Donkey hard bone glue characteristic polypeptide and method for identifying donkey hard bone glue doping in animal glue product

Abstract

The invention belongs to the technical field of medicine and food detection, and relates to a donkey hard bone glue characteristic polypeptide and a method for identifying donkey hard bone glue doping in an animal glue product by using the donkey hard bone glue characteristic polypeptide. Specifically, one donkey hard bone glue characteristic peptide fragment is obtained, the amino acid sequence is shown as SEQ ID NO: 1, the donkey hard bone glue characteristic peptide fragment has very high specificity, and rapid identification and detection of donkey hard bone glue components in animal glue products are achieved; according to the donkey hard bone glue characteristic peptide fragment and the detection method thereof, the whole method is simple to operate and accurate in judgment, and donkey hard bone glue and animal glue products can be accurately distinguished; meanwhile, the donkey hard bone glue characteristic peptide fragment and the detection method thereof provided by the invention are beneficial to quality control of animal glue products, and provide an effective technical means for identifying whether donkey hard bone glue is doped in the animal glue products or not.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and food testing technology, specifically relating to a characteristic polypeptide of donkey bone glue and a method for identifying donkey bone glue adulteration in animal glue products. Background Technology

[0002] With the improvement of national economic level and health consumption concept, the market demand for animal glue products, especially donkey-hide gelatin products, continues to grow, leading to a tight supply of raw donkey hides and high product prices. Driven by high profits, some merchants adulterate the products with cheap animal hides and bones such as horses, cattle, pigs, and mules, or even inferior donkey bones, seriously damaging consumer rights, disrupting market order, and affecting the reputation of the donkey-hide gelatin industry.

[0003] To address the issue of adulteration with animal-derived ingredients, the national market supervision department has issued several supplementary testing methods based on characteristic peptide mass spectrometry analysis technology, effectively curbing adulteration with skin from different species. However, since donkey bone and donkey skin are both from the donkey and their type I collagen amino acid sequences are highly consistent, existing detection methods struggle to distinguish between donkey skin glue and donkey bone glue, making donkey bone adulteration a blind spot in current quality supervision.

[0004] Currently, although some studies have reported methods for identifying characteristic peptides in donkey bone glue, our project team has experimentally verified that most of these peptides actually originate from type II collagen in donkey cartilage (such as ear cartilage). Donkey-hide gelatin production typically uses whole donkey hides, inevitably including small amounts of cartilage tissue such as donkey ear cartilage. Using these "donkey cartilage characteristic peptides" as detection indicators could lead to false positives for legitimate products, failing to accurately identify intentionally added donkey bone components, thus causing misjudgments.

[0005] Therefore, in order to make up for the shortcomings of existing technologies in the identification of donkey cartilage adulteration and avoid false positives caused by cartilage residues, it is urgent to discover the characteristic polypeptides specific to donkey cartilage glue and establish reliable and sensitive detection methods to support the accurate identification of donkey cartilage glue adulteration in donkey-hide gelatin and other related animal glue products, so as to ensure product quality and the standardized development of the market. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides characteristic peptides of donkey bone glue and a method for identifying donkey bone glue adulteration in animal glue products. The aim is to establish a detection method for donkey bone glue components in animal glue products. By utilizing the high specificity of the characteristic peptides of donkey bone glue, the detection method is simple to operate and highly sensitive. It can not only quickly identify whether donkey bone glue is present, but also accurately quantify it.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a characteristic polypeptide of donkey bone glue, wherein the amino acid sequence of the polypeptide is Gly-Leu-(Hyp)-Gly-Thr-Ala-Gly-Leu-(Hyp)-Gly-Met-Lys.

[0008] Secondly, the present invention provides a polypeptide positive control, wherein the polypeptide positive control contains the characteristic polypeptide of donkey bone glue as described in the first aspect.

[0009] Thirdly, the present invention provides a detection kit comprising the polypeptide as described in the first aspect or the polypeptide positive control as described in the second aspect.

[0010] Fourthly, the present invention provides the application of the characteristic polypeptide of donkey-hide gelatin described in the first aspect in the preparation of a formulation for detecting whether donkey-hide gelatin has been added to animal gelatin products.

[0011] Furthermore, the animal glue products mentioned include donkey bone glue, donkey-hide gelatin, yellow gelatin, pigskin glue, turtle shell glue, and donkey-hide gelatin cake.

[0012] Fifthly, the present invention provides a method for detecting whether donkey bone glue has been added to animal glue products, characterized in that the method comprises: 1) Sample preparation; 2) Detect whether the sample contains the polypeptides described in the first aspect using liquid chromatography-mass spectrometry (LC-MS); If an ion peak with the same retention time as the polypeptide ion pair described in the first aspect is detected, then the sample to be tested contains donkey bone glue component.

[0013] Further, the sample preparation method in step 1) is as follows: the sample to be tested is dissolved in 1% w / v NH4HCO3 solution, trypsin is added for enzymatic hydrolysis, and the solution is filtered through a 0.22μm microporous membrane to obtain the enzymatic hydrolysate; preferably, the enzymatic hydrolysis conditions are 35~38℃ for 10~16 hours.

[0014] Furthermore, the detection conditions for the liquid chromatography-mass spectrometry (LC-MS) instrument in step 2) are as follows: Liquid chromatography conditions: C18 reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A was 0.1% v / v formic acid solution, mobile phase B was acetonitrile, flow rate 0.3 mL / min; gradient elution: 0 min–5 min, linear gradient of phase A from 90% to 80%; 5 min–10 min, linear gradient of phase A from 80% to 70%; 10 min–12 min, linear gradient of phase A from 70% to 10%; 12 min–15 min, linear gradient of phase A maintained at 10%. Mass spectrometry conditions: electrospray ionization source (ESI+), multiple reaction monitoring (MRM), selected m / z 565.9 (double charge) → 448.3 (quantitative ion pair) and m / z 565.9 (double charge) → 618.3 (qualitative ion pair) as detection ion pairs.

[0015] In a sixth aspect, the present invention provides the application of the method described in the fifth aspect in detecting whether donkey-hide gelatin has been added to pharmaceuticals, food or health products; preferably, the pharmaceuticals, food or health products include donkey-hide gelatin, yellow gelatin, pigskin gelatin, turtle shell gelatin, and donkey-hide gelatin cake; most preferably, the pharmaceuticals, food or health products include donkey-hide gelatin.

[0016] The beneficial effects of this invention include: (1) The present invention obtains a characteristic peptide segment of donkey bone glue by liquid chromatography-mass spectrometry. This characteristic peptide segment of donkey bone glue has high specificity, realizing the rapid identification and detection of donkey bone glue components in animal glue products.

[0017] (2) The characteristic peptides of donkey bone glue and their detection method provided by the present invention are simple to operate, accurate in judgment, and can accurately distinguish the characteristic peptides of donkey bone glue.

[0018] (3) The characteristic peptides of donkey bone glue and their detection method provided by the present invention are beneficial to the quality control of animal glue products and provide an effective technical means for identifying whether donkey bone glue is added to animal glue products. Attached Figure Description

[0019] Figure 1 Mass spectra of donkey cartilage glue, donkey cartilage glue characteristic polypeptide positive control, donkey cartilage glue, and donkey-hide gelatin monitored by selected ion pairs m / z 565.9 (double charge) → 448.3 in MRM scanning mode; Figure 2 Mass spectra of donkey-hide gelatin (containing 5% donkey bone glue), donkey-hide gelatin, yellow gelatin (containing 5% donkey bone glue), yellow gelatin, pigskin glue (containing 5% donkey bone glue), pigskin glue, turtle shell glue (containing 5% donkey bone glue), and turtle shell glue (containing 5% donkey bone glue) monitored by selected ion pair m / z 565.9 (double charge) → 448.3 in MRM scanning mode.

[0020] Figure 3This is a mass spectrum of Ejiao cake containing 1.25% donkey bone glue, monitored by selected ion pair m / z 565.9 (double charge) → 448.3 in MRM scanning mode. Detailed Implementation

[0021] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0022] Example 1 1. Materials and Reagents Samples to be tested: donkey bone glue and donkey cartilage glue, which were obtained by decoction and concentration of donkey bone and donkey ear cartilage, respectively; the characteristic polypeptide positive control of donkey bone glue was purchased from Hubei Qiangyao Biotechnology Co., Ltd.; donkey-hide gelatin was purchased from Dong-E-E-Jiao Co., Ltd.

[0023] Reagents: Ammonium bicarbonate was purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 1066-33-7; trypsin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 9002-07-7, with an enzyme activity of 1000-2000 U / mg.

[0024] 2. Detection Methods Take 0.05g of the sample to be tested into a 25mL volumetric flask, dissolve and dilute to volume with 1% w / v NH4HCO3 solution; accurately measure 2mL, add 1mL of 2mg / mL trypsin solution, place in a 5mL volumetric flask, dilute to volume with 1% w / v NH4HCO3 solution, incubate at 37℃ for 12h, filter through a 0.22μm microporous membrane to obtain the enzymatic hydrolysate.

[0025] Take 5 μl of the enzymatic digestion solution and analyze it using a liquid chromatography-mass spectrometry (LC-MS) system. LC conditions: C18 reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% v / v formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0 min–5 min, linear gradient of phase A from 90% to 80%; 5 min–10 min, linear gradient of phase A from 80% to 70%; 10 min–12 min, linear gradient of phase A from 70% to 10%; 12 min–15 min, linear gradient of phase A maintained at 10%. Mass spectrometry conditions: electrospray ionization (ESI+), multiple reaction monitoring (MRM), selected m / z 565.9 (double charge) → 448.3 (quantitative ion pair) and m / z 565.9 (double charge) → 618.3 (qualitative ion pair) as detection ion pairs.

[0026] See results Figure 1 Only donkey bone glue and donkey bone glue characteristic polypeptide positive control were found to have corresponding ion peaks, while others were not detected. This shows that the method can specifically detect donkey bone glue components and distinguish them from donkey-hide gelatin and donkey cartilage glue.

[0027] Example 2 1. Materials and Reagents Sample to be tested: The donkey-hide gelatin containing 5% donkey bone glue was made by adding 5% w / w of donkey bone glue from Example 1 to the donkey-hide gelatin from Example 1; Yellow gelatin containing 5% donkey bone glue was made by adding 5% w / w of donkey bone glue from Example 1 to the yellow gelatin from Example 2. The pigskin glue containing 5% donkey bone glue was made by adding 5% w / w of donkey bone glue from Example 1 to the pigskin glue of Example 2. The turtle shell glue containing 5% donkey bone glue was made by adding 5% w / w of donkey bone glue from Example 1 to the turtle shell glue from Example 2. Yellow gelatin was purchased from Dong-E-E-Jiao Co., Ltd.; turtle shell gelatin was purchased from Henan Furentang Pharmaceutical Co., Ltd.; pigskin gelatin was obtained by boiling and concentrating pigskin.

[0028] Reagents: Ammonium bicarbonate was purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 1066-33-7; trypsin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 9002-07-7, with an enzyme activity of 1000-2000 U / mg.

[0029] 2. Detection Methods Take 0.05g of the sample to be tested into a 25mL volumetric flask, dissolve and dilute to volume with 1% w / v NH4HCO3 solution; accurately measure 2mL, add 1mL of 2mg / mL trypsin solution, place in a 5mL volumetric flask, dilute to volume with 1% w / v NH4HCO3 solution, incubate at 37℃ for 12h, filter through a 0.22μm microporous membrane to obtain the enzymatic hydrolysate.

[0030] Take 5 μl of the enzymatic digestion solution and analyze it using a liquid chromatography-mass spectrometry (LC-MS) system. LC conditions: C18 reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% v / v formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0 min–5 min, linear gradient of phase A from 90% to 80%; 5 min–10 min, linear gradient of phase A from 80% to 70%; 10 min–12 min, linear gradient of phase A from 70% to 10%; 12 min–15 min, linear gradient of phase A maintained at 10%. Mass spectrometry conditions: electrospray ionization (ESI+), multiple reaction monitoring (MRM), selected m / z 565.9 (double charge) → 448.3 (quantitative ion pair) and m / z 565.9 (double charge) → 618.3 (qualitative ion pair) as detection ion pairs.

[0031] See results Figure 2 The corresponding ion peaks were detected in donkey-hide gelatin (Ejiao), yellow gelatin (Huangmingjiao), pigskin gelatin (Zhupijiao), and turtle shell gelatin (Tuijiajiao) containing 5% donkey-hide gelatin, but not in Ejiao, yellow gelatin, pigskin gelatin, or turtle shell gelatin. This demonstrates that this method can specifically detect Ejiao, yellow gelatin, pigskin gelatin, and turtle shell gelatin mixed with donkey-hide gelatin.

[0032] Example 3 1. Materials and Reagents Sample to be tested: The donkey-hide gelatin cake containing 1.25% donkey bone glue was made by adding 1.25% w / w of donkey bone glue from Example 1 to the donkey-hide gelatin cake of Example 3; The donkey-hide gelatin cake was purchased from Dong-E-E-Jiao Co., Ltd.

[0033] Reagents: Ammonium bicarbonate was purchased from Sinopharm Chemical Reagent Co., Ltd., CAS No. 1066-33-7; trypsin was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 9002-07-7, with an enzyme activity of 1000-2000 U / mg.

[0034] 2. Detection Methods Take 50g of the sample to be tested, add 500mL of water, stir and sonicate until fully dissolved to obtain a sample suspension; take 2mL of the sample suspension into a 25mL volumetric flask, dissolve and dilute to volume with 1% w / v NH4HCO3 solution; accurately measure 2mL, add 1mL of 2mg / mL trypsin solution, place in a 5mL volumetric flask, dilute to volume with 1% w / v NH4HCO3 solution, incubate at 37℃ for 12h, filter through a 0.22μm microporous membrane to obtain the enzymatic hydrolysate.

[0035] Take 5 μl of the enzymatic digestion solution and analyze it using a liquid chromatography-mass spectrometry (LC-MS) system. LC conditions: C18 reversed-phase column (2.1 mm × 100 mm, 1.8 μm), mobile phase A: 0.1% v / v formic acid solution, mobile phase B: acetonitrile, flow rate: 0.3 mL / min; gradient elution: 0 min–5 min, linear gradient of phase A from 90% to 80%; 5 min–10 min, linear gradient of phase A from 80% to 70%; 10 min–12 min, linear gradient of phase A from 70% to 10%; 12 min–15 min, linear gradient of phase A maintained at 10%. Mass spectrometry conditions: electrospray ionization (ESI+), multiple reaction monitoring (MRM), selected m / z 565.9 (double charge) → 448.3 (quantitative ion pair) and m / z 565.9 (double charge) → 618.3 (qualitative ion pair) as detection ion pairs.

[0036] See results Figure 3 Only donkey-hide gelatin cakes containing 5% donkey bone glue showed the corresponding ion peaks; none were detected in other donkey-hide gelatin cakes. This demonstrates that this method can specifically detect donkey-hide gelatin cakes mixed with donkey bone glue.

[0037] In summary, this invention is based on the characteristic polypeptide Gly-Leu-(Hyp)-Gly-Thr-Ala-Gly-Leu-(Hyp)-Gly-Met-Lys of donkey bone glue, selecting m / z 565.9 (double charge) → 448.3 and m / z 565.9 (double charge) → 618.3 as detection ion pairs, with m / z 565.9 (double charge) → 448.3 selected as the quantitative ion pair. Trypsin digestion and liquid chromatography-mass spectrometry (LC-MS) are used to detect donkey-hide gelatin products. This method can not only accurately distinguish between animal gelatin products and donkey bone glue, but also accurately quantify the components of donkey bone glue.

[0038] It should be noted that the above embodiments are only used to illustrate the technical solutions of the invention and not to limit it. Although the invention has been described with reference to the preferred embodiments of the invention, various modifications or alterations made by those skilled in the art do not depart from the spirit and scope of the invention, and these equivalent forms also fall within the scope of the invention.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A characteristic polypeptide of donkey cartilage glue, characterized in that, The amino acid sequence of the polypeptide is Gly-Leu-(Hyp)-Gly-Thr-Ala-Gly-Leu-(Hyp)-Gly-Met-Lys (SEQ ID NO:1).

2. A polypeptide positive control, characterized in that, The standard contains the characteristic polypeptide of donkey bone glue as described in claim 1.

3. A test kit, characterized in that, The kit includes the polypeptide as described in claim 1 or the polypeptide positive control as described in claim 2.

4. The use of the characteristic polypeptide of donkey-hide gelatin as described in claim 1 in the preparation of a formulation for detecting whether donkey-hide gelatin has been added to animal gelatin products.

5. The application according to claim 4, characterized in that, The animal glue products mentioned include donkey bone glue, donkey-hide gelatin, yellow gelatin, pigskin glue, turtle shell glue, and donkey-hide gelatin cake.

6. A method for detecting whether donkey bone glue has been added to animal glue products, characterized in that, The method includes: (1) Sample preparation; (2) Detect whether the sample contains the polypeptide described in claim 1 using a liquid chromatography-mass spectrometry (LC-MS) instrument; If an ion peak with the same retention time as the polypeptide ion pair described in claim 1 is detected, then the sample to be tested contains donkey bone glue component.

7. The method according to claim 6, characterized in that, The sample preparation method in step (1) is as follows: the sample to be tested is dissolved in 1% w / v NH4HCO3 solution, trypsin is added for enzymatic hydrolysis, and the solution is filtered through a 0.22μm microporous membrane to obtain the enzymatic hydrolysate.

8. The method according to claim 7, characterized in that, The conditions for trypsin hydrolysis are 35-38℃ for 10-16 hours.

9. The method according to claim 6, characterized in that, The detection conditions for the liquid chromatography-mass spectrometry (LC-MS) instrument in step (2) are as follows: Liquid chromatography conditions: C18 reversed-phase column, mobile phase A is 0.1% v / v formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3 mL / min; gradient elution: 0 min-5 min, linear gradient of phase A from 90% to 80%; 5 min-10 min, linear gradient of phase A from 80% to 70%; 10 min-12 min, linear gradient of phase A from 70% to 10%; 12 min-15 min, linear gradient of phase A maintained at 10%; mass spectrometry conditions: electrospray ionization source ESI+, multiple reaction monitoring, m / z 565.9→448.3 as quantitative ion pair, m / z 565.9→618.3 as qualitative ion pair for detection.

10. The method according to any one of claims 6 to 9 is used in detecting whether donkey bone glue has been added to food, medicine or health products.

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