Hemoglobin peptide capable of promoting iron absorption and preparation method thereof

Abstract

A hemoglobin peptide and preparation method thereof that can promote iron absorption, the mass content of polypeptide in the hemoglobin peptide is greater than 90%, containing at least one of FERF and FDGL;Preparation method includes: S1, after adding trisodium citrate to fresh animal blood, centrifugation adds NaCl solution to wash, after preparing solution, high pressure homogenization broken wall 2 5 times, obtain hemoglobin solution;S2, after adding 10 200 U / mL neutral protease heat treatment, then add 5 50 U / mL flavor protease enzymolysis, deactivate enzyme to obtain enzymolysis solution;S3, ultrafiltration membrane filtering, collect permeate;S4, after adjusting pH, add in anion exchange chromatography column, NaCl solution elution, collect eluate;S5, desalination, concentration, obtain hemoglobin peptide after drying;S6, mass spectrometry detection, bioactive peptide screening, obtain short peptide with promoting iron absorption activity. The hemoglobin peptide obtained by the preparation method of the present invention has good iron chelating ability and promotes iron absorption ability, and can significantly improve the bioavailability of iron ions.

Description

Technical Field

[0001] The present invention relates to the technical field of biological products, and in particular to a hemoglobin peptide capable of promoting iron absorption and a preparation method thereof. Background Art

[0002] Iron-deficiency anemia occurs when the body's iron stores are insufficient to meet the needs of normal red blood cell production. It is generally caused by decreased hemoglobin synthesis due to insufficient iron intake, reduced absorption, impaired iron utilization, or excessive iron loss. Anemia patients often experience symptoms such as pale complexion, fatigue, lethargy, poor appetite, dizziness, and tinnitus. Severe cases may also experience chest tightness, palpitations, a rapid heart rate, and mental abnormalities such as irritability, temper tantrums, difficulty concentrating, developmental delay, and intellectual disability. Iron-deficiency anemia has become one of the most common nutritional deficiencies in China. The "2015-2017 China Residents Nutrition and Health Status Report" indicates that the prevalence of anemia among adolescents, 12.5% ​​among the elderly, and 19.1% among pregnant women in my country is 15.4%.

[0003] Peptides of specific origin and structure are considered to be very effective metal chelators. They can firmly complex metal ions in the peptide chain structure, effectively avoiding the precipitation or antagonistic effects of substances such as phytic acid on metal ions in the gastrointestinal tract. They are rapidly absorbed by small intestinal cells through the peptide absorption system, and have many advantages such as improving the absorption rate and bioavailability of trace elements. Some studies have shown that ( Malison A, Arpanutud P, Keeratipibul S. Chicken foot broth byproduct: A new source for highly effective peptide-calcium chelate. Food Chem. 2021 May 30;345:128713. ) A peptide with high calcium chelating ability was isolated from chicken feet broth. The peptide was rich in Glu, Asp, Lys, Gly and Leu, and showed stronger calcium absorption in chelate-treated Caco-2 cells relative to CaCl2 at calcium ion concentrations of 0-10 mM.

[0004] Hemoglobin is a protein that contains trace amounts of iron, accounting for 75%-80% of the total protein in animal blood. It contains eight essential amino acids for the human body. Its nutritional value lies in its role as a key oxygen transport protein in humans and animals, transporting oxygen from the lungs to various tissues and organs throughout the body. Hemoglobin peptides and heme iron can be used as iron supplements to help prevent conditions such as anemia. CN 115010803 A discloses a method for preparing hemoglobin peptides rich in heme iron. Hemoglobin is enzymatically hydrolyzed, membrane-concentrated, and then dried to produce the hemoglobin peptide product. CN 118005774 A discloses a hemoglobin ferrous iron chelate peptide and its preparation method. Hemoglobin is hydrolyzed by protease, membrane-separated, and then chelated with iron ions to produce the hemoglobin ferrous iron chelate peptide. However, current technologies often rely on simple hemoglobin hydrolysis without further purification, and the hemoglobin peptide content is not clearly defined. This makes it impossible to effectively enrich specific peptide segments that chelate iron, resulting in a limited enhancement of iron absorption. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art by providing a hemoglobin peptide that promotes iron absorption and a method for its preparation. The hemoglobin peptide product contains at least 90% of the peptide and contains at least one of the amino acid sequences FERF and FDGL. The hemoglobin peptide product prepared by this method can be applied to various foods and promotes iron absorption in people at risk of iron deficiency.

[0006] The present invention is achieved through the following technical solutions:

[0007] A hemoglobin peptide capable of promoting iron absorption, wherein the peptide mass content is greater than 90%. The amino acid sequence of the hemoglobin peptide contains at least one of the sequences Phe-Glu-Arg-Phe (FERF) and Phe-Asp-Gly-Leu (FDGL), wherein Phe represents phenylalanine, Glu represents glutamic acid, Arg represents arginine, Asp represents aspartic acid, Gly represents glycine, and Leu represents leucine. The two sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0008] A method for preparing the above-mentioned hemoglobin peptide capable of promoting iron absorption comprises the following steps:

[0009] S1. High-pressure homogenization pretreatment of blood cells: After adding 0.1%-1.0% trisodium citrate to fresh animal blood, the blood cell pellet is collected by centrifugation. The blood cell pellet is then washed with an equal volume of 0.5%-1.0% NaCl solution and centrifuged. Repeat the washing process 2-3 times to obtain pure red blood cells. Then, water is added to prepare a 5%-15% red blood cell solution. The hemoglobin solution is then broken by high-pressure homogenization at a pressure of 500-1500 bar for 2-5 times to obtain a hemoglobin solution.

[0010] S2. Enzymatic hydrolysis: The hemoglobin solution obtained in step S1 is adjusted to a pH of 5.0-7.0, and 10-200 U / mL of neutral protease is added and incubated at 30-60°C for 0.5-4 hours. 5-50 U / mL of flavor protease is then added and enzymatically hydrolyzed for 4-24 hours. The enzyme is then inactivated at 70-100°C for 30 minutes to obtain an enzymatic hydrolyzate.

[0011] S3, membrane filtration: centrifuge the enzymatic hydrolyzate obtained in step S2, collect the supernatant, and then filter it using an ultrafiltration membrane with a molecular weight cutoff of 3-20 kD, and collect the permeate;

[0012] S4. Ion exchange chromatography: After adjusting the pH of the permeate collected in step S3 to 5.0-6.0, 2-5 column volumes of the permeate are added to a weak anion exchange chromatography column. Impurities, such as polysaccharides, are eluted with 2-5 column volumes (CV) of deionized water. After discarding the eluate, 3-6 column volumes of 0.05-0.5 mol / L NaCl solution are used for elution, and the eluate is collected.

[0013] S5, concentration and drying: desalting, concentrating and drying the eluate collected in step S4 to obtain hemoglobin peptides;

[0014] The hemoglobin peptides obtained in steps S6 and S5 were identified as active peptides using nanoliquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). Bioactive peptide screening and prediction of the toxicity, solubility, sensitization, and absorption characteristics of the active peptides in the human intestine were performed using ToxinPred, INNOVAGEN, Allergen FP, and ADMET Lab 3.0 tools, respectively. Finally, two safe short peptide sequences, Phe-Glu-Arg-Phe (FERF) and Phe-Asp-Gly-Leu (FDGL), with the activity of promoting iron absorption were screened out.

[0015] Furthermore, the specific method of step S6 is:

[0016] The hemoglobin peptide sample was dissolved in 7 μL of 0.1% formic acid aqueous solution mobile phase A, with an injection volume of 3 μL and a flow rate of 10 μL / min. The sample was trapped on the trapping column for 3 min, followed by gradient elution chromatography on a nanoliter analytical column. The acquisition was automatically switched between MS and MS / MS in data-dependent mode.

[0017] A full-scan MS was performed using an Orbitrap primary scan with a scan range of m / z 150-1600 and a resolution of 120,000. The raw data was processed and analyzed using PEAKS software. Peptides with an average local confidence (ALC) score exceeding 95% were further scored using PeptideRanker, with a score threshold of 0.8, to identify high-potential bioactive peptides. These high-potential bioactive peptides were then functionally screened against the BIOPEP database, ultimately identifying several peptides with high iron / zinc / calcium binding activity. PeptideRanker, as a tool for predicting bioactivity, can be used to predict the activity of new peptides; higher scores indicate higher bioactivity. The BIOPEP database allows for functional analysis of input peptide sequences to identify peptides with suitable functions.

[0018] ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools were used to predict the toxicity, solubility, sensitization and absorption characteristics in the human intestine of peptides with iron / zinc / calcium binding activity, respectively. Finally, two short peptide sequences, Phe-Glu-Arg-Phe (FERF) and Phe-Asp-Gly-Leu (FDGL), were screened out as safe and iron-absorbing peptides.

[0019] The ToxinPred tool can be used to predict the toxicity of peptides in batches. The INNOVAGEN software can predict the molecular weight, solubility, and isoelectric point of peptides. The Allergen FP (Allergen FingerPrint) tool predicts and evaluates the potential allergenicity of target sequences by aligning them with sequences in a database of known allergens. The ADMET Lab 3.0 tool can predict and evaluate the ADMET properties (absorption, distribution, metabolism, excretion, and toxicity) of compounds.

[0020] More preferably, in step S1, the mass content of the added NaCl solution is 0.9%, and the mass concentration of the red blood cell solution prepared by adding water is 8%-12%.

[0021] More preferably, in step S2, the temperature of the hemoglobin solution is 40-55° C. when the neutral protease is added, and the enzymatic hydrolysis time after the flavor protease is added is 8-12 h.

[0022] More preferably, the molecular weight cut-off of the ultrafiltration membrane in step S3 is 5-10 KD.

[0023] Further preferably, in step S5, the desalting of the eluate is performed using a nanofiltration membrane with a molecular weight cut-off of 300-600 Da, and the drying is performed by freeze drying.

[0024] More preferably, in step S4, the filler of the weak anion exchange chromatography column is DEAE 52 cellulose.

[0025] More preferably, the centrifugal speed in step S1 is 5000 r / min and the centrifugal time is 15 min; the centrifugal speed in step S3 is 5000 r / min and the centrifugal time is 15 min. The fresh animal blood in step S1 is bovine blood, sheep blood, pig blood or chicken blood.

[0026] Furthermore, the hemoglobin peptide obtained in step S5 is subjected to Fc 2+ Determination of chelating capacity and Fe 2+ In vitro bioavailability determination was used to further evaluate the performance of hemoglobin peptide in promoting iron absorption. 2+ The chelating capacity was determined by UV-visible spectrophotometry. 2+ The in vitro bioavailability was determined using the Caco-2 cell model.

[0027] Furthermore, the Caco-2 cell model method was used to determine the Fe 2+ In vitro bioavailability, the specific steps include:

[0028] Establishment of Caco-2 monolayer cell model: resuspend cells in complete culture medium; before inoculating cells, pre-wet the Transwell chamber with DMEM culture medium, and then plate Caco-2 cells at a concentration of 1×10 5 cell / cm 2 Cells were seeded into a 12-well Transwell plate. 0.5 mL of cell suspension was added to each well on the AP side of the chamber, and 1.5 mL of complete culture medium was added to the BL side of the chamber. After seeding, the culture medium was changed every 6-16 hours, and then on both sides every other day. The cell culture cycle was 21 days, until cell membrane differentiation was complete. The transmembrane resistance of the cells was measured using a resistance meter, and the measurement was recorded every other day to ensure that the transepithelial resistance was greater than 200 Ω·cm at 21 days. 2 ;

[0029] Preparation of donor solution: Dissolve the hemoglobin peptide in deionized water to obtain a 2 mg / mL solution, and incubate at 37°C in a water bath for 10 minutes to fully dissolve it. Take 10 mL of the fully dissolved peptide solution, add 1 mL of 0.01 mol / L FeSO4, and incubate for 30 minutes. Centrifuge at 2268g for 20 minutes to remove the precipitate, add 6 volumes of anhydrous ethanol to the supernatant, collect the precipitate, and freeze-dry to obtain the hemoglobin peptide-Fe complex. Dissolve ferrous sulfate and the hemoglobin peptide-Fe complex in DMEM medium, with an Fe content of 100 μg / mL in each, and preheat to 37°C to use as the donor solution.

[0030] Caco-2 monolayer transport assay: Change the culture medium 12-24 hours before the experiment to prevent the cells from depleting essential nutrients. Transfer the cell monolayer to a new 12-well plate containing 1.5 mL of HBSS buffer per well on the BL side and carefully add 0.5 mL of HBSS buffer to the AP side. Incubate at 37°C for 15-20 min.

[0031] AP-BL direction experiment: carefully aspirate the HBSS buffer on the BL side, add new HBSS buffer, pour the HBSS buffer on the AP side, add 0.5 mL of donor solution to the AP side, add 1.0 mL of HBSS buffer to the BL side, and immediately take a sample at t = 0 to measure the iron content to prevent part of the compound from being transported before sampling is completed;

[0032] The Transwell plate used for the cell transport experiment was placed in a cell culture incubator. The transport time was set to 2 h. The solutions on the AP and BL sides were collected, and the solution volume and ferrous iron content were measured. After 2 h of cell transport and absorption, the in vitro bioavailability of ferrous iron was calculated as follows:

[0033]

[0034] in:

[0035] C0 is the initial ferrous iron concentration on the AP side, μg / mL;

[0036] V0 is the volume of the AP side solution at the initial time, mL;

[0037] C 2h is the concentration of ferrous iron on the AP side 2 hours after transport, μg / mL;

[0038] V 2h The volume of the AP side solution after 2 hours of transport, mL.

[0039] The preparation method of the present invention adopts high-pressure homogenization + composite protease hydrolysis, membrane separation + ion exchange chromatography purification and other technologies to hydrolyze and obtain a specific peptide segment with the activity of promoting iron absorption. The product polypeptide content is more than 90%, and its amino acid sequence contains at least one of FERF and FDGL. The obtained hemoglobin peptide product has good iron chelating ability and iron absorption promoting ability. The IC of iron ion chelation is 50 The Caco-2 cell model showed that it significantly improved the bioavailability of iron ions at a concentration below 1 mg / mL, and a new method for evaluating the performance of promoting iron absorption was proposed. The peptides in hemoglobin peptides were evaluated and screened using nanoliter liquid chromatography-tandem mass spectrometry NanoLC-MS / MS, ALC scoring, Peptide Ranker scoring, BIOPEP database comparison analysis and other technical means. The relevant properties of the peptides were predicted using tools such as ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab3.0. By combining multiple methods, peptides with excellent comprehensive performance were screened to meet the requirements. This played a significant role in the screening of FERF and FDGL peptide sequences that can promote iron absorption, providing innovative ideas for research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the mass spectrum of the polypeptide FERF in hemoglobin peptide.

[0041] Figure 2 This is the mass spectrum of the polypeptide FDGL in hemoglobin peptide.

[0042] Figure 3 This is the interface diagram for toxicity prediction of FDGL and FDGL in hemoglobin peptides using ToxinPred.

[0043] Figure 4 Schematic diagram of the Caco-2 monolayer cell model grown on a permeable filtration scaffold in the test of iron bioavailability by hemoglobin peptides in an embodiment of the present invention. DETAILED DESCRIPTION

[0044] Example 1

[0045] A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps:

[0046] S1. Blood cell pretreatment by high-pressure homogenization: Fresh bovine blood was added with 0.5% trisodium citrate and centrifuged at 5000 rpm for 15 minutes to collect the blood cell pellet. The pellet was then washed with an equal volume of 0.9% NaCl solution and centrifuged at 5000 rpm for 15 minutes. This was repeated twice to obtain pure red blood cells. The red blood cells were then added to water to prepare a 10% solution. The hemoglobin solution was then broken down by high-pressure homogenization twice at 1000 bar to obtain the hemoglobin solution.

[0047] S2. Enzymatic hydrolysis: Adjust the pH of the hemoglobin solution to 6.0, add 100 U / mL of neutral protease, and keep it in a 45°C water bath for 2 h. Then, add 20 U / mL of flavor protease, continue to keep it in a water bath for 8 h, and then heat it to 90°C and inactivate the enzyme for 30 min to obtain the enzymatic hydrolyzate.

[0048] S3. Membrane filtration: Centrifuge the enzymatic hydrolysate (5000 rpm, 10 min) and collect the supernatant. Then filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10 KD and collect the permeate.

[0049] S4. Ion exchange chromatography: After adjusting the pH of the permeate to 5.0, add 2 column volumes (i.e., 2CV, CV stands for column volume) of the permeate to a weak anion exchange chromatography column (filler DEAE 52 cellulose). Use 5CV of deionized water to elute impurities such as polysaccharides. After discarding the eluate, elute with 4CV of 0.1 mol / L NaCl solution and collect the eluate.

[0050] S5. Concentration and drying: The collected eluate is desalted and concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da, and then freeze-dried to obtain hemoglobin peptides.

[0051] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0052] Comparative Example 1

[0053] In this comparative example, the same hemoglobin peptide preparation method as in Example 1 was adopted, except that in step S2, papain was used for the enzymatic hydrolysis in this comparative example.

[0054] Comparative Example 2

[0055] In this comparative example, the same hemoglobin peptide preparation method as in Example 1 was adopted, except that step S3 in Example 1 was omitted in this comparative example, that is, an ultrafiltration membrane was not used to perform membrane separation on the polypeptide.

[0056] Comparative Example 3

[0057] In this comparative example, the same hemoglobin peptide preparation method as in Example 1 was adopted, except that step S4 in Example 1 was omitted in this comparative example, that is, the hemoglobin peptide was not subjected to ion exchange chromatography using an exchange chromatography column.

[0058] Example 2

[0059] A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps:

[0060] S1. Blood cell pretreatment by high-pressure homogenization: Fresh bovine blood was added with 0.5% trisodium citrate and centrifuged at 5000 rpm for 15 minutes to collect the blood cell pellet. The pellet was then washed with an equal volume of 0.9% NaCl solution and centrifuged at 5000 rpm for 15 minutes. This was repeated twice to obtain pure red blood cells. The red blood cells were then added to water to a 10% solution. The hemoglobin solution was then broken down by high-pressure homogenization twice at 1500 bar to obtain the hemoglobin solution.

[0061] S2. Enzymatic hydrolysis: Adjust the pH of the hemoglobin solution to 7.0, add 200 U / mL of neutral protease, and keep it in a 45°C water bath for 4 h. Then, add 50 U / mL of flavor protease, continue to keep it in a water bath for 4 h, and then heat it to 90°C and inactivate the enzyme for 30 min to obtain the enzymatic hydrolyzate.

[0062] S3. Membrane filtration: Centrifuge the enzymatic hydrolysate (5000 rpm, 10 min) and collect the supernatant. Then filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 5 kD and collect the permeate.

[0063] S4. Ion exchange chromatography: After adjusting the pH of the permeate to 5.0, add 2 column volumes (2CV) of the permeate to a weak anion exchange chromatography column (filler: DEAE 52 cellulose). Elute the impurities with 5CV of deionized water. After discarding the eluate, elute with 4CV of 0.05 mol / L NaCl solution and collect the eluate.

[0064] S5. Concentration and drying: The collected eluate is desalted and concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da, and then freeze-dried to obtain hemoglobin peptides.

[0065] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0066] Example 3

[0067] A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps:

[0068] S1. Blood cell pretreatment by high-pressure homogenization: Fresh pig blood was added with 0.5% trisodium citrate and centrifuged at 5000 rpm for 15 minutes to collect the blood cell pellet. The pellet was then washed with an equal volume of 0.9% NaCl solution and centrifuged at 5000 rpm for 10 minutes. This was repeated twice to obtain pure red blood cells. The red blood cells were then added to water to prepare a 10% solution. The hemoglobin solution was then broken down by high-pressure homogenization twice at 500 bar to obtain the hemoglobin solution.

[0069] S2. Enzymatic hydrolysis: adjust the pH of the hemoglobin solution to 5.0, add 50 U / mL neutral protease, keep it in a 55℃ water bath for 4 h, then add 5 U / mL flavor protease, continue to keep it in a water bath for 24 h, then heat it to 90℃ and inactivate the enzyme for 30 min to obtain the enzymatic hydrolyzate.

[0070] S3. Membrane filtration: Centrifuge the enzymatic hydrolysate (5000 rpm, 10 min) and collect the supernatant. Then filter the supernatant using an ultrafiltration membrane with a molecular weight cutoff of 10 KD and collect the permeate.

[0071] S4. Ion exchange chromatography: After adjusting the pH of the permeate to 6.0, add 5 column volumes (5 CV) of the permeate to a weak anion exchange chromatography column (filler: DEAE 52 cellulose). Elute the impurities with 5 CV of deionized water. After discarding the eluate, elute with 4 CV of 0.2 mol / L NaCl solution and collect the eluate.

[0072] S5. Concentration and drying: The collected eluate is desalted and concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da, and then freeze-dried to obtain hemoglobin peptides.

[0073] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0074] Structure identification and property determination:

[0075] 1. Identification and screening of active peptides in hemoglobin peptides

[0076] The prepared hemoglobin peptide samples were identified using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). The sample was dissolved in 7 μL of mobile phase A (containing 0.1% formic acid in water), injected 3 μL at a flow rate of 10 μL / min, and trapped on a trapping column for 3 minutes. Gradient elution chromatography was then performed on a nano-analytical column, with automatic switching between MS and MS / MS acquisition in data-dependent mode. Full-scan MS was performed using an Orbitrap primary scan with a scan range of m / z 150-1600 and a resolution of 120,000. PEAKS software was used for data processing and spectral interpretation.

[0077] ALC (%) stands for Average Local Confidence, a measure of confidence in de novo sequencing data. A score greater than 80% is generally considered reliable, and greater than 95% is considered very reliable. The 2,627 peptides with ALC scores exceeding 95% were further evaluated using PeptideRanker, with a score threshold of 0.8. This resulted in the identification of 178 peptides with high potential for bioactivity. These 178 peptides were then functionally screened in the BIOPEP database, ultimately identifying 63 peptides with potential for high iron / zinc / calcium binding activity.

[0078] The safety, water solubility and absorbability of active peptides have an important impact on their application value. Therefore, the toxicity, solubility, sensitization and absorption characteristics of the above 63 peptides were predicted by using ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools. Finally, two short peptides with excellent characteristics, Phe-Glu-Arg-Phe (FERF) and Phe-Asp-Gly-Leu (FDGL), were screened out. These two short peptides have excellent performance in safety, solubility, absorption in the human intestine and promoting iron absorption activity. The mass spectra of FERF and FDGL are shown in Figure 2. Figure 1 and Figure 2 As shown in Table 1, where Phe represents phenylalanine, Glu represents glutamic acid, Arg represents arginine, Asp represents aspartic acid, Gly represents glycine, and Leu represents leucine. The relative contents of FERF and FDGL in the hemoglobin peptides of each embodiment were calculated by area normalization. The results are shown in Table 1. Taking toxicity as an example, the prediction results of these two short peptide sequences are shown in Table 1. Figure 3 .

[0079] 2. Hemoglobin peptide Fe 2+ Determination of chelating capacity

[0080] Hemoglobin peptides were diluted to different concentrations using MES buffer (10 mM, pH 5.5) and filtered through a 0.22 μm cellulose acetate membrane. 20 μL of FeSO4 solution (0.2 mM) was then added to 140 μL of hemoglobin peptide solutions of different concentrations in a 96-well plate. The plate was then incubated in a constant temperature chamber at 37°C for 3 h. After incubation, 40 μL of ferrozine (5 mM) solution was added to terminate the reaction. After the plate was placed at room temperature for 10 minutes, the absorbance at 562 nm was read using a microplate reader. Ferrozine-Fe 2+ The percentage inhibition of complex formation (Fe 2+ The chelating capacity) is calculated as follows:

[0081]

[0082] in:

[0083] A0 is the absorbance of the blank;

[0084] A s is the absorbance of the sample.

[0085] IC 50 Defined as 50% inhibition of ferrozine-Fe 2+ Peptide concentration for complex formation.

[0086] 3. Caco-2 Cell Model for Fe Determination 2+ In vitro bioavailability

[0087] Establishment of Caco-2 monolayer cell model:

[0088] Resuspend the cells in complete culture medium. Before seeding the cells, pre-wet the Transwell chamber with DMEM medium. Then, seed the Caco-2 cells at a concentration of 1×10 5 cell / cm 2 Inoculate into 12-well Transwell plates (eg Figure 4 ), add 0.5mL of cell suspension to each well on the top (AP) side of the chamber, and add 1.5mL of complete culture medium to the basolateral (BL) side. After inoculation, replace the culture medium every 6-16 hours, and then replace the culture medium on both sides every other day. The cell culture cycle is 21 days, until the cell membrane differentiation is complete. Use a resistance meter to measure the transmembrane resistance of the cells, measure and record it every other day, and ensure that the transepithelial resistance (TEER value) is greater than 200Ω·cm at 21 days. 2 .

[0089] Donor solution preparation:

[0090] Dissolve the hemoglobin peptide in deionized water to a 2 mg / mL solution and incubate at 37°C for 10 minutes to fully dissolve. Add 1 mL of FeSO₄ (0.01 mol / L) to 10 mL of the fully dissolved peptide solution and incubate for 30 minutes. Centrifuge at 2268 g (3000 rpm) for 20 minutes to remove the precipitate. Add 6 volumes of anhydrous ethanol to the supernatant, collect the precipitate, and freeze-dry to obtain the hemoglobin peptide-Fe complex. Dissolve ferrous sulfate (Fe content 100 μg / mL) and the hemoglobin peptide-Fe complex (Fe content 100 μg / mL) in DMEM medium and preheat to 37°C to prepare the donor solution.

[0091] Caco-2 monolayer cell transport assay:

[0092] Change the culture medium 12-24 hours before the experiment to prevent the cells from depleting essential nutrients. Transfer the cell monolayer to a new 12-well plate containing 1.5 mL of HBSS buffer per well on the BL side and carefully add 0.5 mL of HBSS buffer to the AP side. Incubate at 37°C for 15-20 minutes.

[0093] AP-BL direction experiment: Carefully aspirate the HBSS buffer on the BL side, add new HBSS buffer, pour the HBSS buffer on the AP side, add 0.5 mL of donor solution to the AP side, add 1.0 mL of HBSS buffer to the BL side, and immediately sample at t = 0 to determine the ferrous content to prevent some compound from being transported before sampling is completed.

[0094] The Transwell plate used for the cell transport experiment was placed in a cell culture incubator and the transport time was set to 2 hours. The solutions on the AP and BL sides were collected and the solution volume and ferrous iron content were measured. After 2 hours of cellular transport and absorption, the in vitro bioavailability of ferrous iron was calculated as follows:

[0095]

[0096] in:

[0097] C0: initial AP side ferrous iron concentration (μg / mL);

[0098] V0: initial volume of the AP side solution (mL);

[0099] C 2h : ferrous iron concentration on the AP side 2 h after transport (μg / mL);

[0100] V 2h : AP side solution volume after 2 h of transport (mL).

[0101] 4. Peptide Content Determination Method

[0102] Weigh 500 mg of hemoglobin peptide sample into a 25 mL volumetric flask, dilute to 25 mL with 15% trichloroacetic acid solution, centrifuge and take the supernatant. Determine the protein content in the supernatant according to the Kjeldahl nitrogen method in GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food" to obtain the acid-soluble protein content. X 1.

[0103] Weigh 400 mg of hemoglobin peptide and dilute to 10 mL with 5% trichloroacetic acid solution. Determine the content of free amino acids in hemoglobin peptide according to the determination method of free amino acids in GB 22492-2008 "Soybean Peptide Powder". X 2.

[0104] Polypeptide content (g / 100g) = X 1 - X 2;

[0105] Where, X 1 represents the content of acid-soluble protein, in g / 100g;

[0106] X 2 represents the content of free amino acids, in g / 100g.

[0107] 5. Test results

[0108] The above test scheme was used to test the polypeptide content, iron chelating ability (ferrozine-Fe 2+ Complex inhibition IC 50 ), iron bioavailability, the test results are shown in Table 1.

[0109] Table 1 Test and application results of each sample

[0110]

[0111] Note: The test data of FERF sample and FDGL sample were obtained by using the two pure synthesized polypeptide sequences as samples according to the above test method. The test data of FeSO4 sample were obtained by using ferrous sulfate dissolved in DMEM medium as the donor solution.

[0112] The test results show that the content of polypeptides in the hemoglobin peptides obtained in the examples is above 90%, which is greater than the content of polypeptides in the comparative examples; the content of FERF and FDGL sequences in the hemoglobin peptides obtained in the examples is much higher than that in the comparative examples; the hemoglobin peptides obtained in the examples have a ferrozine-Fe 2+ Complex inhibition IC 50All were lower than 1 mg / mL, much lower than the IC of the control group. 50 That is, the hemoglobin peptide prepared by the preparation method of the present invention can inhibit 50% of the ferrozine-Fe 2+ The formation of the complex indicates that it has a strong affinity for Fe 2+ The chelating ability is strong, and it has a good ability to promote iron absorption; the iron bioavailability of the hemoglobin peptides obtained in the examples is higher than 20%, which is much higher than that of the comparative example, indicating that the hemoglobin peptides obtained by the method of the present invention can significantly improve Fe 2+ The bioavailability of the hemoglobin peptide-Fe complex formed by the invention is higher than that of the conventional hemoglobin peptide. The iron in the hemoglobin peptide-Fe complex formed by the invention is more bioavailable. Therefore, the preparation method of the invention can obtain a hemoglobin peptide with excellent comprehensive performance, especially excellent performance in promoting iron absorption.

[0113] The above detailed description is a specific description of a feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the patent scope of this case.

Claims

1. A hemoglobin peptide composition capable of promoting iron absorption, characterized in that: The mass content of the polypeptide in the hemoglobin peptide composition is greater than 90%. The hemoglobin peptide composition contains at least one of amino acid FERF and amino acid FDGL. FERF and FDGL are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. A method for preparing the hemoglobin peptide composition capable of promoting iron absorption according to claim 1, characterized in that: The steps include: S1. High-pressure homogenization pretreatment of blood cells: After adding 0.1%-1.0% trisodium citrate to fresh animal blood, the blood cell pellet is collected by centrifugation; then, the same volume of 0.5%-1.0% NaCl solution is added for washing, followed by centrifugation. This washing process is repeated 2-3 times to obtain pure red blood cells; then, water is added to prepare a 5%-15% red blood cell solution, which is then subjected to high-pressure homogenization at 500-1500 bar for 2-5 cycles to obtain a hemoglobin solution; S2, enzymatic hydrolysis: the hemoglobin solution obtained in step S1 is adjusted to a pH of 5.0-7.0, 10-200 U / mL of neutral protease is added and incubated at 30-60°C for 0.5-4 hours, 5-50 U / mL of flavor protease is then added and enzymatically hydrolyzed for 4-24 hours, and the enzyme is inactivated at 70-100°C for 30 minutes to obtain an enzymatic hydrolyzate; S3, membrane filtration: centrifuge the enzymatic hydrolyzate obtained in step S2, collect the supernatant, and then filter it using an ultrafiltration membrane with a molecular weight cutoff of 3-20 kD, and collect the permeate; S4, ion exchange chromatography: After adjusting the pH of the permeate collected in step S3 to 5.0-6.0, 2-5 column volumes of the permeate are added to a weak anion exchange chromatography column, and impurities are eluted with 2-5 column volumes of deionized water. After discarding the eluate, 3-6 column volumes of 0.05-0.5 mol / L NaCl solution are used for elution, and the eluate is collected; S5, concentration and drying: desalting, concentrating and drying the eluate collected in step S4 to obtain a hemoglobin peptide composition; The hemoglobin peptide composition obtained in steps S6 and S5 was used to identify active peptides by nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS). Bioactive peptide screening and prediction of the toxicity, solubility, sensitization and absorption characteristics of the active peptides in the human intestine were performed using ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools, respectively. Finally, two safe short peptide sequences, Phe-Glu-Arg-Phe and Phe-Asp-Gly-Leu, with the activity of promoting iron absorption were screened out.

3. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: The specific method of step S6 is: The hemoglobin peptide composition sample was dissolved in 7 μL of mobile phase A containing 0.1% formic acid in water, with an injection volume of 3 μL and a flow rate of 10 μL / min. The sample was trapped on the trapping column for 3 minutes, followed by gradient elution chromatography on a nanoliter analytical column. The acquisition was automatically switched between MS and MS / MS in data-dependent mode. Full-scan MS was performed using an Orbitrap primary scan with a scan range of m / z 150-1600 and a resolution of 120,000. The resulting raw data was processed and analyzed using PEAKS software. Peptides with an average local confidence (ALC) score exceeding 95% were further scored using PeptideRanker, with a score threshold of 0.8, to identify high-potential bioactive peptides. These high-potential bioactive peptides were then functionally screened against the BIOPEP database, identifying several peptides with high iron / zinc / calcium binding activity. ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools were used to predict the toxicity, solubility, sensitization and absorption characteristics in the human intestine of peptides with iron / zinc / calcium binding activity, respectively. Finally, two short peptide sequences, Phe-Glu-Arg-Phe and Phe-Asp-Gly-Leu, which are safe and have the activity of promoting iron absorption, were screened out.

4. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: In step S1, the mass concentration of the added NaCl solution is 0.9%, and the mass concentration of the red blood cell solution prepared by adding water is 8%-12%.

5. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: In step S2, the temperature of the hemoglobin solution is 40-55° C. when the neutral protease is added, and the enzymatic hydrolysis time after the flavor protease is added is 8-12 h.

6. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: The molecular weight cut-off of the ultrafiltration membrane in step S3 is 5-10KD.

7. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: In step S5, the eluate is desalted using a nanofiltration membrane with a molecular weight cutoff of 300-600 Da, and the drying is performed by freeze drying; in step S4, the filler of the weak anion exchange chromatography column is DEAE 52 cellulose.

8. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: The centrifugal speed in step S1 is 5000 r / min and the centrifugal time is 15 min; the centrifugal speed in step S3 is 5000 r / min and the centrifugal time is 15 min; the fresh animal blood in step S1 is cow blood, sheep blood, pig blood or chicken blood.

9. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 2, characterized in that: The hemoglobin peptide composition obtained in step S5 is also subjected to Fe 2+ Determination of chelating capacity and Fe 2+ In vitro bioavailability determination was used to further evaluate the performance of the hemoglobin peptide composition in promoting iron absorption. 2+ The chelating capacity was determined by UV-visible spectrophotometry. 2+ The in vitro bioavailability was determined using the Caco-2 cell model.

10. The method for preparing a hemoglobin peptide composition capable of promoting iron absorption according to claim 9, characterized in that: Determination of Fe of hemoglobin peptide compositions using Caco-2 cell model 2+ In vitro bioavailability, the specific steps include: Establishment of Caco-2 monolayer cell model: resuspend cells in complete culture medium; before inoculating cells, pre-wet the Transwell chamber with DMEM culture medium, and then plate Caco-2 cells at a concentration of 1×10 5 cell / cm 2 Cells were seeded into a 12-well Transwell plate. 0.5 mL of cell suspension was added to each well on the AP side of the chamber, and 1.5 mL of complete culture medium was added to the BL side of the chamber. After seeding, the culture medium was changed every 6-16 hours, and then on both sides every other day. The cell culture cycle was 21 days, until cell membrane differentiation was complete. The transmembrane resistance of the cells was measured using a resistance meter, and the measurement was recorded every other day to ensure that the transepithelial resistance was greater than 200 Ω·cm at 21 days. 2 ; Preparation of donor solution: Dissolve the hemoglobin peptide composition in deionized water to obtain a 2 mg / mL solution, and incubate in a 37°C water bath for 10 minutes to fully dissolve it; take 10 mL of the fully dissolved peptide solution, add 1 mL of 0.01 mol / L FeSO4, and incubate for 30 minutes; centrifuge at 2268g for 20 minutes to remove the precipitate, add 6 volumes of anhydrous ethanol to the supernatant, collect the precipitate, and freeze-dry to obtain the hemoglobin peptide-Fe complex; dissolve ferrous sulfate and the hemoglobin peptide-Fe complex in DMEM medium, each with an Fe content of 100 μg / mL, and preheat to 37°C to use as the donor solution; For Caco-2 monolayer transport experiments: Change the culture medium 12-24 hours before the experiment to prevent the cells from depleting essential nutrients. Transfer the cell monolayer to a new 12-well plate containing 1.5 mL of HBSS buffer per well on the BL side and carefully add 0.5 mL of HBSS buffer to the AP side. Incubate at 37°C for 15-20 minutes. AP-BL direction experiment: carefully aspirate the HBSS buffer on the BL side, add new HBSS buffer, pour the HBSS buffer on the AP side, add 0.5 mL of donor solution to the AP side, add 1.0 mL of HBSS buffer to the BL side, and immediately take a sample at t = 0 to measure the ferrous content to prevent part of the compound from being transported before sampling is completed; The Transwell plate used for the cell transport experiment was placed in a cell culture incubator. The transport time was set to 2 hours. The solutions on the AP and BL sides were collected, and the solution volume and ferrous iron content were measured. After 2 hours of cell transport and absorption, the in vitro bioavailability of ferrous iron was calculated as follows: in: C0 is the initial ferrous iron concentration on the AP side, μg / mL; V0 is the volume of the AP side solution at the initial time, mL; C 2h is the concentration of ferrous iron on the AP side 2 hours after transport, μg / mL; V 2h The volume of the AP side solution after 2 hours of transport, mL.

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