CN104710525B - Tuna bone collagen source zinc chelated collagen peptide and preparation method and application thereof - Google Patents
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- WGIWBXUNRXCYRA-UHFFFAOYSA-H trizinc;2-hydroxypropane-1,2,3-tricarboxylate Chemical compound [Zn+2].[Zn+2].[Zn+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O WGIWBXUNRXCYRA-UHFFFAOYSA-H 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a preparation method of zinc chelated collagen peptide of tuna fishbone collagen, which specifically comprises the steps of taking tuna fishbone as a raw material, extracting collagen by an acid method, carrying out enzymolysis on the collagen by pepsin and trypsin, separating and purifying by ultrafiltration, macroporous resin column chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain the zinc chelated collagen peptide Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTGWPG), detecting the molecular weight of ESI/MS (electronic signature/mass spectrometry) 701.73 Da.
Description
Technical Field
The invention relates to tuna fish bone collagen source zinc chelated collagen peptide and a preparation method and application thereof, belonging to the technical field of deep processing of aquatic products.
Background
Collagen peptide is favored by more and more consumers due to the unique effect of the collagen peptide in the application of medicines, health products, foods, cosmetics and the like, so that the collagen peptide becomes a focus of competitive attention in the food industry, the health product industry and the daily chemical industry. Zinc is a trace element essential to human body, and the deficiency of zinc can cause the symptoms of poor growth and development of children, lagged intelligence development, reduced immunity and the like. The traditional zinc supplement (such as zinc gluconate and zinc citrate) has poor stability, low absorption and utilization rate and large intestine and stomach stimulation, and is not easy to be taken for a long time. The chelated salt formed by the zinc and the metal chelated peptide can promote the absorption of trace elements and meet the requirements of organisms on active peptides, amino acids and the like, thereby gaining wide attention of scholars at home and abroad.
Tuna is an important fish in ocean fishery, and the annual capture amount exceeds 600 million tons. In the processing process of tuna, most of fishbones accounting for 25-30% of the total weight are processed into fishbone powder or are discarded as waste, so that precious fishery resources are wasted. Through retrieval, no report is found on the preparation of the zinc chelated collagen peptide by using the tuna bone collagen. Based on the research status, the invention provides the collagen peptide with metal zinc chelating activity and the preparation method thereof according to the research status of the zinc chelating collagen peptide and the tuna bone.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide a zinc chelated collagen peptide derived from tuna bone collagen in view of the above technical status quo.
The second technical problem to be solved by the invention is to provide a preparation method of zinc chelated collagen peptide derived from tuna bone collagen.
The invention adopts the technical scheme that the zinc chelated collagen peptide derived from tuna fish bone collagen is characterized in that the amino acid sequence of the collagen peptide is Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTPG), and the molecular weight of GWESI/MS is 701.73 Da.
The technical solution adopted by the present invention to solve the second technical problem is: a preparation method of zinc chelated collagen peptide derived from tuna bone collagen is characterized by comprising the following steps:
1) pretreating tuna bones, namely adding 0.1 mol/L NaOH solution into the tuna bones according to the feed-liquid ratio of 1g: 10-15 m L, soaking at 4 ℃ for 3-5 hours to remove non-collagen, repeatedly washing the treated tuna bones with distilled water until the pH value is 6.5-7.0, draining, adding 0.5 mol/L EDTA solution (pH value is 7.4) according to the feed-liquid ratio of 1g: 5-10 m L, soaking at 4 ℃ for 3-5 days, replacing the EDTA solution 1 time every day, removing calcium in the tuna bones, drying and crushing to obtain the decalcified tuna bones.
2) The preparation method of the tuna fishbone collagen comprises the steps of adding 0.5 mol/L of acetic acid solution into decalcified fishbone powder according to the feed-liquid ratio of 1g: 3-5 m L, soaking for 2-3 days at 4 ℃, centrifuging for 30 min at 12000 g, taking supernatant, adding NaCl until the final concentration of the solution is 0.8-1.0 mol/L, standing for 30-60 min, centrifuging for 25-30 min at 15000 g to obtain precipitate, and freeze-drying to obtain the tuna fishbone collagen.
) Enzymolysis of tuna bone collagen, namely adding ultrapure water into the collagen according to the feed-liquid ratio of 1g to 15-20 m L, adjusting the pH of the solution to 1.5-2.5 by using HCl, and adding pepsin (the enzyme activity is more than or equal to 1.6 × 10) into the collagen solution according to the mass of 1.5-2.0% of the collagen5U/g), performing enzymolysis for 3-5 h, heating the solution to 90-95 ℃, keeping the temperature for 10-15 min, cooling to room temperature, adjusting the pH of the solution to 7.5-8.5 by using NaOH, and adding trypsin (the enzyme activity is more than or equal to 2.5 × 10) into the solution according to 1.2-1.5% of the mass of the collagen4U/g) and carrying out enzymolysis for 3-4 h at the temperature of 35-45 ℃ to obtain an enzymolysis product;
4) the preparation method of the tuna bone zinc chelate collagen peptide comprises the steps of carrying out ultrafiltration treatment on a prepared collagen enzymolysis product by using a 3 kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3 kDa to obtain an ultrafiltration enzymolysis liquid, adding the ultrafiltration enzymolysis liquid into a chromatographic column filled with 10-15 times of D101 macroporous resin according to the volume ratio, washing with 3-5 times of column volume to remove impurities, eluting with 5-8 times of column volume of 95% ethanol, carrying out low-pressure rotary evaporation on ethanol eluent at the temperature of below 50 ℃ to remove ethanol, carrying out freeze drying to obtain a collagen peptide mixture, and purifying the collagen peptide mixture by using gel column chromatography and reverse phase high performance liquid chromatography (RP-HP L C) in sequence to obtain the tuna bone zinc chelate collagen peptide.
Preferably, the specific processes of gel column chromatography and RP-HP L C purification in the step 4) are as follows:
performing gel column chromatography, namely dissolving the collagen peptide mixture in double distilled water to prepare a solution with the concentration of 10-20 mg/m L, performing column chromatography separation on the solution through sephadex G-25, eluting the solution by using the double distilled water, and collecting an elution component according to an absorbance curve under 214 nm, wherein the peak with the highest Zn chelating activity is a gel chromatography zymolyte;
and (3) RP-HP L C purification, namely preparing the gel chromatography zymolyte into a solution of 45-55 mu g/m L by using double distilled water, purifying by using RP-HP L C, and obtaining 1 collagen peptide with high Zn chelating activity, namely Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTGGWGG), according to the chelating activity to Zn.
Preferably, the RP-HP L C has the conditions of sample injection amount of 15-20 mu L, chromatographic column Zorbax C18, mobile phase of 30% acetonitrile, elution speed of 0.8-1.0 m L/min and ultraviolet detection wavelength of 214 nm.
The collagen peptide with high Zn chelation activity is prepared by taking tuna bones as raw materials and controlling the enzymolysis conditions of pepsin and trypsin based on the theoretical basis of polypeptide and metal ion chelation and the unique effect of a polypeptide-Zn chelate (polypeptide/amino acid and Zn can be supplemented simultaneously). The invention provides a technical support for the development of zinc-supplementing health-care food and medicines, and also provides a new idea for the high-valued utilization of tuna bones.
Drawings
FIG. 1 is a Sephadex G-25 chromatogram of the invention.
FIG. 2 is an analysis chart of RP-HP L C of the substrate prepared from Sephadex G-25 of the present invention.
FIG. 3 shows the mass spectrum of Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTGWPG) according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
A process for preparing the zinc chelated collagen peptide from the collagen of tuna bone includes such steps as extracting the collagen from tuna bone, enzymolyzing, ultrafiltering, purifying by macroreticular resin, gel filtering, and chromatography (RP-HP L C).
Example (b):
1) pretreating tuna bones, namely adding 0.1 mol/L NaOH solution into tuna (bonito) bones according to a feed-liquid ratio of 1g to 15m L, soaking at 4 ℃ for 5 hours to remove non-collagen, repeatedly washing the treated bones with distilled water until the pH value is 7.0, draining, adding 0.5 mol/L EDTA solution (pH value is 7.4) according to a feed-liquid ratio of 1g to 5m L, soaking at 4 ℃ for 3 days, replacing the EDTA solution 1 time per day, removing calcium in the bones, drying and crushing to obtain the decalcified bone meal.
2) The preparation method of tuna fishbone collagen comprises the steps of adding 0.5 mol/L acetic acid solution into decalcified fishbone powder according to the feed-liquid ratio of 1g to 3 m L, soaking for 3 d at 4 ℃, centrifuging for 30 min at 12000 g, taking supernatant, adding NaCl until the final concentration of the solution is 0.9 mol/L, standing for 60 min, centrifuging for 30 min at 15000 g to obtain precipitate, and freeze-drying to obtain the fishbone collagen.
) Enzymolysis of tuna bone collagen, namely adding ultrapure water into collagen according to a feed-liquid ratio of 1g to 15m L, adjusting the pH of the solution to 2.0 by using HCl, and adding pepsin (the enzyme activity is more than or equal to 1.6 × 10) into the collagen solution according to 1.5 percent of the mass of the collagen5U/g), performing enzymolysis for 3-5 h, heating the solution to 90-95 ℃, keeping the temperature for 10 min, cooling to room temperature, adjusting the pH of the solution to 8.0 by using NaOH, adding trypsin (the enzyme activity is more than or equal to 2.5 × 10) into the solution according to 1.5 percent of the mass of the collagen4U/g) and carrying out enzymolysis for 4 hours at the temperature of 37 ℃ to obtain an enzymolysis product;
4) the preparation method of the tuna bone zinc chelate collagen peptide comprises the following steps of carrying out ultrafiltration treatment on a prepared collagen enzymolysis product by using a 3 kDa ultrafiltration membrane, collecting a part with the molecular weight less than 3 kDa to obtain ultrafiltration enzymolysis liquid, adding the ultrafiltration enzymolysis liquid into a chromatographic column filled with 15 times of D101 macroporous resin according to the volume ratio, washing by using 3-5 times of column volume to remove impurities, eluting by using 95% ethanol with 6 times of column volume, carrying out low-pressure rotary evaporation on ethanol eluent at the temperature of below 50 ℃ to remove ethanol, carrying out freeze drying to obtain a collagen peptide mixture, and purifying the collagen peptide mixture by using gel column chromatography and reversed-phase high-performance liquid chromatography (RP-HP L C) in sequence to obtain the tuna bone zinc chelate collagen peptide, wherein the structure of the tuna bone zinc chelate collagen peptide is determined by using amino acid sequence analysis and mass spectrum, and the specific process:
① performing gel column chromatography, dissolving the collagen peptide mixture in double distilled water to obtain solution with concentration of 10-20 mg/m L, performing Sephadex G-25 column chromatography separation, eluting with double distilled water, and collecting eluate according to absorbance curve at 214 nm, wherein the peak with highest Zn chelating activity is gel chromatography zymolyte (F3) (FIG. 1);
② RP-HP L C purification, namely preparing the gel chromatography zymolyte into a 45-55 mu g/m L solution by using double distilled water, and purifying by using RP-HP L C (the conditions of the RP-HP L C are that the sample amount is 15-20 mu L, a chromatographic column is Zorbax C18, a mobile phase is 30% acetonitrile, the elution speed is 0.8 m L/min, the ultraviolet detection wavelength is 214 nm), so that 1 collagen peptide with high Zn chelating activity is obtained according to the chelating activity to Zn (figure 2).
③ structure detection, Zn chelated collagen peptide is detected as single peak, amino acid sequence determined by protein/polypeptide sequence analyzer is Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTGWPG), ESI/MS detection molecular weight is 701.73 Da (figure 3).
The chelating effect of Zn-chelated collagen peptide on zinc ions is determined by EDTA titration method, 100 mg of chelate is taken to be put into a small beaker with the thickness of 100 m L, 50 m L of water is added, a plurality of drops of HCl (6 mol/L) are dropped, the mixture is shaken up and heated on water bath to be completely dissolved, the volume is determined to be l00 m L after the mixture is cooled, l0m L is sucked from the mixture to be put into a triangular flask, 3 parts are paralleled, and NH with the pH value of 10 is added3-NH4Cl buffer l0m L, chrome black T indicator in appropriate amount, then 0.01 mol/L Na2EDTA droplets were fixed to blue; the amount of EDTA consumed in milliliters was recorded and the zinc content of the chelate calculated.
The determination result shows that the Zn-chelated collagen peptide Gly-L ys-Thr-Gly-Trp-Pro-Gly (GKTGWPG) obtained by purification has the chelating capacity of 83.56 mu g/mg for zinc ions, and the chelating capacity of Zn is greatly improved compared with that of a collagen enzymolysis product (30.12 mu g/mg).
Finally, it should be noted that the above-mentioned list is only one specific embodiment of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
SEQUENCE LISTING
<110> Zhejiang ocean academy
<120> tuna fish bone collagen source zinc chelating collagen peptide, and preparation method and application thereof
<130>zjou-wb-201507
<160>1
<170>PatentIn version 3.5
<210>1
<211>7
<212>PRT
<213> Artificial Synthesis
<400>1
Gly Lys Thr Gly Trp Pro Gly
1 5
Claims (1)
1. The zinc chelated collagen peptide derived from tuna fish bone collagen is characterized in that the amino acid sequence of the zinc chelated collagen peptide is Gly-L ys-Thr-Gly-Trp-Pro-Gly, and the molecular weight is 701.73 Da through ESI/MS detection.
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| CN105112481A (en) * | 2015-07-30 | 2015-12-02 | 上海市水产研究所 | Method for extracting undenatured type II collagen from squid cartilage |
| CN105087728B (en) * | 2015-08-14 | 2018-09-14 | 浙江省海洋开发研究院 | A kind of preparation method of tuna fish bone collagen protein sources selenium chelating collagen peptide |
| CN105087729B (en) * | 2015-08-14 | 2019-02-12 | 浙江省海洋开发研究院 | A kind of preparation method of tuna fish bone collagen protein peptides |
| CN107698780B (en) * | 2017-10-19 | 2021-07-02 | 中国水产科学研究院黄海水产研究所 | Preparation method and application of collagen peptide chelated ferrous hydrogel |
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| CN108935913A (en) * | 2018-06-27 | 2018-12-07 | 华中农业大学 | Sequestering pig bone collagen peptide of calcium and preparation method thereof |
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| CN114703245A (en) * | 2022-04-14 | 2022-07-05 | 甘肃中肽生物科技有限公司 | Preparation process of bovine bone protein peptide chelated zinc |
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