Characteristic Peptides of Donkey Bone Glue and Method for Identifying Adulteration of Donkey Bone

Abstract

The present invention discloses donkey bone glue characteristic peptide segments and a method for identifying donkey bone adulteration, belonging to the technical field of medical and food detection. Through a liquid chromatography-mass spectrometry instrument, the present invention screens out donkey bone glue characteristic peptide segments with the sequences of LLESPAVALDDIR and LVVGPVVVAAPQLPPPVHR. These two donkey bone glue characteristic peptide segments have high specificity and can be used to identify products containing donkey bone glue. The donkey bone glue characteristic peptide segments and the detection method provided by the present invention screen out the optimal chromatographic conditions through a liquid chromatography-mass spectrometry instrument. The whole method is simple to operate, accurate in judgment, and can accurately distinguish donkey bone glue characteristic peptide segments. The method of the present invention has the characteristics of obvious features, high sensitivity, and simple operation, providing an effective technical means for detecting donkey bone glue components in animal skin glue and its products.

Description

Technical Field

[0001] The present invention relates to characteristic peptide segments of donkey bone glue and a method for identifying adulteration of donkey bones, and particularly to a method for detecting the component of donkey bone glue in animal skin glue and its products. The present invention belongs to the technical field of medical and food detection. Background Art

[0002] With the development of social economy, the market demand and supply of Ejiao and related products are increasing day by day. However, due to the limited resources of donkey skins, some enterprises use alternative raw materials such as cow skins, horse skins, sheep skins, pig skins, etc. to manufacture Ejiao, and even use donkey bones instead of donkey skins to make fake Ejiao. Such behavior seriously affects the public's food safety and medication safety.

[0003] The main component of Ejiao is type I collagen. At present, the main technology for quality control of Ejiao is to select specific peptide segments or polypeptides in Ejiao as detection indicators and use liquid chromatography-mass spectrometry technology for identification. For example, the identification method in the quality standard of Ejiao in the first part of the Chinese Pharmacopoeia 2015 edition uses a liquid chromatography-triple quadrupole mass spectrometer for qualitative identification. However, subsequent research and actual use have found that this specific peptide segment can also be detected in horse skin glue and mule skin glue, so it lacks specificity. In addition, the type I collagen sequences of Ejiao and donkey bone glue are almost exactly the same, making it difficult to effectively distinguish and identify them. It can be seen that the current technology has limited qualitative identification of donkey bone glue and is difficult to ensure the uniqueness of the identified donkey bone glue peptides. Therefore, there is an urgent need to propose a method that can quickly and accurately detect the content of donkey bone glue in animal skin glue and its products to achieve the authenticity identification of Ejiao products. Summary of the Invention

[0004] Aiming at the above deficiencies of the prior art, the present invention provides characteristic peptide segments of donkey bone glue and a method for identifying adulteration of donkey bones, aiming to establish a detection method for the component of donkey bone glue in animal skin glue and its products. By using the strong specificity of donkey bone glue characteristic peptide segments, the detection method is simple to operate, has high sensitivity, and can quickly identify whether it contains donkey bone glue components.

[0005] The first technical solution provided by the present invention is a characteristic polypeptide of donkey bone glue, and its amino acid sequence is LLESPAVALDDIR (SEQ ID NO: 1) or LVVGPVVVAAPQLPPPVHR (SEQ ID NO: 2).

[0006] The second technical solution provided by the present invention is a composition containing the characteristic polypeptide of donkey bone glue described in the first technical solution.

[0007] The third technical solution provided by the present invention is a detection kit containing the characteristic polypeptide of donkey bone glue described in the first technical solution or the composition described in the second technical solution.

[0008] The fourth technical solution provided by the present invention is the application of the characteristic polypeptide of donkey bone glue described in the first technical solution, or the composition described in the second technical solution, or the detection kit described in the third technical solution in detecting the donkey bone glue component in animal hide glue and its products.

[0009] In some embodiments, the characteristic polypeptide of bone glue is detected by a liquid chromatography-mass spectrometry instrument to determine whether the donkey bone glue component is contained in animal hide glue and its products.

[0010] The fifth technical solution provided by the present invention is a method for detecting the donkey bone glue component in animal hide glue and its products, comprising the following steps:

[0011] After subjecting the animal hide glue sample to be detected to enzymatic digestion with trypsin, the enzymatic hydrolysate is injected into a liquid chromatography-mass spectrometry instrument. Using the characteristic polypeptide of donkey bone glue as a reference substance, in the multiple reaction monitoring mode of the second-level mass spectrometry, the selected conditions are as follows: m / z706.39 double charge → 872.5, DP = 40, CE = 28; m / z 973.59 double charge → 943.5, DP = 40, CE = 28; if the above ion pairs are detected, and the retention time of the ions is consistent with that of the reference substance, and the product ions are consistent with those of the reference substance, then the sample to be detected contains the donkey bone glue component.

[0012] In some embodiments, the enzymatic digestion comprises the following steps:

[0013] 1) Pretreat the dry animal hide glue and its products with a traditional Chinese medicine grinder to obtain pretreated animal product powder;

[0014] 2) Treat the pretreated animal product powder in step 1) with trypsin: accurately weigh 0.25 g (±0.01 g) of the sample, ultrasonically dissolve it with 1% ammonium bicarbonate solution, and then make up the volume to 25 mL with 1% ammonium bicarbonate solution. Take 1.0 mL of the above solution, filter it through a 0.22 μm filter membrane, accurately measure 200 μL of the filtrate into a 2 mL ultrafiltration centrifugal tube filter membrane tube, add 20 μL of trypsin solution (1 mg / mL), mix well, enzymatically hydrolyze at 37°C for 16 h, centrifuge at 15,000×g and 4°C for 10 min, and collect the lower-layer filtrate.

[0015] In some embodiments, in step 1), the pretreatment conditions of the traditional Chinese medicine grinder are: the crushing amount is 100 g - 500 g, and the intermittent treatment time is 2 - 30 min.

[0016] In some embodiments, the centrifugation in step 2) includes one or more of pressure filtration, horizontal screw centrifugation, disc centrifugation, and fixed-angle rotor centrifugation.

[0017] In some embodiments, the liquid phase conditions detected by the liquid chromatography-mass spectrometry instrument are as follows: the liquid chromatography column is filled with octadecylsilyl-bonded silica gel; 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%) is used as mobile phase B. The relevant liquid phase gradient settings are as follows: 0 min - 50 min, the linear gradient of mobile phase B ranges from 4% to 50%; 50 min - 54 min, the linear gradient of mobile phase B ranges from 50% to 100%; 54 min - 60 min, mobile phase B is maintained at 100%.

[0018] In some embodiments, the animal skin glue includes donkey-hide gelatin, yellow gelatin, and pigskin glue.

[0019] In some embodiments, the animal skin glue products include foods, health products, or drugs made from animal skin glue.

[0020] Beneficial effects

[0021] (1) Through the liquid chromatography-mass spectrometry instrument, the present invention obtains 2 characteristic peptide segments of donkey bone glue. These 2 characteristic peptide segments of donkey bone glue have high specificity, realizing the rapid identification and detection of donkey bone glue components in animal skin glue and its products.

[0022] (2) The characteristic peptide segments of donkey bone glue and the detection method provided by the present invention are simple to operate and accurate in judgment, and can accurately distinguish the characteristic peptide segments of donkey bone glue.

[0023] (3) The characteristic peptide segments of donkey bone glue and the detection method provided by the present invention are beneficial to the quality control of animal skin glue and its products, and provide an effective technical means for detecting the presence of donkey bone glue components in animal skin glue. Description of the drawings

[0024] Figure 1 It is the XIC diagram of the characteristic polypeptide of donkey bone glue with the amino acid sequence shown in SEQ ID NO:1.

[0025] Figure 2 It is the XIC diagram of the characteristic polypeptide of donkey bone glue with the amino acid sequence shown in SEQ ID NO:2. Specific embodiments

[0026] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0027] Raw materials used in the examples:

[0028] 1. Ammonium bicarbonate was purchased from Sinopharm Chemical Reagent Co., Ltd. with a CAS number of 1066-33-7; trypsin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. with a CAS number of 9002-07-7 and an enzyme activity of 1000-2000 U / mg.

[0029] 2. Donkey skin, donkey bones and donkey-hide gelatin blocks are all purchased from Shandong Gubentang Health Industry Development Group Co., Ltd.

[0030] Example 1

[0031] A donkey bone gelatin characteristic peptide detection, such as Figure 1 As shown, the preparation steps are as follows:

[0032] (1) Samples to be tested: various pure glues, including donkey bone glue and donkey hide glue, obtained by steaming pure donkey bones and pure donkey hides respectively; donkey hide glue blocks, donkey hide glue blocks with donkey bone glue added. The reference substance is donkey bone glue.

[0033] (2) Detection method:

[0034] 1) Pre-treat 300 g of dried animal hide glue and its products with a traditional Chinese medicine grinder for 5 min to obtain pre-treated animal product powder;

[0035] 2) Treat the pre-treated animal product powder in step 1) with trypsin: accurately weigh 0.25 g (±0.01 g) of the sample, dissolve it with 1% ammonium bicarbonate solution by ultrasonication, and then dilute it to 25 mL with 1% ammonium bicarbonate solution. Take 1.0 mL of the above solution, filter it through a 0.22 μm filter membrane, accurately measure 200 μL of the filtrate into a 2 mL ultrafiltration centrifuge filter membrane tube, add 20 μL of trypsin solution (1 mg / mL), mix well, and perform enzymolysis at 37°C for 16 h, centrifuge at 15,000×g, 4°C for 10 min, and collect the lower filtrate.

[0036] 3) After the sample was treated according to step 2), it was ultrafiltered (10KD) and desalted before LC-MS detection: the liquid chromatography column was filled with octadecylsilane bonded silica gel; 0.1% formic acid aqueous solution was used as mobile phase A, 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 84%) was used as mobile phase B, and the relevant liquid phase gradient was set as follows: 0 min-50 min, liquid B linear gradient from 4% to 50%; 50 min-54 min, liquid B linear gradient from 50% to 100%; 54 min-60 min, liquid B was maintained at 100%. The secondary mass spectrometry mode was used, and m / z 706.39 double charged → 872.5, DP = 40, CE = 28; m / z 973.59 double charged → 943.5, DP = 40, CE = 28 were selected as detection ion pairs.

[0037] ResultsFigure 1 At 50 - 51 minutes, corresponding ion peaks were only detected in the colla corii asini and the ejiao blocks added with colla corii asini, and not detected in others. It can be seen that this method can specifically detect the colla corii asini component in ejiao products.

[0038] Example 2

[0039] Detection of characteristic peptides of colla corii asini is as follows Figure 2 shown, and its preparation steps are specifically as follows:

[0040] (1) Test samples: Various pure glue products, including colla corii asini and colla corii asini, were obtained by steaming pure donkey bones and pure donkey skins respectively; ejiao blocks, ejiao blocks added with colla corii asini. The reference substance is colla corii asini.

[0041] (2) Detection method:

[0042] 1) Pretreat 300 g of dry animal glue and its products with a traditional Chinese medicine grinder for 5 minutes to obtain pretreated animal product powder;

[0043] 2) Treat the pretreated animal product powder in step 1) with trypsin: Accurately weigh 0.25 g (±0.01 g) of the sample, dissolve it ultrasonically with 1% ammonium bicarbonate solution, and then make up the volume to 25 mL with 1% ammonium bicarbonate solution. Take 1.0 mL of the above solution, filter it through a 0.22 μm filter membrane, accurately measure 200 μL of the filtrate into a 2 mL ultrafiltration centrifugal tube filter membrane tube, add 20 μL of trypsin solution (1 mg / mL), mix well, enzymatically hydrolyze at 37°C for 16 h, centrifuge at 15,000×g at 4°C for 10 minutes, and collect the lower layer filtrate.

[0044] 3) After the sample is treated according to step 2), ultrafiltration (10KD), desalting, and then liquid chromatography - mass spectrometry detection: The liquid chromatography column is filled with octadecylsilane - bonded silica gel; 0.1% formic acid aqueous solution is used as mobile phase A, and 0.1% formic acid acetonitrile aqueous solution (acetonitrile is 84%) is used as mobile phase B. The relevant liquid phase gradient settings are as follows: 0 min - 50 min, the linear gradient of mobile phase B is from 4% to 50%; 50 min - 54 min, the linear gradient of mobile phase B is from 50% to 100%; 54 min - 60 min, mobile phase B is maintained at 100%. The secondary mass spectrometry mode is adopted, and m / z 706.39 double - charge → 872.5, DP = 40, CE = 28; m / z 973.59 double - charge → 943.5, DP = 40, CE = 28 are selected as the detection ion pairs.

[0045] The results are shown in Figure 2 , at 53 - 54 minutes, corresponding ion peaks were only detected in the colla corii asini and the ejiao blocks added with colla corii asini, and not detected in others. It can be seen that this method can specifically detect the colla corii asini component in ejiao products.

[0046] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Characteristic polypeptide of donkey bone glue, characterized in that, The amino acid sequence is LLESPAVALDDIR or LVVGPVVVAAPQLPPPVHR.

2. Use of the characteristic polypeptide of donkey bone glue described in claim 1 in detecting the donkey bone glue component in animal hide glue and its products, characterized in that, The animal hide glue and its products are donkey hide glue or ejiao.

3. The application according to claim 2, wherein Detect the characteristic polypeptide of the bone glue by a liquid chromatography-mass spectrometry instrument, so as to determine whether the donkey bone glue component is contained in the animal hide glue and its products.

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