Crocodile bone collagen peptide powder for improving bone density and preparation method and application thereof

Abstract

This invention provides a crocodile bone collagen peptide powder for improving bone density, its preparation method, and its application, relating to the field of biopharmaceutical technology. The preparation method includes the following steps: crocodile bone is pulverized and mixed with sodium chloride solution, followed by a first separation to obtain bone residue; water is added and mixed, followed by a second separation to obtain defatted bone residue; the defatted bone residue is mixed with water and a deodorizing agent, stirred, and separated to obtain deodorized bone residue; the deodorized bone residue is added to water, and ultrasonic-assisted extraction is performed, separating and collecting the supernatant; the supernatant is mixed with collagenase and trypsin for a first enzymatic hydrolysis; carboxypeptidase A and proline endopeptidase are added for a second enzymatic hydrolysis; the supernatant is separated, and transglutaminase is added to react, separating and obtaining the enzymatic hydrolysate; the enzymatic hydrolysate is separated using a multi-stage tangential flow membrane separation system, collecting the retentate from a 500 Da nanofiltration membrane; the final product is concentrated and dried. The crocodile bone collagen peptide powder obtained by this invention through a specific preparation method significantly improves taste, enhances stability, and strengthens the effect of improving bone density and bone calcium.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a crocodile bone collagen peptide powder for improving bone density, its preparation method, and its application. Background Technology

[0002] Bone health is a crucial foundation for overall health. Bone mineral density (BMD), a core indicator of bone strength, directly impacts bone load-bearing capacity and fracture resistance. With the increasing aging of the global population and changes in modern lifestyles, decreased bone mineral density and osteoporosis have become global public health issues, seriously threatening the quality of life for middle-aged and elderly individuals. Furthermore, factors such as prolonged sitting, lack of exercise, nutritional imbalances, and declining estrogen levels further accelerate bone loss, leading to a trend of bone health problems affecting younger people.

[0003] Currently, products and technologies on the market that improve bone density and prevent osteoporosis mainly have the following shortcomings:

[0004] 1. The effect of calcium supplementation alone is limited, and the effect on improving bone density and bone calcium is also limited: Traditional calcium supplements are mainly inorganic calcium (such as calcium carbonate). Although they have a high calcium content, they are highly dependent on stomach acid and can easily cause gastrointestinal discomfort such as bloating and constipation. In addition, they lack absorption-promoting factors. Calcium is easily combined with oxalic acid and phytic acid in the intestine to form insoluble salts, resulting in serious loss and low bioavailability. Therefore, their effect on improving bone density and bone calcium is limited, and they cannot fundamentally solve the problem of "calcium supplementation without calcium retention".

[0005] 2. Limited Raw Material Sources, Constraining Activity and Efficacy: Existing bone health products mostly use common raw materials such as bovine and pork bones. The amino acid composition, bioactivity, and human compatibility of these collagen peptides are limited, and they are easily affected by factors such as farming environment and disease, resulting in insufficient stability of efficacy. Furthermore, traditional extraction processes often involve simple hydrolysis, yielding peptides with large molecular weights that are difficult for the human body to absorb and utilize efficiently.

[0006] 3. Outdated technology and poor product stability: Some products use physical crushing and simple enzymatic hydrolysis processes, which cannot accurately control the molecular weight of peptides. This results in problems such as loss of active ingredients, easy aggregation, and off-odors, leading to low absorption efficiency and insufficient efficacy in the body.

[0007] The relevant patents and documents retrieved are as follows: This patent, published in China (CN105463046A) on March 17, 2020, discloses a method for preparing crocodile bone collagen peptide powder. The method includes: A. High-pressure cooking of crocodile bones (with muscle and fat removed) in a solvent to concentrate the crocodile bone glue; B. Treating the obtained crocodile bone glue with a protease to obtain an enzymatic hydrolysate; C. Centrifuging the enzymatic hydrolysate, drying the supernatant, and obtaining crocodile bone collagen peptide powder. While this method has demonstrated the crocodile bone collagen peptide powder's effects on anti-oxidation, in vitro DNA damage resistance, and whitening, it does not focus on its impact on improving bone density and calcium, product taste, or stability.

[0008] The patent, published in China (CN113398241A) on September 17, 2021, discloses a bone peptide composition comprising vitamin K2 powder and collagen peptide powder. The collagen peptide powder is prepared by hydrolyzing collagen, and is bovine collagen powder. The ratio of vitamin K2 powder to collagen peptide powder is 1:40.

[0009] The journal is titled *Clinical Rational Drug Use*, and the article is titled "Efficacy of Salmon Calcitonin Combined with Calcium Carbonate D3 in the Treatment of Primary Osteoporosis in the Elderly," Volume 18, Issue 15, published on May 25, 2025. This article discloses that calcium carbonate D3 is a fundamental drug in the clinical treatment of osteoporosis, as calcium carbonate provides calcium. D3 is an active form of vitamin D that enhances intestinal calcium absorption, promotes calcium deposition in bones, and regulates calcium and phosphorus metabolism to maintain bone health. The article, titled "Efficacy of Salmon Calcitonin Combined with Calcium Carbonate D3 in the Treatment of Primary Osteoporosis in the Elderly," discusses this topic.

[0010] As a "living fossil," the crocodile's skeleton possesses unique physiological characteristics and medicinal value. The Compendium of Materia Medica records that crocodile shell has the effects of breaking up blood stasis, reducing swelling, and promoting tissue regeneration. Modern research confirms that crocodile bone is rich in active calcium, phosphorus, and high-quality collagen, possessing the effects of strengthening muscles and bones, replenishing essence and marrow, and can be used to prevent and treat osteoporosis and rickets in children. Compared to ordinary bone-derived collagen, crocodile bone collagen peptides have three core advantages: (1) Amino acid composition adapted to bone matrix: rich in glycine, proline, hydroxyproline and other characteristic amino acids of bone collagen, which can directly participate in bone collagen synthesis and provide a high-quality organic framework for bones.

[0011] (2) Small molecules are easily absorbed: Crocodile bone collagen peptides can be obtained by controlling the preparation method to obtain protein peptides with small molecular weight. They can be absorbed by the intestines without complicated digestion and can quickly reach bone tissue to exert their effects.

[0012] (3) Unique bioactivity: Crocodile bone peptides can regulate the balance between osteoblasts and osteoclasts, activate osteoblast activity, promote bone matrix mineralization, and moderately inhibit excessive activation of osteoclasts, thus slowing down bone loss from the root.

[0013] However, the development and utilization of crocodile bone resources is still in its early stages. Research on crocodile bone collagen peptides is limited both domestically and internationally, particularly lacking systematic research and efficient preparation processes specifically targeting bone density improvement. Furthermore, existing crocodile bone-related products are mostly crudely processed bone powder or simple extracts, suffering from uneven molecular weight, low content of effective ingredients, and poor taste. These products fail to fully realize the unique value of crocodile bone collagen and cannot meet the market demand for highly effective, safe, and easily absorbed bone density-enhancing products.

[0014] In summary, there is an urgent need for a bone density enhancement product based on high-quality natural raw materials, which combines the dual benefits of supplementing collagen and promoting calcium absorption. By optimizing the preparation method, the shortcomings of crocodile bone collagen peptides in terms of poor taste and stability can be overcome, providing a brand-new solution for osteoporosis prevention and bone health maintenance, which has significant market value and social significance. Summary of the Invention

[0015] To address the aforementioned problems, this invention provides a crocodile bone collagen peptide powder for improving bone density, its preparation method, and its application. Through a specific preparation process, crocodile bone collagen peptide powder with a specific molecular weight is obtained, which is odorless, has good stability, and has a better effect on improving bone density and bone calcium.

[0016] Terminology Explanation: The term "enhancement" refers to the significant enhancement of the content, activity, stability, or bioavailability of a target ingredient during the preparation process through physical, chemical, or biological means, in order to optimize product efficacy and quality. It is often used to describe the improvement in extraction rate, purity, activity retention rate, or absorption efficiency.

[0017] The term "collagen peptide powder" refers to a bioactive powder rich in collagen peptides extracted from animal bones through enzymatic hydrolysis, separation, and drying processes. It has the characteristics of easy absorption and high bioavailability and is mainly used in the preparation of functional foods or medicines related to joint repair, skin anti-aging, and bone health.

[0018] The term "defatting" refers to the process of removing fat from raw materials (such as animal bones) using organic solvents or physical centrifugation to reduce the risk of oxidative deterioration and improve the purity and stability of subsequent extractions.

[0019] The term "crocodile bone" refers to the skeletal tissue derived from crocodilians. It is rich in type I collagen, calcium and phosphorus minerals, and active bone peptides, making it one of the high-quality raw materials for preparing bone-derived biological agents.

[0020] The term "crushing" refers to the process of breaking large aggregates into fine particles using mechanical force to increase the specific surface area and promote the dissolution efficiency of active ingredients in subsequent extraction processes.

[0021] The term "mixing" refers to the operation of uniformly blending two or more materials in a predetermined ratio to ensure consistent component distribution. It is often used in the formulation feeding and intermediate preparation stages.

[0022] The term "stirring" refers to a process in which a liquid or slurry is made to flow and shear through a mechanical device in order to achieve uniform mass transfer, heat transfer, or accelerate a dissolution reaction.

[0023] The term "separation" refers to the technique of separating different components in a mixed system based on differences in physical properties (such as density, particle size, and solubility), and is commonly used in solid-liquid separation or protein fractionation processes.

[0024] The term "bone residue" refers to the insoluble solid residue remaining after enzymatic hydrolysis and filtration during the extraction of bone protein. Its main components are incompletely hydrolyzed bone matrix and inorganic salts.

[0025] The term "deodorization" refers to the process steps of removing odorous substances (such as trimethylamine and short-chain fatty acids) from animal-derived extracts through physical adsorption, chemical reactions, or biological enzymatic methods to improve the sensory quality of the product.

[0026] The term "deodorizing agent" refers to functional additives used to eliminate or mask unpleasant odors in biological extracts, including activated charcoal, β-cyclodextrin, plant polyphenols, or specific flavor enzyme preparations.

[0027] The term "non-decalcification low-temperature extraction" refers to a technique that extracts active ingredients under mild conditions of 40-50℃ without damaging the natural calcium and phosphorus structure of bone tissue. This technique preserves the integrity of bone minerals and enhances biocompatibility.

[0028] The term "ultrasound-assisted extraction" refers to an enhanced extraction method that utilizes the cavitation effect of ultrasound to disrupt the structure of cells or bone matrix, accelerate the release of target components, improve extraction rate and speed, and reduce energy consumption and solvent usage.

[0029] The term "supernatant" refers to the clear portion that lies on the upper layer of the liquid after centrifugation or settling. It typically contains soluble proteins, peptides, or small molecule active substances and is the primary target for subsequent concentration and purification.

[0030] The term "enzymatic hydrolysis" refers to the biochemical process in which bone protein is hydrolyzed in a targeted manner using proteases under specific temperature and pH conditions to generate small molecule peptides and amino acids. It is the core step in the preparation of collagen peptides.

[0031] The term "pH" refers to the negative logarithm of the hydrogen ion concentration in a solution. It is used to characterize the acidity or alkalinity of a reaction system and directly affects enzyme activity, protein solubility, and stability. It is a key control parameter in the enzymatic hydrolysis process.

[0032] The term "enzyme inactivation" refers to terminating enzymatic reactions by raising the temperature, adjusting the pH, or adding inhibitors to prevent excessive hydrolysis that could lead to product degradation or increased bitterness, thus ensuring consistent product quality.

[0033] The term "enzymatic hydrolysate" refers to the mixed liquid containing collagen peptides, amino acids, and soluble nutrients formed after bone protein has undergone enzymatic hydrolysis. It serves as the initial raw material for subsequent separation and purification.

[0034] The term "multi-stage tangential flow membrane separation system" refers to a continuous staged purification device composed of ultrafiltration membranes and nanofiltration membranes connected in series. It reduces membrane fouling through tangential flow and achieves gradient separation and concentration of large, medium and small molecules.

[0035] The term "ultrafiltration membrane" refers to a semi-permeable membrane with a molecular weight cutoff of 1-100 kDa, used to remove large molecular impurities from enzymatic hydrolysates or enrich target peptides to improve product purity.

[0036] The term "nanofiltration membrane" refers to membrane materials with smaller pore sizes (approximately 1 kDa or less), which can further desalinate and concentrate small molecule peptides, and remove inorganic ions and pigments, making them suitable for the purification of high-purity peptide products.

[0037] The term "retained liquid" refers to the concentrated liquid retained by the membrane material during membrane separation. It is rich in target macromolecules or medium-molecular-weight active ingredients and is usually used as input material for the next process.

[0038] The term "drying" refers to the removal of moisture from the enzyme hydrolysate concentrate by spray drying or freeze drying to obtain a free-flowing powdered product that is easy to store, transport, and process into formulations.

[0039] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing crocodile bone collagen peptide powder to improve bone density, comprising the following steps: S1: Degreasing: After crushing the crocodile bone, mix it with a 2-5 wt% sodium chloride solution and stir. Separate once to obtain bone residue; mix the bone residue with water and separate a second time to obtain degreased bone residue; S2: Deodorization: Mix defatted bone meal with water, add deodorizing agent, stir, separate, and obtain deodorized bone meal; S3: Non-decalcified low-temperature extraction: Mix deodorized bone residue with water, and extract with ultrasound assistance at 40-50℃ for 20-40 minutes. Separate and collect the supernatant. S4: Enzymatic hydrolysis: Mix the supernatant with collagenase and trypsin, control the pH to 7.8-8.0 and the temperature to 40-45℃ for the first enzymatic hydrolysis; adjust the pH to 7.0-7.2 and the temperature to 50-55℃, add carboxypeptidase A and proline endopeptidase (PEP) for the second enzymatic hydrolysis; inactivate the enzymes, separate the supernatant, adjust the pH to 6.0-6.5, add transglutaminase (TGase) for the third enzymatic hydrolysis, inactivate the enzymes, separate, and obtain the enzymatic hydrolysate; S5: The enzymatic hydrolysate is separated by a multi-stage tangential flow membrane separation system, passing sequentially through a 1000 Da ultrafiltration membrane and a 500 Da nanofiltration membrane. The retentate from the 500 Da nanofiltration membrane is collected, concentrated, and dried to obtain the final product.

[0040] Preferably, in step S1, the crocodile bone is fresh artificially farmed crocodile bone (after removing muscle, fat and connective tissue), cleaned, and cut into 1-2cm pieces; Preferably, in step S1, the pulverization includes the following steps: homogenizing the pulverized material 1-3 times under a pressure of 20-25 MPa.

[0041] Any point value or sub-range value within the range of 20-25MPa is applicable to this invention, including but not limited to 20MPa, 21MPa, 22MPa, 23MPa, 24MPa, and 25MPa.

[0042] Any point value or sub-range value within the range of 1 to 3 is applicable to this invention, including but not limited to 1st, 2nd, and 3rd times.

[0043] Preferably, in step S1, the mass ratio of the crocodile bone to the sodium chloride solution is 1:4-8.

[0044] Any point value or sub-range value within the range of 1:4-8 is applicable to this invention, including but not limited to 1:4, 1:5, 1:6, 1:7, and 1:8.

[0045] Preferably, in step S1, the stirring time is 3-5 hours and the temperature is 30-40°C.

[0046] Any point value or sub-range value within the range of 3-5h is applicable to this invention, including but not limited to 3h, 3.5h, 4h, 4.5h, and 5h.

[0047] Any point value or sub-range value within the range of 30-40℃ is applicable to this invention, including but not limited to 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, and 40℃.

[0048] Preferably, in step S1, both the primary and secondary separations are centrifugation, with a rotation speed of 3000-5000 r / min and a time of 5-15 min.

[0049] Any point value or sub-range value within the range of 3000-5000 r / min is applicable to this invention, including but not limited to 3000 r / min, 3100 r / min, 3200 r / min, 3300 r / min, 3400 r / min, 3500 r / min, 3600 r / min, 3700 r / min, 3800 r / min, 3900 r / min, 4000 r / min, 4100 r / min, 4200 r / min, 4300 r / min, 4400 r / min, 4500 r / min, 4600 r / min, 4700 r / min, 4800 r / min, 4900 r / min, and 5000 r / min.

[0050] Any point value or sub-range value within the range of 5-15 min is applicable to this invention, including but not limited to 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0051] Preferably, in step S1, the mass ratio of bone residue to water is 1:4-8.

[0052] Any point value or sub-range value within the range of 1:4-8 is applicable to this invention, including but not limited to 1:4, 1:5, 1:6, 1:7, and 1:8.

[0053] Preferably, in step S2, the mass ratio of the defatted bone residue to water is 1:5-10.

[0054] Any point value or sub-range value within the range of 1:5-10 is applicable to this invention, including but not limited to 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10.

[0055] Preferably, in step S2, the deodorizing agent is a mixture of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin.

[0056] Preferably, the mass ratio of the tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin is 1-3:0.5-1.5:0.1-0.3.

[0057] Any point value or sub-range value within the range of 1-3:0.5-1.5:0.1-0.3 is applicable to this invention, including but not limited to 1:0.5:0.1, 1:0.6:0.1, 1:0.7:0.1, 1:0.8:0.1, 1:0.9:0.1, 1:1.0:0.1, 1:1.1:0.1, 1:1.2:0.1, 1:1.3:0.1, 1:1.4:1, 1:1.5:0.1, 2:0.5:0.1, 2:0.6:0.1, 2:0.7:0.1, 2:0.8:0.1, 2:0.9:0.1, 2:1.0:0.1, 2:1.2:0.1, 2:1.2:0.1, 2:1.3:0.1, 2:1.4 :1, 2:1.5:0.1, 3:0.5:0.1, 3:0.6:0.1, 3:0.7:0.1, 3:0.8:0.1, 3:0.9:0.1, 3:1.0:0.1, 3:1.3:0.1, 3:1.2:0.1, 3:1.3:0.1, 3:1.4:1, 3:1.5:0.1, 1:0. 5:0.2, 1:0.6:0.2, 1:0.7:0.2, 1:0.8:0.2, 1:0.9:0.2, 1:1.0:0.2, 1:1.1:0.2, 1:1.2:0.2, 1:1.3:0.2, 1:1.4:1, 1:1.5:0.2, 2:0.5:0.2, 2:0.6:0.2, 2 :0.7:0.2, 2:0.8:0.2, 2:0.9:0.2, 2:1.0:0.2, 2:1.2:0.2, 2:1.2:0.2, 2:1.3:0.2, 2:1.4:1, 2:1.5:0.2, 3:0.5:0.2, 3:0.6:0.2, 3:0.7:0.2, 3:0.8:0 2, 3:0.9:0.2, 3:1.0:0.2, 3:1.3:0.2, 3:1.2:0.2, 3:1.3:0.2, 3:1.4:1, 3:1.5:0.2, 1:0.5:0.3, 1:0.6:0.3, 1:0.7:0.3, 1:0.8:0.3, 1:0.9:0.3, 1:1. 0:0.3, 1:1.1:0.3, 1:1.2:0.3, 1:1.3:0.3, 1:1.4:1, 1:1.5:0.3, 2:0.5:0.3, 2:0.6:0.3, 2:0.7:0.3, 2:0.8:0.3, 2:0.9:0.3, 2:1.0:0.3, 2:1.2:0.3, 2 :1.2:0.3, 2:1.3:0.3, 2:1.4:1, 2:1.5:0.3, 3:0.5:0.3, 3:0.6:0.3, 3:0.7:0.3, 3:0.8:0.3, 3:0.9:0.3, 3:1.0:0.3, 3:1.3:0.3, 3:1.2:0.3, 3:1.3:0.3, 3:1.4:1, 3:1.5:0.3.

[0058] More preferably, the mass ratio of the tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin is 2:1:0.2.

[0059] Preferably, the molecular weight of the chitosan oligosaccharide is 1000-2000 Da.

[0060] Any point value or sub-range value within the range of 1000-2000Da is applicable to this invention, including but not limited to 1000Da, 1100Da, 1200Da, 1300Da, 1400Da, 1500Da, 1600Da, 1700Da, 1800Da, 1900Da, and 2000Da.

[0061] Preferably, in step S2, the mass of the deodorizing agent is 0.5%-1.5% of the mass of the defatted bone residue.

[0062] Any point value or sub-range value within the range of 0.5%-1.5% is applicable to this invention, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%.

[0063] Preferably, in step S2, the separation is centrifugation at a speed of 3000-5000 r / min for 5-15 min.

[0064] Any point value or sub-range value within the range of 3000-5000 r / min is applicable to this invention, including but not limited to 3000 r / min, 3100 r / min, 3200 r / min, 3300 r / min, 3400 r / min, 3500 r / min, 3600 r / min, 3700 r / min, 3800 r / min, 3900 r / min, 4000 r / min, 4100 r / min, 4200 r / min, 4300 r / min, 4400 r / min, 4500 r / min, 4600 r / min, 4700 r / min, 4800 r / min, 4900 r / min, and 5000 r / min.

[0065] Any point value or sub-range value within the range of 5-15 min is applicable to this invention, including but not limited to 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0066] Preferably, in step S3, the mass ratio of the deodorized bone residue to water is 1:3-6.

[0067] Any point value or sub-range value within the range of 1:3-6 is applicable to this invention, including but not limited to 1:3, 1:4, 1:5, and 1:6.

[0068] Preferably, in step S3, the power of the ultrasound-assisted extraction is 200W-600W.

[0069] Any point value or sub-range value within the range of 200W-600W is applicable to this invention, including but not limited to 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, 300W, 310W, 320W, 330W, 340W, 350W, 360W, 370W, 380W, 390W, 400W, 410W, 420W, 430W, 440W, 450W, 460W, 470W, 480W, 490W, 500W, 510W, 520W, 530W, 540W, 550W, 560W, 570W, 580W, 590W, and 600W.

[0070] Preferably, in step S4, the mass ratio of collagenase to trypsin is 1:5-8.

[0071] Any point value or sub-range value within the range of 1:5-8 is applicable to this invention, including but not limited to 1:5, 1:6, 1:7, and 1:8.

[0072] Preferably, in step S4, the mass of the collagenase and trypsin is 1%-3% of the mass of the crocodile bone in step S1.

[0073] Any point value or sub-range value within the range of 1%-3% is applicable to this invention, including but not limited to 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, and 3.0%.

[0074] Preferably, in step S4, the first enzymatic hydrolysis takes 2-4 hours.

[0075] Any point value or sub-range value within the range of 2-4h is applicable to this invention, including but not limited to 2h, 2.5h, 3h, 3.5h, and 4h.

[0076] Preferably, in step S4, the mass ratio of carboxypeptidase A to proline endonuclease is 1-3:1.

[0077] Any point value or sub-range value within the range of 1-3:1 is applicable to this invention, including but not limited to 1:1, 2:1, and 3:1.

[0078] Preferably, in step S4, the mass of carboxypeptidase A and proline endonuclease is 0.5%-1.5% of the mass of crocodile bone in step S1.

[0079] Any point value or sub-range value within the range of 0.5%-1.5% is applicable to this invention, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%.

[0080] Preferably, in step S4, the second enzymatic hydrolysis takes 1-2 hours.

[0081] Any point value or sub-range value within the range of 1-2h is applicable to this invention, including but not limited to 1h, 1.5h, and 2h.

[0082] Preferably, in step S4, the mass of the transglutaminase is 0.1%-0.5% of the mass of the crocodile bone in step S1.

[0083] Any point value or sub-range value within the range of 0.1%-0.5% is applicable to this invention, including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%.

[0084] Preferably, in step S4, the third enzymatic hydrolysis takes 30-60 minutes and is carried out at a temperature of 40-50°C.

[0085] Any point value or sub-range value within the range of 30-60 min is applicable to this invention, including but not limited to 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min.

[0086] Any point value or sub-range value within the range of 40-50℃ is applicable to this invention, including but not limited to 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, and 50℃.

[0087] Preferably, in step S4, the enzyme inactivation operation is as follows: heat to 90-95℃ to inactivate the enzyme for 10-20 minutes.

[0088] Any point value or sub-range value within the range of 90-95℃ is applicable to this invention, including but not limited to 90℃, 91℃, 92℃, 93℃, 94℃, and 95℃.

[0089] Any point value or sub-range value within the range of 10-20 min is applicable to this invention, including but not limited to 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, and 20 min.

[0090] Preferably, in step S5, the transmembrane pressure of the separation is 0.15-0.3 MPa.

[0091] Any point value or sub-range value within the range of 0.15-0.3MPa is applicable to this invention, including but not limited to 0.15MPa, 0.16MPa, 0.17MPa, 0.18MPa, 0.19MPa, 0.20MPa, 0.21MPa, 0.22MPa, 0.23MPa, 0.24MPa, 0.25MPa, 0.26MPa, 0.27MPa, 0.28MPa, 0.29MPa, and 0.3MPa.

[0092] Secondly, the present invention provides crocodile bone collagen peptide powder prepared by the above preparation method.

[0093] Preferably, the molecular weight of the crocodile bone collagen peptide powder is 600 Da-800 Da.

[0094] Thirdly, this invention demonstrates the application of crocodile bone collagen peptide powder prepared by the above-mentioned preparation method in the preparation of products that improve bone density.

[0095] The technical feature "2-5 wt% sodium chloride solution" is derived from the corresponding technical features of 3 wt% sodium chloride solution, 2 wt% sodium chloride solution, and 5 wt% sodium chloride solution in the foregoing explanation and / or Examples 1-3, summarized by the common feature "2-5 wt% sodium chloride solution". Therefore, those skilled in the art can reasonably infer that the technical feature 2-5 wt% sodium chloride solution, the subordinate concept of 2-5 wt% sodium chloride solution, the basically equivalent technical means of 2-5 wt% sodium chloride solution, and the technical means that can replace 2-5 wt% sodium chloride solution based on the existing technical level and within the scope of conventional technical means and common knowledge should all fall within the protection scope of this invention.

[0096] The technical feature “the mass ratio of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin is 1-3:0.5-1.5:0.1-0.3” is summarized from the corresponding technical features in the foregoing explanation and / or Examples 1-3, such as the mass ratio of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin being 2:1:0.2, 1:1.5:0.1, and 3:0.5:0.3, which are derived from the common feature “1-3:0.5-1.5:0.1-0.3”. Therefore, those skilled in the art can reasonably presume that the technical features of the mass ratio of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin being 1-3:0.5-1.5:0.1-0.3, their subordinate concepts, their essentially equivalent technical means, and technical means that can replace the mass ratio of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin being 1-3:0.5-1.5:0.1-0.3 based on existing technical levels and conventional technical means and common knowledge, should all fall within the protection scope of this invention.

[0097] The technical feature "ultrasound-assisted extraction at 40-50℃" is derived from the aforementioned explanations and / or the corresponding technical features of ultrasound-assisted extraction at 45℃, 40℃, and 50℃ in Examples 1-3, summarized by the common feature "ultrasound-assisted extraction at 40-50℃". Therefore, those skilled in the art can reasonably infer that the technical feature "ultrasound-assisted extraction at 40-50℃", its subordinate concepts, its substantially equivalent technical means, and technical means that can replace ultrasound-assisted extraction at 40-50℃ based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention.

[0098] The technical feature "the mass ratio of collagenase to trypsin is 1:5-8" is derived from the corresponding technical features in the foregoing explanation and / or Examples 1-3, such as collagenase and trypsin with a mass ratio of 1:6, 1:5, and 1:8, through the common feature "the mass ratio of collagenase to trypsin is 1:5-8". Therefore, those skilled in the art can reasonably infer that the technical feature of the mass ratio of collagenase to trypsin being 1:5-8, its subordinate concepts, its substantially equivalent technical means, and technical means that can replace the mass ratio of collagenase to trypsin being 1:5-8 within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0099] The technical feature "the mass ratio of carboxypeptidase A to proline endonuclease is 1-3:1" is derived from the corresponding technical features in Examples 1-3 with a mass ratio of 2:1, 3:1, and 1:1, summarized by the common feature "the mass ratio of carboxypeptidase A to proline endonuclease is 1-3:1". Therefore, those skilled in the art can reasonably infer that the technical feature "the mass ratio of carboxypeptidase A to proline endonuclease is 1-3:1", its subordinate concepts, its essentially equivalent technical means, and technical means that can replace the mass ratio of carboxypeptidase A to proline endonuclease of 1-3:1 within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0100] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention employs non-decalcification low-temperature ultrasound-assisted extraction, which maximizes the preservation of the natural active ingredients of crocodile bone. No decalcification treatment is performed during the extraction process, avoiding the damage of hydroxyapatite, collagen, and active peptides in the bone matrix caused by strong acids and alkalis. This allows the organic calcium and phosphorus in the crocodile bone to form a natural complex structure with collagen. Simultaneously, the 40-50℃ low-temperature ultrasound-assisted extraction is gentle and efficient, improving the collagen dissolution rate without damaging the original activity of the peptides, laying the foundation for the subsequent preparation of highly active bone health peptides.

[0101] 2. This invention employs a stepwise, targeted enzymatic hydrolysis combined with transglutaminase modification to obtain functional peptides with concentrated molecular weight and strong bone targeting. The three-step segmented enzymatic hydrolysis system—initial hydrolysis with collagenase and trypsin, precise cleavage with carboxypeptidase A and proline endonuclease, and modification with transglutaminase—allows for precise control of the hydrolysis sites, significantly improving the yield of small-molecule active peptides. Further separation using a multi-stage membrane system of 1000 Da ultrafiltration and 500 Da nanofiltration yields highly active peptides in the 600-800 Da range. This molecular weight range is easily absorbed and utilized by the human body, enabling direct action on bone tissue to promote osteoblast proliferation and inhibit osteoclast activity, thereby significantly increasing bone density.

[0102] 3. The product prepared by this invention has low fishy odor, good solubility, and high stability, making it suitable for long-term consumption. Through specific defatting and deodorizing pretreatment, fat and odorous substances in crocodile bones are effectively removed, resulting in a product without fishy odor and with a mild taste. After appropriate cross-linking with transglutaminase, the product's solubility, thermal stability, and storage stability are significantly improved, making it less prone to precipitation and deterioration. It can be directly made into powders, tablets, or capsules, suitable for long-term consumption by people with osteoporosis, the elderly, and those who need bone health maintenance, and has good industrialization prospects and application value. Detailed Implementation

[0103] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0104] Collagenase: Purchased from Jiangsu Caiwei Biotechnology Co., Ltd., enzyme activity 100,000 U / g.

[0105] Trypsin: purchased from Shanghai Kanglang Biotechnology Co., Ltd., enzyme activity 1000U / mg.

[0106] Carboxypeptidase A: Purchased from Shanghai Yuanye Biotechnology Co., Ltd., enzyme activity 200,000 U / g.

[0107] Proline endonuclease: purchased from Shaanxi Mufan Biotechnology Co., Ltd., enzyme activity 50U / mg.

[0108] Transglutaminase: purchased from Chongqing Tianrun Biological Products Co., Ltd., enzyme activity 100U / mg.

[0109] Example 1 A crocodile bone collagen peptide powder for improving bone density and its preparation method: S1: Degreasing: Fresh artificially bred crocodile bones, after removing muscle, fat and connective tissue, are cleaned, cut into 1-2cm bone pieces, crushed, homogenized twice under 22MPa pressure, and then mixed with 3wt% sodium chloride solution at a mass ratio of 1:6. The mixture is stirred at 35℃ for 4 hours, filtered, and the bone residue is obtained. The bone residue is mixed with water, filtered, and the degreased bone residue is obtained. S2: Deodorization: Defatted bone meal and water are mixed at a mass ratio of 1:8. A deodorizing agent of 1% by mass of the defatted bone meal is added. The deodorizing agent is a mixture of tangerine peel extract, chitosan oligosaccharide (molecular weight of 1500 Da), and hydroxypropyl-β-cyclodextrin in a mass ratio of 2:1:0.2. The mixture is stirred, centrifuged at 4000 r / min for 10 min to obtain deodorized bone meal. S3: Non-decalcified low-temperature extraction: Deodorized bone residue and water are mixed at a mass ratio of 1:5, and ultrasonically assisted extraction is performed at 45℃ for 30 min. The supernatant is then separated and collected. S4: Enzymatic hydrolysis: The supernatant is mixed with collagenase and trypsin at a mass ratio of 1:6 (the mass of collagenase and trypsin is 2% of the mass of crocodile bone in step S1), and the pH is controlled at 7.8 and the temperature at 40℃. The first enzymatic hydrolysis is carried out for 3 hours. The pH is adjusted to 7.2 and the temperature is 55℃. Carboxypeptidase A and proline endopeptidase at a mass ratio of 2:1 (the mass of carboxypeptidase A and proline endopeptidase is 1% of the mass of crocodile bone in step S1) are added. The second enzymatic hydrolysis is carried out for 1.5 hours. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The supernatant is separated. The pH is adjusted to 6.2. Transglutaminase (TGase) is added for the third enzymatic hydrolysis reaction for 45 minutes at 45℃. The mass of transglutaminase is 0.3% of the mass of crocodile bone in step S1. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The mixture is separated to obtain the enzymatic hydrolysate. S5: The enzymatic hydrolysate is separated by a multi-stage tangential flow membrane separation system, passing sequentially through a 1000 Da ultrafiltration membrane and a 500 Da nanofiltration membrane, with the transmembrane pressure controlled at 0.2 MPa. The retentate from the 500 Da nanofiltration membrane is collected, concentrated, and dried to obtain the final product.

[0110] Example 2 A crocodile bone collagen peptide powder for improving bone density and its preparation method: S1: Degreasing: Fresh artificially bred crocodile bones, after removing muscle, fat and connective tissue, are cleaned, cut into 1-2cm bone pieces, crushed, homogenized 3 times under 20MPa pressure, and then mixed with 5wt% sodium chloride solution at a mass ratio of 1:4. The mixture is stirred at 40℃ for 3 hours, filtered, and the bone residue is obtained. The bone residue is mixed with water, filtered, and the degreased bone residue is obtained. S2: Deodorization: Defatted bone meal is mixed with water at a mass ratio of 1:5. A deodorizing agent of 0.5% by mass of the defatted bone meal is added. The deodorizing agent is a mixture of tangerine peel extract, chitosan oligosaccharide (molecular weight of 2000 Da), and hydroxypropyl-β-cyclodextrin in a mass ratio of 1:1.5:0.1. The mixture is stirred, centrifuged at 5000 r / min for 5 min to obtain deodorized bone meal. S3: Non-decalcified low-temperature extraction: Deodorized bone residue and water are mixed at a mass ratio of 1:3, and extracted with ultrasound at 40℃ for 40 min. The supernatant is then separated and collected. S4: Enzymatic hydrolysis: The supernatant is mixed with collagenase and trypsin at a mass ratio of 1:5 (the mass of collagenase and trypsin is 1% of the mass of crocodile bone in step S1), and the pH is controlled at 8.0 and the temperature is 45℃. The first enzymatic hydrolysis is carried out for 2 hours. The pH is adjusted to 7.0 and the temperature is 50℃. Carboxypeptidase A and proline endopeptidase at a mass ratio of 3:1 (the mass of carboxypeptidase A and proline endopeptidase is 1.5% of the mass of crocodile bone in step S1) are added. The second enzymatic hydrolysis is carried out for 1 hour. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The supernatant is separated. The pH is adjusted to 6.5. Transglutaminase (TGase) is added for the third enzymatic hydrolysis reaction for 605 minutes at a temperature of 40℃. The mass of transglutaminase is 0.1% of the mass of crocodile bone in step S1. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The mixture is separated to obtain the enzymatic hydrolysate. S5: The enzymatic hydrolysate is separated by a multi-stage tangential flow membrane separation system, passing sequentially through a 1000 Da ultrafiltration membrane and a 500 Da nanofiltration membrane, with the transmembrane pressure controlled at 0.15 MPa. The retentate from the 500 Da nanofiltration membrane is collected, concentrated, and dried to obtain the final product.

[0111] Example 3 A crocodile bone collagen peptide powder for improving bone density and its preparation method: S1: Degreasing: Fresh artificially bred crocodile bones, after removing muscle, fat and connective tissue, are cleaned, cut into 1-2cm bone pieces, crushed, homogenized once under 25MPa pressure, and then mixed with 2wt% sodium chloride solution at a mass ratio of 1:8. The mixture is stirred at 30℃ for 5 hours, filtered, and the bone residue is obtained. The bone residue is mixed with water, filtered, and the degreased bone residue is obtained. S2: Deodorization: Defatted bone meal is mixed with water at a mass ratio of 1:10. A deodorizing agent of 1.5% by mass of the defatted bone meal is added. The deodorizing agent is a mixture of tangerine peel extract, chitosan oligosaccharide (molecular weight of 1000 Da), and hydroxypropyl-β-cyclodextrin in a mass ratio of 3:0.5:0.3. The mixture is stirred, centrifuged at 3000 r / min for 15 min to obtain deodorized bone meal. S3: Non-decalcified low-temperature extraction: Deodorized bone residue and water are mixed at a mass ratio of 1:6, and ultrasonically assisted extraction is performed at 50℃ for 20 min. The supernatant is then separated and collected. S4: Enzymatic hydrolysis: The supernatant is mixed with collagenase and trypsin at a mass ratio of 1:8 (the mass of collagenase and trypsin is 3% of the mass of crocodile bone in step S1), and the pH is controlled at 7.8 and the temperature at 40℃. The first enzymatic hydrolysis is carried out for 4 hours. The pH is adjusted to 7.2 and the temperature is 55℃. Carboxypeptidase A and proline endopeptidase at a mass ratio of 1:1 (the mass of carboxypeptidase A and proline endopeptidase is 0.5% of the mass of crocodile bone in step S1) are added. The second enzymatic hydrolysis is carried out for 2 hours. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The supernatant is separated. The pH is adjusted to 6.0. Transglutaminase (TGase) is added for a third enzymatic hydrolysis reaction for 30 minutes at a temperature of 50℃ (the mass of transglutaminase is 0.5% of the mass of crocodile bone in step S1). The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes. The mixture is separated to obtain the enzymatic hydrolysate. S5: The enzymatic hydrolysate is separated by a multi-stage tangential flow membrane separation system, passing sequentially through a 1000 Da ultrafiltration membrane and a 500 Da nanofiltration membrane, with the transmembrane pressure controlled at 0.3 MPa. The retentate from the 500 Da nanofiltration membrane is collected, concentrated, and dried to obtain the final product.

[0112] Comparative Example 1 Crocodile bone collagen peptide powder was prepared according to Example 2 described in Chinese patent application CN105463046A.

[0113] Comparative Example 2 A crocodile bone collagen peptide powder for improving bone density and its preparation method: Compared with Example 1, only the deodorizing agent was replaced with activated carbon; otherwise, it was the same as Example 1.

[0114] Comparative Example 3 A crocodile bone collagen peptide powder for improving bone density and its preparation method: Compared with Example 1, only step S4 is changed, and the rest is the same as Example 1.

[0115] S4: Enzymatic hydrolysis: The supernatant is mixed with collagenase and trypsin at a mass ratio of 1:5 (the mass of collagenase and trypsin is 1% of the mass of crocodile bone in step S1), and the pH is controlled at 8.0 and the temperature is 45℃. The first enzymatic hydrolysis is carried out for 2 hours; the mixture is then separated to obtain the enzymatic hydrolysate. Comparative Example 4 A crocodile bone collagen peptide powder for improving bone density and its preparation method: Compared with Example 1, only step S4 is changed, and the rest is the same as Example 1.

[0116] S4: Enzymatic hydrolysis: The supernatant is mixed with collagenase and trypsin (the mass of collagenase is 2% of the mass of crocodile bone in step S1), and the pH is controlled at 7.8 and the temperature at 40℃. The first enzymatic hydrolysis is carried out for 3 hours. The pH is adjusted to 7.2 and the temperature is 55℃. Proline endonuclease (the mass of proline endonuclease is 1% of the mass of crocodile bone in step S1) is added, and the second enzymatic hydrolysis is carried out for 1.5 hours. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes, and the supernatant is separated. The pH is adjusted to 6.2, and trypsin is added for the third enzymatic hydrolysis reaction for 45 minutes at 45℃. The mass of trypsin is 0.3% of the mass of crocodile bone in step S1. The temperature is raised to 95℃ to inactivate the enzyme for 15 minutes, and the mixture is separated to obtain the enzymatic hydrolysate.

[0117] Comparative Example 5 A crocodile bone collagen peptide powder for improving bone density and its preparation method: Compared with Example 1, only step S5 is removed, and the rest is the same as Example 1.

[0118] Test Example 1 (1) Bone mineral density measurement: Refer to the national method for assessing bone density function in health food products.

[0119] Forty healthy female Sprague-Dawley (SD) rats (60.0±10.0g, 3 weeks old) were purchased from Harbin Medical University, China. The rats were housed in accordance with animal management and usage guidelines, with three rats per cage. Standard cages allowed free access to food and water. The ambient temperature was maintained between 22±2℃ and 45±5℃, and the light-out / darkness cycle was 12 hours. Rats were randomly divided into 8 groups, each group was fed with crocodile bone collagen peptides from Examples 1-3 and Comparative Examples 1-5 at a dose of 200 mg / kg BW for 9 weeks. During this period, the activity of the rats was observed. After the last feeding, the rats were sacrificed, and the left and right femurs were quickly dissected. The femurs were then soaked in physiological saline and stored at -40°C for later testing. For the test, the left femur was thawed and the surface water was removed. It was then evenly arranged on a muscle simulation plate, and the entire left femur was scanned using a LUNAR Prodigy dual-energy X-ray absorptiometry (DXA) bone densitometer. The average bone mineral density was used to represent the bone mineral density. The data were analyzed using GE Lunar software and are shown in Table 1.

[0120] (2) Determination of bone calcium content: The determination method was carried out according to the national standard GB / T5009.92-2003 using a SpectrAA-220FS atomic absorption spectrophotometer, as detailed in Table 1.

[0121] Table 1

[0122] Note: Compared with Example 1, # P < 0.05 ## P < 0.01.

[0123] As shown in Table 1, the crocodile bone collagen peptides prepared by the specific preparation method of the present invention have a good effect on increasing bone density and bone calcium content. Compared with Example 1, Comparative Examples 1-5 changed the deodorizing agent, enzymatic hydrolysis, molecular weight control and overall preparation method, and the effect of the products on increasing bone density and bone calcium content was significantly reduced.

[0124] Test Example 2 Determination of the content and corresponding molecular weight of crocodile bone collagen peptides The detection method for crocodile bone collagen peptides in Examples 1-3 and Comparative Examples 1-5 was performed according to Appendix B of GB / T 22492-2008; the determination method for the relative molecular weight distribution of the crocodile bone collagen peptides was performed according to Appendix A of GB / T 22492-2008, and the test results are shown in Table 2. Table 2

[0125] As shown in Table 2, the crocodile bone collagen peptides prepared by the specific preparation method of the present invention have a high collagen peptide content, and are concentrated in the small molecular weight range of 600-800 Da, which is conducive to absorption. Compared with Example 1, Comparative Examples 4-5, in terms of molecular weight control and overall preparation method, although the collagen peptide content of their products is still acceptable, they cannot obtain crocodile bone collagen peptides with a specific molecular weight of 600-800 Da.

[0126] Test Example 3 Taste test: Test Method: The products prepared in Examples 1-3 and Comparative Examples 1-5 were used as the research subjects. The irritation, bitterness, and acceptability after trial were scored. In this trial, Examples 1-3 and Comparative Examples 1-5 were divided into 8 groups, with 5 people selected from each group for testing. The participants were healthy individuals aged 18-45 years with no history of smoking or alcohol abuse. The scoring criteria are shown in Table 3. Irritation was scored from 1 to 10, with higher scores indicating stronger irritation; bitterness was scored from 1 to 10, with higher scores indicating stronger bitterness; and acceptability was scored from 1 to 10, with higher scores indicating stronger acceptability. The average score was calculated, and the final trial results are shown in Table 4. Table 3

[0127] Table 4

[0128] As shown in Table 4, the collagen peptides prepared by the specific preparation method of this invention have significantly improved taste, are non-irritating and bitter, and are more acceptable.

[0129] Test Example 4 stability (1) Testing standards: Refer to the "Stability Test Procedures and Methods for Health Foods" and GB / T 22492-2008 "Collagen Peptides"; (2) Test conditions: Sensory indicators, moisture content and collagen peptide content of Examples 1-3 and Comparative Examples 1-5 were tested at 0 months and 3 months at 37±2℃ and 75%±5% relative humidity. Three independent batches of samples were taken from each group for testing.

[0130] Detection indicators and methods: Sensory indicators: color, odor, texture (GB / T 22492-2008).

[0131] Collagen peptide content: Appendix B of GB / T 22492-2008.

[0132] The specific results are shown in Table 5.

[0133] Table 5

[0134] As shown in Table 5, the collagen peptides prepared by the specific preparation method of the present invention have significantly improved stability, no sensory changes after 3 months of storage, and a higher retention rate of collagen peptides.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing crocodile bone collagen peptide powder to improve bone density, characterized in that, Includes the following steps: S1: Degreasing: After crushing the crocodile bone, mix it with a 2-5 wt% sodium chloride solution and stir. Separate once to obtain bone residue; mix the bone residue with water and separate a second time to obtain degreased bone residue; S2: Deodorization: Mix defatted bone meal with water, add deodorizing agent, stir, separate, and obtain deodorized bone meal; S3: Non-decalcified low-temperature extraction: Mix deodorized bone residue with water, and extract with ultrasound assistance at 40-50℃ for 20-40 minutes. Separate and collect the supernatant. S4: Enzymatic hydrolysis: Mix the supernatant with collagenase and trypsin, control the pH to 7.8-8.0 and the temperature to 40-45℃ for the first enzymatic hydrolysis; adjust the pH to 7.0-7.2 and the temperature to 50-55℃, add carboxypeptidase A and proline endopeptidase for the second enzymatic hydrolysis; inactivate the enzymes, separate the supernatant, adjust the pH to 6.0-6.5, add transglutaminase for the third enzymatic hydrolysis, inactivate the enzymes, separate, and obtain the enzymatic hydrolysate; S5: The enzymatic hydrolysate is separated by a multi-stage tangential flow membrane separation system, passing sequentially through a 1000 Da ultrafiltration membrane and a 500 Da nanofiltration membrane. The retentate from the 500 Da nanofiltration membrane is collected, concentrated, and dried to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In step S1, the pulverization includes the following steps: homogenizing 1-3 times under a pressure of 20-25 MPa after pulverization; the mass ratio of crocodile bone to sodium chloride solution is 1:4-8; the stirring time is 3-5 h and the temperature is 30-40℃; the primary and secondary separations are both centrifugation at a speed of 3000-5000 r / min for 5-15 min; the mass ratio of bone residue to water is 1:4-8.

3. The preparation method according to claim 1, characterized in that, In step S2, the mass ratio of defatted bone residue to water is 1:5-10; the deodorizing agent is a mixture of tangerine peel extract, chitosan oligosaccharide, and hydroxypropyl-β-cyclodextrin in a mass ratio of 1-3:0.5-1.5:0.1-0.3; the molecular weight of the chitosan oligosaccharide is 1000-2000 Da; the mass of the deodorizing agent is 0.5%-1.5% of the mass of the defatted bone residue; the separation is performed by centrifugation at a speed of 3000-5000 r / min for 5-15 min.

4. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of the deodorized bone residue to water is 1:3-6; the power of the ultrasound-assisted extraction is 200W-600W.

5. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of collagenase to trypsin is 1:5-8; the mass of collagenase and trypsin is 1%-3% of the mass of crocodile bone in step S1.

6. The preparation method according to claim 1, characterized in that, In step S4, the mass ratio of carboxypeptidase A to proline endonuclease is 1-3:1; the mass of carboxypeptidase A and proline endonuclease is 0.5%-1.5% of the mass of crocodile bone in step S1.

7. The preparation method according to claim 1, characterized in that, In step S4, the mass of the transglutaminase is 0.1%-0.5% of the mass of the crocodile bone in step S1.

8. The preparation method according to claim 1, characterized in that, In step S4, the third enzymatic hydrolysis takes 30-60 minutes.

9. Crocodile bone collagen peptide powder prepared by the preparation method according to any one of claims 1-8.

10. The use of crocodile bone collagen peptide powder prepared by the preparation method according to any one of claims 1-8 in the preparation of products that improve bone density.

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