O-glycan marker for identifying cubilose and application of O-glycan marker

Abstract

The invention provides a series of cubilose specific markers, a use method of the specific markers and a method for identifying whether cubilose exists in a sample or not. Whether a detected sample contains the cubilose or not can be identified by detecting one or more specific O-glycan markers of the cubilose. The method can be used for identifying cubilose raw materials, related products and the like, and can also be used for distinguishing white cubilose and grass cubilose. The invention also provides a separation method of the O-glycan marker and a method for screening a specific marker for identifying the cubilose.

Description

TECHNICAL FIELD

[0001] The present invention relates to various glycan markers discovered in grass bird’s nest and the use of these glycan markers in the identification of bird’s nest raw materials and related products. The methods described in the present invention can be applied to qualitatively or quantitatively identify grass bird’s nest, and / or distinguish white bird’s nest and grass bird’s nest. Qualitative and quantitative analysis of glycan markers in the identification of bird’s nest raw materials and related products. O- O- The present invention relates to various glycan markers discovered in grass bird’s nest and the use of these glycan markers in the identification of bird’s nest raw materials and related products. The methods described in the present invention can be applied to qualitatively or quantitatively identify grass bird’s nest, and / or distinguish white bird’s nest and grass bird’s nest. Qualitative and quantitative analysis of glycan markers in the identification of bird’s nest raw materials and related products. O- BACKGROUND

[0002] Bird’s nest (Edible Bird’s Nest, EBN) is a precious food from Southeast Asia produced by swiftlets that is rich in carbohydrates and proteins. Traditionally, bird’s nest is highly valued for its effects on moistening the lung, reducing phlegm, and relieving cough, and it has now been verified to have pharmacological effects on enhancing immunity, anti-aging, and anti-virus. These findings have driven the growing demand for bird’s nest, and it is expected that the bird’s nest industry will reach millions of dollars by 2030. However, the huge market demand and high price pose a severe challenge to its quality control.

[0003] On the market, bird’s nest is divided into three types, including grass bird’s nest, hair bird’s nest, and white bird’s nest of different grades, with different prices. Counterfeiting and adulteration are two sources of poor quality EBN. Not only are there counterfeit or adulterated products on the market, but mixing grass bird’s nest (often mistaken for the more expensive white bird’s nest) into the product also creates another low-quality EBN, which is a key issue affecting its quality.

[0004] Bird’s nest is mainly composed of glycoproteins, which are secretions of several species of Aerodramus saliva glands in the Apodidae family (Lee, T. H., et al. (2017). Recent advances in the identification and authentication methods of edible bird’s nest. Food Research International, Vol 100, 14-27. https: / / doi.org / 10.1016 / j.foodres.2017.07.036). The identified grass bird’s nest is derived from the Pacific swiftlet (Apus pacificus) of the Apodidae family, which costs 40 dollars per gram, while the official / hair bird’s nest derived from the Aerodramus genus costs 160 dollars per gram. Pacificus Aerodramus

[0005] ​​​​Generally, the two kinds of bird's nest can be distinguished by visual inspection in intact condition. However, after the raw materials are washed, broken, processed into pieces or made into final products, it is difficult to identify the adulterated grass bird's nest with white bird's nest by microscopic identification and morphological identification.

[0006] As a method for identifying true and false bird's nest, microscopes and morphological identification, as well as chemical analysis including sugar composition, amino acid determination and oligosaccharide marker detection are commonly used, but these methods cannot distinguish grass bird's nest from white bird's nest. It is still a difficult problem to identify whether there is grass bird's nest in the relatively broken bird's nest raw materials and prepared products (products).

[0007] Currently, gene identification based on cytochrome c oxidase b gene sequences in mitochondrial DNA has shown good prospects in the identification of grass bird's nest; however, the difficulty of extracting high-quality DNA and the complexity of subsequent steps such as PCR and sequencing analysis hinder its wide application. Proteomics analysis also faces similar challenges.

[0008] Therefore, there is still a need for a reliable and effective method to identify the quality of bird's nest raw materials and finished products, such as whether they contain or are adulterated with grass bird's nest, especially in the food industry. SUMMARY

[0009] The present application provides a new liquid chromatography-mass spectrometry (LC-MS) method for identifying grass bird's nest by screening a series of grass bird's nest-specific O- glycans from the chemically deglycosylated O- glycan products of grass bird's nest labeled with 4-aminobenzoic acid ethyl ester (ABEE) or its analogues, and comparing with the chemically deglycosylated O- glycan products of white bird's nest labeled with ABEE or its analogues. O- The present application establishes glycan markers for detecting and qualitatively or quantitatively identifying grass bird's nest, and / or distinguishing white bird's nest and grass bird's nest, as well as the separation and use methods of these markers.

[0010] Specifically, the present application deglycosylates a plurality of batches of white bird's nest and grass bird's nest by β-elimination reaction, uses ABEE derivatization for the alkaline hydrolysis products, and performs chemical analysis on the sugar profile. By using liquid chromatography-diode array detector-quantitative time-of-flight mass spectrometry (LC-DAD-qTOF-MS) analysis, a series of O- hexasaccharides (hereinafter also referred to as O- glycans) specific to grass bird's nest are revealed, which O- do not exist in other bird's nests and common adulterants, and can be used as specific markers to identify the presence of grass bird's nest.

[0011] This series of grass bird's nest-specificO- The structure of the glycan markers was confirmed by mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy.

[0012] This invention provides a method for screening specific biomarkers for qualitative analysis and identification of bird's nest.

[0013] This invention relates to an ABEE-derived... O- A method for identifying grass-fed bird's nest using polysaccharide markers. This method can successfully identify grass-fed bird's nest in raw bird's nest and commercially available products using liquid chromatography-mass spectrometry.

[0014] This invention provides ABEE-labeled products generated from bird's nest through a β-elimination reaction. O- Polysaccharide markers, and their separation and application methods. O- Glycan markers can be used by using O- Polysaccharide markers are used for qualitative and quantitative analysis of bird's nest raw materials and products, or products or traditional Chinese medicine preparations suspected of containing bird's nest.

[0015] The present invention further provides a method for identifying bird's nest raw materials and related products, including identifying whether a test sample contains grass bird's nest components.

[0016] The present invention includes the following aspects.

[0017] A first aspect of the invention provides a method for screening specific markers for identifying bird's nest, the method comprising: S1) Chemically deglycosylate the grass bird's nest samples, other bird's nest samples, and adulterants to release the sugars in the grass bird's nest samples, other bird's nest samples, and adulterants. O- polysaccharides; S2) Derivatization (labeling) of the substance using ethyl 4-aminobenzoate (ABEE) or an analogue thereof. O- polysaccharides; S3) Analyze the labeled samples using LC-MS. O- polysaccharides, to obtain O- Glycan profile; S4) Regarding the above O- The glycan profile was analyzed, and the most relevant data obtained from LC-MS measurements that were expressed only in bird's nest were selected. O- Polysaccharides serve as potential specific biomarkers; S5) Compare the original LC-MS intensity of potential specific biomarkers with the set LC-MS intensity threshold to screen for specific biomarkers of bird's nest and identify the structure of the isolated biomarkers.

[0018] The term "most relevant" can be extracted, for example, through supervised (partial least squares discriminant analysis) PLS-DA analysis. O- the contribution of glycans as variables to distinguish the classification of the grass and white bird's nest, so as to obtain variable projection importance (VIP) score for judgment, and the components with VIP value greater than or equal to 3 are regarded as the most relevant.

[0019] In one embodiment of the first aspect, the specific marker screened for identifying the grass bird's nest is selected from one or more of: O - glycan (GBM101); ABEE-labeled with retention time of 6.898 min and mass-to-charge ratio of 901.3294 determined by LC-MS O - glycan (GBM102); ABEE-labeled with retention time of 17.007 min and mass-to-charge ratio of 943.3436 determined by LC-MS O - glycan (GBM103); ABEE-labeled with retention time of 10.506 min and mass-to-charge ratio of 490.1941 determined by LC-MS O - glycan (GBM107); ABEE-labeled with retention time of 11.100 min and mass-to-charge ratio of 739.2767 determined by LC-MS O - glycan (GBM108); ABEE-labeled with retention time of 9.335 min and mass-to-charge ratio of 739.2767 determined by LC-MS O - glycan (GBM109).

[0020] Preferably, the specific marker screened for identifying the grass bird's nest is GBM101 and / or GBM102. Wherein, the GBM101 has the structure Gal α (1→4) Gal β (1→4) Gal β (1→3) GalNAc; and the GBM102 has the structure Gal α (1→4) Gal β (1→4) Gal β (1→4) GalNAc.

[0021] In one embodiment of the first aspect, the method comprises one or more of the following steps: B1) a step of contacting the test sample with an alkaline hydrolysis agent, so that alkaline hydrolysis occurs to form an alkaline hydrolysis sample; B2) contacting the alkaline hydrolysis sample with an acidic neutralizing agent to neutralize, obtaining a neutralized sample, centrifuging the neutralized sample to obtain a supernatant sample, and using 4-aminobenzoic acid ethyl ester (ABEE) or its analogs to label the O- polysaccharides are derivatized to obtain labeled O- polysaccharides.

[0022] In one embodiment, the method comprises B3) separating the labeled O polysaccharides obtained in B2) to obtain separated labeled O polysaccharides.

[0023] In certain embodiments, the structure of the separated markers is elucidated by mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy analysis.

[0024] A second aspect of the invention provides a method for identifying a bird's nest product comprising grass bird's nest.

[0025] The method comprises: A1) chemically deglycosylating a test sample to release O- polysaccharides in the test sample; A2) derivatizing (labeling) the O- polysaccharides using 4-aminobenzoic acid ethyl ester (ABEE) or an analogue thereof; A3) analyzing the labeled O- polysaccharide sample using LC-MS to obtain a labeled O- polysaccharide profile of the test sample; A4) comparing the labeled O- polysaccharide profile obtained in A3) with a polysaccharide profile comprising one or more specific markers for identifying grass bird's nest as described in the first aspect, to determine whether the labeled O- polysaccharide sample obtained in A2) comprises the specific markers for grass bird's nest, and thereby identifying whether the bird's nest product comprises grass bird's nest. O

[0026] In one embodiment of the second aspect, the one or more specific markers for grass bird's nest comprises the specific markers for identifying grass bird's nest screened out in the first aspect.

[0027] In one embodiment, the test sample is identified to comprise grass bird's nest when the labeled O- polysaccharide profile from the test sample has peaks corresponding to one or more of the O- polysaccharides determined by LC-MS with retention time, mass-to-charge ratio selected from: ABEE-labeled O polysaccharide (GBM101) with retention time of 8.369 min and mass-to-charge ratio of 901.3294; ABEE-labeled O polysaccharide (GBM102) with retention time of 6.898 min and mass-to-charge ratio of 901.3294; ABEE-labeled O polysaccharide (GBM103) with retention time of 17.007 min and mass-to-charge ratio of 943.3436.- Glycan (GBM 103); ABEE labeled with a retention time of 10.506 min and a mass to charge ratio of 490.1941 O - Glycan (GBM 107); ABEE labeled with a retention time of 11.100 min and a mass to charge ratio of 739.2767 O - Glycan (GBM 108); ABEE labeled with a retention time of 9.335 min and a mass to charge ratio of 739.2767 O - one or more of Glycan (GBM 109).

[0028] Preferably, when the labeled O- Glycan profile from the test sample has a peak corresponding to one or more of the retention time, mass to charge ratio determined by LC-MS selected from: O- - Glycan (GBM 103); ABEE labeled with a retention time of 10.506 min and a mass to charge ratio of 490.1941 O - Glycan (GBM 107); ABEE labeled with a retention time of 11.100 min and a mass to charge ratio of 739.2767 O - Glycan (GBM 108); ABEE labeled with a retention time of 9.335 min and a mass to charge ratio of 739.2767

[0029] In one embodiment, the O- Glycan profile can be represented by chromatographic peaks in the total ion chromatogram (TIC), extracted ion chromatogram (EIC) or UV chromatogram.

[0030] The bird's nest product can be selected from one or more of raw material, treated raw material, downstream product prepared from raw material, etc.

[0031] In one embodiment of the second aspect, the O- Glycan is GBM 101, having the following structure:

[0032] Formula I Galα(1→4) Galβ (1→4) Galβ(1→3) GalNAc; The O- Glycan is GBM 102, having the following structure:

[0033] Formula II Galα(1→4) Galβ (1→4) Galβ(1→4) GalNAc.

[0034] The third aspect of the invention provides specific markers for identifying the presence of the Edible-nest bat roosts for use in the first aspect of the invention. The specific markers comprise one or more ABEE or its analogues labelled O - one or more glycans selected from the group consisting of GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109; preferably one or more selected from GBM101, GBM102.

[0035] In one embodiment of the third aspect, the specific markers are one or more glycans labelled (derivatized) with ABEE or its analogues. O- glycans.

[0036] The fourth aspect of the invention provides a method of using the specific markers for identifying the presence of the Edible-nest bat roosts of the third aspect, in particular for use in quantitatively or qualitatively identifying the Edible-nest bat roosts, preferably the Edible-nest bat roosts, including identifying the presence of the Edible-nest bat roosts in Edible-nest bat roost products, or distinguishing the white Edible-nest bat roosts from the green Edible-nest bat roosts, etc.

[0037] The Edible-nest bat roost products are selected from one or more of the group consisting of raw materials, processed raw materials, downstream products prepared from the raw materials.

[0038] The fifth aspect of the invention provides a method of isolating the specific markers for identifying the presence of the Edible-nest bat roosts of the third aspect from the materials containing the Edible-nest bat roosts, the method comprising: B1) chemically deglycosylating a sample of the raw material containing the Edible-nest bat roosts to obtain a sample of the Edible-nest bat roosts O- glycan mixture; B2) reacting the sample of the Edible-nest bat roosts O- glycan mixture with ABEE or its analogues to obtain a sample of the labelled Edible-nest bat roosts O- glycan mixture; B3) separating the sample of the labelled Edible-nest bat roosts O- glycan mixture on a preparative chromatographic column, collecting and combining to obtain a purified sample of the O- glycan marker.

[0039] Advantages of the invention The invention has discovered a series of new specific O glycan markers for the Edible-nest bat roosts, providing a method for identifying the Edible-nest bat roosts and related products. The O- glycan markers can be detected by liquid chromatography-mass spectrometry analysis.

[0040] The invention provides a method for identifying the presence of the Edible-nest bat roosts by labelling O- glycans with a derivatizing agent, and detecting the retention time and mass-to-charge ratio of the O- glycans in the sample to be tested by liquid chromatography-mass spectrometry analysis to determine whether the sample contains the specific O-The method for determining whether the sample contains grass bird's nest by using glycan markers. Thus, the challenge that the current bird's nest identification method is difficult to distinguish white bird's nest and grass bird's nest is overcome, so that the grass bird's nest in the bird's nest raw materials and products can be identified by the method of liquid chromatography-mass spectrometry analysis.

[0041] The experiments prove that the above grass bird's nest specificity O- The glycan markers can be used as reliable indicators for detecting grass bird's nest, and the identification method has high practicability and reliable results, and enhances the traceability and authenticity of bird's nest products on the market. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Screening of grass bird's nest O- The flowchart of glycan markers.

[0043] Figure 2 The result figure of screening the differential markers of white bird's nest and grass bird's nest by PLS-DA analysis.

[0044] Among them, Figure 2 A is the typical total ion flow chromatogram (TIC) of grass bird's nest and white bird's nest, the upper is grass bird's nest, and the lower is white bird's nest; Figure 2 B is the PLS-DA score plot of liquid chromatography-mass spectrometry data, the first component accounts for 80.1%, the second component accounts for 3.8%, and the third component accounts for 2.4%, the left red represents grass bird's nest, and the right green represents white bird's nest, R 2 = 0.9231, Q 2 =0.9095 precision = 1.0; Figure 2 C is the top 20 components in the variable projection importance (VIP) value in PLS-DA, and the components that are significantly different in grass bird's nest are highlighted by asterisks O- Glycan GBM101-GBM109; Figure 2 D is O- The original LC-MS intensity plot of glycan GBM101-GBM109, the left is the grass bird's nest sample, and the right is the white bird's nest sample.

[0045] Figure 3 Verification and further analysis of grass bird's nest markers in different samples. Figure 3 A is the ultraviolet chromatogram of sugar spectrum labeled by ABEE of grass bird's nest, white bird's nest and agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder and tremella (from top to bottom), λ = 305 nm; Figure 3B is the total extracted ion chromatogram (TIC) (top) and extracted ion chromatogram (EIC) (bottom) of GBM101, GBM102, GBM103, GBM107, GBM108, GBM109 labeled with ABEE. The asterisks show GBM101 and GBM102. The EIC was obtained by extracting the ion m / z ± 0.050 ESI-; Figure 3 C is the corresponding case of GBM101, GBM102, GBM103, GBM107, GBM108, GBM109 in the EIC plot of different samples.

[0046] Figure 4 For the verification and further analysis of the marker of the grass swallow nest in different samples. Figure 4 A is the total extracted ion chromatogram (TIC) (top) and extracted ion chromatogram (EIC) (bottom) of GBM104, GBM105 and GBM106 labeled with ABEE in the grass swallow nest. Figure 4 B is the EIC plot of the grass swallow nest, white swallow nest and agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder and tremella (from top to bottom) labeled with ABEE, showing the corresponding case of GBM104, GBM105 and GBM106 therein. The EIC was obtained by extracting the ion m / z ± 0.050 ESI-.

[0047] Figure 5 For the chemical structure characterization of GBM102 labeled with ABEE. Figure 5 A is the chemical structure diagram of GBM102; Figure 5 B (from top to bottom) is the 1 HNMR, 13 CNMR and DEPT135 spectrum of GBM102, 1 H and 13 CNMR spectrum was recorded at 400 MHz and 100 MHz, respectively; Figure 5 C is the HSQC spectrum of GBM102; Figure 5 D is the 1 H- 1 HCOSY spectrum of GBM102; E is the HMBC spectrum of GBM102.

[0048] Figure 6 For the chemical structure characterization of GBM101 labeled with ABEE. Figure 6 A is the chemical structure diagram of GBM101; Figure 6 B (from top to bottom) is the 1 H-NMR, 13 C-NMR and DEPT135 spectrum of GBM101, 1 H and 13CNMR spectra were recorded at 400 MHz and 100 MHz, respectively; Figure 6 C is the HSQC spectrum of GBM101; Figure 6 D is GBM101 1 H- 1 H COSY spectrum; Figure 6 E is the HMBC spectrum of GBM101.

[0049] Figure 7 Peaks of ABEE-labeled GBM101 and GBM102 detected in multiple batches of bird's nest samples (LC-qToF-MS method). Figure 7 A represents the result in the raw material fragments. Figure 7 B represents the result in the product. Detailed Implementation

[0050] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0051] The term “exemplary” as used herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments. Although various aspects of embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated otherwise.

[0052] Furthermore, the technical features involved in the different embodiments of this application described below can be combined with each other as long as they do not conflict with each other.

[0053] the term O- polysaccharides O- polysaccharides (O The basic building blocks of N-glycans are a core structure composed of GalNAc (N-acetylgalactosamine) residues. Compared to N-glycans, analysis... O- Glycans are challenging because of the lack of enzymes that promote the release of N-glycans from mucins, and the limited databases available for structural analysis.

[0054] N Polysaccharides are oligosaccharide structures in which N-acetylglucosamine and asparagine residues of proteins are linked by N-glycosidic bonds on their side chains. They are products of N-glycosylation modification of proteins. Mucins are cell surface or secreted proteins with high... O- Glycosylated glycoproteins.

[0055] In this article, " O- "Glycan profile" or "glycospectrum" refers to the glycan content in biological samples. O -Linked polysaccharides (O - the qualitative, quantitative or relative abundance profile obtained after mass spectrometry / chromatography detection, for example able to be provided in the form of a mass spectrum, of a total ion chromatogram (TIC), of a chromatographic peak in an ultraviolet chromatogram.

[0056] The term "beta-elimination reaction" refers to a reaction in which two substituents (usually a hydrogen atom and a leaving group such as a halogen, a hydroxyl group, etc.) on two adjacent carbon atoms (alpha and beta carbons) in an organic molecule are removed simultaneously or sequentially, wherein the leaving group (X) is removed from the alpha carbon and the hydrogen atom (H) is removed from the beta carbon, thereby forming a new double bond (usually an alkene) between the alpha and beta carbons, and simultaneously producing a small molecule (such as HX, H2O, etc.).

[0057] The term "labeling", "derivitization", "derivatization" as used herein refers to a method of converting a compound into a similar chemical structure using chemical transformation. The purpose of derivatization of a sample is mainly to convert a difficult-to-analyze substance into a similar chemical structure but easy-to-analyze substance, which is convenient for quantification and separation. When the substance to be detected is not easy to be detected, such as no ultraviolet absorption, etc., it can be treated, such as adding a chromophore, etc., to generate a detectable substance. Derivatization method is widely used in instrumental analysis. The general chemical derivatization method mainly has the following purposes: improving the sensitivity of sample detection; improving the separation degree of sample mixture; suitable for further structural identification, such as mass spectrometry, infrared or nuclear magnetic resonance, etc.

[0058] The term "most relevant", for example, can be extracted by supervised PLS-DA analysis O The contribution of glycan as a variable to the classification of distinguishing grass bird's nest from white bird's nest, so as to obtain variable projection importance (VIP) score for judgment, and the components with VIP value greater than or equal to 3 are considered to be the most relevant.

[0059] The term "raw LC-MS intensity" as used herein refers to the ion signal intensity recorded directly by the detector without normalization, correction or any data processing, also known as raw ion abundance.

[0060] The term "mass-to-charge ratio" as used herein refers to the ratio of the mass of an ion (in units of relative atomic mass) to the charge it carries (in units of electronic charge), written as m / z.

[0061] The "retention time", "mass-to-charge ratio", "molecular weight", "peak intensity", "raw LC-MS intensity" and other indicators for characterizing markers as used herein are indicators detected by LC-MS technology, unless otherwise specified.

[0062] It is to be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. Furthermore, any method, apparatus and material similar or equivalent to those described herein can be used in the practice or testing of the present application, with the preferred methods, apparatus and materials described below.

[0063] The technical features involved in different embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0064] In a first embodiment, a method for screening specific markers for identifying grass-edible-nest, the method comprises the following steps: S1) chemically deglycosylating the grass-edible-nest sample and other edible-nest samples, adulterants to release O- glycans in the grass-edible-nest sample and other edible-nest samples, adulterants.

[0065] In one embodiment, the chemical deglycosylation can be selected from the group consisting of: β-elimination reaction, hydrazinolysis, preferably β-elimination reaction, more preferably base hydrolysis catalyzed β-elimination reaction.

[0066] The other edible-nest samples can be selected from the group consisting of: white edible-nest, hair edible-nest and the like.

[0067] The adulterants (adulterants) can be listed as agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder and tremella and the like, but are not limited thereto, and can include other substances used to imitate edible-nest.

[0068] The release refers to the release of glycosyl substances connected to amino acids in glycoproteins from glycoproteins.

[0069] S2) using 4-aminobenzoic acid ethyl ester (ABEE) or its analogs to label the O- glycans released in S1).

[0070] The ABEE analogs have the following structure:

[0071] wherein R 1 is hydrogen or alkyl; R 2 is hydrogen, alkyl, cyano, alkoxy, hydroxyl, dialkylamine, ester, amide, urea; and R 3 is hydrogen, alkyl, cycloalkyl, heterocycloalkyl aryl, heteroaryl, amine, amide, urea or CO2R 4 , wherein R 4 is hydrogen, alkyl, cycloalkyl heterocycloalkyl aryl or heteroaryl.

[0072] S3) analyzing the labeled O- glycans obtained in S2) using LC-MS, liquid chromatography-ultraviolet detection (LC-UV), liquid chromatography-diode array detection (LC-DAD), glycan chip technology or nanopore technology to obtain labeled O- glycan profiles of the bird's nest samples, the other bird's nest samples and the adulterants.

[0073] In one embodiment, the LC-MS analysis is selected from LC-qTOF-MS. In some embodiments, the LC-MS method comprises ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS / MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS / MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QgQ-MS / MS), electro- repulsion hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC / LC-MS / MS). In some preferred embodiments, the liquid chromatography-mass spectrometry method is liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) or liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS / MS). In some more preferred embodiments, the liquid chromatography-mass spectrometry method is liquid chromatography-diode array detector (DAD)-time-of-flight mass spectrometry (LC-DAD-QTOF-MS).

[0074] In one embodiment, the LC-MS method in the present specification is performed by standard techniques well known in the art.

[0075] O- The glycan profile comprises: total extracted ion chromatogram (TIC), extracted ion chromatogram (EIC), chromatographic peaks in the ultraviolet chromatogram.

[0076] In one embodiment, O- The glycan profile can be obtained from a data matrix comprising sample information, mass-to-charge ratio (m / z), retention time (Rt) and molecular weight.

[0077] S4) performing PLS-DA analysis on the O- glycan profiles obtained in S3), extracting O - the degree of contribution of glycans as variables to the classification of the bird's nest samples into the grass bird's nest and white bird's nest categories, and the glycans with the most relevant degree of contribution, O-glycans as potential specific markers for identifying the grass-edible bird's nest.

[0078] The contribution degree may be expressed, for example, by the VIP value of the glycan. O - the VIP value of the glycan.

[0079] The term "VIP value" or "VIP score" refers to Variable Importance in Projection, which is an index commonly used in PLS-DA to evaluate the importance of variables. The VIP value is used to measure the predictive ability of each independent variable on the dependent variable. The greater the value, the greater the importance of the corresponding variable for the prediction of the model.

[0080] The term "most relevant" may be determined, for example, by the VIP value obtained by supervised PLS-DA analysis, and the components with a VIP value greater than or equal to 3 are considered to be the most relevant.

[0081] In one embodiment, S4) comprises selecting the glycans that have a statistically significant difference between the grass-edible bird's nest and the white-edible bird's nest O- glycans as potential specific markers. The criteria for determining a statistically significant difference may be, for example, that the Variable Importance in Projection (VIP) score in the PLS-DA analysis is greater than 3, and the significance level P <0.001.

[0082] S5) comparing the original LC-MS intensity of the potential specific marker for identifying the grass-edible bird's nest with a set LC-MS intensity threshold to determine the specific marker specific to the grass-edible bird's nest.

[0083] In one embodiment, S5) further comprises: selecting the specific marker with an original LC-MS signal intensity ≥ 10 4 as a specific marker for identifying the grass-edible bird's nest.

[0084] In the first embodiment, the specific marker for identifying the grass-edible bird's nest can be selected from GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109.

[0085] The retention time of the GBM101 labeled by ABEE determined by LC-MS is 8.369 min, the mass-to-charge ratio is 901.3294, and the molecular weight is 707.3294; The retention time of the GBM102 labeled by ABEE determined by LC-MS is 6.898 min, the mass-to-charge ratio is 901.3294, and the molecular weight is 707.3294; The retention time of the GBM103 labeled with ABEE determined by LC-MS is 17.007 min, the mass-to-charge ratio is 943.3436, and the molecular weight is 749.3436; The retention time of the GBM107 labeled with ABEE determined by LC-MS is 10.506 min, the mass-to-charge ratio is 490.1941, and the molecular weight is 296.1941; The retention time of the GBM108 labeled with ABEE determined by LC-MS is 11.100 min, the mass-to-charge ratio is 739.2767, and the molecular weight is 545.3436; The retention time of the GBM109 labeled with ABEE determined by LC-MS is 9.335 min, the mass-to-charge ratio is 739.2767, and the molecular weight is 545.3436.

[0086] In one embodiment, the GBM101 has the following structure:

[0087] Formula I Galα(1→4) Galβ (1→4) Galβ(1→3) GalNAc.

[0088] wherein the GBM102 has the following structure:

[0089] Formula II Galα(1→4) Galβ (1→4) Galβ(1→4) GalNAc.

[0090] wherein the GBM105 has the following structure:

[0091] Formula III Gal β (1→4) GalNAc.

[0092] wherein the GBM109 has the following structure:

[0093] Formula IV Gal β (1→4) Gal β (1→4) GalNAc.

[0094] In one embodiment, S5) further comprises: screening specific markers for identifying the bird's nest according to the response and retention time in LC-MS analysis.

[0095] In one embodiment, the glycans are selected to have shorter retention times on the chromatographic column and to be better separated from adjacent O- glycans. O- In one embodiment, the glycans are selected to have shorter retention times on the chromatographic column and to be better separated from adjacent glycans.

[0096] In one embodiment, the method can comprise one or more of the following steps C1) - C4): C1) contacting the test sample with an alkaline hydrolysis agent to allow a beta-elimination reaction to occur to form O a mixture of glycans; In one embodiment, the alkaline hydrolysis agent is selected from one or more of: NaOH, KOH, LiOH, Na2C03, K2C03, NH4OH. In one embodiment, the alkaline hydrolysis agent is selected from NaOH.

[0097] In one embodiment, the conditions for the alkaline hydrolysis can be selected from: 0.05% NaOH, 0.1% NaOH, 0.25% NaOH, 0.5% NaOH, 1.0% NaOH.

[0098] C2) contacting O the mixture of glycans with an acidic neutralization agent to allow neutralization, obtaining a neutralized sample, centrifuging the neutralized sample, obtaining a supernatant sample; In one embodiment, the acidic neutralization agent can be used those commonly used in the art without particular limitation and can be selected from: acetic acid, hydrochloric acid, perchloric acid.

[0099] The pH range of the neutralized sample: 5.0-8.0.

[0100] The centrifugation speed range: 10,000-15,000 revolutions per minute.

[0101] C3) derivatizing the glycans in the supernatant sample with 4-aminobenzoic acid ethyl ester (ABEE) or an analogue thereof, obtaining labeled O glycans. O- glycans.

[0102] The analogue of ABEE is as described in step S2) above.

[0103] Preferably, the labeled O- glycans can comprise one or more of the ABEE or analogue thereof labeled O glycans selected from: GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109; preferably one or more selected from GBM101, GBM102.

[0104] C4) separating the labeled glycan obtained in step C3) by chromatography to obtain separated (purified) labeled O- glycans. O - glycans.

[0105] In one embodiment, in the method of the present application, the column of the liquid chromatography in the chromatography is selected from a silica-based column, a polymer-based column, or other inorganic packing column. Preferably, a C18 octadecyl-bonded silica liquid chromatography column, a C8 octane-bonded silica liquid chromatography column, a NH2 amino-bonded silica column, a diol-based bonded silica column, a phenyl-bonded silica column, an ion exchange column, or an amide-bonded silica column is used; more preferably, an ACQUITY UPLC BEH C8 column or a Waters UPLC XBridge BEH Amide column is used.

[0106] The mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, isooctane, n-hexane, n-decane, cyclohexane, carbon disulfide, carbon tetrachloride, benzene, xylene, toluene, chlorobenzene, dichloromethane, tetrahydrofuran, ethyl acetate, chloroform, aniline, pyridine, acetone, methanol, ethanol, isopropanol, n-propanol, or a combination thereof; preferably, the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, or a combination thereof; more preferably, the mobile phase of the liquid chromatography is 0.1%~0.5% formic acid in water and 0.1%~0.5% formic acid in acetonitrile.

[0107] Preferably, the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, or a combination thereof. More preferably, the mobile phase of the liquid chromatography is 0.1%~0.5% formic acid in water and 0.1%~0.5% formic acid in acetonitrile.

[0108] In some embodiments, the mobile phase (eluent) of the liquid chromatography is aqueous formic acid and aqueous formic acid in acetonitrile. In some specific embodiments, the eluent is 0.1-0.5% aqueous formic acid A and 0.1-0.5% formic acid in acetonitrile B. In some specific embodiments, the eluent A is 0.1-0.4% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.3% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.2% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.2% aqueous formic acid. In some other specific embodiments, the eluent B is 0.1-0.4% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.1-0.3% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.1-0.2% formic acid in acetonitrile. In some specific embodiments, the eluent A is 0.5% aqueous formic acid. In some specific embodiments, the eluent A is 0.4% aqueous formic acid. In some specific embodiments, the eluent A is 0.3% aqueous formic acid. In some specific embodiments, the eluent A is 0.2% aqueous formic acid. In some specific embodiments, the eluent A is 0.1% aqueous formic acid. In some other specific embodiments, the eluent B is 0.5% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.4% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.3% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.2% formic acid in acetonitrile. In some other specific embodiments, the eluent B is 0.1% formic acid in acetonitrile.

[0109] In some embodiments, the eluent flow rate ranges from 0.3 ± 0.05 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.04 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.03 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.02 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.01 mL / min. In some preferred embodiments, the eluent flow rate is 0.3 mL / min.

[0110] In some embodiments, the column temperature of the liquid chromatography column is 30-50 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 30-45 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 30-40 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 30-35 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 50 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 45 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 40 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 35 °C. In some specific embodiments, the column temperature of the liquid chromatography column is 30 °C.

[0111] In some embodiments, the detection wavelength of the diode array detector (DAD) is 190-760 nm, preferably 305 nm.

[0112] In one embodiment, the above method further comprises a step of preparing the bird's nest sample into a sample solution before performing the sample identification, preferably, the sample solution is prepared using an aqueous solvent, more preferably, the aqueous solvent is methanol or water, and most preferably, the aqueous solvent is a methanol:water aqueous solvent with a volume ratio of 0-70% water.

[0113] In one embodiment, the above method, the step of labeling or derivatization is preferably performed using a derivatization reagent with a chromophore, such as 2-AA, 2-AB, PMP, 2-AP, ABP, ABME, HOA, AMAC, 3-(acetylamino)-6-amino-acridine (AA-Ac), ANTS, phenylhydrazine, dansylhydrazine, etc., and more preferably, using ABEE or its analogs.

[0114] In some embodiments, the structure of the isolated marker is elucidated by mass spectrometry and one-dimensional / two-dimensional nuclear magnetic resonance spectroscopy.

[0115] In one embodiment, the grass bird's nest sample, white bird's nest sample, and adulterant are samples identified by known methods as containing grass bird's nest, containing white bird's nest, and containing adulterant.

[0116] The second embodiment provides a method for identifying a bird's nest product containing grass bird's nest, the method comprising: A1) chemically deglycosylating a sample (test sample) from a bird's nest product to release O- glycans in the test sample.

[0117] The chemical deglycosylation is as described in the first embodiment above. The bird's nest product is as described in the first embodiment above. Unless otherwise specified, all descriptions in the first embodiment apply to this embodiment and the other embodiments described below.

[0118] In some embodiments, the method for identifying a sample or the method for screening a marker specific to Edible Bird's Nest comprises extracting the marker prior to identifying the sample, wherein the extraction of the marker comprises preparing an Edible Bird's Nest sample into a sample solution with an aqueous solvent prior to identifying the sample, centrifuging, and taking the supernatant for use.

[0119] The form of the Edible Bird's Nest product is not limited, and various forms such as nest fragments, nest cups, nest strips, nest corners, and nest filaments can be listed, and can include one or more of raw materials, processed raw materials, and downstream products prepared from raw materials. Preferably, the Edible Bird's Nest comprises white nest cups, yellow nest cups, blood nest cups, or hair nest cups. The Edible Bird's Nest product can include instant Edible Bird's Nest, thick Edible Bird's Nest, or Edible Bird's Nest drinks, etc.

[0120] In some embodiments, the Edible Bird's Nest sample to be detected is an Edible Bird's Nest raw material, and the preparation of the sample solution comprises adding an aqueous solvent to a dry Edible Bird's Nest sample powder, vortexing to mix, and then ultrasonicating. In some embodiments, the Edible Bird's Nest sample to be detected is an Edible Bird's Nest product, and the preparation of the sample solution comprises directly taking a solution in the Edible Bird's Nest product or directly dissolving the Edible Bird's Nest product in an aqueous solvent.

[0121] In some embodiments, the aqueous solvent is an aqueous alcohol solution. In some embodiments, the aqueous solvent is a methanol aqueous solution. In some embodiments, the aqueous solvent is an ethanol aqueous solution. In some embodiments, the aqueous solvent is a propanol aqueous solution. In some embodiments, the aqueous solvent is a butanol aqueous solution. In some embodiments, the aqueous solvent is a pentanol aqueous solution. In some embodiments, the aqueous solvent is a methanol:water volume ratio of 0% to 70%.

[0122] A2) using 4-aminobenzoic acid ethyl ester (ABEE) or an analogue thereof to label the O- glycan sample obtained in A1), to obtain a labelled O glycan sample.

[0123] “Derivatization” using ABEE or an analogue thereof, and “labeling”, “labelled”, etc. are used herein with the same meaning.

[0124] In some embodiments, the derivatization reaction temperature is 60°C to 90°C.

[0125] In some embodiments, the derivatization reaction time is 1 to 30 minutes.

[0126] A3) using LC-MS to detect the labelled O- glycan sample obtained in A2), to obtain data characteristic of the O glycan of the test sample.

[0127] The LC-MS is as described above in the first embodiment.

[0128] A4) determining whether the labeled O - glycan sample obtained in A2) comprises one or more O - glycan markers, wherein the one or more O - glycan markers have the following characteristics: retention time of 8.369 min, mass to charge ratio of 901.3294 of ABEE labeled O - glycans; retention time of 6.898 min, mass to charge ratio of 901.3294 of ABEE labeled O - glycans; retention time of 17.007 min, mass to charge ratio of 943.3436 of ABEE labeled O - glycans; retention time of 10.506 min, mass to charge ratio of 490.1941 of ABEE labeled O - glycans; retention time of 11.100 min, mass to charge ratio of 739.2767 of ABEE labeled O - glycans; retention time of 9.335 min, mass to charge ratio of 739.2767 of ABEE labeled O - glycans.

[0129] In one embodiment, determining whether the labeled O - glycan sample obtained in A2) comprises one or more O - glycan markers comprises determining whether the one or more O - glycan markers have the predicted retention time, mass to charge ratio, determining whether the labeled O - glycan sample comprises one or more of the specific markers for Edible-nest bat roosts as described in the first embodiment and thereby identifying whether the Edible-nest product comprises Edible-nest bat roosts.

[0130] In one embodiment, the presence of Edible-nest bat roosts in the test sample is identified when the labeled O- glycan profile from the test sample has one or more peaks corresponding to one or more of the specific markers for identifying Edible-nest bat roosts of the first embodiment. Preferably, the presence of Edible-nest bat roosts in the test sample is identified when the labeled O- glycan profile from the test sample has at least the peaks corresponding to GBM101, GBM102.

[0131] In one embodiment, the one or more O - glycan markers having the peak characteristics are selected from: retention time of 8.369 min, mass to charge ratio of 901.3294 of ABEE labeledO - Glycan; ABEE labeled with retention time of 6.898 min and mass to charge ratio of 901.3294 by LC-MS O - Glycan.

[0132] In one embodiment, the bird's nest product is selected from the group consisting of raw material, treated raw material, downstream product prepared from raw material, and the like.

[0133] In one embodiment, the O- Glycan profile is the same as described in the first embodiment.

[0134] In one embodiment, the LC-MS analysis is selected from the group consisting of LC-DAD-qTOF-MS, liquid chromatography diode array (LC-DAD). In one embodiment, LC-UV can be used to obtain O- Glycan profile.

[0135] In one embodiment, the chromatographic condition is using C18 octadecyl bonded silica liquid chromatography column: the mobile phase of the liquid chromatography is 0.1%~0.5% formic acid in water and 0.1%~0.5% formic acid in acetonitrile.

[0136] In one embodiment, the mass spectrometry can include: quadrupole mass spectrometer (QMS), triple quadrupole mass spectrometer (QQQ), time-of-flight mass spectrometer (TOF), ion trap mass spectrometer (IT), orbitrap mass spectrometer (Orbitrap) analysis.

[0137] In one embodiment, the mass spectrometry is Q-TOF mass spectrometry.

[0138] The LC-MS analysis includes providing extracted ion chromatogram (EIC), primary and secondary mass spectrometry as the O- Glycan profile.

[0139] In one embodiment, the predicted retention time, mass to charge ratio, and / or the O- Glycan profile of the first embodiment for identifying specific markers of grass bird's nest can be obtained from detection data from other methods determined as grass bird's nest samples, and the like.

[0140] In one embodiment, the method comprises a method for identifying grass bird's nest based on ABEE derived O- Glycan markers.

[0141] It is understood that although methods of derivatization are enumerated, the identification methods of the present application also include cases where no derivatization is performed. In one embodiment, the identification method comprises the following steps: (1) preparing a sample solution, wherein the sample solution is directly used in the subsequent steps without derivatization; (2) separating and detecting the sample using liquid chromatography-mass spectrometry to obtain the retention time and / or mass-to-charge ratio of the sample; (3) comparing the retention time and / or mass-to-charge ratio of the sample with the markers to identify the sample.

[0142] The third embodiment provides a method for qualitatively or quantitatively identifying the O glycan markers and structural analogs thereof, which are used to identify the O The glycan markers are specific markers for identifying the

[0143] In one embodiment, the specific markers for identifying the

[0144] In one embodiment, the specific markers for identifying the O The glycan markers GBM101 and GBM102 have the structural formula and corresponding peak characteristics as described in the above embodiments.

[0145] The fourth embodiment provides the use and method of using the specific markers for identifying the

[0146] The specific markers for identifying the

[0147] The fifth embodiment provides a method for separating the specific markers for identifying the

[0148] The method comprises one or more of the following steps: C1) chemically deglycosylating the test sample, preferably by contacting the test sample with an alkaline hydrolysis agent to undergo a β-elimination reaction to form O a glycan mixture sample; In one embodiment, the basic hydrolysis agent can use those commonly used in the art without particular limitation, for example, can be selected from one or more of: NaOH, KOH, LiOH, Na2CO3, K2CO3, NH4OH.

[0149] In one embodiment, the conditions of the basic hydrolysis can be selected from: 0.05% NaOH, 0.1% NaOH, 0.25% NaOH, 0.5% NaOH, 1.0% NaOH.

[0150] C2) contacting O - the glycan mixture sample with an acidic neutralizing agent to neutralize, obtaining a neutralized sample, centrifuging the neutralized sample, obtaining a supernatant sample; In one embodiment, the acidic neutralizing agent can use those commonly used in the art without particular limitation, for example, can be selected from: acetic acid, hydrochloric acid, perchloric acid.

[0151] The pH range of the neutralized sample: 5.0-8.0.

[0152] The centrifugation speed range: 10,000-15,000 revolutions.

[0153] C3) derivatizing the O glycans in the supernatant sample with 4-aminobenzoic acid ethyl ester (ABEE) or its analogues, obtaining labeled O- glycans.

[0154] The analogues of ABEE are as described in the S2) step above.

[0155] Preferably, the labeled O- glycans can comprise one or more of the ABEE or its analogue labeled O glycans selected from: GBM101, GBM102, GBM103, GBM107, GBM108, and GBM109; preferably one or more selected from GBM101, GBM102; C4) separating the labeled O- glycans obtained in the C3) step by chromatography, obtaining separated labeled O glycans.

[0156] In one embodiment, the method comprises: separating the ABEE labeled O glycan mixture sample on a liquid chromatography column, collecting and combining the mixture containing ABEE labeled O glycan markers; wherein preferably 10%, 11%, 12%, 13%, 14%, 15%, and 16% of acetonitrile are eluted per column volume, respectively; The combined mixture is re-separated on a liquid chromatography column using 12% and 13% acetonitrile respectively in one embodiment, and the target mixture is collected; The re-chromatography separation is performed on a liquid chromatography column; and The purified ABEE-labeled O glycan markers are obtained using 12% acetonitrile solution containing 0.1% formic acid.

[0157] In one embodiment, the purified ABEE-labeled O glycan markers are selected from one or more of ABEE-labeled Gal α (1→4) Gal β (1→4) Gal β (1→3) GalNAc (GBM101) and / or ABEE-labeled Gal α (1→4) Gal β (1→4) Gal β (1→4) GalNAc (GBM102), and ABEE-labeled BNM103.

[0158] In a seventh embodiment, a separation method for standard samples of O- glycan markers for qualitative and quantitative detection is provided, the method comprising: B1) chemically deglycosylating a crude sample containing Herba Ophiopogonis to obtain a Herba Ophiopogonis O- glycan mixture sample; B2) reacting the Herba Ophiopogonis O- glycan mixture sample with ABEE or an analog thereof to obtain a labeled Herba Ophiopogonis O- glycan sample; B3) separating the labeled Herba Ophiopogonis O- glycan mixture sample on a chromatography column, collecting and combining to obtain purified O- glycan markers.

[0159] The O- glycan markers can be selected from one or more of Gal α (1→4) Gal β (1→4) Gal β (1→3) GalNAc (GBM101) and / or Gal α (1→4) Gal β (1→4) Gal β (1→4) GalNAc (GBM102), and BNM103.

[0160] Embodiments The present application will be further illustrated by the following examples. The features and advantages of the present application will become more apparent from these descriptions.

[0161] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

[0162] Moreover, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0163] Materials and methods used in the examples Chemicals and materials ABEE, sodium cyanoborohydride, glacial acetic acid, sodium hydroxide, and reference standards of glucose (Glc), galactose (Gal), mannose (Man), N-acetylgalactosamine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol for liquid chromatography-mass spectrometry were purchased from RCI Labscan Limited (Thailand). Water was purified using a Millipore Milli-Q purification system. Other reagents were HPLC grade.

[0164] Edible bird's nest A total of 160 batches of edible bird's nest (EBN) raw materials were purchased, including 13 batches of grass EBN (n = 13), 107 batches of white EBN (n = 107), and 40 batches of EBN fragments (n = 40), all from the Chinese market. To confirm the biological origin of the EBN samples, representative batches of grass EBN and white EBN were identified as coming from Apus pacificus (grass EBN) and Aerodramus maximus (white EBN), respectively, by genetic identification. pacificus ) and Aerodramus fuciphagus ) respectively.

[0165] In addition, as EBN products, 69 batches of EBN products from 42 different brands from Vietnam and China were purchased. The EBN raw materials collected were identified as EBN by previously published marker BNM001 (Li LF, Cheng HY, Lang J, et al. An oligosaccharide marker for rapid authentication of edible bird's nest. Food Chem . 2023; 409: 135334. doi:10.1016 / j.foodchem.2022.135334).

[0166] Adulterants Agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder, and wood ear were from Chinese suppliers.

[0167] Example 1 O Preparation of polysaccharide mixture samples The main component of bird's nest is mucin. Mucin is highly... O Glycosylated glycoproteins are primarily composed of proteins and oligosaccharide chains. Mucins, in particular, contain a large number of oligosaccharide chains... O- Glycoside bonds are linked to serine and threonine residues on the protein backbone, while oligosaccharide side chains linked by N-glycoside bonds are less common.

[0168] The following methods were used to prepare bird's nest from grass, white bird's nest, agar, egg white, gelatin, milk powder, pig skin, rice flour, fish maw, and snow fungus. O - Polysaccharide mixture sample.

[0169] The method of this invention uses mild alkaline hydrolysis to remove the impurities in bird's nest. O - The polysaccharide is released.

[0170] The sample preparation method was performed according to the method reported in (Bilgi, Berna & Çelik, Süeda. (2004). Solubility and emulsifying properties of barley protein concentrate. European Food Research and Technology. 218. 437-441. 10.1007 / s00217-004-0895-4.), with modifications.

[0171] 1. Accurately weigh 10 mg of sample (when the test sample is an instant bird's nest product, the sample amount is 25 mg).

[0172] 2. Add 500 μL of 0.25% (w / w) NaOH solution and heat the mixture in an 80°C water bath for 40 minutes.

[0173] 3. Add acetic acid to neutralize excess alkali. After neutralization, the pH should be 5-8. Centrifuge and collect the supernatant. The supernatant is the solution. O - Polysaccharide mixture sample.

[0174] 4. Take 100 μL O - Polysaccharide mixture samples were subsequently subjected to ABEE derivatization (ABEE labeling).

[0175] Example 2 Preparation of ABEE-derived O - Polysaccharide sample Derivatization reagents: 400 μL 0.6 mol / L ABEE, 80 μL glacial acetic acid and 80 μL 1.4 mol / L sodium cyanoborohydride.

[0176] The method for ABEE derivatization of glycans in the sample is reported in the literature (Hui-Yuan Cheng, Li-Feng Li, Wen-Jie Wu, et al. Qualitative and quantitative analysis of agar inedible bird's nest and related products based on a daughter oligosaccharide-marker approach using LC-QTOF-MS, Food Control, Volume 132, 2022, 108514, ISSN 0956-7135, https: / / doi.org / 10.1016 / j.foodcont.2021.108514.) and ABEE derivatized O glycan samples were prepared from grass swallow nest, white swallow nest, agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder and snow ear, respectively. O

[0177] The method for ABEE derivatization of glycans is as follows: O - glycans is as follows: The supernatant prepared in Example 1, 100 μL, was mixed with 560 μL of derivatization reagent and incubated at 80°C for 5 minutes. After cooling, 700 μL of water and diethyl ether were added to the solution, and after mixing, centrifuged at 15,000 rpm for 5 minutes. The upper liquid (containing ABEE solution) was discarded, and the remaining aqueous phase was extracted with diethyl ether three times. The remaining aqueous phase was dried and redissolved in 200 μL of 70% (v / v) methanol solution to obtain the ABEE derivatized O glycan sample; The ABEE derivatized O glycan sample was subjected to LC-qTOF-MS analysis.

[0178] Example 3 LC-DAD-qTOF-MS analysis to obtain glycan profile The ABEE derivatized O glycan samples from grass swallow nest, white swallow nest, agar, egg white, gelatin, milk powder, pigskin, rice powder, swim bladder and snow ear samples were subjected to LC-qTOF-MS analysis using the following instruments and parameters.

[0179] ​Separation was performed using an Agilent 1290 UHPLC system (Agilent Technologies). This system is equipped with a binary pump, a temperature-controlled column, an autosampler, a degasser, and a diode array detector (DAD). The system was controlled by Mass Hunter B.06 software.

[0180] The chromatographic column was an ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA), eluted with a linear gradient of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) at a flow rate of 0.3 mL / min and an elution temperature of 30 °C.

[0181] The solvent gradient program was as follows: 0-3 min, 13% B; 3-22 min, 13-16% B; 22-25 min, 16-25% B; 25-28 min, 25-100% B; 28-30 min, 100% B; 30-30.1 min, 100-13% B; 30.1-34 min, 13% B. The injection volume was 2 μL.

[0182] MS data were collected using an Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a quadrupole time-of-flight (Q-TOF) mass spectrometer and a JetStream electrospray ionization (ESI) source. Data acquisition was controlled using MassHunter B.06 software (Agilent Technologies).

[0183] The operating parameters in negative ion mode are as follows: atomizing gas (N2) flow rate, 7.0 L / min; atomizing gas temperature, 300℃; atmospheric pressure gas flow rate, 8 L / min; sheath gas temperature, 350℃; nebulizer, 40 psi; capillary, 3000V; sampling cone, 65V; Oct RFV, 600 V; pyrolysis voltage 150 V; mass scan range: m / z 100~2000; mass resolution 20 000; DAD detection wavelength 305 nm.

[0184] Obtain the raw liquid chromatography-mass spectrometry (LC-MS) file.

[0185] 1. Use MassHunter B.06 software to process the raw liquid chromatography-mass spectrometry (LC-MS) file into net cumulative distribution function (CDF) format.

[0186] 2. Transfer the CDF format file to R-XC-MS (XCMS (X Chromatography Mass Spectrometry) in R language) for automatic peak detection, retention time comparison and peak matching.

[0187] 3. The generated data matrix was used for statistical analysis. The data matrix included sample information, mass-to-charge ratio (m / z), retention time (Rt) and peak intensity, and the information of chromatogram was obtained.

[0188] Example 4 O- Screening and validation of glycan markers In this example, glycan markers capable of distinguishing between O- Cordyceps sinensis and Baiyunia sinensis were screened and validated.

[0189] Firstly, for the data matrix of Cordyceps sinensis and Baiyunia sinensis obtained in Example 3, online platform MetaboAnalyst 5.0 (https: / / www.metaboanalyst.ca) was used to perform analysis by partial least squares discriminant analysis (PLS-DA). Glycans that met the criteria of having statistically significant differences between Cordyceps sinensis and Baiyunia sinensis were selected as potential markers. O-

[0190] A total of 120 samples were analyzed by LC-qTOF-MS, including 13 Cordyceps sinensis and 107 Baiyunia sinensis. O- Glycan profiles were represented by chromatographic peaks in total ion chromatogram (TIC) (A). Figure 2 Supervised PLS-DA analysis was performed using 1856 data points, and the results are shown in Figure 2 .

[0191] Statistical difference criteria: variable importance on projection (VIP) score in PLS-DA analysis was more than 3, and significance level P <0.001.

[0192] Figure 2 B shows that there is a clear separation between Cordyceps sinensis and Baiyunia sinensis samples. Two different clusters were determined in the PLS-DA score plot. The two components cumulatively explained 86.3% of the total variation, of which the first component (80.1%), the second component (3.8%) and the third component (2.4%) explained the variation between Cordyceps sinensis and Baiyunia sinensis samples. The cumulative value of PLS-DA was R 2 = 0.9231, Q 2 = 0.9095, accuracy = 1.0, indicating that the model fits well.

[0193] The most relevant O - glycans in LC-MS response in the above data were extracted by projection variable importance (VIP) method, and the top 20 VIP value components are shown in Figure 2 C.

[0194] ​Figure 2 C shows that among the top 20 VIP components, there are 9 O - Sugars are abundant in the grass nest, with 11 O - Glycans are abundant in the white nest. The 9 O - Glycans are: GBM101~GBM109. The above 9 O - Glycans are screened as potential specific markers for identifying grass nest.

[0195] The inventors preliminarily determined the structures of the 9 O - Sugar mass fragment information and searched the Glycopedia (https: / / glycopedia.eu / ) and GlycoMass (https: / / glycomass.com / ) databases to preliminarily determine the structures of the 9

[0196] Example 5 O- Further screening and verification of glycan markers First, the inventors further screened the 9 candidate markers determined in Example 4, including comparing their original LC-MS signal intensities, which are shown in Figure 2 D.

[0197] Further screening of grass nest O - Standard for glycan markers: 1) The signal of the marker only exists in the grass nest, but not in the white nest and other adulterants, and the "not" means that its level is lower than the detection limit LOD; 2) The signal intensity should be high enough, for example, signal intensity ≥ 10 4 to ensure the best sensitivity; 3) The structural characteristics of the marker should be clear.

[0198] As Figure 2 D, Figure 3 A, Figure 3 C, Table 1, after comparing the original LC-MS intensities of these markers, GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109 were identified to meet the above standards.

[0199] Subsequently, in view of the fact that grass nest is easy to be adulterated, this embodiment detected the performance of the above grass nest O- Glycan markers in the nest product and potential nest adulterants, and confirmed that the above grass nest O- Glycan markers have good specificity.

[0200] Specifically, the inventors compared the extracted ion chromatograms of the ABEE-labeled natural sugar profiles of the samples of grass bird's nest, white bird's nest and other potential adulterants, thereby verifying the specificity of these identified markers. The results are shown in Figure 3 Table 1.

[0201] Table 1. Detection results of nine candidate markers in different samples of grass bird's nest, white bird's nest, etc.

[0202]

[0203] Figure 3 A shows the chromatograms with different patterns between grass bird's nest, white bird's nest and adulterants. By the extracted ion chromatograms (EIC) of grass bird's nest, white bird's nest and adulterants, it is confirmed that ABEE-labeled GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109 can be used as markers of grass bird's nest, with high specificity. The peaks of each sample that match in mass-to-charge ratio (m / z) and retention time (Rt) were identified. As shown in Figure 4 B, these 6 markers are unique to grass bird's nest, with high specificity.

[0204] Figure 4 The mass-to-charge ratio and retention time of GBM104, GBM105 and GBM106, and the detection results in the extracted ion chromatograms (EIC) of grass bird's nest, white bird's nest and adulterants are shown. GBM104, GBM105 and GBM106 also appeared peaks in the corresponding positions in white bird's nest (B), suggesting that GBM104, GBM105 and GBM106 are not unique markers in grass bird's nest. Figure 5 B).

[0205] Therefore, through further screening and verification, it is confirmed that these 6 O glycans GBM101, GBM102, GBM103, GBM107, GBM108 and GBM109 can be used as markers to distinguish grass bird's nest and white bird's nest.

[0206] More preferred markers From the above-mentioned markers unique to grass bird's nest, more preferred markers are further screened.

[0207] First, in terms of sensitivity, it is desirable that the marker used for detection is sufficiently abundant (high content) to ensure reliable detection even in the presence of only a small amount of substance. In contrast, the 6 O- In the glycan, the response of ABEE-labeled GBM107, GBM108, and GBM109 is lower, while the response of GBM101, GBM102, and GBM103 is higher, and the sensitivity is better in the liquid chromatography-mass spectrometry (LC-MS) analysis. In addition, the peak symmetry of ABEE-labeled GBM108 and GBM109 is poor, which may affect the sensitivity.

[0208] Secondly, considering that these markers are newly discovered by the inventors, there is a lack of commercially available standards, and therefore it is more preferable to use markers that are easier to separate relatively pure compounds from the grass swallow nest. Compared with ABEE-labeled GBM103, ABEE-labeled GBM101 and GBM102 have shorter retention times on the C18 column, and are adjacent to O- The glycan is better separated, and it is easier to separate relatively pure compounds from the grass swallow nest. ABEE-labeled GBM101 and GBM102 can be preferably used as specific markers for identifying the grass swallow nest.

[0209] In summary, O - The glycan GBM101, GBM102, GBM103, GBM107, GBM108, and GBM109 all exhibit high grass swallow nest specificity and can be used as markers for detecting the grass swallow nest; among them, GBM101 and GBM102 are more preferably used for identification of the grass swallow nest.

[0210] Example 6 O- Separation of glycan markers In this example, the same method as in Examples 1 and 2 was used to prepare ABEE-derivatized O glycan samples from 100 grams of grass swallow nest (EBN) powder. O - Six glycan markers, GBM101, GBM102, GBM103, GBM107, GBM108, and GBM109, were separated.

[0211] Experimental procedures First, the above ABEE-derivatized O glycan samples from the grass swallow nest were separated on an open C18 column (ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA) with 10%, 11%, 12%, 13%, 14%, 15%, and 16% acetonitrile (ACN) eluted per column volume, respectively; According to the retention time and peak intensity, the components containing the above six O glycan markers were collected and combined, and then re-separated on an open C18 column (same as above) with 12% and 13% acetonitrile, respectively; The collected target components were subjected to further chromatographic separation on an Alltima C18 analytical column (2.6 x 250 mm, 5 μm); The separated ABEE derivatized markers were obtained using 12% acetonitrile solution containing 0.1% formic acid. The purity of the separated products was monitored using liquid chromatography-mass spectrometry (LC-MS) analysis.

[0212] The purified markers were lyophilized for further structural analysis.

[0213] Example 7 O- Structural analysis of glycan markers The purified markers obtained in Example 6 were subjected to structural characterization using mass spectrometry and nuclear magnetic resonance (NMR). Specifically, the purified markers prepared in Example 6 were analyzed by LC-qTOF-MS / MS to obtain their molecular weights and fragmentation information. O- Glycans, the molecular weights and fragmentation information.

[0214] The markers were dissolved in D2O with TMS (Trimethylsilyl) as an internal standard. The purified markers were subjected to1H and13C NMR spectroscopy using a Bruker Avance 400 spectrometer (Bruker, Germany) at 25 °C at 400 and 100 MHz frequencies, respectively. The chemical shifts were expressed in ppm and calibrated by reference shifts: O- 1H and13C NMR spectra. The chemical shifts were expressed in ppm and calibrated by reference shifts: 1 1H NMR (D2O) at 4.80 ppm, 13 13C NMR (TMS) at 0 ppm, 13 13C NMR (TMS) at 0 ppm, 1 1H NMR (D2O) at 4.80 ppm.

[0215] All data were evaluated using MestReNova. Standard Bruker pulse sequences were also used for 1 1H- 1 1H Correlation NMR spectroscopy (COSY), 1 1H- 13 13C Heteronuclear Multiple Bond Correlation NMR spectroscopy (HMBC), and 1 1H- 13 13C Heteronuclear Single Quantum Coherence spectroscopy (HSQC).

[0216] The structural characterization of GBM102 is shown in Figure 6 , and the structural characterization of GBM101 is shown in Figure 5 .

[0217] Chemical structure analysis The inventors isolated and purified ABEE-labeled GBM101 and GBM102 by the method of Example 6. To accurately identify the structures of the markers GBM101 and GBM102, GBM105 and GBM109 were also isolated.

[0218] The inventors identified GBM101 and GBM102 as GalNAc(Gal)3 isomers based on mass spectrometry, database searching, and published literature on mucin glycan and monosaccharide composition analysis (Tung et al., 2008) (Van Zelst et al., 2019; Wishart et al., 2009). Meanwhile, GBM105 and GBM109 were predicted as GalNAcGal and GalNAc(Gal)2, respectively. More structural details including sugar unit sequence, glycosidic linkage, and anomeric configuration type were obtained by nuclear magnetic resonance spectroscopy. The structures of ABEE-labeled GBM101 and GBM102 were further confirmed by chemical shift assignment of H-NMR and C-NMR spectra. 1 H-NMR and 13 Chemical shift assignment of C-NMR spectra is shown in Table 2.

[0219] The inventors first analyzed GBM105, which is the simplest in structure. As shown in Table 2, 13 C-NMR and 1 Characteristic signals in H-NMR spectra have been completely assigned by DEPT 135, HSQC, 1 H- 1 H COSY and HMBC spectra. ABEE-labeled GBM105 was identified as Gal β (1→4) GalNAc-ABEE. GBM109 was identified as a homolog of GBM105, predicted to be ABEE-labeled Gal β (1→4) Gal β (1→4) GalNAc.

[0220] As Figure 6 shown, based on the structures of GBM105 and GBM109, GBM102 was identified as ABEE-labeled Gal α (1→4) Gal β (1→4) Gal β (1→4) GalNAc. Unlike GBM105 and GBM109, GBM102 presented α anomeric configuration. This configuration was indicated by J values of 4.0 and chemical shifts at C-1 protons 4.99 ppm.

[0221] As Figure 7As shown, due to the same mass-to-charge ratio, the isomer of GBM101 was identified as ABEE-labeled Gal α (1→4)Gal β (1→4)Gal β (1→3)GalNAc.

[0222] The main difference between GBM102 and GBM101 is the linkage between Gal and GalNAc, the rest of the components of GBM101 are consistent with ABEE-labeled GBM102. According to 1 H- 1 H COSY analysis showed that the signal at δH 4.36 was associated with the 2nd carbon atom in the GalNAc-ABEE residue, and was associated with δH 3.93. This signal was further associated with δC 81.7 in HSQC, indicating the presence of (1→3)-linked D-galactopyranose. The correlation between δH 4.36 and δC 81.7 in HMBC further supported this analysis.

[0223] Table 2 Chemical shifts assignment based on ABEE-labeled GBM101 and GBM102 1 H-NMR and 13 C-NMR spectra

[0224] Example 8 Identification of bird’s nest raw material pieces and products ABEE-derivatized glycan samples were prepared from 40 batches of bird’s nest raw material pieces (RM-001~RM-040) and 69 batches of products (P-01~P-69) collected using the same method as in Example 1 and Example 2. O - glycan samples.

[0225] These samples were detected by LC-qTOF-MS using the same method as in Example 3-5, and the raw material pieces and products containing ABEE-labeled GBM101 and GBM102 were identified as containing grass bird’s nest. The determination results are shown in Figure 7 and Table 3. As shown in Figure 7 A and Table 3, 12 batches of samples in the raw materials contained grass bird’s nest.

[0226] Table 3

[0227]

[0228] Table 4

[0229]

[0230] The tested bird's nest products mostly claimed to be made of or contain white bird's nest, but there were also cases of adulteration with grass bird's nest. ​ As shown in Table 4, characteristic peaks of GBM101 and GBM102 were detected in 15 out of 69 samples of bird's nest products. These 15 bird's nest products actually contained grass bird's nest.

[0231] The method and markers of the present application can be used to identify bird's nest raw materials and products, and detect whether they contain grass bird's nest components.

[0232] Industrial applicability The present application relates to a series of quality control markers derived from the alkali hydrolysis-released O- glycans of grass bird's nest, as well as methods for screening these markers, and related methods for qualitatively and quantitatively identifying bird's nest raw materials and products using these O- glycan markers.

[0233] The above description of the present application is made in combination with preferred embodiments, but these embodiments are only exemplary and serve only to illustrate the present application. On this basis, various substitutions and improvements can be made to the present application, and these all fall within the scope of protection of the present application.

Claims

1. A method for identifying a bird's nest product comprising cubilago, the method comprising: A1) subjecting a sample from a bird's nest product to a chemical deglycosylation to release the O glycans therein, obtaining O a glycans mixture sample; A2) contacting the O- glycan mixture sample with 4-aminobenzoic acid ethyl ester (ABEE) or an analogue thereof, to obtain a labeled O- glycan sample; A3) detecting the labeled glycan samples obtained in A2) using liquid chromatography-mass spectrometry (LC-MS) to obtain data characteristic of the O- glycans of the bird's nest product O- glycans; A4) subjecting the product obtained in step A3) to O- The data characterizing the glycans are compared with data of the specific markers of the edible bird's nest and it is determined whether the sample from the bird's nest product comprises the specific markers of the edible bird's nest and thereby it is identified whether the bird's nest product comprises the edible bird's nest, wherein the cubilago-specific marker comprises one or more of the following: - Glycan; ABEE labeled with retention time 8.369 min and mass to charge ratio 901.3294 determined by LC-MS O - Glycan; ABEE labeled with retention time 6.898 min and mass to charge ratio 901.3294 determined by LC-MS O - Glycan; ABEE labeled with retention time 17.007 min and mass to charge ratio 943.3436 determined by LC-MS O - Glycan; ABEE labeled with retention time 10.506 min and mass to charge ratio 490.1941 determined by LC-MS O - Glycan; ABEE labeled with retention time 11.100 min and mass to charge ratio 739.2767 determined by LC-MS O - Glycan; ABEE labeled with retention time 9.335 min and mass to charge ratio 739.2767 determined by LC-MS O - Glycan.

2. The method of claim 1, wherein, the chemical deglycosylation comprises subjecting the sample to a beta-elimination reaction, preferably subjecting the sample to a beta-elimination reaction comprises hydrolyzing the sample with an alkaline hydrolysis agent, preferably the alkaline hydrolysis agent is selected from the group consisting of NaOH, KOH, LiOH, Na2CO3, K2CO3, NH4OH.

3. The method of claim 2, wherein, the alkaline hydrolysis agent is selected from NaOH.

4. The method of any one of claims 1-3, wherein, the liquid chromatography-mass spectrometry technique in step A3) is selected from LC-DAD-qTOF-MS.

5. The method according to claims 1-4, wherein, the data obtained in step A3) comprises one or more of the following: retention time, mass to charge ratio, molecular weight, preferably at least retention time, mass to charge ratio.

6. The method of claims 1-5, wherein, step A4) comprises determining whether the retention time, mass to charge ratio corresponds to the retention time, mass to charge ratio of the cubilago-specific marker.

7. The method of any one of claims 1-6, wherein, The said grass swallow nest specific markers comprise: ABEE labeled with a retention time of 8.369 min and a mass-to-charge ratio of 901.3294 determined by LC-MS O - polysaccharides; ABEE labeled with a retention time of 6.898 min and a mass-to-charge ratio of 901.3294 determined by LC-MS O - at least one of the polysaccharides.

8. The method of any one of claims 1-7, wherein, the bird's nest product is selected from one or more of the following: raw material, processed raw material, downstream product prepared from raw material.

9. Use of the cubilago-specific marker of claim 1 for identifying a bird's nest product.

10. The use according to claim 9, wherein the grass swallow nest specific markers comprise: at least one of - glycans (GBM101); ABEE labeled with a retention time of 6.898 min and a mass to charge ratio of 901.3294 determined by LC-MS O - glycans (GBM101); ABEE labeled with a retention time of 6.898 min and a mass to charge ratio of 901.3294 determined by LC-MS O - glycans (GBM102) determined by LC-MS. wherein said GBM101 has the structure Gal α (1→4) Gal β (1→4) Gal β (1→3) GalNAc; said GBM102 has the structure Gal α (1→4) Gal β (1→4) Gal β (1→4) GalNAc.

11. A method for screening for a specific marker for identifying cubilago, comprising the steps of: S1 ) chemically deglycosylating the grass bird's nest sample, the white bird's nest sample and the adulterant, respectively, to release O - polysaccharides, wherein the chemical deglycosylation is preferably selected from the group consisting of beta-elimination reaction; S2) labelling said glycan with ABEE or an analogue thereof; O - a glycan is labelled with ABEE or an analogue thereof; S3) analyzing the labeled O glycans of said raw bird's nest sample, said white bird's nest sample and said adulterant by LC-MS to obtain labeled O glycan profiles of said raw bird's nest sample, said white bird's nest sample and said adulterant; S4) performing a partial least squares discriminant analysis (PLS-DA) on the O - the PLS-DA analysis extracting O - the contribution of the glycans as variables to the discrimination of the grass bird's nest and white bird's nest classes, said contribution being expressed as O - the VIP values of the glycans, The O - Glycan profile VIP values greater than 3 and expressed only in grass swallow nests O - Glycans as potential specific markers for identifying grass swallow nests; and S5) comparing the raw LC-MS intensities of potential specific markers for identifying cubilago to determine a cubilago-specific marker.

12. The method of claim 11, wherein the grass swallow's nest specific marker is selected from one or more of: an ABEE labeled -glycan with a retention time of 8.369 min and a mass to charge ratio of 901.3294 determined by LC-MS O an ABEE labeled -glycan with a retention time of 6.898 min and a mass to charge ratio of 901.3294 determined by LC-MS O an ABEE labeled -glycan with a retention time of 17.007 min and a mass to charge ratio of 943.3436 determined by LC-MS O an ABEE labeled -glycan with a retention time of 10.506 min and a mass to charge ratio of 490.1941 determined by LC-MS O an ABEE labeled -glycan with a retention time of 11.100 min and a mass to charge ratio of 739.2767 determined by LC-MS O an ABEE labeled -glycan with a retention time of 9.335 min and a mass to charge ratio of 739.2767 determined by LC-MS O an ABEE labeled -glycan with a retention time of 9.335 min and a mass to charge ratio of 739.2767 determined by LC-MS 13. The method of claim 11 or 12, wherein the method further comprises the step of S6) further screening for a specific marker for identifying cubilago based on the response and retention time in the LC-MS analysis.

14. The method according to any one of claims 11-13, wherein the specific marker selected for identifying grass bird’s nest is selected from: an ABEE labeled -glycan with a retention time of 8.369 min and a mass to charge ratio of 901.3294 determined by LC-MS O an ABEE labeled -glycan with a retention time of 6.898 min and a mass to charge ratio of 901.3294 determined by LC-MS O an ABEE labeled -glycan.

15. The method of any one of claims 11-14, wherein the method comprises one or more of the following steps; B1) contacting the grass bird's nest sample, the white bird's nest sample and the adulterant with an alkaline hydrolysis agent to undergo a beta-elimination reaction to form O - a step of the glycan mixture sample; B2) contacting O - contacting the glycan mixture sample with an acidic neutralizing agent to neutralize, obtaining a neutralized sample, centrifuging the neutralized sample, discarding the pellet, obtaining a supernatant O - a glycan sample, The supernatant is collected O - the glycan sample is labeled with 4-aminobenzoic acid ethyl ester (ABEE) or an analogue thereof O - the glycan is labeled, obtaining a labeled O - glycan; B3) isolating the labeled glycan of B2) by chromatography O - the labeled glycan is isolated by chromatography O - the labeled glycan is isolated by chromatography Preferably, said supernatant obtained under B2) is O - the glycan sample comprises one or more of the following O - the glycan: ABEE labeled with a retention time of 8.369 min and a mass to charge ratio of 901.3294 determined by LC-MS O - the glycan: ABEE labeled with a retention time of 6.898 min and a mass to charge ratio of 901.3294 determined by LC-MS O - the glycan: ABEE labeled with a retention time of 17.007 min and a mass to charge ratio of 943.3436 determined by LC-MS O - the glycan: ABEE labeled with a retention time of 10.506 min and a mass to charge ratio of 490.1941 determined by LC-MS O - the glycan: ABEE labeled with a retention time of 11.100 min and a mass to charge ratio of 739.2767 determined by LC-MS O - the glycan: ABEE labeled with a retention time of 9.335 min and a mass to charge ratio of 739.2767 determined by LC-MS O - the glycan.

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