Pneumatophorus japonicus tetrapeptide-calcium nano-composite as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological material preparation, and particularly discloses a mackerel tetrapeptide-calcium nano-composite as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing mackerel flesh homogenate with an organic degreasing fluid, stirring in a constant-temperature water bath, carrying out suction filtration, air-drying and powdering to obtain mackerel protein powder; preparing a reaction solution, heating, cooling, preserving heat, carrying out alkaline enzymolysis, inactivating protease, centrifuging, taking supernatant, and freeze-drying to obtain lyophilized powder of the mackerel protein hydrolysate; identifying a polypeptide sequence, and screening a mackerel tetrapeptide amino acid sequence; the mackerel tetrapeptide is obtained by solid-phase synthesis of macromolecular resin and amino acid. The preparation method comprises the following steps: dissolving mackerel tetrapeptide in ultrapure water, adding a CaCl2 solution, heating under an alkaline condition, adding absolute ethyl alcohol, centrifuging and freeze-drying to obtain the mackerel tetrapeptide-calcium nanocomposite. The invention discloses a mackerel tetrapeptide-calcium nano-composite as well as a preparation method and application thereof. The mackerel tetrapeptide-calcium nano-composite has a self-assembly capability and a calcium absorption promoting effect.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial preparation technology, specifically relating to a mackerel tetrapeptide-calcium nanocomposite, its preparation method, and its application. Background Technology

[0002] Calcium is an essential inorganic element for the human body, accounting for 1.5% to 2.2% of total body weight, and plays a crucial role in vital activities such as bone development, nerve conduction, and muscle contraction. Although dietary supplementation is the main route of calcium intake, factors such as oxalic acid and phytic acid in the diet easily bind with calcium to form precipitates, significantly reducing calcium bioavailability. Therefore, there is an urgent need to develop functional supplements that can efficiently promote calcium absorption.

[0003] Mackerel, an important economic fish species in the East China Sea and Yellow Sea of ​​my country, is rich in nutrients such as protein, calcium, and vitamins. The amino acids in its protein, such as glutamic acid and aspartic acid, have potential calcium-binding activity, making it an excellent source for developing functional calcium supplements. However, the current utilization of mackerel resources is mainly focused on primary products such as fishmeal, frozen fish fillets, and pickled products, with insufficient processing depth and a lack of high-value-added products, thus limiting its industrial value-added potential.

[0004] In recent years, the combination of bioactive peptides and polypeptide self-assembly technology has provided a new direction for the development of functional foods. Polypeptide self-assembly can form nanostructures, which, as carriers, can enhance the cellular absorption of active ingredients. Their self-assembly behavior is closely related to physicochemical properties such as amino acid composition, charge distribution, and hydrophilicity / hydrophobicity. Studies have shown that calcium ions can induce some polypeptides to self-assemble into nanocomplexes. These complexes can maintain the solubility of calcium in the gastrointestinal environment, potentially solving the problem of low calcium absorption efficiency and becoming a research hotspot for calcium supplements. However, no specific peptide derived from mackerel has yet been discovered that possesses both clear self-assembly ability and high calcium absorption-promoting efficacy. This research gap limits the high-value utilization of mackerel protein resources and the development of novel calcium supplements. Therefore, developing an active peptide extracted from mackerel protein that possesses both self-assembly ability and calcium absorption-promoting efficacy is of great significance for increasing the added value of mackerel resources and enriching the variety of functional calcium supplements. Summary of the Invention

[0005] The present invention aims to provide a mackerel tetrapeptide-calcium nanocomposite, its preparation method and application. The mackerel tetrapeptide-calcium nanocomposite has both self-assembly ability and calcium absorption promotion effect, and can be used in the preparation of functional health products or nutritionally fortified foods.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing a mackerel tetrapeptide-calcium nanocomposite includes the following steps: S1. Mix the mackerel meat homogenate with the organic defatting liquid, place it in a constant temperature water bath for magnetic stirring, filter, collect the precipitate, air dry at room temperature, grind into powder, and sieve to obtain mackerel protein powder. S2. The mackerel protein powder obtained in S1 was prepared into a reaction solution with a constant substrate protein concentration of 1.5% to 2.5%. The solution was heated with boiling water, cooled, kept warm, and the pH was adjusted to alkaline. Protease was added for enzymatic hydrolysis. The pH was kept constant during the enzymatic hydrolysis. After the reaction was completed, the pH was adjusted to neutral, the protease was inactivated by heating, the supernatant was collected by centrifugation, and the solution was freeze-dried to obtain lyophilized mackerel protein hydrolysate powder. S3. The lyophilized powder of mackerel protein hydrolysate obtained in S2 was subjected to peptide sequence identification, and the amino acid sequence of mackerel tetrapeptide was screened out. S4. The mackerel tetrapeptide described in S3 was obtained by solid-phase synthesis using a polymer resin and amino acids, and then purified by high-performance liquid chromatography. S5. Dissolve the purified mackerel tetrapeptide obtained in S4 in ultrapure water, add CaCl2 solution, heat the mixture under alkaline conditions, and after the reaction is complete, add anhydrous ethanol to make the ethanol concentration in the mixture 90%. Centrifuge, collect the precipitate, freeze dry, and obtain mackerel tetrapeptide-calcium nanocomposite.

[0007] Preferably, in S1, the mass ratio of the mackerel meat homogenate to the organic degreasing liquid is 1:10~12, and the organic degreasing liquid is a mixture of n-hexane and ethanol at a volume ratio of 2~5:1.

[0008] Preferably, in S1, the temperature for magnetic stirring in a constant temperature water bath is 40~55℃, the magnetic stirring speed is 200~400rpm, and the time is 4~8h. The specific air-drying conditions are: drying at room temperature until the water content of the precipitate is 5%~10%, and the sieve mesh size is 80~100 mesh.

[0009] Preferably, in step S2, the boiling water is heated for 10-15 minutes, cooled to 50-55°C, kept warm, and the pH is adjusted to 8.0-8.5.

[0010] Preferably, in S2, the specific conditions for enzymatic hydrolysis are as follows: add 3000 U / g alkaline protease for 3 hours of enzymatic hydrolysis, adjust the pH to maintain a constant value during the reaction with 0.5 mol / L NaOH or 0.5 mol / L HCl, adjust the pH to neutral after the reaction, and heat at 95~100℃ for 10~15 minutes to inactivate the alkaline protease.

[0011] Preferably, in S2, the centrifugation temperature is 4℃, the rotation speed is 8000~10000rpm, and the time is 15~20min. The specific freeze-drying process parameters are: cold trap temperature is -75~-55℃, vacuum degree is 2~10Pa, and time is 48~72h.

[0012] Preferably, in S4, the polymer resin is 2-chlorotrimethylbenzenechlororesin (2-CTC).

[0013] Preferably, in S5, the specific conditions for the heating reaction under alkaline conditions are: temperature 45~50℃, pH 8.0~8.5, and time 60min; the centrifugation temperature is 4℃, the rotation speed is 10000rpm, and the time is 5~10min; the specific process parameters for the freeze-drying are: cold trap temperature -75~-55℃, vacuum degree 2~10Pa, and time 48~72h.

[0014] The present invention also provides a mackerel tetrapeptide-calcium nanocomposite prepared by the preparation method described above.

[0015] This invention also provides the application of the mackerel tetrapeptide-calcium nanocomposite as described above in the preparation of calcium supplements or calcium-fortified foods.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a mackerel tetrapeptide-calcium nanocomposite, its preparation method, and its application. The prepared mackerel tetrapeptide exhibits stable self-assembly ability, and its interaction with Ca... 2+ The carboxyl groups of Glu residues can form stable bonds with calcium ions, and self-assemble into nanocomposites with a particle size of about 98.3 nm. This structure has high stability and provides a reliable carrier for calcium delivery.

[0017] Furthermore, this mackerel tetrapeptide-calcium nanocomposite can effectively improve the bioavailability of calcium. In the simulated gastrointestinal digestion process, even in the presence of factors that easily lead to calcium precipitation, its calcium solubility is still significantly higher than that of CaCl2, which can reduce the precipitation of calcium in the digestive environment. Moreover, through the Caco-2 cell monolayer model, its calcium transport capacity is significantly better than that of CaCl2, which can effectively promote the absorption of calcium by small intestinal epithelial cells.

[0018] This invention not only realizes the high-value utilization of mackerel resources, but also provides a core component for functional calcium supplements that combines self-assembly carrier function and absorption-promoting activity, thus having significant application value.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 The molecular structural formula of the mackerel tetrapeptide prepared in Example 1 is shown below. Figure 2 The liquid chromatogram of the mackerel tetrapeptide prepared in Example 1; Figure 3The mass spectrum of the mackerel tetrapeptide prepared in Example 1; Figure 4 The mackerel tetrapeptide and Ca prepared in Example 1 2+ Molecular docking model; Figure 5 The particle size distribution curves of the mackerel tetrapeptide and the mackerel tetrapeptide-calcium nanocomposite prepared in Example 1 are shown. Figure 6 The solubility of calcium in the mackerel tetrapeptide-calcium nanocomposite prepared in Example 1 with or without oxalic acid during simulated gastrointestinal digestion is measured. Figure 7 The statistical results of calcium transport in the mackerel tetrapeptide-calcium nanocomposite and CaCl2 digest obtained in Example 1 are shown. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] In this invention, unless otherwise specified, all other test materials and instruments are conventional test materials in the field and can be purchased through commercial channels.

[0024] Example 1 A method for preparing a mackerel tetrapeptide-calcium nanocomposite includes the following steps: S1. Mix the mackerel meat homogenate with an organic defatting liquid (hexane to ethanol volume ratio 3:1) at a mass ratio of 1:10, place it in a 50℃ constant temperature water bath and magnetically stir at 200rpm for 6h. Repeat the stirring operation, filter, collect the precipitate, air dry at room temperature until the water content of the precipitate is 5%, grind into powder, and pass through an 80-mesh sieve to obtain mackerel protein powder. S2. The mackerel protein powder obtained in S1 was prepared into a reaction solution with a constant substrate protein concentration of 2%. The solution was heated in boiling water for 10 min, cooled to 50°C, kept warm, and the pH was adjusted to 8.0. 3000 U / g alkaline protease was added for enzymatic hydrolysis for 3 h. During the enzymatic hydrolysis, the pH was adjusted to a constant value using 0.5 mol / L NaOH or 0.5 mol / L HCl. After the reaction was completed, the pH was adjusted to neutral, and the protease was inactivated by heating at 100°C for 10 min. The supernatant was collected by centrifugation at 10000 rpm for 20 min at 4°C. The supernatant was then freeze-dried at -70°C and 2 Pa under vacuum for 72 h to obtain lyophilized mackerel protein hydrolysate powder. S3. The lyophilized powder of mackerel protein hydrolysate obtained in S2 was subjected to peptide sequence identification, and the tetrapeptide amino acid sequence of mackerel was screened out, specifically as follows: Mackerel protein hydrolysate was desalted by solid-phase extraction: (1) 100% acetonitrile and 1 mL of 0.1% TFA aqueous solution were added to the C18 column in sequence, and centrifuged at 600g for 1 minute at room temperature to activate and equilibrate the C18 column; (2) Sample loading: the peptide sample was dissolved in 0.1% TFA aqueous solution and loaded into the equilibrated C18 column, and centrifuged at 600g for 1 minute at room temperature until all samples were loaded; (3) Sample washing: 1 mL of 0.1% TFA aqueous solution was added and centrifuged at 600g for 1 minute at room temperature; (4) Sample elution: 250 μL of elution buffer (such as 70% acetonitrile / 0.1% TFA solution) was added to elute the sample, and centrifuged at 200g for 1 minute at room temperature; (5) The eluted sample was stored at -80℃ until further use.

[0025] Mackerel protein hydrolysate was desalted and then analyzed by mass spectrometry. The instrument used was an ultra-high performance liquid chromatograph (UHPLC) with a C18 column. The HPLC conditions were as follows: mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile containing 0.1% formic acid (acetonitrile content 84%). The elution conditions were: flow rate 0.5 mL / min, temperature 40℃, and gradient conditions: 0–2.5 min, maintain 99% solution A and 1% solution B; 2.5–5 min, increase solution B from 1% to 5%, decrease solution A from 99% to 95%; 5–10 min, increase solution B from 5% to 10%, decrease solution A from 95% to 90%; 10–30 min, increase solution B from 10% to 25%, decrease solution A from 90% to 75%; 31–35 min, increase solution B from 25% to 40%, decrease solution A from 75% to 60%.

[0026] Mass spectrometry conditions: The mass spectrometer was a Thermo QE Orbitrap. Ion mode was ESI. + The mass range is 50~2000 m / z; the capillary voltage is 3.0 kV; the sampling cone voltage is 35.0 V; the ion source temperature is 105℃; the desolventizing temperature is 350℃; the cone orifice gas flow rate is 50.0 L / h; the desolventizing gas flow rate is 600.0 L / Hr; the collision energy is 6.0 eV; the collision gas flow rate is 0.6 mL / min; the scan time is 0.26 sec; and the internal scan time is 0.02 sec.

[0027] Data retrieval: MaxQuant software 1.5.5.1 was used to retrieve the raw data from the mass spectrometry test and the database, and finally the identification and quantitative analysis results of the peptides were obtained.

[0028] Experimental results: A mackerel tetrapeptide with a molecular weight of 504.49 Da, containing three consecutive Glu residues and one Val residue, was identified. Its amino acid sequence is Val-Glu-Glu-Glu, abbreviated as VEEE. Its structural diagram is shown below. Figure 1 It contains potential calcium ion binding sites.

[0029] S4. The mackerel tetrapeptide described in S3 was obtained by solid-phase synthesis using a polymer resin and amino acids, and purified by high-performance liquid chromatography. The specific experimental protocol is as follows: 2-CTC (2-chlorotrimethylbenzenechloropolymer) resin (Hefei Saimanno Biotechnology Co., Ltd.) was selected as the synthetic route. Following the characteristic of the amino acid sequence Val-Glu-Glu-Glu, the carboxyl group of Glu-4 was first covalently linked to the resin. Then, the amino group of Glu-4 and the carboxyl group of Glu-3 underwent a condensation reaction. Glu-2 was then added, and amino acids were added sequentially from right to left until the last amino acid, Val, was linked. Finally, the resin was cleaved to obtain the target polypeptide. The specific synthetic route is as follows: (1) Calculate the weight of each raw material according to the weight of the target polypeptide, put the resin into a 150mL reactor, and add 50mL of DCM to soak for 2 hours; (2) Wash the resin with DMF, and then dry it. Repeat this process four times to dry the resin; (3) Weigh Fmoc-Glu(Otbu)-OH (the first amino acid at the C-terminus) + DCM + DIEA and add it to the reactor. Then place the reactor in a shaker at 30℃ and react for 2 hours; (4) Seal with methanol solution (methanol:DIEA:DCM=1:1:2) for half an hour, then wash with DMF four times and dry it; (5) Add 20% piperidine solution to the reactor to remove the Fmoc protecting group. After deprotection, wash with DMF four times and then dry; (6) Take a small amount of resin and test it with ninhydrin. If the resin is colored, it means that the deprotection was successful; (7) Weigh Fmoc-Glu(Otbu)-OH (the second amino acid at the C-terminus) + HOBT + DIC and add it to the reactor. Then place the reactor in a shaker at 30°C and react for 1 hour; (8) Take a small amount of resin and test it with ninhydrin. If the resin is colored, it means that the condensation is incomplete and the reaction should continue. If the resin is colorless, it means that the reaction is complete; after the reaction is complete, wash the resin with DMF four times and then dry; (9) Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash four times with DMF, then dry and check whether the protection has been removed. Take a small amount of resin and test it with ninhydrin. If the resin is colored, it means that the deprotection was successful. (10) Connect amino acids in sequence according to steps (7)-(9), use a cutting reagent to remove all the peptide protecting groups, cut the resin and purify it.

[0030] Purification was performed using high-performance liquid chromatography (HPLC) with a VYDAC-C18 column (4.6 mm). 250 nm; mobile phase A was acetonitrile containing 0.1% (v / v) trifluoroacetic acid; mobile phase B was water containing 0.1% (v / v) trifluoroacetic acid; elution conditions were: 0-20.0 min: mobile phase A increased from 22.0% to 32.0%; 20.0-20.1 min: mobile phase A increased from 32.0% to 100.0%; flow rate 1.0 mL / min; detection wavelength 220 nm. The sample was flash-frozen in liquid nitrogen, freeze-dried, and the structure was identified by ESI-MS.

[0031] Experimental Results: VEEE was synthesized using solid-phase synthesis technology. The results of liquid phase purity identification and molecular weight mass spectrometry identification are as follows: Figure 2 and Figure 3 As shown in the test report, the synthesized VEEE has a purity of 95.61% and a molecular weight of 504.49 Da, which meets the requirements for subsequent experiments.

[0032] Mackerel tetrapeptide (VEEE) and Ca 2+ Molecular docking: Obtaining Ca from PubChem 2+ The structure of VEEE was constructed using ChemDraw Academic Edition, and the connection between VEEE and Ca was completed using Discovery Studio software. 2+ The molecular docking was performed, and the final step used PyMol 2.5 to optimize the molecular docking results.

[0033] Experimental results are as follows Figure 4 As shown, Ca 2+ It binds to Glu-3 and Glu-4 bonds in VEEE. The Ca-O bond length in VEEE is between 2.3 and 5.5 kcal, with a binding energy of -7.2825 kcal / mol. The docking conformation of VEEE shows that VEEE forms four bonds with calcium ions, indicating that VEEE has a strong affinity for calcium ions, and Glu provides a more stable binding site for calcium ions.

[0034] S5. The purified VEEE obtained in S4 was dissolved in ultrapure water at a concentration of 2.5 mg / mL. CaCl2 solution was added to make the molar ratio of VEEE to CaCl2 1:6. The mixture was heated at 50℃ for 60 min under pH 8.0 conditions. After the reaction was completed, anhydrous ethanol was added to make the ethanol concentration in the mixture 90% to remove free calcium ions. The mixture was centrifuged at 10000 rpm for 10 min at 4℃, and the precipitate was collected. The precipitate was freeze-dried at -70℃ and 2 Pa vacuum for 72 h to obtain mackerel tetrapeptide-calcium nanocomposite.

[0035] The mackerel tetrapeptide-calcium nanocomposite prepared in Example 1 was tested as follows: The particle size of the mackerel tetrapeptide-calcium nanocomposite prepared in Example 1 was determined, and the results are as follows: Figure 5 As shown.

[0036] Depend on Figure 5 It can be seen that the average particle size of VEEE is approximately 121.6 nm. In the interaction between VEEE and Ca... 2+ After binding, they self-assemble to form a stable nanocomposite with a particle size of 98.3 nm.

[0037] The specific experimental methods for simulating gastrointestinal digestive stability are as follows: A two-stage digestion process was performed on the mackerel tetrapeptide-calcium nanocomposite provided in Example 1. 40 mg of pepsin was dissolved in 1 mL of 0.1 N HCl to prepare a simulated gastric digestion solution. 20 mg of trypsin and 120 mg of sodium taurocholate were dissolved in 10 mL of 0.1 M NaHCO3 to prepare a simulated intestinal digestion solution. 0.2 g of the mackerel tetrapeptide-calcium nanocomposite and CaCl2 were dissolved in 60 mL of ultrapure water and incubated at 37 °C for 30 min. The pH was adjusted to 2.0, and 0.05 mL of simulated gastric juice was added. Simulated gastric digestion was then performed at 37 °C. At digestion times of 0, 5, 10, 30, 60, and 90 min, 4 mL of the solution was removed, heated at 100 °C for 5 min to inactivate the enzymes, and centrifuged at 10,000 rpm for 10 min to obtain the supernatant for later use. After 90 min of simulated gastric digestion, the pH was adjusted to 7.5, and 0.17 mL of simulated intestinal juice was added. Simulated intestinal digestion was continued at 37 °C. When digestion reaches 0, 5, 10, 30, 60, 90 and 150 min, 4 mL of solution is taken out, heated in an 80℃ water bath for 5 min to inactivate the enzyme, centrifuged at 10000 rpm for 10 min, and the supernatant is collected to determine the calcium content in the supernatant.

[0038] Mackerel tetrapeptide-calcium nanocomposite and CaCl2 were dissolved separately in ultrapure water, and 5 mL of 2% oxalic acid was added. The mixture underwent a two-stage digestion process as described above. After centrifugation at 10,000 rpm for 10 min at 4 °C, the supernatant was collected for calcium content determination.

[0039] Using CaCl2 as a control, the calcium solubility results of the mackerel tetrapeptide-calcium nanocomposite after simulated gastrointestinal digestion are as follows: Figure 6 As shown. By Figure 6 It is known that both mackerel tetrapeptide-calcium nanocomposite and CaCl2 have high calcium solubility during gastrointestinal digestion, but the solubility decreases after entering the intestinal digestion stage. This may be because trypsin hydrolyzes mackerel tetrapeptide-calcium nanocomposite, causing the peptide and calcium to separate, and the pH is alkaline, which easily forms Ca(OH)2 precipitate.

[0040] The addition of oxalic acid to mackerel tetrapeptide-calcium nanocomposite and CaCl2 solution decreased calcium solubility. Upon reaching the gastric digestion endpoint and adjusting the pH to 7.5, calcium solubility significantly decreased, then stabilized during intestinal digestion. Furthermore, under the influence of oxalic acid, the calcium solubility in the mackerel tetrapeptide-calcium nanocomposite was significantly higher than that in CaCl2 (…). P <0.01). These results indicate that, under simulated gastric digestion conditions, the mackerel tetrapeptide-calcium nanocomposite is beneficial in improving calcium solubility, and under weakly alkaline simulated intestinal digestion conditions, the mackerel tetrapeptide-calcium nanocomposite can reduce calcium solubility to a certain extent. 2+ Even with the presence of oxalic acid, the mackerel tetrapeptide-calcium nanocomposite maintains high calcium solubility, which is beneficial for the absorption of calcium by intestinal epithelial cells.

[0041] The experimental protocol for studying calcium transport in an in vitro Caco-2 cell monolayer model is as follows: Caco-2 cells were cultured in Eagle's MEM medium containing 20% ​​fetal bovine serum and 1% penicillin / streptomycin antibiotics at 37°C, 5% CO2, and 90% relative humidity. Cells at passages 25-30 were seeded into 12-well Transwell plates (12 wells, 0.4 μm, 1.12 cm). 2 On the surface, the inoculation density was 1.0 × 10⁻⁶. 5 Cells / mL, culture medium was changed every 2 days after inoculation. The integrity of the cell monolayer was assessed by measuring transepithelial electrical resistance (TEER) values ​​using the Millicell-ERS system every other day.

[0042] Caco-2 cell monolayers were gently washed twice with Hank's balanced salt solution (HBSS, calcium and magnesium-free), then transferred to fresh plates and incubated with 1.5 mL of HBSS buffer for 30 min, followed by transport experiments. The HBSS buffer was removed and replaced with 0.5 mL of sample (dissolved in HBSS) on the AP side and 1.5 mL of fresh HBSS buffer on the BL side. Cells were incubated at 37°C in 5% CO2 for 2 h. 1.0 mL of sample from the BL side at different digestion times was extracted, and then 1.0 mL of fresh HBSS buffer was added to the BL side to maintain a constant volume. Calcium content was determined using an atomic absorption spectrophotometer, and calcium uptake was calculated. The results are shown below. Figure 7 As shown.

[0043] Both the mackerel tetrapeptide-calcium nanocomposite digest and the CaCl2 digest exhibited certain calcium transport capabilities. At different transport time points, the calcium transport effect of the digested mackerel tetrapeptide-calcium nanocomposite was significantly better than that of the digested CaCl2, and the calcium transport capacity increased with time, reaching a maximum of 78.73±3.94 μg / well at 120 min. These results indicate that compared with CaCl2-mimicked gastrointestinal digestion products, the digestion products of the mackerel tetrapeptide-calcium nanocomposite have a good calcium absorption-promoting effect, effectively promoting calcium absorption by small intestinal epithelial cells. The mackerel tetrapeptide-calcium nanocomposite prepared in this invention can maintain calcium solubility in the gastrointestinal environment and effectively promote calcium absorption by Caco-2 cells.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions 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 mackerel tetrapeptide-calcium nanocomposite, characterized in that, Includes the following steps: S1. Mix the mackerel meat homogenate with the organic defatting liquid, place it in a constant temperature water bath for magnetic stirring, filter, collect the precipitate, air dry at room temperature, grind into powder, and sieve to obtain mackerel protein powder. S2. Prepare a reaction solution with a constant substrate protein concentration of 1.5% to 2.5% by preparing the mackerel protein powder obtained in S1. Heat the solution in boiling water, cool it, keep it warm, adjust the pH to alkaline, add protease for enzymatic hydrolysis, keep the pH constant during the enzymatic hydrolysis, adjust the pH to neutral after the reaction is completed, heat the solution to inactivate the protease, centrifuge the supernatant, freeze dry it to obtain lyophilized mackerel protein hydrolysate powder. S3. The lyophilized powder of mackerel protein hydrolysate obtained in S2 was subjected to peptide sequence identification, and the amino acid sequence of mackerel tetrapeptide was screened out. S4. The mackerel tetrapeptide described in S3 was obtained by solid-phase synthesis using a polymer resin and amino acids, and then purified by high-performance liquid chromatography. S5. Dissolve the purified mackerel tetrapeptide obtained in S4 in ultrapure water, add CaCl2 solution, heat the mixture under alkaline conditions, and after the reaction is complete, add anhydrous ethanol to make the ethanol concentration in the mixture 90%. Centrifuge, collect the precipitate, freeze dry, and obtain mackerel tetrapeptide-calcium nanocomposite.

2. The preparation method according to claim 1, characterized in that, In S1, the mass ratio of the mackerel meat homogenate to the organic degreasing liquid is 1:10~12, and the organic degreasing liquid is a mixture of n-hexane and ethanol in a volume ratio of 2~5:

1.

3. The preparation method according to claim 1, characterized in that, In S1, the temperature for magnetic stirring in a constant temperature water bath is 40~55℃, the magnetic stirring speed is 200~400rpm, and the time is 4~8h. The specific air-drying conditions are: drying at room temperature until the water content of the precipitate is 5%~10%, and the sieve mesh size is 80~100 mesh.

4. The preparation method according to claim 1, characterized in that, In S2, the boiling water is heated for 10-15 minutes, cooled to 50-55°C, kept warm, and the pH is adjusted to 8.0-8.

5.

5. The preparation method according to claim 1, characterized in that, In S2, the specific conditions for enzymatic hydrolysis are as follows: add 3000 U / g alkaline protease for 3 hours of enzymatic hydrolysis, adjust the pH to a constant level using 0.5 mol / L NaOH or 0.5 mol / L HCl during the reaction, adjust the pH to neutral after the reaction, and heat at 95~100℃ for 10~15 minutes to inactivate the alkaline protease.

6. The preparation method according to claim 1, characterized in that, In S2, the centrifugation temperature is 4℃, the rotation speed is 8000~10000rpm, and the time is 15~20min. The specific freeze-drying process parameters are: cold trap temperature is -75~-55℃, vacuum degree is 2~10Pa, and time is 48~72h.

7. The preparation method according to claim 1, characterized in that, In S4, the polymer resin is 2-chlorotrimethylbenzene chloride resin.

8. The preparation method according to claim 1, characterized in that, In S5, the specific conditions for the heating reaction under alkaline conditions are: temperature 45~50℃, pH 8.0~8.5, and time 60min; the centrifugation temperature is 4℃, the rotation speed is 10000rpm, and the time is 5~10min; the specific process parameters for the freeze-drying are: cold trap temperature -75~-55℃, vacuum degree 2~10Pa, and time 48~72h.

9. The mackerel tetrapeptide-calcium nanocomposite prepared by the preparation method according to any one of claims 1-8.

10. The application of the mackerel tetrapeptide-calcium nanocomposite as described in claim 9 in the preparation of calcium supplements or calcium-fortified foods.

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