CN111751476B - Duck-derived characteristic III-type collagen peptide and application thereof in detection of collagen hydrolysate and products thereof - Google Patents
Duck-derived characteristic III-type collagen peptide and application thereof in detection of collagen hydrolysate and products thereof Download PDFInfo
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
A duck-derived characteristic III-type collagen peptide and application thereof in detection of collagen hydrolysate and products thereof belong to the technical field of detection. The invention relates to duck-derived characteristic III type collagen peptide Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg, and whether duck-derived components are contained can be determined by detecting the characteristic III type collagen peptide. Specifically, the characteristic type III collagen peptide is dissociated by carrying out pancreatin enzyme digestion on protein components in a sample, and then whether the characteristic type III collagen peptide is contained or not is detected by a liquid chromatograph-mass spectrometer to judge whether duck-derived components are contained or not. The method has the advantages of strong characteristics, high sensitivity and simple operation, and can be used for measuring duck-origin components in collagen hydrolysate and products thereof.
Description
Technical Field
The invention relates to duck-derived characteristic III-type collagen peptide and application thereof in detection of collagen hydrolysate and products thereof, belonging to the field of medicine and food detection.
Background
Collagen is present in animal bodies in very high amounts, mainly in connective tissues such as bones, skins, tendons and tendons of animals. Gelatin is a degradation product of collagen, is prepared by properly hydrolyzing animal bones, skins, tendons and the like serving as raw materials, has excellent physicochemical properties, and is widely applied to the fields of food, medicine and the like. The gelatin is mostly made of skins or bones of pigs, cattle and sheep, but the gelatin has important significance for tracing animal-derived components due to different safety risks, muslims and religious beliefs possibly caused by diseases of poultry and livestock.
The main component of the gelatin is collagen polypeptide, and the animal source component can be traced by detecting the polypeptide by using a liquid chromatograph-mass spectrometer, however, whether the selected polypeptide can be used as a tracing marker or not is related to the success of tracing because the polypeptide has the phenomena of mutation and the like in the same species. The selection of the characteristic collagen peptide tracing duck-derived components not only overcomes the influence caused by gene mutation, but also needs to be contained in ducks and not contained in other animals. The selection of non-mutated characteristic collagen peptide in duck becomes the key of duck source component tracing. The above difficulties cannot be overcome by directly adopting a liquid chromatography-mass spectrometer to detect and analyze a sample. Starting from genes, the analysis for searching for the characteristic collagen peptide is a feasible path.
Disclosure of Invention
The invention aims at the problems and provides a duck-origin characteristic collagen peptide obtained from a gene perspective and a detection method of duck-origin components in collagen hydrolysate and products thereof.
The technical scheme of the invention is as follows:
a duck-derived characteristic III-type collagen peptide and application thereof in detection of collagen hydrolysate and products thereof are disclosed, wherein a duck-based III-type collagen sequence comprises the characteristic collagen peptide, and the amino acid sequence of the characteristic collagen peptide is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg, wherein Hyp is 4 hydroxyproline or 3 hydroxyproline. The method is characterized in that a characteristic mRNA sequence of a duck which stably exists is found out from mRNA of COL3A1 protein in cattle, pigs, sheep, chickens and ducks, the mRNA sequence is translated into a protein sequence, and then the protein sequence is detected by using a liquid chromatography-mass spectrometry technology, and finally selected characteristic collagen peptide is obtained.
The application of the characteristic collagen peptide in the detection of collagen hydrolysate and products thereof comprises the following steps:
(1) performing enzyme digestion on a sample to be detected by using trypsin, or extracting protein components in the sample to be detected and performing enzyme digestion by using trypsin;
(2) putting into a liquid chromatography-mass spectrometer, taking characteristic collagen peptide or duck gelatin subjected to trypsin enzyme digestion as a reference substance, and selecting parent ion m/z676.5 and daughter ions thereof for monitoring; if the retention time of the detected ion is consistent with that of a reference substance and the daughter ion of the detected ion is consistent with that of the reference substance, the collagen hydrolysate or the product thereof contains duck-origin components, and the content of the duck-origin components can be calculated by peak areas in the reference substance and the detected sample; if the ion is not consistent with the retention time of the reference substance, the duck-origin component is not contained.
The collagen hydrolysate is gelatin prepared by hydrolyzing animal skin, bone or scale, and the collagen hydrolysate product is food, health product or medicine containing gelatin.
By adopting the method, whether the collagen hydrolysate and the products thereof contain duck-origin components can be rapidly detected. Although the animal has gene mutation to cause protein sequence difference, the characteristic collagen peptide gene has strong conservation in ducks and is peculiar to ducks, and through the characteristic collagen peptide, the duck-derived components in collagen hydrolysate and products thereof can be traced and detected.
Drawings
FIG. 1 is a scanning mass spectrum of the ion of the eigen collagen peptide in duck gelatin (parent ion m/z676.5, scanning range m/z 200-1400 of the ion);
FIG. 2 is a mass spectrum of selected ion pair m/z676.5 → 604.4, 519.1 monitoring under various animal gelatin SRM scanning modes;
FIG. 3 is a graph of the relationship between the mass spectrometric peak areas (m/z676.5 → 604.4) of characteristic collagen peptides corresponding to different contents of duck gelatin;
FIG. 4 is a mass spectrum of duck gelatin and gelatin preparation (QQ sugar as an example) monitored by selective ion pair m/z676.5 → 604.4, 519.1 in SRM scanning mode.
FIG. 5 shows the partial mRNA sequence of COL3A1 protein of chicken, duck, cattle, pig and sheep and their corresponding comparison.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and substitutions are intended to be within the scope of the invention.
Example 1
1 materials and reagents
Materials: gelatin from different animal sources is respectively derived from chicken, duck, cattle, pig and sheep, and is respectively extracted from chicken skin, duck skin, cow leather, pig skin and sheep skin.
Reagent: ammonium bicarbonate (analytically pure), trypsin (sequence pure, purchased from the chinese institute for drug and biological product assay).
2 screening and determination of characteristic collagen peptides
Through duck skin detection, a large amount of COL3A1 protein is found in duck skin, and the protein has certain conservation, so that a section of peptide can be screened from COL3A1 protein to serve as the characteristic III type collagen peptide of duck source. In order to screen the genes for stable characteristic collagen peptides for detection, the following conditions should be satisfied: (a) three bases adjacent to the mRNA sequence corresponding to the peptide segment are arginine or lysine after translation, and 1-3 bases in the mRNA sequence corresponding to the peptide segment are proline after translation; (b) the content of the base C, G in the mRNA sequence corresponding to the peptide fragment is more than 60%; (c) the mRNA corresponding to at least one amino acid in the peptide section meets any one of the following conditions: if the first base is C or G, the first base in the three bases of the codon corresponding to the amino acid is G or C correspondingly in chicken, cattle, pigs or sheep; if the second base is A or T, the second base should be T or A in chicken, cattle, swine or sheep, respectively; if the second base is C or T, the second base should be T or C in chicken, cattle, swine or sheep, respectively; (d) detecting whether the screened peptide segment has post-translational modification (namely whether proline is hydroxylated) by utilizing mass spectrometry, and if not, determining that the screened peptide segment is the characteristic collagen peptide; if the posttranslational modification exists, the posttranslational modified polypeptide is the characteristic collagen peptide; (e) there should be no other interfering signals detected by mass spectrometry. To this end we performed the following experiments:
(1) primary screen for characteristic collagen peptide
The mRNA sequences corresponding to COL3A1 protein of chicken, duck, cattle, pig and sheep were aligned, and a part of the sequences are shown in FIG. 5 (note: the mRNA is transcribed from DNA in the nucleus, and sometimes the sequence of the mRNA is expressed by the base sequence on the DNA strand). Wherein the mRNA sequence "GGC AGT CCA GGT CCC CCT GGC CCA AGT GGA CCT GCA GGA GAA CGT" of duck in the wireframe of FIG. 5 satisfies the above-mentioned conditions (a), (b), and (c). The polypeptide corresponding to the sequence after translation is 'Gly-Ser-Pro-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg', and whether the polypeptide is a characteristic collagen peptide of the duck needs to be further determined to determine whether the post-translational modification exists (namely whether proline is hydroxylated or not), so that the next mass spectrum detection test is carried out to determine whether the post-translational modification exists.
(2) Determination of characteristic collagen peptides
a) Taking 0.05g gelatin sample in a 25ml measuring flask, adding a small amount of 1% NH4HCO3Soaking in the solution for 10min, heating at 60 deg.C to dissolve, and dissolving with 1% NH4HCO3Diluting the solution to scale, shaking, filtering with microporous membrane, precisely taking 0.2ml of the subsequent filtrate, adding 20 μ l of 2mg/ml trypsin solution, and performing enzymolysis at 37 deg.C for 12 hr.
b) And (3) putting 5 mul of duck gelatin enzymolysis solution into a liquid chromatograph-mass spectrometer to detect whether the polypeptide is subjected to hydroxylation modification. Liquid phase conditions: c18Reversed phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3 ml/min; gradient elution: 0-25 min, 98% → 80% mobile phase a, 2% → 20% mobile phase B. Mass spectrum conditions: electrospray positive ion mode (ESI)+) The m/z660.5, 668.5 and m/z676.5 were selected as parent ions for full daughter ion scans, respectively. As a result: (1) the mass spectrogram of a full scan of a daughter ion taking m/z660.5 as a parent ion does not accord with the target polypeptide Gly-Ser-Pro-Gly-Pro-Pro-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg; (2) the mass spectrogram of a full scanning of a daughter ion taking m/z668.5 as a parent ion does not accord with the proline hydroxylation of the target polypeptide; (3) the mass spectrogram of the full scan of the daughter ion taking m/z676.5 as the parent ion is consistent with the hydroxylation of 2 prolines of the target polypeptide, and is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg (shown in figure 1). The result shows that 2 amino acids in the peptide translated by the duck mRNA sequence 'GGC AGT CCA GGT CCC CCT GGC CCA AGT GGA CCT GCA GGA GAA CGT' are modified, namely 2 prolines in the polypeptide are hydroxylated, and the hydroxylated polypeptide sequence is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg.
c) And (3) putting 5 mu l of enzymolysis solution into a liquid chromatograph-mass spectrometer for detection. Liquid phase conditions: c18Reverse phase chromatography column (2.1 mm. times.100 mm, 1.8. mum), the mobile phase A is 0.1 percent formic acid solution, the mobile phase B is acetonitrile, and the flow rate is 0.3 ml/min; gradient elution: 0-25 min, 98% → 80% mobile phase a, 2% → 20% mobile phase B. Mass spectrum conditions: electrospray positive ion mode (ESI)+) The SRM detection was chosen, and m/z676.5 → 604.4, 519.1 was chosen as the detection ion pair. The results are shown in FIG. 2, only the corresponding ion peak is detected in duck gelatin at 7.5min, and none of the other peaks is detected.
In conclusion, the polypeptide Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg can be used as the characteristic collagen peptide III of duck source.
Example 2
1 materials and reagents
Materials: mixed gelatin samples (including pig gelatin with 5% duck gelatin, pig gelatin with 10% duck gelatin, pig gelatin with 20% duck gelatin, pig gelatin with 40% duck gelatin, pig gelatin with 60% duck gelatin, purified duck gelatin) were prepared by precision mixing of the gelatins of example 1.
Reagent: ammonium bicarbonate (analytically pure), trypsin (sequence pure, purchased from the chinese institute for drug and biological product assay).
2 detection method
(1) Taking 0.05g gelatin sample in a 25ml measuring flask, adding a small amount of 1% NH4HCO3Soaking in the solution for 10min, heating at 60 deg.C to dissolve, and dissolving with 1% NH4HCO3Diluting the solution to scale, shaking, filtering with microporous membrane, precisely taking 0.2ml of the subsequent filtrate, adding 20 μ l of 2mg/ml trypsin solution, and performing enzymolysis at 37 deg.C for 12 hr.
(2) 5 mul of enzymolysis solution is put into a liquid chromatograph-mass spectrometer for detection. Liquid phase conditions: c18Reversed phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3 ml/min; gradient elution: 0-25 min, 98% → 80% mobile phase a, 2% → 20% mobile phase B. Mass spectrum conditions: electrospray positive ion mode (ESI)+) SRM detection was selected, and m/z676.5 → 604.4, 519.1 was selected as the detection ion pair.
The concentration of duck gelatin was plotted on the abscissa (X) and the peak area (m/z676.5 → 604.4) on the ordinate (Y). KnotIf the result is shown in FIG. 3, the standard curve Y is 1183997X-12550, and the linear correlation coefficient R is20.9970, the linearity is good. Therefore, the method can be used for detecting the content of the duck-origin components.
Example 3
1 materials and reagents
Materials: duck gelatin, QQ sugar added with cattle gelatin (self-made), and QQ sugar added with duck gelatin (self-made);
reagent: ammonium bicarbonate (analytically pure), trypsin (sequence pure, purchased from the chinese institute for drug and biological product assay).
2 detection method
(1) Respectively putting 0.05-0.25 g of duck gelatin, QQ sugar added with bovine gelatin and QQ sugar added with duck gelatin into 25ml measuring bottles, and adding a small amount of 1% NH4HCO3Soaking in the solution for 10min, heating at 60 deg.C to dissolve, and dissolving with 1% NH4HCO3Diluting the solution to scale, shaking, filtering with microporous membrane, precisely taking 0.2ml of the subsequent filtrate, adding 20 μ l of 2mg/ml trypsin solution, and performing enzymolysis at 37 deg.C for 12 hr.
(2) And (3) putting 5 mu l of enzymolysis solution into a liquid chromatograph-mass spectrometer for detection. Liquid phase conditions: c18Reversed phase chromatographic column (2.1mm × 100mm, 1.8 μm), mobile phase A is 0.1% formic acid solution, mobile phase B is acetonitrile, flow rate is 0.3 ml/min; gradient elution: 0-25 min, 98% → 80% mobile phase a, 2% → 20% mobile phase B. Mass spectrum conditions: electrospray positive ion mode (ESI)+) SRM detection was selected, and m/z676.5 → 604.4, 519.1 was selected as the detection ion pair.
The results are shown in FIG. 4, corresponding ion peaks are detected at 7.5min for duck gelatin and QQ sugar added into duck gelatin, and no QQ sugar added into bovine gelatin is detected, so that the method can specifically detect duck-derived components in gelatin products.
Claims (5)
1. A duck-derived characteristic collagen peptide III is characterized in that the amino acid sequence of the duck-derived characteristic collagen peptide III is Gly-Ser-Hyp-Gly-Pro-Hyp-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Glu-Arg, wherein Hyp is 4-hydroxyproline or 3-hydroxyproline.
2. The duck-derived characteristic collagen type III peptide according to claim 1, wherein the characteristic collagen peptide is finally selected by finding out a characteristic mRNA sequence of a duck which stably exists from mRNA of COL3A1 protein in cattle, pigs, sheep, chickens and ducks, translating the mRNA sequence into a protein sequence, and detecting the protein sequence by using a liquid chromatography-mass spectrometry technology.
3. The use of duck-derived characteristic collagen type III peptide according to claim 1, wherein the duck-derived characteristic collagen type III peptide is used for the source-tracing identification and content determination of duck skin-derived components in collagen hydrolysate and products thereof.
4. The use of duck-derived characteristic collagen type III peptide according to claim 3, comprising the steps of:
(1) performing enzyme digestion on a sample to be detected by using trypsin, or extracting protein components in the sample to be detected and performing enzyme digestion by using trypsin;
(2) putting into a liquid chromatography-mass spectrometer, taking characteristic collagen peptide or duck gelatin subjected to trypsin enzyme digestion as a reference substance, and selecting parent ion m/z676.5 and daughter ions thereof for monitoring; if the retention time of the ions is consistent with that of a reference substance and the daughter ions of the ions are consistent with that of the reference substance, the collagen hydrolysate or the product thereof contains duck skin-derived components, and the content of the duck skin-derived components can be calculated by peak areas of the reference substance and a detection sample; if the ions are not retained for the same time as the control, no duck skin derived component is contained.
5. The use of a duck-derived characteristic collagen type III peptide according to claim 3, wherein said collagen hydrolysate is gelatin obtained by hydrolyzing animal skin, bone or scale, and the collagen hydrolysate product is a food, health product or pharmaceutical product containing gelatin in the raw material.
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