Hemoglobin peptide capable of promoting iron absorption and preparation method thereof

Abstract

The invention relates to a hemoglobin peptide capable of promoting iron absorption and a preparation method thereof, the mass content of polypeptide in the hemoglobin peptide is greater than 90%, and the hemoglobin peptide contains at least one of FERF and FDGL; the preparation method comprises the following steps: S1, adding trisodium citrate into fresh animal blood, centrifuging, adding a NaCl solution for washing, preparing the solution, and performing high-pressure homogenization wall breaking for 2-5 times to obtain a hemoglobin solution; s2, adding 10-200 U / mL of neutral protease, carrying out heat preservation treatment, then adding 5-50 U / mL of flavourzyme, carrying out enzymolysis, and carrying out enzyme deactivation to obtain an enzymatic hydrolysate; s3, filtering with an ultrafiltration membrane, and collecting permeate; s4, adjusting the pH value, adding into an anion exchange chromatographic column, eluting with a NaCl solution, and collecting the eluent; s5, desalting, concentrating and drying to obtain hemoglobin peptide; and S6, carrying out mass spectrometric detection, and screening bioactive peptides to obtain the oligopeptide with the activity of promoting iron absorption. The hemoglobin peptide obtained by the preparation method disclosed by the invention has relatively good iron chelation capability and iron absorption promoting capability, and the bioavailability of iron ions can be remarkably improved.

Description

Technical Field

[0001] The 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 is anemia caused by the inability of the body's iron storage to meet the needs of normal red blood cell production. It is generally caused by insufficient iron intake, reduced absorption, impaired iron utilization or excessive loss, which leads to reduced hemoglobin synthesis. Anemia patients often have symptoms such as pale complexion, physical fatigue, fatigue, poor appetite, dizziness, tinnitus, etc. In severe cases, they may also experience chest tightness, palpitations, increased heart rate and mental abnormalities, such as irritability, temper tantrums, inattention, developmental delay, and mental retardation. Iron deficiency anemia has become one of the most common nutritional deficiency diseases in China. The "2015-2017 China Residents Nutrition and Health Status Report" pointed out that the incidence of anemia in Chinese adolescents is 15.4%, the incidence in the elderly is 12.5%, and the incidence in pregnant women is 19.1%.

[0003] Peptides of specific sources and structures 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 effect 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. ) isolated a peptide with high calcium chelating ability from chicken feet broth. The peptide was rich in Glu, Asp, Lys, Gly and Leu. The chelate-treated Caco-2 cells showed a significant increase in calcium ion concentration compared with CaCl at 0-10 mM. 2 Shows stronger absorption.

[0004] Hemoglobin is a binding protein containing trace iron, accounting for 75%-80% of the total protein in animal blood. It contains 8 essential amino acids for the human body. Its nutritional value lies in that it is an important protein responsible for transporting oxygen in humans and animals, and can transport oxygen from the lungs to various tissues and organs of the body. Hemoglobin peptides and heme iron can be used as iron supplements to help prevent diseases such as anemia. CN 115010803 A discloses a method for preparing a hemoglobin polypeptide rich in heme iron, and hemoglobin is enzymatically hydrolyzed, membrane-concentrated, and then dried to obtain a hemoglobin peptide product. CN 118005774 A discloses a hemoglobin ferrous chelate peptide and a preparation method thereof, and hemoglobin is enzymatically hydrolyzed by protease, membrane-separated, and then chelated with iron ions to prepare a hemoglobin ferrous chelate peptide. However, the current technology is mostly a simple hydrolysis of hemoglobin, and it is not further purified, and the content of hemoglobin peptides is not clear, and specific peptide segments with chelated iron cannot be effectively enriched, and the promotion effect on iron absorption is not obvious. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a hemoglobin peptide that can promote iron absorption and a preparation method thereof. The hemoglobin peptide product has a polypeptide content of more than 90% and contains at least one of the amino acid sequences FERF and FDGL. The hemoglobin peptide product prepared by the method can be applied to various foods and has the function of promoting iron absorption for people at risk of iron deficiency.

[0006] The present invention is achieved through the following technical solutions: A hemoglobin peptide capable of promoting iron absorption, wherein the mass content of the polypeptide is greater than 90%, and the amino acid sequence of the hemoglobin peptide contains at least one of the sequences of Phe-Glu-Arg-Phe (abbreviated as FERF) and Phe-Asp-Gly-Leu (abbreviated as FDGL), wherein Phe represents phenylalanine, Glu represents glutamic acid, Arg represents arginine, Asp represents aspartic acid, Gly represents glycine, and Leu represents leucine. The above two sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0007] A method for preparing the above-mentioned hemoglobin peptide capable of promoting iron absorption comprises the following steps: S1. Pretreatment of blood cells by high-pressure homogenization: After adding trisodium citrate with a mass concentration of 0.1%-1.0% to fresh animal blood, the blood cell precipitate is collected by centrifugation; then the same volume of NaCl solution with a mass concentration of 0.5%-1.0% is added for washing and centrifugation, and the washing is repeated 2-3 times to obtain pure red blood cells; then, water is added to prepare the red blood cells into a solution with a mass concentration of 5%-15%, and the red blood cells are broken by high-pressure homogenization at a pressure of 500-1500 bar for 2-5 times to obtain a hemoglobin solution; S2, enzymatic hydrolysis: the pH of the hemoglobin solution obtained in step S1 is adjusted to 5.0-7.0, 10-200 U / mL of neutral protease is added at a temperature of 30-60°C for heat treatment for 0.5-4h, 5-50 U / mL of flavor protease is added for enzymatic hydrolysis for 4-24h, and the enzyme is inactivated at 70-100°C for 30 min to obtain an enzymatic hydrolyzate; S3, membrane filtration: centrifuge the enzymatic hydrolysate obtained in step S2, collect the supernatant, and then filter it using an ultrafiltration membrane with a molecular weight cutoff of 3-20KD to collect the permeate; S4, ion exchange chromatography: After the pH of the permeate collected in step S3 is adjusted to 5.0-6.0, 2-5 column volumes of the permeate are added to a weak anion exchange chromatography column, and 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; S5, concentration and drying: desalting, concentrating and drying the eluate collected in step S4 to obtain hemoglobin peptides; The hemoglobin peptides obtained in steps S6 and S5 were identified as active peptides by nanoliquid chromatography-tandem mass spectrometry (NanoLC-MS / MS), and bioactive peptides were screened. The toxicity, solubility, sensitization and absorption characteristics of the active peptides in the human intestine were predicted 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), were screened out with the activity of promoting iron absorption.

[0008] Furthermore, the specific method of step S6 is: 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, and then gradient elution chromatography was performed on a nanoliter analytical column. The data-dependent mode was used to automatically switch between MS and MS / MS acquisition. The full scan MS uses Orbitrap for primary scanning, with a scanning range of m / z 150-1600 and a resolution of 120,000. The raw data obtained are processed and analyzed using PEAKS software. Peptides with an average local confidence ALC score of more than 95% are further scored by PeptideRanker, with a score threshold of 0.8, from which high-potential bioactive peptides are screened; high-potential bioactive peptides are functionally screened in the BIOPEP database, and finally several peptides with high iron / zinc / calcium binding activity are screened. PeptideRanker, as a tool for predicting biological activity, can be used to predict the activity of new peptides. The higher the score, the higher the biological activity. The BIOPEP database can perform functional analysis on the input peptide sequence and screen out peptides with suitable functions.

[0009] 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, and finally two safe short peptide sequences, Phe-Glu-Arg-Phe (FERF) and Phe-Asp-Gly-Leu (FDGL), were screened out.

[0010] The ToxinPred tool can be used to predict in batches whether peptides are toxic. 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 comparing them with sequences in a known allergen database. The ADMET Lab 3.0 tool can predict and evaluate the ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of compounds.

[0011] 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%.

[0012] 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.

[0013] More preferably, the molecular weight cut-off of the ultrafiltration membrane in step S3 is 5-10 KD.

[0014] Further preferably, in step S5, the desalting of the eluate is performed using a nanofiltration membrane with a molecular weight cutoff of 300-600 Da, and the drying is performed by freeze drying.

[0015] More preferably, in step S4, the filler of the weak anion exchange chromatography column is DEAE 52 cellulose.

[0016] 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 cow blood, sheep blood, pig blood or chicken blood.

[0017] Further, the hemoglobin peptide obtained in step S5 is subjected to Fe 2+ Determination of chelating capacity and Fe 2+ In vitro bioavailability determination was used to further evaluate the performance of hemoglobin peptides 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.

[0018] Furthermore, the Fe of hemoglobin peptides was determined using the Caco-2 cell model method. 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 inoculate Caco-2 cells at 1×10 5 cell / cm 2 Inoculate into a 12-well Transwell plate, add 0.5 mL of cell suspension to each well on the AP side of the chamber top, and add 1.5 mL of complete culture medium to the BL side of the base end; 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 completed; use a resistor meter to measure the transmembrane resistance of the cells, measure and record every other day, and ensure that the transepithelial resistance is greater than 200Ω·cm at 21 days. 2 ; Preparation of donor solution: Dissolve the hemoglobin peptide in deionized water to obtain a 2 mg / mL solution, and place it in a 37°C water bath for 10 min to fully dissolve it; take 10 mL of the fully dissolved peptide solution and add 1 mL of 0.01 mol / L FeSO 4, incubate for 30 minutes; centrifuge at 2268g for 20 minutes, 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 hemoglobin peptide-Fe complex in DMEM culture medium, the Fe content of each is 100μg / mL, and preheat to 37°C as donor solution; Caco-2 monolayer cell transport experiment: Change the culture medium 12-24 h before the experiment to prevent the cells from consuming necessary nutrients; transfer the cell monolayer to a new 12-well plate containing 1.5 mL HBSS buffer per well on the BL side, and carefully add 0.5 mL HBSS buffer to the AP side; incubate at 37°C for 15-20 min; 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 0.5mL of donor solution to the AP side, add 1.0mL 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; The Transwell plate for cell transport experiment was placed in a cell culture incubator, and the transport time was set to 2 h. The solutions on the AP side and BL side were collected respectively, 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:

[0019] in: C 0 is the initial AP side ferrous iron mass concentration, μg / mL; V 0 is the initial volume of the AP side solution, mL; C 2h is the mass concentration of ferrous iron on the AP side after 2 hours of transport, μg / mL; V 2h The volume of the AP side solution after 2 hours of transport, mL.

[0020] The preparation method of the present invention adopts high-pressure homogenization + composite protease hydrolysis, membrane separation + ion exchange chromatography purification and other technologies, which can hydrolyze to 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, and the IC of iron ion chelation is 50The concentration of ferrous iron in hemoglobin was less than 1 mg / mL, and the Caco-2 cell model showed that it significantly improved the bioavailability of iron ions, and a new method for evaluating the performance of promoting iron absorption was proposed; the peptides in hemoglobin peptides were evaluated and screened through nanoliter liquid chromatography-tandem mass spectrometry NanoLC-MS / MS, ALC scoring, Peptide Ranker scoring, BIOPEP database comparison analysis and other technical means, and the relevant properties of peptides were predicted through tools such as ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab3.0. A variety of methods were combined to screen out peptides with excellent comprehensive performance to meet the requirements, which played a significant role in the screening of FERF and FDGL peptide sequences that can promote iron absorption, and provided innovative ideas for research in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the mass spectrum of the polypeptide FERF in the hemoglobin peptide.

[0022] Figure 2 This is the mass spectrum of the polypeptide FDGL in hemoglobin peptide.

[0023] Figure 3 This is the interface diagram for toxicity prediction of FDGL and FDGL in hemoglobin peptides using ToxinPred.

[0024] Figure 4 Schematic diagram of the Caco-2 monolayer cell model grown on a permeable filtration support in the test of hemoglobin peptide on iron bioavailability in the embodiment of the present invention. DETAILED DESCRIPTION

[0025] Example 1 A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps: S1. Pretreatment of blood cells by high-pressure homogenization: fresh bovine blood was taken, and trisodium citrate with a mass concentration of 0.5% was added, and the blood cell sediment was collected at 5000 r / min for 15 min. Then the blood cell sediment was collected, and the same volume of NaCl solution with a mass concentration of 0.9% was added for washing, and the blood cell sediment was washed at 5000 r / min for 15 min. The blood cell sediment was washed twice, and the ...

[0026] S2. Enzymatic hydrolysis: adjust the pH of the hemoglobin solution to 6.0, add 100 U / mL of neutral protease, keep warm in a 45°C water bath for 2 h, then add 20 U / mL of flavor protease, continue to keep warm and hydrolyze for 8 h, then raise the temperature to 90°C and inactivate the enzyme for 30 min to obtain the enzymatic solution.

[0027] S3. Membrane filtration: Centrifuge the enzymatic hydrolysate (5000rpm, 10min) and collect the supernatant. Then filter it using an ultrafiltration membrane with a molecular weight cutoff of 10 KD and collect the permeate.

[0028] 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, discard the eluate, and then elute with 4CV of 0.1 mol / L NaCl solution, and collect the eluate.

[0029] 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.

[0030] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0031] Comparative Example 1 In this comparative example, the same hemoglobin peptide preparation method as in Example 1 was used, except that in step S2, papain was used for enzymatic hydrolysis in this comparative example.

[0032] Comparative Example 2 In this comparative example, the same hemoglobin peptide preparation method as in Example 1 was used, except that step S3 in Example 1 was omitted in this comparative example, that is, no ultrafiltration membrane was used to perform membrane separation on the polypeptide.

[0033] Comparative Example 3 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, an exchange chromatography column was not used to perform ion exchange chromatography on the hemoglobin peptide.

[0034] Example 2 A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps: S1. Pretreatment of blood cells by high-pressure homogenization: fresh bovine blood was taken, and trisodium citrate with a mass concentration of 0.5% was added, and the blood cell sediment was collected at 5000 r / min for 15 min. Then the blood cell sediment was collected, and the same volume of NaCl solution with a mass concentration of 0.9% was added for washing, and the blood cell sediment was washed at 5000 r / min for 15 min. The blood cell sediment was washed twice, and the ...

[0035] S2. Enzymatic hydrolysis: adjust the pH of the hemoglobin solution to 7.0, add 200 U / mL of neutral protease, keep warm in a 45°C water bath for 4 hours, then add 50 U / mL of flavor protease, continue to keep warm and hydrolyze for 4 hours, then heat to 90°C and inactivate the enzyme for 30 minutes to obtain the enzymatic solution.

[0036] S3. Membrane filtration: After centrifuging the enzymatic hydrolysate (5000 rpm, 10 min), collect the supernatant, and then filter it using an ultrafiltration membrane with a molecular weight cutoff of 5KD to collect the permeate.

[0037] 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), use 5CV of deionized water to elute the impurities, discard the eluate, and then elute with 4CV of 0.05 mol / L NaCl solution, and collect the eluate.

[0038] 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.

[0039] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0040] Example 3 A method for preparing a hemoglobin peptide capable of promoting iron absorption comprises the following steps: S1. Pretreatment of blood cells by high-pressure homogenization: fresh pig blood was taken, and trisodium citrate with a mass concentration of 0.5% was added, and the blood cell sediment was collected at 5000 r / min for 15 min. Then the blood cell sediment was collected, and the same volume of NaCl solution with a mass concentration of 0.9% was added for washing, and the blood cell sediment was washed at 5000 r / min for 10 min. The blood cell sediment was washed twice, and the ...

[0041] S2. Enzymatic hydrolysis: adjust the pH of the hemoglobin solution to 5.0, add 50 U / mL of neutral protease, keep it in a 55°C water bath for 4 h, then add 5 U / mL of flavor protease, continue to keep it in a warm and enzymatic hydrolysis for 24 h, then raise the temperature to 90°C and inactivate the enzyme for 30 min to obtain the enzymatic hydrolyzate.

[0042] S3. Membrane filtration: Centrifuge the enzymatic hydrolysate (5000 rpm, 10 min) and collect the supernatant. Then filter it using an ultrafiltration membrane with a molecular weight cutoff of 10 KD and collect the permeate.

[0043] 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), use 5 CV of deionized water to elute the impurities, discard the eluate, and then elute with 4 CV of 0.2 mol / L NaCl solution, and collect the eluate.

[0044] 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.

[0045] S6. Through nano-LC-MS / MS detection and bioactive peptide screening, short peptides with the activity of promoting iron absorption were screened out.

[0046] Structure identification and property determination: 1. Identification and screening of active peptides in hemoglobin peptides The prepared hemoglobin peptide samples were identified by 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 aqueous solution), with an injection volume of 3 μL and a flow rate of 10 μL / min. It was trapped on the trapping column for 3 minutes, followed by gradient elution chromatography separation on the nano-level analytical column, and the data-dependent mode was used to automatically switch between MS and MS / MS. The full scan MS used Orbitrap for primary scanning, with a scanning range of m / z 150-1600 and a resolution set to 120,000. The raw data obtained was processed and analyzed using PEAKS software.

[0047] ALC(%) stands for Average Local Confidence, which is the confidence of the results of de novo sequencing data. Generally, it is more reliable if it is greater than 80%, and very reliable if it is greater than 95%. The 2627 peptides with ALC scores exceeding 95% were further scored by PeptideRanker, and the score threshold was set to 0.8, from which 178 bioactive peptides with high potential were screened. These 178 peptides were functionally screened in the BIOPEP database, and finally 63 peptides with high iron / zinc / calcium binding activity were screened.

[0048] The safety, water solubility and absorbability of active peptides have an important impact on their application value. Therefore, ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools were used to predict the toxicity, solubility, sensitization and absorption characteristics of the above 63 peptides in the human intestine. 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, 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 .

[0049] 2. Hemoglobin peptide Fe 2+ Determination of chelating capacity 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. Then, 20 μL of FeSO 4 The solution (0.2 mM) was added to 140 μL of hemoglobin peptide solutions of different concentrations. The plate was then incubated in a constant temperature room 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 min, 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:

[0050] in: A 0 is the absorbance of the blank; A s is the absorbance of the sample.

[0051] IC 50 Defined as 50% inhibition of ferrozine-Fe 2+ Peptide concentration for complex formation.

[0052] 3. Caco-2 cell model for Fe determination 2+ In vitro bioavailability Establishment of Caco-2 monolayer cell model: Resuspend the cells in complete medium. Before inoculating the cells, pre-wet the Transwell chamber with DMEM medium. Then, inoculate Caco-2 cells at a rate 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 basal (BL) side. After inoculation, replace the culture medium every 6-16h, and then replace the culture medium on both sides every other day. The cell culture cycle is 21 days until cell membrane differentiation is complete. Use a resistor to measure the transmembrane resistance of the cells, measure and record every other day to ensure that the transepithelial resistance (TEER value) is greater than 200Ω·cm at 21 days. 2 .

[0053] Donor solution preparation: Dissolve the hemoglobin peptide in deionized water to obtain a 2 mg / mL solution and place it in a 37°C water bath for 10 min to fully dissolve it. Take 10 mL of the fully dissolved peptide solution and add 1 mL of FeSO 4 (0.01 mol / L), incubate for 30 min. Centrifuge at 2268 g centrifugal force (3000 rpm) for 20 min 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 hemoglobin peptide-Fe complex (Fe content 100 μg / mL) in DMEM culture medium and preheat them to 37°C to obtain the donor solution.

[0054] Caco-2 monolayer cell transport assay: Change the medium 12-24 h before the experiment to avoid the cells from consuming necessary nutrients. Transfer the cell monolayer to a new 12-well plate containing HBSS buffer (1.5 mL per well) on the BL side and carefully add 0.5 mL HBSS buffer to the AP side. Incubate at 37°C for 15-20 min.

[0055] 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, 1.0 mL of HBSS buffer to the BL side, and immediately take a sample at t=0 to determine the ferrous content to prevent part of the compound from being transported before sampling is completed.

[0056] The Transwell plate for cell transport experiment was placed in a cell culture incubator, and the transport time was set to 2 h. The AP and BL side solutions 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:

[0057] in: C 0 : Initial AP side ferrous iron mass concentration (μg / mL); V 0 : Initial volume of AP side solution (mL); C 2h : ferrous iron mass concentration on AP side after 2 h of transport (μg / mL); V 2h : AP side solution volume after 2 h of transport (mL).

[0058] 4. Peptide content determination method 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 by the Kjeldahl nitrogen method in accordance with GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food" to obtain the acid-soluble protein content. X 1 .

[0059] 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 .

[0060] Peptide content (g / 100g) = X1 - X 2 ; In the formula, X 1 Indicates the content of acid-soluble protein, in g / 100g; X 2 Indicates the content of free amino acids in g / 100g.

[0061] 5. Test results 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.

[0062] Table 1 Test and application results of each sample

[0063] Note: The test data of FERF sample and FDGL sample are obtained by using the two synthesized pure polypeptide sequences as samples according to the above test method. 4 The test data corresponding to the sample is obtained by dissolving ferrous sulfate in DMEM culture medium as the donor solution.

[0064] It can be seen from the test results 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 ferrozine-Fe 2+ Complex inhibition IC 50 All were lower than 1 mg / mL, far 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 ferrozine-Fe by only a smaller concentration than that of the comparative example. 2+ The formation of the complex indicates that it is Fe 2+ The chelating ability is strong and 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, and the iron in the complex is more bioavailable. Therefore, the preparation method of the invention can obtain a hemoglobin peptide with excellent comprehensive performance, especially having excellent performance in promoting iron absorption.

[0065] 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 deviate from the present invention should be included in the patent scope of this case.

Claims

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

2. A method for preparing a hemoglobin peptide capable of promoting iron absorption as claimed in claim 1, characterized in that: The steps include: S1. Pretreatment of blood cells by high-pressure homogenization: After adding trisodium citrate with a mass concentration of 0.1%-1.0% to fresh animal blood, the blood cell precipitate is collected by centrifugation; then the same volume of NaCl solution with a mass concentration of 0.5%-1.0% is added for washing and centrifugation, and the washing is repeated 2-3 times to obtain pure red blood cells; then, water is added to prepare the red blood cells into a solution with a mass concentration of 5%-15%, and the red blood cells are broken by high-pressure homogenization at a pressure of 500-1500 bar for 2-5 times to obtain a hemoglobin solution; S2, enzymatic hydrolysis: the pH of the hemoglobin solution obtained in step S1 is adjusted to 5.0-7.0, 10-200 U / mL of neutral protease is added at a temperature of 30-60°C for heat treatment for 0.5-4h, 5-50 U / mL of flavor protease is added for enzymatic hydrolysis for 4-24h, and the enzyme is inactivated at 70-100°C for 30 min to obtain an enzymatic hydrolyzate; S3, membrane filtration: centrifuge the enzymatic hydrolysate obtained in step S2, collect the supernatant, and then filter it using an ultrafiltration membrane with a molecular weight cutoff of 3-20 KD to collect the permeate; S4, ion exchange chromatography: after adjusting the pH of the permeate collected in step S3 to 5.0-6.0, add 2-5 column volumes of the permeate to a weak anion exchange chromatography column, use 2-5 column volumes of deionized water to elute the impurities, discard the eluate, and then elute with 3-6 column volumes of 0.05-0.5 mol / L NaCl solution, and collect the eluate; S5, concentration and drying: desalting, concentrating and drying the eluate collected in step S4 to obtain hemoglobin peptides; The hemoglobin peptides obtained in steps S6 and S5 were identified as active peptides by nanoliter liquid chromatography-tandem mass spectrometry NanoLC-MS / MS, and bioactive peptides were screened. The toxicity, solubility, sensitization and absorption characteristics of the active peptides in the human intestine were predicted using ToxinPred, INNOVAGEN, Allergen FP and ADMETLab 3.0 tools, respectively. Finally, two safe short peptide sequences Phe-Glu-Arg-Phe and Phe-Asp-Gly-Leu with iron absorption-promoting activity were screened out.

3. The method for preparing a hemoglobin peptide capable of promoting iron absorption according to claim 2, characterized in that: The specific method of step S6 is: 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, and then gradient elution chromatography was performed on a nanoliter analytical column. The data-dependent mode was used to automatically switch between MS and MS / MS acquisition. The full scan MS was performed using Orbitrap for primary scanning, with a scanning range of m / z 150-1600 and a resolution of 120,000. The raw data obtained were processed and analyzed using PEAKS software. Peptides with an average local confidence ALC score of more than 95% were further scored using PeptideRanker, with a score threshold of 0.8, to screen out high-potential bioactive peptides. The high-potential bioactive peptides were functionally screened in the BIOPEP database, and several peptides with high iron / zinc / calcium binding activity were screened out. 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, and finally two safe short peptide sequences Phe-Glu-Arg-Phe and Phe-Asp-Gly-Leu with iron absorption-promoting activity were screened out.

4. The method for preparing a hemoglobin peptide 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 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 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 5-10 KD.

7. The method for preparing a hemoglobin peptide capable of promoting iron absorption according to claim 2, characterized in that: In step S5, the desalting of the eluate is performed by 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 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 capable of promoting iron absorption according to claim 2, characterized in that: The hemoglobin peptide 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 hemoglobin peptides 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 capable of promoting iron absorption according to claim 9, characterized in that: Determination of Fe of Hemoglobin Peptides 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 inoculate Caco-2 cells at 1×10 5 cell / cm 2 Inoculate into a 12-well Transwell plate, add 0.5 mL of cell suspension to each well on the AP side of the chamber top, and add 1.5 mL of complete culture medium to the BL side of the base end; 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 completed; use a resistor meter to measure the transmembrane resistance of the cells, measure and record every other day, and ensure that the transepithelial resistance is greater than 200Ω·cm at 21 days. 2 ; Preparation of donor solution: Dissolve the hemoglobin peptide in deionized water to obtain a 2 mg / mL solution, and place it in a water bath at 37°C for 10 minutes to fully dissolve it; take 10 mL of the fully dissolved peptide solution, add 1 mL of FeSO4 with a concentration of 0.01 mol / L, and incubate for 30 minutes; centrifuge at 2268g for 20 minutes to remove the precipitate, add 6 times the volume of anhydrous ethanol to the supernatant, collect the precipitate, and freeze-dry to obtain the hemoglobin peptide-Fe complex; use DMEM culture medium to dissolve ferrous sulfate and hemoglobin peptide-Fe complex respectively, the Fe content of both is 100 μg / mL, and preheat to 37°C as the donor solution; Caco-2 monolayer cell transport assay: Change the culture medium 12-24 h before the experiment to prevent the cells from consuming necessary nutrients; transfer the cell monolayer to a new 12-well plate containing 1.5 mL HBSS buffer per well on the BL side, and carefully add 0.5 mL HBSS buffer to the AP side; incubate at 37°C for 15-20 min; 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 0.5mL of donor solution to the AP side, 1.0mL 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 for cell transport experiment was placed in a cell culture incubator, and the transport time was set to 2 h. The solutions on the AP side and BL side were collected respectively, 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: in: C0 is the initial mass concentration of ferrous iron on the AP side, μg / mL; V0 is the volume of the AP side solution at the initial time, mL; C 2h is the mass concentration of ferrous iron on the AP side after 2 hours of transport, μg / mL; V 2h The volume of the AP side solution after 2 hours of transport, mL.

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