Food Research International xxx (xxxx) xxx

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

A comprehensive review of edible bird’s nest

Yuwei Dai, Jie Cao, Yuye Wang, Yuejuan Chen, Lin Jiang *
School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China

A R T I C L E I N F O A B S T R A C T

Keywords: Edible bird’s nest (EBN) is built by seven species of Aerodramus and Collocalia (Apodidae), using salivary gland
Edible bird’s nest secretion mixed with feathers or grass during the breeding. Its rich nutritional values such as anti-aging activity,
Composition immunomodulatory and antioxidant activity make consumers flock to it. Consumers’ pursuit, on the one hand,
Pharmacological action
aroused the arrogance of counterfeiters, which eventually leads to food safety problems. On the other hand, it
Authenticity identification
Quantitation
promotes the in-depth studies of EBN in all aspects, such as compositions, biological activities, authenticity
identification, quality control, and so on. This paper presented the origins and classifications of EBN and the
current situation of EBN industry in detail; reviewed the nutritional compositions, pharmacological actions,
identification, inspection and content determination of EBN comprehensively; and prospected the future research
directions to provide suggestions for the further study.

1. Introduction
Edible bird’s nest (EBN) is basically produced in Southeast Asian
countries, with the characteristics of rare quantity, delicious taste and
high nutritional value. As early as Tang Dynasty, it has been regarded as
high-grade health food (Jiang, 2016) and a symbol of the status of
ancient dignitaries. In the Yuan Dynasty, Ming Jia described the nature
of EBN in Food Instructions, and people were already eating EBN at that
time (Feng, 2015). According to literature research, the record of the
curative effect of EBN was first seen in Essential of Materia Medica by
Ang Wang of Qing Dynasty, while the most detailed record of its efficacy
was in A Supplement to Compendium of Materia Medica by Xuemin
Zhao (Jiang, 2016). Traditional Chinese medicine believes that EBN is
neutral in nature and sweet, acts on lung, stomach and kidney merid­
ians; and has the properties of moisturizing the lung, resolving phlegm
and stopping coughing. For a long time, Chinese people believes that
eating EBN can promote health and it has become a health habit. So,
with the gradual improvement of living standard, people’s demand for
EBN is increasing. But disappointingly, the market is full of adulterants
and low quality EBN due to the less production and huge profits. The
lack of regulations and standards on EBN quality at home and abroad
makes it difficult to effectively monitor the market; endless illegal means
such as adulteration or dyeing by some greedy distributors make the
market fall into a vicious competition circle; consumers’ lack of common
sense in EBN makes them easy to be misled by unscrupulous busi­
nessman and irresponsible public opinions. Therefore, it is of great
significance to sort out the basic knowledge of EBN, the current situation
of the industry and the latest researches. It can not only popularize the
elementary knowledge of EBN for the consumers, but also provide sci­
entific and technological reference for EBN’s quality control, provide
theoretical basis for the formulation of laws and regulations, and pro­
vide a scientific basis for basic and applied research.
2. The swiftlets that produce EBN and the classifications of EBN
According to the observation and analysis, the growth and repro­
duction of swiftlets need appropriate environmental conditions: hu­
midity about 90%, temperature 28 ~ 30 ℃ and enough food sources (Li,
Zhang, Li, Xiao, Liu, Gu, & Zhang, 2018). Therefore, swiftlets are only
distributed in areas with suitable conditions, such as Indonesia,
Malaysia, Thailand, Vietnam, etc. The main producing countries and
regions of EBN are shown in the Table 1.
Taxonomists try to classify the birds of Apodidae on the basis of their
morphological characters, behaviors, genetic characteristics and other
information. However, no consistent results have been obtained so far.
Swiftlets are small and light with long wingtips and short legs. When
their wings fold, the tips of the wings go beyond the tips of the tails.
Their short and curved toes as well as small and thin limb muscles are
unable to support their body weight (Zuki et al., 2012), so they cannot
stand, just like their family name Apodidae — means “without feet” in
Greek. Swiftlets are fast and nimble and they spend most of their time
flying. Their short beaks with wide gaps and echolocation are

* Corresponding author.
E-mail address: jiangln@mail.sysu.edu.cn (L. Jiang).

https://doi.org/10.1016/j.foodres.2020.109875
Received 4 May 2020; Received in revised form 27 October 2020; Accepted 28 October 2020
Available online 3 November 2020
0963-9969/© 2020 Published by Elsevier Ltd.

Please cite this article as: Yuwei Dai, Food Research International, https://doi.org/10.1016/j.foodres.2020.109875
Y. Dai et al. Food Research International xxx (xxxx) xxx

guarantees for catching aerial insects (Brinkløv et al., 2013). Table 2

Collocaliini Tribe includes 36 species of swiftlets. They are divided Classification of the EBN.
into four genera, namely Aerodramus, Hydrochous, Schoutedenapus and Classification Category Description
Collocalia. Only seven species of swiftlets from Aerodramus and Collo­ basis
calia produce EBN (Jiang, 2016). They are: C. esculenta and C. linchi of Nest Site Cave EBN Picked from caves
Collocalia; A. fuciphagus, A. germani, A. maximus, A. unicolor and House EBN Picked from swiftlet houses
A. francicus of Aerodramus. Except for special instructions, all “swiftlet” Color White EBN The color of EBN is white
mentioned in this paper refers to the swiftlet that can produce EBN. Yellow EBN The color of EBN is yellow
Red EBN The color of EBN is red
Swiftlets reproduce only once in dry season due to food shortage. Red corner Only two corners are red
When the rainy season comes and the food becomes abundant, the EBN
swiftlet groups will enter an active breeding period and reproduce twice. Quality Imperial EBN Most of the nest’s ingredients are edible
Every time before laying eggs, swiftlet couples will build new nests. Feather EBN Most of the nest’s ingredients are feathers
Grass EBN Most of the nest’s ingredients are grasses
Therefore, taking away the used nests not only cause no harm, but also
Shape EBN cup The shape is complete and is half-bowl; be built
provide them a sustainable nesting environment. However, the nests on the edge of the top of a wall
must be picked after the young birds leave. Some greedy farmers take Triangular The shape is triangular; be built at the corner
advantage of this habit and pick away the newly built nests, forcing the EBN between the ceiling and the top of walls
birds to build nests again in a short time. This kind of unrestrained Model EBN Be shaped into different shapes by models
bar-like EBN The complete EBN cup is crushed during
picking behavior affects the quality of EBN, furthermore, it damages the transportation or processing, unable to
health of swiftlets and hinders the sustainable development of EBN in­ maintain the half-bowl shape and becomes
dustry. To protect the ecological environment, some countries of origin strips
have restricted the picking of cave EBN — which is built in caves, but no EBN corners Corners of EBN; the “load-bearing beam” of the
EBN
regulations for the picking of house EBN — which is built in man-made
EBN Small fragments from the crushed EBN, which
structures. fragments contains all parts of the EBN
The complete EBN is half-bowl shaped and translucent, with unique Density Dense EBN The filaments of EBN are evenly distributed and
fragrance and slightly fishy smell. In general, EBN are classified ac­ the gaps are not obvious
cording to various basis, as shown in Table 2. Sparse EBN The filaments of EBN are unevenly distributed
and the gaps are obvious
Impurities dry process Picking out impurities without using water
3. Industry of EBN removal semi-dry Picking out impurities after sprinkling water
process locally
3.1. Processing of EBN wet process Picking up impurities after soaking in water

Before eating, EBN has to undergo a series of processing. Different

solutions. After that, the EBN will be shaped with a half-bowl mold and
types of EBN products need different processing methods. According to
dried with fans in the air-drying room. Shaping and air drying will
the development of technology, processing technology is divided into
sometimes repeat several times to get a good shape. Finally, EBN will be
primary, deep and biotechnological processing, which can remove
sterilized before packaging and leaving the factory. Sterilization is
harmful substances, retain nutrition, increase flavor and control content.
usually achieved by ultraviolet irradiation, ozone or high temperature.
Primary processing is a necessary step of most EBN products. After
Deep processing technology expands the market by developing
harvesting, natural EBN will be divided into different categories ac­
diversified ready-to-eat products and lowering the price. The most
cording to the feather content, shape, size or other indicators. Then, they
common kind is the processing of rock candy ready-to-eat EBN: soak the
need to go through a series of initial treatments. First, the natural EBN
clean EBN in water and pick out a small amount of tiny impurities, sub-
will be washed with water and this step can not only remove impurities
pack the wet EBN in bottles, add rock candy, cover lid and use autoclave
such as swiftlet dropping and dirt, but also remove part of nitrite. Then,
to ripen and sterilize simultaneously. The processing of other deep
small feathers in EBN will be picked out manually. This is the most
processing products like candy, jelly, drink, effervescent tablet, nano-
tedious step and it determines the cleanliness of EBN — which is one of
sized EBN particles and oral liquid is more complex. In addition, some
the determinants of EBN’s price. Due to labor dependence, human error
of them may add Ejiao, ginseng or other components so as to achieve a
and inconsistency, difficulties exist in the cleanliness detection. To
variety of tastes and rich nutrition (Shan, Y. J. & Chen, Z. K, 2019; Liu, Y.
improve the contrast between EBN and impurities, Gwee, Cheng and
S. 2019; Lai et al., 2016).
Yen (2019) studied the structure, angle, wavelength and intensity of the
Biotechnology in the processing of EBN is mainly the application of
light source. The contrast was increased by 36.2% through adjusting to
enzymatic hydrolysis, extraction, separation and other means to obtain
the best illumination condition. This result helps to develop an efficient
some specific nutritional components in EBN. Biological enzyme hy­
impurity segmentation and detection algorithm for future automation
drolysis technology has a broad application prospect. Using ultrasonic or

Table 1
The main producing countries and regions of EBN.
Country Main regions Mainly Types of EBN

Indonesia Kalimantan, Sumatra, Java, Sulawesi Cave EBN、House EBN

Malaysia Sarawak (e.g. Niah cave) and Sabah (e.g. Gomantong cave) Cave EBN
East and west coasts of Malay Peninsula (e.g. Kuala Lumpur) House EBN
Thailand South (e.g. Chumphon, Changwat Songkhla, Krabi) Cave EBN
Central part House EBN
Vietnam Hoi An, Nha Trang, Quy Nhon Cave EBN
The Philippines Palawan island Grass EBN
Burma Tanintharyi House EBN
Combodia Preah Sihanouk, Koh Kong, Kampot House EBN
China Huaiji County, Zhaoqing City, Guangdong Province; Yanzidong, Jianshui County, Honghe Prefecture, Yunnan Province Grass EBN
Dazhou Island, Hainan Cave EBN

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microwave-assisted protease to extract sialic acid (SA) and protein can

increase the extraction rate (Fan et al., 2015; Li et al., 2017). Making the
EBN extract obtained under the optimum technology into freeze-dried
EBN hydrolysis powder can keep the content of protein and saccha­
rides, and reduce the content of fat (Amin, Din, & Hui, 2019). Using
alkaline protease to hydrolyze the insoluble protein in EBN can trans­
form it into a water-soluble polypeptide, which was beneficial to
digestion and absorption of human body (Zheng et al., 2017). Besides,
the EBN extract digested by protease has stronger whitening and oste­
ogenic activity than the EBN water extract (Wong, Chan, Wu, Poon,
et al., 2018), meaning that the active EBN extract obtained by
biotechnology can be used to develop new EBN products, like health
food and skin care. Using combinative enzyme is a good way to get
different active ingredient at the same time and so as to maximize the
use of EBN. Fig. 1. Trend of EBN annual legal imports from 2014 to 2019.
The processing of EBN is tedious and meticulous. Enterprises or in­
dividuals without business ethics may manipulate any step of the pro­ Approving the import of feather EBN is conducive to the Chinese market
cessing to obtain high profits at low cost. Their illegal methods are to strengthen the quality control of EBN, and it is of great significance for
multifarious, especially in the primary processing, some of which are EBN to penetrate the Chinese market.
shown in the Table 3. Deep-processed EBN derivatives face similar The quality control of EBN also needs the restriction and guidance of
problems, such as fake raw materials and false label content. These food laws and quality standards. Recently, China Agriculture Wholesale
safety problems encumber the healthy development of the industry and Market Association claimed that the national standard of EBN drafting
endanger the safety of consumers. under the leadership of China National Center for Food Safety Risk
Assessment had been finalized (Wu, 2020). But just as important is the
establishment of industry standards and enterprise internal standards, as
3.2. Industry actuality well as the government’s supervision of the implementation of
standards.
The increasing demand and high economic value of EBN breed the
greed of illegal traders. In the context of food adulteration and fraud
4. Compositions of EBN
becoming a global problem, EBN market also faces crises in its largest
consumer countries. To protect the health of consumers and property
4.1. Main compositions in EBN
safety, the concerned departments of the state took active actions.
Through joint assessment with Malaysian food safety experts in April
Studying the compositions of EBN is important to understand its
2012, Ministry of Health of the PRC stipulated that the temporary limit
biological activity as a drug or functional food. Its main compositions in
of nitrite in dry EBN should be 30 mg/kg. The raw materials used for
order of content from low to high are fat, ash, carbohydrate and protein.
derivatives shall also be in accordance with the above provisions.
The content of fat is less than 0.5%, indicating that EBN is a low-fat
As of 2019, 59 overseas EBN product processing enterprises regis­
food. The triglyceride of EBN is rich in poly-unsaturated fatty acids
tered in China, including 34 from Malaysia, 23 from Indonesia and 2
(48.43%) dominated by linoleic acid with 47.15%, followed by satu­
from Thailand. China Academy of Inspection and Quarantine (CAIQ)
rated fatty acids (25.35%) represented by palmitic acid with a relative
reported the legal import volumes of dry EBN in China (Development
abundance of 21.33%, then mono-unsaturated fats (24.74%) among
Report of Imported Bird’s Nest in 2019, 2019), as shown in Fig. 1. The
which oleic acid is the preponderant component with 21.97% (Lee et al.,
import and consumption volumes showed a blowout. It was noteworthy
2019).
that these data did not include a large number of EBN that entered the
The ash content of clean EBN is about 5%, while that of feather EBN
country through informal channels. On August 16, 2018, General
is a little higher. The difference between EBN with different cleanliness
Administration of Customs approved Malaysian feather EBN — meeting
is due to the presence of feathers and other foreign matters. After
relevant requirements — to enter China. But the feather EBN is only used
analyzing 18 kinds of mineral elements in EBN, people found that
for deep processing and not for retail market. On September 24, 2019,
macroelement, essential microelement and heavy metals can be detec­
the General Administration of Customs announced that the first
ted. The contents of Na, Mg, K and Ca were generally high (You et al.,
Malaysian feather EBN processing enterprise was registered in China
2012; Quek et al., 2018a). But the contents of essential microelement
and would trade with China on November 20. On the same day of
such as Fe, Cu and heavy metals such as Hg and As varied in samples,
trading, the first import ceremony of feather EBN was held in Guangxi
which may be related to swiftlets’ foraging environment and nesting
China-Malaysia Industrial Park, marking that Chinese factories can carry
environment (Zha et al., 2011; Mei et al., 2020).
out legal processing of feather EBN on their own land henceforth.
Carbohydrate content is next to protein content, including sialic acid
(SA), mannose (Man), glucosamine (GlcN), galactosamine (GalN),
Table 3 galactose (Gal) and fucose (Fuc) (You et al., 2012). SA is the most
The Illegal Processing of EBN and the Purposes. outstanding component in EBN, with the content of about 10%. It is also
Illegal treatments Purposes known as “EBN acid” because it has the highest content in EBN
Use chemicals to remove swiftlet feathers To reduce labor costs compared to other natural products. SA refers to a series of carboxylated
Bleach (hydrogen peroxide, sulfur dioxide, To beautify the appearance and monosaccharide acylated derivatives containing 9 carbon atoms, which
sulfur trioxide, etc.) make EBN look bright and thick often exists in the form of oligosaccharides, glycolipids or glycoproteins.
Spray water To increase the weight
According to the different connecting groups on the number 5 carbon,
Add preservatives To prevent the corruption
Adulterate (agar, Tremella, pigskin, fish gum To reduce costs SA can be divided into four groups: N-acetylneuraminic acid (Neu5Ac or
powder, eggs, gum, starch processed NANA), N-hydroxyacetylneuraminic acid (Neu5Gc), deamination neu­
products and low-quality EBN, etc.) raminic acid (KDN) and neuraminic acid (Neu). The first two are the
Dye red (fumigation with bird droppings or To forge red EBN chief forms (Liu et al., 2010). SA in EBN refers specifically to Neu5Ac.
nitrites, etc.)
The protein content is above 50%, which is the highest composition

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Y. Dai et al.
in EBN. But EBN may be not the good source of high-quality protein.
Because EBN cannot improve the malnutrition of rats caused by
consuming protein with limiting factors, while a small amount of whey
protein or flaxseed powder can. Besides, the digestion speed of EBN
protein is not as fast as that of boiled chicken protein (Wang, 1921).
Peptides are one of the most important components in EBN. Different
extraction methods will affect the kinds and biological activities of
peptides (Gao, Yao, & Zhang, 2019). Researches on amino acids showed
that its kinds were the same and its proportion was different (Lin, Zhou,
& Wang, 2013; Chen, Bao, Huang, & Xu, 1997; Hou et al., 2007; Cao
et al., 2011; Noor, Babji, & Lim, 2018). This may be related to the
experimental equipment and technology.
A recent study analyzed 22 kinds of amino acids in 10 kinds of EBN.
A total of 20 amino acids were detected, including 8 essential amino
acids (Shangguan et al., 2018). Hydroxyproline (Hyp) and sarcosine
(Sar) were not detected, indicating that EBN had no collagen. The other
20 amino acids were asparticacid (Asp), glutamicacid (Glu), serine (Ser),
histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine
(Ala), tyrosine (Tyr), cysteine (Cys), valine (Val), methionine (Met),
phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys) Proline
(Pro), asparagine (Asn), glutamine (Gln) and tryptophan (Trp). It was
noteworthy that Trp, Cys, Asn and Gln have not been detected in some
studies, which may be because Trp and Cys were completely hydrolyzed
in the acid hydrolysis, while Asn and Gln were deamidated into aspartic
acid and glutamine. The total essential amino acid found in EBN samples
(17.8 g/100 g) was appreciably greater than in other protein-rich foods
such as egg (4.7–7.0 g/100 g) and milk (1.1 g/100 g), meaning that EBN
was a potential source for essential amino acid (Quek et al., 2018a). EBN
also contains water and the water content of EBN in different regions has
no obvious difference. But to protect the integrity, the water content of
EBN in transportation is usually higher than that of commercial EBN
(Lin, Zhou, & Wang, 2013). High water content is good for bacterial
growth and bad for preservation, while low water content makes EBN
dry and fragile and inconvenient for transportation. According to the
standards of Malaysia, Indonesia and Thailand, the water content of
commercial EBN should be controlled below 15% (Chen, Yang, & Lin,
2015).
It is worth noting that EBN contains sensitizing substances, which
often cause consumer concerns. A clinical trial conducted by National
University of Singapore revealed the presence of IgE-mediated in EBN,
which cause the anaphylaxis in children (Goh et al, 2001). This allergen
is a 66kD protein, also found in eggs. The sensitization of EBN from
different geographic location are different. EBN also contains trace
hormone components, including testosterone, estradiol, progesterone,
luteinizing hormone, follicle stimulating hormone and prolactin (Jiang,
2016).
4.2. Compositions in different EBN
The compositions of natural EBN is influenced by many factors.
Scholars studied the differences of compositions in different kinds of
EBN, among which the dissimilarities in EBN from various geographical
location are shown in the Table 4.
We can see the contradictions in the table, and similar situation is
also found in the compositional study of EBN with different colors (Zha
et al., 2011; You et al., 2012; Cao et al., 2011). By analyzing the samples
used in the experiment, we can know that the lack of consistency may be
attributable to the limited number of samples and that some experi­
menters did not strictly control the single variable of the target factors
when selecting the samples.
The species and nesting areas of swiftlets also affect the compositions
of EBN. The total amino acids in A. fuciphagus EBN was found to be 23%
higher than in A. maximus EBN. And EBN from swiftlet houses,
A. fuciphagus and Peninsular Malaysia had higher SA while those from
cave, A. maximus and East Malaysia contained more minerals like Ca and
Mg (Quek et al., 2018a). Besides, the difference of Tyr and Glu content
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Food Research International xxx (xxxx) xxx
Table 4
Compositional dissimilarities in EBN of different origins.
Compositions Differences
Proteins Thailand > Indonesia (Zha et al., 2011)
Indonesia > Malaysia (You et al., 2012)
No significant difference between Peninsular Malaysia and East
Malaysia (Quek et al., 2018a)
No significant difference among Indonesia, Malaysia, Thailand and
the Philippines (Hamzah et al., 2013)
SA Indonesia > Thailand (Zha et al., 2011)
Indonesia > Malaysia (You et al., 2012)
No significant difference among Indonesia, Malaysia and Thailand (
Lu et al., 2016)
Carbohydrate Philippines > Malaysia, Indonesia and Thailand (Hamzah et al.,
2013)
Amino acid No significant difference in the total amount of amino acids in
different geographical locations (Quek et al., 2018a; Hou et al.,
2007)
The proportion of each amino acid was different (Shangguan et al.,
2018)
was suggested to distinguish house and cave EBN (Shangguan et al.,
2018; Seow et al., 2016).
Although the compositional study has been carried out since the
beginning of last century, no systematic and comprehensive result
comes out up to now. Researches on the influences from swiftlets’ spe­
cies, food resources and nesting seasons are also limited. So, we suggest
to establish composition distribution maps of EBN in different
geographical locations in the future.
4.3. Composition changes in processed EBN
Processing partially changes the compositions in EBN. Feather EBN
(unprocessed EBN) seems more nutritional and beneficial compared to
the commercialized EBN (processed EBN). Because after soaking,
removing impurities and sterilizing, although the retention rate of SA in
EBN was higher than 95% (Lian, Fan, & Li, 2017), the protein content
decreased from 55% to 46.6% (Zulkifli et al., 2019). The stewing process
does not destroy the main nutrients in EBN. After stewing, the content of
Cys and Na, K, Zn decreased, but the content of the main nutrients like
SA, protein and total sugar hardly changed (Jian et al., 2016).
5. Pharmacological action of EBN
Traditional Chinese medicine believes that EBN has the functions of
moistening lung, nourishing stomach, relieving liver, clearing eyes and
tonifying heart. Eating EBN is a kind of health culture in China and
recorded in prescriptions of medicine, dietotherapy and folk experience
in medical books of past dynasties. It has not been listed in the drug and
food homology catalogue of the Ministry of health, nor in the Pharma­
copoeia of the people’s Republic of China. But it can be seen in the local
Chinese medicine standards of many provinces (autonomous regions),
such as Jiangsu, Guangxi, Yunnan, Shandong, etc. Modern medicine
researches usually concentrate on component research, authenticity
identification, component determination, pharmacological action
research and traceability research. In recent years, a large number of
studies on the pharmacological action and mechanism of EBN emerged,
which mainly focused on the antiviral effect, immunoregulation,
learning and memory enhancement, neurodegenerative disease
improvement and antioxidant effect. Studies on the mechanism of action
showed that the biological action of EBN is mainly due to the presence of
SA. And many reviews expounded the research progress and biological
activity of SA, including its preparation, separation, purification,
detection methods, biological activity and application status (Liu et al.,
2010; Wei et al., 2011; Chen et al., 2014).
Y. Dai et al.
5.1. Anti influenza virus and inhibitory hemagglutination
SA, which is at the terminal of glycoprotein, can inhibit some
important antigen sites and recognition markers on the cell surface
effectively, thus protecting these glycoproteins from being recognized
and degraded by the surrounding immune system. It is also the receptor
of influenza virus and the binding sites of influenza virus in mucous
cells.
After trypsin hydrolysis, EBN can neutralize the influenza virus
infecting MCDK cells, inhibit the coagulation of red blood cells (Guo
et al., 2006), improve the peripheral blood routine and body weight of
aplastic anemia (AA) mice, and raise the survival rate of AA mice
infected with influenza virus (Guo et al., 2017). The antiviral activity of
EBN from different origins is not the same, it is positively related to the
content of acetylated SA in EBN (Haghani et al., 2016). The antiviral
activities of cave EBN and house EBN are also different, and further
experiments are needed to prove the reasons. Interestingly, Guo and
colleagues (2006) showed that the enzyme hydrolysate of EBN could not
inhibit the sialidase activity of influenza virus, while Haghani and col­
leagues (2016) showed that the enzyme hydrolysate of EBN had the
same high sialidase inhibition as oseltamivir phosphate in vitro and in
vivo. The contradiction may be attributed to the difference between the
two methods of dealing with EBN. The designed derivatives of SA, 4-
amino- and 4-guanidino-2-deoxy-2,3-di-dehydro-D-N-acetylneuraminic
acid, have been reported to have the most effective anti-influenza A
and B activities in vitro and in vivo (Von Itzstein et al., 1993). This was a
successful case of rational, computer-assisted drug design, which pro­
vides useful clues for the development of new anti influenza drugs.
5.2. Immunoregulation
Cell research showed that EBN could not directly promote the
lymphocyte transformation of rat mesenteric lymph nodes, but could
promote the lymphocyte transformation induced by low concentration
Con A. Yellow EBN, red EBN, Huaiji EBN, unprocessed EBN and ready-
to-eat EBN all showed similar properties, among which the unprocessed
EBN had the strongest effect. Enzymatic hydrolysis of EBN with trypsin
or pepsin could enhance the promoting effect (Hou et al., 2010). Animal
experiments showed that feeding pearl-EBN extract to mice could pro­
mote T-lymphocyte transformation and increase the content of IgM in
serum (Zhang, Wang, & Wang, 1994). Zhao and colleagues (2016) used
cyclophosphamide to establish immunosuppressive mice model and
found that EBN can promote the proliferation and activation of B cells,
and enhance the secretion of B cell antibodies, thereby reducing the
intestinal immune damage of model mice. Cao and colleagues (2012)
used hydrocortisone to establish immunosuppressed model mice, and
found that EBN can significantly improve the spleen index and thymus
index, improve the content of serum hemolysin and the level of delayed
allergy, promote the phagocytosis rate and phagocytosis index of peri­
toneal macrophages, thus significantly improved the humoral immune,
cellular immune and nonspecific immune of the model mice, and
regulated the immune function of the body. SA, as the pivotal compo­
sition of EBN, plays a indispensable role in maintaining the fetal-
maternal immune homeostasis during pregnancy: it can protect the
fetus against attack by the maternal innate immune system (Abeln et al.,
2019).
5.3. Improving intelligence and memory
SA is an important nutrient for brain development in infants. It is a
component of brain gangliosides and of polysialic acid, which is linked
to oligosaccharide chains of nerve cell adhesion factor (Jozsef & Gene­
vieve, 1997). Brain gangliosides and polysialylation nerve cell adhesion
factors play important roles in ensuring synaptic connections, memory
formation and neuronal growth. SA is synthesized from the liver. Infants
are not able to synthesize enough SA by themselves due to that their
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Food Research International xxx (xxxx) xxx
livers are not well developed (Duncan et al., 2009). So, they have to get
SA from their mother. After delivery, the levels of SA in mothers tend to
decrease with the passage of time (Wei et al., 2011). Therefore, the
continuous intake of SA by mothers during pregnancy and lactation can
provide sufficient SA for the child and help to maintain their own SA
levels.
Newborn mice with inadequate SA intake during lactation performed
worse on memory tests than those with normal SA intake significantly
(Oliveros et al., 2018). By oral administration of EBN to female mice
during the pregnancy or lactation period, their offspring can improve
the learning and memory functions. The possible mechanism is to in­
crease the activities of superoxide dismutase and choline acetyl­
transferase, as well as decrease the levels of malondialdehyde and
activities of acetylcholinesterase (Xie et al., 2018).
5.4. Improvement of neurodegenerative diseases
Neurodegenerative diseases (such as Alzheimer’s disease (AD) and
Parkinson’s disease (PD)) are caused by the loss of neurons and / or their
myelin sheath, which will worsen over time and lead to dysfunction.
Oxidative stress, inflammation, mitochondrial dysfunction and produc­
tion of excitatory toxins are the common causes.
EBN can significantly enhance memory and play a neuroprotective
role by inhibiting the process of neuroinflammation and oxidative stress
(Careena et al., 2018), suggesting that EBN may be a viable nutraceu­
tical option to improve neurodegenerative diseases. EBN might confer
neuroprotective effect against 6-OHDA-induced degeneration of dopa­
minergic neurons, particularly through inhibition of apoptosis. A total of
29 proteins identified from its extract had potential roles in immunity,
extracellular matrix formation, neurodevelopment, cell survival and
apoptosis, cell proliferation and migration, antioxidation and common
cell processes (Yew et al., 2014). In animal experiments, EBN extract
was shown to have neurotrophic properties by promoting proliferation
and migration of the neural stem cells model and embryonic mouse
neuroectodermal cells, and significantly improved locomotor activity of
PD mice in terms of travel distance and balancing through the
enhancement of antioxidant enzyme activity and the inhibition of
microglia activation, nitric oxide formation and lipid peroxidation (Yew
et al., 2018; Yew et al., 2019).
Menopause causes cognitive and memory dysfunction due to
impaired neuronal plasticity in the hippocampus. EBN and estrogen can
enhance spatial learning and memory and increased serum estrogen and
hippocampal SIRT1 expression in ovariectomized rats. And EBN did not
show as much toxicity to the liver as estrogen (Hou et al., 2017). EBN
was neuroprotective against estrogen deficiency-induced damage,
which was evidenced by the decreased serum AGEs, reduced hippo­
campal and frontal cortical caspase 3 protein and malondialdehyde
levels, and balanced activities of anti-oxidant enzymes in the hippo­
campus and frontal cortex of ovariectomized female rats. These findings
suggest that EBN may serve as an attractive candidate and novel strategy
for clinical treatment of neurodegenerative diseases in menopause (Hou,
Imam, Ismail, Ismail, et al., 2015).
5.5. Promoting cell division
EBN can promote cell proliferation. Low concentration of EBN syn­
ergistically induced corneal cell proliferation, suggesting that EBN
extract is expected to be used in the preparation of eye drops for the
treatment of corneal injury (Zainal Abidin et al., 2011). EBN extract
could also strongly promote the proliferation of human adipose derived
stem cells, which can be used for stem cell therapy. This proliferation
promoting effect was achieved by activating the production of
interleukin-6 and vascular endothelial growth factor (VEGF) induced by
activator protein-1 and nuclear factor-κβ (Roh et al., 2011). In addition,
EBN could improve fertility and embryo implantation rate by promoting
the proliferation and differentiation of uterine structure, which was
Y. Dai et al.
proved by the increased expressions of steroid receptor, epidermal
growth factor (EGF), EGF receptor, VEGF and proliferating cell nulear
antigen in uterus (Albishtue, Yimer, Zakaria, Haron, Babji, Abubakar, &
Almhanawi, 2019).
5.6. Anti-oxidant, anti-inflammatory and anti-aging effects
Aging is inevitable, but it can be delayed. EBN can inhibit the
expression of matrix metalloproteinase-1 via down-regulation of the
extracellular signal-regulated protein kinase / c-Jun N-terminal kinases
and transcription factor activator protein-1 pathway, thus showing the
anti-aging properties (Kim et al., 2012). EBN, especially the components
with molecular weight less than 3 kDa, can increase antioxidant enzyme
activities and decrease content of lipid peroxidation products in
drosophila melanogaster, and then delay the senescence of Drosophila
(Ghassem et al., 2017). Through the compatibility of pearl powder, the
EBN mixture significantly reduced the lipid peroxidation in brain tissue
of mice and improved the level of superoxide dismutase in red blood
cells of rats, and thereby delay aging (Zhang, Wang, & Wang, 1994).
The triglycerides of EBN are rich in unsaturated fatty acids, which
have an important contribution to its antioxidant effect (Lee et al.,
2019). EBN could increase the antioxidant and total antioxidant ca­
pacities and reduced the oxidative stress levels in non-pregnant rats
(Albishtue et al., 2018). Also, it is capable of protecting and preventing
alterations in the reproductive system histomorphology and function
due to Lead acetate toxicity through an integrated mechanism of
maintaining antioxidant-reactive oxygen species balance and regulation
of related genes (Albishtue, Yimer, Zakaria, Haron, Babji, Abubakar,
Baiee, et al., 2019). EBN can inhibit the formation of tumor necrosis
factor-α and nitric oxide (Vimala et al., 2012), and also partly attenuated
high fat diet - induced oxidative stress and inflammation through tran­
scriptional regulation of hepatic antioxidant and inflammation-related
genes, better than Simvastatin (Zhang, Imam, Ismail, Hou, et al., 2015).
5.7. Improving bone strength, increase dermal thickness and promote
chondrocyte regeneration
The bone strength and dermis thickness of ovariectomized rats were
increased by feeding them with appropriate concentration of EBN, while
the serum estradiol concentration was not affected. So, EBN may not
increase the risk of breast cancer and may be used to prevent osteopo­
rosis in postmenopausal women (Matsukawa et al., 2011). Cell culture
experiments show that EBN can also effectively control the deterioration
of arthritis and promote the regeneration of chondrocytes, suggesting
that EBN may be used for the treatment of osteoarthritis (Chua et al.,
2013).
5.8. Improve cardiovascular diseases
EBN had a high ratio of unsaturated fatty acids to saturated fatty
acids, which was conducive to reducing serum cholesterol and athero­
sclerosis and preventing heart disease (Lee et al., 2019).
The drying methods and types of protease had effects on the bio­
logical activity of EBN hydrolysates: Compared with spray dried EBN
hydrolysates, freeze-dried EBN hydrolysates had better antihypertensive
activity; and compared with alkaline protease, trypsin and papain,
bromelain hydrolysates had the highest antihypertensive activity
(Ramachandran, Babji, & Sani, 2018).
EBN could attenuate high fat diet-induced hypercholesterolemia and
coagulation and prevent the worsening of metabolic indices and tran­
scriptional changes in insulin signaling genes. The mechanism of the
former is similar to that of simvastatin, partly through transcriptional
regulation of coagulation-related genes (Zhang, Imam, Ismail, Hou,
et al., 2015). But simvastatin could promote insulin resistance, with
similar mechanism to high fat diet, while EBN could prevent insulin
resistance, meaning that it may be used as functional food to prevent
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insulin resistance (Zhang, Imam, Ismail, Ooi, et al., 2015).
Ovariectomy worsened metabolic indices and disrupted the normal
transcriptional pattern of hepatic insulin signaling genes. EBN improved
the metabolic indices and also produced transcriptional changes in he­
patic insulin signaling genes. These changes tended to enhance insulin
sensitivity and glucose and lipid homeostasis, even better than estrogen.
So, EBN could meliorate estrogen deficiency-associated increase in risk
of cardiometabolic disease in rats (Hou, Imam, Ismail, Ooi, Ideris, &
Mahmud, 2015).
5.9. Other pharmacological effects
The active proteins in EBN can retain its original biochemical activity
after being irradiated by gamma rays with the intensity of up to 2.5
million rad, which could be speculated that EBN has nourishing effect on
patients undergoing X-ray examination and radiation therapy (Chen,
1996). And the antiradical activity of EBN may be due to its high content
of unsaturated fatty acids (Lee et al., 2019).
EBN contains some intermediate and high molecular weight proteins
with anti-digestibility. Its anti-digestibility mechanism is still unclear,
which may be related to sugar chains bound to the end of its peptide
chain. Sugar chains can modify the function of protein by affecting the
whole conformation of protein, such as protein folding, intracellular
localization, antigenicity, cell–cell adhesion and pathogen binding (Tian
& Lu, 2002). The active proteins of EBN in the early studies could avoid
digestion and degradation of gastrointestinal tract and act on intestinal
epithelial cells directly. But the mechanisms of absorption, metabolism
and action of these peptide molecules and macromolecular proteins in
vivo remains to be studied in depth (Xian et al., 2010). Another study
found that EBN can regulate the intestinal flora of normal mice by
supporting the intestinal beneficial bacteria and inhibiting harmful
bacteria. But similarly, the relationship between the regulating effect of
EBN on intestinal microflora and its immune promoting effect needs
further study (Zhao et al., 2014).
6. Identification of EBN
6.1. Morphological identification
A piece of complete EBN is an irregular half-bowl with neat edge. It is
translucent, silvery white, soft and elastic after soaking with water. The
surface color of EBN includes white-like or yellow-white, light red, red,
red-brown, dark red, etc. Its inner side is rough and sunken into a pit; the
bottom and both sides are in the shape of a loofah and even; the outer
side is arched with slightly transverse stripes; the middle part is often
mixed with feathers and colored substances. The dry EBN is hard and
crisp, with a unique fragrance and a slight fishy smell. After the
authentic EBN is stewed, it will have the fragrance of egg white, tasting
delicate, smooth and elastic. In addition, the swelling rate of EBN after
immersion is also the basis for identification (Wang et al., 2013; Ren,
Wang, & Li, 2009; Lai & Lin, 2014).
There are some differences between counterfeit and authentic
products in character. Soak the fake EBN in water for a period of time
and then pull it by hand, it can be found that its elasticity is poor and it
will break as soon as it is pulled. By rubbing the fake EBN with fingers,
you can find that it has no elasticity and can be rubbed into paste. The
expansion rate of counterfeits is very low and it has fishy odor, gelatin
odor, fried odor, chemical odor and so on (Zhang, Lai, & Yang, 2013).
But with the increasing complexity of EBN products on the market, it is
difficult to achieve the purpose of identifying the authenticity of EBN by
sensory organs.
6.2. Physical and chemical identification
The physical and chemical identification methods, such as fluores­
cence, burning, color rendering, precipitation and bubble method, are
Y. Dai et al.
suitable for non-processed EBN (Lai & Lin, 2014). The authentic EBN has
different fluorescence reaction with the counterfeit EBN of different
ingredients under 365 nm ultraviolet lamp. The authentic EBN is yellow-
green, the pigskin is yellow, the Tremella is light blue, and the swim
bladder is dark white (Li, 1996; Liu & Zhang, 1995). When the authentic
EBN is burned, it will burst slightly and foam, with slight smoke and
slight stench of scorch. After burning, the ash is white and can be dis­
solved in dilute hydrochloric acid. When the adulterated EBN is burned,
it will produce intense noise and sparks, and it will not foam. It will
produce much smoke, and the ash is insoluble in dilute hydrochloric
acid (Ma, 1992).
Some specific chemical reagents can identify the authenticity of EBN
to a certain extent, but the specificity and accuracy are insufficient.
When the authentic EBN is boiled with dilute hydrochloric acid, it will
be brown yellow or brown black, with a large number of bubbles, and
the EBN will be dispersed into fragments. But the adulterated EBN
samples treated with the same method will not produce a large number
of bubbles (Ma, 1992). Ninhydrin, a protein identification reagent, can
make EBN blue to distinguish it from non-protein counterfeits such as
agar and starch, but not from pigskin and gelatin containing protein
(Liu, Zhao, & Chen, 2012). Indole quinone reagent can make EBN red-
brown to be distinguished from the pigskin with different amino acids
(Jiang, 2000). Potassium dichromate-dilute hydrochloric acid reagent or
potassium dichromate-tannic acid reagent could precipitate gelatin
protein, but did not react with protein in EBN (Xiong & Zhang, 2013).
The authentic EBN will be dyed yellow by iodine test solution, while the
fake products containing starch will be dyed blue-purple (Jiang, 2000).
Bromothymol can make EBN blue, and copper tartrate can make EBN
produce red precipitation. Both of them react with the polysaccharide
chain in EBN. This reaction can distinguish pigskin, swim bladder and
other non-polysaccharide counterfeits, but it cannot distinguish agar,
seaweed extract and other counterfeits with polysaccharide (Liu, Zhao,
& Chen, 2012). The xanthoprotein reaction can be produced by adding
nitric acid to EBN. So, the color of undoped EBN treated with nitric acid
is darker than that of doped EBN (Marcone, 2005).
Morphological identification and physicochemical identification
technology are a summary of the wisdom and experience of predecessors
in the identification technology. In the case of limited experimental
detection conditions, the identification methods of morphological
characteristics and physicochemical characteristics of EBN are basic
technologies that must be mastered. However, these two methods have
some limitations and cannot accurately identify adulterants.
6.3. Microscopic identification
In the microscopic identification, EBN is mainly identified by the
microscopic characteristics of feathers and the powder microscopic
characteristics of adulterated ingredients such as pig skin, Tremella and
agar. The surface and cross-section of authentic EBN have dense parallel
texture and fine villus, which are the main basis for distinguishing it
from counterfeits. Microscopic identification can be divided into stere­
oscopy and microscopy.
Under stereoscope, the main differences between authentic and
adulterated EBN lie in transparency, surface texture and the presence or
absence of feathers. The authentic EBN is arranged in regular layers
under the stereoscope, with dense parallel texture, stereoscopic and
clear. The layers of adulterated EBN are not clear and its reflection is
slightly strong. There are irregular granules in it, some mixed with gray
and black impurities. Comparing the authentic EBN fragments with
Tremella fragments, it can be found that the former were translucent
with even micro-cracks, while the latter were opaque without cracks
(Lin, Zhou, & Lai, 2006).
Under high power microscopy, the authentic EBN has rectangular,
triangular or irregular crystal blocks. The crystal blocks are translucent
with smooth edge and luster. The surface and cross-section of authentic
EBN have dense textures, which are mostly straight or slightly curved,
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rarely spindle-shaped, and some are radial or arc-shaped. Some blocks
can be seen with crossed transverse stripes and occasionally untextured
patches. Some others have gray, brown or gray-black feathers, which are
natural. The inherent structure of the feathers would not change under
any physical form (Wang & Teng, 1991). Comparing the powders of
EBN, pigskin, Tremella and agar by microscope, significant differences
could be found (Liu and Zhang, 1995). In addition to traditional optical
microscopy, scanning electron microscope (SEM) was also used to study
the identification method of EBN and the results of SEM showed that the
authentic EBN were arranged in regular layers, while the fake products
of carrageenan and pigskin were irregular granular (Liu & Guo, 1996).
EBN is a secretion of salivary gland and does not have the charac­
teristics of tissue or cell, so the reliability of microscopic identification is
not high. And the pretreatment of this method is time-consuming and
laborious. Therefore, microscopic method is often used as an auxiliary
method for rapid detection.
6.4. Chemical instrument identification
6.4.1. Spectral identification
6.4.1.1. Ultraviolet spectroscopy. The main component of EBN is pro­
tein, which has an absorption peak in the ultraviolet region near 280 nm.
Therefore, the authentic and counterfeit EBN can be identified by
detecting the maximum absorption wavelength of ultraviolet. However,
this method is not specific and it is susceptible to residual nucleic acid
and other small molecular impurities in the sample, so it has limitations
in the application of EBN identification.
SA, as a characteristic functional component of EBN, has been used in
authenticity identification. Hydrolyzing EBN by acid to free SA, which
reacted with ninhydrin to form yellow complex for quantitative deter­
mination (Huang et al., 2003). EBN sample has the maximum absorption
under the ultraviolet light of 470 nm, while the counterfeit products
such as pigskin, agar, Tremella, gum and swim bladder do not (Huang,
2007)
6.4.1.2. Infrared spectrometry. The difference of infrared spectra be­
tween authentic EBN and adulterated EBN is mainly reflected in the
absorption peaks of the main nutrients such as protein, amino acid and
polysaccharide. Sun et al. (2001) determined the spectra of five natural
EBN and one commercial EBN directly by Fourier Transform Infrared
spectroscopy (FTIR) with micro-diamond attenuated total reflectance
probe for the first time. They found that different natural species of EBN
had their own characteristic infrared spectra. The authenticity of EBN
could be identified according to the differences of wave numbers, po­
sitions and relative peak intensity of absorption peaks. Deng et al.
(2006) obtained the infrared spectra of 30 kinds of EBN samples by
FTIR. These spectra are different from those of pure EBN in different
degrees, mainly in the absorption peaks of protein, amino acid and
polysaccharide. Guo et al. (2018) and Zhang (2015) analyzed the FTIR
spectrum data of EBN by using the chemometrics methods such as
principal component analysis, linear discriminant analysis and support
vector machine. The results showed that the linear discriminant analysis
and support vector machine models had good classification performance
and could find the differences between genuine and counterfeit products
effectively, which were suitable for rapid identification of adulteration
mode, adulteration proportion and origin and rapid detection of prin­
cipal components.
Adenan and colleagues (2020) innovatively used Mid-Infrared-
Attenuated Total Reflectance (MIR-ATR) spectroscopy combined with
chemometric analysis by Data-Driven Soft Independent Modelling of
Class Analogy (DD-SIMCA) to screen Malaysian EBN for structural
adulterants (namely agar, Karaya gum, bovine gelatin, fish skin, mel­
amine and porcine gelatine) and for confirmation of geographical origin.
The results showed that the sensitivity and specifity values for DD-
Y. Dai et al.
SIMCA models applied to origin determination ranged from 90 to 100%
for production regions from Kelantan, Perak and Malacca. And MIR-ATR
spectroscopy in combination with DD-SIMCA modelling had shown
potential as a rapid screening technique to detect the presence adul­
terants and confirm the provenance of EBN produced in Malaysia.
FTIR is a fast, reliable and nondestructive method for the analysis of
EBN’s quality. It can be applied to the rapid identification of EBN,
especially for the quality supervision department, entry-exit inspection
and quarantine and customs administration. However, FTIR spectros­
copy needs to collect a large amount of data to establish relevant
mathematical models and databases. This detection technology has not
been popularized.
6.4.1.3. Raman spectroscopy. Raman spectroscopy is a molecular
structure characterization technique based on Raman effect, which is
rapid, sensitive, accurate, nondestructive and can be used for qualitative
analysis and identification of unknown chemicals.
Yu (2015) used Raman spectroscopy to determine the authentic EBN
in different regions and brands and obtained six characteristic peaks:
828 cm− 1, 1000 cm− 1, 1237 cm− 1, 1330 cm− 1, 1448 cm− 1 and 1668
cm− 1. The results showed that the characteristic peaks were not affected
by regional and species differences, and the adulterated samples with
5% adulterated amount can be identified.
6.4.2. Chromatographic identification
6.4.2.1. High-performance liquid chromatography (HPLC). The identifi­
cation of EBN by chromatography is mainly through qualitative or
quantitative detection of carbohydrate and amino acid.
Under acidic condition, SA can react with o-phenylenediamine to
form derivatives with strong ultraviolet absorption. By using HPLC to
determine SA in EBN, we can get the spectrum with high characteristic
and high consistency, which can distinguish the authentic EBN, coun­
terfeit EBN and adulterated EBN accurately (Hua, Yang, & Lin, 2010; Lu
et al., 2016).
Lin and colleagues (2013) studied by HPLC and found that the total
amino acid content of genuine Indonesian EBN ranged from 46% to
50%. The total amino acid content of fake EBN containing animal pro­
tein (e.g. pigskin) was higher than that of authentic EBN, while the total
amino acid content of EBN containing non-animal protein (e.g. Tremella)
was much lower than that of authentic EBN. Therefore, the authenticity
of EBN can be distinguished according to the total content of amino
acids. Zhang (2013) used High Performance Thin-Layer Chromatog­
raphy and HPLC to determine the contents of taurine, vitamin B and
biotin in EBN, which served as a reference standard for the determina­
tion of authenticity.
6.4.2.2. Gas chromatography (GC). Chen (1995)used capillary column
GC (CGC) to determine the amino acid composition of EBN and adul­
terants (such as swim bladder, pigskin, Tremella, agar, etc.), and proved
a possible method for EBN identification according to the total amount
and composition of amino acid and the ratio of Val to Ala. Wang, Wang
and Ni (2006) used CGC to find that genuine products have different
monosaccharide chromatographic peaks from pigskin, gelatin and other
counterfeits. Zhao et al. (2015) used GC to determine and identify four
aldoses of EBN imported from Southeast Asia, and found that the GC
spectra of authentic EBN were different from that of counterfeit EBN
significantly. Yu et al. (1998) established a method for the determina­
tion of oligosaccharide chain of SA glycoprotein in EBN and its products
by GC, which can be used for qualitative identification and quantitative
determination at the same time.
6.4.3. Identification by other instruments
Hou et al. (2016) determined 14 inorganic elements in EBN by
Inductively Coupled Plasma Mass (ICP-MS) spectrometry, and made
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principal component analysis and stepwise discriminant analysis by SAS
9.1. After removing five element variables and retaining nine element
variables, they established a discriminant function to discriminate the
adulterated species of counterfeit EBN. The discriminant results were
consistent with the actual adulterated species. The results showed that it
was feasible to identify the adulterated species of EBN by stepwise
discriminant analysis, which can provide a theoretical basis for the
detection of EBN adulteration. Yu (2015) found that HPLC coupled with
Quadrupole Time-of-Flight Mass spectrometry technology was also
applicable to determine the adulteration degree of EBN.
6.5. Molecular biological identification
In recent years, molecular biology technology has a special advan­
tage in the identification of biological species. It can systematically
identify biological species from kingdoms to species, even at subspecies
level. Real-time fluorescent PCR and two-dimensional electrophoresis
(2-DE) are widely used as the industry standard for molecular biology
identification of import and export EBN, with the advantages of high
sensitivity, specificity and stability.
Huang and colleagues (2011) found that precipitating the protein in
the EBN water extract with acetone first and then purifying it by liquid-
phase electro focusing electrophoresis were helpful to obtain a better 2-
DE pattern and greatly shorten the preparation and purification time of
the sample. This method was more suitable for red EBN for its complex
components and low protein extraction rate. However, although this
method could effectively reduce the interference of acid glycoprotein on
2-DE, there were still some protein clusters in the acid end. Moreover,
acetone is a slightly toxic and volatile organic solvent, which is harmful
to human body. Guo and colleagues (2013) optimized the 2-DE tech­
nique to eliminate the aggregation of acid-terminal proteins and ob­
tained 2-DE patterns with good resolution and reproducibility. Jian
(2014) used polyacrylamide gel electrophoresis (SDS-PAGE) to quali­
tatively identify proteins in EBN and its counterfeit products, but the
method can only be used to identify adulterants containing proteins. Xu
and colleagues (2019) used nanocomposites as signal markers, synthe­
sized EBN specific antibodies as recognition biomolecules, and estab­
lished a side flow sandwich immunoassay based on Gold-nano-particle-
decorated silica nanorod for rapid detection of EBN specific glycopro­
tein. This method can detect EBN’s specific glycoprotein sensitively and
specifically within 30 min without any instrument and complex opera­
tion. It can realize the rapid identification and verification of EBN
products’ geographical sources, and be used for the field identification
and quality evaluation of EBN. Wong, Chan, Wu, Lam and colleagues
(2018) produced monoclonal antibodies to recognize acidic mammalian
chitinase-like protein in EBN by hybridoma technology and verified by
immunoprecipitation followed by LC-MS/MS analysis. The results of
western blot and ELISA showed that chitinase-like protein in EBN was
specific and could be used as an authentication marker.
Wu et al. (2010) used two-dimensional gel electrophoresis technol­
ogy and PCR technology based on DNA to identify the authenticity of
EBN. The former was used to determine the proportion of EBN protein in
the total protein, while the latter was used to evaluate the presence of
EBN in the products. PCR technology had high sensitivity and could
detect 0.5% EBN components in the mixture of Tremella and EBN.
Moreover, the technology based on DNA molecule has high specificity
and is more suitable for the identification of EBN in complex environ­
ment. In recent years, DNA molecular technology has been developed in
the research of EBN identification.
In 2009, Lin et al. (2009) published the first technical report on ge­
netic identification of EBN. The technique they developed was based on
sequences of cytochrome-b (cytb) gene in mitochondrial DNA, and the
sample sequences together with the sequences of swiftlets in GenBank
were used to construct phylogenetic trees for genetic identification. This
method was applied to 11 samples and the results showed that all the
EBN samples (including the ready-to-eat EBN products) were built by
Y. Dai et al.
A. fuciphagus while the Huaiji EBN sample was built by Apus nipalensis.
Then, the team optimized the DNA extraction method (Lin et al., 2010).
SDS with high salt was used to crack EBN samples, chloroform and CTAB
were used for deproteinization, and isopropanol was used to precipitate
DNA at low temperature. This DNA extraction technology could get rid
of the interference of sialoglycoprotein and the pollution of exogenous
DNA, so as to obtain high-quality DNA for PCR and sequencing. The
results showed that the DNA obtained by this method could be used to
establish genetic tree of EBN, and the origin of the EBN could be
determined successfully. Guo et al. (2014) designed five sets of primers
and TaqMan probes to detect and distinguish dopants in EBN (such as
Tremella, agar, pigskin and egg white), and the minimum detection
limits were 0.001%, 0.5%, 0.001% and 1%, respectively. Chen (2019)
innovatively applied multiplex real-time PCR detection technology. She
established a dual and triple real-time PCR detection system for the main
components and adulterants of EBN by synthesizing primers and probes
specific to the derived materials of EBN, Tremella and red seaweed.
Thomassen et al. (2005) analyzed the mitochondrial genes of giant
swiftlet (Hydrochous gigas) and swiftlets of the genus Aerodramus. The
results showed that the differences of NADH dehydrogenase subunit 2
(ND2) and cytb sequences could be used to distinguish these two species
of swiftlets. Cranbrook and colleagues (2013) studied the morphology
and genes of wild and house-farm populations in Indonesia, Malaysia
and Thailand. The results of morphological studies showed that house-
farm birds of Sarawak resembled neither of the wild species occurring
naturally in the state. Based on the partial cytb sequence, they per­
formed phylogenetic analyses, AMOVA and pairwise FST comparison,
and found that mtDNA cytb gene could be used as a marker to show the
genetic differences between wild species and house-farm ones. This
suggested that genetic differences can also be used to distinguish cave
EBN and house EBN. The TaqMan probes designed by He et al. (2015),
based on the cytb gene sequences in the DNA of Aerodramus, had a low
matching degree with the cytb gene of other birds, showing good
specificity and sensitivity. Wang, Han, & Lai (2015) amplified and
sequenced ND2 gene sequences from 32 EBN samples of different origins
and varieties by DNA barcode technology, analyzed mutation sites and
calculated the genetic distance of Kimura-2 parameters, so as to
construct adjacent phylogenetic tree. This method was suitable for rapid
and accurate identification of biogenic origin of EBN. Chen et al. (2017)
collected 39 experiment samples from Malaysia, Indonesia, Vietnam and
Thailand. They extracted genomic DNA for the PCR reaction and
sequenced the amplified products. The results showed that 39 samples
were from three kinds of EBN. Samples could be distinguished success­
fully by NJ and UPMGA phylogenetic tree, which meant that cytb se­
quences could be used to distinguish the origins of EBN and it was
efficient for tracing the origin species of EBN. Quek Chin, Tan and col­
leagues (2018) used forensically informative nucleotide sequencing
technology based on mitochondrial and nuclear DNA sequences, and
carried out phylogenetic analysis to determine the types and sources of
EBN. The results showed that cytb, ND2, 12S ribosomal RNA and beta-
fibrinogen intron 7 gene markers could effectively distinguish between
A. fuciphagus and A. maximus. Moreover, by sequencing mitochondrial
cytb and ND2 genes, we can effectively distinguish house EBN and cave
EBN.
6.6. Fingerprint of EBN
Fingerprint is a comprehensive and quantifiable identification
method, including chemical fingerprint and DNA fingerprint, which can
be used to evaluate the authenticity, quality and stability of EBN and its
products. Zhuang et al. (2016) analyzed the amino acids of EBN by high
performance automatic amino acid analyzer. They found that the prin­
cipal components of authentic products were concentrated and the
counterfeits were scattered in the distribution map. The parameters of
adulteration content were analyzed as independent variables, and
evaluative scores as the dependent variable for linear regression
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analysis. The final model were R2＞0.98. The determined values and the
predicted values of the tested samples were fitted well with an average
error of 3.25%. The results showed that amino acid fingerprint could
effectively and reliably identify white EBN. Lin et al. (2015) used full-
automatic amino acid analyzer to analyze, detect and compare the
amino acid composition of white EBN, adulterated white EBN and
common adulterated substances. The fingerprint database of different
types of samples was constructed with 17 kinds of amino acid content as
index, and the amino acid composition of 13 samples in the experiment
was analyzed by cluster analysis. It was found that the proportion of
local peak area in amino acid fingerprints was different. The ratios of
essential amino acids to total amino acids, essential amino acids to
nonessential amino acids in white EBN were different from those of
adulterated EBN and counterfeits, which can be used as symbolic in­
dicators for identifying. The cluster analysis results conformed to the
classification of samples. These studies suggest that amino acid finger­
print identification is an effective and reliable method for identifying
white EBN. Ma et al. (2019) found that EBN contained trace rare earth
elements and rich mineral elements, and most of the elements were
distributed in a skewed way with a large coefficient of variation. It was
feasible to trace the origin of EBN and its harvesting methods based on
multi-element analysis. If we use chemometrics technology to build the
traceability model of EBN origins and collection methods based on
different elements, and collect more EBN samples from different sources
to test the prediction accuracy of the model, it is helpful to establish the
fingerprint of inorganic elements. Quek and colleagues (2018b) estab­
lished the classification model of EBN source recognition by three
pattern recognition methods: principal component analysis, hierarchical
clustering analysis and linear discriminant analysis. The classification
ability of the cross-validation method was 100% and the prediction
ability was 92%. They found that the key chemical markers of produc­
tion origin differentiation were the content of total phenol, Zn, Val and
Ca, while the key chemical markers of species origin differentiation were
SA, Ser, Phe and Val. The results showed that the nutritional and
chemical characteristics combined with pattern recognition analysis can
effectively identify and classify EBN.
DNA fingerprint relies on DNA molecular markers. Genetic markers
(molecular markers), which are based on nucleotide sequence variation
among individuals, directly detect individual differences at DNA level.
Its detection will not be affected by tissue specificity, development stage
and detection environment, and has the advantages of large number of
markers, high polymorphism and genetic stability. Moreover, the tech­
nology is simple, fast and easy to automate. Although there are few
reports on the detection and identification of EBN’s DNA at present, with
the improvement of DNA extraction technology and sequencing tech­
nology, it is believed that more efforts will be devoted to the estab­
lishment of EBN’s DNA fingerprint in the future.
6.7. Combination of multiple methods
With the diversification of EBN products and the continuous devel­
opment of counterfeiting methods, many identification methods have
been unable to identify EBN effectively. In order to improve the reli­
ability of EBN identification, a variety of methods are needed to be used
together.
In order to increase the reliability of identification, Zha et al. (2010)
used character analysis, electrophoresis, ultraviolet spectroscopy and
liquid chromatography to analyze EBN. Yang et al. (2014) used GC–mass
(GC–MS), electron microscopy and western blotting to determine the
authenticity of EBN samples, identifying them from chemical, physical
and biochemical perspectives. Zhu et al. (2007) used both ultraviolet
spectroscopy and amino acid analysis to identify EBN. The results were
confirmed by LC-MS. It was found that the maximum ultraviolet ab­
sorption wavelength of EBN was 280 nm, and the contents of Asp, Leu,
Glu, Val, Ser, Phe in the genuine EBN were higher than that in the fake
one.
Y. Dai et al.
7. Inspection of EBN
Before leaving the factory, the inspection and quarantine de­
partments will inspect the processed EBN. Upon arrival in China, EBN
will be spot checked for component content, including items like SA,
nitrite, sulfur dioxide, water, heavy metals and microorganisms.
7.1. Nitrite
Nitrite is an important inspection item, because it can not only reflect
the management level in the production and processing of EBN, but also
indirectly reflect whether the EBN has been forged.
Where do nitrite come from in EBN? One source is natural trans­
formation. EBN was affected by the climate in Southeast Asia. The
specific humidity and temperature could transform amino acids into
nitrites. The interior of swiftlet houses is perennially humid, light-proof
and poor in air circulation. Under the influence of volatile substances in
bird’s dung, the contents of nitrate and nitrite in EBN increased corre­
spondingly (Li et al., 2010). In the clean swiftlet houses, the nitrite
content in EBN can be controlled in the range of less than 30 ppm, which
will cause no harm to human body (Shao et al., 2018).
Artificial addition is another source of nitrite. The color of EBN was
related to the contents of nitrite and nitrate (Paydar et al., 2013). Under
weak acidic conditions, sodium nitrite can turn white EBN into red one
(But, Jiang, & Shaw, 2013). The nitration of the tyrosyl residue to the 3-
nitrotyrosyl residue in the glycoprotein and the oxidation of iron ions
are part of the causes of the red color (Shim and Lee, 2018; Wong, Chan,
Dong, et al., 2018). Nitrite is a kind of food additive, which has the
function of coloring and antisepsis (Chen, 2013). But it is not allowed to
be added to EBN. The nitrite in the artificial red EBN will exceed the
national management limit. If it is not handled properly when eating
EBN, it will cause great harm to human body. Experiments showed that
the nitrite in EBN can be reduced by soaking in water (Paydar et al.,
2013; Gao, 2013; Chan et al., 2013). The longer the soaking time is, the
more nitrite content will decrease. After 4 h, the decrease rate was more
than 90% (Zhao et al., 2013). So, consumers should soak and wash EBN
properly before eating it.
Sodium nitrite is an acute toxic substance. The owners of swiftlet
houses must implement the management strictly and do a good job in
cleaning the raw EBN. Just as important, the quality of commercial EBN
on the market should be scrutinized. Cheng, Zhu and Wan (2015)
designed a rapid nitrite detection kit, and established a convenient
method to detect nitrite content in EBN. The kit is fast, convenient,
inexpensive, accurate and sensitive with the minimum detection con­
centration of nitrite can reach 0.1 mg/kg. It can be used for rapid
detection of nitrite contents in all kinds of EBN products.
7.2. Sulfite
In order to have a better appearance, some white EBN will be
fumigated with sulfur. Long-term excessive intake of sulfite may cause
adverse reactions in human body, resulting in dyspnea. Sulfite also binds
to calcium, leading to the loss of bone calcium. Jiang et al. (2019)
established an ion chromatography method for the determination of
sulfite in EBN. Sulfite was unstable, so the sample was extracted with 40
mmol/L NaOH solution and formaldehyde was used as stabilizer. This
method had high accuracy, and the operation was simple and fast.
7.3. Heavy metals
Heavy metal content is also an important factor affecting the quality
of EBN. Heavy metals can interact strongly with proteins and enzymes in
human body, making them inactive, and may accumulate in some or­
gans, causing chronic poisoning. At present, the main detection methods
for heavy metals are electronic absorption spectrometry. According to
the different principles, it can be divided into four categories: Atomic
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Food Research International xxx (xxxx) xxx
Fluorescence Spectrometry (AFS), ICP-MS, Flame Atomic Absorption
Spectrometry (FAAS) and Inductively Coupled Plasma Atomic Emission
Spectrometry (ICP-AES). FAAS and AFS cannot detect the isotope ele­
ments effectively. ICP-AES can detect many elements at the same time,
but it still cannot meet the requirements of detecting some elements.
Therefore, ICP-MS is the most effective method for the detection of
heavy metals in food (Zhao, 2018). ICP-MS, with the characteristics of
little interference, high precision, wide linear range and fast analysis
speed, is one of the fastest developing inorganic trace analysis tech­
niques in recent years, which can be used for simultaneous determina­
tion of multiple elements. It has been widely used in trace element
analysis of traditional Chinese medicine, and can also be used for the
determination of heavy metals in EBN (Zou, Lai, & Xu, 2012; Guo, Sun,
& Song, 2016).
7.4. Microorganisms
Feather EBN may also contain pathogenic bacteria and other mi­
croorganisms. For those produced in the epidemic area of avian influ­
enza, they may even carry avian influenza virus. Chen et al. (2015)
determined the levels of fungal contamination in EBN using molecular
techniques. The results showed that there was a significant difference (p
less than 0.05) in the number of types of fungi isolated from raw and
commercial EBN, but no significant difference in the reduction of the
number of types of fungi after boiling the EBN (p greater than 0.05). The
types of fungi isolated from the unboiled raw EBN were mainly soil,
plant and environmental fungi, while the types of fungi isolated from the
boiled raw EBN, unboiled and boiled commercial EBN were mainly
environmental fungi. Some of these fungi were mycotoxin producers and
caused opportunistic infections in humans. For safe consumption,
further studies to determine the mycotoxin levels and methods to pre­
vent or remove these contaminations from EBN are necessary.
Factory managers are responsible for strict supervision of key control
points and making HACCP plan to ensure the quality of EBN (Chen et al.,
2018). Also, the establishment and implementation of stringent regu­
lations for the standards of EBN should be regularly updated and
monitored to improve the quality of the EBN.
8. Content determination of EBN
8.1. Determination of amino acids
The total amino acid content is about 45–50% (Chen, Bao, Huang, &
Xu, 1997; Hou et al., 2007; Cao et al., 2011). Its high amount can pro­
vide more building blocks for protein synthesis and increase the rate of
protein synthesis, thus directly contribute to the growth effects. Lin,
Zhou and Wang (2013) determined the content of amino acid in EBN by
HPLC method to provide the basis for quality evaluation of EBN of
Indonesia. Jian and colleagues (2016) determined the amino acid con­
tent in dry EBN and stewed EBN by amino acid automatic analyzer. The
results showed that the contents of the two were 49.68% and 49.49%,
respectively.
8.2. Determination of carbohydrates
Carbohydrate is the second most abundant component in EBN,
whose composition and content relate closely to the biological function
of EBN. Yu et al. (2000) developed a specific GC detection method for
EBN based on the composition of the oligosaccharide chain combined
with glycoprotein in samples. Five monoses (D-mannitose, D-galactose,
N-acetyl-Dgalactosamine, N-acetyl-D-glucosamine, and N-acetyl-neu­
raminate) that constituted the oligosaccharide chain were detected
using GC and GC–MS spectrometry techniques. Large quantities of
protein and fatty acid present in the samples did not interfere with GC
detection. The GC method has good resolution, high sensitivity, and
strong intuition and consistency. Wang, Wang, Ni (2006) and Zhao et al.
Y. Dai et al.
(2015) determined EBN carbohydrates by CGC. You et al. (2012)
analyzed the monosaccharide components of six species of EBN by Pre-
column Derivatization HPLC. The monosaccharide derivatives were well
separated by hydrolysis of trifluoroacetic acid and derivation of PMP. Li,
Liang and colleagues (2014) established a simple and reliable method
for the determination of polysaccharides in EBN by phenol sulfate
method, using ultraviolet spectrophotometer. Tung et al. (2008)
employed High Performance ion liquid chromatography with pulsed
amperometric detector to analyze the saccharide profiles of EBN and its
processed products. Their method could estimate the content of
authentic EBN contained in commercially dried powder with effect.
8.3. Determination of SA
The determination of SA was used for the identification of EBN in the
beginning. The main form of SA in EBN is Neu5Ac, in which there is less
chromophore. Under acidic conditions, Neu5Ac can react with o-phe­
nylenediamine or ninhydrin to form derivatives with strong ultraviolet
absorption. According to this characteristic, the SA can be detected
quantitatively by ultraviolet detector. In 2004, Huang and colleagues
(2003) established a spectrophotometry for the detection of SA in EBN.
The average value of SA content in 12 standard EBN was 10.75%, which
was used as the standard to judge the authenticity of EBN. Subsequently,
the biological activity of SA has been paid more and more attention. The
content of SA has been used to evaluate the nutritional value of EBN. The
detection method of SA has developed rapidly and diversely. Besides
spectrophotometry, there are also GC, ion chromatography, HPLC and
GC–MS.
Chromatography is an important method for the determination of
SA. In 2014, HPLC became the national standard for the determination
of SA in EBN and its products (GB/T 30636-2014, 2014). Wang, Ni and
Wang (2006) used o-phenylenediamine hydrochloride to obtain SA de­
rivatives with strong ultraviolet absorption, and determined the con­
tents of SA derivatives by RP-HPLC at 230 nm wavelength. Feng et al.
(2010) derivatized SA hydrolyzed from EBN and identified it by a pre-
column derivatization RP-HPLC method with detection with a photo­
diode array detector or a fluorescecse detector. Jiang et al. (2009) hy­
drolyzed EBN protein with phosphoric acid and determined SA content
by HPLC at 192 nm. This method omitted the derivatization process of
SA and was easy to operate, but it was absorbed at the end of the ul­
traviolet band and required high quality mobile phase. Li, ASANTE and
colleagues (2014) established an ion chromatography-integral pulse
amperometric method for the determination of SA in EBN with high
sensitivity and good repeatability. Hou et al. (2013) extracted SA with
phosphoric acid and separated it by solid phase extraction column. The
content was determined by ultra-high-performance liquid
chromatography-mass spectrometry. Similarly, no derivatization of SA
was needed. Lacomba et al. (2010) reviewed the determination methods
of SA, including spectral analysis, liquid chromatography, GC, Matrix-
Assisted Laser Desorption/Ionization Time of Flight Mass Spectrom­
etry, current biosensor and mobility.
8.4. Determination of EBN products
EBN products are mixtures, with complex detection environments. In
the process of content determination, it is necessary to remove the in­
terferences in the sample first, or choose a method that is not interfered
by complex environment. Hu et al. (1996) measured the absorbance of
common interferences in ready-to-eat EBN, and analyzed the elimina­
tion methods of common interferences. It was found that ammonium
sulfate precipitation method had a good effect on the removal of adul­
terants such as Tremella, pigskin, swim bladder, gelatin, agar, carra­
geenan and egg white, while ammonium sulfate precipitation-activated
carbon adsorption method had a good effect on the removal of ginseng.
Lai et al. (2015) established a HPLC-MS method for the determination of
SA in rock candy EBN products. The samples were separated by column
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Food Research International xxx (xxxx) xxx
chromatography after acid hydrolysis, then ionized by electrospray
ionization source, and detected by negative ion mode and selective ion
mode. Hua et al. (2010) used o-phenylenediamine hydrochloride to
derive SA from EBN products, then separated it with C18 column and
determined the content at 201 nm. This method had high sensitivity and
good repeatability, and can determine the content of SA in health
products of EBN accurately. Yu et al. (1998) proposed a new GC method
based on the unique molecular structure of EBN, which had strong
discrimination and recognition ability in the complex mixed system, and
could detect 0.01% of EBN ingredients in EBN products. The method can
detect the feeding ratio of raw EBN according to the sugar component
quantitatively and observe the extent of the destruction of the EBN
structure, so as to judge the rationality of processing technology and
monitor the quality of EBN products.
9. Conclusions and prospects
EBN is a nonnegligible part of Chinese food culture. It used to be
regarded as a symbol of social status. In modern times, it is still regarded
as a good health food. In recent years, there have been several scandals
in EBN industry, and the adulteration of EBN with counterfeit has been
banned repeatedly. However, with the joint efforts of many parties, EBN
industry has gradually recovered its prosperity. From the trend of EBN
import in China in recent years, we can see that the demand for EBN has
increased dramatically. The vigorous development of the industry pro­
motes the development of science and technology. Researchers have
paid great attention to the authenticity, safety and effectiveness of EBN,
and the relevant technologies are constantly innovating. In sorting out
the research work in recent years, we have some views on the future
researches, which are listed as follows:
1. Introduction of swiftlets
The distribution of swiftlets that produces EBN is quite wide. It starts
from the Seychelles islands in the Indian Ocean in the west to the
Marquesas Islands in the Pacific Ocean in the East and from Sichuan
Province in China in the north to Queensland in Australia in the south.
However, only Southeast Asian countries have developed EBN agricul­
ture. Therefore, the introduction of swiftlets and the design of swiftlet
houses should be studied in depth, so as to establish swiftlet houses and
expand the production of EBN in areas suitable for swiftlets, such as
China.
2. Establish the gene bank
The taxonomic studies of swiftlets are mainly based on morpholog­
ical, behavioral and nest evidence. Scholars have different opinions and
no consensus has yet been reached on the classification. Studying the
literature, we find that contradictions exist in several different re­
searches. Most of the studies on active ingredients and properties of EBN
did not specify the exact species of their producers, which reduced the
reliability and repeatability of the experimental results. The wide
application of DNA sequencing technology in the classification and
phylogenetic studies provides enlightenments for solving the above
problems. DNA technology won’t be affected by DNA degradations and
glycoprotein interferences caused by environmental exposure. It can
identify origins of EBN at species or even subspecies level. Therefore, the
establishment of gene bank is of great significance to the classification of
swiftlets and the in-depth study of EBN.
3. Establish EBN fingerprint
SA is a characteristic component of EBN, which not only plays an
important role in nutrition, but also in quality control. However, it has a
wide range of sources and can be synthesized artificially, so it is not
appropriate to use SA as the primary indicator of quality control. In
Y. Dai et al.
order to control the quality effectively, it is necessary to establish the
fingerprint of EBN by thoroughly studying the quality of EBN in different
producing areas, selecting more indexes (such as glycoprotein, amino
acid, inorganic elements, etc.), and using various detection methods
(such as GC, HPLC, PCR, etc.). Considering that EBN products have
complex detection environments, the detection methods that can avoid
interferences to the greatest extent should be selected.
4. Studies on extraction, separation, purification and synthesis of EBN’s
components
There are many extraction methods of EBN, such as water extraction,
enzyme extraction, ultrasonic extraction, microwave extraction, etc.
However, on the one hand, none of these methods can obtain the stan­
dard EBN extract (in fact, no one has reported the requirements for the
standard EBN extract); on the other hand, it is important to extract some
components of EBN with emphasis. The researches on extraction tech­
nology are not only helpful to the development of EBN products, but also
to their quality control. The technology of separation and purification of
components in EBN is developing. However, most of the researches
focus on macromolecular compounds such as glycoprotein, and seldom
on small molecular compounds or monomer compounds. In a recent
experiment, two novel peptides with antioxidant activity were obtained,
indicating that there are still some new functional compounds in EBN to
be found (Ghassem et al., 2017). Therefore, efforts can be put into
finding functional components, even new compounds in EBN. At pre­
sent, SA can be synthesized artificially and the structural modification of
it is also in full swing. Many effective drugs have been developed based
on the structure of SA. It’s not hard to think that the same can be done
with other compounds in EBN.
5. Study on the pharmacological action
The outbreak of COVID-19 in 2019 has not been effectively
controlled so far. It has become an urgent need to improve human viral
resistance and prevent the spread of the virus. Ancient medical books
show that EBN has certain therapeutic effects and has been included in
the scope of traditional Chinese medicine. Previous studies have shown
that EBN has a very good effect on lung, stomach, liver, kidney, heart,
eye and skin; modern researches have proved that EBN has the effects of
anti-recognition, anti influenza virus, anti-inflammatory, strengthening
human immunity and so on. In this context, further development of EBN
antiviral potential is expected to contribute to human health. In addi­
tion, animal experiments are often used to directly prove the functions of
EBN, while many mechanisms are unclear, that makes it difficult to
explain how EBN works in human body. Moreover, some functional
differences between cave EBN and house EBN have not been explained.
Future researches should be carried out in these places.
6. Compatibility of EBN
EBN is widely regarded as a functional food and not a drug. One of
the reasons is that the effects of EBN take a long time to play. Sun pre­
pared Bird’s Nest Sangjie Particles, which were made from EBN, Platy­
codon grandiflorum, mulberry leaves and other medicinal materials (Wu
et al., 2018). It can be used for the treatment of patients with throat
inflammation due to “lung Yin deficiency”, and has the function of
clearing the throat and moistening the lung. This example reminds us
that EBN can be compatible with other medicinal materials to achieve
more rapid, safe and effective effects, which is conducive to improving
people’s acceptance of EBN. Therefore, it is possible to develop health
products for different groups of people, such as pregnant women, weak
men, malnourished children, etc. In addition, the possibility of combi­
nation of EBN and other therapeutic drugs could be considered due to its
functions of improving immunity, anti-recognition, improving inflam­
mation and so on.
12
Food Research International xxx (xxxx) xxx
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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