Antarctic krill calcium-chelating peptide, preparation method and application thereof

Abstract

The present application provides a euphausia superba calcium chelating peptide, which is an enzymatic polypeptide prepared by enzymolysis of euphausia superba meat with neutral protease and papain; a specific amino acid sequence thereof is EEDLER or IVELEEE. The present application also provides a calcium chelating peptide, which is prepared by chelating calcium ions with the polypeptide. The euphausia superba calcium chelating peptide provided by the present application combines with calcium ions to form a calcium peptide chelate, so that the calcium ions can always remain in a soluble state during digestion, especially in the intestinal tract, effectively relieving the precipitation of calcium ions in the intestinal tract, and overcoming the shortcomings of inorganic calcium, such as strong irritation, low absorption and utilization rate, and poor absorption effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioactive products, and particularly relates to a Euphausia superba calcium-chelating peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Calcium is an important mineral nutrient element, which is essential for the formation of human structure and the maintenance of normal physiological functions. The content of calcium in the human body is about 1.5%-2.2% of the body weight, and most of it exists in the form of calcium phosphate in the bone, which helps to improve the toughness of the bone. The remaining calcium found in soft tissues, extracellular fluid and blood exists in ionic form, which is in balance with bone calcium. Calcium is not only essential for bone growth, blood clotting and maintaining heart function, but also plays a crucial role in physiological functions such as signal transduction, nerve impulse transmission, muscle contraction, secretory activity, cell metabolism, immune response and enzyme activation. A series of metabolic bone diseases, including childhood rickets and osteoporosis in the elderly, can be caused by a lack of calcium. In addition, calcium deficiency is related to various diseases such as osteoporosis, kidney stones, hypertension, obesity and colon cancer. Only the calcium intake in the diet can provide the amount of calcium for the body's bones, although the absorption of calcium from the bone can maintain normal serum calcium levels, but the absorption of calcium is interfered by various factors, including food sources, individual metabolism and physiological conditions. For example, after the intake of a diet rich in phytic acid, oxalic acid and phosphoric acid, these substances will produce insoluble phytic acid, oxalic acid and phosphoric acid deposits under the pH conditions in the intestinal tract, thereby reducing the absorption and utilization of calcium by the body.

[0003] With the steady improvement of modern living standards and the increasing awareness of public health, calcium supplementation has gradually become an important part of people's pursuit of a healthy life. Therefore, choosing calcium supplements has gradually become a health trend, and the variety of calcium supplements on the market is increasingly rich, mainly divided into four categories. First-generation calcium supplements are mainly composed of inorganic calcium, which is widely available and inexpensive. However, it is insoluble in water and needs to be dissociated into calcium ions in stomach acid before it can be absorbed by the body. This can affect normal digestive function, and unabsorbed calcium is prone to depositing in the kidneys, increasing the burden on the kidneys and potentially leading to kidney stones, thus adversely affecting human health. Second-generation calcium supplements are mainly composed of traditional organic calcium. Their advantage is better solubility, and compared to first-generation calcium supplements, they are less irritating to the gastrointestinal tract, but the calcium content is relatively low. Furthermore, calcium ions easily interact with oxalic acid, phytic acid, and other substances in food, leading to precipitation, thus hindering the body's effective absorption and utilization of calcium. Third-generation calcium supplements are mainly composed of amino acid chelated calcium, which has a high calcium content, is easily digested and absorbed by the human body, and has good stability. However, their production costs are high, there are no comprehensive quality testing standards, and their mechanisms of action and metabolic pathways are not yet clear, posing potential safety concerns. Fourth-generation calcium supplements are mainly composed of peptide chelated calcium, which is rapidly digested and absorbed, has high nutritional value, and possesses various biological activities while supplementing calcium. Currently, the preparation process is not yet mature, and the peptide-calcium chelation rate is relatively low, requiring further research.

[0004] Current methods for purifying calcium chelating peptides have certain limitations: long processing times, low throughput, unsuitability for industrial production, and low calcium chelating activity in the resulting fractions. Therefore, there is an urgent need to develop an efficient targeted preparation technique to address this issue. Summary of the Invention

[0005] This invention provides an Antarctic krill calcium chelating peptide and its preparation method, the prepared product having better calcium chelating activity.

[0006] This invention provides an Antarctic krill polypeptide and its preparation method, the prepared product having better calcium chelation activity.

[0007] The present invention first provides a polypeptide with calcium chelating activity, wherein the polypeptide is an enzymatically hydrolyzed polypeptide prepared by enzymatic hydrolysis of Antarctic krill meat using neutral protease and papain.

[0008] Preferably, the mass ratio of the neutral protease to the papain is 1:1.

[0009] The enzymatic hydrolysis, wherein the hydrolysis is carried out at 50°C for 4 hours;

[0010] Furthermore, the above-mentioned enzymatically hydrolyzed peptides were purified, including the following steps:

[0011] 1) Affinity chromatography of Antarctic krill peptides

[0012] affinity chromatography of the enzymatic polypeptide using hydroxyapatite filler, gradient elution of the enzymatic polypeptide using Buffer B, and collection of the eluate; the Buffer B is 280 mM phosphate buffer with 100 mM sodium chloride added;

[0013] 2) Sephadex G-15 gel filtration chromatography

[0014] The eluate component obtained by separation and purification of hydroxyapatite is further separated and purified using a Sephadex G-15 gel column, elution is performed using ultrapure water at a flow rate of 0.5 mL / min, detection of absorbance is performed at a wavelength of 220 nm, and components with peak time of 55-105 min are collected for reverse phase high performance liquid chromatography;

[0015] 3) reverse phase high performance liquid chromatography

[0016] The reverse phase high performance liquid chromatography is performed using a C18 semi-preparative column, elution is performed under the following conditions: 0-10 min, 6% acetonitrile; 10-30 min, 6-30% acetonitrile; 30-40 min, 30%-6% acetonitrile, the mobile phase A is ultrapure water, the mobile phase B is acetonitrile, the flow rate is 1 mL / min, the column temperature is 35°C, the ultraviolet detection wavelength is 220 nm, and the elution peak with peak time of 12.2-13.0 min is collected as the purified polypeptide.

[0017] Further, the purified polypeptide has an amino acid sequence of EEDLER or IVELEEE.

[0018] The application also provides a calcium-chelating peptide, which is prepared by chelating calcium ions with the above polypeptide.

[0019] More specifically, the calcium-chelating peptide is prepared by adding a solution of Antarctic krill peptide to activated hydroxyapatite, combining at room temperature, eluting and removing unbound Antarctic krill peptide using a phosphate buffer, and then eluting the Antarctic krill calcium-chelating peptide specifically bound to the hydroxyapatite using a 280 mM phosphate buffer, collecting the eluate, concentrating, dialyzing, desalting, and freeze-drying to obtain the Antarctic krill calcium-chelating peptide.

[0020] The Antarctic krill calcium-chelating peptide provided by the application can be used to prepare a calcium supplement preparation.

[0021] The Antarctic krill calcium-chelating peptide provided by the application combines with calcium ions to form a calcium peptide chelate, so that the calcium ions remain in a soluble state during digestion, especially in the intestinal tract, effectively alleviating the precipitation of calcium ions in the intestinal tract and overcoming the shortcomings of inorganic calcium, such as strong irritation, low absorption and utilization rate, and poor absorption effect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 4 is a chart of calcium chelating activity of Euphausia superba single enzyme hydrolysate,

[0023] Figure 2 Figure 5 is a chart of calcium chelating activity of Euphausia superba complex enzyme hydrolysate,

[0024] Figure 3 Figure 6 is a chart of calcium chelating activity of hydroxyapatite affinity chromatography,

[0025] Figure 4 Figure 7 is a chart of calcium chelating activity of Sephadex G-15 gel filtration chromatography,

[0026] Figure 5 Figure 8 is a chart of calcium chelating activity of reverse phase high performance liquid chromatography,

[0027] Figure 6 Figure 9 is a chart of calcium chelating activity of EEDLER, IVELEEE,

[0028] Figure 7 Figure 10 is a chart of calcium chelating conditions optimization of Euphausia superba peptide. DETAILED DESCRIPTION

[0029] The detection method involved in the present application can adopt the method commonly used in the field, and in specific embodiments, the related method is as follows:

[0030] 1) Flame atomic absorption method for calcium content determination

[0031] The calcium content is determined according to the operation steps of the national standard GB / T 5009.92-2016, and the Ca 2+ content is determined by flame atomic absorption spectrometry (FAAS).

[0032] The reagents and standard curve preparation operation are as follows:

[0033] Preparation method of 1000mg / L calcium standard stock solution: accurately weigh 2.4963g of calcium carbonate, accurate to 0.0001g, dissolve with 6mol / L hydrochloric acid solution, transfer to a 1000mL volumetric flask, and add ultrapure water to the mark, mix well.

[0034] Preparation method of 100mg / L calcium standard intermediate solution: accurately pipette 10mL from the calcium standard stock solution (1000mg / L) into a 100mL volumetric flask, and add 95% nitric acid solution to the mark, mix well.

[0035] Preparation method of calcium standard series solution: 0, 0.500, 1.00, 2.00, 4.00 and 6.00 mL of calcium standard intermediate solution were taken into 100 mL volumetric flasks, 5 mL of lanthanum oxide solution with a concentration of 20 g / L was added into each volumetric flask, and 95% nitric acid solution was added to constant volume at the scale, and mixed uniformly. The mass concentration of calcium in this calcium standard series solution was 0, 0.500, 1.00, 2.00, 4.00 and 6.00 mg / L, respectively.

[0036] The specific operation parameters of atomic absorption spectrophotometer are shown in Tables 1-3. The detection wavelength is 422.7 nm, the slit is 1.3 nm, visible light is used as the light source, the height of the combustion head is 3 mm, the flame is air-acetylene, the lamp current is 5-15 mA, and the standard solution concentration of calcium is 0-6.00 mg / L.

[0037] 2) Antarctic krill peptide calcium chelation activity determination method

[0038] Peptide calcium chelation: 3 mg of freeze-dried sample of enzyme hydrolysate was taken into a test tube, 2 mL of CaCl2 solution (5 mM) was added and mixed thoroughly, the temperature of the water bath shaker was set to 25°C, and the shaking reaction was carried out for 20 min, then 4 mL of phosphate buffer with a concentration of 20 mM was added and mixed thoroughly, the temperature of the water bath shaker was set to 37°C, and the shaking reaction was carried out for 30 min, and then centrifugation was carried out at 4000 x g for 20 min, the supernatant was passed through a 0.22 μm filter membrane, at this time calcium ions and polypeptides formed chelates and dissolved in the supernatant, while calcium ions not combined with peptides formed phosphate precipitate and were removed.

[0039] Sample digestion: 2 mL of sample on the filter membrane was taken into a glass digestion tube, 10 mL of nitric acid was added, and the digestion tube was placed in the digestion hole after shaking. The digestion instrument was programmed to heat to 100°C and continue heating for 30 min; the digestion instrument was heated to 130°C and continue heating for 60 min; the digestion instrument was heated to 150°C and continue heating for 60 min; the digestion instrument was heated to 190°C, and the acid was discharged to about 1 mL of sample remaining in the digestion tube, and attention should be paid that the acid cannot be completely discharged during this process. A blank control was also prepared.

[0040] Constant volume: the digestion tube was taken out, cooled, 500 μL of lanthanum oxide with a concentration of 20 g / L was taken into a 10 mL volumetric flask, the sample was transferred to the volumetric flask with a pipette, and the constant volume was set to the scale with ultrapure water, and then the sample was transferred to a 10 mL centrifuge tube after passing through a 0.22 μm filter membrane for measurement.

[0041] The calcium content was determined by atomic absorption spectrophotometer, and the calculation formula of calcium chelation amount in the sample was as follows:

[0042]

[0043] X——sample calcium chelation amount (μg / mg)

[0044] p - mass concentration of calcium in the sample to be tested (mg / L) ;

[0045] p0 - mass concentration of calcium in the blank solution (mg / L) ;

[0046] f - dilution multiple of the sample digestion solution;

[0047] V - constant volume of the sample digestion solution (L) ;

[0048] m - sample mass (mg).

[0049] The application will be described in detail below in conjunction with specific examples and drawings.

[0050] Example 1 Preparation of Antarctic krill peptide AKP

[0051] First, Antarctic krill peptides with calcium chelation activity are prepared based on hydroxyapatite, including the following steps:

[0052] Antarctic krill meat is homogenized, distilled water is added according to a certain solid-liquid ratio, then single enzymes or compound enzymes are added at a proportion of 0.4%, 0.8%, 1.2%, 1.6%, 2.0% of the substrate, the pH value of the system is adjusted to the optimum pH of each enzyme, and the system is incubated at the enzymolysis temperature for hydrolysis; after enzymolysis, the enzymes are inactivated by boiling, and the supernatant is obtained by centrifugation to obtain Antarctic krill enzymolysis solution; the enzymolysis solution is concentrated and dried to obtain Antarctic krill peptide dry powder.

[0053] 1) Antarctic krill single-enzyme enzymolysis

[0054] Five proteases, namely neutral protease, alkaline protease, flavor protease, papain and trypsin, are selected for single-enzyme enzymolysis, and the enzyme addition amount is 0.4%, 0.8%, 1.2%, 1.6%, 2.0% of the substrate content. Enzymolysis is carried out according to the following process: 5.00 g of homogenized Antarctic krill meat is placed in a conical flask → distilled water is added according to a solid-liquid ratio of 1:5 → the pH of each enzyme is adjusted → the protease is added → the enzymolysis temperature is set to 50°C and the enzymolysis time is 4 h → the enzymes are inactivated by heating at 100°C for 10 min after enzymolysis → centrifugation at 8000 r / min for 15 min → the supernatant is taken as the prepared enzymolysis solution.

[0055] 2) Antarctic krill compound enzymolysis process optimization

[0056] Two proteases, namely neutral protease and papain, are selected for compound enzymolysis, and the enzyme addition amount is 1.6% of the substrate content, and the two enzymes are added at the same time for enzymolysis, and the ratio of neutral protease to alkaline protease is 1:5, 1:3, 1:1, 3:1 and 5:1. The specific enzymolysis steps and conditions are the same as 1.2.3.

[0057] Neutral protease, alkaline protease, flavour protease, papain and trypsin were selected to carry out enzymatic hydrolysis experiment in this embodiment to obtain E. superba hydrolysate, and the results are shown in Figure 1 As the enzyme amount increased, the calcium chelation amount of E. superba peptide showed a fluctuating trend, and the calcium chelation amount of different kinds of hydrolysates was different under the same enzyme amount. Under the conditions of neutral protease and papain with an enzyme amount of 1.6%, more peptide segments with calcium chelation activity were produced, which led to the exposure of chelation sites and higher calcium chelation activity. Papain and neutral protease were selected to carry out complex enzymolysis, and the addition amount of the two was 1.6% of the substrate. The results are shown in Figure 2 Compared with single enzyme hydrolysis, the calcium chelation amount of the complex enzyme hydrolysate increased, and when the addition amount of papain and neutral protease was 1:1, i.e. the addition amount of the two enzymes was 0.8%, the calcium chelation amount of the hydrolysate was the highest, reaching 3.13±0.09 μg / mg.

[0058] Example 2: Purification and sequencing of E. superba peptide with calcium chelation activity

[0059] 1. Affinity chromatography of E. superba peptide

[0060] Activated hydroxyapatite filler was added to a 500 mL centrifuge cup, and then E. superba peptide prepared in Example 1 was added. The mixture was placed in a water bath shaker and shaken, and unbound components were repeatedly centrifuged (1000 r / min) and eluted with 60 mL of Buffer A (5 mM phosphate buffer + 100 mM sodium chloride) for 3 times. The eluate was collected and the volume was recorded. The calcium chelation peptide was gradient eluted with 60 mL of Buffer B (280 mM phosphate buffer + 100 mM sodium chloride), and the eluate was collected and the volume was recorded. The HAP filler was repeatedly washed and centrifuged with 0.5 M, pH 6.8 phosphate buffer for 3 times to fully activate, and then balanced with Buffer A by centrifugal elution for 3 times. Finally, it was stored in 0.1 M NaOH solution. The collected components were concentrated, dialyzed, desalted and freeze-dried.

[0061] Because each hydroxyapatite molecule is composed of five positively charged calcium, the reaction between hydroxyapatite molecules and calcium chelation peptide has strong metal affinity. Elution of calcium chelation peptide requires phosphate solution, and the phosphate concentration of Buffer B solution is much higher than that of Buffer A. Therefore, peptides with high calcium chelation activity are enriched in Buffer B components, and the measured calcium chelation amount of Buffer B is 6.41±0.20 μg / mg, which is increased by 45.17% compared with AKP.

[0062] 2. Sephadex G-15 gel filtration chromatography

[0063] The Buffer B fraction obtained from the hydroxyapatite separation and purification was further purified using a Sephadex G-15 gel column (1.6cm × 60cm). The fraction collected in the previous step was prepared into a 50 mg / mL solution with ultrapure water, filtered through a 0.22 μm filter, and loaded with 1 mL of ultrapure water at a flow rate of 0.5 mL / min. The absorbance was measured at 220 nm, and elution peaks were collected every 5 min. Elution chromatograms were plotted, and the fractions were collected, combined, rotary evaporated, and lyophilized. Based on the elution time sequence, they were named G1, G2, and G3, respectively. The calcium chelating activity of each collected fraction was determined using FAAS.

[0064] In this experiment, a Sephadex G-15 gel column was used to separate and purify the Buffer B fraction obtained from hydroxyapatite affinity chromatography. Three fractions were obtained within the 0-180 min time range, with peak times between 55-105 min, 105-128 min, and 128-150 min, respectively, and were named G1, G2, and G3 according to their elution times. The effect of the three fractions on calcium chelating activity was determined. The calcium chelating activity of fraction G1 was 7.17 ± 0.06 μg / mg, significantly higher than that of fractions G2 and G3 (P < 0.05). Fraction G3 eluted last, possibly containing peptides or free amino acids with very small molecular weights, thus exhibiting lower calcium chelating activity. The calcium chelating activity of G1 was 11.90% higher than that of Buffer B. Therefore, fraction G1 was selected for subsequent separation and purification by C-18 reversed-phase high-performance liquid chromatography after rotary evaporation and lyophilization.

[0065] 3. Reversed-phase high-performance liquid chromatography

[0066] To further separate and purify calcium-chelating peptides, the fraction with the highest calcium-chelating activity, screened by G-15 gel chromatography, was further subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 semi-preparative column (Zorbax SB-C18, 9.4 × 250 mm, 5 μm). The HPLC elution conditions were: 0–10 min, 6% B; 10–30 min, 6–30% B; 30–40 min, 30%–6% B. Mobile phase A was ultrapure water, mobile phase B was acetonitrile, the flow rate was 1 mL / min, the column temperature was 35 °C, and the UV detection wavelength was 220 nm. Each peak fraction was collected, rotary evaporated, and lyophilized. Based on their elution time, they were named C1, C2, C3, C4, C5, and C6, respectively. The calcium-chelating activity of each collected fraction was determined by FAAS.

[0067] G1 was separated and purified from the fraction collected by gel filtration chromatography. Linear elution with 6%–30% acetonitrile yielded six elution peaks within the 0–35 min time range, with elution times between 9.3–12.1 min, 12.2–13.0 min, 13.1–15.1 min, 15.2–20.1 min, 20.2–26.4 min, and 26.5–28.2 min, respectively. These peaks were named C1, C2, C3, C4, C5, and C6 according to their elution times. The calcium chelating activity of the six collected fractions was determined. C2 showed the highest chelating activity (9.38 ± 2.04 μg / mg), significantly higher than the last eluted fraction C6 (P < 0.05), indicating that the net charge and amount of hydrophilic groups of AKP affect its ability to chelate calcium ions. Compared to unisolated and purified AKP, the calcium chelating activity of fraction C2 increased by 112.60%. Therefore, fraction C2 was selected for collection and sequence identification.

[0068] 4. Mass spectrometry identification of high-calcium chelated bioactive peptides from Antarctic krill

[0069] The C2 fraction collected by reversed-phase high-performance liquid chromatography was used to prepare a sample solution with ultrapure water at a concentration of 1 mg / mL. The solution was then filtered through a 0.22 μm aqueous filter membrane before subsequent operations.

[0070] Liquid chromatography conditions for the samples: An ACQUITY UPLC Peptide CSH C18 column was used, with a flow rate of 0.15 mL / min and a column temperature of 40℃. Mobile phase A was 0.1% acetonitrile, and mobile phase B was 0.1% ultrapure water. Elution conditions were as follows: 0-2 min, 2% A; 2-27 min, 2-10% A; 27-37 min, 10-25% A; 37-39 min, 25%-80% A; 39-42 min, 80% A; 42-43 min, 80%-2% A; 43-50 min, 2% A. The injection volume was 10 μL.

[0071] Mass spectrometry conditions for the sample: mass spectrometry scan range 50-1200 m / z, reaction mode positive ion mode, spray voltage set to 3.60 kV, capillary temperature set to 320 °C, signal intensity threshold 1.6 eΩ. 5 .

[0072] Mass spectrometry analysis of the C2 fraction was performed, and the peptide sequences were identified using the de novo sequencing algorithm. Peptide sequences with confidence intervals >95% were selected, resulting in the identification of 45 peptides. These peptides were concentrated between 6 and 13 amino acids, with molecular weights ranging from 300 to 1000 Da. The amino acid composition revealed that the peptides contained acidic amino acids aspartic acid (D) and glutamic acid (E), as well as basic amino acids lysine (K), histidine (H), and arginine (R).

[0073] Table 1: Identification of C2 component peptide sequences

[0074]

[0075]

[0076] 5. Analysis of the calcium chelating activity of EEDLER and IVELEEE

[0077] The calcium chelating activities of the two synthesized peptides, EEDLER and IVELEEE, were verified. The results showed that their calcium chelating activities were 12.06±1.32 μg / mg and 5.11±0.52 μg / mg, respectively. Compared with the C2 fraction collected by liquid phase, the calcium chelating activity of EEDLER was significantly increased, while that of IVELEEE was decreased, and the difference was significant (P<0.05). Furthermore, the significant difference in calcium chelating activities between EEDLER and IVELEEE (P<0.05) may be related to the presence of the hydrophobic amino acid Ile in the IVELEEE sequence, which reduces its solubility. During the chelation reaction with calcium ions, a small amount of undissolved peptides were present in the prepared reaction system, which did not participate in the chelation reaction, thus leading to the decreased calcium chelating activity.

[0078] Example 3: Preparation of Antarctic krill calcium chelate peptides

[0079] The Antarctic krill peptide solution prepared in Example 1 was added to activated hydroxyapatite and bound at room temperature for 3 hours. Unbound Antarctic krill peptides were repeatedly eluted with 5 mM phosphate buffer to remove them. Then, the Antarctic krill calcium chelate peptides specifically bound to hydroxyapatite were eluted with 280 mM phosphate buffer. The eluent was collected, concentrated, dialyzed to desalt, and freeze-dried to obtain Antarctic krill calcium chelate peptides.

[0080] To further improve the calcium chelating activity of AKP, a single-factor experiment was conducted on five factors: CaCl2 concentration, AKP concentration, chelation pH, chelation temperature, and chelation time (Table 2).

[0081] Table 2: Optimization Orthogonal Experiment Table for Antarctic Krill Peptide-Calcium Chelation

[0082]

[0083] Based on the results of the single-factor experiments, three factors that significantly affected the calcium chelation capacity of AKP—CaCl2 concentration (A), AKP concentration (B), and chelation pH (C)—were selected for a three-factor, three-level orthogonal experiment to optimize the calcium chelation conditions. Therefore, the optimal calcium chelation conditions for AKP were determined to be: CaCl2 concentration of 5 mM, AKP concentration of 1.5 mg / mL, chelation temperature of 45℃, chelation time of 30 min, and chelation pH of 8.

Claims

1. A polypeptide with calcium chelating activity, characterized in that, The amino acid sequence of the polypeptide is EEDLER or IVELEEE.

2. The use of the polypeptide according to claim 1 in the preparation of calcium chelating peptides.

3. A calcium chelating peptide, characterized in that, The calcium chelating peptide is prepared by chelating calcium ions using the polypeptide described in claim 1.

4. The calcium chelating peptide as described in claim 3, characterized in that, The calcium chelate peptide is prepared by adding the polypeptide described in claim 1 to activated hydroxyapatite, binding it at room temperature, then eluting with phosphate buffer to remove unbound Antarctic krill peptides, and then eluting the Antarctic krill calcium chelate peptides specifically bound to hydroxyapatite with 280 mM phosphate buffer. The eluent is collected, concentrated, dialyzed for desalting, and freeze-dried to obtain the Antarctic krill calcium chelate peptide.

5. The use of the calcium chelating peptide according to claim 3 in the preparation of calcium supplements.

6. A calcium supplement, characterized in that, The calcium supplement contains a pharmacologically effective concentration of the calcium chelating peptide described in claim 3.

Images