Phosphorylated oil tea meal peptide calcium chelate and application thereof in calcium enhancer

Abstract

The invention belongs to the technical field of biology, and particularly relates to a phosphorylated oil tea meal peptide calcium chelate and application thereof in a calcium enhancer. Oil-tea meal is used as a raw material to extract protein, oil-tea meal active peptide is prepared through a biological enzymolysis method, oil-tea meal polypeptide AHW is screened out through bioinformatics analysis, and the calcium chelation rate of the oil-tea meal polypeptide AHW is 34.8% + / -1.65%. The chelating effect of phosphorylated oil-tea meal polypeptide P-AHW and Ca is further explored, and when the mass ratio of P-AHW to Ca is 1: 6, the chelating rate of the chelate P-AHW-Ca is increased to 61.67% + / -0.71%. The invention also verifies that the P-AHW-Ca chelate has good stability under the influence of different pH values, different temperatures and different food additives, and simulates gastric juice and intestinal juice to prove that the P-AHW-Ca chelate has good gastrointestinal tract adaptability. The invention is applied to preparation of calcium absorption promoting products, or preparation of calcium enhancer products, or preparation of calcium nutritional supplement products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a phosphorylated camellia oil meal peptide calcium chelate and application thereof in a calcium enhancer. Background Art

[0002] Calcium is an essential mineral for the human body and plays a vital role in maintaining numerous life activities. While calcium is primarily obtained through daily dietary intake, various factors, including dietary structure and absorption efficiency, can lead to insufficient calcium intake or reduced calcium utilization. Therefore, the development and application of calcium fortifiers has become a key research area in the field of nutrition and health.

[0003] Currently, the main calcium fortifier products include inorganic calcium, organic calcium, and polypeptide calcium chelates. Inorganic calcium easily forms insoluble precipitates with other anions in the intestinal environment, reducing calcium absorption efficiency and consuming more gastric acid, making it unsuitable for long-term intake. Organic calcium also suffers from intestinal precipitation and low absorption efficiency. Polypeptide calcium chelates have the characteristics of good solubility, good bioavailability, and high absorption rate, which can better meet the body's calcium needs.

[0004] Modification can enhance its ability to bind metal ions by introducing specific groups. Phosphorylation refers to the introduction of negatively charged phosphate groups into the peptide chain. Calcium is a positively charged ion, and the electrostatic attraction between the phosphate group and the calcium ion enhances the chelation ability of the peptide and calcium. Phosphorylated modified peptides chelate calcium as a new type of calcium supplement. For example, studies have found that phosphorylation of collagen peptides significantly enhances their calcium binding capacity; another study showed that the prepared phosphorylated soybean peptide calcium chelate has enhanced stability and can promote osteoblast proliferation and differentiation.

[0005] Camellia oleifera Abel belongs to the genus Camellia of the Theaceae family. It is known as one of the four major woody edible oils in the world, along with oil palm, coconut, and olive. Camellia oleifera meal is a by-product of oil extraction from camellia fruit. It is rich in protein and various minerals and is a high-quality source of plant protein. Camellia oleifera meal protein is rich in serine (Ser), threonine (Thr), histidine (His), and other amino acid residues that contain hydroxyl groups or can serve as phosphorylation sites. This application will use camellia oleifera meal as raw material to extract bioactive peptides in order to obtain polypeptides with strong calcium binding ability. The phosphorylation modification of the polypeptide will evaluate the effect of phosphorylation on the calcium chelation rate, and further explore the stability of the prepared phosphorylated camellia oleifera meal peptide calcium chelate. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention uses tea seed meal protein as raw material, prepares tea seed meal active peptides by bioenzymatic hydrolysis, uses bioinformatics analysis to screen out small molecule peptides with calcium chelation potential, and further enhances the calcium binding ability through phosphorylation modification to prepare phosphorylated peptide calcium chelates, and further explores their stability and application.

[0007] One of the technical solutions provided by the present invention is a camellia oil seed meal polypeptide having the amino acid sequence Ala-His-Trp, abbreviated as AHW. The camellia oil seed meal polypeptide AHW is obtained by extracting protein from camellia oil seed meal through enzymatic hydrolysis, ultrafiltration, liquid chromatography-mass spectrometry (LC-MS / MS), and molecular docking screening.

[0008] The second technical solution provided by the present invention is the use of the camellia oil meal polypeptide AHW described in the first technical solution, particularly in the preparation of products promoting calcium absorption, or in the preparation of calcium fortifier products, or in the preparation of calcium nutritional supplement products;

[0009] The products include but are not limited to food, health products or medicines.

[0010] The third technical solution provided by the present invention is a peptide calcium chelate P-AHW-Ca comprising phosphorylated camellia oil meal polypeptide P-AHW;

[0011] Furthermore, the chelate contains phosphorylated camellia oil meal polypeptide P-AHW as an active ingredient;

[0012] Furthermore, the chelate contains phosphorylated camellia oil meal polypeptide P-AHW and Ca;

[0013] Furthermore, the mass ratio of phosphorylated camellia oil meal polypeptide P-AHW and Ca in the chelate is 1:0.6-60;

[0014] Preferably, the concentration of phosphorylated camellia oil meal polypeptide P-AHW in the chelate is 10 mg / mL, the concentration of calcium chloride solution is 60 mg / mL, and the peptide-calcium mass ratio is 1:6 (w / w), achieving the best chelation effect, with a chelation rate of 61.67%±0.71%.

[0015] The fourth technical solution provided by the present invention is a method for preparing the P-AHW-Ca chelate described in the third technical solution;

[0016] Further, the steps are as follows:

[0017] 1g of AHW peptide powder was added to a 3% (w / v) sodium trimetaphosphate (STMP) solution at 50°C and pH 8.0 for 3 hours. The mixture was dialyzed and freeze-dried in a vacuum oven to obtain P-AHW. A 0.1% (w / v) P-AHW solution was prepared and added to a 0.6% (w / v) calcium chloride solution. The pH was adjusted to 7.0, and the solution was ultrasonically treated at 240W for 5 minutes. The solution was then freeze-dried in a centrifugal vacuum oven to obtain P-AHW-Ca.

[0018] The fifth technical solution provided by the present invention is the use of the phosphorylated camellia oil meal peptide calcium chelate described in the third and fourth technical solutions, particularly in the preparation of products promoting calcium absorption, or in the preparation of calcium fortifier products, or in the preparation of calcium nutritional supplement products;

[0019] The products include but are not limited to food, health products or medicines.

[0020] Beneficial effects:

[0021] (1) The present invention uses camellia oleifera meal as raw material and obtains camellia oleifera meal polypeptide AHW by biological enzymatic hydrolysis and separation, and its calcium chelation rate reaches 34.80%±1.65%.

[0022] (2) The present invention explored the phosphorylation-modified camellia meal polypeptide P-AHW, which significantly enhanced its calcium binding ability, with the chelation rate increasing from 34.80%±1.65% before modification to 61.67±0.71%.

[0023] (3) The present invention verifies that the P-AHW-Ca chelate has good stability and is suitable for conventional additive systems. Simulated gastric juice and intestinal juice prove that it has good gastrointestinal adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the peptide calcium chelation rate of different molecular weight components after ultrafiltration.

[0025] Figure 2 This is the total ion chromatogram of the peptide by LC-MS / MS.

[0026] Figure 3 This is the mass spectrometry analysis of AHW.

[0027] Figure 4 Docking diagram of the interaction between AHW and integrin α5β1.

[0028] Figure 5 Docking diagram of the interaction between AHW and integrin α5β3.

[0029] Figure 6Fourier transform infrared spectra of AHW, P-AHW and P-AHW-Ca.

[0030] Figure 7 Scanning electron micrographs of AHW, P-AHW, AHW-Ca and P-AHW-Ca.

[0031] Figure 8 This is a picture of the freeze-dried powder of P-AHW-Ca chelate.

[0032] Figure 9 The effects of pH and temperature on the stability of P-AHW-Ca chelate.

[0033] Figure 10 This is the effect of ascorbic acid and potassium sorbate on the stability of P-AHW-Ca chelate.

[0034] Figure 11 This is the simulated digestion stability diagram of P-AHW-Ca chelate in gastric juice and intestinal fluid. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of this patent more clear, the following is a further detailed description of this patent in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain this patent and are not intended to limit the present invention.

[0036] The present invention relates to a method for determining the chelation rate of phosphorylated camellia meal chelate as follows:

[0037] (1) Plotting of peptide calcium chelation standard curve

[0038] Calcium standard solutions of different concentrations were prepared according to the kit instructions and added to a 96-well plate. 50 μL of calcium standard solution was added to each well, followed by 150 μL of colorimetric reagent. After oscillation and mixing, the reaction was allowed to stand at room temperature for 6 minutes. After the reaction, the absorbance value was read using a microplate reader at a wavelength of 575 nm. Each group was measured in parallel three times. The peptide calcium chelation standard curve was drawn, and the regression equation was y = 1.3876x + 0.1732, R 2 =0.9987.

[0039] (2) Determination of chelation rate

[0040] Prepare a 5 mmol / L calcium chloride solution and mix it with 0.2 mol / L sodium phosphate buffer at a ratio of 1:2 (v / v). Add 500 μL of a 1 mg / mL phosphorylated peptide solution and react at 40°C for 50 minutes. Centrifuge at 6000 rpm for 10 minutes, and measure the calcium content in the supernatant. Perform the assay three times for each sample, and calculate the calcium chelation rate from the supernatant of each sample.

[0041]

[0042] Where, W1 is the calcium chelation rate, %; M1 is the calcium content in the supernatant, μg; M0 is the total amount of calcium added, μg.

[0043] The present application will be further explained below through specific examples.

[0044] Example 1 Preparation of Camellia oleifera meal polypeptide

[0045] (1) Pretreatment of tea seed meal

[0046] Take an appropriate amount of defatted camellia oil meal, add 20 times the volume of 70% ethanol to dissolve it, then centrifuge at 6000r / min for 10min, remove the supernatant, and dry it in an oven at 50℃ for later use.

[0047] (2) Extraction of camellia oil meal protein by alkali dissolution and acid precipitation

[0048] Degreased and desoaped camellia meal was dissolved in distilled water at a ratio of 1:20 (w / v). The pH was adjusted to 12.0 with 1 mol / L NaOH solution, heated in a 40°C water bath, and magnetically stirred for 90 minutes. The mixture was centrifuged at 6000 rpm for 15 minutes to remove the precipitate. The pH of the supernatant was adjusted to 3.5-4.0 with 1 mol / L citric acid, and impurities were removed by centrifugation at 6000 rpm for 15 minutes. The protein precipitate was collected. The camellia meal protein powder was freeze-dried in vacuo for 48 hours and stored at -20°C.

[0049] (3) Enzymatic hydrolysis to obtain tea seed meal polypeptides

[0050] 3g of camellia oil meal protein was placed in a beaker and dissolved in 100mL of distilled water. Alkaline protease (10,667U / g) was added at a rate of 5.33% (w / w) of the substrate mass at 50°C and a pH of 10. The reaction was allowed to proceed for 2.5 hours. The enzyme was then boiled to inactivate the enzyme and cooled to room temperature. Papain (5,333U / g) was then added at a rate of 2.67% (w / w) of the substrate mass at 65°C and a pH of 7.0. The reaction continued for another 2.5 hours before cooling to room temperature. The enzymatic hydrolysis lasted 5 hours. 1mol / L citric acid was then added to adjust the solution's pH to 4.5. The supernatant was centrifuged at 6,000 rpm for 15 minutes, and the pH adjusted to 7.0.

[0051] (4) Ultrafiltration of Camellia oleifera meal polypeptide liquid

[0052] The camellia oil meal polypeptide hydrolysate was centrifuged at 6000 rpm for 15 minutes to obtain the supernatant. The supernatant was then finely filtered through a 0.45 μm microfiltration membrane and separated using a 5 kDa ultrafiltration membrane at 4°C. The retentate and filtrate were retained. The filtrate was then separated through a 3 kDa ultrafiltration membrane to obtain three camellia oil meal polypeptide fractions with molecular weights of <3 kDa, 3-5 kDa, and >5 kDa. Each polypeptide sample was freeze-dried in a vacuum and stored at -20°C until further use.

[0053] The results of peptide calcium chelation rates of different molecular weight components after ultrafiltration are as follows: Figure 1 As shown, there are significant differences in calcium ion chelation ability among peptide components of different molecular weight ranges (P<0.05). The smaller the molecular weight of the short peptide, the stronger the peptide calcium chelation effect. Among them, the calcium chelation rate of low molecular weight peptides <3KDa was 32.03%±1.47%, which was higher than that of peptide components with molecular weights of 3-5KDa and >5KDa.

[0054] (5) LC-MS / MS identification

[0055] The polypeptide components of tea seed meal with molecular weight less than 3KDa were separated by chromatography and identified by mass spectrometry according to relevant operations. The results were analyzed and identified using the MaxQuant database to obtain the corresponding polypeptide sequences. A total of 933 polypeptide sequences were obtained through LC-MS analysis, and their total ion currents are shown in the figure below. Figure 2 shown.

[0056] (6) Bioinformatics analysis of prediction of non-toxic and non-allergenic active peptides

[0057] Target peptides were screened using camellia oil meal protein sequences downloaded from the NCBI website. Peptides with activity scores greater than 0.5 were identified using the Peptide Ranker online software. The peptides were then analyzed for non-toxicity and allergenicity using the ToxinPred and AllerTOP online websites. Combining these three screening criteria, the tripeptide AHW, characterized by high activity, non-toxicity, and low allergenicity, was selected as a candidate functional peptide. Its amino acid sequence was then compared with known functional peptide sequences in the BIOPEP and AHTPDB databases to ensure the novelty of the peptides.

[0058] (7) Molecular docking

[0059] Ligands and receptors must be processed before docking. Peptides screened for activity, toxicity, and sensitization were entered into an online platform to obtain 3D peptide structures and processed as ligands. The crystal structures of integrin αvβ3 (PDB: 1L5G) and α5β1 (PDB: 3VI4) were downloaded from the PDB database (www.rcsb.org). The structures were processed using the AutoDock tool, including removal of crystal water and non-standard peptide chains, merging non-polar hydrogens, adding all hydrogen atoms, and recalculating Gasteiger charges. The structures were then saved as pdbqt format files for future use. AutoDock software was used to perform molecular docking of the ligand AHW with integrins αvβ3 and α5β1, respectively, to obtain docking conformations with optimal binding energy. The docking results were then imported into PyMol software for visualization and analysis.

[0060] The results showed that the negative binding energy indicated that the target bioactive peptide could form a stable complex with integrin. The relevant properties of the peptide AHW with the lowest binding energy are shown in Table 1. The mass spectrometry analysis of AHW is shown in Figure 3 shown.

[0061] Table 1 Related properties of peptide AHW

[0062]

[0063] Through computer molecular simulation, the docking diagram of the interaction between AHW and integrin α5β1 is shown in Figure 2. Figure 4 As shown, pink represents AHW and blue represents integrin α5β1. It can be seen from the figure that the bond lengths of AHW and integrin α5β1 are and The two hydrogen bonds and bond lengths are and Three hydrophobic interactions and a bond length of The salt bridge of AHW; the docking diagram of the interaction between AHW and integrin α5β3 is shown in Figure 5 As shown, the binding of AHW to integrin αvβ3 mainly depends on three hydrogen bonds, of which the bond length is and And both bond lengths are Hydrophobic interactions. Generally, shorter hydrogen bonds have stronger interactions. Docking results showed that AHW had the lowest docking energy and the highest binding capacity with integrins, leading to the selection of AHW for artificial synthesis and further study.

[0064] Example 2 P-AHW-Ca chelate

[0065] (1) Preparation of P-AHW

[0066] Nanjing Yuanpeptide Biotechnology Co., Ltd. was commissioned to synthesize the peptide AHW based on the amino acid sequence of AHW. One gram of AHW peptide powder was added to a 3% (w / v) STMP solution. The reaction was carried out at 50°C, pH 8.0 for 3 hours. The peptide was then dialyzed using a dialysis bag at 4°C for 24 hours, with deionized water replaced every 6 hours. After dialysis, the peptide was freeze-dried in a vacuum oven to obtain the phosphorylated peptide P-AHW.

[0067] (2) Preparation of P-AHW-Ca chelate

[0068] A 0.1% (w / v) P-AHW solution was prepared and added to a calcium chloride solution at a peptide-calcium mass ratio of 1:6 (w / w). The mixture was adjusted to pH 7.0 and treated with an ultrasonic power of 240 W for 5 min. After chelation at 55°C, the solution was centrifuged at 6000 r / min for 15 min. The supernatant was precipitated with 6 times the volume of anhydrous ethanol, allowed to stand and centrifuged again. The precipitate was collected and vacuum freeze-dried to obtain the phosphorylated peptide calcium chelate P-AHW-Ca.

[0069] (3) Fourier transform infrared spectroscopy (FTIR)

[0070] 1 mg of each of AHW, P-AHW and P-AHW-Ca samples were mixed with 100 mg of dry potassium bromide and ground. After tableting, the samples were observed at a wave number of 4000-400 cm -1 Scan within the range. Figure 6 As shown, after phosphorylation, AHW -1 A new phosphate absorption peak appeared at 3147.85 cm -1 and 3059.71cm -1 The original OH and NH stretching vibration peaks at the position disappeared, indicating that the phosphate group was successfully introduced into the hydroxyl and amino groups of the peptide chain. 2+ After chelation, 993.98 cm -1 、1458.55cm -1 and 1531.45cm -1 The absorption peaks at 1673.28cm -1 The C=O peak shifted to 1637.96 cm -1 , indicating that the carboxyl and phosphate groups are involved in the calcium coordination. In addition, 1293.90 cm -1 and 1101.17cm -1 Also to 1287.10cm -1 and 1166.10cm -1 This suggests that the structure of amide III has changed, further proving that the -CH2 group may also be indirectly involved in the coordination.

[0071] (4) Scanning electron microscopy (SEM) analysis

[0072] The micromorphology of AHW, P-AHW, AHW-Ca and P-AHW-Ca samples was observed using a TESCAN MIRA LMS scanning electron microscope. Figure 7 As shown, the surface of AHW (A) is dense and smooth, while that of P-AHW (B) becomes loose and irregular, with fine cracks. The surface of the AHW-Ca sample (C) is rough, with granular deposits, presumably representing the surface structure of the peptide chain bound to calcium salts. The surface of the P-AHW-Ca sample (D) exhibits numerous clusters of particles, suggesting that the introduction of phosphate groups promotes a tighter coordination between the peptide chain and the calcium ion.

[0073] A comparison found that phosphorylation modification has advantages in enhancing the binding ability between peptides and calcium ions, and can effectively promote the structural aggregation and stable formation of peptide calcium chelates, showing better chelation properties than unphosphorylated peptides.

[0074] Example 3 Physical Properties of P-AHW-Ca Chelate Powder

[0075] This example uses the P-AHW-Ca chelate powder prepared in Example 2 as the research object. Figure 8 As shown, the P-AHW-Ca chelate is a white, loose powder after vacuum freeze-drying.

[0076] (1) Hygroscopicity determination

[0077] Place 0.05 g of vacuum freeze-dried P-AHW-Ca chelate powder in a weighed aluminum box and record the initial total mass. Then place the aluminum box in a sealed constant humidity container containing saturated sodium chloride solution and maintain the relative humidity at approximately 75%. Let it stand at 37°C for 7 days, take it out and weigh it, and calculate the hygroscopicity:

[0078]

[0079] In the formula, m1 is the mass of the aluminum box, g; m2 is the initial mass of the aluminum box + sample, g; m3 is the mass of the aluminum box + sample after moisture absorption, g.

[0080] The results showed that the hygroscopicity of P-AHW-Ca chelate was 23.13%±0.65%, and it was easily affected by moisture. During storage, it was necessary to keep it sealed away from moisture to avoid agglomeration or performance degradation.

[0081] (2) Determination of bulk density, tap density and compression

[0082] First, determine the mass of the dry, empty graduated cylinder and record it as M1. Next, add a certain amount of P-AHW-Ca chelate powder to the graduated cylinder, read the volume of the chelate V1, and then accurately measure the total mass of the graduated cylinder and the chelate after addition, M2, to calculate the bulk density using the following formula:

[0083]

[0084] Where, ρ bulk —Bulk density, g / cm 3 ; M1—mass of the measuring cylinder, g; V1—volume of the chelate in the measuring cylinder, cm 3 ; M2—mass of the measuring cylinder after adding the chelate, g.

[0085] The graduated cylinder containing the chelate powder is continuously shaken until its volume stabilizes and no longer changes. The volume value V2 is then read and the tap density of the chelate is calculated using the following formula:

[0086]

[0087] Where, ρ tap —Tap density, g / cm 3 ; M1—mass of the measuring cylinder, g; V2—volume of the chelate in the measuring cylinder after shaking, cm 3 ; M2—mass of the measuring cylinder after adding the chelate and shaking, g.

[0088] According to the above-mentioned bulk density and tap density, the compressibility of the powder is calculated as follows:

[0089]

[0090] Where, CI—compression degree, %; ρ tap —Tap density, g / cm 3 ρ bulk —Bulk density, g / cm 3 .

[0091] The results showed that the bulk density of P-AHW-Ca chelate was 0.39 g / cm 3 ±0.02g / cm 3 ; Tap density is 0.57g / cm 3 ±0.07g / cm 3 The compression degree is 32.12% ± 4.73%. The chelate powder has loose packing, good compressibility, large interparticle spaces and high volume variability, and is suitable for solid preparations such as tablet compression or capsule filling.

[0092] (3) Wettability determination

[0093] Weigh 0.05g of P-AHW-Ca chelate powder and add it to 8mL of deionized water. The mixture was left to stand and observed, and the time required for complete wetting was recorded. "Complete wetting" refers to the complete submersion of the powder below the liquid surface, with no dry powder residue or visible floating particles on the surface. The measured wetting time was 154s ± 3s, demonstrating good wettability and liquid affinity.

[0094] Example 4 Stability of P-AHW-Ca Chelate Powder

[0095] In this example, the P-AHW-Ca chelate powder prepared in Example 2 was used as a research object to test its stability under various environmental conditions.

[0096] (1) Acid-base stability determination

[0097] Prepare a 10 mg / mL P-AHW-Ca chelate solution and adjust the pH to 3.0, 5.0, 7.0, 9.0, and 11.0 using 0.1 mol / L dilute hydrochloric acid or sodium hydroxide, respectively. Heat the sample in a 37°C water bath for 1 hour, centrifuge at 6000 rpm for 15 minutes, and dilute the supernatant 50-fold before measuring the calcium concentration. Calcium retention is calculated using the following formula:

[0098]

[0099] Wherein, X is calcium retention rate, %; C3 is chelate mass concentration, mg / mL; C4 is supernatant calcium ion mass concentration, mg / mL; P is dilution factor (50).

[0100] The results are as follows Figure 9 As shown in the results, the P-AHW-Ca chelate has good pH stability under the conditions of pH 7.0-9.0, and the retention rate is as high as 93.5%±1.21% at pH 9.0. It is suitable for application requirements in neutral to weakly alkaline environments and has good functional retention ability.

[0101] (2) Temperature stability measurement

[0102] A 10 mg / mL P-AHW-Ca chelate solution was heated in a water bath at five temperatures: 60°C, 70°C, 80°C, 90°C, and 100°C for 1 hour, centrifuged at 6000 r / min for 15 minutes, and the supernatant was diluted 50 times to determine the calcium ion concentration and calculate the calcium retention rate under each temperature condition. Figure 9 As shown in the figure, the P-AHW-Ca chelate can maintain a high calcium retention rate in the range of 60-100 °C. At 100 °C, the P-AHW-Ca chelate still maintains a calcium retention rate of 71.4% ± 1.11%, indicating that it has good thermal stability and is suitable for application in hot processing environments.

[0103] (3) Impact of food additives

[0104] Prepare a 10 mg / mL P-AHW-Ca chelate standard solution, add ascorbic acid at concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% to five groups of samples, and add potassium sorbate food additive at concentrations of 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% to another five groups of samples. After standing at room temperature for 24 hours, the calcium retention rate was measured. Figure 10 As shown, the calcium retention rate at an ascorbic acid concentration of 0.2% was significantly higher than that of the other groups, indicating that the chelate has good stability under low ascorbic acid concentration conditions. At potassium sorbate concentrations of 0.2% to 0.4%, the calcium retention rate remained at a high level, indicating that the chelate structure is stable within this concentration range. The P-AHW-Ca chelate exhibits moderate stability at low concentrations of ascorbic acid and potassium sorbate, suggesting potential for application in food systems containing conventional additives.

[0105] (4) Simulated gastrointestinal digestion stability

[0106] The chelate was added to simulated gastric fluid (containing pepsin, NaCl, pH 3.0) and simulated intestinal fluid (containing bile salts, pancreatic enzymes, pH 7.0), digested at 37°C for 2 hours, and samples were taken every 0.5 hours for testing. Figure 11 As shown in the figure, the calcium retention rate of P-AHW-Ca in gastric juice is maintained at about 50%, and the calcium retention rate in intestinal juice is maintained at about 80%, indicating that it has good gastrointestinal stability.

[0107] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that, without departing from the concept of this patent, a person skilled in the art would be able to make various variations, combinations, and improvements to the above-described embodiments, all of which fall within the scope of protection of this patent. Therefore, the scope of protection of this patent shall be determined by the claims.

Claims

1. A camellia oil seed meal polypeptide, characterized in that The camellia oil seed meal polypeptide is AHW, and its amino acid sequence is: Ala-His-Trp.

2. The use of the camellia oil seed meal polypeptide AHW according to claim 1, particularly in the preparation of products promoting calcium absorption, or in the preparation of calcium fortifier products, or in the preparation of calcium nutritional supplement products.

3. A peptide calcium chelate P-AHW-Ca comprising phosphorylated camellia oil seed meal polypeptide, characterized in that: The chelate has P-AHW as an active ingredient, and contains P-AHW and Ca. The mass ratio of P-AHW to Ca in the complex is 1:0.6-60.

4. The method for preparing the phosphorylated camellia oil meal peptide calcium chelate P-AHW-Ca according to claim 3, characterized in that: The steps are as follows: Weigh 1g of AHW peptide powder and add it to a 3% (w / v) sodium trimetaphosphate solution. React at 50°C, pH 8.0 for 3h, dialyze and dry to obtain the phosphorylated peptide P-AHW. Prepare a 0.1% (w / v) P-AHW solution, add it to a calcium chloride solution at a peptide-calcium mass ratio of 1:6 (w / w), adjust the pH to 7.0, and treat with an ultrasonic power of 240W for 5min. After chelation, centrifuge and dry to obtain the phosphorylated peptide calcium chelate P-AHW-Ca.

5. Use of the phosphorylated camellia oil meal peptide calcium chelate P-AHW-Ca according to claim 3, particularly in the preparation of products promoting calcium absorption, or in the preparation of calcium fortifier products, or in the preparation of calcium nutritional supplement products.

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