A casein phosphopeptide having calcium, iron, zinc, manganese and magnesium absorption promoting and bone health improving effects and a preparation method thereof

Abstract

The application provides a casein phosphopeptide with the functions of promoting the absorption of calcium, iron, zinc, manganese and magnesium and improving bone health and a preparation method, and belongs to the technical field of casein phosphopeptide and comprises the following steps: S1, taking casein and adding pepsin for first enzymolysis; S2, taking the first enzymolysis solution, adjusting pH and temperature, and adding a compound enzyme for second enzymolysis; S3, after the second enzymolysis solution is inactivated, the casein phosphopeptide is obtained through treatment. The casein phosphopeptide prepared by the application has the following advantages: 1. can effectively complex mineral elements and reduce ion channel competition inhibition; 2. promotes the absorption of calcium, increases the hardness of the skeleton, and promotes the growth and development of the skeleton; 3. promotes the absorption of magnesium, assists calcium deposition, reduces bone loss, prevents osteoporosis, 3. promotes the absorption of iron, prevents iron deficiency anemia, and improves uncomfortable symptoms such as dizziness and fatigue; 4. promotes the absorption of zinc, enhances the activity of immune cells, improves resistance, and improves appetite. 5. promotes the absorption of manganese, activates the development of the skeleton, accelerates energy metabolism, and has antioxidant effect.

Description

Technical Field

[0001] This invention relates to the field of casein phosphopeptide technology, specifically to a casein phosphopeptide that promotes the absorption of calcium, iron, zinc, manganese, and magnesium and improves bone health, and its preparation method. Background Technology

[0002] Casein phosphopeptide (CPP) is an active polypeptide containing 1-6 phosphoserine residues (Ser-P). It can form soluble complexes with mineral ions in the weakly alkaline environment of the small intestine, promoting the absorption of minerals such as calcium, iron, and zinc. Its relative molecular mass is 1000-5000 Da. It is widely used in the food industry, as well as in health food products that increase bone density, prevent osteoporosis, and promote bone growth.

[0003] The role of calcium chelate polyphosphate (CPP) in promoting calcium absorption is one of the most extensively and thoroughly studied areas. Calcium is a major component of human bones and teeth, and also participates in physiological processes such as nerve transmission and muscle contraction. However, dietary calcium easily forms insoluble precipitates with phytic acid, oxalic acid, and other substances in the neutral intestinal environment, resulting in low absorption rates. CPP effectively inhibits precipitate formation and improves calcium bioavailability by chelating calcium ions to form soluble CPP-Ca complexes.

[0004] Iron is a key component of hemoglobin and many enzymes; iron deficiency can lead to anemia and weakened immune function. Dietary iron is easily oxidized to ferric iron (Fe3+). 3+ And, it forms an insoluble precipitate with hydroxide ions, making it difficult to absorb. CPP promotes iron absorption through two mechanisms: one is reduction, which breaks down Fe into Fe2+ ions and Fe2+ ions. 3+ Reduced to more easily absorbed Fe 2+ Secondly, it has a chelating effect, forming a soluble complex with iron to prevent precipitation.

[0005] Zinc is a component of more than 300 enzymes in the human body, participating in processes such as growth and development, immune regulation, and DNA synthesis. Dietary zinc readily binds with phytic acid to form insoluble salts, resulting in low absorption rates. CPP chelates with zinc to form soluble CPP-Zn complexes, antagonizing the inhibitory effects of phytic acid and other metal ions.

[0006] Besides calcium, iron, and zinc, CPP also promotes the absorption of minerals such as magnesium and manganese, although related research is relatively limited. Magnesium is a key mineral for energy metabolism and muscle function. CPP chelates with magnesium to form a soluble complex, preventing its precipitation. Research on the promoting effect of CPP on manganese is still in its early stages. It is generally believed that CPP improves the bioavailability of these trace elements by preventing their oxidation or precipitation through a similar chelation mechanism.

[0007] For example, Chinese patent CN120699128A, entitled "A Casein Phosphopeptide for Promoting the Absorption of Calcium, Iron, and Zinc Minerals and Improving Bone Health," discloses a method for obtaining casein phosphopeptides containing highly active peptides through optimized processes. Using six mineral elements—calcium, magnesium, copper, iron, zinc, and manganese—as research subjects, the patent first evaluated the utilization of minerals by CPP in a mineral-deficient rat model. Consuming 7 mg / kg / day increased the contents of Ca, Mg, Cu, Fe, Zn, and Mn in the femur by 7.12%, 3.56%, 20.76%, 15.84%, 9.94%, and 67.12%, respectively. Secondly, molecular docking technology was used to screen peptides with calcium absorption-promoting activity from CPP and perform functional verification, revealing oligopeptides that promote mineral element absorption, including: GPFPI, FYPEL, YPVEPF, GPFPIIV, and VAPFPEV.

[0008] However, existing technologies such as the above-mentioned solutions still require the development of new casein phosphopeptides to further improve the bioaccessibility and bioavailability of minerals in organisms. Summary of the Invention

[0009] In view of this, the present invention provides a casein phosphopeptide that promotes the absorption of calcium, iron, zinc, manganese and magnesium and improves bone health, and a method for preparing the same, thereby enabling the novel casein phosphopeptide to improve the bioavailability and bioavailability of minerals in organisms.

[0010] To achieve the above objectives, the present invention provides a method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health, comprising the following steps: S1. Mix casein with water, adjust pH and temperature, add pepsin for the first enzymatic hydrolysis, and obtain the first hydrolysate. S2. Take the first enzymatic hydrolysate, adjust the pH and temperature, add the compound enzyme to carry out the second enzymatic hydrolysis, and obtain the second enzymatic hydrolysate; the compound enzyme includes any three or more combinations of trypsin, chymotrypsin, elastase, pancreatic lipase and bromelain. S3. After inactivating the enzyme in the second enzymatic hydrolysate, the casein phosphopeptide is obtained by multi-stage membrane separation and drying; the casein phosphopeptide includes the following peptide segments: KSVVVQGLYKSMP.

[0011] Optionally, the mass ratio of casein to water is 1:10-15; the amount of pepsin added is 2-4% of the casein mass.

[0012] Optionally, in step S1, the pH and temperature are adjusted to pH 2-3, temperature 35-40℃, and the first enzymatic hydrolysis time is 1.5-2.5h.

[0013] Optionally, in step S2, the pH and temperature are adjusted to pH 7-7.5 and temperature 35-40°C, and the second enzymatic hydrolysis time is 2.5-3.5 h.

[0014] Optionally, the amount of the compound enzyme added is 1 to 2% of the casein protein mass.

[0015] Optionally, the complex enzyme comprises the following components in weight percentage: trypsin 0.8-1.2%, chymotrypsin 0.3-0.7%, elastase 0.1-0.3%, pancreatic lipase 0.05-0.15%, and bromelain 0.04-0.06%.

[0016] Optionally, the multi-stage membrane separation involves passing the second enzymatic hydrolysate sequentially through a 5 kDa ultrafiltration membrane and a 500 Da nanofiltration membrane.

[0017] To achieve the above objectives, the present invention also provides a composition that promotes the absorption of calcium, iron, zinc, manganese, and magnesium and improves bone health, comprising the above-mentioned casein phosphopeptide.

[0018] To achieve the above objectives, the present invention also provides an application of the above-mentioned casein phosphopeptide in promoting the absorption of calcium, iron, zinc, manganese, and magnesium. The above-described technical solution of the present invention has at least the following beneficial effects: 1. In this invention, the treatment of casein follows the sequence of the human digestive process, sequentially using enzymes of the same type found in the human stomach and intestines—including pepsin, amylase, lipase, trypsin, chymotrypsin, and elastase—to perform stepwise enzymatic hydrolysis, degrading it into small peptide fragments to obtain casein phosphopeptides. The preparation method of this invention significantly reduces the sensitizing potential of casein phosphopeptides and is more conducive to human digestion and absorption. It not only achieves a high yield of casein phosphopeptides but also shortens the phosphopeptide sequence length, increasing the distribution density of phosphate groups, thereby further enhancing its effect on promoting calcium absorption and achieving the goal of improving bone health.

[0019] 2. Due to the relatively large molecular size of natural casein (molecular weight range 57–375 kDa), it is easily eluted with other macromolecules during specific adsorption (e.g., chromatography), resulting in poor enrichment. However, in this invention, after two-step enzymatic hydrolysis, 80% of the peptides in the resulting hydrolysate have a molecular weight below 5000 Da, and 50% have a molecular weight below 2000 Da, making it easier to separate and purify from other peptides (especially macromolecular peptides and non-phosphopeptides).

[0020] 3. Experiments show that the casein phosphopeptide obtained in this invention can increase the solubility of calcium by 65-85%; iron by 55-75%; zinc by 60-85%; magnesium by 70-85%; and manganese by 70-85%. Therefore, the casein phosphopeptide obtained in this invention can be used in products that promote the absorption of calcium, iron, zinc, manganese, and magnesium. This casein phosphopeptide is suitable for various food applications, including infant formula, complementary foods, modified milk and milk powder, cheese, beverages, and cereal products. Attached Figure Description

[0021] Figure 1 This is a graph showing the solubility of calcium in the CPP group and the blank group in the experiment of this invention; Figure 2 This is a graph showing the solubility of iron in the CPP group and the blank group in the experiment of this invention; Figure 3 This is a graph showing the solubility of zinc in the CPP group and the blank group in the experiment of this invention; Figure 4 This is a graph showing the solubility of magnesium in the CPP group and the blank group in the experiments of this invention; Figure 5 This is a graph showing the solubility of manganese in the CPP group and the blank group during the experiment of this invention.

[0022] In the figure: NC represents the blank group; CPP represents the casein phosphopeptide group; p < 0.5; p < 0.01; p < 0.001. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figures 1-5 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0024] Example 1 This invention provides a method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health, comprising the following steps: Casein and water were mixed at a mass ratio of 1:12. The pH of the mixture was adjusted to 2.5 and the temperature was set to 37°C. Then, pepsin was added to carry out the first enzymatic hydrolysis. The mass of pepsin was 3% of the casein mass. The first enzymatic hydrolysis time was 2 hours to obtain the first hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 7.2, and the temperature was set to 37°C. Pepsin was inactivated to prepare for the second enzymatic hydrolysis. A compound enzyme was added for the second enzymatic hydrolysis. The mass of the compound enzyme was 1.85% of the casein protein mass, and it included the following components: trypsin 1%, chymotrypsin 0.5%, elastase 0.2%, pancreatic lipase 0.1%, and bromelain 0.05%. The second enzymatic hydrolysis time was 3 hours to obtain the second enzymatic hydrolysate. The second enzymatic hydrolysate was heated at 95°C for 10 minutes to inactivate the enzymes, then rapidly cooled to inactivate all enzymes. It was then passed through a 5 kDa ultrafiltration membrane and a 500 Da nanofiltration membrane, and finally dried to obtain casein phosphopeptides.

[0025] Example 2 This invention provides a method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health, comprising the following steps: Casein and water were mixed at a mass ratio of 1:10. The pH of the mixture was adjusted to 3 and the temperature was set to 35°C. Then, pepsin was added to carry out the first enzymatic hydrolysis. The mass of pepsin was 4% of the casein mass. The first enzymatic hydrolysis time was 2.5 hours to obtain the first hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 7, and the temperature was set to 40°C. Pepsin was inactivated to prepare for the second enzymatic hydrolysis. A compound enzyme was added for the second enzymatic hydrolysis. The mass of the compound enzyme was 1.8% of the casein protein mass, and it included the following components: trypsin 1.2%, chymotrypsin 0.3%, and elastase 0.3%. The second enzymatic hydrolysis time was 2.5 hours to obtain the second enzymatic hydrolysate. The second enzymatic hydrolysate was heated at 95°C for 10 minutes to inactivate the enzymes, then rapidly cooled to inactivate all enzymes. It was then passed through a 5 kDa ultrafiltration membrane and a 500 Da nanofiltration membrane, and finally dried to obtain casein phosphopeptides.

[0026] Example 3 This invention provides a method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health, comprising the following steps: Casein and water were mixed at a mass ratio of 1:15. The pH of the mixture was adjusted to 2 and the temperature was set to 40°C. Then, pepsin was added to carry out the first enzymatic hydrolysis. The mass of pepsin was 2% of the casein mass. The first enzymatic hydrolysis time was 1.5 hours to obtain the first hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 7.5, and the temperature was set to 37°C. Pepsin was inactivated to prepare for the second enzymatic hydrolysis. A compound enzyme was added for the second enzymatic hydrolysis. The mass of the compound enzyme was 1.69% of the casein protein, and it included the following components: trypsin 0.8%, chymotrypsin 0.7%, pancreatic lipase 0.15%, and bromelain 0.04%. The second enzymatic hydrolysis time was 3.5 hours, and the second enzymatic hydrolysate was obtained. The second enzymatic hydrolysate was heated at 95°C for 10 minutes to inactivate the enzymes, then rapidly cooled to inactivate all enzymes. It was then passed through a 5 kDa ultrafiltration membrane and a 500 Da nanofiltration membrane, and finally dried to obtain casein phosphopeptides.

[0027] Example 4 Compared with Example 1, the only difference is that the complex enzyme includes the following components: 0.9% trypsin, 0.3% chymotrypsin, 0.1% elastase, 0.05% pancreatic lipase and 0.06% bromelain; that is, the mass of the complex enzyme is 1.41% of the casein mass.

[0028] The casein phosphopeptides prepared in Examples 1-4 were analyzed by mass spectrometry. The parameters for mass spectrometry analysis were as follows: reversed-phase column, 0.1% formic acid aqueous solution as the aqueous phase, acetonitrile as the organic phase, flow rate of 0.2-1.0 ml / min, column temperature of 25-40℃, ESI spray voltage of 2.5-5 kV, nitrogen gas as the gas, pressure of 1-3 Pa, and collision energy of 10-50 eV. The following core peptide was obtained: KSVVVQGLYKSMP.

[0029] experiment: The casein phosphopeptide prepared in Example 1 was subjected to a dissolution test to detect its effect on dissolving calcium, iron, zinc, manganese, and magnesium.

[0030] 1. Dissolution test 1.1 Experiment to promote calcium ion absorption A control group and a casein phosphopeptide (CPP) group were set up for comparative experiments.

[0031] The blank group consisted of a saturated calcium chloride solution; the CPP group consisted of a certain amount of casein phosphopeptide (CPP) weighed out, dissolved in a saturated calcium chloride solution, and thoroughly mixed.

[0032] The pH of the two mixtures was adjusted to 8.0 using sodium hydroxide solution to simulate the slightly alkaline environment of the human small intestine. The reaction systems were then placed at a constant temperature for a specified time to promote the complexation reaction between CPP and calcium ions, forming a soluble complex.

[0033] After the reaction, both solutions were centrifuged: centrifuged at 4℃ and 4000×g for 10 minutes, the supernatant was carefully discarded, and the precipitate was collected. The obtained precipitate was dried in a vacuum drying oven, and the mass of the precipitate was weighed every 30 minutes until three consecutive weighings showed no significant change (i.e., constant mass), and the final mass value was recorded. Using the theoretical mass of calcium hydroxide as a reference, the actual solubility of the complex was calculated to evaluate the complexation behavior of CPP with calcium ions and the stability of the complex. Figure 1 .

[0034] 1.2 Experiment to promote iron ion absorption A control group and a casein phosphopeptide (CPP) group were set up for comparative experiments.

[0035] The blank group consisted of a saturated ferrous sulfate solution; the CPP group consisted of a certain amount of casein phosphopeptide (CPP) weighed out, dissolved in a saturated ferrous sulfate solution, and thoroughly mixed.

[0036] The pH of the two mixtures was adjusted to 8.0 using sodium hydroxide solution to simulate the weakly alkaline environment of the human small intestine. The reaction system was then placed under constant temperature and allowed to stand for a specified time to promote the complexation reaction between CPP and ferrous ions, forming a soluble complex.

[0037] After the reaction, both solutions were centrifuged: centrifuged at 4℃ and 4000×g for 10 minutes, the supernatant was carefully discarded, and the precipitate was collected. The obtained precipitate was dried in a vacuum drying oven, and the mass of the precipitate was weighed every 30 minutes until three consecutive weighings showed no significant change (i.e., constant mass), and the final mass value was recorded. Using the theoretical mass of ferrous hydroxide as a reference, the actual solubility of the complex was calculated to evaluate the complexation behavior of CPP with iron ions and the stability of the complex. Figure 2 .

[0038] 1.3 Experiment to promote zinc ion absorption A control group and a casein phosphopeptide (CPP) group were set up for comparative experiments.

[0039] The blank group consisted of a saturated zinc sulfate solution; the CPP group consisted of a certain amount of casein phosphopeptide (CPP) weighed out, dissolved in a saturated zinc sulfate solution, and thoroughly mixed.

[0040] The pH of the two mixtures was adjusted to 8.0 using sodium hydroxide solution to simulate the weakly alkaline environment of the human small intestine. The reaction system was then placed under constant temperature and allowed to stand for a specified time to promote the complexation reaction between CPP and zinc ions, forming a soluble complex.

[0041] After the reaction, both solutions were centrifuged: centrifuged at 4℃ and 4000×g for 10 minutes, the supernatant was carefully discarded, and the precipitate was collected. The obtained precipitate was dried in a vacuum drying oven, and the mass of the precipitate was weighed every 30 minutes until three consecutive weighings showed no significant change (i.e., constant mass), and the final mass value was recorded. Using the theoretical mass of zinc hydroxide as a reference, the actual solubility of the complex was calculated to evaluate the complexation behavior of CPP with zinc ions and the stability of the complex. Figure 3 .

[0042] 1.4 Experiment to promote magnesium ion absorption A control group and a casein phosphopeptide (CPP) group were set up for comparative experiments.

[0043] The blank group consisted of a saturated magnesium sulfate solution; the CPP group consisted of a certain amount of casein phosphopeptide (CPP) weighed out, dissolved in a saturated magnesium sulfate solution, and thoroughly mixed.

[0044] The pH of the two mixtures was adjusted to 8.0 using sodium hydroxide solution to simulate the weakly alkaline environment of the human small intestine. The reaction systems were then placed under constant temperature and allowed to stand for a specified time to promote the complexation reaction between CPP and magnesium ions, forming a soluble complex.

[0045] After the reaction, both solutions were centrifuged: centrifuged at 4℃ and 4000×g for 10 minutes, the supernatant was carefully discarded, and the precipitate was collected. The obtained precipitate was dried in a vacuum drying oven, and the mass of the precipitate was weighed every 30 minutes until three consecutive weighings showed no significant change (i.e., constant mass), and the final mass value was recorded. Using the theoretical mass of magnesium hydroxide as a reference, the actual solubility of the complex was calculated to evaluate the complexation behavior of CPP with magnesium ions and the stability of the complex. Figure 4 .

[0046] 1.5 Experiment to promote manganese ion absorption A control group and a casein phosphopeptide (CPP) group were set up for comparative experiments.

[0047] The blank group consisted of a saturated manganese sulfate solution; the CPP group consisted of a certain amount of casein phosphopeptide (CPP) weighed out, dissolved in a saturated manganese sulfate solution, and thoroughly mixed.

[0048] The pH of the two mixtures was adjusted to 8.0 using sodium hydroxide solution to simulate the weakly alkaline environment of the human small intestine. The reaction system was then placed under constant temperature and allowed to stand for a specified time to promote the complexation reaction between CPP and manganese ions, forming a soluble complex.

[0049] After the reaction, both solutions were centrifuged: centrifuged at 4℃ and 4000×g for 10 minutes, the supernatant was carefully discarded, and the precipitate was collected. The obtained precipitate was dried in a vacuum drying oven, and the mass of the precipitate was weighed every 30 minutes until three consecutive weighings showed no significant change (i.e., constant mass), and the final mass value was recorded. Using the theoretical mass of manganese hydroxide as a reference, the actual solubility of the complex was calculated to evaluate the complexation behavior of CPP with manganese ions and the stability of the complex. Figure 5 .

[0050] 2. Cell transport experiment 2.1 Calcium transport experiment Cell culture: The Caco-2 cell line (passages 50-60) was used as an intestinal epithelial model in calcium transport experiments. Cells were cultured in complete Duchenne modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotics, and 1% non-essential amino acids. Cells were cultured at 3 × 10⁶ cells / year. 5 Cells were seeded at a density of 100 cells / mL on Transwell culture plates with a polycarbonate membrane. Transepithelial resistance (TEER) was measured using a Millicell ERS-2 epithelial voltmeter before the calcium transport assay. The TEER of the cell monolayer was approximately 260 Ω / cm². The culture medium was discarded, and the monolayer was immediately washed twice with Hanks balanced salt solution. The monolayer was then transferred to new wells containing 2 mL of HBSS buffer. 2 mL of HBSS buffer was also added to the top well before the calcium transport assay and the cells were incubated at 37°C, 5% CO₂ for 30 min. After 30 min of incubation, casein phosphopeptide was added to the top well containing calcium solution (300 μg / well). The samples were premixed with the calcium solution before the transport assay. The control group without CPP received only calcium solution (300 μg / well) without CPP or HPLC-separated peak components. At different time points (20, 40, 60, 90, 120, 180, and 240 minutes), 1 mL of HBSS buffer was collected from the basal side to measure calcium ion concentration. Simultaneously, 1 mL of fresh HBSS buffer was added to the basal side to maintain a constant volume.

[0051] The final calcium concentration at each time point was determined by inductively coupled plasma mass spectrometry (ICP-MS). The total amount of calcium transported to the substrate side of each well was calculated according to the following formula 1:

[0052] In the formula, represents the total amount of calcium transported in the 2 mL HBSS buffer on the base side of each well at each selected time point (20, 40, 60, 90, 120, 180, and 240 minutes) (unit: μg / well); 2 is a constant representing the 2 mL HBSS buffer on the base side of each well; An represents the calcium ion concentration in the HBSS buffer on the base side of each well at different time points as determined by ICP-MS (unit: μg / mL); 1 is also a constant representing the 1 mL HBSS buffer collected from the base side of each well for calcium ion concentration determination; n is an independent variable, which in this study can be 1, 2, 3, 4, 5, 6, or 7, representing time points of 20, 40, 60, 90, 120, 180, and 240 minutes, respectively. All measurements were performed in triplicate, and results are expressed as mean ± standard deviation (SD).

[0053] 2.2 Iron Transfer Experiment Casein phosphopeptides (CPPs, 300 μg / well) were added to the top side of the iron-containing solution (150 μg / well). The samples were premixed with the iron solution before the start of the transport study. The control group, which did not contain CPPs, was supplemented only with magnesium solution (150 μg / well). All other conditions (e.g., time points) and analyses (e.g., Equation 1) in the iron transport study were the same as those described in the calcium transport study.

[0054] 2.3 Zinc transport experiment Casein phosphopeptides (CPPs, 300 μg / well) were added to the top side of the zinc-containing solution (150 μg / well). The samples were premixed with the zinc solution before the start of the transport study. The control group without CPPs was treated with only zinc solution (150 μg / well). All other conditions (e.g., time points) and analyses (e.g., Equation 1) in the zinc transport study were the same as those described in the calcium transport study.

[0055] 2.4 Magnesium transport experiment Casein phosphopeptides (CPPs, 300 μg / well) were added to the top side of the magnesium-containing solution (150 μg / well). The samples were premixed with the magnesium solution before the start of the transport study. The control group, which did not contain CPPs, was supplemented only with zinc solution (150 μg / well). All other conditions (e.g., time points) and analyses (e.g., Equation 1) in the magnesium transport study were the same as those described in the calcium transport study.

[0056] 2.5 Manganese transport experiment Casein phosphopeptides (CPPs, 300 μg / well) were added to the top side of the manganese-containing solution (150 μg / well). The samples were premixed with the manganese solution before the start of the transport study. The control group, which did not contain CPPs, was treated with only manganese solution (150 μg / well). All other conditions (e.g., time points) and analyses (e.g., Equation 1) in the manganese transport study were the same as those described in the calcium transport study.

[0057] The transport experiments 2.1 to 2.5 were performed twice each, and the results are shown in Table 1.

[0058] Table 1. Results of calcium, iron, zinc, and manganese transport experiments on casein phosphopeptides.

[0059] 3. Experimental Results 3.1 Experiment to promote absorption 3.1.1 Experiment to promote calcium ion absorption like Figure 1 As shown, in a simulated alkaline environment in the small intestine, the calcium ion solubility rate in the blank control group was 9.59%, while in the experimental group with added casein phosphopeptide (CPP), the calcium ion solubility rate significantly increased to 75.19%. This result indicates that casein phosphopeptide can effectively maintain the dissolved state of calcium ions in the liquid phase and significantly inhibit their precipitation, thereby contributing to the promotion of calcium ion bioavailability and absorption.

[0060] 3.1.2 Experiment to promote iron ion absorption like Figure 2 As shown, in a simulated alkaline environment in the small intestine, the iron ion solubility rate in the blank control group was 9.93%, while in the experimental group with added casein phosphopeptide (CPP), the iron ion solubility rate significantly increased to 65.19%. This result indicates that casein phosphopeptide can effectively maintain the dissolved state of iron ions in the liquid phase and significantly inhibit their precipitation, thereby contributing to the promotion of iron ion bioavailability and absorption.

[0061] 3.1.3 Experiment to promote zinc ion absorption like Figure 3 As shown, in a simulated alkaline environment in the small intestine, the solubility of zinc ions in the blank control group was 5.38%, while in the experimental group with added casein phosphopeptide (CPP), the solubility of zinc ions significantly increased to 73.60%. This result indicates that casein phosphopeptide can effectively maintain the dissolved state of zinc ions in the liquid phase and significantly inhibit their precipitation, thereby contributing to the promotion of zinc ion bioavailability and absorption.

[0062] 3.1.4 Experiment to promote magnesium ion absorption like Figure 4As shown, in a simulated alkaline environment in the small intestine, the solubility of magnesium ions in the blank control group was 15.74%, while in the experimental group with added casein phosphopeptide (CPP), the solubility of magnesium ions significantly increased to 78.61%. This result indicates that casein phosphopeptide can effectively maintain the dissolved state of magnesium ions in the liquid phase and significantly inhibit their precipitation, thereby contributing to the promotion of magnesium ion bioavailability and absorption.

[0063] 3.1.5 Experiment to promote manganese ion absorption like Figure 5 As shown, in a simulated alkaline environment in the small intestine, the solubility of manganese ions in the blank control group was 13.64%, while in the experimental group with added casein phosphopeptide (CPP), the solubility of manganese ions significantly increased to 78.16%. This result indicates that casein phosphopeptide can effectively maintain the dissolved state of manganese ions in the liquid phase and significantly inhibit their precipitation, thereby contributing to the promotion of bioavailability and absorption of manganese ions.

[0064] 3.2 Experiments to promote transport 3.2.1 Experiment to promote calcium ion transport As shown in Table 1, in the in vitro Caco-2 cell transport experiment, after 240 minutes of intervention with casein phosphopeptide, the ion transport efficiency was significantly improved, with a cumulative transport rate of 45.1%. This result indicates that casein phosphopeptide can effectively promote the solubility and transmembrane transport of magnesium ions, possibly by inhibiting the precipitation tendency of magnesium ions in the alkaline environment of the intestine through chelation, thereby enhancing its bioavailability in vivo.

[0065] 3.2.2 Experiments to promote iron ion transport As shown in Table 1, in the in vitro Caco-2 cell transport experiment, after 240 minutes of intervention with casein phosphopeptide, a significant increase in ion transport efficiency was observed, with a cumulative transport rate reaching 32.2%. This result indicates that casein phosphopeptide can effectively promote the solubility and transmembrane transport of magnesium ions, possibly by inhibiting the tendency of magnesium ions to precipitate in the alkaline environment of the intestine through chelation, thereby enhancing its bioavailability in vivo.

[0066] 3.2.3 Experiment to promote zinc ion transport As shown in Table 1, in the in vitro Caco-2 cell transport experiment, after 240 minutes of intervention with casein phosphopeptide, the ion transport efficiency was significantly improved, with a cumulative transport rate of 31.3%. This result indicates that casein phosphopeptide can effectively promote the solubility and transmembrane transport of magnesium ions, possibly by inhibiting the precipitation tendency of magnesium ions in the alkaline environment of the intestine through chelation, thereby enhancing its bioavailability in vivo.

[0067] 3.2.4 Experiment to promote magnesium ion transport As shown in Table 1, in the in vitro Caco-2 cell transport experiment, after 240 minutes of intervention with casein phosphopeptide, a significant increase in ion transport efficiency was observed, with a cumulative transport rate reaching 39.1%. This result indicates that casein phosphopeptide can effectively promote the solubility and transmembrane transport of magnesium ions, possibly by inhibiting the tendency of magnesium ions to precipitate in the alkaline environment of the intestine through chelation, thereby enhancing its bioavailability in vivo.

[0068] 3.2.5 Experiment to promote manganese ion transport As shown in Table 1, in the in vitro Caco-2 cell transport experiment, after 240 minutes of intervention with casein phosphopeptide, a significant increase in ion transport efficiency was observed, with a cumulative transport rate reaching 28.4%. This result indicates that casein phosphopeptide can effectively promote the solubility and transmembrane transport of magnesium ions, possibly by inhibiting the precipitation tendency of magnesium ions in the alkaline environment of the intestine through chelation, thereby enhancing its bioavailability in vivo.

[0069] In summary, the technical solution provided by this invention provides a novel casein phosphopeptide and improves the bioaccessibility and bioavailability of minerals in organisms.

[0070] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health, characterized in that, Includes the following steps: S1. Mix casein with water, adjust pH and temperature, add pepsin for the first enzymatic hydrolysis, and obtain the first hydrolysate. S2. Take the first enzymatic hydrolysate, adjust the pH and temperature, add the compound enzyme to carry out the second enzymatic hydrolysis, and obtain the second enzymatic hydrolysate; the compound enzyme includes any three or more combinations of trypsin, chymotrypsin, elastase, pancreatic lipase and bromelain. S3. After inactivating the enzyme in the second enzymatic hydrolysate, the casein phosphopeptide is obtained by multi-stage membrane separation and drying; the casein phosphopeptide includes the following peptide segments: KSVVVQGLYKSMP.

2. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, The mass ratio of casein to water is 1:10~15; the amount of pepsin added is 2~4% of the casein mass.

3. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, In step S1, the pH and temperature are adjusted to pH 2-3 and temperature 35-40℃, and the first enzymatic hydrolysis time is 1.5-2.5h.

4. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, In step S2, the pH and temperature are adjusted to pH 7-7.5 and temperature 35-40℃, and the second enzymatic hydrolysis time is 2.5-3.5h.

5. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, The amount of the compound enzyme added is 1-2% of the casein protein content.

6. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, The complex enzyme comprises the following components in weight percentage: trypsin 0.8-1.2%, chymotrypsin 0.3-0.7%, elastase 0.1-0.3%, pancreatic lipase 0.05-0.15%, and bromelain 0.04-0.06%.

7. The method for preparing casein phosphopeptides that promote the absorption of calcium, iron, zinc, manganese, and magnesium and improve bone health according to claim 1, characterized in that, The multi-stage membrane separation involves passing the second enzymatic hydrolysate through a 5 kDa ultrafiltration membrane and a 500 Da nanofiltration membrane sequentially.

8. A composition that promotes the absorption of calcium, iron, zinc, manganese, and magnesium and improves bone health, characterized in that, Includes the casein phosphopeptide as described in claim 1.

9. The application of the casein phosphopeptide as described in claim 1 in promoting the absorption of calcium, iron, zinc, manganese, and magnesium.

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