Use of a detection reagent for a biomarker in the preparation of a product for authenticating the genuineness of dairy products

Abstract

The present invention belongs to the technical field of dairy product detection, and particularly relates to the application of detection reagents for biomarkers in the preparation of products for identifying the authenticity of dairy products. The dairy products are sheep milk, goat milk and / or cow milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein. APOH protein and ANPEP protein are biomarkers for identifying sheep milk, A1BG protein and FGA protein are biomarkers for identifying goat milk, and ANG1 protein and SERPING1 protein are biomarkers for identifying cow milk. The research of the present invention will help improve consumers' understanding of the composition and functions of various dairy products, and provide a meaningful theoretical basis for the high-quality development of professional dairy products. In addition, it will also promote the establishment of methods for identifying dairy product adulteration, thus supporting a major challenge faced by the global dairy industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of dairy product detection, and particularly relates to the application of a detection reagent for biomarkers in the preparation of a product for identifying the authenticity of dairy products. Background Art

[0002] Dairy products are rich in nutrients and are an important source of protein in the human diet. Currently, the demand for high-quality dairy products by consumers is increasing, and the supply of high-quality milk from small ruminants, especially sheep milk and goat milk, has risen to meet this market demand. Although cow's milk currently has a large market share, most people in the world are lactose intolerant. Compared with cow's milk, sheep milk and goat milk have higher nutritional value, a relatively higher dry matter content, and milk fat in the form of smaller spherical diameters, which is beneficial to digestion and metabolism. However, the prices of sheep milk and goat milk are high, the yields are low, and some merchants choose to adulterate.

[0003] About 80% of the casein and 20% of the whey protein in milk, including various functional proteins such as lactoferrin and immunoglobulins. In different types of milk, the contents of these proteins vary, and the resulting differences in the functions of dairy products have attracted increasing attention. By comparing the proteomic analysis of different types of milk, specific protein molecules can be identified, and these protein molecules may be potential biomarkers for milk from different species.

[0004] Therefore, analyzing the differences in the components of sheep milk, goat milk and cow's milk and identifying potential biomarkers for different milk types will help improve the understanding of specialty milk and provide a theoretical basis for dairy adulteration monitoring. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides the application of a detection reagent for biomarkers in the preparation of a product for identifying the authenticity of dairy products.

[0006] The present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides the application of a detection reagent for biomarkers in the preparation of a product for identifying the authenticity of dairy products, wherein the dairy products are sheep milk, goat milk and / or cow's milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein.

[0008] To study the differences in protein composition among sheep milk, goat milk, and cow milk and thus identify potential biomarkers, this invention uses Astral-DIA proteomics to analyze differentially expressed proteins and the parallel reaction monitoring (PRM) method to verify the results. Using bioinformatics methods, through Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the functions of characteristic proteins and related biological processes were predicted. A total of 4,316 differentially expressed proteins were identified. Among them, Beta-2-glycoprotein 1 (APOH) and aminopeptidase (ANPEP) can be used as biomarkers for sheep milk, Fibrinogen alpha chain (FGA) and Alpha-1-B glycoprotein (A1BG) can be used as biomarkers for goat milk, and angiogenin-1 (ANG1) and Serpin family G member 1 (SERPING1) can be used as biomarkers for cow milk. Functional analysis shows that these different proteins are enriched through different pathways such as the complement and coagulation cascades. These data reveal the differences in protein content and physiological functions, providing an important basis for the study of dairy product nutrition and adulteration identification.

[0009] Furthermore, the reagent includes a reagent for quantitatively detecting the biomarker using DIA proteomics technology. In the chromatographic separation of DIA proteomics technology, mobile phase A is an aqueous formic acid solution with a volume fraction of 0.09% - 0.11%, mobile phase B is a mixture of formic acid and an aqueous acetonitrile solution, and the volume fraction of acetonitrile in the aqueous acetonitrile solution is 79% - 81%. In mobile phase B, the volume fraction of formic acid is 0.09% - 0.11%.

[0010] Furthermore, the reagent includes a reagent for quantitatively detecting the biomarker using parallel reaction monitoring technology (PRM). In the chromatographic separation of parallel reaction monitoring technology PRM, buffer A is an aqueous formic acid solution with a volume fraction of 0.09% - 0.11%, buffer B is a mixture of formic acid and an aqueous acetonitrile solution, and the volume fraction of acetonitrile in the aqueous acetonitrile solution is 94% - 96%. In buffer B, the volume fraction of formic acid is 0.09% - 0.11%.

[0011] In a second aspect, this invention provides a reagent for detecting the biomarker, and the reagent is used to detect the content of APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein, and / or SERPING1 protein in dairy products.

[0012] Furthermore, the reagent includes a reagent for quantitatively detecting APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein in the dairy product by DIA proteomics technology and / or parallel reaction monitoring technology (PRM); in the chromatographic separation of DIA proteomics technology, mobile phase A is an aqueous formic acid solution with a volume fraction of 0.09% to 0.11%, mobile phase B is a mixed solution of formic acid and an aqueous acetonitrile solution, and the volume fraction of acetonitrile in the aqueous acetonitrile solution is 79% to 81%. In mobile phase B, the volume fraction of formic acid is 0.09% to 0.11%; in the chromatographic separation of parallel reaction monitoring technology PRM, buffer A is an aqueous formic acid solution with a volume fraction of 0.09% to 0.11%, buffer B is a mixed solution of formic acid and an aqueous acetonitrile solution, and the volume fraction of acetonitrile in the aqueous acetonitrile solution is 94% to 96%. In buffer B, the volume fraction of formic acid is 0.09% to 0.11%. Within this range, the corresponding separation effects can be achieved.

[0013] In the third aspect, the present invention provides a kit containing the above-mentioned reagent.

[0014] In the fourth aspect, the present invention provides the application of the above-mentioned kit in identifying the authenticity of dairy products, wherein the dairy products are sheep milk, goat milk and / or cow milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein.

[0015] The present invention has the following beneficial effects:

[0016] Dairy products are of great benefit to human health, and the nutritional differences between different dairy products have attracted people's attention. The biomarkers of the present invention will help improve consumers' understanding of the composition and functions of various dairy products and provide a meaningful theoretical basis for the high-quality development of professional dairy products. In addition, it will also promote the establishment of methods for identifying dairy product adulteration, thus supporting a major challenge faced by the global dairy industry. Description of the Drawings

[0017] Figure 1 It is a Venn diagram of proteins in sheep, goat and cow milk.

[0018] Figure 2 It is a PCA score plot.

[0019] Figure 3 It is a hierarchical clustering of significantly different proteins in sheep milk, goat milk and cow milk.

[0020] Figure 4 It is the difference in proteins in the sheep milk - goat milk group (S - G), sheep milk - cow milk group (S - C), and goat milk - cow milk group (G - C), where:

[0021] A is the difference in proteins in the sheep milk - goat milk group (S - G).

[0022] B is the difference in proteins in the sheep milk - cow milk group (S - C).

[0023] C is the difference in proteins in the goat milk - cow milk group (G - C).

[0024] Figure 5 is the GO enrichment analysis of differential proteins in sheep milk, goat milk and cow milk based on biological processes.

[0025] Figure 6 is the GO enrichment analysis of differential proteins in sheep milk, goat milk and cow milk based on cellular components.

[0026] Figure 7 is the GO enrichment analysis of differential proteins in sheep milk, goat milk and cow milk based on molecular functions.

[0027] Figure 8 is the KEGG pathway analysis of differentially expressed proteins in the sheep milk - goat milk group (S - G).

[0028] Figure 9 is the KEGG pathway analysis of differentially expressed proteins in the sheep milk - cow milk group (S - C).

[0029] Figure 10 is the KEGG pathway analysis of differentially expressed proteins in the goat milk - cow milk group (G - C).

[0030] Figure 11 is the PPI network analysis of differentially expressed proteins in the sheep milk - goat milk group (S - G). Red nodes represent up - regulated proteins; green nodes represent down - regulated proteins.

[0031] Figure 12 is the PPI network analysis of differentially expressed proteins in the sheep milk - cow milk group (S - C). Red nodes represent up - regulated proteins; green nodes represent down - regulated proteins.

[0032] Figure 13 is the PPI network analysis of differentially expressed proteins in the goat milk - cow milk group (G - C). Red nodes represent up - regulated proteins; green nodes represent down - regulated proteins.

[0033] Figure 14 are the results of DIA and PRM for detecting APOH, A1BG, and ANG1 in sheep milk, goat milk and cow milk, where:

[0034] A is the result of DIA for detecting APOH in sheep milk, goat milk and cow milk.

[0035] B is the result of DIA detection of A1BG in sheep milk, goat milk and cow milk.

[0036] C is the result of DIA detection of ANG1 in sheep milk, goat milk and cow milk.

[0037] D is the result of PRM detection of APOH in sheep milk, goat milk and cow milk.

[0038] E is the result of PRM detection of A1BG in sheep milk, goat milk and cow milk.

[0039] F is the result of PRM detection of ANG1 in sheep milk, goat milk and cow milk.

[0040] Figure 15 are the results of DIA and PRM detections of ANPEP, FGA, and SERPING1 in sheep milk, goat milk and cow milk, where:

[0041] A is the result of DIA detection of ANPEP in sheep milk, goat milk and cow milk.

[0042] B is the result of DIA detection of FGA in sheep milk, goat milk and cow milk.

[0043] C is the result of DIA detection of SERPING1 in sheep milk, goat milk and cow milk.

[0044] D is the result of PRM detection of ANPEP in sheep milk, goat milk and cow milk.

[0045] E is the result of PRM detection of FGA in sheep milk, goat milk and cow milk.

[0046] F is the result of PRM detection of SERPING1 in sheep milk, goat milk and cow milk. Detailed implementation mode

[0047] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0048] Example 1: Screening and verification of biomarkers related to dairy product variety identification

[0049] 1 Test method

[0050] 1.1 Sample and protein preparation

[0051] Raw East Friesian sheep milk samples were obtained from Yuansheng Agriculture and Animal Husbandry Technology Co., Ltd. (Jinchang, Gansu, China). Saanen goat milk and Holstein cow milk samples were obtained from Heshi Dairy Co., Ltd. (Shaanxi, China). Forty samples were collected from each species at approximately 15-30, 60, 120, and 180 days after lambing. The original samples were randomly mixed into four biological replicates (each replicate contained 10 samples) for protein extraction. After freeze-drying, SDT lysis solution (4% SDS, 100mMTris-HCl, pH 7.6) was added to each sample. The samples were then transferred to EP tubes, boiled in a water bath, and ultrasonically disrupted for 2 min. The supernatant was taken and quantitatively analyzed using a BCA protein detection kit (BeyoTime, China).

[0052] For each sample, dithiothreitol (DTT) (100mM) was added, boiled in a water bath for 5min, and then cooled to 25°C. Then, 200μL UA buffer (8M urea, 150mM Tris-HCl, pH 8.0) was added and transferred to a 10KD ultrafiltration centrifuge tube. Then, 100μL IAA (50mM IAA in UA) was added, shaken at 600rpm for 1min, and stored at room temperature in the dark for 30min. UA buffer (100μL) and ammonium bicarbonate buffer (Sigma) (100μL) were added and centrifuged at 14000g for 10min. 40μL trypsin buffer (6μg trypsin in 40μL ammonium bicarbonate buffer) was added and continued at 600rpm for 1min. The filtrate was collected and an appropriate 0.1% TFA solution was added. After enzymatic hydrolysis, the peptides were desalted with a C18 cartridge and lyophilized under vacuum. The peptide concentration was determined by LC-MS.

[0053] 1.2LC-MS / MS analysis

[0054] LC-MS / MS was performed using an Orbitrap Elite mass spectrometer coupled to a new UHPLC system (Thermo Fisher Scientific). The RP-HPLC mobile phase A was an aqueous solution of 0.1% formic acid by volume, and the mobile phase B was an aqueous solution of acetonitrile containing 0.1% formic acid with a volume fraction of 80%. Peptides were eluted with a linear gradient of 1.25 μL / min for 8 min. The linear gradient was set as follows: 0 - 0.1 min, the linear gradient was 4% - 6% buffer B; 0.1 - 1.1 min, the linear gradient was 6% - 12%; 1.1 - 4.3 min, 12% - 2.3 - 6.1 min, the linear gradient of 25% - 45% was 6.5 min, the linear gradient was 45% - 99% buffer B; 6.5 - 8 min, buffer B was maintained at 99%. The DIAMS / MS scan was performed using Astral, 150 - 2000 m / z, the isolation window was 2 m / z, the AGC target was 500%, and the injection time was 3 ms. The normalized collision energy was set to 25, and the cycle time was 0.6 s.

[0055] 1.3 Bioinformatics

[0056] Principal component analysis was performed on the quantitative protein data to visualize the relationships between samples from different heights. Biostatistical analysis was performed using Excel 2016 and R statistical computing software. Fisher's exact test was used for GO and KEGG, and FDR correction was performed for multiple tests. GO was divided into three categories: biological process (BP), molecular function (MF), and cellular component (CC). A protein-protein interaction (PPI) network was constructed using the String database and Cytoscape software.

[0057] 1.4 LC-PRM / MS analysis

[0058] LC-PRM / MS analysis was performed on the polypeptide (2 μg). Chromatographic separation was carried out using a nano-flow Easy nLC 1200 chromatographic system (Thermo Scientific). Buffer A was an aqueous solution of 0.1% formic acid by volume, and buffer B was an aqueous solution of 95% acetonitrile containing 0.1% formic acid. It was equilibrated with 95% buffer A. The sample was poured into a trap column (100 μm * 20 mm, 5 μm, C18, Maisch GmbH), and then gradient separation was performed on a chromatographic column (75 μm * 150 mm, 3 μm, C18, Maisch GmbH). These peptides were separated and subjected to targeted PRM mass spectrometry analysis using a QExactive HF-X mass spectrometer (Thermo Scientific). The analysis time was 60 min, the detection mode was positive ion, the parent ion scan range was 300 - 1200 m / z, the primary mass spectrometry resolution: 60000 @ m / z 200, AGC target: 3e6, the maximum IT of the primary mass spectrometry: 50 ms. Secondary mass spectrometry analysis of the peptides was performed: resolution was 30000 @ m / z 200, AGC target: 1e6, the maximum IT of the secondary mass spectrometry: 100 ms, MS2 activation type: HCD, isolation window: 1.6 Th. The obtained RAW mass spectrometry files were analyzed using Skyli software.

[0059] 1.5 Data analysis

[0060] All mass spectrometry data were combined using DIA-nn (version 1.8.1) software to complete database searching of DIA mass spectrometry data and quantitative analysis of DIA proteins. The main software parameters were: trypsin was used as the enzyme, the false discovery rate (FDR) at both the peptide spectrum match (PSM) and protein levels was 0.01, and the fixed modification was carbamidomethyl (C). The databases were sheep (9940), goat (9925), and cattle (9913) (https: / / www.uniprot.org / taxonomy / 9904(9925)(9913)). To determine the significant differences between proteins, a t-test combined with the Fold change (FC) method was used to analyze between two groups, and proteins with significant differences were screened out (P value < 0.05, FC >= 1.5 or <= 1 / 1.5). One-way analysis of variance (ANOVA) was used for comparisons between groups. All data visualization was performed using GraphPad Prism 8.

[0061] 2 Experimental results

[0062] 2.1 Identification of proteins in sheep milk, goat milk, and cow milk

[0063] A total of 4316 proteins were identified in sheep milk, goat milk, and cow milk. The Venn diagram shows the differences in the identified proteins among different species, as Figure 1As shown in the figure. 3310, 3859, and 2998 proteins were detected in sheep milk, goat milk, and cow milk, respectively. There were 2373 common proteins (54.98%) in sheep milk, goat milk, and cow milk. 116, 458, and 264 unique proteins were identified in sheep milk, goat milk, and cow milk, respectively. In addition, there were 744 proteins common to sheep milk and goat milk, 77 proteins common to sheep milk and cow milk, and 284 proteins common to goat milk and cow milk. These data revealed the differences in protein levels in sheep, goat, and cow milk, laying a foundation for further screening of potential biomarkers.

[0064] 2.2 Differentially Expressed Proteins (DEPs) in Sheep Milk, Goat Milk, and Cow Milk

[0065] Principal component analysis of the identified proteins showed that the samples in this study were divided into sheep milk, goat milk, and cow milk. Figure 2 The PCA score plot in shows that, according to the PC1 direction, goat milk is different from the other two types (sheep milk and cow milk). PC1 and PC2 accounted for 41.17% and 23.97% of the protein variation among different species, respectively. In this study, P < 0.05 and FC ≥ 1.50 or ≤ 1 / 1.50 were considered to be important DEPs. As Figure 3 shown by the clustering results of, three main different protein clusters were observed in the heatmap, and there were differences in the patterns among the samples of the three species. LBP, TUBB4A, ATP1B1, TUBB2A, and TUBA1D were the most abundant proteins, but their proportions were different in sheep milk, goat milk, and cow milk. Figure 4 The volcano plot shows the differential expression of proteins in the sheep milk - goat milk group (S - G), sheep milk - cow milk group (S - C), and goat milk - cow milk group (G - C). Each point represents a protein. These results provide a basis for further studying the functions of differentially expressed proteins.

[0066] 2.3 GO Enrichment Analysis of DEPs in Sheep, Goat, and Cow Milk

[0067] Using GO enrichment analysis, the DEPs in sheep milk, goat milk, and cow milk were divided into three categories: BP, CC, and MF. The results showed that the different proteins identified in S - G were significantly enriched in peptide metabolic process and peptide biosynthesis process in BP, significantly enriched in ribosome and intracellular anatomical structure in CC, and significantly enriched in ribosome structural component and ligase activity in MF ( Figures 5 - 7)。The different proteins identified in S-C were significantly enriched in BP, the extracellular region and proteolysis in the extracellular space, secretion, and the endopeptidase regulatory activity in MF. The different proteins identified in G-C were significantly enriched in translation, the peptide biosynthetic process in BP, ribosomes and endopeptidase complexes in CC, and ribosomal structural components in MF. These results predicted the functions of the differentially enriched proteins in sheep milk, goat milk, and cow milk.

[0068] 2.4 KEGG pathway enrichment analysis of DEPs in sheep, goat, and cow milk

[0069] Figures 8 - 10 A chord diagram of the top 15 pathways related to DEPs in different types of milk is shown. According to KEGG analysis, many proteins are involved in multiple disease pathways. In the S-G group, the differential proteins were mainly involved in processes such as coronavirus disease-COVID-19, ribosomes, etc., while in the S-C group, the differential proteins were mainly involved in complement and coagulation cascades, lysosomes, etc. In the G-C group, the differentially expressed proteins were mainly involved in coronavirus disease-COVID-19, complement and coagulation cascades. These differentially expressed proteins are mainly involved in immune and disease-related signaling pathways and play important roles in these areas.

[0070] 2.5 PPI network analysis of DEPs in sheep, goat, and cow milk

[0071] PPI analysis of the differentially expressed proteins in the S-G group showed 406 nodes. The top 50 differentially expressed proteins with the highest connectivity are shown as Figure 11 follows. Among them, Large ribosomal subunit protein eL19 (RPL19) interacted with 26 proteins and was the node with the highest degree of interaction. Small ribosomal subunit protein eS28 (RPS28), Small ribosomal subunit protein uS3 (RPS3), Large ribosomal subunit protein, differential proteins such as uL14 (RPL23) and Elongation factor 2 (EEF2) were related to multiple proteins. In the SC group, 167 differentially expressed protein nodes were identified by PPI analysis, and the top 50 differentially expressed proteins with the highest connectivity are shown as Figure 12As shown, Alpha-2-HS-glycoprotein (AHSG) interacts with 16 proteins and is the node with the highest degree of interaction. Differential proteins such as Plasminogen (PLG), Beta-2-glycoprotein 1 (APOH), Apolipoprotein A-I (APOA1), and Albumin (ALB) are related to many proteins. PPI analysis of differentially expressed proteins in the GC group showed 279 nodes. The top 50 differentially expressed proteins with the highest connectivity are as Figure 13 shown. Among them, Large ribosomal subunit protein eL19 (RPL19) interacts with 17 proteins and is the node with the highest degree of interaction. Small ribosomal subunit protein uS3 (RPS3), Large ribosomal subunit protein uL14 (RPL23), Elongation factor 2 (EEF2), actin, and differential proteins such as Cytoplasmic 1 (ACTB) are related to multiple proteins. These results reflect the internal relationships among sheep, goat, and milk proteins.

[0072] 2.6 PRM analysis of proteins in sheep, goats, and milk

[0073] In the PRM experiment, we verified two potential functional proteins in each of the three groups and found that PRM analysis accurately characterized the differentially expressed proteins in sheep, goats, and milk. As Figure 14 and Figure 15 shown, DIA analysis indicated that among the six proteins. Beta-2-glycoprotein 1 (APOH) and Aminopeptidase (ANPEP) are highly enriched proteins in sheep milk. Fibrinogen alpha chain (FGA) and alpha-1-b glycoprotein (A1BG) are highly enriched proteins in goat milk, and Angiogenin-1 (ANG1) and Serpin family G member 1 (SERPING1) are highly enriched proteins in milk. The results of PRM analysis were generally similar to those of DIA analysis, confirming the accuracy of the identification results.

[0074] Table 1 Information on six biomarkers

[0075] Gene Name Protein Name Gene ID APOH Beta-2-glycoprotein 1 W5Q268 ANPEP Aminopeptidase W5PQH0 FGA Fibrinogen alpha chain A0A452F4L3 A1BG Alpha-1-B glycoprotein A0A452G9T9 ANG1 Angiogenin-1 P10152 SERPING1 Serpin family G member 1 E1BMJ0

[0076] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. The use of a biomarker detection reagent in the preparation of a product for identifying the authenticity of dairy products, characterized in that: The dairy product is sheep milk, goat milk and / or cow milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein.

2. The use of the biomarker detection reagent according to claim 1 in the preparation of a product for identifying the authenticity of dairy products, characterized in that: APOH protein and ANPEP protein are biomarkers for identifying sheep milk.

3. The use of the biomarker detection reagent according to claim 1 in the preparation of a product for identifying the authenticity of dairy products, characterized in that: A1BG protein and FGA protein are biomarkers for identifying goat milk.

4. The use of the biomarker detection reagent according to claim 1 in the preparation of a product for identifying the authenticity of dairy products, characterized in that: ANG1 protein and SERPING1 protein are biomarkers for identifying milk.

5. The use of the biomarker detection reagent according to claim 1 in the preparation of a product for identifying the authenticity of dairy products, characterized in that: The reagents include reagents for quantitatively detecting the biomarkers using the DIA proteomics technology. In the chromatographic separation using the DIA proteomics technology, mobile phase A is a formic acid aqueous solution with a volume fraction of 0.09% to 0.11%, and mobile phase B is a mixture of formic acid and acetonitrile aqueous solutions, wherein the volume fraction of acetonitrile in the acetonitrile aqueous solution is 79% to 81%, and the volume fraction of formic acid in the mobile phase B is 0.09% to 0.11%.

6. Use of the biomarker detection reagent according to claim 1 in preparing a product for identifying the authenticity of dairy products, characterized in that: The reagents include reagents for quantitatively detecting the biomarkers using the parallel reaction monitoring technology PRM. In the chromatographic separation of the parallel reaction monitoring technology PRM, buffer A is a formic acid aqueous solution with a volume fraction of 0.09% to 0.11%, buffer B is a mixture of formic acid and acetonitrile aqueous solutions, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 94% to 96%, and the volume fraction of formic acid in the buffer B is 0.09% to 0.11%.

7. A reagent for detecting the biomarker according to claim 1, characterized in that: The reagent is used for detecting the contents of APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein in dairy products.

8. The reagent according to claim 7, characterized in that The reagents include reagents for quantitatively detecting APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and / or SERPING1 protein in the dairy product by DIA proteomics technology and / or parallel reaction monitoring technology PRM; in the chromatographic separation of DIA proteomics technology, mobile phase A is a formic acid aqueous solution with a volume fraction of 0.09% to 0.11%, mobile phase B is a mixed solution of formic acid and acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 79% to 81%, and the volume fraction of formic acid in the mobile phase B is 0.09% to 0.11%; in the chromatographic separation of parallel reaction monitoring technology PRM, buffer A is a formic acid aqueous solution with a volume fraction of 0.09% to 0.11%, buffer B is a mixed solution of formic acid and acetonitrile aqueous solution, the volume fraction of acetonitrile in the acetonitrile aqueous solution is 94% to 96%, and the volume fraction of formic acid in the buffer B is 0.09% to 0.11%.

9. A kit comprising the reagent according to claim 7 or claim 8.

10. Use of the kit according to claim 9 in identifying the authenticity of dairy products, characterized in that: The dairy product is sheep milk, goat milk and / or cow milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein.

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