A holothurian flower octapeptide with selenium absorption promoting efficacy, and a preparation method and application thereof

Abstract

The present application relates to the technical fields of polypeptide biomaterial and its synthesis, and particularly relates to a sea cucumber flower octapeptide with selenium absorption promoting effect, and a preparation method and application thereof.The sea cucumber flower octapeptide with selenium absorption promoting effect provided by the present application is an active peptide derived from sea cucumber flower protein, which can self-assemble and efficiently bind selenium, has self-assembling characteristics and high selenium chelating capacity, can construct a peptide-selenium complex, and form a sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption promoting effect, and has important scientific and application values for realizing full-efficiency utilization of sea cucumber by-products and promoting development of safe and efficient selenium supplements.The sea cucumber flower octapeptide-selenium nanocomposite provided by the present application can effectively resist gastrointestinal digestion, and has self-assembling capacity and selenium absorption promoting effect, and the peptide stability after simulated gastrointestinal digestion is far higher than that of pure peptide, and can effectively enhance selenium delivery capacity, and can be used in the fields of selenium supplements and the like.

Description

Technical Field

[0001] This invention relates to the field of polypeptide biomaterials and their synthesis technology, and in particular to a sea cucumber flower octapeptide with selenium absorption-promoting effect, its preparation method and application. Background Technology

[0002] Selenium, an essential trace element for the human body, plays an indispensable role in physiological processes such as antioxidant defense, immune regulation, thyroid hormone metabolism, and anti-cancer activity, despite being required in very small amounts. Large areas of the world are selenium-deficient, and insufficient dietary selenium intake is directly related to endemic diseases such as Keshan disease and Kashin-Beck disease, and generally affects immune and reproductive health. Currently, selenium supplements on the market mainly include inorganic selenium (such as sodium selenite) and organic selenium (such as selenomethionine). However, inorganic selenium has low bioavailability and a narrow safety window, while the stability and absorption efficiency of organic selenium also need improvement. Therefore, developing a new, efficient, stable, and bio-friendly selenium delivery system is an important need in the field of nutritional fortification.

[0003] Sea cucumber florets, a major byproduct of sea cucumber processing, are rich in high-quality protein, phospholipids, and various bioactive substances, making them a valuable resource for developing bioactive peptides. Their protein has a balanced amino acid composition and contains numerous potential metal ion binding sites. However, currently, sea cucumber floret resources are largely underutilized, often being discarded or treated only for low-value purposes, resulting in serious resource waste and loss of industrial value.

[0004] In recent years, the use of bioactive peptides as trace element carriers has become a research frontier. Peptide segments with specific sequences can self-assemble into nanoscale or microscale structures. These structures can not only stably bind with trace elements such as selenium through coordination bonds to form organic complexes, but also protect selenium from degradation in the gastrointestinal environment and promote its intestinal transport through specific peptide absorption pathways, thereby significantly improving the bioavailability of selenium and reducing its potential toxicity. Although the advantages of peptide-selenium complexes have been preliminarily recognized, there are currently no reports of specific peptide segments with both self-assembly properties and highly efficient selenium chelating ability being prepared from sea cucumber flower protein through targeted enzymatic hydrolysis. Summary of the Invention

[0005] In view of this, the present invention provides a sea cucumber flower octapeptide with selenium absorption-promoting effect, its preparation method and application. The sea cucumber flower octapeptide with selenium absorption-promoting effect provided by the present invention has both self-assembly characteristics and efficient selenium chelation ability.

[0006] This invention provides a sea cucumber flower octapeptide with selenium absorption-promoting effects, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a method for preparing sea cucumber flower octapeptide with selenium absorption-promoting effect as described in the above-mentioned scheme, including method one or method two; Method 1 includes the following steps: 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, centrifuging to collect the precipitate, freeze-drying to obtain sea cucumber flower protein powder, mixing the sea cucumber flower protein powder with water, simulated gastric juice and simulated intestinal juice (referred to as the first mixture) for enzymatic hydrolysis, inactivating enzymes, centrifuging to collect the supernatant, freeze-drying to obtain sea cucumber flower octapeptide with selenium absorption promotion effect; Method 2 includes the following steps: according to the amino acid sequence Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val, the polymer resin is sequentially subjected to condensation reaction and cleavage reaction with Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)-OH to obtain the sea cucumber flower octapeptide with selenium absorption promotion effect.

[0008] Preferably, in method one, the first mixing includes the following steps: mixing the sea cucumber flower protein powder and water to obtain sea cucumber flower protein liquid, then mixing the sea cucumber flower protein liquid with simulated gastric juice, adjusting the pH value to 2.0 to carry out an enzymatic hydrolysis reaction (referred to as the first enzymatic hydrolysis reaction), and then mixing the obtained reactants with simulated intestinal juice, adjusting the pH value to 7.0 to carry out an enzymatic hydrolysis reaction (referred to as the second enzymatic hydrolysis reaction).

[0009] Preferably, in method one, the temperature of the first enzymatic hydrolysis reaction is 35~39℃, and the holding time is 1.8~2.2h; the pH value of the first enzymatic hydrolysis reaction is constant at 2.0; the temperature of the second enzymatic hydrolysis reaction is 35~39℃, and the holding time is 1.8~2.2h; the pH value of the second enzymatic hydrolysis reaction is constant at 7.0.

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

[0011] The present invention also provides a sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect, comprising a sea cucumber flower octapeptide with selenium absorption-promoting effect, and selenium ions bound to the Asp-4 amino acid of the sea cucumber flower octapeptide with selenium absorption-promoting effect; the sea cucumber flower octapeptide with selenium absorption-promoting effect is the sea cucumber flower octapeptide with selenium absorption-promoting effect described in the above scheme or the sea cucumber flower octapeptide with selenium absorption-promoting effect obtained by the preparation method described in the above scheme.

[0012] Preferably, the selenium ion is a divalent selenium ion (Se). 2- ).

[0013] This invention also provides a method for preparing the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect as described in the above-mentioned scheme, comprising the following steps: Sea cucumber flower octapeptide with selenium absorption-promoting effect, selenium salt solution and water are mixed and reacted to obtain the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect.

[0014] Preferably, the selenium salt solution is a sodium selenite solution; the concentration of the selenium salt solution is 0.45~0.55 mol / L.

[0015] Preferably, the reaction temperature is 55~60℃, and the holding time is 18~25min; the reaction is carried out under alkaline conditions; the pH range of the alkaline conditions is 8.5~9.

[0016] Compared with existing technologies, the sea cucumber flower octapeptide with selenium absorption-promoting effects, its preparation method, and its application provided by this invention have achieved the following beneficial effects: The sea cucumber flower octapeptide (SEDDKLIV) provided by this invention is an active peptide derived from sea cucumber flower protein that can self-assemble and efficiently bind selenium. It has both self-assembly properties and efficient selenium chelation ability, and can construct peptide-selenium complexes to form sea cucumber flower octapeptide-selenium nanocomplexes with selenium absorption-promoting effects. It has important scientific and application value for realizing the full utilization of sea cucumber by-products and promoting the development of safe and efficient selenium supplements.

[0017] The sea cucumber flower octapeptide-selenium nanocomposite (SEDDKLIV-Se) provided by this invention has the effect of promoting selenium absorption. It can effectively resist gastrointestinal digestion and has both self-assembly ability and selenium absorption promotion effect. The peptide stability after simulated gastrointestinal digestion reaches 86.37%±0.17%, which is much higher than the 60.40%±0.23% of pure peptide. It can also effectively enhance the selenium delivery ability. It can be used in the field of selenium supplements, realizing the high-value development of sea cucumber flower resources and the development of a new generation of selenium supplements.

[0018] The method for preparing sea cucumber flower octapeptide with selenium absorption-promoting effect provided by the present invention adopts the standard Fmoc synthesis method, which is mature and stable, compatible with existing industrial production lines, and has controllable cost, making it suitable for industrial-scale production. 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 This is a schematic diagram of the potential binding model between sea cucumber flower octapeptide, which has the effect of promoting selenium absorption, and selenium; in this diagram, yellow spheres represent selenium, light blue chains represent sea cucumber flower octapeptide with the effect of promoting selenium absorption, dark blue segments represent the N-terminus, and red segments represent the oxygen terminus. Figure 2 The images show the liquid chromatogram and mass spectrum of sea cucumber flower octapeptide with selenium absorption-promoting effect; where a is the liquid chromatogram of sea cucumber flower octapeptide with selenium absorption-promoting effect and b is the mass spectrum of sea cucumber flower octapeptide with selenium absorption-promoting effect. Figure 3 SEM images of sea cucumber flower octapeptide with selenium absorption-promoting effect and sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect (scale bar: 1000x = 10μm; 3500x = 3μm; 8000x = 1μm). Figure 4 The image shows the elemental surface distribution of the energy-dispersive X-ray spectroscopy of sea cucumber flower octapeptide and the sea cucumber flower octapeptide-selenium nanocomposite, both of which have the effect of promoting selenium absorption. Image A shows the elemental surface distribution of the energy-dispersive X-ray spectroscopy of sea cucumber flower octapeptide with the effect of promoting selenium absorption, with a scale bar of 2.5 μm. Image B shows the elemental surface distribution of the energy-dispersive X-ray spectroscopy of sea cucumber flower octapeptide-selenium nanocomposite, both of which have the effect of promoting selenium absorption, with a scale bar of 2.5 μm. Figure 5 The images show the infrared spectra and secondary structure analysis diagrams of sea cucumber flower octapeptide and sea cucumber flower octapeptide-selenium nanocomplex, both of which promote selenium absorption. Image a shows the infrared spectra of the sea cucumber flower octapeptide and sea cucumber flower octapeptide-selenium nanocomplex, and image b shows the secondary structure analysis diagram of the sea cucumber flower octapeptide and sea cucumber flower octapeptide-selenium nanocomplex. Figure 6 The digestive stability analysis diagrams are shown for sea cucumber flower octapeptide and sea cucumber flower octapeptide-selenium nanocomposite, which have the effect of promoting selenium absorption. Among them, a is the digestive stability analysis diagram of sea cucumber flower octapeptide with the effect of promoting selenium absorption, and b is the digestive stability analysis diagram of sea cucumber flower octapeptide-selenium nanocomposite, which have the effect of promoting selenium absorption. Figure 7 Total ion current chromatogram (TIC) for the spectroscopic identification of the simulated gastrointestinal digestive products of sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect based on Nano-LC-ESI-MS / MS. Figure 8 The SEDDKLIV image shows the spectroscopic identification of the gastrointestinal digestive products of sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects based on Nano-LC-ESI-MS / MS. Figure 9 EDDKLIV image of simulated gastrointestinal digestion products of sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect based on Nano-LC-ESI-MS / MS. Figure 10 SEDDKL plot of simulated gastrointestinal digestion products of sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect based on Nano-LC-ESI-MS / MS; Figure 11 The DDKLIV image shows the spectroscopic identification of the gastrointestinal digestive products of sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects based on Nano-LC-ESI-MS / MS. Figure 12 shows the statistical results of selenium transport in the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect prepared in Example 1 and the Na2SeO3 digest. Detailed Implementation

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

[0022] The sea cucumber flower octapeptide provided by this invention has the amino acid sequence Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val (SEDDKLIV) and has the effect of promoting selenium absorption.

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

[0024] The present invention also provides a method for preparing sea cucumber flower octapeptide with selenium absorption-promoting effect as described in the above-mentioned scheme, including method one or method two; Method 1 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, centrifuging and collecting the precipitate, freeze-drying to obtain sea cucumber flower protein powder, mixing the sea cucumber flower protein powder with water, simulated gastric juice and simulated intestinal juice for enzymatic hydrolysis, inactivating enzymes, centrifuging and collecting the supernatant, freeze-drying to obtain sea cucumber flower octapeptide with selenium absorption promotion effect; Method 2 includes the following steps: according to the amino acid sequence Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val, the polymer resin is sequentially subjected to condensation reaction and cleavage reaction with Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)-OH to obtain the sea cucumber flower octapeptide with selenium absorption promotion effect.

[0025] In Method 1, the present invention mixes sea cucumber flower powder and water, adjusts the pH value to alkaline, heats and mixes, centrifuges and collects the supernatant, adjusts the pH value of the supernatant to acidic, centrifuges and collects the precipitate, freeze-dries it, and obtains sea cucumber flower protein powder.

[0026] In this invention, the water is preferably pure water.

[0027] In this invention, the mass ratio of sea cucumber flower powder to water is preferably 1:10~15, more preferably 1:11~14, and even more preferably 1:12~13.

[0028] In this invention, the reagent used to adjust the pH value to alkaline is preferably a NaOH solution; the pH value used to adjust the pH value to alkaline is preferably 8.5 to 9.0, more preferably 8.7.

[0029] In this invention, the heating and mixing temperature is preferably 40~55℃, more preferably 45~50℃, and the heat preservation time is preferably 4~8h, more preferably 6h; the heating and mixing is preferably heating and stirring; the heating and stirring speed is preferably 200~400rpm, more preferably 300rpm.

[0030] In this invention, the centrifugation speed for collecting the supernatant is preferably 8000~10000 rpm, and the time is preferably 10~20 min.

[0031] In this invention, the reagent used to adjust the pH of the supernatant to acidic is preferably hydrochloric acid; the pH value used to adjust the pH of the supernatant to acidic is preferably 3.5 to 4.0, more preferably 3.8.

[0032] In this invention, the preferred speed for centrifuging to collect the precipitate is 8000~10000 rpm, and the preferred time is 10~20 min.

[0033] 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.

[0034] 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) for enzymatic hydrolysis, inactivates enzymes, collects the supernatant by centrifugation, and freeze-dries it to obtain sea cucumber flower octapeptide with selenium absorption promotion effect.

[0035] In this invention, the mass ratio of the sea cucumber flower protein powder to the volume of water is preferably (25~35) mg:1 mL, more preferably 30 mg:1 mL.

[0036] In this invention, the simulated gastric juice preferably comprises 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 comprises 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.

[0037] 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 pancreatic enzymes is preferably 1:0.5 to 1.5, more preferably 1:1; the intestinal electrolyte solution preferably comprises: 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 bile in the simulated intestinal fluid is preferably 150 to 170 mmol / L.

[0038] In this invention, the first mixing preferably includes the following steps: mixing the sea cucumber flower protein powder and water to obtain sea cucumber flower protein liquid, then mixing the sea cucumber flower protein liquid with simulated gastric juice, adjusting the pH value to 2.0 to carry out an enzymatic hydrolysis reaction (referred to as the first enzymatic hydrolysis reaction), and then mixing the obtained reactants with simulated intestinal juice, adjusting the pH value to 7.0 to carry out an enzymatic hydrolysis reaction (referred to as the second enzymatic hydrolysis reaction).

[0039] In this invention, the volume ratio of the simulated gastric juice to the sea cucumber flower protein liquid is preferably 1:9~11, more preferably 1:10.

[0040] In this invention, the volume ratio of the simulated intestinal fluid to the sea cucumber flower protein solution is preferably 1:9~11, more preferably 1:10.

[0041] In this invention, the temperature of the first enzymatic hydrolysis reaction is preferably 35~39℃, more preferably 37℃, and the holding time is preferably 1.8~2.2h, more preferably 2h; the pH value is kept stable during the first enzymatic hydrolysis reaction.

[0042] In this invention, the temperature of the second enzymatic hydrolysis reaction is preferably 35~39℃, more preferably 37℃, and the holding time is preferably 1.8~2.2h, more preferably 2h; the pH value is kept stable during the second enzymatic hydrolysis reaction. This invention uses a hydrochloric acid solution (1mol / L) to maintain a stable pH value during both the first and second enzymatic hydrolysis reactions.

[0043] In this invention, the enzyme inactivation method is preferably heating; the target temperature for heating is preferably 90~100℃, and the holding time is preferably 15~20min, more preferably 17min.

[0044] In this invention, the temperature of the supernatant collected by centrifugation is preferably 3~5℃, more preferably 4℃, the centrifugation speed is preferably 8000~10000rpm, more preferably 9000rpm, and the centrifugation time is preferably 10~15min, more preferably 13min.

[0045] 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 6 Pa, and the preferred heat preservation time is 48 to 72 hours, more preferably 60 hours.

[0046] In Method 2, according to the amino acid sequence Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val, the polymer resin is sequentially subjected to condensation reactions and cleavage reactions with Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)-OH to obtain the sea cucumber flower octapeptide with selenium absorption-promoting effect.

[0047] In this invention, the polymer resin is preferably 2-chlorotrimethylbenzene chloride resin; the polymer resin is preferably pretreated before use; the pretreatment includes the following steps: soaking the polymer resin in dichloromethane (DCM) and then washing and drying it sequentially, repeating the washing and drying process more than 4 times.

[0048] In this invention, the soaking time is preferably 2 hours or more, and more preferably 2 to 3 hours.

[0049] In this invention, the washing reagent is preferably N,N-dimethylformamide.

[0050] In this invention, the polymer resin undergoes condensation reactions sequentially with Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Ser(tBu)-OH, which are referred to as the first condensation reaction, the second condensation reaction, the third condensation reaction, the fourth condensation reaction, the fifth condensation reaction, the sixth condensation reaction, the seventh condensation reaction, and the eighth condensation reaction.

[0051] In this invention, the first condensation reaction preferably includes the following steps: mixing a polymer resin, Fmoc-Val-OH, dichloromethane and N,N-diisopropylethylamine (DIEA) to carry out the first condensation reaction.

[0052] In this invention, the mass ratio of the polymer resin to Fmoc-Val-OH is preferably 1:1 to 1.2; the mass-to-volume ratio of Fmoc-Val-OH to dichloromethane is preferably 1 g:(8 to 12) mL; and the volume ratio of N,N-diisopropylethylamine to dichloromethane is preferably 1:1.8 to 2.2, more preferably 1:2.

[0053] In this invention, the temperature of the first condensation reaction is preferably 28~32℃, more preferably 30℃, and the holding time is preferably 1.8~2.2h, more preferably 2h; the first condensation reaction is preferably carried out in a shaker.

[0054] In this invention, the first condensation reaction preferably includes post-processing of the obtained product; the post-processing preferably includes sequential sealing with methanol, washing and drying, and repeating washing and drying more than 4 times.

[0055] In this invention, the volume ratio of methanol to N,N-diisopropylethylamine is preferably 0.9 to 1.1:1, more preferably 1:1.

[0056] In this invention, the sealing time is preferably 25-35 minutes, more preferably 30 minutes.

[0057] In this invention, the washing reagent is preferably N,N-dimethylformamide (DMF).

[0058] In this invention, the post-treatment preferably further includes mixing the obtained product and piperidine solution and sequentially performing deprotection, washing, and drying.

[0059] In this invention, the volume concentration of the piperidine solution is preferably 18-22%, more preferably 20%; the mass ratio of the piperidine solution to methanol is preferably 1:8-12. This invention removes the Fmoc protecting group using a piperidine solution. A sample of 2-chlorotrimethylbenzenechloro polymer resin is taken and tested using the ninhydrin method. If the 2-chlorotrimethylbenzenechloro polymer resin shows color, it indicates successful deprotection.

[0060] In this invention, the washing reagent is preferably N,N-dimethylformamide; and the washing and drying are preferably performed four or more times.

[0061] In this invention, the second condensation reaction preferably includes the following steps: mixing the product obtained from the first condensation reaction with Fmoc-Ile-OH, 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC) to carry out a second condensation reaction.

[0062] In this invention, the preferred mass ratio of the polymer resin to Fmoc-Ile-OH is 3~5:1; the preferred mass ratio of Fmoc-Ile-OH to 1-hydroxybenzotriazole is 1:1~1.2; and the preferred mass ratio of 1-hydroxybenzotriazole to N,N'-diisopropylcarbodiimide is 1:1~1.5.

[0063] In this invention, the temperature of the second condensation reaction is preferably 28~32℃, more preferably 30℃, and the holding time is preferably 0.8~1.2h, more preferably 1h; the second condensation reaction is preferably carried out in a shaker. After the second condensation reaction, a sample of the 2-chlorotrimethylbenzenechloro polymer resin is tested using the ninhydrin method. If the 2-chlorotrimethylbenzenechloro polymer resin is colored, it indicates that the condensation reaction is incomplete, and the condensation reaction should continue. If the 2-chlorotrimethylbenzenechloro polymer resin is colorless, it indicates that the condensation reaction is complete.

[0064] In this invention, the second condensation reaction preferably includes post-processing of the obtained product; the post-processing preferably includes washing and drying in sequence, and repeating washing and drying more than 4 times.

[0065] In this invention, the washing reagent is preferably N,N-dimethylformamide.

[0066] In this invention, the post-treatment preferably further includes mixing the obtained product and piperidine solution and sequentially performing deprotection, washing, and drying.

[0067] In this invention, the volume concentration of the piperidine solution is preferably 18-22%, more preferably 20%; the volume ratio of the piperidine solution to N,N-dimethylformamide is preferably 1:3-5, more preferably 1:4.

[0068] In this invention, the deprotection is preferably carried out by shaking on a decolorizing shaker; the deprotection time is preferably 18-25 minutes, more preferably 20 minutes. This invention removes the Fmoc protecting group from 2-chlorotrimethylbenzenechloro polymer resin through deprotection. This invention removes the Fmoc protecting group using a piperidine solution. A sample of the 2-chlorotrimethylbenzenechloro polymer resin is taken and tested using the ninhydrin method. If the 2-chlorotrimethylbenzenechloro polymer resin shows color, it indicates successful deprotection.

[0069] In this invention, the washing reagent is preferably N,N-dimethylformamide; and the washing and drying are preferably performed four or more times.

[0070] In this invention, the methods for the third, fourth, fifth, sixth, seventh, and eighth condensation reactions are preferably the same as those for the second condensation reaction, except that they are replaced with the target amino acid, which will not be described in detail here.

[0071] In this invention, the cutting reagent used to cut the polymer resin is preferably a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TI) and water (H2O); the volume ratio of the trifluoroacetic acid, triisopropylsilane and water is preferably 95:2.5:2.5.

[0072] In this invention, the process of removing the polymer resin preferably includes post-processing of the resulting product; the post-processing preferably includes purification, quick freezing, pre-freezing and freeze drying in sequence.

[0073] In this invention, the purification is preferably carried out by high-performance liquid chromatography (HPLC). The parameters for HPLC purification preferably include: a VYDAC-C18 column with a size of 4.6 × 250 mm; mobile phase A being acetonitrile containing 0.1% (v / v) trifluoroacetic acid; mobile phase B being water containing 0.1% (v / v) trifluoroacetic acid; elution conditions: 0–20.0 min: mobile phase A increases from 22.0% (v / v) to 32.0% (v / v); 20.0–20.1 min (excluding 20.0 min): mobile phase A increases from 32.0% (v / v) to 100.0% (v / v); flow rate is 1.0 mL / min; and detection wavelength is 220 nm.

[0074] In this invention, the quick-freezing is preferably quick-freezing with liquid nitrogen; the holding time for quick-freezing is preferably 5 to 10 minutes, more preferably 7 to 8 minutes.

[0075] In this invention, the quick-freezing preferably includes the following steps: after collecting the main peak fraction, the purified product is immediately placed in a 1.5 mL EP tube (the liquid volume does not exceed 400 μL) and quick-frozen in liquid nitrogen.

[0076] In this invention, the pre-freezing temperature is preferably -85~-75℃, more preferably -80℃, and the heat preservation time is preferably 120~150min, more preferably 135min.

[0077] In this invention, the freeze-drying time is preferably 48-60 hours, more preferably 55 hours; the freeze-drying preferably includes sequentially performing a first freeze-drying, a second freeze-drying, and a third freeze-drying; the temperature of the first freeze-drying is preferably -40 to -10°C, more preferably -30 to -20°C, and the holding time is preferably 2-3 hours, more preferably 2.5 hours; the temperature of the second freeze-drying is preferably -40 to -10°C, more preferably -30 to -20°C, the pressure is preferably 0 to 20 Pa, more preferably 10 to 15 Pa, and the holding time is preferably 1-2 hours, more preferably 1.5 hours; the temperature of the third freeze-drying is preferably 0 to 25°C, more preferably 10 to 15°C, and the pressure is preferably 0 to 20 Pa, more preferably 10 to 15 Pa.

[0078] The present invention also provides the application of the sea cucumber flower octapeptide with selenium absorption-promoting effect described in the above-described scheme or the sea cucumber flower octapeptide with selenium absorption-promoting effect obtained by the preparation method described in the above-described scheme as a selenium carrier.

[0079] The sea cucumber flower octapeptide provided by this invention, which promotes selenium absorption, can be used as a carrier of selenium, including but not limited to selenium ions, especially divalent selenium ions, for use in selenium supplements and other fields, and has broad application prospects.

[0080] The present invention also provides a sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect, comprising a sea cucumber flower octapeptide with selenium absorption-promoting effect, and selenium ions bound to the Asp-4 amino acid of the sea cucumber flower octapeptide with selenium absorption-promoting effect; the sea cucumber flower octapeptide with selenium absorption-promoting effect is the sea cucumber flower octapeptide with selenium absorption-promoting effect described in the above scheme or the sea cucumber flower octapeptide with selenium absorption-promoting effect obtained by the preparation method described in the above scheme.

[0081] In this invention, the selenium ion is preferably a divalent selenium ion (Se). 2- ).

[0082] The sea cucumber flower octapeptide-selenium nanocomposite provided by this invention has a peptide stability of up to 86.37%±0.17% after simulated gastrointestinal digestion, which is much higher than the 60.40%±0.23% of pure sea cucumber flower octapeptide with selenium absorption promotion effect. It can also effectively enhance selenium delivery capacity and can be used in selenium supplements and other selenium-fortified products.

[0083] This invention also provides a method for preparing the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect as described in the above-mentioned scheme, comprising the following steps: Sea cucumber flower octapeptide with selenium absorption-promoting effect, selenium salt solution and water are mixed and reacted to obtain the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect.

[0084] In this invention, the selenium salt solution is preferably a sodium selenite solution; the concentration of the selenium salt solution is preferably 0.45~0.55 mol / L, more preferably 0.5 mol / L.

[0085] In this invention, the mass ratio of the sea cucumber flower octapeptide with selenium absorption-promoting effect to selenium salt is preferably 1:6~8, more preferably 1:7.

[0086] In this invention, the water is preferably ultrapure water, specifically Milli-Q water.

[0087] In this invention, the preferred mass-to-volume ratio of the sea cucumber flower octapeptide with selenium absorption-promoting effect to water is (4~8) mg:1 mL, more preferably 5 mg:1 mL.

[0088] In this invention, the reaction temperature is preferably 55~60℃, more preferably 58℃, and the holding time is preferably 18~25min, more preferably 20min; the reaction is preferably carried out under alkaline conditions; the pH range of the alkaline conditions is preferably 8.5~9, more preferably 8.8.

[0089] In this invention, the reaction preferably further includes mixing the resulting product with ethanol, centrifuging, collecting the precipitate, and freeze-drying.

[0090] In this invention, the ethanol is more preferably anhydrous ethanol; the mass ratio of the ethanol to the reaction product is preferably 8~20:1, more preferably 8~10:1.

[0091] In this invention, the centrifugation temperature is preferably 3~5℃, more preferably 4℃; the centrifugation speed is preferably 8000~10000rpm, more preferably 9000rpm; and the centrifugation time is preferably 5~10min, more preferably 8min.

[0092] 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 6 Pa, and the preferred heat preservation time is 48 to 72 hours, more preferably 60 hours.

[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 simulated gastric juice is composed of a gastric electrolyte solution and pepsin; the volume ratio of the gastric electrolyte solution to pepsin is 1:1; the composition of the gastric electrolyte solution is as follows: 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 400 mL distilled water; the concentration of pepsin is 2000 U / mL.

[0097] The simulated intestinal fluid consisted of intestinal electrolyte solution, pancreatic enzymes, and bile; the volume ratio of intestinal electrolyte solution to pancreatic enzymes was 1:1; the intestinal electrolyte solution consisted of 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 400 mL distilled water; the concentration of bile in the simulated intestinal fluid was 160 mmol / L.

[0098] Example 1: This embodiment describes a method for preparing a sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects. The specific steps are as follows: S1. Dissolve the sea cucumber flower powder in pure water at a mass ratio of 1:13. Adjust the pH value to 8.5-9.0 with NaOH solution. Stir at 45℃ and 300 rpm for 6 hours. Centrifuge at 9000 rpm for 15 minutes and collect the supernatant. Adjust the pH value of the supernatant to 3.5-4.0 with hydrochloric acid. Centrifuge at 9000 rpm for 15 minutes and collect the precipitate. Freeze-dry the precipitate at -65℃ and vacuum degree controlled at 2-10 Pa for 60 hours to obtain sea cucumber flower protein powder.

[0099] S2. Prepare a 30 mg / mL sea cucumber flower protein solution by mixing the sea cucumber flower protein powder with simulated gastric juice at a volume ratio of 1:10. Adjust the pH to 2.0 and enzymatically hydrolyze the solution at 37°C for 2 hours. Then, add simulated intestinal juice at a volume ratio of 1:10 and adjust the pH to 7.0. Enzymatically hydrolyze the solution at 37°C for 2 hours, maintaining a stable pH of 7.0 throughout the hydrolysis process. Centrifuge and collect the supernatant. Freeze-dry the solution at -65°C and a vacuum of 2-10 Pa for 60 hours to obtain sea cucumber flower polypeptide containing sea cucumber flower octapeptide with selenium absorption-promoting effects.

[0100] S3. The prepared sea cucumber flower polypeptide was subjected to polypeptide sequence identification, and the amino acid sequence of the sea cucumber flower octapeptide with selenium absorption-promoting effect was screened out. The specific steps are as follows: Sea cucumber flower peptides were desalted by solid phase extraction: (1) 100% acetonitrile and 1 mL of 0.1% (volume percentage) TFA aqueous solution were added to the C18 column in sequence, and centrifuged at 600g for 1 min at room temperature to activate and equilibrate the C18 column; (2) Sample loading: The sea cucumber flower peptide sample was dissolved in 0.1% (volume percentage) TFA aqueous solution and loaded into the equilibrated C18 column, and centrifuged at 600g for 1 min at room temperature until the sea cucumber flower peptide sample was loaded; (3) Sample washing: 1 mL of 0.1% (volume percentage) TFA aqueous solution was added, and centrifuged at 600g for 1 min at room temperature; (4) Sample elution: 250 μL of elution buffer (acetonitrile and TFA aqueous solution with a volume ratio of 70:30, of which the volume percentage of TFA in the TFA aqueous solution is 0.1%) was added to elute the sample, and centrifuged at 200g for 1 min at room temperature; (5) The eluted sample was stored at -80℃ for later use.

[0101] Mass spectrometry detection of desalted sea cucumber flower peptides. The instrument was an ultra-high performance liquid chromatograph (UHPLC) with a C18 column. The HPLC conditions were as follows: mobile phase A was a 0.1% (v / v) formic acid aqueous solution, and mobile phase B was a 0.1% (v / v) formic acid-acetonitrile aqueous solution (acetonitrile volume percentage 84%). Elution conditions were: flow rate 0.5 mL / min, temperature 40℃, and gradient conditions: 0–2.5 min, maintaining 99% (v / v) of solution A and 1% (v / v) of solution B; 2.5–5 min (excluding 2.5 min), solution B increased from 1% (v / v) to 5% (v / v), and solution A decreased from 99% (v / v) to 95% (v / v); 5–10 min (excluding 5 min), solution B increased from 5% (v / v) to 10% (v / v). For each volume percentage, solution A is reduced from 95% to 90%; from 10 to 30 minutes (excluding 10 minutes), solution B is increased from 10% to 25% and solution A is reduced from 90% to 75%; from 30 to 35 minutes (excluding 30 minutes), solution B is increased from 25% to 40% and solution A is reduced from 75% to 60%.

[0102] Mass spectrometry conditions: The mass spectrometer was a Thermo QE Orbitrap. Ion mode was ESI+, mass range 50–2000 m / z; capillary voltage was 3.0 kV; sampling cone voltage was 35.0 V; ion source temperature was 105 °C; desolventizing temperature was 350 °C; cone gas flow rate was 50.0 L / h; desolventizing gas flow rate was 600.0 L / Hr; collision energy was 6.0 eV; collision gas flow rate was 0.6 mL / min; scan time was 0.26 sec; internal scan time was 0.02 sec.

[0103] Data retrieval: MaxQuant software 1.5.5.1 was used to retrieve the raw data from the mass spectrometry test and the database, and finally the identification and quantitative analysis results of the peptides were obtained.

[0104] Experimental results: A sea cucumber flower octapeptide with a molecular weight of 918.02 Da and the function of promoting selenium absorption was identified. Its amino acid sequence is Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val. It was later verified that it contains potential selenium ion binding sites.

[0105] S4. Using molecular docking technology, a binding model of the interaction between sea cucumber flower octapeptide with selenium absorption-promoting effect and selenium ions was established to evaluate its potential for binding with selenium ions.

[0106] The structure of sea cucumber flower octapeptide with selenium absorption-promoting effects was constructed using ChemDraw Academic Edition. The structure of the sea cucumber flower octapeptide with selenium absorption-promoting effects and Se were then synthesized using Discovery Studio software. 2- Molecular docking was performed, and the results were optimized using PyMol 2.5, as shown in the following figure. Figure 1 As shown.

[0107] according to Figure 1 It can be seen that the Asp-4, Lys-5, and Leu-6 amino acids of the sea cucumber flower octapeptide with selenium absorption-promoting effect combine to form a selenium-O bond length between 3.1 and 4.3 kcal / mol in the sea cucumber flower octapeptide-selenium nanocomposite, with a binding energy of -5.77 kcal / mol. The docking conformation of the sea cucumber flower octapeptide with selenium absorption-promoting effect indicates that its possible binding method is the combination of selenium ions and the negatively charged carboxyl group (Asp-4). The sea cucumber flower octapeptide with selenium absorption-promoting effect forms three binding bonds with selenium ions, with selenium located in the middle of the sea cucumber flower octapeptide, indicating that the sea cucumber flower octapeptide with selenium absorption-promoting effect has a strong affinity for selenium ions.

[0108] S5. Using 2-chlorotrimethylbenzenechloro polymer resin, a solid-phase synthesis with amino acids was performed to obtain sea cucumber flower octapeptide with selenium absorption-promoting effects, followed by high-performance liquid chromatography purification. The carboxyl group of the C-terminal amino acid Val-8 (valine) was covalently linked to 2-chlorotrimethylbenzenechloropolymer resin and immobilized on a solid support. The protecting group of the Val-8 amino group was removed, and the carboxyl group of Ile-7 (isoleucine) was activated, causing it to undergo a condensation reaction with the Val-8 amino group to form a peptide bond. Subsequent amino acids were added sequentially, and the resin was excised using a cleavage reagent, removing the side chain protecting groups to obtain a sea cucumber flower octapeptide with selenium absorption-enhancing effects. The specific steps are as follows: (1) Calculate the weight of the raw material based on the weight of the sea cucumber flower octapeptide that promotes selenium absorption. Put 5g of 2-chlorotrimethylbenzenechloro polymer resin into a 150mL reactor and add 50mL of DCM to soak for 2h; (2) Wash the 2-chlorotrimethylbenzenechloro polymer resin with DMF and then dry it. Repeat this process 4 times to dry the 2-chlorotrimethylbenzenechloro polymer resin; (3) Weigh Fmoc-Val-OH, DCM and DIEA and add them to the reactor. The mass ratio of 2-chlorotrimethylbenzenechloro polymer resin to Fmoc-Val-OH is 1. :1.1, the mass-volume ratio of Fmoc-Val-OH and dichloromethane is 1g:10mL, the volume ratio of N,N-diisopropylethylamine and dichloromethane is 1:2, and the reactor is placed in a shaker at 30℃ for 2h; (4) it is sealed with methanol solution (the volume ratio of methanol, DIEA and DCM is 1:1:2) for 30min, then washed with DMF 4 times and dried; (5) a 20% volume concentration piperidine solution is added to the reactor, the mass ratio of piperidine solution and methanol in step (4) is 1:10, and the Fmoc protecting group is removed. After deprotection, wash with DMF 4 times and then dry; (6) Take a sample of 2-chlorotrimethylbenzyl chloride polymer resin and test it with ninhydrin. If the 2-chlorotrimethylbenzyl chloride polymer resin is colored, it indicates that the deprotection was successful; (7) Weigh Fmoc-Ile-OH, HOBt and DIC and add them to the reactor. The mass ratio of 2-chlorotrimethylbenzyl chloride polymer resin to Fmoc-Ile-OH is 4:1, the mass ratio of Fmoc-Ile-OH to 1-hydroxybenzotriazole is 1:1.1, and the mass ratio of 1-hydroxybenzotriazole to N,N'-diisopropylcarbodiimide is 1:1.25. React in a shaker at 30℃ for 1 h; (8) Take a sample of 2-chlorotrimethylbenzyl chloride polymer resin and test it with ninhydrin. If the 2-chlorotrimethylbenzyl chloride polymer resin is colored, it indicates that the condensation is incomplete and the reaction should continue. If the 2-chlorotrimethylbenzene chloride polymer resin is colorless, it indicates that the reaction is complete. After the reaction is complete, wash the 2-chlorotrimethylbenzene chloride polymer resin with DMF 4 times and then dry it. (9) Add a 20% volume concentration piperidine solution (the volume ratio of piperidine solution to DMF is 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the 2-chlorotrimethylbenzene chloride polymer resin.After deprotection, wash with DMF 4 times, then dry and check whether the protection has been removed. Take a sample of 2-chlorotrimethylbenzenechloro polymer resin and test it with ninhydrin method. If the 2-chlorotrimethylbenzenechloro polymer resin is colored, it means that the deprotection is successful. (10) Connect amino acids Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)-OH in sequence according to steps (7) to (9). Use a cleavage reagent (a mixed solution of trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.5) to remove all the polypeptide protecting groups. Then purify the cleaved 2-chlorotrimethylbenzenechloro polymer resin by high performance liquid chromatography.

[0109] (11) Purification was performed using high performance liquid chromatography. The column model was VYDAC-C18 with a size of 4.6×250mm. Mobile phase A was acetonitrile containing 0.1% (v / v) trifluoroacetic acid; mobile phase B was water containing 0.1% (v / v) trifluoroacetic acid. The elution conditions were as follows: 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); the flow rate was 1.0 mL / min, and the detection wavelength was 220 nm. After collecting the main peak fraction, the purified product was immediately placed in a 1.5 mL EP tube (volume not exceeding 400 μL), flash-frozen in liquid nitrogen for 8 min, pre-frozen at -80℃ for 135 min, freeze-dried at -30℃ for 2.5 h, freeze-dried at -20℃ and 15 Pa for 1.5 h, and freeze-dried at 15℃ and 15 Pa for 51 h, for a total freeze-drying time of 55 h, to obtain sea cucumber flower octapeptide with selenium absorption-promoting effects. The structure was identified by ESI-MS, and the results are as follows: Figure 2 As shown in Table 1.

[0110] Table 1. HPLC chromatographic peak values ​​of sea cucumber flower octapeptide with selenium absorption-promoting effects:

[0111] according to Figure 2 As can be seen from Table 1, the present invention synthesized sea cucumber flower octapeptide with selenium absorption-promoting effect through solid-phase synthesis technology; the purity of the sea cucumber flower octapeptide with selenium absorption-promoting effect prepared by the present invention is 98.698%, and the molecular weight is 918.02 Da, which meets the requirements for subsequent preparation.

[0112] S6. Sea cucumber flower octapeptide powder with selenium absorption-promoting effects was dissolved in Milli-Q water to obtain a 5 mg / mL solution of sea cucumber flower octapeptide with selenium absorption-promoting effects. The pH was adjusted to 9.0 using 1 mol / L hydrochloric acid and 1 mol / L NaOH solution. Subsequently, the sea cucumber flower octapeptide with selenium absorption-promoting effects and a 0.5 mol / L sodium selenite solution were mixed at a volume ratio of 2:1 and stirred for 20 min at 60℃ and pH 9. After the reaction was completed, ethanol with a final concentration of 95 wt% was added to the reactants. After standing for 1 h, the mixture was centrifuged at 8000 rpm for 5 min, and the precipitate was collected. The precipitate was freeze-dried at -70℃ and 2 Pa vacuum for 72 h to obtain the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects.

[0113] Test Example 1: The microstructure of the sea cucumber flower octapeptide with selenium absorption-promoting effect and the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect prepared in Example 1 were determined. The specific steps are as follows: Sea cucumber flower octapeptide and sea cucumber flower octapeptide-selenium nanocomposite, both exhibiting selenium absorption-promoting effects, were 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 magnified at different magnifications (1000x, 3500x, and 8000x) using a Zeiss Gemini 500 field emission scanning electron microscope to obtain SEM and EDS images, observing their microstructure. The results are as follows: Figure 3 and Figure 4 As shown.

[0114] according to Figure 3 As can be seen, the microstructure of the sea cucumber flower octapeptide with selenium absorption-promoting effect exhibits a smooth and dense sheet-like structure, while the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect has a rough and loose surface, exhibiting a wrinkled structure of nearly spherical particles. This may be because during the formation of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect, the interaction between the sea cucumber flower octapeptide and selenium disrupts its original dense surface structure. Simultaneously, some non-covalent interactions, such as hydrophobic interactions, electrostatic interactions, van der Waals forces, and aromatic π-π interactions, may induce the self-assembly behavior of the sea cucumber flower octapeptide with selenium absorption-promoting effect, thereby leading to the aggregation of the sea cucumber flower octapeptide with selenium absorption-promoting effect into a nanostructure.

[0115] Further testing using EDS-mapping revealed the internal distribution of chemical elements in the sea cucumber flower octapeptide and the sea cucumber flower octapeptide-selenium nanocomposite, both of which promote selenium absorption. The results are as follows: Figure 4 As shown.

[0116] according to Figure 4 As can be seen, the EDS-mapping pattern of the sea cucumber flower octapeptide with selenium absorption-promoting effect shows a weak Se signal, while the selenium metal signal of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect is stronger. Scanning electron microscopy images and EDS results confirm the successful preparation of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect.

[0117] Test Example 2: Fourier transform infrared spectroscopy analysis was performed on the sea cucumber flower octapeptide with selenium absorption-promoting effect prepared in Example 1 and the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect. The steps are as follows: Thoroughly dry the sample to be tested. Accurately weigh 2 mg of the sample and mix it with the dried KBr. Grind evenly and then compress into tablets. Instrumental testing: Set the scanning range to 4000~400 cm⁻¹. -1 The resolution is 4cm. -1 The number of scans was 64. The potential interaction between sea cucumber flower octapeptide (which promotes selenium absorption) and selenium nanoparticles was analyzed by observing peak shifts, the appearance of new peaks, or the disappearance or weakening of existing peaks. The test results are as follows: Figure 5 As shown.

[0118] according to Figure 5 As can be seen from 'a' in the image, some characteristic peaks in the infrared spectrum of sea cucumber flower octapeptide, which promotes selenium absorption, changed before and after binding with selenium ions. The sea cucumber flower octapeptide with selenium absorption-promoting effect showed a change at 3448.46 cm⁻¹. -1 The absorption peaks at this point are for -OH and NH, but shift to 3440.56 cm⁻¹ after binding with selenium ions. -1 This suggests that the binding of selenium ions may be related to hydroxyl and amino groups; amide I band (1630~1690cm) -1 A redshift occurred, from 1632.66 cm. -1 Offset to 1601.05cm -1 This is related to the C=O stretching vibration. After sea cucumber flower octapeptide, which promotes selenium absorption, binds to selenium ions, its characteristic peak changes from 1352.94 cm⁻¹. -1 It changed to 1357.68cm -1 This is caused by the out-of-plane bending vibration of the CH plane; the CH out-of-plane bending vibration region (1000~650cm) on the octapeptide spectrum of sea cucumber flower, which has the effect of promoting selenium absorption. -1 The length also changes; after combining with selenium ions, it increases from 777.69 cm. -1 The location has moved to 772.95cm. -1 The above results indicate that the sea cucumber flower octapeptide, which promotes selenium absorption, mainly binds to selenium ions through the carboxyl and amino groups on its peptide chain.

[0119] according to Figure 5 As shown in b, after selenium ions bind to the sea cucumber flower octapeptide with selenium absorption-promoting effects, the content of α-helix and random coil structures significantly decreases (p<0.05), while the content of β-sheet and β-turn structures increases. These results indicate that the α-helix and random coil structures of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects are transformed into β-sheet and β-turn structures, exhibiting a more ordered structure. Previous studies have shown that when peptides bind to mineral ions, hydrophobic groups may be exposed, leading to the disruption of intramolecular hydrogen bonds and a reduction in α-helices. Therefore, the more ordered structure of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects may be attributed to the folding of the sea cucumber flower octapeptide with selenium absorption-promoting effects induced by selenium ion interactions, and the stabilization of its folded conformation.

[0120] Test Example 3: The gastrointestinal stability of sea cucumber flower octapeptide, which promotes selenium absorption, was determined using RP-HPLC, as follows: (1) Sea cucumber flower octapeptide with selenium absorption-promoting effect and sea cucumber flower octapeptide-selenium nanocomplex with selenium absorption-promoting effect simulate gastrointestinal digestion: The sea cucumber flower octapeptide-selenium nanocomposite, which promotes selenium absorption, was incubated in a 37°C 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 of digestion. After digestion, 2 mL of the simulated gastric digestion product was collected, and the enzyme was inactivated in a 100°C water bath for 5 min. The mixture was then centrifuged at 10000g and 4°C for 10 min, and the supernatant was collected. Subsequently, the pH of the supernatant was adjusted to 7.5, and simulated intestinal digestion fluid was added. The mixture was then subjected to simulated intestinal digestion at 37°C 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°C water bath for 5 min. The mixture was then centrifuged at 10000g and 4°C for 10 min, and the supernatant was collected for subsequent tests.

[0121] (2) The gastrointestinal stability of sea cucumber flower octapeptide, which has the effect of promoting selenium absorption, was analyzed by RP-HPLC: Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect the changes in the content of sea cucumber flower octapeptide (which promotes selenium absorption) and sea cucumber flower octapeptide-selenium nanocomposite (which promotes selenium absorption) during simulated digestion. The supernatant obtained in step (1) was filtered through a 0.22 μm filter and placed in a liquid chromatography sample vial 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 min, 10–35% (v / v) mobile phase B; 25.0–35 min, 35% (v / v) 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.

[0122] Table 2. Peptide retention rates of sea cucumber flower octapeptide with selenium absorption-promoting effects and sea cucumber flower octapeptide-selenium nanocomplex after simulated gastrointestinal digestion:

[0123] according to Figure 6 It can be seen that the sea cucumber flower octapeptide with selenium absorption-promoting effect eluted at around 16.2 min, and only one elution peak was observed for this product. This indicates that the sea cucumber flower octapeptide with selenium absorption-promoting effect remained relatively stable after gastrointestinal digestion. Table 2 shows the retention rates of the sea cucumber flower octapeptide with selenium absorption-promoting effect in various digestion stages within the sea cucumber flower octapeptide-selenium nanocomposite and the sea cucumber flower octapeptide-selenium nanocomposite. Table 2 shows that the sea cucumber flower octapeptide with selenium absorption-promoting effect underwent a certain degree of degradation during simulated gastrointestinal digestion. In the gastric digestion stage, the peptide retention rate was 83.96% ± 0.14%, and after further intestinal digestion, the peptide retention rate was only about 60.40% ± 0.23%. After the addition of inorganic selenium, its resistance to enzymatic digestion was improved; the peptide retention rate was above 91.08% ± 0.30% in the gastric digestion stage and reached 86.37% ± 0.17% in the intestinal digestion stage. This may be because after selenium ions bind to the sea cucumber flower octapeptide that promotes selenium absorption, the spatial structure of the peptide chain of the sea cucumber flower octapeptide that promotes selenium absorption is altered. Selenium ions induce the sea cucumber flower octapeptide that promotes selenium absorption to fold, and the enzyme cleavage site is hidden, thereby reducing the probability of being digested and decomposed.

[0124] Test Example 4: The digestate of the sea cucumber flower octapeptide-selenium nanocomplex, which has the effect of promoting selenium absorption, was identified by mass spectrometry using Nano-LC-ESI-MS / MS, as follows: (1) Sample preparation: Add 1 mg of sea cucumber flower octapeptide dry powder with selenium-enhancing effects or 100 μL of sea cucumber flower octapeptide solution with selenium-enhancing effects (6 mg / mL) to 100 μL of 0.1% (v / v) trifluoroacetic acid (TFA) aqueous solution, sonicate to homogenize, centrifuge at 14000g for 10 minutes, collect the supernatant, and ultrafilter the obtained supernatant using a 10KD ultrafiltration tube (PALL, OD010C35), centrifuge at 13500g for 10 minutes, and use a nanodrop 2000C UV-Vis spectrophotometer to quantify peptides. 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% (v / v) TFA, load the ultrafiltered sample solution, and elute the peptides with 70% (v / v) acetonitrile aqueous solution.

[0125] (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 formic acid-acetonitrile aqueous solution with a formic acid concentration of 0.1% (v / v) and an acetonitrile concentration of 84% (v / v). The column was equilibrated with 95% (v / v) of 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 (Thermoscientific 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, the linear gradient of liquid B is 35~100% (volume percentage); 58~60 min, liquid B is maintained at 100% (volume percentage).

[0126] (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 resolution was 70,000 at m / z 200, 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-charge ratio of peptides and peptide fragments was acquired using the following method: 10 fragment spectra were acquired after each full scan (MS2 scan), the secondary mass spectrometry activation mode was high-energy collisional dissociation (HCD), the isolation window was 2 m / z, the secondary mass spectrometry resolution was 17,500 at a mass-charge ratio of 200, the normalized collision energy was 30 eV, and the underflow ratio was 0.1%.

[0127] (4) Database retrieval: The mass spectrometry test raw file was retrieved from the corresponding database using MaxQuant software 1.5.5.1, and the results are as follows: Figures 7-11 As shown in Tables 3 to 7.

[0128] Table 3. Identification of gastrointestinal digestive products of sea cucumber flower octapeptide-selenium nanocomplex with selenium absorption-promoting effects:

[0129] Table 4 Figure 8 Summary of structured data in:

[0130] Table 5 Figure 9 Summary of structured data in:

[0131] Table 6 Figure 10 Summary of structured data in:

[0132] Table 7 Figure 11 Summary of structured data in:

[0133] according to Figures 7-11As shown in Tables 3-7, the initial SEDDKLIV peptide sequence was also identified during simulated gastrointestinal digestion and partially remained stable. Furthermore, peptide sequences such as EDDKLIV, SEDDKL, and DDKLIV were also identified. Based on these results, this invention discovers that the sea cucumber flower octapeptide (SEDDKLIV), which promotes selenium absorption, possesses a certain degree of digestive stability. Despite partial degradation, the polypeptide fragments generated from simulated gastrointestinal digestion still contain aspartic acid or leucine residues and have the potential to bind to selenium ions.

[0134] Test Example 5: This test case demonstrates selenium transport assays using an in vitro Caco-2 cell monolayer model. The assay method is as follows: Caco-2 cells were cultured in Eagle's MEM medium containing 20% ​​(v / v) fetal bovine serum and 1% (v / v) a mixture of penicillin and streptomycin antibiotics at 37°C, 5% (v / v) 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 ), 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.

[0135] 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 then incubated at 37°C in 5% (v / v) 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. Selenium content was determined using hydride generation-atomic fluorescence spectrometry, and selenium uptake was calculated. Results are as follows: Figure 12 As shown.

[0136] according to Figure 12It can be seen that both the digests of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects and the digests of Na2SeO3 exhibit certain selenium transport capabilities. Furthermore, at different transport time points, the selenium transport effect of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects is significantly better than that of the digested Na2SeO3. Moreover, the amount of selenium transported increases with time, reaching a maximum at 120 min (112.05 ± 3.55 μg / well). In summary, compared with the Na2SeO3 simulated gastrointestinal digestion products, the digests of the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects provided by this invention have a good selenium absorption-promoting effect and can effectively promote the absorption of selenium by small intestinal epithelial cells. The sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effects provided by this invention can maintain the solubility of selenium in the gastrointestinal environment and effectively promote the absorption of selenium by Caco-2 cells.

[0137] 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 octapeptide with selenium absorption-promoting effects, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing a sea cucumber flower octapeptide with selenium absorption-promoting effect, wherein the sea cucumber flower octapeptide with selenium absorption-promoting effect is the sea cucumber flower octapeptide with selenium absorption-promoting effect described in claim 1, characterized in that, The process includes the following steps: following the amino acid sequence Ser-Glu-Asp-Asp-Lys-Leu-Ile-Val, polymer resin is sequentially condensed with Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH and Fmoc-Ser(tBu)-OH to obtain the sea cucumber flower octapeptide with selenium absorption-promoting effect.

3. The application of a sea cucumber flower octapeptide with selenium absorption-promoting effect in the preparation of selenium carrier products, characterized in that, The sea cucumber flower octapeptide with selenium absorption-promoting effect is the sea cucumber flower octapeptide with selenium absorption-promoting effect described in claim 1 or the sea cucumber flower octapeptide with selenium absorption-promoting effect obtained by the preparation method described in claim 2.

4. A sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect, characterized in that, The product includes a sea cucumber flower octapeptide with selenium absorption-promoting effects, and selenium ions bound to the Asp-4 amino acid of the sea cucumber flower octapeptide with selenium absorption-promoting effects; the sea cucumber flower octapeptide with selenium absorption-promoting effects is the sea cucumber flower octapeptide with selenium absorption-promoting effects described in claim 1 or the sea cucumber flower octapeptide with selenium absorption-promoting effects obtained by the preparation method described in claim 2; the selenium ions are divalent selenium ions.

5. A method for preparing a sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect, wherein the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect is the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect as described in claim 4, characterized in that... Includes the following steps: Sea cucumber flower octapeptide with selenium absorption-promoting effect, selenium salt solution and water are mixed and reacted to obtain the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect; the selenium salt solution is sodium selenite solution.

6. The method for preparing the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect according to claim 5, characterized in that, The concentration of the selenium salt solution is 0.45~0.55 mol / L.

7. The method for preparing the sea cucumber flower octapeptide-selenium nanocomposite with selenium absorption-promoting effect according to claim 5, characterized in that, The reaction temperature is 55~60℃, and the holding time is 18~25min; The reaction is carried out under alkaline conditions, wherein the pH value of the alkaline conditions is in the range of 8.5 to 9.

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