Extraction method and application of ferrous chelated heptapeptide

Abstract

The invention discloses an extraction method and application of ferrous chelated heptapeptide, and belongs to the technical field of biology. The heptapeptide disclosed by the invention is clear and definite in structure, can be prepared by adopting a solid-phase chemical synthesis method, and also can be obtained by separating and purifying fermented scallop skirt hydrolysate. The heptapeptide provided by the invention has a remarkable improvement effect on symptoms caused by iron-deficiency anemia. Experimental results show that the heptapeptide can promote hemoglobin synthesis and regulate expression of related genes of anemia individuals. The method is suitable for developing novel iron-rich foods and iron supplements, and can also be compounded with other health-care products or food additives for use.

Description

Technical Field

[0001] The present invention relates to a method for extracting ferrous chelated heptapeptide and its application, belonging to the field of biotechnology. Background Art

[0002] Iron is one of the essential trace elements in the human body and is involved in important physiological processes such as oxygen transport, DNA synthesis, and muscle metabolism. In particular, during early life, adolescence, and pregnancy, the human body's demand for iron increases significantly, and iron deficiency problems are prone to occur. The World Health Organization has pointed out that iron deficiency and iron deficiency anemia are global public health challenges, affecting the health of approximately 2 billion people and causing problems such as anemia, child developmental retardation, and memory loss.

[0003] Ferrous chelated peptides, as a new generation of dietary supplements, have attracted much attention due to their characteristics of high absorption efficiency, low-energy consumption metabolism, rapid transport, and non-saturation. These peptides can enter intestinal mucosal cells through the small peptide transport system, providing a stable iron supply and reducing metal antagonism. Existing reports mostly use enzymatic hydrolysis methods to prepare bioactive peptides and further separate and purify them to extract the target peptide segments. However, the peptide sequence richness obtained by enzymatic hydrolysis methods is insufficient, and the purified peptide segments have limited sequences, unable to fully explore all potential active ingredients in the substrate. The microbial fermentation method is a technology that uses proteases produced by microorganisms during their growth to hydrolyze substrate proteins to prepare bioactive peptides. Using the microbial fermentation method, a variety of bioactive peptides can be efficiently produced from natural substrates without the need to add additional enzymes or carbon sources. This method not only simplifies the production process but also improves the diversity and yield of peptide segments.

[0004] Scallop skirts, as by-products of seafood processing, contain rich proteins and are ideal raw materials for preparing polypeptides with high nutritional value. However, there are few studies on directly preparing and extracting iron chelated peptides from fermented scallop skirts, especially technical solutions to achieve this goal without the assistance of additional enzymes.

[0005] The present invention proposes a brand-new method of using Bacillus subtilis to ferment scallop skirts in order to isolate heptapeptides with strong ferrous chelating ability from the hydrolysate. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides a method for extracting ferrous chelated heptapeptide and its application, aiming to solve the technical problem of the method for directly preparing and extracting iron chelated peptides from fermented scallop skirts without the assistance of additional enzymes in the existing technologies.

[0007] The first technical solution provided by the present invention is a ferrous chelated heptapeptide, and its amino acid sequence is Glu-Glu-Glu-Trp-Asp-Arg-Glu (EEEWDRE).

[0008] The second technical solution provided by the present invention is a preparation method of a ferrous chelated heptapeptide, comprising the following steps:

[0009] (1) Sample pretreatment: Take the scallop skirt, wash it, remove impurities, dry it in an oven at 50 °C for 8 hours, crush it, and pass it through a 100-mesh sieve twice.

[0010] (2) Preparation of the sample to be fermented: Mix the scallop skirt powder obtained in step (1) with water and sterilize it under high temperature and high pressure to obtain a sterilized scallop skirt liquid.

[0011] (3) Preparation of the fermented scallop skirt crude peptide liquid: Add a Bacillus subtilis suspension to the cooled sterilized scallop skirt liquid, carry out fermentation culture, and freeze-dry the fermentation supernatant to obtain the fermented scallop skirt crude peptide liquid.

[0012] (4) Purification and identification of the fermented scallop skirt hydrolyzate: Use an ultrafiltration centrifugal tube to ultrafilter and separate the hydrolyzate. The specifications of the ultrafiltration centrifugal tube are 1KDa, 3KDa, 5KDa, and 10KDa. Further select the component with the highest ferrous ion chelation rate, and use 5 column volumes of 0.3 mol / L FeSO4·7H 2 O to fill the iron ion metal chelating affinity chromatography column (IMAC). A 2 mL sample of the fermented scallop skirt hydrolyzate with a concentration of 5 mg / mL is loaded onto the IMAC column, and the column is left standing at room temperature for 120 minutes. The first component (F1), that is, the non-iron chelated peptide, is eluted with the equilibrium buffer (20 mmol / L sodium acetate, 0.5 mol / L NaCl, pH 5.0); subsequently, the iron chelated peptide (F2) is eluted with the elution buffer (20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 8.0), and the elution rate is set at 2 mL / min. Monitor the absorbance of the eluate at 220 nm. Collect the F2 component, and further separate it by Sephadex G-25 gel filtration chromatography. The eluent is deionized water, the flow rate is 0.3 mL / min, and the elution peak is monitored at 220 nm; collect the component with the highest metal chelating activity, and identify its peptide sequence by mass spectrometry. The results confirm that it contains the heptapeptide of the present invention.

[0013] In some embodiments, in step (2), the material-liquid ratio of the scallop skirt powder to deionized water is 1:5 (g / mL); the sterilization conditions are 90 °C for 20 min.

[0014] In certain embodiments, in step (3), the Bacillus subtilis is Bacillus subtilis M17-b7, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 10, 2023, with the deposit number CGMCC No. 27847; the fermentation conditions are as follows: the final concentration of the bacterial solution is 1×10 7 CFU / mL, placed in a shaker at 37°C for shaking culture, the shaker speed is 200 rpm, and the culture time is 36 h; the centrifugation conditions are centrifugation at 10,000 rpm for 10 min.

[0015] The third technical solution provided by the present invention is a ferrous-heptapeptide chelate, and the preparation method of the chelate is to adjust the pH of the heptapeptide described in the first technical solution to 6.5 - 7.0, then mix it with ferrous sulfate, vortex and oscillate for 30 min, and freeze-dry it by spray drying or freeze drying to obtain the ferrous-heptapeptide chelate.

[0016] The fourth technical solution provided by the present invention is the application of the heptapeptide described in the first technical solution, or the ferrous-heptapeptide chelate described in the third technical solution in the preparation of iron-supplementing drugs or health products.

[0017] The fifth technical solution provided by the present invention is the application of the heptapeptide described in the first technical solution, or the ferrous-heptapeptide chelate described in the third technical solution in the preparation of drugs for relieving and / or treating iron deficiency anemia.

[0018] In certain embodiments, the application at least includes one of the following effects:

[0019] (a) Improving the abnormalities of HGB, HCT, and MCH caused by iron deficiency anemia in an individual;

[0020] (b) Downregulating the expression of duodenal TFR1, DMT1, ZIP8, ZIP14, PCBP2, and FPN1 genes and the expression of small intestine ZO-1 and PepT1 genes in anemic individuals, and upregulating the expression of liver FTL, FTH, Ferritin, and Hamp genes in anemic individuals.

[0021] The sixth technical solution provided by the present invention is a polynucleotide encoding the heptapeptide described in the first technical solution.

[0022] The seventh technical solution provided by the present invention is a recombinant vector carrying the polynucleotide described in the sixth technical solution.

[0023] The eighth technical solution provided by the present invention is a recombinant cell expressing the heptapeptide described in the first technical solution, or containing the polynucleotide described in the sixth technical solution, or transformed with the recombinant vector described in the seventh technical solution.

[0024] The ninth technical solution provided by the present invention is a product, and the product contains the heptapeptide described in the first technical solution.

[0025] In some embodiments, the product includes food, medicine or health products.

[0026] The technical effects of the present invention are as follows:

[0027] 1. The structure of the heptapeptide of the present invention is clear and definite. It can be prepared by solid-phase chemical synthesis method, or can be isolated and purified from the hydrolyzate of fermented scallop skirts.

[0028] 2. The heptapeptide of the present invention has a significant improvement effect on the symptoms caused by iron deficiency anemia. The experimental results show that the heptapeptide can promote hemoglobin synthesis and regulate the expression of genes related to anemic individuals. It is suitable for the development of new iron-rich foods and iron supplements, and can also be used in combination with other health products or food additives.

[0029] Biological material preservation

[0030] Bacillus subtilis M17-b7, with the taxonomic name of Bacillus subtilis, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 10, 2023. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 27847. Description of the drawings

[0031] Figure 1 It is the mass spectrometry identification result of the heptapeptide. Detailed implementation manners

[0032] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0033] Testing method:

[0034] (1) Establishment of a mouse model of iron deficiency anemia

[0035] Thirty 3-week-old male ICR mice were weaned and acclimated in a barrier system for 1 week, and then randomly divided into 5 groups according to body weight: normal group (Control), positive control (Pos), negative control (Neg), low-dose (Low), and high-dose (High) groups. The normal group was fed with maintenance feed, and the other groups were fed with low-iron feed (5 ppm Fe). All groups had free access to water. The hemoglobin levels of the mice in each group were monitored weekly. After the hemoglobin content of the mice in each group was lower than 100 g / L, 2 mL / 1000 g bw of the sample was administered by gavage: the normal and negative control groups were gavaged with ultrapure water, the positive control group was gavaged with ferrous sulfate at 4 mg Fe / kg bw, and the low- and high-dose groups were gavaged with heptapeptide-ferrous chelate at 1 and 4 mg Fe / kg bw, respectively.

[0036] (2) Determination of blood routine indexes of mice

[0037] After the experiment, the mice were anesthetized, and 0.5 mL of blood was collected from the orbital cavity. The blood routine indexes of the mice were measured using a fully automatic five-class hematology analyzer. The measured indexes included hemoglobin concentration HGB, hematocrit HCT, and mean corpuscular hemoglobin content MCH.

[0038] (3) Determination of the expression levels of iron homeostasis-related genes

[0039] 1. Extraction of RNA from mouse liver, duodenum, and small intestine

[0040] The mouse liver, duodenum, and small intestine were separated and the surface bloodstains were washed with DEPC water. 100 mg of the sample was taken, 1 mL of Trizol reagent was added, and the sample was homogenized thoroughly in an ice bath. The homogenized sample was allowed to stand at room temperature for 5 minutes, 200 μL of chloroform was added, and the mixture was vortexed vigorously and then allowed to stand at room temperature for 3 minutes. The mixture was centrifuged at 4 °C and 12,000 g for 15 minutes, and the upper layer liquid was transferred to a new enzyme-free EP tube. 500 μL of isopropanol was added, and the mixture was inverted and mixed well and then allowed to precipitate at room temperature for 10 minutes. The mixture was centrifuged at 4 °C and 12,000 g for 10 minutes, and the RNA precipitate could be seen at the bottom of the tube. The RNA precipitate was washed with 75% pre-cooled ethanol in a 1:1 ratio, centrifuged at 4 °C and 12,000 g for 5 minutes, and the supernatant was discarded. The RNA precipitate was air-dried after spinning at 4 °C and 12,000 g for 5 minutes, and 20 μL of DEPC water was added to resuspend it. The RNA concentration was detected using a micro-spectrophotometer.

[0041] 2. Preparation of reverse transcription system and reaction conditions

[0042] (1) Reaction for removing genomic DNA

[0043]

[0044] The mixed system was reacted in a 42 °C water bath for 2 minutes.

[0045] (2) Reverse transcription reaction

[0046]

[0047] The PCR program is 37 °C for 15 minutes; 85 °C for 5 seconds.

[0048] (3) Preparation of real-time fluorescence quantitative PCR (RT-qPCR) system and reaction conditions

[0049]

[0050]

[0051] The PCR program is: 95 °C for 30 seconds; 95 °C for 5 seconds, 60 °C for 5 seconds, and this process undergoes 40 cycles. The primer sequences are shown in the following table:

[0052]

[0053] The relative gene expression level is calculated according to the following formula:

[0054] Relative gene expression level = 2 -ΔΔCt

[0055] Among them, ΔCt is the difference in the number of cycles between the target gene and the internal reference gene, and ΔΔCt is the difference in ΔCt between the sample group gene and the control gene. β-actin is used as the internal reference gene.

[0056] Raw materials used in the examples:

[0057] 1. A strain of Bacillus subtilis M17-b7, which has been deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit number is CGMCC No. 27847.

[0058] 2. ICR male mice: Male SPF-grade ICR mice (3 weeks old, weighing 12 - 15 g), purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning Experimental Animal Resource Center, license number: SCXK(Liao)2015 - 0001), and raised in the animal room 401 of the National Engineering Research Center for Marine Food of Dalian Polytechnic University (license number: SYXK(Liao)2017 - 0005). The environmental temperature is 22 ± 2 °C, the humidity is 65 ± 5%, and the lights in the animal room alternate between light and dark for 12 hours.

[0059] 3. Liquid medium formulation: The LB Broth (LB) solid / liquid medium for activating strains was purchased from Qingdao Haibo Biotechnology Co., Ltd. The main components of the liquid medium are (g / L): tryptone 10.0, yeast extract powder 5.0, sodium chloride 10.0. The pH value is 7.0 ± 0.1. The formulation of the solid medium contains 15.0 g / L agar in addition to the above components.

[0060] Example 1 Preparation of Chelated Heptapeptide

[0061] I. Preparation of Fermented Scallop Skirt Coarse Peptide Solution

[0062] (1) Raw material preparation: Wash the scallop skirt, remove impurities, dry it in an oven at 50 °C for 8 hours, crush it, and sieve it through a 100-mesh sieve twice to obtain scallop skirt dry powder for standby;

[0063] (2) Scallop skirt sterilized solution: Mix the scallop skirt powder prepared in step (1) with a certain amount of water, and the material-liquid ratio is 1:5 (g / mL) to obtain a mixed solution; Sterilize it at 121 °C for 20 min to obtain a sterilized scallop skirt solution;

[0064] (3) Fermented scallop skirt hydrolyzate: Inoculate Bacillus subtilis M17-b7 into the LB liquid medium, place it in a shaking incubator at 37 °C for 18 h for activation. After activation, centrifuge to collect the bacterial cells, and add sterile deionized water to prepare a bacterial suspension. After the sterilized scallop skirt solution prepared in step (2) is cooled, add the Bacillus subtilis suspension to make the final concentration of the bacterial solution 10 7 CFU / mL, shake and culture the mixed solution in a shaking table for 24 h; Centrifuge the mixed solution, and the centrifugation conditions are centrifugation at 8000 rpm for 20 min. Collect the supernatant, and freeze-dry the supernatant to obtain the fermented scallop skirt hydrolyzate.

[0065] II. Separation and Purification of Heptapeptide

[0066] Use an ultrafiltration centrifugal tube to ultrafilter and separate the fermented scallop skirt coarse peptide solution. The specifications of the ultrafiltration centrifugal tube are 1KDa, 3KDa, 5KDa, 10KDa. Further select the component with the highest chelation rate of ferrous ions, and use 5 column volumes of 0.3 mol / L FeSO4·7H 2O - loaded immobilized metal affinity chromatography (IMAC) column. 2 mL of ultrafiltered fermented scallop skirt hydrolysate with a concentration of 5 mg / mL was loaded onto the IMAC column, and the column was left standing at room temperature for 120 minutes. The first fraction (F1), namely non - iron - chelated peptides, was eluted with the equilibration buffer (20 mmol / L sodium acetate, 0.5 mol / L NaCl, pH 5.0); subsequently, the iron - chelated peptides (F2) were eluted with the elution buffer (20 mmol / L sodium phosphate, 0.5 mol / L NaCl, pH 8.0), and the elution rate was set at 2 mL / min. The absorbance of the eluate was monitored at 220 nm. The F2 fraction was collected and further separated by Sephadex G - 25 gel filtration chromatography. The eluent was deionized water, the flow rate was 0.3 mL / min, and the elution peak was monitored at 220 nm; the fraction with the highest metal - chelating activity was collected to obtain the target heptapeptide. The peptide sequence was identified by liquid phase and mass spectrometry, and the results confirmed that it contained the heptapeptide of the present invention. The mass spectrometry identification results of the heptapeptide are as Figure 1 shown.

[0067] Example 2 Preparation of heptapeptide - ferrous chelate

[0068] After adjusting the pH of the heptapeptide in Example 1 to 6.5 - 7.0, it was mixed with ferrous sulfate. The mass ratio of the heptapeptide to ferrous ions was 1:3, and it was vortex - oscillated for 30 min, and then freeze - dried by spray drying or freeze - drying method to obtain the ferrous - heptapeptide chelate.

[0069] Example 3 Improvement effect of heptapeptide on blood routine indexes of iron - deficient anemia mice

[0070] The blood routine indexes of five groups of iron - deficient anemia mice were measured.

[0071] Table 1 Results of blood routine index determination of mice

[0072]

[0073]

[0074] Different lowercase letters indicate significant differences between different groups for the same index (p < 0.05).

[0075] The test results are shown in Table 1. Mice in the negative control group (without iron supplementation) showed significant decreases in hemoglobin concentration (HGB), hematocrit (HCT), and mean corpuscular hemoglobin content (MCH), indicating that the iron-deficiency anemia model had been successfully established. For the low-dose and high-dose heptapeptide-ferrous chelate treatment groups, key indicators such as HGB, HCT, and MCH in mice gradually returned to normal levels, with the effect being particularly significant in the high-dose group. Specifically, the parameters in the high-dose group not only showed statistically significant differences from the negative control group (p < 0.05), but also almost reached the levels of the normal group. This result indicates that heptapeptide-ferrous chelate has an obvious improvement effect on iron-deficiency anemia in mice, and this effect shows an obvious dose-dependent relationship. In summary, our results confirm the effectiveness of heptapeptide-ferrous chelate in improving iron-deficiency anemia in mice, and heptapeptide-ferrous chelate is expected to be used as a new type of iron supplement.

[0076] Example 4 Effect of Heptapeptide-Ferrous Chelate on the Expression Levels of Genes Related to Iron Homeostasis in Mice

[0077] Liver, duodenum, and small intestine tissues of five groups of mice were obtained, and the surface bloodstains were washed with DEPC water. After washing, the determination of the expression levels of genes related to iron homeostasis was carried out.

[0078] Table 2 Relative Expression Levels of Iron Homeostasis Genes in Mice of Different Treatment Groups

[0079]

[0080] As shown in Table 2, intragastric administration of high-dose heptapeptide-ferrous chelate down-regulated the expression of TFR1, DMT1, ZIP8, ZIP14, PCBP2, and FPN1 genes in the duodenum of anemic mice, and up-regulated the expression of FTL, FTH, Ferritin, and Hamp genes in the liver, restoring iron homeostasis in mice. In the negative control group, compared with the normal group, genes such as Ferritin, FTH, and FTL were down-regulated by approximately 76%, 64%, and 84% respectively, while Hamp was almost completely inhibited; FPN1 was significantly up-regulated by approximately 213%. In contrast, in the high-dose group, the change ranges of most genes were small, and even some gene expressions were close to normal levels or slightly up-regulated. And the expression levels of ZO-1 and PepT1 genes in the small intestine of mice were significantly up-regulated, indicating that in addition to being absorbed in the duodenum, part of the iron was absorbed into the body in the form of iron-chelated peptides through the paracellular transport and with the help of dipeptide and tripeptide transporters. In summary, the heptapeptide of the present invention can improve iron-deficiency anemia in mice by regulating the expression of genes related to anemic individuals.

[0081] Comparative Example 1

[0082] I. Improvement effect of polypeptide-ferrous chelate extracted from fermented scallop skirts on blood routine indexes of iron-deficiency anemia mice

[0083] In addition to the heptapeptide described in Example 1, another 8 polypeptides identified from the crude peptide of fermented scallop skirts were selected and synthesized by chemical synthesis method to prepare the corresponding polypeptide-ferrous chelates. Anemic mice were gavaged with the same dosage as the high-dose group, and their blood routine indexes were measured.

[0084] Table 3 Results of determination of blood routine indexes of mice

[0085]

[0086] As shown in Table 3, the improvement effects of the ferrous chelates prepared by gavaging other peptide segments purified from fermented scallops on the hemoglobin concentration, hematocrit and mean corpuscular hemoglobin content indexes of mice were all lower than those of the heptapeptide-ferrous chelate proposed in this patent. This indicates that the heptapeptide-ferrous chelate proposed in this patent may have higher efficiency in improving iron-deficiency anemia.

[0087] II. Effect of polypeptide-ferrous chelate extracted from fermented scallop skirts on the expression levels of genes related to iron homeostasis in mice

[0088] Liver, duodenum and small intestine tissues of mice were taken, and the surface blood stains were washed with DEPC water. After washing, the expression levels of genes related to iron homeostasis were measured.

[0089] Table 4 Relative expression levels of iron homeostasis genes in mice with different polypeptide-ferrous chelates

[0090]

[0091]

[0092] As shown in Table 4, the relative expression levels of each gene in mice gavaged with the ferrous chelates prepared from other polypeptides purified from fermented scallops were comparable to those in the anemic mouse group. This indicates that these polypeptide-ferrous chelates did not show significant effects in improving the expression of anemia-related genes, probably because these peptide segments do not possess the specific biological activities or structural characteristics of the heptapeptide proposed in this patent and cannot effectively regulate the expression of genes related to anemia.

[0093] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A ferrous chelated heptapeptide, characterized in that: Its amino acid sequence is EEEWDRE.

2. The method for preparing the ferrous chelated heptapeptide according to claim 1, characterized in that: The steps include: (1) preparing a fermented scallop skirt crude peptide liquid: adding a Bacillus subtilis suspension to a scallop skirt liquid for fermentation, and freeze-drying the fermentation supernatant to obtain a fermented scallop skirt crude peptide liquid; (2) Purification and identification of fermented scallop skirt hydrolyzate: The hydrolyzate was ultrafiltered and separated using an ultrafiltration centrifuge tube, and the filtrate was collected. A metal chelate affinity chromatography column was filled with iron ions to further select the component with the highest ferrous ion chelation rate. The component was further separated using Sephadex G-25 gel filtration chromatography to collect the component with the highest metal chelation activity. The peptide sequence was identified using mass spectrometry to obtain the heptapeptide.

3. The preparation method according to claim 2, characterized in that: In step (1), the preparation method of scallop skirt liquid is as follows: the scallop skirt is washed, impurities are removed, and it is dried in a 50°C oven for 8 hours, and then crushed and sieved through a 100-mesh sieve twice; the scallop skirt powder and water are mixed according to a solid-liquid ratio of 1g:5mL and then sterilized by high temperature and high pressure to obtain scallop skirt liquid; the Bacillus subtilis is Bacillus subtilis M17-b7, which has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 10, 2023, with a deposit number of CGMCC No.27847; the fermentation conditions are: the final concentration of the bacterial liquid is 1×10 7 CFU / mL, placed in a 37°C shaker with a shaking speed of 200 rpm for 36 h; In step (2), the specifications of the ultrafiltration centrifuge tube are 1KDa, 3KDa, 5KDa, and 10KDa, and 5 column volumes of 0.3mol / LFeSO4·7H2O iron ions are used to fill the metal chelate affinity chromatography column. The process of the metal chelate affinity chromatography is as follows: 2mL of ultrafiltered fermented scallop skirt hydrolyzate with a concentration of 5mg / mL is loaded onto the IMAC column, and the column is allowed to stand at room temperature for 120 minutes. The first component, i.e., the non-iron chelating peptide, is eluted with pH 5.0, 20mmol / L sodium acetate, 0.5mol / L NaCl as an equilibrium buffer; subsequently, the second component, i.e., the iron chelating peptide, is eluted with pH 8.0, 20mmol / L sodium phosphate, 0.5mol / L NaCl was used as the elution buffer for elution, the elution rate was set to 2 mL / min, and the absorbance of the eluent at 220 nm was monitored. The Sephadex G-25 gel filtration chromatography conditions were as follows: the eluent was deionized water, the flow rate was 0.3 mL / min, and the elution peak was monitored at 220 nm.

4. A ferrous iron-heptapeptide chelate, characterized in that: The preparation method of the chelate is as follows: the pH of the heptapeptide according to claim 1 is adjusted to 6.5-7.0, and then mixed with ferrous sulfate, vortexed for 30 minutes, and freeze-dried by spray drying or freeze drying to obtain the ferrous-heptapeptide chelate.

5. Use of the heptapeptide according to claim 1 or the ferrous iron-heptapeptide chelate according to claim 4 in the preparation of medicines or health products for iron supplementation.

6. Use of the heptapeptide according to claim 1 or the ferrous iron-heptapeptide chelate according to claim 4 in the preparation of a medicament for alleviating and / or treating iron deficiency anemia, characterized in that: The application includes at least one of the following functions: (a) Improve the abnormalities of HGB, HCT and MCH caused by individual iron deficiency anemia; (b) Down-regulated the expression of TFR1, DMT1, ZIP8, ZIP14, PCBP2 and FPN1 genes in the duodenum and ZO-1 and PepT1 genes in the small intestine of anemic individuals, and up-regulated the expression of FTL, FTH, Ferritin and Hamp genes in the liver of anemic individuals.

7. A polynucleotide encoding the heptapeptide according to claim 1.

8. A recombinant vector carrying the polynucleotide according to claim 7.

9. A recombinant cell expressing the heptapeptide of claim 1, or containing the polynucleotide of claim 7, or transformed with the recombinant vector of claim 8.

10. A product, characterized in that The product contains the heptapeptide according to claim 1.

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