Application of biomarker detection reagent in preparation of product for identifying authenticity of dairy product

Abstract

The invention belongs to the technical field of dairy product detection, and particularly relates to application of a biomarker detection reagent in preparation of a product for identifying authenticity of dairy products. The dairy product is sheep milk, goat milk and / or milk, and the biomarkers are APOH protein, ANPEP protein, A1BG protein, FGA protein, ANG1 protein and SERPING1 protein. The APOH protein and the ANPEP protein are biomarkers for identifying sheep milk, the A1BG protein and the FGA protein are biomarkers for identifying goat milk, and the ANG1 protein and the SERPING1 protein are biomarkers for identifying milk. The research of the invention is helpful for improving the understanding of consumers on the components and functions of various dairy products, and provides a significant theoretical basis for the high-quality development of professional dairy products. In addition, the method can promote establishment of a method for identifying adulteration of dairy products, so that a major challenge faced by the global dairy product industry is supported.

Description

Technical Field

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

[0002] Dairy products are rich in nutrients and are an important source of protein in the human diet. Currently, consumer demand for high-quality dairy products is growing, and the supply of high-quality small ruminant milk, especially sheep milk and goat milk, has risen to meet this market demand. Although cow milk currently has a large market share, most people in the world are lactose intolerant. Compared with cow milk, sheep milk and goat milk have higher nutritional value, relatively higher dry matter content, and milk fat is in a spherical shape with a smaller diameter, which is conducive to digestion and metabolism. However, the price of sheep milk and goat milk is high, the output is low, and some merchants choose to adulterate.

[0003] Milk is about 80% casein and 20% whey protein, including a variety of functional proteins such as lactoferrin and immunoglobulins. The content of these proteins varies in different types of milk, and the resulting differences in dairy product function are increasingly attracting attention. By comparing proteomic analysis of different types of milk, specific protein molecules can be identified that may be potential biomarkers for milk from different species.

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

[0005] In order to solve the above technical problems, the present invention provides the use of a biomarker detection reagent in the preparation of a product for identifying the authenticity of dairy products.

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

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

[0008] In order to study the differences in protein composition in sheep milk, goat milk and cow milk, and thus determine potential biomarkers, the present invention uses Astral-DIA proteomics to analyze differentially expressed proteins, and uses parallel reaction monitoring (PRM) method to verify the results. Using bioinformatics methods, the functions of characteristic proteins and related biological processes were predicted through gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and a total of 4316 differential proteins were identified, among which 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 milk. Functional analysis shows that these different proteins are enriched through different pathways such as complement and coagulation cascades. These data reveal differences in protein content and physiological functions, providing important basis for research on 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 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 solution, 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%.

[0010] Furthermore, the reagent includes a reagent for quantitatively detecting the biomarker by parallel reaction monitoring technology (PRM). 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 mixture 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 buffer B is 0.09% to 0.11%.

[0011] In a second aspect, the present invention provides a reagent for detecting the biomarker, wherein 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] Further, the reagents include reagents 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 a formic acid aqueous solution with a volume fraction of 0.09% to 0.11%, mobile phase B is a mixture 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 mixture 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%. Within this range, the corresponding separation effect can be achieved.

[0013] In a third aspect, the present invention provides a kit comprising the reagent.

[0014] In a fourth aspect, the present invention provides use of the 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 have great benefits for 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 to identify dairy adulteration, thus supporting a major challenge facing the global dairy industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A Venn diagram of proteins in sheep, goat, and milk.

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

[0019] Figure 3 Hierarchical clustering of significantly different proteins in sheep, goat, and cow milk.

[0020] Figure 4 The difference in protein between sheep milk-goat milk group (SG), sheep milk-cow milk group (SC), goat milk-cow milk group (GC), among which:

[0021] A is the difference in protein between sheep milk and goat milk group (SG).

[0022] B is the difference in protein between sheep milk and cow milk group (SC).

[0023] C is the difference in protein between goat milk and cow milk group (GC).

[0024] Figure 5 To conduct GO enrichment analysis on differentially expressed proteins in sheep milk, goat milk and cow milk based on biological processes.

[0025] Figure 6 To conduct GO enrichment analysis of differentially expressed proteins in sheep milk, goat milk and cow milk based on cellular components.

[0026] Figure 7 To conduct GO enrichment analysis of differentially expressed proteins in sheep milk, goat milk and cow milk based on molecular functions.

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

[0028] Fig. 9 KEGG pathway analysis of differentially expressed proteins in sheep milk-cow milk group (SC).

[0029] Fig.10 KEGG pathway analysis of differentially expressed proteins in goat milk-cow milk group (GC).

[0030] Fig.11 Figure 2 shows the PPI network analysis of differentially expressed proteins in the sheep milk-goat milk group (SG). Red nodes represent up-regulated proteins, while green nodes represent down-regulated proteins.

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

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

[0033] Fig.14 The results of DIA and PRM testing of APOH, A1BG, ANG1 in sheep milk, goat milk and cow milk, among which:

[0034] A is the result of DIA detection of 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] Fig.15 Results of DIA and PRM testing of ANPEP, FGA, SERPING1 in sheep milk, goat milk and cow milk, including:

[0041] A is the result of DIA testing 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 shows the results 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 DESCRIPTION

[0047] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

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

[0049] 1 Test methods

[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 Orbital Astro mass spectrometer coupled to a Conquest Neo UHPLC system (Thermo Fisher Scientific). The RP-HPLC mobile phase A was 0.1% formic acid in water, and the mobile phase B was 80% acetonitrile in water with 0.1% formic acid. The 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, 4% to 6% buffer B; 0.1-1.1 min, 6% to 12%; 1.1-4.3 min, 12% to 2.3-6.1 min, 25% to 45% linear gradient, 6.5 min, 45% to 99% buffer B; 6.5-8 min, buffer B was maintained at 99%. DIAMS / MS scans were performed using Astral, 150-2000 m / z, isolation window of 2 m / z, AGC target of 500%, and injection time of 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. GO and KEGG were analyzed using Fisher's exact test, and FDR correction was performed for multiple tests. GO was divided into three categories: biological process (BP), molecular function (MF), and cellular component (CC). Protein-protein interaction (PPI) networks were constructed using the String database and Cytoscape software.

[0057] 1.4LC-PRM / MS analysis

[0058] Peptides (2 μg) were subjected to LC-PRM / MS analysis. Chromatographic separation was performed using a nano-flow Easy nLC 1200 chromatography system (Thermo Scientific). Buffer A was a 0.1% formic acid solution in water, and buffer B was a 95% acetonitrile solution in water containing 0.1% formic acid. Equilibration was performed with 95% buffer A. The sample was poured into a trap column (100 μm*20 mm, 5 μm, C18, Maisch GmbH) and then gradient separated on a chromatographic column (75 μm*150 mm, 3 μm, C18, Maisch GmbH). The 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 spectrum resolution was 60000@m / z200, the AGC target was 3e6, and the primary mass spectrum maximum IT was 50 ms. The peptide was subjected to secondary mass spectrometry analysis: the resolution was 30000@m / z200, the AGC target was 1e6, the secondary mass spectrum maximum IT was 100 ms, the MS2 activation type was HCD, and the separation window was 1.6 Th. The resulting RAW mass spectrum files were analyzed using Skyli software.

[0059] 1.5Data analysis

[0060] DIA-nn (version 1.8.1) software was used to combine all mass spectrometry data, complete database retrieval of DIA mass spectrometry data and quantitative analysis of DIA proteins. The main software parameters were: trypsin was used as the enzyme, the FDR of peptide spectrum matching (PSM) and protein level was 0.01, and the modification was fixed to amino group (C). The databases were sheep (9940), goat (9925) and cattle (9913) (https: / / www.uniprot.org / taxonomy / 9904(9925)(9913)). To determine the significant difference between proteins, the t-test combined with the Fold change (FC) method was used to analyze the 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 to compare the groups. All data visualizations were performed using GraphPad Prism 8.

[0061] 2 Test results

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

[0063] A total of 4316 proteins were identified in sheep, goat, and cow milk. The Venn diagram shows the differences in identified proteins between species, such as Figure 1As shown. 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 in sheep milk and goat milk, 77 proteins in sheep milk and cow milk, and 284 proteins in goat milk and cow milk. These data reveal the differences in protein levels in sheep, goat and cow milk, laying the foundation for further screening of potential biomarkers.

[0064] 2.2 Differentially expressed proteins (DEPs) in sheep, goat, and cow milk

[0065] Principal component analysis of the identified proteins showed that the samples in this study were classified into sheep milk, goat milk, and cow milk. Figure 2 The PCA score plot in Figure 2 shows that goat milk is different from the other two types (sheep milk and cow milk) according to the direction of PC1. PC1 and PC2 account for 41.17% and 23.97% of the protein variation between different species, respectively. In this study, P < 0.05 and FC ≥ 1.50 or ≤ 1 / 1.50 were considered to be significant DEPs. Figure 3 As shown in the clustering results, three major distinct protein clusters were observed in the heat map, and the patterns differed between samples of the three species. LBP, TUBB4A, ATP1B1, TUBB2A, and TUBA1D were the most abundant proteins, but in different proportions in sheep, goat, and cow milk. Figure 4 The volcano plot shows the differential expression of proteins in the sheep milk-goat milk group (SG), sheep milk-cow milk group (SC), and goat milk-cow milk group (GC), and 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, goats and milk

[0067] Using GO enrichment analysis, 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 SG were significantly enriched in peptide metabolic processes and peptide biosynthesis processes in BP, ribosomes and intracellular anatomical structures in CC, and ribosome structural components and ligase activity in MF ( Figure 5 to Figure 7). The different proteins identified in SC were significantly enriched in BP in CC, proteolysis in the extracellular region and extracellular space, secretion, and endopeptidase regulatory activity in MF. The different proteins identified in GC were significantly enriched in translation, peptide biosynthesis processes in BP, ribosome and endopeptidase complexes in CC, and ribosome 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, goats and milk

[0069] Figure 8 to Figure 10 The chord diagrams of the top 15 pathways associated with DEP in different types of milk are shown. According to KEGG analysis, many proteins are involved in multiple disease pathways. In the SG group, the differential proteins were mainly involved in processes such as coronavirus disease-COVID-19, ribosome, etc., while in the SC group, the differential proteins were mainly involved in processes such as complement and coagulation cascades, lysosome, etc. In the GC 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, goats and milk

[0071] PPI analysis of differentially expressed proteins in the SG group showed 406 nodes. The top 50 differentially expressed proteins with the highest connectivity were as follows: Fig.11 As shown. Among them, Large ribosomal subunit protein eL19 (RPL19) interacts with 26 proteins and is 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) are associated with multiple proteins. In the SC group, 167 differentially expressed protein nodes were identified by PPI analysis. The top 50 differentially expressed proteins with the highest connectivity are as follows Fig.12As shown in the figure. 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 AI (APOA1) and Albumin (ALB) are associated with many proteins. PPI analysis of differentially expressed proteins in the GC group showed that there were 279 nodes. The top 50 differentially expressed proteins with the highest connectivity are shown in the figure. Fig.13 As shown. Among them, Large ribosomal subunit protein eL19 (RPL19) interacted with 17 proteins and was the node with the highest degree of interaction. Smallribosomal subunit protein uS3 (RPS3), Large ribosomal subunit protein uL14 (RPL23), Elongation factor 2 (EEF2), actin and differential proteins such as Cytoplasmic 1 (ACTB) were associated with multiple proteins. These results reflect the intrinsic relationship between sheep, goat and milk proteins.

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

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

[0074] Table 1 Information of six biomarkers

[0075] Gene name Protein name Gene ID APOH Beta-2-glycoprotein 1 W5Q ANPEP Aminopeptidase W5PQ 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, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and 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 equivalents, 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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