Application of Gallus gallus endometrium specific peptides in identification of Gallus gallus endometrium

Abstract

The present invention relates to the field of analytical chemistry, and in particular, to the application of a chicken gizzard lining specific polypeptide in identifying chicken gizzard lining. The present invention uses high-resolution liquid-mass spectrometry technology and combines proteomics theory to screen out characteristic polypeptides of chicken gizzard lining, and the characteristic polypeptides can be used to quickly and accurately identify chicken gizzard lining components in chicken gizzard lining samples (e.g., chicken gizzard lining standard decoction), and can quickly identify whether the chicken gizzard lining standard decoction is mixed with avian gizzard lining such as duck gizzard lining, goose gizzard lining and pigeon gizzard lining.

Description

[0001] This application is a divisional application with application number 202010715915.6, application date July 23, 2020, and invention name “Chicken Gizzard Specific Polypeptide and Its Application in Identification of Chicken Gizzard Specific Polypeptide”. Technical Field

[0002] The invention relates to the field of analytical chemistry, and in particular to a chicken gizzard lining specific polypeptide and application thereof in identifying chicken gizzard lining. Background Art

[0003] Chicken gizzard lining is the dried inner wall of the gizzard of the pheasant Gallus gallus domesticus. After killing the chicken, take out the gizzard, immediately peel off the inner wall, wash and dry it. Chicken gizzard lining was originally called chicken gizzard lining yellow skin, chicken gizzard, chicken gizzard lining yellow skin, chicken food skin, chicken zygote, chicken gold, fossil gallbladder, bone-dissolving gallbladder, etc. It was first recorded in the Materia Medica "Shennong's Herbal Classic" as "yellow skin in the gizzard lining of chicken canthus is used to treat diarrhea."

[0004] Chicken's gizzard lining is sweet and neutral in nature. It enters the spleen, stomach, small intestine, and bladder meridians. It has the effects of strengthening the stomach and digesting food, astringing sperm and stopping nocturnal emission, and clearing stranguria and dissolving stones. It is often used for indigestion, vomiting and diarrhea, malnutrition in children, nocturnal emission, nocturnal emission, stone stranguria, pain, gallbladder distension and flank pain. Modern clinical studies have also shown that chicken's gizzard lining can well improve the clinical symptoms of diabetes and its complications.

[0005] However, the quality standard of chicken gizzard lining is not perfect. In the 2015 edition of the Chinese Pharmacopoeia, only the properties, moisture, total ash and extracts of chicken gizzard lining are controlled, and there is no identification method. Since the dried chicken gizzard lining is brittle and easy to break, it is difficult to distinguish it from the gizzard lining of duck, goose, pigeon and other poultry in appearance. After being boiled in water or made into formula granules, the decoction loses its original properties, making it even more difficult to distinguish with the existing control methods.

[0006] The applicant searched for literature reports on methods for identifying chicken gizzard lining and other poultry. Traditional techniques mainly distinguish them based on their properties, such as Zou Honglai et al.'s "Identification of Chicken Gizzard lining and Duck Gizzard lining and Goose Gizzard lining" and Liu Xiaozhuan's "How to Identify Chicken Gizzard lining and Duck Gizzard lining". Chen Zhende et al. used protein polypeptide high-performance capillary electrophoresis to identify chicken gizzard lining and duck gizzard lining, and showed that there were differences between the two by the difference in the HPCE spectra of chicken gizzard lining and duck gizzard lining; Lv Wuqing et al. showed the differences between chicken, duck, and goose gizzard lining by the activity of amylase and protease, and the differences in amino acid and trace element content. However, the above methods are relatively complicated, and for the decoction of chicken gizzard lining, the identification methods in the above documents are not applicable or not specific to distinguish chicken gizzard lining from duck gizzard lining, goose gizzard lining, and pigeon gizzard lining. Summary of the invention

[0007] The invention aims to provide a chicken gizzard lining specific polypeptide and application thereof in identifying chicken gizzard lining; the polypeptide has good specificity and can be distinguished from goose gizzard lining, duck gizzard lining and pigeon gizzard lining.

[0008] The present invention relates to a chicken gizzard lining specific polypeptide, which is selected from one or more polypeptides shown in SEQ ID NOs: 1-4.

[0009] According to yet another aspect of the present invention, the present invention also relates to the use of the Gallus gallus endometrium-specific polypeptide as described above in identifying Gallus gallus endometrium.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The present invention uses high-resolution liquid-mass spectrometry technology and combines proteomics theory to screen out characteristic polypeptides of chicken's gizzard lining. The characteristic polypeptides can be used to quickly and accurately identify the chicken's gizzard lining components in a chicken's gizzard lining sample (e.g., a chicken's gizzard lining standard decoction), and can quickly identify whether the chicken's gizzard lining standard decoction is mixed with duck's gizzard lining, goose's gizzard lining, pigeon's gizzard lining, and other poultry gizzard linings. This method has strong specificity and can provide a scientific experimental basis for the quality evaluation research of chicken's gizzard lining samples, thereby achieving specific identification of chicken's gizzard lining samples in terms of quality, and can make up for the defect of inaccurate identification caused by the lack of the appearance characteristics of the decoction pieces in the standard decoction, reduce the adverse effects of counterfeit products on the traditional Chinese medicine market, and ensure that the public uses high-quality, authentic chicken's gizzard lining preparation products. At the same time, it also provides a new means for improving the quality standards of chicken's gizzard lining medicinal material decoction pieces. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=379.21 are extracted in one embodiment of the present invention;

[0014] Figure 2 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=785.41 are extracted in one embodiment of the present invention;

[0015] Figure 3 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=487.24 are extracted in one embodiment of the present invention;

[0016] Figure 4The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=607.98 extracted in one embodiment of the present invention;

[0017] Figure 5 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=683.32 extracted in one embodiment of the present invention;

[0018] Figure 6 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=362.72 are extracted in one embodiment of the present invention;

[0019] Figure 7 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=426.70 extracted in one embodiment of the present invention;

[0020] Figure 8 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=400.22 extracted in one embodiment of the present invention;

[0021] Fig. 9 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=415.74 are extracted in one embodiment of the present invention;

[0022] Fig.10 The mass spectra of the gizzard lining of chicken, duck, goose and pigeon gizzard lining at m / z=421.76 are extracted in one embodiment of the present invention;

[0023] Fig.11 The secondary ion comparison mass spectra of the chicken gizzard polypeptide m / z=379.21 and the artificially synthesized polypeptide in one embodiment of the present invention are shown;

[0024] Fig.12 The secondary ion comparison mass spectra of the chicken gizzard polypeptide m / z=785.41 and the artificially synthesized polypeptide in one embodiment of the present invention are shown in FIG.

[0025] Fig.13 The secondary ion comparison mass spectra of the chicken gizzard polypeptide m / z=487.24 and the artificially synthesized polypeptide in one embodiment of the present invention are shown;

[0026] Fig.14 The secondary ion comparison mass spectra of the chicken gizzard polypeptide m / z=607.98 and the artificially synthesized polypeptide in one embodiment of the present invention are shown;

[0027] Fig.15 This is a secondary ion comparison mass spectrum of the duck and goose endogenous gold polypeptide m / z=683.32 and the artificially synthesized polypeptide in one embodiment of the present invention;

[0028] Fig.16This is a secondary ion comparison mass spectrum of duck and goose endogenous gold polypeptide m / z=362.72 and artificially synthesized polypeptide in one embodiment of the present invention;

[0029] Fig.17 This is a secondary ion comparison mass spectrum of the duck and goose endogenous gold polypeptide m / z=426.70 and the artificially synthesized polypeptide in one embodiment of the present invention;

[0030] Fig.18 This is a secondary ion comparison mass spectrum of the pigeon gold polypeptide m / z=400.22 and the artificially synthesized polypeptide in one embodiment of the present invention;

[0031] Fig.19 This is a secondary ion comparison mass spectrum of the pigeon gold polypeptide m / z=415.74 and the artificially synthesized polypeptide in one embodiment of the present invention;

[0032] Fig. 20 This is a secondary ion comparison mass spectrum of the pigeon gold polypeptide m / z=421.76 and the artificially synthesized polypeptide in one embodiment of the present invention;

[0033] Fig.21 This is a mass spectrum of the components of the mixed chicken gizzard lining slices in one embodiment of the present invention;

[0034] Fig. 22 This is a mass spectrum of the components of the mixed chicken gizzard lining standard decoction of chicken gizzard lining in one embodiment of the present invention;

[0035] Fig.23 This is a mass spectrum of the components of mixed chicken gizzard lining slices (5%), duck gizzard lining and goose gizzard lining in one embodiment of the present invention;

[0036] Fig.24 This is a mass spectrum of the pigeon gizzard lining component detected in a mixed chicken gizzard lining decoction piece (5% ratio) in one embodiment of the present invention;

[0037] Fig.25 This is a mass spectrum of the components of duck gizzard lining and goose gizzard lining mixed with chicken gizzard lining standard decoction (5%) in one embodiment of the present invention;

[0038] Fig.26 This is a mass spectrum of the pigeon gizzard lining component detected in a mixed chicken gizzard lining standard decoction (5% ratio) in one embodiment of the present invention. DETAILED DESCRIPTION

[0039] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0040] Therefore, it is intended that the present invention covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are obvious from the following detailed description. Those of ordinary skill in the art will appreciate that this discussion is only a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention.

[0041] The present invention relates to a chicken gizzard lining specific polypeptide, which is selected from one or more polypeptides shown in SEQ ID NOs: 1-4.

[0042] According to yet another aspect of the present invention, the present invention also relates to the use of the Gallus gallus endometrium-specific polypeptide as described above in identifying Gallus gallus endometrium.

[0043] In some embodiments, the application is used to distinguish chicken gizzard lining from other avian gizzard linings, wherein the other avian gizzard linings are selected from one or more of goose gizzard lining, duck gizzard lining, and pigeon gizzard lining.

[0044] In some embodiments, the application is used to identify whether chicken gizzard lining is mixed with other poultry lining, wherein the other poultry lining is selected from one or more of goose gizzard lining, duck gizzard lining and pigeon gizzard lining;

[0045] Wherein, the presence of goose gizzard lining and / or duck gizzard lining is determined based on the presence or absence of at least one of the polypeptides shown in SEQ ID NOs: 5 to 7;

[0046] The presence or absence of pigeon gold in the object to be detected is determined based on the presence or absence of at least one of the polypeptides shown in SEQ ID NOs: 8 to 10.

[0047] The identification method of the specific polypeptide can be selected by methods well known to those skilled in the art, such as antibodies and antibody fragments, lectins or aptamers that can specifically bind to the specific polypeptide. Specific binding means that the binding agent can have at least 10 7 l / mol affinity, or 10 8 l / mol, or more preferably 10 9l / mol affinity. It can also be performed by biological mass spectrometry, etc. In view of the fact that the objects to be identified in the present invention are usually complex in composition, the preferred identification method is a method based on chromatography-mass spectrometry.

[0048] In some embodiments, the application is based on a chromatography-mass spectrometry method to identify the object to be detected, and the parent ion and daughter ion m / z of the chicken gizzard lining specific polypeptide in multiple reaction monitoring (MRM) in electrospray ionization (ESI) positive ion mode are as follows:

[0049]

[0050] In some embodiments, the parent ion and product ion m / z of goose gizzard lining, duck gizzard lining and pigeon gizzard lining specific polypeptide in multiple reaction monitoring (MRM) in electrospray ionization (ESI) positive ion mode are as follows:

[0051]

[0052]

[0053] In some embodiments, when performing chromatographic determination of the analyte, gradient elution is performed with mobile phase A and mobile phase B;

[0054] The mobile phase A is acetonitrile;

[0055] The mobile phase B is a 0.07 v / v% to 0.13 v / v% formic acid aqueous solution.

[0056] In some embodiments, the conditions of the gradient elution are:

[0057]

[0058] In some embodiments, the flow rate of the gradient elution is 0.3-0.5 mL / min, and 0.4 mL / min can also be selected.

[0059] In some embodiments, the main functional group of the stationary phase of the chromatographic column used in the chromatography is a C18 functional group.

[0060] In some embodiments, the chromatographic column is a Waters ACQUITY UPLC BEH C18 chromatographic column.

[0061] In some embodiments, the chromatographic column has a length of 100 to 200 mm, an inner diameter of 1.0 to 3.0 mm, and a filler particle size of 1.4 to 2.0 μm.

[0062] In some embodiments, the chromatographic column has a length of 150 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm.

[0063] In some embodiments, the column temperature of the chromatographic column is 25°C to 35°C, and can also be 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C or 34°C.

[0064] In some embodiments, the object to be detected is filtered through a 0.20-0.24 μm microporous filter membrane, preferably a 0.22 μm microporous filter membrane.

[0065] In some embodiments, the mass spectrometry conditions are:

[0066] Electrospray positive ion mode, sheath gas flow rate: 30 L / min; auxiliary gas flow rate: 10 L / min; spray voltage: 3.30 kV; capillary temperature: 320 °C; auxiliary gas temperature: 320 °C;

[0067] The floating range of each numerical parameter involved in the above conditions is ±10% of the numerical parameter.

[0068] In some embodiments, the object to be detected is chicken gizzard lining decoction or slices.

[0069] The decoction pieces can be original medicinal material decoction pieces and decoction pieces that have been cut and / or processed. The processing methods include but are not limited to steaming, boiling, frying, roasting, roasting, roasting, calcining, soaking, and flying.

[0070] Chicken gizzard lining decoction can be obtained by decocting the original medicinal material or decoction pieces of chicken gizzard lining; it usually also includes removing the residue and obtaining the juice, for example, filtering with a 320-380 mesh sieve.

[0071] In some embodiments, when the object to be detected is a decoction piece, the object to be detected is subjected to trypsin enzymatic hydrolysis. Further, the enzymatic hydrolysis time is 8 to 16 hours, and 12 hours can also be selected.

[0072] In some embodiments, when the object to be detected is a decoction piece, the object to be detected is ground and / or ultrasonically treated; further, the ultrasonic power is 200-300W, the frequency is 40-60kHz, and the ultrasonic time is 20-40 minutes.

[0073] After pretreatment, the object to be detected can effectively remove impurities and release specific peptides, thereby increasing the specificity and sensitivity of the detection.

[0074] The embodiments of the present invention will be described in detail below with reference to examples.

[0075] Example 1 Screening and artificial synthesis verification of specific polypeptides

[0076] 1 Preparation of test solution

[0077] Take an appropriate amount of sample, grind it into powder, take about 0.1g, add 50mL of 1% ammonium bicarbonate solution, ultrasonically treat (power 250W, frequency 50kHz) for 30 minutes, filter with 0.22μm microporous filter membrane, accurately transfer 100μL into a microinjection bottle, add 10μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1mg per 1mL, prepare it before use), shake well, and perform enzymolysis at 37℃ for 12 hours.

[0078] 2. Preparation of control medicinal material solution

[0079] Weigh 0.5 g of chicken gizzard lining control medicinal material powder, add 50 mL of 1% ammonium bicarbonate solution, heat and reflux for 30 minutes, shake well, filter with a 0.22 μm microporous filter membrane, take 100 μL of the filtrate, put it in a microinjection bottle, add 10 μL of trypsin solution (take trypsin, add 1% ammonium bicarbonate solution to dissolve it, make a solution containing 1 mg per 1 mL, prepare it before use), shake well, and perform enzymolysis at 37°C for 12 hours.

[0080] 3 Chromatographic conditions

[0081] Waters ACQUITY UPLC BEH C18 column (150 mm×2.1 mm, 1.7 μm); mobile phase was acetonitrile-0.1% formic acid aqueous solution for gradient elution; column temperature was 30° C., flow rate was 0.3 mL / min, and 2 μL was injected.

[0082] Gradient elution table

[0083]

[0084] 4 Mass spectrometry conditions

[0085] Electrospray positive ion mode (HESI+), sheath gas flow rate: 30 L / min; auxiliary gas flow rate: 10 L / min; spray voltage: 3.30 kV; capillary temperature: 320°C; auxiliary gas temperature: 320°C; perform Full-MS-ddms2 mode scanning.

[0086] 5 Determination

[0087] Accurately pipette 2 μL of the control medicinal material solution and the test sample solutions of chicken gizzard lining, duck gizzard lining, goose gizzard lining, and pigeon gizzard lining, inject into the liquid chromatograph, and measure and collect the mass spectrum according to the above conditions.

[0088] 6 Data processing

[0089] The collected mass spectrometry data were imported into Proteome Discoverer 2.4 analysis software, and the Uniprot-Aves database was used for searching the database to match the characteristic peptides unique to chicken gizzard lining, duck gizzard lining, goose gizzard lining, and pigeon gizzard lining. The specific peptide sequences and corresponding mass-to-charge ratios in each gizzard lining are as follows:

[0090]

[0091] The species specificity of the selected mass-to-charge ratio can be further determined by comparing the mass spectra. Figures 1 to 4 The four characteristic peptides of chicken gizzard lining only exist in chicken gizzard lining, but not in goose gizzard lining, duck gizzard lining and pigeon gizzard lining. Figures 5 to 7 The three characteristic peptides of goose gizzard lining and duck gizzard lining were the same, and were not found in chicken gizzard lining and pigeon gizzard lining. Figures 8 to 10 The three characteristic peptides of pigeon gizzard lining only exist in pigeon gizzard lining, and are not found in chicken gizzard lining, duck gizzard lining and goose gizzard lining.

[0092] 7. Sequence Synthesis Verification of Selected Peptides

[0093] The above-screened sequences were commissioned to Jier Biochemical (Shanghai) Co., Ltd. for artificial synthesis, and then the same chromatographic and mass spectrometric conditions as the samples were used for determination. By comparing the retention time and secondary ion fragments of the synthetic peptides and the sample screening peptides at the same mass-to-charge ratio, the results showed that the retention time and secondary ion fragments of all specific peptides in chicken gizzard lining, duck gizzard lining, goose gizzard lining and pigeon gizzard lining corresponded one to one with the artificial synthesis, indicating that the sequences of the screened peptides were all correct sequences. Figures 11 to 20 .

[0094] Example 2 Detection of mixed chicken gizzard lining slices and standard decoctions containing duck gizzard lining, goose gizzard lining and pigeon gizzard lining

[0095] The chicken gizzard lining polypeptide 2, duck and goose gizzard lining polypeptide 6, and pigeon gizzard lining polypeptide 9 in Example 1 were selected, and appropriate secondary ions were selected to detect chicken gizzard lining pieces and standard decoction samples mixed with duck gizzard lining, goose gizzard lining, and pigeon gizzard lining by PRM.

[0096] The details are as follows:

[0097] 1. Preparation of mixed slices of chicken gizzard lining

[0098] Weigh an appropriate amount of chicken gizzard lining coarse powder, and mix it with duck gizzard lining, goose gizzard lining and pigeon gizzard lining powder at 10%, 5% and 2% of the weight of chicken gizzard lining to obtain three mixed gizzard lining samples.

[0099] 2. Preparation of mixed decoction (standard decoction)

[0100] Take an appropriate amount of chicken gizzard lining and place it in a ceramic medicine pot, mix duck gizzard lining, goose gizzard lining and pigeon gizzard lining at 10%, 5% and 2% of the weight of chicken gizzard lining to obtain 3 mixed gizzard lining samples. Add water and decoct twice. The first decoction is directly added to 10 times of boiling water. After boiling with high heat (500W), keep the slow fire (200W) slightly boiling for 30 minutes. The decoction is filtered while hot through a 350 mesh screen, and the filtrate is rapidly cooled with cold water. Add 8 times of water for the second time, heat and boil with high heat, keep the slow fire slightly boiling for 10 minutes, filter the decoction with a 350 mesh screen while hot, and cool the filtrate rapidly with cold water. Combine the two decoctions to obtain the final product.

[0101] 3 Preparation of test solution

[0102] Take 0.1g of mixed chicken gizzard lining slices and an appropriate amount of mixed decoction (approximately equivalent to 0.1g of chicken gizzard lining slices) into different 50mL volumetric flasks, add 1% ammonium bicarbonate solution to the scale, ultrasonically treat (power 250W, frequency 50kHz) for 30 minutes, filter with a 0.22μm microporous filter membrane, accurately transfer 100μL and place in a microinjection bottle, add 10μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution to make a solution containing 1mg per 1mL, prepare it before use), shake well, and perform enzymolysis at 37℃ for 12 hours to obtain the test solution of each mixed slice and mixed decoction.

[0103] 4. Preparation of reference solution

[0104] Take appropriate amounts of artificial synthetic peptide reference substances corresponding to polypeptide sequences SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 9, accurately weigh them, and add methanol to prepare solutions containing 10 μg of each polypeptide reference substance per 1 mL.

[0105] 5 Chromatographic conditions

[0106] Waters ACQUITY UPLC BEH C18 chromatographic column (150 mm×2.1 mm, 1.7 μm); the mobile phase was acetonitrile-0.1% formic acid aqueous solution, and the elution was performed according to the gradient in the table below. The column temperature was 30° C. and the flow rate was 0.3 mL / min.

[0107] Gradient elution table

[0108]

[0109] 6 Mass spectrometry conditions

[0110] Electrospray positive ion mode (ESI+), sheath gas flow rate: 30 L / min; auxiliary gas flow rate: 10 L / min; spray voltage: 3.30 kV; capillary temperature: 320°C; auxiliary gas temperature: 320°C; PRM mode scanning was performed according to the following ion pairs.

[0111]

[0112] 7 Determination

[0113] Accurately pipette 2 μL of the reference medicinal material solution and each mixed decoction test solution, inject them into the liquid chromatograph, and measure and collect the mass spectrum according to the above conditions.

[0114] 8 Results and Analysis

[0115] The test results showed that the mixed pieces and the mixed decoction simultaneously presented a chromatographic peak with the same chromatographic retention time as the artificial synthetic peptide reference substance corresponding to the peptide SEQ ID NO: 2 ( Fig.21 , Fig. 22 ), indicating that the method can accurately detect the components of Gallus gallus endometrium;

[0116] In the 5% mixed decoction piece, chromatographic peaks with the same chromatographic retention time as the artificial synthetic peptide reference substance corresponding to the peptide segment SEQ ID NO: 6 and the peptide segment SEQ ID NO: 9 can be simultaneously presented ( Fig.23 , Fig.24 ), that is, when 5% of duck gizzard lining, goose gizzard lining and pigeon gizzard lining are mixed in the chicken gizzard lining decoction sample, the components of duck gizzard lining, goose gizzard lining and pigeon gizzard lining can still be detected.

[0117] In the 5% mixed decoction, chromatographic peaks with the same chromatographic retention time as the artificial synthetic peptide reference substances corresponding to peptide SEQ ID NO: 6 and peptide SEQ ID NO: 9 can be simultaneously presented ( Fig.25 , Fig.26 ), that is, when 5% of duck gizzard lining, goose gizzard lining and pigeon gizzard lining were mixed into the standard decoction sample of chicken gizzard lining, the components of duck gizzard lining, goose gizzard lining and pigeon gizzard lining could still be detected.

[0118] The results showed that the method has high sensitivity and can effectively control the adulteration of chicken gizzard lining.

[0119] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of chicken gizzard lining specific peptides in the identification of chicken gizzard lining; It is used to distinguish chicken gizzard lining from other poultry gizzard linings, wherein the other poultry gizzard linings are selected from one or more of goose gizzard lining, duck gizzard lining and pigeon gizzard lining. The Gallus gallus domesticus-specific polypeptide is the polypeptide shown in SEQ ID NO:

3.

2. The use according to claim 1, wherein the Gallus gallus domesticus-specific polypeptide further comprises the polypeptide shown in SEQ ID NO:

2.

3. The application according to claim 1, which is used to identify whether the gizzard lining of chicken is mixed with the gizzard lining of other poultry; in, Determine whether goose gizzard lining and / or duck gizzard lining are present in the test object according to the presence or absence of at least one of the polypeptides shown in SEQ ID NOs: 5 to 7; The presence or absence of pigeon gold in the object to be detected is determined based on the presence or absence of at least one of the polypeptides shown in SEQ ID NOs: 8 to 10.

4. The use according to any one of claims 1 to 3, wherein the object to be detected is identified based on a chromatography-mass spectrometry method, and the parent ion and daughter ion m / z of the chicken gizzard lining specific polypeptide in multiple reaction monitoring (MRM) mode of electrospray ionization (ESI) positive ion mode are as follows:

5. According to the use of claim 3, the parent ion and daughter ion m / z of the specific polypeptides of goose gizzard lining, duck gizzard lining and pigeon gizzard lining in multiple reaction monitoring (MRM) mode of electrospray ionization (ESI) positive ion mode are as follows:

6. The use according to claim 4, wherein when performing chromatographic determination of the substance to be detected, gradient elution is performed using mobile phase A and mobile phase B; The mobile phase A is acetonitrile; The mobile phase B is a 0.07 v / v% to 0.13 v / v% formic acid aqueous solution.

7. The use according to claim 6, wherein the conditions of the gradient elution are:

8. The use according to claim 7, wherein the flow rate of the gradient elution is 0.3-0.5 mL / min; The main functional group of the stationary phase of the chromatographic column used in the chromatography is a C18 functional group; The column temperature of the chromatographic column is 25°C to 35°C.

9. The use according to any one of claims 5, 6, 7 and 8, wherein the mass spectrometry conditions are: Electrospray positive ion mode, sheath gas flow rate: 30 L / min; auxiliary gas flow rate: 10 L / min; spray voltage: 3.30 kV; capillary temperature: 320 °C; auxiliary gas temperature: 320 °C; The floating range of each numerical parameter involved in the above conditions is ±10% of the numerical parameter.

10. The use according to any one of claims 3, 5, 6, 7 and 8, wherein the object to be detected is a chicken gizzard lining decoction or a decoction piece.

11. The use according to claim 10, when the object to be detected is a decoction piece, the object to be detected is subjected to trypsin enzymatic hydrolysis.

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