Calcium ion sequestering peptides, methods of making and using the same, peptide calcium chelates, compositions

Abstract

The present application relates to the technical field of bioactive peptides, in particular to calcium ion chelating peptides, a preparation method and application thereof, peptide calcium chelates and compositions. The present application uses gluten as raw material, and through enzymatic purification and separation, low oligopeptides GVDDQPLFH and PQFEE are identified, both of which have high calcium ion chelating activity, higher than 75%, excellent calcium loading capacity, and potential for developing new biological calcium supplements. The experiment verifies that the peptide calcium chelate prepared from the separated and identified low oligopeptide GVDDQPLFH has excellent calcium supplement effect and higher bioavailability compared with traditional calcium supplement reagents, and can be used for preparing calcium supplement drugs or health foods.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, specifically to calcium ion chelating peptides, their preparation methods and applications, peptide-calcium chelates, and compositions. Background Technology

[0002] Calcium is an essential nutrient for the human body, playing a vital role in various physiological and metabolic activities, such as muscle contraction, blood clotting, nerve transmission, and bone development. Insufficient calcium intake often leads to metabolic bone diseases, such as rickets, osteomalacia, osteoarthritis, and osteoporosis. Calcium exists in ionic form and easily forms precipitates during gastrointestinal digestion, resulting in significant deficiencies in calcium absorption and bioavailability. Long-term use may produce side effects and increase the burden on the body; therefore, there is a need for calcium supplements that are more easily absorbed and have higher bioavailability. Peptide-calcium chelates, as a novel calcium supplement, possess independent chelation and transport systems, preventing phytate precipitation in the intestines and promoting its absorption by the body. This makes them an excellent carrier for calcium absorption and transport. Peptide-calcium chelates have dual nutritional functions, serving as both a calcium supplement and a source of bioactive peptides for the body. Casein phosphopeptides were the first calcium ion chelating peptides reported and studied. Subsequently, bioactive peptides with calcium ion chelating capabilities have been isolated from proteins from various sources, including grains, animal bones, and seafood.

[0003] In recent years, research on calcium chelating peptides from plant sources, especially agricultural byproducts, has become increasingly popular. Wheat oligopeptides possess various physiological functions and are characterized by low cost, good water solubility, easy absorption, strong bioactivity, and good processability. Wheat oligopeptides typically consist of 2-6 amino acid molecules with a molecular weight below 1000 Da and a balanced amino acid composition. Reports on wheat oligopeptides often indicate their effects on enhancing immunity, anti-oxidation, and regulating the immune system. Natural oligopeptides prepared from wheat are a good source for preparing chelated calcium. Studies have also investigated the isolation of calcium-chelating peptides from wheat protein; however, these typically involve directly chelating a mixture of enzymatically hydrolyzed peptides with calcium ions, resulting in wheat peptides with low calcium chelation rates. Therefore, it is necessary to screen for and prepare oligopeptides with high calcium chelating activity. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide calcium ion chelating peptides with high calcium chelation activity.

[0005] The second objective of this invention is to provide a method for preparing calcium ion chelating peptides that is simple to operate and suitable for industrial application.

[0006] The third objective of this invention is to provide a peptide-calcium chelate, which has excellent calcium supplementation effect, high bioavailability, and can be used as a calcium supplement.

[0007] The fourth objective of this invention is to provide a calcium supplement composition comprising the peptide calcium chelate provided by this invention, which has enhanced absorption and bioavailability.

[0008] The fifth objective of this invention is to provide the application of the calcium ion chelating peptide provided by this invention in the preparation of calcium supplements.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] Calcium ion chelating peptides, including oligopeptide 1 and / or oligopeptide 2; wherein the amino acid sequence of oligopeptide 1 is GVDDQPLFH, as shown in SEQ ID NO.1 of the sequence listing; and the amino acid sequence of oligopeptide 2 is PQFEE, as shown in SEQ ID NO.2 of the sequence listing.

[0011] The preparation method of calcium ion chelating peptides includes the following steps:

[0012] 1) Using gluten powder as raw material, the enzyme hydrolysate was prepared by sequentially hydrolyzing it with neutral protease and alkaline protease;

[0013] 2) The enzymatic hydrolysate was purified to obtain enzymatic hydrolysate with a molecular weight ≤1kDa;

[0014] 3) Oligopeptides with high calcium chelating activity were isolated and identified from the enzymatic hydrolysate prepared in step 2).

[0015] Optionally, the specific method for enzymatic hydrolysis in step 1) is as follows: First, prepare a gluten powder suspension, heat it to 45-50℃, adjust the pH to 7.0, add neutral protease, and after enzymatic hydrolysis, heat it to 55-65℃, adjust the pH to 8.5, add alkaline protease, and after enzymatic hydrolysis, inactivate the enzyme, centrifuge and take the supernatant to obtain the enzymatic hydrolysate.

[0016] In a specific embodiment of the present invention, the mass-volume concentration of the gluten powder suspension is 5-10%; the specific preparation method of the gluten powder suspension is as follows: gluten powder is added to deionized water to dissolve and stir until uniform, ultrasonically mixed at 400W for 20 minutes to fully disperse, and magnetically stirred for 1 hour to form a uniformly dispersed suspension.

[0017] In a specific embodiment of the present invention, the amount of neutral protease is 3000-4000 U / g; the amount of alkaline protease is 6000-8000 U / g; the enzymatic hydrolysis time of neutral protease is 2-3 h; and the enzymatic hydrolysis time of alkaline protease is 3-4 h.

[0018] Optionally, step 2) involves purifying the enzymatic hydrolysate using a filter membrane with a molecular weight cutoff of 1 kDa, at a processing pressure of 1.0-2.0 MPa and a processing capacity of 2-5 L / h, followed by spray drying to obtain the enzymatic hydrolysate.

[0019] Optionally, step 3) involves the following method for separating and identifying oligopeptides with high calcium chelating activity: LC-MS / MS technology is used for separation and identification. The liquid chromatography conditions are as follows: buffer solution A is a 0.1% formic acid aqueous solution, and solution B is a mixed solution of 0.1% formic acid, 80% acetonitrile, and water; the column is equilibrated with 100% solution A; after sample injection, gradient separation is performed through the chromatographic analysis column at a flow rate of 300 nmol / min; the liquid chromatography separation gradient is as follows: 0-2 min, linear gradient of solution B from 2% to 5%; 2-44 min, linear gradient of solution B from 5% to 28%; 44-51 min, linear gradient of solution B from 28% to 40%; 51-53 min, linear gradient of solution B from 40% to 100%; 53-60 min, solution B is maintained at 100%.

[0020] The calcium chelating activities of the isolated peptides were compared and screened to identify the oligopeptides with high calcium chelating activities.

[0021] Before liquid chromatography injection separation, the sample preparation process for the enzymatic hydrolysate to be separated also includes desalting using C18 StageTip, vacuum drying, redissolving with 0.1% (v / v) formic acid, and filtration through a 0.45 μm membrane.

[0022] The calcium ion chelating peptide provided by this invention can be used to prepare calcium supplement drugs or health foods, including first chelating the calcium ion chelating peptide with calcium ions to prepare a peptide-calcium chelate, and then using it as the core active ingredient to prepare a calcium supplement composition.

[0023] Beneficial effects of this invention:

[0024] 1) This invention uses gluten powder as raw material to isolate and identify oligopeptides with calcium ion chelating activity, namely GVDDQPLFH (oligopeptide 1) and PQFEE (oligopeptide 2), with calcium ion chelation rates of 77.81% and 75.05%, respectively. Oligopeptide 1 has a better calcium loading capacity than oligopeptide 2.

[0025] 2) In the specific embodiments of the present invention, the peptide-calcium chelate prepared from the oligopeptides with calcium ion chelating activity isolated and identified by the present invention has excellent calcium absorption effect and higher bioavailability, and can be used as a raw material source for the preparation of novel calcium supplements through in vitro simulation experiments.

[0026] 3) The preparation method of this invention uses a combination of two enzymes for enzymatic hydrolysis, combined with molecular weight screening and liquid chromatography-mass spectrometry to separate and identify oligopeptides with high calcium ion chelating activity. The operation is simple and easy to promote and apply industrially. Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the comparison of calcium chelation rates of chelated calcium 1, chelated calcium 2, and chelated calcium 3 in Example 1.

[0028] Figure 2 A schematic diagram of the molecular docking simulation results between GVDDQPLFH and calcium;

[0029] Figure 3 A schematic diagram of the molecular docking simulation results between PQFEE and calcium;

[0030] Figure 4 The liquid chromatogram of solid-phase synthesized GVDDQPLFH;

[0031] Figure 5 The liquid chromatogram of PQFEE synthesized in the solid phase;

[0032] Figure 6 The mass spectrum of solid-phase synthesized GVDDQPLFH;

[0033] Figure 7 The mass spectrum of solid-phase synthesized PQFEE;

[0034] Figure 8 A schematic diagram showing the comparison of bone volume fraction (BV / TV) of the tibia in mice from different experimental groups;

[0035] Figure 9 A schematic diagram showing the comparison of trabecular bone thickness (Tb.Th) in the tibia of mice in different experimental groups;

[0036] Figure 10 Micro-CT images of the tibia of mice in different experimental groups. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available.

[0038] The evaluation method for calcium ion chelation activity in the following examples is as follows:

[0039] Weigh the oligopeptide and anhydrous calcium chloride to be tested at a mass ratio of 2:1, mix them, add 10 times their mass of water, mix thoroughly to dissolve, adjust the pH to 8.5, and place in a 65℃ constant temperature water bath with continuous stirring for 3 hours for chelation reaction. After cooling to room temperature, centrifuge at 4000 r / min for 10 min to remove the precipitate, and obtain the supernatant as peptide chelated calcium solution. Put the reaction solution into a 200 Da dialysis bag and dialyze for 24 hours to remove free calcium ions from the product, changing the pure water 3 times during the process. Concentrate the dialysate under reduced pressure and spray dry to obtain wheat oligopeptide chelated calcium, and determine the chelation rate.

[0040] The peptide-chelated calcium was prepared into an aqueous solution of 0.2 mg / mL. The calcium content was determined by flame atomic absorption spectrometry according to GB 5009.92-2016, and the chelation rate was calculated using the following formula:

[0041]

[0042] In the formula:

[0043] W-chelation rate, in %;

[0044] Calcium content in m1-peptide calcium chelate, in mg;

[0045] The amount of calcium added during the m2- reaction, expressed in mg.

[0046] Example 1

[0047] This embodiment provides a method for preparing calcium ion chelating peptides, and the specific operation steps are as follows:

[0048] 1) Enzymatic hydrolysis:

[0049] Add wheat gluten powder to deionized water to dissolve and stir to prepare an 8% (w / v) mixture. Sonicate at 400W for 20 minutes to disperse wheat protein in water and stir magnetically for 1 hour to form a uniform suspension.

[0050] First stage: Heat the water bath to 45℃ and adjust the pH to 7.0. Add 4000 U / g of neutral protease according to the substrate mass and enzymatically hydrolyze for 2.5 hours.

[0051] Second stage: Heat to 60℃ and adjust pH to 8.5. Add 8000 U / g alkaline protease according to the substrate mass, enzymatically hydrolyze for 3 h, inactivate enzyme in boiling water bath for 10 min, cool and centrifuge at 4000 r / min for 10 min, take the supernatant to obtain the enzymatic hydrolysate. According to the above evaluation method for calcium ion chelation activity, the enzymatic hydrolysate is subjected to a preliminary chelation reaction with anhydrous calcium chloride to obtain chelated calcium 1.

[0052] 2) Purification process:

[0053] The enzymatic hydrolysate with a molecular weight cutoff of 1 kDa was purified using a filter membrane at a processing pressure of 1.5 MPa and a processing flow rate of 3 L / h. The retentate was collected and spray-dried to obtain enzymatic hydrolysates with a molecular weight ≤ 1 kDa. Enzymatic hydrolysates with a molecular weight ≥ 1 kDa were also collected.

[0054] According to the above evaluation method for calcium ion chelation activity, the enzymatic hydrolysates with molecular weight ≤1kDa and molecular weight ≥1kDa were chelated with anhydrous calcium chloride to obtain chelated calcium 2 and chelated calcium 3.

[0055] The comparative analysis results of the chelation rates of chelated calcium 1, chelated calcium 2, and chelated calcium 3 are as follows: Figure 1As shown, enzymatic hydrolysates with a molecular weight ≤1kDa have higher calcium chelation activity;

[0056] 3) Identification and screening of amino acid sequences with high calcium chelating activity:

[0057] The enzymatic hydrolysate with a molecular weight ≤1kDa purified and separated in step 2) was desalted using a C18 StageTip, vacuum dried, and the dried peptide fragments were reconstituted with 0.1% (v / v) formic acid and filtered through a 0.45μm membrane for detection.

[0058] LC-MS / MS detection liquid chromatography conditions: Chromatographic separation was performed using a nano-flow rate Easy nLC 1200 chromatography system (ThermoScientific). Buffer solutions: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a mixed solution of 0.1% formic acid, acetonitrile, and water (acetonitrile comprising 80%). The column was equilibrated with 100% solution A. Samples were injected into a trap column (100 μm * 20 mm, 5 μm, C18, Dr. Maisch GmbH) and then subjected to gradient separation using an analytical column (75 μm * 150 mm, 3 μm, C18, Dr. Maisch GmbH) at a flow rate of 300 nL / min.

[0059] The liquid phase separation gradients are as follows: 0-2 min, linear gradient of liquid B from 2% to 5%; 2-44 min, linear gradient of liquid B from 5% to 28%; 44-51 min, linear gradient of liquid B from 28% to 40%; 51-53 min, linear gradient of liquid B from 40% to 100%; 53-60 min, liquid B is maintained at 100%.

[0060] After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF mass spectrometer (Thermo Scientific). The analysis time was 60 min, and the detection mode was positive ion. The precursor ion scan range was 350-1800 m / z, the primary mass spectrometry resolution was 120,000 m / z 200, the secondary mass spectrometry resolution was 15,000 m / z 200, and the collision energy was CE 28 eV.

[0061] Two peptides with high calcium chelating activity and good biocompatibility were selected: PQFEE (oligopeptide 2) and GVDDQPLFH (oligopeptide 1). The physicochemical properties of the selected peptides are shown in Table 1.

[0062] Table 1 shows the sequence information and physicochemical properties of peptides with potential high calcium chelating activity.

[0063] peptide sequence hydrophilic toxicity electric charge pI molecular weight 1 PQFEE 0.74 Non-toxic -2.00 3.80 648.28 2 GVDDQPLFH -0.01 Non-toxic -1.50 4.20 1026.48

[0064] It should be noted that in the preparation and screening process of the oligopeptides with calcium ion chelating activity of the present invention, the parameters in the enzymatic hydrolysis and purification steps can be adjusted within a certain range, and the oligopeptides described in the present invention can still be isolated and identified. Specifically, the mass concentration of the gluten powder solution is 5%-10%; the parameters such as temperature, enzyme dosage, and hydrolysis time are as follows: in the first stage, after heating to 45-50℃, add neutral protease at a dosage of 3000-4000 U / g and hydrolyze for 2-3 hours; in the second stage, heat to 55-65℃ and add alkaline protease at a dosage of 6000-8000 U / g and hydrolyze for 3-4 hours; filter membrane purification is performed at a processing pressure of 1.0-2.0 MPa and a processing volume of 2-5 L / h.

[0065] Example 2 Performance Evaluation

[0066] 1. Evaluation of the stability of calcium ion chelate molecular structure:

[0067] The peptide structure was optimized using Chem 3D software. After minimizing the peptide energy using the MM2 algorithm, batch molecular docking simulations of GVDDQPLFH (oligopeptide 1) and PQFEE (oligopeptide 2) with calcium were performed using Autodock software. Peptides with lower docking energies were screened based on docking energy. The results were processed using PyMol software. The molecular docking results are shown below. Figure 2 (GVDDQPLFH) and Figure 3 As shown in (PQFEE).

[0068] Molecular docking was used to predict the calcium chelation mode. The results showed that PQFEE-Ca chelates via the carboxyl oxygen atom of Gln2, belonging to the monodentate mode of the calcium chelation model. GVDDQPLFH-Ca connects to the carboxyl oxygen atom of Val2 via the amino nitrogen atom of Asp4, belonging to the α mode of the chelation model. The α mode of GVDDQPLFH chelates with calcium via two chelate bonds to the amino acid residues of the peptide; the bond length is shorter, the bond energy is stronger, and the binding is more robust than the monodentate mode.

[0069] 2. Calcium ion chelation activity:

[0070] Solid-phase synthesis and activity verification of active sequences

[0071] Based on the screening results in Example 1, Nanjing Jietai Biotechnology Co., Ltd. was commissioned to synthesize peptides GVDDQPLFH and PQFEE using a solid-phase synthesis method. The liquid-phase characterization results are as follows: Figure 4 (GVDDQPLFH) Figure 5 (PQFEE) is shown; the mass spectrometry characterization results are as follows. Figure 6 (GVDDQPLFH) Figure 7 As shown in (PQFEE).

[0072] The calcium chelation rate of the synthesized peptides was detected according to the detection method provided in the specific embodiments of the present invention. The results are shown in Table 2. Compared with the chelated calcium 1 prepared from the preliminary enzymatic hydrolysate, the calcium chelation rate of the purified and screened peptides was significantly improved. Among them, the peptide GVDDQPLFH had the highest calcium chelation activity.

[0073] Table 2 Calcium chelation rate of synthesized peptide sequences

[0074] Serial Number peptide sequence Chelation rate (%) 1 GVDDQPLFH 77.81±0.21 2 PQFEE 75.05±0.34 3 Chelated calcium 1 35.72±0.18

[0075] 3. Evaluation of the application value of calcium supplement preparation:

[0076] (1) Preparation of peptide-chelated calcium aqueous solution:

[0077] Weigh out oligopeptide 1GVDDQPLFH and anhydrous calcium chloride at a mass ratio of 2:1, mix them, add 10 times their mass of water, mix thoroughly to dissolve, adjust the pH to 8.5, and place in a 65℃ constant temperature water bath with continuous stirring for 3 hours for chelation reaction. After cooling to room temperature, centrifuge at 4000 r / min for 10 min to remove the precipitate, and obtain the supernatant as a peptide chelated calcium aqueous solution.

[0078] (2) Experimental procedure:

[0079] Four-week-old male KM mice were purchased from Henan Skbes Biotechnology Co., Ltd. After one week of acclimatization, 10 mice were divided into 6 groups. After 4 weeks of feeding, gavage treatment was started. The experimental groups are shown in Table 3.

[0080] Table 3 Grouping of Animal Experiments

[0081]

[0082]

[0083] To investigate the ameliorative effect of peptide-calcium chelates on osteoporosis, 3D images of the tibia were stained using micro-CT to determine the microstructure of the trabecular bone. The distal femur was scanned using a micro-CT system under specific conditions of 70 kV voltage and 114 μA current. The scan covered 3 mm of the distal femur bone at a resolution of 10 μm. Based on the micro-CT images (e.g., ...), the microstructure was analyzed. Figure 10 (As shown) Calculate morphological measurement parameters, specifically including trabecular bone volume percentage (BV / TV) (e.g.) Figure 8 As shown), trabecular bone thickness (Tb.Th) (as shown) Figure 9 (As shown).

[0084] The results showed that the osteoporosis model group fed with a low-calcium diet exhibited sparse interconnectedness of bone trabeculae. After intervention with peptide-calcium chelates, the bone loss symptoms in mice were alleviated. At the same time, the bone volume fraction and trabecular thickness increased in the peptide-calcium chelate treatment group. In summary, it had a more significant calcium supplementation effect than the calcium chloride drug control group, indicating that the oligopeptide with high calcium ion chelating activity isolated and identified in this invention can be used as a key material to prepare peptide-calcium chelates for the preparation of calcium supplement drugs or health foods.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A calcium ionophore peptide characterized in that, The oligopeptide 1 and / or the oligopeptide 2; wherein the amino acid sequence of the oligopeptide 1 is GVDDQPLFH, as shown in the sequence table SEQ ID NO. 1; and the amino acid sequence of the oligopeptide 2 is PQFEE, as shown in the sequence table SEQ ID NO.

2.

2. The method of preparing calcium ionophore peptide according to claim 1, characterized in that, The method comprises the following steps: 1) using gluten meal as raw material, sequentially treating with neutral protease and alkaline protease to prepare an enzyme hydrolysate; 2) purifying the enzyme hydrolysate to obtain an enzyme hydrolysate with a molecular weight of less than 1 kDa; 3) separating and identifying the oligopeptide with high calcium chelating activity from the enzyme hydrolysate prepared in step 2); In step 1), the specific method of enzyme hydrolysis treatment is as follows: first, prepare a gluten meal suspension, heat to 45-50℃, adjust pH to 7.0, add neutral protease, hydrolyze, then heat to 55-65℃, adjust pH to 8.5, add alkaline protease, hydrolyze, then inactivate the enzyme, centrifuge to obtain the supernatant, and obtain the enzyme hydrolysate; the amount of neutral protease is 3000-4000 U / g; the amount of alkaline protease is 6000-8000 U / g; the hydrolysis time of neutral protease is 2-3 h; the hydrolysis time of alkaline protease is 3-4 h; In step 2), the specific method of purifying the enzyme hydrolysate is as follows: purify with a filter membrane with a molecular weight cutoff of 1 kDa, the treatment pressure is 1.0-2.0 MPa, the treatment volume is 2-5 L / h, and spray drying is performed to obtain the enzyme hydrolysate; In step 3), the specific method of separating and identifying the oligopeptide with high calcium chelating activity is as follows: use LC-MS / MS technology for separation and identification, the liquid chromatography conditions are as follows: buffer solution: A solution is 0.1% formic acid aqueous solution, B solution is 0.1% formic acid, 80% acetonitrile and water mixed solution; the chromatographic column is equilibrated with 100% A solution; the sample is injected into the chromatographic column for gradient separation at a flow rate of 300 nl / min; the liquid separation gradient is as follows: 0-2 min, B solution linear gradient from 2% to 5%; 2-44 min, B solution linear gradient from 5% to 28%; 44-51 min, B solution linear gradient from 28% to 40%; 51-53 min, B solution linear gradient from 40% to 100%; 53-60 min, B solution maintains at 100%; Compare the calcium chelating activity of each separated peptide segment to screen the oligopeptide with high calcium chelating activity; The amino acid sequence of the oligopeptide 1 is GVDDQPLFH, as shown in the sequence table SEQ ID NO. 1; and the amino acid sequence of the oligopeptide 2 is PQFEE, as shown in the sequence table SEQ ID NO.

2.

3. The method for preparing calcium ion chelating peptide as described in claim 2, characterized in that, The mass concentration of the gluten meal suspension is 5-10%; the specific preparation method of the gluten meal suspension is as follows: add gluten meal into deionized water, dissolve and stir to mix uniformly, ultrasonic at a power of 400 W for 20 min, disperse thoroughly, and magnetically stir for 1 h to form a uniformly dispersed suspension.

4. The use of the calcium ion chelating peptide according to claim 1 in the preparation of a calcium supplement drug or health food.

5. A peptide calcium chelate characterized in that, Prepared from the calcium ion chelating peptide according to claim 1 and calcium ions.

6. A calcium supplement composition characterized in that, The peptide calcium chelate according to claim 5.

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