CN104710511B - Iron chelating peptide derived from hairtail fish protein and preparation method and application thereof - Google Patents
Iron chelating peptide derived from hairtail fish protein and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a preparation method of iron chelating peptide derived from hairtail fish meat protein, in particular to a method for preparing the iron chelating peptide derived from hairtail fish meat protein by taking hairtail fish meat as a raw material, carrying out enzymolysis on the hairtail fish meat protein by using papain and trypsin, and separating and purifying by adopting ultrafiltration, macroporous resin column chromatography, gel column chromatography and reversed-phase high performance liquid chromatography to obtain the iron chelating peptide Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met, wherein the molecular weight is 1045.18 Da when ESI/MS is used for determination. The hairtail fish protein iron chelating peptide has high absorption and utilization rate due to the unique chelating and transporting mechanism, can supplement polypeptide/amino acid and iron at the same time, and is an ideal iron supplementing substance.
Description
Technical Field
The invention relates to a hairtail fish meat protein iron chelating peptide and a preparation method thereof, belonging to the technical field of deep processing of aquatic products.
Background
The bioactive peptides refer to peptides with special physiological activity, and are hot spots of current medicine and health care product research. At present, active peptides produced by proteolysis are mainly derived from land protein sources, the living environment of marine organisms is obviously different from the land environment, the amino acid composition or sequence of the proteins of the active peptides are greatly different from that of the land biological proteins, and the species and the quantity of the active peptides are far more than those of the land protein resources. Therefore, by using the enzyme engineering technology developed by the land bioactive peptide, the marine bioprotein resource is taken as a raw material, and through comprehensive research on the preparation process, a series of natural, efficient and novel bioactive peptides which cannot be produced by the land protein source and chemical synthesis can be developed.
Iron is an essential trace element for human beings and is vital to the growth and development of infants and children. Although the macromolecular protein can also be combined with iron ions, the iron-chelating protein has a large molecular weight, is difficult to pass through intestinal mucosa, and is not beneficial to the absorption of iron ions. The existing research shows that the iron chelating peptide can greatly improve the absorption and utilization rate of iron ions, and meanwhile, the polypeptide also has multiple remarkable biological activities and shows double effects.
Based on the research status, the invention provides the iron chelating peptide derived from the hairtail fish protein and the preparation method of the iron chelating peptide.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide an iron chelating peptide derived from hairtail fish meat protein in view of the above technical status.
The second technical problem to be solved by the invention is to provide a preparation method of iron chelating peptide derived from hairtail fish protein.
The technical scheme adopted by the invention for solving the first technical problem is as follows: an iron chelating peptide derived from fish protein of hairtail, which is characterized in that the amino acid sequence of the polypeptide is Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met (APPENGMAMM), and the molecular weight of ESI/MS is 1045.18 Da.
The technical solution adopted by the present invention to solve the second technical problem is: a preparation method of iron chelating peptide derived from hairtail fish protein is characterized by comprising the following steps:
1) pre-treating hairtail flesh: putting the cleaned and boneless hairtail flesh with bones and skins removed into phosphate buffer solution (0.02 mol/L, pH 6.5) according to the solid-to-liquid ratio of 1g:2 mL-3 mL, processing the mixture into homogenate by using a high-speed tissue triturator, then putting the homogenate into ether according to the volume ratio of 1: 3-5 mL, degreasing for 4-6 h, centrifuging at 4 ℃, 5000-6000 rpm for 15-20 min, removing ether, and freeze-drying the precipitate to obtain the degreased hairtail flesh.
) Enzymolysis of hairtail fish protein, namely adding a phosphate buffer solution (0.02 mol/L, pH 6.5) into defatted hairtail fish according to the feed-liquid ratio of 1g to 20-25 mL, and adding papain (the enzyme activity is more than or equal to 2.0 × 10) into the solution according to the mass of 1.5-2.5% of the defatted hairtail fish4U/g), performing enzymolysis at the temperature of 55-65 ℃ for 4-6 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 NaOH, and adding trypsin (the enzyme activity is more than or equal to 2.5 × 10) into the solution according to the mass of 1.5-2.0% of the protein4U/g) and carrying out enzymolysis for 3-4 h at the temperature of 35-45 ℃ to obtain an enzymolysis product;
3) preparing hairtail fish meat protein iron chelating peptide: carrying out ultrafiltration treatment on the prepared hairtail fish flesh protease hydrolysate 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 10-15 times of AB-8 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 the ethanol, freezing and drying to obtain a polypeptide mixture, and purifying the polypeptide mixture by using gel column chromatography and reverse phase high performance liquid chromatography (RP-HPLC) in sequence to obtain the hairtail fish flesh protein iron chelating peptide.
Preferably, the hairtail in the step 1) is Japanese hairtail (A)Trichiurus japonicus）。
Preferably, the specific processes of gel column chromatography and RP-HPLC purification in the step 4) are as follows:
gel column chromatography: dissolving the polypeptide mixture in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, performing column chromatography separation by sephadex LH-20, eluting by using double distilled water, and collecting an elution component according to an absorbance curve under 220 nm, wherein a peak with the highest iron chelating activity is gel chromatography zymolyte;
RP-HPLC purification: preparing the gel chromatography zymolyte into a solution of 80-100 mu g/mL by using double distilled water, purifying by using RP-HPLC, and obtaining 1 high-iron chelating active peptide Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met (APPENGMAQM) according to the iron chelating activity.
More preferably, the RP-HPLC conditions are: the sample injection amount is 10-15 mu L; chromatographic column Kromasil C18(250 mm × 4.6.6 mm, 5 μm), mobile phase 20% acetonitrile, elution speed 0.8-1.0 mL/min, ultraviolet detection wavelength 220 nm.
The invention is based on the theoretical basis of polypeptide and metal ion chelation and the unique effect of polypeptide-iron chelate (polypeptide/amino acid and iron can be supplemented simultaneously), takes hairtail fish as raw material, and prepares the polypeptide with high iron chelation activity by controlling the enzymolysis conditions of papain and trypsin. The invention provides a technical support for the development of iron-supplementing health-care food and medicines.
Drawings
FIG. 1 is a Sephadex LH-20 chromatogram of the invention.
FIG. 2 is an RP-HPLC analysis chart of the substrate prepared from Sephadex LH-20 of the present invention.
FIG. 3 is a mass spectrum of Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met (APPENGMAQM) of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
A preparation method of iron chelating peptide derived from hairtail fish protein comprises the following steps: the ' degreasing ' enzymolysis ' zymolyte of the hairtail fish meat is ' ultrafiltered ' AB-8 macroporous resin purified ' gel filtration chromatography ' RP-HPLC to prepare ' iron chelating peptide '.
Example (b): 1) pre-treating hairtail flesh: washing Japanese hairtail (bone and skin removed) ((Trichiurus japonicus) And (3) putting the fish meat into a buffer solution according to the solid-to-liquid ratio of 1g to 3mL, processing the fish meat into homogenate by using a high-speed tissue triturator, degreasing the fish meat in diethyl ether for 5 hours according to the volume ratio of 1 to 5mL, centrifuging the mixture at 4 ℃ and 6000 rpm for 15min to remove the diethyl ether, and freeze-drying the precipitate to obtain the degreased hairtail fish meat.
2) Enzymolysis of hairtail fish protein: adding the mixture into defatted hairtail meat according to the feed-liquid ratio of 1g to 25 mLAdding phosphate buffer solution (0.02 mol/L, pH 6.5), adding papain (enzyme activity is greater than or equal to 2.0 × 10) to obtain solution with a mass of 2.5% of the fish meat of defatted hairtail4U/g), performing enzymolysis at 60 deg.C for 6 hr, heating the solution to 95 deg.C, maintaining the temperature for 10 min, cooling to 37 deg.C, adjusting pH to 8.0 with NaOH, adding trypsin (enzyme activity is 2.5 × 10 or more) into the solution according to 2.0% of protein mass4U/g) and carrying out enzymolysis for 4 hours at the temperature of 37 ℃ to obtain an enzymolysis product;
3) preparing hairtail fish meat protein iron chelating peptide: carrying out ultrafiltration treatment on the prepared hairtail fish flesh protease hydrolysate 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 AB-8 macroporous resin according to the volume ratio, washing with 5 times of column volume water to remove impurities, eluting with 95% ethanol with 6 times of column volume, removing the ethanol by rotary evaporation at a low pressure below 50 ℃, freezing and drying to obtain a polypeptide mixture, purifying the polypeptide mixture by gel column chromatography and reversed phase high performance liquid chromatography (RP-HPLC) in sequence to obtain hairtail fish flesh protein iron chelating peptide, and determining the structure by using an amino acid sequence analyzer and a mass spectrum, wherein the specific process comprises the following steps:
gel column chromatography: dissolving the polypeptide mixture in double distilled water to prepare a solution with the concentration of 25-30 mg/mL, performing column chromatography separation by sephadex LH-20, eluting by the double distilled water, and collecting an elution component according to an absorbance curve at 220 nm, wherein a peak with the highest iron chelating activity is gel chromatography zymolyte (F2) (figure 1);
② RP-HPLC purification, preparing the gel chromatography zymolyte into a solution of 80-100 μ g/mL with double distilled water, and purifying by RP-HPLC (sample size of 10-15 μ L; chromatographic column Kromasil C18(250 mm × 4.6.6 mm, 5 μm), mobile phase 20% acetonitrile, elution speed 0.8-1.0 mL/min, ultraviolet detection wavelength 220 nm), and obtaining 1 high-iron chelating active peptide according to the chelating activity to iron (figure 2).
Structure detection: the iron chelating peptide is detected as a single peak, the amino acid sequence determined by a protein/polypeptide sequence analyzer is Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met (APPENGMAMM), and the molecular weight determined by ESI/MS is 1046.17 Da.
And (3) determining the chelation of the hairtail fish protein source chelating peptide on iron ions by adopting a phenanthroline colorimetric method. The sample of 0.05g is placed in a 100 mL beaker, 2 mL of concentrated hydrochloric acid is added, and after the sample is completely dissolved, the volume is determined to be 100 mL of volumetric flask by distilled water. Accurately sucking 5mL of sample liquid into a 50 mL volumetric flask, adding 1mL of 1 mol/L HCl solution, 1mL of 10% hydroxylamine hydrochloride and 1mL of 0.12% phenanthroline, then adding 5mL of 10% sodium acetate, diluting with water to a scale, and shaking up. And (3) taking a reagent blank solution without iron as a reference solution, measuring the absorbance at the wavelength of 510 nm, and substituting the value into a standard curve to calculate the iron binding capacity.
And (3) preparing a standard curve: sucking 10 mu g/mL of iron standard solution 0, 2.0, 4.0, 6.0, 8.0 and 10.0 mL, respectively placing the solution in a 50 mL volumetric flask, adding L mol/L of HCl solution 1mL, 10% hydroxylamine hydrochloride L mL and 0.12% o-phenanthroline 1mL, then adding 10% sodium acetate 5mL, diluting with water to the scale, and shaking up. Taking a reagent blank solution without iron as a reference solution, measuring the absorbance at the wavelength of 510 nm, drawing a standard curve, and obtaining a standard curve formula as follows: y = 0.1873x +0.011, R2=0.9998。
The measurement result shows that: the iron chelating peptide Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met (APPENGMAMM) obtained by purification has the iron ion chelating capacity of 74.69 mu g/mg, and compared with a proteolysis product (33.52 mu g/mg), the iron chelating capacity is greatly improved.
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> an iron chelating peptide derived from fish protein of hairtail, preparation method and application thereof
<130>zjou-wb201503
<160>1
<170>PatentIn version 3.5
<210>1
<211>10
<212>PRT
<213> Artificial Synthesis
<400>1
Ala Pro Pro Glu Asn Gly Met Ala Gln Met
1 5 10
Claims (1)
1. The amino acid sequence of the iron chelating peptide is Ala-Pro-Pro-Glu-Asn-Gly-Met-Ala-Gln-Met, and the molecular weight is 1045.18 Da when ESI/MS is used for preparing iron supplement medicines and health care products.
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| CN105601706B (en) * | 2015-12-11 | 2020-11-27 | 浙江海洋学院 | Antibacterial hairtail tetrapeptide and preparation method thereof |
| CN105420323A (en) * | 2015-12-23 | 2016-03-23 | 吉林大学 | Phytochelatin iron supplementation powder containing iron and preparation method of phytochelatin iron supplementation powder |
| CN106579064B (en) * | 2016-10-21 | 2020-10-30 | 广东省农业科学院蚕业与农产品加工研究所 | Fish meat snack food capable of enhancing iron absorption and preparation method thereof |
| CN107058437B (en) * | 2017-05-05 | 2021-02-05 | 浙江海洋大学 | Hairtail minced fillet protein powder for pizza embryo and preparation method thereof |
| CN107245508A (en) * | 2017-06-21 | 2017-10-13 | 兰溪市沉默生物科技有限公司 | The hairtail activity extract repaired for Bones and joints |
| CN108047325A (en) * | 2017-10-26 | 2018-05-18 | 浙江海洋大学 | A kind of reducing blood lipid pentapeptide for coming from the brown croaker flesh of fish and its application |
| CN107840869A (en) * | 2017-11-08 | 2018-03-27 | 金华市飞凌生物科技有限公司 | Alleviate the preparation method of the active peptides of knee osteoarthritis |
| CN108030101A (en) * | 2017-11-08 | 2018-05-15 | 金华市飞凌生物科技有限公司 | The method of abstraction function small peptide from low value ocean fish |
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| CN107936113B (en) * | 2017-12-12 | 2020-11-10 | 浙江海洋大学 | Mullet scale iron chelating peptide and preparation method thereof |
| CN114262728A (en) * | 2021-12-20 | 2022-04-01 | 海南三元星生物科技股份有限公司 | Preparation method of hairtail protein peptide chelated ferrous iron |
| CN114317656B (en) * | 2021-12-29 | 2024-03-08 | 宁波盈前科技有限公司 | Bioactive hairtail peptide microelement chelate and preparation method thereof |
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| CN103388014A (en) * | 2012-05-11 | 2013-11-13 | 浙江海洋学院 | Preparation method for ferrous chelated antibacterial peptide |
| CN103467568A (en) * | 2013-05-23 | 2013-12-25 | 浙江海洋学院 | Sepia esculenta protein antioxidant peptide, and preparation method and use thereof |
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| CN103388014A (en) * | 2012-05-11 | 2013-11-13 | 浙江海洋学院 | Preparation method for ferrous chelated antibacterial peptide |
| CN103467568A (en) * | 2013-05-23 | 2013-12-25 | 浙江海洋学院 | Sepia esculenta protein antioxidant peptide, and preparation method and use thereof |
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