Method for identifying Putian black duck and Liancheng white duck eggs based on metabonomics
Through metabolomics technology, differential metabolites were screened to distinguish between the duck eggs of Putian black duck and Liancheng white duck, solving the problem of low identification accuracy in the existing technology, and achieving accurate identification of duck eggs and supporting food traceability.
Patent Information
- Application Number
- CN202510284115.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-06-27
AI Technical Summary
The prior art is difficult to accurately distinguish between the duck eggs of Putian black duck and Liancheng white duck. The traditional method has low accuracy and is difficult to distinguish similar varieties.
Using a metabolomics-based method, the metabolites components of duck egg white samples were analyzed by liquid chromatography-secondary mass spectrometry combination technology, and differential metabolites were screened to achieve accurate identification of varieties.
It has achieved the precise identification of Putian Black Duck and Liancheng White Duck Duck Eggs, provided an excellent food traceability, quality control and variety identification method, and has broad application prospects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of duck egg variety identification, and particularly relates to a method for identifying Putian black duck and Liancheng white duck eggs based on metabolomics. Background Art
[0002] Poultry eggs contain all the nutrients necessary for maintaining life, including proteins, lipids, minerals, vitamins, etc., and are one of the most important sources of animal nutrition for humans. There are certain differences in the nutrient composition of different poultry eggs, thus presenting different flavors and textures. For example, the hardness and chewiness of the egg white gel of Liancheng white duck eggs are lower than those of Putian black duck eggs, so Putian black duck eggs do not taste as smooth and tender as Liancheng white duck eggs; and Liancheng white duck eggs not only meet the selenium-rich standard, but also are rich in 17 kinds of amino acids, and the protein and trace element contents are also extremely rich.
[0003] In the market, the identification of duck egg varieties is of great significance for food quality control and brand protection. Traditional identification methods rely on morphological characteristics or protein analysis, but these methods have low accuracy and are difficult to distinguish similar varieties. Metabolomics is a newly developed discipline following genomics and proteomics, which can comprehensively analyze small molecule metabolites in biological samples and has the characteristics of high throughput, short required time, and high precision. Among them, untargeted metabolomics helps to more comprehensively reflect the differences in metabolites, and the precise identification of varieties can be achieved by detecting the differences in metabolites of different varieties of duck eggs, laying a foundation for the precise evaluation of food quality and traceability. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for identifying Putian black duck and Liancheng white duck eggs based on metabolomics, so as to provide technical support for the precise identification of Putian black duck and Liancheng white duck eggs.
[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0006] The present invention provides a method for identifying Putian black duck and Liancheng white duck eggs based on metabolomics, comprising the following steps:
[0007] (1) Pretreat the duck egg white to obtain a test sample;
[0008] (2) Extract and mix the solution of each test sample obtained in step (1) to obtain a quality control sample;
[0009] (3) Inject the test sample and the quality control sample, and analyze the metabolite components of the sample by liquid chromatography-tandem mass spectrometry technology;
[0010] The liquid chromatography is carried out in positive and negative ion modes; positive ion mode: mobile phase A is formic acid, and mobile phase B is methanol; negative ion mode: mobile phase A is ammonium acetate, and mobile phase B is methanol; the elution gradient is: at 0 min, 98% A, 2% B; at 1.5 min, 98% A, 2% B; at 3 min, 15% A, 85% B; at 10 min, 0% A, 100% B; at 10.1 min, 98% A, 2% B; at 11 min, 98% A, 2% B; at 12 min, 98% A, 2% B;
[0011] The mass spectrometry signals are collected in positive and negative ion scanning modes; the scanning range is 100 - 1500 m / z; the spray voltage: 3.3 - 3.8 kV; the sheath gas flow rate: 30 - 40 psi; the auxiliary gas flow rate: 8 - 12 L / min; the ion transfer tube temperature: 300 - 340 °C; the ion introduction RF level: 50 - 70; the auxiliary gas heater temperature: 330 - 370 °C; MS / MS second-level scanning: ddMS 2 ;
[0012] (4) Process and analyze the data obtained in step (3) to screen for differential metabolites in the egg white samples of Putian black ducks and Liancheng white ducks;
[0013] (5) Identify the eggs of Putian black ducks and Liancheng white ducks based on the differential metabolites obtained in step (4).
[0014] Preferably, the pretreatment method in step (1) is: after shelling the duck eggs, collect the egg white liquid, and after homogenization, treat the egg white liquid by the methanol extraction method;
[0015] The steps of treating by the methanol extraction method are: mix the egg white liquid with a methanol aqueous solution and perform ice bath treatment, after solid-liquid separation, take the liquid part, adjust the methanol content to 50 - 55% and then perform solid-liquid separation again, take the liquid part to obtain the test sample.
[0016] Preferably, the concentration of the methanol aqueous solution is 75 - 85%; the volume ratio of the egg white liquid to the methanol aqueous solution during mixing is 80 - 120:300 - 500; the mixing method is vortex oscillation, and the frequency during vortex oscillation is 2500 - 3500 g, and the time is 10 - 30 s;
[0017] The time of the ice bath treatment is 3 - 7 min; the method of solid-liquid separation is centrifugation, the temperature during centrifugation is 3 - 5 °C, the rate is 13000 - 17000 g, and the time is 15 - 25 min.
[0018] Preferably, the chromatographic column used in the liquid chromatography in step (3) is a Hypesil Gold column, 1.9 μm, 2.1 mm × 100 mm; the column temperature is 35 - 45 °C, and the flow rate is 0.15 - 0.25 mL / min;
[0019] The concentration of the formic acid is 0.08 - 0.12%, and the concentration of the ammonium acetate is 4 - 6 mmol / L.
[0020] Preferably, the method for data processing in step (4) is as follows: Use CompoundDiscoverer 3.1 data processing software to perform integration and calibration of chromatographic peaks on the raw data downloaded from the instrument. Extract chromatographic peaks according to the set mass deviation of 4 - 6 ppm, signal intensity deviation of 28 - 32%, and signal-to-noise ratio of 3. Among them, the peak area Area of each chromatographic peak represents the relative content of the corresponding substance; then predict the molecular formula through the molecular ion peak and fragment ions and compare it with the database, remove background ions with the blank sample, and perform normalization processing on the original quantitative results. Finally, obtain the identification and relative quantitative results of metabolites; among them, the blank sample is an aqueous solution of 50 - 55% methanol instead of the sample;
[0021] The method for the analysis is as follows: Use the KEGG database and the LIPID MAPS database to annotate the identified metabolites; perform principal component analysis and partial least squares discriminant analysis on the data after conversion through the metabolomics data processing software metaX to obtain the VIP value of each metabolite; detect the significance level of each metabolite between the two groups based on the t-test and calculate the FC value of the metabolite between the two groups;
[0022] The criteria for screening differential metabolites are: VIP > 1, P < 0.05, and the ratio of metabolite expression levels between Putian Black Duck and Liancheng White Duck egg samples is greater than 1.5 or less than 0.67.
[0023] Preferably, in order to streamline the number of differential metabolites, the fold change of each differential metabolite in the two groups of samples of the egg white of Putian Black Duck and Liancheng White Duck was also compared. Taking Log2 as the base, the calculation results were sorted, and differential metabolites with |Log2FC| greater than 2.5 were selected.
[0024] Preferably, the differential metabolites with |Log2 FC| greater than 2.5 include Caffeine, 5,6-dimethyl-4-oxo-4H-pyran-2-carboxylic acid, Homogentisic Acid, 6-Aminonicotinamide, Dl-3,4-Dihydroxymandelic Acid, 5,6-Dimethylbenzimidazole, 4-acetyl-4-(ethoxycarbonyl)heptanedioic acid, Guanethidine Monosulfate, 2-Hydroxy-2-methyl-3-buten-1-ylbeta-D-glucopyranoside, Prostaglandin A1, (2S)-4-Oxo-2-phenyl-3,4-dihydro-2H-chromen-7-yl beta-D-glucopyranoside, alpha-Ketoglutaric acid, N-Acetylsphingosine, 3-Methylcrotonylglycine, and 6-anilino-1,3-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione.
[0025] Preferably, in step (5), if the differential metabolites Caffeine, 5,6-dimethyl-4-oxo-4H-pyran-2-carboxylic acid, Homogentisic Acid, 6-Aminonicotinamide, Dl-3,4-Dihydroxymandelic Acid, 5,6-Dimethylbenzimidazole, 4-acetyl-4-(ethoxycarbonyl)heptanedioic acid, Guanethidine Monosulfate, and 2-Hydroxy-2-methyl-3-buten-1-ylbeta-D-glucopyranoside are extremely significantly highly expressed, it is identified as Liancheng white duck eggs;
[0026] If the differential metabolites Prostaglandin A1, (2S)-4-Oxo-2-phenyl-3,4-dihydro-2H-chromen-7-yl beta-D-glucopyranoside, alpha-Ketoglutaric acid, N-Acetylsphingosine, 3-Methylcrotonylglycine, and 6-anilino-1,3-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione are extremely significantly highly expressed, it is identified as Putian Black Duck eggs.
[0027] The present invention provides an application of differential metabolites in identifying Putian Black Duck and Liancheng White Duck eggs, and the differential metabolites are selected from any one or several of the differential metabolites screened by the described method.
[0028] The present invention also provides a differential metabolite composition for identifying Putian Black Duck and Liancheng White Duck eggs, and the differential metabolite composition is selected from any two or more of the differential metabolites screened by the described method.
[0029] Compared with the prior art, the present invention has the following beneficial effects:
[0030] The present invention provides a method for screening differential metabolites for identifying Putian Black Duck and Liancheng White Duck eggs by using non-targeted metabolomics technology, and the method can clearly reveal the differences between Putian Black Duck and Liancheng White Duck eggs. The differential metabolites screened by the present invention are significantly different in the egg whites of Putian Black Duck and Liancheng White Duck eggs and can be used to accurately identify Putian Black Duck and Liancheng White Duck eggs. The present invention provides an excellent method for food traceability, quality control, and variety identification of Putian Black Duck and Liancheng White Duck eggs and has broad application prospects. Description of the Drawings
[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.
[0032] Figure 1 It is the correlation analysis result of the quality control samples in the positive (left in the figure) and negative (right in the figure) ion modes in Example 1;
[0033] Figure 2 It is the PCA score plot of the egg white differential metabolites in the positive (left in the figure) and negative (right in the figure) ion modes in Example 1;
[0034] Figure 3 It is the partial least squares discriminant analysis result of differential metabolites in positive (left in the figure) and negative (right in the figure) ion modes in Example 1;
[0035] Figure 4 It is the clustering heat map of differential metabolites screened in the positive ion mode in Example 1;
[0036] Figure 5 It is the clustering heat map of differential metabolites screened in the negative ion mode in Example 1. Detailed implementation manners
[0037] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.
[0038] The experimental instruments and reagents used in the following embodiments are shown in Table 1 and Table 2.
[0039] Table 1 Experimental instruments
[0040]
[0041] Table 2 Experimental reagents
[0042] Name Purity CAS No. Water LC-MS Grade 7732-18-5 Methanol LC-MS Grade 67-56-1 Formic Acid LC-MS Grade 64-18-6 Ammonium Acetate LC-MS Grade 631-61-8
[0043] Example 1
[0044] A method for identifying Putian black duck and Liancheng white duck eggs based on metabolomics, the steps are as follows:
[0045] (1) Preparation of test samples and quality control samples
[0046] After the duck eggs are shelled, the egg white liquid is collected, impurities are removed and homogenized, the test samples are processed using the methanol extraction method, and quality control samples are set.
[0047] Test sample processing method: Take 100 μL of egg white liquid and place it in an EP tube, add 400 μL of 80% methanol aqueous solution, vortex at 3000 g for 20 s, let it stand in an ice bath for 5 min, centrifuge at 4 °C and 15000 g for 20 min, take the supernatant and dilute it with mass spectrometry-grade water until the methanol content is 53%, centrifuge at 4 °C and 15000 g for 20 min, collect the supernatant to obtain the test sample.
[0048] Equal-volume samples are taken from each test sample and mixed to obtain the quality control sample.
[0049] (2) Sample detection
[0050] Inject the test samples and quality control samples obtained in step (1), and analyze the metabolite components of the samples by liquid chromatography-tandem mass spectrometry to obtain the original data of the sample metabolites.
[0051] Among them, the liquid chromatography conditions are shown in Table 3. The sample mass spectrometry signals are collected using positive and negative ion scanning modes: scanning range: 100 - 1500 m / z; spray voltage: 3.5 kV; sheath gas flow rate: 35 psi; auxiliary gas flow rate: 10 L / min; ion transfer tube temperature: 320 °C; ion introduction RF level: 60; auxiliary gas heater temperature: 350 °C; MS / MS second-level scan: ddMS 2 .
[0052] Table 3 Liquid Chromatography Conditions
[0053]
[0054] (3) Metabolite Identification
[0055] Import the original data obtained in step (2) into CompoundDiscoverer 3.1 (CD 3.1) software for chromatographic peak integration and calibration. Extract chromatographic peaks according to the set mass deviation of 5 ppm, signal intensity deviation of 30%, and signal-to-noise ratio of 3. Among them, the peak area Area of each chromatographic peak represents the relative content of the corresponding substance; then predict the molecular formula through the molecular ion peak and fragment ions and compare it with the database, remove background ions with a blank sample (replacing the test sample with a 53% methanol aqueous solution, and the pretreatment process is the same as that of the test sample), and standardize the original quantitative results. Finally, obtain the identification and relative quantification results of the metabolites.
[0056] (4) Data Processing
[0057] Use the KEGG database (https: / / www.genome.jp / kegg / pathway.html) and the LIPID MAPS database (http: / / www.lipidmaps.org / ) to annotate the metabolites identified in step (3).
[0058] Multivariate statistical analysis: After converting the data through the metabolomics data processing software metaX, perform principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to obtain the VIP value of each metabolite.
[0059] In univariate analysis, the significance level (P-value) of each metabolite between the two groups was detected based on the t-test, and the fold change (FC) value of the metabolite between the two groups was calculated. The screening criteria for differential metabolites were VIP > 1, P < 0.05, and FC > 1.5 or FC < 0.67.
[0060] The clustering heatmap (heatmap) was drawn using the R package Pheatmap, and the metabolite data was normalized using z-score.
[0061] The correlation analysis diagrams of quality control samples in positive and negative ion modes are as Figure 1 shown, and the PCA score diagram of differential metabolites in egg white is as Figure 2 shown, and the partial least squares discriminant analysis results of differential metabolites are as Figure 3 shown, and the heatmap diagrams of differential metabolites are as Figure 4 and Figure 5 shown.
[0062] It can be seen from Figure 1 that the correlation coefficients between QC samples are all greater than 0.99 and close to 1, indicating good instrument stability and reliable data.
[0063] To understand the variability between and within sample groups, PCA analysis was performed on the test sample data. According to Figure 2 shown, in positive and negative ion modes, the egg white samples of Putian black ducks and Liancheng white ducks were significantly separated, indicating significant metabolite differences between the two groups of samples.
[0064] PLS-DA statistical analysis was performed on the egg white samples of Putian black ducks and Liancheng white ducks, and permutation tests were performed on the model parameters R2 and Q2, with the number of tests set to 200 times. According to Figure 3 it can be seen that in the positive ion mode for the egg white samples of Putian black ducks and Liancheng white ducks, R2Y = 0.99, Q2Y = 0.89, R2Y > Q2Y, and the intercept of Q2 with the Y-axis is less than 0. In the negative ion mode, R2Y = 0.99, Q2Y = 0.88, R2Y > Q2Y, and the intercept of Q2 with the Y-axis is less than 0. The results in both positive and negative ion modes indicate high model quality, strong reliability in screening differential metabolites, no "overfitting", and clear separation between the two breeds in the PC1 direction, indicating differences in the metabolomes of the Putian black duck group and the Liancheng white duck group samples.
[0065] Figure 4 and Figure 5It is a clustering correlation heat map of differential metabolites. Changing from red to blue indicates a down-regulation trend, and the overall change trend of differential metabolites can be seen from the figure. Under positive and negative ion modes, the egg white samples of Putian black ducks and Liancheng white ducks are clustered into two categories according to breed, indicating that there is little difference in the metabolome within the breed, but there are differences between breeds; the hierarchical clustering of the relative quantitative values of metabolites in different groups is significantly distinguishable, showing obvious expression differences. From Figure 4 and Figure 5 it can be seen that by using VIP>1.0, P<0.05 and FC>1.5 or FC<0.67 as the criteria to screen differential metabolites between groups, for the analysis of differential metabolites in the egg white of Putian black ducks and Liancheng white ducks, 93 differential metabolites were screened out under positive ion mode, and 69 differential metabolites were identified in total under negative ion mode.
[0066] At the same time, in order to streamline the number of differential metabolites, the change multiples of each differential metabolite in the two groups of samples of the egg white of Putian black ducks and Liancheng white ducks were compared, and the calculation results were sorted with Log2 as the base, and differential metabolites with |Log2 FC| greater than 2.5 were selected, and a total of 15 differential metabolites were obtained. The results are shown in Table 4. It can be seen from Table 4 that 9 metabolites were extremely significantly highly expressed in the egg white of Liancheng white ducks, and 6 metabolites were extremely significantly low expressed, which can clearly distinguish the duck eggs of Liancheng white ducks and Putian black ducks.
[0067] Table 4 Differential metabolites between the egg white groups of Putian black ducks and Liancheng white ducks
[0068]
[0069]
[0070] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A method for identifying duck eggs from Putian black duck and Liancheng white duck based on metabolomics, characterized in that: The steps include: (1) Pre-treating duck egg white to obtain a test sample; (2) extracting and mixing the test sample solutions obtained in step (1) to obtain a quality control sample; (3) Inject test samples and quality control samples, and analyze the metabolite components of the samples by liquid chromatography-tandem mass spectrometry; The liquid chromatography was performed in positive and negative ion modes; in positive ion mode, mobile phase A was formic acid and mobile phase B was methanol; in negative ion mode, mobile phase A was ammonium acetate and mobile phase B was methanol; the elution gradient was: 0 min, 98% A, 2% B; 1.5 min, 98% A, 2% B; 3 min, 15% A, 85% B; 10 min, 0% A, 100% B; 10.1min, 98% A, 2% B; 11min, 98% A, 2% B; 12min, 98% A, 2% B; The mass spectrometry signal was collected in positive and negative ion scanning mode; the scanning range was 100-1500 m / z; the spray voltage was 3.3-3.8 kV; the sheath gas flow rate was 30-40 psi; the auxiliary gas flow rate was 8-12 L / min; the ion transfer tube temperature was 300-340 °C; the ion introduction radio frequency level was 50-70; Auxiliary gas heater temperature: 330~370℃; MS / MS secondary scan: ddMS 2 ; (4) processing and analyzing the data obtained in step (3) to screen for differential metabolites in egg white samples of Putian black duck and Liancheng white duck; (5) Identify the eggs of Putian black duck and Liancheng white duck based on the differential metabolites obtained in step (4).
2. The method according to claim 1, characterized in that The pretreatment method in step (1) is: shelling duck eggs, collecting egg white liquid, homogenizing the egg white liquid and then extracting it with methanol; The steps of the methanol extraction method are: mixing the egg white liquid and the methanol aqueous solution and then treating them in an ice bath, taking the liquid part after solid-liquid separation, adjusting the methanol content to 50-55%, and then performing solid-liquid separation again, taking the liquid part, and obtaining the test sample.
3. The method according to claim 2, characterized in that The concentration of the methanol aqueous solution is 75-85%; the volume ratio of the egg white solution and the methanol aqueous solution when mixed is 80-120:300-500; the mixing method is vortex oscillation, the frequency of the vortex oscillation is 2500-3500g, and the time is 10-30s; The ice bath treatment time is 3 to 7 minutes; the solid-liquid separation method is centrifugation, the temperature during the centrifugation is 3 to 5° C., the speed is 13000 to 17000 g, and the time is 15 to 25 minutes.
4. The method according to claim 1, characterized in that The chromatographic column used for liquid chromatography in step (3) is a Hypesil Gold column, 1.9 μm, 2.1 mm×100 mm; the column temperature is 35 to 45° C., and the flow rate is 0.15 to 0.25 mL / min; The concentration of the formic acid is 0.08-0.12%, and the concentration of the ammonium acetate is 4-6 mmol / L.
5. The method according to claim 1, characterized in that The data processing method in step (4) is as follows: the raw data off the machine is integrated and corrected by using Compound Discoverer 3.1 data processing software, and the chromatographic peaks are extracted according to the set mass deviation of 4 to 6 ppm, signal intensity deviation of 28 to 32%, and signal-to-noise ratio of 3, wherein the peak area Area of each chromatographic peak represents the relative content of the corresponding substance; then the molecular formula is predicted by the molecular ion peak and the fragment ion and compared with the database, the background ions are removed by using the blank sample, and the original quantitative results are standardized, and finally the identification and relative quantitative results of the metabolites are obtained; wherein the blank sample is a 50 to 55% methanol aqueous solution instead of the sample; The analysis method is as follows: annotating the identified metabolites using the KEGG database and the LIPID MAPS database; performing principal component analysis and partial least squares discriminant analysis after data conversion using the metabolomics data processing software metaX to obtain the VIP value of each metabolite; detecting the significance level of each metabolite between the two groups based on the t-test, and calculating the FC value of the metabolite between the two groups; The criteria for screening differential metabolites were: VIP>1, P<0.05, and the ratio of metabolite expression between Putian black duck and Liancheng white duck egg samples was greater than 1.5 or less than 0.
67.
6. The method according to claim 5, characterized in that In order to streamline the number of differential metabolites, the fold changes of each differential metabolite in the egg white samples of Putian black duck and Liancheng white duck were compared. The calculation results were sorted based on Log2, and differential metabolites with |Log2FC| greater than 2.5 were selected.
7. The method according to claim 6, characterized in that |Log2 FC|Differential metabolites greater than 2.5 include Caffeine, 5,6-dimethyl-4-oxo-4H-pyran-2-carboxylic acid, Homogene ntisic Acid, 6-Aminonicotinamide, Dl-3,4-Dihydroxymandelic Acid, 5,6-Di methylbenzimidazole, 4-acetyl-4-(ethoxycarbonyl)heptanedioic acid, Guanethi dine Monosulfate, 2-Hydroxy-2-methyl-3-buten-1-yl beta-D-glucopyranoside, Prostaglandin A1, (2S)-4-Oxo-2-phenyl-3,4-dihydro-2H-chromen-7-yl beta-D-glucopyranoside, alpha-Ketoglutaric acid, N-Acetylsphingosine, 3-Methylcrot onylglycine and 6-anilino-1,3-dimethyl-1,2,3,4-tetrahydropyrimidine-2,4-dione.
8. The method according to claim 7, characterized in that In step (5), if the differential metabolites Caffeine, 5,6-dimethyl-4-oxo-4H-pyran-2-carboxylic acid, Homogentisic Acid, 6-Aminonicotinamide, Dl-3,4-Dihydroxymandelic Acid, 5,6-Dimethylbenzimidazole, 4-acetyl-4-(ethoxycarbonyl)heptanedioic acid, Guanethidine Monosulfate and 2-Hydroxy-2-methyl-3-buten-1-yl beta-D-glucopyranoside are extremely significantly highly expressed, the duck eggs are identified as Liancheng white duck eggs; If the differential metabolites Prostaglandin A1, (2S)-4-Oxo-2-phenyl-3,4-dihydro-2H-chloromen-7-yl beta-D-glucopyranoside, alpha-Ketoglutaric acid, N-Acetylsphingosine, 3-Methylcrotonylglycine and 6-anilino-1,3-dimethyl-1,2,3,4-tetrahydropyri midine-2,4-dione are extremely significantly overexpressed, then the eggs are identified as Putian black duck eggs.
9. Application of differential metabolites in identifying eggs of Putian black duck and Liancheng white duck, characterized in that: The differential metabolites are selected from any one or more of the differential metabolites screened according to the method of any one of claims 1 to 7.
10. A differential metabolite composition for identifying eggs of Putian black duck and Liancheng white duck, characterized in that: The differential metabolite composition is selected from any two or more of the differential metabolites screened according to the method of any one of claims 1 to 7.