An iron-chelating peptide, its preparation method and use in preparing an iron supplement

Abstract

This invention belongs to the field of bioactive peptide preparation, specifically relating to an iron chelating peptide, its preparation method, and its application in the preparation of iron supplements. This study used soybean protein as raw material and screened four iron chelating peptides—EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR—through enzymatic hydrolysis, purification, identification, and molecular docking techniques. Using a Caco-2 small intestinal epithelial cell model, it was found that all of the above iron chelating peptides significantly promoted iron transport and absorption, and significantly promoted Fe2+ absorption. 2+ Retention in the Caco-2 cell monolayer. The novel iron chelating peptides screened from soybean protein hydrolysates in this invention hold promise as a safe and effective iron supplement.

Description

An iron chelating peptide, its preparation method, and its application in the preparation of iron supplements. Technical Field

[0001] This invention belongs to the field of active peptide preparation, specifically relating to an iron chelate peptide, its preparation method, and its application in the preparation of iron supplements. Background Technology

[0002] Iron is a vital trace element for the human body, playing a central role in many biological processes, including oxygen transport, DNA synthesis, and electron transport. Nutritional iron deficiency occurs when the iron absorbed from the daily diet is insufficient to meet physiological needs. In severe cases, it can reduce work capacity, impair physical function, affect children's cognitive development, and delay motor development.

[0003] Bioactive peptides are specific protein fragments composed of amino acids linked by covalent bonds, which have a positive impact on bodily functions and conditions. In recent years, an increasing number of food-derived bioactive peptides with iron-chelating activity have been reported, and their significant role in promoting intestinal iron transport and absorption has been found. Iron-chelating peptides and their peptide-iron chelates are becoming a hot topic of attention and research. For example, Wu et al. (WuW,Li B,HouH,etal.Identification of iron-chelating peptides from pacific cod skin gelatin and the possible binding mode[J].Journal of Functional Foods,2017,35:418-427.) purified enzymatic hydrolysates of Pacific cod skin gelatin using immobilized metal affinity chromatography and RP-HPLC, and identified three novel iron-chelating peptides by LC-HRMS / MS: GPAGPHGPPGKDGR, AGPHGPPGKDGR, and AGPAGPA. These peptides exhibit iron-chelating activity against Fe. 2+It exhibits 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.) obtained high affinity from tilapia skin collagen hydrolysate by immobilized metal affinity chromatography (IMAC-Fe 2+ Iron chelate peptides from tilapia skin collagen were obtained. After RP-HPLC purification, GLPGPSGEEGKR identified in the peptides increased the iron content in Caco-2 cells. 2+ Bioavailability.

[0004] When a large number of peptide sequences are identified from isolated and purified fractions, rapid screening becomes a major challenge. Current screening methods for metal-chelating peptides primarily rely on extensive 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 isolated and purified mung bean protein fractions. They selected and synthesized 10 peptide sequences with high leucine, isoleucine, and aspartic acid content, finding 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 a nanocarrier of iron through the gastrointestinal tract[J]. Food Bioscience, 2020, 36: 100657.) identified more than 1000 peptide sequences from Antarctic krill iron chelate peptides and focused on three peptide sequences: all containing three glutamic acid residues, but with different positions of the glutamic acid residues on the peptide chain. Therefore, they synthesized these three bioactive peptides and found that they all possessed strong iron chelating activity, but with differences. With the development of small molecule bioactive peptides and the interdisciplinary application of computer science and bioinformatics, more and more studies are applying 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 umamipeptides from chicken soup by molecular docking[J]. Food Chemistry, 2023, 404(A): 134414.) used molecular docking technology to screen 20 potential umami peptides from 208 peptides identified in chicken soup, among which PPQEAAQF had a high 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 four ACE-inhibiting peptides from 710 peptide sequences identified in douchi using molecular docking technology. In the study of metal chelate peptides, molecular docking technology is often used to study the binding sites and interactions between metals and bioactive peptides; however, there are few reports on the use of molecular docking technology to assist in the rapid screening of bioactive peptides.

[0005] Soybeans are the most widely cultivated legume and oilseed crop globally, with abundant resources and a high protein content (40-50%). Processing soybean protein through enzymatic hydrolysis and other methods helps reduce the content of anti-nutrients. Furthermore, the released soybean protein peptides possess various biological activities, such as lipid-lowering activity, antioxidant activity, and antihypertensive activity. Summary of the Invention

[0006] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide an iron chelating peptide.

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

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

[0009] The objective of this invention is achieved through the following technical solution:

[0010] An iron chelating peptide having an amino acid sequence of 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 chelate peptide includes the following steps:

[0016] Iron chelates can be prepared by solid-phase synthesis or by enzymatic hydrolysis and purification of soy protein isolate using papain.

[0017] The preferred specific operation for the papain enzymatic hydrolysis is as follows:

[0018] Soy protein isolate solution and papain were mixed for enzymatic hydrolysis, then inactivated in a water bath, cooled, centrifuged, and the supernatant was collected and further freeze-dried to obtain soy protein hydrolysate;

[0019] The preferred percentage of soy protein isolate in the soy protein isolate solution is 7% by mass.

[0020] The preferred conditions for enzymatic hydrolysis are: enzymatic hydrolysis at pH 5.5 and 60°C for 3.5 hours;

[0021] The enzyme substrate content of the enzymatic hydrolysis is 0.25% (m / m).

[0022] The preferred conditions for water bath inactivation are 95°C water bath for 10 minutes;

[0023] The purification process includes steps such as ultrafiltration and liquid chromatography.

[0024] The specific operation of ultrafiltration separation is as follows:

[0025] Soybean protein hydrolysates were separated using ultrafiltration tubes, and fractions with molecular weight <3kDa were collected.

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

[0027] High-performance liquid chromatography (HPLC) was used to separate and purify the <3kDa ultrafiltration fraction of soybean protein hydrolysate using a Prep150 preparative liquid chromatography system; the active fraction was collected, freeze-dried, and iron chelate peptides were obtained.

[0028] The preferred conditions for the liquid chromatography are:

[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, all percentages are volume percentages;

[0030] Application of the iron chelating peptide in the preparation of iron supplements;

[0031] The application of the iron chelate peptide in the fields of pharmaceuticals and health products;

[0032] An iron supplement comprising at least one of the following as an active ingredient: the iron chelate peptide, a soy protein isolate containing the iron chelate peptide, a papain hydrolysate, and a hydrolysate containing the iron chelate peptide.

[0033] The iron supplement also contains a pharmaceutically acceptable carrier or excipient;

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

[0035] A pharmaceutical or health product comprising at least one of the following as an active ingredient: the above-mentioned iron chelate peptide, a soy protein isolate containing the above-mentioned iron chelate peptide, and a hydrolysate containing the above-mentioned iron chelate peptide.

[0036] The present invention has the following advantages and effects compared with the prior art:

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

[0038] (2) This invention further investigated the iron-chelating capacity of the screened iron chelating peptides and their iron-promoting and retention activities in a Caco-2 cell monolayer model. The results showed that in the Caco-2 small intestinal epithelial cell model, after 180 min of transport, the total iron transport of the four iron chelating peptides increased by 69.36%, 29.48%, 54.17%, and 51.37% respectively compared to the control group, with EEPPFNLRSR showing the strongest activity. Further research was conducted on EEPPFNLRSR, which exhibited the strongest iron-promoting activity. It was found that compared to the control group, the Fe concentration in the Caco-2 cell monolayer of the EEPPFNLRSR group was significantly higher. 2+ The retention rate increased by 113.10%, significantly promoting iron absorption. In studies on the transmembrane transport of bioactive peptides, it was found that 24 derived peptides of EDEPFNLRSR, after degradation, could achieve transmembrane transport within the Caco-2 cell monolayer.

[0039] (3) This invention reveals the iron transport-promoting behavior of novel iron chelating peptides in the intestine and their transmembrane transport behavior, providing new information for understanding the mechanism by which bioactive peptides promote intestinal iron absorption and providing a theoretical basis for developing dietary iron supplements using soybean protein. Attached Figure Description

[0040] Figure 1 shows the evaluation results of iron chelating activity of soybean protein hydrolysate and ultrafiltration fraction. Different letters indicate significant differences (p<0.05).

[0041] Figure 2 shows the chromatograms of the ultrafiltration fraction (<3kDa) further separated by RP-HPLC and the results of the iron chelating activity evaluation of each fraction. In the figure, A: RP-HPLC separation chromatogram of the ultrafiltration fraction (<3kDa), B: iron chelating activity evaluation of the liquid phase fractions F1-F22, and different letters indicate significant differences (p<0.05).

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

[0043] Figure 4 shows the relationship between four soybean protein iron chelating peptides (EDEPFNLRSR, EDEPFNLR, DDLERLKKIR, and EEQPLEVR) and Fe. 2+ Analysis of molecular docking results.

[0044] Figure 5 shows the results of iron chelating capacity analysis of enzymatic hydrolysate, ultrafiltration fraction (<3kDa), and soybean protein iron chelating peptides (EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR). Different letters indicate significant differences (p<0.05).

[0045] Figure 6 is an analysis of the results of the cytotoxicity study of ultrafiltration components (<3kDa) and soybean protein iron chelate peptides (EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR).

[0046] Figure 7 shows the results of iron transport at different transport times for ultrafiltration components (<3kDa) and soybean protein iron chelate peptides (EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR). Different letters indicate significant differences (p<0.05).

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

[0048] Figure 9 shows the RP-HPLC chromatograms of EEPPFNLRSR transported across the small intestinal epithelial cell model. In the figure, A: chromatograms of EEPPFNLRSR and the solution at the top of the Transwell plate at each time point, and B: chromatograms of reagent blank (HBSS) and the solution at the outer side of the Transwell plate at each time point. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0050] Materials and reagents used in the examples: Soy protein isolate, Shanghai Yuanye Biotechnology Co., Ltd.; FeCl2·4H2O, Tianjin Baishi Chemical Co., Ltd.; phenanthroxazine and thiazolyl blue (MTT), Shanghai Maclean 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), USA; FerroOrange fluorescent probe was purchased from Dongren Chemical Technology (Shanghai) Co., Ltd. All other chemicals and solvents were analytical grade.

[0051] Data analysis in this embodiment: 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 ANOVA. A difference was considered statistically significant when p < 0.05.

[0052] Example 1: Isolation, purification, and iron chelating activity of soybean protein iron chelating peptides

[0053] 1. Experimental Methods

[0054] 1.1 Preparation of soybean protein hydrolysate

[0055] A certain amount of soy protein isolate was weighed and added to distilled water to obtain a solution with a soy protein isolate content of 7%. The pH of the system was adjusted to 5.5, and papain solution was added at a ratio of 0.25% (m / m) to the enzyme base. The mixture was enzymatically hydrolyzed at 60°C for 3.5 h. The resulting mixture was placed in a 95°C water bath for 10 min to inactivate the protease, and then centrifuged at 4500 rpm for 20 min. The supernatant was collected to obtain the soy protein hydrolysate.

[0056] 1.2 Ultrafiltration Separation

[0057] Soybean protein hydrolysate was separated using an ultrafiltration membrane (3kDa, Merck Millipore, Billerica, MA) to obtain two fractions (>3kDa and <3kDa). The iron chelating activities of the two fractions were then measured to identify the most active fraction for further separation and purification.

[0058] 1.3 RP-HPLC separation

[0059] The ultrafiltrate fraction with good iron chelating activity was filtered through a 0.22 μm filter membrane and then used to prepare a liquid phase system via a Prep150 (reverse phase column: SunFire Prep C18 OBDT). TM Further separation was performed using a 19mm x 250mm, 5μm (Waters) sample. 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 wavelengths: 214 and 280nm; Gradient elution: 0-10min: 10-30% mobile phase B, 30-60min: 60-80% mobile phase B, 70min: 90% mobile phase B (all percentages are volume percentages). Each elution peak was collected, frozen, concentrated, and the iron chelating activity of each component was determined.

[0060] 1.4 Determination of iron chelating activity

[0061] The iron chelating activity was determined using the phenanthroxazine colorimetric method, which was performed with slight modifications based on the method described in the reference (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.). The specific method is as follows:

[0062] The samples (soybean protein hydrolysate, ultrafiltration fractions, or fractions separated by RP-HPLC) were dissolved in sodium acetate solution (50 mM, pH 5.0) to a final concentration of 1 mg / mL. 220 μL of the sample solution was then transferred to a 96-well plate, and FeSO4·7H2O was added and the mixture was shaken and incubated at 37 °C for 20 min. The reaction was then terminated by adding phenanthridine solution, and the absorbance was measured at 562 nm after 10 min of incubation. A blank control group was also included, which received only sodium acetate solution and underwent the same procedures as the treatment group.

[0063] 2. Experimental Results

[0064] 2.1 Ultrafiltration Separation

[0065] Soy protein isolate was hydrolyzed with papain to obtain soy protein hydrolysate. To evaluate the effect of different molecular weights on the iron-chelating activity of soy peptides, we used a 3 kDa ultrafiltration membrane to separate the soy protein hydrolysate into two components and compared their activity differences. The results are shown in Figure 1. At the same concentration, all components could chelate iron, while the <3 kDa group had the strongest iron-chelating ability, reaching 38.83%. These results indicate that the lower molecular weight active peptides in the soy protein hydrolysate have a stronger iron-binding capacity.

[0066] 2.2 RP-HPLC separation

[0067] RP-HPLC is widely used for the separation and purification of bioactive peptides, primarily utilizing the differences in their hydrophobicity. To obtain soybean protein peptides with strong iron-chelating activity, the <3kDa ultrafiltration fraction was further separated. After RP-HPLC separation, 22 fractions were obtained from the <3kDa ultrafiltration fraction, labeled F1-F22 (Figure 2A), and the iron-chelating activity of each fraction was measured, as shown in Figure 2B. Among them, F3 exhibited the strongest iron-chelating activity, reaching 91.91%, which is 2.37 times that of the <3kDa ultrafiltration fraction at the same concentration. The results indicate that RP-HPLC can effectively separate bioactive peptides from soybean protein, obtaining highly active soybean protein iron-chelated peptides. Therefore, fraction F3 was enriched for mass spectrometry analysis to determine its peptide information.

[0068] Example 2: Identification and molecular docking screening of soybean protein iron chelate peptides

[0069] 1. Experimental Methods

[0070] 1.1 Identification of the amino acid sequence of iron chelate peptides by LC-MS / MS

[0071] 1.1.1 Preprocessing

[0072] The liquid fraction F3, which exhibited the best iron-chelating activity in Example 1, was lyophilized and dissolved in washing buffer (0.1% FA (formic acid), 2% CAN (acetonitrile)). Subsequently, desalting was performed using a C18 desalting column, followed by elution with elution buffer (0.1% FA, 60% CAN) to a new centrifuge tube. All percentages are 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] 2 μL of sample was loaded onto a nanoViper C18 pre-column (3 μm), washed and desalted, and then separated by the analytical column. The program settings were as follows: gradient elution: mobile phase B (80% acetonitrile, 0.1% formic acid) increased from 5% to 38% within 60 min (all percentages are volume percentages); spray voltage: 1.9 kV; ion transfer tube heating temperature: 275 °C; mass spectrometry mode: information-dependent acquisition mode (DDA, Data Dependent Analysis); primary mass spectrometry resolution: 70000; scan range: 100-1500 m / z; maximum injection time: 100 ms; secondary mass spectrometry maximum ion injection time: 50 ms; collision chamber energy (high-energy collision-induced dissociation, HCD): 28 eV, applicable to all precursor ions; dynamic exclusion setting: 6 s.

[0076] 1.1.3 Database Retrieval and Result Analysis

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

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

[0079] Using Discovery Studio 2019 software, a three-step docking method was employed to screen monomeric 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.). 3D models of the small peptides were built in Discovery Studio, and the ligand structures were optimized and energy minimization was performed. 2+ The 3D structure was downloaded from the PUBCHEM database and imported. Binding sites were then defined, and molecular docking was performed using the Dock Ligands (CDOCKER) protocol tool to calculate the binding energy.

[0080] 2 Experimental Results

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

[0082] The liquid phase component F3 was identified using LC-MS / MS, and its total ion chromatogram is shown in Figure 3. Based on the criteria of -10lgP ≥ 40 and peptide chain length ≤ 10, a total of 104 monomeric peptides without modified 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 simulations can be used to explore the interaction between metal ions and iron chelate peptides, and to predict their binding affinity and stability. Therefore, we identified 104 iron chelate peptides that interact with Fe... 2+ The top four docking sites with the strongest binding capacity that were not previously reported are shown in Table 1.

[0084] Table 1. The four iron chelating peptides with the lowest molecular docking binding energies.

[0085]

[0086]

[0087] 2.2 Prediction of peptide iron binding sites and interaction analysis

[0088] We selected four iron chelates (EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR) with the strongest binding energy and which had not been previously reported, for further investigation. Their molecular docking results are shown in Figure 4. Among them, EDEPFNLRSR had the lowest iron binding energy (-91.7953 kcal / mol), indicating that, based on the predicted results, the EDEPFNLRSR-iron chelate structure is the most stable. Furthermore, EDEPFNLRSR and Fe... 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 [missing information]. Asn-6 and Fe 2+ Metal-acceptor interactions were formed, with Leu-7 interacting with Fe. 2+ Van der Waals forces were formed between them. Simultaneously, EDEPFNLR, DDLERLKKIR, and EEQPLEVR interacted with Fe. 2+The binding sites are located at Glu-3, Glu-4, and Gln-3, and all of them form metal-acceptor interactions. Specifically, Glu-3 and Glu-4 on the EDEPFNLR and DDLERKKIR peptide chains also bind to Fe. 2+ Charge interactions were formed. Notably, EDEPFNLR and DDLELKKIR interacted with Fe. 2+ The bonding mode is called the "bidental" mode, i.e., Fe 2+ It bonds to the two oxygen atoms on the carboxylic acid group; while EDEPFNLRSR and EEQPLEVR bond to Fe. 2+ Combined in a "single-tooth" mode.

[0089] In general, the iron-binding sites on soybean protein iron chelate peptides are mainly located at Glu, Gln, and Asn, with Fe-O bond lengths ranging from [value missing]. The main types of interactions between ligands and receptors include receptor-metal interactions, van der Waals forces, and charge interactions. The results indicate that Glu, Gln, and Asn play important roles in the binding of soybean protein iron chelating peptides to iron.

[0090] Example 3 Synthesis of Monomeric Peptides

[0091] The monomeric peptides (EDEPFNLRSR, EDEPFNLR, DDLELKKIR, EEQPLEVR) screened in Example 2 were synthesized using the Fmoc synthesis principle and a solid-phase synthesis method. The monomeric peptides in this study were synthesized by Nanjing Jietai Co., Ltd. (Nanjing, China). LC-MS analysis confirmed that the purity of the monomeric peptides was above 98%.

[0092] Example 4: Iron chelating capacity of soybean protein iron chelating peptides

[0093] 1. Experimental Methods

[0094] The determination of the iron chelating capacity of monomeric peptides 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.), with slight modifications. The specific method is as follows:

[0095] The sample powder (enzymatic hydrolysate, <3kDa ultrafiltration fraction, soybean protein iron chelate peptides EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR) was dissolved in FeSO4·7H2O solution containing 1mM Na2S2O4 (to prevent oxidation of iron ions) (molar ratio of monomeric peptide to iron was 1:2, final concentration of monomeric peptide was 1mg / mL), and reacted at pH 6.0 and 25℃ for 1h. After the reaction was completed, 9 volumes of ethanol solution were added, and the mixture was shaken thoroughly and allowed to stand for 60min. Then it was 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.

[0096] 2. Experimental Results

[0097] To evaluate the iron-chelating ability of four monomeric peptides screened using molecular docking technology, peptide-iron chelates were prepared in this example, and the iron content per mg of chelate was determined. The results are shown in Figure 5. The iron contents in the chelates of the enzymatic hydrolysate, the ultrafiltration fraction (<3 kDa), and the soybean protein iron-chelating peptides EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR were 85.20, 122.06, 129.75, 68.82, 110.08, and 80.94 μg / mg, respectively. The iron-chelating ability was in the order of EDEPFNLRSR > ultrafiltration fraction (<3 kDa) > DDLELKKIR > enzymatic hydrolysate > EEQPLEVR > EDEPFNLR. Among them, EDEPFNLRSR, with the lowest iron-binding energy from molecular docking, had the strongest iron-chelating ability. In addition, although EDEPFNLRSR and EDEPFNLR have very similar structures, their iron-chelating abilities differ. This is likely because, besides the type and sequence of amino acids playing a crucial role in the iron-chelating ability of bioactive peptides, the spatial effect—that is, the spatial distribution of amino acid functional groups—also influences the iron-binding capacity of bioactive peptides. The increase or decrease of amino acids may affect the folding pattern, three-dimensional structure, and hydrophobic / hydrophilic properties of the peptide chain, thus affecting the spatial effect of the bioactive peptide. The spatial effect of bioactive peptides is one of the foundations for peptide interactions with various substances; therefore, although EEPPFNLRSR and EEPPFNLR have very similar amino acid sequences, their iron-chelating abilities differ. Furthermore, we observed that the iron-binding capacity of the ultrafiltrate was second only to EEPPFNLRSR and higher than the other three monomeric peptides, which may be due to a synergistic effect of the peptide mixture in the ultrafiltrate.

[0098] Example 5: Study on the cytotoxicity of soybean protein iron chelating peptides and their iron transport and retention in the intestine.

[0099] 1. Test Methods

[0100] 1.1 Cytotoxicity assay

[0101] 1.1.1 Cell Culture

[0102] The Caco-2 cells used in the experiment were purchased from the American Type Culture Collection (Rockville, MD, USA), with a cell passage number of 20-30. After seeding the cells in culture flasks, 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 reached confluence in about 3-5 days. When the cell coverage reached 80-90%, the cells were digested with 0.25% trypsin solution for 3-5 minutes, then pipetted off the cells for subculturing and plating.

[0103] 1.2.2 MTT Experiment

[0104] The effects of EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR on Caco-2 cell viability were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazole bromide) toxicity assay. The specific methods are as follows:

[0105] In a 96-well plate, at 5×10 4 Cells were seeded at a density of 10 cells / well and the complete culture medium was removed after 24 h of incubation. Then, different final concentrations of EEPPFNLRSR, EEPPFNLR, DDLELKKIR, and EEQPLEVR (0.05, 0.1, 0.2, 0.25, 0.5, and 1 mg / mL) were added, and incubation continued for another 24 h. Subsequently, the sample was aspirated, 100 μL of 0.5 mg / mL MTT solution was added, and the sample was incubated in an incubator for 4 h. The solution was then replaced with DMSO. After shaking for 10 min, the absorbance was measured at 490 nm, and cell viability was calculated.

[0106] 1.2 Iron transport and retention of soybean protein iron chelating peptides in the intestine

[0107] Construction of the Caco-2 small intestinal epithelial cell model: Caco-2 cells were used at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in 12-well Transwell nested plates (3460, Corning, USA) and incubated for 21 days. During this period, the medium was changed every other day, and transepithelial resistivity (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 were incubated. 2 Only a single cell monolayer can be used for subsequent iron transport and retention experiments.

[0108] 1.2.1 Iron Transfer Experiment

[0109] The iron transport experiment was performed using our previously published method (Lao L, He J, Liao W, et al. Casein 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:

[0110] Mix EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR thoroughly with the pre-prepared FeCl2·4H2O (Fe 2+ The final concentration is 80 μg / mL, and the final concentration of the monomeric peptide is 568 μg / mL. The mixture is reacted in an incubator for 1 hour to obtain the peptide-iron mixture.

[0111] Before the transport experiment officially began, the medium in the Transwell nested plate was changed, and the nest was transferred to a new 12-well plate. Then, a peptide-iron mixture and a control group (FeCl2·4H2O only) were added to the top of the Transwell nested plate. After 30, 60, 120, and 180 min of transport, 1 mL of culture medium was collected from the outside of the substrate to determine the amount of iron transported (graphite furnace atomic absorption spectrometry), while 1 mL of culture medium was added to keep the volume of the outside of the substrate constant.

[0112] 1.2.2 Fluorescence microscopy monitoring of cellular iron retention

[0113] Before the experiment, DMSO was added to a centrifuge tube containing the fluorescent probe FerroOrange and mixed by pipetting to prepare a 1 mmol / L FerroOrange solution, which was then diluted to 1 μmol / L with HBSS. After the experiment began, the culture medium was aspirated, and the Transwell nested plate and cells were washed three times with HBSS. Subsequently, pre-mixed peptide-iron mixture (Fe) was added to the top of the Transwell nested plate. 2+The final concentration was 80 μg / mL, and the final concentration of the monomeric peptide was 568 μg / mL. The specific preparation method is as described in the iron transport experiment. A control group (FeCl2·4H2O was added only) was also included. After 60 min and 180 min of sample treatment, the sample solution was aspirated, and then 1 μmol / L FerroOrange working solution was added to the top. The Transwell plate was then incubated in a 37℃, 5% CO2 incubator for 30 min (Ye S, Xu M, Zhu T, et al. Cytoglobin promotes sensitivity to ferroptosis by regulating p53-yap1axis in colon cancer cells[J]. Journal of Cellular and Molecular Medicine, 2021, 25(7):3300-3311.). The cells were then observed and photographed under a fluorescence microscope. The obtained fluorescence images were statistically analyzed using ImageJ software.

[0114] 2. Test Results

[0115] Iron absorption mainly occurs in the duodenum and upper jejunum. Therefore, 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.

[0116] 2.1 Cytotoxicity Analysis

[0117] This study used the MTT assay to evaluate the effect of bioactive peptides on the survival rate of Caco-2 cells. The results are shown in Figure 6. The lowest survival rate of Caco-2 cells (97.39%, >90%) was observed after treatment with ultrafiltration fraction (<3kDa), EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR. This indicates that the bioactive peptide samples had no toxic effect on Caco-2 cells and did not affect normal cell proliferation.

[0118] 2.2 Effects of soybean protein iron chelate peptides on iron transport in Caco-2 cells

[0119] Caco-2 is a human colon adenocarcinoma cell line. After differentiation, its structure and function are very similar to intestinal cells, and it is often used to study intestinal mineral uptake and transport. In this embodiment, after successfully constructing a Caco-2 small intestinal epithelial cell model, samples from each group were applied to the apex, and the iron content on the lateral side of the basal layer was measured at various time points. As shown in Figure 7, the total iron transport in each group increased continuously over time. After 180 min of transport, compared with the control group (total iron transport: 14.62 μg / well), the total iron transport of the ultrafiltration fraction (<3 kDa), iron chelating peptide EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR groups was significantly increased (P<0.05), at 19.72, 24.76, 18.93, 22.54, and 22.13 μg / well, respectively, representing increases of 34.88%, 69.36%, 29.48%, 54.17%, and 51.37%, respectively, all demonstrating good iron transport-promoting ability.

[0120] The iron transport-promoting capacity, in descending order, was EDEPFNLRSR > DDLELKKIR > EEQPLEVR > ultrafiltration fraction (<3kDa) > EDEPFNLR. Interestingly, this trend is very similar to the trend in iron chelation capacity (EDEPFNLRSR > ultrafiltration fraction (<3kDa) > DDLELKKIR > EEQPLEVR > EDEPFNLR). Notably, although DDLELKKIR and EEQPLEVR have weaker iron chelation capacity than the ultrafiltration fraction (<3kDa), their iron transport-promoting capacity is stronger than that of the ultrafiltration fraction (<3kDa). This may be because molecular weight is an important factor affecting the iron transport capacity of active peptides. Studies have reported that when exogenous iron is added, iron chelating peptides with a molecular weight <1kDa are more effective in promoting the absorption of iron ions in the intestine. The molecular weights of DDLERLKKIR and EEQPLEVR are 1284.7513 Da and 998.5032 Da, respectively, while the molecular weight distribution of the ultrafiltration components is 0-3 kDa. The absorption modes of small molecule oligopeptides in small intestinal epithelial cells are mainly: (1) peptide transporter 1 pathway (carrier of dipeptides and tripeptides), (2) endocytosis (intracellular vesicle-mediated transport system), and (3) passive diffusion near the cell. In addition, after the active peptide binds 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 iron absorption pathways, thus improving the bioavailability of iron.

[0121] 2.2 Effect of soybean protein iron chelating peptides on iron retention in Caco-2 cells

[0122] Studying iron retention in cells helps to understand the effects of iron chelating peptides on improving iron bioavailability in small intestinal epithelial cells and their influence on iron transport pathways. Therefore, in this study, EEPPFNLRSR, which has the strongest iron transport-promoting activity, was selected as a representative peptide to further investigate its effects on Fe in Caco-2 cells. 2+ The effect of retention. During intestinal transport and absorption, for iron ions, Fe... 3+ It must first be reduced to Fe by reducing agents such as duodenal pigment beta. 2+ Subsequently, it is internalized by intestinal cells via apical divalent metallotransferase 1 (DMT 1). After treating Caco-2 cells with the sample for a certain period, a fluorescent probe was added to detect Fe within the live cells. 2+ Fluorescence imaging was performed, and the results are shown in Figure 8. It was found that Fe in the cells of each group increased over time. 2+ The content of Fe in all samples increased to varying degrees. However, compared with the control group, the DDLERLKKIR group showed significantly higher levels of Fe in cells after treatment for 60 min and 180 min. 2+ The fluorescence intensity was increased by 17.23% and 113.10%, respectively. These results indicate that EDEPFNLRSR promotes Fe 2+ Retention was observed in Caco-2 cells. This is likely due to their interaction with the cell membrane, promoting iron uptake and transport to the cytoplasm. Iron absorption pathways primarily include the paracellular pathway (a non-saturated diffusion mode) and transcellular transport. 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 via endocytosis. Therefore, it is possible that EDEPFNLRSR promotes the relative expression level of DMT1, thereby promoting iron uptake. 2+ It enters intestinal cells via DMT 1; or the EEPPFNLRSR-iron chelate may enter cells via endocytosis through specific binding to receptors on the cell surface and in vesicle form. The specific mechanism needs further investigation.

[0123] Example 6: Transmembrane transport of soybean protein iron chelate peptides in Caco-2 cells

[0124] 1. Test Methods

[0125] Understanding the transmembrane transport behavior of iron chelating peptides is beneficial for comprehending the pathways by which they promote iron transport and absorption. Therefore, the monomeric peptide with the strongest iron-promoting capacity (EDEPFNLRSR) was selected as a representative to further investigate its transmembrane transport within the small intestinal epithelial cell monolayer. The pretreatment method for transmembrane transport of iron chelating peptides was the same as in the iron transport experiment in Example 51.2.1. Solutions from the apical and basal sides were collected separately for analytical reversed-phase high-performance liquid chromatography (RP-HPLC) detection and mass spectrometry identification.

[0126] 1.1 Liquid-phase monitoring of monomeric peptide transmembrane transport

[0127] The collected solutions from the top and outer substrate were filtered through a 0.22 μm filter membrane and then analyzed using an LC-10A analytical high-performance liquid chromatography (C18 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 wavelengths: 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. All percentages are volume percentages.

[0128] 1.2 Mass spectrometry analysis of transmembrane transport of iron chelate peptides

[0129] The permeation and degradation of iron chelating peptides in a small intestinal epithelial cell model were further investigated using solutions from the apical and basal outer surfaces transported for 180 min. The specific methods are as follows:

[0130] (1) After desalting the sample using a C18 desalting column, it was analyzed by LC-MS / MS (ThermoFisher Scientific, MA, USA) equipped with an online nano-jet ion source. Gradient elution started with 4% mobile phase B (acetonitrile), increased to 50% in 53.6 min with a non-linear gradient, increased to 95% within 40 s, and maintained for 5.6 min. All percentages are volume percentages. The parameter settings are as follows: Sample loading volume: 3 μL (analytical column: Acclaim PepMap C18, 75 μm x 25 cm); Column flow rate: 300 nL / min; Column temperature: 40℃; Electrospray voltage: 2 kV; MS scan range: 350-1500 m / z; Resolution: 120000; Normalized AGC target (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.

[0131] (2) Database search: Tandem mass spectra were analyzed using PEAKS Studio software. Search parameters: Fragment ion mass tolerance: 0.02 Da; Precursor ion mass tolerance: 10 ppm; Protein card value: containing at least unique peptide; Peptide card value: -10lgP ≥ 20.

[0132] 2. Test Results

[0133] Iron chelating peptides serve as crucial carriers for iron ion entry into small intestinal cells, making their absorption and metabolic mechanisms in the intestine of significant importance. Various peptidases involved in the transport process, such as brush border membrane peptidases at the apex of small 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, EEPPFNLRSR, which exhibits the strongest iron transport-promoting activity, was selected as a representative to investigate its degradation and permeation absorption within the small intestinal epithelial cell monolayer.

[0134] 2.1 Liquid chromatography detection of transmembrane transport of soybean protein iron chelate peptides

[0135] The solutions at the top and outer sides of the Transwell plate after a certain transport time were analyzed using RP-HPLC, and the HPLC results are shown in Figure 9. In the chromatogram of the top solution, EEPPFNLRSR showed an absorption peak at approximately 9.45 min of elution. The peak area decreased continuously with increasing transport time, reaching 70.85% after 180 min of transport. Combined with the mass spectrometry results in Table 2, it was found that EEPPFNLRS abundance was highest after 180 min of transport. This may be because EEPPFNLRSR degrades after interacting with brush edge membrane peptidase. However, since the detection principle of HPLC is mainly based on hydrophobic interactions, the removal of one polar amino acid, arginine (R), does not change the overall hydrophobic characteristics. Therefore, the elution time and peak shape of the corresponding HPLC chromatogram after EEPPFNLRSR degradation to EEPPFNLRS did not change significantly. Furthermore, new absorption peaks began to appear in the HPLC chromatogram after 30 min of transport, which may be due to the degradation of EEPPFNLRSR into new fragments.

[0136] Figure 9B shows the liquid chromatograms of the substrate-external solution after transport for 30, 60, 120, and 180 min. It can be observed that new absorption peaks (elution peaks 1 and 2) appear after 30 min of transport, and the peak areas gradually increase with increasing transport time. This indicates that with prolonged transport time, the derived peptides from the degradation of EEPPFNLRSR are transported to the substrate-external region and accumulate continuously.

[0137] 2.2 Mass spectrometry analysis of transmembrane transport of soybean protein iron chelate peptides

[0138] The top and outer substrate solutions were collected after 180 min of transport and subjected to mass spectrometry identification. The results are shown in Tables 2 and 3. A total of 18 monomeric peptides were identified in the top solution, while 24 monomeric peptides were identified in the outer substrate solution, including the 18 peptides found in the top solution. This indicates that the 24 derived peptides after EEPPFNLRS degradation can be completely absorbed by small intestinal epithelial cells. Based on the peptide peak area, the abundance of EEPPFNLRS was highest in both the top and outer substrate solutions, indicating that the EEPPFNLRS (1105.5024 Da) structure is relatively stable and not easily degraded. Furthermore, due to its relatively high molecular weight, it may be due to Fe... 2+ The main carrier for entering intestinal cells via endocytosis. Furthermore, we noted that the degradation of EEPPFNLRSR primarily occurs at both ends of the peptide chain, while, based on molecular docking predictions, its iron-binding site is mainly located at Asn-6 in the middle of the peptide chain. Therefore, the structure of the peptide-iron chelate may not be destroyed by brush border membrane peptidase, and it can still promote iron transport in the Caco-2 cell monolayer.

[0139] Notably, EEPPFNLR was detected in both the top solution and the solution outside the substrate. This indicates that EEPPFNLRSR degrades to generate EEPPFNLR, and that EEPPFNLR can be transported intact to the outside of the substrate. EEPPFNLR is one of the four novel iron chelating peptides we screened and synthesized. According to iron transport assays, the total iron transport capacity of the EEPPFNLR group was 16.09 μg / well after 60 min of transport, an increase of 87.57% compared to the control group. These results demonstrate that peptides generated from the degradation of EEPPFNLRSR by enzymes, such as EEPPFNLR, also possess iron-promoting capabilities and are essential for iron transport. 2+ An important carrier for entering intestinal cells. Based on this, we infer that among the four isolated monomers, EDEPFNLRSR has the strongest iron-promoting ability, possibly because: (1) EDEPFNLRSR has the strongest iron-binding ability, which increases Fe 2+ Solubility at the apex of small intestinal epithelial cells effectively promotes Fe 2+ (2) EDEPFNLRSR and Fe 2+ After binding, an EEPPFNLRSR-Fe chelate is formed. At the top of the small intestinal epithelial cell model, it is hydrolyzed into EEPPFNLRS by brush border membrane peptidase. It then enters the Caco-2 cell monolayer intact through endocytosis in the form of the EEPPFNLRS-Fe complex, promoting iron absorption; (3) Peptides degraded by enzymes, such as EEPPFNLR, also have the ability to promote iron transport.

[0140] Table 2. Peptide sequences identified from the top solution.

[0141]

[0142]

[0143] Table 3. Peptide sequences identified from the substrate-external solution.

[0144]

[0145] This study used soybean protein as raw material and screened four novel iron chelating peptides—EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR—through enzymatic hydrolysis, purification, identification, and molecular docking techniques. Using a Caco-2 small intestinal epithelial cell model, it was found that EDEPFNLRSR, EDEPFNLR, DDLELKKIR, and EEQPLEVR all significantly promoted iron transport and absorption. For EDEPFNLRSR, which exhibited the strongest iron chelating capacity and iron transport-promoting activity, its intestinal iron retention-enhancing activity and transmembrane transport behavior were further investigated. It was found that EDEPFNLRSR significantly promoted Fe... 2+ Retention in the monolayer of Caco-2 cells. Novel iron chelating peptides screened from soybean protein hydrolysates hold promise as a safe and effective iron supplement. In future studies, we will delve into the molecular mechanisms by which they enhance intestinal iron transport and simultaneously validate the iron transport and absorption-promoting activity of soybean protein iron chelating peptides in animal experiments.

[0146] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An iron chelate peptide, characterized in that... Its amino acid sequence is: DDLERLKKIR.

2. The method for preparing the iron chelate peptide according to claim 1, characterized in that... The process includes the following steps: preparing iron chelate peptides through solid-phase synthesis or obtaining iron chelate peptides by enzymatic hydrolysis and purification using soy protein isolate as raw material and papain.

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

4. The method for preparing the iron chelate peptide according to claim 2, characterized in that: The purification process includes ultrafiltration and liquid chromatography steps.

5. The use of the iron chelating peptide according to claim 1 in the preparation of iron supplements.

6. The use of the iron chelating peptide according to claim 1 in the preparation of iron supplement pharmaceuticals and iron supplement health products.

7. An iron supplement, characterized in that it comprises the iron chelate peptide of claim 1.

8. The iron supplement according to claim 7, characterized in that: the iron supplement further comprises pharmaceutically acceptable excipients.

9. The iron supplement according to claim 8, characterized in that: the pharmaceutically acceptable excipients include solvents.

10. The iron supplement according to claim 8, characterized in that: the pharmaceutically acceptable excipients include at least one of solubilizers, humectants, surfactants, matrixes, emulsifiers, and preservatives.

11. A pharmaceutical or health product, characterized in that it comprises the iron chelate peptide as described in claim 1.

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