A bovine milk beta-casein-derived intestinal zinc absorption enhancing peptide and its use in the preparation of a zinc supplement

Abstract

The present application relates to a kind of bovine milk beta-casein source intestinal zinc absorption promoting peptide and its application in the preparation of zinc supplement.The sequence of the intestinal zinc absorption promoting peptide is YPVEPF, which is not only non-toxic, high safety, but also can be combined with Zn 2+ Specific binding, strong zinc chelating ability, can significantly improve the zinc transport amount in Caco-2 small intestinal epithelial cell model, thereby having good activity of improving intestinal zinc absorption.The intestinal zinc absorption promoting peptide can be obtained by solid phase synthesis method, so it can be produced on a large scale, and the synthesized intestinal zinc absorption promoting peptide not only has high purity but also stable peptide quality, which can meet the current market demand.

Description

Technical Field

[0001] The present invention relates to the field of small molecule peptides, and more particularly 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 essential nutrients for human health. Zinc is a structural and functional component of numerous transcription factors and enzymes that regulate cell growth, gene expression, and immune responses. Zinc's physiological functions depend on its bioavailability in tissues, which in turn depends on intestinal absorption of dietary zinc. Zinc ions form insoluble complexes with dietary fiber and phytic acid, hindering intestinal absorption and reducing zinc bioavailability. Therefore, improving zinc absorption and bioavailability is essential.

[0003] Existing methods for improving intestinal zinc absorption include adding zinc salts to foods, using complex mineral supplements, or degrading phytic acid in foods. However, these methods may present the following challenges: adding zinc salts to foods can alter their sensory and physicochemical properties; complex mineral supplements have low intestinal stability and can cause intestinal problems; and, in practical processing, it is difficult to treat all foods containing phytic acid.

[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 can bind to divalent metal ions such as zinc and zinc ions to form complexes that have 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 research on zinc chelate peptides is still relatively limited.

[0005] Therefore, more zinc chelating 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 current problem of the lack of zinc chelating peptides and provide a bovine milk β-casein-derived intestinal zinc absorption-promoting peptide. The intestinal zinc absorption-promoting peptide is not only non-toxic and highly safe, but also can be combined 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 adjuvant for zinc supplements.

[0009] Another object of the present invention is to provide a 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 purpose of the present invention is achieved through the following technical solutions:

[0012] A bovine milk β-casein-derived peptide for promoting intestinal zinc absorption, wherein the peptide for promoting intestinal zinc absorption has the 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 is non-toxic and highly safe, and 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 and separation of bovine casein, or by solid-phase synthesis. Since the solid-phase synthesis method is available, large-scale production is possible. Furthermore, the synthesized intestinal zinc absorption-promoting peptide has high purity and stable quality, meeting 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 scope of protection 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] More 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, glycyrrhizic zinc, 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 to 1000 μg / mL.

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

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

[0026] More 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 peptide that promotes intestinal zinc absorption from bovine milk β-casein. The peptide is non-toxic and highly safe and can be used in combination 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 and separation of bovine casein, or by solid-phase synthesis. Since the solid-phase synthesis method is available, large-scale production is possible. Furthermore, the synthesized intestinal zinc absorption-promoting peptide has high purity and stable quality, meeting 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 This is a graph showing the results 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] Bovine milk casein was dissolved in deionized water to a concentration of 7%. The initial pH was adjusted to 8.5, and trypsin was added at 0.6% of the substrate mass. Enzymatic hydrolysis was performed in a water bath at 50°C for 3 hours. After the hydrolysis was completed, the solution was heated at 90°C for 10 minutes to inactivate the enzyme, cooled to room temperature, and the pH was adjusted to 4.6 to precipitate the unhydrolyzed casein. The solution was then centrifuged at 4000 rpm for 20 minutes. The supernatant was freeze-dried to obtain the casein hydrolysate.

[0039] 1.2 Separation of casein hydrolysate

[0040] Casein hydrolysates were separated by IMAC. 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 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 lower hose of the column (200mm×10mm) was clamped and loaded with IDA-Sepharose 6B. The column was allowed to precipitate until distinct layers were separated. The clamp was released to allow the aqueous layer to flow out of the column. The column was then washed with 5 times the volume of the medium using a constant flow pump using deionized water. This was followed by a rinse with 5 times the volume of the medium using a ZnSO4 solution (0.2mol / L), and then with 5 times the volume of the medium using deionized water. Casein hydrolysate was dissolved in deionized water to obtain a 15mg / mL casein hydrolysate solution. 2mL of the casein hydrolysate solution was then loaded onto the column. The column was first eluted with deionized water at pH 5.5 at a rate of 1mL / min. The absorbance of the eluted fraction at a wavelength of 214nm was monitored until the unbound peptide fraction was completely eluted with deionized water at pH 5.5. The unbound peptide fraction was designated F1. Subsequently, the bound peptide fraction was eluted with deionized water at pH 2.0 at a flow rate of 1 mL / min, the eluted fractions were collected, concentrated, and lyophilized, and the bound peptide fraction was named F-IMAC. Deionized water of different pH values ​​was used as the mobile phase during the elution process because the peptide with zinc chelating activity under neutral conditions could 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, followed by elution with elution buffer (0.1% FA, 60% ACN) into a new centrifuge tube. The sample solution was centrifuged and concentrated to dryness. After desalting, the sample was redissolved in Nano-LC mobile phase A (0.1% FA / water). 2μL of the sample was loaded onto a nanoViper C18 pre-column (3μm), washed and desalted, and then separated on an analytical column (Acclaim PepMap C18, 75μm x 25cm). F-IMAC peptides were identified using an OrbitrapFusion Lumos mass spectrometer coupled 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: data-dependent acquisition mode (DDA); primary mass spectrometry scanning resolution was 70,000, the scanning range was 100-1500 m / z, and the maximum injection time was 100 ms; the maximum injection time of secondary mass spectrometry ions was 50 ms; collision cell energy (high-energy collision-induced dissociation, HCD): 28 eV, applicable to all precursor ions; dynamic exclusion setting: 6 s.

[0043] PEAKS Studio 8.5 software was used to process and search the raw mass spectrometry files. The database used was the Bos taurus (Bovine) casein protein database downloaded from Uniprot. The search parameters were set as follows: a 10 ppm mass tolerance for the primary mass spectrometry and a 0.05 Da mass tolerance for the secondary mass spectrometry.

[0044] A total of 50 F-IMAC peptides were identified from the F-IMAC components. Based on the MS / MS spectra of each peptide and the comparison with the database, the F-IMAC peptides were found to be 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 50 identified peptides ranged in length from 6 to 23 amino acids, and all had molecular weights less than 3 kDa.

[0045] Example 2 Molecular docking

[0046] Molecular docking was used to simulate the binding of peptides to zinc ions. Using docking binding energy as a metric, casein zinc chelate peptides with stable zinc ion chelation were further screened. First, a new zinc ion model was created using Discovery Studio Client 2019. Next, the peptide sequence was imported into a new window and hydrogenated and minimized using the Protein Preparation tool. The zinc ion was then selected as the ligand and the peptide as the receptor, with the entire peptide defined as the docking site. Finally, the Calculate Docking Binding Energy function in the Docking module was used to predict the binding energy of the interaction.

[0047] The CDOCKER module in the Discovery Studio Client 2019 docking simulation was used to complete the docking of the selected highly active zinc chelating peptides with Zn 2+ The docking of the zinc chelation mechanism can be predicted by determining the amino acid site of action, the type of binding force, the bond length and other information.

[0048] The 50 peptides were ranked according to the molecular docking binding energy. The lower the binding energy, the better the peptide is at binding to Zn. 2+ 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 glutamic acid (Glu4). Specifically, the carbonyl oxygen atom in proline binds to Zn through the Metal-Acceptor interaction. 2+ Specific binding, forming a Zn-O bond with a bond length of The carboxyl oxygen atom in glutamic acid 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 Bovine Milk β-Casein-Derived Intestinal Zinc Absorption-Promoting Peptides

[0053] 3.1 Verification of zinc chelating activity

[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 greater 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 sample in 40 mM HEPES-KOH buffer, take 175 μL of 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, and react at 37°C for 30 minutes, then add 100 μL of 2 mmol / L PAR solution to terminate the reaction. After 10 minutes, measure the absorbance at 500 nm using a microplate reader. EDTA was selected as the positive control. HEPES-KOH buffer was selected as the blank group, and each sample was repeated three times. 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; *** indicates p < 0.001, significant difference). The zinc ion chelation rate of YPVEPF was 55.81 ± 0.83%, which is higher than the zinc chelation rates of some existing zinc chelating peptides, such as the zinc chelation rate of wheat germ protein-derived zinc chelating peptide (Asn-Ala-Pro-Leu-Pro-Pro-Pro-Leu-Lys-His) of approximately 15.2% (see literature DOI: 10.1016 / j.jff.2014.10.030). This indicates that the intestinal zinc absorption-promoting peptide of the present invention has good zinc chelation activity.

[0058] 3.2 Cytotoxicity determination by MTT assay

[0059] Caco-2 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were seeded 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 from passages 15 to 30 were selected for subsequent experiments. The complete culture medium was prepared from 79% EMEM basal medium, 20% fetal bovine serum, and 1% antibiotics (penicillin and streptomycin). The cells were cultured at 37°C, 5% CO2, and 95% relative humidity, and the complete culture medium was replaced every 2 days.

[0060] The MTT assay was used to determine whether the intestinal zinc absorption promoting peptide had toxicity 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 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated in complete medium for 24 hours to allow complete cell attachment. The complete medium was then removed, and 100 μL of YPVEPF sample solution (dissolved in complete medium) at different concentrations (25, 50, 100, 250, 500, 1000 μg / mL) was added. The complete medium was used as a blank control group and the cells were placed in an incubator for 24 hours. The sample solution was removed, and 100 μL of 0.5 mg / mL MTT solution was added and treated 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 was calculated as follows: Cell viability (%) = (absorbance of sample group / absorbance of 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 Bovine Milk β-Casein-Derived Intestinal Zinc Absorption-Promoting Peptides

[0063] Before cell inoculation, 0.5 mL and 1.5 mL of complete culture 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 hour to allow equilibrium. After 1 hour, the complete culture medium in the upper chamber was removed. When the cell confluence reached 80% of the T25 culture flask, 4 × 10 5Cells were seeded into the upper chamber of a 12-well polyester Transwell 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 assessed by measuring the transmembrane electrical resistance (TEER). When the TEER value was greater than 500 Ω / 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 began, a new 12-well culture plate was prepared and 1.5 mL of complete culture medium was added. The Caco-2 monolayer 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 were 2+ The concentration was 50 μg / mL. After transport began, 1 mL of culture medium was removed from the lower chamber of the Transwell plate at 30, 60, 120, and 180 minutes, and 1 mL of culture medium was quickly added to maintain the volume of the transport system constant. The zinc concentration in the lower chamber culture medium at each time point was determined by flame atomic absorption spectroscopy. The formula for calculating zinc transport 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 means of different letters (a to f) in each column are significantly different (p < 0.05)). 2+) and the sample group (YPVEPF) showed an increasing trend with the increase of 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 its activity in 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 a 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 the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Use of an intestinal zinc absorption promoting peptide in the preparation of a product capable of promoting zinc absorption, characterized in that: The peptide promoting intestinal zinc absorption is the sequence shown in SEQ ID NO:

1.

2. Use of an intestinal zinc absorption promoting peptide in the preparation of an adjuvant for zinc supplements, characterized in that: The peptide promoting intestinal zinc absorption is the sequence shown in SEQ ID NO:

1.

3. Use of a combination of an intestinal zinc absorption promoting peptide and a zinc agent in the preparation of a zinc supplement, characterized in that: The peptide promoting intestinal zinc absorption is the sequence shown in SEQ ID NO:

1.

4. The use according to claim 3, characterized in that The zinc agent is a zinc salt.

5. The use according to claim 4, characterized in that The zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

6. The use according to claim 5, characterized in that The inorganic zinc salt is at least one of zinc sulfate, zinc chloride or zinc nitrate.

7. The use according to claim 5, characterized in that The organic zinc salt is at least one of zinc gluconate, glycyrrhizic zinc, zinc acetate, zinc citrate, amino acid zinc or zinc lactate.

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