Milk protein peptide capable of promoting bone growth as well as preparation method and application of milk protein peptide

Abstract

The invention discloses a milk protein peptide for promoting bone growth as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The preparation method specifically comprises the following steps: (1) adding milk protein into a sodium hydroxide aqueous solution, and homogenizing through a colloid mill; (2) adjusting the pH value to 7.8, carrying out enzymolysis, carrying out enzyme inactivation, and cooling; (3) ultrafiltration and collection; (4) centrifugally filtering, taking supernate, and concentrating; and (5) carrying out spray drying. The milk protein peptide can significantly promote proliferation and differentiation of osteoblasts MC3T3-E1, up-regulate expression of osteogenesis-related genes ALP, promote mineralization of extracellular matrixes, promote proliferation of chondrocytes C28 / I2, and contribute to thickening of cartilage growth plates so as to promote bone growth. The compound can be used for preparing medicines and foods for promoting bone growth, preventing or assisting in treating osteoporosis and promoting bone health, and has the advantages of simple preparation process, low cost and definite components and activity.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and more specifically to a milk protein peptide that promotes bone growth, its preparation method, and its application. Background Technology

[0002] Bone growth and development is a complex physiological process involving multiple factors such as heredity, nutrition, and environment. It is mainly achieved by promoting osteoblast proliferation, inhibiting osteoclast differentiation, and promoting chondrocyte differentiation and proliferation. Insulin-like growth factor (IGF) helps promote chondrocyte proliferation and differentiation. Sufficient and balanced nutrition is the material basis for bone growth.

[0003] As a core nutrient, the quality of protein intake and its utilization efficiency directly affect children's bone growth potential. Studies have found that the human body absorbs short peptides derived from protein digestion more effectively than free amino acids and complete proteins. These peptides may also promote mineral absorption and participate in bone metabolism regulation, thus promoting bone growth. However, currently available commercially available nutritional supplements for children's bone growth are primarily multi-component mixtures containing complete proteins (such as whey protein, casein, and colostrum basic protein (CBP)) or hydrolyzed protein peptides (such as hydrolyzed egg yolk powder), which may affect children's intestinal absorption and bone growth promotion effects. Furthermore, existing patented technologies mostly focus on nutrient combinations (such as the peptide-containing nutritional composition disclosed in CN202411120826.1), but lack the development and application of single, structurally defined, and functionally specific short peptide components. Meanwhile, osteoporosis, caused by increased osteoclast activity and decreased osteogenic activity, leads to decreased bone mass, damage to bone tissue microstructure, increased bone fragility, and a high risk of fractures, affecting the bone health and mobility of middle-aged and elderly populations. Currently, bisphosphonates and estrogens are the main treatment drugs, but long-term use may be accompanied by adverse reactions such as gastrointestinal discomfort and osteonecrosis of the mandible. Therefore, it is of great significance to develop safe and effective food-derived active ingredients to promote bone growth and prevent and slow down osteoporosis and other bone diseases.

[0004] Bioactive peptides are small molecular fragments produced from proteins through enzymatic hydrolysis or fermentation. They typically consist of 2-20 amino acids and possess various physiological regulatory functions, such as antioxidation, blood pressure reduction, and promotion of mineral absorption. Milk-derived proteins, due to their comprehensive nutrition and abundant availability, have become important raw materials for academic research and industrial development of bioactive peptides. Existing studies have shown that milk-derived peptides, such as casein phosphopeptides (CPPs), indirectly promote bone growth by enhancing calcium absorption. However, small molecular peptides, especially short peptides, with high specificity, well-defined activity, and direct action on osteoblasts and chondrocytes to promote their proliferation and differentiation still require further exploration.

[0005] Therefore, how to develop a bioactive peptide with bone growth-promoting function is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a milk protein peptide that promotes bone growth, its preparation method and application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a bone-growth-promoting milk protein peptide, wherein the bone-growth-promoting milk protein peptide is a pentapeptide with the amino acid sequence Leu-Pro-Leu-Pro-Leu, abbreviated as LPLPL (as shown in SEQ ID NO.1 of the sequence listing), and a molecular weight of 551.3682 Da. The preparation method specifically includes the following steps: (1) Add milk protein to an aqueous sodium hydroxide solution, and homogenize it by colloid milling to obtain a homogenized milk protein solution; (2) Adjust the pH of the milk protein homogenate to 7.8, add trypsin, alkaline protease and papain in sequence, perform enzymatic hydrolysis, inactivate enzymes, cool, and obtain milk protein hydrolysate. (3) Ultrafiltration was performed on the milk protein hydrolysate, and the components with a molecular weight <2kDa were collected to obtain the milk protein hydrolysate ultrafiltrate; (4) Centrifuge and filter the milk protein hydrolysate, take the supernatant, concentrate it, and obtain milk protein hydrolysate concentrate; (5) Spray dry the milk protein hydrolysate concentrate to obtain the bone growth promoting milk protein peptide.

[0008] Furthermore, in step (1) above, the pH value of the sodium hydroxide aqueous solution is 8.0; the mass ratio of milk protein to sodium hydroxide aqueous solution is 1:10; the homogenization treatment conditions are 80 MPa and 1 cycle.

[0009] Furthermore, in step (2) above, the reagent for adjusting the pH value is citric acid; the amount of trypsin, alkaline protease, and papain added is 0.5% of the milk protein mass; the temperature of enzymatic hydrolysis is 50-55℃, and the time is 6h; the temperature of enzyme inactivation is 80-90℃, and the time is 30min. Among them, trypsin is added first at 0h for enzymatic hydrolysis, alkaline protease is added at 3h, and papain is added at 5h.

[0010] Furthermore, in step (3) above, the molecular weight cutoff of ultrafiltration is 2 kDa.

[0011] Furthermore, in step (4) above, the centrifugal filtration speed is 12000 r / min; the concentration temperature is 60-70℃, and the concentration is 24%-28% to Brix.

[0012] Furthermore, in step (5) above, the spray drying temperature is 135-150℃.

[0013] This invention also claims protection for the use of the aforementioned bone-growth-promoting milk protein peptide in the preparation of products that promote bone growth. Here, "promoting bone growth" refers to promoting the proliferation and / or differentiation of osteoblasts and chondrocytes, thereby promoting bone growth and health.

[0014] The present invention also claims protection for the use of the above-mentioned bone growth-promoting milk protein peptide in the preparation of products for the prevention or adjunctive treatment of osteoporosis.

[0015] Furthermore, the aforementioned products are either pharmaceuticals or food. Among them, food includes functional foods such as health foods and nutritional foods, such as cereal products, dairy products, beverages, or special dietary foods.

[0016] Furthermore, the aforementioned drugs also contain pharmaceutically and food-grade carriers or excipients, such as diluents, binders, lubricants, and disintegrants. The dosage forms of these drugs include tablets, capsules, granules, oral dosage forms (oral solutions), injectable dosage forms (injectable solutions), or inhalation dosage forms.

[0017] Furthermore, the aforementioned foods also contain food-grade carriers or excipients. The dosage forms of these foods include tablets, solid beverages, capsules, drinks, or gelatinous candies, including liquid milk, yogurt, milk powder, cheese, protein bars, beverages, or foods for special medical purposes.

[0018] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention uses milk protein as raw material and prepares a milk protein peptide LPLPL through a compound enzyme hydrolysis process. It can significantly promote the proliferation and differentiation of osteoblasts MC3T3-E1, upregulate the expression of osteogenic-related gene ALP and promote extracellular matrix mineralization, promote the proliferation of chondrocytes C28 / I2, help thicken the cartilage growth plate and thus promote bone growth. It can be used to prepare medicines and foods that promote bone growth, prevent or assist in the treatment of osteoporosis and promote bone health. It has the advantages of simple preparation process, low cost and clear composition and activity.

[0019] 2. This invention identifies and develops a specific amino acid sequence LPLPL pentapeptide from milk protein that has significant activity in promoting the proliferation and differentiation of osteoblasts and chondrocytes, which is of great value for the development of medicines and foods related to bone health.

[0020] 3. This invention is the first to discover and confirm that the milk protein peptide, specifically the pentapeptide LPLPL, possesses significant activity in promoting the proliferation and differentiation of osteoblasts and chondrocytes. This pentapeptide has a small molecular weight, is easily absorbed by the human body, and is derived from food protein, thus exhibiting high biosafety. This pentapeptide can be obtained through enzymatic hydrolysis of inexpensive and readily available milk protein, making it abundant in raw materials, low in cost, and convenient for industrial production. Furthermore, this pentapeptide exhibits bone-growth-promoting activity even at low concentrations, providing new active ingredients and ideas for developing novel bone health-related drugs and foods. Attached Figure Description

[0021] Figure 1 This is a high-performance liquid chromatography (HPLC) chromatogram of milk protein peptides from Example 1. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1 The preparation method of milk protein peptides that promote bone growth specifically includes the following steps: (1) Take milk protein and add 10 times the mass of sodium hydroxide aqueous solution with a pH of 8.0 according to the material-liquid ratio. After processing with a colloid mill for 10 min, it is then homogenized under the condition of 80 MPa for one cycle to obtain milk protein homogenate. (2) Adjust the pH of the milk protein homogenate to 7.8 with citric acid. First, add trypsin at 0.5% of the milk protein content. Add 0.5% alkaline protease at 3h of enzymatic hydrolysis. Add 0.5% papain at 5h of enzymatic hydrolysis. Perform enzymatic hydrolysis at 55℃ for 6h, then inactivate the enzyme at 90℃ for 30min. Cool to obtain milk protein hydrolysate. (3) Use an ultrafiltration device with a molecular weight cutoff of 2kDa to ultrafilter the milk protein hydrolysate, collect the components with a molecular weight <2kDa, and obtain the milk protein hydrolysate ultrafiltrate. (4) The milk protein hydrolysate was centrifuged and filtered at 12000 r / min. The supernatant was collected and concentrated at 70°C to a Brix value of 26% to obtain the milk protein hydrolysate concentrate. (5) The milk protein hydrolysate concentrate was spray-dried at 145°C to obtain milk protein peptides (powder) that promote bone growth. (6) Perform LC-MS / MS peptide spectrometry analysis on milk protein peptide powder to obtain the peptide segments contained in milk protein peptide. Screen the obtained peptide segments using bioinformatics methods, perform molecular docking on the dominant peptide segments, and determine the theoretically effective peptide segments based on the scoring results.

[0024] The specific analysis process is as follows: S1. Sample pretreatment: The crude peptide was purified by preparative high performance liquid chromatography and lyophilized to obtain a high-purity (>95%) white milk protein peptide powder. 100 μL of 10 mg / mL milk protein peptide (weigh 10 mg of milk protein peptide powder and dissolve it in 1 mL of ultrapure water) + 300 μL of acetonitrile, stood at 4℃ for 1-2 h / ice bath for 40 min, centrifuged at 4℃, 10000 g for 15 min, and 250 μL of the supernatant was diluted with 1000 μL of 0.1% formic acid water to 0.5 mg / mL. The solution was passed through a brown membrane into a liquid chromatography vial for sample detection.

[0025] S2. Milk protein peptides were analyzed by LC-MS: They were identified using a Waters Acquity ultra-high performance liquid chromatography (UPLC) tandem with a Bruker Impact II quadrupole time-of-flight (QTOF) mass spectrometer.

[0026] The mass spectrometry parameters were as follows: ESI ion source in positive ion mode; drying gas flow rate and temperature were 8 L / min and 200℃, respectively; nebulizer drying pressure was 1.5 bar; mass spectrometer scanning range and frequency were 50-1500 m / z and 5 Hz, respectively. Separation was performed using a Waters ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, 130 Å). The liquid chromatography conditions were as follows: mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0-2 min, 0% B; 2-12 min, 0%-30% B; 12-14 min, 30%-90% B; 14-16 min, 90% B; 16-16.5 min, 90%-0% B; 16.5-20 min, 0% B. The mobile phase flow rate was 0.2 mL / min, the injection volume was 2 μL, and the sample loading concentration was 0.5 mg / mL.

[0027] The raw file generated from the LC-MS / MS data of milk protein peptides was processed to obtain the HPLC chromatogram, as shown in the figure. Figure 1 657 peptide segments were obtained.

[0028] S3: Utilize PeptdieRanker scoring and CPPpred tools to predict the cell membrane penetration ability of peptides, thereby obtaining active, non-toxic peptides with good cell membrane penetration ability.

[0029] S4: After screening to obtain suitable peptides, molecular docking was performed with OPG and IGFBP protein receptors. The active peptides of milk protein were obtained by docking score screening. The crystal structures of OPG and IGFBP were downloaded from the PDB database, with IDs of 3URF and 7WRQ, respectively.

[0030] S5: Select the peptide with the best activity for cell testing on osteoblast and chondrocyte proliferation.

[0031] 150 peptides were selected based on a PeptdieRanker score ≥0.5. Further analysis using the CPPpred tool to predict the cell membrane penetration ability (≥0.5) of these peptides revealed 7 dominant peptides (Table 1). The amino acid sequence of LPLPL is shown in SEQ ID NO.1, and the amino acid sequence of MALPK is shown in SEQ ID NO.2. Molecular docking was then performed with OPG and IGFBP3 protein receptors. The optimal milk protein peptide, LPLPL, was selected based on docking score, hydrogen bond number, and amino acid residue selection, and then used for cell testing (Tables 2 and 3).

[0032] Table 1. Peptide sequence information of milk protein peptides

[0033] Table 2. Results of docking of milk protein peptide fragments with OPG protein receptor molecules

[0034] Table 3. Results of docking of milk protein peptide fragments with IGFBP3 protein receptor molecules.

[0035] Example 2 Effects of milk protein pentapeptide LPLPL on osteoblast proliferation Mouse osteoblast precursor cells MC3T3-E1 (purchased from the Cell Bank of the Chinese Academy of Sciences) were used in experiments. Cells were cultured in α-MEM medium containing 10% fetal bovine serum. MC3T3-E1 cells were cultured at 5 × 10⁶ cells / year. 3Cells were seeded at a density per well in 96-well plates. After 24 hours of culture and adherence, the medium was replaced with medium containing different concentrations (0, 1, 10, 50, 100 μg / mL) of milk protein pentapeptide LPLPL, and a blank control group was set up. After 24 hours and 48 hours of further culture, CCK-8 solution was added to each well, and after 2 hours of incubation, the absorbance (OD value) at 450 nm was measured, and the cell proliferation rate was calculated as (OD of experimental group / OD of blank group) × 100%.

[0036] Table 4. Proliferation rate of osteoblasts after 48 hours of culture.

[0037] Note: Compared with the control group, ***p < 0.001.

[0038] The results are shown in Table 4. Compared with the blank control group, milk protein pentapeptide LPLPL can significantly promote the proliferation of MC3T3-E1 cells in the concentration range of 10-100 μg / mL (p<0.001), and the proliferation-promoting effect is most significant at a concentration of 50 μg / mL.

[0039] Example 3 Effects of milk protein pentapeptide LPLPL on chondrocyte proliferation The human chondrocyte cell line C28 / I2 (purchased from Merck, Germany) was used in experiments. C28 / I2 cells were cultured in DMEM / F12 medium containing 10% FBS. Cells were sputtered at a rate of 2 × 10⁶ cells / year. 3 Cells were seeded at a density per well in 96-well plates. After 24 hours of culture and adherence, the medium was replaced with medium containing different concentrations (0, 1, 10, 50, 100 μg / mL) of milk protein pentapeptide LPLPL, and a blank control group was set up. After 24 hours and 48 hours of further culture, CCK-8 solution was added to each well, and after 2 hours of incubation, the absorbance (OD value) at 450 nm was measured, and the cell proliferation rate was calculated as (OD of experimental group / OD of blank group) × 100%.

[0040] Table 5. Proliferation rate of chondrocytes cultured for 48 hours

[0041] Note: Compared with the control group, *p<0.05, ***p<0.001.

[0042] The results are shown in Table 5. Compared with the blank control group, the milk protein pentapeptide LPLPL significantly promoted chondrocyte proliferation in the concentration range of 10-100 μg / mL (p<0.05 & 0.001), and the proliferative effect was most significant at a concentration of 100 μg / mL. Combined with the docking results with IGFBP3, it can be concluded that the active ingredient of the milk protein peptide in this invention may promote chondrocyte proliferation by increasing IGFBP3 expression, which is beneficial to bone growth.

[0043] Example 4 Effects of milk protein pentapeptide LPLPL on osteoblast differentiation (ALP activity assay) MC3T3-E1 cells were loaded at 1×10 5 Cells were seeded at a density of 12-well plates and cultured in osteogenic induction medium (containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate). Experimental groups included those supplemented with 50 and 100 μg / mL milk protein pentapeptide LPLPL and 100 μg / mL commercially available casein peptide powder (with a molecular weight less than 1000 Da and a content greater than 70%). A control group was established using induction medium without peptides. The medium was changed every 2 days. On day 7 of intervention culture, cells were collected, and ALP activity was measured using an alkaline phosphatase (ALP) assay kit. Total protein concentration was determined and calibrated using the BCA method.

[0044] Table 6. Alkaline phosphatase (ALP) activity in osteoblasts

[0045] Note: **Compared with commercially available casein peptide powder, p < 0.01 The results are shown in Table 6. Compared with the commercially available casein peptide powder group, the ALP activity of the 100 μg / mL milk protein pentapeptide LPLPL treatment group was significantly increased (p<0.01), indicating that milk protein pentapeptide LPLPL can effectively promote the differentiation and maturation of osteoblasts.

[0046] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a milk protein peptide that promotes bone growth, characterized in that, The amino acid sequence of the bone-growth-promoting milk protein peptide is LPLPL. The preparation method specifically includes the following steps: (1) Add milk protein to an aqueous sodium hydroxide solution, and homogenize it by colloid milling to obtain a homogenized milk protein solution; (2) Adjust the pH of the milk protein homogenate to 7.8, add trypsin, alkaline protease and papain in sequence, perform enzymatic hydrolysis, inactivate enzymes, cool, and obtain milk protein hydrolysate. (3) Ultrafiltration was performed on the milk protein hydrolysate, and the components with a molecular weight <2kDa were collected to obtain the milk protein hydrolysate ultrafiltrate; (4) Centrifuge and filter the milk protein hydrolysate, take the supernatant, concentrate it, and obtain milk protein hydrolysate concentrate; (5) Spray-dry the milk protein hydrolysate concentrate to obtain the bone growth promoting milk protein peptide with the amino acid sequence LPLPL.

2. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (1), the pH value of the sodium hydroxide aqueous solution is 8.0; the mass ratio of milk protein to sodium hydroxide aqueous solution is 1:

10.

3. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (1), the homogenization process is performed at 80 MPa for one cycle.

4. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (2), the pH-adjusting reagent is citric acid; the amount of trypsin, alkaline protease and papain added is 0.5% of the milk protein content; the enzymatic hydrolysis treatment temperature is 50-55℃ and the time is 6h; the enzyme inactivation treatment temperature is 80-90℃ and the time is 30min.

5. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (3), the molecular weight cutoff of the ultrafiltration is 2 kDa.

6. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (4), the centrifugal filtration speed is 12000 r / min; the concentration temperature is 60-70℃, and the concentration is 24%-28% at the Brix level.

7. The method for preparing a bone-growth-promoting milk protein peptide according to claim 1, characterized in that, In step (5), the spray drying temperature is 135-150℃.

8. The use of the bone-growth-promoting milk protein peptide as described in claim 1 in the preparation of bone-growth-promoting products.

9. The use of the bone-growth-promoting milk protein peptide as described in claim 1 in the preparation of products for the prevention or adjunctive treatment of osteoporosis.

10. The application according to claim 8 or 9, characterized in that, The product is either a pharmaceutical or a food product.

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