An egg yolk-derived oligopeptide with the function of promoting chondrocyte differentiation and application thereof

Abstract

This invention discloses an egg yolk-derived oligopeptide that promotes chondrocyte differentiation and its application, belonging to the field of bioactive peptide technology. The egg yolk-derived oligopeptide that promotes chondrocyte differentiation is any one or more of the following amino acid sequences and their derivatives: (1) Ser-Leu-Pro-Val-Gly-Pro-Arg; (2) Ala-Ala-pSer-Trp-Pro-Lys, where pSer represents phosphorylated serine residues; (3) Asn-Gly-Val-Trp-Pro-Arg; or a polypeptide with more than 80% homology to the above amino acid sequences and possessing chondrocyte differentiation-promoting activity. This invention employs a chondrocyte differentiation model, screens for active peptides that promote chondrocyte differentiation from egg yolk protein hydrolysates, further screens for digestible active peptides through simulated in vitro gastrointestinal digestion, and systematically verifies their effects on promoting longitudinal bone growth in chondrocyte proliferation, differentiation, mineralization, and adolescent animal models.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an egg yolk-derived oligopeptide that promotes chondrocyte differentiation and its applications. Background Technology

[0002] Longitudinal bone growth during childhood and adolescence is primarily driven by the proliferation, hypertrophy, and differentiation of growth plate chondrocytes, and is a key factor determining adult height and long-term bone health. Approximately 40%-60% of adult bone mass is accumulated during early adolescence. Furthermore, about 90% of peak bone mass is reached before age 18, and higher peak bone mass is closely associated with a reduced risk of osteoporosis in adulthood. Therefore, adolescence is a critical window for bone development, and interventions during this stage can yield lasting health benefits.

[0003] Currently, recombinant human growth hormone (rhGH) therapy is widely used clinically to promote bone growth in children with growth disorders. However, mounting evidence suggests that rhGH treatment may be associated with potential safety issues. Studies have indicated that children receiving rhGH treatment have a higher incidence of type 2 diabetes compared to the general population. Furthermore, overstimulation of longitudinal bone growth may increase the sensitivity of the growth plate to shear forces, thereby increasing the risk of slipped epiphysis of the femoral head. Therefore, there is an urgent need to develop safer and more effective intervention strategies to meet the demand for promoting longitudinal bone growth.

[0004] In recent years, bioactive peptides derived from food have shown promising potential in promoting longitudinal bone growth. Egg yolk protein is considered an excellent source of bioactive peptides, typically possessing advantages such as easy absorption, low immunogenicity, high safety, and minimal toxicity. Existing technologies have reported various osteogenic peptides derived from egg yolk protein. For example, patent CN117143189A discloses the heptapeptide VPLWMAR, which can be isolated and purified from the enzymatic hydrolysate of defatted egg yolk powder and can promote osteoblast proliferation, differentiation, and mineralization; patent CN117859870A reports the octapeptide TVVEPADR, which can upregulate osteogenic-related indicators, increase bone mineral density, and improve the osteoporosis phenotype; patent CN119241643A discloses the small-molecule tetrapeptide EPGF derived from egg yolk protein; and patent CN119241640A reports the tripeptide VPF, which significantly increases the secretion levels of IGF-1, TGF-β1, and PINP by promoting osteoblast proliferation and differentiation, thereby enhancing bone formation capacity.

[0005] The aforementioned studies used an osteoblast model, and their results suggest that these bioactive peptides may play a role in enhancing bone density and alleviating osteoporosis. These findings demonstrate the potential of egg yolk-derived peptides in regulating osteogenic metabolism; however, longitudinal bone growth determines final height and is more closely related to adolescent growth. Longitudinal bone growth occurs due to skeletal activity at the growth plates located proximal and distal to the epiphysis, near the ends of long bones. Growth plate development and the proliferation and gene expression of chondrocytes within them play crucial roles. Furthermore, existing research still has certain limitations: most osteopromoting bioactive peptides are obtained directly through enzymatic hydrolysis, lacking in vitro simulated gastrointestinal digestion evaluation, making it difficult to guarantee their resistance to digestive enzyme degradation and maintenance of their biological activity after oral administration into the gastrointestinal tract.

[0006] Therefore, it is necessary to develop novel bioactive peptides that are resistant to digestive enzyme degradation and can regulate chondrocyte proliferation and differentiation, and to further verify their biological effects in promoting longitudinal bone growth in animal models, thereby providing a theoretical basis for the development of health foods or nutritional supplements that promote longitudinal bone growth. Summary of the Invention

[0007] The technical problem to be solved: Addressing the issue that existing technologies using chondrocyte differentiation-promoting active peptides promote osteoblast proliferation and differentiation but are intolerant to digestive enzyme digestion, the purpose of this invention is to provide an egg yolk-derived oligopeptide that promotes chondrocyte differentiation and its applications. This invention employs a chondrocyte differentiation model, screens for chondrocyte differentiation-promoting active peptides from egg yolk protein hydrolysates, further screens for digestibility-resistant active peptides through simulated in vitro gastrointestinal digestion, and systematically verifies its effect on promoting longitudinal bone growth in chondrocyte proliferation and differentiation and adolescent animal models, elucidating its molecular mechanism of action (pharmacological activity mechanism).

[0008] Technical solution: An egg yolk-derived oligopeptide that promotes chondrocyte differentiation, wherein the egg yolk-derived oligopeptide that promotes chondrocyte differentiation is any one or more of the following amino acid sequences and their derivatives: (1)Ser-Leu-Pro-Val-Gly-Pro-Arg (SLPVGPR); (2) Ala-Ala-pSer-Trp-Pro-Lys (AApSWPK), where pSer represents phosphorylated serine residues; (3) Asn-Gly-Val-Trp-Pro-Arg (NGVWPR); Or a polypeptide that has more than 80% homology with the above amino acid sequence and has the activity of promoting chondrocyte differentiation.

[0009] Furthermore, the C-terminus of the egg yolk-derived oligopeptide that promotes chondrocyte differentiation contains a Pro-X structural feature, wherein X is a basic amino acid.

[0010] Furthermore, the yolk-derived oligopeptide that promotes chondrocyte differentiation exhibits good digestibility and stability, and did not degrade under simulated digestion conditions in vitro.

[0011] Furthermore, the method for preparing the egg yolk-derived oligopeptide that promotes chondrocyte differentiation includes obtaining it by enzymatic hydrolysis, in vitro simulated gastrointestinal digestion, and separation and purification of defatted egg yolk powder; or by solid-phase chemical synthesis.

[0012] Furthermore, the oligopeptide activates the BMP / Smad signaling pathway by binding to BMPR1A and / or activates the FAK / ERK signaling pathway by acting on Integrin β1. Both signaling pathways work together to promote ATDC5 cell differentiation.

[0013] This invention also provides the application of egg yolk-derived oligopeptides that promote chondrocyte differentiation in the preparation of products that promote longitudinal bone growth.

[0014] Furthermore, the product that promotes longitudinal bone growth is a functional food, health food, or medicine that enhances the development of the tibial growth plate and increases the length of the femur and tibia by promoting chondrocyte differentiation.

[0015] Furthermore, the product for promoting longitudinal bone growth also contains any one or more of the following ingredients: functional additives and any acceptable excipients.

[0016] The present invention also provides a mimic digest containing an egg yolk protease hydrolysate that promotes chondrocyte differentiation, the mimic digest containing the aforementioned egg yolk-derived oligopeptide that promotes chondrocyte differentiation.

[0017] The present invention also provides a method for preparing the above-mentioned egg yolk protease hydrolysate with the function of promoting chondrocyte differentiation, comprising the following steps: (1) Pass the defatted egg yolk powder through a 60-mesh sieve and mix it with deionized water. Then, sonicate it to dissolve it completely to obtain a mixed solution. (2) Adjust the pH of the mixture to 7.0-7.5 and add pancreatic enzymes for enzymatic hydrolysis; (3) Adjust the pH to 9.5-10 and add alkaline protease for enzymatic hydrolysis; (4) Add keratinase directly and continue enzymatic hydrolysis to obtain the hydrolysate; (5) Heat the enzyme hydrolysate to above 90°C for 10-20 min to inactivate the enzyme, remove the precipitate, and obtain the supernatant; (6) Concentrate the supernatant and freeze-dry it to obtain egg yolk protein hydrolysate; (7) Dissolve the egg yolk protein hydrolysate in deionized water (w / v = 1:80) and adjust the pH to 2.0 with HCl; (8) Add pepsin at an enzyme to substrate ratio (E / S) of 2% and hydrolyze at 37°C for 2 h to simulate gastric digestion; (9) Then, the pH was adjusted to 7.0 with NaOH, and pancreatic enzyme was added at E / S = 2% and enzymatically hydrolyzed at 37°C for 2 h to simulate intestinal digestion; (10) After the enzymatic hydrolysis is completed, the reaction is terminated by heating in a boiling water bath for 10 min. After cooling to room temperature, the pH is adjusted to 7.0. The supernatant is collected by centrifugation, and after C18 desalting, it is freeze-dried to obtain the simulated digest of egg yolk protein hydrolysate.

[0018] Furthermore, the mass of the deionized water mentioned in step (1) is 9-10 times the mass of the defatted egg yolk powder.

[0019] Furthermore, the amount of pancreatic enzyme added in step (2) is 1.8-2.1% of the weight of defatted egg yolk powder; the enzymatic hydrolysis temperature is 36-38℃, and the enzymatic hydrolysis time is 2.5-3.5h.

[0020] Furthermore, in step (3), the amount of alkaline protease added is 1.4%-1.6% of defatted egg yolk powder, the enzymatic hydrolysis temperature is 50-55℃, and the enzymatic hydrolysis time is 2-3h.

[0021] Furthermore, in step (4), the amount of keratinase added is 1.6-2.0% of defatted egg yolk powder, the enzymatic hydrolysis temperature is 48-50℃, and the enzymatic hydrolysis time is 1-2h.

[0022] Furthermore, the egg yolk protein hydrolysate that promotes chondrocyte differentiation can promote longitudinal bone growth in adolescent animals, manifested as increased body length, increased femur and tibia length, enhanced tibial growth plate development, and increased serum IGF-1 and BALP levels.

[0023] The present invention also provides a composition, characterized in that the composition comprises the above-mentioned oligopeptide or digestive mimic.

[0024] The present invention also provides a product that promotes longitudinal bone growth, the product comprising the above-mentioned oligopeptide, digestive mimic, or composition. Beneficial effects

[0025] 1. This invention, for the first time, screened three chondrogenic peptides—SLPVGPR, AAPSWPK, and NGVWPR—from egg yolk protein hydrolysates using a growth plate chondrocyte differentiation model (ATDC5 cells). This breakthrough overcomes the conventional approach of screening osteogenic peptides using osteoblast models in existing technologies. Existing technologies all use osteoblast models to evaluate the efficacy of active peptides, and their results suggest effects on increasing bone density and alleviating osteoporosis, but they cannot directly reflect the promoting effect on longitudinal bone growth. Longitudinal bone growth is mainly driven by the proliferation, hypertrophy, and differentiation of growth plate chondrocytes, and is a key factor determining the height of adolescents. This invention uses a chondrocyte differentiation model as the core screening platform, more precisely targeting the biological needs for promoting longitudinal bone growth from the functional evaluation point.

[0026] 2. The three bioactive peptides provided in this invention are all derived from dietary egg yolk protein, are safe and have no toxic side effects, and exhibit good digestibility stability as verified by in vitro simulated gastrointestinal digestion. Specifically, the heptapeptide SLPVGPR and hexapeptide AAPSWPK did not degrade after simulated gastrointestinal digestion, with a retention rate close to 100%; the hexapeptide NGVWPR achieved a retention rate of 85.02% after simulated digestion, with most remaining in its original form. This invention further screens the peptides through simulated gastrointestinal digestion, ensuring that the obtained bioactive peptides can reach their target sites intact in vivo to exert their biological effects under oral administration, significantly improving the feasibility of practical applications.

[0027] 3. The three bioactive peptides provided by this invention exhibit significant chondrocyte differentiation and mineralization-promoting activities: Within the concentration range of 10-40 μM, SLPVGPR, AAPSWPK, and NGVWPR can all increase the alkaline phosphatase (ALP) activity of ATDC5 cells. Among them, NGVWPR achieved a relative ALP activity of 127% at 20 μM, while SLPVGPR and AAPSWPK showed more significant differentiation-promoting effects, with maximum ALP activities reaching 152% and 157%, respectively. Simultaneously, all three peptides promoted the formation of mineralized nodules in ATDC5 cells at concentrations of 20 μM and 40 μM, exhibiting a dose-dependent effect, indicating their ability to promote terminal differentiation of chondrocytes.

[0028] 4. The active peptides provided by this invention exhibit excellent effects in promoting longitudinal bone growth in a adolescent rat model, with multiple indicators superior to the rhGH positive control. After 21 days of intervention in the high-dose group, body length, femur length, and tibia length all increased significantly, with tibia length (41.15 mm, an increase of 4.35%) being superior to the rhGH group (40.52 mm). The maximum tibial load increased by 18.99% compared to the normal group (only 4.16% in the rhGH group), and bone mineral content increased by 83.68% (only 15.40% in the rhGH group). The growth plate thickness increased to 145.61%, significantly superior to the rhGH group (110.66%). Bone microstructure parameters (BV / TV, trabecular bone number, trabecular bone spacing) and serum IGF-1 and BALP levels were significantly improved and superior to those in the rhGH group. These results fully demonstrate that the active peptides of this invention have significant and comprehensive biological effects in promoting longitudinal bone growth.

[0029] 5. The bioactive peptides provided by this invention have good safety. Analysis of organ index in adolescent rats showed no significant differences in the relative weights of important organs such as the liver, kidneys, spleen, heart, and thymus among the groups (P>0.05), indicating that oral administration of egg yolk bioactive peptides did not adversely affect important metabolic, excretory, immune, or circulatory systems. Organ development and body growth maintained good synchronicity, providing strong support for oral safety. Compared to the potential safety risks associated with rhGH treatment, such as increased incidence of type 2 diabetes and slipped adipose tissue, the food-derived bioactive peptides provided by this invention have a significant advantage in terms of safety.

[0030] 6. The active peptides provided by this invention are naturally sourced, orally effective, highly safe, and have superior effects in improving bone microstructure and promoting growth plate development. They can be used as active ingredients in functional foods, health foods, or pharmaceuticals to promote longitudinal bone growth in adolescents, and have broad application prospects. Attached Figure Description

[0031] Figure 1 The effect of egg yolk protein hydrolysate and its mimic digests on the proliferation activity of ATDC5 cells in Example 1; Figure 2 The effects of egg yolk protein hydrolysate and its simulated digests on alkaline phosphatase secretion and mineralization in ATDC5 cells are shown in Example 1; where A represents relative ALP activity and B represents mineralization activity. Figure 3 This is the total ion chromatogram of the simulated digest of egg yolk protein hydrolysate analyzed by mass spectrometry in Example 2; Figure 4 This is the primary mass spectrum of the simulated digest of egg yolk protein hydrolysate analyzed by mass spectrometry in Example 2. The arrows point to the ion proton peaks corresponding to the three active peptides.

[0032] Figure 5 The following are secondary mass spectra of peptides SLPVGPR, AAPSWPK, and NGVWPR in Example 2; Figure 6 The effects of peptides SLPVGPR, AAPSWPK, and NGVWPR on the proliferation activity of ATDC5 cells in Example 3; Figure 7 The effects of peptides SLPVGPR, AAPSWPK, and NGVWPR on alkaline phosphatase secretion and mineralization in ATDC5 cells are shown in Example 3; where A represents relative ALP activity and B represents mineralization activity. Figure 8 The results are HPLC analysis of the egg yolk-derived oligopeptide fragments that promote chondrocyte differentiation in Example 4 before and after in vitro simulated digestion; where A is SLPVGPR; B is AAPSWPK; and C is NGVWPR.

[0033] Figure 9 This is an example of the effect of SLPVGPR on Smad1 / 5 phosphorylation and Runx2 protein expression in ATDC5 cells. A shows the Western Blot bands of Smad1 / 5, p-Smad1 / 5, Runx2, and β-actin protein expression; B shows the grayscale analysis of p-Smad1 / 5 / Smad1 / 5 expression levels; and C shows the grayscale analysis of Runx2 protein expression levels. Figure 10 This section shows the effect of the BMP receptor inhibitor LDN-193189 on the differentiation-promoting effect of SLPVGPR in Example 5. A represents the Western Blot bands of Smad1 / 5, p-Smad1 / 5, Runx2, and β-actin protein expression; B represents the grayscale analysis of p-Smad1 / 5 expression levels; C represents the grayscale analysis of Runx2 protein expression levels; and D represents the results of alkaline phosphatase secretion detection in each group. Figure 11 The results of DARTS identification of SLPVGPR target in Example 5 are shown; where A is a protein electrophoresis image stained with Coomassie Brilliant Blue; B is the enzyme-resistant proteins verified by Western Blot as BMPR1A and Integrin β1. Figure 12 The image shows the effect of SLPVGPR on the phosphorylation levels of FAK and ERK1 / 2 in ATDC5 cells in Example 5. A represents the Western Blot bands of FAK, p-FAK, ERK1 / 2, and p-ERK1 / 2 protein expression; B represents the grayscale analysis of p-FAK / FAK expression levels; and C represents the grayscale analysis of p-ERK1 / 2 / ERK1 / 2 protein expression levels. Figure 13 This is the effect of egg yolk active peptides on rat body length in Example 6; where A is the body length growth curve of rats in each group over 21 days; B is the statistical result of the body length of rats in each group on day 21. Figure 14 The effects of egg yolk active peptides on the growth of isolated femurs and tibias of rats in Example 6 are shown in Figure 6. A represents the statistical results of femur length; B represents a representative image of the femur; C represents the statistical results of tibia length; and D represents a representative image of the tibia. Figure 15 The effects of egg yolk active peptides on bone strength (A), bone mineral content (B), bone mineral density (C), and bone microstructure (DH) in rats, as shown in Example 6. Figure 16 The images show the statistical analysis results and representative H&E staining images of the tibial growth plate thickness in rats in Example 6; where A represents the relative height of the growth plate; and B represents a representative H&E staining image. Figure 17 The values ​​represent the concentrations of IGF-1 and BALP in the serum of rats in each group in Example 6; where A represents IGF-1 in serum and B represents BALP in serum. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0035] The following pancreatic enzymes have an activity of 200,000 U / g; alkaline protease has an activity of 200,000 U / g (purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., China); keratinase is food grade with an activity of 100,000 U / g (purchased from Henan Sanhua Biotechnology Co., Ltd., Bacillus subtilis source).

[0036] Example 1: Chondrogenic activity of egg yolk protein hydrolysate and its mimic digestion products 1. The preparation method of egg yolk protein hydrolysate, the steps are as follows: (1) Dissolve 5 g of defatted egg yolk powder in 50 mL of deionized water and sonicate the egg yolk liquid (300W, 20 min, sonicate for 3 s and stop for 3 s). (2) Adjust the pH of the solution to 7.3, add 0.1 g of trypsin (enzyme to substrate mass ratio (E / S) = 2%), start the enzymatic hydrolysis reaction at 37℃, and hydrolyze for 3 h; (3) Adjust the pH to 10.0 and add 0.075 g of alkaline protease (E / S=1.5%), and continue enzymatic hydrolysis at 50℃ for 2.5 h; (4) Without adjusting the pH, add 0.1 g of keratinase (E / S=2%), hydrolyze for 1.5 h, inactivate the enzyme with boiling water for 10 min, cool to room temperature, and adjust the pH to 7.0; (5) Centrifuge at 5000 rpm for 15 min, take the supernatant, and freeze dry to obtain egg yolk protein hydrolysate, hereinafter referred to as enzymatic hydrolysate (EYH).

[0037] 2. In vitro simulated digestion method (1) Dissolve the prepared enzymatic hydrolysate (EYH) in deionized water (w / v = 1:80) and adjust the pH to 2.0 with HCl; (2) Add pepsin at an enzyme to substrate ratio (E / S) of 2% and enzymatically hydrolyze at 37°C for 2 h to simulate gastric digestion; (3) Then, the pH was adjusted to 7.0 with NaOH, and pancreatic enzyme was added at E / S = 2%. The enzyme was enzymatically hydrolyzed at 37 °C for 2 h to simulate intestinal digestion. (4) After the enzymatic hydrolysis is completed, the reaction is terminated by heating in a boiling water bath for 10 min. After cooling to room temperature, the pH is adjusted to 7.0. The supernatant is collected by centrifugation, and after C18 desalting, it is freeze-dried to obtain the simulated digest of egg yolk protein hydrolysate, hereinafter referred to as simulated digest (HSD).

[0038] 3. Determination of chondrocyte proliferation-promoting activity Logarithmic growth phase ATDC5 cells were seeded at an appropriate density in 96-well plates and cultured in complete medium (DMEM + 10% FBS + 1% PS) until the density reached 80%. The medium was then replaced with complete medium containing different samples and cultured for 24 h. Afterward, each well was replaced with complete medium containing 0.5 mg / mL MTT and cultured in a cell culture incubator for 4 h. The medium was discarded, and 150 μL LDMSO was added to dissolve the purple crystals. The absorbance at 490 nm was measured using a microplate reader. The survival rate of the control group was set at 100%, and the following formula was used to calculate: Cell viability (%) = (OD experimental group / OD control group) × 100%.

[0039] The results are as follows Figure 1As shown, within the range of 25-150 μg / mL, the cell proliferation activities of both the egg yolk protein hydrolysate group and the in vitro simulated digestion group were significantly lower than those of the control group, exhibiting a dose-dependent effect. Generally, when cell proliferation activity is higher than 80%, the sample can be considered to have no adverse effect on cells. Specifically, the cell proliferation activities of the egg yolk protein hydrolysate at 100 μg / mL and 150 μg / mL were 78.92% and 78.27%, respectively, suggesting that high concentrations of egg yolk protein hydrolysate inhibited cell proliferation. This may be due to the influence of a small amount of salt (approximately 4%) generated during the preparation of the egg yolk protein hydrolysate. In contrast, the in vitro simulated digestion, after desalting during preparation, showed proliferation activities of 87.36% and 88.74% at the same concentration, respectively, indicating a relatively smaller impact on cell growth.

[0040] 4. Determination of chondrocyte differentiation-promoting activity: The chondrocyte differentiation-promoting activities of the prepared enzymatic hydrolysate (EYH) and simulated digestate (HSD) were measured. The specific steps were as follows: ATDC5 cells in logarithmic growth phase were seeded at an appropriate density in 12-well plates and cultured in complete medium (DMEM + 10% FBS + 1% PS) until cell confluence reached 80%-90%. Then, the medium was replaced with differentiation medium containing different samples (DMEM / F12 + 5% FBS + 1% PS + 50 μg / mL ascorbic acid + 1% ITS), and cultured for another 24 h. The medium was discarded, and cell lysates were prepared after washing twice with PBS. ALP activity was measured using an alkaline phosphatase (ALP) kit, and protein content was corrected using the BCA method. Relative activity was calculated with the control group's ALP activity as 100%.

[0041] ALP enzymes are markers of cell differentiation. Experimental results show (e.g.) Figure 2 As shown in Figure A), both the egg yolk protein hydrolysate and the simulated digest can increase the ALP activity of ATDC5 cells in the concentration range of 25-100 μg / mL, and this effect is dose-dependent. At a concentration of 100 μg / mL, the relative ALP activities of the hydrolysate and the simulated digest are increased to 146.6% and 131.5%, respectively, indicating that both the hydrolysate and the simulated digest have biological activities that promote chondrocyte differentiation, but the ALP activity decreases after digestion.

[0042] 5. Assay for chondrocyte mineralization activity The chondrogenic activity of the prepared enzymatic hydrolysate (EYH) and simulated digestate (HSD) was evaluated. Specifically, ATDC5 cells were cultured in complete medium until confluence reached 80%-90%. The original medium was discarded, and the cells were replaced with differentiation medium containing different concentrations of the sample for induction culture. The medium was changed every 2 days for a total of 21 days. After culture, Alizarin Red staining was used to detect the formation of mineralized nodules in the cells, and imaging was recorded.

[0043] The experimental results show (e.g.) Figure 2 As shown in Figure B, within the concentration range of 25-100 μg / mL, both the enzymatic hydrolysate and its simulated digestion products can promote the formation of mineralized nodules in ATDC5 cells, and this effect is dose-dependent. The enzymatic hydrolysate showed the most significant promoting effect under the treatment condition of 100 μg / mL. After simulated digestion, the promoting effect of egg yolk protein hydrolysate on mineralized nodules was reduced.

[0044] The above results indicate that although egg yolk protein hydrolysates still exhibit good chondrocyte differentiation and mineralization activity after simulated gastrointestinal digestion, their activity decreases. This is because some active peptides in the egg yolk protein hydrolysates are degraded during digestion. Therefore, isolating and identifying digestible egg yolk active peptides and elucidating their mechanism of action has important application value.

[0045] Example 2: Identification of bioactive peptides in simulated digests 1. Separation and purification based on the hydrophobicity of peptides. To improve the resolution of peptide identification, the simulated digest (HSD) obtained in Example 1 was separated and purified. C18 resin (YMC*GEL ODS) was thoroughly wetted and packed into a chromatography column (1 cm × 10 cm). The column bed was washed with distilled water until no significant change was observed under 220 nm UV detection. The simulated digest was prepared into a 50 mg / mL solution, and 1 mL was loaded onto the chromatography column for separation. A peristaltic pump was used to control the flow rate at 1 mL / min, and gradient elution was performed sequentially with deionized water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and anhydrous ethanol as eluents, eluting for 1.5 column volumes with each eluent. The fractions eluted with 60%–80% ethanol were collected based on UV detection results. The in vitro chondrocyte differentiation-promoting activity of each fraction was evaluated using the same method as in Example 1, and the fraction with the highest activity was screened for subsequent analysis.

[0046] 2. Separate and purify peptides based on their molecular weight. The collected fractions were further separated and purified using a Sephadex C25 chromatography column. The Sephadex C25 packing material was fully swollen in 0.02 M sodium acetate buffer (pH 4.0) and packed onto a 1.0 cm × 20 cm column, which was then equilibrated with the same buffer. Two mL of a 20 mg / mL sample solution was loaded, and gradient elution was performed using sodium acetate buffers with different NaCl concentrations at a flow rate of 2 mL / min, with each gradient elution lasting 10 min. The elution process was monitored in real-time using 220 nm UV absorption, and each eluted fraction was collected.

[0047] 3. The in vitro chondrocyte differentiation-promoting activity of each component was evaluated using the same method as in Example 1. The component with the highest activity was screened for subsequent analysis.

[0048] 4. Identification was performed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) to obtain the total ion chromatogram (e.g., Figure 3 (As shown). The raw mass spectrometry data were processed using Xcalibur software, and protein sequences of *Gallus gallus* species were retrieved from the Uniprot database (version 2025, containing 18370 entries) using PEAKS Studio 10.6 software to identify and analyze peptide sequences. A total of 2648 peptides were identified, among which those with an abundance greater than 1×10⁻⁶ were selected. 8 A total of 169 peptides were identified, and the identified peptides were mainly medium- and short-chain polypeptides, with hexapeptides and heptapeptides being the most numerous, at 36 and 33 respectively.

[0049] 5. Molecular docking analysis of the identified peptides with the BMPR1A receptor was performed using AutoDock Vina software. Based on the peptide abundance information and the molecular docking results with the BMPR1A receptor, three highly active peptides were screened, as shown in Table 1. The peptides SLPVGPR, AAPSWPK, and NGVWPR all originated from vitellogenin II and had high relative abundance. The molecular docking results showed that these three peptides exhibited low binding energies with the BMPR1A receptor, indicating their good binding potential.

[0050] Table 1. Potentially bioactive peptides obtained by docking with BMPR1A receptor molecules SLPVGPR 725.43 Vitellogenin II (1546-1552) <![CDATA[2.14×10 9 ]]> -5.7 kcal / mol AApSWPK 739.32 Vitellogenin II (1365-1370) <![CDATA[8.61×10 8 ]]> -7 kcal / mol NGVWPR 729.37 Vitellogenin II (81-86) <![CDATA[7.68×10 8 ]]> -6.2 kcal / mol 6. The primary mass spectra corresponding to the above three peptide segments are as follows: Figure 4 As shown, it exists in the same isolated fraction of egg yolk peptides; its secondary mass spectrum is shown below. Figure 5As shown, fragment ion analysis revealed continuous proton peaks of both b and y ions in each peptide segment, covering the main sequence fragments and demonstrating the accuracy of the identified peptide sequences.

[0051] The amino acid sequence of the peptide SLPVGPR is Ser-Leu-Pro-Val-Gly-Pro-Arg; The amino acid sequence of the peptide AAPSWPK is Ala-Ala-pSer-Trp-Pro-Lys, where pSer represents phosphorylated serine residues; The amino acid sequence of the peptide NGVWPR is Asn-Gly-Val-Trp-Pro-Arg.

[0052] Example 3: Determination of the chondrocyte differentiation-promoting and mineralization activity of egg yolk-derived oligopeptides that promote chondrocyte differentiation. 1. The three polypeptide sequences SLPVGPR, AAPSWPK, and NGVWPR identified in Example 2 were prepared using solid-phase chemical synthesis, with a purity of over 98%. The in vitro chondrocyte differentiation-promoting activity of the active peptides was determined, following the same experimental procedures as in Example 1.

[0053] Effects on chondrocyte proliferation activity, such as Figure 6 As shown in the figure, SLPVGPR, AAPSWPK, and NGVWPR did not significantly affect the proliferation activity of ATDC5 cells within the concentration range of 20-80 μM. Among them, SLPVGPR and AAPSWPK showed a certain proliferative trend at concentrations of 20, 40, and 80 μM. At 80 μM, the cell proliferation activities of the SLPVGPR and AAPSWPK treatment groups reached 111.97% and 117.62%, respectively. In contrast, with increasing NGVWPR concentration, the activity of ATDC5 cells showed a slight decreasing trend, with a cell proliferation activity of 91.29% at 80 μM, but the difference compared with the control group was not statistically significant (P>0.05).

[0054] Effects on chondrocyte differentiation, such as Figure 7 As shown in Figure A, all three peptides can enhance ALP activity in ATDC5 cells, exhibiting a promoting effect in the range of 10-40 μM. NGVWPR showed the highest activity at 20 μM, at 127%. SLPVGPR and AAPSWPK showed more significant differentiation-promoting effects, with maximum activities reaching 152% and 157%, respectively, indicating that all three active peptides have the activity of promoting chondrocyte differentiation.

[0055] 2. The chondrocyte mineralization activity of the above-mentioned active peptides was determined, and the specific steps were the same as step 4 of Example 1.

[0056] The results are as follows Figure 7 As shown in B, SLPVGPR, AAPSWPK, and NGVWPR peptides can all promote the formation of mineralized nodules in ATDC5 cells at concentrations of 20 μM and 40 μM, and the promoting effect increases with increasing concentration, showing a certain dose-dependent effect.

[0057] The above results indicate that the three identified polypeptide sequences, SLPVGPR, AAPSWPK, and NGVWPR, all possess good chondrocyte differentiation and mineralization-promoting activities.

[0058] Example 4: Evaluation of the digestibility of egg yolk-derived oligopeptides that promote chondrocyte differentiation 1. Dissolve SLPVGPR, AAPSWPK, and NGVWPR separately in water and adjust the pH to 2.0. Add pepsin at an enzyme-to-substrate ratio (E / S) of 1:50 and digest at 37 °C for 2 h. Then adjust the pH to 7.0, add trypsin at an enzyme-to-substrate ratio (E / S) of 1:50, and continue digestion at 37 °C for 2 h. After digestion, inactivate the enzymes in a boiling water bath for 10 min, centrifuge, and collect the supernatant for subsequent analysis.

[0059] 2. HPLC was used to analyze untreated peptides and peptides after in vitro simulated gastrointestinal digestion. A ZORBAX SB-C18 column (5 μm, 4.6 × 250 mm) was used. Mobile phase A was an aqueous solution containing 0.1% TFA, and mobile phase B was acetonitrile containing 0.1% TFA. The gradient elution program was as follows: 0–25 min, phase B linearly increased from 10% to 35%; 25.0–25.1 min, phase B increased to 100%; then maintained until 30.0 min. The flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL.

[0060] The results are as follows Figure 8 As shown, heptapeptide SLPVGPR ( Figure 8 A) and hexapeptide AAPSWPK ( Figure 8 B) The presence of a single characteristic peak after simulated gastrointestinal digestion in vitro indicates no degradation after digestion. Although a degradation product peak was detected in the hexapeptide NGVWPR after simulated digestion, the retention rate of the original peptide reached 85.02%, with most of it entering the target cells in vivo in its original form. These results indicate that all three bioactive peptides have good resistance to digestion.

[0061] Example 5: Study on the target and molecular mechanism of action of egg yolk-derived oligopeptides that promote chondrocyte differentiation. Taking the heptapeptide SLPVGPR as an example, this study reveals the target and molecular mechanism of action of the yolk peptide that promotes chondrogenic differentiation.

[0062] 1. BMP / Smad signaling pathway activation and BMP receptor inhibition experiments ATDC5 cells with a confluence of 80-90% were cultured in differentiation medium containing SLPVGPR peptide for 2 hours. The medium was then discarded, and RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added to extract total cell protein. The protein concentration was determined using the BCA method.

[0063] Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking at room temperature for 1 h, primary antibodies p-Smad1 / 5 (1:1000) and β-actin (1:3000) were added and incubated overnight at 4 °C. Then, the membrane was incubated with HRP-labeled secondary antibody for 1 h. The membrane was then developed by ECL chemiluminescence and protein band signals were acquired using an imaging system. The membrane was then regenerated using antibody stripping buffer and incubated with the corresponding total protein primary antibodies, including Smad1 / 5 (1:1000) and Runx2 (1:1000), overnight at 4 °C. After incubation with HRP-labeled secondary antibody for 1 h, the membrane was developed by ECL and images were acquired. ImageJ software was used to perform grayscale analysis of the protein bands, and β-actin was used as an internal control for normalization.

[0064] To further verify the role of BMP receptor in the differentiation-promoting effect of SLPVGPR, ATDC5 cells were subjected to serum starvation and then incubated for 1 h with differentiation medium containing 500 nM LDN-193189 (a BMPR1A receptor inhibitor) to inhibit BMPR1A receptor activity. Subsequently, SLPVGPR was added to a final concentration of 40 μM and the cells were cultured for another 2 h. After culture, total cellular protein was extracted, and protein concentration was determined using the BCA method. The expression levels of p-Smad1 / 5, Smad1 / 5, Runx2, and β-actin were detected using the same method. ALP activity was also measured in each treatment group.

[0065] 2. Identification and validation experiments of target proteins using DARTS technology. ATDC5 cells were cultured to approximately 80%-90% confluence, washed with PBS, and lysed with lysis buffer containing protease inhibitors. The cells were lysed on ice for 15 min, centrifuged at 4 °C, and the supernatant was collected and the protein concentration adjusted to 4-6 μg / μL. An equal volume of cell lysate was co-incubated with different concentrations of SLPVGPR peptide for 45 min, with corresponding solvent treatment as a control. Subsequently, different concentrations of Pronase were added for enzymatic digestion for 10 min, and SDS loading buffer was added and the reaction was terminated by boiling in a water bath. The treated samples were subjected to SDS-PAGE electrophoresis, stained with Coomassie Brilliant Blue R250 and destained, and the changes in protein bands were observed and recorded. Simultaneously, the proteins separated by SDS-PAGE were transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 1 h, and then incubated overnight at 4 °C with BMPR1A antibody (1:1000) and Integrin β1 antibody (1:1000), respectively. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 1 h. h, the sample was developed using ECL chemiluminescence and then imaged for analysis.

[0066] 3. Expression of key proteins in the Integrin / FAK signaling pathway The specific steps are the same as step 1 in Example 5. After blocking the PVDF membrane, primary antibodies p-FAK (1:1000) and p-ERK1 / 2 (1:1000) were added and incubated overnight at 4 °C. Then, the membrane was incubated with HRP-labeled secondary antibody for 1 h. After ECL chemiluminescence development, protein band signals were acquired using an imaging system. The membrane was then regenerated using antibody stripping buffer and incubated with the corresponding total protein primary antibodies FAK (1:1000) and ERK1 / 2 (1:1000) for 1 h at 4 °C. After ECL development and image acquisition, the protein bands were analyzed using ImageJ software.

[0067] Experimental results are as follows Figure 9 As shown, 20-40 μM SLPVGPR treatment can promote the phosphorylation level of Smad1 / 5 and the expression level of Runx2 protein in a dose-dependent manner. Specifically, under 40 μM treatment, the phosphorylation level of Smad1 / 5 and the expression level of Runx2 increased by approximately 1.37-fold and 1.71-fold, respectively, compared to the control group, indicating that SLPVGPR can activate the BMP / Smad signaling pathway and promote the expression of the chondrogenic differentiation-related transcription factor Runx2.

[0068] Further analysis using the BMP receptor inhibitor LDN-193189 verified the mediating role of the BMP / Smad signaling pathway in the SLPVGPR-induced ATDC5 cell differentiation process, and the results are as follows: Figure 10As shown, under LDN-193189 treatment, SLPVGPR-induced Smad1 / 5 phosphorylation levels were significantly inhibited, and there was no significant difference compared with the LDN-193189-only treatment group. Figure 10 (B) suggests that the promoting effect of SLPVGPR on Smad1 / 5 is mainly mediated by the BMP receptor. Under inhibitor conditions, although SLPVGPR-induced Runx2 expression was significantly decreased, it was still significantly higher than in the inhibitor-only treatment group (B). Figure 10 C); In addition, LDN-193189 also failed to completely inhibit the SLPVGPR-induced increase in ALP activity ( Figure 10 (D) indicates that the promoting effect of SLPVGPR on Runx2 expression and ALP activity is not entirely dependent on BMP receptor mediation, suggesting that SLPVGPR may exert its differentiation-promoting effect through multiple signaling pathways.

[0069] DARTS results are as follows Figure 11 As shown, under 1:100 (E / S) Pronase digestion conditions, after co-incubation with ATDC5 cell lysis buffer using 0.2 mM and 0.4 mM SLPVGPR, distinct enzyme-resistant bands were observed in the 100-70 kDa, 70-55 kDa, and 55-35 kDa regions. Figure 11 A) suggests that SLPVGPR may have multiple potential binding targets. The 70-55 kDa range closely matches the theoretical molecular weight of BMPR1A (60 kDa), and Western blotting confirmed that this enzyme-resistant band corresponds to BMPR1A (A). Figure 11 B). The 100-70 kDa range matches the theoretical molecular weight of Integrin β1, which is approximately 88 kDa. Therefore, further Western blotting was performed on this band, confirming that the corresponding receptor protein is Integrin β1. Figure 11 B).

[0070] To further verify whether Integrin β1-related downstream signals were activated, the FAK / ERK signaling pathway was detected, and the results are as follows: Figure 12 As shown, within the concentration range of 20-40 μM, SLPVGPR treatment significantly promoted the phosphorylation levels of FAK and ERK1 / 2; among them, the promoting effect on FAK phosphorylation was most significant at 20 μM, approximately 1.48 times that of the control group, while the promoting effect on ERK1 / 2 phosphorylation increased to approximately 1.42 times at 40 μM.

[0071] The above results indicate that SLPVGPR activates the BMP / Smad signaling pathway by binding to BMPR1A and activates the FAK / ERK signaling pathway by acting on Integrin β1. Both signaling pathways are involved in promoting ATDC5 cell differentiation.

[0072] Example 6: Evaluation of the efficacy of egg yolk bioactive peptides in promoting longitudinal bone growth (animal experiment) 1. Preparation of egg yolk active peptide components The simulated digest (HSD) was prepared according to Example 1. Based on the hydrophobicity of the peptides, separation and purification were performed. To achieve large-scale preparation of the target active component, a macroporous adsorption resin chromatography column was used instead of a C18 column for sample separation and purification. XAD-1600 macroporous adsorption resin was thoroughly wetted and packed into a chromatography column (2.5 cm × 40 cm). The column bed was washed with distilled water until no significant change was observed under 220 nm UV detection. The simulated digest (HSD) obtained in Example 1 was prepared into a 300 mg / mL solution, and 10 mL was loaded onto the chromatography column for separation. A peristaltic pump was used to control the flow rate at 3 mL / min. Gradient elution was performed sequentially using deionized water, 20% ethanol, 40% ethanol, 60% ethanol, 80% ethanol, and anhydrous ethanol as eluents, with 1.5 column volumes eluted with each eluent. The 60%-80% ethanol eluent fraction was collected based on UV detection results.

[0073] 2. Animal grouping Twenty-four 3-week-old male SD rats were housed in an SPF-grade environment (temperature 22±2℃, humidity 55±5%, 12-hour light-dark cycle) with free access to food and water. After one week of acclimatization, the rats were randomly divided into four groups of six (3 rats / cage) each, and administered the drug for three consecutive weeks. The normal control group received an equal volume of distilled water by gavage; the positive control group received subcutaneous injection of recombinant human growth hormone (20 μg / kg bw); the experimental groups received the active component obtained in step 1 by gavage at doses of 1 mg / kg bw (low-dose group) and 3 mg / kg bw (high-dose group), respectively, once daily. During the experiment, the rats had free access to standard feed and water. Body length (distance from nose tip to anus) was measured weekly using calipers. The results are as follows: Figure 13 As shown.

[0074] 3. Collection and processing of animal samples At the end of the experiment (3 weeks later), blood was collected via the abdominal aorta under moderate anesthesia. Blood was collected in anticoagulant-free blood collection tubes, allowed to stand at room temperature for 2 hours, and then centrifuged at 4 ℃ and 3000 r / min for 10 min. The supernatant serum was collected, aliquoted, and frozen at -80℃ for later use in subsequent biochemical assays. After blood collection, the rats were euthanized, and the heart, liver, spleen, kidneys, and thymus were quickly dissected. Residual blood and connective tissue on the surface of the organs were rinsed with pre-cooled physiological saline, and surface moisture was blotted dry with filter paper. The wet weight of each organ was measured, and the organ index (g / 100g) was calculated. Organ Index = Organ Weight (g) / Body Weight (100 g) × 100% Bilateral hind limbs of rats were separated, soft tissues were removed, and care was taken to protect the bones and epiphyses; residual tissue on the bone surface was rinsed with pre-cooled physiological saline, and the water was blotted dry with filter paper to obtain isolated tibias, which were then placed on ice for later use.

[0075] 4. Measurement of femur and tibia length Skeletal lengths were measured using vernier calipers. Tibial length was defined as the straight-line distance from the proximal to the distal epiphysis; femoral length was defined as the straight-line distance from the femoral head to the intercondylar fossa. The results are as follows: Figure 14 As shown.

[0076] 5. Measurement of bone strength Place the tibia on the support frame of the mechanical testing instrument, adjust the support distance to a position greater than the bone length, and tighten the sample by rotating the fixing knob counterclockwise. After covering with the protective cover, start the testing program, record the maximum load value of the tibia, and use it as an indicator of bone strength. The results are as follows: Figure 15 As shown in Figure A.

[0077] 6. Bone mineral density and bone microstructure analysis The fixed tibia was scanned using the Hiscan In Vivo Micro-CT tomographic system. The proximal trabecular bone region of the tibia was selected as the region of interest (ROI) in HiscanAnalyzer software, and quantitative analysis was performed under uniform threshold conditions. Bone microstructural parameters measured included bone mineral content (BMC), bone mineral density / bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp), such as... Figure 15 As shown in BH. All samples used the same scanning parameters, reconstruction algorithm, and threshold criteria to ensure the reproducibility of the results and the comparability between groups.

[0078] 7. H&E staining analysis of tibial growth plate Tibial bones from each group of rats were fixed with 4% paraformaldehyde for 24 h, then decalcified in 10% EDTA solution until the bone tissue was completely softened. After decalcification, the bones were dehydrated with a gradient of ethanol, cleared with xylene, and embedded in paraffin to prepare paraffin sections. After dewaxing to water, the sections were stained with hematoxylin and eosin (H&E), undergoing hematoxylin staining, differentiation, blueing, and counterstaining with eosin. After dehydration and clearing, the sections were mounted. The structure of the tibial growth plate was observed under an optical microscope, and the thickness of the growth plate was measured. The results are as follows: Figure 16 As shown.

[0079] 8. Serum biochemical analysis The levels of bone alkaline phosphatase (BALP) and insulin-like growth factor-1 (IGF-1) in serum were determined using enzyme-linked immunosorbent assay (ELISA). The specific procedure was as follows: Serum samples were appropriately diluted according to the kit instructions and added to the wells of an ELISA plate pre-coated with antibodies. After incubation at 37 °C for a certain period, the plate was washed. Enzyme-labeled antibody was added, and incubation continued, followed by washing. Then, the chromogenic substrate solution was added for the reaction. After the reaction was complete, stop solution was added to terminate the reaction. The absorbance of each well was measured at the specified wavelength using an ELISA reader. The contents of BALP and IGF-1 in the sample were calculated based on the standard curve. The results are shown below. Figure 17 As shown.

[0080] 9. Results Analysis The results showed that, compared with the normal group, gavage administration of the active peptide component of egg yolk promoted skeletal growth and development in adolescent rats, manifested as a significant increase in height (body length) and longitudinal bone growth (including femur and tibia). After 21 days of intervention, compared with the normal group, the average body length of the high-dose group significantly increased from 21.53 cm to 22.33 cm. Figure 13 The positive control group (growth hormone injection group) showed a significant increase in body length to 22.55 cm. Femur and tibia length measurements showed ( Figure 14 Both low- and high-dose groups of egg yolk active peptide administered by gavage increased the length of the femur and tibia, showing a certain dose-response relationship. The high-dose group achieved a femur length of 35.19 mm and a tibia length of 41.15 mm, which were 2.43% and 4.35% higher than the normal group, respectively. The effect was also better than that of the growth hormone group (femur length of 34.96 mm and tibia length of 40.52 mm).

[0081] In addition, the active peptide component of egg yolk can effectively enhance the bone strength of adolescent rats. Figure 15(A) Compared with the normal group (39.20 N), the maximum tibial load of adolescent rats in all intervention groups showed an increasing trend, with a certain dose-dependent effect. The maximum load in the growth hormone group increased to 40.83 N. The increase was more significant in the low-dose and high-dose groups of the active component, with maximum loads reaching 43.21 N and 46.65 N, respectively, representing increases of 10.23% and 18.99% compared with the normal group.

[0082] For bone mineral content ( Figure 15 B) and bone mineral density ( Figure 15 (C) Both showed a similar trend, with the active peptide components of egg yolk effectively promoting bone mineralization and bone mass accumulation. Taking bone mineral content as an example, the normal group was 0.435 mg, while the growth hormone group, low-dose group, and high-dose group increased to 0.502, 0.563, and 0.799 mg, respectively, with the high-dose group showing an 83.68% increase compared to the normal group (p<0.05).

[0083] Furthermore, the bone microstructure of rats in all intervention groups was improved. Figure 15 EH). This is manifested in a significant increase in bone volume fraction (BV / TV). Figure 15 E, the high-dose group showed a 71.22% increase compared to the normal group, and a reduction in trabecular spacing ( Figure 15 F decreased by 27.37% and 39.30% in the low-dose group and the high-dose group, respectively, and trabecular thickness remained unchanged. Figure 15 G), but the number of trabeculae increased significantly ( Figure 15 The levels of H in the low-dose and high-dose active peptide groups were increased by 40.87% and 56.73% respectively compared to the normal group. This indicates that the active peptide component can improve bone microstructure by increasing the number of trabeculae and reducing the intertrabecular gaps. Notably, the bone microstructure data after oral administration of egg yolk active peptide were superior to those in the growth hormone injection group (positive control group), demonstrating significant advantages in its application prospects.

[0084] The results of growth plate staining showed that ( Figure 16 Compared with the normal group (100%), the growth hormone group showed a 110.66% increase in growth plate thickness, while the egg yolk active peptide group exhibited a good dose-dependent effect, with the low-dose and high-dose groups increasing to 128.86% and 145.61%, respectively, both significantly better than the positive control group (P<0.05), indicating a superior effect in promoting tibial growth plate development. Serum biochemical analysis results showed ( Figure 17 The active ingredient can increase the levels of IGF-1 and BALP. In the high-dose group, IGF-1 increased to 1149.59 ng / mL and BALP increased to 10.01 ng / mL, which were 29.37% and 19.24% higher than those in the normal group, respectively, and the effect was better than that in the growth hormone group.

[0085] 10. Analysis of rat organ indices The organ index results are shown in Table 2. There were no significant differences in the relative weights of important organs such as the liver, kidneys, spleen, heart, and thymus among the groups. From a toxicological perspective, the stability of the organ index suggests that the experimental intervention did not induce pathological changes in the organs, ruling out direct toxic effects on important metabolic (liver), excretory (kidneys), immune (spleen and thymus), and circulatory (heart) systems. This indicates that the experimental intervention did not have a significant adverse effect on the development and function of rat organs, providing strong support for the safety of orally administered egg yolk active peptides. Regarding the coordination of growth and development, the organ index showed a consistent trend with changes in body weight and body length, indicating that organ development and body growth in rats maintained good synchronicity during the experimental period, and no organ-body growth imbalance caused by the intervention was observed.

[0086] Table 2. Effects of different intervention groups on organ indices in rats normal group 3.24±0.26 0.85±0.05 0.21±0.01 0.42±0.04 0.28±0.02 low-dose group 3.24±0.30 0.83±0.06 0.21±0.03 0.41±0.02 0.26±0.04 High-dose group 3.31±0.30 0.87±0.11 0.22±0.04 0.42±0.05 0.29±0.04 Growth hormone group 3.22±0.13 0.84±0.06 0.21±0.03 0.43±0.03 0.28±0.05 Note: There were no significant differences in the same indicator among different intervention groups (P>0.05). In summary, the three egg yolk active peptides SLPVGPR, AAPSWPK, and NGVWPR described in this invention not only possess bioactivity that promotes chondrocyte differentiation and mineralization, but also exhibit good digestibility, allowing them to exert their effects in vivo through the gastrointestinal tract. Molecular mechanism studies have shown that the heptapeptide SLPVGPR can simultaneously act on BMPR1A and Integrin β1 receptors on chondrocytes, jointly regulating chondrocyte differentiation by activating the BMP / Smad and Integrin / FAK signaling pathways. In vivo experiments with enriched active peptide components in adolescent rats further validated their excellent effect in promoting longitudinal bone growth. Compared with the positive control group (injected with growth hormone), high-dose active peptide components showed superior effects in increasing body length, femur and tibia length, increasing tibial growth plate thickness, and serum biomarkers IGF-1 and BALP levels, especially demonstrating a significant advantage in promoting tibial growth plate development, indicating stronger bone growth potential. Considering that the active peptide sequence provided by this invention is derived from food-derived egg yolk protein hydrolysate, it is safe and has no toxic side effects. The highly active components containing active peptides can be used as functional foods or nutritional supplements related to bone development in adolescents.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An oligopeptide derived from egg yolk that promotes chondrocyte differentiation, characterized in that, The yolk-derived oligopeptide that promotes chondrocyte differentiation is any one or more of the following amino acid sequences and their derivatives: (1)Ser-Leu-Pro-Val-Gly-Pro-Arg; (2) Ala-Ala-pSer-Trp-Pro-Lys, where pSer represents phosphorylated serine residues; (3) Asn-Gly-Val-Trp-Pro-Arg; Or a polypeptide that has more than 80% homology with the above amino acid sequence and has the activity of promoting chondrocyte differentiation.

2. The yolk-derived oligopeptide for promoting chondrocyte differentiation according to claim 1, characterized in that, The C-terminus of the egg yolk-derived oligopeptide that promotes chondrocyte differentiation contains a Pro-X structural feature, where X is a basic amino acid.

3. The yolk-derived oligopeptide for promoting chondrocyte differentiation according to claim 1, characterized in that, The method for preparing the egg yolk-derived oligopeptide that promotes chondrocyte differentiation includes obtaining it by enzymatic hydrolysis, in vitro simulated gastrointestinal digestion, and separation and purification of defatted egg yolk powder; or by solid-phase chemical synthesis.

4. The egg yolk-derived oligopeptide with chondrocyte differentiation-promoting properties according to claims 1-3, characterized in that, The oligopeptide activates the BMP / Smad signaling pathway by binding to BMPR1A and / or activates the FAK / ERK signaling pathway by acting on Integrin β1. Both signaling pathways work together to promote ATDC5 cell differentiation.

5. The application of the egg yolk-derived oligopeptide with chondrocyte differentiation-promoting properties as described in claims 1-3 in the preparation of products that promote longitudinal bone growth.

6. The application according to claim 5, characterized in that, The product that promotes longitudinal bone growth is a functional food, health food, or medicine that enhances the development of the tibial growth plate and increases the length of the femur and tibia by promoting chondrocyte differentiation.

7. The application according to claim 5, characterized in that, The product promoting longitudinal bone growth also contains any one or more of the following ingredients: functional additives and any acceptable excipients.

8. A yolk protein that promotes chondrocyte differentiation, characterized in that, The simulated digest contains the egg yolk-derived oligopeptide as described in claim 1, which promotes chondrocyte differentiation.

9. A composition, characterized in that, The composition comprises the oligopeptide of claim 1 or the mimic digestive material of claim 8.

10. A product that promotes longitudinal bone growth, characterized in that, The product comprises the oligopeptide of claim 1, the digestive mimic of claim 8, or the composition of claim 9.

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