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Method for identifying nile tilapia skin collagens

A technology of fish skin collagen and Nile tilapia, which is applied in the field of protein analysis, can solve the problems of lack of technical support and deceiving consumers in the rapid detection of products

Active Publication Date: 2018-03-06
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, due to the traditional analytical methods such as electrophoresis, amino acid analysis, infrared spectroscopy, and X-ray diffraction, it is impossible to distinguish collagen from different sources, and the rapid detection of related products lacks technical support, resulting in many unscrupulous merchants shoddy , to deceive consumers, so there is a need for an efficient and accurate method for identifying collagen from different sources

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining the Specific Peptide Group of Nile Tilapia Skin Collagen

[0035] 1) Take the extracted and purified Nile tilapia skin collagen and dissolve it with 0.25mol / L acetic acid at a concentration of 4mg / mL, and use ultrasonic-assisted dissolution method with a frequency of 25-40KHz for 10 minutes;

[0036] 2) Mix the collagen solution with 0.25mol / L NH 4 HCO 3 The solution was mixed evenly according to the volume ratio of 1:1, treated at 50°C for 3 hours, and then centrifuged at 12000×g for 20 minutes to remove insoluble collagen;

[0037] 3) Take 100 μL of centrifuged supernatant, add 1 mg / mL trypsin solution (enzyme: substrate = 1:25, w / w), and perform enzymatic hydrolysis reaction at 37° C. for 20 h;

[0038] 4) The enzymolysis solution is filtered with a 0.22 μm microporous membrane and then filtered with Zip Desalting and concentrating the micro-chromatography column, to be analyzed by liquid chromatography-tandem mass spectrometry;

[0039] 5) L...

Embodiment 2

[0047] The detection and analysis of embodiment 2 Nile tilapia skin fish skin collagen

[0048] 1) Take 3 copies of Nile tilapia fish skin collagen for experiments respectively, take the freeze-dried collagen and dissolve it with 0.01mol / L acetic acid, the concentration is 4mg / mL, and use the ultrasonic-assisted dissolution method, the frequency is 25-40KHz, Time 5min;

[0049] 2) Mix the collagen solution with 0.1mol / L NH 4 HCO 3 The solution is mixed evenly according to the volume ratio of 1:1, treated at 50°C for 2-3 hours, and then centrifuged at 12000×g for 10-20 minutes to remove insoluble collagen;

[0050] 3) Take 100-500 μL of centrifuged supernatant, add 1 mg / mL trypsin solution (enzyme: substrate = 1:50, w / w), and perform enzymatic hydrolysis at 37°C for 18 hours;

[0051] 4) The enzymolysis solution is filtered with a 0.22 μm microporous membrane and then filtered with Zip Desalting and concentrating the micro-chromatography column, to be analyzed by liquid chr...

Embodiment 3

[0060] The detection and analysis of embodiment 3 cod skin collagen

[0061] 1) Take the freeze-dried cod skin collagen and dissolve it with 0.05mol / L acetic acid, the concentration is 2mg / mL, and use the ultrasonic-assisted dissolution method, the frequency is 25-40KHz, and the time is 5min;

[0062] 2) Mix the collagen solution with 0.05mol / L NH 4 HCO 3 The solution was mixed evenly according to the volume ratio of 1:1, treated at 50°C for 3 hours, and then centrifuged at 12000×g for 10 minutes to remove insoluble collagen;

[0063] 3) Take 100 μL of centrifuged supernatant, add 1 mg / mL trypsin solution (enzyme: substrate = 1:25, w / w), and perform enzymatic hydrolysis at 37°C for 16 hours;

[0064] 4) The enzymolysis solution is filtered with a 0.22 μm microporous membrane and then filtered with Zip Desalting and concentrating the micro-chromatography column, to be analyzed by liquid chromatography-tandem mass spectrometry;

[0065] 5) Liquid chromatography-tandem mass ...

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Abstract

The invention relates to a method for identifying nile tilapia skin collagens by using specific peptide fragment groups. The peptide fragment groups comprise polypeptides with sequences of SEQ ID NO:1-10. The method comprises the following steps: performing pretreatment on collagens, and performing liquid chromatography-tandem mass spectrometry detection and analysis, thereby obtaining the sequences of the fragment groups after enzymolysis. By virtue of amino acid sequence alignment of nile tilapia skin collagens in a database, the method is proved to be capable of identifying most of peptidefragment sequences. Repeated experimental results show that the peptide fragment sequences SEQ ID NO:1-10 exist in the nile tilapia skin collagens. Therefore, the specific peptide fragment groups of the nile tilapia skin collagens are obtained, and the problem that the previous collagen source is difficult to identify is solved.

Description

technical field [0001] The invention belongs to the technical field of protein analysis, and in particular relates to a method for identifying Nile tilapia skin collagen. Background technique [0002] Collagen exists in the skin, bones, and blood vessels of multicellular animals in the form of insoluble fibers. It has biocompatibility, biodegradability, and low antigenicity. Its extracted products have been widely used in medicine, health care, food processing, cosmetics, etc. Numerous fields. For a long time, collagen is mainly extracted from the skin of some terrestrial mammals such as cattle and pigs. In recent years, people have gradually shifted their focus to the research on fish collagen. Fish skin is the part with a relatively high collagen content in the by-products of aquatic products, mainly type I collagen, which can be used as a safe cosmetic and medical biomaterial. [0003] Compared with fish skin collagen from different sources, some have good thermal stab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K14/00C12P21/06G01N30/02G01N30/72
CPCG01N30/02G01N30/72C12P21/06C07K7/06C07K7/08C07K14/00Y02A40/81
Inventor 李八方侯虎张燕
Owner OCEAN UNIV OF CHINA
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