A bone repair and growth composition and its preparation method

Abstract

The present invention relates to a composition for bone repair and growth and a preparation method thereof, belonging to the field of medical technology. The composition includes a peptide extracted from the gelatinous layer of sand jellyfish and a bamboo yellow-Grifola frondosa composite bacterial peptide; the peptide extracted from the gelatinous layer of sand jellyfish contains a product between 500Da and 2000Da obtained by sequentially hydrolyzing the gelatinous layer of sand jellyfish with lumbrokinase, serrapeptase, and transglutaminase; the bamboo yellow-Grifola frondosa composite bacterial peptide contains a product between 500Da and 2000Da obtained by co-enzymatic hydrolysis of bamboo yellow mushrooms and Grifola frondosa mushrooms with cellulase and chitinase, then co-enzymatic hydrolysis with nattokinase and serrapeptase, and finally co-enzymatic hydrolysis with aminopeptidase and transglutaminase. The present invention adopts a special method to prepare the peptide extracted from the gelatinous layer of sand jellyfish and the bamboo yellow-Grifola frondosa composite bacterial peptide, and the combined use achieves good biological activity, stability, and absorption utilization rate, overcomes the defects of existing bone repair and growth drugs, and effectively promotes bone repair and growth.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a bone repair and growth composition and a preparation method thereof. Background Art

[0002] Bone health is crucial to the human body, not only supporting its structure but also participating in numerous physiological functions. However, due to a variety of factors, bone injuries and pathologies, such as fractures, osteoporosis, and bone tumors, are very common. These problems seriously impact patients' quality of life and can even be life-threatening. Therefore, the research and development of technologies and products that promote bone repair and growth has always been a key focus and hot topic in the fields of medicine and biomaterials.

[0003] Fractures are common bone injuries, and their healing is a complex physiological process, which is divided into the hematoma-inflammation-organization phase, the original callus formation phase, and the bone plate formation and plasticity phase. However, the self-repair ability of bones is limited and slow, which often leads to delayed healing or even non-healing of fractures in patients, causing bone nonunion, which brings great physical and mental pain to patients. Especially for elderly fracture patients, due to the decline in bone repair ability, it is difficult to quickly reduce swelling, relieve pain, and form calluses at the fracture site by relying solely on early rehabilitation exercises after surgery. The swelling and pain caused by rehabilitation treatment and the long-term non-healing of calluses make it difficult for many patients to adhere to rehabilitation treatment, further delaying the healing speed of the fracture site. Osteoporosis is a common disease that affects the health of the people. The repair of bone defects caused by complications such as fractures, tumors, trauma, and inflammation faces huge challenges. The disease impairs the body's osteogenesis / angiogenesis and bone remodeling capabilities, resulting in limited efficacy of traditional bone repair materials.

[0004] Currently, there are a variety of products and methods on the market to improve bone health, such as calcium supplements, vitamin D, and specific medications. However, these methods have limitations. Long-term use may cause side effects, such as gastrointestinal discomfort and an increased risk of stone formation in calcium supplements; excessive vitamin D intake can be toxic; and long-term use of some medications can affect liver and kidney function. Furthermore, these ingredients have low absorption rates, and their absorption varies greatly between individuals, limiting their applicability to specific populations. For example, those with certain medical conditions or specific physical conditions may not be able to use these products or experience poor results. To address these issues, some studies have utilized tissue engineering techniques to combine mesenchymal stem cells with scaffold materials and induce osteoblastic differentiation. However, mesenchymal stem cells are susceptible to local microenvironmental regulation, and inflammatory cytokines can inhibit their osteoblast differentiation, impairing bone regeneration and repair. Furthermore, stem cell proliferation carries the risk of tumorigenesis. Alternatively, percutaneous injection of bone growth factors is less invasive and has a wide range of indications. However, the extraction and purification process is complex, and bone growth factors are prone to loss of efficacy during purification, storage, and use. Their effectiveness is short-lived, requiring continuous injections for effectiveness. As research deepens on the mechanisms of bone repair and growth, several bioactive substances and materials have been identified that have a positive effect on promoting bone repair and growth. Such as milk calcium, colostrum alkaline protein, etc., but they are also affected by absorption and preparation costs, and their utilization rate is not high.

[0005] In summary, the existing methods and products for promoting bone repair and growth have many shortcomings. Therefore, there is a need to continuously develop safe, efficient and widely applicable bone repair and growth drugs. Summary of the Invention

[0006] To address the shortcomings of existing bone repair and growth drugs, such as poor safety, absorption rate, and stability, which affect the effectiveness of bone repair and growth, the present invention provides a bone repair and growth composition and its preparation method. A special method is used to prepare peptides extracted from the gelatinous layer of sand jellyfish and a bamboo yellow-Grifola frondosa composite fungal peptide. Their combined use achieves excellent biological activity, stability, and absorption rate, overcoming the shortcomings of existing bone repair and growth drugs and effectively promoting bone repair and growth. The specific technical solution is as follows:

[0007] A composition for bone repair and growth, comprising a peptide extracted from the gelatinous layer of a sand jellyfish and a bamboo yellow-Grifola frondosa composite peptide; the peptide extracted from the gelatinous layer of a sand jellyfish comprises a product with a Da of 500 to 2000 obtained by sequentially reacting the gelatinous layer of the sand jellyfish with lumbrokinase, serrapeptase, and transglutaminase; the bamboo yellow-Grifola frondosa composite peptide comprises a product with a Da of 500 to 2000 obtained by first co-enzymatically hydrolyzing bamboo yellow mushrooms and Grifola frondosa mushrooms with cellulase and chitinase, then co-enzymatically hydrolyzing them with nattokinase and serrapeptase, and finally co-reacting with aminopeptidase and transglutaminase.

[0008] Furthermore, the preparation method of the peptide extracted from the gelatinous layer of the sand jellyfish includes the following steps: taking the dehydrated gelatinous layer of the sand jellyfish and crushing it, adding a phosphate buffer solution at a material-liquid mass ratio of 1:(10-15), ultrasonically treating it to obtain an extract; adding lumbrokinase to the extract, enzymatically hydrolyzing it at 37°C to 40°C for 2h to 2.5h, heat-inactivating the enzyme, cooling it to 45°C to 50°C and keeping it constant, adding serrapeptase, enzymatically hydrolyzing it for 1.5h to 2h, heat-inactivating the enzyme, cooling it to 37°C to 40°C and keeping it constant, adding glutamine transaminase, reacting it for 1h to 1.5h, and heat-inactivating the enzyme; filtering it through a mesh, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying it to obtain the peptide extracted from the gelatinous layer of the sand jellyfish.

[0009] In the preparation method of peptides extracted from the gelatinous layer of sand jellyfish, the pH value of the phosphate buffer solution is 7.0-7.4; the power of the ultrasound is 200W-300W, the temperature of the ultrasound is 30°C-35°C, and the time of the ultrasound is 30min-45min.

[0010] In the preparation method of peptides extracted from the gelatinous layer of sand jellyfish, the added amount of lumbrokinase is 0.5% to 1.0% of the mass of the gelatinous layer of sand jellyfish; the added amount of serrapeptase is 0.8% to 1.5% of the mass of the gelatinous layer of sand jellyfish; and the added amount of glutamine transaminase is 1.0% to 1.5% of the mass of the gelatinous layer of sand jellyfish.

[0011] In the preparation method of peptide extracted from the gelatinous layer of sand jellyfish, the temperature of heat-inactivating enzyme is 85-90° C., the time of heat-inactivating enzyme is 10-15 minutes, and the mesh number of the sieve is 250-325 meshes.

[0012] Furthermore, the preparation method of the bamboo yellow-Grifola frondosa composite fungus peptide comprises the following steps: mixing and crushing bamboo yellow mushroom and Grifola frondosa mushroom in a mass ratio of (3-4): (1-2) to obtain fungus powder; adding acetic acid-sodium acetate buffer solution in a material-liquid mass ratio of 1: (10-15), adding cellulase and chitinase, ultrasonic enzymolysis, heat-inactivating enzyme, cooling to 45°C-50°C and maintaining constant temperature, adjusting the pH value to 7.0-7.5, adding nattokinase and serrapeptase, enzymolysis for 2h-3h, heat-inactivating enzyme, cooling to 40°C-45°C, adjusting the pH value to 7.5-8.0, adding aminopeptidase and glutamine transaminase, reacting for 1.5h-2h, heat-inactivating enzyme, filtering through a mesh, ultrafiltration of the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying to obtain the bamboo yellow-Grifola frondosa composite fungus peptide.

[0013] In the preparation method of the bamboo yellow-Grifola frondosa composite peptide, the pH value of the acetic acid-sodium acetate buffer solution is 5.0-5.5; the added amount of the cellulase is 1.0%-1.5% of the mass of the bacterial powder; and the added amount of the chitinase is 0.3%-0.6% of the mass of the bacterial powder.

[0014] In the preparation method of the bamboo yellow-Grifola frondosa composite peptide, the ultrasonic enzymatic hydrolysis has an ultrasonic power of 250W to 350W, a temperature of 40°C to 45°C, and a time of 3h to 4h.

[0015] In the preparation method of the bamboo yellow-Grifola frondosa composite bacterial peptide, the added amount of the nattokinase and serrapeptase is 0.5% to 1.0% of the mass of the bacterial powder; the added amount of the aminopeptidase is 0.5% to 0.8% of the mass of the bacterial powder; and the added amount of the glutamine transaminase is 0.5% to 1.0% of the mass of the bacterial powder.

[0016] In the preparation method of bamboo yellow-Grifola frondosa composite peptide, the temperature of the heat inactivation enzyme is 85° C. to 90° C., the time of the heat inactivation enzyme is 10 min to 15 min, and the mesh number of the sieve is 250 mesh to 325 mesh.

[0017] The preparation method of the above-mentioned bone repair and growth composition comprises the following steps: mixing peptides extracted from the gelatinous layer of sand jellyfish: bamboo yellow-Grifola frondosa composite fungus peptide = (15-20): (8-10) according to the mass ratio to obtain the composition.

[0018] The above-mentioned bone repair and growth composition is mixed with drugs and / or drug excipients, and after sterilization, an oral preparation or an injection is prepared for bone repair and growth.

[0019] The present invention provides a bone repair and growth composition and a preparation method thereof, which have the following beneficial effects:

[0020] The gelatinous layer of sand jellyfish is rich in collagen, mucopolysaccharides, and bioactive peptides. Its three-dimensional fibrous network structure is similar to that of the bone matrix, providing a biomimetic structural foundation for bone repair. This invention employs a step-by-step enzymatic hydrolysis strategy: pretreatment with lumbrokinase opens collagen fiber bundles and specifically cleaves them through fibrinolysis, exposing internal active sites; secondary enzymatic hydrolysis with serrapeptase specifically cleaves the carboxyl termini of hydrophobic amino acids (including leucine and phenylalanine), releasing chemotactic oligopeptide fragments; and modification with transglutaminase catalyzes the crosslinking of γ-glutamyl groups with the ε-amino group of lysine, forming a stable three-dimensional network structure and extending the half-life of the active peptides. Peptides between 500 and 2000 Da exhibit optimal transmembrane transport efficiency and can effectively activate signaling pathways, promoting osteoblast differentiation.

[0021] Second, the synergistic effect of the bamboo yellow-Grifola frondosa composite mycopeptide: cellulase and chitinase jointly break down the mycelial cell wall, synergistically decomposing the mycelial cell wall and increasing the release rate of intracellular active substances. A combination of nattokinase and serrapeptase: the former specifically cleaves the carboxyl termini of lysine and arginine, while the latter acts on hydrophobic sites, producing short peptides with pro-angiogenic activity. Aminopeptidase modification removes N-terminal hydrophobic amino acid residues to improve water solubility and bioavailability. Glutamine transaminase modification catalyzes the cross-linking of γ-glutamyl groups with lysine ε-amino groups, forming a stable three-dimensional network structure and extending the half-life of the active peptide. The resulting product can effectively regulate the balance between osteoclasts and osteoblasts, promote the absorption of components such as calcium ions, and enhance calcium influx-driven osteoblast activation; it can effectively regulate cell proliferation, differentiation, and extracellular matrix synthesis, promoting fracture healing.

[0022] 3. Synergistic mechanism of rationally proportioned combination: Sand jellyfish peptide activates FAK / PI3K / Akt pathways through integrin receptors, while mushroom peptide activates cAMP / PKA pathways through GPCRs. Different pathways synergistically promote the expression of Runx2 transcription factor. Some peptides in mushroom peptide reverse the inhibitory effect of inflammatory microenvironment on osteogenic differentiation of mesenchymal stem cells. Sand jellyfish peptide provides sites (through the carboxyl group of Glu / Gln residues and Ca²² +Chelation), the ingredients in mushroom peptides inhibit osteoclast acid secretion, regulate immunity, fight inflammation, and promote the absorption of calcium ions and other ingredients. Through the synergistic effect of various ingredients, they achieve excellent biological activity, stability, and absorption utilization, overcoming the shortcomings of existing bone repair and growth drugs and promoting bone repair and growth. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to specific implementation cases, but the present invention is not limited to these embodiments.

[0024] Example 1: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the gelatinous layer of the sand jellyfish: the bamboo yellow-Grifola frondosa complex peptide = 15:8.

[0025] The preparation of peptides extracted from the gelatinous layer of sand jellyfish includes: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution with a pH value of 7.0 at a material-liquid mass ratio of 1:10, and ultrasonically treating it at an ultrasonic power of 200 W and a temperature of 30°C for 30 minutes to obtain an extract; adding 0.5% lumbrokinase by weight of the gelatinous layer of sand jellyfish to the extract, enzymatically hydrolyzing it at 37°C for 2 hours, heat-inactivating the enzyme at 85°C for 10 minutes, cooling it to a constant temperature of 45°C, adding 0.8% serrapeptidase by weight of the gelatinous layer of sand jellyfish, enzymatically hydrolyzing it for 1.5 hours, heat-inactivating the enzyme at 85°C for 10 minutes, cooling it to a constant temperature of 37°C, adding 1.0% glutamine transaminase by weight of the gelatinous layer of sand jellyfish, reacting it for 1 hour, and heat-inactivating the enzyme at 85°C for 10 minutes; filtering it through a 250-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying it.

[0026] The preparation of the bamboo yellow-Grifola frondosa composite peptide comprises the following steps: mixing and crushing bamboo yellow mushrooms and Grifola frondosa mushrooms in a mass ratio of 3:1 to obtain fungus powder; adding an acetic acid-sodium acetate buffer solution with a pH value of 5.0 in a material-liquid mass ratio of 1:10, adding 1.0% cellulase by mass of the fungus powder and 0.3% chitinase by mass of the fungus powder, performing ultrasonic enzymolysis at an ultrasonic power of 200 W and a temperature of 40° C. for 3 h, performing heat inactivation at 85° C. for 10 min, cooling to a constant temperature of 45° C., and adjusting the pH. The pH value was adjusted to 7.0, 0.5% nattokinase by weight of the bacterial powder and 0.5% serrapeptase by weight of the bacterial powder were added, the enzyme was hydrolyzed for 2 h, the enzyme was inactivated by heat at 85 ° C for 10 min, the temperature was lowered to 40 ° C, 0.5% aminopeptidase by weight of the bacterial powder and 0.5% glutamine transaminase by weight of the bacterial powder were added, the reaction was continued for 1.5 h, the enzyme was inactivated by heat at 85 ° C for 10 min, the solution was filtered through a 250-mesh sieve, the filtrate was ultrafiltered through an ultrafiltration membrane, the components between 500 Da and 2000 Da were collected, and the components were freeze-dried.

[0027] Example 2: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the gelatinous layer of the sand jellyfish: the bamboo yellow-Grifola frondosa complex peptide = 15:10.

[0028] The preparation of peptides extracted from the gelatinous layer of sand jellyfish includes: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution with a pH value of 7.4 at a material-liquid mass ratio of 1:10, and ultrasonically treating it at an ultrasonic power of 200 W and a temperature of 35°C for 30 minutes to obtain an extract; adding 1.0% lumbrokinase by weight of the gelatinous layer of sand jellyfish to the extract, performing enzymatic hydrolysis at 37°C for 2.5 hours, heat-inactivating the enzyme at 85°C for 15 minutes, cooling to a constant temperature of 45°C, adding 1.5% serrapeptidase by weight of the gelatinous layer of sand jellyfish, performing enzymatic hydrolysis for 1.5 hours, heat-inactivating the enzyme at 90°C for 10 minutes, cooling to a constant temperature of 40°C, adding 1.0% glutamine transaminase by weight of the gelatinous layer of sand jellyfish, reacting for 1.5 hours, and heat-inactivating the enzyme at 85°C for 15 minutes; filtering through a 250-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying.

[0029] The preparation of the bamboo yellow-Grifola frondosa composite peptide comprises the following steps: mixing and crushing bamboo yellow mushrooms and Grifola frondosa mushrooms at a mass ratio of 4:1 to obtain fungus powder; adding an acetic acid-sodium acetate buffer solution with a pH value of 5.0 at a material-liquid mass ratio of 1:15, adding 1.5% of cellulase by mass of the fungus powder and 0.3% of chitinase by mass of the fungus powder, performing ultrasonic enzymolysis at an ultrasonic power of 250W and a temperature of 40°C for 4 hours, performing heat inactivation at 85°C for 15 minutes, cooling to a constant temperature of 45°C, and adjusting the pH. The value was raised to 7.5, 0.5% of nattokinase by weight of bacterial powder and 1.0% of serrapeptase by weight of bacterial powder were added, enzymatic hydrolysis was carried out for 2 hours, enzyme was inactivated by heat at 90°C for 10 minutes, the temperature was lowered to 45°C, 0.5% of aminopeptidase by weight of bacterial powder and 1.0% of glutamine transaminase by weight of bacterial powder were added, reaction was carried out for 1.5 hours, enzyme was inactivated by heat at 90°C for 10 minutes, filtered through a 325-mesh sieve, the filtrate was ultrafiltered through an ultrafiltration membrane, components between 500Da and 2000Da were collected, and freeze-dried.

[0030] Example 3: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the gelatinous layer of the sand jellyfish: the bamboo yellow-Grifola frondosa complex peptide = 17.5:9.

[0031] The preparation of peptides extracted from the gelatinous layer of sand jellyfish includes: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution with a pH value of 7.2 at a material-liquid mass ratio of 1:12, and ultrasonically treating it at an ultrasonic power of 250W and a temperature of 32°C for 40 minutes to obtain an extract; adding 0.8% lumbrokinase by weight of the gelatinous layer of sand jellyfish to the extract, enzymatically hydrolyzing it at 38°C for 2 hours, heat-inactivating the enzyme at 88°C for 12 minutes, cooling it to a constant temperature of 48°C, adding 1.2% serrapeptidase by weight of the gelatinous layer of sand jellyfish, enzymatically hydrolyzing it for 1.5 hours, heat-inactivating the enzyme at 88°C for 12 minutes, cooling it to a constant temperature of 38°C, adding 1.2% glutamine transaminase by weight of the gelatinous layer of sand jellyfish, reacting it for 1 hour, and heat-inactivating the enzyme at 88°C for 12 minutes; filtering it through a 325-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying it.

[0032] The preparation of bamboo yellow-Grifola frondosa composite peptide comprises the following steps: mixing and crushing bamboo yellow mushroom and Grifola frondosa mushroom in a mass ratio of 3.5:1.5 to obtain fungus powder; adding acetic acid-sodium acetate buffer solution with a pH value of 5.2 in a material-liquid mass ratio of 1:12, adding 1.2% cellulase by mass of the fungus powder and 0.5% chitinase by mass of the fungus powder, ultrasonically enzymolyzing at an ultrasonic power of 220W and a temperature of 42°C for 3.5h, heat-inactivating the enzyme at 88°C for 12min, cooling to a constant temperature of 48°C, and adjusting The pH value was raised to 7.2, 0.8% of nattokinase by weight of the bacterial powder and 0.8% of serrapeptase by weight of the bacterial powder were added, and the enzymatic hydrolysis was carried out for 2.5 hours. The enzyme was heat-inactivated at 88°C for 12 minutes. The temperature was lowered to 43°C, and 0.6% of aminopeptidase by weight of the bacterial powder and 0.7% of glutamine transaminase by weight of the bacterial powder were added. The reaction was carried out for 1.5 hours. The enzyme was heat-inactivated at 88°C for 12 minutes. The mixture was filtered through a 325-mesh sieve, and the filtrate was ultrafiltered through an ultrafiltration membrane to collect the components between 500Da and 2000Da, and freeze-dried.

[0033] Example 4: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the gelatinous layer of the sand jellyfish: the bamboo yellow-Grifola frondosa complex peptide = 20:9.

[0034] The preparation of peptides extracted from the gelatinous layer of sand jellyfish includes: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution with a pH value of 7.4 at a material-liquid mass ratio of 1:15, and ultrasonically treating it at an ultrasonic power of 300 W and a temperature of 35°C for 45 minutes to obtain an extract; adding 1.0% lumbrokinase by weight of the gelatinous layer of sand jellyfish to the extract, performing enzymatic hydrolysis at 40°C for 2.5 hours, heat-inactivating the enzyme at 90°C for 15 minutes, cooling to 50°C and maintaining the constant temperature, adding 1.5% serrapeptidase by weight of the gelatinous layer of sand jellyfish, performing enzymatic hydrolysis for 2 hours, heat-inactivating the enzyme at 90°C for 15 minutes, cooling to 40°C and maintaining the constant temperature, adding 1.5% glutamine transaminase by weight of the gelatinous layer of sand jellyfish, reacting for 1.5 hours, and heat-inactivating the enzyme at 90°C for 15 minutes; filtering through a 325-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying.

[0035] The preparation of bamboo yellow-Grifola frondosa composite peptide comprises the following steps: mixing and crushing bamboo yellow mushrooms and Grifola frondosa mushrooms in a mass ratio of 4:1.5 to obtain fungus powder; adding acetic acid-sodium acetate buffer solution with a pH value of 5.5 in a material-liquid mass ratio of 1:15, adding 1.5% cellulase by mass of the fungus powder and 0.6% chitinase by mass of the fungus powder, ultrasonically hydrolyzing at an ultrasonic power of 250W and a temperature of 45°C for 4h, heat-killing the enzyme at 90°C for 15min, cooling to a constant temperature of 50°C, and adjusting The pH value was raised to 7.5, 1.0% nattokinase by weight of the bacterial powder and 1.0% serrapeptase by weight of the bacterial powder were added, and enzymatic hydrolysis was carried out for 3 hours. The enzyme was heat-inactivated at 90°C for 15 minutes. The temperature was lowered to 45°C, and 0.8% aminopeptidase by weight of the bacterial powder and 1.0% glutamine transaminase by weight of the bacterial powder were added. The reaction was carried out for 2 hours. The enzyme was heat-inactivated at 90°C for 15 minutes. The mixture was filtered through a 325-mesh sieve, and the filtrate was ultrafiltered through an ultrafiltration membrane to collect components between 500Da and 2000Da, and freeze-dried.

[0036] Example 5: The mixing mass ratio of the composition for bone repair and growth is the peptide extracted from the gelatinous layer of the sand jellyfish: the bamboo yellow-Grifola frondosa complex peptide = 20:8.

[0037] The preparation method of peptides extracted from the gelatinous layer of sand jellyfish includes: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution with a pH value of 7.0 at a material-liquid mass ratio of 1:15, and ultrasonically treating it at an ultrasonic power of 300 W and a temperature of 30°C for 45 minutes to obtain an extract; adding 0.5% lumbrokinase by weight of the gelatinous layer of sand jellyfish to the extract, performing enzymatic hydrolysis at 40°C for 2 hours, heat-inactivating the enzyme at 90°C for 10 minutes, cooling to a constant temperature of 50°C, adding 0.8% serrapeptidase by weight of the gelatinous layer of sand jellyfish, performing enzymatic hydrolysis for 2 hours, heat-inactivating the enzyme at 85°C for 15 minutes, cooling to a constant temperature of 37°C, adding 1.5% glutamine transaminase by weight of the gelatinous layer of sand jellyfish, reacting for 1 hour, and heat-inactivating the enzyme at 90°C for 10 minutes; filtering through a 325-mesh sieve, ultrafiltering the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying.

[0038] The preparation of the bamboo yellow-Grifola frondosa composite peptide comprises the following steps: mixing and crushing bamboo yellow mushrooms and grifola frondosa mushrooms in a mass ratio of 3:2 to obtain fungus powder; adding an acetic acid-sodium acetate buffer solution with a pH value of 5.5 in a material-liquid mass ratio of 1:10, adding 1.0% cellulase by mass of the fungus powder and 0.6% chitinase by mass of the fungus powder, performing ultrasonic enzymolysis at an ultrasonic power of 200 W and a temperature of 45° C. for 3 h, performing heat inactivation at 90° C. for 10 min, cooling to a constant temperature of 50° C., and adjusting the pH value. The H value was raised to 7.0, 1.0% of nattokinase by weight of the bacterial powder and 0.5% of serrapeptase by weight of the bacterial powder were added, and the enzymatic hydrolysis was carried out for 3 hours. The enzyme was heat-inactivated at 85°C for 15 minutes. The temperature was lowered to 40°C, and 0.8% of aminopeptidase by weight of the bacterial powder and 0.5% of glutamine transaminase by weight of the bacterial powder were added. The reaction was carried out for 2 hours. The enzyme was heat-inactivated at 85°C for 15 minutes. The mixture was filtered through a 250-mesh sieve. The filtrate was ultrafiltered through an ultrafiltration membrane to collect components between 500Da and 2000Da, and freeze-dried.

[0039] The bone repair and growth composition prepared in the above embodiments is mixed with drugs and / or pharmaceutical excipients, and after sterilization, an oral preparation or an injection is prepared for bone repair and growth.

[0040] In the above embodiments, the enzyme activity of lumbrokinase is 20,000 U / g, which is sourced from Xi'an Muguo Biotechnology Co., Ltd. The enzyme activity of serrapeptase is 200,000 U / g, which is sourced from Shenzhen Hengsheng Biotechnology Co., Ltd. The enzyme activity of transglutaminase is 100,000 U / g, which is sourced from Shenzhen Hengsheng Biotechnology Co., Ltd. The enzyme activity of cellulase is 100,000 U / g, which is sourced from Shandong Yaqiu Biotechnology Co., Ltd. The enzyme activity of chitinase is 100,000 U / g, which is sourced from Guangdong Yuanfeng Chemical Reagent Co., Ltd. The enzyme activity of nattokinase is 40,000 U / g, which is sourced from Shaanxi Yunhe Biotechnology Co., Ltd. The enzyme activity of aminopeptidase is 20,000 U / g, which is sourced from Shandong Xinxiong Biotechnology Co., Ltd.

[0041] Comparative Example 1

[0042] The bone repair and growth composition has a mixing ratio of 8:15 of peptide extracted from the gelatinous layer of sand jellyfish to bamboo yellow-Grifola frondosa complex peptide. Other methods and parameters are the same as those in Example 1.

[0043] Comparative Example 2

[0044] The bone repair and growth composition has a mixing ratio of peptide extracted from the gelatinous layer of sand jellyfish to bamboo yellow-Grifola frondosa complex peptide = 15:4. Other methods and parameters are the same as those in Example 1.

[0045] Comparative Example 3

[0046] The gelatinous layer of the sand jellyfish was replaced by the gelatinous layer of the surface jellyfish; other methods and parameters were the same as those in Example 1.

[0047] Comparative Example 4

[0048] In the preparation of the composite peptide of bamboo yellow fungus and maitake mushroom, bamboo yellow fungus and maitake mushroom are mixed and crushed in a mass ratio of 1:3; other methods and parameters are the same as those in Example 1.

[0049] Comparative Example 5

[0050] In the preparation of peptides extracted from the gelatinous layer of sand jellyfish: lumbrokinase was replaced by nattokinase; other methods and parameters were the same as in Example 1.

[0051] Comparative Example 6

[0052] In the preparation of peptides extracted from the gelatinous layer of sand jellyfish: serrapeptase was replaced by nattokinase; other methods and parameters were the same as in Example 1.

[0053] Comparative Example 7

[0054] In the preparation of peptides extracted from the gelatinous layer of sand jellyfish: no transglutaminase was added to the reaction; other methods and parameters were the same as in Example 1.

[0055] Comparative Example 8

[0056] In the preparation of the bamboo yellow-Grifola frondosa composite peptide: the reaction was carried out without adding glutamine transaminase; other methods and parameters were the same as in Example 1.

[0057] Comparative Example 9

[0058] In the preparation of the bamboo yellow-Grifola frondosa composite peptide: the reaction was carried out without adding aminopeptidase; other methods and parameters were the same as in Example 1.

[0059] Comparative Example 10

[0060] In the preparation of the bamboo yellow-Grifola frondosa composite peptide: aminopeptidase and transglutaminase were not used simultaneously for the reaction; other methods and parameters were the same as those in Example 1.

[0061] Comparative Example 11

[0062] In the preparation of the bamboo yellow-Grifola frondosa composite peptide, bean kinase and serrapeptase were replaced by bromelain (the enzymatic activity of bromelain was 200,000 U / g); other methods and parameters were the same as those in Example 1.

[0063] 1. Safety testing:

[0064] 1. Acute Toxicity Test: Animal Model: ICR mice (half male and half female, 10 mice per group); Dosage: Single intraperitoneal injection of 5 mg / kg, 10 mg / kg, or 20 mg / kg; Blank control group: Injection of an equal volume of normal saline. Observe and monitor for signs of toxicity. 2. Subacute Toxicity Test: Animal Model: SD rats (half male and half female, 10 mice per group); Daily intramuscular injection of 4 mg / kg for 28 consecutive days; Observe and monitor for signs of toxicity. 3. Hypersensitivity Test (Guinea Pig Maximization Test): Guinea pigs (male, 6 mice per group) were subcutaneously injected with the composition (5 mg / kg) on days 0 and 7, and intra-auricularly injected with the composition (1 mg / kg) on day 21. Observe and monitor for signs of allergic reactions. 4. Genotoxicity Test (Ames Test): Strains: TA97, TA98, TA100, TA1535 (+ / - S9 metabolic activation); Dose: 1.0 mg / dish; Criterion: Positive if the number of revertant colonies is ≥2 times that of the negative control. The above safety test results of the compositions of the embodiments and comparative examples were all non-toxic, non-allergenic, and negative in the Ames test (non-mutagenic).

[0065] 2. Mechanism research:

[0066] 1. Signaling pathway activation experiment:

[0067] 1.1 FAK / PI3K / Akt pathway detection (effect of sand jellyfish peptide):

[0068] Detection method: Western Blot.

[0069] step:

[0070] Cell model: Mouse mesenchymal stem cells (C3H10T1 / 2).

[0071] Group treatment: control group: serum-free culture medium; sand jellyfish peptide group (150 μg / mL); combined group (sand jellyfish peptide 150 μg / mL + mushroom peptide 80 μg / mL); inhibitor control group: sand jellyfish peptide 150 μg / mL + PI3K inhibitor LY294002 (10 μM).

[0072] Processing time: 60min.

[0073] Protein extraction: Total protein was extracted using RIPA lysis buffer.

[0074] Detection target:

[0075] Phosphorylated FAK (p-FAK Tyr397).

[0076] Phosphorylates PI3K p85 (Tyr458).

[0077] Phosphorylates Akt (Ser473).

[0078] Antibody dilution ratio: 1:1000 (phosphoantibody), 1:2000 (total protein antibody).

[0079] 1.2 cAMP / PKA pathway detection (mushroom peptide effect):

[0080] Detection method: ELISA+Western Blot.

[0081] step:

[0082] Cell model: Mouse mesenchymal stem cells (C3H10T1 / 2).

[0083] Group treatment: control group: serum-free medium; mushroom peptide group (80 μg / mL); combined group (sand jellyfish peptide 150 μg / mL + mushroom peptide 80 μg / mL); inhibitor control group: mushroom peptide 80 μg / mL + PKA inhibitor H89 (10 μM);

[0084] cAMP detection: After 30 minutes of treatment, the intracellular cAMP level was detected using an ELISA kit.

[0085] PKA detection: Western blot detection of phosphorylated PKA substrate (CREB Ser133).

[0086] 2 Runx2 transcriptional regulation experiment:

[0087] 2.1 Runx2 protein and mRNA expression

[0088] Detection method: qRT-PCR+Western Blot.

[0089] step:

[0090] Cells and treatment: Mouse mesenchymal stem cells (C3H10T1 / 2), treated for 48 hours.

[0091] Group treatment: control group: serum-free medium; sand jellyfish peptide group (150 μg / mL); mushroom peptide group (80 μg / mL); combined group (sand jellyfish peptide 150 μg / mL + mushroom peptide 80 μg / mL);

[0092] qRT-PCR:

[0093] Primer: Runx2

[0094] F:5'-CCGCACCGACAACCGCACCAT-3', R:5'-CGCTCCGGCCCACAAATCTC-3'.

[0095] Internal reference: GAPDH.

[0096] Western Blot: anti-Runx2 antibody (1:1000).

[0097] 2.2 Runx2 transcriptional activity

[0098] Detection method: dual-luciferase reporter system.

[0099] step:

[0100] Plasmid transfection: Cells were transfected with a Runx2 response element (OSE2)-driven luciferase reporter plasmid.

[0101] Treatment: Grouping is the same as before, and treatment is carried out for 24 hours.

[0102] Assay: Luciferase activity (Firefly / Renilla ratio).

[0103] 3. Regulation of inflammatory microenvironment (effects of mushroom peptides):

[0104] 3.1 Inhibition of inflammatory factor secretion:

[0105] Detection method: LPS-induced inflammation model + ELISA.

[0106] step:

[0107] Cell model: Macrophages (RAW264.7) were co-cultured with hBMSCs.

[0108] Inflammation induction: Macrophages were stimulated with LPS (1 μg / mL) for 24 h.

[0109] Treatment groups: LPS group; LPS + mushroom peptide group (80 μg / mL); LPS + combined peptide group (sand jellyfish peptide 150 μg / mL + mushroom peptide 80 μg / mL).

[0110] Detection: ELISA was used to detect the levels of TNF-α and IL-6 in the supernatant.

[0111] 3.2 Osteogenic differentiation rescue experiment:

[0112] Detection method: ALP staining + qRT-PCR.

[0113] step:

[0114] Co-culture system: LPS-activated macrophages were co-cultured with hBMSCs in Transwell.

[0115] Treatment: A peptide combination (150 μg / mL of sand jellyfish peptide and 80 μg / mL of mushroom peptide) was added to the co-culture system and osteogenic induction was performed for 7 days.

[0116] Detection:

[0117] ALP activity (BCIP / NBT staining).

[0118] Osteogenic gene (ALP, OCN) mRNA expression.

[0119] 4. Calcium metabolism regulation:

[0120] 4.1 Calcium ion chelating ability (action of jellyfish peptide):

[0121] Detection method: Pyrene red colorimetric method (Calcium Binding Assay).

[0122] step:

[0123] Sample: Sand jellyfish peptide solution (1 mg / mL).

[0124] Control: BSA control group (1 mg / mL).

[0125] Reaction: Add CaCl2 (final concentration 2 mM) and incubate at 37°C for 30 min.

[0126] Detection: Add pyrene red (50 μM) and measure the absorbance at OD540 nm.

[0127] 4.2 Promotion of mineralized nodule formation:

[0128] Detection method: Alizarin red staining.

[0129] step:

[0130] Cells and treatments: hBMSCs were induced to osteogenetically for 21 days and divided into three groups: BSA control group (1 mg / mL), sand jellyfish peptide group (1 mg / mL), and combination group (peptide combination 15:8; 1 mg / mL).

[0131] Staining: Fix with 4% PFA and stain with 2% Alizarin Red (pH 4.2) for 30 min.

[0132] Quantification: 10% CPC dissolved dye, measured OD562nm.

[0133] Table 1 Test results (5 parallel samples)

[0134]

[0135] Key Results Description:

[0136] 1. Pathway Synergy: FAK / PI3K / Akt Pathway: p-Akt expression in the combination group was 4-fold higher than in the control group (3-fold higher with jellyfish peptide alone), and LY294002 completely inhibited activation. cAMP / PKA Pathway: cAMP levels in the combination group were 3.8-fold higher than in the control group (3-fold higher with mushroom peptide alone), with p-CREB also upregulated.

[0137] 2. Runx2 cascade amplification: Combined treatment increased Runx2 transcriptional activity (luciferase) by 4 times and mRNA by more than 3 times, which was significantly higher than that of the single peptide group (p<0.05).

[0138] 3. Inflammation reversal ability: Mushroom peptides reduced LPS-induced TNF-α secretion by 60% (*p<0.01), and osteogenic differentiation markers (ALP, OCN) returned to 80% of normal levels under combined treatment.

[0139] 4. Dual pathways of calcium metabolism: Ca of sand jellyfish peptide 2+ The chelation rate reached 65% (*p<0.01 vs BSA); the amount of mineralized nodules formed in the combined group increased by 250%, confirming the synergistic effect of promoting bone mineralization.

[0140] Notes: 1. Data are mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA and Tukey's test (*p < 0.05, **p < 0.01, ***p < 0.001). 2. NS: No significant difference. ↑ / ↓: Increase / decrease by fold or percentage compared to the control group. 3. The inhibitor control group verifies pathway specificity (e.g., LY294002 does not increase p-Akt after PI3K inhibition). 4. In the above tests, the combination group used a 15:8 mass ratio of sand jellyfish peptide to mushroom peptide. All the above-mentioned drug components were prepared using the method in Example 1.

[0141] 3. Animal Experimental Test on Bone Repair and Growth:

[0142] Experimental animals: 102 healthy SD rats (half male and half female) aged 8 weeks, weighing 200g-260g, were selected and fed adaptively for 1 week before the experiment.

[0143] Fracture model construction: Rats were anesthetized with 3 wt % sodium pentobarbital (30 mg / kg) intraperitoneally, and a closed fracture model was created in the right forelimb humerus.

[0144] Drug Administration: Rats were randomly divided into 17 groups (1 blank control group, 5 experimental groups according to the example, and 11 comparative groups), with 6 rats per group (half male and half female). The experimental groups received intramuscular injections of the corresponding composition solution (5 mg / mL) around the fracture site at a dose of 0.2 mL per injection. The blank control group received an equal volume of PBS buffer. Subsequent injections were administered every two days. Rats were maintained on a regular basis, and their diets were supplemented with nutrients and trace elements, including vitamin C and zinc gluconate.

[0145] Experimental Testing: Bone density was measured at the humeral fracture site of the rats' right forelimbs using a bone densitometer on rearing days 0, 14, 28, and 42. Fracture healing was assessed by radiographic examination of the right forelimbs on rearing days 15, 18, 21, 24, 27, 30, 33, 36, 39, and 42. Fracture healing was considered complete when the fracture line was blurred and a continuous callus crossed the fracture line. The fracture healing time was recorded. The results are shown in Table 2 below.

[0146] Table 2 Bone repair growth test results (average value)

[0147]

[0148] From the above results, it can be seen that the combination of Examples 1 to 5 has a better effect in promoting bone repair and growth, and effectively shortens the fracture healing time.

[0149] In Comparative Example 1, the relatively low content of peptides extracted from the gelatinous layer of sand jellyfish prevented optimal synergy with the bamboo-growing fern complex peptides. Bone repair is a complex process involving multiple cellular activities and signaling pathways. The two types of biological peptides act on different aspects of the process (the peptides extracted from the gelatinous layer of sand jellyfish promote osteoblast proliferation, while the bamboo-growing fern complex peptides regulate bone extracellular matrix synthesis). When the amount of peptides extracted from the gelatinous layer of sand jellyfish is insufficient, osteoblast proliferation is restricted, affecting the formation of new bone tissue, resulting in slow bone density growth and prolonged fracture healing.

[0150] Comparative Example 2 contained too little bamboo yellow-Grifola frondosa peptide, which provided insufficient anti-inflammatory peptides and failed to effectively inhibit the interference of the local inflammatory microenvironment on osteogenic differentiation. Bone extracellular matrix synthesis was inhibited, failing to provide a good attachment and growth environment for osteoblasts, affecting the osteogenesis process. Furthermore, the inability to efficiently regulate the cytokine balance in the local microenvironment was detrimental to the resolution of the inflammatory response and tissue repair during fracture healing, resulting in a lack of significant bone density increase and delayed fracture healing.

[0151] In Comparative Example 3, the collagen type in the midgut of jellyfish was different, and the content of callus-promoting peptides generated after enzymatic hydrolysis was significantly reduced. The midgut of jellyfish differs from that of sand jellyfish in chemical composition, and these differences lead to differences in the amino acid composition, sequence, and spatial structure of the extracted peptides. The structure of the peptide determines its function, including its ability to bind to osteogenesis-related receptors. Peptides extracted from the midgut of jellyfish are less effective than peptides extracted from the midgut of sand jellyfish in activating relevant signaling pathways in osteoblasts (including the mitogen-activated protein kinase (MAPK) signaling pathway, and sand jellyfish peptides activate the FAK / PI3K / Akt pathway through the integrin α2β1 receptor). These pathways are crucial for the proliferation, differentiation, and survival of osteoblasts, affecting the increase in bone density and the rate of fracture healing.

[0152] In Comparative Example 4, the mass ratio of bamboo yellow mushrooms to maitake mushrooms was changed to 1:3, altering the composition of the composite peptide. Peptides produced by different mushrooms have varying bioactivities, playing different roles in promoting angiogenesis, regulating immune responses, and providing antioxidant benefits to bone repair. Peptides extracted from bamboo yellow mushrooms are particularly effective in promoting vascular endothelial growth factor (VEGF) expression, activating the cAMP / PKA pathway through GPCRs, while peptides extracted from maitake mushrooms are more critical in regulating immune cells. This altered ratio leads to insufficient expression of VEGF and the Runx2 transcription factor, reduced angiogenesis at the fracture site, limited nutrient and oxygen supply, and impaired osteoblast metabolism and function, ultimately resulting in slower bone density improvement and prolonged fracture healing.

[0153] In Comparative Example 5, lumbrokinase was replaced with nattokinase. Nattokinase has poor specificity for the cleavage site on collagen, resulting in a deviation in the molecular weight distribution of the resulting peptides and changes in the amino acid sequence, affecting their binding efficiency to integrin receptors. During the preparation of peptides extracted from the gelatinous layer of sand jellyfish, lumbrokinase can specifically cleave certain peptide bonds, producing peptides with specific structures and activities. After being replaced by nattokinase, the structure and sequence of the peptides produced by enzymatic cleavage were altered. These new peptides were unable to effectively activate osteogenesis-related signaling pathways, thereby affecting bone density growth and fracture healing.

[0154] Comparative Example 6: Serrapeptase is replaced with nattokinase. Serrapeptase has a unique three-dimensional substrate recognition ability and can release specific anti-inflammatory peptides; nattokinase cannot replace this function, resulting in insufficient suppression of local inflammation. Serrapeptase participates in the modification and generation of peptides at specific steps. It acts on specific amino acid residues of peptides extracted from the gelatinous layer of sand jellyfish, affecting the final structure and function of the peptides. After being replaced by nattokinase, the unique modification effect of serrapeptase cannot be completed, which reduces the affinity of the extracted peptides to the surface receptors of osteoblasts, hinders intracellular signal transduction, and is not conducive to the differentiation of osteoblasts and the synthesis of bone matrix, resulting in poor bone density improvement and delayed fracture healing.

[0155] In the preparation of peptides extracted from the gelatinous layer of sand jellyfish in Comparative Example 7, transglutaminase was absent. This lack of transglutaminase-mediated peptide cross-linking resulted in decreased peptide chain stability, shortened half-life, decreased hydrophilicity, decreased absorption and utilization, and a reduced duration of effective action. Transglutaminase catalyzes the cross-linking reaction between glutamine and lysine residues during peptide preparation, forming stable isopeptide bonds. Its absence prevents the extracted peptides from forming specific higher-order structures, impairing their biological activity. For example, it is unable to effectively bind to and activate insulin-like growth factor (IGF), which plays a crucial role in promoting osteoblast proliferation and bone matrix synthesis. Impaired IGF activity slows osteoblast metabolic activity, slows bone density growth, and prolongs fracture healing.

[0156] In the preparation of the bamboo-Grifola frondosa composite peptide in Comparative Example 8, transglutaminase was absent. Transglutaminase also plays a role in forming stable structural and functional peptides. Its absence destabilizes the composite peptide structure, preventing it from effectively regulating the balance between osteoclasts and osteoblasts. This limits osteoblast function and prevents timely bone replenishment, impairing bone density and fracture healing.

[0157] In the preparation of the bamboo yellow-Grifola frondosa composite peptide in Comparative Example 9, the absence of aminopeptidase results in N-terminal hydrophobic amino acid residues (including Leu and Phe), which inhibit the peptide's interaction with calcium channels (including Cav1.2) and reduce calcium influx-driven osteoblast activation. The absence of aminopeptidase prevents the amino acid sequence of the composite peptide from being properly modified, impairing its recognition and binding to cell surface receptors. This includes an inability to activate the fibroblast growth factor (FGF) signaling pathway, leading to decreased bone repair capacity, slowed bone density growth, and delayed fracture healing.

[0158] In the preparation of the bamboo yellow-Grifola frondosa composite peptide in Comparative Example 10, both the aminopeptidase and the glutamine transaminase reactions were missing. The double deletion resulted in the peptide chain being unable to form a stable structure and unable to undergo correct amino acid modification, resulting in a residual inhibitory N-terminus, which synergistically weakened the bone regeneration signaling pathway; during the fracture healing process, it was unable to effectively regulate cell proliferation, differentiation, and extracellular matrix synthesis, resulting in almost no significant increase in bone density and a significant extension of the fracture healing time.

[0159] In Comparative Example 11, bromelain preferentially cleaves basic amino acids (including Lys and Arg), destroying functional peptides and resulting in poorer efficacy. The enzymatic specificity and mechanism of action of bromelain differ from those of nattokinase and serrapeptase. Using bromelain produces a peptide mixture distinct from that produced by the normal preparation process. These peptides exhibit varying charge distributions, spatial conformations, and bioactivities. They have difficulty activating key intracellular signaling pathways associated with bone repair, including the bone morphogenetic protein (BMP) signaling pathway. Furthermore, they are unable to effectively promote osteoblast differentiation and bone tissue remodeling, resulting in slow bone density growth and prolonged fracture healing.

Claims

1. A composition for bone repair and growth, characterized in that: The composition comprises peptides extracted from the gelatinous layer of sand jellyfish and a bamboo yellow-Grifola frondosa composite peptide, wherein the mass ratio of the peptides extracted from the gelatinous layer of sand jellyfish to the bamboo yellow-Grifola frondosa composite peptide is (15-20):(8-10); the peptides extracted from the gelatinous layer of sand jellyfish comprise a product with a mass of 500Da to 2000Da obtained by sequentially hydrolyzing the gelatinous layer of sand jellyfish with lumbrokinase, serrapeptase, and transglutaminase; the bamboo yellow-Grifola frondosa composite peptide comprises a product with a mass of 500Da to 2000Da obtained by first co-hydrolyzing bamboo yellow mushrooms and Grifola frondosa mushrooms with cellulase and chitinase, then co-hydrolyzing with nattokinase and serrapeptase, and finally co-reacting with aminopeptidase and transglutaminase.

2. A bone repair and growth composition according to claim 1, characterized in that: The preparation method of the peptide extracted from the gelatinous layer of sand jellyfish comprises the following steps: taking the dehydrated gelatinous layer of sand jellyfish and crushing it, adding a phosphate buffer solution at a mass ratio of material to liquid of 1:(10-15), ultrasonically treating it to obtain an extract; adding lumbrokinase to the extract, performing enzymatic hydrolysis at 37-40°C for 2-2.5 hours, inactivating the enzyme by heat, cooling to a constant temperature of 45-50°C, adding serrapeptase, performing enzymatic hydrolysis for 1.5-2 hours, inactivating the enzyme by heat, cooling to a constant temperature of 37-40°C, adding glutamine transaminase, reacting for 1-1.5 hours, and inactivating the enzyme by heat; filtering through a mesh, ultrafiltration of the filtrate through an ultrafiltration membrane, collecting components with a mass between 500Da and 2000Da, and freeze-drying the components to obtain the peptide extracted from the gelatinous layer of sand jellyfish.

3. A bone repair and growth composition according to claim 2, characterized in that: The pH value of the phosphate buffer solution is 7.0-7.4; the power of the ultrasound is 200W-300W, the temperature of the ultrasound is 30°C-35°C, and the time of the ultrasound is 30min-45min.

4. The bone repair and growth composition according to claim 2, characterized in that: The amount of lumbrokinase added is 0.5% to 1.0% of the mass of the gelatinous layer of the sand jellyfish; the amount of serrapeptase added is 0.8% to 1.5% of the mass of the gelatinous layer of the sand jellyfish; and the amount of transglutaminase added is 1.0% to 1.5% of the mass of the gelatinous layer of the sand jellyfish.

5. The bone repair and growth composition according to claim 1, characterized in that: The preparation method of the bamboo yellow-Grifola frondosa composite fungus peptide comprises the following steps: mixing and crushing bamboo yellow mushrooms and Grifola frondosa mushrooms in a mass ratio of (3-4): (1-2) to obtain fungus powder; adding acetic acid-sodium acetate buffer solution in a mass ratio of material to liquid of 1: (10-15), adding cellulase and chitinase, performing ultrasonic enzymolysis, heat-inactivating the enzyme, cooling to a constant temperature of 45-50°C, adjusting the pH value to 7.0-7.5, adding nattokinase and serrapeptase, performing enzymolysis for 2-3 hours, heat-inactivating the enzyme, cooling to 40-45°C, adding aminopeptidase and glutamine transaminase, reacting for 1.5-2 hours, heat-inactivating the enzyme, filtering through a mesh, ultrafiltration of the filtrate through an ultrafiltration membrane, collecting components between 500Da and 2000Da, and freeze-drying to obtain the bamboo yellow-Grifola frondosa composite fungus peptide.

6. The bone repair and growth composition according to claim 5, characterized in that: The pH value of the acetic acid-sodium acetate buffer solution is 5.0-5.5; the amount of the cellulase added is 1.0%-1.5% of the mass of the bacterial powder; and the amount of the chitinase added is 0.3%-0.6% of the mass of the bacterial powder.

7. The bone repair and growth composition according to claim 5, characterized in that: The ultrasonic enzymatic hydrolysis has an ultrasonic power of 250W to 350W, a temperature of 40°C to 45°C, and a time of 3h to 4h.

8. The bone repair and growth composition according to claim 5, characterized in that: The addition amount of the nattokinase and serrapeptase is 0.5% to 1.0% of the mass of the bacterial powder; the addition amount of the aminopeptidase is 0.5% to 0.8% of the mass of the bacterial powder; and the addition amount of the glutamine transaminase is 0.5% to 1.0% of the mass of the bacterial powder.

9. A bone repair and growth composition according to claim 2 or 5, characterized in that: The temperature for heat inactivation of enzymes is 85° C. to 90° C., and the time for heat inactivation of enzymes is 10 min to 15 min. The mesh number of the sieve is 250 mesh to 325 mesh.

10. A method for preparing the bone repair and growth composition according to claim 1, characterized in that: The method comprises the following steps: mixing peptides extracted from the gelatinous layer of sand jellyfish and bamboo yellow-grifola frondosa composite peptides according to a mass ratio of (15-20): (8-10) to obtain a composite material.

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