A polypeptide derived from lactadherin and uses thereof

Abstract

This invention relates to a polypeptide derived from lactoferrin and its uses. The amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1 to SEQ ID NO:3. The polypeptide of this invention is not only non-toxic and highly safe, but also exhibits good osteogenic activity, specifically promoting osteoblast proliferation, differentiation, and mineralization, and increasing the calcium ion content within osteoblasts. Therefore, the polypeptide of this invention has a positive effect on bone development due to its good osteogenic activity and can be used in the preparation of food (e.g., infant formula).

Description

Technical Field

[0001] This invention belongs to the fields of food manufacturing, biopharmaceutical manufacturing, gene-engineered drugs, and vaccine manufacturing technology. More specifically, it relates to a polypeptide derived from lactoferrin and its uses. Background Technology

[0002] Bone growth and development are particularly important in the early stages of life. With the accelerated bone turnover and steady increase in bone mass during this stage, the bone mass accumulation in early life (infancy and early childhood) plays a decisive role in an individual's peak bone mass and is of great significance for developing a strong skeletal system and reducing the risk of future bone diseases.

[0003] Dietary nutrients in natural foods have great potential in actively regulating bone metabolism: Studies have confirmed that food components such as minerals, polysaccharides, proteins, and polyphenols have positive regulatory effects on bone health, with short peptides containing ≤10 amino acid residues showing particularly significant effects in enhancing bone health and promoting bone development. Hwang et al. (see *The in vitro and in vivo effects of the low molecular weight fucoidan on the boneosteogenic differentiation properties*) investigated the potential effects of fucoidan on bone health and found that low molecular weight fucoidan not only stimulates osteogenic differentiation in vitro but also promotes bone mineralization in vivo. Wang et al. (see *Cell Proliferation Stimulation Ability and Osteogenic Activity of Low Molecular Weight Peptides Derived from Bovine Gelatin Hydrolysates*) showed that culturing MC3T3-E1 cells in a medium containing low molecular weight peptides derived from bovine bone gelatin hydrolysates significantly enhanced cell differentiation and mineralization capabilities.

[0004] Further development of more bioactive peptides that can promote bone growth to meet current applications in the development of infant formula or nutritional products. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a polypeptide derived from lactoferrin.

[0006] A second object of the present invention is to provide the use of the above-mentioned polypeptide in the preparation of products that promote osteogenic formation.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: A polypeptide derived from lactoferrin, wherein the amino acid sequence of the polypeptide is any one of SEQ ID NO:1 to SEQ ID NO:3.

[0008] The amino acid sequence of the polypeptide of the present invention is as follows: SEQ ID NO:1: FIPTESAN; SEQ ID NO:2: WLKPDPSQK; SEQ ID NO:3: NNKYPDAVA.

[0009] The polypeptides of this invention are derived from lactoapontin, which can be obtained either by mimicking gastrointestinal digestion (enzymatic hydrolysis) and purification of lactoapontin (milk-derived osteopontin, LPN, also known as mammary osteopontin), or by solid-phase synthesis. Among these methods, the polypeptides obtained by solid-phase synthesis can be mass-produced with high purity and stable quality, which can meet market demand.

[0010] The inventors of this invention have discovered that the polypeptide of this invention is not only non-toxic and highly safe, but also exhibits excellent osteogenic activity, specifically by promoting osteoblast proliferation, differentiation, and mineralization, as well as increasing the calcium ion content within osteoblasts. Therefore, the polypeptide of this invention, due to its excellent osteogenic activity, has a positive effect on bone development and can be used in the preparation of food products (e.g., infant formula).

[0011] Preferably, the sixth amino acid at the N-terminus of the sequence shown in SEQ ID NO:1 is further phosphorylated, i.e., FIPTESpAN, where Sp represents phosphorylated serine.

[0012] The structural formula of phosphorylated serine is shown below: .

[0013] Preferably, the amino acid sequence of the polypeptide is either SEQ ID NO:1 or SEQ ID NO:3.

[0014] In terms of inducing osteogenic differentiation and mineralization, SEQ ID NO:1 and SEQ ID NO:3 showed higher activity than SEQ ID NO:2.

[0015] The use of the above-mentioned polypeptides in the preparation of products that promote osteogenic formation.

[0016] Preferably, the product is at least one of food and medicine.

[0017] More preferably, the food is at least one of supplementary food, formula food, or health food.

[0018] More preferably, the supplementary food is an infant supplementary food.

[0019] More preferably, the formulated food is at least one of infant formula and special medical purpose formula.

[0020] More preferably, the infant formula is at least one of infant formula, follow-up formula, toddler formula, and infant formula for special medical purposes.

[0021] More preferably, the food is at least one of liquid food, semi-liquid food, solid food, or semi-solid food.

[0022] More preferably, the food is a dairy product.

[0023] More preferably, the dairy product is at least one of fermented milk and modified milk powder.

[0024] More preferably, the drug is a drug for the prevention and / or treatment of osteoporosis.

[0025] The polypeptides of this invention have a positive effect on the prevention and treatment of osteoporosis due to their good osteogenic activity, and can be used to prepare drugs.

[0026] Preferably, the osteogenic process promotes cell proliferation.

[0027] Preferably, the osteogenic process promotes cell differentiation.

[0028] Preferably, the osteogenic process promotes cell mineralization.

[0029] Preferably, the osteogenic process involves increasing the intracellular calcium ion content.

[0030] The present invention has the following beneficial effects: The polypeptides of this invention are not only non-toxic and highly safe, but also exhibit excellent osteogenic activity, specifically promoting osteoblast proliferation, differentiation, and mineralization, as well as increasing the calcium ion content within osteoblasts. Therefore, the polypeptides of this invention, due to their excellent osteogenic activity, have a positive effect on bone development and can be used in the preparation of food products (e.g., infant formula). Attached Figure Description

[0031] Figure 1 This is a chromatogram of the high-performance liquid chromatography analysis of the Mw < 3kDa component in Example 1.

[0032] Figure 2 This is the secondary mass spectrum of peptide 1.

[0033] Figure 3 This is the secondary mass spectrum of peptide 2.

[0034] Figure 4 This is the secondary mass spectrum of peptide 3.

[0035] Figure 5 The figure shows the effect of peptides 1-3 on cell proliferation activity at different time points.

[0036] Figure 6 The figure shows the effect of peptides 1-3 on cell differentiation activity.

[0037] Figure 7 The figure shows the effect of peptides 1-3 on cell mineralization activity.

[0038] Figure 8 The figure shows the effect of peptides 1-3 on intracellular calcium ion content. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] All experiments were repeated three times. One-way ANOVA was performed with a significance level set at p < 0.05. Data are expressed as mean ± standard deviation.

[0042] Example 1: Preparation, Identification and Synthesis of Polypeptides I. Preparation of Polypeptides 1.1 Dynamic simulation of gastrointestinal digestion In the entire system, the pepsin activity in the gastric simulated digestive fluid was 525 U / mL, the pancreatic enzyme activity in the intestinal simulated digestive fluid was 20 U / mL (based on the trypsin activity), and the bile mass concentration was 9.8 mol / L.

[0043] After the dynamic in vitro human gastrointestinal system (DHSI-IV, Xiaodong Yijian Instrument Equipment Co., Ltd.) was installed, 6 mL of fasting gastric juice was added to the stomach. Then, simulated gastric digestive juice, simulated intestinal digestive juice, acid, and alkali were introduced into the corresponding secretion devices on the side of the instrument. Air was expelled from the tubing, and the ambient heating was turned on for preheating, maintaining the system temperature at 37℃ during the experiment. After preheating, 6 g of L-OPN (lactoadenosine, provided by Biostime (Changsha) Nutritional Food Co., Ltd.) was dissolved in 120 mL of deionized water to obtain a lactoadenosine solution. The lactoadenosine solution was then poured into the funnel above the instrument, and simulated digestion was started using the system's recommended parameters. After 180 min of digestion, the collected digestive products were subjected to high-temperature enzyme inactivation and centrifugation to obtain the in vitro digestive products.

[0044] 1.2 Separation and Purification Active peptides from the dynamic digestion products were enriched using DA201C macroporous resin. They were then separated using a 3 kDa ultrafiltration tube (Merck Millipore, Billerica, MA, USA). W >3kda and M W The ultrafiltration fraction <3kDa was freeze-dried, and its effect on cell proliferation activity was determined. Data showed that M... W The <3kDa component showed a significantly better effect on promoting osteoblast proliferation than the M component. W The >3kDa component exhibits a more prominent advantage in proliferation activity.

[0045] M obtained by ultrafiltration W The fraction <3kDa was filtered through a 0.45µm filter membrane and separated by preparative liquid chromatography using a C18 reversed-phase column (20 mm × 450 mm, 10µm). Chromatographic conditions were as follows: Mobile phase A: double-distilled water containing 0.1% trifluoroacetic acid (TFA); Mobile phase B: methanol containing 0.1% TFA; Elution gradient: 0–25 min, 12%–30% mobile phase B; 25–49 min, 30%–42% mobile phase B; 49–73 min, 42%–50% mobile phase B; 73–78 min, 50%–70% mobile phase B; 78–93 min, 50%–70% mobile phase B; 93–115 min, 70%–90% mobile phase B; 115–165 min, 90%–90% mobile phase B. Injection volume: 5 mL; Flow rate: 5 mL / min. The detection wavelength was 214 nm. The elution peaks were collected, concentrated, and freeze-dried to prepare solutions with a concentration of 50 μg / mL. The cell proliferation activity of different components was then measured.

[0046] Eight sub-active components (F1, F2, F3, F4, F5, F6, F7, F8) were obtained by RP-HPLC separation, as follows: Figure 1 As shown, the concentration of each sub-active component was uniformly adjusted to 50 μg / mL, and the promoting activity and timeliness of each component on osteoblast proliferation were systematically evaluated. Cell proliferation activity assays showed that at 24h, 48h, and 72h culture time points, osteoblasts treated with the other seven sub-active components, except for component F7, did not show toxic damage. Proliferation activity analysis indicated that at 24h and 48h of culture, components F4 and F6 showed significant advantages in promoting proliferation; at 48h, the proliferation activity of component F4 was significantly increased by 40.78% compared to the blank control group, and that of component F6 was increased by 32.93%; by 72h, component F4 still maintained a highly efficient proliferative capacity, with a proliferation rate increased by 34.98% compared to the blank group, and significantly higher than all other components (…). P<0.05), exhibiting stable and potent osteoblast-promoting properties. Data from various time points showed that all five components (F1, F2, F3, F4, and F6) positively regulated osteoblast proliferation, with component F4 exhibiting the most prominent activity. Further analysis of chromatographic elution patterns revealed that the relatively bioactive components F1-F4 eluted within the 13–44 min range, while component F6 eluted within the 60–66 min range. The proliferative activity of different components fluctuated significantly with changes in the mobile phase ratio. In conclusion, component F4, purified by RP-HPLC, exhibited the strongest and most sustained osteoblast-promoting activity, and was therefore selected as the target for subsequent peptide composition identification and in-depth investigation of its osteogenic mechanism.

[0047] 1.3 Mass Spectrometry Identification Methods for Peptides Peptides were identified using LC-MS / MS. The identification conditions were as follows: the entire system consisted of a QExactive mass spectrometer connected to an EASY-nanoLC1200. A co-injection of 10 μL of sample was used, employing an Acclaim PepMap C18 column (75 μm × 25 cm) at a flow rate of 300 nL / min, maintaining a column temperature of 40℃, and an electrospray voltage of 2 kV. A binary gradient elution program was used, with the mobile phases being: Phase A (0.1% formic acid aqueous solution) and Phase B (acetonitrile solution containing 0.1% formic acid). The chromatographic gradient settings were as follows: 0–3 min, 2%–6% Phase B; 3–42 min, 6%–20% Phase B; 42–47 min, 20%–35% Phase B; 47–48 min, 35%–100% Phase B; 48–100 min, 100%–100% Phase B. The mass spectrometry parameters were set as follows: (1) MS: scan range (m / z) 200~1500; resolution 70000; AGC target value 3e6; maximum injection time 60 ms; scan charge 1~7; (2) HCD-MS / MS (top10): resolution 17500; isolation window 2.2 m / z; AGC target value 5e4; maximum injection time 50 ms; collision energy 27; dynamic exclusion time 20 s.

[0048] A total of 294 peptides were identified in fraction F4, with molecular weights ranging from 299.1845 to 3204.2058 Da and sequence lengths between 3 and 27 amino acids. Based on the screening criteria of "amino acid number < 10, confidence level > 40", 69 target peptide sequences were initially screened from fraction F4. After constructing the three-dimensional structure of each peptide sequence using Discovery Studio software, a batch docking strategy was adopted to obtain peptides with stable binding and optimal activity: First, three rounds of parallel molecular docking were performed on the 69 peptides, and the top 25% (18 peptides) of docking activity were selected; then, these 18 peptides were subjected to three more rounds of parallel docking, and finally, three peptides with the best docking activity were selected. Polypeptide 1: A polypeptide with an amino acid sequence as shown in SEQ ID NO:1, wherein SEQ ID NO:1: FIPTESAN, and its N-terminal 6th amino acid (serine) is also phosphorylated, i.e. FIPTESpAN. Polypeptide 2: A polypeptide with an amino acid sequence as shown in SEQ ID NO:2, wherein SEQ ID NO:2: WLKPDPSQK; Polypeptide 3: A polypeptide with an amino acid sequence as shown in SEQ ID NO:3, wherein SEQ ID NO:3: NNKYPDAVA.

[0049] The molecular docking energies are shown in Table 1, and the secondary mass spectra are shown in Table 2. Figures 2-4 As shown.

[0050] Table 1

[0051] II. Synthesis of Polypeptides Peptides 1-3 were synthesized by Nanjing Jietai Biotechnology Co., Ltd. using solid-phase synthesis, with a purity greater than 98%, and were used for subsequent testing.

[0052] Example 2: Culture of MC3T3-E1 cells MC3T3-E1 cells (Chinese Academy of Sciences Cell Bank) were seeded in α-MEM medium containing 10% (v / v) serum and 1% antibiotics (streptomycin and penicillin), and then cultured in a cell culture incubator at 37 ℃ and 5% CO2 until the cells covered 80% of the bottom area of ​​the flask (the medium was replaced with fresh medium every 2 days during the culture process). After digestion with 0.25% trypsin (P7545, enzyme activity 6.4 U / mg, purchased from Sigma, USA) for 2 min, the cells were passaged to the logarithmic growth phase.

[0053] Example 3: Effects of peptides 1-3 on cell proliferation activity I. Testing Methods MC3T3-E1 cells were loaded at 5×10 4 Cells were seeded at a density of 1:1 in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. Control and test groups were established. The test group cells were then cultured for 24, 48, and 72 h in peptide solutions of different concentrations. After removing the culture medium, 100 µL of 0.5 mg / mL MTT solution was added to each well, and the cells were incubated for 4 h. The MTT solution was then removed, and 150 µL of DMSO solution was added to each well. After gentle mixing, the absorbance (A) was measured at 570 nm using a microplate reader. The cell proliferation rate (%) was then calculated according to the formula: A = (Cells * Cell ... 测试组 / A 对照组 The cell proliferation rate is calculated by multiplying the percentage by 100%.

[0054] II. Test Results The results are as follows Figure 5 As shown, where, Figure 5 In the figure, A represents the effect of peptides 1-3 on cell proliferation activity at 24 h. Figure 5 In the figure, B represents the effect of peptides 1-3 on cell proliferation activity at 48 h. Figure 5 C in the figure represents the effect of peptides 1-3 on cell proliferation activity at 72 h.

[0055] visible: (1) Compared with the blank control group, at 24 h, 48 h and 72 h of culture, the three peptides FIPTESpAN, WLKPDPSQK and NNKYPDAVA had no obvious toxic effects on osteoblasts in the concentration range of 12.5~50 μg / mL, and the cell survival rate remained at the normal level.

[0056] (2) In the concentration range of 12.5~50 μg / mL, all three peptides showed significant osteocyte proliferation-promoting effects and there was a clear concentration-effect relationship: after 48 h of treatment with FIPTESpAN and WLKPDPSQK, the proliferation activity of the 50 μg / mL concentration group reached its peak, which was significantly increased by 56.43% and 60.12% respectively compared with the blank control group (P<0.05); while the optimal proliferation-promoting concentration of NNKYPDAVA was 25 μg / mL, and the proliferation rate was 49.01% higher than that of the blank group after 48 h of culture (P<0.05).

[0057] Example 4: Effects of peptides 1-3 on cell differentiation activity I. Testing Methods MC3T3-E1 cells in logarithmic growth phase were used at a rate of 3 × 10⁻⁶. 5Cells / wells were placed in 12-well plates and cultured at 37°C for 24 h. Then, they were treated for 7 days with differentiation medium (α-MEM medium supplemented with 10% fetal bovine serum, 50 µg / mL L-ascorbic acid, and 10 mM β-glycerophosphate) containing different concentrations of peptides (0, 1, 10, 50 µg / mL). The medium was changed every two days. After 7 days, the medium was aspirated, and the bottom of the wells was washed with PBS. After washing, the cells were fixed in 4 wt% paraformaldehyde for 30 min. After fixation, the fixative was aspirated, and 0.8 mL of BCIP / NBT staining solution was added to each well. The cells were incubated in the dark for 3 h, then washed twice with distilled water. The culture dish is shown in the image below. Figure 6 As shown in Figure A, the photograph under the microscope (40X) is as follows. Figure 6 As shown in B, staining intensity analysis and normalization were performed using ImageJ software. Figure 6 C).

[0058] II. Test Results visible: The staining intensity of cells in the FIPTESpAN and WLKPDPSQK groups increased with increasing sample concentration—the ALP activity of the cells reached its highest level at a concentration of 50 μg / mL, increasing by 144.28% and 103.15% respectively compared to the control group; the ALP activity of the NNKYPDAVA group was highest at a concentration of 25 μg / mL, increasing by 162.19% compared to the control group. In conclusion, treatment with FIPTESpAN, WLKPDPSQK, and NNKYPDAVA can significantly increase the ALP activity of MC3T3-E1 cells and promote cell differentiation.

[0059] Based on the cell morphology characteristics observed in this study, it is indicated that FIPTESpAN, WLKPDPSQK, and NNKYPDAVA possess the potential to promote osteoblast mineralization.

[0060] Example 5: Effects of peptides 1-3 on cell mineralization activity Mineralization is a core step in bone formation, referring to the process by which osteoblasts deposit inorganic hydroxyapatite onto an organic collagen matrix. Its activity directly reflects the strength of osteogenic function. This process is crucial for the development, formation, and homeostasis of healthy bone; therefore, exploring the regulatory role of bioactive peptides in osteoblast mineralization levels has significant theoretical and applied value. Calcium deposition is a key indicator for assessing osteoblast mineralization levels; higher calcium deposition indicates more complete cellular mineralization.

[0061] I. Testing Methods MC3T3-E1 cells in logarithmic growth phase were used at a rate of 3 × 10⁻⁶. 5Cells / wells were placed in 12-well plates and cultured at 37 °C and 5% CO2 for 24 h. Then, they were treated for 21 days with differentiation medium (α-MEM medium supplemented with 10% fetal bovine serum, 50 µg / mL L-ascorbic acid, and 10 mM β-glycerophosphate) containing different concentrations of peptides (0, 1, 10, 50 µg / mL). The medium was changed every two days. After 21 days, the medium was aspirated, and the bottom of the wells was washed with PBS. After washing, the cells were fixed in 4 wt% paraformaldehyde for 30 min and rinsed with deionized water. Cells were stained with Alizarin Red dye for 30 min, rinsed several times with distilled water, and the Alizarin Red staining results were photographed using a microscope (40X) equipped with a digital camera (Canon DS126201, Japan). Calcium nodules were quantified by dissolving stained calcium nodules in 10% (w / v) hexadecylpyridine chloride for 30 min, and the absorbance (A) of the extracted stained calcium nodules was measured at 562 nm using a microplate reader. The mineralization rate (%) was then calculated as follows: 测试组 / A 对照组 The cell mineralization rate is calculated by multiplying the percentage by 100%.

[0062] II. Test Results Cellular alizarin red staining results as follows Figure 7 As shown in Figure A, the mineralization rate results are as follows: Figure 7 As shown in Figure B, the number of calcium nodules formed by osteoblast mineralization and the intensity of Alizarin Red staining were significantly increased. The mineralization degree in the FIPTESpAN and WLKPDPSQK groups increased gradient with increasing peptide concentration, reaching its peak at a concentration of 50 μg / mL, representing increases of 97.27% and 58.66% respectively compared to the control group. The optimal mineralization concentration for the NNKYPDAVA group was 25 μg / mL, at which point the number of calcium nodules significantly increased by 119.35% compared to the control group, demonstrating a strong mineralization-promoting effect.

[0063] Example 6: Effects of peptides 1-3 on intracellular calcium ion content Calcium, as the most abundant inorganic element in the human body, participates in regulating various metabolic-related biological processes throughout the body, playing an irreplaceable core role, especially in bone formation, mineralization, and homeostasis maintenance. Existing research has shown that increased intracellular calcium ion concentration is a key signal promoting osteoblast mineralization: on the one hand, calcium ions, as a core raw material for the mineralization process, directly provide a material basis for hydroxyapatite deposition when their content increases; on the other hand, increased intracellular calcium ion concentration can regulate the Wnt / β-catenin pathway and the TGF-β1 / Smad2 / 3 pathway, thereby activating the expression of osteogenic-related genes and enhancing osteogenic activity. The Fluo-4 calcium ion fluorescent probe detection method is a commonly used and reliable method for detecting intracellular calcium ion concentration levels.

[0064] I. Testing Methods MC3T3-E1 cells in logarithmic growth phase were used at a rate of 3 × 10⁻⁶. 5 Cells / wells were placed in 12-well plates and cultured at 37 °C and 5% CO2 for 24 h. Then, they were treated with differentiation medium containing peptide 1, peptide 2, or peptide 3 (FIPTESpAN: 50 μg / mL; WLKPDPSQK: 50 μg / mL; NNKYPDAVA: 25 μg / mL) and cultured at 37 °C for 12 h. After washing MC3T3-E1 cells three times with PBS, the calcium ion concentration level in MC3T3-E1 cells was detected according to the instructions of the Fluo-4 AM calcium ion fluorescent probe kit (purchased from Wuhan Yilairuit Biotechnology Co., Ltd., China).

[0065] II. Test Results Test results as follows Figure 8 As shown, it can be seen that: (1) Compared with the blank control group, the intensity and distribution area of ​​calcium-specific fluorescence in osteoblasts were significantly increased after treatment with FIPTESpAN, WLKPDPSQK and NNKYPDAVA, indicating that the intracellular calcium ion level was significantly increased.

[0066] (2) The intracellular fluorescence intensity in the FIPTESpAN group was 1.17 times higher than that in the control group, the WLKPDPSQK group was 0.54 times higher, and the NNKYPDAVA group showed the most significant increase, reaching 1.92 times. All three groups showed statistical differences. P The value <0.05 indicates that all three peptides can effectively increase the calcium ion content in osteoblasts, with NNKYPDAVA showing the best regulatory effect.

[0067] In summary, FIPTESpAN, WLKPDPSQK, and NNKYPDAVA can significantly enhance osteogenic activity by upregulating intracellular calcium ion concentration in osteoblasts, thus providing key raw materials for the mineralization process and strengthening osteogenic regulation by activating related signaling pathways, which is beneficial for promoting bone health.

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide derived from lactoferrin, characterized in that, The amino acid sequence of the polypeptide is any one of SEQ ID NO:1 to SEQ ID NO:

3.

2. The polypeptide according to claim 1, characterized in that, The sixth amino acid at the N-terminus of the sequence shown in SEQ ID NO:1 is also phosphorylated.

3. Use of the polypeptide according to any one of claims 1 to 2 in the preparation of products that promote osteogenic formation.

4. The use according to claim 3, characterized in that, The product is at least one of food or medicine.

5. The use according to claim 4, characterized in that, The food is at least one of supplementary food, formula food, or health food.

6. The application according to claim 4, characterized in that, The food is at least one of liquid food, semi-liquid food, solid food, or semi-solid food.

7. The use according to claim 3, characterized in that, Osteogenesis is the process of promoting cell proliferation.

8. The use according to claim 3, characterized in that, Osteogenesis is the process of promoting cell differentiation.

9. The use according to claim 3, characterized in that, Osteogenesis promotes cell mineralization.

10. The use according to claim 3, characterized in that, Osteogenesis involves increasing the intracellular calcium ion content.

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