A high-Fe resistant to degradation by gastrointestinal enzymes 2+ Combined active peptides and their applications

Abstract

The invention discloses a high Fe 2+ The invention relates to the field of biotechnology by combining active peptides and their applications. The invention obtains a high Fe peptide resistant to gastrointestinal enzyme degradation by precisely phosphorylating specific amino acids in the GL-Hyp-GPSGEEGKR peptide chain and performing specific folic acid modification on key sites in the peptide chain. 2+ Combined with active peptides, which have higher Fe 2+ The present invention also clarifies that the obtained peptide exerts high Fe 2+ The potential mechanism of binding activity and resistance to gastrointestinal degradation is high Fe 2+ It provides a new approach for the preparation of combined active peptides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a high Fe 2+ Combined active peptides and their applications. Background Art

[0002] Iron ions are essential trace nutrients for the formation of human tissues and the maintenance of normal physiological functions. They are components of hemoglobin and most enzymes, participate in the metabolism of body cells, and maintain normal hematopoietic function. Iron ions in the human body cannot be synthesized in the body, so they must be obtained from the daily diet. The Chinese Residents' Dietary Trace Element Reference Intake indicates that people need to consume about 20 mg of iron per day. In pregnant women, this demand increases by 1-2 times. Iron deficiency (ID) is a global problem that affects more than 2 billion people worldwide. Prevalence forecasts show that by 2050, another 1.4 billion children under 5 years old and women of childbearing age may be at risk of ID. When the body lacks iron, it is easy to cause anemia and iron deficiency anemia; it is easy to cause premature birth during pregnancy; it is easy to cause developmental delay in childhood; it is easy to feel weak and tired in adulthood, which affects normal life. Iron deficiency may be caused by a variety of factors, such as insufficient dietary iron intake, low iron bioavailability, or a high demand for iron in a certain period of time. Iron deficiency and iron deficiency anemia are global public health issues that have a significant impact on personal health, quality of life, and society. Therefore, in order to solve the problems of insufficient dietary iron absorption, low bioavailability, and high demand, and to reduce the incidence of iron deficiency, iron fortifiers are often used. However, conventional iron fortifiers are prone to cause gastrointestinal diseases due to their unstable properties.

[0003] Fe 2+ Binding activity peptide refers to the ability to bind to Fc 2+ Active peptide substances are mainly derived from plant proteins, terrestrial animal proteins and aquatic proteins. They are characterized by small molecular weight and rich in characteristic amino acids such as Glu and Asp. 2+ The peptide-Fe 2+ Conjugate, which can increase Fe in the intestine 2+ solubility and convert Fe 2+ Bring it to the surface of intestinal cells, promoting the transport of Fe by transporters 2+ , or through peptide transport pathways, endocytosis pathways, etc. 2+ Transported into cells, increasing Fe 2+ Bioavailability of Peptide-Fe 2+ The conjugate has the advantages of fast transport and absorption, high stability, not easy to be saturated and can reduce the antagonism between metals, and is a potential dietary Fe 2+Supplements. In addition, in addition to Glu and Asp in the peptide chain, Fe 2+ In addition, after phosphorylation, the phosphate group of Ser in the peptide chain can also bind to Fe 2+ , thereby increasing the Fe 2+ Binding activity.

[0004] However, oral peptide biologics need to pass through multiple barriers in the gastrointestinal tract to exert their biological activity. That is, the peptide first reaches the stomach through the oral esophagus, where there is an acidic environment and pepsin. The cleavage site of pepsin is mainly hydrophobic amino acids. After passing through the stomach, the peptide reaches the small intestine. The intestinal lumen also contains various enzymes (such as trypsin). The cleavage site of trypsin is mainly the carboxyl end of lysine. The enzymes in the gastrointestinal tract work in a synergistic manner, degrading peptide substances and affecting the biological activity of peptide biologics. Therefore, it is crucial to explore how peptide biologics can overcome the gastrointestinal enzyme barrier for the oral use of peptide biologics. Summary of the invention

[0005] The purpose of the present invention is to provide a high Fe 2+ The active peptide and its application are combined to solve the problems existing in the above-mentioned prior art. 2+ Binding active peptides to increase the Fe 2+ The binding activity can also improve the peptide's resistance to gastrointestinal enzymatic hydrolysis, thereby increasing Fe 2+ Stability of bound active peptides during gastrointestinal digestion.

[0006] Strategies to overcome the gastrointestinal enzyme barrier include: N-acetylation or C-amidation of the peptide terminal, or coupling of appropriate compounds to key sites to protect the amino acids at the cleavage sites of the peptide from degradation by gastrointestinal enzymes. 2+ The pepsin cleavage site of the binding active peptide (GL-Hyp-GPSGEEGKR) is a Leu residue, and the trypsin cleavage site is a Lys residue. In addition, the inventors found in their previous studies that the Glu-Glu in GL-Hyp-GPSGEEGKR is Fe 2+ Therefore, according to the cleavage sites of gastrointestinal enzymes and the amino acid sequence characteristics of the peptide chain, without affecting Fe 2+ In the case of binding activity, folic acid is coupled to the N-terminus (-NH2) and Lys side chain (-NH2) of the peptide chain to resist degradation by gastrointestinal enzymes, improve the enzymatic resistance of the peptide, and thus increase the Fe 2+ Combining oral bioactivity and gastrointestinal absorption stability of active peptides.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a high Fe 2+ Combined with active peptide, its chemical structure is as follows

[0009]

[0010] The present invention also provides a high Fe 2+ Method for preparing combined active peptides, comprising combining folic acid with GL-Hyp-GPS (PO4) GEEGKR is coupled to obtain the high Fe 2+ The step of combining active peptides;

[0011] The GL-Hyp-GPS (PO4) The chemical structure of GEEGKR is as follows:

[0012]

[0013] Furthermore, the coupling reaction step comprises:

[0014] The activated folic acid is obtained by reacting folic acid, a carbodiimide reagent and N-hydroxysuccinimide;

[0015] The GL-Hyp-GPS (PO4) GEEGKR reacts with activated folic acid to obtain the high Fe 2+ Combined active peptides.

[0016] Furthermore, the carbodiimide reagent is dicyclohexylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, carbodiimide hydrochloride or N,N-carbonyldiimidazole.

[0017] Furthermore, the molar ratio of the folic acid, the carbodiimide reagent and the N-hydroxysuccinimide is 1:(1-5):(1-5).

[0018] Furthermore, the GL-Hyp-GPS (PO4) The molar ratio of GEEGKR to activated folic acid is 1:(1-5).

[0019] The present invention also provides the above-mentioned high Fe resistant to gastrointestinal enzyme degradation 2+ Combined with active peptides in the preparation of peptide-Fe 2+ Application in combination.

[0020] The present invention also provides a peptide-Fe 2+ The peptide-Fe conjugate 2+ The conjugate is a high Fe 2+ Binding of active peptides to Fe 2+The combination reaction is carried out.

[0021] The present invention also provides the above-mentioned high Fe resistant to gastrointestinal enzyme degradation 2+ Binding active peptide or peptide-Fe 2+ Conjugates in the preparation of dietary Fe 2+ Application in supplements.

[0022] The present invention also provides a dietary Fe 2+ Supplements, the active ingredients of which include a high-Fe compound as described above that is resistant to degradation by gastrointestinal enzymes 2+ Binding active peptide or peptide-Fe 2+ Conjugate.

[0023] The present invention discloses the following technical effects:

[0024] (1) Based on GL-Hyp-GPSGEEGKR, the present invention obtains a high Fe-rich protein that is resistant to gastrointestinal enzyme degradation through precise phosphorylation of specific amino acids and specific folic acid modification of key sites. 2+ Binding active peptide - Folate-GL-Hyp-GPS (PO4) GEEGKR, in increasing the Fe 2+ The binding activity can also improve the peptide's resistance to gastrointestinal enzymatic hydrolysis, thereby increasing Fe 2+ Stability of bound active peptides during gastrointestinal digestion.

[0025] (2) Precise phosphorylation of specific amino acids in the GL-Hyp-GPSGEEGKR peptide chain resulted in a peptide with a higher Fe 2+ Binding activity. Specific folic acid modification of key sites in the peptide chain makes the obtained peptides more resistant to gastrointestinal enzymatic hydrolysis and clarifies the role of the obtained peptides in exerting high Fe 2+ The potential mechanism of binding activity and resistance to gastrointestinal degradation is high Fe 2+ It provides a new approach for the preparation of combined active peptides.

[0026] (3) Folate-GL-Hyp-GPS provided by the present invention (PO4) GEEGKR can be used as a Fe supplement 2+ / Fe 2+ Absorbable ingredients for dietary oral Fe supplementation 2+ The product has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 For GL-Hyp-GPS (PO4) The chemical structure of GEEGKR;

[0029] Figure 2 For Folate-GL-Hyp-GPS (PO4) The structural formula of GEEGKR;

[0030] Figure 3 is the Fe of peptide before and after degradation in simulated gastrointestinal tract 2+ Binding rate;

[0031] Figure 4 is the molecular weight distribution of the peptide before and after degradation in the simulated gastrointestinal tract;

[0032] Figure 5 For GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR and pepsin;

[0033] Figure 6 For GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR and trypsin;

[0034] Figure 7 For Folate-GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR and pepsin;

[0035] Figure 8 For Folate-GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR and trypsin. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Synthesis of Folate-GL-Hyp-GPS (PO4) GEEGKR

[0043] (1) Solid-phase synthesis of GL-Hyp-GPS (PO4)GEEGKR: First, perform operation 1 on the Fmoc-Arg(pbf)-OH resin, i.e., add 20% Piperidine (Remove the N-terminal Fmoc group with piperidine) to react for 20 minutes, and use a detection reagent to detect the color to be blue. Perform operation 2, i.e., add HBTU+DIEA to Fmoc-Lys(Boc)-OH to react for 1 hour, and use a detection reagent to detect the color to be colorless. Perform operation 1 again, i.e., add 20% Piperidine (Remove the N-terminal Fmoc group with piperidine) to react for 20 minutes, and use a detection reagent to detect the color to be blue. Perform operation 2, i.e., add HBTU+DIEA to Fmoc-Gly-OH to react for 1 hour, and use a detection reagent to detect the color to be colorless. Perform operation 1 again, and so on, until the last amino acid Gly is de-Fmoc, then the resin is drained and cut to obtain a crude peptide. The crude peptide is sent to HPLC for purification, and GL-Hy p-GPS is obtained after freeze-drying. (PO4) GEEGKR pure product. Among them, GL-Hyp-GPS (PO4) During the synthesis process, GEEGKR directly uses phosphoserine as the raw material for the synthesis of the serine part to ensure accurate phosphorylation of serine. The phosphoserine is provided by Shanghai Jier Biochemical Co., Ltd.

[0044] GL-Hyp-GPS (PO4) The chemical structure of GEEGKR is as follows Figure 1 shown.

[0045] (2) Activation of folic acid: Mix folic acid, carbodiimide reagent - 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (NHS) in a molar ratio of 1:2:2, and react at room temperature in the dark for 6 hours to obtain activated folic acid (Folate-NHS). Carbodiimide reagents can also be selected from dicyclohexylcarbodiimide, carbodiimide hydrochloride or N,N-carbonyldiimidazole. Activated folic acid can be prepared by reacting folic acid, carbodiimide reagent and N-hydroxysuccinimide in a molar ratio of 1:(1-5):(1-5).

[0046] (3)GL-Hyp-GPS (PO4) GEEGKR coupled to folic acid: GL-Hyp-GPS (PO4) GEEGKR and Folate-NHS were mixed in a molar ratio of 1:2 and reacted at room temperature in the dark for 2 h to obtain folic acid-modified GL-Hyp-GPS. (PO4) GEE GKR is Folate-GL-Hyp-GPS (PO4)GEEGKR, chemical structure is as follows Figure 2 GL-Hyp-GPS (PO4) GEEGKR and Folate-NHS react at a molar ratio of 1:(1-5) to obtain Folate-GL-Hyp-GPS (PO4) GEEGKR.

[0047] (4) Molecular docking verification: Using the molecular docking software Autodock Vina, pepsin and trypsin were docked with the peptide GL-Hyp-GPS. (PO4) GEEGKR and Folate-GL-Hyp-GPS (PO4) GEEGKR was simulated and docked to analyze and compare the sites where the enzyme interacted with the main peptide chain.

[0048] 2. Detection Experiment

[0049] 2.1 Related determination methods

[0050] (1) Simulate gastrointestinal digestion: Folate-GL-Hyp-GPS (PO4) The pH of GEEGKR was adjusted to 2.0, and artificial gastric juice was added. The mass ratio of pepsin to substrate in the artificial gastric juice was 1:50. The reaction was carried out at 37°C for 2 hours. The pH was adjusted to 6.8, and artificial intestinal juice was added. The mass ratio of trypsin to substrate in the artificial intestinal juice was 1:50. The reaction was carried out at 37°C for 2 hours. The enzyme was inactivated in a boiling water bath for 10 minutes, and the mixture was freeze-dried to obtain Folate-GL-Hyp-GPS after simulated gastrointestinal digestion. (PO4) GEEGKR. Artificial gastric juice (article number: R22155-500ml) and artificial intestinal juice (article number: R22156-500ml) were provided by Shanghai Yuanye Biotechnology Co., Ltd.

[0051] (2) The Fe content of peptides before and after phosphorylation, phosphorylated peptides before and after folic acid modification, and before and after simulated gastrointestinal digestion was determined by the phenanthroline method. 2+ The binding rate is as follows:

[0052] GL-Hyp-GPSGEEGKR and GL-Hyp-GP S were prepared using sodium acetate buffer (0.2 mol / L, pH 5). (PO4) GEEGKR sample solution (1 mg / mL), take 700 μL of peptide sample solution, add 100 μL of ferrous sulfate solution (0.2 mol / L, pH 5), carry out binding reaction at room temperature for 1.5 h, add 200 μL of phenanthroline solution (5 mmol / L), incubate at room temperature for 10 min, and read the absorbance at 562 nm using an enzyme reader. GL-Hyp-GPS was prepared using sodium acetate buffer (0.2 mol / L, pH 5). (PO4)GEEGKR sample solution (1 mg / mL), take 700 μL of peptide sample solution, add folic acid-NHS (peptide: folic acid molar ratio 1:2) and react at room temperature in the dark for 2 hours, add 100 μL of ferrous sulfate solution (0.2 mol / L, pH 5), after the binding reaction at room temperature for 1.5 hours, add 200 μL of phenanthroline solution (5 mmol / L), incubate at room temperature for 10 minutes, and read the absorbance at 562 nm with an enzyme reader. GL-Hyp-GPS was prepared using sodium acetate buffer (0.2 mol / L, pH 5). (PO4) GEEGKR sample solution (1 mg / mL), take 700 μL of peptide sample solution, add 100 μL of ferrous sulfate solution (0.2 mol / L, pH 5), carry out binding reaction at room temperature for 1.5 h, add folic acid-NHS (peptide: folic acid molar ratio 1:2) and react at room temperature in the dark for 2 h, then add 200 μL of phenanthroline solution (5 mmol / L), incubate at room temperature for 10 min, and read the absorbance at 562 nm with an enzyme reader. Fe after gastrointestinal digestion 2+ The binding rate refers to the above experimental group, before adding 200 μL of phenanthroline solution, first performing gastrointestinal digestion simulation, then adding 200 μL of phenanthroline solution (5 mmol / L), incubating at room temperature for 10 min, and reading the absorbance at 562 nm with an enzyme reader.

[0053] Fe 2+ Binding rate (%) = (A0-A1) / A0×100, where A0 is the absorbance value after adding an equal volume of water, and A1 is the absorbance value after adding the sample.

[0054] (3) Detection of Folate-GL-Hyp-GPS by UV spectrophotometry (PO4) The conjugated amount of folic acid in GEEGKR was determined by preparing a series of folic acid solutions, placing them in a 10 mm quartz cuvette, and measuring the photometric value at 281 nm using a UV spectrophotometer. A folic acid standard curve was prepared based on the concentration and UV photometric value. (PO4) GEEGKR was prepared into a 1 mg / mL solution, placed in a 10 mm quartz cuvette, and the UV light value was measured at 281 nm. Folate-GL-Hyp-GPS was calculated based on the standard curve. (PO4) Conjugated amount of folic acid in GEEGKR.

[0055] (4) High-performance size exclusion chromatography was used to detect the enzymatic resistance of peptides before and after simulated gastrointestinal digestion. The peptide sample was prepared into a 2 mg / mL solution, filtered through a 0.22 μm microporous filter membrane, and placed in an injection bottle. The mobile phase was A (acetonitrile, containing 0.1% trifluoroacetic acid): B (water, containing 0.1% trifluoroacetic acid) = 20:80 (V / V), the detection wavelength was 214 nm, the injection volume was 20 μL (2 mg / mL), the flow rate was 0.5 mL / min, and the elution time was 40 min. The sample molecular weight distribution curve was prepared with the relative retention time (t) as the horizontal axis and the fluorescence response value as the vertical axis, and the molecular weight distribution of the peptide chain after simulated gastrointestinal digestion was obtained.

[0056] 2.2 Test results

[0057] Figure 3 is the Fe of peptide before and after degradation in simulated gastrointestinal tract 2+ The binding rate is Figure 3 It can be seen that GL-Hyp-GPSGEEGKR, GL-Hyp-GPS (PO4) GEEGKR, Folate-GL-Hyp-GPS (PO4) GEEGKR, its Fe 2+ The binding rates were 43.60%, 72.16%, 74.75% and GL-Hyp-GPS (PO4) GEEGKR and Folate-GL-Hyp-GPS (PO4) GEEGKR Fe 2+ The binding rate was significantly higher than that of GL-Hyp-GPSGEEGKR, indicating that phosphorylation can improve the Fe 2+ After simulated gastrointestinal digestion, GL-Hyp-GPSGEEGKR, GL-Hyp-GPS (PO4) GE EGKR, Folate-GL-Hyp-GPS (PO4) GEEGKR Fe 2+ The binding rates were 10.68%, 22.51%, 65.99% and Folate-GL-Hyp-GPS (PO4) GEEGKR Fe 2+ The binding rate was significantly higher than that of GL-Hyp-GPSGEEGKR and GL-Hyp-GPS (PO4) GEEGKR, indicating that folate modification significantly increased Folate-GL-Hyp-GPS (PO4) GEEG KR is resistant to degradation by gastrointestinal enzymes and is different from GL-Hyp-GPSGEEGKR and GL-Hyp-GPS (PO4) Compared with GEEGKR, it effectively maintains its Fe 2+ Binding activity. In addition, Figure 3(Folate-GL-Hyp-GPS (PO4) GEEGKR)+Fe 2+ Indicates GL-Hyp-GPS (PO4) GEEGKR first undergoes folic acid modification to generate Folate-GL-Hyp-GPS (PO4) GEEGKR and then Fe 2+ Fe 2+ Binding rate. Folate-(GL-Hyp-GPS (PO4) GEEGKR+Fe 2+ ) means GL-Hyp-GPS (PO4) GEEGKR first reacts with Fe 2+ After the binding reaction, folic acid modification was performed. The results showed that (Folate-GL-Hyp-GPS (PO4) GEEGKR)+Fe 2+ Fe 2+ The binding rate is higher than Folate-(GL-Hyp-GPS (PO4) GEEG KR+Fe 2+ ), Folate-(GL-Hyp-GPS (PO4) GEEGKR+Fe 2+ ) 2+ Binding rate and GL-Hyp-GPS (PO4) GEEGKR+Fe 2+ There is no significant difference. This shows that the order of folic acid modification and addition reaction is critical. Folic acid first modifies GL-Hyp-GPS (PO4) GEEGKR and then Fe 2+ reaction, and its binding rate was higher than that of GL-Hyp-GPS (PO4) GEEGKR and Fe 2+ After the reaction, folic acid was added for modification.

[0058] Figure 4 Peptide and its peptide-Fe 2+ The molecular weight distribution of the conjugates before and after simulated gastrointestinal digestion. Liquid phase detection after simulated digestion in artificial gastrointestinal fluid revealed that GL-Hyp-GPSGEEGKR and GL-Hyp-GPS (PO4) The liquid phase spectra of GEEGKR showed degradation peaks, indicating that both of them are easily degraded by gastrointestinal enzymes. (PO4) The liquid phase spectra of GEEGKR were basically the same, and no degradation peak appeared, indicating that Folate-GL-Hyp-GP S (PO4) GEEGKR has a certain resistance to gastrointestinal enzymes, that is, folate modification can increase Folate-GL-Hyp-GPS(PO 4) GEEGKR is resistant to enzymatic degradation by gastrointestinal enzymes. 2+ and GL-Hyp-GPS (PO4) GEEGKR-Fe 2+ A degradation peak appeared, and (Folate-GL-Hyp-GPS (PO4) GEEGKR)-Fe 2+ There is no degradation peak. The results show that GL-Hyp-GPS (PO4) Specific folate modification at specific sites in GEEGKR can improve resistance to degradation by gastrointestinal enzymes, and GL-Hyp-GPS (PO4) GEEGKR first reacts with folic acid to be modified with folic acid and then reacts with Fe 2+ The combined anti-degradation ability is stronger than GL-Hyp-GPS (PO4) GEEGKR first reacts with Fe 2+ After conjugation, folic acid was added for folate modification.

[0059] Figure 5 and Figure 6 GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR with pepsin and trypsin. Figure 5 It can be seen that pepsin and GL-Hyp-GPS (PO4) In GEEGKR, there are hydrogen bonds between the Hyp3 residue -OH, Gly7 main chain C=O, Gly8 main chain -NH and C=O, Glu8 side chain -COOH, Glu9 main chain -NH, Gly10 main chain -NH, Lys11 main chain -NH, Lys11 side chain -NH2, and Arg12 side chain -NH, and the number of hydrogen bonds that interact is 10. Figure 6 It can be seen that trypsin and GL-Hyp-GPS (PO4) In GEEGKR, there are hydrogen bonds between -NH2 at the N-terminus of Gly1, C=O in the main chain of Pro5, -OH at Ser6, C=O and -NH on the main chain of Glu8, -OH and main chain C=O of Glu9 side chain, C=O on the main chain of Gly10, and -NH2 on the side chain of Lys11. The number of hydrogen bonds that interact is 9. Therefore, GL-Hyp-GPS (PO4) GEEGKR has many direct interaction sites with pepsin and trypsin, and is very likely to be degraded during gastrointestinal digestion.

[0060] Figure 7 and Figure 8 Folate-GL-Hyp-GPS (PO4) Molecular docking diagram of GEEGKR with pepsin and trypsin. Figure 7 It can be seen that pepsin and Folate-GL-Hyp-GPS (PO4) There are hydrogen bonds between the pteridine ring of folic acid, the side chain -OH of Hyp3, the main chain -NH and C=O of Glu8, the side chain -COOH, the main chain C=O and the side chain -COOH of Glu9 in GEEGKR. The number of hydrogen bonds is 18, of which only 7 are directly formed by pepsin with Folate-GL-Hyp-GPS. (PO4) Peptide-host interactions of GEEGKR. Figure 8 It can be seen that trypsin and Folate-GL-Hy p-GPS (PO4) There are 16 hydrogen bonds between the pteridine ring of the coupled folic acid in GEEGKR, the main chain -NH and C=O of Gly4, the side chain -COOH of Glu8, and the main chain C=O of Gly10, of which only 7 are directly bonded between trypsin and Folate-GL-Hyp-GPS. (PO4) The peptide-host interaction of GEEGKR. (PO4) GEEGKR forms Folate-GL-Hyp-GPS (PO4) GEEGKR inhibits pepsin and trypsin from acting directly on GL-Hyp-GPS (PO4) The main body of GEEGKR has a greater impact. Pepsin and trypsin tend to interact with the hydrophobic pteridine ring in folic acid. Folic acid hinders the protease from GL-Hyp-GPS through steric hindrance. (PO4) The direct action of GEEGKR main body, thereby playing a role in resisting gastrointestinal enzyme degradation.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-Fe product that resists degradation by gastrointestinal enzymes 2+ The binding active peptide is characterized in that Its chemical structure is as follows: 。 2. A high Fe resistant to gastrointestinal enzyme degradation as claimed in claim 1 2+ The method for preparing a binding active peptide is characterized in that: Includes folic acid and GL-Hyp-GPS (PO4) GEEGKR is coupled to obtain the high Fe 2+ The step of combining active peptides; The GL-Hyp-GPS (PO4) The chemical structure of GEEGKR is as follows: 。 3. The preparation method according to claim 2, characterized in that: The steps of the coupling reaction include: The activated folic acid is obtained by reacting folic acid, a carbodiimide reagent and N-hydroxysuccinimide; The GL-Hyp-GPS (PO4) GEEGKR reacts with activated folic acid to obtain the high Fe 2+ Combined active peptides.

4. The preparation method according to claim 3, characterized in that: The carbodiimide reagent is dicyclohexylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, carbodiimide hydrochloride or N,N-carbonyldiimidazole.

5. The preparation method according to claim 3, characterized in that: The molar ratio of the folic acid, the carbodiimide reagent and the N-hydroxysuccinimide is 1:(1-5):(1-5).

6. The preparation method according to claim 3, characterized in that: The GL-Hyp-GPS (PO4) The molar ratio of GEEGKR and activated folic acid is 1:(1~5).

7. A high Fe resistant to gastrointestinal enzyme degradation as claimed in claim 1 2+ Combined with active peptides in the preparation of peptide-Fe 2+ Application in combination.

8. A peptide-Fe 2+ A combination, characterized in that The peptide-Fe 2+ The conjugate is a high Fe 2+ Binding of active peptides to Fe 2+ The combination reaction is carried out.

9. A high Fe resistant to gastrointestinal enzyme degradation as claimed in claim 1 2+ Binding active peptide or peptide-Fc according to claim 8 2+ Conjugates in the preparation of dietary Fe 2+ Application in supplements.

10. A dietary Fe 2+ A supplement, characterized in that The active ingredient comprises the high Fe resistant to gastrointestinal enzyme degradation as claimed in claim 1 2+ Combined active peptides.

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