CN-122011114-A - Donkey-hide gelatin characteristic polypeptide and method for identifying donkey-hide gelatin doping in animal gelatin product

Abstract

The invention belongs to the technical field of medicine and food detection, and relates to a donkey-hide gelatin characteristic polypeptide and a method for identifying donkey-hide gelatin doping in an animal gelatin product. Specifically, the donkey hard gelatin characteristic peptide fragment is obtained, the amino acid sequence of the donkey hard gelatin characteristic peptide fragment is shown as SEQ ID NO. 1, the donkey hard gelatin characteristic peptide fragment has high specificity, the rapid identification and detection of donkey hard gelatin components in animal gelatin products are realized, the whole method is simple to operate and accurate to judge, and donkey hard gelatin and animal gelatin products can be accurately distinguished.

Inventors

• Zhai Changqi
• DUAN XIAOBO
• LIU HAIBIN
• GE NAN
• TIAN LEYUAN
• ZHANG YING
• Jia Fangxin
• LIU BENYU
• TIAN XIAO

Assignees

• 东阿阿胶股份有限公司

Dates

Publication Date

20260512

Application Date

20260105

Claims (10)

1. 1. A donkey-hide gelatin characteristic polypeptide is 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. 2. A polypeptide positive control, characterized in that the standard comprises the donkey-hide gelatin characteristic polypeptide of claim 1.
3. 3. A test kit comprising the polypeptide of claim 1 or the polypeptide positive control of claim 2.
4. 4. Use of a polypeptide characteristic of donkey-hide gelatin as defined in claim 1 in the preparation of a formulation for detecting whether or not donkey-hide gelatin is added to an animal gelatin preparation.
5. 5. The use according to claim 4, wherein the animal glue product comprises donkey-hide gelatin, yellow gelatin, pork skin gelatin, turtle shell gelatin, and donkey-hide gelatin cake.
6. 6. A method for detecting whether donkey-hide gelatin is added to an animal gelatin product, the method comprising: (1) Preparing a sample; (2) Detecting whether the sample contains the polypeptide in claim 1 by a liquid chromatograph/mass spectrometer; if an ion peak which is consistent with the polypeptide ion pair and the retention time is detected, the donkey hard gelatin component is added into the sample to be detected.
7. 7. The method of claim 6, wherein the sample preparation in step (1) is performed by dissolving the sample to be tested in 1% w/vNH 4 HCO 3 solution, adding trypsin for enzymolysis, and passing through a 0.22 μm microporous filter membrane to obtain an enzymolysis solution.
8. 8. The method of claim 7, wherein the trypsin is enzymatically hydrolyzed at 35-38 ℃ for 10-16 hours.
9. 9. The method of claim 6, wherein the detection conditions of the lc in step (2) are: The liquid phase condition is C18 reversed phase chromatographic column, mobile phase A is 0.1% v/v formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3mL/min, gradient elution is 0min-5min, A phase linear gradient is 90% -80%, 5min-10min, A phase linear gradient is 80% -70%, 10min-12min, A phase linear gradient is 70% -10%, 12min-15min, A phase linear gradient is maintained at 10%, mass spectrum condition is electrospray ion source ESI+, multi-reaction monitoring, m/z 565.9-448.3 is selected as quantitative ion pair, and m/z 565.9-618.3 is selected as qualitative ion pair for detection.
10. 10. The use of the method according to any one of claims 6-9 for detecting whether donkey-hide gelatin is added to food, pharmaceutical or health care products.

Description

Donkey-hide gelatin characteristic polypeptide and method for identifying donkey-hide gelatin doping in animal gelatin product Technical Field The invention belongs to the technical field of medicine and food detection, and particularly relates to a donkey-hide gelatin characteristic polypeptide and a method for identifying donkey-hide gelatin doping in an animal gelatin product. Background Along with the improvement of national economy level and healthy consumption concept, the market demand of animal glue products, especially donkey-hide glue products, is continuously increased, so that the raw donkey-hide glue is in tension supply and the product price is high. Under the drive of high profit, some merchants blend low-cost animal skin bones such as horses, cattle, pigs, mules and the like, even inferior donkey bones are inflated for a second time, so that the rights and interests of consumers are seriously damaged, the market order is disturbed, and the reputation of the donkey-hide gelatin industry is influenced. In order to solve the problem of adulteration of animal source components, the national market supervision authorities have issued a plurality of supplementary inspection methods based on characteristic peptide mass spectrometry technology, so that the adulteration behavior of the heterogeneous animal skin source is effectively restrained. However, as the donkey bone and the donkey skin belong to the same donkey source, the amino acid sequence of the type I collagen is highly consistent, and the donkey skin gelatin and the donkey bone gelatin are difficult to distinguish by the existing detection method, so that the donkey bone adulteration becomes a blind area of current quality supervision. At present, although research has been reported on a characteristic peptide identification method of donkey bone gelatin, experiments prove that the project group finds that most peptide fragments are actually derived from type II collagen in donkey cartilage (such as ear bone). The donkey-hide gelatin is usually produced by feeding the whole donkey hide, and a small amount of cartilage tissues such as donkey ears are inevitably carried. If such "donkey cartilage characteristic peptides" are used as detection indexes, false positive judgment may be generated on the synthetic product, and the artificial deliberately added donkey hard bone component cannot be accurately identified, so that misjudgment is caused. Therefore, in order to make up the defects of the prior art in the aspect of donkey hard bone doping identification, avoid the false positive problem caused by cartilage residue, the development of special characteristic polypeptides of donkey hard bone gelatin is urgent, and a reliable and sensitive detection method is established so as to support the accurate identification of donkey hard bone gelatin doping behaviors in donkey gelatin and other related animal gelatin products and ensure the product quality and the development of market specifications. Disclosure of Invention Aiming at the defects of the prior art, the invention provides the donkey-hide gelatin characteristic peptide fragment and the method for identifying the donkey-hide gelatin doping in the animal gelatin product, and aims to establish a detection method aiming at donkey-hide gelatin components in the animal gelatin product. In order to achieve the above purpose, the technical scheme of the invention is realized as follows: in a first aspect, the invention provides a polypeptide characteristic of donkey-hide gelatin, wherein the amino acid sequence of the polypeptide is Gly-Leu- (Hyp) -Gly-Thr-Ala-Gly-Leu- (Hyp) -Gly-Met-Lys. In a second aspect, the present invention provides a polypeptide positive control, which contains the polypeptide characteristic of donkey sclerosteosis in the first aspect. In a third aspect, the present invention provides a detection kit comprising the polypeptide according to the first aspect or the polypeptide positive control according to the second aspect. In a fourth aspect, the invention provides the use of a polypeptide characteristic of donkey-hide gelatin according to the first aspect in the preparation of a formulation for detecting whether or not donkey-hide gelatin is added to an animal gelatin preparation. Further, the animal glue product comprises donkey hard bone glue, donkey-hide gelatin, yellow gelatin, pig skin glue, turtle shell glue and donkey-hide gelatin cake. In a fifth aspect, the present invention provides a method for detecting whether donkey-hide gelatin is added to an animal gelatin product, the method comprising: 1) Preparing a sample; 2) Detecting whether the sample contains the polypeptide in the first aspect by a liquid chromatograph/mass spectrometer; If an ion peak consistent with the polypeptide ion pair and consistent with the retention time is detected, the donkey sclerosteosis component is added into the sample to be detected. The sample preparation method in the step