Characterization of Bird’s Saliva Soup by peptide fingerprinting with principal component analysis

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.

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