

spectra were calibrated with the 520 cm-1 silicon peak, and the spectra were triple

204

accumulation collected for 60 s. All Raman spectra were obtained at room temperature (25

205

°C). To avoid a subjective decision, principal component analysis (PCA), was utilized to

206

reveal the possible relationship between Raman spectra and EBN color. PCA was

207

performed by using SIMCA 13 (Umetrics, Sweden) in ‘PCA-X’ mode. The Wire 4.0 software

208

was used to make the baseline correction, spectra smoothing and peak area integration.

209

The peak areas at 490 cm-1 and 590 cm-1 in Raman spectra were analyzed by PCA.

210 211

Data analysis. Statistical tests were performed by using one-way ANOVA provided in 11 ACS Paragon Plus Environment


212

GraphPad Prism 6.0. Statistically significant changes were classed as [*] where p < 0.05, [**]

213

where p < 0.01 and [***] where p < 0.001.

214 215

■ RESULTS

216

Extraction of EBN proteins. Ten EBN samples, including different colors, were

217

collected from Hong Kong market (Table S1). To ensure the complete solubility of EBN

218

protein, the extraction was done by the over-stewing method, as described previously.15 The

219

EBN samples were sent to a food laboratory for protein analysis by Kjeldahl method.14 The

220

average protein content of EBNs (from sample 1-10) was 52 ± 4.2% of the total dried weight,

221

which was close to the reported value, i.e. 50%.15,16 This average value was thereby utilized

222

as a denominator in estimating the extraction rate. Having the over-stewing method, the

223

extracted protein was over 80% from White and Yellow EBNs, whereas around 67% was

224

found for Red EBNs. The discrimination of protein extraction rate among EBNs was in

225

consonance with the historical practice of longer stewing time for Red EBN.13,15

226 227

Total Fe content in EBN. Traditionally, EBN was mainly classified by its color, including

228

white, yellow and red (Figure 1A); but the color origin of EBN has not been revealed.

229

Previous studies have suggested two possibilities for the color of EBN, i.e. Fe content and

230

nitrification of aromatic amino acids.10,12 To reveal the cause of EBN color, we tested the

231

total Fe content in EBN by ICP-OES. Total Fe content of the extractives from White, Yellow 12 ACS Paragon Plus Environment

Page 12 of 42

Page 13 of 42


232

and Red EBN did not show obvious difference having a range from 30 to 40 mg/kg (Figure

233

1B). In addition, no free Fe ion, both Fe(II) and Fe(II), was detected in EBN extracts (Figure

234

1B). The red color intensity of EBN extract was monitored by photo-spectrometry with

235

absorbance at 405 nm. Red EBN showed the highest absorbance at 405 nm, whereas

236

White EBN showed the least (Figure 1C). Therefore, the color of EBN was not positively

237

related to its total Fe content.

238

The N-O (~1406 to 1408 cm−1) and C-N (~873 to 874 cm−1) stretching intensity of EBN

239

samples were monitored by FT-IR before/after the chemical-induced color change. White

240

EBN was changed to red color after treating with NaNO2/HCl (an oxidizing agent); Red EBN

241

was changed to yellow color with the addition of H2O2 (a reducing agent). No significant

242

correlation was found between the stretching intensity of N-O and C-N between White and

243

Red EBNs (Figure 1D and 1E). The results suggested that the assumption of nitrification of

244

aromatic amino acids could not be fully accounted for color change of EBN.

245 246

The color of EBN extract is reversible. In order to test the origin of EBN color, EBN

247

extract was directly treated with various chemicals. White EBN extract was changed to red

248

color after treating with NaNO2/HCl and then back to white color by adding H2O2 (Figure 2A).

249

In contrast, Red EBN extract, both from cave and house, was changed to white color by

250

adding H2O2 and then back to red color after treating with NaNO2/HCl (Figure 2A). This

251

color change of Red EBN showed no difference between the EBN from house or from cave 13 ACS Paragon Plus Environment


252

at their production sites. This finding demonstrated that the color of EBN could be altered by

253

chemicals, reversibly. Moreover, cold acetone precipitation was performed here with Red

254

EBN extract: the red color was precipitated at the bottom suggesting an association of red

255

color to the precipitate, e.g. protein (Figure 2B). The color change of EBN extract after cold

256

acetone precipitation was monitored by a spectrometer. The red color intensity of EBN

257

extract was significantly decreased after cold acetone precipitation (Figure 2B).

258 259

Structural properties of EBN before/ after the color change. In order to evaluate the

260

structural difference between White and Red EBN, the EBN extracts were analyzed by NMR

261

and CD. The 1H-NMR spectrums of White EBN and Red EBN were highly similar except at

262

3.3-3.9 ppm of the spectrum (Figure 3A). The spectrums were also highly similar to

263

White/Red EBN being treated by NaNO2/HCl or H2O2. In addition, the secondary structures

264

of various forms of EBN were revealed by CD spectrum. The secondary structure of EBN

265

extracts was closed to a structure of poly type II helix (Figure 3B). A minor difference in CD

266

absorbance between White and Red EBNs was identified. The maximum CD absorbance of

267

NaNO2-treated White EBN was increased: this profile showed a closer similarity to that of

268

Red EBN. High tension (HT) voltage was roughly proportional to absorbance. The HT

269

voltage below 600 indicated the detector was not saturated during measurement (Figure

270

3C). Having the data from NMR and CD spectrum, the secondary structure of EBN did not

271

alter significantly in regard to their color types. In the analysis of Red EBN produced from 14 ACS Paragon Plus Environment

Page 14 of 42

Page 15 of 42


272

the cave, the physical parameters, generated by NMR and CD, were very similar to that of

273

parameters from the house Red EBN, i.e. similar chemical nature between the two sources

274

(Figure S3).

275 276

Identification of protein responsible for the color change. Red EBN could turn to

277

peal white by treatment with H2O2 (Figure 4A). In Red EBN digests, HPLC chromatograph

278

showed distinct peaks at 280 nm, which were named as peaks 1 to 4, as shown in Figure

279

4B. Peak 1 being the major fraction contained only a single peptide, as shown in a sizing

280

HPLC chromatogram (Figure 4B insert). After H2O2 treatment, the peaks 1 and 2 were

281

disappeared, while peaks 3 and 4 were retained (Figure 4B). Assuming the red color of

282

EBN is closely linked with protein, the peptide fractions of digested EBN were collected and

283

monitored by a spectrometer with 405 nm absorbance for red color. In Red EBN

284

chromatograph, higher 405 absorbance was detected in peaks 1 and 2 (Figure 4C). After

285

treatment with 5% H2O2, the red color intensity of peaks 1 and 2 was decreased, suggesting

286

the possible red color-associated peptides. By using a monoclonal antibody against EBN

287

AMCase-like protein CFW-12,16 the peptides within the peaks 1 and 2 were recognized

288

robustly by the antibody in an ELISA assay (Figure 4D). In parallel, the content of Fe was

289

determined in each fraction, and the peaks 1 and 2 showed Fe-containing fractions (Figure

290

4E). In contrast, total Fe content in Red EBN, before or after H2O2 treatment, remained

291

unchanged (Figure 4E insert). 15 ACS Paragon Plus Environment


292

White EBN was turned into red color in the treatment of NaNO2/HCl (Figure 5A). HPLC

293

chromatogram of digested White EBN showed a profile distinction as compared to Red EBN,

294

having only peaks 3 and 4 as the dominant peaks (Figure 5B). The digest of NaNO2-treated

295

White EBN showed a HPLC profile similar to that of Red EBN, i.e. appearance of peaks 1

296

and 2: these two peaks showed robust absorbance at 405 nm (Figure 5C). By ELISA using

297

the anti-AMCase-like antibody, the peak 5 in White EBN digest could be recognized, slightly,

298

by the antibody (Figure 5D). The content of Fe was determined in each fraction, and the

299

peaks 3, 4 and 5 showed Fe-containing fractions (Figure 5E). Total Fe content in White EBN,

300

before or after NaNO2 treatment, did not vary during this process (Figure 5E insert). The

301

peaks (1 to 5) from the digested EBN were collected and subjected to ion-trap LC-MS/MS

302

analysis. None of the proteins identified from EBN extracts matched with Aerodramus, likely

303

because the genome sequence of swiftlet was not completed. We then searched the protein

304

sequences from C. pelagica, a species in the same family as A. fuciphagus. Only the

305

fragments of acidic mammalian chitinase-like, lysyl oxidase homolog 3 and Muc-5AC-like

306

fragment were identified in peak 1-5 (Table 1).

307

308

Determination of total Fe, Fe (II) and Fe (III) in EBN extract. Here, we examined the

309

possible association of EBN color with Fe ions. As shown in Figure S1, White EBN was

310

changed to red gradually with the addition of Fe3+ after a day, and White EBN was changed


Page 16 of 42

Page 17 of 42


311

to blue when Cu2+ was added. The color remained at EBN after sonication, and which

312

suggested metallic binding sites could be available in EBN. Furthermore, the Fe ion state in

313

EBN extract was examined by HPLC-DAD with post-column devitalization. All the tested

314

EBN did not show significant difference in their total Fe content, and the free Fe(II) or Fe(III)

315

were not detected in the EBN (Figure 1B). Thus, Fe ion should be tightly bonded to EBN.

316

The iron-oxygen bond could be detected by Raman spectroscopy in hemoglobin,

317

transferrin, ferritin and even oxygenated complex of the nitric-oxide synthase.17,18 Here, the

318

distinctive peaks of Fe-O and Fe-NO were measured at 490 cm-1 and 590 cm-1 in Raman

319

spectra, respectively. In EBN Raman spectroscopy, the patterns of all EBN samples were

320

highly similar (Figure 6A). In all samples, there were two noticeable peaks at 490 cm-1 and

321

590 cm-1. The peak at 490 cm-1 (Fe-O) was slightly higher in Red EBN, as compared to

322

White EBN. The peak at 490 cm-1 was increased in White EBN after the treatment of NaNO2;

323

whereas it was comparable between White EBN and Yellow EBN. Besides, no

324

distinguishable peak was found around 530 cm-1 representing Fe-O2. The peak at 590 cm-1

325

could be Fe-NO bond.19 The Raman spectra suggested that Red EBN should have more

326

Fe-O than White EBN, and Fe bond in EBN could be mainly in a form of Fe-O and Fe-NO

327

bond.

328

By using the resulted Raman spectra, PCA analysis was performed. Red EBN and

329

NaNO2-treated White EBN was clustered together, and which was in distinction to White



330

EBN, Yellow EBN, and H2O2-treated Red EBN (Figure 6B). The two clusters were utilized

331

first two principal components, PC1 (t1) and PC2 (t2), accounting for 100% of the total

332

variation. The values of fitness of data (R2) and predictive ability (Q2) were 100% and 100%,

333

respectively, in the model. The analysis showed no sample being outside Hotelling T2 95%

334

confidence ellipse that could influence the outcomes. Briefly, the result reflected that Fe-O

335

bond intensity was significantly different between White EBN and Red EBN.

336

337

■ DISCUSSION

338

White EBN changed to Red EBN in the present of NaNO2/2% HCl, or the soil collected

339

from swiftlets’ house.11–13 The soil in EBN producing sites contains swiftlets’ droppings and

340

high content of NO2. Thus, the key player in directing EBN color is NO2. However, the

341

chemical nature of EBN during the color change has not been fully investigated. Here,

342

various analytical instruments were utilized to investigate the chemical nature of color

343

change in EBN. Nitro-fixation on aromatic acids of EBN proteins has been postulated as one

344

of the reasons for the color of EBN: because a difference of C-N and N-O bond stretching

345

has been reported in raw White EBN and Red EBN.12 However, the difference in C-N and

346

N-O bond stretching has not been measured for White EBN and Red EBN before/after the

347

color change. Here, we analyzed the same EBN sample before/after the color change. In

348

our analysis, the raw EBN was soaked and washed with distilled water, which removed the


Page 18 of 42

Page 19 of 42


349

impurities and salts, i.e. sodium nitrate, sodium nitrite, citric acid. Thus, this discrepancy of

350

our measurement as compared to that of Paydar et al. (2013) could be just the cause of

351

sample variation.12

352

The color of EBN was proposed to be related to Fe content: because the Fe content of

353

White EBN was found at 30 ppm whereas cave Red EBN was at 60 ppm.10 In line with this

354

study, our results showed that around 30 ppm of Fe was found in White, Yellow and Red

355

EBN samples, as determined by ICP-OES. The cave EBN is generated in natural habitat,

356

and which is expected to contain more minerals, as compared to those produced in house.

357

Besides, the time of nesting in cave EBN is usually much longer than that of in swiftlet house,

358

resulting in higher Fe content. However, our results in measuring various physical

359

parameters have suggested that the chemical nature between house and cave EBN could

360

be very similar, as well as the content of Fe.

361

According to the results in peptide fingerprint, CD and NMR, the chemical nature of

362

White Yellow and Red EBN should be very close, except slight variation in the peptide HPLC

363

fingerprint. As observed in HPLC chromatograms, the major peptide peak in Red EBN (peak

364

1) was more polar than White EBN (i.e. at peak 4). The fingerprint of White EBN after

365

treatment of sodium nitrite resembled Red EBN: but the fingerprint of Red EBN after

366

treatment of H2O2 was different from White EBN. This result suggested the chemical change

367

is favorable from White to Red, even though the color of the EBN extract could be changed

368

reversibly. There was a difference in chemical shift between 3.3-3.9 ppm in 1H NMR graph of 19 ACS Paragon Plus Environment


369

Red and White EBN. Normally, this range is referring to the glycan part of glycoprotein,

370

which therefore suggests that the “red” color may associate with the glycan part of EBN.

371

Thus, we hypothesized that the “red” element could react with the glycan part of

372

AMCase-like protein in EBN. This notion is fully supported by the protein identification by

373

mass spectroscopy and antibody recognition, as reported here.

374

White EBN was gradually changed to red color in addition of NaNO2 in acidic medium.

375

NO was observed when NaNO2 was dissolved in hydrochloric acid. Eventually, a brown gas,

376

nitrogen dioxide (NO2) appeared on top of the reaction mixture, and thus oxidation has

377

occurred in EBN. In the absence of EBN, the reaction mixture remained clear. This

378

NaNO2/HCl chemical reaction is expected to generate NaNO3. Supporting this notion, NO3

379

content in Red EBN was 10-700 fold higher than NO2 content, whereas only 2-10 fold

380

difference was observed in White EBN, as reported previously.13 In natural conversion of

381

White to Red EBN, we believed that the accumulated NaNO3 was increased according to

382

the time of EBN being deposited: NaNO3 could turn into NaNO2 in the present of nitrate

383

reductase, an enzyme being identified in EBN.13 In contrast, hydrogen peroxide is a redox

384

reagent, which could reverse the aforementioned reaction triggered by NaNO2.

385

The market price of Red EBN is often higher than White EBN due to immensely scared

386

supply. 13 Some unethical merchants changed White EBN to red color by using NaNO2, and

387

sold that at higher price. From the analytical results of NMR, CD, FTIR and HPLC fingerprint,

388

the NaNO2/HCl-treated White EBN closely resembled with that of house Red EBN. Besides, 20 ACS Paragon Plus Environment

Page 20 of 42

Page 21 of 42


389

the solubility of EBN protein is a problem for consumption, in particular for Red EBN, and

390

moreover the functional distinction of Red EBN, as compared to other types of EBN, was not

391

identified. The functional assays on White EBN and Red EBN, e.g. osteogenic, inflammatory,

392

estrogenic and anti-oxidant properties, were measured (Data not shown), and the results

393

suggested that White and Red EBN shared similar biological effects. Thus, our current result

394

is contradicting to the historical record of Chinese medicine literature.11

395

Swiftlets usually build their nest either in cave or swiftlet house, named as cave EBN or

396

house EBN, respectively.11 The characteristics of cave EBN is affected by its natural climate

397

and environment, and thus the texture tends to be stiffer and harder, with a darker color and

398

less appealing appearance. Having the reduced yield of cave EBN, house EBN is being

399

considered to be a replacement. The internal setting of the birds’ house is controlled by the

400

farmer; therefore, less impurity is expected in house EBN. The house EBN is cleaner with a

401

complete shape like a boat or a spoon. Normally, the price of cave EBN is at least 40%

402

higher than house EBN, owing to the limited supply. According to our current physical

403

measurements, house EBN and cave EBN share similar chemical nature.

404

The major component in nest cement is sialic acid-rich glycoproteins.20 Indeed, acidic

405

mammalian chitinase-like, a sialoglycoprotein, has been reported to be a major protein in

406

EBN.15,16,,21,22,23 Chitinase is an enzyme that breaks down glycosidic bonds in chitin, which

407

is an abundant glycopolymer in insect exoskeletons and fungal cell walls.24,25 Arthropods are

408

the main food sources of Aerodramus swiftlets, thus chitinase protein is useful for them to 21 ACS Paragon Plus Environment


409

digest chitin, especially during breeding seasons. Nevertheless, the enzymatic activity of

410

chitinase has not been assayed in EBN. Herein, this is the first report showing AMCase-like

411

protein could bind to Fe and change to red color. From our findings, Fe turned White EBN to

412

red. We speculated NO2 could react with Fe in parts of the AMCase-like protein, resulting in

413

more organometallic bond is being formed on the protein, and leading to stronger and tighter

414

structure that is more resistant to hydrolysis and enzymatic digestion, i.e. Red EBN is hard

415

to be digested.15 From Raman spectra, more Fe-O was observed in EBN in red color, and

416

the major bond is Fe-O instead of Fe-O2. The proposed mechanism of EBN color change

417

was illustrated in Figure S2. Herein, the present study suggested that Fe in EBN was

418

oxidized during the color change. This is always challenging to analyze the oxidation state of

419

metal ions in food substances. Further investigation could be carried out to reveal the

420

oxidation state of Fe in EBN.

421

Here, the chemical nature and color origin were revealed for EBN. The red color could

422

be reversible with chemicals, but it was less favorable than that from white to red. There was

423

a binding site(s), provided by AMCase-like protein, of EBN for Fe that directs the colors of

424

EBN. The amount of NaNO2 in EBN could play as an oxidizing agent to change the color of

425

the EBN, possibly via the oxidized/reduced status of Fe. During the site visit to swiftlet house

426

in Malaysia and Indonesia, there are rooms with lots of bird droppings, some of the White

427

EBN gradually changed to Red EBN. The droppings provided NaNO2 and weak acidic

428

medium, and the presence of nitrate reductase in EBN could catalyze the reaction.13 Thus, 22 ACS Paragon Plus Environment

Page 22 of 42

Page 23 of 42


429

the formation of Red EBN in swiftlet houses is very likely due to the environment condition

430

therein. This study could provide a comprehensive picture of Red EBN, which could be

431

valuable for future study on Red EBN such as quality control and bio-functions.

432 433

■ AUTHOR INFORMATION

434

Corresponding Author

435

*Phone: +852- 2358 7332; fax: +852- 2358 1552; e-mail: [email protected].

436 437

Notes

438

The authors declare no competing financial interest.

439 440

■ ABBREVIATIONS USED

441

AMCase-like, acidic mammalian chitinase-like; CD, circular dichroism; EBN, edible bird’s

442

nest; DAD, diode array detector; FT-IR, fourier transform infrared; HPLC, high performance

443

liquid chromatography; ICP-OES, inductively coupled plasma optical emission spectrometry;

444

LC−MS/MS, liquid chromatography−tandem mass spectrometry; PCA, principal component

445

analysis; Muc-5AC-like, Mucin-5AC-like; NMR, Nuclear magnetic resonance; SGF,

446

simulated gastric fluid.

447 448

■ ACKNOWLEDGMENTS 23 ACS Paragon Plus Environment


Page 24 of 42

449

This study was supported by Hong Kong RGC Theme-based Research Scheme

450

(T13-607/12R), Innovation Technology Fund (UIM/288, UIM/302, UIM/340, UIT/137,

451

ITS/022/16FP), TUYF15SC01, Shenzhen Science and Technology Committee Research

452

Grant (JCYJ20,160,229,205,726,699, JCYJ20,160,229,205,812,004, JCYJ20,160,229,210,

453

027,564,

454

707,281,432,317 and 20,170,326). Z.W. received a scholarship from HK JEBN Limited. The

455

authors express their gratitude to technical support provided by Pui-Shuen Wong (Bio-CRF

456

of HKUST) for protein identification.

CKFW2,016,082,916,015,476,

JCYJ20,170,413,173,747,440,

457


ZD,SYS,201,

Page 25 of 42


458

■ REFERENCES

459

(1) Kong, Y. C.; Keung, W. M.; Yip, T. T., Ko; K. M.; Tsao, S. W.; Ng, M. H. Evidence that

460

epidermal growth factor is present in swiftlet's (Collocalia) nest. Comp. Biochem.

461

Physiol. B Biochem. Mol. Biol. 1987, 87B, 221–226.

462

(2) Kong, Y. C.; Tsao, S. W.; Song, M. E.; Ng, M. H.; Lin, Z. Potentiation of mitogenic

463

response by extracts of the swiftlet's (Apus) nest collected from Huai-Ji. Acta Zool Sin.

464

1989, 35, 429–435.

465

(3) Guo, C. T.; Takahashi, T.; Bukaw, W.; Takahashi, N.; Yagi, H.; Kato, K.; Hidari, K. I. J.;

466

Miyamoto, D.; Suzuki, T.; Suzuki, Y. Edible bird’s nest extract inhibits influenza virus

467

infection. Antivir. Res. 2006, 70, 140–146.

468 469

(4) Aswir, A. R.; Wan Nazaimoon, W. M. Effect of edible bird’s nest on cell proliferation and tumor necrosis factor- alpha (TNF-α) release in vitro. IFRJ 2011,18, 1123–1127.

470

(5) Matsukawa, N.; Matsumoto, M.; Bukawa, W.; Chiji, H.; Nakayama, K.; Hara, H.;

471

Tsukahara, T. Improvement of bone strength and dermal thickness due to dietary edible

472

bird’s nest extract in ovariectomized rats. Biosci. Biotechnol. Biochem. 2011, 75, 590–

473

592.

474

(6) Yew, M. Y.; Koh, R. Y.; Chye, S. M.; Othman, I.; Ng, K. Y. Edible bird's nest ameliorates

475

oxidative stress-induced apoptosis

476

BMC complem. Altern. M. 2014, 14, 391.

477

in

SH-SY5Y human

neuroblastoma cells.

(7) Chan, G. K.; Wong, Z. C.; Lam, K. Y.; Cheng, L. K.; Zhang, L. M.; Lin, H.Q.; Dong, T. T.; 25 ACS Paragon Plus Environment


Page 26 of 42

478

Tsim, K. W. Edible bird’s nest, an Asian health food supplement, possesses skin

479

lightening activities: identification of n-acetylneuraminic acid as active ingredient.

480

JCDSA 2015, 5, 262–274.

481

(8) Zhang, Y.; Imam, M. U.; Ismail, M.; Hou, Z. P.; Abdullah, M. A.; Ideris, A.; Norharina,

482

Ismail. Edible Bird’s Nest attenuates high fat diet-induced oxidative stress and

483

inflammation

484

BMC complem. Altern. M. 2015,15, 310.

via regulation

of

hepatic

antioxidant

and inflammatory

genes.

485

(9) Kang, N.; Hails, C. J.; Sigurdsson, J. B. Nest construction and egg-laying in edible-nest

486

swiftlets Aerodramus spp. and the implications for harvesting. IBIS 1991, 133, 170–177.

487 (10) Marcone, M. F. Characterization of the edible bird’s nest the “Caviar of the East”. Food 488

Res. Int. 2005, 38, 1125–1134.

489 (11) But, P. P.; Jiang, R. W.; Shaw, P. C., Edible bird's nests--how do the red ones get red? J. 490

Ethnopharmacol. 2013, 145, 378–80.

491 (12) Paydar, M.; Wong, Y. L.; Wong, W. F.; Ahmed Hamdi, O. A.; Kadir, N. K.; Looi, C. Y. 492

Prevalence of nitrite and nitrate contents and its effect on edible bird nest’s color. J.

493

Food Sci. 2013, 78, 1940–1947.

494 (13) Chan, G. K. L.; Zhu, K. Y.; Chou, D. J. Y.; Guo, A. J. Y.; Dong, T. T. X.; Tsim, K. W. K., 495

Surveillance of nitrite level in cubilose: Evaluation of removal method and proposed

496

origin of contamination. Food Control 2013, 34, 637–644.

497 (14) Codex Alimentarius International Food Standards, Recommended Methods of Analysis 26 ACS Paragon Plus Environment

Page 27 of 42

498


and Sampling. 1999, CODEX STAN 234.

499 (15) Wong, C. F.; Chan, G. K. L.; Zhang, M. L.; Yao, P.; Lin, H. Q.; Dong, T. T. X.; Li, G.; Lai, 500

X. P.; Tsim, K. W. K. Characterization of edible bird’s nest by peptide fingerprinting with

501

principal component analysis. Food Quality and Safety 2017, 1, 83–92.

502 (16) Wong, Z. C. F.; Chan, G. K. L.; Wu, L.; Lam, H. H. N.; Yao, P.; Dong, T. T. X.; Tsim, K. 503

W. K. A comprehensive proteomics study on edible bird’s nest using new monoclonal

504

antibody approach and application in quality control. J. Food Compost. Anal. 2018, 66,

505

145–151.

506 (17) Das, T. K.; Couture, M.; Ouellet, Y.; Guertin, M.; Rousseau, D. L. Simultaneous 507

observation of the O—O and Fe—O2 stretching modes in oxyhemoglobins. Proc. Natl.

508

Acad. Sci. U. S. A. 2001, 98, 479–484.

509 (18) Ashton, L.; Brewster, V. L.; Correa, E.; Goodacre, R. M. Detection of glycosylation and 510

Fe-binding protein modifications using Raman spectroscopy. Analyst 2017, 142, 808–

511

814.

512 (19) Hunt, A. P.; Lehnert, N. Heme-Nitrosyls: Electronic Structure Implications for Function in 513

Biology. Acc. Chem. Res. 2015, 48, 2117–2125.

514 (20) Kathan, R. H.; Weeks, S. I. Structure studies of collocalia mucoid: I. Carbohydrate and 515

amino acid composition. Arch. Biochem. Biophys. 1969, 134, 572–576.

516 (21) Liu, X. Q.; Lai, X. T.; Zhang, S. W.; Huang, X. L.; Lan Q. X.; Li, Y.; Li, B. F.; Chen, W.; 517

Zhang, Q. L.; Hong, D. Z.; Yang, G. W. Proteomic profile of edible bird’s nest proteins. J. 27 ACS Paragon Plus Environment


518

Agric. Food Chem. 2012, 60, 12477–12481.

519 (22) You, Y. Y.; Cao, Y.; Guo, S.; Xu, J.; Li, Z. J.; Wang, J. F.; Xue, C. H. Purification and 520

identification of α 2–3 linked sialoglycoprotein and α 2–6 linked sialoglycoprotein in

521

edible bird’s nest. Eur. Food Res. Technol. 2015, 240, 389–397.

522 (23) Kong, H. K.; Wong, K. H.; Lo, S. C. L. Identification of peptides released from hot water 523

insoluble fraction of edible bird's nest under simulated gastro-intestinal conditions. Food

524

Res. Int. 2016, 85, 19–25.

525 (24) Boot, R. G.; Blommaart, E. F. C.; Swart, E.; Vlugt, K. G. D.; Bijl,N.; Moe, C.; Place, A.; 526

Aerts, J. M. F. G. Identification of a novel acidic mammalian chitinase distinct from

527

chitotriosidase. J. Biol. Chem. 2001, 276, 6770–6778.

528 (25) Bussink, A. P.; Speijer, D.; Aerts, J. M. F. G.; Boot, R. G. Evolution of mammalian 529

chitinase (-like) members of family 18 glycosyl hydrolases. Genetics 2007, 177, 959–

530

970.

531


Page 28 of 42

Page 29 of 42


532

Figure Legends:

533

Figure 1. The Fe content, red color intensity and infrared (IR) spectroscopy of EBN

534

extract

535

(A) Photos of White, Yellow and Red EBN. Bar = 1 cm. (B) Different types of EBN were

536

extracted to molten form by stewing for 8-40 hours. The contents of total Fe in EBN extract

537

was determined by ICP-OES; free Fe (II) and free Fe(III) were examined with post-column

538

derivatization/HPLC. (C) The color intensity of EBN extract measured at the visible

539

wavelength 405 nm by a spectrometer. Fe content and absorbance values are expressed as

540

Mean ± SEM, n = 3. The color of EBN could be changed by adding chemicals. N-O

541

(approximately 1406 to 1408 cm−1) and C-N (approximately 873 to 874 cm−1) stretching

542

intensity of EBN samples were monitored by FT-IR. (D) White EBN was treated with 0.15 M

543

NaNO2/ 2%HCl at 25 oC for 3 days. (E) Red EBN was treated with 5% H2O2 at 25 oC for 3

544

days.

545

Figure 2. The color of White and Red EBN extracts could be reversed by chemicals.

546

(A) White EBN extract was treated with 0.15M NaNO2/ 2%HCl at 25 oC for 2 hours, followed

547

by the treatment of 5% H2O2 at 80oC for 2 hours. The tube without EBN was served as a

548

control. Red EBN extract (from house or cave) was treated with of 5% H2O2 at 80 oC for 2

549

hours, followed by the treatment of 0.3 M NaNO2/ 2%HCl at 25 oC for 2 hours. The color

550

change of EBN extract was monitored by a spectrometer with 405 nm absorbance.



Page 30 of 42

551

Arrowheads indicated the treatment time. (B) Red EBN extract was treated with cold

552

acetone and centrifuged. The red color of EBN was precipitated at the bottom (left panel).

553

Bar: 1 cm. The color change of EBN extract was monitored by a spectrometer with 405 nm

554

absorbance (right panel). Values are expressed as Mean ± SEM, n = 3.

555

Figure 3. H-NMR and circular dichroism spectrum of EBN extract before/ after the

556

color change induced by chemicals.

557

(A) White EBN was firstly treated with 0.15 M NaNO2/ 2%HCl at 25 C for 3 days; Red EBN

558

was treated with of 5% H2O2 for 3 days. The samples were rinsed and stewed until

559

completely molten. The samples were filtered, lyophilized and re-suspended in D2O. The

560

structural change of EBN extract was monitored by 1H-NMR. The secondary structure

561

change of EBN extract was monitored by circular dichroism. (B) CD spectrum (C) High

562

tension (HT) voltage spectrum. n = 4.

563

Figure 4. Fractions responsible for the color change of Red EBN digest

564

Red EBN was treated with of 5% H2O2 for 3 days. The samples were extracted, digested,

565

and analyzed by HPLC. The fractions of EBN fingerprint were collected (1 min/ fraction) by a

566

fraction collector. (A) Morphology of Red EBN before/ after the color change was captured.

567

(B) The stewed EBN lyophilized powder (50 mg) was digested with a 5 mL SGF (pH 2) for 1

568

hour at 37 oC. The digest was neutralized with sodium hydroxide and filtered before

569

subjected to HPLC analysis. The upper panel refers to Red EBN digest while the lower

o


Page 31 of 42


570

panel refers to digest of Red EBN treated with H2O2. Peak 1 was collected and further

571

separated by size exclusion column under 214 nm absorbance, and the chromatogram was

572

shown in the insert. (C) Digest of Red EBN was collected, from (B) above, and the

573

absorbance of red color was measured at 405 nm (white bar); digest of Red EBN treated

574

with H2O2 and the color intensity was measured (black bar). (D) Immunochemical analysis

575

of Red EBN fractions, from (B), with anti-AMCase-like (EBN; CFW-12) antibody by ELISA

576

test. The absorbance of ELISA was measured at 450 nm. (E) Total Fe content of EBN

577

fractions was analyzed by ICP-OES. The total Fe content of Red EBN before/ after the color

578

change was shown in the insert. Values are expressed as Mean ± SEM, n = 3.

579

Figure 5. Fractions responsible for the color change of White EBN digest

580

White EBN was treated with of 0.15 M NaNO2/ HCl for 3 days. The samples were extracted,

581

digested, and analyzed by HPLC. The digestion of EBN was identical as in Figure 4. The

582

fractions of EBN fingerprint were collected (1 min/fraction) by a fraction collector. (A)

583

Morphology of White EBN before/ after the color change was captured. (B) Protein

584

fingerprint of the EBN digest: the upper panel refers to White EBN digest while the lower

585

panel refers to digest of White EBN treated with NaNO2/ HCl. (C) Digest of White EBN was

586

collected, and the absorbance of red color was measured at 405 nm (white bar); the digest

587

of White EBN was treated with NaNO2/HCl, and the color intensity was measured (black

588

bar). (D) Immunochemical analysis of White EBN fractions with anti-AMCase-like (EBN;



589

CFW-12) antibody by ELISA test. The absorbance of ELISA was measured at 450 nm. (E)

590

Total Fe content of EBN fractions was analyzed by ICP-OES. The total Fe content of White

591

EBN before/ after the color change was shown in the insert. Values are expressed as Mean

592

± SEM, n = 3.

593

Figure 6. Raman spectroscopy of White and Red EBN extracts before/after color

594

change induced by chemicals.

595

(A) White EBN was firstly treated with 0.15 M NaNO2/ 2% HCl at 25 oC for 3 days; Red EBN

596

was treated with of 5% H2O2 for 3 days. The samples were rinsed and stewed until complete

597

molten. The samples were filtered and lyophilized into dry powders. The samples were

598

analyzed by Raman micro-spectrometer with an excitation laser wavelength of 785 nm. The

599

laser power was set at 25 mW, and the exposure time was set at 60s. The area of dotted

600

lines of 490 cm-1 and 590 cm-1 was enlarged. (B) The peak area of the spectrum at 490 cm-1

601

and 590 cm-1 was used in PCA. The score plot of EBN samples was shown, and the ellipse

602

Hotelling T2 was at 95% confidence.

603


Page 32 of 42

Page 33 of 42


604

Supporting Information

605

Figure S1. Color change induced by metal ions.

606

White EBN changed to red gradually with the addition of Fe3+ after a day. The solution was

607

removed and Cu2+ was added to “red EBN”, the red color remained unchanged; White EBN

608

changed to blue when Cu2+was added. The solution was removed and Fe3+ was added to

609

“blue EBN”, it eventually changed to “red”. Bar = 1 cm. n = 3.

610

Figure S2. Proposed color change origin of EBN

611

AMCase-like protein, associated with a significant amount of Fe, was identified as the color

612

origin in EBN. The red color could be reversible with chemicals, but it was less favorable

613

than that from white to red. There was also binding site of EBN for transition metals that

614

could induce colors on EBN. During the color change, oxygen is bonded to Fe in

615

AMCase-like protein, and eventually which induces the red color of EBN.

616 617

Figure S3. Chemical nature of cave Red EBN

618

Cave Red EBN was extracted by the over-stewing method, and the extract was filtered and

619

lyophilized as dry powder. (A) The secondary structure of cave Red EBN was monitored by

620

circular dichroism. (B) The samples were re-suspended in D2O and monitored by 1H-NMR.

621

(C) Cave Red EBN was digested with SGF for 1 hour and then analyzed by HPLC. The

622

fingerprint of the EBN digest was shown. n = 3. 33 ACS Paragon Plus Environment


Page 34 of 42

623

Tables

624

Table 1. Protein identification by ion-trap liquid chromatography–tandem mass

625

spectrometry (LC–MS/MS).

626 Peaka

Protein b

Accession c

Major fragments

Score

1

AMCase-like

XP_010005363.1

DMDDF

31

2

unknown

Lysyl oxidase 3

XP_010006484.1 LKGGAKVGEGRVEVLR

71

homolog 3

4

Mucin-5AC-like

XP_009994736

MWDKKTSIF

41

5

AMCase-like

XP_010005363.1

AIGGWNFGTAKF

55

627 628 629 630 631 632

a

Peaks 1-5 in fingerprint of EBN were collected and subjected to LC–MS/MS for protein

identification. n = 3. b

The peptide sequences were matched with Chaetura pelagica (Chimney swift) protein

database in NCBI. c

Accession number was reference to C. pelagica in NCBI database


Page 35 of 42


633 634

Table S1. Information of Edible bird’s nest (EBN) samples.

635 country of sample

production

color a

origin

site

1

white

Indonesia

house

2

white

Vietnam

cave

3

white

Malaysia

house

4

white

Thailand

cave

5

yellow

Indonesia

house

6

yellow

Indonesia

house

7

yellow

Indonesia

house

8

red

Indonesia

house

9

red

Indonesia

house

10

red

Malaysia

house

a

636 637

a

The production sites of EBN were provided by the merchants from local market.

638



639 640

TABLE OF CONTENTS GRAPHICS

641 NaNO2/H

Fe

H2O2

Fe-O


Page 36 of 42

Page 37 of 42


A

C

90

Fe(II) & Fe(III)

75

Total Fe

0.20

45 30

FTIR Absorbance (x10-2)

0.15 0.10

15

0.05

0

0

White EBN White EBN + NaNO2/H+ 10 8 6 4 2 0

2500 2000 W avenumber cm-1

1800

1500

1600

1400

1200

1000

1000

800

Wavenumber (cm-1)

W1 W 1-C

E FTIR Absorbance (x10-2)

Page 1/1

3000

0.25

60

D

3000

0.30

A405 nm

Fe Content (µg/g)

B

10 8 6 4 2 0

1800

1500

1600

1400

1200

1000

1000

Wavenumber (cm-1)

R1-C

8/17/2015

Red EBN Red EBN + H2O2

2500 2000 W avenumber cm-1

R1

500

8/17/2015

800

500

8/17/2015 8/17/2015

Page 1/1

Fig. 1 ACS Paragon Plus Environment

Wong et al 2018


A

White EBN

0.8 0.7

Red EBN

0.6

H2O2

Page 38 of 42

House Cave

0.5

0.4

0.5

A405 nm

A405 nm

0.6

0.4 0.3

H2O2

0.3

0.2

0.2 NaNO2/H+

0.1

0.1

0

NaNO2/H+

0 1

2

3

4

1

Time (hour)

2

3

4

Time (hour)

B A405 nm

0.4

Red EBN Extract

Cold acetone precipitation

0.3 0.2 0.1

0


Wong et al 2018

Page 39 of 42


30 unit

A

White EBN

Intensity

Yellow EBN

Red EBN

White EBN + NaNO2/H+

Red EBN + H2O2 8

Ɛ (mol-1 dm-3 cm-1)

B

7

6

5

4

3

ppm

2

1

0

-1

0 -3 -6 -9

Blank White EBN Yellow EBN Red EBN White EBN + NaNO2/H+ Red EBN + H2O2

-12 -15 -18 -21 -24 200

C

220

240

280

300

320

800 700

Blank White EBN Yellow EBN Red EBN White EBN + NaNO2/H+ Red EBN + H2O2

600 500

HT

260

Wave length (nm)

400 300 200 100 0 200

220

240

260

280

300

320

Wave length (nm) ACS Paragon Plus Environment

Fig. 3 Wong et al 2018


A

Page 40 of 42

Red EBN

H2O2

1

60 mAU

Red EBN

A214 nm

B

1

Time (min)

A280 nm

2 3

4

3

4

Red EBN + H2O2 0

10

20

30

Time (min)

A405 nm

0.05 unit

C

0

Red EBN Red EBN + H2O2

1 2

10

20

30

Time (min)

A450 nm

0.05 unit

D

0

1

Red EBN + CFW-12 2

10

20

30

Fe (ng/mL)

10 unit

E

Fe (µg/g)

Time (min) Red EBN

1

10

15 0 4

2 0

30

20

30

Time (min)


Wong et al 2018

Page 41 of 42


A

White EBN

NaNO2/H+

B

4

60 mAU

White EBN

3

A280 nm

5

2

4 White EBN + NaNO2/H+

1 0

10

20

30

Time (min)

A405 nm

0.05 unit

C

0

White EBN White EBN + NaNO2/H+

10

20

30

Time (min)

A450 nm

0.05 unit

D

5

White EBN + CFW-12

3 4

0

10

20

30

Fe (ng/mL)

10 unit

E

Fe (µg/g)

Time (min) White EBN

30 15 0 5

3 4 0

10

20

30

Time (min)

ACS Paragon Plus Environment

Fig. 5 Wong et al 2018

A

White EBN

Intensity

490

590

Red EBN

490

590

Yellow EBN

Page 42 of 42

490

590

3000 unit


White EBN+NaNO2/H+

1600

1200

490

590

2000

490

590

Red EBN+H2O2

800

Raman shift

B

White EBN Yellow EBN Red EBN

400

(cm-1)

White EBN+NaNO2/H+ Red EBN+H2O2

1.4

PC2

0.7 0

-0.7 -1.4

-4

-2

0

2

4

PC1 R2X[1]=0.776 R2X[2]=0.224 Ellipse: Hotelling’s T2 (95%)


Wong et al 2018

Origin of Red Color in Edible Bird's Nests Directed by the Binding of

Recommend Documents