A walnut polypeptide and application thereof in preparation of calcium chelating agent

Abstract

The application discloses a walnut polypeptide, and the amino acid sequence of the walnut polypeptide is Phe-Phe-Asp-Gln-Gln-Glu. The walnut polypeptide FFDQQE can form a stable peptide calcium chelate. It is proved through structural characterization and cytotoxicity experiments that the calcium chelating capacity of the walnut polypeptide FFDQQE can be further enhanced by ultrasonic-assisted chelation. The walnut polypeptide FFDQQE can be used in the preparation of a calcium chelating agent, and the walnut polypeptide FFDQQE has no toxic side effects on the body and is safe and edible.

Description

Technical Field

[0001] This invention relates to a walnut polypeptide and its application in the preparation of calcium chelating agents, belonging to the field of active peptide technology. Background Technology

[0002] Calcium is an essential element for the human body, and almost all life activities require its participation. Maintaining a balanced calcium metabolism is crucial for maintaining human health. Multiple national dietary surveys have shown that calcium intake is generally low across different age groups and occupations in my country, with an average daily intake of only 400mg per person, just 50% of the recommended intake, indicating a serious calcium deficiency. Calcium deficiency can cause bodily abnormalities and increase susceptibility to diseases. For example, it can affect the development of infants and children, increase the risk of gestational hypertension in pregnant women, and lead to osteoporosis in middle-aged and elderly individuals. Severe calcium deficiency can even damage the immune system, becoming a serious public health problem. While traditional calcium supplements on the market (such as inorganic calcium salts, organic acid calcium salts, and amino acid calcium) have some effect in preventing and combating calcium deficiency, they generally suffer from low bioavailability, poor stability, and gastrointestinal discomfort, making them unsuitable for long-term use.

[0003] Peptide-chelated calcium has been proven to have excellent calcium absorption and bioavailability, making it a promising novel calcium supplement. Peptides are essential for maintaining life activities, and due to their high activity, high selectivity, and minimal side effects, peptide-chelated calcium has become a research hotspot in calcium supplementation. However, a major limitation in the application of peptide-calcium chelates lies in their stability in the gastrointestinal tract. Calcium readily forms calcium phosphate precipitates under the alkaline conditions of the intestine, thus reducing its bioavailability. To overcome this problem, ultrasound-assisted chelation has emerged as a novel strategy to improve the efficiency and stability of peptide-metal chelation. The ultrasonic cavitation effect generates localized high temperature and pressure, promoting peptide chain unfolding and exposing binding sites. This not only enhances calcium binding capacity but also alters spatial conformation and intermolecular interactions, thereby strengthening the thermal stability, pH stability, and digestive stability of the chelate.

[0004] Walnuts are rich in high-quality protein, but their utilization through deep processing is insufficient. The in-depth development and utilization of walnut protein and the development of new products are current research hotspots, with a significant amount of research focusing on the enzymatic hydrolysis of walnut protein to prepare bioactive peptides. Bioactive peptides typically exhibit different biological activities due to their varying structures. Summary of the Invention

[0005] Based on this, the present invention provides a walnut polypeptide with the amino acid sequence Phe-Phe-Asp-Gln-Gln-Glu (FFDQQE). The walnut polypeptide of the present invention has the ability to chelate metal calcium and can be used to prepare calcium chelating agents.

[0006] The objective of this invention is achieved through the following technical solution: 1. Weigh 10g of walnut protein powder, add 190mL of ultrapure water and stir well. Place the mixture in an ultrasonic instrument for pretreatment. After ultrasonication, adjust the pH of the mixture to 7.0, add trypsin for enzymatic hydrolysis, remove the mixture and inactivate the enzyme in a boiling water bath. Centrifuge and collect the supernatant. Centrifuge the supernatant again through a 1kDa ultrafiltration centrifuge tube to obtain the enzymatic hydrolysis product with a molecular weight <1kDa. Freeze-dry the product and store it at -20℃ for later use.

[0007] 2. Using an ultra-high performance liquid chromatography-quadrupole-orbitrap-mass spectrometer (UPLC-Q-rbitrap-MS) 2 To identify the polypeptide sequences in enzymatic digests with molecular weights <1 kDa, Autodock Vina 1.5.7 software was used to correlate the identified polypeptide sequences with Ca2+. 2+ and Ca 2+ Molecular docking was performed on the channel protein TRPV6. Based on the docking results, the three peptides with the highest absolute binding energies were selected as peptides with high chelation potential. After experimental verification, the peptide with the strongest chelation ability, FFDQQE, was selected. A large amount of this peptide was artificially synthesized for subsequent experiments. The molecular weight of the peptide FFDQQE is 813.35 Da.

[0008] 3. Verification of the calcium chelating ability of peptide FFDQQE (1) Add walnut peptide FFDQQE to ultrapure water and stir to mix. Adjust the pH to 8.0. Add CaCl2 at a peptide-calcium mass ratio of 3:1 and then use ultrasound to assist chelation to obtain a chelation solution. (2) Place the chelate in a 40℃ constant temperature water bath and stir magnetically for 30 min. After the reaction is complete, slowly add anhydrous ethanol, let stand overnight and then centrifuge to obtain the chelate precipitate. After centrifugation, wash the chelate precipitate repeatedly with anhydrous ethanol, freeze dry, and then obtain the walnut polypeptide-calcium chelate. Store at -20℃ for later use. The beneficial effects of this invention are: (1) The walnut polypeptide FFDQQE provided by the present invention has a high calcium chelating ability, and ultrasound-assisted chelation can enhance the calcium chelating ability of FFDQQE. (2) The polypeptide provided by the present invention not only has a good calcium binding capacity but also does not produce toxic side effects on the body, making it an excellent choice for preparing calcium supplements. Attached Figure Description

[0009] Figure 1 The ultraviolet spectra of FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca are shown. Figure 2 The fluorescence spectra of FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca are shown. Figure 3Infrared spectra of FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca; Figure 4 The energy dispersive spectrum (top) and SEM (bottom) of FFDQQE are shown. Figure 5 The energy dispersive spectrum (top) and SEM (bottom) of FFDQQE-Ca are shown. Figure 6 The energy dispersive spectrum (top) and SEM (bottom) of U-FFDQQE-Ca are shown. Figure 7 X-ray diffraction patterns of FFDQQE-Ca and U-FFDQQE-Ca; Figure 8 Figure A shows the temperature (Figure A) and acid-base stability (Figure B) of FFDQQE-Ca and U-FFDQQE-Ca. Figure 9 The results show the cytotoxicity assays for FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca. Figure 10 Results of calcium transport in Caco-2 cells. Detailed Implementation

[0010] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the content described herein; In the examples described below, cell culture was performed in DMEM medium (containing 10% fetal bovine serum), and the cells were cultured in an incubator containing 5% CO2 at 37 °C, and passaged every 24 h.

[0011] Example 1: Obtaining walnut polypeptide FFDQQE 1. Weigh 10g of walnut protein powder, add 190mL of ultrapure water and stir well. Adjust the pH to 7.0 with 1mol / L HCl and NaOH. Add trypsin and incubate with magnetic stirring at 40℃ for 4h. Remove the product and inactivate the enzyme in a boiling water bath for 10min. Centrifuge at 8000r / min for 10min and collect the supernatant. Filter the supernatant through a 0.22μm filter membrane and add it to a 1kDa ultrafiltration centrifuge tube. Centrifuge at 5000r / min for 30min to obtain the enzymatic hydrolysis product with a molecular weight <1kDa. Freeze-dry and store at -20℃ for later use.

[0012] 2. Use UPLC-Q-rbitrap-MS 2Identification steps (1) were performed on the polypeptide sequences in the enzymatic hydrolysis products with a molecular weight <1 kDa, under the following conditions: mobile phase A was acetonitrile containing 0.1% formic acid, and mobile phase B was ultrapure water containing 0.1% formic acid; the flow rate was 0.2 mL / min, the elution gradient was 5-95% A, and the duration was 30 minutes; the mass spectrometry conditions were positive ion scanning mode (ESI+); the spray voltage was 3.2 kV; the capillary and drying temperatures were set to 350 °C; the data acquisition range was 200-2000 m / z; and the mass spectrometry data scanning mode was Full MS-dd MS. 2 The resolution of the first-stage mass spectrometer is 70,000, and the resolution of the second-stage mass spectrometer is 35,000.

[0013] 3. The identified polypeptide sequences were compared with Ca using Autodock Vina 1.5.7 software. 2+ and Ca 2+ Molecular docking was performed on the channel protein TRPV6, and the binding energy was calculated to screen out three peptides with high chelation potential. After experimental verification, the peptide with the strongest chelation ability was selected. The results showed that the peptide FFDQQE had the strongest chelation ability of 68.72±0.44μg / mg.

[0014] 4. Chelation experiment of peptide FFDQQE with calcium ions (1) Add walnut peptide FFDQQE to ultrapure water at a concentration of 1 mg / mL and stir to mix. Adjust the pH to 8.0. Add CaCl2 at a concentration of 5 mmol / L at a peptide-calcium mass ratio of 3:1. Then, sonicate for 5 min at 300 W and 4 °C to obtain chelate solution. (2) After placing the chelating solution in a 40℃ constant temperature water bath and stirring magnetically for 30 min, stop the chelation reaction, slowly add 8 times its volume of anhydrous ethanol to the chelating solution, let stand overnight, centrifuge at 8000 r / min for 10 min to obtain the chelate precipitate, wash the chelate precipitate repeatedly with anhydrous ethanol, freeze dry, and obtain the walnut polypeptide-calcium chelate U-FFDQQE-Ca; At the same time, walnut polypeptide-calcium chelate FFDQQE-Ca was prepared using the same method as above, except that ultrasonic chelation was not performed in step (1). The results of calcium binding capacity of walnut polypeptide-calcium chelate are shown in the table below. The results show that walnut polypeptide FFDQQE has a high calcium chelating capacity, and ultrasound-assisted chelation can enhance the calcium chelating capacity of FFDQQE. Table 1 Calcium binding capacity .

[0015] Example 2: Characterization of FFDQQE, FFDQQE-Ca and U-FFDQQE-Ca (1) Ultraviolet-visible spectral scanning FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca powders were dispersed in ultrapure water to prepare solutions with a concentration of 0.5 mg / mL. The ultraviolet-visible absorption spectrophotometer was used to scan the spectra, with a scanning interval of 1 nm and a wavelength range of 200–420 nm. The absorbance of ultrapure water was used as a blank control. The results are as follows: Figure 1 As shown, ultraviolet spectroscopy reveals that ultrasound further alters the chemical structure and promotes the exposure of carboxyl oxygen at the metal-binding sites of calcium ions and aspartic acid and glutamic acid in the peptide, thereby promoting the chelation reaction.

[0016] (2) Fluorescence spectroscopy scanning The fluorescence spectrophotometer was set to an excitation wavelength of 280 nm and an emission wavelength of 230–400 nm. FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca powders were dissolved in ultrapure water at a concentration of 0.05 mg / mL, and the fluorescence intensity was measured. The results are as follows: Figure 2 As shown, fluorescence quenching is mainly attributed to the coordination binding of calcium ions with the carboxyl oxygen and N-terminal amino groups of the aspartic acid (D) and glutamic acid (E) side chains in the peptide chain. The spectral changes of the ultrasound-assisted chelate samples compared to those chelated by conventional methods are more significant, further demonstrating that ultrasound can significantly enhance calcium chelation ability.

[0017] (3) Infrared spectral scanning 1 mg each of FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca, along with 200 mg of dry KBr, were added to a mortar and ground thoroughly multiple times. The resulting powder was then pressed into discs. The wavenumber range for each sample was set to 4000 cm⁻¹. -1 Up to 400cm -1 Fourier transform infrared spectrometer was used at a focal length of 4 cm⁻¹. -1 Infrared spectra were obtained by performing 32 scans at high resolution, and the results are as follows: Figure 3 As shown, this confirms that calcium ions coordinate with the carboxyl oxygen and amino nitrogen atoms on the peptide chain. Compared with conventional methods, sonication exposes more of the key functional groups (NH, C=O, -COOH), thereby promoting the chelation reaction.

[0018] (4) Scanning electron microscope The microstructures of FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca were analyzed using scanning electron microscopy. After gold plating, the powder samples were then photographed and tested using a Zeiss Sigma 300 scanning electron microscope. The accelerating voltage for morphology photography was 3 kV, and the accelerating voltage for energy spectrum mapping photography was 15 kV. The detector was an SE2 secondary electron detector. The results are as follows Figure 4-6 As shown, ultrasonic treatment makes the chelate more homogeneous and smaller in size, indicating that ultrasound promotes structural remodeling and enhances the homogeneity of the complex.

[0019] X-ray diffraction FFDQQE, FFDQQE-Ca, and U-FFDQQE-Ca powders were placed on a glass slide. The test range was 10-80°, the scan rate was 2° / min, the light source was Cu-Kα rays, the tube voltage was 40kV, and the current was 15mA. The measurement results are as follows: Figure 7 As shown, with Ca 2+ After chelation, FFDQQE-Ca exhibited sharp and narrow diffraction peaks, while the U-FFDQQE-Ca diffraction peaks were in almost the same position, but with varying relative intensities. These observations indicate that calcium incorporation enhances the crystallinity of the peptide, confirming the effect of FFDQQE on calcium... 2+ The effective combination of these properties, and the ability of ultrasound to form crystal nuclei more uniformly.

[0020] (6) Temperature and acid-base stability analysis FFDQQE-Ca and U-FFDQQE-Ca solutions with a concentration of 5 mg / mL were reacted at different temperatures (20, 40, 60, 80, 100 ℃) and different pH values ​​(2, 4, 6, 8, 10) for 2 h. Then, the mixture was centrifuged at 8000 rpm for 15 min, and the supernatant was mixed with 8 times its volume of anhydrous ethanol. After precipitation for 1 hour, the mixture was centrifuged at 8000 rpm for 15 min. The Ca content in the precipitate was... 2+ The quality was determined using the o-cresolphthalein-tyrosol colorimetric method, Ca 2+ The retention rate is calculated using the following formula.

[0021]

[0022] Where m1 is the mass of calcium ions in the precipitate (μg) and m2 is the mass of calcium ions in the sample solution (μg). The results are as follows Figure 8 As shown, the chelation effect of walnut peptide FFDQQE significantly enhances the Ca2+ chelation. 2+ The retention rates under different temperature and pH conditions were as follows: at 20-100℃, the calcium retention rate was above 80%; at pH 6-10, the calcium retention rate was above 60%. Ultrasound promoted the chelate to have better temperature and acid-base stability.

[0023] (7) Cytotoxicity Cell viability was determined using the CCK-8 assay. Caco-2 cells in the logarithmic growth phase were inoculated at 5 × 10⁻⁶ cells / cells. 4Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were incubated for 24 h. The old culture medium was discarded, and complete culture medium of different concentrations of the test samples was added to each well and incubated for 24 h. Then, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. Cell viability was calculated by measuring the absorbance at 490 nm using a microplate reader, and cell survival rate was calculated. A control group was set up, which only added complete culture medium and CCK-8. ; The results are as follows Figure 9 As shown, at concentrations of 100-500 μg / mL, the cell viability of the walnut polypeptide-calcium chelate treatment group was higher than that of the inorganic calcium salt treatment group and the peptide group, indicating that the walnut polypeptide-calcium chelate had no toxicity to cells.

[0024] (8) Study on calcium transport in Caco-2 cells Based on cytotoxicity experiments, an appropriate drug concentration was selected to investigate its effect on calcium transport. The peptide-calcium chelate used was at a concentration of 200 μg / mL. After 21 days of incubation, the culture medium was discarded, and the monolayer cells were immediately washed twice with Hanks' balanced salt solution (HBSS, calcium and magnesium-free). 0.5 mL of HBSS was added to the AP side and 1.5 mL to the BL side. After incubation for 20 min, the HBSS was discarded. 0.5 mL of each drug group was added to the AP side, and 1.5 mL of HBSS was added to the BL side. At different time points (30, 60, 120, and 180 min), 1 mL of HBSS was collected from the lateral side of the substrate to determine calcium content. Simultaneously, 1 mL of fresh HBSS buffer was added to the lateral side of the substrate to maintain a constant volume. Results are as follows: Figure 10 As shown, the calcium transport effect in the ultrasonic treatment group was significantly stronger than that in the other groups.

Claims

1. A walnut polypeptide having the amino acid sequence Phe-Phe-Asp-Gln-Gln-Glu.

2. The application of the walnut polypeptide according to claim 1 in the preparation of calcium chelating agents.

3. The application according to claim 1, characterized in that: Calcium chelating agents were prepared by ultrasound-assisted chelation.

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