Iron chelating peptide, preparation method thereof and application of iron chelating peptide in preparation of iron supplement

Abstract

The invention belongs to the field of active peptide preparation, and particularly relates to an iron chelating peptide, a preparation method thereof and application of the iron chelating peptide in preparation of an iron supplement. According to the research, soybean protein is taken as a raw material, and four iron chelating peptides, namely EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR, are screened out by virtue of enzymolysis, purification identification and molecular docking technologies. A Caco-2 small intestine epithelial cell model finds that the iron chelating peptide can obviously promote the transport and absorption of iron and obviously promote the retention of Fe < 2 + > in a Caco-2 cell monomolecular layer. The novel iron chelating peptide screened from soybean protein hydrolysate is expected to become a safe and effective iron supplement.

Description

Technical Field

[0001] The invention belongs to the field of active peptide preparation, and specifically relates to an iron chelating peptide and a preparation method thereof and application thereof in the preparation of an iron supplement. Background Art

[0002] Iron is a trace element that is essential to the human body. It plays a central role in many biological processes, including oxygen transport, DNA synthesis, and electron transport. When the iron absorbed from the daily diet cannot meet physiological needs, nutritional iron deficiency occurs. In severe cases, it can reduce work ability, affect physical function, affect children's cognitive development, and delay motor development.

[0003] Bioactive peptides refer to specific protein fragments formed by amino acids connected by covalent bonds, which have a positive effect on body functions and conditions. In recent years, more and more bioactive peptides with food-derived iron chelating activity have been reported, and it has been found that they have a significant effect on promoting intestinal iron transport and absorption. Iron-chelating peptides and their peptide iron chelates are becoming a hot topic of attention and research. For example, Wu et al. (Wu W, Li B, Hou H, et al. Identification of iron-chelating peptides from pacific cod skin gelatin and the possible binding mode [J]. Journal of Functional Foods, 2017, 35: 418-427.) used immobilized metal affinity chromatography and RP-HPLC to purify Pacific cod skin gelatin hydrolysate, and identified three new iron-chelating peptides by LC-HRMS / MS, namely GPAGPHGPPGKDGR, AGPHGPPGKDGR and AGPAGPAGAR, which have a strong affinity for Fe. 2+It showed high affinity. Lin et al. (Lin S, Hu X, Li L, et al. Preparation, purification and identification of iron-chelating peptides derived from tilapia (Oreochromis Niloticus) skin collagen and characterization of the peptide-iron complexes [J]. Food Science & Technology, 2021, 149: 111796.) (Lin S, Hu X, Yang X, et al. GLPGPSGEEGKR: Fe2+ chelating characterization and potential transport pathways for improving Fe2+ bioavailability in Caco-2 cells [J]. Food Bioscience, 2022, 48: 101806.) from tilapia skin collagen hydrolysate, by immobilized metal affinity chromatography (IMAC-Fe 2+ ) and obtained tilapia skin collagen iron chelating peptide. After RP-HPLC purification, GLPGPSGEEGKR was identified from it and increased the Fe 2+ bioavailability.

[0004] When there are many peptide sequences identified from the separated and purified components, how to quickly screen them becomes a major problem. The current screening of metal chelating peptides mainly relies on a lot of synthesis and chemical experience. For example, Budseekoad et al. (Budseekoad S, Yupanqui CT, Sirinupong N, et al. Structural and functional characterization of calcium and iron-binding peptides from mung bean protein hydrolysate [J]. Journal of Functional Foods, 2018, 49: 333-341.) identified 133 potential metal chelating peptides from the separated and purified components of mung bean protein, and selected and synthesized 10 peptide sequences with high leucine, isoleucine and aspartic acid content, and found that AIVIL and HADAD had the strongest calcium chelating ability, while PAIDL had the strongest iron chelating ability. Wang et al. (Wang T, Lin S, Cui P, et al. Antarctic krill derived peptide as ananocarrier of iron through the gastrointestinal tract [J]. Food Bioscience, 2020, 36: 100657.) identified more than 1,000 peptide sequences from Antarctic krill iron chelating peptides and focused on three of them: all of which contained three glutamic acids, but the positions of glutamic acids on the peptide chain were different. Therefore, these three active peptides were synthesized and found to have strong iron chelating activity and differences. With the development of small molecule bioactive peptides and the cross-application of computer science and bioinformatics science, more and more studies have applied molecular docking technology to the screening of bioactive peptides. Zhang et al. (Zhang J, Zhang J, Liang L, et al. Identification and virtual screening of novel umami peptides from chicken soup by molecular docking [J]. Food Chemistry, 2023, 404 (A): 134414.) screened out 20 potential umami peptides from 208 peptides identified in chicken soup through molecular docking technology, among which PPQEAAQF had a higher umami intensity.Guo et al. (Guo W, Xiao Y, Fu X, et al. Identification of novel α-glucosidase and ACE inhibitory peptides from douchi using peptidomics approach and molecular docking [J]. Food Chemistry: X, 2023, 19: 100779.) screened out 4 ACE inhibitory active peptides from 710 peptide sequences identified from Douchi by molecular docking technology. In the study of metal chelating peptides, molecular docking technology is often used to study the binding sites and interactions between metals and active peptides, but there are few reports on the use of molecular docking technology to assist in the rapid screening of active peptides.

[0005] Soybean is the most planted legume and oilseed plant in the world. It is rich in resources and rich in protein (40-50%). Soy protein is treated by enzymatic hydrolysis and other processes to reduce the anti-nutrient content in the protein. In addition, the released soy protein peptides have different biological activities, such as hypolipidemic activity, antioxidant and anti-hypertensive activity. Summary of the invention

[0006] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide an iron chelating peptide.

[0007] Another object of the present invention is to provide a method for preparing the above iron-chelating peptide.

[0008] Another object of the present invention is to provide the application of the above iron-chelating peptide.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] An iron chelating peptide, the amino acid sequence of which is at least one of the following sequences a)-d):

[0011] a)EDEPFNLRSR;

[0012] b) EDEPFNLR;

[0013] c)DDLERLKKIR;

[0014] d) EEQPLEVR;

[0015] The method for preparing the iron chelating peptide comprises the following steps:

[0016] The iron chelate peptide is prepared by solid phase synthesis or by using soy protein isolate as raw material and hydrolyzing and purifying the iron chelate peptide by papain;

[0017] The specific operation of the papain enzymolysis is preferably:

[0018] The soy protein isolate solution and papain are mixed for enzymolysis, then inactivated in a water bath, cooled, centrifuged, the supernatant is collected, and further freeze-dried to obtain a soy protein hydrolysate;

[0019] The mass percentage of soy protein isolate in the soy protein isolate solution is preferably 7%;

[0020] The enzymatic hydrolysis conditions are preferably: enzymatic hydrolysis at pH 5.5 and 60° C. for 3.5 h;

[0021] The enzyme substrate mass ratio of the enzymatic hydrolysis is 0.25% (m / m);

[0022] The water bath inactivation condition is preferably placed in a 95°C water bath for 10 minutes;

[0023] The purification comprises the steps of ultrafiltration separation and liquid chromatography separation;

[0024] The specific operation of the ultrafiltration separation is:

[0025] Ultrafiltration tubes were used to separate soybean protein hydrolysates and fractions with molecular weight <3 kDa were collected;

[0026] The specific operation of the liquid chromatography separation is:

[0027] The ultrafiltration fraction of soybean protein hydrolysate <3 kDa was separated and purified by high performance liquid chromatography using the Prep150 preparative liquid phase system; the active fraction was collected and freeze-dried to obtain the iron chelating peptide;

[0028] The conditions of the liquid chromatography are preferably:

[0029] Mobile phase A: distilled water + 0.1% trifluoroacetic acid; mobile phase B: methanol + 0.1% trifluoroacetic acid; injection volume: 8 mL; flow rate: 10 mL / min; detection wavelength: 214 and 280 nm; gradient elution: 0-10 min: 10-30% mobile phase B, 30-60 min: 60-80% mobile phase B, 70 min: 90% mobile phase B, the above percentages are all volume percentages;

[0030] Use of the iron chelating peptide in the preparation of an iron supplement;

[0031] Application of the iron chelating peptide in the fields of food, medicine and health products;

[0032] An iron supplement comprising at least one of the above-mentioned iron chelate peptide, papain hydrolyzate of soy protein isolate containing the above-mentioned iron chelate peptide, and hydrolyzate containing the above-mentioned iron chelate peptide as an active ingredient;

[0033] The iron supplement further comprises a pharmaceutically acceptable carrier or auxiliary material;

[0034] The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a humectant, a surfactant, a matrix, an emulsifier, a preservative and a solvent;

[0035] A food, medicine or health product, comprising at least one of the above-mentioned iron chelate peptide, papain hydrolyzate of soy protein isolate containing the above-mentioned iron chelate peptide and hydrolyzate containing the above-mentioned iron chelate peptide as an active ingredient;

[0036] Compared with the prior art, the present invention has the following advantages and effects:

[0037] (1) The present invention uses soy protein isolate as raw material, and undergoes papain enzymatic hydrolysis, ultrafiltration separation and purification, RP-HPLC separation and purification, and HPLC-MS / MS identification and molecular docking technology to screen high-activity peptides. A total of 4 new iron chelating peptides EDEPFNLRSR (1261.6051Da), EDEPFNLR (1018.4719Da), DDLERLKKIR (1284.7513Da) and EEQPLEVR (998.5032Da) are screened out, and the iron contents in the formed chelates are 129.75, 68.82, 110.08 and 80.94μg / mg, respectively.

[0038] (2) The present invention further studies the iron chelating ability of the screened iron chelating peptides and their iron transport and retention activities in the Caco-2 cell monolayer model. The results show that in the Caco-2 small intestinal epithelial cell model, after 180 minutes of transport, the total iron transport amounts of the four iron chelating peptides were increased by 69.36%, 29.48%, 54.17% and 51.37% respectively compared with the control group, with EDEPFNLRSR having the strongest activity. EDEPFNLRSR, which had the strongest iron transport activity, was selected for further study, and it was found that compared with the control group, the Fe content in the Caco-2 cell monolayer in the EDEPFNLRSR group was 2+ The retention amount increased by 113.10%, significantly promoting the absorption of iron. In the study of transmembrane transport of active peptides, it was found that 24 peptides derived from EDEPFNLRSR after degradation could achieve transmembrane transport in the Caco-2 cell monolayer.

[0039] (3) The present invention reveals the role of the novel iron chelating peptide in promoting iron transport in the intestine and its own transmembrane transport behavior, providing new information for understanding the mechanism by which bioactive peptides promote intestinal iron absorption and providing a theoretical basis for the development of dietary iron supplements using soy protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is an analysis chart of the evaluation results of iron chelating activity of soybean protein hydrolysate and ultrafiltration fractions, where different letters indicate significant differences (p<0.05).

[0041] Figure 2 It is a chromatogram of the ultrafiltration component (<3kDa) further separated by RP-HPLC and an analysis chart of the iron chelating activity evaluation results of each component, wherein, A: RP-HPLC separation chromatogram of the ultrafiltration component (<3kDa), B: iron chelating activity evaluation of F1-F22 liquid phase components, different letters indicate significant differences (p<0.05).

[0042] Figure 3 This is the total ion current diagram of liquid phase component F3 identified by LC-MS / MS.

[0043] Figure 4 It is 4 kinds of soy protein iron chelate peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR, EEQPLEVR) and Fe 2+ Molecular docking result analysis diagram.

[0044] Figure 5 This is an analysis chart of the iron chelating ability of the enzymatic hydrolysate, ultrafiltration fraction (<3kDa), and soy protein iron chelating peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR, and EEQPLEVR), where different letters indicate significant differences (p<0.05).

[0045] Figure 6 It is an analysis chart of the results of cytotoxicity study on ultrafiltration fractions (<3 kDa) and soybean protein iron chelating peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR).

[0046] Figure 7 This is an analysis chart of the iron transport results of the ultrafiltration fraction (<3kDa) and soy protein iron chelating peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR) at different transport times, where different letters indicate significant differences (p<0.05).

[0047] Figure 8The results of the analysis of the effect of EDEPFNLRSR on iron retention in Caco-2 cells are shown in Figure 2. 2+ Effect of influx into Caco-2 cells, B: Fluorescence intensity statistics, different letters indicate significant differences (p<0.05).

[0048] Fig. 9 This is the RP-HPLC analysis chromatogram of EDEPFNLRSR transport across the small intestinal epithelial cell model, where A: chromatogram of EDEPFNLRSR and the top solution of the Transwell plate at each time point, B: chromatogram of the reagent blank (HBSS) and the basolateral solution of the Transwell plate at each time point. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0050] Materials and reagents in the examples: soy protein isolate, Shanghai Yuanye Biotechnology Co., Ltd.; FeCl2·4H2O, Tianjin Baishi Chemical Co., Ltd.; phenanthroline, thiazolyl blue (MTT), Shanghai McLean Biotechnology Co., Ltd.; penicillin-streptomycin solution, fetal bovine serum and 0.25% trypsin were purchased from Gibco; EMEM basal medium and Caco-2 cells were purchased from ATCC (American type culture collection), and the fluorescent probe FerroOrange was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. Other chemicals and solvents were of analytical grade.

[0051] Data analysis in the examples: Each experiment was repeated at least three times. Data analysis was performed using Microsoft Excel 2021 software. SPSS software was used to perform one-way analysis of variance (ANOVA). When p < 0.05, the difference was considered statistically significant.

[0052] Example 1 Isolation, purification and iron chelating activity of soybean protein iron chelate peptide

[0053] 1 Test method

[0054] 1.1 Preparation of soybean protein hydrolysate

[0055] A certain amount of soy protein isolate was weighed and added into distilled water to obtain a solution with a mass percentage of 7% of soy protein isolate; the pH of the system was adjusted to 5.5, and a papain solution was added according to an enzyme substrate mass ratio of 0.25% (m / m), and enzymolysis was performed at 60°C for 3.5h; the obtained mixture was placed in a 95°C water bath for 10min to inactivate the protease, and then centrifuged at 4500rpm for 20min, and the supernatant was collected to obtain a soy protein hydrolysate.

[0056] 1.2 Ultrafiltration separation

[0057] The soybean protein hydrolysate was separated using an ultrafiltration membrane (3 kDa, Merck Millipore, Billerica, MA) to obtain two components (>3 kDa and <3 kDa). The iron chelating activity of the two components was then determined to determine the most active component for subsequent further separation and purification.

[0058] 1.3 RP-HPLC separation

[0059] The ultrafiltrate fraction with better iron chelating activity was filtered through a 0.22 μm filter membrane and then purified by Prep150 preparative liquid phase system (reversed phase column: SunFire Prep C18 OBDT TM 19mm x 250mm, 5μm, Waters) for further separation. The program was set as follows: mobile phase A: distilled water + 0.1% trifluoroacetic acid; mobile phase B: methanol + 0.1% trifluoroacetic acid; injection volume: 8mL; flow rate: 10mL / min; detection wavelength: 214 and 280nm; gradient elution: 0-10min: 10-30% mobile phase B, 30-60min: 60-80% mobile phase B, 70min: 90% mobile phase B, the above percentages are volume percentages. Collect each elution peak, freeze-concentrate and measure the iron chelating activity of each component.

[0060] 1.4 Determination of iron chelating activity

[0061] The determination of iron chelating activity adopts the phenanthroline colorimetric method, which is operated according to the reference method (Miao J, Liao W, Pan Z, et al. Isolation and identification of iron-chelating peptides from casein hydrolysates [J]. Food & Function, 2019, 1 (5): 2372-2381.) and slightly modified. The specific method is as follows:

[0062] The sample (soybean protein hydrolysate, ultrafiltration fraction or each component separated by RP-HPLC) was dissolved in sodium acetate solution (50mM, pH 5.0) to a final concentration of 1mg / mL; then 220μL of the sample solution was added to a 96-well plate, and FeSO4·7H2O was added and shaken, and then reacted at 37°C for 20min; then phenanthroline solution was added to terminate the reaction, and the absorbance value was measured at 562nm after 10min. At the same time, a blank control group was set up, in which only sodium acetate solution was added, and other operations were the same as those of the treatment group.

[0063] 2 Test results

[0064] 2.1 Ultrafiltration separation

[0065] Soy protein isolate was enzymatically hydrolyzed by papain to obtain soybean protein hydrolysate. In order to evaluate the effect of different molecular weights on the iron chelating activity of soybean peptides, we used a 3kDa ultrafiltration membrane to separate the soybean protein hydrolysate into two components and compared their activity differences. Figure 1 As shown in the figure, at the same concentration, all components can chelate iron, and the iron chelation ability of the <3kDa group is the strongest, reaching 38.83%. These results show that the active peptides with lower molecular weight in soybean protein hydrolysate have stronger iron binding ability.

[0066] 2.2 RP-HPLC separation

[0067] RP-HPLC is widely used for separation and purification of active peptides, mainly using the difference in hydrophobicity of active peptides. In order to obtain soybean protein peptides with strong iron chelating activity, the ultrafiltration fraction <3kDa was further separated. After the ultrafiltration fraction <3kDa was separated by RP-HPLC, 22 fractions were obtained, labeled F1-F22 (such as Figure 2 A), and the iron chelating activity of each component was measured respectively. The results are shown in Figure 2 As shown in B. Among them, F3 has the strongest iron chelating activity, reaching 91.91%, which is 2.37 times that of the ultrafiltration fraction with the same concentration <3kDa. The results show that RP-HPLC can effectively separate the active peptides in soy protein and obtain highly active soy protein iron chelating peptides. Therefore, the F3 component was enriched for mass spectrometry analysis to determine its peptide information.

[0068] Example 2 Identification and Molecular Docking Screening of Soy Protein Iron Chelating Peptides

[0069] 1 Test method

[0070] 1.1 LC-MS / MS identification of amino acid sequence of iron chelating peptide

[0071] 1.1.1 Pre-processing

[0072] The liquid phase component F3 with the best iron chelating activity in Example 1 was freeze-dried and dissolved in a washing buffer (0.1% FA (formic acid), 2% CAN (acetonitrile)). Subsequently, desalting was performed using a C18 desalting column, and then the sample was eluted into a new centrifuge tube using an elution buffer (0.1% FA, 60% CAN), and the above percentages are all volume percentages. Finally, centrifugation, concentration and drying were performed to complete the pretreatment.

[0073] 1.1.2 LC-MS / MS analysis

[0074] After desalting, the sample was redissolved in Nano-LC mobile phase A (0.1% formic acid / water, volume percentage), and then loaded for online LC-MS analysis. The specific method is as follows:

[0075] The sample was loaded onto a nanoViper C18 precolumn (3 μm) at a loading volume of 2 μL, and then separated by an analytical column after washing and desalting. The program was set as follows: gradient elution: mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 60 min, and the above percentages are all volume percentages; spray voltage: 1.9 kV; ion transfer tube heating temperature: 275 ° C; mass spectrometry scanning mode: information-dependent acquisition working mode (DDA, Data Dependent Analysis), primary mass spectrometry scanning resolution: 70000; scanning range: 100-1500 m / z; maximum injection time: 100 ms; secondary mass spectrometry ion maximum injection time: 50 ms; collision cell energy (high energy collision induced dissociation, HCD): 28 eV, applicable to all precursor ions; dynamic exclusion setting: 6 s.

[0076] 1.1.3 Database search and result analysis

[0077] The original raw spectrum files collected by LC-MS / MS were processed and searched using PEAKS Studio 8.5 (Bioinformatics Solutions Inc. Waterloo, Canada). The database used was the Glycine max species protein database downloaded by Uniprot. The search parameters were set as follows: the mass tolerance of the primary mass spectrometry was 10 ppm, and the secondary mass spectrometry was 0.05 Da.

[0078] 1.2 Molecular docking screening of highly active iron-chelating peptides

[0079] Using Discovery Studio 2019 software, a three-step docking method was used to screen out monomer peptides with strong iron chelating activity (Ding X, Li H, Xu M, et al. Peptide composition analysis, structural characterization, and prediction of iron binding modes of small molecular weight peptides from mung bean [J]. Food Research International, 2024, 175: 113735.). A 3D model of the small molecule peptide was established in Discovery Studio, and the ligand structure was optimized and energy minimized. Fe 2+ The 3D structure of was downloaded from the PUBCHEM database and imported. Subsequently, the binding site was defined, and the Dock Ligands (CDOCKER) protocol tool was used for molecular docking and calculation of binding energy.

[0080] 2 Experimental results

[0081] 2.1 Identification and molecular docking screening of soybean protein iron chelating peptides

[0082] The liquid phase component F3 was identified by LC-MS / MS, and its total ion current was as follows: Figure 3 According to -10lgP ≥ 40 and peptide chain length ≤ 10, a total of 104 monomeric peptides without modification groups were identified.

[0083] Molecular docking technology, as a theoretical simulation method based on bioinformatics, is widely used to predict the interaction between ligands and receptors. Computer simulation can be used to explore the interaction between metal ions and iron chelating peptides and predict their binding ability and stability. 2+ After docking, the top four molecules with the strongest binding energies that have not been reported are shown in Table 1.

[0084] Table 1 The top four iron-chelating peptides with the lowest molecular docking binding energy

[0085]

[0086] 2.2 Prediction of peptide iron binding sites and interaction analysis

[0087] We selected the four iron-chelating peptides with the strongest binding energy and which have not been reported (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR) for further study. The molecular docking results are shown in Figure 4 Among them, EDEPFNLRSR has the lowest iron binding energy (-91.7953 kcal / mol), indicating that according to the prediction results, the EDEPFNLRSR-iron chelate structure is the most stable. 2+ The binding site between them is located at Asn-6 on the peptide chain, and the Fe-O bond length in the EDEPFNLRSR-iron chelate is Asn-6 and Fe 2+ Forming a metal-receptor interaction, Leu-7 and Fe 2+ At the same time, EDEPFNLR, DDLERLKKIR and EEQPLEVR interact with Fe 2+ The binding sites of EDEPFNLR and DDLERLKKIR are located at Glu-3, Glu-4 and Gln-3, and all of them form metal-receptor interactions. Among them, Glu-3 and Glu-4 on the peptide chains of EDEPFNLR and DDLERLKKIR also interact with Fe 2+ It is noteworthy that EDEPFNLR and DDLERLKKIR interact with Fe 2+ The binding mode is called the "bidentate" mode, that is, Fe 2+ binds to the two oxygen atoms of the carboxylic acid group; whereas EDEPFNLRSR and EEQPLEVR bind to Fe 2+ Combined in "single tooth" mode.

[0088] In general, the iron binding sites on soy protein iron chelate peptides are mainly located at Glu, Gln and Asn, and the Fe-O bond length is The main types of interactions between ligands and receptors are receptor-metal interaction, van der Waals force and charge interaction. The results showed that Glu, Gln and Asn played an important role in the binding of soybean protein iron chelate peptide to iron.

[0089] Example 3 Synthesis of Monomeric Peptides

[0090] The monomer peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR, EEQPLEVR) screened in Example 2 were synthesized by solid phase synthesis using the Fmoc synthesis principle. The monomer peptides in this study were synthesized by Nanjing Jie Peptide Co., Ltd. (Nanjing, China). LC-MS was used to determine that the purity of the monomer peptides was above 98%.

[0091] Example 4 Iron chelating ability of soybean protein iron chelate peptide

[0092] 1 Test method

[0093] The determination of monomeric peptide iron chelating ability was based on the method of Hu et al. (Hu S, Lin S, Wang D, et al. Antarctic krill-derived peptides with consecutive Glu residues enhanced iron binding, solubility, and absorption [J]. Food & Function, 2021, 12 (18): 8615-8625.) and slightly modified. The specific method is as follows:

[0094] The powder of the samples (enzyme hydrolysate, ultrafiltration fraction <3kDa, soy protein iron chelate peptides EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR) was dissolved in a FeSO4·7H2O solution containing 1mM Na2S2O4 (to avoid oxidation of iron ions) (the molar ratio of monomer peptide to iron was 1:2, and the final concentration of monomer peptide was 1mg / mL), and allowed to react at pH 6.0 and 25°C for 1h; after the reaction was completed, 9 times the volume of ethanol solution was added, the mixture was shaken thoroughly and allowed to stand for 60min; then centrifuged at 4500rpm for 20min; after washing 3 times, the precipitate was collected and freeze-dried to obtain the peptide iron chelate, and its iron content was determined by flame atomic absorption spectrophotometry.

[0095] 2 Test results

[0096] In order to evaluate the iron chelating ability of four monomeric peptides screened by molecular docking technology, peptide iron chelates were prepared in this example, and the iron content per mg of the chelate was determined. Figure 5As shown in the figure. The iron contents in the hydrolysate, ultrafiltration fraction (less than 3 kDa), and the chelate of soybean protein iron chelate peptides EDEPFNLRSR, EDEPFNLR, DDLERLKKIR, and EEQPLEVR were 85.20, 122.06, 129.75, 68.82, 110.08, and 80.94 μg / mg, respectively. The iron chelating ability was EDEPFNLRSR>ultrafiltration fraction (<3 kDa)>DDLERLKKIR>hydrolysate>EEQPLEVR>EDEPFNLR. Among them, EDEPFNLRSR, which had the lowest molecular docking iron binding energy, had the strongest iron chelating ability. In addition, although EDEPFNLRSR and EDEPFNLR have very similar structures, they have different iron chelating abilities. This may be because, in addition to the important role of amino acid type and sequence in the iron chelating ability of active peptides, the spatial effect, that is, the spatial distribution of amino acid functional groups, also affects the iron binding ability of active peptides. The increase or decrease of amino acids may affect the folding mode, three-dimensional structure and hydrophobic / hydrophilic properties of the peptide chain, and thus affect the spatial effect of the active peptide. The spatial effect of the active peptide is one of the bases for the interaction between the peptide and various substances. Therefore, although the amino acid sequences of EDEPFNLRSR and EDEPFNLR are very similar, there are differences in their iron chelating ability. At the same time, we also observed that the iron binding capacity of the ultrafiltrate is second only to EDEPFNLRSR and is higher than the other three monomeric peptides. This may be due to the synergistic effect of the peptide mixture in the ultrafiltrate.

[0097] Example 5 Study on cytotoxicity of soy protein iron chelate peptide and iron transport and retention in the intestine

[0098] 1. Test methods

[0099] 1.1 Cytotoxicity assay

[0100] 1.1.1 Cell culture

[0101] The Caco-2 cells used in the experiment were purchased from American Type Culture Collection (Rockville, MD, USA), with a cell generation of 20-30; after the cells were inoculated into culture bottles, they were placed in an incubator (37°C, 5% CO2) and cultured, with the medium changed every two days. The complete culture medium used was formulated as follows: 79% EMEM, 1% penicillin-streptomycin solution, and 20% fetal bovine serum. Caco-2 cells generally grow in about 3-5 days. When the cell coverage rate reaches 80-90%, they are digested with a 0.25% pancreatic enzyme solution for 3-5 minutes, and then the cells are blown off for passage and plating.

[0102] 1.2.2 MTT experiment

[0103] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) toxicity test was used to evaluate the effects of EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR on the survival rate of Caco-2 cells. The specific method is as follows:

[0104] In a 96-well plate, 5 × 10 4 The cells were inoculated at a density of 100 cells / well, and the complete medium was removed after incubation for 24 hours; then different final concentrations of EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR (0.05, 0.1, 0.2, 0.25, 0.5, 1 mg / mL) were added and incubated for another 24 hours; then, the sample was aspirated, 100 μL of 0.5 mg / mL MTT solution was added, and the cells were placed in an incubator for incubation for 4 hours, and then replaced with DMSO; after oscillation for 10 minutes, the absorbance was measured at 490 nm, and the cell survival rate was calculated.

[0105] 1.2 Iron transport and retention of soy protein iron chelate peptide in the intestine

[0106] Construction of Caco-2 small intestinal epithelial cell model: Caco-2 cells were cultured at 1×10 5 The cells were seeded at a density of 1000 Ω / well in a 12-well Transwell insert plate (3460, Corning, USA) and incubated for 21 days. During this period, the medium was changed every other day, and the transepithelial electrical resistance (TEER) was measured on days 8, 16, and 21 to ensure the integrity of the cell monolayer. Only cells with a resistance exceeding 500 Ω / cm 2 The cell monolayer can be used for subsequent iron transport and retention experiments.

[0107] 1.2.1 Iron transport assay

[0108] The iron transport experiment was performed according to our previously published method (Lao L, He J, Liao W, et al. Case in calcium-binding peptides: preparation, characterization, and promotion of calcium uptake in Caco-2 cell monolayers [J]. Process Biochemistry (1991), 2023, 130: 78-86.) with slight modifications. The specific method is as follows:

[0109] Thoroughly mix EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR with the pre-prepared FeCl2·4H2O (Fe 2+The final concentration is 80 μg / mL, and the final concentration of the monomer peptide is 568 μg / mL), and the mixture is reacted in an incubator for 1 h to obtain a peptide-iron mixture;

[0110] Before the formal start of the transport experiment, the medium of the Transwell nested plate was changed and the nested plate was transferred to a new 12-well plate. Then, the peptide iron mixture and the control group (only FeCl2·4H2O) were added to the top of the Transwell nested plate; after 30, 60, 120 and 180 minutes of transport, 1 mL of culture medium was collected from the basolateral side to determine the amount of iron transport (graphite furnace atomic absorption spectrometry), and 1 mL of culture medium was added to keep the basolateral volume unchanged.

[0111] 1.2.2 Monitoring cellular iron retention by fluorescence microscopy

[0112] Before the experiment, DMSO was added to the centrifuge tube containing the fluorescent probe FerroOrange and mixed by pipetting to prepare a 1mmol / L FerroOrange solution, which was then diluted to 1μmol / L with HBSS. After the experiment officially started, the culture medium was aspirated and the Transwell nested plate and cells were washed 3 times with HBSS. Then, the pre-mixed peptide iron mixture (Fe 2+ The final concentration was 80 μg / mL, and the final concentration of the monomer peptide was 568 μg / mL. The specific preparation method refers to the iron transport experiment) and the control group (only FeCl2·4H2O was added). After 60 min and 180 min of sample treatment, the sample solution was aspirated, and then 1 μmol / L of FerroOrange working solution was added to the top, and the Transwell plate was placed in a 37 ° C, 5% CO2 incubator for 30 min (Ye S, Xu M, Zhu T, et al. Cytoglobin promotes sensitivity to ferroptosis by regulating p53-yap1 axis in colon cancer cells [J]. Journal of Cellular and Molecular Medicine, 2021, 25 (7): 3300-3311.). The cells were then observed under a fluorescence microscope and photographed. The resulting fluorescent photos were statistically analyzed with the help of Image J software.

[0113] 2. Test results

[0114] Iron absorption mainly occurs in the duodenum and upper jejunum, so studying the behavior of soy protein iron chelate peptides in promoting the transport, retention and absorption of iron in the intestine is of great significance for the development of iron supplements.

[0115] 2.1 Cytotoxicity analysis

[0116] In this study, MTT colorimetry was used to evaluate the effect of active peptides on the survival rate of Caco-2 cells. Figure 6 As shown, the survival rate of Caco-2 cells treated with ultrafiltration fraction (<3 kDa), EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR was the lowest at 97.39% (>90%), indicating that the active peptide samples had no toxic effect on Caco-2 cells and did not affect the normal proliferation of cells.

[0117] 2.2 Effect of soybean protein iron chelate peptide on iron transport in Caco-2 cells

[0118] Caco-2 is a human colon adenocarcinoma cell line. After differentiation, its structure and function are very similar to those of intestinal cells and are often used in the study of intestinal uptake and transport of minerals. In this example, after successfully constructing the Caco-2 small intestinal epithelial cell model, each group of samples was applied to the apex and the iron content on the basolateral side was measured at various time points. Figure 7 As shown in the figure, the total iron transport of each group continued to increase with the increase of time. After 180 minutes of transport, compared with the control group (total iron transport: 14.62 μg / well), the total iron transport of the ultrafiltration fraction (<3kDa), iron chelating peptide EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR groups was significantly increased (P<0.05), which were 19.72, 24.76, 18.93, 22.54 and 22.13 μg / well, respectively, and increased by 34.88%, 69.36%, 29.48%, 54.17% and 51.37%, respectively, all showing good ability to promote iron transport.

[0119] The order of iron transport ability is EDEPFNLRSR>DDLERLKKIR>EEQPLEVR>ultrafiltration component (<3kDa)>EDEPFNLR. Interestingly, this trend is very similar to the trend of iron chelation ability (EDEPFNLRSR>ultrafiltration component (<3kDa)>DDLERLKKIR>EEQPLEVR>EDEPFNLR). It is worth noting that although the iron chelation ability of DDLERLKKIR and EEQPLEVR is not as good as that of the ultrafiltration component (<3kDa), their iron transport ability is stronger than that of the ultrafiltration component (<3kDa). This may be because molecular weight is an important factor affecting the iron transport ability of active peptides. According to research reports, when exogenous iron is added, iron chelating peptides with a molecular weight of <1kDa are more conducive to promoting the absorption of iron ions in the intestine. The molecular weights of DDLERLKKIR and EEQPLEVR are 1284.7513Da and 998.5032Da, respectively, while the molecular weight distribution of the ultrafiltration fraction is 0-3kDa. The absorption modes of small molecule oligopeptides in intestinal epithelial cells are mainly: (1) peptide transporter 1 pathway (carrier of dipeptides and tripeptides), (2) endocytosis (intracellular vesicle-mediated transport system), and (3) paracellular passive diffusion. Moreover, after the active peptides bind to iron, some iron ions can also be absorbed through the peptide absorption pathway. As oligopeptides, DDLERLKKIR and EEQPLEVR have relatively small molecular weights. After binding to iron, in addition to increasing the solubility of ferrous ions, they also increase the absorption pathways of iron, thus increasing the bioavailability of iron.

[0120] 2.2 Effect of soy protein iron chelate peptide on iron retention in Caco-2 cells

[0121] Studying the amount of iron retained in cells is helpful to understand the effect of iron chelating peptides on improving the bioavailability of iron in small intestinal epithelial cells and their effects on iron transport pathways. Therefore, in this study, EDEPFNLRSR, which has the strongest iron transport activity, was selected as a representative to further study its effect on Fe 2+ In the process of intestinal transport and absorption, for iron ions, Fe 3+ Must first be reduced to Fe by a reducing agent such as duodenal pigment b 2+ After that, it is internalized by intestinal cells through the apical divalent metal transporter 1 (DMT 1). After the sample is treated with Caco-2 cells for a certain period of time, the fluorescent probe is added and the Fe 2+ Fluorescence imaging was performed, and the results were Figure 8 As shown. It was found that with the increase of time, the Fe 2+ Compared with the control group, the intracellular Fe2+ The fluorescence intensity of EDEPFNLRSR increased by 17.23% and 113.10%, respectively. These results show that EDEPFNLRSR promotes the 2+ retained in Caco-2 cells. It is possible that they interact with the cell membrane to promote the uptake and transport of iron ions to the cytoplasm. The absorption pathways of iron mainly include the paracellular pathway (a non-saturated diffusion mode) and transcellular transport. Among them, cellular transport mainly involves two mechanisms: vesicle transport and endocytosis. Many low molecular weight iron chelating peptides and their peptide iron chelates can be absorbed by endocytosis. Therefore, it is possible that EDEPFNLRSR promotes the relative expression level of DMT 1, thereby promoting Fe 2+ Entering intestinal cells through DMT 1; or EDEPFNLRSR-iron chelate may specifically bind to receptors on the cell surface and enter the cell through endocytosis in the form of vesicles. The specific mechanism needs to be further studied.

[0122] Example 6 Transmembrane transport of soy protein iron chelate peptide in Caco-2 cells

[0123] 1. Test methods

[0124] Understanding the transmembrane transport behavior of iron chelating peptides is helpful to understand the ways in which iron chelating peptides promote iron transport and absorption. Therefore, the monomeric peptide (EDEPFNLRSR) with the strongest ability to promote iron transport was selected as a representative to further study its transmembrane transport in the small intestinal epithelial cell monolayer. The preliminary treatment method for transmembrane transport of iron chelating peptides is the same as the iron transport test in Example 5 1.2.1, and the apical and basolateral solutions are collected separately for analytical reversed-phase high-performance liquid chromatography detection and mass spectrometry identification.

[0125] 1.1 Monitoring the transmembrane transport of monomeric peptides in liquid phase

[0126] The collected apical and basolateral solutions were filtered through a 0.22 μm filter membrane and then detected by LC-10A analytical high performance liquid chromatography (C 18 column, 5 μm, 4.6 mm×250 mm). The parameters were set as follows: mobile phase A: distilled water (containing 0.1% trifluoroacetic acid); mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid); detection wavelength: 214 and 280 nm; injection volume: 20 μL; gradient elution: 0.01-3 min: 10%-25% mobile phase B, 10-15 min: 30%-35% mobile phase B; 18-20 min: 50%-70% mobile phase B, 22-32 min: 90% mobile phase B, and the above percentages are all volume percentages.

[0127] 1.2 Mass spectrometry analysis of iron chelating peptide transmembrane transport

[0128] The apical and basolateral solutions transported for 180 min were selected to further study the permeation and degradation of the iron chelating peptide in the small intestinal epithelial cell model. The specific method is as follows:

[0129] (1) After desalting with a C18 desalting column, the samples were analyzed by LC-MS / MS (ThermoFisher Scientific, MA, USA) equipped with an online nanospray ion source. Gradient elution started with 4% mobile phase B (acetonitrile), increased to 50% at 53.6 min, increased to 95% within 40 s, and maintained for 5.6 min. The above percentages are volume percentages. The parameters were set as follows: sample volume: 3 μL (analytical column: Acclaim PepMap C18, 75 μm x 25 cm); column flow rate: 300 nL / min; column temperature: 40°C; electrospray voltage: 2 kV; MS scan range: 350-1500 m / z; resolution: 120000; Normalized AGCtarget (MS): 200%; maximum injection time: 50 ms; resolution: 15000; Normalized AGC target (HCD-MS / MS): 100%; maximum injection time: 25 ms; collision energy: 25%, 30%, 35%; dynamic exclusion time: 30 s.

[0130] (2) Database search: Tandem mass spectra were analyzed using PEAKS Studio software. The search parameters were as follows: fragment ion mass tolerance: 0.02 Da; parent ion mass tolerance: 10 ppm; protein card value: at least one unique peptide; peptide card value: -101 gP ≥ 20.

[0131] 2. Test results

[0132] Iron chelating peptides are important carriers for iron ions to enter intestinal cells, and it is of great significance to understand their absorption and metabolic mechanisms in the intestine. Various peptidases during transportation, such as brush edge membrane peptidases at the top of intestinal epithelial cells and cytosol hydrolases in intestinal cells, catalyze the digestion of bioactive peptides into smaller peptides and amino acids, affecting their degradation and absorption. Therefore, in this study, EDEPFNLRSR, which has the strongest iron transport activity, was selected as a representative to study its degradation and permeation absorption in the monolayer of intestinal epithelial cells.

[0133] 2.1 Liquid phase detection of transmembrane transport of soybean protein iron chelate peptide

[0134] RP-HPLC was used to analyze the top and basolateral solutions of the Transwell plate after a certain period of transport. The liquid phase results are as follows: Fig. 9As shown. In the chromatogram of the top solution, EDEPFNLRSR has an absorption peak at about 9.45min of elution. As the transport time increases, the peak area of ​​the absorption peak continues to decrease, and after 180min of transport, the peak area drops to 70.85%. Combined with the mass spectrometry results in Table 2, it was found that the abundance of EDEPFNLRS was the highest after 180min of transport. This may be because EDEPFNLRSR is degraded after the action of brush edge membrane peptidase. However, since the detection principle of HPLC is mainly based on hydrophobic interactions, reducing a polar amino acid, namely arginine (R), will not change the overall hydrophobic characteristics. Therefore, after EDEPFNLRSR is degraded into EDEPFNLRS, the corresponding liquid phase spectrum has no obvious changes in peak time and peak shape. In addition, after 30min of transport, new absorption peaks began to appear in the liquid phase spectrum, which may be due to the degradation of EDEPFNLRSR into new fragments.

[0135] The liquid chromatograms of the basolateral solution after 30, 60, 120 and 180 min of transport are shown in Figure 2. Fig. 9 As shown in Figure B. It can be found that after 30 minutes of transport, new absorption peaks (elution peaks 1 and 2) appeared, and the peak area gradually increased with the increase of transport time. This shows that as the transport time increases, the derivative peptides after EDEPFNLRSR degradation are transported to the basolateral side and continue to accumulate.

[0136] 2.2 Mass spectrometry analysis of transmembrane transport of soybean protein iron chelating peptide

[0137] The apical solution and basolateral solution were collected after 180 minutes of transport for mass spectrometry identification, and the results are shown in Tables 2 and 3. A total of 18 monomeric peptides were identified in the apical solution, and a total of 24 monomeric peptides were identified in the basolateral solution, including 18 peptides in the apical solution. This indicates that the 24 derivative peptides after EDEPFNLRSR degradation can be completely absorbed in the small intestinal epithelial cells. According to the peptide peak area, it was found that the abundance of EDEPFNLRS was the highest in both the apical and basolateral solutions, indicating that the structure of EDEPFNLRS (1105.5024Da) is relatively stable and not easily degraded. And because of the relatively high molecular weight, it may be Fe 2+ The main carrier that enters the intestinal cells through endocytosis. In addition, we noticed that the degradation of EDEPFNLRSR mainly occurred in the two segments of the peptide chain, while according to the prediction results of molecular docking, its iron binding site was mainly located at Asn-6 in the middle of the peptide chain. Therefore, under the action of brush edge membrane peptidases, the structure of the peptide iron chelate may not be destroyed and can still promote iron transport in Caco-2 cell monolayers.

[0138] It is worth noting that EDEPFNLR was detected in both the apical solution and the basolateral solution. This shows that EDEPFNLR is generated after the degradation of EDEPFNLRSR, and EDEPFNLR can be transported intact to the basolateral side. And EDEPFNLR is one of the four new iron chelating peptides we screened and synthesized. According to the iron transport experiment, the total iron transport amount of the EDEPFNLR group was 16.09 μg / well after 60 minutes of transport, which was an increase of 87.57% compared with the control group. These results show that the peptides produced by the degradation of EDEPFNLRSR and enzymes, such as EDEPFNLR, also have the ability to promote iron transport and are Fe 2+ It is an important carrier for entering intestinal cells. Based on this, we infer that among the four isolated monomers, EDEPFNLRSR has the strongest ability to promote iron transport, which may be due to: (1) EDEPFNLRSR has the strongest iron binding ability, which increases the Fe 2+ Solubility at the top of the intestinal epithelial cells, effectively promoting Fe 2+ Entering into intestinal cells through the cell apex; (2) EDEPFNLRSR and Fe 2+ After binding, EDEPFNLRSR-Fe chelate is formed, which is hydrolyzed into EDEPFNLRS after the action of brush edge membrane peptidase at the top of the small intestinal epithelial cell model. It enters the Caco-2 cell monolayer intact through endocytosis in the form of EDEPFNLRS-Fe complex, promoting iron absorption; (3) Peptides degraded after enzyme action, such as EDEPFNLR, also have the ability to promote iron transport.

[0139] Table 2 Peptide sequences identified from the apical solution

[0140]

[0141] Table 3 Peptide sequences identified from basolateral solution

[0142]

[0143]

[0144] In this study, soybean protein was used as raw material, and four new iron chelating peptides, EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR, were screened out through enzymatic hydrolysis, purification identification and molecular docking technology. Through the Caco-2 small intestinal epithelial cell model, it was found that EDEPFNLRSR, EDEPFNLR, DDLERLKKIR and EEQPLEVR could significantly promote the transport and absorption of iron. For EDEPFNLRSR, which had the strongest iron chelating ability and the activity of promoting iron transport, its intestinal iron retention activity and the transmembrane transport behavior of the peptide itself were further studied. It was found that EDEPFNLRSR significantly promoted Fe 2+ Retention in the Caco-2 cell monolayer. The new iron chelate peptide screened from soybean protein hydrolysate is expected to become a safe and effective iron supplement. In subsequent studies, we will conduct in-depth research on its molecular mechanism of enhancing intestinal iron transport, and verify the iron transport and absorption activity of soybean protein iron chelate peptide in animal experiments.

[0145] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An iron chelating peptide, characterized in that Its amino acid sequence is at least one of the following sequences a) to d): a)EDEPFNLRSR; b)EDEPFNLR; c)DDLERLKKIR; d)EEQPLEVR.

2. The method for preparing the iron-chelating peptide according to claim 1, characterized in that The following steps are included: The iron chelate peptide is prepared by solid phase synthesis or by using soy protein isolate as raw material and hydrolyzing and purifying it by papain.

3. The method for preparing the iron-chelating peptide according to claim 2, wherein: The specific operation of the papain enzymolysis: The soy protein isolate solution and papain are mixed for enzymatic hydrolysis, and then inactivated in a water bath, cooled, centrifuged, and the supernatant is collected and further freeze-dried to obtain a soy protein hydrolysate.

4. The method for preparing the iron-chelating peptide according to claim 2, wherein: The purification comprises ultrafiltration separation and liquid chromatography separation steps.

5. Use of the iron chelating peptide according to claim 1 in the preparation of an iron supplement.

6. Use of the iron chelating peptide according to claim 1 in the fields of food, medicine and health care products.

7. An iron supplement, characterized by comprising at least one of the iron chelate peptide according to claim 1, a papain hydrolyzate of soy protein isolate containing the iron chelate peptide according to claim 1, and a hydrolyzate containing the iron chelate peptide according to claim 1 as an active ingredient.

8. The iron supplement according to claim 7, characterized in that: The iron supplement further comprises a pharmaceutically acceptable carrier or auxiliary material.

9. The iron supplement according to claim 8, characterized in that: The pharmaceutically acceptable carrier or excipient includes at least one of a cosolvent, a moisturizer, a surfactant, a matrix, an emulsifier, a preservative and a solvent.

10. A food, medicine or health product, characterized by comprising at least one of the iron chelate peptide according to claim 1, a papain hydrolyzate of soy protein isolate containing the iron chelate peptide according to claim 1, and a hydrolyzate containing the iron chelate peptide according to claim 1 as an active ingredient.

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