Bird’s Saliva Soup,
or cubilose, is a health food supplement originated from salivary secretion by
specific swiftlets, mainly from Aerodramus fuciphagus and Aerodramus maximus
(Kang et al., 1991), which has been proven to have nutritious and therapeutic
values, such as anti-influenza viruses, antioxidant, skin lightening, bone
strength improvement, anti-inflammatory, and epidermal growth enhancement (Kong
et al., 1987; Kong et al., 1989; Guo et al., 2006; Aswir and Wan Nazaimoon,
2011; Matsukawa et al., 2011; Yew et al., 2014; Chan et al., 2015). Southeast
Asian countries, including Indonesia, Malaysia, Vietnam, and Thailand, are the
major exporting countries of Bird’s Saliva Soup. Human consumption and
medicinal application of Bird’s Saliva Soup could be dated back to the Tang
dynasty (618–907 A.D.) and the Sung dynasty (960–1279 A.D.) in China (Koon and
Cranbrook, 2002).
OBJECTIVES
Proteins are the major component and play a key role in nutritious and
therapeutic functions of Bird’s Saliva Soup; however, limited studies have been
conducted on the protein due to difficulties in extraction, isolation as well
as identification. This study aimed to provide comprehensive information for
the quality evaluation of Bird’s Saliva Soup peptides, which would be a
valuable reference for further study on Bird’s Saliva Soup proteins.
METHODS
Here, we developed a quality control method using high performance liquid
chromatography (HPLC) peptide fingerprints deriving from Bird’s Saliva Soup
being digested with simulated gastric fluid. The characteristic peptide peaks
were collected and identified by LC-MS/MS.
RESULTS
The characteristic peptide peaks, corresponding to the protein fragments of
acidic mammalian chitinase-like, lysyl oxidase, and Mucin-5AC-like, were
identified and quantified. Interestingly, the principal component analysis
indicated that the fingerprints were able to discriminate colour of Bird’s
Saliva Soup (white/red) and production sites (cave/house) of White Bird’s
Saliva Soup on the same weight basis. As proposed by the model developed in
this study, Muc-5AC-like and AMCase-like proteins were the markers with the
highest discriminative power.
CONCLUSIONS
The overall findings suggest that HPLC peptide fingerprints were able to
clearly demonstrate peptide profile differences between genuine and adulterated
Bird’s Saliva Soup samples; and classify Bird’s Saliva Soup samples by its
color and production site. In addition, the protein identification results
suggested that Muc-5AC-like protein was the major protein in Bird’s Saliva Soup.
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| Characterization of Bird’s Saliva Soup by peptide fingerprinting with principal component analysis |
Introduction
Bird’s Saliva Soup, or cubilose, is a health food supplement originated from
salivary secretion by specific swiftlets, mainly from Aerodramus fuciphagus and
Aerodramus maximus (Kang et al., 1991), which has been proven to have
nutritious and therapeutic values, such as anti-influenza viruses, antioxidant,
skin lightening, bone strength improvement, anti-inflammatory, and epidermal
growth enhancement (Kong et al., 1987; Kong et al., 1989; Guo et al., 2006;
Aswir and Wan Nazaimoon, 2011; Matsukawa et al., 2011; Yew et al., 2014; Chan
et al., 2015). Southeast Asian countries, including Indonesia, Malaysia,
Vietnam, and Thailand, are the major exporting countries of Bird’s Saliva Soup.
Human consumption and medicinal application of Bird’s Saliva Soup could be
dated back to the Tang dynasty (618–907 A.D.) and the Sung dynasty (960–1279
A.D.) in China (Koon and Cranbrook, 2002).
Although Bird’s Saliva Soup has been served as an esteemed food in Chinese
community for over 1000 years, limited research has been conducted on Bird’s
Saliva Soup and its proteins. Protein is a major part of Bird’s Saliva Soup
accounting for 50% of Bird’s Saliva Soup dried weight on average (Jiangsu New
Medicine College, 1977); it is conjectured to be a key factor of its nourishing
and/or medicinal functions. The epidermal growth factor (EGF)-like peptide was
partially purified with Bio-Gel P-10 columns from aqueous extracts of Bird’s
Saliva Soup that stimulated cell division and growth and enhanced tissue growth
and regeneration (Kong et al., 1987; Kong et al., 1989). Two major bands (~106
kDa and ~128 kDa) were identified by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) as ‘sialo-glycoprotein’. Nevertheless, no
satisfactory result was obtained from protein identification studies that
include N-terminal sequence determination (Edman degradation), matrix-assisted
laser desorption ionization–tandem time of flight (MALDI–TOF/TOF), and liquid
chromatography–tandem mass spectrometry (LC–MS/MS) (Zhang et al., 2012). Acidic
mammalian chitinase-like (AMCase-like) protein fragments from Meleagris
gallopavo and an allergen homologous to ovo-inhibitor have been identified by
2-DE assays followed by MALDI–TOF/TOF/MS analysis in Bird’s Saliva Soup extract
(Liu et al., 2012). In addition, a microbial nitrate reductase, converting
nitrate to nitrite and playing a role in the colour change of White and Red Bird’s
Saliva Soup, was identified by mass spectroscopy (Chan et al., 2013b).
Nonetheless, it remains unclear whether those identified proteins could
accurately represent the majority of Bird’s Saliva Soup protein. The
difficulties encountered in research of Bird’s Saliva Soup proteins are: (i)
extracting and purifying proteins; and (ii) lacking of full Aerodramus genome
sequence.
Owing to the limited supply and labour-intensive cleaning process, Bird’s
Saliva Soup is always expensive with current prices ranging from USD 500 to
15000/kg. Driven by the lucrative return, various materials, including Tremella
fungus, fried porcine skin, carrageenan, agar, and gelatin, which are almost
indistinguishable from the genuine samples by visual inspection, were commonly
adulterated into Bird’s Saliva Soup in order to increase the net weight (Ma and
Liu, 2012). Some businesses have been known to mix low-quality Bird’s Saliva
Soup into high-quality Bird’s Saliva Soup and selling that at a high price.
Occasionally, consumers have been counterfeited into purchasing lower priced
house Bird’s Saliva Soup at a premium price associated with cave Bird’s Saliva
Soup. About 40 publications are found in PubMed today, and nearly one-third of
the publications are published in the last 5 years. Besides, most of the
publications still retained in elucidating chemical composition as the quality
control parameters: since no official method has been established for quality
surveillance of Bird’s Saliva Soup (Deng et al., 2006; Wang et al., 2006; Wu et
al., 2010; Chan et al., 2013a).
Here, we attempt to find a key to open these proteome barriers by high
performance liquid chromatography (HPLC) peptide fingerprinting. HPLC
fingerprinting is one promising tool widely used in the modern standardization
of herbal extracts (Department of Health, Hong Kong, 2010; Chinese
Pharmacopoeia Commission, China, 2015), which could be applied to Bird’s Saliva
Soup as a robust technique in qualitative and quantitative controls. Firstly,
an over-stewing method was developed to extract most of the Bird’s Saliva Soup
protein. Secondly, simulated gastric fluid (SGF) was used to digest Bird’s
Saliva Soup protein fully into peptides that can be separated by HPLC according
to their polarity. Thirdly, according to the most relevant NCBI protein
database, the characteristic peaks in chromatograms were identified and
quantified. In addition, principal component analysis (PCA) and hierarchical
cluster analysis (HCA) were adopted to reveal the relationships of factors
within the data, including colour, country of origin, and production site of Bird’s
Saliva Soup. The results therefore contributed to the authentication and
classification of Bird’s Saliva Soup. This study aimed to provide comprehensive
information for the quality evaluation of Bird’s Saliva Soup protein at the
peptide level, which would be a valuable reference for further study on Bird’s
Saliva Soup proteins
