Method for evaluating authenticity and identifying adulteration of duck blood

Abstract

The present application relates to the technical field of food detection, and particularly relates to a duck blood authenticity evaluation and adulteration identification method. The method comprises the following steps: extracting protein of a duck blood sample to be detected, and performing enzymolysis; detecting the enzymolysis product, and realizing identification of the duck blood sample to be detected according to a detection result of a characteristic peptide segment combination, wherein the characteristic peptide segment combination is composed of the following characteristic peptide segments: MFIAYPQTK, GYETLLEK, MFTTYPPTK, GYESLLEK, MFLGFPTTK, ADFTEISK, AAVTAFWGK, LVNELTEFAK, AAVTGFWGK and YLYEVAR. In the present application, hemoglobin and plasma albumin are used as target detection marker proteins, different characteristic peptide segments are screened, and based on the detection result, the authenticity evaluation and adulteration identification of duck blood can be accurately and stably realized, thereby providing technical support for quality control of duck blood products.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for evaluating the authenticity of duck blood and identifying adulteration. Background Technology

[0002] Duck blood, a traditional nutritious food, is popular among consumers due to its smooth and delicate texture, unique flavor, and high nutritional value. Compared to other livestock and poultry blood products, duck blood has a relatively low fat content and its protein is easily digested and absorbed. Its unique taste and flavor characteristics result in high market demand and commercial value. However, the limited production of duck blood and the growing contradiction between supply and demand have led to a large number of adulterated duck blood products on the market, seriously disrupting the food market order and harming consumer rights. Currently, duck blood adulteration takes many forms, including directly using whole blood from other livestock and poultry such as chicken, pig, cow, and sheep to impersonate or mix with other types of blood; pre-treating other livestock and poultry blood through physical processing methods such as fat separation and centrifugal concentration to impersonate duck blood; adding serum protein powder obtained from the deep processing of other livestock and poultry blood to duck blood to create high-quality adulterated products; and even using gelatin and a small amount of duck blood powder to prepare fake duck blood. These adulteration phenomena, especially highly processed ones, are closer to real duck blood in composition and taste, making them difficult to identify using conventional methods. These adulteration practices not only disrupt market order and damage corporate reputation, but more importantly, they directly threaten consumers' right to know and right to safety, seriously impacting the healthy development of the food industry.

[0003] Currently, the identification of adulterated duck blood mainly employs PCR technology based on DNA polymorphism, which distinguishes different livestock and poultry by amplifying species-specific genes. However, this technology has significant limitations. PCR is essentially a qualitative identification method; it can only determine the presence of adulterants but cannot quantitatively analyze the adulteration ratio, making it difficult to accurately assess the authenticity of the product. Because blood products undergo high-temperature and high-pressure processing, DNA is easily degraded and damaged. In older or heavily processed samples, DNA integrity is low, leading to decreased detection sensitivity. Furthermore, PCR technology is less effective against carefully concealed trace adulteration and highly processed adulterated products, making it difficult to effectively combat increasingly sophisticated adulteration methods. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides a method for evaluating the authenticity of duck blood and identifying adulteration. It accurately identifies whether other similar animal-derived components are added to duck blood products by utilizing characteristic peptides of hemoglobin and plasma albumin.

[0005] In a first aspect, the present invention provides a method for evaluating the authenticity and identifying adulteration of duck blood, comprising: Protein was extracted from the duck blood sample to be tested and then enzymatically digested. The enzymatic hydrolysis products are detected, and the duck blood sample is identified based on the detection results of a combination of characteristic peptides. This combination of characteristic peptides consists of the following peptides: MFIAYPQTK, GYETLLEK, MFTTYPPTK, GYESLLEK, MFLGFPTTK, ADFTEISK, AAVTAFFWGK, LVNELTEFAK, AAVTAFFWGK, and YLYEVAR. Hemoglobin and plasma albumin are the most abundant proteins in whole blood of livestock and poultry, accounting for approximately 60%-70% and 10%-15% of total proteins, respectively. In normal duck blood samples, the content and relative proportion of these two proteins remain relatively constant. When other exogenous substances or blood from other livestock and poultry are added, the absolute content and relative proportion of these two proteins will inevitably change. Therefore, the content of these two proteins is highly correlated with the authenticity of livestock and poultry blood. Moreover, there are certain species differences in the amino acid sequences of hemoglobin and plasma albumin among different livestock and poultry species. Therefore, this invention targets species-specific peptides in duck blood and other livestock and poultry blood for detection, which can accurately identify whether duck blood samples are mixed with other livestock and poultry blood components such as chicken blood, pig blood, cow blood, and sheep blood, thus achieving accurate identification of adulterated components.

[0006] Furthermore, in the combination of characteristic peptides, MFIAYPQTK, MFTTYPPTK, MFLGFPTTK, AAVTAFWGK, and AAVTGFWGK are used for quantitative determination. GYETLLEK, GYESLLEK, ADFTEISK, LVNELTEFAK, and YLYEVAR are used for qualitative judgment.

[0007] Furthermore, in the combination of characteristic peptides: MFIAYPQTK and GYETLLEK are used for the identification of duck blood components; MFTTYPPTK and GYESLLEK are used for the identification of chicken blood components; MFLGFPTTK and ADFTEISK are used for the identification of components in pig blood; AAVTAFWGK and LVNELTEFAK are used for bovine blood component identification; AAVTGFWGK and YLYEVAR are used for the identification of sheep blood components.

[0008] Furthermore, the identification includes any of the following: (1) When the duck blood sample to be tested contains MFIAYPQTK or GYETLLEK, it is determined that the duck blood sample to be tested contains real duck blood components. (2) Detect the ion peak of MFIAYPQTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the duck blood component content in the duck blood sample to be tested based on the comparison result; (3) When the duck blood sample to be tested contains MFTTYPPTK or GYESLLEK, it is determined that it contains adulterated chicken blood. (4) Detect the ion peak of MFTTYPPTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of chicken blood adulterants in the duck blood sample to be tested based on the comparison result. (5) When the duck blood sample to be tested contains MFLGFPTTK or ADFTEISK, it is determined that it contains adulterated pig blood components; (6) Detect the ion peak of MFLGFPTTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of pig blood adulterants in the duck blood sample to be tested based on the comparison result. (7) When the duck blood sample to be tested contains AAVTAFFGK or LVNELTEFAK, it is determined that it contains adulterated bovine blood. (8) Detect the ion peak of AATVAFTWGK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of bovine blood adulterants in the duck blood sample to be tested based on the comparison result; (9) When the duck blood sample to be tested contains AAVTGFWGK or YLYEVAR, it is determined that it contains adulterated sheep blood. (10) Detect the ion peak of AAVTGFWGK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of sheep blood adulterants in the duck blood sample to be tested based on the comparison result.

[0009] The standard curve described in this invention is plotted based on the peak area detection results of different proportions of adulterated samples (such as chicken blood, cow blood, and other blood) in the experimental sample (duck blood).

[0010] Furthermore, proteins were extracted from the duck blood samples to be tested using the following method: The duck blood sample to be tested was cut into small pieces, ground, and then defatted. The defatted product was placed in the extraction solution and extracted at 55-65℃ for 2-4 hours, followed by centrifugation and retention of the supernatant. Enzymatic hydrolysis is performed using the following method: After reducing and alkylating the product of the supernatant, it was enzymatically hydrolyzed with trypsin at 35-40°C for 10-15 hours and then purified.

[0011] Furthermore, the extraction solution comprises: 10-15 g / mL urea and 0.03-0.06 M Tris-HCl buffer.

[0012] Furthermore, the characteristic peptides were detected by liquid chromatography-tandem mass spectrometry or liquid chromatography-tandem high-resolution mass spectrometry.

[0013] Furthermore, in the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions include: A C18 column was used, the column oven temperature was 38~42℃, the mobile phase A was 0.05~0.15% formic acid water, the mobile phase B was 0.05~0.15% formic acid acetonitrile, and the liquid phase flow rate was 0.2~0.4 mL / min. Separation was performed using a shorter gradient elution program (injection volume of 3–8 μL): 0-0.5 min, 5% B; 0.5-3.0 min, 5-25% B; 3-7 min, 25-45% B; 7-7.5 min, 45-100% B; 7.5-9.0 min, 100% B; 9.0-9.1 min, 100-5% B; 9.1-10.0 min, 5% B.

[0014] The conditions for mass spectrometry include: using an electrospray ionization source, scanning mode of positive ion scanning, monitoring mode of multiple reaction monitoring, ion spray voltage of 3000~4000 V, nebulizing gas pressure of 25~35 psi, drying gas temperature of 260~300℃, drying gas flow rate of 10~13 L / min, sheath gas temperature of 300~350℃, and sheath gas flow rate of 8~12 L / min; In the liquid chromatography-tandem high-resolution mass spectrometry method, the liquid chromatography conditions include: A C18 column was used, with 0.05–0.15% formic acid in water as mobile phase A and 0.05–0.15% formic acid in acetonitrile as mobile phase B. The flow rate of the liquid phase was 0.2–0.4 mL / min. Separation was performed using a gradient elution process: 0-2.0 min, 10% B; 2.0-13.0 min, 10-40% B; 13.0-18.4 min, 40-90% B; 18.4-18.5 min, 90-10% B; 18.5-20.0 min, 10% B.

[0015] The injection volume is 3~8μL.

[0016] The conditions for high-resolution mass spectrometry include: sheath gas flow rate of 40-50 L / min, auxiliary gas flow rate of 8-12 L / min, baffle gas flow rate of 0 L / min, electrospray voltage of 3-4 kV, ion tube temperature of 300-340 °C, S-lens RF level of 40-80, and ion source temperature of 320-380 °C.

[0017] The targeted protein quantification method based on liquid chromatography-mass spectrometry (LC-MS / MS) technology overcomes the aforementioned technical bottlenecks and has significant technical advantages. Proteins, as the target analyte, have better thermal stability than DNA and are less likely to be completely inactivated by high temperatures and pressures during food processing. This allows for reliable detection signals even in old or heavily processed samples, providing a reliable material basis for adulteration identification. Furthermore, unlike the qualitative results of PCR, LC-MS / MS can accurately determine the relative content of each adulterant component, enabling quantitative assessment of the adulteration ratio. It can detect trace adulteration below 1%, which is particularly important for identifying highly concealed adulterated products after fine processing. This technology has high species specificity and detection specificity, effectively avoiding false positive results caused by individual differences.

[0018] The quantitative detection method and discrimination data model established based on LC-MS / MS have high repeatability and stability. The resulting detection standards and operating procedures are highly universal and easy to promote, and can be effectively promoted and applied in different laboratories. This provides scientific, accurate and efficient technical support for enterprises' raw material procurement and quality control, ensures the reliability of the "real duck blood" product claim, and enhances consumers' trust in the product.

[0019] In a second aspect, the present invention provides a characteristic peptide composition comprising the following characteristic peptides: MFIAYPQTK, GYETLLEK, MFTTYPPTK, GYESLLEK, MFLGFPTTK, ADFTEISK, AAVTAFWGK, LVNELTEFAK, AAVTGFWGK and YLYEVAR.

[0020] Thirdly, the present invention provides the aforementioned identification method, or the application of the aforementioned characteristic peptide composition in any of the following: (1) Quality control of duck blood raw material procurement; (2) Evaluation of the authenticity of duck blood products; (3) Identification of adulteration of duck blood products.

[0021] The present invention has the following beneficial effects: 1. This invention breaks through the bottleneck of traditional PCR technology, which is difficult to accurately quantify. By targeting and detecting hemoglobin characteristic peptides that are strongly correlated with blood content, and combining them with the standard curve method, it can accurately determine the adulteration ratio of various exogenous blood, providing direct scientific data for judging the severity of adulteration.

[0022] 2. This invention can detect extremely low levels of adulteration, with a limit of quantification (LOQ) of less than 0.5% for commonly adulterated blood, which is far superior to existing methods.

[0023] 3. The characteristic peptide combination provided by this invention can simultaneously perform qualitative and quantitative detection of multiple animal-derived components (chicken, pig, cow, sheep, etc.) that may be mixed in duck blood in a single analysis, which significantly improves detection efficiency and reduces the cost of a single detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 These are the chromatographic analysis results of ducks and chickens provided in the embodiments of the present invention.

[0026] Figure 2 These are the chromatographic analysis results of pigs and cattle provided in the embodiments of the present invention.

[0027] Figure 3 These are the chromatographic analysis results of sheep provided in the embodiments of the present invention.

[0028] Figure 4 This is a secondary mass spectrum of the duck characteristic peptide provided in this embodiment of the invention.

[0029] Figure 5 This is a secondary mass spectrum of the characteristic chicken peptides provided in this embodiment of the invention.

[0030] Figure 6 This is a secondary mass spectrum of the porcine characteristic peptide provided in an embodiment of the present invention.

[0031] Figure 7 This is a secondary mass spectrum of bovine characteristic peptides provided in an embodiment of the present invention.

[0032] Figure 8 This is a secondary mass spectrum of sheep characteristic peptides provided in an embodiment of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0034] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0035] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0036] The instruments and reagents used in the following examples are commercially available, for example: The mass spectrometer (Q-Exactive) was purchased from Thermo Fisher Scientific, USA.

[0037] The benchtop low-temperature centrifuge (Microfuge 22R Centrifuge) was purchased from BeckMAN Coulter, Inc., USA.

[0038] The electronic analytical balance (PL203) was purchased from METTLERTOLEDO GmbH, Germany.

[0039] The evaporation concentrator (Speed-Vac System, RVC2-18) was purchased from MarinChrist GmbH, Germany.

[0040] The ultra-low temperature freezer (MDF-U3286S) was purchased from SANYO Corporation of Japan.

[0041] The 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometer was purchased from Agilent Technologies, USA.

[0042] Dithiothreitol (DTT) was purchased from Solarbio, a Chinese company.

[0043] Iodoacetamide (IAA) was purchased from Solarbio, a Chinese company.

[0044] The Bradford method protein quantification kit was purchased from Solarbio, a Chinese company.

[0045] Ammonium bicarbonate (NH4HCO3) was purchased from Sigma-Aldrich, Inc.

[0046] The vortex meter (G560E) was purchased from Scientific Industries, Inc., USA.

[0047] Tris-HCL was purchased from Solarbio, a Chinese company.

[0048] Example 1: Evaluation of duck blood authenticity and identification of adulteration based on LC-HRMS method 1. Sample source.

[0049] Purchase genuine blood samples from ducks, chickens, pigs, cattle, and sheep from markets or farmers.

[0050] 2. Experimental procedures.

[0051] (1) Solution preparation.

[0052] 40 mM NH4HCO3 solution: Weigh 0.316 g NH4HCO3, dilute to 100 mL with ultrapure water, and store at 4℃ for later use.

[0053] 100 mM dithiothreitol solution: Weigh 0.617 g of DTT, dilute to 40 mL with 40 mM NH4HCO3 solution, and vortex thoroughly. Aliquot into 2 mL tubes and store at -20℃ for later use.

[0054] 100 mM iodoacetamide solution: Weigh 0.925 g of IAA, dilute to 50 mL with 40 mM NH4HCO3 solution, and vortex thoroughly. Aliquot into 1.5 mL tubes and store at -20℃ for later use.

[0055] 100 μg / mL Trypsin solution: Dissolve 10 mg of Trypsin in 40 mM NH4HCO3, bring the volume to 10 mL, and vortex thoroughly. The final concentration is 1000 mg / mL. Store at -20℃ for later use. Further dilute to 100 µg / mL and store at 4℃ until use.

[0056] Extraction solution: 12.01 g urea and 4 mL 1 M Tris-HCl buffer were diluted to 100 mL with ultrapure water and stored at 4℃ until use.

[0057] 0.1% Formic Acid-Water: Transfer 100 µL of formic acid, dilute with ultrapure water and bring the volume to 100 mL, and store at 4℃ until use.

[0058] Activation solution: ACN, store at 4℃.

[0059] Equilibration solution: 50 μL TFA, diluted to 50 mL with ultrapure water, stored at 4 °C.

[0060] Eluent: 70% ACN, 30% equilibration, store at 4°C.

[0061] Reconstituted solution: 10 μL FA, 500 μL ACN, diluted to 10 mL with ultrapure water, stored at 4 °C.

[0062] (2) Pretreatment of blood samples.

[0063] ① Sample preparation and degreasing.

[0064] Wash the solid sample with distilled water and drain. Take 500 mg of the sample, cut it into small pieces and grind it with liquid nitrogen. Weigh 2 g of the well-mixed sample and place it in a 50 mL centrifuge tube. Add 10 mL of n-hexane, sonicate for 3 min, vortex for 3 min, and then centrifuge the sample (4℃, 8000 r / min) to remove the upper organic phase and retain the precipitate. Repeat the above steps twice and dry the sample with nitrogen.

[0065] ② Protein extraction.

[0066] The above samples were added to 20 mL of extraction buffer (vortexed for 5 min, sonicated for 3 min, and heated in a water bath at 60℃ for 3 h; the extract was then centrifuged at 8000 r / min for 15 min, and the supernatant was used as the protein extract). The protein concentration in the supernatant was determined using the Bradford assay kit (Coomassie Brilliant Blue method).

[0067] ③ Reductive alkylation.

[0068] Transfer 500 μL of protein extract and add 2 mL of 40 mmol / L ammonium bicarbonate solution (50 mmol / L ammonium bicarbonate contains 2 mol / L urea) and mix thoroughly. Add 250 μL of 100 mM DTT, mix well, and react at 60 °C for 40 min, then cool to room temperature; add 750 μL of 100 mM IAA, mix well, and react in the dark at room temperature for 30 min.

[0069] ④ Enzymatic hydrolysis.

[0070] Trypsin was added at a ratio of m(sample):m(enzyme) = 40:1. After thorough mixing, the mixture was incubated at 37°C for 12 h. The enzymatic hydrolysate was then removed, and 2 μL of formic acid was added to terminate the hydrolysis. The hydrolyzed peptide solution was purified by solid-phase extraction, concentrated under vacuum, and then reconstituted with 200 μL of 0.1% formic acid solution. The reconstituted sample was then analyzed by mass spectrometry.

[0071] (3) Preparation of adulterated samples.

[0072] One or more blood samples from ducks, chickens, pigs, cattle, and sheep are randomly selected and added to pure duck blood at a fixed ratio of 5% or 10%, and then pre-treated to create adulterated blood samples.

[0073] (4) Mass spectrometry analysis of blood samples.

[0074] Q Exactive plus was used to test pure blood samples and adulterated blood samples from various livestock and poultry.

[0075] The chromatographic conditions were as follows: a C18 column was used. A gradient elution program was employed, with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B: 0–2.0 min, 10% B; 2.0–13.0 min, 10–40% B; 13.0–18.4 min, 40–90% B; 18.4–18.5 min, 90–10% B; 18.5–20.0 min, 10% B; the flow rate was 0.30 mL / min; and the injection volume was 5.0 μL.

[0076] Ion source parameters: sheath gas flow rate 45; auxiliary gas flow rate 10; baffle gas flow rate 0; electrospray voltage 3.5kV; ion conduit temperature 320 ℃; S-lens RF level set to 60; ion source temperature 350 ℃.

[0077] The acquisition mode was Full MS-ddMS in positive ion mode. 2 : The specific parameter settings for Full MS are as follows: Resolution: 70000; AGC Target: 3e6; Maximum IT: 250 ms; Scan range: 300-1500 Da; Spectrum data: Centroid. (dd-MS...) 2 The specific parameter settings are as follows: Resolution: 17500; AGC Target: 1e6; Maximum IT: 120 ms; Loop count: 5; Isolationwindow: 1.0 m / z; NCE: 20, 40, 60; Spectrum data: Centroid. In the dd settings, Minimum AGC: 8.0e3; Apex trigger: 2-6 s; Exclude isotope: on; Dynamic exclus: 5.0 s.

[0078] Mass spectrometry data is collected and stored using Xcalibur software. The raw data acquired by mass spectrometry is analyzed using Xcalibur's Qualitative Browser, and combined with mass fragmentation spectra (MS / MS), unique species-specific peptides are screened and verified.

[0079] The characteristic peptides of ducks, chickens, pigs, cattle, and sheep were finally identified as follows: The hemoglobin characteristic peptides include the parent ion of the detection signal generated by duck (MFIAYPQTK) with a mass-to-charge ratio of m / z: 549.7863; and daughter ions with mass-to-charge ratios of 279.1156, 820.4550, and 707.3710.

[0080] The precursor ion of the detection signal generated by chicken (MFTTYPPTK) has a mass-to-charge ratio of m / z: 543.2704; it includes daughter ions with mass-to-charge ratios of 251.1213, 807.4245, and 442.2650.

[0081] The precursor ion of the detection signal generated by pigs (MFLGFPTTK) has a mass-to-charge ratio of m / z: 521.2755; it includes daughter ions with mass-to-charge ratios of 251.1209, 763.4342, and 279.1158.

[0082] The precursor ion of the detection signal generated by the bovine (AAVTAFWGK) has a mass-to-charge ratio of m / z: 475.7584; it includes daughter ions with mass-to-charge ratios of 143.0814, 115.0868, and 204.1342.

[0083] The precursor ion of the detection signal generated by the sheep (AAVTGFWGK) has a mass-to-charge ratio of m / z: 468.7505; it includes daughter ions with mass-to-charge ratios of 143.0815, 695.3511, and 594.3035.

[0084] The characteristic peptides of plasma albumin include the precursor ion of the detection signal generated by duck (GYETLLEK) with a mass / charge ratio of m / z: 476.7529; and daughter ions with mass / charge ratios of 732.4138, 603.3457, and 221.1276.

[0085] The precursor ion of the detection signal generated by the chicken (GYESLLEK) has a mass-to-charge ratio of m / z: 469.7451; it includes daughter ions with mass-to-charge ratios of 199.1805, 789.3890, and 227.1754.

[0086] The precursor ion of the detection signal generated by the pig (ADFTEISK) has a mass-to-charge ratio of m / z: 455.7295; it includes daughter ions with mass-to-charge ratios of 724.3876, 577.3192, and 234.1448.

[0087] The precursor ion of the detection signal generated by the bovine (LVNELTEFAK) has a mass-to-charge ratio of m / z: 582.3190; it includes daughter ions with mass-to-charge ratios of 951.4785, 595.3073, and 708.3927.

[0088] The precursor ion of the detection signal generated by the sheep (YLYEVAR) has a mass-to-charge ratio of m / z: 457.2425; it includes daughter ions with mass-to-charge ratios of 637.3299, 175.1192, and 345.2250.

[0089] Therefore, when 5% or 10% of other animal blood is artificially added to duck blood, characteristic peptide signals of adulterants (such as pig or sheep) are detected in the chromatogram, showing a clear difference from the chromatogram of pure duck blood. By comparing the retention time and the signal intensity and peak area of ​​these peptides, adulterants can be effectively identified. Figures 1-8 The chromatograms and mass spectra of the aforementioned characteristic peptides are shown in detail for precise extraction.

[0090] Example 2: Evaluation of duck blood authenticity and identification of adulteration based on LC-MS / MS method 1. Sample source.

[0091] Purchase genuine blood samples from ducks, chickens, pigs, cattle, and sheep from markets or farmers.

[0092] 2. Experimental procedures.

[0093] (1) Solution preparation.

[0094] Same as Example 1.

[0095] (2) Pretreatment of blood samples.

[0096] Same as Example 1.

[0097] (3) Preparation of adulterated blood samples.

[0098] Adding chicken blood, pig blood, cow blood, and sheep blood at mass fractions of 0.5%, 1%, 3%, 10%, 20%, and 50% respectively to duck blood, and then pre-treating the samples, creates adulterated samples.

[0099] (4) MRM condition optimization.

[0100] The purpose of establishing the MRM method is to achieve accurate quantification; therefore, it is necessary to optimize various mass spectrometry conditions to improve detection sensitivity. Table 1 shows in detail the optimized MRM parameters for various peptides.

[0101] (5) Establishment of the standard curve.

[0102] Chicken blood, pig blood, cow blood, and sheep blood were mixed with duck blood at mass fractions of 0.5%, 1%, 3%, 10%, 20%, and 50%, respectively, to create standard curves. Standard curves were constructed with the mixing ratio (mass fraction) as the x-axis (X, %) and the peak areas (Y) of the characteristic peptides from the selected chicken blood, pig blood, cow blood, and sheep blood as the y-axis.

[0103] Table 1. Characteristic peptide parameters (sequence, retention time, parent ion, collision energy, theoretical fragment ion) of duck, chicken, pig, cow, and sheep blood.

[0104]

[0105] Notes: a represents a quantitative peptide; * represents a stable isotope-labeled peptide; b represents a quantitative daughter ion.

[0106] (6) Mass spectrometry analysis of blood samples.

[0107] The processed blood samples were analyzed using a 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometer.

[0108] The chromatographic conditions were as follows: In liquid chromatography, a short C18 column was used for separation; the column oven temperature was 40℃; 0.1% formic acid in water was used as mobile phase A; 0.1% formic acid in acetonitrile was used as mobile phase B; and a short gradient elution program was employed. 0-0.5 min, 5% B; 0.5-3.0 min, 5-25% B; 3-7 min, 25-45% B; 7-7.5 min, 45-100% B; 7.5-9.0 min, 100% B; 9.0-9.1 min, 100-5% B; 9.1-10.0 min, 5% B; liquid phase flow rate is 0.30 mL / min; The injection volume was 5.0 μL.

[0109] The mass spectrometry conditions for LC-MS / MS were as follows: electrospray ionization source (ESI); scanning mode: positive ion scan; monitoring mode: multiple reaction monitoring (MRM); ion spray voltage: 3500 V; nebulizer gas pressure: 30 psi; drying gas temperature: 280 ℃; drying gas flow rate: 11 L / min; sheath gas temperature: 325 ℃; sheath gas flow rate: 10 L / min.

[0110] (7) Quantitative judgment.

[0111] When testing an unknown duck blood sample, LC-MS / MS detects the signals of these characteristic peptides. If characteristic peptide signals from other livestock or poultry are detected, the intensity (peak area) of that signal is substituted into the standard curve of its characteristic peptide to calculate the content of other livestock or poultry blood samples mixed in with the original sample.

[0112] (8) Limit of Detection (LOD) and Limit of Quantification (LOQ): The limits of detection (LOD) and quantitation (LOQ) of the method were determined by using a signal-to-noise ratio (S / N) of 3 and 10 times, respectively. The LOD and LOQ for chicken blood, pig blood, cow blood, and sheep blood were finally determined to be 0.2% and 0.5%, respectively.

[0113] (9) Recovery rate and precision.

[0114] The adulterated samples prepared above were subjected to mass spectrometry detection, and the spiked recovery was calculated quantitatively using a standard curve. The average spiked recovery rate was 83.6%–107.2%, the intra-day precision (RSD) was less than 9.3%, and the inter-day RSD was less than 12.8%, demonstrating that the method has high accuracy and stability. This method can reliably determine the content of four adulterated blood components (chicken blood, pig blood, bovine blood, and sheep blood) in duck blood samples.

[0115] Example 3: Investigation into the identification of adulteration of duck blood in commercially available boxed duck blood products 1. Sample source.

[0116] Thirty portions of finished duck blood products were randomly purchased from the market and online shopping platforms.

[0117] 2. Experimental procedures.

[0118] (1) Solution preparation.

[0119] Same as Example 1.

[0120] (2) Pretreatment of blood samples.

[0121] Same as Example 1.

[0122] (3) Mass spectrometry analysis of blood samples.

[0123] The processed blood samples were analyzed using LC-Q Exactive and 1290 Infinity liquid chromatography-6495 triple quadrupole mass spectrometry.

[0124] The chromatographic and mass spectrometry conditions were the same as in Examples 1 and 2.

[0125] (4) Qualitative and quantitative judgment.

[0126] When testing an unknown duck blood sample, LC-MS / MS detects the signals of these characteristic peptides. If characteristic peptide signals from other livestock or poultry are detected, the intensity (peak area) of that signal is substituted into the standard curve of its characteristic peptide to calculate the content of other livestock or poultry blood samples mixed in with the original sample.

[0127] (5) Survey results.

[0128] To verify the applicability of the established analytical method, this invention tested 30 samples of finished duck blood products randomly purchased from the market and online shopping platforms using the method proposed in this invention. Table 2 shows the test results in detail. Ten of the 30 samples had problems: two samples contained no duck blood components, indicating they were entirely made of pig blood; two samples contained mixed pig blood; one sample contained both chicken and pig blood; and five samples, while containing duck blood components, had significantly lower levels of duck blood protein compared to the labeled levels, accounting for only 9.4%-35.7% of the labeled duck blood protein content, suggesting that these duck blood samples may have been mixed with gelatin or other ingredients. This invention further used PCR for comparison, and the adulteration detection results were consistent with this method, further proving the applicability of the method provided in this invention. Moreover, compared to PCR, the method provided in this invention can accurately detect the proportion of duck blood components, while PCR is only a qualitative analysis.

[0129] Table 2. Detection of adulteration with chicken, pig, cow, and sheep blood in 30 duck blood samples.

[0130]

[0131] Note: ND indicates that it was not detected.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the authenticity and identifying adulteration of duck blood, characterized in that, include: Protein was extracted from the duck blood sample to be tested and then enzymatically digested. The enzymatic hydrolysis products are detected, and the duck blood sample to be tested is identified based on the detection results of the characteristic peptide combination. The characteristic peptide combination consists of the following characteristic peptides: MFIAYPQTK, GYETLLEK, MFTTYPPTK, GYESLLEK, MFLGFPTTK, ADFTEISK, AAVTAFFWGK, LVNELTEFAK, AAVTAFFWGK, and YLYEVAR.

2. The method for evaluating the authenticity and identifying adulteration of duck blood according to claim 1, characterized in that, In the combination of the characteristic peptides MFIAYPQTK, MFTTYPPTK, MFLGFPTTK, AAVTAFWGK, and AAVTGFWGK are used for quantitative determination. GYETLLEK, GYESLLEK, ADFTEISK, LVNELTEFAK, and YLYEVAR are used for qualitative judgment.

3. The method for evaluating the authenticity and identifying adulteration of duck blood according to claim 1, characterized in that, In the combination of characteristic peptides: MFIAYPQTK and GYETLLEK are used for the identification of duck blood components; MFTTYPPTK and GYESLLEK are used for the identification of chicken blood components; MFLGFPTTK and ADFTEISK are used for the identification of components in pig blood; AAVTAFWGK and LVNELTEFAK are used for bovine blood component identification; AAVTGFWGK and YLYEVAR are used for the identification of sheep blood components.

4. The method for evaluating the authenticity and identifying adulteration of duck blood according to claim 1, characterized in that, The identification includes any of the following: (1) When the duck blood sample to be tested contains MFIAYPQTK or GYETLLEK, it is determined that the duck blood sample to be tested contains real duck blood components. (2) Detect the ion peak of MFIAYPQTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the duck blood component content in the duck blood sample to be tested based on the comparison result; (3) When the duck blood sample to be tested contains MFTTYPPTK or GYESLLEK, it is determined that it contains adulterated chicken blood. (4) Detect the ion peak of MFTTYPPTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of chicken blood adulterants in the duck blood sample to be tested based on the comparison result. (5) When the duck blood sample to be tested contains MFLGFPTTK or ADFTEISK, it is determined that it contains adulterated pig blood components; (6) Detect the ion peak of MFLGFPTTK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of pig blood adulterants in the duck blood sample to be tested based on the comparison result. (7) When the duck blood sample to be tested contains AAVTAFFGK or LVNELTEFAK, it is determined that it contains adulterated bovine blood. (8) Detect the ion peak of AATVAFTWGK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of bovine blood adulterants in the duck blood sample to be tested based on the comparison result; (9) When the duck blood sample to be tested contains AAVTGFWGK or YLYEVAR, it is determined that it contains adulterated sheep blood. (10) Detect the ion peak of AAVTGFWGK in the duck blood sample to be tested, compare the detection result of the peak area with the standard curve, and determine the content of sheep blood adulterants in the duck blood sample to be tested based on the comparison result.

5. The method for evaluating the authenticity and identifying adulteration of duck blood according to any one of claims 1-4, characterized in that, Protein was extracted from the duck blood sample to be tested using the following method: The duck blood sample to be tested was cut into small pieces, ground, and then defatted. The defatted product was placed in the extraction solution and extracted at 55-65℃ for 2-4 hours, followed by centrifugation and retention of the supernatant. Enzymatic hydrolysis is performed using the following method: After reducing and alkylating the product of the supernatant, it was enzymatically hydrolyzed with trypsin at 35-40°C for 10-15 hours and then purified.

6. The method for evaluating the authenticity and identifying adulteration of duck blood according to any one of claims 1-4, characterized in that, Characteristic peptides were detected by liquid chromatography-tandem mass spectrometry or liquid chromatography-tandem high-resolution mass spectrometry.

7. The method for evaluating the authenticity and identifying adulteration of duck blood according to claim 6, characterized in that, In the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions include: A C18 column was used, the column oven temperature was 38~42℃, the mobile phase A was 0.05~0.15% formic acid water, the mobile phase B was 0.05~0.15% formic acid acetonitrile, and the liquid phase flow rate was 0.2~0.4 mL / min. The conditions for mass spectrometry include: using an electrospray ionization source, scanning mode of positive ion scanning, monitoring mode of multiple reaction monitoring, ion spray voltage of 3000~4000 V, nebulizing gas pressure of 25~35 psi, drying gas temperature of 260~300 ℃, drying gas flow rate of 10~13 L / min, sheath gas temperature of 300~350 ℃, and sheath gas flow rate of 8~12 L / min; In the liquid chromatography-tandem high-resolution mass spectrometry method, the liquid chromatography conditions include: A C18 column was used, with 0.05–0.15% formic acid in water as mobile phase A and 0.05–0.15% formic acid in acetonitrile as mobile phase B. The flow rate of the liquid phase was 0.2–0.4 mL / min. The conditions for high-resolution mass spectrometry include: sheath gas flow rate of 40-50 L / min, auxiliary gas flow rate of 8-12 L / min, baffle gas flow rate of 0 L / min, electrospray voltage of 3-4 kV, ion tube temperature of 300-340 °C, S-lens RF level of 40-80, and ion source temperature of 320-380 °C.

8. A characteristic peptide composition, characterized in that, It is composed of the following characteristic peptide segments: MFIAYPQTK, GYETLLEK, MFTTYPPTK, GYESLLEK, MFLGFPTTK, ADFTEISK, AAVTAFWGK, LVNELTEFAK, AAVTGFWGK and YLYEVAR.

9. The method for evaluating the authenticity and identifying adulteration of duck blood according to any one of claims 1-7, or the application of the characteristic peptide composition according to claim 8 in any one of the following: (1) Quality control of duck blood raw material procurement; (2) Evaluation of the authenticity of duck blood products; (3) Identification of adulteration of duck blood products.

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