A sea cucumber flower heptapeptide with iron absorption-promoting effects, its preparation method and application

Abstract

This invention relates to the field of biopeptide synthesis technology, and particularly to a sea cucumber flower heptapeptide with iron absorption-promoting effects, its preparation method, and its applications. The sea cucumber flower heptapeptide with iron absorption-promoting effects provided by this invention exhibits good digestive stability after gastrointestinal digestion, providing a reliable carrier for iron delivery. The sea cucumber flower heptapeptide with iron absorption-promoting effects provided by this invention can be formulated into a sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects, which can promote iron absorption and is suitable for use in the field of iron supplements, enriching the variety of functional iron supplements. The sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects provided by this invention has the ability to effectively resist digestion by gastrointestinal proteases. The results of the examples show that after simulated gastrointestinal digestion, its peptide stability is significantly higher than that of the sea cucumber flower heptapeptide with iron absorption-promoting effects; at the same time, its iron transport capacity is significantly better than that of FeCl2, effectively promoting the absorption of iron by small intestinal epithelial cells.

Description

Technical Field

[0001] This invention relates to the field of biopeptide synthesis technology, and in particular to a sea cucumber flower heptapeptide with iron absorption-promoting effects, its preparation method, and its application. Background Technology

[0002] Iron is an essential trace element for the human body, and iron deficiency leads to iron-deficiency anemia. Generally, the human body cannot produce the iron it needs through metabolism and must obtain it from external sources. However, due to individual differences in absorption and the influence of dietary iron absorption inhibitors (such as phytic acid and tannins), iron deficiency is prevalent, especially among infants and pregnant women. Iron supplementation is the primary means of addressing this problem. Currently, the most representative iron supplements fall into two categories: inorganic iron (such as ferrous sulfate) and organic iron (such as ferrous fumarate). However, their low bioavailability and gastrointestinal side effects severely limit their widespread applicability.

[0003] Sea cucumber flowers, a byproduct of sea cucumber processing, are often discarded during traditional processing. However, their dried product contains up to 60% protein and 19 amino acids (mainly glutamic acid and aspartic acid). These functional amino acids possess potential iron-binding activity, making them an excellent source for developing functional iron supplements. Currently, the utilization of sea cucumber flowers is mainly focused on extraction process research, with a scarcity of refined processed products. Studies have shown that iron ions can induce the self-assembly of some peptides to form nanocomposites. These nanocomposites can maintain the solubility of iron in the gastrointestinal environment, potentially solving the problem of low iron absorption efficiency and becoming a research hotspot for iron supplements. However, no specific peptides derived from sea cucumber flowers have yet been found that possess both digestive stability and high iron absorption efficiency. This research gap limits the high-value utilization of sea cucumber flower protein resources and the development of new iron supplements. Summary of the Invention

[0004] In view of this, the present invention provides a sea cucumber flower heptapeptide with iron absorption-promoting effect, its preparation method and application. The sea cucumber flower heptapeptide with iron absorption-promoting effect provided by the present invention can form a sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect, and has excellent iron absorption-promoting effect.

[0005] This invention provides a sea cucumber flower heptapeptide with iron absorption-promoting effects, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides a method for preparing the sea cucumber flower heptapeptide with iron absorption-promoting effect described in the above-mentioned scheme, including method one and method two; The method includes the following steps: according to the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys, 2-chlorotrimethylbenzenechloro resin (2-CTC) polymer resin is covalently linked to Fmoc-Lys(Boc)-OH, and then sequentially condensed with Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH and Fmoc-Glu(OtBu)-OH, and then the 2-chlorotrimethylbenzenechloro resin polymer resin is removed to obtain the sea cucumber flower heptapeptide with iron absorption promoting effect; Method 2 includes the following steps: mixing sea cucumber flower powder and water, adjusting the pH value to alkaline, heating and mixing, centrifuging and collecting the supernatant, adjusting the pH value of the supernatant to acidic, and centrifuging and collecting the precipitate to obtain sea cucumber flower protein powder; mixing the sea cucumber flower protein powder with water, simulated gastric juice and simulated intestinal juice, performing simulated digestion, inactivating protease, centrifuging and collecting the supernatant, and freeze-drying to obtain the sea cucumber flower heptapeptide with iron absorption promotion effect.

[0007] Preferably, in method one, the covalent linkage includes the following steps: mixing 2-chlorotrimethylbenzenechloro resin polymer, Fmoc-Lys(Boc)-OH, dichloromethane and N,N-diisopropylethylamine (DIEA) for a covalent linkage reaction, blocking with methanol, N,N-diisopropylethylamine and dichloromethane, and deprotecting with piperidine solution.

[0008] Preferably, in method two, the simulated gastric juice includes a gastric electrolyte solution (SGF) and pepsin; the volume ratio of the gastric electrolyte solution to pepsin is 1:0.5~1.5; the gastric electrolyte solution includes the following components: 6.9 mmol / L KCl, 0.9 mmol / L KH2PO4, 25 mmol / L NaHCO3, 47.2 mmol / L NaCl, 0.1 mmol / L MgCl2(H2O)6, 0.5 mmol / L (NH4)2CO3, 15.6 mmol / L HCl and distilled water; the concentration of the pepsin is 2000 U / mL.

[0009] Preferably, the simulated intestinal fluid comprises an intestinal electrolyte solution (SIF), pancreatic enzymes, and bile; the volume ratio of the intestinal electrolyte solution to the pancreatic enzymes is 1:0.5~1.5; the intestinal electrolyte solution comprises the following components: 6.8 mmol / L KCl, 0.8 mmol / L KH2PO4, 85 mmol / L NaHCO3, 38.4 mmol / L NaCl, 0.33 mmol / L MgCl2(H2O)6, 8.4 mmol / L HCl, and distilled water; the concentration of the pancreatic enzymes is 100 U / mL; and the concentration of bile in the simulated intestinal fluid is 150~170 mmol / L.

[0010] The present invention also provides the application of the sea cucumber flower heptapeptide with iron absorption-promoting effect described in the above-described scheme or the sea cucumber flower heptapeptide with iron absorption-promoting effect obtained by the preparation method described in the above-described scheme as an iron carrier.

[0011] This invention also provides a sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects, comprising sea cucumber flower heptapeptide with iron absorption-promoting effects and iron ions bound to the Asp-3, Asp-4, and Arg-5 amino acids of the sea cucumber flower heptapeptide with iron absorption-promoting effects; the sea cucumber flower heptapeptide with iron absorption-promoting effects is the sea cucumber flower heptapeptide with iron absorption-promoting effects described in the above scheme or the sea cucumber flower heptapeptide with iron absorption-promoting effects obtained by the preparation method described in the above scheme.

[0012] This invention also provides a method for preparing the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects described in the above-mentioned scheme, comprising the following steps: Sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, was mixed with water to obtain a sea cucumber flower heptapeptide solution. After adjusting the pH value of the sea cucumber flower heptapeptide solution to 6.0~7.0, it was mixed with FeSO4·4H2O and Na2S2O4 to react and obtain the sea cucumber flower heptapeptide-iron nanocomposite with the effect of promoting iron absorption.

[0013] Preferably, the molar ratio of the sea cucumber flower heptapeptide with iron absorption-promoting effect to FeSO4·4H2O is 1:4~8.

[0014] Preferably, the reaction is carried out under stirring conditions; the reaction temperature is 20~28℃, the pH value is 6.0~7.0, and the holding time is 60~90min.

[0015] Compared with existing technologies, the sea cucumber flower heptapeptide with iron absorption-promoting effects, its preparation method, and its application provided by this invention have achieved the following beneficial effects: This invention is the first synthesis of a sea cucumber flower heptapeptide with iron absorption-promoting effects. The amino acid sequence of the sea cucumber flower heptapeptide with iron absorption-promoting effects is: Glu-Val-Asp-Asp-Arg-Trp-Lys (abbreviated as EVDDRWK), with a purity of 97.84% and a molecular weight of 947.02 Da.

[0016] The sea cucumber flower heptapeptide with iron-enhancing effects provided by this invention maintains its integrity well after gastrointestinal digestion, exhibiting good digestive stability and providing a reliable carrier for iron delivery. This sea cucumber flower heptapeptide with iron-enhancing effects can be formulated into a sea cucumber flower heptapeptide-iron nanocomplex, which can promote iron absorption and is suitable for use in the field of iron supplements, enriching the variety of functional iron supplement products.

[0017] This invention not only provides iron supplements with core components that possess both gastrointestinal digestive stability and iron absorption-promoting activity, but also realizes the high-value utilization of sea cucumber resources, enhances the added value of sea cucumber flower resources, and has significant application value, with remarkable economic and social benefits.

[0018] The sea cucumber flower heptapeptide-iron nanocomposite (denoted as EVDDRWK-Fe) provided by this invention possesses the ability to effectively resist digestion by gastrointestinal proteases, significantly improving iron delivery efficiency on the one hand and promoting iron absorption on the other. The results of the examples show that after simulated gastrointestinal digestion, its peptide stability is 89.69%±0.45%, significantly higher than that of sea cucumber flower heptapeptide with iron absorption-promoting effects (63.75%±0.02%); simultaneously, its iron transport capacity is significantly better than FeCl2, effectively promoting the absorption of iron by small intestinal epithelial cells. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of this invention, the accompanying drawings used in the embodiments of this invention or in the prior art are briefly described below. For those skilled in the art, other drawings can be derived from the following drawings without creative effort, and all such drawings are within the protection scope of this invention.

[0020] Figure 1 The sea cucumber flower heptapeptide and Fe provided by this invention have the effect of promoting iron absorption. 2+ Potential combination model; where the brown sphere is Fe 2+ The light blue chain is sea cucumber flower heptapeptide, which promotes iron absorption; the dark blue segment is the N-terminus; and the red segment is the oxygen-terminus. Figure 2The liquid chromatogram and mass spectrum of the sea cucumber flower heptapeptide with iron absorption promoting effect prepared in Example 1 are shown below; wherein, a is the liquid chromatogram of the sea cucumber flower heptapeptide with iron absorption promoting effect prepared in Example 1; b is the mass spectrum of the sea cucumber flower heptapeptide with iron absorption promoting effect prepared in Example 1. Figure 3 SEM images of the sea cucumber flower heptapeptide with iron absorption-promoting effect prepared in Example 1 and the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect; wherein, a is the SEM image of the sea cucumber flower heptapeptide with iron absorption-promoting effect (scale bar: 1000x is 10μm; 3500x is 3μm; 8000x is 1μm), and b is the SEM image of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect. Figure 4 EDS-mapping images of the sea cucumber flower heptapeptide with iron absorption-promoting effect prepared in Example 1 and the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect; wherein, a is the EDS-mapping image of the sea cucumber flower heptapeptide with iron absorption-promoting effect (scale bar is 2.5 μm) and b is the EDS-mapping image of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect. Figure 5 The image shows the elemental composition analysis of the sea cucumber flower heptapeptide with iron absorption-promoting effect and its iron absorption-promoting sea cucumber flower heptapeptide-iron nanocomposite prepared in Example 1; wherein, A is the XPS spectrum of the sea cucumber flower heptapeptide with iron absorption-promoting effect and the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect, and B is the Fe content of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect. 2p High-resolution chromatogram, C represents the C6 of sea cucumber flower heptapeptide, which promotes iron absorption. 1s High-resolution spectrum, D represents C of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect. 1s High-resolution chromatogram, E represents N of sea cucumber flower heptapeptide, which has iron absorption-promoting effects. 1s High-resolution spectrum, F represents N of sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect. 1s High-resolution chromatogram, G represents the O of sea cucumber flower heptapeptide, which has iron absorption-promoting effects. 1s High-resolution spectrum, H represents the O of sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect. 1s High-resolution images; Figure 6 Stability analysis of the sea cucumber flower heptapeptide with iron absorption-promoting effect prepared in Example 1 and the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect was determined by RP-HPLC: where a is the digestive stability analysis of the sea cucumber flower heptapeptide with iron absorption-promoting effect, and b is the digestive stability analysis of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect. Figure 7 The image shows the spectral identification of the iron-absorbing sea cucumber flower heptapeptide-iron nanocomposite by simulated gastrointestinal digestion based on Nano-LC-ESI-MS / MS; where A is the iron-absorbing sea cucumber flower heptapeptide EVDDRWK and B is VDDRWK. Figure 8 The image shows the spectral identification of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects after simulated gastrointestinal digestion based on Nano-LC-ESI-MS / MS; where A is EVDDRW and B is DDRWK. Figure 9 The image shows the spectral identification of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects after simulated gastrointestinal digestion based on Nano-LC-ESI-MS / MS; where A is DRWK and B is DDRW. Figure 10 The image shows the spectral identification of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects after simulated gastrointestinal digestion based on Nano-LC-ESI-MS / MS; where A is VDDR and B is RWK. Figure 11 The statistical results of iron transport in the sea cucumber flower heptapeptide-iron nanocomposite and FeCl2 digest prepared in Example 1, which have the effect of promoting iron absorption. Detailed Implementation

[0021] This invention provides a sea cucumber flower heptapeptide with iron absorption-promoting effects, the amino acid sequence of which is shown in SEQ ID NO.1.

[0022] The sea cucumber flower heptapeptide provided by this invention has the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys (EVDDRWK) and has the effect of promoting iron absorption.

[0023] In this invention, the structure of the sea cucumber flower heptapeptide with iron absorption-promoting effect is shown in Formula I: Formula I.

[0024] In this invention, the purity of the sea cucumber flower heptapeptide with iron-enhancing effects is 97.84%, and the molecular weight is 947.02 Da. The sea cucumber flower heptapeptide with iron-enhancing effects provided by this invention has a certain ability to resist digestion by gastrointestinal proteases and to bind iron.

[0025] The present invention also provides a method for preparing the sea cucumber flower heptapeptide with iron absorption-promoting effect described in the above-mentioned scheme, including method one and method two; The method includes the following steps: according to the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys, 2-chlorotrimethylbenzenechloro resin (2-CTC) polymer resin is covalently linked to Fmoc-Lys(Boc)-OH, and then sequentially condensed with Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH and Fmoc-Glu(OtBu)-OH, and then the 2-chlorotrimethylbenzenechloro resin polymer resin is removed to obtain the sea cucumber flower heptapeptide with iron absorption promoting effect; Method 2 includes the following steps: mixing sea cucumber flower powder and water, adjusting the pH value to alkaline, heating and mixing, centrifuging and collecting the supernatant, adjusting the pH value of the supernatant to acidic, and centrifuging and collecting the precipitate to obtain sea cucumber flower protein powder; mixing the sea cucumber flower protein powder with water, simulated gastric juice and simulated intestinal juice, performing simulated digestion, inactivating protease, centrifuging and collecting the supernatant, and freeze-drying to obtain the sea cucumber flower heptapeptide with iron absorption promotion effect.

[0026] In Method 1, 2-chlorotrimethylbenzenechloropolymer resin is covalently linked to Fmoc-Lys(Boc)-OH according to the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys, and then sequentially condensed with Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH and Fmoc-Glu(OtBu)-OH. Finally, the 2-chlorotrimethylbenzenechloropolymer resin is removed to obtain the sea cucumber flower heptapeptide with iron absorption promoting effect.

[0027] In this invention, the 2-chlorotrimethylbenzenechloro resin polymer is preferably pretreated before use; the pretreatment preferably includes: soaking the 2-chlorotrimethylbenzenechloro resin polymer in dichloromethane (DCM), then washing with N,N-dimethylformamide (DMF) and filtering.

[0028] In this invention, the soaking time is preferably 1.8 to 2.2 hours, and more preferably 2 hours.

[0029] In this invention, the washing is preferably performed 3 times or more, more preferably 4 times; the filtration is preferably performed by desiccation.

[0030] In this invention, the covalent linkage preferably includes the following steps: mixing 2-chlorotrimethylbenzenechloro resin, Fmoc-Lys(Boc)-OH, dichloromethane (DCM, denoted as first dichloromethane) and N,N-diisopropylethylamine (DIEA, denoted as first N,N-diisopropylethylamine) for a covalent linkage reaction, blocking with methanol, N,N-diisopropylethylamine (DIEA, denoted as second N,N-diisopropylethylamine) and dichloromethane (DCM, denoted as second dichloromethane), and deprotecting with piperidine solution.

[0031] In this invention, the molar ratio of the 2-chlorotrimethylbenzene chloride resin polymer to Fmoc-Lys(Boc)-OH is preferably 1:1 to 1:1.2.

[0032] In this invention, the mass-to-volume ratio of Fmoc-Lys(Boc)-OH to dichloromethane is preferably 1g:(8~12)mL, more preferably 1g:10mL.

[0033] In this invention, the molar ratio of the first N,N-diisopropylethylamine to Fmoc-Lys(Boc)-OH is preferably 2 to 5:1, more preferably 3:1.

[0034] In this invention, the temperature of the covalent bonding reaction is preferably 28~32℃, more preferably 30℃, and the holding time is preferably 1.8~2.2h, more preferably 2h; the covalent bonding reaction is preferably carried out in a shaker.

[0035] In this invention, the volume ratio of methanol, second N,N-diisopropylethylamine and second dichloromethane is preferably 2:1:15~18, more preferably 2:1:17.

[0036] In this invention, the preferred ratio of the total volume of methanol, second N,N-diisopropylethylamine, and second dichloromethane to the mass of 2-chlorotrimethylbenzene chloride resin is 4 mL: 1 g.

[0037] In this invention, the sealing time is preferably 25-35 minutes, more preferably 30 minutes.

[0038] In this invention, the sealing process preferably further includes washing the resulting product with N,N-dimethylformamide and then filtering it; the washing is preferably performed 4 times or more; and the filtering is preferably performed by vacuum drying.

[0039] In this invention, the volume percentage of piperidine in the piperidine solution is preferably 19-21%, more preferably 20%; the volume ratio of methanol to piperidine solution is preferably 1:8-12, more preferably 1:10.

[0040] In this invention, the deprotection temperature is preferably 23~27℃, and the time is preferably 5~25min.

[0041] In this invention, the deprotection process preferably further includes washing the obtained product with N,N-dimethylformamide and then filtering it; the washing is preferably performed at least four times; and the filtering is preferably performed by vacuum drying. After deprotection, the present invention uses the ninhydrin method to detect the sample; the 2-chlorotrimethylbenzenechloro resin polymer shows color, indicating successful deprotection.

[0042] In this invention, the condensation reactions of Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH and Fmoc-Glu(OtBu)-OH are sequentially referred to as the first condensation reaction, the second condensation reaction, the third condensation reaction, the fourth condensation reaction, the fifth condensation reaction and the sixth condensation reaction.

[0043] In this invention, the first condensation reaction preferably includes the following steps: mixing the covalently linked product with Fmoc-Trp-OH, 1-hydroxybenzotriazole (HOBT) and N,N'-diisopropylcarbodiimide (DIC) to carry out a condensation reaction.

[0044] In this invention, the molar ratio of Fmoc-Lys(Boc)-OH to Fmoc-Trp-OH is preferably 3~5:1.

[0045] In this invention, the molar ratio of Fmoc-Trp-OH to 1-hydroxybenzotriazole is preferably 1:1 to 1.2.

[0046] In this invention, the volume ratio of 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide is preferably 1:0.8~1.5.

[0047] In this invention, the temperature of the first condensation reaction is preferably 28~32℃, more preferably 30℃, and the holding time is preferably 0.9~1.1h, more preferably 1h; the first condensation reaction is preferably carried out in a shaker. A sample of the 2-chlorotrimethylbenzenechloro resin is tested using the ninhydrin method. If the 2-chlorotrimethylbenzenechloro resin is colored, it indicates that the condensation reaction is incomplete, and the condensation reaction should be continued. If the 2-chlorotrimethylbenzenechloro resin is colorless, it indicates that the condensation reaction is complete.

[0048] In this invention, the first condensation reaction preferably includes washing the resulting product with N,N-dimethylformamide and then filtering it; the washing is preferably performed 4 times or more, more preferably 4 times; the filtering is preferably performed by vacuum drying.

[0049] In this invention, it is preferable to perform deprotection by mixing the product obtained from the first condensation reaction with a piperidine solution before the second condensation reaction.

[0050] In this invention, the volume percentage of piperidine in the piperidine solution is preferably 19-21%, more preferably 20%; the volume ratio of N,N-dimethylformamide to piperidine solution is preferably 3.5-4.5:1, more preferably 4:1.

[0051] In this invention, the deprotection temperature is preferably 23~27°C, and the time is preferably 18~23 min, more preferably 20 min. This invention removes the Fmoc protecting groups from the 2-CTC polymer resin through deprotection.

[0052] In this invention, the deprotection process preferably further includes washing the obtained product with N,N-dimethylformamide and then filtering it; the washing is preferably performed at least four times; and the filtering is preferably performed by vacuum drying. After deprotection, the present invention uses the ninhydrin method to detect the sample; the 2-chlorotrimethylbenzenechloro resin polymer shows color, indicating successful deprotection.

[0053] In this invention, the methods for the second, third, fourth, fifth, and sixth condensation reactions are preferably the same as those for the first condensation reaction, and will not be described again here. This invention sequentially connects the amino acids Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, and Fmoc-Glu(OtBu)-OH through the first to the sixth condensation reactions.

[0054] In this invention, the cutting reagent used to cut the 2-chlorotrimethylbenzenechloro resin polymer is preferably a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water (H2O); the volume ratio of the trifluoroacetic acid, triisopropylsilane, and water is preferably 95:2.5:2.5; the amount of the cutting reagent is preferably 10~20 mL / g resin, more preferably 15 mL / g resin; the temperature of the cutting reaction is preferably 23~26℃, and the time is preferably 2~4 h, more preferably 3 h.

[0055] In this invention, after removing the 2-chlorotrimethylbenzene chloride resin polymer, the process preferably further includes purifying, quick-freezing, and freeze-drying the resulting product in sequence.

[0056] In this invention, the purification is preferably carried out by high-performance liquid chromatography (HPLC); the chromatographic column used for HPLC purification is preferably a VYDAC-C18 with a size of 4.6 mm × 250 mm; the mobile phase A for HPLC purification is preferably acetonitrile containing 0.1% (v / v) trifluoroacetic acid; the mobile phase B is preferably water containing 0.1% (v / v) trifluoroacetic acid; the elution conditions for HPLC purification are preferably: 0~20.0 min: mobile phase A increases from 22.0% (v / v) to 32.0% (v / v); 20.0~20.1 min (excluding 20 min): mobile phase A increases from 32.0% (v / v) to 100.0% (v / v); the flow rate is 1.0 mL / min, and the detection wavelength is 220 nm.

[0057] In this invention, the quick-freezing is preferably carried out in liquid nitrogen; the holding time for quick-freezing is preferably 10 to 30 minutes.

[0058] In this invention, the freeze-drying temperature is preferably -70~-50℃, more preferably -55℃, the vacuum degree is preferably 1~20Pa, more preferably 10Pa, and the heat preservation time is preferably 48~72h, more preferably 60h.

[0059] In Method 2, the present invention mixes sea cucumber flower powder and water, and sequentially adjusts the pH value to alkaline, heats and mixes, centrifuges and collects the supernatant, adjusts the pH value of the supernatant to acidic, and centrifuges and collects the precipitate to obtain sea cucumber flower protein powder; the sea cucumber flower protein powder is then mixed with water, simulated gastric juice and simulated intestinal juice, and sequentially subjected to simulated digestion, inactivation of protease, centrifugation and collection of the supernatant, and freeze-drying to obtain the sea cucumber flower heptapeptide with iron absorption promoting effect.

[0060] This invention involves mixing sea cucumber flower powder and water, adjusting the pH value to alkaline, heating and mixing, centrifuging to collect the supernatant, adjusting the pH value of the supernatant to acidic, and centrifuging to collect the precipitate to obtain sea cucumber flower protein powder.

[0061] In this invention, the water is preferably deionized water; the mass ratio of the sea cucumber flower powder to the volume of the water is preferably 1g:(8~12)mL, more preferably 1g:10mL.

[0062] In this invention, the reagent used to adjust the pH value to alkaline is preferably a sodium hydroxide solution; the concentration of the sodium hydroxide solution is preferably 2.8~3.2 mol / L, more preferably 3 mol / L.

[0063] In this invention, the pH value of the alkaline substance is preferably 8.5 to 9.0, and more preferably 8.7 to 8.8.

[0064] In this invention, the heating and mixing is preferably carried out under stirring conditions; the stirring conditions are preferably magnetic stirring; the heating and mixing temperature is preferably 38~45℃, more preferably 40~42℃, and the holding time is preferably 3~4h, more preferably 3.5h.

[0065] In this invention, the centrifugation speed for collecting the supernatant is preferably 3000~4000 rpm, and the centrifugation time is preferably 10~15 min.

[0066] In this invention, the reagent used to adjust the pH value of the supernatant to acidic is preferably a hydrochloric acid solution; the concentration of the hydrochloric acid solution is preferably 2.8~3.2 mol / L, more preferably 3 mol / L; the acidic pH value is preferably 4.0~6.5, more preferably 4.5~6.

[0067] In this invention, the centrifugation speed for collecting the precipitate is preferably 3000~4000 rpm, and the centrifugation time is preferably 10~15 min.

[0068] In this invention, the centrifugation and collection of the precipitate preferably further includes washing the obtained precipitate and then freeze-drying it; the washing is preferably water washing; the water used for water washing is preferably distilled water; the cold trap temperature for freeze-drying is preferably -75~-55℃, more preferably -65℃, the vacuum degree is preferably 2~10Pa, more preferably 5Pa, and the holding time is preferably 48~72h, more preferably 60h.

[0069] After obtaining sea cucumber flower protein powder, the present invention mixes the sea cucumber flower protein powder with water, simulated gastric juice and simulated intestinal juice (referred to as the first mixture), and sequentially performs simulated digestion, inactivates protease, centrifuges to collect the supernatant and freeze-drys, to obtain the sea cucumber flower heptapeptide with iron absorption promotion effect.

[0070] In this invention, the mass ratio of the sea cucumber flower protein powder to the volume ratio of water is preferably (10~50) mg:1 mL, more preferably (20~40) mg:1 mL, and even more preferably 30 mg:1 mL.

[0071] In this invention, the simulated gastric juice preferably includes a gastric electrolyte solution (SGF) and pepsin; the volume ratio of the gastric electrolyte solution to pepsin is preferably 1:0.5~1.5, more preferably 1:1; the gastric electrolyte solution preferably includes the following components: 6.9 mmol / L KCl, 0.9 mmol / L KH2PO4, 25 mmol / L NaHCO3, 47.2 mmol / L NaCl, 0.1 mmol / L MgCl2(H2O)6, 0.5 mmol / L (NH4)2CO3, 15.6 mmol / L HCl and distilled water; the concentration of the pepsin is preferably 2000 U / mL.

[0072] In this invention, the volume ratio of the simulated gastric juice to the total volume of sea cucumber flower protein powder and water is preferably 1~2:10, more preferably 1.5:10.

[0073] In this invention, the simulated intestinal fluid preferably comprises an intestinal electrolyte solution (SIF), pancreatic enzymes, and bile; the volume ratio of the intestinal electrolyte solution to the pancreatic enzymes is preferably 1:0.5 to 1.5, more preferably 1:1; the intestinal electrolyte solution preferably comprises the following components: 6.8 mmol / L KCl, 0.8 mmol / L KH2PO4, 85 mmol / L NaHCO3, 38.4 mmol / L NaCl, 0.33 mmol / L MgCl2(H2O)6, 8.4 mmol / L HCl, and distilled water; the concentration of the pancreatic enzymes is preferably 100 U / mL; the concentration of bile in the simulated intestinal fluid is preferably 150 to 170 mmol / L, more preferably 160 mmol / L.

[0074] In this invention, the volume ratio of the simulated intestinal fluid to the total volume of sea cucumber flower protein powder and water is preferably 1~2:10, more preferably 1.5:10.

[0075] In this invention, the first mixing preferably includes the following steps: mixing sea cucumber flower protein powder with water to obtain a reaction solution; adding simulated gastric juice to the reaction solution and adjusting the pH value to 2.0~5.0; stirring at 200~400 rpm for 30~150 min at a temperature of 30~40℃ to obtain a gastric juice reaction solution; adding simulated intestinal juice to the gastric juice reaction solution and adjusting the pH value to 7.0~9.0; stirring at 200~400 rpm for 20~120 min at a temperature of 35~38℃.

[0076] In this invention, the inactivated protease preferably includes the following steps: keeping the reaction system obtained from simulated digestion in a boiling water bath at 90~95℃ for 10~15 min.

[0077] In this invention, the centrifugation to collect the supernatant is preferably carried out at 4°C, the centrifugation speed is preferably 8000~10000 rpm, more preferably 9000 rpm, and the centrifugation time is preferably 5~10 min, more preferably 7 min.

[0078] In this invention, the preferred temperature of the freeze-drying cold trap is -70 to -50°C, more preferably -55°C, the preferred vacuum degree is 1 to 20 Pa, more preferably 10 Pa, and the preferred time is 48 to 72 hours, more preferably 60 hours.

[0079] The present invention also provides the application of the sea cucumber flower heptapeptide with iron absorption-promoting effect described in the above-described scheme or the sea cucumber flower heptapeptide with iron absorption-promoting effect obtained by the preparation method described in the above-described scheme as an iron carrier.

[0080] The sea cucumber flower heptapeptide provided by this invention has iron-binding capacity and can be used as a iron carrier in various fields. After digestion in the gastrointestinal tract, the sea cucumber flower heptapeptide with iron-enhancing effects maintains good peptide stability, providing a reliable carrier for iron delivery and can be applied in fields such as iron supplements.

[0081] The present invention also provides a sea cucumber flower heptapeptide-iron nanocomposite with iron absorption promoting effect, comprising sea cucumber flower heptapeptide with iron absorption promoting effect and iron ions bound to the Asp-3, Asp-4 and Arg-5 amino acids of the sea cucumber flower heptapeptide with iron absorption promoting effect.

[0082] In this invention, the iron ions are preferably divalent iron ions (Fe2+). 2+ ).

[0083] This invention also provides a method for preparing the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects described in the above-mentioned scheme, comprising the following steps: Sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, is mixed with water to obtain a sea cucumber flower heptapeptide solution. After adjusting the pH value of the sea cucumber flower heptapeptide solution to 6.0~7.0, it is mixed with FeSO4·4H2O and Na2S2O4 (to obtain a reaction solution) to react and obtain the sea cucumber flower heptapeptide-iron nanocomposite with the effect of promoting iron absorption.

[0084] In this invention, the water is preferably ultrapure water; the ultrapure water is preferably Milli-Q water.

[0085] In this invention, the concentration of the sea cucumber flower heptapeptide solution with iron absorption-promoting effect is preferably 3~8 mg / mL, more preferably 4~7 mg / mL, and even more preferably 5~6 mg / mL.

[0086] In this invention, the molar ratio of the sea cucumber flower heptapeptide with iron absorption-promoting effect to FeSO4·4H2O is preferably 1:4~8, more preferably 1:5~7, and even more preferably 1:6.

[0087] In this invention, the concentration of Na₂S₂O₄ in the reaction solution is preferably 80-120 mmol / L, more preferably 100 mmol / L. The addition of Na₂S₂O₄ in this invention is to prevent the oxidation of ferrous ions.

[0088] In this invention, the reaction is preferably carried out under stirring conditions; the reaction temperature is preferably 20~28℃, more preferably 24℃, the pH value is preferably 6.0~7.0, and the holding time is preferably 60~90min, more preferably 70~80min.

[0089] In this invention, the reaction preferably includes collecting and precipitating the resulting product and then freeze-drying it; the collection of the precipitate is preferably carried out by mixing the reaction product and ethanol, allowing them to stand, centrifuging, and collecting the precipitate in sequence; the proportion of ethanol to the total mass of the reaction product and ethanol is preferably 90-95 wt%; the standing time is preferably 60-120 min, more preferably 80-100 min; the relative centrifugal force of the centrifugation is preferably 10000-12000 g, and the centrifugation time is preferably 5-10 min, more preferably 7 min.

[0090] In this invention, the preferred temperature of the freeze-drying cold trap is -75 to -55°C, more preferably -65°C, the preferred vacuum degree is 2 to 10 Pa, more preferably 5 to 6 Pa, and the preferred heat preservation time is 48 to 72 hours, more preferably 60 hours.

[0091] The sea cucumber flower heptapeptide prepared in this invention, which promotes iron absorption, possesses a certain ability to resist digestion by gastrointestinal proteases and bind with iron ions. In the sea cucumber flower heptapeptide with iron absorption-promoting effect:Fe 2+ When the molar ratio is 1:6, the peptide stability of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect reaches 89.69%±0.45% after simulating the gastrointestinal digestion endpoint, which is significantly higher than the peptide stability of the sea cucumber flower heptapeptide with iron absorption-promoting effect (63.75%±0.02%).

[0092] The sea cucumber flower heptapeptide-iron nanocomplex provided by this invention can maintain the solubility of iron in the gastrointestinal environment and effectively promote the absorption of iron by Caco-2 cells. When simulating the end point of gastrointestinal digestion, its iron transport capacity can reach 79.15±1.86μg / well. The iron transport effect is significantly better than that of FeCl2 after digestion. It can be applied to iron supplements and other fields.

[0093] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments.

[0094] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0095] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0096] In a specific embodiment of the present invention, the preparation steps of some of the raw materials used are as follows: SGF (Saline-Gastrointestinal Electrolyte Solution): SGF was prepared by dissolving 6.9 mmol / L KCl, 0.9 mmol / L KH2PO4, 25 mmol / L NaHCO3, 47.2 mmol / L NaCl, 0.1 mmol / L MgCl2(H2O)6, 0.5 mmol / L (NH4)2CO3 and 15.6 mmol / L HCl in 400 mL of distilled water.

[0097] SIF (Self-Idled Electrolyte Solution): Dissolve 6.8 mmol / L KCl, 0.8 mmol / L KH2PO4, 85 mmol / L NaHCO3, 38.4 mmol / L NaCl, 0.33 mmol / L MgCl2(H2O)6 and 8.4 mmol / L HCl in 400 mL of distilled water to obtain SIF.

[0098] The simulated gastric juice was prepared by mixing SGF and pepsin (2000 U / mL) in a volume ratio of 1:1. The simulated intestinal fluid was prepared by mixing SIF and pancreatic enzyme (100 U / mL) at a volume ratio of 1:1, and then adding 160 mmol / L bile (the volume ratio of simulated intestinal fluid to bile was 4:0.5).

[0099] Example 1: This embodiment prepared a sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects. The specific steps involved are as follows: S1. Sea cucumber flower protein extraction: Sea cucumber flower freeze-dried powder and water were mixed at a ratio of 1g:10mL to obtain a freeze-dried powder solution. A 3mol / L NaOH solution was added to the prepared freeze-dried powder solution to adjust the pH value to 9.0. The solution was magnetically stirred at 40℃ for 3.5h and centrifuged at 3500rpm for 10min. The precipitate was discarded to obtain the supernatant. A 3mol / L HCl solution was added to adjust the pH value of the supernatant to 4.8. The solution was centrifuged at 3500rpm for 10min, and the precipitate was collected. The precipitate was washed with distilled water and freeze-dried at -55℃ and 10Pa vacuum for 72h to obtain sea cucumber flower protein powder.

[0100] S2. Preparation of sea cucumber flower polypeptide: The sea cucumber flower protein powder obtained in S1 was prepared into a reaction solution with a substrate concentration of 30 mg / mL. Simulated gastric juice was added, with a volume ratio of simulated gastric juice to reaction solution of 1:10. The pH of the reaction solution was adjusted to 2.0. The mixture was stirred at 300 rpm for 120 min at 37℃. Simulated intestinal juice was then added, with a volume ratio of simulated intestinal juice to reaction solution of 1:10. The pH of the reaction solution was adjusted to 7.0. The mixture was stirred at 300 rpm for 120 min at 37℃. During digestion, the pH was kept stable with 1 mol / L HCl. After the reaction, the pH was adjusted to 7.0. The reaction solution was quickly placed in a boiling water bath at 95℃ for 10 min. The mixture was then centrifuged at 8000 rpm for 10 min at 4℃. The supernatant was collected and freeze-dried in a -55℃ cold trap under a vacuum of 10 Pa for 72 h to obtain sea cucumber flower polypeptide. The polypeptide was stored at -20℃ for later use.

[0101] S3. Identification of the peptide sequence with iron absorption-promoting effect: First, desalting was performed using Pierce C18 spin tips (Pierce C18 Spin Tips, Thermo Fisher Scientific, MA, USA). 10 μL of a 1.0 mg / mL sea cucumber flower peptide solution was loaded into a capture column (Column Technology Inc. 0.15 mm × 150 mm, RP-C18), with a flow rate of 10 μL / min, and linear eluted in an analytical column (Zorbax 300SB-C18 peptide capture device). Mobile phase A was water containing 0.1% (v / v) formic acid, and mobile phase B was acetonitrile containing 0.1% (v / v) formic acid. Elution conditions were as follows: 0–50 min, 4–50% (v / v) mobile phase B; 50–54 min (excluding 50 min), 50–100% (v / v) mobile phase B; 54–60 min (excluding 54 min), 100% (v / v) mobile phase B. Peptides of sea cucumber flower polypeptide were separated using Nano-LC and identified by QExactive mass spectrometry. Raw peptide sequence data were processed using Data Analysis 4.0 software and searched using the MaxQuant server.

[0102] Analysis and identification revealed it to be related to Fe 2+ The relevant binding sequence is Glu-Val-Asp-Asp-Arg-Trp-Lys (EVDDRWK), which contains consecutive Asp residues and Glu residues and may have multiple iron-binding sites.

[0103] S4, Molecular docking model: First, Fe was obtained from PubChem. 2+The structure of EVDDRWK was constructed using ChemDraw Academic Edition, and the connection between EVDDRWK and Fe was completed using Discovery Studio software. 2+ Molecular docking was performed, and then the results were optimized using PyMol 2.5 to evaluate its compatibility with Fe. 2+ The ability to combine, the result is as follows Figure 1 As shown.

[0104] according to Figure 1 As can be seen, this invention establishes a molecular docking technique between the peptide chain EVDDRWK and Fe... 2+ The combination model shows that Fe 2+ It can bind to the Asp-3, Asp-4 and Arg-5 amino acids of the peptide chain EVDDRWK, forming four stable binding bonds with bond lengths between 2.7 and 3.9 kcal and binding energies of -7.2825 kcal / mol and -6.3984 kcal / mol, respectively, and has potential iron-binding ability.

[0105] S5. Solid-phase synthesis of sea cucumber flower heptapeptide with iron absorption-promoting effect: Using 2-chlorotrimethylbenzenechloropolymer resin (Hefei Saimanno Biotechnology Co., Ltd.), following the characteristic of the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys, the carboxyl group of Fmoc-Lys(Boc)-OH was first covalently linked to the 2-CTC polymer resin. Then, the amino group of Fmoc-Lys(Boc)-OH and the carboxyl group of Fmoc-Trp-OH underwent a condensation reaction. Next, Fmoc-Arg(Pbf)-OH was added, followed by the sequential addition of amino acids Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, and Fmoc-Glu(OtBu)-OH, from right to left, until the last amino acid Fmoc-Glu(OtBu)-OH was added. Finally, the 2-CTC polymer resin was removed, yielding the sea cucumber flower heptapeptide with iron absorption-promoting effect. Specific steps are as follows: (1) Calculate the weight of each raw material based on the weight of sea cucumber flower heptapeptide with iron absorption-promoting effect. Put 5g of 2-CTC polymer resin into a 150mL reactor and add 50mL of DCM to soak for 2h. (2) Wash the 2-CTC polymer resin with DMF and then dry it. Repeat this process 4 times. (3) Weigh Fmoc-Lys(Boc)-OH (the first amino acid at the C-terminus), DCM and DIEA and add them to the reactor. The molar ratio of 2-CTC polymer resin to Fmoc-Lys(Boc)-OH is 1:1.1 and the mass-volume ratio of Fmoc-Lys(Boc)-OH to DCM is 1g. :10mL, the molar ratio of DIEA and Fmoc-Lys(Boc)-OH is 3:1, and then the reactor is placed in a shaker at 30℃ for 2h; (4) use methanol, DIEA and DCM as blocking liquid for blocking (the volume ratio of methanol, DIEA and DCM used in this step is 2:1:17, and the amount of blocking liquid and 2-CTC polymer resin is 4.0mL:1g) for 30min, then wash with DMF 4 times and dry; (5) add 20% piperidine solution by volume percentage to the reactor according to the volume ratio of methanol and piperidine solution of 1:10, deprotect at 25℃ for 15min, and remove the Fmoc protecting group. After deprotection, wash with DMF 4 times and then dry; (6) Take 10 mg of 2-CTC polymer resin and test it with ninhydrin method. If the 2-CTC polymer resin is colored, it indicates that the deprotection is successful; (7) According to the molar ratio of Fmoc-Lys(Boc)-OH and Fmoc-Trp-OH being 4:1, the molar ratio of Fmoc-Trp-OH and 1-hydroxybenzotriazole being 1:1.1, and the molar ratio of 1-hydroxybenzotriazole and N,N The volume ratio of '-diisopropylcarbodiimide is 1:1.2. Weigh Fmoc-Trp-OH (the second amino acid at the C-terminus), 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide and add them to the reactor. Then place the reactor in a shaker at 30°C and react for 1 hour. (8) Take 10 mg of 2-CTC polymer resin for testing. Use the ninhydrin method to test. If the 2-CTC polymer resin is colored, it indicates that the condensation is incomplete and the reaction should continue. If the 2-CTC polymer resin is colorless, it indicates that the reaction is complete. After the reaction is complete, wash the 2-CTC polymer resin with DMF 4 times and then dry it. (9) Add 20% piperidine solution (the volume ratio of piperidine solution and DMF is 1:4) to the reactor and shake it on a decolorizing shaker at 25°C for 20 minutes to remove the Fmoc protecting group on the 2-CTC polymer resin.After deprotection, wash with DMF 4 times, then dry and check whether the protection has been removed. Take 10mg of 2-CTC polymer resin and test it with ninhydrin method. If the 2-CTC polymer resin is colored, it means that the deprotection is successful. (10) Connect amino acids Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH and Fmoc-Glu(OtBu)-OH in sequence according to steps (7) to (9). Cut with cutting reagent (a mixture of trifluoroacetic acid, triisopropylsilane and water with a volume ratio of 95:2.5:2.5, the amount of which is 15mL / g resin) at 24℃ for 3h to remove all the peptide protecting groups. After cutting the 2-CTC polymer resin, purify it by high performance liquid chromatography.

[0106] (11) Purification was performed using high-performance liquid chromatography (HPLC). The chromatographic column was a VYDAC-C18, 4.6 × 250 mm. Mobile phase A was acetonitrile containing 0.1% (v / v) trifluoroacetic acid; mobile phase B was water containing 0.1% (v / v) trifluoroacetic acid. Elution conditions were: 0–20.0 min: mobile phase A increased from 22.0% (v / v) to 32.0% (v / v); 20.0–20.1 min (excluding 20.0 min): mobile phase A increased from 32.0% (v / v) to 100.0% (v / v); flow rate was 1.0 mL / min; detection wavelength was 220 nm. The sample was rapidly frozen in liquid nitrogen for 20 min, then freeze-dried (temperature -55℃, vacuum 10 Pa, holding time 60 h), and the structure was identified by ESI-MS. The results are shown in Table 1 and [Table data missing]. Figure 2 As shown.

[0107] Table 1. HPLC chromatographic peak values ​​of sea cucumber flower heptapeptide with iron absorption-promoting effects:

[0108] As shown in Table 1, this invention synthesizes sea cucumber flower heptapeptide with iron absorption-promoting effects through solid-phase synthesis technology, and its molecular structure is shown in Formula I; the results of liquid phase purity identification and molecular weight mass spectrometry identification are as follows. Figure 2 As shown. According to Figure 2 It can be seen that the purity of the sea cucumber flower heptapeptide with iron absorption-promoting effect synthesized in this embodiment is 97.84%, and the molecular weight is 947.02 Da, which meets the requirements of subsequent experiments.

[0109] S6. Dissolve sea cucumber flower heptapeptide powder with iron absorption-promoting effects in Milli-Q water to obtain a 5 mg / mL solution of sea cucumber flower heptapeptide with iron absorption-promoting effects. Adjust the pH to 6.0 with 1 mol / L HCl and 1 mol / L NaOH solutions. Then, mix the sea cucumber flower heptapeptide with iron absorption-promoting effects and FeSO4·4H2O at a molar ratio of 1:6. Add 100 mmol / L Na2S2O4 to prevent oxidation of ferrous ions. Stir and react for 1 h at 25 °C and pH 6.0. Add 90% ethanol to the reactants, let stand for 1 h, centrifuge at 12000g for 5 min, collect the precipitate, and freeze-dry at -70 °C and 2 Pa vacuum for 72 h to obtain the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects.

[0110] Test Example 1: The microstructure of the sea cucumber flower heptapeptide with iron absorption-promoting effect prepared in Example 1 and its iron absorption-promoting sea cucumber flower heptapeptide-iron nanocomplex was determined. The specific steps are as follows: Sea cucumber flower heptapeptide, which promotes iron absorption, and its iron-enhancing nanocomposite, were respectively sputter-coated with gold at a current of 15 mA to a thickness of 15 nm. The samples were observed under an accelerating voltage of 5 kV, and SEM images were obtained using a Zeiss Gemini 500 field emission scanning electron microscope at different magnifications (1000x, 3500x, and 8000x) to observe their microstructure. The results are as follows: Figure 3 As shown.

[0111] according to Figure 3 As can be seen, in the microstructure of the sea cucumber flower heptapeptide with iron absorption-promoting effect and its iron-promoting sea cucumber flower heptapeptide-iron nanocomplex, the sea cucumber flower heptapeptide with iron absorption-promoting effect exhibits an irregular sheet-like structure with a smooth and dense surface. Its interaction with Fe... 2+ After combining, they exhibit a spherical, wrinkled structure with a loose texture. This may be due to the combination of sea cucumber flower heptapeptide, which promotes iron absorption, and Fe. 2+ The interaction between them disrupts the original dense structure on the surface of the polypeptide.

[0112] Further analysis of the elemental distribution of sea cucumber flower heptapeptide and its iron-absorbing nanocomplex with iron-enhancing effects was performed using EDS-mapping. The results are as follows: Figure 4 As shown.

[0113] according to Figure 4 Fe can be clearly seen2+ Based on the changes in the distribution of iron before and after, the sea cucumber flower heptapeptide with iron absorption-promoting effect only showed an extremely weak iron signal, while the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect showed a stronger iron signal and a more uniform distribution, indicating that the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect was successfully prepared.

[0114] Test Example 2: X-ray photoelectron spectroscopy (XPS) was performed on the sea cucumber flower heptapeptide with iron absorption-promoting effect prepared in Example 1 and its iron absorption-promoting sea cucumber flower heptapeptide-iron nanocomposite. The specific steps are as follows: Sea cucumber flower heptapeptide, which promotes iron absorption, and its lyophilized powder, a sea cucumber flower heptapeptide-iron nanocomposite, were fixed onto an XPS sample holder using adhesive tape. XPS spectra were then recorded using an X-ray photoelectron spectroscopy (Thermo Scientific K-Alpha, USA) equipped with an Al Kα X-ray source (1486.6 eV). All XPS measurements were performed at room temperature using the following parameters: pass energy 23.5 eV, limiting pressure 3 × 10⁻⁶. -10 Pa, takeoff angle 90°. Binding energy was calibrated using an Al Kα excitation source; excitation voltage was 1486.6 eV, C1s was 284.8 eV. Results are as follows: Figure 5 As shown.

[0115] according to Figure 5 As can be seen from A in the figure, the XPS spectrum of sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, has three obvious peaks at 284 eV, 399 eV, and 532 eV, corresponding to C1s, N1s, and O1s, respectively. Figure 5 As shown in B, the XPS spectrum of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects reveals, in addition to the peaks of C, N, and O elements in the sea cucumber flower heptapeptide with iron absorption-promoting effects, a photoelectron peak of Fe 2p is also observed at 709 eV. This indicates that the sea cucumber flower heptapeptide with iron absorption-promoting effects reacts with Fe... 2+ A sea cucumber flower heptapeptide-iron nanocomplex with iron-enhancing effects was successfully formed. Furthermore, a photoelectron peak of FeO was observed at 710.9 eV, indicating that Fe... 2+ It was bound to sea cucumber flower heptapeptide, which has iron-enhancing effects, via the O atom. According to... Figure 5 As can be seen from C and D, in the high-resolution spectrum of C1s, the photoelectron peaks of the CC, CN, CO, and OC=O bonds are observed at 284.6 eV, 285.3 eV, 286.3 eV, and 288.2 eV, respectively. According to... Figure 5As can be seen from E and F in the high-resolution N 1s spectrum, an NH bond at 399.3 eV and an amide nitrogen at 400.3 eV are observed. However, it is noteworthy that a new Fe-N bond at 399.1 eV is also observed in the high-resolution N 1s spectrum of the sea cucumber flower heptapeptide-iron nanocomposite with iron-enhancing effects, indicating that the amino nitrogen atom of the sea cucumber flower heptapeptide reveals the coordination significance of iron. Furthermore, in Figure 5 In the G and H, it can be found that the sea cucumber flower heptapeptide with iron absorption-promoting effect and the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect contain OH (530.9eV), C=O (531.8eV) and C-OH (532.8eV).

[0116] Test Example 3: The gastrointestinal stability of the sea cucumber flower heptapeptide with iron absorption-promoting effect and its iron absorption-promoting nanocomplex prepared in Example 1 was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The specific steps are as follows: (1) Sea cucumber flower heptapeptide with iron absorption-promoting effect and its sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effect simulate gastrointestinal digestion: Sea cucumber flower heptapeptide-iron and its nanocomposite having iron-enhancing effects were incubated in a 37℃ water bath for 30 min. After the system stabilized, the pH was adjusted to 2.0, and simulated gastric digestion fluid was added for 120 min. After digestion, 2 mL of the simulated gastric digestion product was collected, and the enzyme was inactivated in a 100℃ water bath for 5 min. The supernatant was then collected by centrifugation at 10000g and 4℃ for 10 min. Subsequently, the pH was adjusted to 7.5, and simulated intestinal digestion fluid was added, followed by simulated intestinal digestion at 37℃ for 120 min. After the reaction, 2 mL of the simulated gastrointestinal digestion product was collected, and the enzyme was inactivated by heating in a 100℃ water bath for 5 min. The supernatant was then collected for subsequent experiments.

[0117] (2) Analysis of the gastrointestinal stability of sea cucumber flower heptapeptide, which has the effect of promoting iron absorption: RP-HPLC was used to detect the changes in the content of heptapeptide from sea cucumber flower with iron-promoting effects and the heptapeptide-iron nanocomposite with iron-promoting effects during simulated digestion. The supernatant sample obtained in step (1) was filtered through a 0.22 μm filter and placed in a liquid sample bottle for analysis using a high-performance liquid chromatograph (Thermo Scientific, Waltham, MA, USA). The detection conditions were as follows: Column: C18 column (250 mm × 4.6 mm, 5 μm); Mobile phase A: aqueous solution containing 0.1% (v / v) trifluoroacetic acid; Mobile phase B: acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid; Elution conditions: 0.01–25.0 min, 10–35% mobile phase B; 25.0–35 min (excluding 25.0 min), 35% mobile phase B; Flow rate: 1.0 mL / min; Detection wavelength: 220 nm; Injection volume: 10 µL. Results are as follows: Figure 6 As shown in Table 2, the peptide stability of sea cucumber flower heptapeptide, which promotes iron absorption, at each digestion stage is shown in Table 2.

[0118] Table 2. Stability of EVDDRWK and EVDDRWK-Fe peptides after simulated gastrointestinal digestion:

[0119] according to Figure 6 It can be seen that the heptapeptide from sea cucumber flower with iron absorption-promoting effects eluted at around 9.9 min, and only one elution peak was observed for this heptapeptide. This indicates that the heptapeptide from sea cucumber flower with iron absorption-promoting effects remains relatively stable after gastrointestinal digestion. Table 2 shows that the heptapeptide from sea cucumber flower with iron absorption-promoting effects underwent some degradation during simulated gastrointestinal digestion. During gastric digestion, the peptide stability was 83.75% ± 0.37%, but after further intestinal digestion, the peptide stability was only 63.75% ± 0.02%. After the addition of ferrous ions, the heptapeptide from sea cucumber flower with iron absorption-promoting effects showed improved resistance to enzymatic digestion. During gastric digestion, the peptide stability was above 94.12% ± 0.16%, and during intestinal digestion, the stability increased compared to the pure peptide, reaching 89.69% ± 0.45%. The reason may be that ferrous ions induce the binding of sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, causing the peptide chain to fold, thus hiding the enzyme cleavage site of sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, and reducing enzyme degradation.

[0120] Test Example 4: The digest of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects was identified by Nano-LC-ESI-MS / MS.

[0121] (1) Sample preparation: Add 1 mg of peptide powder or 100 μL of solution to 100 μL of 0.1% trifluoroacetic acid (TFA) aqueous solution, homogenize by sonication, centrifuge at 14000g for 10 min, collect the supernatant, and ultrafilter the supernatant using a 10KD ultrafiltration tube (PALL, OD010C35), centrifuge at 13500g for 10 min, and quantify the peptides using a nanodrop 2000C UV-Vis spectrophotometer. Desalt the filtrate using a C18 solid-phase extraction column (66871, Sigma). The specific steps are as follows: first, wash the extraction column with acetonitrile, then equilibrate the extraction column with 0.1% TFA, load the ultrafiltered sample solution, and elute the peptides with 70% acetonitrile aqueous solution.

[0122] (2) Chromatographic separation: Each fractionated sample was separated using a nanoliter flow rate HPLC system, Easy nLC. Buffer solutions: Solution A was a 0.1% (v / v) formic acid aqueous solution, and Solution B was a 0.1% (v / v) formic acid-acetonitrile aqueous solution (acetonitrile volume percentage 84%). The column was equilibrated with 95% (v / v) solution A. Samples were loaded via an autosampler onto the loading column (Thermo Scientific EASY column, 100 μm × 2 cm, 5 μm, C18), and then separated onto the analytical column (Thermo Scientific EASY column, 75 μm × 10 cm, 3 μm, C18) at a flow rate of 250 nL / min. The relevant liquid phase gradients are as follows: 0~50 min, the linear gradient of liquid B is 0~35% (volume percentage); 50~58 min (excluding 50 min), the linear gradient of liquid B is 35~100% (volume percentage); 58~60 min (excluding 58 min), liquid B is maintained at 100% (volume percentage).

[0123] (3) Mass spectrometry identification: After chromatographic separation, the samples were analyzed by mass spectrometry using a Q-Exactive HF-X mass spectrometer. The analysis time was 60 min, the detection mode was positive ion, the precursor ion scan range was 300–1800 m / z, the primary mass spectrometry was performed at a mass-to-charge ratio of 200 with a resolution of 70,000, the automatic gain control (AGC) target value was 3e6, the maximum injection time was 10 ms, and the dynamic exclusion time was 40.0 s. The mass-to-charge ratio of peptides and peptide fragments was acquired using the following method: 10 fragment spectra were acquired after each full scan (MS2scan), the secondary mass spectrometry activation mode was high-energy collisional dissociation (HCD), the isolation window was 2 m / z, the secondary mass spectrometry was performed at a mass-to-charge ratio of 200 with a resolution of 17,500, the normalized collision energy was 30 eV, and the underflow ratio was 0.1%.

[0124] (4) Database retrieval: The mass spectrometry test raw files were retrieved from the relevant database using MaxQuant software 1.5.5.1. The results are as follows: Figures 7-10 As shown in Tables 3-11.

[0125] Table 3. Identification of gastrointestinal digestive products of sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects:

[0126] Table 4 Figure 7 Structural coordinate data of A in the middle:

[0127] Table 5 Figure 7 Structural coordinate data of B in the middle:

[0128] Table 6 Figure 8 Structural coordinate data of A in the middle:

[0129] Table 7 Figure 8 Structural coordinate data of B in the middle:

[0130] Table 8 Figure 9 Structural coordinate data of A in the middle:

[0131] Table 9 Figure 9 Structural coordinate data of B in the middle:

[0132] Table 10 Figure 10 Structural coordinate data of A in the middle:

[0133] Table 11 Figure 10 Structural coordinate data of B in the middle:

[0134] according to Figures 7-10As shown in Tables 3-11, undigested sea cucumber flower heptapeptides with iron-enhancing effects can still be detected in a simulated gastrointestinal environment, indicating that they possess partial resistance to digestion. Secondary peptides produced during digestion, including VDDRWK, EVDDRW, DDRWK, DRWK, DDRW, VDDR, and RWK, undergo pepsin cleavage primarily at the C-terminal tryptophan (Trp) residue, while also exhibiting some trypsin dissociation at the N-terminal glutamate (Glu) site. Notably, these degradation fragments retain iron-coordinating residues such as aspartic acid (Asp) or valine, maintaining their affinity for Fe. 2+ The above results indicate that sea cucumber flower heptapeptide, which promotes iron absorption, binds with Fe. 2+ After the formation of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects, its digestibility was improved. Although the sea cucumber flower heptapeptide with iron absorption-promoting effects is partially degraded during gastrointestinal digestion, the resulting short-chain peptide fragments may continue to exert iron-carrying functions due to the retention of potential coordinating amino acids.

[0135] Test Example 5: The specific steps for iron transport assays using an in vitro Caco-2 cell monolayer model are as follows: Caco-2 cells were cultured in Eagle's MEM medium containing 20% ​​fetal bovine serum and 1% penicillin / streptomycin antibiotics at 37°C, 5% CO2, and 90% relative humidity. Cells at passages 25-30 were seeded into 12-well Transwell plates (12 wells, 0.4 μm, 1.12 cm). 2 On the surface, the inoculation density was 1.0 × 10⁻⁶. 5 Cells / mL, culture medium was changed every 2 days after inoculation. The integrity of the cell monolayer was assessed by measuring transepithelial electrical resistance (TEER) values ​​every other day using the Millicell-ERS system.

[0136] Caco-2 cell monolayers were gently washed twice with Hank's balanced salt solution (HBSS), then transferred to new plates and incubated with 1.5 mL of HBSS buffer for 30 min, followed by transport experiments. The HBSS buffer was removed and replaced with 0.5 mL of sample (dissolved in HBSS) on the AP side and 1.5 mL of fresh HBSS buffer on the BL side. Cells were incubated at 37°C and 5% CO2 for 2 h. 1.0 mL of sample from the BL side at different digestion times was extracted, and then 1.0 mL of fresh HBSS buffer was added to the BL side to maintain a constant volume. Iron content was determined using an atomic absorption spectrophotometer, and iron absorption was calculated. The results are shown below. Figure 11 As shown.

[0137] according to Figure 11It can be seen that both the digestate of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects and the FeCl2 digestate exhibit certain iron transport capabilities. Furthermore, at different transport time points, the iron transport effect of the digested sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects is significantly better than that of the digested FeCl2. Moreover, the amount of iron transported increases with time, reaching a maximum at 120 min (79.15±1.86 μg / well). In summary, compared with FeCl2-mimicked gastrointestinal digestion products, the digestate of the sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects provided by this invention has a good iron absorption-promoting effect and can effectively promote the absorption of iron by small intestinal epithelial cells. The sea cucumber flower heptapeptide-iron nanocomplex with iron absorption-promoting effects provided by this invention can maintain the solubility of iron in the gastrointestinal environment and effectively promote the absorption of iron by Caco-2 cells.

[0138] The embodiments of the present invention have been described above; however, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the above embodiments of the present invention without inventive effort are within the protection scope of the present invention.

Claims

1. A sea cucumber flower heptapeptide with iron absorption-promoting effects, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing sea cucumber flower heptapeptide with iron absorption-promoting effects, characterized in that, The sea cucumber flower heptapeptide with iron-promoting effects mentioned above is the sea cucumber flower heptapeptide with iron-promoting effects described in claim 1. The process includes the following steps: Following the amino acid sequence Glu-Val-Asp-Asp-Arg-Trp-Lys, 2-chlorotrimethylbenzenechloropolymer resin is covalently linked to Fmoc-Lys(Boc)-OH, and then sequentially condensed with Fmoc-Trp-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Val-OH, and Fmoc-Glu(OtBu)-OH. Finally, the 2-chlorotrimethylbenzenechloropolymer resin is cleaved to obtain the sea cucumber flower heptapeptide with iron-promoting effects.

3. The preparation method of sea cucumber flower heptapeptide with iron absorption-promoting effect according to claim 2, characterized in that, The covalent connection includes the following steps: A covalent bonding reaction was carried out by mixing 2-chlorotrimethylbenzene chloride resin, Fmoc-Lys(Boc)-OH, dichloromethane, and N,N-diisopropylethylamine. The mixture was blocked with methanol, N,N-diisopropylethylamine, and dichloromethane, and deprotected with piperidine solution.

4. The application of a sea cucumber flower heptapeptide with iron absorption-promoting effects as a iron carrier, characterized in that, The sea cucumber flower heptapeptide with iron absorption promoting effect is the sea cucumber flower heptapeptide with iron absorption promoting effect described in claim 1 or the sea cucumber flower heptapeptide with iron absorption promoting effect described in any one of claims 2 to 3, which is obtained by the preparation method of the sea cucumber flower heptapeptide with iron absorption promoting effect.

5. A sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects, characterized in that, It includes sea cucumber flower heptapeptide with iron-enhancing effects and iron ions bound to the Asp-3, Asp-4, and Arg-5 amino acids of the sea cucumber flower heptapeptide with iron-enhancing effects; the iron ions are Fe. 2+ ; The sea cucumber flower heptapeptide with iron absorption promoting effect is the sea cucumber flower heptapeptide with iron absorption promoting effect described in claim 1 or the sea cucumber flower heptapeptide with iron absorption promoting effect described in any one of claims 2 to 3, which is obtained by the preparation method of the sea cucumber flower heptapeptide with iron absorption promoting effect.

6. A method for preparing a sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effects, characterized in that, The sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect is the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect as described in claim 5, comprising the following steps: Sea cucumber flower heptapeptide, which has the effect of promoting iron absorption, was mixed with water to obtain a sea cucumber flower heptapeptide solution. After adjusting the pH value of the sea cucumber flower heptapeptide solution to 6.0~7.0, it was mixed with FeSO4·4H2O and Na2S2O4 to react and obtain the sea cucumber flower heptapeptide-iron nanocomposite with the effect of promoting iron absorption.

7. The preparation method of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect according to claim 6, characterized in that, The molar ratio of the sea cucumber flower heptapeptide, which promotes iron absorption, to FeSO4·4H2O is 1:4~8.

8. The preparation method of the sea cucumber flower heptapeptide-iron nanocomposite with iron absorption-promoting effect according to claim 6, characterized in that, The reaction is carried out under stirring conditions, at a temperature of 20-28°C, a pH of 6.0-7.0, and a holding time of 60-90 min.

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