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Prevalence of Nitrite and Nitrate Contentsand Its Effect on Edible Bird Nest’s ColorMohammadjavad Paydar, Yi Li Wong, Won Fen Wong, Omer Abdalla Ahmed Hamdi, Noraniza Abd. Kadir,and Chung Yeng Looi

Abstract: Edible bird nests (EBNs) are important ethnomedicinal commodity in the Chinese community. Recently, Butand others showed that the white EBNs could turn red by vapors from sodium nitrite (NaNO2) in acidic condition orfrom bird soil, but this color-changing agent remained elusive. The aim of this study was to determine the prevalence ofnitrite and nitrate contents and its affects on EBN’s color. EBNs were collected from swiftlet houses or caves in SoutheastAsia. White EBNs were exposed to vapor from NaNO2 in 2% HCl, or bird soil. The levels of nitrite (NO2

−) and nitrate(NO3

−) in EBNs were determined through ion chromatography analysis. Vapors from NaNO2 in 2% HCl or bird soilstained white bird nests to brown/red colors, which correlated with increase nitrite and nitrate levels. Moreover, naturallyformed cave-EBNs (darker in color) also contained higher nitrite and nitrate levels compared to white house-EBNs,suggesting a relationship between nitrite and nitrate with EBN’s color. Of note, we detected no presence of hemoglobin inred “blood” nest. Using infrared spectra analysis, we demonstrated that red/brown cave-EBNs contained higher intensitiesof C-N and N-O bonds compared to white house-EBNs. Together, our study suggested that the color of EBNs wasassociated with the prevalence of the nitrite and nitrate contents.

Keywords: edible bird nests, ion chromatography, nitrate, nitrite

IntroductionEdible bird’s nest (EBN) is a well-known traditional food, used byancient Chinese due to its nutritious properties. It is also known as“caviar of the East” due to its high market value (Marcone 2005).EBN is made by specific swiftlets of the genera Aerodramus, Apus,and Collocalia (Marshall and Folley 1956; Kong and others 1989;Lim 2002). It usually contains amino acids, carbohydrates, mineralsalts, and also glycoproteins as the major ingredients (Kathan andWeeks 1969). Some studies showed that EBNs impart youthfulappearance, improve immune function, raise the libido, enhancemental focus, and treat respiratory ailments as well as digestiveproblems (Ng and others 1986; Kong and others 1987; Guo andothers 2006; Yagi and others 2008; Abidin and others 2011; Mat-sukawa and others 2011; Vimala and others 2011; Roh and others2012; Zhang and others 2012). A recent study demonstrated theanti-inflammatory effect of EBN, by reducing TNF-α productionin RAW 264.7 cells (Aswir and Wan Nazaimoon 2011).

EBNs are usually white but some are found in caves with dullbrownish or orange red colors (Lim 2002). In the market, redblood nests are much more expensive than white EBNs and theyhave been traditionally claimed to have better health benefits effect.It was thought that the red EBNs are swiftlet blood mixed withsalivas or due to the special type of food swiftlet consumed. Othersbelieve that the caves contain specific minerals and iron whichturn it red. Different explanations have been suggested, but thereis a lack of scientific evidence to support each claim. Recently,

MS 20130675 Submitted 5/31/2013, Accepted 10/14/2013. Authors Paydar,Y. L. Wong, Kadir, and Looi are with Dept. of Pharmacology, Faculty of Medicine,Univ. of Malaya, Kuala Lumpur, 50603 Malaysia. Author W. F Wong is withDept. of Medical Microbiology, Faculty of Medicine, Univ. of Malaya, Kuala Lumpur,50603 Malaysia. Author Hamdi is with Dept. of Chemistry, Faculty of Science,Univ. of Malaya, Kuala Lumpur, 50603, Malaysia. Direct inquiries to author Paydar(E-mail: paydarmj@gmail.com).

But and others reported the vapors from sodium nitrite (NaNO2)dissolved in 2% acid hydrochloric (HCl) or bird soil could turnwhite EBNs red (But and others 2013). They suspected that nitricoxide was involved in this process but nitrite or nitrate levels intheir guano-created red nests were not examined.

In August 2011, the Chinese government imposed a ban onEBN products imported from overseas, due to the high level ofdetected nitrite (NO2

−) in these products (AQSIQ 2011). Thehigh level of nitrite found in EBNs has raised public concern,casting doubt whether these EBNs are truly “edible.” Nitrite hasbeen used as a food preservative and antibotulinal agent in the foodprocessing industry and its level is strictly controlled to preventfood toxicity (DSM 2011). In this study, we set to investigate theprevalence of nitrite and nitrate and its implication on EBN’s color.

Materials and Methods

Samples and chemicalsWhite swiftlet house-derived EBNs (house-EBNs) and bird

soil-weighed 500 g were obtained from local swiflet houses. Redand brown cave derived-EBNs (cave-EBNs) were collected fromvarious parts in Southeast Asia via EBN distributors. Sodium ni-trite (NaNO2) was from Sigma-Aldrich (St. Louis, Mo., U.S.A.)and 36% HCl were from Fisher Scientific (Fair Lawn, N.J., U.S.A.).

Effect of sodium nitrite and bird soil on EBNsNaNO2 (0.2 g) and a piece of aluminum foil support were

placed on the bottom of bottle. Twenty milliliter of distilled wateror 2% HCl was added into the bottle. Small pieces of EBN (1 g)were moistened with distilled water and placed on the support.Experiments were repeated with NaNO2 or 2% HCl alone. Thebottles were sealed and placed in dark at room temperature. Colorof EBNs was monitored for 7 d.

In another setting, 1/3 of the bottle was filled with moistur-ized bird soil. EBN piece (1 g) was put onto the aluminum foil

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support. The bottles were sealed, wrapped with aluminum foil,and incubated at 50 ◦C. The color was observed after 2 wk.

Ion chromatography (IC) analysisEBN samples were dried and sent to MS ISO/IEC 17025 certi-

fied laboratory for analysis of anion content. The test method wasbased on AOAC method 973.31 and Dionex application note 133and application update 131. Briefly, the method involved mixingan accurate weight of grounded bird nests with ultrapure water(1:40 w/v) and placed in warm water bath for 30 min. The mix-ture was cooled and centrifuged. The supernatant fluid was filteredand subjected for IC analysis. An external calibration curves forfluoride, chloride, phosphate, nitrite, and nitrate were constructedand used for quantitative determination.

Hemastix R© hemoglobin content analysisHemoglobin contents of different EBNs were measured us-

ing SIEMENS HemastixR© Blood ID Reagent Strips (SIRCHIE,Youngsville, N.C., U.S.A.). The color changes of the strips wereused to detect the presence of hemoglobin content. Serial dilutionsof the blood from different species were used as positive controland NaCl dissolved in water was used as negative control.

Hemoglobin colorimetric assayThe total hemoglobin concentration in different EBNs was

also determined using Cayman’s Hemoglobin Colorimetric As-say kit. In brief, standard wells were prepared by adding 200 μL ofHemoglobin Standard per well in the designated wells on a 96-wellplate. Sample wells were prepared by adding 20 μL of sonicated,hydrated, and boiled EBNs. Then 180 μL of Hemoglobin Detec-tor was added to each of the sample wells, the plate was coveredand incubated for 15 min at room temperature. The absorbancewas read at 570 to 590 nm and standard curve was plotted based onthe corrected absorbance of standard wells. The hemoglobin con-centration of the samples was calculated using the linear regressionobtained from the plotted standard curve.

Infrared spectra analysisInfrared (IR) spectra of the EBN samples were collected us-

ing PerkinElmer Spectrum 400, FT-IR/FT-FIR spectrometer(PerkinElmer, Waltham, Mass., U.S.A.). About 1 mg of dried EBNsamples was used to collect IR spectra. The spectral measurementwas recorded from 4000 cm−1 to 450 cm−1. The spectral resolu-tion was approximately 4 cm−1, and 16 scans were accumulated.

Results

Yellow, brown, or red EBNs can be derived from white EBNsby sodium nitrite in acidic condition

Marketed EBNs appear white, yellow, brownish to reddish incolors. Yellow, brown, and red EBNs are rare in the market.Among all, red EBNs are highly esteemed and retailed at a priceapproximately 5 times more than the common white EBNs. Arecent study by But and others suggested the marketed red EBNmay be artificially stained since chemical treatment with NaNO2

+ HCl, or with bird soil can turned color of EBN from white tored, however the chemical content remained unknown (But andothers 2013). To further investigate this, we obtained the swift-let house-EBN which presented in clear white color from EBNdistributor (Figure 1A).

A single house-EBN was cut into 4 small pieces for treatmentsusing different methods (Figure 1B). When EBN piece was soaked

in NaNO2 + 2% HCl solution, the white color of EBN turnedyellowish which color remained the same for over 1 wk in thesolution (Figure 1B (i)). Based on the method used by But andothers we foamed white house-EBN pieces with NaNO2 + 2%HCl vapor. A piece of white EBN was placed on top of an alu-minum foil stage fixed inside a sealed bottle to hold the birdnest above the solution. Aluminum foil and the EBN were notin direct contact to the solution, thus allowed us to examine thevapor effect against EBN. We noticed that under this condition,white house-EBN initially displayed yellowish color within 1 dwhich gradually turned darker into brownish red color at day 7(Figure 1B(ii)). On the other hand, the color of white EBN re-mained unchanged when foamed with NaNO2 (without HCl) orwith 2% HCl alone (Figure 1B (iii) and 1B (iv)).

Environmental nitrite and nitrate prevalence could affectcolor of the EBN

Some swiftlet house owners claimed that incubating the EBNin bird soil could induce EBNs’ color changes from white intobrownish or reddish colors. To examine this, we obtained bird soilfrom a local swiftlet house. We filled 1/3 of sealed bottles withthe moisturized bird soil. White house-EBNs were fumigatedwith bird soil in a sealed bottles protected from light. Bottle waswrapped with aluminum foil and incubated at 50 ◦C for 2 wk.Using this method, we observed that the white house-EBNs werechanged to brownish yellow color (Figure 1C).

Color change of white EBNs correlates with increase levelsof nitrite and nitrate

To examine if the brown/red colors of NaNO2 + 2% HClvapored house-EBN was caused by presence of nitrite, originalhouse-EBN (white) and NaNO2 + 2% HCl vapored house-EBN (brownish) were dried and subjected to IC analysis fornitrite, nitrate, and other chemical contents. We detected pres-ence of chloride, nitrite, nitrate, fluoride, and phosphate in EBNs.Both nitrite and nitrate level in the brown/orange red EBNs va-pored by NaNO2 + 2% HCl were significantly higher than theuntreated white house-EBNs (Figure 2). As shown in Table 1,house-EBN (white) contained a low amount of nitrite at 14.3 ±6.7 μg/g sample, whereas the NaNO2 + 2% HCl vapored house-EBN (brownish) showed nearly 30-fold increase of nitrite level at415.4 ± 343.5 μg/g sample. The level of nitrate was also remark-ably higher in NaNO2 + 2% HCl vapored EBN at 29094.5 ±8497.3 μg/g sample, compared to only 43.9 ± 36.2 μg/g samplein the original white house-EBN. Other anions such as chloride,fluoride, and phosphate were not significantly changed in these 2groups of EBNs (data not shown). For bird soil vapored EBNs,nitrite and nitrate levels were at 297.58 ± 23.9 μg and 1341.4 ±389.9 μg per g of sample, respectively.

Natural brown or red cave-EBNs possess high levels ofnitrite and nitrate

EBNs from swiftlet houses are generally white, while thosefrom caves usually appear brownish or reddish. To compare theprevalence of nitrite and nitrate contents in these EBNs fromdifferent habitats, we collected cave- or house-EBNs from differentsources for measurement of nitrite and nitrate levels. Three house-EBNs which presented in white color and 4 cave-EBNs presentedin either brownish or reddish color were collected from differentvendors and were labeled as house-EBNs (Source I–III) and cave-EBNs (Source I–IV) (Figure 3).

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Figure 1–Color changes of white house-EBNinduced by NaNO2 or bird soil. (A) Picture of atypical white house-EBN. (B) Color of thehouse-EBN after different treatments. A whitehouse-EBN was divided into small pieces andplaced in bottles containing (i) NaNO2 + HCl,(ii) NaNO2 + HCl, (iii) HCl, and (iv) NaNO2. Inbottle (i) and (ii), EBN pieces were dipped intothe solution. For bottle (ii), (iii), and (iv), EBNpieces were placed on top of aluminum foil toobserve the vapor effect. (C) Color ofhouse-EBN after vapored with bird soil.

Table 2 shows that all house-EBNs demonstrated low amountof nitrite (14.3 ± 6.7 μg/g sample) and nitrate (43.9 ± 36.2 μg/gsample), whereas 4 cave-EBNs demonstrated a varied amount ofnitrite and nitrate levels. In general, the nitrite level of cave-EBNsregardless of brown or red colors showed high reading of nitritelevel (83.5 ± 75.4 μg/g sample), 5-fold higher than those in thehouse-EBNs (Table 2). Strikingly, the nitrate levels were extremelyhigh in the cave-EBNs (13077.6 ± 11870.4 μg/g sample). We alsonoted that for cave-EBNs, the nitrite and nitrate levels appearedcorrelated to each other in a negative pattern in which EBNswith high nitrite level demonstrated lower nitrate level, and viceversa. Although the levels of nitrite and nitrate in the cave-EBNswere both significantly higher than house-EBNs, we noticed thatthe readings were widely dispersed among samples in each group.As a result, the standard deviation values were high, especiallyin cave EBNs. We suspected that these variations of nitrite andnitrate levels among cave EBNs derived from different sourcesmight be due to distinct environment, humidity, pH, and climateof the habitat. Besides, the age of the EBNs before collection

may also be a crucial factor in determining the nitrite and nitratelevels.

Hemoglobin content analysisSIEMENS HemastixR© Blood ID Reagent Strips were used to

measure the hemoglobin contents of various EBNs, based on thecolor change. The efficiency of the HemastixR© strips was exam-ined by positive (diluted/nondiluted blood from different species)and negative controls (NaCl dissolved in water). As shown inFigure 4A, all of the blood samples, including diluted and nondi-luted samples, were highly positive (Large +++), which indicateshigh sensitivity of the strips. On the contrary, the results ob-tained from the strips for control NaCl were negative. None ofthe tested EBNs, including red samples, showed positive results onHemastixR© (Figure 4A).

Hemoglobin colorimetric assayTo confirm the results of HemastixR©, we used another test

(Cayman’s Hemoglobin Colorimetric Assay kit) to determine the

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Figure 2–Nitrite (NO2−) and nitrate (NO3

−) levels in house-EBNs. Un-treated white house-EBN and house-EBN treated with NaNO2 + HCl vaporor bird soil vapor were analyzed using ion chromatography. Bar chart showsconcentrations of nitrite (NO2

−) and nitrate (NO3−) in each house-EBNs.

Data are mean ± standard deviation (SD).

presence of hemoglobin. In this test, rat blood (10000 × diluted)were used as positive control. As shown in Figure 4B, diluted ratblood resulted in color change from colorless to yellow. Again,white or red EBNs showed negative result (colorless), which in-dicate that they did not contain any hemoglobin.

Infrared spectra analysisTo examine the chemical content in the EBN samples, we pre-

pared 1 mg of dried EBN samples for IR spectra analysis. Nitrogenbonding are structurally different and exist in the form of -NH2

(primary amine), NH- (secondary amine), N- (tertiary amine)

Table 1–Nitrite and nitrate levels of untreated house-EBNs(white) and house-EBNs stained with NaNO2 + 2% HCl vapor(brown/orange red). N = 7 for untreated house-EBNs; n = 2 fortreated chemical or bird soil treated house-EBNs. SD, standarddeviation.

Concentrations (mean ± SD)μg per g of sample

Nitrite NitrateEBN samples Appearance (NO2)− (NO3)−

House-EBN White 14.3 ± 6.7 43.9 ± 36.2House-EBN

(NaNO2 +2% HCl)

Brown/red 415.4 ± 343.5 29094.5 ± 8497.3

House-EBN(bird soil)

Brown 9.8 ± 5.9 23.9 ± 11.0

Figure 3–Representative picture of a house-EBN (white) in comparison to2 cave-EBNs (red or brown).

as well as in the form of hydrogen bondings like N-H . . . O,O-H . . . .N, N-H . . . N. The diverse nitrogen binding can be de-tected by IR spectrophotometric method due to absorption differ-ence between each functional group. The IR spectra revealed thepresence of NH2- at vibrations 3281.91 cm−1 (red), 3274.7 cm−1

(white), and 3284.01 cm−1 (brown) EBNs (Figure 5A and 5B).Besides, we found the presence of N-O stretching at vibrations1408.64, and 1406.47 cm−1 in the red and brown cave-EBNs,respectively (Figure 5A and 5B), whereas the peak was not sig-nificant in the IR spectra of white EBNs (Jones and Sandorfy1956; Nakanishi 1977; Colthup and others 1990). These data were

Table 2–Nitrite and nitrate levels of house-EBNs (white) and cave-EBNs (brown or red) derived from different sources. N = 2 to 3for each group. Data were presented as mean ± standard deviation (SD) and statistical significances were compared to house-EBNs,using Student’s t-test. Statistically significant (P < 0.05), NS, statistically not significant (P > 0.05).

Concentration (μg per g of sample)

Nitrite (NO2)− Nitrate (NO3)−

EBN samples mean ± SD P value mean ± SD P value

House 14.3 ± 6.7 43.9 ± 36.2House Source I White 12.9 ± 1.4 NS 23.7 ± 4.5 NSHouse Source II White 22.0 ± 3.2 NS 20.4 ± 0.5 NSHouse Source III White 7.9 ± 2.6 NS 87.5 ± 28.2 NS

Cave 83.5 ± 75.4 0.0453 13077.6 ± 11870.4 0.019Cave Source I Red 39.2 ± 3.0 0.0026 30016.7 ± 1149.8 <0.0001Cave Source II Brown 47.44 ± 15.1 0.0088 12168.2 ± 6600.7 0.0008Cave Source III Red 65.0 ± 4.35 <0.0001 3482.5 ± 1143.9 0.0001Cave Source IV Brown 212.9 ± 32.4 <0.0001 2128.6 ± 325.3 <0.0001

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Figure 4–Absence of hemoglobin in EBNs. (A)Diluted/nondiluted blood samples from variousspecies were used as positive controls, to test thesensitivity of the Hemastix R© strips for hemoglobindetection. Right panel showed blood before andafter dilution. NaCl dissolved in waster was used asnegative control, to test the specificity of theHemastix R© strips for hemoglobin detection.Red/white EBNs were hydrated or sonicated andtested using the Hemastix R© strips. (B) Totalhemoglobin concentration as examined bycolorimetric assay. Standard curve was preparedbased on the manufacturer’s instructions and ratblood was used as positive control.

consistent with our IC data which suggested that red/brown colorsof the cave-EBNs might be due to the existence of N-O stretch-ing. Comparison of the IR results obtained from brown or redcave-EBNs also indicated that these EBNs contained higher per-centages of C-N bond in its structure (approximately 873), whilethe peaks were less visible in the white EBNs (Figure 5A and 5B).

DiscussionNitrite and nitrate issue has been a subject of contentment over

the past decades. Study showed that the cancer risk of nitriteand nitrate is related to the formation of N-nitroso compounds(Eichholzer and Gutzwiller 1998). In this study, we found thatprocessed red/brown EBN still contains high level of nitrite and

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Figure 5–Infrared (IR) spectroscopy of brown cave-EBN (A) or red cave-EBN (B) overlapped with white house-EBN. (Red line: red/brown cave-EBN; blackline: white house-EBN). The IR results which showed significant differences between cave and house EBNs have been highlighted and labeled as area(1) (N-O stretching at vibrations approximately 1406 to 1408 cm−1) and area (2) (C-N stretching at vibrations approximately 873 to 874 cm−1). Thedotted lines indicate enlarged areas from the original plot.

nitrate compared to the processed white EBN, indicating thatmore rigorous and efficient food processing method is needed.However, we observed that nitrite and nitrate in unprocessed whiteEBN from the swiftlet houses can be reduced significantly byovernight soaking or brief sonication in water (data not shown).

Coincidentally, it is a common practice for the Chinese to soakEBNs for several hours, or even overnight, and washing themthoroughly with clean water (Ma and Liu 2012). This empiricalknowledge may be ethnopharmacologically important, as it cansubstantially reduce nitrite levels in EBNs as nitrites are highly

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water soluble (Weast 1979; Lide 2005). Nevertheless, the publicshould discard the water used for soaking EBNs before cookingto avoid consumption of excessive nitrite or nitrate.

The present study shows that white EBNs overall containedlow amounts of nitrite and nitrate. Here we provided evidencethat foaming EBNs with NaNO2 + HCl (But and others 2013)or bird soil increased the levels of nitrate and nitrate that maychange the color of white EBNs. Enhancing nitrite level by addingNaNO2 alone demonstrated no effect on white EBNs, indicatingthat chemical reaction is necessary for the color change. Sodiumnitrite mixed with strong acid such as HCl could form nitrousacid which can bind with proteins in EBNs through a processknown as xanthoproteic reaction. However, we found that thepH of bird soil is near neutral and the reaction probably is notanalogy to xanthoproteic reaction. We hypothesize that the sourcesof nitrite and nitrate could have been derived from ammoniathrough anaerobic fermentation by the bacteria (Percheron andothers 1999). Although cave-EBNs have higher nitrite contentcompared to white EBNs, the nitrate content in cave-EBNs wasmore than 100-fold higher than the house-EBNs. This may bedue to nitrate is a more stable form and it can be derived viaoxidixation of nitrite (Eichholzer and Gutzwiller 1998). Throughobservation, we noticed that the color of EBNs foamed withsodium nitrite in acidic condition turned from white to yellow,followed by brownish red. However, the nitrite and nitrate levelsin some brown EBNs are higher than red EBNs, indicating otherfactors could be involved in the reddening process. The ancientChinese community believed that the red EBNs could be a productof blood-vomiting process by the swiftlets, thus naming them“blood” nest. We found this to be a misnomer as 2 independenthemoglobin tests did not reveal the presence of heme in the EBNs.Other researchers suggested that ovotransferrin, an albumin, maybe contributing to the red appearance of EBNs (Marcone 2005).Thus, further analysis is needed to analyze whether ovotransferrinis responsible for the reddening effect in natural cave-EBNs, apartfrom nitrite or nitrate.

In this study, we found that the microenvironment of the EBNs(swiftlet house or the caves) plays a crucial role in the prevalenceof nitrite and nitrate. Foaming white EBNs with nitrite enrichedbird soil could turn them into brownish yellow, but not red asderived from sodium nitrite in acidic condition. This discrep-ancy is probably due to the nitrite and nitrate levels in the vapor.Longer incubation time is needed to generate nitrite or nitratefrom the soil as fermentation is a slower process compare to thedirect chemical reaction of sodium nitrite with HCl. The sourceof bird soil used in our study is also a contributing factor as swiflethouse bird soil has a lower nitrite and nitrate contents compared tocave guanos that contain bird or bat droppings mixed with otherorganic materials rich in nitrite and nitrate. Thus, we speculatedthat EBN’s color is greatly affected by the nitrite and nitrate con-tents present in the environment. Our data provided evidence thatEBNs from the caves generally contained higher nitrite and nitratelevels compared to those from swiftlet houses. This is because mostoperators clean their swiftlet houses frequently by removing thebird soil. In addition, swiflet houses usually have better ventilationcompared to the caves, which do not favor the fermentation pro-cess due to the open environment. In contrast, the caves, wherethe EBNs were harvested, has a pool of guano underneath it andthe place is filled with strong ammonia smell, according to someEBN collectors. Therefore, EBNs from swiflet houses are usuallywhite whereas the brown or red or brown EBNs are commonlyderived from caves due to the environmental nitrite and nitrate

content. However, this generalization is not necessarily true as thebrown/red EBNs could also derived from swiftlet houses if thebird soil is not removed and the nests are left for a long periodof time. Thus, cleaning the bird soil frequently, ensuring ampleventilation and harvesting the bird nests promptly once formed,are feasible ways to control amount of nitrite and nitrate in EBNsby swiflet house operators.

EBNs are reported to contain protein (62% to 63%), carbohy-drate (25.62% to 27.26%), ash (2.1%), and lipid (0.14% to 1.28%)(Marcone 2005; Aswir and Wan Nazaimoon 2011). The aminoacids found in EBNs are aspartic acid (Asp), threonine (Thr),serine (Ser), glutamic acid (Glu), proline (Pro), glycine (Gly), ala-nine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine(Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine(Lys), tryptophan (Trp), and arginine (Arg) (Marcone 2005; Maand Liu 2012; Teo and others 2013). Among them, aromaticamino acids (tyrosine, phenylalanine, tryptophan) possess phenylrings that could react with nitric acid/nitrous acid (derived fromHCl and sodium nitrate reaction or bacterial fermentation of birdsoil) to form yellow EBNs through formation of aryl-C-N andNO2 side group (Figure S1). The presence of salt such as sodiumor calcium in natural habitat with the existence of water formsodium or calcium hydroxide (base). We hypothesized that pHincrement in the presence of activated aromatic rings may furtherpromote color changing process (brown or red). However, a moredetailed chemical explanation of the color change requires furtherinvestigations. The present study could bring public awareness onthe existence of different types of EBN (white, yellow, brown,red), so that the consumers will have a better knowledge on se-lecting a good EBN by taking into consideration the prevalenceof nitrate and nitrite in these EBNs.

In conclusion, the color of EBNs is affected by the prevalenceof the nitrite and nitrate content. The cave-EBNs are generallydarker in color and contain higher nitrite and nitrate contentscompare to house-EBNs. More scientific researches are needed tounderstand the underlying chemistry on how nitrite and nitratebind to EBNs, which could provide scientific explanation on theorigin of yellow, brown, or red EBNs that have mystified theChinese community for centuries.

AcknowledgmentsThis work was supported by Univ. of Malaya research grant

(RG434-12HTM).

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Supporting InformationAdditional Supporting Information may be found in the onlineversion of this article at the publisher’s website:

Figure S1. Model showing color change of aromatic amino acidthrough xanthoproteic reaction.

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