Prevalence of Nitrite and Nitrate Contents and Its Effect on Edible



Prevalence of Nitrite and Nitrate Contents and Its Effect on Edible
Prevalence of Nitrite and Nitrate Contents
and Its Effect on Edible Bird Nest’s Color
Mohammadjavad Paydar, Yi Li Wong, Won Fen Wong, Omer Abdalla Ahmed Hamdi, Noraniza Abd. Kadir,
and Chung Yeng Looi
Edible bird nests (EBNs) are important ethnomedicinal commodity in the Chinese community. Recently, But
and others showed that the white EBNs could turn red by vapors from sodium nitrite (NaNO2 ) in acidic condition or
from bird soil, but this color-changing agent remained elusive. The aim of this study was to determine the prevalence of
nitrite and nitrate contents and its affects on EBN’s color. EBNs were collected from swiftlet houses or caves in Southeast
Asia. 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 soil
stained white bird nests to brown/red colors, which correlated with increase nitrite and nitrate levels. Moreover, naturally
formed 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 in
red “blood” nest. Using infrared spectra analysis, we demonstrated that red/brown cave-EBNs contained higher intensities
of C-N and N-O bonds compared to white house-EBNs. Together, our study suggested that the color of EBNs was
associated with the prevalence of the nitrite and nitrate contents.
Keywords: edible bird nests, ion chromatography, nitrate, nitrite
Edible bird’s nest (EBN) is a well-known traditional food, used by
ancient 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, mineral
salts, and also glycoproteins as the major ingredients (Kathan and
Weeks 1969). Some studies showed that EBNs impart youthful
appearance, improve immune function, raise the libido, enhance
mental focus, and treat respiratory ailments as well as digestive
problems (Ng and others 1986; Kong and others 1987; Guo and
others 2006; Yagi and others 2008; Abidin and others 2011; Matsukawa and others 2011; Vimala and others 2011; Roh and others
2012; Zhang and others 2012). A recent study demonstrated the
anti-inflammatory effect of EBN, by reducing TNF-α production
in RAW 264.7 cells (Aswir and Wan Nazaimoon 2011).
EBNs are usually white but some are found in caves with dull
brownish or orange red colors (Lim 2002). In the market, red
blood nests are much more expensive than white EBNs and they
have been traditionally claimed to have better health benefits effect.
It was thought that the red EBNs are swiftlet blood mixed with
salivas or due to the special type of food swiftlet consumed. Others
believe that the caves contain specific minerals and iron which
turn it red. Different explanations have been suggested, but there
is a lack of scientific evidence to support each claim. Recently,
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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 with
Dept. 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: [email protected]).
Journal of Food Science r Vol. 78, Nr. 12, 2013
But and others reported the vapors from sodium nitrite (NaNO2 )
dissolved in 2% acid hydrochloric (HCl) or bird soil could turn
white EBNs red (But and others 2013). They suspected that nitric
oxide was involved in this process but nitrite or nitrate levels in
their guano-created red nests were not examined.
In August 2011, the Chinese government imposed a ban on
EBN products imported from overseas, due to the high level of
detected nitrite (NO2 − ) in these products (AQSIQ 2011). The
high level of nitrite found in EBNs has raised public concern,
casting doubt whether these EBNs are truly “edible.” Nitrite has
been used as a food preservative and antibotulinal agent in the food
processing industry and its level is strictly controlled to prevent
food toxicity (DSM 2011). In this study, we set to investigate the
prevalence of nitrite and nitrate and its implication on EBN’s color.
Materials and Methods
Samples and chemicals
White swiftlet house-derived EBNs (house-EBNs) and bird
soil-weighed 500 g were obtained from local swiflet houses. Red
and brown cave derived-EBNs (cave-EBNs) were collected from
various parts in Southeast Asia via EBN distributors. Sodium nitrite (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 EBNs
NaNO2 (0.2 g) and a piece of aluminum foil support were
placed on the bottom of bottle. Twenty milliliter of distilled water
or 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. The
bottles were sealed and placed in dark at room temperature. Color
of EBNs was monitored for 7 d.
In another setting, 1/3 of the bottle was filled with moisturized bird soil. EBN piece (1 g) was put onto the aluminum foil
C 2013 Institute of Food Technologists
Journal of Food Science No claim to original US government works
doi: 10.1111/1750-3841.12313
Further reproduction without permission is prohibited
Nitrite and nitrate affect bird nest color . . .
support. The bottles were sealed, wrapped with aluminum foil, in NaNO2 + 2% HCl solution, the white color of EBN turned
and incubated at 50 ◦ C. The color was observed after 2 wk.
yellowish which color remained the same for over 1 wk in the
solution (Figure 1B (i)). Based on the method used by But and
others we foamed white house-EBN pieces with NaNO2 + 2%
Ion chromatography (IC) analysis
EBN samples were dried and sent to MS ISO/IEC 17025 certi- HCl vapor. A piece of white EBN was placed on top of an alufied laboratory for analysis of anion content. The test method was minum foil stage fixed inside a sealed bottle to hold the bird
based on AOAC method 973.31 and Dionex application note 133 nest above the solution. Aluminum foil and the EBN were not
and application update 131. Briefly, the method involved mixing in direct contact to the solution, thus allowed us to examine the
an accurate weight of grounded bird nests with ultrapure water vapor effect against EBN. We noticed that under this condition,
(1:40 w/v) and placed in warm water bath for 30 min. The mix- white house-EBN initially displayed yellowish color within 1 d
ture was cooled and centrifuged. The supernatant fluid was filtered which gradually turned darker into brownish red color at day 7
and subjected for IC analysis. An external calibration curves for (Figure 1B(ii)). On the other hand, the color of white EBN refluoride, chloride, phosphate, nitrite, and nitrate were constructed mained unchanged when foamed with NaNO2 (without HCl) or
with 2% HCl alone (Figure 1B (iii) and 1B (iv)).
and used for quantitative determination.
Hemastix hemoglobin content analysis
Hemoglobin contents of different EBNs were measured using SIEMENS Hemastix Blood ID Reagent Strips (SIRCHIE,
Youngsville, N.C., U.S.A.). The color changes of the strips were
used to detect the presence of hemoglobin content. Serial dilutions
of the blood from different species were used as positive control
and NaCl dissolved in water was used as negative control.
Environmental nitrite and nitrate prevalence could affect
color of the EBN
Some swiftlet house owners claimed that incubating the EBN
in bird soil could induce EBNs’ color changes from white into
brownish or reddish colors. To examine this, we obtained bird soil
from a local swiftlet house. We filled 1/3 of sealed bottles with
the moisturized bird soil. White house-EBNs were fumigated
with bird soil in a sealed bottles protected from light. Bottle was
Hemoglobin colorimetric assay
wrapped with aluminum foil and incubated at 50 ◦ C for 2 wk.
The total hemoglobin concentration in different EBNs was Using this method, we observed that the white house-EBNs were
also determined using Cayman’s Hemoglobin Colorimetric As- changed to brownish yellow color (Figure 1C).
say kit. In brief, standard wells were prepared by adding 200 μL of
Hemoglobin Standard per well in the designated wells on a 96-well Color change of white EBNs correlates with increase levels
plate. Sample wells were prepared by adding 20 μL of sonicated, of nitrite and nitrate
hydrated, and boiled EBNs. Then 180 μL of Hemoglobin DetecTo examine if the brown/red colors of NaNO2 + 2% HCl
tor was added to each of the sample wells, the plate was covered
vapored house-EBN was caused by presence of nitrite, original
and incubated for 15 min at room temperature. The absorbance
house-EBN (white) and NaNO2 + 2% HCl vapored housewas read at 570 to 590 nm and standard curve was plotted based on
EBN (brownish) were dried and subjected to IC analysis for
the corrected absorbance of standard wells. The hemoglobin connitrite, nitrate, and other chemical contents. We detected prescentration of the samples was calculated using the linear regression
ence of chloride, nitrite, nitrate, fluoride, and phosphate in EBNs.
obtained from the plotted standard curve.
Both nitrite and nitrate level in the brown/orange red EBNs vapored by NaNO2 + 2% HCl were significantly higher than the
Infrared spectra analysis
untreated white house-EBNs (Figure 2). As shown in Table 1,
Infrared (IR) spectra of the EBN samples were collected us- house-EBN (white) contained a low amount of nitrite at 14.3 ±
ing PerkinElmer Spectrum 400, FT-IR/FT-FIR spectrometer 6.7 μg/g sample, whereas the NaNO + 2% HCl vapored house2
(PerkinElmer, Waltham, Mass., U.S.A.). About 1 mg of dried EBN EBN (brownish) showed nearly 30-fold increase of nitrite level at
samples was used to collect IR spectra. The spectral measurement 415.4 ± 343.5 μg/g sample. The level of nitrate was also remarkwas recorded from 4000 cm−1 to 450 cm−1 . The spectral resolu- ably higher in NaNO + 2% HCl vapored EBN at 29094.5 ±
tion was approximately 4 cm−1 , and 16 scans were accumulated. 8497.3 μg/g sample, compared to only 43.9 ± 36.2 μg/g sample
in the original white house-EBN. Other anions such as chloride,
fluoride, and phosphate were not significantly changed in these 2
Yellow, brown, or red EBNs can be derived from white EBNs groups of EBNs (data not shown). For bird soil vapored EBNs,
nitrite and nitrate levels were at 297.58 ± 23.9 μg and 1341.4 ±
by sodium nitrite in acidic condition
Marketed EBNs appear white, yellow, brownish to reddish in 389.9 μg per g of sample, respectively.
colors. Yellow, brown, and red EBNs are rare in the market.
Among all, red EBNs are highly esteemed and retailed at a price Natural brown or red cave-EBNs possess high levels of
approximately 5 times more than the common white EBNs. A nitrite and nitrate
recent study by But and others suggested the marketed red EBN
EBNs from swiftlet houses are generally white, while those
may be artificially stained since chemical treatment with NaNO2 from caves usually appear brownish or reddish. To compare the
+ HCl, or with bird soil can turned color of EBN from white to prevalence of nitrite and nitrate contents in these EBNs from
red, however the chemical content remained unknown (But and different habitats, we collected cave- or house-EBNs from different
others 2013). To further investigate this, we obtained the swift- sources for measurement of nitrite and nitrate levels. Three houselet house-EBN which presented in clear white color from EBN EBNs which presented in white color and 4 cave-EBNs presented
distributor (Figure 1A).
in either brownish or reddish color were collected from different
A single house-EBN was cut into 4 small pieces for treatments vendors and were labeled as house-EBNs (Source I–III) and caveusing different methods (Figure 1B). When EBN piece was soaked EBNs (Source I–IV) (Figure 3).
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Nitrite and nitrate affect bird nest color . . .
Figure 1–Color changes of white house-EBN
induced by NaNO2 or bird soil. (A) Picture of a
typical white house-EBN. (B) Color of the
house-EBN after different treatments. A white
house-EBN was divided into small pieces and
placed in bottles containing (i) NaNO2 + HCl,
(ii) NaNO2 + HCl, (iii) HCl, and (iv) NaNO2 . In
bottle (i) and (ii), EBN pieces were dipped into
the solution. For bottle (ii), (iii), and (iv), EBN
pieces were placed on top of aluminum foil to
observe the vapor effect. (C) Color of
house-EBN after vapored with bird soil.
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Table 2 shows that all house-EBNs demonstrated low amount
of nitrite (14.3 ± 6.7 μg/g sample) and nitrate (43.9 ± 36.2 μg/g
sample), whereas 4 cave-EBNs demonstrated a varied amount of
nitrite and nitrate levels. In general, the nitrite level of cave-EBNs
regardless of brown or red colors showed high reading of nitrite
level (83.5 ± 75.4 μg/g sample), 5-fold higher than those in the
house-EBNs (Table 2). Strikingly, the nitrate levels were extremely
high in the cave-EBNs (13077.6 ± 11870.4 μg/g sample). We also
noted that for cave-EBNs, the nitrite and nitrate levels appeared
correlated to each other in a negative pattern in which EBNs
with high nitrite level demonstrated lower nitrate level, and vice
versa. Although the levels of nitrite and nitrate in the cave-EBNs
were both significantly higher than house-EBNs, we noticed that
the readings were widely dispersed among samples in each group.
As a result, the standard deviation values were high, especially
in cave EBNs. We suspected that these variations of nitrite and
nitrate levels among cave EBNs derived from different sources
might be due to distinct environment, humidity, pH, and climate
of the habitat. Besides, the age of the EBNs before collection
T1942 Journal of Food Science r Vol. 78, Nr. 12, 2013
may also be a crucial factor in determining the nitrite and nitrate
Hemoglobin content analysis
SIEMENS Hemastix Blood ID Reagent Strips were used to
measure the hemoglobin contents of various EBNs, based on the
color change. The efficiency of the Hemastix strips was examined by positive (diluted/nondiluted blood from different species)
and negative controls (NaCl dissolved in water). As shown in
Figure 4A, all of the blood samples, including diluted and nondiluted samples, were highly positive (Large +++), which indicates
high sensitivity of the strips. On the contrary, the results obtained from the strips for control NaCl were negative. None of
the tested EBNs, including red samples, showed positive results on
Hemastix (Figure 4A).
Hemoglobin colorimetric assay
To confirm the results of Hemastix , we used another test
(Cayman’s Hemoglobin Colorimetric Assay kit) to determine the
Nitrite and nitrate affect bird nest color . . .
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 for
treated chemical or bird soil treated house-EBNs. SD, standard
Concentrations (mean ± SD)
μg per g of sample
EBN samples
(NaNO2 +
2% HCl)
(bird soil)
(NO2 )−
(NO3 )−
14.3 ± 6.7
415.4 ± 343.5
43.9 ± 36.2
29094.5 ± 8497.3
9.8 ± 5.9
23.9 ± 11.0
Figure 2–Nitrite (NO2 − ) and nitrate (NO3 − ) levels in house-EBNs. Untreated white house-EBN and house-EBN treated with NaNO2 + HCl vapor
or bird soil vapor were analyzed using ion chromatography. Bar chart shows
concentrations of nitrite (NO2 − ) and nitrate (NO3 − ) in each house-EBNs.
Figure 3–Representative picture of a house-EBN (white) in comparison to
Data are mean ± standard deviation (SD).
2 cave-EBNs (red or brown).
presence of hemoglobin. In this test, rat blood (10000 × diluted)
were used as positive control. As shown in Figure 4B, diluted rat
blood resulted in color change from colorless to yellow. Again,
white or red EBNs showed negative result (colorless), which indicate that they did not contain any hemoglobin.
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 detected by IR spectrophotometric method due to absorption difference between each functional group. The IR spectra revealed the
presence 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).
Infrared spectra analysis
Besides, we found the presence of N-O stretching at vibrations
To examine the chemical content in the EBN samples, we pre- 1408.64, and 1406.47 cm−1 in the red and brown cave-EBNs,
pared 1 mg of dried EBN samples for IR spectra analysis. Nitrogen respectively (Figure 5A and 5B), whereas the peak was not sigbonding are structurally different and exist in the form of -NH2 nificant in the IR spectra of white EBNs (Jones and Sandorfy
(primary amine), NH- (secondary amine), N- (tertiary amine) 1956; 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 3
for 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)
mean ± SD
EBN samples
House Source I
House Source II
House Source III
Cave Source I
Cave Source II
Cave Source III
Cave Source IV
Nitrate (NO3 )−
P value
mean ± SD
43.9 ±
23.7 ±
20.4 ±
87.5 ±
13077.6 ±
30016.7 ±
12168.2 ±
3482.5 ±
2128.6 ±
P value
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Nitrite (NO2 )−
Nitrite and nitrate affect bird nest color . . .
Figure 4–Absence of hemoglobin in EBNs. (A)
Diluted/nondiluted blood samples from various
species were used as positive controls, to test the
sensitivity of the HemastixR strips for hemoglobin
detection. Right panel showed blood before and
after dilution. NaCl dissolved in waster was used as
negative control, to test the specificity of the
HemastixR strips for hemoglobin detection.
Red/white EBNs were hydrated or sonicated and
tested using the HemastixR strips. (B) Total
hemoglobin concentration as examined by
colorimetric assay. Standard curve was prepared
based on the manufacturer’s instructions and rat
blood was used as positive control.
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consistent with our IC data which suggested that red/brown colors
of the cave-EBNs might be due to the existence of N-O stretching. Comparison of the IR results obtained from brown or red
cave-EBNs also indicated that these EBNs contained higher percentages of C-N bond in its structure (approximately 873), while
the peaks were less visible in the white EBNs (Figure 5A and 5B).
T1944 Journal of Food Science r Vol. 78, Nr. 12, 2013
Nitrite and nitrate issue has been a subject of contentment over
the past decades. Study showed that the cancer risk of nitrite
and nitrate is related to the formation of N-nitroso compounds
(Eichholzer and Gutzwiller 1998). In this study, we found that
processed red/brown EBN still contains high level of nitrite and
Nitrite and nitrate affect bird nest color . . .
nitrate compared to the processed white EBN, indicating that
more rigorous and efficient food processing method is needed.
However, we observed that nitrite and nitrate in unprocessed white
EBN from the swiftlet houses can be reduced significantly by
overnight soaking or brief sonication in water (data not shown).
Coincidentally, it is a common practice for the Chinese to soak
EBNs for several hours, or even overnight, and washing them
thoroughly with clean water (Ma and Liu 2012). This empirical
knowledge may be ethnopharmacologically important, as it can
substantially reduce nitrite levels in EBNs as nitrites are highly
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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; black
line: 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 ). The
dotted lines indicate enlarged areas from the original plot.
Nitrite and nitrate affect bird nest color . . .
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water soluble (Weast 1979; Lide 2005). Nevertheless, the public
should discard the water used for soaking EBNs before cooking
to avoid consumption of excessive nitrite or nitrate.
The present study shows that white EBNs overall contained
low amounts of nitrite and nitrate. Here we provided evidence
that foaming EBNs with NaNO2 + HCl (But and others 2013)
or bird soil increased the levels of nitrate and nitrate that may
change the color of white EBNs. Enhancing nitrite level by adding
NaNO2 alone demonstrated no effect on white EBNs, indicating
that chemical reaction is necessary for the color change. Sodium
nitrite mixed with strong acid such as HCl could form nitrous
acid which can bind with proteins in EBNs through a process
known as xanthoproteic reaction. However, we found that the
pH of bird soil is near neutral and the reaction probably is not
analogy to xanthoproteic reaction. We hypothesize that the sources
of nitrite and nitrate could have been derived from ammonia
through anaerobic fermentation by the bacteria (Percheron and
others 1999). Although cave-EBNs have higher nitrite content
compared to white EBNs, the nitrate content in cave-EBNs was
more than 100-fold higher than the house-EBNs. This may be
due to nitrate is a more stable form and it can be derived via
oxidixation of nitrite (Eichholzer and Gutzwiller 1998). Through
observation, we noticed that the color of EBNs foamed with
sodium nitrite in acidic condition turned from white to yellow,
followed by brownish red. However, the nitrite and nitrate levels
in some brown EBNs are higher than red EBNs, indicating other
factors could be involved in the reddening process. The ancient
Chinese community believed that the red EBNs could be a product
of blood-vomiting process by the swiftlets, thus naming them
“blood” nest. We found this to be a misnomer as 2 independent
hemoglobin tests did not reveal the presence of heme in the EBNs.
Other researchers suggested that ovotransferrin, an albumin, may
be contributing to the red appearance of EBNs (Marcone 2005).
Thus, further analysis is needed to analyze whether ovotransferrin
is responsible for the reddening effect in natural cave-EBNs, apart
from 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 prevalence
of nitrite and nitrate. Foaming white EBNs with nitrite enriched
bird soil could turn them into brownish yellow, but not red as
derived from sodium nitrite in acidic condition. This discrepancy is probably due to the nitrite and nitrate levels in the vapor.
Longer incubation time is needed to generate nitrite or nitrate
from the soil as fermentation is a slower process compare to the
direct chemical reaction of sodium nitrite with HCl. The source
of bird soil used in our study is also a contributing factor as swiflet
house bird soil has a lower nitrite and nitrate contents compared to
cave guanos that contain bird or bat droppings mixed with other
organic materials rich in nitrite and nitrate. Thus, we speculated
that EBN’s color is greatly affected by the nitrite and nitrate contents present in the environment. Our data provided evidence that
EBNs from the caves generally contained higher nitrite and nitrate
levels compared to those from swiftlet houses. This is because most
operators clean their swiftlet houses frequently by removing the
bird soil. In addition, swiflet houses usually have better ventilation
compared to the caves, which do not favor the fermentation process due to the open environment. In contrast, the caves, where
the EBNs were harvested, has a pool of guano underneath it and
the place is filled with strong ammonia smell, according to some
EBN collectors. Therefore, EBNs from swiflet houses are usually
white whereas the brown or red or brown EBNs are commonly
derived from caves due to the environmental nitrite and nitrate
T1946 Journal of Food Science r Vol. 78, Nr. 12, 2013
content. However, this generalization is not necessarily true as the
brown/red EBNs could also derived from swiftlet houses if the
bird soil is not removed and the nests are left for a long period
of time. Thus, cleaning the bird soil frequently, ensuring ample
ventilation and harvesting the bird nests promptly once formed,
are feasible ways to control amount of nitrite and nitrate in EBNs
by swiflet house operators.
EBNs are reported to contain protein (62% to 63%), carbohydrate (25.62% to 27.26%), ash (2.1%), and lipid (0.14% to 1.28%)
(Marcone 2005; Aswir and Wan Nazaimoon 2011). The amino
acids found in EBNs are aspartic acid (Asp), threonine (Thr),
serine (Ser), glutamic acid (Glu), proline (Pro), glycine (Gly), alanine (Ala), valine (Val), methionine (Met), isoleucine (Ile), leucine
(Leu), tyrosine (Tyr), phenylalanine (Phe), histidine (His), lysine
(Lys), tryptophan (Trp), and arginine (Arg) (Marcone 2005; Ma
and Liu 2012; Teo and others 2013). Among them, aromatic
amino acids (tyrosine, phenylalanine, tryptophan) possess phenyl
rings that could react with nitric acid/nitrous acid (derived from
HCl and sodium nitrate reaction or bacterial fermentation of bird
soil) to form yellow EBNs through formation of aryl-C-N and
NO2 side group (Figure S1). The presence of salt such as sodium
or calcium in natural habitat with the existence of water form
sodium or calcium hydroxide (base). We hypothesized that pH
increment in the presence of activated aromatic rings may further
promote color changing process (brown or red). However, a more
detailed chemical explanation of the color change requires further
investigations. The present study could bring public awareness on
the existence of different types of EBN (white, yellow, brown,
red), so that the consumers will have a better knowledge on selecting a good EBN by taking into consideration the prevalence
of nitrate and nitrite in these EBNs.
In conclusion, the color of EBNs is affected by the prevalence
of the nitrite and nitrate content. The cave-EBNs are generally
darker in color and contain higher nitrite and nitrate contents
compare to house-EBNs. More scientific researches are needed to
understand the underlying chemistry on how nitrite and nitrate
bind to EBNs, which could provide scientific explanation on the
origin of yellow, brown, or red EBNs that have mystified the
Chinese community for centuries.
This work was supported by Univ. of Malaya research grant
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Supporting Information
Additional Supporting Information may be found in the online
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