Method for constructing a standard database for the provenance of bird's nest based on proteomics analysis and application

Abstract

The application relates to a method for constructing a bird's nest origin traceability standard database based on proteomics analysis and application; characteristic protein information in bird's nest samples of known origin is extracted by using a proteomics analysis method, an extraction system containing specific ionic liquid, subcritical water and a reducing agent is combined with inert gas and ultrasonic treatment, an alkylation, ultrafiltration and enzymolysis process is used to promote the sufficient release of glycoproteins in the bird's nest, rich characteristic protein information is retained, characteristic protein data extraction is conducted in combination with liquid chromatography tandem mass spectrometry and a protein database, a bird's nest origin traceability standard database corresponding to characteristic protein information with high coverage and high abundance and origin information is formed, and the bird's nest origin traceability standard database is constructed; the database is suitable for different origin bird's nest samples, can accurately and comprehensively reflect the characteristic protein information difference of different origin bird's nests, realizes rapid, accurate and objective tracing of the origin of unknown origin bird's nest samples, and has important significance for promoting the sustainable development of the bird's nest industry.

Description

Technical Field

[0001] This application relates to the field of bird's nest origin traceability technology, and in particular to a method and application for constructing a standard database for bird's nest origin traceability based on proteomics analysis. Background Technology

[0002] Bird's nest is mainly the saliva secretion of swiftlets during their breeding season. It is a precious tonic. Bird's nests from different places of origin vary in quality, including nutritional components, efficacy, and taste. Therefore, identifying the origin of bird's nest is one of the keys to ensuring its quality.

[0003] Traditional methods for identifying the origin of bird's nests primarily rely on sensory evaluation. This involves observing the nest's appearance, such as its shape, color, and texture, as well as smelling and tasting it to roughly determine its origin. However, this method is highly subjective, and different experts may arrive at significantly different conclusions. Furthermore, it struggles to accurately distinguish bird's nests from similar origins. Secondly, chemical analysis methods are used for bird's nest origin identification, such as measuring the sialic acid content and amino acid composition. While this improves accuracy to some extent, the chemical composition of bird's nests fluctuates due to environmental factors during their formation, making precise traceability difficult using only chemical analysis. Therefore, currently, there is no system or method that can achieve accurate and reliable traceability of bird's nest origins. Summary of the Invention

[0004] Based on this, this application provides a method for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis. This method utilizes the differences in characteristic bird's nest protein information in bird's nest samples from different known origins to distinguish bird's nests from different origins, constructs a traceability standard database corresponding to characteristic protein information and origin, and enables accurate traceability of bird's nest samples from unknown origins, providing a reliable basis for the quality of bird's nests and related products.

[0005] Firstly, this application provides a method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis, including the following steps:

[0006] Bird's nest samples from known different origins were collected, and proteomics analysis was performed independently on each of the bird's nest samples from known different origins to obtain characteristic protein data and corresponding origin information, forming a standard database for bird's nest origin traceability.

[0007] The proteomics analysis includes the following steps:

[0008] A mixture was prepared by mixing bird's nest samples with an extract containing ionic liquid, subcritical water, and a reducing agent.

[0009] An inert gas is introduced into the mixture to form inert gas microbubbles, and the mixture is subjected to ultrasonic treatment. The supernatant is then separated and subjected to alkylation treatment to prepare a protein solution.

[0010] The protein solution was subjected to ultrafiltration and enzymatic hydrolysis to prepare bird's nest peptides;

[0011] Characteristic protein data of the bird's nest peptides were extracted using liquid chromatography-tandem mass spectrometry and a protein database.

[0012] The ionic liquid is selected from 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt; the reducing agent is selected from tris(3-hydroxypropyl)phosphine.

[0013] In some embodiments, the mass ratio of the ionic liquid, the subcritical water, and the reducing agent is (0.5-1.5):(0.5-1.5):(0.01-0.03); and / or, the mass-to-volume ratio of the bird's nest sample to the extract is 1g:(10-50)mL; and / or, the temperature of the subcritical water is 150℃-200℃, and the pressure is 2MPa-15MPa.

[0014] In some embodiments, the content of the inert gas microbubbles in the mixture is 1 × 10⁻⁶. 5 Cells / mL - 1×10 7 per mL; and / or, the inert gas includes one or more of nitrogen, argon and helium.

[0015] In some embodiments, the ultrasonic treatment includes pulsed focused ultrasound treatment;

[0016] The frequency of the pulsed focused ultrasound treatment is 20 kHz-50 kHz, preferably 35 kHz, and the power is 200 W-500 W, preferably 320 W. The pulsed focused ultrasound treatment process consists of a 10-second operation followed by a 5-second pause, and the process includes at least 15 cycles.

[0017] In some embodiments, the ultrafiltration includes at least one molecular weight fractionation ultrafiltration, wherein the ultrafiltration employs an ultrafiltration membrane with a molecular weight cutoff of 3 kDa-30 kDa;

[0018] In the ultrafiltration process, the protein solution is mixed with a buffer salt solution and then ultrafiltration is performed; the ultrafiltration membrane is a PEG-modified ultrafiltration membrane, and the molecular weight of the PEG in the PEG-modified ultrafiltration membrane is 1000 Da-2000 Da.

[0019] In some embodiments, the ultrafiltration employs at least two-stage molecular weight fractional ultrafiltration, wherein the first stage of ultrafiltration employs an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the second stage of ultrafiltration employs an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0020] or,

[0021] Ultrafiltration employs at least three molecular weight fractionation ultrafiltration stages, wherein the first stage of ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, the second stage of ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, and the third stage of ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0022] In some embodiments, the alkylation reagent used in the alkylation step includes one or more of iodoacetamide, N-ethylmaleimide and 4-vinylpyridine; and / or, the enzyme used for enzymatic hydrolysis includes trypsin; and / or, the amount of enzyme added during enzymatic hydrolysis is 2%-5% of the mass of the protein in the ultrafiltrate obtained after the ultrafiltration step.

[0023] In some embodiments, the bird's nest sample is prepared by grinding and / or pressurizing the raw bird's nest;

[0024] The grinding process uses liquid nitrogen grinding.

[0025] The pressure for the pressurization process is 100 MPa-2000 MPa, the temperature is 20℃-30℃, and the time is 1 min-10 min.

[0026] In some embodiments, the step of extracting characteristic protein data of the bird's nest peptide by liquid chromatography-tandem mass spectrometry and a protein database includes: performing liquid chromatography-tandem mass spectrometry analysis on the bird's nest peptide to obtain mass spectrometry data, importing the mass spectrometry data into Proteome Discoverer software for analysis, and extracting characteristic protein data of the bird's nest peptide.

[0027] The parameters of the liquid chromatography-tandem mass spectrometry include liquid chromatography parameters and mass spectrometry parameters:

[0028] The liquid chromatography parameters included: a C18 column, a column temperature of 37℃, a flow rate of 0.2 mL / min-0.3 mL / min, a detection wavelength of 214 nm, mobile phase A being an aqueous solution containing 0.1% formic acid and 0.5 mM ammonium formate, and mobile phase B being an acetonitrile solution containing 0.1% formic acid. Gradient elution was used, with the following program: 0 min-5 min, maintaining mobile phase B at 3% volume; 5 min-20 min, increasing mobile phase B from 3% to 15% volume; 20 min-40 min, increasing mobile phase B from 15% to 30% volume; 40 min-50 min, increasing mobile phase B from 30% to 90% volume; and 50 min-55 min, maintaining mobile phase B at 90% volume.

[0029] The mass spectrometry parameters include: an electrospray ionization source in positive ion mode and DIA scanning mode, wherein the first-stage scanning range is 200 m / z-2000 m / z, the second-stage scanning range is 100 m / z-1800 m / z, and the resolution is 60000-120000; the ion source parameters include a spray voltage of 2.0 kV-2.5 kV, a capillary temperature of 300℃-350℃, a sheath gas flow rate of 30 arb-40 arb, and an auxiliary gas flow rate of 10 arb-15 arb.

[0030] Secondly, this application also provides a method for accurate traceability of bird's nest origin using a standard database for bird's nest origin traceability, including the following steps:

[0031] Collect bird's nest samples from unknown origins, and use the proteomics analysis method in the method for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis provided in the first aspect to perform proteomics analysis on the bird's nest samples from unknown origins, and obtain characteristic protein data of the bird's nest samples from unknown origins.

[0032] The method provided in the first aspect for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis analyzes the characteristic protein data of bird's nest samples from unknown origins to obtain the origin information of the bird's nest samples from unknown origins, thereby achieving origin tracing of the bird's nest samples from unknown origins.

[0033] Compared with traditional technologies, the beneficial effects of the technical solution in this application include:

[0034] This application develops a method for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis. It utilizes proteomics analysis to extract characteristic protein information from bird's nest samples from different known origins, forming a standard database for tracing the origin of bird's nests that corresponds to the characteristic protein information and the origin information. Compared to traditional techniques, the standard database for tracing the origin of bird's nests constructed in this application has high sensitivity and accuracy, enabling efficient and accurate tracing of bird's nest origins.

[0035] This application aims to improve the richness and accuracy of characteristic protein information extraction from bird's nest samples. A specific proteomics analysis method is developed, employing an extraction system comprising "ionic liquid + subcritical water + reducing agent" to extract bird's nest samples, promoting the full release of glycoproteins. Combined with inert gas dispersion and ultrasonic treatment, extraction efficiency is enhanced. Following alkylation, ultrafiltration, and enzymatic hydrolysis, protein peptides retaining rich characteristic protein information are prepared. Characteristic protein information is extracted from these protein peptides using liquid chromatography-tandem mass spectrometry and a protein database. Adapted to bird's nest samples from different origins, this method accurately and comprehensively reflects the differences in characteristic protein information from different origins, achieving a one-to-one correspondence between characteristic protein information and origin information. This forms a high-coverage and high-abundance standard database for bird's nest origin traceability, enabling rapid and accurate traceability of bird's nest samples from unknown origins. The technical solution of this application solves the technical problems of low accuracy, susceptibility to human interference, and long testing cycle of traditional bird's nest origin identification methods (such as morphological identification and DNA barcoding) in deep-processed bird's nest. Attached Figure Description

[0036] Figure 1 The flowchart of the proteomics analysis in this application is shown.

[0037] Figure 2 This document presents a flowchart illustrating the origin traceability process for bird's nests in this application.

[0038] Figure 3 The mass spectrum of the bird's nest peptide sample No. 1 obtained in Application Example 1 of this application is shown. Detailed Implementation

[0039] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0041] As used herein, "optional," "optional," and "optional" refer to either "with" or "without" parallel options. If multiple "optional" entries appear in a technical solution, each "optional" entry is independent unless otherwise specified and there are no contradictions or mutual constraints. The term "and / or" as used herein includes any and all combinations of one or more related listed items. Unless otherwise specified, "multiple," "multiple," etc., as used herein refer to a quantity greater than 2 or equal to 2; for example, "one or more" indicates one, two, or more than two. In open-ended technical features or solutions described herein using words such as "containing," "including," and "comprising," unless otherwise specified, additional members beyond the listed members are not excluded. This can be considered as providing both a closed-ended feature or solution consisting of the listed members and an open-ended feature or solution that includes additional members beyond the listed members.

[0042] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0043] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0044] In this application, inert gas microbubbles refer to microbubbles, also known as micro-nano bubbles, which are tiny bubbles with diameters ranging from tens of nanometers to tens of micrometers. The International Organization for Standardization (ISO) defines them as having a particle size of less than 100 μm. In this application, inert gas microbubbles refer to tiny bubbles with a particle size of 100 nm to 100 μm formed by dispersing chemically inert gases (such as nitrogen, argon, and helium) through a microbubble generator. These microbubbles are chemically stable in the extraction system, do not react with the components in the system, and can generate a cavitation effect under ultrasonic action, which assists in the cleavage and release of bird's nest protein.

[0045] Traditional methods of identifying the origin of bird's nests using sensory evaluation and chemical methods have proven ineffective. Previous research employed molecular biology techniques to trace the origin of bird's nests, analyzing specific gene fragments to determine their species origin and thus infer their place of origin. However, the application of this method is limited by the fact that bird's nest DNA is easily degraded during processing, and there may be cross-species interaction between swiftlets from different origins. Furthermore, proteomics, as the executor of functions within organisms, is regulated by both genetic and environmental factors. Bird's nests from different origins exhibit variations in protein composition and expression levels due to differences in the swiftlets' living environment and food sources. The applicant's extensive research revealed that existing protein extraction technologies are ineffective for extracting proteins from bird's nest. For example, traditional urea and SDS extraction systems for glycoprotein extraction from bird's nest still result in a large amount of poorly soluble matter remaining in the samples, failing to effectively disrupt the complex glycoprotein cross-linking structure. This leads to extremely low protein extraction efficiency, the loss of numerous low-soluble proteins with distinctive geographical origin fingerprints, and a limited number of peptides obtainable after enzymatic hydrolysis. Consequently, the number of identifiable proteins in a single sample is typically low, making it difficult to establish stable and reproducible geographical origin fingerprints. Furthermore, while strong surfactants like SDS can improve the solubility of bird's nest, they severely interfere with subsequent enzymatic hydrolysis and mass spectrometry signals, causing a decrease in resolution. Conversely, mild extraction solutions are incapable of dissolving bird's nest.

[0046] Therefore, this application aims to efficiently extract rich characteristic bird's nest protein information from bird's nest samples from different origins, thereby utilizing the differences in characteristic protein information to form a standard database for tracing the origin of bird's nests from different origins, so as to achieve accurate, objective and efficient tracing of bird's nest samples from unknown origins.

[0047] Firstly, this application provides a method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis, including the following steps:

[0048] Bird's nest samples from known origins were collected, and proteomics analysis was performed independently on each sample to obtain characteristic protein data and corresponding origin information, thus forming a standard database for bird's nest origin traceability.

[0049] See appendix Figure 1 As shown, proteomics analysis includes the following steps:

[0050] S1. Mix the bird's nest sample with an extract containing ionic liquid, subcritical water and reducing agent to prepare a mixture.

[0051] S2. Inert gas is introduced into the mixture to form inert gas microbubbles, and the mixture is subjected to ultrasonic treatment. The supernatant is separated and alkylated to prepare protein solution.

[0052] S3. The protein solution is subjected to ultrafiltration and enzymatic hydrolysis to prepare bird's nest peptides.

[0053] S4. Characteristic protein data of bird's nest peptides were extracted by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and protein database.

[0054] The ionic liquid is selected from 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ([HMIM][Tf2N]); the reducing agent is selected from tris(3-hydroxypropyl)phosphine (THP).

[0055] It should be noted that after preparing bird's nest peptides, the characteristic protein data of bird's nest peptides are extracted. This process mainly involves reorganizing the information of bird's nest proteins. In this way, the obtained characteristic protein data of bird's nest can form a high-abundance standard database for tracing the origin of bird's nest.

[0056] Traditional methods for constructing databases using proteomics analysis present significant technical challenges. Bird's nest is not a simple protein mixture; it is rich in highly glycosylated glycoproteins (sialic acid glycoproteins) with an extremely dense structure and numerous disulfide bonds and hydrophobic interactions between molecules. This unique structure results in extremely poor solubility of bird's nest in water and conventional protein lysis solutions (such as urea and SDS). Therefore, directly applying traditional proteomics pretreatment methods to bird's nest often leads to low protein extraction rates, incomplete extraction, and even biased protein extraction (only extracting readily soluble proteins while losing crucial poorly soluble characteristic proteins). Consequently, it is impossible to obtain bird's nest protein peptide information, compromising the accuracy and reliability of bird's nest origin traceability.

[0057] Based on this, this application develops a specific proteomics analysis method for the efficient and thorough extraction of insoluble glycoprotein matrix from bird's nest. The extraction system, comprising "specific ionic liquid + subcritical water + reducing agent," has a strong penetrating effect on the mucin structure in bird's nest, fully releasing the highly glycosylated and complex network structure of insoluble glycoproteins. This solves the technical problem that conventional extraction agents have difficulty penetrating mucin, providing favorable conditions for preserving the rich bird's nest protein information in bird's nest peptides and realizing the extraction of bird's nest protein information.

[0058] The ionic liquid used in this application contains both anions and cations, possessing an amphiphilic structure capable of forming hydrogen bonds with proteins, thereby competitively disrupting the intramolecular and intermolecular hydrogen bond network of bird's nest protein. Specifically, the alkyl imidazoline in [HMIM][Tf2N] serves as the cationic portion of the ionic liquid. The alkyl chain further disrupts the protein's folding structure through hydrophobic interactions, allowing the bird's nest protein to be fully released and dissolved from the dense matrix. Furthermore, the medium-chain alkyl group used in this ionic liquid is more easily removed by ultrafiltration than the long-chain alkyl group, avoiding residual interference. The fluorosulfonylimide anion is used as the anionic portion of the ionic liquid. The fluorosulfonylimide anion has a stronger hydrogen bond-breaking ability, specifically dissociating the intermolecular hydrophobic interactions of bird's nest glycoprotein. Furthermore, subcritical water is used as an auxiliary solvent to soften the dense glycoprotein network structure of bird's nest, forming a "dissolution-penetration" synergistic effect with a specific ionic liquid, significantly improving the extraction rate of poorly soluble glycoproteins. Simultaneously, the synergistic reducing agent efficiently and synergistically enhances the reduction of disulfide bonds without affecting subsequent enzymatic hydrolysis. Therefore, this application, through a synergistic extraction system of "specific ionic liquid + subcritical water + reducing agent," significantly enhances the release of glycoproteins, obtains abundant bird's nest protein information from bird's nest samples, and solves the technical problems of low content, low coverage, and low abundance of characteristic protein information in the process of constructing databases for proteomics analysis methods, achieving efficient and accurate traceability of bird's nest origin.

[0059] In some embodiments, the reducing agent is selected from tris(3-hydroxypropyl)phosphine (THP). Compared with DTT, THP is less susceptible to oxidation and more stable. Compared with TCEP, THP does not produce precipitates and is more compatible with ionic liquids.

[0060] In some embodiments, the mass ratio of ionic liquid, subcritical water and reducing agent is (0.5-1.5): (0.5-1.5): (0.01-0.03).

[0061] In some embodiments, the mass-to-volume ratio of bird's nest sample to extract is 1g: (10-50)mL.

[0062] Subcritical water, due to its unique state and microstructure, possesses excellent chemical activity and solubility. This application uses subcritical water as an auxiliary solvent, in conjunction with ionic liquids and reducing agents, to significantly improve the dissolution and release efficiency of highly glycosylated and complex network-structured insoluble glycoproteins in mucin. More preferably, the parameters of subcritical water within a temperature range of 150℃-200℃ and a pressure range of 2MPa-15MPa better achieve the extraction effect.

[0063] This application utilizes inert gas microbubbles combined with pulsed focused ultrasound to achieve cavitation effect. In conjunction with a specific extraction system, this rapidly, efficiently, and fully releases insoluble glycoproteins, improving the extraction efficiency of characteristic protein information from bird's nest. Specifically, the content of inert gas microbubbles in the mixture is 1×10⁻⁶. 5 Cells / mL - 1×10 7 A concentration of 100 cells / mL can achieve better extraction results.

[0064] In some embodiments, the inert gas includes one or more of nitrogen, argon, and helium.

[0065] The preparation method of this application employs ultrasonic-assisted treatment to further promote the penetration of the extract and the release of glycoproteins. As a non-limiting example, the ultrasonic treatment includes pulsed focused ultrasound treatment; the frequency of the pulsed focused ultrasound treatment is 20 kHz-50 kHz, and the power is 200 W-500 W. More preferably, the frequency of the pulsed focused ultrasound treatment is 35 kHz, and the power is 320 W. The pulsed focused ultrasound treatment process consists of a 10-second operation followed by a 5-second pause, and the process includes at least 15 cycles.

[0066] In some embodiments, ultrafiltration includes at least one stage of molecular weight fractional ultrafiltration, wherein the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to 30 kDa. Further, the ultrafiltration employs at least two stages of molecular weight fractional ultrafiltration, wherein the first stage of ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, and the second stage of ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

[0067] In some preferred embodiments, ultrafiltration employs at least three-stage molecular weight gradient ultrafiltration. The first stage uses an ultrafiltration membrane with a molecular weight cutoff of 30 kDa, the second stage uses a membrane with a molecular weight cutoff of 10 kDa, and the third stage uses a membrane with a molecular weight cutoff of 3 kDa. This at least three-stage gradient ultrafiltration process achieves the technical effects of stepwise removal of macromolecular impurities, enrichment of target proteins, and deep desalination. Preferably, the pre-stage ultrafiltration collects the permeate to remove macromolecular impurities, and the final stage ultrafiltration collects the retentate to enrich bird's nest proteins or peptides within the target molecular weight range, avoiding the "co-retention of impurities" problem of single-stage ultrafiltration. Using at least three stages of ultrafiltration better achieves the reduction of impurities while maintaining rich characteristic protein information, thereby improving the resolution of bird's nest peptide characteristics.

[0068] In some embodiments, the ultrafiltration membrane is a PEG-modified ultrafiltration membrane, wherein the molecular weight of the PEG in the PEG-modified ultrafiltration membrane is 1000 Da-2000 Da. It is understood that the PEG-modified ultrafiltration membrane is modified with PEG (polyethylene glycol) having a molecular weight of 1000 Da-2000 Da. Further, the molecular weight of the PEG is 2000 Da, and PEG-2000 indicates that the molecular weight of the polyethylene glycol is 2000.

[0069] In some embodiments, the preparation method of the PEG-modified ultrafiltration membrane includes immersing an initial ultrafiltration membrane in a polyethylene glycol (PEG) solution. It should be noted that introducing PEG, a hydrophilic polymer, into the surface or structure of the ultrafiltration membrane alters its physicochemical properties. By forming a surface resistant to protein adsorption, PEG modification ensures that most target proteins, after being trapped due to size, are stably retained in the concentrate, rather than being wasted by being "stuck" on the membrane. The non-specific adsorption of proteins by unmodified membranes leads to the direct loss of a large amount of target proteins (such as drugs, enzymes, etc.) onto the membrane, reducing product yield. This application utilizes a PEG-modified ultrafiltration membrane, which significantly reduces protein adsorption by the ultrafiltration membrane, minimizing protein loss and achieving a protein recovery rate increase of over 95%.

[0070] In some embodiments, the ultrafiltration step includes adding a buffer salt solution to the protein solution, mixing, and then performing ultrafiltration. As a non-limiting example, the buffer salt solution includes one or more of ammonium bicarbonate (NH4HCO3) solution, Tris-HCl buffer, and phosphate buffered saline (PBS).

[0071] In some embodiments, the alkylating agent used in the alkylation step includes one or more of iodoacetamide (IAA), N-ethylmaleimide (NEM), and 4-vinylpyridine (4-VP).

[0072] In some embodiments, the alkylation step includes: adding a reducing agent to the supernatant and treating it at 50°C-60°C, then adding an alkylating reagent and treating it to prepare a protein solution. Optionally, during the alkylation process, the pH of the solution is 8.0.

[0073] In some embodiments, the reducing agent in the alkylation step comprises an organophosphorus reducing agent. Further, the organophosphorus reducing agent comprises trihydroxyalkyl-substituted phosphine. As a non-limiting example, trihydroxyalkyl-substituted phosphine comprises tris(3-hydroxypropyl)phosphine (THP).

[0074] In some embodiments, during the alkylation step, the amount of reducing agent added is 0.2%-0.8% of the mass of the supernatant.

[0075] In some embodiments, the amount of alkylating agent added is 1%-2% of the mass of the supernatant.

[0076] In some embodiments, prior to the alkylation step, the mixture after ultrasonic treatment is further subjected to heat treatment at 80°C-95°C and centrifuged to obtain the supernatant. Heat treatment can precipitate and remove impurities such as extraneous proteins and insoluble macromolecular impurities from the mixture, allowing the bird's nest protein to fully denature and expose its thiol groups, thereby improving the efficiency of subsequent reduction and alkylation reactions. The heat treatment time is 20-40 minutes.

[0077] In some embodiments, the enzymatic hydrolysis step includes: adding an enzyme to the ultrafiltrate collected after ultrafiltration for treatment.

[0078] In some embodiments, the enzyme used for enzymatic hydrolysis includes one or more of trypsin, alkaline protease, and flavor protease. Trypsin is preferred.

[0079] In some embodiments, the amount of enzyme added during enzymatic hydrolysis is 2%-5% of the mass of the protein in the ultrafiltrate obtained after the ultrafiltration step.

[0080] In some embodiments, the bird's nest sample is prepared by grinding and / or pressurizing the raw bird's nest.

[0081] As a non-limiting example, the grinding process employs liquid nitrogen grinding. Liquid nitrogen grinding refers to grinding the bird's nest raw material under a liquid nitrogen atmosphere. The ultra-low temperature (-196℃) of liquid nitrogen can instantly suppress the heat generation effect during the grinding process, preventing the denaturation, degradation, or loss of activity of heat-sensitive proteins in the bird's nest, thus preserving the natural protein structure to the greatest extent. It also significantly increases the brittleness of the bird's nest tissue, allowing for rapid grinding into a fine powder with uniform particle size. This avoids the problems of uneven particle size and protein agglomeration caused by grinding at room temperature, providing a good mass transfer basis for subsequent extraction. Grinding is a key step in achieving sample homogenization, efficient mass transfer, and process adaptation. As a non-limiting example, the particle size of the ground bird's nest sample is <100 mesh.

[0082] As a non-limiting example, the pressure of the pressurization treatment is 100 MPa-2000 MPa, the temperature is 20℃-30℃, and the time is 1 min-10 min.

[0083] In some preferred embodiments, liquid nitrogen grinding combined with pressure treatment can make the particle size distribution of bird's nest sample powder more uniform, providing a better pre-state for the subsequent full dissolution of proteins.

[0084] In some embodiments, the step of extracting characteristic protein data of bird's nest peptides by liquid chromatography-tandem mass spectrometry (LC-MS / MS) and a protein database includes: performing LC-MS / MS analysis on bird's nest peptides to obtain mass spectrometry data, importing the mass spectrometry data into Proteome Discoverer software for analysis, and extracting characteristic protein data of bird's nest peptides.

[0085] In some embodiments, the parameters of liquid chromatography-tandem mass spectrometry (LC-MS / MS) include liquid chromatography parameters and mass spectrometry parameters:

[0086] The liquid chromatography parameters included: a C18 column, a column temperature of 37℃, a flow rate of 0.2 mL / min-0.3 mL / min, a detection wavelength of 214 nm, mobile phase A being an aqueous solution containing 0.1% formic acid and 0.5 mM ammonium formate, and mobile phase B being an acetonitrile solution containing 0.1% formic acid. Gradient elution was used, with the following program: 0 min-5 min, maintaining mobile phase B at 3% volume; 5 min-20 min, increasing mobile phase B from 3% to 15% volume; 20 min-40 min, increasing mobile phase B from 15% to 30% volume; 40 min-50 min, increasing mobile phase B from 30% to 90% volume; and 50 min-55 min, maintaining mobile phase B at 90% volume.

[0087] The mass spectrometry parameters include: positive ion mode using an electrospray ionization source, DIA scanning mode, with a first-stage scan range of 200 m / z-2000 m / z, a second-stage scan range of 100 m / z-1800 m / z, and a resolution of 60000-120000; the ion source parameters include a spray voltage of 2.0 kV-2.5 kV, a capillary temperature of 300℃-350℃, a sheath gas flow rate of 30-40 arb, and an auxiliary gas flow rate of 10 arb-15 arb.

[0088] As a non-limiting example, the C18 column includes a core-shell C18 column or a fully porous particulate C18 column. A core-shell C18 column is preferred.

[0089] As a non-limiting example, the mass spectrometry in liquid chromatography-tandem mass spectrometry employs Orbitrap high-resolution mass spectrometry.

[0090] In some embodiments, before acquiring characteristic protein data, the method further includes separating and enriching bird's nest peptides using a high-pH chromatographic separation mode to achieve bird's nest peptide grading. Grading can significantly improve the separation degree and detection coverage of the first bird's nest peptide, enrich characteristic peptide segments from bird's nests of different origins, enhance the degree of difference in protein information, and thus enhance the distinguishing ability and accuracy of the bird's nest origin traceability standard database.

[0091] This application utilizes a specific proteomics analysis process to promote the full release of proteins in bird's nest, retain rich characteristic protein information, obtain in-depth data support for the complete protein spectrum of bird's nest, form a high-abundance standard database for bird's nest origin traceability, construct a high-resolution standard database for bird's nest origin traceability, and then achieve accurate and reliable traceability of bird's nest origin based on this standard database, providing a theoretical basis for the application of bird's nest.

[0092] Secondly, this application also provides a standard database for tracing the origin of bird's nest, which is constructed using the method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis provided in the first aspect.

[0093] Thirdly, this application also provides a method for accurate traceability of bird's nest origin using a standard database for bird's nest origin traceability, including the following steps (see appendix). Figure 2 (as shown)

[0094] T1. Construct a standard database for tracing the origin of bird's nest using the method provided in the first aspect, which is based on proteomics analysis.

[0095] T2. Collect bird's nest samples from unknown origins, and use the proteomics analysis method provided in the first aspect of the method for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis to perform proteomics analysis on the bird's nest samples from unknown origins, and obtain characteristic protein data of bird's nest samples from unknown origins.

[0096] T3. Using the method provided in the first aspect for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis, the characteristic protein data of bird's nest samples from unknown origins are analyzed to obtain the origin information of bird's nest samples from unknown origins, thereby realizing the origin traceability of bird's nest samples from unknown origins.

[0097] Fourthly, this application also provides a terminal device, including a memory, a processor, and a computer program stored in the memory and executable on the processor. The processor executes the computer program to implement the steps of the method for accurate traceability of bird's nest origin using the bird's nest origin traceability standard database provided in the third aspect.

[0098] Fifthly, this application also provides a computer-readable storage medium storing a computer program, which is executed by a processor to implement the steps of the method for accurate traceability of bird's nest origin using the bird's nest origin traceability standard database provided in the third aspect.

[0099] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0100] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0101] The specific embodiments of this application involve the following reagents and raw materials, including their types and sources:

[0102] Preparation of PEG-modified (30 kDa molecular weight cutoff) filter membrane: The initial filter membrane (30 kDa molecular weight cutoff) was further immersed in 2 wt% PEG (PEG-2000 was used in this application) solution for 2 h to obtain the PEG-modified 30 kDa filter membrane. PEG-2000 modification can reduce the non-specific adsorption of proteins on the membrane surface and improve the recovery rate of target proteins and peptides.

[0103] Preparation of PEG-modified (10 kDa molecular weight cutoff) filter membrane: The initial filter membrane (10 kDa molecular weight cutoff) was soaked in 2wt% PEG (PEG-2000 was used in this application) solution for 2 h to prepare the PEG-modified 10 kDa filter membrane.

[0104] Preparation of PEG-modified (3 kDa molecular weight cutoff) filter membrane: The initial filter membrane (3 kDa molecular weight cutoff) was soaked in 2 wt% PEG (PEG-2000 was used in this application) solution for 2 h to prepare the PEG-modified 3 kDa filter membrane.

[0105] Example 1

[0106] Sample pretreatment: Collect 5g of bird's nest sample, remove impurities such as bird feathers, and grind it into powder with a particle size of less than 100 mesh using liquid nitrogen.

[0107] Weigh out bird's nest powder and pre-treat it under ultra-high pressure (400 MPa, 25℃, 5 min). Then, take the pre-treated bird's nest powder and mix it with the extraction system ([HMIM][Tf2N] ionic liquid + subcritical water (temperature 150 ℃, pressure 5MPa) + 50mM THP reducing agent, the mass ratio of the three is 1:1:0.02) at a material-to-liquid ratio of 1g:20mL to form a uniform suspension.

[0108] Subsequently, nitrogen inert gas microbubbles were introduced into the suspension using a microbubble generator, controlling the final concentration of microbubbles in the suspension to be approximately 1 × 10⁻⁶. 6 Proteins were analyzed at a concentration of 1000 / mL using pulsed focused ultrasound (35 kHz, 320 W, 10 s operation + 5 s pause as one cycle, 15 cycles). The protein was then denatured at 95°C for 30 min to promote protein structure development and improve subsequent reduction and enzymatic digestion efficiency. After treatment, the material was centrifuged (16000 g, 4°C, 10 min), and the supernatant was used to determine the protein concentration.

[0109] Add THP to the supernatant to a final concentration of 50 mM (1.04% of the supernatant mass), treat at 56°C for 30 min, cool, add IAA to a final concentration of 80 mM (1.48% of the supernatant mass), alkylate at room temperature in the dark for 25 min, and adjust the pH to 8.0 with 1M Tris-HCl during the process.

[0110] Ammonium bicarbonate washing: Take the above sample liquid (equivalent to 175 μg protein), add 120 μL of 75 mM ammonium bicarbonate aqueous solution, mix thoroughly, transfer to a PEG-modified filter membrane, and perform three-stage gradient ultrafiltration sequentially. The ultrafiltration steps are as follows:

[0111] First-stage ultrafiltration: The mixture was transferred to a PEG-2000 modified 30 kDa filter membrane and centrifuged at 14000 g and 20℃ for 25 min. The filtrate (i.e., permeate) was collected to remove macromolecular impurities. 150 μL of 75 mMNH4HCO3 aqueous solution was added to the filter membrane, and the membrane was centrifuged and washed again. The two filtrates were combined.

[0112] Second-stage ultrafiltration: The filtrate obtained from the first-stage ultrafiltration was transferred to a PEG-2000 modified 10 kDa filter membrane and centrifuged at 14000 g and 20℃ for 25 min. The filtrate (i.e., permeate) was collected. After washing with 150 μL of 75 mM NH4HCO3 aqueous solution, the filtrates were centrifuged again and combined.

[0113] Third-stage ultrafiltration: The filtrate obtained from the second-stage ultrafiltration is transferred to a PEG-2000 modified 3 kDa filter membrane and centrifuged at 14000 g and 20℃ for 25 min. The retentate (i.e. the membrane retention liquid) is collected. The retentate obtained is the target protein component with a molecular weight of 3kDa-10 kDa.

[0114] Enzymatic hydrolysis: The collected retentate after the third-stage ultrafiltration was transferred to a new PEG-2000 modified 3kDa membrane to reduce the impact of residual impurities from the preceding ultrafiltration process on the enzymatic hydrolysis. Trypsin was added at a protein:trypsin mass ratio of 30:1. After sealing, the membrane was incubated at 37°C with shaking for 4 h. After hydrolysis, the reaction was terminated with 1% formic acid. The 3kDa membrane was centrifuged at 14000 g at 20°C for 10 min, and the filtrate (which included bird's nest peptides) was collected.

[0115] Peptide merging: The filter membrane was rinsed twice with 50 μL of ultrapure water, and centrifuged at 14000 g and 20℃ for 10 min each time. The two filtrates were combined with the previous filtrate to obtain the total peptide solution.

[0116] Example 2

[0117] The preparation method is basically the same as that in Example 1, except that the extraction system is [HMIM][Tf2N] + subcritical water (temperature 150℃, pressure 5MPa) + 50mM THP with a mass ratio of 0.5:1.5:0.02. The rest of the preparation method is the same as that in Example 1.

[0118] Example 3

[0119] The preparation method is basically the same as that in Example 1, except that the extraction system is [HMIM][Tf2N] + subcritical water (temperature 150℃, pressure 5MPa) + 50mM THP with a mass ratio of 1.5:0.5:0.02. The rest of the preparation method is the same as that in Example 1.

[0120] Example 4

[0121] The preparation method is basically the same as that in Example 1, except that pulseless conventional ultrasound (frequency 35 kHz, power 320 W) is used instead of pulsed focused ultrasound pyrolysis for the same duration. All other preparation methods are the same as in Example 1.

[0122] Example 5

[0123] The preparation method is basically the same as that in Example 1, except that conventional physical grinding is used instead of liquid nitrogen grinding in the pretreatment process, and the particles are ground to the same size. The rest of the preparation methods are the same as those in Example 1.

[0124] Example 6

[0125] The preparation method is basically the same as that in Example 1, except that ultra-high pressure treatment is not performed during the pretreatment process, while the rest of the preparation methods are the same as those in Example 1.

[0126] Example 7

[0127] The preparation method is basically the same as that in Example 1, except that only the third stage of ultrafiltration is used in the ultrafiltration process, and the first and second stage ultrafiltration steps are omitted. That is, the sample after washing with ammonium bicarbonate is directly transferred to the PEG-modified 3 kDa filter membrane for ultrafiltration. The rest of the preparation method is the same as that in Example 1.

[0128] Example 8

[0129] The preparation method is basically the same as that in Example 1, except that only the first and third stages of ultrafiltration are used in the ultrafiltration process, and the second stage of ultrafiltration is omitted. The rest of the preparation methods are the same as those in Example 1.

[0130] Example 9

[0131] The preparation method is basically the same as that in Example 1, except that PEG-1000 modified ultrafiltration membranes are used in the three-stage gradient ultrafiltration process. The rest of the preparation methods are the same as those in Example 1.

[0132] Example 10

[0133] The preparation method is basically the same as in Example 1, except that the tertiary ultrafiltration does not use PEG modification and is performed directly. All other preparation methods are the same as in Example 1.

[0134] Example 11

[0135] The preparation method is basically the same as in Example 1, except that a fully porous particle C18 column (EASY-Spray PepMap RSLC C18 model, catalog number ES801, Thermo Fisher Scientific) is used instead of a core-shell C18 column in the LC-MS / MS analysis process, and the mass spectrometry uses DDA scanning mode.

[0136] Comparative Example 1

[0137] The preparation method is basically the same as that in Example 1, except that 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imine salt ionic liquid is used in place of [HMIM][Tf2N] in equal amounts. The rest of the preparation and construction methods are the same as those in Example 1.

[0138] Comparative Example 2

[0139] The preparation method is basically the same as that in Example 1, except that 1-octyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt ionic liquid is used in equal amounts to replace [HMIM][Tf2N]. All other preparation methods are the same as in Example 1.

[0140] Comparative Example 3

[0141] The preparation method is basically the same as that in Example 1, except that an equal amount of 1-dodecyl-3-methylimidazolium chloride ionic liquid is used instead of [HMIM][Tf2N]. All other preparation methods are the same as in Example 1.

[0142] Comparative Example 4

[0143] The preparation method is basically the same as that in Example 1, except that 1-dodecyl-3-methylimidazolium chloride ionic liquid is used to replace [HMIM][Tf2N] in equal amounts, and TCEP (tris(2-carboxyethyl)phosphine) is used to replace THP in equal amounts.

[0144] Comparative Example 5

[0145] The preparation method is basically the same as that in Example 1, except that TCEP (tris(2-carboxyethyl)phosphine) is used to replace THP in equal amounts.

[0146] Comparative Example 6

[0147] The preparation method is basically the same as that in Example 1, except that an equal amount of pure water is used instead of subcritical water in the extraction system (only the types of pure water and subcritical water are replaced, and the pure water treatment conditions are: temperature 100°C, pressure normal pressure (because pure water cannot remain liquid at 150°C and normal pressure)). The rest of the preparation methods are the same as those in Example 1.

[0148] Comparative Example 7

[0149] The preparation method is basically the same as that in Example 1, except that no inert gas is introduced and no inert gas microbubbles are formed in the mixture. All other preparation methods are the same as those in Example 1.

[0150] Comparative Example 8

[0151] The preparation method is basically the same as that in Example 1, except that inert gas microbubbles are not introduced and pulsed focused ultrasound lysis is not used. All other preparation methods are the same as those in Example 1.

[0152] Comparative Example 9

[0153] The preparation method is basically the same as in Example 1, except that pulsed focused ultrasound lysis is not used; instead, nitrogen inert gas microbubbles are introduced into the suspension through a microbubble generator (controlling the final concentration of microbubbles to 1×10⁻⁶). 6 (pcs / mL), without ultrasonic treatment, the rest of the preparation method is the same as in Example 1.

[0154] Experimental Example 1

[0155] The protein content, protein quantity, and peptide quantity in the bird's nest peptides prepared in Examples 1-11 and Comparative Examples 1-9 were analyzed using PD software. The specific results are shown in Table 1.

[0156] Table 1: Analysis results of protein quality, protein quantity, and peptide quantity in bird's nest peptides

[0157]

[0158] As shown in Table 1, the protein content, number of proteins, and relative proportion of peptides in the bird's nest peptides prepared in the examples are higher than those in the comparative examples. This indicates that the proteomics analysis method provided in this application can release proteins in bird's nest samples more fully, solving the problem of low protein extraction rate in bird's nest. The high protein count indicates that the technical solution of this application extracts a wider variety of proteins from bird's nest and can cover more specific proteins in bird's nest more broadly. The high peptide count indicates that the proteolytic activity of bird's nest in the technical solution of this application is more complete and the peptide coverage is more comprehensive, providing richer molecular feature information for subsequent mass spectrometry analysis.

[0159] Furthermore, the results of Example 7 show that when only 3 kDa single-stage ultrafiltration is used, the number of proteins and peptides decreases significantly, indicating that single-stage ultrafiltration easily leads to co-retention of impurities and loss of target proteins. The results of Example 8 show that although two-stage ultrafiltration can improve the enrichment effect of target proteins to a certain extent, it still has the problem of insufficient protein coverage compared with three-stage gradient ultrafiltration, indicating that three-stage gradient ultrafiltration is more conducive to achieving a balance between the removal of macromolecular impurities, enrichment of target proteins, and removal of small molecule impurities.

[0160] The results of Examples 1 and 9 show that ultrafiltration membranes modified with PEG-1000 to PEG-2000 can effectively reduce non-specific protein adsorption on the membrane surface and improve the recovery rate of target proteins. Among them, the PEG-2000 modified ultrafiltration membrane exhibits superior overall performance in terms of protein quantity, peptide quantity, and membrane flux. The results of Example 10 show that compared with PEG-modified ultrafiltration membranes, the number of proteins and peptides obtained by filtration without PEG modification is lower, because some substances are adsorbed onto the filter membrane.

[0161] The results of Example 4 and Comparative Examples 7-9 indicate that there is a synergistic enhancement effect between inert gas microbubbles and pulsed focused ultrasound, which can further promote the destruction and release of insoluble glycoprotein structures in bird's nest, thereby increasing the number of proteins and peptides detected in subsequent proteomics analysis.

[0162] Application Example 1: Constructing a Standard Database for Traceability of Bird's Nest Origin

[0163] Collection and Proteomics Analysis: Fourteen bird's nest samples from different known origins were collected, namely from West Kalimantan, East Kalimantan, Central Kalimantan, Semarang, Sulawesi, Kotaraya, Sungai Danau, Kaiteng, Ogowele, Maby, Labean, Lumut, Malaysia, and Thailand. The corresponding bird's nest peptide samples from these fourteen origins were prepared using the proteomics analysis procedure described in Example 1. Three parallel experiments were performed on each sample during the experiment.

[0164] Peptide separation: The above bird's nest peptide sample was filtered through a 0.22 μm filter membrane to obtain bird's nest peptide filtrate.

[0165] Mass spectrometry data acquisition: The bird's nest peptide filtrate was analyzed by LC-MS / MS using a core-shell C18 reversed-phase column (1.7 μm, 100 μm × 100 mm, ACQUITY UPLC M-Class Peptide CSH C18 Column, Waters Corporation). Mass spectrometry data of 14 bird's nest peptide samples from different origins were independently acquired. In this application, the mass spectrometry data of bird's nest sample No. 1 is used as an example. See Appendix. Figure 3 The parameters involved in the analysis are as follows:

[0166] Liquid chromatography conditions: column temperature 37℃, flow rate 0.25 mL / min, detection wavelength 214 nm, mobile phase A was 0.1% formic acid + 0.5 mM ammonium formate aqueous solution, mobile phase B was 0.1% formic acid - 90% acetonitrile aqueous solution; gradient elution and elution program (v / v): 0 min - 5 min, maintain mobile phase B volume percentage at 3%; 5 min - 20 min, increase mobile phase B volume percentage from 3% to 15%; 20 min - 40 min, increase mobile phase B volume percentage from 15% to 30%; 40 min - 50 min, increase mobile phase B volume percentage from 30% to 90%; 50 min - 55 min, maintain mobile phase B volume percentage at 90%.

[0167] Mass spectrometry conditions: Orbitrap Fusion Lumos mass spectrometer, using electrospray ionization (ESI) in positive ion mode and DIA scanning mode. The first-stage scan range was 200 m / z–2000 m / z (resolution 120,000), and the second-stage scan range was 100 m / z–1800 m / z (resolution 60,000). Ion source parameters included a spray voltage of 2.5 kV, a capillary temperature of 350 °C, a sheath gas flow rate of 40 arb, and an auxiliary gas flow rate of 15 arb.

[0168] Construction of a standard database for tracing the origin of bird's nest: The mass spectrometry data of bird's nest peptides from 14 different origins obtained above were searched in the target protein database to obtain the characteristic protein data and protein content corresponding to each origin, and a standard database for tracing the origin of bird's nest was constructed.

[0169] It should be noted that the method for constructing the standard database for tracing the origin of bird's nest may specifically include the following steps:

[0170] (1) Based on the mass spectrometry data of a certain bird's nest sample, the protein database was searched and identified in multiple protein databases, and the initial protein database was selected based on the search and identification results.

[0171] (2) Optimize the search parameters of the initial protein database to obtain the target protein database.

[0172] Because the same protein in different species of birds within the Apodidae family shows high similarity, homologous proteins belonging to different species are simultaneously presented in the protein identification results. Based on this consideration, the proteomic data downloaded from the UniProt database does not use the Apodidae protein database; the scope of species included in the database needs to be narrowed. The following four protein databases were initially selected: ① Java Swiftlet: *Aerodramus fuciphagus* (number of proteins: 161), ② *Aerodramus* (number of proteins: 387), ③ Chimney Swiftlet: *Chaetura pelagica* (number of proteins: 3214), ④ Crested Swiftlet: *Hemiprocne* (number of proteins: 13840).

[0173] In this embodiment of the application, the protein databases corresponding to the bird's nest peptide samples are searched and identified from multiple protein databases to obtain the search and identification results. Based on the search and identification results, the initial protein database is selected as the Crested Swiftlet Database. The search and identification results are shown in Table 2 below.

[0174] The Java Swiftlet Database and the Swiftlet Genus Database contain too few proteins, resulting in no proteins being identified. A comparison was made with the Chimney Swiftlet Database and the Crested Swiftlet Database, the latter identifying more peptides (PRPs), proteins (PROs), and peptide map matching numbers (PSMs). Sequence similarity was also compared between the Java Swiftlet and Crested Swiftlet databases: the similarity for the classic protein Cytochrome b was 92.8%, and the similarity for the classic protein NADH-ubiquinone oxidoreductase chain 2 was 85.8%. These results indicate high sequence conservation of homologous proteins between the two species. Therefore, using the more comprehensive Crested Swiftlet Database can effectively cover peptides from the Java Swiftlet, which is one reason why the Java Swiftlet Database failed to identify any proteins.

[0175] Table 2: Identification results from multiple protein database searches

[0176]

[0177] Note: "-" indicates that no protein, peptide, or peptide spectrum match was identified.

[0178] However, the applicant found that even when using the Crested Swift database for data searching, the search results for some samples were still unsatisfactory. Through research and analysis, the following conclusion was reached: because bird's nests are composed of bird saliva, which contains various proteases that can enzymatically break down proteins, resulting in the production of non-specific enzymatically hydrolyzed peptides.

[0179] Therefore, in the Proteome Discoverer search parameter settings, the enzyme parameter was changed from Trypsin (full) to Trypsin (semi). Trypsin (semi) allows one side of the digested peptide to deviate from the classic enzyme digestion rules of Trypsin, in order to increase the identification efficiency of peptides generated by non-specific enzyme digestion.

[0180] Taking sample 1 as an example, mass spectrometry data from three biological replicates were searched, and two different enzymatic digestion parameter settings were selected for each. The results showed that, compared with the Trypsin (full) setting, although the Trypsin (semi) setting did not significantly increase the number of proteins identified, it significantly increased the number of peptides identified (46%-66%) and the number of peptide spectrum matches (PSM, 44%-59%), which is very beneficial for improving the accuracy of protein quantification information (Table 3).

[0181] Table 3: Identification results of Trypsin parameter selection (full) and (semi) (taking sample 1 as an example)

[0182]

[0183] To demonstrate that the aforementioned improvement in mass spectrometry information was not universal but rather due to auto-digestion in the bird's nest samples, 293T cells were used as a comparative experiment. Bird's nest, as a secretion, is rich in proteases and readily undergoes non-specific auto-digestion. 293T cells, on the other hand, are a widely studied and used human embryonic kidney cell line with well-defined protein expression profiles and physiological characteristics. Therefore, 293T cells were used as a negative control in proteomics analysis.

[0184] Mass spectrometry data from three independent bird's nest samples were obtained using the exact same proteomics analysis workflow. The results showed that, compared to the Trypsin (full) setting, the Trypsin (semi) setting slightly increased the number of identified peptides (6.8-7.7%) and the number of peptide chromatogram matches (PSM, 5.4-6.8%). This indicates that when there is virtually no non-specific enzymatic digestion in the sample, the two enzymatic digestion parameter settings do not significantly affect the number of peptides and the number of peptide chromatogram matches, indirectly proving the presence of considerable non-specific enzymatic digestion in bird's nest.

[0185] Therefore, by adjusting the search parameters of the Crested Swift database, the optimized Crested Swift database was obtained as the target protein database.

[0186] (3) PD software was used to analyze mass spectrometry data, match target protein database, obtain characteristic protein data corresponding to bird's nest samples from different origins, and construct a standard database for bird's nest origin traceability based on characteristic protein data and corresponding origin information from each origin.

[0187] In this embodiment, MaxQuant (MQ) and PD software were used to search the databases of 42 sets of mass spectrometry data and match the target protein databases. When using MQ, the number of proteins identified in the samples ranged from 51 to 77. When using PD, the number of proteins identified in the samples ranged from 49 to 107 (see Table 4 for specific results). Overall, compared with MQ, PD can identify more proteins, and the protein composition of bird's nest samples from different origins has distinguishable characteristic information.

[0188] Table 4: Data analysis and database search results of bird's nest samples from different origins based on PD.

[0189]

[0190] As shown in Table 4, the parallel test data are within the same range, with no significant differences between them. This indicates that the bird's nest peptide preparation method and LC-MS / MS analysis method provided in this application are reproducible and highly consistent, further demonstrating the high reliability of the bird's nest origin traceability standard data constructed in this application. The above analysis shows that preliminary analysis of the origin attribution of different samples can be conducted based on protein data.

[0191] (4) Import the LC-MS / MS mass spectrometry data of bird's nest peptides from different origins into PD software for analysis to obtain characteristic protein data from different origins. The characteristic protein data includes the name of the characteristic protein and the content of the characteristic protein (see Table 5 for specific results). The characteristic protein data from different origins and the origin information together form the bird's nest origin traceability standard database.

[0192] After mixing samples from a specific production area, further sample processing, mass spectrometry analysis, and target protein database search analysis were performed, identifying a total of 271 proteins. Based on the number of times each protein was identified (i.e., PSM count), quantitative analysis was conducted on the identified proteins, and the list of the top-ranked proteins is shown in Table 5.

[0193] Table 5: Characteristic proteins and protein content of a certain production area

[0194]

[0195] In addition, cluster analysis can be performed based on the characteristic protein data of 14 different origins in the bird's nest origin traceability standard database, and the accuracy of the bird's nest origin traceability standard database can be further verified based on the cluster analysis results.

[0196] In summary, the bird's nest peptides prepared using the specific proteomics analysis process described in this application contain abundant characteristic protein data, which can fully reflect the characteristic protein information of bird's nest samples from different origins. Further mass spectrometry analysis of the bird's nest peptides and extraction of characteristic protein data can construct a standard database for bird's nest origin traceability. This database highly covers the characteristic protein information of bird's nests from different origins, effectively reflects the origin characteristics of bird's nest samples, and can be used to achieve the analysis and traceability identification of bird's nest origin.

[0197] This application constructed a high-resolution standard database for tracing the origin of bird's nests by screening a specific database of swiftlet-specific proteins, optimizing the search parameters of haptenase to suit the self-digestion characteristics of bird's nests, and using MQ and PD dual software for cross-validation of extracted components and modification information. It was precisely because key technical problems such as low species coverage and difficulties in non-specific enzyme digestion identification were solved during the database construction process that accurate traceability of bird's nest origins was ultimately achieved.

[0198] Application Example 2: Using the bird's nest origin traceability standard database to trace the origin of unknown bird's nests.

[0199] Bird's nest peptides were extracted from the bird's nest sample of unknown origin using the same proteomics analysis method as in Application Example 1. LC-MS / MS data of these peptides were obtained and imported into the target protein database in PD software to acquire characteristic protein data. This characteristic protein data was then analyzed using the established bird's nest origin traceability standard database to obtain the origin information of the bird's nest sample of unknown origin. Furthermore, cluster analysis can be performed between the characteristic protein data of the bird's nest sample of unknown origin and the characteristic protein data of 14 different origins in the bird's nest origin traceability standard database. If the cluster analysis results are consistent with the origin information of the bird's nest sample of unknown origin obtained above, the accuracy of the traceability standard database in tracing the origin information of the unknown bird's nest can be further confirmed.

[0200] Specifically, for example: bird's nest samples from unknown origins were collected, and bird's nest peptide samples were prepared according to the proteomics analysis method in Example 1. Characteristic protein data were extracted from the samples: LC-MS / MS analysis was used to obtain the characteristic protein data of the samples, in which the relative content of characteristic protein Chia (chitinase) was 23.32 wt%, the relative content of Muc5ac (mucin 5AC) was 17.16 wt%, and the Indonesian Sulawesi-specific characteristic peptide Atp5f1b (relative content 0.72 wt%) was also detected.

[0201] Source traceability analysis: The protein data of the sample to be verified was imported into the established bird's nest origin traceability standard database and compared with the characteristic protein data of the "Sulawesi, Indonesia" origin in the database. This revealed the presence of Chia and Muc5ac content, as well as the specific peptide Atp5f1b, thus directly obtaining the origin information of the bird's nest sample. To further verify the accuracy of this origin traceability, the sample was further validated using the origin clustering branch. The results showed a complete match with the characteristics of this origin clustering branch. Furthermore, through cluster distance calculation, the similarity between the verified sample and the "Sulawesi, Indonesia" branch in the bird's nest origin traceability standard database was 93.2%, significantly higher than the similarity with other origin branches (<70.1%).

[0202] Verification results: The bird's nest sample from an unknown origin (actually a known verified sample) was accurately identified by the database as originating from Sulawesi, Indonesia, with a verification accuracy rate of over 95%, further demonstrating the high-resolution identification capability of this traceability standard database for bird's nests from different Indonesian production areas.

[0203] Verification Example: Origin Traceability Verification of Bird's Nest from East Kalimantan, Indonesia

[0204] The verification sample was prepared by collecting bird's nest raw material (No.: V-001) from East Kalimantan Province, Indonesia, which is known to be the place of origin.

[0205] The bird's nest peptides were prepared and detected by processing the sample according to the preparation method in Example 1:

[0206] LC-MS / MS analysis was used to obtain the characteristic protein data of this sample. The relative content of the characteristic protein Chia (chitinase) was 22.14 wt%, the relative content of Muc5ac (mucin 5AC) was 19.37 wt%, and the East Kalimantan-specific characteristic peptide Pxy1p1 (relative content 3.28 wt%) was also detected.

[0207] The traceability comparison analysis imported the characteristic protein data of the verification sample into the established bird's nest origin traceability standard database. The data was compared with the characteristic protein data of the "East Kalimantan, Indonesia" origin in the database, revealing the presence of Chia, Muc5ac content, and the specific peptide Pxy1p1. Therefore, the origin information of this bird's nest sample could be directly obtained. To further verify the accuracy of the origin traceability, the clustering branch of this origin was used for verification. The results showed a complete match with the characteristics of this clustering branch. Cluster distance calculation showed that the similarity between the verification sample and the "East Kalimantan, Indonesia" branch in the database was 97.1%, significantly higher than the similarity with other origin branches (<68%).

[0208] The verification results showed that the bird's nest sample from an unknown origin (actually a known verified sample) was accurately identified by the database as originating from East Kalimantan Province, Indonesia, with an accuracy rate of over 95%. This further demonstrates the high-resolution identification capability of this traceability standard database for bird's nests from different Indonesian production areas.

[0209] Based on this, the method for constructing standard data for bird's nest origin traceability provided in this application can obtain comprehensive and rich characteristic protein information of bird's nest peptides, form a database of characteristic proteins with high coverage, fully analyze the characteristic peptide differences of bird's nest proteins, significantly improve the accuracy and resolution of bird's nest origin traceability, and realize bird's nest origin traceability.

[0210] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0211] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis, characterized in that, Includes the following steps: Bird's nest samples from known different origins were collected, and proteomics analysis was performed independently on each of the bird's nest samples from known different origins to obtain characteristic protein data and corresponding origin information, forming a standard database for bird's nest origin traceability. The proteomics analysis includes the following steps: A mixture was prepared by mixing bird's nest samples with an extract containing ionic liquid, subcritical water and reducing agent; An inert gas is introduced into the mixture to form inert gas microbubbles, and the mixture is subjected to ultrasonic treatment. The supernatant is then separated and subjected to alkylation treatment to prepare a protein solution. The protein solution is subjected to ultrafiltration and enzymatic hydrolysis to prepare bird's nest peptides. The ultrafiltration adopts at least three-stage molecular weight fractional ultrafiltration, wherein the first stage ultrafiltration adopts an ultrafiltration membrane with a molecular weight cutoff of 30kDa, the second stage ultrafiltration adopts an ultrafiltration membrane with a molecular weight cutoff of 10kDa, and the third stage ultrafiltration adopts an ultrafiltration membrane with a molecular weight cutoff of 3kDa. Characteristic protein data of the bird's nest peptides were extracted using liquid chromatography-tandem mass spectrometry and a protein database. The ionic liquid is 1-hexyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt; the reducing agent is tris(3-hydroxypropyl)phosphine.

2. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, The mass ratio of the ionic liquid, the subcritical water, and the reducing agent is (0.5-1.5):(0.5-1.5):(0.01-0.03); and / or, the mass-to-volume ratio of the bird's nest sample to the extract is 1g:(10-50)mL; and / or, the temperature of the subcritical water is 150℃-200℃, and the pressure is 2MPa-15MPa.

3. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, The content of the inert gas microbubbles in the mixture is 1×10⁻⁶. 5 Cells / mL - 1×10 7 per mL; and / or, the inert gas includes one or more of nitrogen, argon and helium.

4. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, Ultrasonic treatment includes pulsed focused ultrasound treatment; The pulsed focused ultrasound treatment has a frequency of 20 kHz-50 kHz and a power of 200 W-500 W. The pulsed focused ultrasound treatment process consists of a 10-second working cycle followed by a 5-second pause, and includes at least 15 cycles.

5. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, During the ultrafiltration process, the protein solution is mixed with a buffer salt solution before ultrafiltration.

6. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, The ultrafiltration membrane is a PEG-modified ultrafiltration membrane, and the molecular weight of the PEG in the PEG-modified ultrafiltration membrane is 1000 Da-2000 Da.

7. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to claim 1, characterized in that, In the alkylation step, the alkylating agent used includes one or more of iodoacetamide, N-ethylmaleimide and 4-vinylpyridine; and / or, the enzyme used for enzymatic hydrolysis includes trypsin; and / or, the amount of enzyme added during enzymatic hydrolysis is 2%-5% of the mass of the protein in the ultrafiltrate obtained after the ultrafiltration step.

8. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to any one of claims 1 to 7, characterized in that, The bird's nest sample was prepared by grinding and / or pressurizing the raw bird's nest material; The grinding process uses liquid nitrogen grinding. The pressure for pressurization is 100 MPa-2000 MPa, the temperature is 20℃-30℃, and the time is 1 min-10 min.

9. The method for constructing a standard database for tracing the origin of bird's nest based on proteomics analysis according to any one of claims 1 to 7, characterized in that, The steps for extracting characteristic protein data of the bird's nest peptide by liquid chromatography-tandem mass spectrometry and protein database include: performing liquid chromatography-tandem mass spectrometry analysis on the bird's nest peptide to obtain mass spectrometry data, importing the mass spectrometry data into Proteome Discoverer software for analysis, and extracting characteristic protein data of the bird's nest peptide. The parameters of the liquid chromatography-tandem mass spectrometry include liquid chromatography parameters and mass spectrometry parameters: The liquid chromatography parameters included: a C18 column, a column temperature of 37℃, a flow rate of 0.2 mL / min-0.3 mL / min, a detection wavelength of 214 nm, mobile phase A being an aqueous solution containing 0.1% formic acid and 0.5 mM ammonium formate, and mobile phase B being an acetonitrile solution containing 0.1% formic acid. Gradient elution was used, with the following program: 0 min-5 min, maintaining mobile phase B at 3% volume; 5 min-20 min, increasing mobile phase B from 3% to 15% volume; 20 min-40 min, increasing mobile phase B from 15% to 30% volume; 40 min-50 min, increasing mobile phase B from 30% to 90% volume; and 50 min-55 min, maintaining mobile phase B at 90% volume. The mass spectrometry parameters include: an electrospray ionization source in positive ion mode and DIA scanning mode, wherein the first-stage scanning range is 200 m / z-2000 m / z, the second-stage scanning range is 100 m / z-1800 m / z, and the resolution is 60000-120000; the ion source parameters include a spray voltage of 2.0 kV-2.5 kV, a capillary temperature of 300℃-350℃, a sheath gas flow rate of 30 arb-40 arb, and an auxiliary gas flow rate of 10 arb-15 arb.

10. A method for precise traceability of bird's nest origin, characterized in that, Includes the following steps: Collect bird's nest samples from unknown origins, and use the proteomics analysis method in the method for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis as described in any one of claims 1 to 9 to perform proteomics analysis on the bird's nest samples from unknown origins, and obtain characteristic protein data of the bird's nest samples from unknown origins. The method described in any one of claims 1 to 9 for constructing a standard database for tracing the origin of bird's nests based on proteomics analysis analyzes the characteristic protein data of bird's nest samples from unknown origins to obtain the origin information of the bird's nest samples from unknown origins, thereby achieving origin tracing of the bird's nest samples from unknown origins.

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