A milk protein peptide with zinc chelating activity and its application in preparing zinc supplements

Abstract

The present invention relates to a milk protein peptide with zinc chelating activity and its application in the preparation of zinc supplements. The sequence of the milk protein peptide is: EMPFPK (Glu-Met-Pro-Phe-Pro-Lys) and / or EAMAPKHKEMPFPK (Glu-Ala-Met-Ala-Pro-Lys-His-Lys-Glu-Met-Pro-Phe-Pro-Lys). The milk protein peptide not only has a strong zinc chelating ability (chelating rate reaches more than 64%), but also can increase the transport amount of zinc by human intestinal adenocarcinoma cell Caco-2 cell monolayer, and has a strong intestinal zinc absorption promoting activity. In addition, the milk protein peptide of the present invention is derived from milk, is non-toxic, and has high safety.

Description

Technical Field

[0001] The present invention relates to the field of small molecule peptides, and more particularly to a milk protein peptide with zinc chelating activity and application thereof in the preparation of zinc supplements. 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 affects human health and nutrition by participating in various biological processes. For example, zinc deficiency can lead to growth retardation and impaired immune function. Zinc's role in the body depends on its bioavailability in tissues, which is influenced by the efficiency of intestinal absorption of zinc from food. However, zinc ions readily bind to dietary fiber and phytic acid to form insoluble complexes. This binding interferes with intestinal zinc absorption, thereby reducing zinc bioavailability. Therefore, improving zinc absorption and bioavailability is essential.

[0003] While multimineral supplements can be used as a means of zinc supplementation, their poor intestinal stability and potential risk of intestinal disease have limited their widespread use. Another approach to improving zinc absorption is to degrade phytic acid in foods using commercial phytase or through fermentation, grinding, and other methods. However, in actual food processing, such treatment of all phytic acid-containing foods is difficult, limiting the widespread application and development of this approach. Furthermore, food formulations using zinc salts such as zinc sulfate as raw materials have the potential to improve zinc absorption, but this approach may alter the original sensory and physicochemical properties of the food, thereby affecting consumer acceptance.

[0004] Food protein-derived peptides are dietary ingredients with multiple biological activities. Among them, some metal chelate peptides can serve as transport carriers for metal ions such as zinc ions during intestinal absorption, thereby improving the bioavailability of dietary zinc and reducing the risk of zinc deficiency. 2+ The combination forms a complex that has high solubility, good stability, and high safety in the intestinal environment. For example, Meng et al. (DOI: 10.1016 / j.foodchem.2021.129043) prepared and characterized zinc chelating peptides from tilapia skin collagen, but research on zinc chelating peptides is still relatively limited.

[0005] Therefore, it is of great significance to develop more peptides with zinc chelating activity. Summary of the Invention

[0006] The primary objective of the present invention is to overcome the current shortage of zinc chelating peptides by providing a milk protein peptide with zinc chelating activity. This milk protein peptide not only exhibits strong zinc chelation (achieving a chelation rate exceeding 64%) but also enhances zinc transport by human intestinal adenocarcinoma cell monolayers (Caco-2 cells), demonstrating strong intestinal zinc absorption-promoting activity. Furthermore, this milk protein peptide is derived from milk, is non-toxic, and is highly safe.

[0007] A further object of the present invention is to provide the use of the above-mentioned milk protein peptide in the preparation of a product capable of promoting zinc absorption.

[0008] Another object of the present invention is to provide the use of the above-mentioned milk protein peptide in preparing an adjuvant for zinc supplements.

[0009] Another object of the present invention is to provide a use of the composition of the milk protein peptide and zinc agent in the preparation of a zinc supplement.

[0010] Another object of the present invention is to provide a composition comprising the above-mentioned milk protein peptide.

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

[0012] A milk protein peptide with zinc chelating activity, wherein the milk protein peptide is the sequence shown in SEQ ID NO: 1 and / or the sequence shown in SEQ ID NO: 2.

[0013] The milk protein peptide of the present invention is extracted from casein in cow's milk. The amino acid sequence of the milk protein peptide is as follows: EMPFPK (Glu-Met-Pro-Phe-Pro-Lys) and / or EAMAPKHKEMPFPK (Glu-Ala-Met-Ala-Pro-Lys-His-Lys-Glu-Met-Pro-Phe-Pro-Lys).

[0014] The inventors of the present invention have found that the milk protein peptide of the present invention can be combined with Zn 2+ EMPFPK binds specifically to Zn through the amide bond in phenylalanine and the carbonyl group of proline. 2+ The zinc binding site of EAMAPKHKEMPFPK is the amide bond in lysine.

[0015] The milk protein peptide of the present invention not only has a strong zinc chelating ability (chelating rate reaches more than 64%), but also can enhance the zinc transport capacity of human intestinal adenocarcinoma cell Caco-2 cell monolayer, and has a strong intestinal zinc absorption promoting activity.

[0016] In addition, the milk protein peptide of the present invention is derived from milk, is non-toxic and highly safe.

[0017] The milk protein peptides 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 can be used, batch production is possible, and the synthesized milk protein peptides have high purity, stable product quality, and market competitiveness.

[0018] Preferably, the milk protein peptide is the sequence shown in SEQ ID NO: 2.

[0019] The present invention also protects the use of the milk protein peptide in preparing a product capable of promoting zinc absorption.

[0020] The present invention also protects the use of the milk protein peptide in preparing an adjuvant for zinc supplements.

[0021] The use of the above-mentioned combination of milk protein peptide and zinc agent in the preparation of zinc supplements is also within the scope of protection of the present invention.

[0022] Preferably, the zinc agent is a zinc salt.

[0023] More preferably, the zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

[0024] More preferably, the inorganic zinc salt is at least one of zinc sulfate, zinc chloride or zinc nitrate.

[0025] 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.

[0026] A composition comprising the above-mentioned milk protein peptide.

[0027] Preferably, the content of milk protein peptide in the composition is 25-1000 μg / mL.

[0028] Preferably, the composition further comprises a zinc agent.

[0029] More preferably, the zinc agent is a zinc salt.

[0030] More preferably, the zinc salt is one or both of an inorganic zinc salt and an organic zinc salt.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention provides a milk protein peptide with zinc chelating activity. This milk protein peptide not only exhibits strong zinc chelation (achieving a chelation rate exceeding 64%) but also enhances zinc transport by human intestinal adenocarcinoma cell monolayers (Caco-2 cells), demonstrating strong intestinal zinc absorption-promoting activity. Furthermore, the milk protein peptide is derived from milk, making it non-toxic and highly safe.

[0033] The milk protein peptides 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 can be used, batch production is possible, and the synthesized milk protein peptides have high purity, stable product quality, and market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Milk protein peptide and Zn 2+ Theoretical binding mode diagram, where Figure A is EMPFPK-Zn 2+ Diagram of the complex and its action site; Figure B is EAMAPKHKEMPFPK-Zn 2+ Diagram of the complex and its action site.

[0035] Figure 2 This is a graph showing the zinc chelation rate of the milk protein peptide of the present invention.

[0036] Figure 3 This is a graph showing the results of the cytotoxicity assay of the milk protein peptide of the present invention.

[0037] Figure 4 This is a graph showing the results of the zinc transport activity of the milk protein peptide of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1 Enzymatic Hydrolysis, Separation, Peptide Identification, and Molecular Docking of Bovine Milk Casein

[0040] 1.1 Enzymatic hydrolysis and separation of casein

[0041] Bovine milk casein was dissolved in deionized water to obtain a casein solution having a mass concentration of 7% bovine milk casein. The pH of the solution was then adjusted to 8.5, and trypsin was added at a mass ratio of 0.6:100 to substrate for enzymatic hydrolysis at 50°C for 3 hours. The solution was then heated to 90°C and kept warm for 10 minutes to inactivate the enzyme. After cooling to room temperature, the pH was adjusted to 4.6 to precipitate the unhydrolyzed casein. The solution was then centrifuged (4000 rpm for 20 minutes), and the supernatant was freeze-dried to obtain a casein hydrolysate.

[0042] Immobilized metal ion affinity chromatography (IMAC) was used to separate casein hydrolysates. Sepharose 6B was added to NaOH solution (1 mol / L) and epichlorohydrin, activated at 40°C for 3.5 h, filtered, and washed with deionized water 7 times. 6 g of dicarboxymethylamine was added to 40 mL of Na2CO3 (2 mol / L) solution, and the pH was adjusted to 13.0 with 6 mol / L NaOH to obtain a dicarboxymethylamine solution. The activated sepharose 6B was mixed with the dicarboxymethylamine solution in a volume ratio of 1:2, stirred at 60°C at a speed of 120 r / min for 8 h, and a dicarboxymethylamine-sepharose 6B solution was obtained. IMAC-Zn 2+ The lower end of a column (200 mm × 10 mm) was clamped with a rubber hose and loaded with dicarboxymethylamine-Sepharose 6B solution. The solution was allowed to settle until distinct layers separated. The clamp was released to allow the aqueous layer to flow off the column. The column was then washed with 5 times the medium volume of deionized water using a constant flow pump, followed by 5 times the medium volume of 0.2 mol / L ZnSO₄ solution, and then 5 times the medium volume of deionized water. 2 mL of a 15 mg / mL casein hydrolysate solution (solvent: water) was then loaded onto the column. The column was initially eluted with deionized water at pH 5.5 at a rate of 1 mL / min. The absorbance of the eluted fractions at 214 nm was monitored until the unbound peptide fraction was completely eluted by the pH 5.5 deionized water. This unbound peptide fraction was designated F1. Subsequently, the bound peptide fraction was eluted with deionized water at pH 2.0 at a rate of 1 mL / min. The eluted fractions were collected, concentrated, and lyophilized. The bound peptide fraction was designated F-IMAC. Deionized water with different pH values was used as the mobile phase during the elution process because the zinc chelating peptide could bind to IMAC-Zn under neutral conditions. 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.

[0043] 1.2 HPLC-MS / MS identification of the peptide composition of F-IMAC

[0044] 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. The sample was eluted with elution buffer (0.1% FA, 60% ACN) into a new centrifuge tube, centrifuged, dried, and then redissolved in Nano-LC mobile phase A, which consisted of 0.1% formic acid solution. 2μL of the sample was loaded onto a nanoViper C18 pre-column (3μm), washed and desalted, and then separated on an Acclaim PepMap C18 analytical column (75μm x 25cm). The F-IMAC peptides were identified using an Orbitrap FusionLumos 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 cell energy (high-energy collision-induced dissociation, HCD): 28 eV, applicable to all precursor ions; dynamic exclusion setting: 6 s.

[0045] PEAKS Studio 8.5 software was used to process and search the raw mass spectrometry files. The database used for this analysis was the Bos taurus (Bovine) casein protein database. 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.

[0046] 1.3 Molecular docking

[0047] Molecular docking technology was used to simulate the interaction between casein peptides and zinc ions, and docking energy was used as the evaluation criterion to screen out casein zinc chelate peptides that bind stably to zinc ions. The specific steps are as follows: First, a new zinc ion model was created using DiscoveryStudio Client 2019 software. Secondly, the peptide sequence was imported into the software and hydrogenated and energy minimized using the protein preparation tool. The zinc ion was set as the ligand, the peptide molecule was set 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.

[0048] The CDOCKER module in the Discovery Studio Client 2019 docking simulation was used to complete the docking of the screened high-activity 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.

[0049] HPLC-MS / MS identified 50 distinct F-IMAC peptides in the F-IMAC fraction. Comparison of the MS / MS spectra of each peptide with database information revealed that these peptides were primarily distributed across four casein subtypes: 20 peptides from αs2-casein, 15 from αs1-casein, nine from β-casein, and six from κ-casein. These peptides ranged in length from 6 to 23 amino acids, with molecular weights below 3 kDa.

[0050] The 50 peptides were ranked according to the binding energy of molecular docking. The lower the binding energy, the better the peptide is at binding to Zn. 2+ The more stable the binding, the better. Three highly active zinc chelating peptides were screened, two of which were EMPFPK and EAMAPKHKEMPFPK. Both of these milk protein peptides are derived from β-casein in bovine milk casein. The sequence information and molecular docking results of the casein zinc chelating peptides identified by NanoLC-MS / MS are shown in Table 1.

[0051] Table 1 Sequence information and molecular docking results of casein zinc chelating peptides identified by NanoLC-MS / MS

[0052]

[0053] Milk protein peptides and Zn 2+ Theoretical combination model such as Figure 1 As shown, Figure A is EMPFPK-Zn 2+ Diagram of the complex and its action site; Figure B is EAMAPKHKEMPFPK-Zn 2+ Complex and action site diagram. 2+ In the complex, Zn 2 + Through the Metal-Acceptor function, it not only combines with the amide bond of phenylalanine (Phe4) to form a Zn-O bond with a bond length of It also combines with the carbonyl oxygen atom in proline (Pro5) to form a Zn-O bond with a bond length of In EAMAPKHKEMPFPK-Zn 2+ In the complex, Zn 2+It combines with the amide bond of lysine (Lys8) through the Metal-Acceptor function to form a Zn-O bond with a bond length of The longer the bond length, the more stable the structure. Therefore, in EMPFPK, Zn 2+ The binding to phenylalanine may be more stable than to proline.

[0054] Example 2 Zinc chelating activity and cytotoxicity

[0055] 2.1 Verification of zinc chelating activity

[0056] The milk protein peptides EMPFPK and EAMAPKHKEMPFPK obtained above were 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.

[0057] The method for determining the zinc ion chelation rate is as follows: dissolve 1 mg / mL of sample in HEPES-KOH buffer (40 mM), 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 ZnSO4 solution (250 μmol / L) to each well, shake and mix, and react at 37°C for 30 minutes, then add 100 μL of PAR solution (2 mmol / L) to terminate the reaction. After 10 minutes, measure the absorbance at 500 nm. EDTA was selected as the positive control, HEPES-KOH buffer was selected as the blank group, and three parallel samples were set for each sample. The formula for calculating the zinc ion chelation rate is as follows:

[0058] 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 group sample, Abs 样品 is the absorbance value of the sample.

[0059] 2.2 MTT assay for cytotoxicity

[0060] Caco-2 cells are human intestinal adenocarcinoma cells that share biochemical and morphological characteristics with intestinal epithelial cells. They have proven to be a valuable tool for studying zinc and other mineral absorption. Caco-2 cells (American Type Culture Collection, Rockville, MD, USA) were seeded into T25 culture flasks. When the cell density reached 70% to 80%, 0.25% EDTA-trypsin was added for digestion at 37°C for 3 minutes. Cells were passaged at a 1:3 ratio. Cells from passages 15 to 30 were used for subsequent experiments. Complete culture medium consisted of 79% EMEM basal medium, 20% fetal bovine serum, and 1% antibiotics (penicillin and streptomycin). Cells were cultured at 37°C, 5% CO2, and 95% relative humidity, with complete culture medium replaced every two days.

[0061] The MTT assay was used to determine whether milk protein 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 seeded in 96-well plates (at a density of 5 × 10 4 / well), and incubate in complete culture medium for 24 hours to allow the cells to fully adhere. Then remove the complete culture medium, add 100 μL of milk protein peptide sample solution (dissolved in complete culture medium) of different concentrations (25, 50, 100, 250, 500, 1000 μg / mL) respectively, use complete culture medium as blank control group, and put it in the incubator for 24 hours. Remove the sample solution, add 100 μL of MTT solution (0.5 mg / mL) and treat for 4 hours, and finally remove the MTT solution. Next, add 100 μL of DMSO to dissolve the purple crystalline product, and measure the absorbance at 490 nm. The cell viability calculation formula is as follows: Cell viability (%) = (absorbance value of sample group / absorbance value of blank control group) × 100%.

[0062] The test results of zinc chelation activity are as follows Figure 2 As shown (values are expressed as mean ± standard deviation, n = 3; there are significant differences (p < 0.05) between the means in each column with different labels (a to c). Figure 2 It can be seen that the zinc ion chelation rate of EMPFPK is 64.29±0.27%, and the zinc ion chelation rate of EAMAPKHKEMPFPK is 69.74±0.05%, both of which are higher than the iron chelation rates of some existing iron chelate peptides, such as rapeseed-derived zinc chelate peptides Ala-Arg (34.7%) and Gly-Lys-Arg (54.5%) (see literature DOI: 10.1016 / j.foodchem.2014.10.030). This shows that the milk protein peptides of the present invention have good zinc chelation activity.

[0063] Cytotoxicity test results Figure 3 As shown, the results of the cytotoxicity test showed that EMPFPK and EAMAPKHKEMPFPK had no toxic effects on Caco-2 cells within the concentration range of 25 to 1000 μg / mL, and subsequent zinc transport activity experiments were performed within this concentration range.

[0064] Example 3 Determination of zinc transport activity

[0065] Before cell inoculation, 0.5 mL of complete medium was added to the upper chamber of a 12-well polyester Transwell membrane culture plate (PET, Corning, 3460), and 1.5 mL of complete medium was added to the lower chamber. The plates were placed in an incubator for 1 hour to allow them to equilibrate. After 1 hour, the complete medium in the upper chamber was removed. When the cell confluence reached 80% of the T25 culture flask, 4 × 10 5 Cells 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.

[0066] The milk protein peptide sample was mixed with the 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 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 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:

[0067]

[0068] 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 nrepresents 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.

[0069] Zinc transport activity test results Figure 4 As shown (values are expressed as mean ± standard deviation, n = 3. The mean values of different letters (a to i) in each column are significantly different (p < 0.05)). Within 180 min, the control group (only Zn was added) 2+ ) and sample groups (EMPFPK and EAMAPKHKEMPFPK) gradually increased their zinc transport. At 30 minutes of transport, the zinc transport of the sample group was significantly higher than that of the control group. Among them, EMPFPK increased by 300.41% compared with the control group, and EAMAPKHKEMPFPK increased by 882.81% compared with the control group. This indicates that EMPFPK and EAMAPKHKEMPFPK quickly exerted their zinc transport-promoting activity after the onset of transport, increasing zinc absorption by Caco-2 cell monolayers. During the transport process, the zinc transport capacity of EAMAPKHKEMPFPK was always significantly higher (p < 0.05) than that of EMPFPK. At 60 minutes of transport, the zinc transport capacity of EMPFPK and EAMAPKHKEMPFPK increased by 185.23% and 318.49% respectively compared with the control group. At 120 minutes of transport, the zinc transport capacity of EMPFPK and EAMAPKHKEMPFPK increased by 71.74% and 145.45% respectively compared with the control group. At 180 minutes of transport, the zinc transport amounts of EMPFPK and EAMAPKHKEMPFPK were increased by 67.40% and 123.37% respectively compared with the control group. The above shows that the milk protein peptide of the present invention has an excellent effect in enhancing intestinal zinc absorption activity.

[0070] 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 milk protein peptide in the preparation of a product capable of promoting zinc absorption, characterized in that: The milk protein peptide is the sequence shown in SEQ ID NO:

2.

2. The use of milk protein peptide in the preparation of zinc supplements, characterized in that: The milk protein peptide is the sequence shown in SEQ ID NO:

2.

3. The use of milk protein peptide in the preparation of an adjuvant for zinc supplements, characterized in that: The milk protein peptide is the sequence shown in SEQ ID NO:

2.

4. Use of a composition of milk protein peptide and zinc agent in the preparation of a zinc supplement, characterized in that: The milk protein peptide is the sequence shown in SEQ ID NO:

2.

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

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