Lactopontin-derived polypeptide for promoting osteoblast proliferation and differentiation and use thereof

Abstract

The present invention belongs to the technical field of bioactive peptides. Disclosed are a lactopontin-derived polypeptide for promoting osteoblast proliferation and differentiation and the use thereof. Provided in the present invention is a milk-derived bioactive peptide WLKPDPS. The peptide has an extremely high biosafety and the function of promoting osteoblast proliferation and differentiation. The milk-derived bioactive polypeptide WLKPDPS of the present invention has a significant effect in promoting osteoblast proliferation, and can significantly promote osteoblast mineralization, thus holding promise for use in the preparation of a drug for promoting osteoblast proliferation and preventing osteoporosis. The milk-derived bioactive polypeptide WLKPDPS of the present invention is convenient to synthesize, can be produced on an industrial scale, and has good application prospects in fields such as food, medicine and cosmetics.

Description

A lactobridged protein-derived polypeptide that promotes osteoblast proliferation and differentiation and its application Technical Field

[0001] This invention relates to a lactoadenosine-derived polypeptide that promotes osteoblast proliferation and differentiation and its applications, belonging to the field of bioactive peptide technology. Background Technology

[0002] Bone mineral density (BMD) refers to the density of bone minerals per unit volume of bone, primarily reflecting the strength and resilience of bones. Improving BMD is crucial for maintaining bone health, especially in preventing osteoporosis. Clinically, osteoporosis is still mainly treated with chemical drugs, but these often carry certain risks, such as long-term use potentially causing gastrointestinal problems and cardiovascular diseases. Therefore, the search for safer, food-sourced natural alternatives that can promote bone formation and reverse bone structural damage is receiving increasing attention.

[0003] Peptides are protein degradation products, and various bioactive peptides have been reported to have functions such as regulating immunity, preventing cardiovascular disease, and promoting bone health. These peptides can be obtained through microbial fermentation, digestive enzymatic hydrolysis, artificial synthesis via genetic engineering, and chemical synthesis. Most bioactive peptides consist of 2-20 amino acid residues and are easily digested and absorbed by the human body. Milk-derived peptides are abundant, but research on their relationship to bone health is relatively limited. In 2007, Huttunen et al. reported that the lactotripeptide IPP promotes osteoblast proliferation, differentiation, and signal transduction; however, compared to the abundance of milk-derived peptides, research reports are still relatively limited. Lactopontin (LPN) is an important bioactive protein from mammalian milk. LPN can promote bone development in growing mice, but whether it produces osteogenic bioactive peptides that regulate bone health after gastrointestinal digestion remains unclear.

[0004] Therefore, it is of great significance to explore bioactive peptides from LPN sources that promote bone health. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a milk-derived bioactive peptide, WLKPDPS, and NE(pS)PEQTDDL, which exhibit extremely high biocompatibility and the ability to promote osteoblast proliferation and differentiation.

[0006] The first objective of this invention is to provide a milk-derived bioactive peptide, wherein the amino acid sequence of the milk-derived bioactive peptide is WLKPDPS (SEQ ID NO: 1) or NESPEQTDDL (SEQ ID NO: 2).

[0007] In one embodiment, the serine in the milk-derived bioactive peptide NESPEQTDDL can also be a phosphate-modified serine.

[0008] In one embodiment, the milk-derived bioactive peptide is WLKPDPS or NE(pS)PEQTDDL.

[0009] In one embodiment, the preparation method of the milk-derived bioactive peptides includes solid-phase synthesis, enzymatic hydrolysis, and microbial expression.

[0010] A second object of the present invention is to provide a food, medicine, health product or nutritional product containing an effective dose of any of the above-mentioned milk-derived bioactive peptides.

[0011] Optionally, the food, medicine, health product, or nutritional product may also contain any of the above-mentioned milk-derived bioactive peptides; the derivatives of the milk-derived bioactive peptides refer to milk-derived bioactive peptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification, or glycosylation of the amino acid side chain groups, amino terminus, or carbonyl terminus of the milk-derived bioactive peptides.

[0012] In one embodiment, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0013] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0014] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0015] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0016] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0017] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

[0018] In one embodiment, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages;

[0019] The food products mentioned also include special dietary foods;

[0020] The health products also contain acceptable excipients.

[0021] In one embodiment, the health product includes gel candy.

[0022] In one embodiment, the gummies are gel candies, and the preparation steps are as follows:

[0023] (1) Add gelatin, starch, oligosaccharides, edible glycerin and water to a mixing bowl and stir for 20-40 minutes to obtain a gelatin liquid;

[0024] (2) Add WLKPDPS or NE(pS)PEQTDDL polypeptide powder, fruit flavoring, phospholipids and sunflower seed oil to a mixing tank and stir for 30 minutes to obtain sandwich liquid.

[0025] The mass ratio of gelatin, starch, oligosaccharides, edible glycerin, water, polypeptide powder, fruit flavoring, phospholipids, and sunflower seed oil is 2–6:0.5–2:0.5–2:8–12:4–8:0.1–1:0.1–0.5:0.3–0.8:5–10.

[0026] (3) The gel liquid and filling liquid are formed by candy machinery and cooled at 20-25℃ for 60-80 minutes to obtain polypeptide gel candy.

[0027] A third object of the present invention is to provide the use of any of the above-mentioned milk-derived bioactive peptides in the preparation of food, pharmaceuticals, health products or nutritional products for promoting calcium absorption.

[0028] In one embodiment, the milk-derived bioactive peptides can also be used to prepare products with antioxidant, anti-inflammatory, cartilage and tissue regeneration repair (e.g., promoting bone matrix mineralization, increasing bone density, accelerating bone tissue growth, and shortening fracture healing period), metabolic and immune function regulation (e.g., regulating enzyme activity and enhancing immunity), dental caries prevention, and digestive function improvement.

[0029] In one embodiment, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages;

[0030] The food products mentioned also include special dietary foods;

[0031] The health products also contain acceptable excipients.

[0032] In one embodiment, the food, medicine, health product, or nutritional product is used to promote calcium absorption.

[0033] In one embodiment, the drug further contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

[0034] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, croscarmellose, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0035] Optionally, the dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form;

[0036] Optionally, the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions;

[0037] Optionally, the injectable dosage form includes, but is not limited to, injection solution and injection powder for injection;

[0038] Optionally, the inhalation dosage form includes, but is not limited to, aerosols and powder inhalers;

[0039] Optionally, the food includes, but is not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; and the health products also contain acceptable excipients.

[0040] In one embodiment, the health product includes gel candy.

[0041] Beneficial effects of the present invention

[0042] The milk-derived bioactive polypeptides WLKPDPS and NE(pS)PEQTDDL of this invention have significant effects on promoting osteoblast proliferation and can significantly promote osteoblast mineralization, and are expected to be used to prepare drugs that promote osteoblast proliferation and prevent osteoporosis.

[0043] The milk-derived bioactive polypeptides WLKPDPS and NE(pS)PEQTDDL of the present invention are easy to synthesize and can be industrially produced, showing good application prospects in the fields of food, medicine and cosmetics. Attached Figure Description

[0044] Figure 1 shows the HPLC detection results of milk-derived bioactive peptide WLKPDPS;

[0045] Figure 2 shows the LC-MS detection results of milk-derived bioactive peptide WLKPDPS;

[0046] Figure 3 shows the HPLC detection results of milk-derived bioactive peptide NE(pS)PEQTDDL;

[0047] Figure 4 shows the LC-MS detection results of milk-derived bioactive peptide NE(pS)PEQTDDL;

[0048] Figure 5 shows the effects of milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on osteoblast proliferation.

[0049] Figure 6 shows the effects of milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on osteoblast differentiation;

[0050] Figure 7 shows the effects of milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL on osteoblast-related pathways.

[0051] Figure 8 shows the effect of milk-derived bioactive peptide WLKPDPS on the growth performance of rats.

[0052] Figure 9 shows the effect of milk-derived bioactive peptide WLKPDPS on apparent parameters of rat femur.

[0053] Figure 10 shows the effect of milk-derived bioactive peptide WLKPDPS on the mechanical properties of rat femur.

[0054] Figure 11 shows the results of three-dimensional reconstruction of rat femur using Micro-CT using milk-derived bioactive peptide WLKPDPS.

[0055] Figure 12 shows the effect of milk-derived bioactive peptide WLKPDPS on the microstructure of rat bones. Detailed Implementation

[0056] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0057] Raw materials used in the examples:

[0058] LPN was isolated from fresh milk. For specific methods, please refer to the literature (Ma Ping, Sun Jie, Liu Ning. Isolation, purification and identification of osteopontin in milk [J]. Food and Fermentation Industries, 2008, (06): 135-139.).

[0059] Pepsin, pancreatin, and bile salts were purchased from Sigma-Aldrich, USA, while potassium chloride, sodium chloride, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0060] The milk-derived bioactive peptides used in the examples were all synthesized by Shanghai Ketai Biotechnology Co., Ltd.

[0061] Test method:

[0062] 1. HPLC method

[0063] High-performance liquid chromatography (HPLC) was used with a Kromasil 100-5-C18 column (4.6 × 250 mm × 5 μm). The mobile phases were 0.1% trifluoroacetic acid aqueous solution (A) and 0.1% trifluoroacetic acid acetonitrile solution (B), with a total flow rate of 1 mL / min and a detection wavelength of 214 nm. Samples were dissolved in ultrapure water, with an injection volume of 7 μL. The gradient elution program was as follows:

[0064] 2. LC-MS method

[0065] An electrospray ionization (ESI) interface was used, with the detector voltage set to -0.2 kV, CDL temperature to 250 °C, CDL voltage to 0 V, block temperature to 200 °C, nebulizing gas flow rate to 1.5 L / min, preset bias to +4.5 kV, and T-Flow to 0.2 ml / min. Samples were dissolved in a mixed solvent of 15% acetonitrile (ACN) and 85% water (H₂O) at an injection volume of 0.2 μL.

[0066] Example 1: Preparation of milk-derived bioactive peptides

[0067] The preparation of milk-derived bioactive peptides and the in vitro simulated digestion using an infant model are as follows:

[0068] (1) Stomach digestion stage:

[0069] 10 mg / mL LPN solution was added to the enzyme reactor and preheated, and the pH was adjusted to 5.3. Then it was mixed with simulated gastric juice (pH 5.3, 724.3 U pepsin / mL, 13 mM potassium chloride, 94 mM sodium chloride) at a ratio of 63:37 (v / v). The reaction was carried out at 37 °C and pH 5.3 for 60 min. The pH was adjusted to 7.0 with 2 M sodium hydroxide to terminate this stage of digestion, and the gastric digestion stage product was obtained.

[0070] (2) Intestinal digestion stage:

[0071] The pH of the gastric digestion products was adjusted to 6.6, and then mixed with simulated intestinal fluid (pH 6.6, 42.1 U pancreatic enzyme / mL, 8.2 mM bile salts, 10 mM potassium chloride, 249 mM sodium chloride) at a ratio of 62:38 (v / v). Digestion was carried out at 37°C and pH 6.6 for 60 min, and intestinal digestion was stopped at 95°C for 10 min to obtain the final digestion products.

[0072] (3) Identification of polypeptide composition:

[0073] The final digestion products were analyzed by LC-MS / MS using an online nanospray ionization source. The entire system was an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, MA, USA) with a tandem EASY-nanoLC 1200. A total of 5 μL of sample was loaded (analytical column: Acclaim PepMap C18, 75 μm x 25 cm), and the sample was separated using a gradient over 60 min. The column flow rate was controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray voltage at 2 kV. The gradient started at 4% B phase, increased nonlinearly to 50% within 53 min 40 sec, increased to 95% within 40 sec, and was maintained for 5 min 40 sec. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0074] The mass spectrometry parameters were set as follows: (1) MS: Scan range (m / z): 100-1500; Resolution: 120,000; Normalized AGC target: 200%; Maximum injection time: 100ms; (2) HCD-MS / MS: Resolution: 50,000; Normalized AGC target: 200%; Maximum injection time: 86ms; Collision energy: 25%, 30%, 35%; Dynamic exclusion time: 30s. The tandem mass spectra were analyzed using PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada). The database was Uniprot-Bos taurus (version 2024, 26635 entries). Library search parameters: Fragment ion mass tolerance: 0.02 Da, precursor ion mass tolerance: 10 ppm, maximum missed cuts: 2, fixed modifications: Carbamidomethylation 57.02, variable modifications: Oxidation (M) 15.99, Deamidation (NQ) 0.98, Acetylation (Protein N-term) 42.01, Phosphorylation (STY) 79.97. Protein card value is 1% FDR, containing at least 1 unique peptide; peptide card value is 1% FDR.

[0075] 242 peptides derived from LPN were identified. Molecular docking of the peptides was performed with BMPR1A as the target. Based on the binding energy, potential milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL were screened.

[0076] Shanghai Ketai Biotechnology Co., Ltd. was commissioned to synthesize milk-derived bioactive peptides WLKPDP (SEQ ID NO: 3), WLKPDPS (SEQ ID NO: 1), and NE(pS)PEQTDDL.

[0077] Example 2: Detection of bioactive peptides from milk

[0078] The results of testing the milk-derived bioactive peptides WLKPDPS and NE(pS)PEQTDDL prepared in Example 1 are as follows:

[0079] (1) HPLC detection results

[0080] The HPLC detection results are shown in Figure 1 (WLKPDPS) and Figure 3 (NE(pS)PEQTDDL).

[0081] (2) LC-MS detection results

[0082] The LC-MS detection results are shown in Figure 2 (WLKPDPS) and Figure 4 (NE(pS)PEQTDDL).

[0083] (3) Promoting effect on osteoblasts

[0084] The effects of milk-derived bioactive peptides VFTP (VP4), WLKPDP (WP6), WLKPDPS (WS7), and NE(pS)PEQTDDL (NL10) on osteoblast proliferation were investigated using the following steps:

[0085] MC3T3-E1 Subclone14 cells were seeded at a density of 5000 cells / well in 96-well plates. After incubation for 24 h, the samples were dissolved in α-MEM medium, filtered through a 0.22 μm membrane for sterilization, and medium containing 100 μg / mL IPP, 1, 10, and 100 μg / mL VP4, WP6, WS7, and NL10 was added to the wells. After incubation for 24 h, cell counts were determined using CCK-8 assay.

[0086] The results are shown in Figure 5. The results indicate that none of the samples had a significant effect on cell viability, indicating that 100 μg / mL IPP, 1, 10, 100 μg / mL VP4, WP6, WS7, and NL10 were not cytotoxic to MC3T3-E1 Subclone14 cells.

[0087] (4) Effects on osteoblast differentiation

[0088] The effect of milk-derived bioactive peptide WLKPDPS on osteoblast differentiation was investigated using the following steps:

[0089] 1×10 5 Cells were seeded per well in 12-well plates, with 1 mL of culture medium added to each well. After 24 hours, the culture medium was replaced with induction medium containing 100 μg / mL IPP, 100 μg / mL VP4, WP6, WS7, and NL10, respectively, and the medium was changed every two days. After 3 and 7 days of intervention, the cell culture supernatant was collected, and the ALP activity in the supernatant was measured using an ALP kit.

[0090] The results are shown in Figure 6. The results indicate that, after 3 days (left figure) and 7 days (right figure) of induced differentiation, the activity of alkaline phosphatase in the cell culture supernatant was significantly increased compared with the control group and the IPP group (different lowercase letters indicate significant differences between groups, p<0.05). On day 3, the ALP activity in the WS7 group was 803.06±22.19 U / mL, and that in the NL10 group was 802.43±53.39 U / mL, which were 39.7% and 39.6% higher than that in the IPP group, respectively. Similar results were observed on day 7.

[0091] (5) Effects on osteoblast-related pathways

[0092] The effect of milk-derived bioactive peptide WLKPDPS on osteoblast differentiation was investigated using the following steps:

[0093] 1×10 5 Cells were seeded per well in 12-well plates, with 1 mL of culture medium added to each well. After 24 h, the culture medium was replaced with induction medium containing 100 μg / mL WLKPDPS. The culture medium was changed every two days. After 3 days of intervention, cellular RNA was extracted using an animal RNA extraction kit, reverse transcribed into cDNA, and the expression levels of related genes in the BMP / Smad pathway were determined by qPCR.

[0094] The results are shown in Figure 7. The results indicate that, compared with the control group, WLKPDPS significantly upregulated the mRNA expression levels of related Bmpr, Smad1, Smad5 and Runx2, increasing them by 558.9%, 58.2%, 30.1% and 81.4%, respectively. NE(pS)PEQTDDL significantly upregulated the mRNA expression levels of related Bmpr, Smad1, Smad5 and Runx2, increasing them by 533.9%, 32.3%, 10.6% and 151.8%, respectively.

[0095] Example 3: Effects of milk-derived bioactive peptides on bone growth and development in rats

[0096] The milk-derived bioactive peptide WLKPDPS prepared in Example 1 was used for in vivo animal experiments. Thirty-two 3-week-old male SPF-grade Sprague-Dawley (SD) rats were purchased from Vital River Laboratory Animal Technology Co., Ltd. They were provided with free access to food and water, and the ambient temperature was 20℃-26℃, with a relative humidity of 40%-70%.

[0097] After adapting to the environment, the rats were randomly divided into four groups:

[0098] (1) Control group (n=8) was given deionized water by gavage daily;

[0099] (2) LPN group (LPN, n=8), 15 mg / kg BW LPN was administered by gavage daily;

[0100] (3) WS7 low-dose group (WS7-L, n=8), 0.41 mg / kg BW polypeptide WLKPDPS was administered by gavage daily;

[0101] (4) The high-dose WS7 group (WS7-H, n=8) was administered 4.1 mg / kg BW peptide WLKPDPS by gavage daily. The rats' body weight was recorded weekly, and the experiment was terminated after 4 weeks of continuous gavage.

[0102] The effects of milk-derived bioactive peptides on rats were investigated, as follows:

[0103] 1. Effects on growth performance of rats

[0104] The effect of milk-derived bioactive peptide WLKPDPS on the growth performance of rats was investigated using the following steps: After anesthetizing rats with isoflurane, the distance from the tip of the nose to the base of the tail was measured with a ruler, which was taken as the body length.

[0105] The results are shown in Figure 8. The growth curves of rats in each group showed high consistency during the experimental period, and there was no significant difference in final body weight among the groups (p>0.05). The high-dose WS7 (WS-7) group had the highest body weight, upregulated by 9.83% compared to the control group. After intervention with LPN and low and high doses of WS7, the body length of rats increased by 6.78%, 9.83%, and 7.45%, respectively (p<0.05), and there was no significant difference in growth-promoting effect among the intervention groups (p>0.05).

[0106] 2. Effects on apparent parameters of rat femur

[0107] The effects of milk-derived bioactive peptide WLKPDPS on apparent parameters of rat femur were investigated using the following steps: rat femurs were isolated, muscles were removed from the bones, and femur parameters were measured using calipers.

[0108] As shown in Figure 9, the femoral length, wet weight, and dry weight of rats in the LPN intervention group increased significantly by 5.84%, 21.31%, and 14.33%, respectively, compared with the control group (p<0.05). Low-dose WS7 produced an equivalent bone growth-promoting effect: the increases in length (+5.45%, p<0.05), wet weight (+28.27%, p<0.05), and dry weight (+16.09%, p<0.05) were not statistically different from those in the LPN group (p>0.05). High-dose WS7 (WS7-H) showed a positive trend but no significant difference (p>0.05), suggesting a dose threshold effect. All treatments had no effect on femoral width and thickness (p>0.05).

[0109] 3. Effects on the mechanical properties of rat bones

[0110] The effects of milk-derived bioactive peptide WLKPDPS on the mechanical properties of rat bones were investigated using the following steps: A three-point bending test was performed on the femur using a TA.XT plus analyzer with a 12mm support span (HDP / 3PB probe) and a crosshead speed of 0.6mm / min. The load was consistently applied to the mid-femur region. Load-displacement curves for each femur were used to determine the elastic load, maximum load (N), and displacement (mm) under the maximum load.

[0111] As shown in Figure 10, both LPN and WS7-H interventions significantly increased the maximum femoral load (p<0.05), increasing it by 12.57% and 18.43% respectively compared to the control group, while WS7-L did not show a significant promoting effect (p>0.05). Regarding bone toughness, there was no significant difference in maximum deflection among the groups (p>0.05). This indicates that WS7 can only replicate the bone strength-improving effect of LPN at high doses.

[0112] 4. Effects on the microstructure of rat skeleton

[0113] The effects of milk-derived bioactive peptide WLKPDPS on the microstructure of rat bones were investigated using the following steps: Isolated femurs were fixed in 4% paraformaldehyde solution for 24-48 hours. The samples were then rinsed three times with PBS and stored in 75% ethanol solution at 4°C for later use. The treated femurs were scanned using a Micro-CT (Bruker SkyScan 1276) small animal imaging system under the following conditions: resolution 18 μm, voltage 65 kV, current 385 μA, and a 1 mm Al filter. The scanning mode was high resolution, and the scan time was 4 minutes. Three-dimensional reconstruction was performed using the scan data, and the microstructural parameters of the target bone tissue were analyzed using system analysis software, including measurements of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

[0114] The results, as shown in Figures 11 and 12, significantly improved the femoral microstructure: bone mineral density increased by 21.11% and 28.78% respectively compared to the control group, and bone volume fraction increased by 20.41% and 49.62% respectively (p<0.05). The bone volume fraction in the WS7-H group was also significantly higher than that in the LPN group (p<0.05). Both treatments simultaneously increased the number of trabeculae (+21.76% and 39.70%, p<0.05) and decreased trabecular separation (-26.66% and 23.44%, p<0.05). Three-dimensional reconstruction also showed a reduction in the volume of the femoral cavity. WS7-L only showed a significantly better effect on trabecular separation than the control group (p<0.05). Furthermore, there was no statistically significant difference in trabecular thickness among the groups (p>0.05). Changes in femoral microstructure are highly correlated with the aforementioned increase in bone strength. These results indicate that high-dose WS7 can completely replicate the effect of LPN on improving bone microstructure and enhance femoral strength by strengthening the network connectivity of trabecular bone.

[0115] In summary, the results indicate that WS7 administered via gavage at doses of 0.41–4.1 mg / kg achieved nearly the same effect on promoting bone and growth performance as 15 mg / kg LPN, while WS7 exhibited better safety, absorption efficiency, and bioavailability.

[0116] Example 4: Preparation of polypeptide gel candies

[0117] The raw materials include the following weights: 4 kg gelatin, 1 kg starch, 1 kg oligosaccharides, 10 kg edible glycerin, 6 kg water, 0.5 kg WLKPDPS or NE(pS)PEQTDDL polypeptide dry powder obtained in Example 1, 0.3 kg fruit flavoring, 0.5 kg phospholipids and 7 kg sunflower seed oil.

[0118] (1) Weigh each raw material according to the above weight;

[0119] (2) Add gelatin, starch, oligosaccharides, edible glycerin and water to a mixing tank, stir for 30 minutes, and cook to obtain a gelatin liquid;

[0120] (3) Add the polypeptide powder, fruit flavoring, phospholipids and sunflower seed oil to the mixing tank and stir for 30 minutes to obtain the sandwich liquid;

[0121] (4) The gel liquid and filling liquid are shaped by candy machinery and cooled at 25°C for 60 minutes to obtain polypeptide gel candy.

[0122] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A milk-derived bioactive peptide, characterized in that, The amino acid sequence of the milk-derived bioactive peptide is WLKPDPS or NESPEQTDDL.

2. The milk-derived bioactive peptide according to claim 1, characterized in that, The serine in the milk-derived bioactive peptide NESPEQTDDL is a phosphate-modified serine.

3. The milk-derived bioactive peptide according to any one of claims 1 to 2, characterized in that, The preparation methods of the milk-derived bioactive peptides include solid-phase synthesis, enzymatic hydrolysis, and microbial expression.

4. A food, medicine, health product, or nutritional product, characterized in that, The food, medicine, health product, or nutritional product contains an effective dose of any of the milk-derived bioactive peptides described in claims 1 to 3.

5. The food, drug, nutraceutical or nutritional product of claim 4, wherein, The food, medicine, health product or nutritional product may also contain any of the milk-derived bioactive peptides described in claims 1 to 3; the derivatives of the milk-derived bioactive peptides refer to milk-derived bioactive peptide derivatives obtained by hydroxylation, carbonylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation of the amino acid side chain groups, amino terminus or carbonyl terminus of the milk-derived bioactive peptides.

6. The food, drug, nutraceutical or nutritional product of claim 4, wherein, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

7. The food, drug, nutraceutical or nutritional product of claim 6, wherein, The excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

8. The food, medicine, health product, or nutritional product according to claim 4, characterized in that, The dosage form of the drug includes, but is not limited to, oral dosage form, injection dosage form, and inhalation dosage form; the oral dosage form includes, but is not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

9. The food, medicine, health product, or nutritional product according to claim 4, characterized in that, The injectable dosage forms include, but are not limited to, injectable solutions and injectable powders for injection.

10. The food, medicine, health product, or nutritional product according to claim 4, characterized in that, The inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

11. The food, medicine, health product, or nutritional product according to claim 4, characterized in that, The food products include, but are not limited to, grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages. The food products mentioned also include special dietary foods; The health products also contain acceptable excipients.

12. The food, medicine, health product, or nutritional product according to claim 4, characterized in that, The health products include gel candies.

13. The application of the milk-derived bioactive peptides according to any one of claims 1 to 3 in the preparation of food, pharmaceuticals, health products, or nutritional products, characterized in that, The food, medicine, health product or nutritional product is used to promote calcium absorption.

14. The application according to claim 13, characterized in that, The drug also contains pharmaceutically acceptable excipients; the excipients refer to conventional drug carriers in the pharmaceutical field.

15. The application according to claim 14, characterized in that, The excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, and mannitol; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbates, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resins, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

16. The application according to claim 13, characterized in that, The dosage form of the drug includes, but is not limited to, oral dosage form, injectable dosage form, and inhaled dosage form.

17. The application according to claim 13, characterized in that, The oral dosage forms include, but are not limited to, tablets, capsules, granules, oral liquids, and oral suspensions.

18. The application according to claim 13, characterized in that, The injectable dosage forms include, but are not limited to, injectable solutions and injectable powders for injection.

19. The application according to claim 13, characterized in that, The inhalation dosage form includes, but is not limited to, aerosols and powder inhalers.

20. The application according to claim 13, characterized in that, The food products include, but are not limited to, gummy candies, cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food products also include special dietary foods; the health products also contain acceptable excipients.

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