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Makara Journal of Science Makara Journal of Science
Volume 26
Issue 1
March
Article 7
3-30-2022
Evaluation of Nitrite Concentration in Edible Bird’s Nest (White, Evaluation of Nitrite Concentration in Edible Bird’s Nest (White,
Yellow, Orange, and Red Blood) Yellow, Orange, and Red Blood)
Siti Gusti Ningrum
Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya 60225, Indonesia
,
sitiningrum@uwks.ac.id
Bagus Uda Palgunad
Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya 60225, Indonesia
Rochiman Sasmita
Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya 60225, Indonesia
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Recommended Citation Recommended Citation
Ningrum, Siti Gusti; Palgunad, Bagus Uda; and Sasmita, Rochiman (2022) "Evaluation of Nitrite
Concentration in Edible Bird’s Nest (White, Yellow, Orange, and Red Blood),"
Makara Journal of Science
:
Vol. 26: Iss. 1, Article 7.
DOI: 10.7454/mss.v26i1.1311
Available at: https://scholarhub.ui.ac.id/science/vol26/iss1/7
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Makara Journal of Science, 26/1 (2022), 68−72
doi: 10.7454/mss.v26i1.1311
68 March 2022 Vol. 26 No. 1
Evaluation of Nitrite Concentration in Edible Bird’s Nest
(White, Yellow, Orange, and Red Blood)
Siti Gusti Ningrum*, Bagus Uda Palgunad, and Rochiman Sasmita
Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya 60225, Indonesia
*E-mail: sitiningrum@uwks.ac.id
Received January 22, 2022 | Accepted March 1, 2022
Abstract
The color of edible bird’s nest is associated with its nitrite concentration, but this relationship remains inconclusive. This
investigation aimed to evaluate the nitrite content in edible bird’s nest of four different colors: white, yellow, orange, and
red blood. Fifty-eight edible bird’s nest samples were obtained from five swiftlet farmhouses in Borneo Island, Indo-
nesia and analyzed for nitrite content using Genesys 30 visible spectrophotometer. Results showed that the dark-colored
edible bird’s nests (yellow, orange, and red blood) had higher nitrite concentrations of 304, 317, and 309 ppm, respec-
tively, compared with the white-colored one (15 ppm). Therefore, the color of edible bird’s nest was associated with its
nitrite concentration. This study provided updated information about the nitrite concentration in edible bird’s nest of
various colors.
Keywords: contamination, public health, food safety
Introduction
During the COVID-19 outbreak, edible bird’s nest (EBN)
has been considered to boost immunity [1]. The main
components of EBN are amino acids, carbohydrates, min-
eral salts, and glycoproteins [2]. Consumer demand for
EBN has expanded significantly in recent years [3]. Ini-
tially, EBN is consumed only by the Chinese. After some
time, the Dayaks people in Borneo Island, Indonesia have
started to consume EBN. As a result, EBN has become
an essential commodity in the export food trade from In-
donesia to China.
In trade, red blood, yellow, and orange EBNs are pricier
than white EBN [4] because of their positive influence on
the health of a particular community. Different explana-
tions have been suggested; one of which is that red blood
EBN represents swiftlet bleeding diluted with saliva [5].
A previous work [6] reported that the color of EBN is
related to its nitrite concentration.
The risk of carcinogenic nitrosamine formation is one of
the adverse effects of high nitrite content in EBN [7]. In-
creased nitrite absorption was observed in patients with
bladder cancer [8], pancreatic cancer, and gastric cancer
[9]. Therefore, the maximum acceptable levels for this
compound have been proposed. In food trading, China
has set the allowable nitrite content value in EBN to be
less than 30 ppm to enter the country; this number is
lower than the 80 ppm Indonesian National Standard No.
8998:2021 [10].
Most EBNs are commonly produced from Borneo Island
in Indonesia [5], where one swiftlet house could produce
111 kg/year [11]. However, almost no data are available
on the nitrite levels of various EBNs from Borneo swift-
let farmhouses. Therefore, this study aimed to evaluate
and compare the nitrite concentration in white, yellow,
orange, and red blood EBNs of Borneo origin to provide
information to consumers, healthcare professionals, and
food manufacturers. This report may improve the future
trading of EBNs.
Methods
Sample preparation. All samples including white, yellow,
orange, and red blood uncleaned EBNs were collected
from five swiftlet farmhouses in South Borneo (Table 1).
The representative EBNs are depicted in Figure 1. All
feathers in the samples were first removed using tweezers.
In brief, 1 g from each cleaned EBN was gently grinded
on a mortar until fragments were obtained. Afterward, 0.5
mg of each sample was added with a 3 mL of saturated
NaCl (Merck, Germany) solution, followed by aquadest
to reach a final volume of 50 mL [12].
Standard curve. A standard curve was constructed by
diluting the nitrite standard solution (1 ppm) (Merck,
Evaluation of Nitrite Concentration in Edible Bird’s Nest 69
Makara J. Sci. March 2022 Vol. 26 No. 1
Table 1. Sample Collection of Edible Bird Nest from Five Swiftlet Farmhouses in South Borneo
Swiftlet Farmhouses
N
White
Yellow
Orange
Red blood
1
15
4
4
4
3
2
7
4
0
0
3
3
16
4
4
4
4
4
13
4
3
3
3
5
7
4
1
1
1
Total n
58
16
12
12
14
Figure 1. Representative Picture of the EBNs: A. White Nest,
B. Yellow Nest, C. Orange Nest, D. Blood Nest
Germany) with 0.6 mL of saturated NaCl (Merck, Ger-
many) solution, 0.5 mL of sulfanilamide (Merck, Ger-
many) solution, 0.5 mL of naphthyl ethylene diamine
(Merck, Germany) solution, and aquadest in six concen-
tration levels (0, 0.2, 0.3, 0.4, 0.5, and 0.6 µg/L). The
standard solutions were allowed to stand for 15 min and
then placed in a cuvette to determine their absorbance us-
ing Genesys 30 visible spectrophotometer (Thermo Sci-
entific, USA) at 541 nm wavelength [13].
Nitrite extraction and spectrophotometry. All samples
were tested using Elmasonic S 30 H (Elma, Germany) at
40 °C for 30 min with occasionally stirring [14]. The
samples were then removed from the sonicator and
cooled down to room temperature. Sample extracts were
prepared using Whatman filter sheets no. 42 (GE
Healthcare, Germany). Nitrite concentration was meas-
ured in 1 mL of each extract using Genesys 30 visible
spectrophotometer (Thermo Scientific, USA) at 541 nm
wavelength [15].
Data analysis. Differences in nitrate levels among various
farms and EBN colors were statistically analyzed using
ANOVA, followed by Tukey HSD test. All statistical
data were processed using SPSS for Windows 23.0.
Results and Discussion
Nitrite is of great concern because its high content in food
can cause bladder cancer [8], pancreatic cancer, and
gastric cancer [9] in humans. Indonesia is the most
important supplier of EBN and the source of around 80%
of the global nest supply to various countries, including
China [15]. Borneo Island, which belongs to Indonesia
Island, has become the most crucial EBN producer
because of its many nesting sites [5, 16]. For EBN export
trading to the People’s Republic of China (PRC), the
hazardous substance content must be below a certain
level. The PRC government has set a limit of not more
than 30 ppm nitrite. With this regulation, the EBN
industry must naturally reduce the nitrite concentration in
EBNs [17].
Nitrite is normally present in any swiftlet nest or cave and
could be synthesized from ammonia by bacteria through
anaerobic fermentation. Nitrite is created in the nest and
absorbed by the swiftlet nesting habitat, particularly from
the decomposing organic debris on the floor [6]. Bird
dropping fermentation and natural ecological elements,
such as air, water, and soil, have caused the penetration
of nitrite into EBNs [7]. The lack of good farming
practices in swiftlet farmers can be a factor in the high
nitrite concentration of raw uncleaned (RUC) EBNs,
which are collected from caves and farms (swiftlet home)
without any cleaning technique, and raw cleaned (RC)
EBNs, which are cleaned by sorting, soaking, feather and
impurity removal, molding, drying, grading, and
packaging [4]. The variations in nitrite levels may be
attributed to a variety of factors, including differences in
the environmental factors of cave and swiftlet farm, such
as humidity, pH, and climate; age of harvested EBNs
(harvesting time); contamination during harvest; and
cleaning processes for the collected EBNs [16].
Appropriate management of swiftlet housing, such as
regularly removing the swiftlet guano while leaving the
cave guano uncleaned, may contribute to the decrease
nitrite content in house nesting. In addition, the proper
ventilation of swiftlet housing contributes to reducing
bacterial anaerobic fermentation and lowering the nitrite
levels [7].
In this work, 58 EBN samples were collected from five
swiftlet farmhouses (Table 1). The linear regression
equation for nitrite was y = 0.9963x–0.0024, and the linear
correlation coefficient for nitrite was 0.99999. The highest
mean of clean white EBN was 19.9400 ± 4.96674 ppm
(Table 2). This value is still below the maximum limit for
nitrite levels set by the PRC government. The nitrite level
in this study was lower than the previous results of 100
ppm for RC EBNs from Hong Kong market [17] and 93
70 Ningrum, et al.
Makara J. Sci. March 2022 Vol. 26 No. 1
ppm for RUC EBNs from South Borneo, Indonesia [18].
By contrast, the nitrite level in the present work agreed
with the previous values of 7.9–22 ppm for RC EBN
from three houses-EBN in Malaysia [6]. The current data
also showed that white EBN had the lowest nitrite
concentration among the groups (Figure 2), indicating
that all the investigated swiftlet farmhouses have applied
good management practices.
Table 2. Multiple Comparisons of Nitrite Levels of Swiftlet Farmhouses-EBN (White) Derived from Different Sources using
Tukey’s HSD test. N = 4 for Each Group
EBN Samples (I)
EBN Samples (J)
Nitrite Concentration (ppm per g of Sample)
Mean
Mean Difference (I-J)
SD
P Value
SF 1
19.9400
4.96674
SF 2
4.60750
2.56890
0.412
SF 3
4.81500
2.56890
0.371
SF 4
6.82250
2.56890
0.109
SF 5
6.36500
2.56890
0.148
SF 2
15.3325
2.25229
SF 1
−4.60750
2.56890
0.412
SF 3
.20750
2.56890
1.000
SF 4
2.21500
2.56890
0.906
SF 5
1.75750
2.56890
0.957
SF 3
15.1250
4.20100
SF 1
−4.81500
2.56890
0.371
SF 2
−.20750
2.56890
1.000
SF 4
2.00750
2.56890
0.932
SF 5
1.55000
2.56890
0.972
SF 4
13.1175
3.57852
SF 1
−6.82250
2.56890
0.109
SF 2
−2.21500
2.56890
0.906
SF 3
−2.00750
2.56890
0.932
SF 5
−.45750
2.56890
1.000
SF 5
13.5750
2.40772
SF 1
−6.36500
2.56890
0.148
SF 2
−1.75750
2.56890
0.957
SF 3
−1.55000
2.56890
0.972
SF 4
.45750
2.56890
1.000
SF: swiftlet farmhouse; HSD: honestly significant difference
Figure 2. Multiple Comparisons of the Nitrite Levels of Swiftlet Farmhouses-EBN (White, Yellow, Red Blood, and Orange)
Derived from Different Sources using Tukey’s HSD Test
Evaluation of Nitrite Concentration in Edible Bird’s Nest 71
Makara J. Sci. March 2022 Vol. 26 No. 1
Some commercial EBNs generally have a white or
yellowish–white color [19]. EBNs also exhibit a variety
of colors such as bright yellow, red blood, and orange,
which have more expensive market price than those with
white color [20–22]. In the food market, red blood EBN
is the most expensive type with cost ranging from US$
1000 to US$ 15 000 per kilogram [4]. According to
historical records from 1700 (Qing dynasty in China), red
blood EBN provides more health benefits than white
EBN [23]. However, this special EBN has a hidden threat.
The nitrite content in EBNs of various colors was also
examined. Figure 2 shows the nitrite levels from the
lowest to the highest as follows: white, yellow, red, and
orange EBN. Tukey HSD test revealed that the nitrite
content in the yellow, red blood, and orange EBNs was
significantly higher by 30 folds compared with that in
white EBN. Moreover, orange EBN contained the
highest nitrite level for this group. The nitrite level in the
orange EBN group was not significantly different from
that in the yellow and red blood EBN groups.
This study compared nitrite concentrations in EBNs of
different colors obtained from five swiftlet farmhouses.
Results showed that orange EBN had higher nitrite
concentration than yellow and red blood EBNs; however,
the difference was not statistically significant. This work
also found that the yellow, orange, or red blood EBN had
significantly higher nitrite yield than white EBN. This
finding agreed with previous works [6, 7, 17, 20].
Therefore, yellow, orange, or red blood EBN should not
be traded to China because of their high nitrite
concentrations. In 2011, Chinese authorities reported
nitrite contamination in Zhejiang Province, thus raising
public concern about the safety of EBN consumption.
The highest reported nitrite concentration in red blood
EBN has reached 11 000 ppm, which led to the
immediate ban on importing EBNs [23].
The color of cave EBN is associated with its nitrite and
nitrate contents [6]. Several studies have conducted tests
in these types of EBN, particularly red blood EBN.
Another work [22] reported that the Fe ion oxidation in
AMCase-like protein plays remarkably in EBN color
change. The other factor affecting the color change of
EBN is the nitration of tyrosyl residue to the 3-
nitrotyrosyl (3-NTyr) residue in the glycoprotein [21]. 3-
Ntyr acts as an indicator of the color change from yellow
to red in acid to red in alkali occurring at around pH 7.
As a result of nitrite accumulation, EBN changes its color
from white to yellow to orange to red [19]. Thus,
environmental conditions play a significant role in the
dark color formation of EBN. The elevated nitrite levels
on EBN may be due to the contaminating nitrate and
microbial nitrate reductase from the environment.
This study suggested that color is an indicator of the
nitrite level in EBNs. The high nitrite concentrations in
dark EBNs (yellow, orange, and red blood EBN) can be
a hidden threat to human health. In addition, this work
successfully proved that dark EBNs from Indonesia have
high nitrite levels, and not all EBNs are good for human
consumption due to their nitrite levels. Therefore, nitrite
concentration for each batch must be controlled at the
industrial level. The results highlight the concerns over
nitrite concentration in EBNs as a novel food for human
consumption and provide information for future research
in the food industry.
Conclusion
Yellow, orange, and red blood EBNs have higher nitrite
concentrations than white EBN and thus require
additional treatment to reach the quality standards.
Despite the nutrition advantages of these types of EBN,
the toxication risk of their high nitrite content can
threaten public health. Good nitrite control in the EBN
industry may mitigate this risk.
Acknowledgements
The authors would like to thank LPPM UWKS for
providing financial support for this study (grant number
of 19 year 2 Maret 2021).
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