CN117024573B

CN117024573B - Collagen peptide with iron chelating activity and preparation method and application thereof

Info

Publication number: CN117024573B
Application number: CN202311008403.6A
Authority: CN
Inventors: 胡杨; 乐薇; 熊喆; 尹晓清; 熊善柏
Original assignee: Hubei Reborn Biotech Co ltd; Huazhong Agricultural University
Current assignee: Hubei Reborn Biotech Co ltd; Huazhong Agricultural University
Priority date: 2023-08-10
Filing date: 2023-08-10
Publication date: 2024-08-20
Anticipated expiration: 2043-08-10
Also published as: CN117024573A

Abstract

The invention belongs to the technical field of deep processing of aquatic products, and particularly relates to a collagen peptide with iron chelating activity, and a preparation method and application thereof. The amino acid sequence of the collagen peptide is shown as SEQ ID NO. 1. The collagen peptide prepared by using the silver carp scales as a raw material has higher iron chelating activity and good capability of promoting iron absorption and transportation, can effectively improve the absorption rate of iron in human intestinal tracts, improves the bioavailability of iron, and is expected to be applied to iron supplements in the fields of foods, medicines and health care products.

Description

Collagen peptide with iron chelating activity and preparation method and application thereof

Technical Field

The invention relates to the technical field of deep processing of aquatic products, in particular to a collagen peptide with iron chelating activity, and a preparation method and application thereof.

Background

Iron is one of the microelements necessary for the human body, is widely distributed in the human body, has important biological activity, and participates in various important biological processes of the human body, such as heme formation, anemia prevention, cytochrome synthesis, tissue respiration regulation and energy metabolism regulation; maintain the immunity and anti-infective ability of the organism. Iron deficiency occurs due to insufficient daily dietary iron intake and low bioavailability of the ingested iron, and iron deficiency in humans is prone to anemia, a phenomenon that is common worldwide. Statistics show that about 30% of the world's people suffer from anemia, about half of which is due to iron deficiency. Therefore, reasonable iron supplement is important.

In order to solve the iron deficiency problem, different kinds of iron supplements are developed in the market, but common iron supplements such as ferrous sulfate, ferrous gluconate, ferrous glycinate and the like have the defects of unstable property, low absorption and utilization rate, large side effect, easy stimulation of gastrointestinal tract, relatively high preparation cost and the like. The research shows that the protein peptide with the iron chelating activity is obtained by enzymolysis of protein, and can be combined with ferrous ions to form peptide iron chelate, the peptide iron chelate can increase the solubility of the iron ions in human intestinal tracts, and increase the absorption rate of the iron ions, and compared with the traditional iron supplement, the peptide iron chelate has the advantages of good bioavailability, strong absorption capacity, good stability, high safety and the like in human bodies, so that the peptide iron chelate has great development potential as a novel iron supplement.

At present, a large amount of leftovers such as fish scales, fish skins, fish fillets and the like are generated in the processing process of the silver carp, and the leftovers contain a large amount of proteins, so that the direct discarding of the leftovers not only can cause resource waste, but also can cause environmental pollution, therefore, comprehensive research and utilization of the silver carp leftovers are more and more paid attention to the past years, and related reports on the chelation of aquatic polypeptides such as sea cucumber polypeptides, antarctic krill polypeptides, tilapia skin collagen polypeptides and the like and ferrous iron are currently available, but the research on the chelation of fish scale active polypeptides of the silver carp and ferrous iron is lacked, and the silver carp fish scale is a protein with certain biological activity and is a high-quality raw material for preparing the active polypeptides, but is not utilized effectively, and is to be developed and explored.

The present application has been made for the above reasons.

Disclosure of Invention

Aiming at the problems that the active polypeptide of the fish scales of the silver carp is not effectively utilized and the iron chelating polypeptide of the fish scales of the silver carp is lacked in the prior art, the invention provides a collagen peptide with the iron chelating activity, a preparation method of the collagen peptide with the iron chelating activity and application of the collagen peptide in preparation of an iron supplementing agent, and aims to solve a part of problems in the prior art or at least relieve a part of problems in the prior art.

In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:

The first aspect of the invention provides an iron-chelating collagen peptide for promoting iron absorption, which comprises DY peptide and/or LR peptide, wherein the amino acid sequence of the DY peptide is shown as SEQ ID NO.1, and the amino acid sequence of the LR peptide is shown as SEQ ID NO. 9.

Further, the iron-chelating collagen peptide includes at least the DY peptide.

In a second aspect the invention provides the use of an iron-sequestered collagen peptide for promoting iron absorption as described above in an iron supplement.

Further, the iron supplement is a food, a medicine and/or a health care product with an iron supplementing function.

Further, the application includes: mixing the iron chelate collagen peptide with an iron ion solution to prepare a peptide iron chelate, and applying the peptide iron chelate to an iron supplement.

Still further, the ferric ion solution is selected from at least one of a ferrous chloride solution, a ferrous sulfate solution, and a ferrous nitrate solution; the molar ratio of the DY peptide to the iron ions in the iron ion solution is 1:0-2, the molar ratio of LR peptide to iron ions in the iron ion solution is 1:0-1.

The third aspect of the present invention provides a method for preparing an iron-chelating collagen peptide for promoting iron absorption, comprising the steps of:

s1, smashing degreased and decalcified silver carp scales to obtain silver carp scale powder;

s2, adding alkaline protease into the silver carp fish scale powder, performing enzymolysis treatment, inactivating enzyme at high temperature after enzymolysis is completed, centrifuging to obtain supernatant, ultrafiltering, collecting components with molecular weight less than 3kDa, concentrating and drying to obtain silver carp fish scale zymolyte;

s3, analyzing polypeptide components in the chub fish scale zymolyte by adopting a liquid chromatography tandem mass spectrometry technology;

S4, constructing a molecular structure model and simulating molecular dynamics of the polypeptide component analyzed in the step S3, and screening to obtain the iron-chelating collagen peptide by combining a root mean square error value, the interaction energy between carboxyl oxygen and ferrous iron and the dynamic distance analysis between iron atoms;

The iron chelating collagen peptide comprises DY peptide and/or LR peptide, wherein the amino acid sequence of the DY peptide is shown as SEQ ID NO.1, and the amino acid sequence of the LR peptide is shown as SEQ ID NO. 9.

Further, in the step S2, the addition amount of the alkaline protease is 400-500U/g, and the enzymolysis time is 4-6h.

Further, in step S4, a molecular structure model is built by Alphofold on-line software, then molecular dynamics simulation is performed by GROMACS2021.4 software, amber force field is selected, water molecules adopt SPC model, ferrous ions and chloride ions are added to a cubic water box to balance charges and perform energy minimization treatment, and root mean square error value, interaction energy between carboxyl oxygen and ferrous and dynamic distance between iron atoms and oxygen atoms are obtained.

Further, the interaction energy between the carboxyl oxygen and ferrous iron includes Lennard-Jones short range energy and Coul-SR short range energy.

The invention has the advantages and positive effects that:

The invention takes the silver carp scales as raw materials, and the prepared DY peptide and LR peptide have higher iron chelating activity and good capability of promoting iron absorption and transportation, can effectively improve the absorption rate of iron in human intestinal tracts, improve the bioavailability of iron, and are expected to be applied to iron supplements in the fields of foods, medicines and health care products.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a total ion flow chromatogram and a first-order mass spectrum of the silver carp fish scale collagen peptide with the molecular weight of less than 3kDa in the embodiment 1;

FIG. 2 shows root mean square error values of 17 silver carp fish scale collagen peptides according to example 1 in an iron ion environment;

FIG. 3 shows the dynamic distances between oxygen atoms and iron ions of 17 kinds of silver carp scale collagen peptides in the molecular dynamics simulation process according to the embodiment 1 of the present invention;

FIG. 4 is a mass spectrum identification chart of the iron chelating collagen peptide DY peptide and the LR peptide of example 1 of the present invention;

FIG. 5 is a liquid chromatogram of the iron-chelating collagen peptide DY peptide and LR peptide of example 1 of the present invention;

FIG. 6 is a graph showing the energy dispersion spectra before and after iron ion chelation of the iron chelating collagen peptide DY peptide and the LR peptide of example 2 of the present invention; the graphs A-D are the energy spectrum test results of DY, DY-Fe, LR and LR-Fe in sequence, the left graph is the range selected on the surface of the sample structure during point scanning, and the right graph is the distribution condition of main elements of the sample obtained through the energy spectrum test;

FIG. 7 is an X-ray diffraction chart before and after iron ion chelation of the iron chelating collagen peptide DY peptide and the LR peptide of example 2 of the present invention;

FIG. 8 is an infrared spectrum of the iron-chelating collagen peptide DY peptide and the LR peptide of example 2 before and after chelating iron ions; wherein, figure A is DY peptide and figure B is LR peptide;

FIG. 9 is a graph of the original isothermal titration of iron ions into DY peptide and LR peptide solutions for thermal analysis in accordance with example 2 of the present invention;

FIG. 10 is a graph showing the effect of varying concentrations of a mixture of ferrous chloride and peptide iron on cell viability in example 3 of the present invention; wherein, figure A is ferrous chloride and figure B is a peptide iron mixture;

FIG. 11 is a schematic diagram showing the structure of a monolayer cell model according to example 3 of the present invention;

FIG. 12 shows the effect of varying concentrations of DY peptide and LR peptide on iron transport in monolayer cells according to example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. The examples described herein are intended to illustrate the invention only and are not intended to limit the invention.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages and other values used in the present invention are to be understood as being modified in all instances by the term "about". The term "about" has its ordinary meaning as used to indicate that a value includes the inherent variation of the error of the device or method used to determine the value, or includes values that are close to the value, e.g., within 10% of the value (or range of values). Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained.

The terms "comprising," "including," "having," and the like are intended to be non-limiting, as other steps and other ingredients not affecting the result may be added. The term "and/or" should be taken to refer to a specific disclosure of each of the two specified features or components with or without the other. For example, "a and/or B" will be considered to encompass the following: (i) A, (ii) B, and (iii) A and B.

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

The embodiment of the invention provides an iron-chelating collagen peptide for promoting iron absorption, which comprises DY peptide and/or LR peptide, wherein the amino acid sequence of the DY peptide and/or LR peptide is as follows:

DY peptide: DTSGGYDEY (see SEQ ID NO. 1);

LR peptide: LQGSNEIEIR (see SEQ ID NO. 9).

The invention takes silver carp scales as raw materials to prepare DY peptide and/or LR peptide, the molecular weight of the DY peptide is 1006.45Da, the molecular weight of the LR peptide is 1156.50Da, the DY peptide and the LR peptide have higher iron chelating activity, can be combined with Fe ²⁺ through carboxyl, amino and nitrogen atoms on amido on peptide chains thereof to autonomously generate complexation reaction, form stable peptide iron chelate, avoid oxidation precipitation of Fe ²⁺, The stoichiometric ratio of DY peptide to Fe ²⁺ was 1 as measured by Isothermal Titration Calorimetry (ITC) technique: 2, stoichiometric ratio of lr peptide to Fe ²⁺ is 1:1. Further researching the iron transport capacity of the polypeptide by a Caco-2 intestinal epithelial cell model, under the condition of incubation at 37 ℃ for 2 hours, the iron transport quantity of Fe-DY peptide or LR peptide chelate across monolayer cells is obviously higher than that of FeCl ₂ group, and the Fe ²⁺ transport quantity is increased along with the increase of the concentration of the polypeptide to a certain extent, when the concentration of FeCl ₂ is 30 mug/mL, When the mass concentration of DY peptide is 0.2mg/mL, the transport amount of Fe ²⁺ is 0.6112 mug/well, when the mass concentration of FeCl ₂ is 30 mug/mL, the mass concentration of LR peptide is 0.4mg/mL, the transport amount of Fe ²⁺ is 0.3175 mug/well, the DY peptide and the LR peptide have the capability of well promoting iron absorption and transportation, and can effectively improve the absorption rate of iron in human intestinal tracts and improve the bioavailability of iron. in addition, through cytotoxicity experiments, the DY peptide and the LR peptide provided by the invention are safe and have no toxic or side effect, so that the DY peptide and the LR peptide can be applied to iron supplements in the fields of foods, medicines and health products, provide theoretical basis for developing high-activity silver carp scale iron supplements, have wide application prospect, improve the utilization rate and the added value of silver carp scales, and provide a new approach for high-value utilization of silver carp scales.

Peptide fragments DY and LR both have the ability to promote iron absorption and DY peptide has a superior effect on promoting iron absorption over LR peptide, therefore, in a preferred embodiment, the iron-chelating collagen peptide comprises at least DY peptide.

Yet another embodiment of the present invention provides a use of an iron-chelating collagen peptide for promoting iron absorption in an iron supplement.

The advantages of the iron-absorption-promoting iron-chelating collagen peptide in the iron supplement are the same as those of the iron-absorption-promoting iron-chelating collagen peptide described above with respect to the prior art, and are not described in detail herein.

Optionally, the iron supplement is a food, a drug and/or a health care product with an iron supplementing function.

Optionally, the application includes: mixing the iron chelate collagen peptide with an iron ion solution to prepare an iron-peptide chelate, and applying the iron-peptide chelate to an iron supplement.

It will be appreciated that ferric ion solutions are generally used to provide ferrous ions, and the present invention is not limited to any particular type of salt that provides ferrous ions, and can be used to effectively form ferrous ions. In a specific embodiment, the ferric ion solution may be selected from at least one of a ferrous chloride solution, a ferrous sulfate solution, and a ferrous nitrate solution.

Preferably, the molar ratio of the DY peptide to the iron ions in the iron ion solution is 1, based on the stoichiometric ratio of polypeptide to Fe ²⁺: 0-2, the molar ratio of LR peptide to iron ions in the iron ion solution is 1:0-1.

Another embodiment of the present invention provides a method for preparing an iron-chelating collagen peptide for promoting iron absorption, comprising the steps of:

S3, analyzing polypeptide components in the chub fish scale zymolyte by adopting a liquid chromatography tandem mass spectrometry (LC-MS/MS) technology;

S4, constructing a molecular structure model and simulating molecular dynamics of the polypeptide component analyzed in the step S3, and screening to obtain the iron chelate collagen peptide by combining a root mean square error (RMSD) value, the interaction energy between carboxyl oxygen and ferrous iron and the dynamic distance analysis between an iron atom and an oxygen atom (FE-O).

According to the invention, degreased and decalcified silver carp scales are taken as raw materials, and are subjected to alkaline protease enzymolysis, LC-MS/MS separation and identification and molecular simulation treatment in sequence, so that small molecular polypeptides are obtained through the alkaline protease enzymolysis treatment, then the amino acid composition of the small molecular polypeptides is obtained through the LC-MS/MS separation and identification, and finally peptide fragments with high iron chelating activity are DTSGGYDEY and LQGSNEIEIR respectively through molecular simulation treatment. Based on the preparation method, when collagen peptide extracted from silver carp scales is chelated with iron, the yield of the obtained chelate is up to more than 60%, and the obtained DY peptide and LR peptide have high iron chelating activity and excellent iron transport promoting capability.

Optionally, in the step S2, the addition amount of the alkaline protease is 400-500U/g, and the enzymolysis time is 4-6h.

Optionally, in step S3, the analysis of the polypeptide component adopts the conventional operation in the art, and the obtained chub fish scale zymolyte is detected by liquid chromatography, and then the characteristic ion proton peak of the chub fish scale zymolyte is subjected to secondary mass spectrometry analysis and identification by tandem mass spectrometry, so as to obtain the amino acid composition of the polypeptide.

Optionally, in step S4, a molecular structure model is built by Alphofold on-line software, then molecular dynamics simulation is performed by GROMACS2021.4 software, amber force field is selected, water molecules adopt SPC model, ferrous ions and chloride ions are added to a cubic water box to balance charges and perform energy minimization treatment, and an RMSD value, interaction energy between carboxyl oxygen and ferrous ions and dynamic distance between FE-O are obtained. The interaction energy between the aforementioned carboxyoxy species and ferrous iron includes Lennard-Jones short range energy (LJ-SR) and Coul-SR short range energy (Coul-SR).

The invention will be further illustrated with reference to specific examples. The experimental methods, which do not address specific conditions in the following examples, are generally in accordance with the conditions recommended by the manufacturer. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 preparation of iron-chelating collagen peptides

The iron chelating collagen peptide is prepared by the following steps:

S1, preprocessing fish scales of silver carp: 200g of dried silver carp scales are taken, and the feed liquid ratio is 1:10, adding 0.1mol/L sodium hydroxide solution, stirring at 25 ℃ for 4 hours, and washing with distilled water to be neutral; then according to the feed liquid ratio of 1:10, 0.5mol/L hydrochloric acid solution is added, decalcification is carried out for 1.5 hours at 25 ℃, and distilled water is used for washing to neutrality; finally, according to the feed liquid ratio of 1:10 is added with 0.5mol/L sodium bicarbonate solution, degreased for 8 hours at 25 ℃, washed to be neutral by distilled water, soaked for 1.5 hours in hot water at 85 ℃, and crushed by a grinder to obtain pretreated silver carp fish scale powder.

S2, proteolysis treatment: adding silver carp fish scale powder into distilled water to adjust the substrate concentration, adjusting the initial pH value to 8.0-8.5 by hydrochloric acid or sodium hydroxide, adding 420U/g alkaline protease, uniformly mixing, performing enzymolysis for 4h in a magnetic stirrer, performing enzyme deactivation in a water bath for 15min, centrifuging, taking supernatant, passing through a 3kDa ultrafiltration membrane, collecting filtrate with molecular weight smaller than 3kDa, vacuum concentrating, and spray-drying to obtain silver carp fish scale zymolyte, wherein the spray-drying condition is that the air inlet temperature is 200 ℃, the air outlet temperature is 90 ℃, and the sample injection amount is 200mL/h.

S3, polypeptide component identification: firstly, detecting the obtained chub fish scale zymolyte by adopting liquid chromatography, comprising the following steps: redissolving the silver carp fish scale zymolyte to about 35 mu L by using buffer A (formula: 5% acetonitrile (CAN) +0.1% Formic Acid (FA)), centrifuging for 10min by 20000g, removing insoluble substances, separating by using a nano liter liquid chromatograph (Shimadzu corporation LC-20 AD), wherein the loading amount is 30 mu L, and the separation column comprises a Trap column and an analysis column, wherein the separation procedure is as follows: the sample was first brought onto the Trap column at a flow rate of 8 μl/min for 4min, followed by the sample being brought into the analytical column at an analytical gradient with a total flow rate of 300nL/min, and the polypeptide components were separated and transferred to a tandem mass spectrometry system. The aforementioned analytical gradient was set as follows: eluting with 5% buffer B (formula: 95% CAN+0.1% FA) for 5min, then linearly increasing the ratio of buffer B from 5% to 35% in 35min, further increasing the ratio to 60% in 5min, increasing the ratio of buffer B to 80% in 2min and maintaining the ratio for 2min, and finally recovering the ratio to 5% in 1min and balancing the ratio for 10min under the condition.

The polypeptide separated in liquid phase was ionized by the nanoESI source and then entered into a tandem mass spectrometer Orbitrap Fusion Lumos (Thermo FISHER SCIENTIFIC, san Jose, CA) for DDA (data-DEPENDENT ACQUISITION) mode detection. The main parameters were set as follows: the ion source voltage is 2kV, the primary mass spectrum scanning range is 350-1500m/z, the resolution is 60000, the secondary mass spectrum initial m/z is fixed to 100, and the resolution is 15000; the screening conditions of the secondary fragmentation parent ions are as follows: charge 2+ to 6+, parent ion with peak intensity exceeding 20000 intensity row of front 30, ion fragmentation mode HCD, fragment ion detection in Orbitrap, dynamic exclusion time set to 30s, agc set to: primary 1E5, secondary 2E4.

280 Peptide fragments are totally resolved by a liquid chromatography tandem mass spectrometry (LC-MS/MS) technology, and a total ion flow chromatogram (TIC) (upper diagram) and a primary mass spectrometry Basepeak (lower diagram) of the obtained silver carp scale collagen peptide are shown in figure 1. Of 280 peptide fragments, gly-X-Y (X and Y are usually proline and hydroxyproline) peptide sequences are found more, and are considered as characteristic sequences of Collagen, while 224 peptide fragments of precursor protein are derived from type I Collagen (Collagen type I) alpha chain, and the identified sample is silver carp fish scale Collagen peptide because the Collagen chain is a main component of silver carp fish scale Collagen.

The molecular weight is an important factor influencing the iron chelating activity, in the previous research, peptide fragments with the molecular weight distribution of 300-1500Da have high iron chelating activity, according to the identified peptide fragments and the research result of the previous people, in the research of collagen peptide, peptide fragments with arginine at the C end have high iron chelating activity, and the content of arginine, aspartic acid, glutamine, histidine, lysine, serine, threonine and tyrosine in the identified iron chelating collagen peptide is rich, so 17 potential iron chelating collagen peptide fragments are screened, and the amino acid sequence and molecular weight information of the peptide fragments are shown in table 1.

TABLE 1 sequence information of potential iron-chelating collagen peptides analyzed by LC-MS/MS

S4, constructing a molecular structure model and simulating Molecular Dynamics (MD) of the 17 analyzed collagen peptides, constructing a molecular structure model through Alphofold on-line software, then simulating molecular dynamics through GROMACS 2021.4 software, selecting Amber force field, adopting SPC model as water molecules, adding ferrous ions and chloride ions into a cube water box to balance charges and minimize energy, and dividing the molecular dynamics into three parts of energy minimization, balance and finished product simulation. The method is characterized in that 5000 steps are operated by using a steepest descent method, 50000 steps are operated by using a speed regulation temperature controller coupling method under a regular ensemble (canonical ensemble, NVT) in an equilibrium stage, the system temperature is kept at 310K, 50000 steps are operated under an isothermal-pressure (NPT) ensemble, the system pressure is kept at 1.0bar by using a Parrinello-Rahman barostat, and 50ns of finished product simulation is operated under the NPT ensemble by using a frog-leaping algorithm. The particle grid algorithm is used, the cut-off distance is 1.0nm, the step length in the molecular dynamics process is 2fs, and all the analyses are screened by using GROMACS self-contained analysis and insertion analysis.

To investigate the conformational changes of collagen peptide chains relative to the initial position during normal temperature simulation, root mean square error (RMSD) values of collagen peptide molecules relative to the initial structural peptide backbone position were calculated, which reflect the conformational changes of collagen peptide molecules in the environment of ferrous ions. FIG. 2 shows the RMSD values of 17 peptide fragments in the Fe ²⁺ environment, and after 50ns molecular dynamics simulation, the RMSD values of 17 peptide fragments all have different amplitude fluctuation, and the vibration frequency and amplitude of the 8 peptide fragments ,1-DTSGGYDEY、7-EKAPDPFRHF、8-IVGLPGQR、9-LQGSNEIEIR、10-IAGPAGPR、11-AQGPIGAR、12-GAQGPIGAR、13-VGPSGPAGAR in the same MD simulation time are smaller in the dynamics simulation process compared with those of the RMSD values of other peptide fragments, so that the structure is more stable in a Fe ²⁺ system.

To evaluate the contribution of the overall binding of carboxyl oxygen to iron on the peptide fragment, two types of short-range energies, lennard-Jones short-range energy (LJ-SR) and Coul-SR short-range energy (Coul-SR), were calculated to determine the interaction energy of the iron atom with the carboxyl oxygen on the peptide fragment throughout the MD track (see Table 2). After 50ns of MD simulation, the total Coul-SR energy of each peptide segment is far higher than that of LJ-SR, which shows that Coul-SR short-range energy of peptide segments 1-DTSGGYDE in each peptide segment is highest, and strong Coul-SR interaction energy of-66.40 kJ/mol is shown to play an important role in electrostatic interaction. In addition, 2-IAQPAEKAPDPF, 9-LQGSNEIEIR also showed higher interaction energies (sum of Coul-SR and LJ-SR energies), respectively, -52.62kJ/mol, -23.64kJ/mol. By comparing the interactions between the carboxyl oxygen and iron on 17 peptide fragments, it is possible that 3 peptide fragments 1-DTSGGYDEY, 2-IAQPAEKAPDPF, 9-LQGSNEIEIR may exhibit higher affinity activity for high iron.

TABLE 2 interaction energy analysis between carboxyoxy and iron on collagen peptides

In order to more accurately find a peptide fragment with potential high-iron chelating activity, the distance track generated in the process of combining Fe ²⁺ with carboxyl oxygen atoms on each peptide fragment is analyzed, and FIG. 3 shows the dynamic distance between Fe (FE in the figure) and oxygen atoms (O) in the molecular dynamics simulation process, wherein the atoms of the chelating sites are the oxygen atoms in the carboxyl groups of Glu, asp, arg residues respectively. With 0.35nm as the boundary of the distance between FE-O, collagen peptides most likely to bind to FE ²⁺ were screened, with the 2 peptide fragments 1-DTSGGYDEY and 9-LQGSNEIEIR being contacted with FE ²⁺ the most often, 16 and 8 times respectively, of the 17 peptide fragments, within the same simulation time, i.e. the iron atoms were more prone to binding reactions with 1-DTSGGYDEY and 9-LQGSNEIEIR.

And (3) synthesizing molecular dynamics simulation results, and screening 1-DTSGGYDEY and 9-LQGSNEIEIR as potential high-iron chelating peptides to perform next synthesis verification.

S5, synthesis and identification: the molecular weight mass spectrum identification results of DTSGGYDEY (DY) nonapeptide and LQGSNEIEIR (LR) decapeptide synthesized by solid phase synthesis technology are shown in fig. 4, and the liquid chromatography purity results are shown in fig. 5.

From the above figures, DY peptide had a purity of 98.73%, a molecular weight of 1006.45Da, LR peptide had a purity of 98.94%, and a molecular weight of 1156.50Da.

EXAMPLE 2 chelate die-closing type investigation between iron-chelate collagen peptide and iron

(1) Microstructure analysis of collagen peptide before and after iron ion chelation

And grinding SCSCP-Fe and SCSCP powder uniformly, taking a proper amount of samples, uniformly dispersing the samples on a double-sided adhesive tape of a sample disk, spraying gold, vacuumizing, obtaining microstructures of SCSCP-Fe and SCSCP under the condition that the accelerating voltage is 15kV, and comparing and analyzing the microstructures, wherein the results are shown in a graph A-D, which are the energy spectrum test results of DY, DY-Fe, LR and LR-Fe respectively, the left graph is the range selected on the surface of the sample structure during point scanning, and the right graph is the distribution condition of main elements of the sample obtained through the energy spectrum test.

As can be seen from the graph, the proportion of C, N, O elements in the DY peptide is 38.22%, 41.12% and 20.66% respectively, and the DY peptide does not contain iron, while the proportion of C, N, O, fe elements in the DY peptide iron chelate (DY-Fe) is 32.35%, 30.33%, 31.66% and 5.66% respectively, and peaks of iron element appear in the energy spectrum result; the percentage change of iron element also occurs in the element distribution results of LR peptide and LR peptide iron chelate (LR-Fe), which indicates that DY peptide and LR peptide are chelated with Fe ²⁺, resulting in more iron element ratio in the element distribution of DY-Fe and LR-Fe. In addition, the percentage of elemental iron in DY-Fe was 5.66%, which is higher than that in LR-Fe by 4.56%, presumably the 9 peptide DY was more capable of binding Fe ²⁺ than the 10 peptide LR.

(2) Analysis of Crystal Structure of collagen peptide before and after iron ion chelation

The crystal morphology analysis of DY peptide, DY peptide iron chelate (DY-Fe), LR peptide and LR peptide iron chelate (LR-Fe) was performed using an X-ray diffractometer, and the results are shown in FIG. 7.

As can be seen from the graph, when LR peptide and DY peptide are compared, strong diffraction peaks appear at about 8 °, 16 °, 19 ° and 23 ° of 2θ, and when DY peptide is chelated with Fe ²⁺, the intensity of LR-Fe diffraction peak decreases and gradually moves to a large glancing angle (8 °, 19 °, 23 °) and even disappears (16 ° of 2θ), one wide scattering diffraction peak appears at about 22 ° of 2θ in the XRD pattern of DY-Fe, and diffraction peaks at other places disappear, that is, diffraction peaks of two peptide fragments before and after chelation appear distinct morphological characteristics, and the diffraction peaks of the two peptide fragments after chelation with Fe ²⁺ even disappear, which is similar to an amorphous spectrum, and belongs to an amorphous structure, indicating that Fe ²⁺ is in a highly dispersed state and is combined with the peptide fragments in a coordination bond form.

(3) Collagen peptide iron binding site assay

The collagenase iron binding site was analyzed by fourier transform infrared spectroscopy, and the results are shown in fig. 8.

From the figure, the differences between the DY peptide and the LR peptide and the characteristic peaks of the infrared spectra before and after chelation of Fe ²⁺ indicate that the addition of Fe ²⁺ affects the original position of the functional group absorption peak. N-H stretching vibration in the peptide fragment is respectively moved from 3298cm ^-1(DY)、3278cm^-1 (LR) to 3308cm ^-1、3290cm^-1, and the peak is widened after Fe ²⁺ is added, which shows that-NH ₂ and-NH groups participate in chelation reaction, N-Fe bonds replace N-OH (hydrogen bonds). The amide I band (1700-1600 cm ^-1) is mainly related to the stretching vibration of C=O on the peptide chain, and the absorption peaks of DY and LR are respectively shifted from 1644cm ^-1、1636cm^-1 to 1660cm ^-1、1632cm^-1, indicating that the peptide fragment binds to Fe ²⁺ and carboxyl group, Amide groups are involved. The characteristic frequency of the amide II band is typically in the range of 1600-1550cm ^-1, resulting from N-H flexural and C-N tensile vibrations. The wavenumbers of DY and LR at the amide II band were 1516cm ^-1 and 1548cm ^-1, respectively, which, in combination with Fe ²⁺, blue shifted to 1519cm ^-1、1558cm^-1, It is possible that the addition of Fe ²⁺ replaces H on the N-H bond to form Fe-N bond. The absorption peaks of DY and LR at 1234cm ^-1 and 1288cm ^-1, respectively, correspond to the amide III band (1320-1220 cm ^-1), which is characteristic of the-COO-group of the peptide fragment, and, after binding to Fe ²⁺, The shift to 1243cm ^-1 and 1236cm ^-1, respectively, may occur due to the fact that the carboxyl oxygen atoms in the DY peptide (aspartic acid, glutamic acid, and N-terminal tyrosine) and the LR peptide (aspartic acid, glutamic acid, and N-terminal arginine) provide free electron pairs to react with Fe ²⁺ in combination to form-COO-Fe. In summary, DY peptides and LR peptides bind Fe ²⁺ mainly through the nitrogen atoms on the carboxyl, amino, and amide groups on their peptide chains.

(4) Isothermal titration quantitative thermal analysis (ITC)

The isothermal titration curves obtained by titrating Fe ²⁺ into the two peptide solutions DTSGGYDEY and LQGSNEIEIR using ITC with 50mM Tris-HCI buffer (pH 7.5) as solvent, 2mM FeCl ₂ solution, 0.1897mM DTSGGYDEY peptide solution and 0.1730mM LQGSNEIEIR peptide solution were prepared, and the thermodynamic parameters of the interactions were summarized as shown in Table 3.

TABLE 3 parameters of binding characteristics of iron ions to DY and LR peptides determined by isothermal titration quantitative thermal analysis

From the isothermal titration curve, fe ²⁺ gradually drops to the two peptide solutions, the heat of which gradually decreases, and the interaction results in negative Δh and Δg values, indicating that the chelation reaction of Fe ²⁺ with DY and LR peptides is an exothermic reaction. On the other hand, positive Δs values for chelate formation indicate that these reactions are entropy driven by an entropy increase, and the main cause of such changes may be solvation effects of the system or free changes in conformation. Research shows that the main acting force type between two molecules has close relation with the three of delta H, delta S and delta G; when Δh > 0, Δs > 0, it means that the forces between the two molecules are mainly hydrophobic interactions; when Δh < 0, Δs < 0, it is mainly manifested as hydrogen bonds and van der waals forces; when ΔH < 0, ΔS > 0, electrostatic interactions are dominant. As can be seen from the data in Table 3, titration of Fe ²⁺ into DY peptide solution resulted in a ΔH of-6.623 kJ/mol and a ΔS of 55.05kJ/mol; titration of Fe ²⁺ to LR peptide solution resulted in a ΔH of-18.41 kJ/mol and a ΔS of 26.74kJ/mol, i.e., ΔH < 0 and ΔS > 0 in both systems, indicating that Fe ²⁺ combined with both peptide fragments primarily exhibited electrostatic interactions, which corresponds to the result of dominant coulomb forces (DY: -66.4024.+ -. 16kJ/mol, LR: -23.5549.+ -. 4.5 kJ/mol) using computer modeling. The interactions lead to negative ΔH values (DY: -6.623kJ/mol, LR: -18.41 kJ/mol) and ΔG values (DY: -23.035kJ/mol, LR: -26.385 kJ/mol), meaning that the complexation of Fe ²⁺ and the two peptide fragments occurs autonomously.

For the DY groups, DTSGGYDEY and Fe ²⁺ Ka and K _d are 1.086×10 ⁴ M and 9.208 ×10 ^-5M^-1, respectively, the stoichiometry can be given directly by the computer when performing ITC titration, and the stoichiometry of DY peptide to Fe ²⁺ is determined to be 1:2 (n=2.160), indicating that the peptide contains two functional iron chelating sites, and on average one DY peptide molecule binds to about 2 iron atoms; for the LR group, K _a and K _d of LR peptide and Fe ²⁺ were 4.195 ×10 ⁴ M and 2.384 ×10 ^-5M^-1, respectively, and the stoichiometric ratio of LR peptide to Fe ²⁺ was 1:1 (n=1.251), indicating that about one iron atom can be bound per LR peptide molecule. Comparing the sizes of K _a and K _d, it can be seen that DY peptide binds to Fe ²⁺ less strongly than LR peptide, and has better dissociation capability than LR peptide.

EXAMPLE 3 in vitro iron absorption Activity study of iron-chelating collagen peptides

(1) Cytotoxicity test

The effect of different concentrations of ferrous chloride (5-50. Mu.g/mL) on survival of human colorectal adenocarcinoma cells (Caco-2 cells) was tested using the CCK-8 method, and different concentrations (0.05-1.2 mg/mL) of DY peptide and LR peptide were mixed with a fixed concentration of ferrous chloride to test their effect on survival of Caco-2 cells.

Fig. 10 shows the effect of varying concentrations of ferrous chloride (panel a) and peptide iron mixtures (panel B) on cell viability. As can be seen from the graph A, the cell viability gradually decreased with increasing FeCl ₂ concentration, but the cell viability remained above 80% all the time under the FeCl ₂ condition of 5-50 μg/mL, no significant effect on the growth of cells, and too low iron concentration could not be selected for convenient measurement of iron transport amount, so 30 μg/mL FeCl ₂ was selected as the fixed concentration for the iron transport experiment. As shown in the graph B, in order to screen the proper peptide concentration range, 30 mug/mL FeCl ₂ is used as a blank control group, 30 mug/mL FeCl ₂ is mixed with DY peptide and LR peptide with different concentrations to be used as an experimental group, after mixed culture for 24 hours, the survival rate of Caco-2 cells is above 85%, which shows that the mixture of DY and LR peptide with proper concentration (0.05-1.2 mg/mL) and 30 mug/mL FeCl ₂ has no toxic effect on cell growth, so that three concentration gradients with small cytotoxicity of 0.1, 0.2 and 0.4mg/mL are selected for further iron transport experiments to evaluate the iron absorption promotion effect of the two peptides.

(2) Evaluation of iron absorption promoting Effect of DY peptide and LR peptide

As shown in FIG. 11, a Caco-2 cell monolayer model was established to evaluate the effect of iron transport in the digestive juice, the number of cell passages used to construct the model was between 20 and 50, cells in the logarithmic phase were made into cell suspensions, and cells were inoculated into 12-well Transwell-insert culture plates at a cell inoculation density of 2X 10 ⁵/mL. 0.5mL of the cell suspension was added to the intestinal lumen side (AP side, upper chamber) of the transfer tank, and 1.5mL of complete medium was added to the basal side (BL side, lower chamber). The liquid is changed every other day in the first week of culture, and the liquid is changed every day after one week. After 22 days of cell growth, the cells were assessed for use.

Caco-2 monolayer cells that passed the integrity assessment were washed 2 times with pre-warmed HBSS buffer and incubated in an incubator for 30min. The HBSS buffer was pipetted, 0.5mL of a sample prepared with the HBSS buffer (30. Mu.g/mL of FeCl ₂, 30. Mu.g/mL of FeCl ₂ +0.1mg/mL of DY/LR, 30. Mu.g/mL of FeCl ₂ +0.2mg/mL of DY/LR, 30. Mu.g/mL of FeCl ₂ +0.4mg/mL of DY/LR) was added to the upper chamber, 1.5mL of the HBSS buffer was added to the lower chamber, after incubation for 2 hours at 37℃0.5mL of the solution was pipetted from the BL side, the collected transport solution was digested by mixing with HNO ₃ and HCIO ₄, the iron content was measured by atomic absorption, and the iron transport amount was calculated.

As shown in FIG. 12, the effect of varying concentrations of the nonapeptide DY and the decapeptide LR on iron transport in monolayer cells was compared by adding 30 μg/mL FeCl ₂ to the upper chamber, and the concentration of DY/LR was able to affect iron transport in Caco-2 monolayer cells. For the LR group, with the increase of the peptide concentration, the iron transport amount gradually increases, when 0.1mg/mL and 0.2mg/mL are added, the iron transport amount is 0.2316 and 0.2349 mug/well respectively, which are improved by 16.69 percent and 18.86 percent respectively compared with the blank group without peptide, when the LR concentration is 0.4mg/mL, the iron absorption promoting activity is the highest, the iron transport amount reaches 0.3175 mug/well, and compared with the blank group without peptide, the iron transport amount is improved by 43.32 percent, and the difference is obvious (p is less than 0.05); for the DY group, with increasing peptide concentration, the iron transport amount is remarkably increased (p < 0.05) compared with that of the blank group without peptide, when DY peptide of 0.1mg/mL, 0.2mg/mL and 0.4mg/mL is added into the upper chamber, the iron transport amounts are 0.3188, 0.6112 and 0.3515 mug/well respectively, the iron transport amounts are respectively increased by 29.33%, 117.7 and 93.87% compared with that of the blank group without peptide, and when the DY concentration is 0.2 mug/mL, the iron absorption promoting activity is highest. Furthermore, at the same peptide concentration, the transport amount of DY group iron was higher than that of LR group.

The results show that the peptide DY and the LR can be combined with a certain amount of Fe ²⁺ to avoid the oxidative precipitation of Fe ²⁺, so that the bioavailability of iron is improved, and the peptide DY and the LR have good iron absorption promoting capability. And the effect of DY peptide in promoting iron absorption is better than LR peptide. From the isothermal titration results, the stoichiometric ratio of LR peptide to Fe is 1:1, and the stoichiometric ratio of DY peptide to Fe is 1:2, and the binding amount of LR peptide to iron is not as large as that of DY peptide, so that the iron transport promotion capability of DY peptide is better than that of the LR peptide.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. The collagen peptide with the iron chelating activity is characterized in that the amino acid sequence of the collagen peptide is shown as SEQ ID NO. 1.

2. The application of collagen peptide with iron chelating activity in preparing iron supplement is characterized in that the collagen peptide is DY peptide or LR peptide, the amino acid sequence of the DY peptide is shown as SEQ ID NO.1, and the amino acid sequence of the LR peptide is shown as SEQ ID NO. 9;

the application comprises: mixing the collagen peptide with an iron ion solution to prepare a peptide iron chelate, and applying the peptide iron chelate to the iron supplement.

3. The use according to claim 2, wherein the iron supplement is a pharmaceutical or health product with iron supplementing function.

4. The use according to claim 3, wherein the ferric ion solution is selected from at least one of ferrous chloride solution, ferrous sulfate solution and ferrous nitrate solution;

The molar ratio of the DY peptide to the iron ions in the iron ion solution is 1:2, the molar ratio of LR peptide to ferric ion in the ferric ion solution is 1:1.

5. A method for preparing a collagen peptide having iron chelating activity, comprising the steps of:

S2, adding alkaline protease into the silver carp fish scale powder for enzymolysis treatment, wherein the addition amount of the alkaline protease is 400-500U/g, the enzymolysis time is 4-6h, after the enzymolysis is finished, inactivating the enzyme at high temperature, centrifuging to obtain supernatant, ultrafiltering, collecting components with molecular weight less than 3kDa, concentrating and drying to obtain silver carp fish scale zymolyte;

S4, constructing a molecular structure model and simulating molecular dynamics of the polypeptide component analyzed in the step S3, and screening to obtain the collagen peptide with iron chelating activity by combining a root mean square error value, the interaction energy between carboxyl oxygen and ferrous iron and the dynamic distance analysis between iron atoms, wherein the amino acid sequence of the collagen peptide is shown as SEQ ID NO. 1.

6. The preparation method according to claim 5, wherein in step S4, a molecular structure model is built by Alphofold on-line software, then molecular dynamics simulation is performed by GROMACS2021.4 software, amber force field is selected, water molecules adopt SPC model, ferrous ions and chloride ions are added to a cubic water box to balance charges and perform energy minimization treatment, and root mean square error value, interaction energy between carboxyl oxygen and ferrous and dynamic distance between iron atoms and oxygen atoms are obtained.

7. The method of claim 6, wherein the interaction energy between the carboxyoxy compound and ferrous iron comprises Lennard-Jones short range energy and Coul-SR short range energy.