Sea cucumber flower self-assembled polypeptide-selenium nano-carrier with antioxidant activity as well as preparation method and application of sea cucumber flower self-assembled polypeptide-selenium nano-carrier

Abstract

The invention discloses a sea cucumber flower self-assembled polypeptide-selenium nano-carrier with antioxidant activity and a preparation method and application thereof, belongs to the technical field of functional protein peptides, and particularly relates to a preparation method of the sea cucumber flower self-assembled polypeptide-selenium nano-carrier with antioxidant activity, which comprises the following steps: heating a sea cucumber flower protein solution; the preparation method comprises the following steps: mixing sea cucumber with simulated gastric juice, reacting for 30-150 minutes, then adding simulated intestinal juice, reacting for 20-120 minutes, carrying out enzyme deactivation treatment, centrifuging, taking supernate, and freeze-drying to obtain sea cucumber flower self-assembled polypeptide; and mixing the sea cucumber flower self-assembled polypeptide with deionized water to prepare a sea cucumber flower self-assembled polypeptide solution, mixing the sea cucumber flower self-assembled polypeptide solution with a sodium selenite solution and a phosphate buffer solution, reacting for 20-60 minutes, washing with an ethanol solution, centrifugally collecting precipitate, and freeze-drying to obtain the sea cucumber flower self-assembled polypeptide-selenium nano-carrier. The sea cucumber flower self-assembled polypeptide-selenium nano-carrier prepared by the invention has good antioxidant activity and potential of effectively delivering selenium to an organism.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional protein peptides, and in particular relates to a sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity, and a preparation method and application thereof. Background Art

[0002] Currently, the sea cucumber processing industry focuses primarily on the development of the sea cucumber body wall, leading to the disposal of byproducts such as sea cucumber viscera and cooking liquid. Research has shown that the nutrient content of sea cucumber byproducts is comparable to that of the sea cucumber body wall, and some even contain higher levels of active ingredients than the body wall. Sea cucumber flower, a byproduct of the sea cucumber processing process, is rich in protein and a good source of functional protein peptides, possessing significant development value.

[0003] Selenium (Se) is a trace element that the human body must ingest from the outside world. It has physiological functions such as antioxidant, anti-tumor, and immunomodulatory properties, and plays a vital role in maintaining human life. Selenium deficiency may lead to certain types of antioxidant stress disorders. Currently, common selenium supplements are mainly divided into two categories: inorganic selenium and organic selenium. Although inorganic selenium has certain antioxidant activity, its high toxicity and low bioavailability limit its scope of application. In contrast, organic selenium has more advantages. It includes organic selenium preparations, natural products produced in selenium-rich areas, and microbial products of artificial biotransformation, such as selenomethionine, selenium-enriched eggs, selenium-enriched yeast, and selenium-enriched spirulina. Organic selenium not only enhances the stability and bioavailability of selenium ions but also significantly improves their antioxidant activity. It can more effectively scavenge free radicals, reduce oxidative damage, and is safer, showing greater potential and application value in the field of antioxidants.

[0004] In order to meet the above challenges, the development of organic selenium supplements is expected to occupy an important position in the future development of selenium supplement preparations. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity and its preparation method and application. The present invention prepares a sea cucumber flower polypeptide with self-assembling properties by simulating the gastrointestinal digestive system in vitro, and further reacts with inorganic selenium to generate a sea cucumber flower self-assembling polypeptide-selenium nanocarrier (HSHP GI -Se), the nanocarrier has good antioxidant activity and the potential to effectively deliver selenium to the body, and can be used in the fields of selenium-fortified foods, functional health products, etc.

[0006] To achieve the above object, the present invention provides a method for preparing a sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity, comprising the following steps:

[0007] 1) heating the sea cucumber flower protein aqueous solution, then mixing it with simulated gastric fluid, and performing a first reaction for 30 to 150 minutes. After the first reaction, adding simulated intestinal fluid, and performing a second reaction for 20 to 120 minutes. After the second reaction, adjusting the pH to 7.0, inactivating the enzyme, centrifuging, collecting the supernatant, and freeze-drying to obtain the sea cucumber flower self-assembling polypeptide;

[0008] 2) The sea cucumber flower self-assembling polypeptide obtained in step 1) is mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution, the sea cucumber flower self-assembling polypeptide solution, sodium selenite aqueous solution and phosphate buffer are mixed, and a third reaction is carried out for 20 to 60 minutes, followed by washing with ethanol aqueous solution, collecting the precipitate by centrifugation, and freeze-drying to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier.

[0009] Preferably, the concentration of the sea cucumber flower protein aqueous solution in step 1) is 10-50 mg / mL, and the sea cucumber flower protein aqueous solution is prepared by dissolving sea cucumber flower protein powder in water; the heating temperature in step 1) is 30-40° C., and the heating time is 10-30 min.

[0010] Further preferably, the preparation method of the sea cucumber flower protein powder is specifically as follows: sea cucumber flowers and deionized water are mixed at a ratio of 1g:0.8~2.0mL, homogenized at 8000~10000rpm for 1~3min to obtain sea cucumber flower homogenate, first vacuum freeze-dried to obtain sea cucumber flower freeze-dried powder, sea cucumber flower freeze-dried powder and water are mixed at a ratio of 1g:8~12mL to obtain the solution to be extracted, the pH of the solution to be extracted is adjusted to 8.5~9.0, stirred at 38~45℃ for 3~4h, then centrifuged for the first time to collect the supernatant, adjusted the pH of the supernatant to 4.0~6.5, then centrifuged for the second time to collect the precipitate, washed with distilled water, and vacuum freeze-dried for the second time to obtain sea cucumber flower protein powder.

[0011] Still further preferably, the temperature of the first vacuum freeze-drying is -50 to -70°C, the vacuum degree is 1 to 20 Pa, and the time is 48 to 72 hours; a 3 mol / L NaOH aqueous solution is used to adjust the pH of the solution to be extracted; the speed of the first centrifugation is 3500 r / min, and the time of the first centrifugation is 10 minutes; a 3 mol / L HCl aqueous solution is used to adjust the pH of the supernatant; the speed of the second centrifugation is 3500 r / min, and the time of the second centrifugation is 10 minutes; the temperature of the second vacuum freeze-drying is -50 to -70°C, the vacuum degree is 1 to 20 Pa, and the time is 48 to 72 hours.

[0012] Preferably, the volume ratio of the simulated gastric fluid and the sea cucumber flower protein aqueous solution in step 1) is 1 to 2:10; the simulated gastric fluid in step 1) is prepared by mixing a gastric electrolyte solution and 2000U / mL of pepsin in a volume ratio of 1:1, and the gastric electrolyte solution is 6.9mL with a concentration of 6.9mmoL -1 0.9 mL of KCl aqueous solution with a concentration of 0.9 mmoL -1 12.5mL of KH2PO4 aqueous solution with a concentration of 25mmoL -1 11.8 mL of NaHCO3 aqueous solution has a concentration of 47.2 mmol / L -1 0.4mL of NaCl aqueous solution with a concentration of 0.1mmoL -1 MgCl2(H2O)6 aqueous solution, 0.5mL concentration of 0.5mmoL -1 of (NH4)2CO3 aqueous solution and 1.3mL of a 15.6mmoL -1 The HCl aqueous solution is dissolved in 400 mL of distilled water to prepare the obtained solution; in step 1), the pH of the first reaction is 2.0 to 5.0, the temperature of the first reaction is 30 to 40° C., the speed of the first reaction is 200 to 400 rpm, and 1 M HCl solution is used to maintain pH stability during the first reaction.

[0013] Preferably, the volume ratio of the simulated intestinal fluid and the sea cucumber flower protein aqueous solution in step 1) is calculated as 1 to 2:10; the simulated intestinal fluid in step 1) is a mixture of an intestinal electrolyte solution and 100U / mL pancreatic enzyme in a volume ratio of 1:1 to obtain a premixed intestinal fluid, and then the premixed intestinal fluid is mixed with bile with a concentration of 160mmol / L in a volume ratio of 4:0.5 to 1.0 to prepare the intestinal electrolyte solution, which is 6.8mL with a concentration of 6.8mmoL -1 0.8 mL of KCl aqueous solution with a concentration of 0.8 mmoL -1 KH2PO4 aqueous solution, 42.5mL concentration of 85mmoL -1 NaHCO3 aqueous solution, 9.6mL concentration of 38.4mmoL -1 1.1mL of NaCl aqueous solution with a concentration of 0.33mmoL -1 MgCl2(H2O)6 aqueous solution and 0.7mL of 8.4mmoL -1 The obtained solution is prepared by dissolving an aqueous solution of HCl in 400 mL of distilled water; in step 1), the pH of the second reaction is 7.0 to 9.0, the temperature of the second reaction is 30 to 40° C., the speed of the second reaction is 200 to 400 rpm, and 1 M NaOH solution is used to maintain pH stability during the second reaction.

[0014] Preferably, the enzyme inactivation treatment in step 1) is specifically: 95-100°C water bath for 10-15 minutes; the centrifugation temperature in step 1) is 4°C, the centrifugation speed is 8000 r / min, and the centrifugation time is 10 minutes.

[0015] Preferably, the freeze-drying temperature in step 1) is -50 to -70°C, the vacuum degree is 1 to 20 Pa, and the time is 48 to 72 hours.

[0016] Preferably, the particle size of the sea cucumber flower self-assembling polypeptide in step 1) is 80-100 mesh.

[0017] Preferably, the volume ratio of the sea cucumber flower self-assembling polypeptide solution, sodium selenite aqueous solution and phosphate buffer mixed in step 2) is 2-3:1:1, the concentration of the sea cucumber flower self-assembling polypeptide solution is 10-60 mg / mL, the molar concentration of the sodium selenite aqueous solution is 0.5-1 M, and the pH of the phosphate buffer is 7.0; the pH of the third reaction in step 2) is 7.0-9.0, and the temperature of the third reaction is 30-60°C.

[0018] Preferably, the volume concentration of the ethanol aqueous solution in step 2) is 85%, and the number of washing times of the ethanol aqueous solution in step 2) is 3 times to remove unbound polypeptides and free selenium ions; the centrifugal temperature in step 2) is 4°C, the centrifugal force of the centrifugation is 12000g, and the centrifugation time is 5min.

[0019] Preferably, the freeze-drying in step 2) is specifically as follows: pre-cooling at -40 to -10°C for 2 to 3 hours, freezing at -40 to -10°C and 0 to 20 Pa for 1 to 2 hours, and freeze-drying at 0 to 25°C and 0 to 20 Pa until completely dry.

[0020] Preferably, the average particle size of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier in step 2) is 221±8.56 nm, and the average potential of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier is -23.22±1.89 mV.

[0021] The present invention also provides the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared by the preparation method.

[0022] The present invention also provides the use of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared by the preparation method in preparing a product having antioxidant activity and capable of effectively delivering selenium.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity prepared by the present invention has good antioxidant activity and the potential to effectively deliver selenium to the body. It can effectively enhance the stability of selenium ions and provide new ideas for the development of selenium-fortified foods, functional health products and other fields.

[0025] The sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity prepared by the present invention has a selenium binding activity of 317.15±5.75 μg / mg and has good selenium binding capacity.

[0026] In the sea cucumber flower self-assembly polypeptide-selenium nanocarrier with antioxidant activity prepared by the invention, the proportion of the molecules with molecular weight less than 3000 Da is more than 70%.

[0027] The sea cucumber flower self-assembly polypeptide-selenium nanocarrier with antioxidant activity prepared by the present invention has excellent antioxidant activity and digestion stability, and its DPPH free radical scavenging rate is as high as 89.65%, and ABTS + The free radical scavenging rate reaches 81.82%; in the process of simulating gastrointestinal digestion by sea cucumber flower self-assembled polypeptide-selenium nanocarrier, the selenium dialysis rate of intestinal digestion can be as high as 23.37±1.03%. It has good digestive stability, can effectively deliver selenium elements, and can be widely used in selenium-fortified foods and functional health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The effect of sea cucumber flower protein prepared in Example 1 before simulated digestion, gastric digestion end point and intestinal digestion end point on selenium binding capacity and hydrolysis degree. In the figure, HSHP represents before simulated digestion, HSHP represents G Stands for gastric digestion end point, HSHP GI represents intestinal digestion endpoint, a, b, and c represent significance analysis;

[0030] Figure 2 The UV spectra, fluorescence spectra, FT-IR spectra and circular dichroism spectra of the sea cucumber flower self-assembling polypeptide prepared in Example 1 and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared with different ratios of sea cucumber flower self-assembling polypeptide solution and Na2SeO3 aqueous solution, wherein A is the UV spectrum, B is the fluorescence spectrum, C is the FT-IR spectrum, D is the circular dichroism spectrum, HSHP in the figure GIThe sea cucumber flower self-assembling polypeptide prepared in Example 1, HSHP GI +50mM Na2SeO3, HSHP GI +100mMNa2SeO3, HSHP GI +200mM Na2SeO3, HSHP GI +300mM Na2SeO3, HSHP GI +400mM Na2SeO3 and HSHP GI +500mM Na2SeO3 represents the sea cucumber flower self-assembled polypeptide-selenium nanocarrier prepared with different ratios of sea cucumber flower self-assembled polypeptide solution and Na2SeO3 aqueous solution, HSHP GI -Se represents the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1;

[0031] Figure 3 The microstructure of the sea cucumber flower self-assembling polypeptide and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, wherein a is the HSHP under the magnification of 500× and 1000× respectively. GI b is the scanning electron microscope image of HSHP at magnifications of 500× and 1000×. GI -Se scanning electron microscopy image, c is HSHP at 5000× magnification. GI Elemental composition analysis diagram, d is HSHP at 5000× magnification GI -Se element composition analysis chart;

[0032] Figure 4 The particle size, potential and relative molecular weight distribution analysis results of the sea cucumber flower self-assembling polypeptide and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, wherein A is the particle size distribution analysis result diagram, B is the potential distribution analysis result diagram, and C is the relative molecular weight distribution analysis result diagram. GI The sea cucumber flower self-assembling polypeptide prepared in Example 1, HSHP GI -Se represents the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, and "*" represents significance analysis;

[0033] Figure 5 The antioxidant activity test results of the sea cucumber flower self-assembling polypeptide and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1 and Comparative Example 1, wherein A is the DPPH free radical scavenging rate of the sea cucumber flower self-assembling polypeptide and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, and B is the ABTS free radical scavenging rate of the sea cucumber flower self-assembling polypeptide and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1. +The scavenging rate is shown in Figure 1. C is a comparison of the DPPH free radical scavenging rate of the sea cucumber flower self-assembling polypeptide prepared in Example 1 and Comparative Example 1. D is a comparison of the DPPH free radical scavenging rate of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1 and Comparative Example 1. E is a comparison of the DPPH free radical scavenging rate of the sea cucumber flower self-assembling polypeptide ABTS prepared in Example 1 and Comparative Example 1. + Comparison of clearance rates, F is the sea cucumber flower self-assembling polypeptide-selenium nanocarrier ABTS prepared in Example 1 and Comparative Example 1 + Clearance comparison chart, HSHP GI The sea cucumber flower self-assembling polypeptide prepared in Example 1, the comparative example HSHP GI The sea cucumber flower self-assembling polypeptide prepared in Comparative Example 1, HSHP GI -Se represents the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, and the comparative example HSHP GI -Se represents the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in comparative example 1, and "*" represents significance analysis;

[0034] Figure 6 The results of the digestion stability test of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier and Na2SeO3 prepared in Example 1 are shown in FIG. GI -Se represents the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Example 1, and "*" represents significance analysis;

[0035] Figure 7 HSHP prepared in Example 1 detected by LC-MS / MS GI Peptide chain length distribution diagram and peptide molecular weight distribution diagram, where A is the peptide chain length distribution diagram and B is the peptide molecular weight distribution diagram. GI The sea cucumber flower self-assembling polypeptide prepared in Example 1. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The sources of the materials used in the present invention: phosphate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sea cucumber flowers were sourced from Shandong Yantai Huakang Biomedicine Technology Co., Ltd.

[0042] Example 1

[0043] The cleaned sea cucumber flowers were mixed with deionized water at a ratio of 1 g:2 mL, homogenized at 8000 rpm for 3 min to obtain a sea cucumber flower homogenate, and the mixture was first vacuum freeze-dried for 72 h (the cold trap temperature was set to -55 ° C and the vacuum degree was 10 Pa) to obtain sea cucumber flower freeze-dried powder. The sea cucumber flower freeze-dried powder was mixed with water at a ratio of 1 g:10 mL to obtain an extraction solution. The pH of the extraction solution was adjusted to 9.0 with a 3 mol / L NaOH aqueous solution, and magnetically stirred at 40 ° C for 3.5 h. Then, the supernatant was collected by first centrifugation at a speed of 3500 r / min for 10 min, and the pH of the supernatant was adjusted to 4.8 with a 3 mol / L HCl aqueous solution. The precipitate was collected by a second centrifugation at a speed of 3500 r / min for 10 min, washed with distilled water, and second vacuum freeze-dried for 72 h (the cold trap temperature was set to -55 ° C and the vacuum degree was 10 Pa) to obtain sea cucumber flower protein (HSH) powder.

[0044] 1) Sea cucumber flower protein powder was dissolved in water to prepare a sea cucumber flower protein aqueous solution with a concentration of 30 mg / mL. The sea cucumber flower protein aqueous solution was heated at 35°C for 20 min and then mixed with simulated gastric fluid (the volume ratio of simulated gastric fluid to sea cucumber flower protein aqueous solution was 1:10). 1M HCl was used to adjust the pH to 2.0, the temperature was 37°C, and the speed was 300 rpm. The first reaction lasted 120 min. During the first reaction, 1M HCl was used to maintain pH stability. After the first reaction, simulated intestinal fluid was added (the amount of simulated intestinal fluid added was calculated based on the volume ratio of simulated intestinal fluid to sea cucumber flower protein aqueous solution being 1:10). 1M NaOH was used to adjust the pH to 7.0, the temperature was 37°C, and the speed was 300 rpm. The second reaction lasted 120 min. During the second reaction, 1M NaOH was added to keep the pH stable. After the second reaction, the pH was adjusted to 7.0, and the enzyme was inactivated (100°C water bath for 15 min). The supernatant was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was freeze-dried for 72 h (the cold trap temperature was set to -55°C and the vacuum degree was 10 Pa). The sea cucumber flower self-assembling polypeptide (HSHP) was obtained. GI ), the particle size is in the range of 80-100 mesh, and it is stored at -20℃ for future use.

[0045] Gastric electrolyte solution (SGF): 6.9 mL KCl (6.9 mmoL -1 ) aqueous solution, 0.9mL KH2PO4 (0.9mmoL -1 ) aqueous solution, 12.5mLNaHCO3(25mmoL -1 ) aqueous solution, 11.8mLNaCl (47.2mmoL -1 ) aqueous solution, 0.4mL MgCl2(H2O)6(0.1mmoL -1 ) aqueous solution, 0.5mL (NH4) 2CO3 (0.5mmoL -1 ) aqueous solution and 1.3 mL HCl (15.6 mmol -1 ) aqueous solution was dissolved in 400 mL of distilled water to prepare a gastric electrolyte solution.

[0046] Intestinal electrolyte solution (SIF): 6.8 mL KCl (6.8 mmol -1 ) aqueous solution, 0.8mLKH2PO4(0.8mmoL -1 ) aqueous solution, 42.5mLNaHCO3(85mmoL -1 ) aqueous solution, 9.6mLNaCl(38.4mmoL -1 ) aqueous solution, 1.1mL MgCl2(H2O)6(0.33mmoL -1 ) aqueous solution and 0.7 mL HCl (8.4 mmol -1) aqueous solution was dissolved in 400 mL of distilled water to prepare an intestinal electrolyte solution.

[0047] Simulated gastric fluid: SGF and pepsin (2000 U / mL) were mixed in a volume ratio of 1:1.

[0048] Simulated intestinal fluid: SIF and pancreatic enzyme (100 U / mL) were mixed at a volume ratio of 1:1 to obtain premixed intestinal fluid, which was then mixed with bile having a concentration of 160 mmol / L at a volume ratio of 4:1 to prepare the obtained fluid.

[0049] 2) The sea cucumber flower self-assembling polypeptide was mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution with a concentration of 50 mg / mL, and 100 mL of the sea cucumber flower self-assembling polypeptide solution, 50 mL of a 0.5 M sodium selenite (Na2SeO3) aqueous solution and 50 mL of a phosphate buffer solution with a pH of 7.0 were mixed, the pH was 8.0, and the temperature was continuously stirred at 30 to 60° C. for a third reaction for 30 min, and then washed three times with an 85% volume concentration of ethanol aqueous solution, centrifuged at 4° C. and 12000 g for 5 min, the precipitate was collected, and freeze-dried (specifically: pre-cooling at -40 to -10° C. for 2 h, freezing at -40 to -10° C. and 0 to 20 Pa for 2 h, and freeze-dried at 0 to 25° C. and 0 to 20 Pa until completely dry) to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier (HSHP GI -Se).

[0050] Example 2

[0051] The cleaned sea cucumber flowers were mixed with deionized water at a ratio of 1 g:0.8 mL, homogenized at 8000 rpm for 1 min to obtain a sea cucumber flower homogenate, and the mixture was first vacuum freeze-dried for 48 h (the cold trap temperature was set to -50 ° C and the vacuum degree was 1 Pa) to obtain sea cucumber flower freeze-dried powder. The sea cucumber flower freeze-dried powder was mixed with water at a ratio of 1 g:8 mL to obtain an extraction solution. The pH of the extraction solution was adjusted to 8.5 with a 3 mol / L NaOH aqueous solution, and magnetically stirred at 38 ° C for 3 h. The supernatant was collected by the first centrifugation at a speed of 3500 r / min for 10 min, and the pH of the supernatant was adjusted to 4.0 with a 3 mol / L HCl aqueous solution. The precipitate was collected by the second centrifugation at a speed of 3500 r / min for 10 min, washed with distilled water, and second vacuum freeze-dried for 48 h (the cold trap temperature was set to -50 ° C and the vacuum degree was 1 Pa) to obtain sea cucumber flower protein powder.

[0052] 1) Sea cucumber flower protein powder was dissolved in water to prepare a sea cucumber flower protein aqueous solution with a concentration of 10 mg / mL. The sea cucumber flower protein aqueous solution was heated at 30°C for 10 min and then mixed with simulated gastric fluid (the volume ratio of simulated gastric fluid to sea cucumber flower protein aqueous solution was 1:10). 1M HCl was used to adjust the pH to 2.0, the temperature was 30°C, and the speed was 200 rpm. The first reaction was carried out for 30 min. 1M HCl was used to maintain pH stability during the first reaction. After the first reaction, simulated intestinal fluid was added (the amount of simulated intestinal fluid added was calculated based on the volume ratio of simulated intestinal fluid to sea cucumber flower protein aqueous solution being 1:10). 1M NaOH was used to adjust the pH to 7.0, the temperature was 30°C, and the speed was 200 rpm. The second reaction was carried out for 20 min. 1M HCl was used during the second reaction. NaOH was used to maintain pH stability. After the second reaction, the pH was adjusted to 7.0, and the enzyme was inactivated (95°C water bath for 15 min). The mixture was centrifuged at 8000 r / min at 4°C for 10 min, and the supernatant was taken and freeze-dried for 48 h (the cold trap temperature was set at -50°C and the vacuum degree was 1 Pa). The sea cucumber flower self-assembling polypeptide was obtained, and its particle size was in the range of 80 to 100 meshes. The polypeptide was stored at -20°C for use.

[0053] Gastric electrolyte solution (SGF): 6.9 mL KCl (6.9 mmoL -1 ) aqueous solution, 0.9mL KH2PO4 (0.9mmoL -1 ) aqueous solution, 12.5mLNaHCO3(25mmoL -1 ) aqueous solution, 11.8mLNaCl (47.2mmoL -1 ) aqueous solution, 0.4mL MgCl2(H2O)6(0.1mmoL -1 ) aqueous solution, 0.5mL (NH4) 2CO3 (0.5mmoL -1 ) aqueous solution and 1.3 mL HCl (15.6 mmol -1 ) aqueous solution was dissolved in 400 mL of distilled water to prepare a gastric electrolyte solution.

[0054] Intestinal electrolyte solution (SIF): 6.8 mL KCl (6.8 mmol -1 ) aqueous solution, 0.8mLKH2PO4(0.8mmoL -1 ) aqueous solution, 42.5mLNaHCO3(85mmoL -1 ) aqueous solution, 9.6mLNaCl(38.4mmoL -1 ) aqueous solution, 1.1mL MgCl2(H2O)6(0.33mmoL -1 ) aqueous solution and 0.7 mL HCl (8.4 mmol -1 ) aqueous solution was dissolved in 400 mL of distilled water to prepare an intestinal electrolyte solution.

[0055] Simulated gastric fluid: SGF and pepsin (2000 U / mL) were mixed in a volume ratio of 1:1.

[0056] Simulated intestinal fluid: SIF and pancreatic enzyme (100 U / mL) were mixed in a volume ratio of 1:1 to obtain premixed intestinal fluid, which was then mixed with bile having a concentration of 160 mmol / L in a volume ratio of 4:0.5 to prepare the obtained fluid.

[0057] 2) The sea cucumber flower self-assembling polypeptide was mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution with a concentration of 10 mg / mL. 100 mL of the sea cucumber flower self-assembling polypeptide solution, 50 mL of a 0.5 M sodium selenite (Na2SeO3) aqueous solution and 50 mL of a phosphate buffer solution with a pH of 7.0 were mixed, the pH was 7.0, and the temperature was continuously stirred at 30-60°C for a third reaction for 20 minutes. The mixture was then washed three times with an 85% volume concentration of ethanol aqueous solution, centrifuged at 12000 g for 5 minutes at 4°C, the precipitate was collected, and freeze-dried (specifically: pre-cooling at -40-10°C for 2 hours, freezing at -40-10°C and 0-20 Pa for 1 hour, and freeze-dried at 0-25°C and 0-20 Pa until completely dry) to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier.

[0058] Example 3

[0059] The cleaned sea cucumber flowers were mixed with deionized water at a ratio of 1 g:2 mL, homogenized at 10,000 rpm for 3 minutes to obtain a sea cucumber flower homogenate, and the mixture was first vacuum freeze-dried for 72 hours (the cold trap temperature was set to -70 ° C and the vacuum degree was 20 Pa) to obtain sea cucumber flower freeze-dried powder. The sea cucumber flower freeze-dried powder was mixed with water at a ratio of 1 g:12 mL to obtain an extraction solution. The pH of the extraction solution was adjusted to 9.0 with a 3 mol / L NaOH aqueous solution, and magnetically stirred at 45 ° C for 4 hours. Then, the supernatant was collected by the first centrifugation at a speed of 3500 r / min for 10 minutes, and the pH of the supernatant was adjusted to 5.6 with a 3 mol / L HCl aqueous solution. The precipitate was collected by the second centrifugation at a speed of 3500 r / min for 10 minutes, washed with distilled water, and second vacuum freeze-dried for 72 hours (the cold trap temperature was set to -70 ° C and the vacuum degree was 20 Pa) to obtain sea cucumber flower protein powder.

[0060] 1) Sea cucumber flower protein powder was dissolved in water to prepare a sea cucumber flower protein aqueous solution with a concentration of 50 mg / mL. The sea cucumber flower protein aqueous solution was heated at 40°C for 30 min and then mixed with simulated gastric fluid (the volume ratio of simulated gastric fluid to sea cucumber flower protein aqueous solution was 2:10). The pH was adjusted to 5.0 with 1M HCl, the temperature was 40°C, and the speed was 400 rpm. The first reaction lasted 150 min. During the first reaction, 1M HCl was used to maintain pH stability. After the first reaction, simulated intestinal fluid was added (the amount of simulated intestinal fluid added was calculated based on the volume ratio of simulated intestinal fluid to sea cucumber flower protein aqueous solution being 2:10). The pH was adjusted to 9.0 with 1M NaOH, the temperature was 40°C, and the speed was 400 rpm. The second reaction lasted 120 min. During the second reaction, 1M NaOH was used to maintain pH stability. After the second reaction, the pH was adjusted to 7.0, and the enzyme was inactivated (100°C water bath for 15 min). The mixture was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was taken and freeze-dried for 72 h (the cold trap temperature was set to -70°C and the vacuum degree was 20 Pa) to obtain the sea cucumber flower self-assembling polypeptide with a particle size in the range of 80 to 100 meshes. The polypeptide was stored at -20°C for future use.

[0061] Gastric electrolyte solution (SGF): 6.9 mL KCl (6.9 mmoL -1 ) aqueous solution, 0.9mL KH2PO4 (0.9mmoL -1 ) aqueous solution, 12.5mLNaHCO3(25mmoL -1 ) aqueous solution, 11.8mLNaCl (47.2mmoL -1 ) aqueous solution, 0.4mL MgCl2(H2O)6(0.1mmoL -1 ) aqueous solution, 0.5mL (NH4) 2CO3 (0.5mmoL -1 ) aqueous solution and 1.3 mL HCl (15.6 mmol -1 ) aqueous solution was dissolved in 400 mL of distilled water to prepare a gastric electrolyte solution.

[0062] Intestinal electrolyte solution (SIF): 6.8 mL KCl (6.8 mmol -1 ) aqueous solution, 0.8mLKH2PO4(0.8mmoL -1 ) aqueous solution, 42.5mLNaHCO3(85mmoL -1 ) aqueous solution, 9.6mLNaCl(38.4mmoL -1 ) aqueous solution, 1.1mL MgCl2(H2O)6(0.33mmoL -1 ) aqueous solution and 0.7 mL HCl (8.4 mmol -1 ) aqueous solution was dissolved in 400 mL of distilled water to prepare an intestinal electrolyte solution.

[0063] Simulated gastric fluid: SGF and pepsin (2000 U / mL) were mixed in a volume ratio of 1:1.

[0064] Simulated intestinal fluid: SIF and pancreatic enzyme (100 U / mL) were mixed at a volume ratio of 1:1 to obtain premixed intestinal fluid, which was then mixed with bile having a concentration of 160 mmol / L at a volume ratio of 4:1 to prepare the obtained fluid.

[0065] 2) The sea cucumber flower self-assembling polypeptide was mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution with a concentration of 60 mg / mL. 150 mL of the sea cucumber flower self-assembling polypeptide solution, 50 mL of a 1 M sodium selenite (Na2SeO3) aqueous solution and 50 mL of a phosphate buffer solution with a pH of 7.0 were mixed, the pH was 9.0, and the temperature was continuously stirred at 30-60°C for a third reaction for 60 minutes. The mixture was then washed three times with an 85% volume concentration of ethanol aqueous solution, centrifuged at 4°C and 12000g for 5 minutes, the precipitate was collected, and freeze-dried (specifically: pre-cooling at -40 to -10°C for 3 hours, freezing at -40 to -10°C and 0 to 20Pa for 2 hours, and freeze-dried at 0 to 25°C and 0 to 20Pa until completely dry) to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier.

[0066] Comparative Example 1

[0067] The cleaned sea cucumber flowers were mixed with deionized water at a ratio of 1 g:2 mL, homogenized at 8000 rpm for 3 min to obtain a sea cucumber flower homogenate, and the mixture was first vacuum freeze-dried for 72 h (the cold trap temperature was set to -55 ° C and the vacuum degree was 10 Pa) to obtain sea cucumber flower freeze-dried powder. The sea cucumber flower freeze-dried powder was mixed with water at a ratio of 1 g:10 mL to obtain an extraction solution. The pH of the extraction solution was adjusted to 9.0 with a 3 mol / L NaOH aqueous solution, and magnetically stirred at 40 ° C for 3.5 h. The supernatant was collected by first centrifugation at a speed of 3500 r / min for 10 min, and the pH of the supernatant was adjusted to 4.8 with a 3 mol / L HCl aqueous solution. The precipitate was collected by a second centrifugation at a speed of 3500 r / min for 10 min, washed with distilled water, and second vacuum freeze-dried for 72 h (the cold trap temperature was set to -55 ° C and the vacuum degree was 10 Pa) to obtain sea cucumber flower protein powder.

[0068] 1) Sea cucumber flower protein powder was dissolved in water to prepare a 30 mg / mL sea cucumber flower protein aqueous solution, the sea cucumber flower protein aqueous solution was heated at 35°C for 20 min, and then mixed with simulated gastric fluid (prepared by dissolving 10 mg pepsin (2000 U / mL) in 1 mL of 0.1 mol / L HCl aqueous solution) (the ratio of the sea cucumber flower protein aqueous solution to the simulated gastric fluid was calculated according to the protein content in the sea cucumber flower protein aqueous solution, and the volume ratio of the protein in the sea cucumber flower protein aqueous solution to the simulated gastric fluid was 1:100), 1M The pH was adjusted to 2.0 with HCl, the temperature was 37°C, the rotation speed was 300 rpm, and the first reaction was carried out for 120 min. After the first reaction, simulated intestinal fluid was added (the simulated intestinal fluid was prepared by dissolving 20 mg trypsin (100 U / mL) and 120 mg bile salt in 10 mL of 0.1 mol / L NaHCO3 aqueous solution. The amount of simulated intestinal fluid added was calculated based on the protein content in the sea cucumber flower protein aqueous solution, with the volume ratio of protein in the sea cucumber flower protein aqueous solution to simulated intestinal fluid being 1:100), 1 M The pH was adjusted to 7.0 with NaOH, the temperature was 37°C, the rotation speed was 300 rpm, and the second reaction was carried out for 120 min. After the second reaction, the pH was adjusted to 7.0, the enzyme was inactivated (100°C water bath for 15 min), and the mixture was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was taken and freeze-dried for 72 h (the cold trap temperature was set to -55°C and the vacuum degree was 10 Pa) to obtain the sea cucumber flower self-assembling polypeptide with a particle size in the range of 80 to 100 mesh, which was stored at -20°C for use.

[0069] 2) The sea cucumber flower self-assembling polypeptide was mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution with a concentration of 50 mg / mL. 100 mL of the sea cucumber flower self-assembling polypeptide solution, 50 mL of a 0.5 M sodium selenite (Na2SeO3) aqueous solution and 50 mL of a phosphate buffer solution with a pH of 7.0 were mixed, the pH was 8.0, and the temperature was continuously stirred at 30 to 60°C for a third reaction for 30 minutes. The mixture was then washed three times with an 85% volume concentration of ethanol aqueous solution, centrifuged at 4°C and 12000g for 5 minutes, the precipitate was collected, and freeze-dried (specifically: pre-cooling at -40 to -10°C for 2 hours, freezing at -40 to -10°C and 0 to 20Pa for 1 hour, and freeze-dried at 0 to 25°C and 0 to 20Pa until completely dry) to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier.

[0070] Experimental Example 1

[0071] Effects of different digestion stages of sea cucumber flower protein (HSH) prepared in Example 1 on selenium binding capacity and hydrolysis degree.

[0072] The selenium content was determined by 3,3'-diaminobenzidine colorimetry. 0.1 g of HSH was weighed at different digestion stages (before digestion, i.e., the sea cucumber flower protein HSHP prepared in Example 1, and the end point of gastric digestion, i.e., ... G The end point of intestinal digestion is the sea cucumber flower self-assembling polypeptide HSHP prepared in Example 1 GI ) The lyophilized powder after the reaction of the product with selenium ions was added to a digestion tube, 12 mL of nitric acid and 3 mL of perchloric acid were added, and the mixture was mixed. A heating program was set with a graphite digester. After digestion until clear and transparent, the mixture was transferred to a beaker, the pH was adjusted to 7.0 with 10 mol / L NaOH, and the volume was adjusted to 50 mL. Secondly, a standard curve was prepared by preparing a selenium standard solution with sodium selenite. 0 mL, 2 mL, 4 mL, 6 mL, 8 mL, and 10 mL of a 5 μg / mL selenium standard solution were taken into a conical flask, diluted to 40 mL with ultrapure water, and the pH was adjusted to 2.0. 2 mL of EDTA-Na2 solution was added, followed by 2 mL of 3,3'-diaminobenzidine solution. The mixture was shaken and reacted in the dark for 50 min. The pH was adjusted to 7.0. 10 mL of toluene was added, and the mixture was vortexed for 2 min. The mixture was allowed to stand. The toluene layer was taken and the absorbance at 420 nm was measured using a UV spectrophotometer. To determine the selenium content in the sample, transfer 10 mL of the digestion solution to a conical flask and follow the standard curve procedure. Measure the absorbance at 420 nm. Calculate the selenium content using the following formula:

[0073] Selenium content (μg / g) = CV / MN;

[0074] Wherein, C is the mass concentration of selenium (μg / mL); V is the sample volume obtained by toluene extraction (mL); M is the mass of the sample (g); and N is the measured sample volume / total volume (v / v).

[0075] The results are as follows Figure 1 As shown, before simulated digestion (HSHP), gastric digestion end point (HSHP G ), intestinal digestive endpoint (HSHP GI ) to determine its selenium binding activity and hydrolysis degree, HSHP GI The selenium binding activity of HSHP can be as high as 317.15±5.52μg / mg, which is significantly higher than that of HSHP and HSHP. G (p<0.05), which is due to the continuous gastrointestinal digestion process GI More selenium binding sites are exposed. The results show that sea cucumber flower self-assembling polypeptides with high selenium ion binding capacity can be prepared by simulating the gastrointestinal digestive system.

[0076] Experimental Example 2

[0077] HSHP prepared in Example 1 GI and HSHPGI -Se spectroscopy analysis.

[0078] (1)HSHP GI and HSHP GI -Se UV and fluorescence spectroscopic determination:

[0079] HSHP GI and HSHP GI -Se was dissolved in deionized water to a final concentration of 0.1 mg / mL, and HSHP was recorded in the wavelength range of 200-500 nm using a UV-Vis spectrophotometer (Perkin Elmer, Salem, MA). GI and HSHP GI UV-Vis spectra of -Se; fluorescence spectroscopy was performed using a Bioteck Synergy H1 multifunctional microplate reader. Assay conditions: excitation wavelength 280 nm, emission wavelength 290-500 nm.

[0080] (2) HSHP GI and HSHP GI FT-IR spectroscopic determination of -Se:

[0081] 1 mg of HSHP GI and HSHP GI -Se was mixed with 100 mg of KBr, and the mixture was ground into fine powder under infrared light and pressed into transparent thin sheets. The Fourier transform infrared spectrometer Nicolet IS50 FT-IR was used for recording, with a scanning range of 4000-400 cm -1 , with a resolution of 4cm -1 All measurements were performed in a dry environment at room temperature (25 ± 1 °C). Peak signals in the spectra were analyzed using OMNIC 9.2 software.

[0082] (3)HSHP GI and HSHP GI -Circular dichroism determination of Se:

[0083] HSHP was measured using a Jasco J-1500 circular dichroism spectrometer at 20 °C with a 1 mm pathlength cell. GI and HSHP GI -Se secondary structure. Each CD spectrum is measured at 1 nm·s -1 The speed is scanned in the range of 190 to 260 nm.

[0084] The results are as follows Figure 2 As shown, Figure 2 As shown in A, HSHP GIAfter combining with selenium ions, the absorption wavelength red-shifts as the concentration of selenium ions increases, which indicates that the spatial structure of the dyeing groups (CO and -COOH) and auxiliary pigments (-OH and -NH2) changes after complexing with selenium ions; the fluorescence absorption spectrum is as follows Figure 2 As shown in B, as the concentration of selenium ions increases, HSHP GI The -Se fluorescence intensity continued to decrease and was accompanied by a red shift. This was because some chromogenic amino acids folded and aggregated during the process of selenium ions inducing sea cucumber flower polypeptides to form self-assembled nanocarriers.

[0085] like Figure 2 Middle C and Figure 2 As shown in D, HSHP GI The FTIR spectrum of the -Se peptide shifted, the negative peak of the CD spectrum dropped sharply, the α-helix and random coil contents decreased by 1.37% and 5.2%, respectively, and the β-turn content increased by 2.27%. These results indicate that selenium ions induce the self-assembly of sea cucumber flower polypeptides to form a new substance with an ordered structure.

[0086] Experimental Example 3

[0087] HSHP prepared in Example 1 GI and HSHP GI- Microstructural determination of Se.

[0088] HSHP GI and HSHP GI -Se was gold-sprayed at a current of 15 mA. The gold thickness was 15 nm. The samples were observed at an accelerating voltage of 5 kV, and SEM and EDS images were obtained at various magnifications using a Zeiss Gemini 500 field-emission scanning electron microscope to examine their microstructure.

[0089] SEM and EDS test results are as follows Figure 3 Middle a, Figure 3 Middle b, Figure 3 Middle C and Figure 3 As shown in middle d, selenium ions induce HSHP GI In the process of HSHP, the original smooth and dense structure of the surface is destroyed to form loose spherical particles; in addition, some non-covalent interactions, such as hydrophobic interaction, electrostatic interaction, van der Waals force and aromatic π-π interaction, may be involved in the HSHP GI The self-assembly process leads to HSHP GI It combines with selenium ions and further self-assembles to form nanocarriers with a certain spherical structure.

[0090] Experimental Example 4

[0091] HSHP prepared in Example 1 GI and HSHPGI- Zeta potential and particle size distribution of Se.

[0092] Determination of HSHP using Zetasizer Nano S90 GI and HSHP GI- Zeta-potential and particle size distribution of Se, HSHP GI and HSHP GI- Se lyophilized powder was dissolved in deionized water to a final concentration of 1 mg / mL. After equilibration at 25°C for 2 minutes, the solution was placed in a sample cell and the zeta potential and particle size distribution were measured three times for each sample, and the average value was calculated.

[0093] HSHP GI and HSHP GI- The particle distribution of Se is as follows Figure 4 As shown in A, the results show that HSHP GI After reacting with selenium ions, the particle size increased from 130.6±1.06nm to 221±8.56nm, which may be due to the HSHP induced by selenium ions. GI The nanoparticles formed by self-assembly undergo a certain degree of aggregation, which increases their particle size.

[0094] HSHP GI and HSHP GI- Potential analysis of Se Figure 4 As shown in B, HSHP GI The zeta potential after binding with selenium ions increased from -42.7±8.42 mV to -23.22±1.89 mV (P<0.05), which may be due to the neutralization of part of the negative charge (-COO-) by selenium ions during the self-assembly process.

[0095] Experimental Example 5

[0096] HSHP prepared in Example 1 GI and HSHP GI- Molecular weight distribution of Se.

[0097] The molecular weight distribution was determined by chromatography using a TSK gel 3000PWXL column. The test conditions were as follows: the mobile phase was acetonitrile: water: trifluoroacetic acid (40:60:0.1, V / V); the detection wavelength was 220 nm; the flow rate was 0.40 mL / min; the column temperature was 30°C; the injection volume was 10 μL; and the standards selected included cytochrome C (12384 Da), aprotinin (6511.51 Da), bacitracin (1422.69 Da), L-oxidized glutathione (612.63 Da), and hydroxyproline (131.13 Da). A relative molecular mass calibration curve was drawn, and HSHP was prepared using the mobile phase at a final concentration of 1 mg / mL. GI and HSHP GI- Se solution and passed through a 0.22 μm organic filter membrane for measurement, and the molecular weight distribution was calculated using the calibration curve.

[0098] The results are as follows Figure 4 As shown in C, HSHP GI The relative content of -Se in the 500~1000Da range is higher than that of HSHP. GI The relative content of the 1000-3000Da component was significantly decreased, while the relative content of the 1000-3000Da component was significantly increased. These results indicate that selenium ions play a role in inducing HSHP. GI The complex formed during the self-assembly process has a larger relative molecular mass; the selenium ion ligand and the polypeptide are connected by chemical bonds, making the structure of the newly formed polypeptide-selenium nanoparticles more complex and the molecular mass also increases accordingly. GI -Se particle size distribution measurement results are consistent.

[0099] Experimental Example 6

[0100] HSHP prepared in Example 1 GI and HSHP GI- Determination of the antioxidant activity of the sea cucumber flower self-assembling polypeptide and sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Se and comparative example 1.

[0101] (1) DPPH free radical scavenging ability:

[0102] Different concentrations of HSHP GI , HSHP GI- Solutions of Se, the sea cucumber flower self-assembling polypeptide prepared in Comparative Example 1, or the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in Comparative Example 1 were mixed with a 0.1 mM DPPH solution at a volume ratio of 1.5:1. Anhydrous ethanol was mixed with the same amount of sample solution and 0.1 mM DPPH solution, respectively. All reactions were carried out in the dark for 30 minutes. Absorbance was measured at 517 nm using a microplate spectrophotometer.

[0103] (2)ABTS + Free radical scavenging ability:

[0104] 7mmol / LABTS + The stock solution was mixed with an equal amount of 2.45 mmol / L potassium persulfate solution to prepare ABTS. + The solution was placed in the dark for 16 h. Subsequently, ABTS was diluted with methanol. + Solution, so that the absorbance at 734nm reaches 0.70±0.02. Take 0.2mL of HSHP GI and HSHP GI- The Se sample solutions were mixed with 3.8 mL of ABTS solution respectively. After mixing for 6 min, the absorbance was measured at 734 nm. Distilled water was used as the blank group.

[0105] The results are as follows Figure 5 China A and Figure 5 As shown in B, ABTS + , DPPH free radical scavenging rate and HSHP GI and HSHP GI- At the same concentration, HSHP GI and HSHP GI- ABTS of Se + There were significant differences in the scavenging rates of DPPH and ABTS free radicals (P<0.05). When the concentration reached 10 mg / mL, the DPPH and ABTS free radicals of sea cucumber flower polypeptide-selenium nanocarriers were significantly different. + The free radical scavenging rates reached the highest values, namely 89.65% and 81.82% respectively; this may be because the presence of selenium ions changes the spatial conformation of the polypeptide, thereby exposing more amino acids in the peptide chain that can provide electrons or protons to free radicals.

[0106] like Figure 5 Middle C, Figure 5 Middle D, Figure 5 Zhong E and Figure 5 As shown in F, HSHP prepared in Example 1 of the present invention GI and HSHP GI -Se DPPH free radical scavenging rate, ABTS + The free radical scavenging rates were significantly higher than those of the sea cucumber flower self-assembling polypeptide prepared in comparative example 1 and the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared in comparative example 1 (p<0.05). This fully demonstrates that the HSHP prepared in Example 1 of the present invention GI and HSHP GI -Se has more excellent antioxidant properties and stronger free radical scavenging ability. It can be used as a highly potential antioxidant and play an important role in the field of functional health foods.

[0107] Experimental Example 7

[0108] HSHP prepared in Example 1 GI- Digestion stability of Se and Na2SeO3.

[0109] Take a certain volume of HSHP GI -Se lyophilized powder was added to water and mixed to prepare an enzymatic hydrolysis reaction solution with a protein concentration of 2 mg / mL. The solution was heated at 35°C for 20 minutes. The pH was adjusted to 2.0 and the temperature was adjusted to 37°C. Based on the protein content in the enzymatic hydrolysis reaction solution, simulated gastric fluid was added at a ratio of 1:100 (v / v). The solution was reacted at 300 rpm for 2 hours, heated at 100°C to inactivate the enzyme for 15 minutes, and centrifuged at 4°C and 8000g for 10 minutes. The supernatant was collected. After the pH of the gastric digestion sample was adjusted to 7.0, simulated intestinal fluid was added at a ratio of 1:100 (v / v). The mixture was then transferred to a dialysis bag and digested in a 37°C water bath with shaking. The selenium content of the dialysate outside the dialysis bag was measured at different times (0, 30, 60, 90, and 120 minutes) to represent the amount of selenium ions that passed through the simulated intestinal digestion. The selenium dialysis rate was calculated as follows:

[0110] Selenium dialysis rate (%) = m3 / m4×100;

[0111] Where m3 and m4 represent the mass (mg) of selenium ions outside the dialysis bag and in the solution, respectively.

[0112] The results are as follows Figure 6 As shown, HSHP GI The selenium dialysis rates of -Se and Na2SeO3 in the simulated intestinal digestion process gradually increased with the increase of intestinal digestion time. When the digestion time was 0-60min, the selenium dialysis rate of Na2SeO3 was higher than that of HSHP. GI -Se. When the digestion time is 60 to 120 minutes, HSHP GI The selenium dialysis rate of -Se increased to 23.37±1.03%, while the selenium dialysis rate of Na2SeO3 only increased slightly to 17.75±0.76; this phenomenon may be due to the instability of inorganic selenium when entering the weak alkaline environment of the intestine, and the rapid increase in the initial selenium dialysis rate; while HSHP GI -Se has a certain digestive stability when entering the intestinal pH range, and the selenium dialysis rate increases rapidly in the later stage. GI -Se can be further decomposed into small molecules under the action of pancreatic enzymes in the small intestine, which is beneficial to promote the absorption of selenium by intestinal cells. GI -Se has a significantly higher selenium dialysis rate than inorganic selenium salt (Na2SeO3) in the simulated intestinal digestion experiment, and is a new type of selenium nutritional enhancer with great potential.

[0113] Experimental Example 8

[0114] HSHP prepared in Example 1 GI and HSHP GI- Peptide sequence identification of Se.

[0115] HSHP was characterized by Nano-LC-ESI-MS / MS GI The peptide sequence was identified, including the following steps: desalting with PierceC18 Spin Tips (Thermo Fisher Scientific, MA, USA). 10 μL HSHP GI The sample solution was loaded onto a capture column (Column Technology Inc., 0.15 mm × 150 mm, RP-C18) at a flow rate of 10 μL / min and linearly eluted from an analytical column (Zorbax 300SB-C18 peptide trap). Mobile phase A consisted of water containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. Elution conditions were as follows: 0–50 min, 4–50% B; 50–54 min, 50–100% B; and 4–60 min, 100% B. HSHP was separated by Nano-LC. GI The peptides were identified using a QExactive mass spectrometer. The raw peptide sequence data were processed using Data Analysis 4.0 software and searched using the MaxQuant server.

[0116] Using Paragon TM Algorithm: 5.0.0.0 (Revision No.: 4767, SCIEX, USA) was used to extract the MS / MS spectra generated by the instrument and search the corresponding database (uniprotkb_Stichopus_japonicus_30507_2024_03_13) using the software MaxQuant 1.5.5.1. Finally, the protein identification and quantitative analysis results were obtained, and some peptide sequences were screened out, as shown in Table 1.

[0117] Table 1 Peptide sequences with potential self-assembly ability and selenium binding activity identified by Nano-LC-ESI-MS / MS

[0118]

[0119]

[0120] like Figure 7 China A and Figure 7 As shown in Figure B, Nano-LC-ESI-MS / MS technology was used to analyze HSHP GIPeptide sequence analysis yielded 3,207 peptide sequences, of which approximately 63% were between 8 and 14 amino acids in length. Molecular weights were primarily below 1,500 Da, with approximately 63% of these peptides below 1,400 Da. Based on this information, a subset of these sequences was selected for further investigation.

[0121] HSHP GI In the process of self-assembly with selenium, the ionic interaction between repeated charged amino acids and the hydrophobic interaction between hydrophobic amino acids are the main driving forces for the formation of ordered supramolecular structures. The hydrophilic surface formed by polar charged amino acids and the hydrophobic surface formed by non-polar uncharged amino acids are distributed on both sides of the main peptide chain of the ion-complementary peptide, respectively. As shown in Table 1, some polypeptide sequences have typical type I (-+ / +-), type II (--++ / ++--) and type III (---++++ or ++++---) charge distributions. The alternating arrangement of hydrophobic amino acids and hydrophilic amino acids can promote the spontaneous formation of β-folded structures of polypeptides, further promoting HSHP. GI -Se self-assembled nanocomposites. In addition, in HSHP GI In the process of binding with selenium, amino acids in the sequence such as methionine, cysteine, leucine, aspartic acid, glycine, valine, etc. can provide binding sites for selenium ions. Among them, acidic amino acids (such as aspartic acid and glutamic acid) are more likely to bind to selenium. The peptide-selenium complex has a high content of hydrophobic amino acids (valine, tryptophan, valine, phenylalanine). These hydrophobic amino acids can bind to selenium through hydrophobic interaction. In summary, the results show that HSHP GI It contains a large number of polypeptide sequences that have both selenium binding ability and self-assembly potential.

[0122] In summary, the present invention prepares sea cucumber flower self-assembling polypeptide-selenium nanocarriers with antioxidant activity, prepares sea cucumber flower polypeptides by simulating the gastrointestinal digestive system, and further induces selenium ions to induce the self-assembly of sea cucumber flower polypeptides to form peptide-selenium nanocarriers with good antioxidant activity and the potential to effectively deliver selenium to the body, which can effectively enhance the digestion stability of selenium ions, and provide new ideas for the development of selenium-fortified foods, functional health products and other fields.

[0123] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a sea cucumber flower self-assembling polypeptide-selenium nanocarrier with antioxidant activity, characterized in that: The following steps are involved: 1) heating the sea cucumber flower protein aqueous solution, then mixing it with simulated gastric fluid, and performing a first reaction for 30 to 150 minutes. After the first reaction, adding simulated intestinal fluid, and performing a second reaction for 20 to 120 minutes. After the second reaction, adjusting the pH to 7.0, inactivating the enzyme, centrifuging, collecting the supernatant, and freeze-drying to obtain the sea cucumber flower self-assembling polypeptide; 2) The sea cucumber flower self-assembling polypeptide obtained in step 1) is mixed with deionized water to prepare a sea cucumber flower self-assembling polypeptide solution, the sea cucumber flower self-assembling polypeptide solution, sodium selenite aqueous solution and phosphate buffer are mixed, and a third reaction is carried out for 20 to 60 minutes, followed by washing with ethanol aqueous solution, collecting the precipitate by centrifugation, and freeze-drying to obtain the sea cucumber flower self-assembling polypeptide-selenium nanocarrier.

2. The preparation method according to claim 1, characterized in that The concentration of the sea cucumber flower protein aqueous solution in step 1) is 10-50 mg / mL, and the sea cucumber flower protein aqueous solution is prepared by dissolving sea cucumber flower protein powder in water; the heating temperature in step 1) is 30-40° C., and the heating time is 10-30 min.

3. The preparation method according to claim 2, characterized in that: The preparation method of the sea cucumber flower protein powder specifically comprises: mixing sea cucumber flowers with deionized water at a ratio of 1g:0.8-2.0mL, homogenizing at 8000-10000rpm for 1-3min to obtain a sea cucumber flower homogenate, performing a first vacuum freeze-drying to obtain sea cucumber flower freeze-dried powder, mixing the sea cucumber flower freeze-dried powder with water at a ratio of 1g:8-12mL to obtain a solution to be extracted, adjusting the pH of the solution to be extracted to 8.5-9.0, stirring at 38-45°C for 3-4h, then performing a first centrifugation to collect the supernatant, adjusting the pH of the supernatant to 4.0-6.5, performing a second centrifugation to collect the precipitate, washing with distilled water, and performing a second vacuum freeze-drying to obtain the sea cucumber flower protein powder.

4. The preparation method according to claim 1, characterized in that The simulated gastric fluid in step 1) is mixed with the sea cucumber flower protein aqueous solution in a volume ratio of 1 to 2:10; the simulated gastric fluid in step 1) is prepared by mixing a gastric electrolyte solution and 2000 U / mL of pepsin in a volume ratio of 1:1, and the gastric electrolyte solution is 6.9 mL with a concentration of 6.9 mmol / L -1 0.9 mL of KCl aqueous solution with a concentration of 0.9 mmoL -1 12.5mL of KH2PO4 aqueous solution with a concentration of 25mmoL -1 11.8 mL of NaHCO3 aqueous solution has a concentration of 47.2 mmol / L -1 0.4mL of NaCl aqueous solution with a concentration of 0.1mmoL -1 MgCl2(H2O)6 aqueous solution, 0.5mL concentration of 0.5mmoL -1 of (NH4)2CO3 aqueous solution and 1.3mL of a 15.6mmoL -1 The HCl aqueous solution is dissolved in 400 mL of distilled water to prepare the obtained solution; in step 1), the pH of the first reaction is 2.0 to 5.0, the temperature of the first reaction is 30 to 40° C., the speed of the first reaction is 200 to 400 rpm, and 1 M HCl solution is used to maintain pH stability during the first reaction.

5. The preparation method according to claim 1, characterized in that: The volume ratio of the simulated intestinal fluid and the sea cucumber flower protein aqueous solution in step 1) is calculated as 1 to 2:10; the simulated intestinal fluid in step 1) is a mixture of an intestinal electrolyte solution and 100U / mL pancreatic enzyme in a volume ratio of 1:1 to obtain a premixed intestinal fluid, and then the premixed intestinal fluid is mixed with bile having a concentration of 160mmol / L in a volume ratio of 4:0.5 to 1.0 to prepare the intestinal electrolyte solution, which is 6.8mL with a concentration of 6.8mmoL -1 0.8 mL of KCl aqueous solution with a concentration of 0.8 mmoL -1 KH2PO4 aqueous solution, 42.5mL concentration of 85mmoL -1 NaHCO3 aqueous solution, 9.6mL concentration of 38.4mmoL -1 1.1mL of NaCl aqueous solution with a concentration of 0.33mmoL -1 MgCl2(H2O)6 aqueous solution and 0.7mL of 8.4mmoL -1 The obtained solution is prepared by dissolving an aqueous solution of HCl in 400 mL of distilled water; in step 1), the pH of the second reaction is 7.0 to 9.0, the temperature of the second reaction is 30 to 40° C., the speed of the second reaction is 200 to 400 rpm, and 1 M NaOH solution is used to maintain pH stability during the second reaction.

6. The preparation method according to claim 1, characterized in that: The particle size of the sea cucumber flower self-assembling polypeptide in step 1) is 80-100 meshes.

7. The preparation method according to claim 1, characterized in that: The volume ratio of the sea cucumber flower self-assembling polypeptide solution, sodium selenite aqueous solution and phosphate buffer mixed in step 2) is 2-3:1:1, the concentration of the sea cucumber flower self-assembling polypeptide solution is 10-60 mg / mL, the molar concentration of the sodium selenite aqueous solution is 0.5-1 M, and the pH of the phosphate buffer is 7.0; the pH of the third reaction in step 2) is 7.0-9.0, and the temperature of the third reaction is 30-60°C.

8. The preparation method according to claim 1, characterized in that: The average particle size of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier in step 2) is 221±8.56 nm, and the average potential of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier is -23.22±1.89 mV.

9. The sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the sea cucumber flower self-assembling polypeptide-selenium nanocarrier prepared by the preparation method according to any one of claims 1 to 8 in preparing a product having antioxidant activity and capable of effectively delivering selenium.

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