Sea cucumber flower self-assembled polypeptide-iron nano-carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of preparation of biological materials, and particularly discloses a sea cucumber flower self-assembled polypeptide-iron nano-carrier as well as a preparation method and application of the sea cucumber flower self-assembled polypeptide-iron nano-carrier. The preparation method comprises the following steps: dissolving freeze-dried sea cucumber flower powder in deionized water, adjusting the pH value to be alkaline, heating and stirring, centrifuging, discarding the precipitate, adjusting the pH value to be acidic, centrifuging, collecting the precipitate, and freeze-drying to obtain sea cucumber flower protein powder; preparing a reaction solution, adding simulated gastric juice and simulated intestinal juice, stirring for reaction, inactivating protease, centrifuging, taking supernatant, and freeze-drying to obtain sea cucumber flower self-assembled polypeptide; the preparation method comprises the following steps: preparing a solution from sea cucumber, adding FeCl2. 4H2O and ascorbic acid, adjusting the pH value to be acidic, carrying out heating reaction, centrifuging, discarding the supernatant, and freeze-drying to obtain the sea cucumber flower self-assembled polypeptide-iron nano-carrier. The invention discloses a sea cucumber flower self-assembled polypeptide-iron nano-carrier as well as a preparation method and application thereof. The carrier can promote absorption of an organism to iron, a relatively high iron ion release rate is maintained in an intestinal environment, and the in-vivo bioavailability of iron ions is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterial preparation, and particularly relates to a sea cucumber flower self-assembling polypeptide-iron nanocarrier and a preparation method and application thereof. Background Art

[0002] Iron is an essential trace element for the human body and is involved in important physiological functions such as hemoglobin synthesis and immune regulation. However, iron deficiency anemia has affected more than 1.2 billion people worldwide, especially in infants, pregnant women and the elderly. Traditional iron supplements (such as ferrous sulfate) easily combine with phytic acid, polyphenols and other ingredients in food, leading to precipitation of iron ions in the intestines, low bioavailability, and possible side effects such as gastrointestinal discomfort. In addition, the poor stability of such iron supplements can easily lead to changes in the sensory properties of food, limiting their use in nutritionally fortified foods.

[0003] Sea cucumbers are an important marine resource, with a total national production of approximately 292,000 tons in 2023. However, by-products such as viscera and cooking liquid produced during the processing are often discarded. Studies have shown that the nutrient content in sea cucumber by-products is comparable to that in the sea cucumber body wall, and some components are even higher than those in the sea cucumber body wall. Currently, the development and utilization of sea cucumber by-products has become a research hotspot. Sea cucumber flower (HSH) is a general term for the mature gonadal organs and intestines of sea cucumbers during the reproductive period. It is a by-product of the sea cucumber processing process, rich in protein, and is a potential source of functional protein peptides, with important development value.

[0004] In recent years, peptide self-assembly technology has provided new insights into the development of iron supplements. Studies have shown that peptides such as abalone and whey protein peptides can form soluble nanocomplexes with iron ions, maintaining the soluble state of iron ions in the intestine and promoting absorption. However, existing technologies for preparing peptide-iron nanocarriers primarily use animal or plant-derived peptides, and the self-assembly application of sea cucumber flower peptides has not been explored. Furthermore, these technologies present challenges such as complex preparation processes and insufficient carrier stability.

[0005] Therefore, how to efficiently utilize sea cucumber flower by-products and develop an iron nanocarrier with high bioavailability and high stability has become a technical problem that needs to be solved urgently in the field of iron nutrition fortification. Summary of the Invention

[0006] The present invention aims to provide a sea cucumber flower self-assembling polypeptide-iron nanocarrier and its preparation method and application. The sea cucumber flower self-assembling polypeptide-iron nanocarrier can promote the body's absorption of iron, maintain a high iron ion release rate in the human intestinal environment, and improve the in vivo bioavailability of iron ions. It can be used to prepare iron-fortified foods or functional health products.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] A method for preparing a sea cucumber flower self-assembling polypeptide-iron nanocarrier comprises the following steps:

[0009] S1. Extraction of sea cucumber flower protein: dissolving sea cucumber flower freeze-dried powder in deionized water, adjusting the pH to alkaline, heating with magnetic stirring, centrifuging, discarding the precipitate, adjusting the pH to acidic, centrifuging again, collecting the precipitate, washing, and freeze-drying to obtain sea cucumber flower protein powder;

[0010] S2. Preparation of self-assembling polypeptide: The sea cucumber flower protein powder obtained in S1 was prepared into a reaction solution with a substrate concentration of 10-50 mg / mL, and simulated gastric fluid and simulated intestinal fluid were added in sequence to react with stirring. After the reaction, the protease was inactivated and the supernatant was centrifuged and freeze-dried to obtain the sea cucumber flower self-assembling polypeptide HSHP. GI , stored at -20℃ for future use;

[0011] S3. Nanocarrier construction: The sea cucumber flower self-assembling polypeptide HSHP obtained in S2 was GI A solution with a substrate concentration of 20-50 mg / mL was prepared, 40-60 mmol / LFeCl2·4H2O and ascorbic acid were added in sequence, the pH was adjusted to acidic, the reaction was heated, and the mixture was washed with 85% ethanol solution by mass fraction, the supernatant was discarded by centrifugation, and lyophilized to obtain the sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP. GI -Fe.

[0012] Preferably, in S1, the sea cucumber flower freeze-dried powder is dissolved in deionized water at a liquid-solid ratio of 8 to 12:1, the pH is adjusted to 8.5 to 9.0, heated at 38 to 45°C with magnetic stirring for 3 to 4 hours, centrifuged, the precipitate is discarded, the pH is adjusted to 4.8, centrifuged again, the precipitate is collected, washed, and freeze-dried to obtain sea cucumber flower protein powder.

[0013] Preferably, in S1, the sea cucumber flower freeze-dried powder is dissolved in deionized water at a liquid-solid ratio of 8 to 12:1, 3 mol / L NaOH solution is added to adjust the pH to 8.5 to 9.0, heated at 38 to 45 ° C and magnetically stirred for 3 to 4 hours, centrifuged at 3000 to 4000 r / min for 10 to 15 minutes, discarded the precipitate, added 3 mol / L HCl solution to adjust the supernatant pH to 4.0 to 6.5, 3000 to 4000 r / min centrifuged again for 10 to 15 minutes, collected the precipitate, washed with distilled water, and freeze-dried to obtain sea cucumber flower protein powder.

[0014] Preferably, in S1, the freeze-drying process parameters are: cold trap -75 to -55°C, vacuum degree 2 to 10 Pa, and time 48 to 72 hours.

[0015] Preferably, in S2, the simulated gastric fluid is prepared by mixing gastric electrolyte solution (SGF) and pepsin (2000 U / mL) at a volume ratio of 1:0.5-1.5;

[0016] The simulated intestinal fluid is prepared by mixing intestinal electrolyte solution (SIF) and pancreatic enzyme (100 U / mL) at a volume ratio of 1:0.5-1.5, and adding 150-170 mmol / L bile.

[0017] Preferably, the SGF comprises the following ingredients: 6.9mmol / L KCl, 0.9mmol / LKH2PO4, 25mmol / LNaHCO3, 47.2mmol / L NaCl, 0.1mmol / LMgCl2(H2O)6, 0.5mmol / L(NH4)2CO3, 15.6mmol / L HCl;

[0018] The SIF includes the following components: 6.8 mmol / L KCl, 0.8 mmol / L KH2PO4, 85 mmol / L NaHCO3, 38.4 mmol / L NaCl, 0.33 mmol / L MgCl2(H2O)6, and 8.4 mmol / L HCl.

[0019] Preferably, in S2, the volume ratio of the simulated gastric fluid, simulated intestinal fluid and reaction solution is 1 to 2:10.

[0020] Preferably, in S2, after adding the simulated gastric fluid, the pH is adjusted to 2.0-5.0, and the mixture is stirred at a speed of 200-400 rpm for 30-150 min at a temperature of 30-40°C; after adding the simulated intestinal fluid, the pH is adjusted to 7.0-9.0, and the mixture is stirred at a speed of 200-400 rpm for 20-120 min at a temperature of 35-38°C.

[0021] Preferably, in S2, the inactivation of protease is specifically as follows: after the reaction is completed, the reaction solution is placed in a boiling water bath at 90-95°C for 10-15 minutes, centrifuged at 8000-10000 r / min at 4°C for 5-10 minutes, and the supernatant is collected.

[0022] Preferably, in S2, the specific process parameters of the freeze-drying are: cold trap -70 to -50°C, vacuum degree 1 to 20 Pa, and freeze-drying time 48 to 72 hours.

[0023] Preferably, in S2, the sea cucumber flower self-assembling polypeptide HSHP GI The mesh size is 80 to 100 meshes.

[0024] Preferably, in S3, the mass ratio of FeCl2·4H2O to ascorbic acid is 0.2-0.5:1.

[0025] Preferably, in S3, the pH is adjusted to 5.0-6.0, the reaction is heated at 25-30°C for 30-40 minutes, washed three times with 85% ethanol solution, centrifuged at 4°C and 10,000-12,000g for 5-10 minutes, and the supernatant is discarded.

[0026] Preferably, in S3, the freeze-drying is carried out in three stages, specifically:

[0027] The first stage of precooling: temperature is -40 to -10°C, time is 2 to 3 hours;

[0028] The second stage of vacuuming: temperature is -40 to -10°C, pressure is 0 to 20 Pa, and time is 1 to 2 hours;

[0029] The third stage of freeze drying: temperature is 0-25°C, pressure is 0-20Pa, and time is 40-50h.

[0030] The present invention also provides a sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP prepared by the preparation method GI -Fe.

[0031] The present invention also provides the sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP GI -Fe in the preparation of iron-fortified foods or functional health products.

[0032] Compared with the prior art, the present invention has the following advantages and technical effects:

[0033] The present invention discloses a sea cucumber flower self-assembling polypeptide-iron nanocarrier, its preparation method, and application. The sea cucumber flower self-assembling polypeptide-iron nanocarrier prepared by this method can promote the body's absorption of iron, maintain a high iron ion release rate in the human intestinal environment, and improve the bioavailability of iron ions in the body. This provides new ideas for the development of iron-fortified foods, functional health products, and other fields.

[0034] In the present invention, the prepared sea cucumber flower self-assembling polypeptide-iron nanocarrier (HSHP GI -Fe) can reach an iron binding rate of 84.73±0.92%, and it can maintain a high iron ion release rate (25.37±1.27%) during the simulated gastrointestinal digestion process, making it easier for it to be absorbed by small intestinal epithelial cells, thereby improving the bioavailability of iron ions in vivo.

[0035] The HSHP of the present invention GI -Fe has a simple preparation process, does not require complex equipment, and has good potential for effectively delivering iron to the body, which improves production efficiency and is suitable for industrial production.

[0036] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The sea cucumber flower self-assembling polypeptide HSHP prepared in Example 1 GI Protein identification and quantitative analysis results, including: Figure 1 A in the figure is the peptide chain length distribution diagram detected by LC-MS / MS. Figure 1 B in the figure is the molecular weight distribution of peptides detected by LC-MS / MS;

[0038] Figure 2 HSHP prepared in Example 1 GI and HSHP GI -Fe structural characterization results, among which, Figure 2 A in is the XRD analysis result, Figure 2 B in the figure is the result of FTIR spectrum analysis;

[0039] Figure 3 HSHP prepared in Example 1 GI and HSHP GI -Fe microstructure measurement results, among which, Figure 3 A in the figure is HSHP at magnifications of 500× and 50000×. GI SEM images of Figure 3 B in the figure is HSHP at magnifications of 500× and 50000×. GI -SEM image of Fe, Figure 3 C in the figure is HSHP at a magnification of 5000×. GI Elemental composition analysis chart, Figure 3 D in the figure is HSHP at a magnification of 5000×. GI -Fe element composition analysis chart;

[0040] Figure 4 HSHP prepared in Example 1 GI -Fe in a simulated gastrointestinal environment 2+ Release rate statistics chart. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0043] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.

[0044] In the present invention, the gastric electrolyte solution (SGF) is prepared as follows: 6.9 mL 6.9 mmol / L KCl, 0.9 mL 0.9 mmol / L KH2PO4, 12.5 mL 25 mmol / L NaHCO3, 11.8 mL 47.2 mmol / L NaCl, 0.4 mL 0.1 mmol / LMgCl2(H2O)6, 0.5 mL 0.5 mmol / L (NH4)2CO3, and 1.3 mL 15.6 mmol / L HCl are dissolved in 400 mL distilled water to prepare SGF.

[0045] The intestinal electrolyte solution (SIF) was prepared as follows: 6.8 mL of 6.8 mmol / L KCl, 0.8 mL of 0.8 mmol / L KH2PO4, 42.5 mL of 85 mmol / L NaHCO3, 9.6 mL of 38.4 mmol / L NaCl, 1.1 mL of 0.33 mmol / L MgCl2(H2O)6, and 0.7 mL of 8.4 mmol / L HCl were dissolved in 400 mL of distilled water to prepare SIF.

[0046] The simulated gastric fluid was prepared by mixing SGF and pepsin (2000 U / mL) in a volume ratio of 1:1;

[0047] Simulated intestinal fluid was prepared by mixing SIF and pancreatic enzyme (100 U / mL) in a volume ratio of 1:1 and adding 160 mmol / L bile (the volume ratio of simulated intestinal fluid to bile was 4:0.5-1.0).

[0048] Example 1

[0049] A method for preparing a sea cucumber flower self-assembling polypeptide-iron nanocarrier comprises the following steps:

[0050] S1. Extraction of sea cucumber flower protein: In S1, sea cucumber flower freeze-dried powder was dissolved in deionized water at a liquid-to-solid ratio of 10:1, 3 mol / L NaOH solution was added to adjust the pH to 9.0, heated at 40°C with magnetic stirring for 3.5 h, centrifuged at 3500 rpm for 10 min, the precipitate was discarded, 3 mol / L HCl solution was added to adjust the pH of the supernatant to 4.8, and centrifuged again at 3500 rpm for 10 min. The precipitate was collected, washed with distilled water, and freeze-dried in a cold trap at -55°C and a vacuum of 10 Pa for 72 h to obtain sea cucumber flower protein powder;

[0051] S2. Preparation of self-assembling polypeptide: The sea cucumber flower protein powder obtained in S1 was prepared into a reaction solution with a substrate concentration of 30 mg / mL, and simulated gastric fluid was added to adjust the pH to 2.0. The mixture was stirred at 300 rpm for 120 min at a temperature of 37°C, and then simulated intestinal fluid was added to adjust the pH to 7.0. The mixture was stirred at 300 rpm for 120 min at a temperature of 37°C. 1M HCl was used to keep the pH stable during the digestion process. After the reaction, the pH was adjusted to 7.0, and the reaction solution was quickly placed in a 95°C boiling water bath for 10 min. The reaction solution was centrifuged at 8000 r / min for 10 min at 4°C, and the supernatant was taken and freeze-dried in a -55°C cold trap under a vacuum of 10 Pa for 72 h to obtain the sea cucumber flower self-assembling polypeptide HSHP. GI , stored at -20°C for future use; wherein, the volume ratio of simulated gastric fluid, simulated intestinal fluid and reaction solution is 1:10.

[0052] S3. Nanocarrier construction: The sea cucumber flower self-assembling polypeptide HSHP obtained in S2 was GI A solution with a substrate concentration of 30 mg / mL was prepared, 50 mmol / L FeCl2·4H2O was added, and ascorbic acid was added to prevent ferrous ion oxidation, wherein the mass ratio of FeCl2·4H2O:ascorbic acid was 0.2:1, the pH was adjusted to 5.0, the reaction was heated at 30°C for 30 minutes, washed three times with an 85% ethanol solution by mass fraction, centrifuged at 4°C and 12000g for 5 minutes, the supernatant was discarded, and freeze-dried in three stages, specifically: the first stage: pre-cooling at a temperature of -20°C for 2 hours; the second stage vacuuming section: vacuuming at a temperature of -40°C and a pressure of 10 Pa for 1 hour; the third stage: freeze-drying at a temperature of 20°C and a pressure of 20 Pa for 48 hours to obtain sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP. GI -Fe.

[0053] The sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP prepared in Example 1 was tested by the following experiments. GI -Fe to verify the effect.

[0054] 1. Determination of HSHP GI -Fe amino acid composition, the experimental scheme is as follows: take 50mg HSHP GI To the lyophilized Fe-Fe powder, 5 mL of 6 mol / L HCl was added, nitrogen was purged, and the mixture was sealed by heat fusion. The mixture was then hydrolyzed in a 110°C oven for 24 h. After deacidification, the volume was diluted with water to the appropriate concentration. The sample was loaded and analyzed using an L-8900 fully automatic amino acid analyzer. The results are shown in Table 1.

[0055] Table 1: HSHP GI -Fe amino acid composition

[0056]

[0057]

[0058] Note: 1 Hydrophobic amino acids include Ala, Phe, Ile, Leu, Val, Met, and Pro; 2 Hydrophilic amino acids include Thr, Ser, Cys, Tyr, His, Lys, Arg, Asp, and Glu; 3 Aromatic amino acids include Phe and Tyr. 4 Positively charged amino acids include His, Lys, and Arg; 5 Negatively charged amino acids include Asp and Glu; 6 Essential amino acids include Lys, Phe, Met, Thr, Ile, Leu, and Val.

[0059] As shown in Table 1, HSHP GI -Fe contains 67.28% hydrophilic amino acids, 32.37% hydrophobic amino acids, 8.07% aromatic amino acids, 10.41% positively charged amino acids, and 29.01% negatively charged amino acids. Furthermore, the amino acids closely related to iron ion binding activity, such as Glu, Cys, and Asp, account for a relatively high proportion, at 16.35%, 15.12%, and 8.86%, respectively. The presence of these amino acids is beneficial to promoting HSHP. GI -Fe formation. In addition, HSHP GI -Fe contains as much as 29.00% of essential amino acids, indicating that HSHP GI -Fe not only serves as a carrier for iron delivery, but also supplements essential amino acids for the body.

[0060] 2. Nano-LC-ESI-MS / MS identification:

[0061] Nano-LC-ESI-MS / MS was used to analyze the HSHP prepared in Example 1. GI The method comprises the following steps:

[0062] First, desalting was performed using Pierce C18 Spin Tips (Thermo Fisher Scientific, MA, USA). 10 μL HSHP GIThe 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 data of the peptide sequences were processed using Data Analysis 4.0 software and searched using the MaxQuant server.

[0063] 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. The results are shown in Figure 2 . Figure 1 and Table 2.

[0064] Table 2 Identification of peptide sequences with potential self-assembly ability and iron binding activity using Nano-LC-ESI-MS / MS

[0065]

[0066]

[0067] Depend on Figure 1 As shown in Table 2, HSHP GI The peptides were mainly composed of 3-4 amino acids, accounting for 71.6%. The molecular weight was mainly distributed below 1000Da. Among them, the number of peptides with a molecular weight below 750Da was the largest, accounting for 73.5%. In addition, 75.8% of the identified peptides contained at least one of Asp, Glu, Val or Lys. Amino acids with carboxyl and imidazole residues can be found in HSHP. GI Studies have shown that Arg, Asn, Asp, Cys, Gln, Glu, His, Lys, Ser, Thr, and Tyr are abundant in the identified iron-binding peptides, and these peptides are responsible for providing iron-binding sites.

[0068] In addition, some of the peptide sequences listed in Table 2 contain a certain proportion of hydrophobic and hydrophilic amino acids, such as VLLLDAGFLP and LLLA. Their hydrophobic ends are composed of several consecutive hydrophobic amino acid residues, while the hydrophilic groups are charged amino acids. They can self-assemble to form nanostructures through hydrophobic interactions and hydrogen bonds. At the same time, the ionic interactions between repeated charged amino acids and the hydrophobic interactions between hydrophobic amino acids are also the main driving forces for the formation of ordered supramolecular structures. The ion-complementary peptides formed, such as ELLH, are typical type I (-+ or +-), which promote the spontaneous formation of β-folded structures. 2+ Will further induce HSHP GI Formation of HSHP GI -Fe self-assembled nanostructures.

[0069] In summary, the use of peptidomics technology in the present invention to identify and screen sea cucumber flower self-assembling polypeptides with both self-assembly function and iron ion binding activity is efficient and feasible; and the sea cucumber flower self-assembling polypeptides are rich in peptide segments, which can provide a large number of polypeptide fragments for screening.

[0070] 3. HSHP provided in Example 1 GI and HSHP GI -Fe was structurally characterized as follows:

[0071] XRD determination: the concentration is 1 mg mL -1 HSHP GI , HSHP GI -Fe was dropped onto a copper grid, air-dried at room temperature, and then analyzed by TEM. It was analyzed using a Shimadzu XRD-7000s X-ray diffractometer. The test conditions were: a Cu target-excited Kα radiation source with an operating voltage and current of 40 kV and 30 mA; a scan range of 2θ = 10 to 70°, and a speed of 4° min-1. -1 , the X-ray diffraction pattern is imaged on an X-ray diffractometer.

[0072] FTIR determination: 1 mg of HSHP GI , HSHP GI -Fe were mixed with 100 mg of KBr, and the mixture was ground into fine powder under infrared lamp and pressed into transparent thin sheets. All Fourier transform infrared spectra were recorded by Fourier transform infrared spectrometer Nicolet IS50 FT-IR 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). The peak signals in the spectrum were analyzed using OMNIC 9.2 software. The structural analysis was performed using XRD and FTIR spectroscopy, and the results were as follows: Figure 2 A and Figure 2 As shown in B.

[0073] Depend on Figure 2 A and Figure 2 From B in HSHP, we can see that GI -Fe showed some new sharp and narrow diffraction peaks in the XRD pattern, and the FTIR spectrum was blue-shifted, indicating that iron ions induced the self-assembly of sea cucumber flower polypeptide to form a new substance with a certain crystal structure.

[0074] Determination of microstructure: HSHP GI and HSHP GI -Fe was gold-sprayed at a current of 15 mA. The thickness of the gold sprayed was 15 nm. The sample was observed at an accelerating voltage of 5 kV and magnified to different magnifications using a Zeiss Gemini 500 field emission scanning electron microscope to obtain SEM and EDS images to observe its microstructure. The results are shown in Figure 2. Figure 3 .

[0075] Depend on Figure 3 It can be seen that iron ions induce HSHP GI 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 formation of HSHP. GI The self-assembly process leads to HSHP GI It combines with iron ions and further self-assembles to form nanocarriers with a certain crystal structure.

[0076] 4.HSHP GI -Fe simulated gastrointestinal digestion stability analysis, the experimental scheme is as follows: HSHP was determined by ferrozine colorimetry GI -Fe was used to evaluate the iron ion release during the simulated gastrointestinal digestion process to evaluate its gastrointestinal digestion stability. GI -Fe and FeSO4·4H2O were dissolved in pure water to prepare a 2 mg / mL reaction solution (50 mL). The pH was adjusted to 2.0 and the temperature was maintained at 37°C. 5 μL of simulated gastric fluid was added to each reaction solution, and the reaction was continued at 300 rpm for 120 min. The pH was adjusted to 7.0 and the temperature was maintained at 37°C. 5 μL of simulated intestinal fluid was added to each solution, and the reaction was continued at 300 rpm for 120 min. During the digestion process, 1 M HCl was used to maintain a stable pH. During the digestion process, 5 mL of the reaction solution was collected at different digestion time points (0, 30, 60, 90, 120, 150, 180, 210, and 240 min) and centrifuged at 8000 g for 10 min at 4°C. The supernatant was collected and the iron content was determined spectrophotometrically. The iron solubility rate was calculated as follows:

[0077]

[0078] HSHP GI -Fe in a simulated gastrointestinal environment 2+ Release rate Figure 4 shown.

[0079] Depend on Figure 4 It can be seen that in the simulated gastric juice digestion process, FeSO4·4H2O and HSHP GI -Fe in Fe 2+ The release rate gradually increased, which may be due to the 2+ The solubility is higher in the pH range of gastric fluid, while HSHP GI -Fe 2+ The release rate was significantly lower than that of FeSO4·4H2O (P<0.05), indicating that HSHP GI -Fe can maintain a stable structure in gastric juice and carry more Fe 2+ Enters the small intestine system. Further, when entering the small intestine digestion stage, when the pH is adjusted to 7.0, FeSO4·4H2O and HSHP GI -Fe 2+ The release rate dropped sharply, which may be due to the 2+ The precipitation was caused by the weak alkaline environment of intestinal fluid; in addition, during the whole simulated small intestinal digestion process, HSHP GI -Fe 2+ The release rate of HSHP was always significantly higher than that of FeSO4·4H2O until the end of intestinal digestion. GI -Fe 2+ The release rate was still as high as 25.37±1.27%.

[0080] In summary, HSHP GI -Fe can maintain a high iron ion release rate in a simulated human intestinal environment, making it easier for it to be absorbed by small intestinal epithelial cells, thereby improving the bioavailability of iron ions in the body.

[0081] The present invention discloses a sea cucumber flower self-assembling polypeptide-iron nanocarrier and its preparation method and application, wherein the self-assembling polypeptide (HSHP) is prepared by hydrolyzing sea cucumber flower protein in a simulated gastrointestinal digestion system. GI ), which can self-assemble to form a soluble HSHP with a stable structure after reacting with iron ions GI -Fe, prepared HSHP GI-Fe has a unique self-assembly mechanism and gastrointestinal digestion stability, and has the potential to effectively deliver iron to the body while supplementing a variety of essential amino acids. This nanocarrier can be used to prepare iron-fortified foods, functional health products, etc.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a sea cucumber flower self-assembling polypeptide-iron nanocarrier, characterized in that: The following steps are involved: S1. Extraction of sea cucumber flower protein: dissolving sea cucumber flower freeze-dried powder in deionized water, adjusting the pH to alkaline, heating with magnetic stirring, centrifuging, discarding the precipitate, adjusting the pH to acidic, centrifuging again, collecting the precipitate, washing, and freeze-drying to obtain sea cucumber flower protein powder; S2. Preparation of self-assembling polypeptide: The sea cucumber flower protein powder obtained in S1 was prepared into a reaction solution with a substrate concentration of 10-50 mg / mL, and simulated gastric fluid and simulated intestinal fluid were added in sequence to react with stirring. After the reaction, the protease was inactivated and the supernatant was centrifuged and freeze-dried to obtain the sea cucumber flower self-assembling polypeptide HSHP. GI , stored at -20℃ for future use; S3. Nanocarrier construction: The sea cucumber flower self-assembling polypeptide HSHP obtained in S2 was GI A solution with a substrate concentration of 20-50 mg / mL was prepared, 40-60 mmol / LFeCl2·4H2O and ascorbic acid were added in sequence, the pH was adjusted to acidic, the reaction was heated, and the mixture was washed with 85% ethanol solution by mass fraction, the supernatant was discarded by centrifugation, and lyophilized to obtain the sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP. GI -Fe.

2. The preparation method according to claim 1, characterized in that In S1, the sea cucumber flower freeze-dried powder is dissolved in deionized water at a liquid-solid ratio of 8 to 12:1, the pH is adjusted to 8.5 to 9.0, heated at 38 to 45° C. with magnetic stirring for 3 to 4 hours, centrifuged, the precipitate is discarded, the pH is adjusted to 4.8, centrifuged again, the precipitate is collected, washed, and freeze-dried to obtain the sea cucumber flower protein powder.

3. The preparation method according to claim 1, characterized in that: In S2, the simulated gastric fluid is prepared by mixing gastric electrolyte solution and pepsin in a volume ratio of 1:0.5-1.5; the simulated intestinal fluid is prepared by mixing intestinal electrolyte solution and pancreatic enzyme in a volume ratio of 1:0.5-1.5, and adding 150-170 mmol / L bile.

4. The preparation method according to claim 3, characterized in that The gastric electrolyte solution includes the following components: 6.9mmol / L KCl, 0.9mmol / L KH2PO4, 25mmol / LNaHCO3, 47.2mmol / L NaCl, 0.1mmol / L MgCl2(H2O)6, 0.5mmol / L(NH4)2CO3, and 15.6mmol / L HCl.

5. The preparation method according to claim 3, characterized in that: The intestinal electrolyte solution includes the following components: 6.8mmol / L KCl, 0.8mmol / L KH2PO4, 85mmol / LNaHCO3, 38.4mmol / L NaCl, 0.33mmol / L MgCl2(H2O)6, and 8.4mmol / LHCl.

6. The preparation method according to claim 1, characterized in that: In S2, after adding the simulated gastric fluid, the pH is adjusted to 2.0-5.0, and the mixture is stirred at a speed of 200-400 rpm for 30-150 min at a temperature of 30-40°C; after adding the simulated intestinal fluid, the pH is adjusted to 7.0-9.0, and the mixture is stirred at a speed of 200-400 rpm for 20-120 min at a temperature of 35-38°C.

7. The preparation method according to claim 1, characterized in that: In S2, the inactivation of protease is specifically as follows: after the reaction is completed, the reaction solution is placed in a boiling water bath at 90-95°C for 10-15 minutes, centrifuged at 8000-10000 r / min for 5-10 minutes at 4°C, and the supernatant is collected.

8. The preparation method according to claim 1, characterized in that: In S3, the pH is adjusted to 5.0-6.0, the reaction is heated at 25-30° C. for 30-40 min, washed three times with 85% ethanol solution, centrifuged at 4° C. and 10,000-12,000 g for 5-10 min, and the supernatant is discarded.

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

10. The sea cucumber flower self-assembling polypeptide-iron nanocarrier HSHP as claimed in claim 9 GI -Fe in the preparation of iron-fortified foods or functional health products.

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