Cow milk beta-casein derived intestinal zinc absorption promoting peptide and application thereof in preparation of zinc supplement

Abstract

The invention relates to a cow milk beta-casein derived intestinal zinc absorption promoting peptide and application thereof in preparation of a zinc supplement. The sequence of the peptide for promoting intestinal zinc absorption is YPVEPF, and the peptide is non-toxic, high in safety, capable of being specifically combined with Zn < 2 + >, high in zinc chelating capacity and capable of remarkably improving the zinc transport amount in a Caco-2 small intestine epithelial cell model, so that the peptide has the good activity of improving intestinal zinc absorption. The peptide capable of promoting intestinal zinc absorption can be obtained through a solid-phase synthesis method, so that large-scale production can be carried out, and the synthesized peptide capable of promoting intestinal zinc absorption is high in purity and stable in quality, and can meet current market requirements.

Description

Technical Field

[0001] The present invention relates to the field of small molecule peptides, and more specifically, to a bovine milk beta-casein-derived peptide for promoting intestinal zinc absorption and application thereof in the preparation of a zinc supplement. Background Art

[0002] Zinc is the second most abundant trace element in the human body and one of the most basic nutrients required for human health. Zinc is a structural and functional component of many transcription factors and enzymes that regulate cell growth, gene expression, and immune response. The physiological function of zinc depends on its bioavailability in tissues, which in turn depends on the intestinal absorption of dietary zinc. Since zinc ions form insoluble complexes with dietary fiber and phytic acid, the absorption of zinc in the intestine is hindered, thereby reducing the bioavailability of zinc. Therefore, it is necessary to improve the absorption and bioavailability of zinc.

[0003] Existing methods to improve intestinal zinc absorption include adding zinc salts to food, using complex mineral supplements, or degrading phytic acid in food. However, these methods may have the following problems: adding zinc salts to food will change the original sensory and physical and chemical properties of the food; complex mineral supplements have low stability in the intestinal environment and may cause intestinal diseases; and it is difficult to process all foods containing phytic acid during actual processing.

[0004] Food protein-derived peptides are dietary ingredients with multiple biological activities. Among them, metal chelating peptides can bind to Zn 2 + , Fe 2+ , Ca 2+ , Cu 2+ The zinc chelate peptides are combined with divalent metal ions to form a complex, which has high solubility, good stability and high safety in the intestinal environment. For example, Liu et al. (DOI: 10.3390 / md17080438) isolated and identified multiple zinc chelate peptides from sea cucumbers, but the research on zinc chelate peptides is still relatively small.

[0005] Therefore, more zinc chelate peptides need to be developed to meet the current demand for zinc supplementation in nutritional supplementation, disease treatment, etc. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the problem that zinc chelating peptides are still relatively scarce, and to provide a pro-intestinal zinc absorption peptide derived from bovine milk β-casein. The pro-intestinal zinc absorption peptide is not only non-toxic and highly safe, but also can react with Zn 2+ It has specific binding and strong zinc chelating ability, and can significantly increase the amount of zinc transport in the Caco-2 small intestinal epithelial cell model, thus having good activity in enhancing intestinal zinc absorption.

[0007] A further object of the present invention is to provide the use of the above-mentioned peptide for promoting intestinal zinc absorption in the preparation of products capable of promoting zinc absorption.

[0008] Another object of the present invention is to provide the use of the above-mentioned peptide for promoting intestinal zinc absorption in the preparation of an auxiliary agent for zinc supplements.

[0009] Another object of the present invention is to provide the use of the above-mentioned combination of the intestinal zinc absorption promoting peptide and zinc agent in the preparation of zinc supplements.

[0010] Another object of the present invention is to provide a composition comprising the above-mentioned peptide for promoting intestinal zinc absorption.

[0011] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0012] A peptide for promoting intestinal zinc absorption derived from bovine milk beta-casein, wherein the peptide for promoting intestinal zinc absorption is a sequence shown in SEQ ID NO: 1.

[0013] The inventors of the present invention extracted a peptide that promotes intestinal zinc absorption from bovine milk β-casein. The amino acid sequence of the peptide is as follows: YPVEPF (Tyr-Pro-Val-Glu-Pro-Phe). The peptide that promotes intestinal zinc absorption is not only non-toxic and highly safe, but also can react with Zn 2+ It has specific binding and strong zinc chelating ability, and can significantly increase the amount of zinc transport in the Caco-2 small intestinal epithelial cell model, thus having good activity in enhancing intestinal zinc absorption.

[0014] The intestinal zinc absorption promoting peptide of the present invention can be obtained by enzymatic hydrolysis, separation and other operations on bovine milk casein, or by solid phase synthesis. Among them, since it can be obtained by solid phase synthesis, it can be mass-produced, and the synthesized intestinal zinc absorption promoting peptide has high purity and stable peptide quality, which can meet the current market demand.

[0015] The present invention also protects the use of the above-mentioned peptide for promoting intestinal zinc absorption in the preparation of products capable of promoting zinc absorption.

[0016] The present invention also protects the use of the above-mentioned peptide for promoting intestinal zinc absorption in preparing an auxiliary agent for zinc supplements.

[0017] The use of the above-mentioned combination of intestinal zinc absorption promoting peptide and zinc agent in the preparation of zinc supplements is also within the protection scope of the present invention.

[0018] Preferably, the zinc agent is a zinc salt.

[0019] More preferably, the zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

[0020] Further preferably, the inorganic zinc salt is at least one of zinc sulfate, zinc chloride or zinc nitrate.

[0021] Further preferably, the organic zinc salt is at least one of zinc gluconate, glycyrrhizin, zinc acetate, zinc citrate, amino acid zinc or zinc lactate.

[0022] A composition comprising the above-mentioned peptide for promoting intestinal zinc absorption.

[0023] Preferably, the content of the peptide promoting intestinal zinc absorption in the composition is 25-1000 μg / mL.

[0024] Preferably, the composition further comprises a zinc agent.

[0025] More preferably, the zinc agent is a zinc salt.

[0026] Further preferably, the zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a milk β-casein-derived peptide for promoting intestinal zinc absorption. The peptide for promoting intestinal zinc absorption is not only non-toxic and highly safe, but also can react with Zn 2+ It has specific binding and strong zinc chelating ability, and can significantly increase the amount of zinc transport in the Caco-2 small intestinal epithelial cell model, thus having good activity in enhancing intestinal zinc absorption.

[0029] The intestinal zinc absorption promoting peptide of the present invention can be obtained by enzymatic hydrolysis, separation and other operations on bovine milk casein, or by solid phase synthesis. Among them, since it can be obtained by solid phase synthesis, it can be mass-produced, and the synthesized intestinal zinc absorption promoting peptide has high purity and stable peptide quality, which can meet the current market demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the source protein of the polypeptide identified by LC-MS / MS analysis in Example 1.

[0031] Figure 2 Peptide for promoting intestinal zinc absorption from bovine milk β-casein and Zn 2+ Theoretical combination model diagram.

[0032] Figure 3 This is a graph showing the zinc chelation rate of YPVEPF of the present invention.

[0033] Figure 4 It is a graph showing the result of the cytotoxicity assay of YPVEPF of the present invention.

[0034] Figure 5This is a graph showing the results of the zinc transport activity of YPVEPF of the present invention. DETAILED DESCRIPTION

[0035] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0036] Example 1 Enzymatic hydrolysis, separation and peptide identification of bovine milk casein

[0037] 1.1 Enzymatic hydrolysis of bovine milk casein

[0038] The bovine milk casein was dissolved in deionized water to make the concentration of the bottom bovine milk casein reach 7%, the initial pH was adjusted to 8.5, trypsin was added, and the amount of trypsin added was 0.6% of the substrate mass. The enzymatic hydrolysis was carried out in a water bath, the temperature was controlled at 50°C, and the enzymatic hydrolysis time was 3h. After the enzymatic hydrolysis was completed, the solution was heated at 90°C for 10min to kill the enzyme, cooled to room temperature, and the pH of the solution was adjusted to 4.6 to precipitate the unenzymatically hydrolyzed casein, followed by centrifugation at 4000r / min for 20min. The supernatant after centrifugation was freeze-dried to obtain the casein enzymatic hydrolysate.

[0039] 1.2 Separation of casein hydrolysate

[0040] Casein hydrolysates were separated by IMAC. The Sepharose 6B matrix was mixed with NaOH solution (1 mol / L) and epichlorohydrin, activated at 40°C for 3.5 h, then filtered with a funnel and washed 7 times with deionized water. 6 g of iminodiacetic acid (IDA) was weighed and dissolved in 40 mL of Na2CO3 (2 mol / L) solution, and the pH was adjusted to 13.0 with 6 mol / L NaOH to obtain an IDA solution. The activated Sepharose 6B matrix was mixed with the IDA solution (volume ratio = 1:2), placed at 60°C and stirred for 8 h (120 r / min) to obtain IDA-Sepharose 6B. IMAC-Zn 2+The rubber hose at the bottom of the column (200mm×10mm) was clamped, and IDA-Sepharose 6B was loaded. After precipitation until obvious stratification, the clamp was released to allow the water layer to flow out of the column. Then, the column was washed with 5 times the volume of medium deionized water using a constant flow pump, followed by rinsing with 5 times the volume of medium ZnSO4 solution (0.2mol / L), and then rinsing with 5 times the volume of medium deionized water. Casein hydrolysate was dissolved in deionized water to obtain a 15mg / mL casein hydrolysate solution, and then 2mL of casein hydrolysate solution was loaded onto the column. First, it was eluted with deionized water at pH 5.5 at an elution rate of 1mL / min. The absorbance of the eluted component at a wavelength of 214nm was monitored until the unbound peptide component had been completely eluted by deionized water at pH 5.5. The unbound peptide component was named F1. Subsequently, the bound peptide components were eluted with deionized water at pH 2.0 at a flow rate of 1 mL / min, and the eluted components were collected, concentrated, and lyophilized. The bound peptide components were named F-IMAC. Deionized water with different pH values ​​was used as the mobile phase during the elution process because the peptides with zinc chelating activity under neutral conditions can bind to IMAC-Zn 2+ The zinc chelating peptide can be eluted under acidic conditions. 2+ The column-bound peptides with zinc chelating activity were mainly present in F-IMAC, so F-IMAC was used for subsequent peptide identification.

[0041] 1.3 HPLC-MS / MS identification of peptide composition of F-IMAC

[0042] The lyophilized F-IMAC was dissolved in a buffer solution (0.1% formic acid (FA), 2% acetonitrile (ACN)). TM-X 33μm Polymeric Reversed Phase, 10mg / 1mL) for desalting, and then eluted the sample into a new centrifuge tube with elution buffer (0.1% FA, 60% ACN). The sample solution was centrifuged and concentrated to dryness, and the sample was redissolved in Nano-LC mobile phase A (0.1% FA / water) after desalting. 2μL of the sample was loaded onto a nanoViper C18 pre-column (3μm), washed and desalted, and then separated by an analytical column (Acclaim PepMap C18, 75μm x 25cm). The F-IMAC peptide was identified using an OrbitrapFusion Lumos mass spectrometer connected to an EASY-nanoLC 1200 liquid chromatograph. Spray voltage: 1.9 kV; ion transfer tube heating temperature: 275 ° C; liquid chromatography elution gradient: mobile phase B (80% ACN, 0.1% FA) increased from 5% to 38% within 60 min; mass spectrometry parameters were set as follows: mass spectrometry scanning mode: information-dependent acquisition working mode (DDA, Data Dependent Analysis); primary mass spectrometry scanning resolution was 70000, scanning range was 100-1500 m / z, and maximum injection time was 100 ms; secondary mass spectrometry ion maximum injection time was 50 ms; collision chamber energy (high energy collision induced dissociation, HCD): 28 eV, applicable to all precursor ions; dynamic exclusion setting: 6s.

[0043] The original raw spectrum files collected by mass spectrometry were processed and searched with the help of PEAKS Studio 8.5 software. The database used was the Bos taurus (Bovine) casein species protein database downloaded by Uniprot. The search parameters were set as follows: the mass tolerance of the primary mass spectrometry was 10 ppm, and the secondary mass spectrometry was 0.05 Da.

[0044] A total of 50 peptides of F-IMAC were identified from the F-IMAC components. Based on the secondary mass spectra of each peptide and the comparison with the database, it was found that the peptides of F-IMAC were distributed in four subtypes of casein, such as Figure 1 Among them, 20 peptides were derived from αs2-casein, 15 from αs1-casein, 9 from β-casein, and 6 from κ-casein. The lengths of the 50 peptides identified ranged from 6 to 23 amino acids, and the molecular weights were all less than 3 kDa.

[0045] Example 2 Molecular docking

[0046] The binding of peptides to zinc ions was simulated by molecular docking technology, and the docking binding energy was used as an indicator to further screen casein zinc chelate peptides that were stable with zinc ions. First, a new zinc ion model was created using Discovery Studio Client2019 software. Secondly, the peptide sequence was imported into a new window, and the protein preparation tool was used to hydrogenate and minimize its energy. After that, the zinc ion was selected as the ligand, the peptide molecule was selected as the receptor, and the entire peptide molecule was defined as the docking site. Finally, the calculation of docking binding energy in the docking module was selected to predict the binding energy of its action.

[0047] The selected high-activity zinc chelating peptides were docked with Zn using the CDOCKER module in the Discovery Studio Client 2019 docking simulation. 2+ The docking of the zinc chelation protein can be used to determine the amino acid site of action, the type of binding force, the bond length and other information, thereby realizing the prediction of the zinc chelation mechanism.

[0048] The 50 peptides were ranked according to the molecular docking binding energy. The lower the binding energy, the better the peptides are bound to Zn 2+ The more stable the binding, the better. A total of three highly active zinc chelating peptides were screened, one of which was the intestinal zinc absorption promoting peptide YPVEPF, which was derived from β-casein in bovine milk casein. The sequence information and molecular docking results of the casein zinc chelating peptide identified by NanoLC-MS / MS are shown in Table 1.

[0049] Table 1 Sequence information and molecular docking results of casein zinc chelating peptides identified by NanoLC-MS / MS

[0050]

[0051] YPVEPF and Zn 2+ Theoretical combination model such as Figure 2 As shown. In YPVEPF-Zn 2+ In the complex, Zn 2+ It binds to proline (Pro2) and glutamate (Glu4). Specifically, the carbonyl oxygen atom in proline binds to Zn through the Metal-Acceptor reaction. 2+ Specific binding, forming a Zn-O bond with a bond length of The carboxyl oxygen atom in glutamate binds to Zn through charge-charge interaction. 2+ Specific binding, forming a Zn-O bond with a bond length of The longer the bond length, the more stable the structure. Therefore, in YPVEPF, Zn 2+ The binding to glutamate may be more stable than to proline.

[0052] Example 3 Determination of zinc chelating activity and cytotoxicity of intestinal zinc absorption promoting peptide derived from bovine milk β-casein

[0053] 3.1 Zinc chelating activity verification

[0054] The intestinal zinc absorption-promoting peptide YPVEPF derived from bovine milk β-casein obtained above was synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. using solid phase synthesis method, with a purity of more than 98%, and then the zinc ion chelating activity was determined.

[0055] The method for determining the zinc ion chelation rate is as follows: dissolve 1 mg / mL of the sample in 40 mM HEPES-KOH buffer, take 175 μL of the sample solution and add it to a 96-well plate, and preheat it at 37°C for 10 minutes. Add 25 μL of 250 μmol / L ZnSO4 solution to each well, shake and mix, react at 37°C for 30 minutes, and then add 100 μL of 2 mmol / L PAR solution to terminate the reaction. After 10 minutes, measure the absorbance at 500 nm using an enzyme reader. EDTA was selected as the positive control. HEPES-KOH buffer was selected as the blank group, and each sample was tested in triplicate. The formula for calculating the zinc ion chelation rate is as follows:

[0056] Zinc ion chelation rate (%) = (Abs 空白 -Abs 样品 ) / Abs 空白 ×100%, where Abs 空白 is the absorbance value of the blank sample, Abs 样品 is the absorbance value of the sample.

[0057] The test results of zinc chelation activity are as follows Figure 3 As shown (values ​​are expressed as mean ± standard deviation, n = 3; *** represents p < 0.001, significant difference). The zinc ion chelation rate of YPVEPF is 55.81 ± 0.83%, which is higher than the zinc chelation rate of some existing zinc chelating peptides, for example, the zinc chelation rate of wheat germ protein-derived zinc chelating peptide (Asn-Ala-Pro-Leu-Pro-Pro-Pro-Leu-Lys-His) is about 15.2% (see document DOI: 10.1016 / j.jff.2014.10.030). This shows that the intestinal zinc absorption promoting peptide of the present invention has good zinc chelation activity.

[0058] 3.2 MTT assay for cytotoxicity

[0059] Caco-2 cells were purchased from American Type Culture Collection (Rockville, MD, USA). The cells were inoculated into T25 culture flasks. When the cell density reached 70% to 80%, 0.25% EDTA-trypsin was added and digested at 37°C for 3 minutes. The cells were passaged at a ratio of 1:3. Cells of passages 15 to 30 were selected for subsequent experiments. The complete culture medium was prepared with 79% EMEM basal culture medium, 20% fetal bovine serum and 1% antibiotics (penicillin and streptomycin). The culture environment of the cells was 37°C, 5% CO2, and 95% relative humidity. The complete culture medium was replaced every 2 days.

[0060] The MTT assay was used to determine whether the intestinal zinc absorption-promoting peptides were toxic to the growth of Caco-2 cells. Caco-2 cells in the logarithmic growth phase were digested with 0.25% EDTA-trypsin and plated at 5×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL / well and incubated in complete medium for 24 hours to allow the cells to fully adhere to the wall. Then the complete medium was removed, and 100 μL of YPVEPF sample solutions (dissolved in complete medium) of different concentrations (25, 50, 100, 250, 500, 1000 μg / mL) were added respectively. The complete medium was used as the blank control group and placed in an incubator for 24 hours. The sample solution was removed, and 100 μL of 0.5 mg / mL MTT solution was added for 4 hours. Finally, the MTT solution was removed, 100 μL of DMSO was added to dissolve the purple crystalline product, and the absorbance was measured at 490 nm using a multifunctional microplate reader. The cell viability calculation formula is as follows: Cell viability (%) = (absorbance of the sample group / absorbance of the blank control group) × 100%.

[0061] Cytotoxicity test results Figure 4 As shown, the MTT results showed that YPVEPF had no toxic effect on Caco-2 cells within the concentration range of 25 to 1000 μg / mL, and the subsequent zinc transport activity experiment was performed within this concentration range.

[0062] Example 4 Determination of zinc transport activity of intestinal zinc absorption promoting peptide derived from bovine milk β-casein

[0063] Before cell inoculation, 0.5 mL and 1.5 mL of complete medium were added to the upper and lower chambers of a 12-well polyester Transwell membrane culture plate (PET, Corning, 3460), respectively, and placed in an incubator for 1 h to allow equilibrium. After 1 h, the complete medium in the upper chamber was removed. When the cell coverage rate reached 80% of the T25 culture flask, 4 × 10 5The cells were inoculated into the upper chamber of a 12-well polyester Transwell membrane culture plate at a density of 100 cells / mL. The cells were cultured every other day for 21 days. The integrity of the cell monolayer was evaluated by measuring the transmembrane electrical resistance (TEER). When the TEER value was greater than 500Ω / cm 2 This indicates that the Caco-2 cell monolayer model was successfully established and can be used for zinc transport activity determination experiments.

[0064] YPVEPF was mixed with ZnSO4 solution and reacted at 37°C for 1 hour to obtain a peptide-zinc mixture. Before the transport experiment, a new 12-well culture plate was prepared and 1.5 mL of complete culture medium was added. The Caco-2 monolayer cell membrane cultured for 21 days was transferred to the new 12-well plate. The pre-mixed peptide-zinc solution (sample group) and ZnSO4 solution (control group) were added to the upper chamber of the transport model. The Zn in the sample group and the control group was 2+ The concentration was 50 μg / mL. After the transport started, 1 mL of culture medium was taken from the lower chamber of the Transwell nested plate at 30, 60, 120, and 180 min, and 1 mL of culture medium was quickly added to maintain the volume of the transport system unchanged. The zinc concentration of the lower chamber culture medium at each time point was determined by flame atomic absorption spectrometry. The formula for calculating the zinc transport amount at each time point is as follows:

[0065]

[0066] In the formula, T n represents the total zinc transport amount at each time point (μg); 1.5 represents the volume of the lower complete culture medium (mL); C n represents the zinc concentration (μg / mL) in the samples obtained at different time points; 0.40 represents the zinc concentration in the complete culture medium itself (μg / mL); 1 represents the volume of the sample obtained from the lower layer (mL); n represents the independent variable, which can be 1, 2, 3, 4, representing 4 different time points, namely 30min, 60min, 120min, and 180min.

[0067] Zinc transport activity test results Figure 5 As shown (values ​​are expressed as mean ± standard deviation, n = 3. The mean values ​​of different letters (a-f) in each column are significantly different (p < 0.05)). Within 180 min, the control group (only Zn was added) 2+) and the sample group (YPVEPF) showed an increasing trend with time. When transported to 30min, the zinc transport amount of YPVEPF was significantly increased by 473.34% compared with the control group, which shows that YPVEPF quickly exerted the activity of promoting zinc transport after the start of transport, and increased the absorption of zinc by the Caco-2 cell monolayer. When transported to 60min and 120min, the zinc transport amount of YPVEPF was increased by 207.10% and 104.63% respectively compared with the control group; when transported to 180min, the zinc transport amount of YPVEPF was increased by 99.80% compared with the control group, and the difference in zinc transport amount between YPVEPF and the control group reached the maximum value. The above shows that the intestinal zinc absorption-promoting peptide derived from bovine milk β-casein of the present invention has good activity in enhancing intestinal zinc absorption.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A bovine milk β-casein-derived peptide for promoting intestinal zinc absorption, characterized in that: The peptide promoting intestinal zinc absorption is the sequence shown in SEQ ID NO:

1.

2. Use of the intestinal zinc absorption promoting peptide according to claim 1 in the preparation of products capable of promoting zinc absorption.

3. Use of the intestinal zinc absorption promoting peptide according to claim 1 in the preparation of zinc supplements.

4. Use of the peptide for promoting intestinal zinc absorption according to claim 1 in preparing an adjuvant for zinc supplements.

5. Use of the composition of the intestinal zinc absorption promoting peptide and zinc agent according to claim 1 in the preparation of zinc supplements.

6. The use according to claim 5, characterized in that The zinc agent is a zinc salt.

7. The use according to claim 6, characterized in that The zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

8. The use according to claim 7, characterized in that The inorganic zinc salt is at least one of zinc sulfate, zinc chloride or zinc nitrate.

9. The use according to claim 7, characterized in that: The organic zinc salt is at least one of zinc gluconate, licorice zinc, zinc acetate, zinc citrate, amino acid zinc or zinc lactate.

10. A composition, characterized in that It comprises the intestinal zinc absorption promoting peptide as claimed in claim 1.

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