King salmon collagen peptide for promoting longitudinal growth of bone and preparation method and application thereof

Abstract

The application provides king salmon bone collagen peptide for promoting longitudinal growth of bone, a preparation method and application, and relates to the technical field of collagen peptides. The preparation method comprises the following steps: (1) treating king salmon bone raw materials by steam explosion to obtain pretreated king salmon bone; then mixing the pretreated king salmon bone with pepsin and performing first enzymolysis under pressure to obtain a first enzymolysis mixture; mixing the first enzymolysis mixture with collagenase and ficin, performing second enzymolysis under pressure, collecting an enzymolysis liquid, and performing ultrafiltration on the enzymolysis liquid to obtain collagen peptide. In the application, pepsin is used for first enzymolysis under pressure, then collagenase and ficin are used for compounding, the synergistic effect of the three kinds of proteases makes the collagen be fully enzymolyzed, the diversity of the cleavage site is improved, the molecular weight of the polypeptide is reduced, the activity of the polypeptide is improved, the longitudinal growth of the bone is promoted, and the bone density is improved.

Description

Technical Field

[0001] This invention belongs to the field of collagen peptides, specifically relating to king salmon bone collagen peptides that promote longitudinal bone growth, their preparation methods, and applications. Background Technology

[0002] Collagen peptides are hydrolyzed products of collagen. They have a smaller molecular weight and are more easily absorbed by the human body than collagen itself, which is primarily absorbed directly in the form of polypeptides after protein digestion. Collagen peptides have various effects, including antioxidant, anti-tumor, blood pressure lowering, liver protection, calcium absorption promotion, hormone level regulation, and skin care benefits.

[0003] Compared to terrestrial animal collagen, marine collagen has less contamination and a smaller molecular weight, making it easier for the human body to absorb. Currently, extensive research has been conducted on the extraction of collagen from marine animals. Chinese invention patent CN105925649A discloses a method for preparing functional active peptides from low-molecular-weight defatted squid protein, including pretreatment, decolorization, and enzymatic hydrolysis under pressure. The pretreatment step involves removing the internal organs of the squid to obtain squid plates; these plates are then cut and crushed to obtain minced squid pieces. The decolorization and fixation step involves ultrasonic decolorization and ultrasonic fixation of the squid pieces, followed by extraction to obtain a squid protein extract. The enzymatic hydrolysis and pressure treatment step involves sequentially enzymatically hydrolyzing and pressurizing the squid protein extract to obtain the active peptides. In the enzymatic hydrolysis process of this invention, the squid protein extract is first adjusted to alkalinity, and alkaline protease is added for enzymatic hydrolysis for 1-3 hours at a temperature of 55-65℃; then the enzyme is inactivated to obtain a squid protease hydrolysate. The molecular weight of most of the active peptides obtained by this method is below 3KD, and the content of oligopeptides is around 80%-85%.

[0004] Chinese invention patent CN112062834A discloses a method for extracting and preparing collagen peptides from deep-sea fish skin, comprising the following steps: S1, pre-treating deep-sea fish skin to remove fishy odor; S2, homogenizing the fish skin treated in step S1 to obtain a deep-sea fish skin homogenate; S3, adding pepsin to the deep-sea fish skin homogenate for enzymatic hydrolysis at a pH of 1.5-3; adding papain for enzymatic hydrolysis at a pH of 5.5-7; and adding trypsin for enzymatic hydrolysis at a pH of 7.5-8.5 to obtain an enzymatic hydrolysis mixture; S4, inactivating, purifying, and drying the enzymatic hydrolysis mixture to obtain deep-sea fish skin collagen peptides. This invention utilizes three enzymes to fully hydrolyze the fish skin, which can significantly reduce the protein content in the fish skin to obtain peptides, thereby improving the final extraction rate of protein peptides.

[0005] Currently, most methods for preparing marine collagen peptides focus on improving the extraction rate or reducing the molecular weight of the peptides, with little attention paid to the impact of marine collagen peptides on bone growth and development. After being absorbed by the body, collagen is broken down into small amino acids, which are the raw materials for synthesizing the body's own collagen. During bone growth, osteoblasts use these raw materials to synthesize new collagen, forming bone matrix fibers, which then combine with minerals such as calcium and phosphorus to deposit as bone, promoting bone growth and development. Alternatively, collagen and its breakdown products (such as peptides) can stimulate the proliferation and differentiation of osteoblasts through signal transduction pathways, enhancing their ability to synthesize bone matrix (such as collagen and osteocalcin), thereby promoting new bone formation. Collagen peptides can also reduce bone resorption and maintain bone metabolic balance by inhibiting osteoclast differentiation and activity.

[0006] The organic matter in bones is mainly composed of proteins (such as collagen). Proteins are essential raw materials for bone growth and repair, and peptide chains or small polypeptides with specific endpoints are more likely to promote bone growth. In addition, the vertebrae and other structures of marine organisms are often considered low-value raw materials, and even if they are enzymatically hydrolyzed, the degree of enzymatic hydrolysis is limited due to their dense structure.

[0007] Therefore, it is necessary to find a marine organism bone and spine raw material rich in high-quality protein, and to perform specific extraction and enzymatic hydrolysis of its protein to obtain collagen peptide products rich in low molecular weight protein peptides, making the protein peptide products easy to absorb and utilize, supporting the synthesis of organic matter in bones, enhancing bone toughness and strength, promoting longitudinal bone growth, and improving bone density. Summary of the Invention

[0008] This invention addresses the problems existing in the prior art by providing a method for preparing and applying a king salmon bone collagen peptide that promotes longitudinal bone growth. The method involves steam-explosion treatment of king salmon bone to break down its dense physical structure, which facilitates protein denaturation and sugar dissolution. Under pressure, pepsin is used for the first enzymatic hydrolysis, followed by compounding with collagenase and fibrinase. The synergistic effect of these three proteases ensures thorough hydrolysis of collagen, increases the diversity of cleavage sites, reduces the molecular weight of the peptide, enhances peptide activity, exposes more active sites, promotes longitudinal bone growth, and significantly improves bone density.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] First, this invention provides a method for preparing king salmon collagen peptides that promote longitudinal bone growth, comprising the following steps:

[0011] (1) The raw material of king salmon bone is subjected to steam explosion treatment and crushed to obtain pretreated king salmon bone;

[0012] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under pressure to obtain the first enzymatic hydrolysis mixture;

[0013] (3) Mix the first enzymatic hydrolysis mixture with collagenase and figase, and carry out a second enzymatic hydrolysis under pressure. Collect the enzymatic hydrolysate, and ultrafilter the enzymatic hydrolysate to obtain collagen peptides.

[0014] The complex protease consists of pepsin, collagenase, and figase.

[0015] Preferably, in step (1), the raw material of king salmon bone is king salmon bone after removing impurities and defatting; the treatment of removing impurities and defatting is a conventional treatment method in the art: after steaming the fish bone, add it to sodium chloride solution, stir overnight at 2-5℃, wash with clean water, and drain; mix the king salmon bone after removing impurities with ethanol solution, stir at 2-5℃ for 12-24h, wash with clean water, and drain.

[0016] Preferably, in step (1), the steam explosion treatment specifically involves maintaining a pressure of 180-210℃ and 1.2-1.8MP for 5-15 minutes, followed by instantaneous pressure release.

[0017] More preferably, in step (1), the steam explosion treatment specifically involves maintaining a pressure of 1.6 MPa at 190°C for 10 minutes, followed by instantaneous depressurization.

[0018] Preferably, in step (1), the particle size of the pulverized material is 100-300 mesh.

[0019] More preferably, in step (1), the particle size of the pulverized material is 200 mesh.

[0020] Preferably, in steps (2) and (3), the pressurization conditions are 5-10 MPa.

[0021] More preferably, in steps (2) and (3), the pressurization condition is 6-8 MPa.

[0022] Preferably, in step (2), the first enzymatic hydrolysis is carried out under the following conditions: the hydrolysis temperature is 35-43℃, the hydrolysis time is 3-6h, the amount of pepsin used is 30-40U / g (pretreated king salmon bone), the solid-liquid ratio is 1g:15-30mL (water), and the hydrolysis pH is 2-3.5.

[0023] More preferably, in step (2), the first enzymatic hydrolysis is carried out under the following conditions: the hydrolysis temperature is 40°C, the hydrolysis time is 5h, the amount of pepsin used is 35U / g, the solid-liquid ratio is 1g:20mL, and the hydrolysis pH is 2.4.

[0024] Preferably, in step (2), during the first enzymatic hydrolysis, an acid is added to adjust the pH, and the acid is selected from at least one of malic acid, citric acid, acetic acid, tartaric acid, sulfuric acid, and hydrochloric acid.

[0025] Preferably, in step (3), the second enzymatic hydrolysis is carried out under the following conditions: the hydrolysis temperature is 40-50℃, the hydrolysis time is 4-10h, the amount of collagenase used is 30-50U / g (pretreated king salmon bone), the solid-liquid ratio is 1g:15-30mL (water), and the hydrolysis pH is 6.0-7.7.

[0026] More preferably, in step (3), the second enzymatic hydrolysis is carried out under the following conditions: the hydrolysis temperature is 42℃, the hydrolysis time is 6h, the amount of collagenase used is 40U / g, the solid-liquid ratio is 1g:20mL, and the hydrolysis pH is 6.8.

[0027] Preferably, in step (3), the amount of fig protease used is 10-25 U / g (weight of pretreated king salmon bone).

[0028] More preferably, in step (3), the amount of fig protease used is 20 U / g.

[0029] Preferably, in the complex protease, the ratio of pepsin, collagenase and figase is 30-40:30-50:10-25, in units of U:U:U.

[0030] More preferably, in the complex protease, the ratio of pepsin, collagenase and figase is 35:40:20, in units of U:U:U.

[0031] Preferably, in step (3), during the second enzymatic hydrolysis, an alkali needs to be added to adjust the pH. The alkali is a commonly used alkali in the art, and is not limited to sodium carbonate, sodium bicarbonate, or sodium hydroxide.

[0032] Preferably, in step (3), the ultrafiltration uses an ultrafiltration membrane to collect collagen peptides below 10 kDa.

[0033] In this invention, the king salmon bone is the bone of the king salmon after removing the skin, meat, and internal organs.

[0034] Then, the present invention provides a collagen peptide prepared by the above-described preparation method.

[0035] Finally, the present invention provides the application of the above-mentioned collagen peptides in the preparation of health foods that promote bone growth, enhance bone toughness and strength, and improve bone density.

[0036] In this invention, "instantaneous pressure release" refers to the instantaneous release of pressure through high-temperature and high-pressure steam, thereby disrupting cell structure.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. This invention uses deep-sea king salmon as the raw material for preparing collagen peptides. King salmon is rich in high-quality protein, and its amino acid composition is close to that of the human body. As a small molecule, collagen peptides are easily absorbed and utilized, which can support the synthesis of organic matter in bones, enhance the toughness and strength of bones, promote the longitudinal growth of bones, and improve bone density.

[0039] 2. This invention employs a steam explosion instantaneous depressurization process, which softens the bone tissue structure and increases its pore size. This makes the denatured collagen easier to mix with proteases, thereby maximizing the enzymatic hydrolysis effect of the proteases. Furthermore, during the hydrolysis process, the pressure applied further compresses and breaks down the triple helix structure of collagen, transforming it into a more easily hydrolyzed random coil structure. Simultaneously, high pressure alters the spatial conformation of the enzyme, enhancing its catalytic activity and thus improving hydrolysis efficiency. It also reduces the molecular weight of collagen peptides, completing the hydrolysis at a lower temperature and ensuring the activity of the collagen peptide products.

[0040] 3. This invention employs three specific enzymes for two enzymatic hydrolysis treatments: pepsin, collagenase, and figase work synergistically to improve hydrolysis efficiency under mild conditions. This ensures a high yield of collagen peptides while reducing their molecular weight, increasing their activity, exposing more active sites, and promoting osteoblast growth and longitudinal bone growth. Detailed Implementation

[0041] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0042] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0044] In the following embodiments of the present invention, the raw material of king salmon bone is the bone of king salmon after removing the skin, meat and internal organs; it is treated to remove impurities and fat: the fish bone is steamed and then added to sodium chloride solution, stirred overnight at 2-5°C, washed with clean water and drained; the king salmon bone with impurities removed is mixed with ethanol solution, stirred at 2-5°C for 12-24 hours, washed with clean water and drained.

[0045] Example 1

[0046] A method for preparing a king salmon collagen peptide that promotes longitudinal bone growth, comprising the following steps:

[0047] (1) The raw material of king salmon bone was subjected to steam explosion treatment: 190℃, 1.6MP pressure for 10min, instantaneous pressure release; then crushed and passed through a 200-mesh sieve to obtain pretreated king salmon bone;

[0048] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1MPa: the hydrolysis temperature was 40℃, the hydrolysis time was 5h, the amount of pepsin was 35U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 2.4 with malic acid; the first hydrolysis mixture was obtained.

[0049] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under a pressure of 7±1MPa: the enzymatic hydrolysis temperature was 42℃, the enzymatic hydrolysis time was 6h, the amount of collagenase was 40U / g (mass of pretreated king salmon bone), the amount of figase was 20U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and sodium hydroxide solution was used for enzymatic hydrolysis at pH 6.8;

[0050] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0051] Example 2

[0052] Unlike Example 1, the amount of complex protease used is different.

[0053] A method for preparing a king salmon collagen peptide that promotes longitudinal bone growth, comprising the following steps:

[0054] (1) The raw material of king salmon bone was subjected to steam explosion treatment: 190℃, 1.6MP pressure for 10min, instantaneous pressure release; then crushed and passed through a 200-mesh sieve to obtain pretreated king salmon bone;

[0055] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1MPa: the hydrolysis temperature was 40℃, the hydrolysis time was 5h, the amount of pepsin was 30U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 2.4 with malic acid; the first hydrolysis mixture was obtained.

[0056] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under a pressure of 7±1MPa: the enzymatic hydrolysis temperature was 42℃, the enzymatic hydrolysis time was 6h, the amount of collagenase was 50U / g (mass of pretreated king salmon bone), the amount of figase was 10U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and sodium hydroxide solution was used for enzymatic hydrolysis at pH 6.8;

[0057] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0058] Example 3

[0059] Unlike Example 1, the amount of complex protease used is different.

[0060] A method for preparing a king salmon collagen peptide that promotes longitudinal bone growth, comprising the following steps:

[0061] (1) The raw material of king salmon bone was subjected to steam explosion treatment: 190℃, 1.6MP pressure for 10min, instantaneous pressure release; then crushed and passed through a 200-mesh sieve to obtain pretreated king salmon bone;

[0062] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1MPa: the hydrolysis temperature was 40℃, the hydrolysis time was 5h, the amount of pepsin was 40U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 2.4 with malic acid; the first hydrolysis mixture was obtained.

[0063] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under a pressure of 7±1MPa: the enzymatic hydrolysis temperature was 42℃, the enzymatic hydrolysis time was 6h, the amount of collagenase was 30U / g (mass of pretreated king salmon bone), the amount of figase was 25U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and sodium hydroxide solution was used for enzymatic hydrolysis at pH 6.8;

[0064] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0065] Example 4

[0066] A method for preparing a king salmon collagen peptide that promotes longitudinal bone growth, comprising the following steps:

[0067] (1) The raw material of king salmon bone was subjected to steam explosion treatment: 180℃, 1.2MP pressure for 15min, instantaneous pressure release; then crushed and passed through a 300-mesh sieve to obtain pretreated king salmon bone;

[0068] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 6±1MPa: the hydrolysis temperature was 35℃, the hydrolysis time was 6h, the amount of pepsin was 35U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:30mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 2 with citric acid; the first hydrolysis mixture was obtained.

[0069] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under a pressure of 6±1MPa: the enzymatic hydrolysis temperature was 50℃, the enzymatic hydrolysis time was 4h, the amount of collagenase was 40U / g (mass of pretreated king salmon bone), the amount of figase was 20U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:30mL (mass of pretreated king salmon bone: volume of water), and sodium hydroxide solution was used for enzymatic hydrolysis at pH 7.7.

[0070] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0071] Example 5

[0072] A method for preparing a king salmon collagen peptide that promotes longitudinal bone growth, comprising the following steps:

[0073] (1) The raw material of king salmon bone was subjected to steam explosion treatment: 210℃, 1.8MP pressure for 5min, instantaneous pressure release; then crushed and passed through a 100-mesh sieve to obtain pretreated king salmon bone;

[0074] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 8±1MPa: the hydrolysis temperature was 43℃, the hydrolysis time was 3h, the amount of pepsin was 35U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:15mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 3.5 with acetic acid; the first hydrolysis mixture was obtained.

[0075] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under a pressure of 8±1MPa: the enzymatic hydrolysis temperature was 40℃, the enzymatic hydrolysis time was 10h, the amount of collagenase was 40U / g (mass of pretreated king salmon bone), the amount of figase was 20U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:15mL (mass of pretreated king salmon bone: volume of water), and sodium carbonate solution was used for enzymatic hydrolysis at pH 6.0;

[0076] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0077] Comparative Example 1

[0078] Unlike Example 1, the steam explosion treatment in step (1) is replaced with ordinary heated steam treatment, specifically:

[0079] The raw king salmon bone was placed in the steam of boiling water and steamed for 1 hour. Then it was crushed and passed through a 200-mesh sieve to obtain the pretreated king salmon bone. Steps (2)-(3) are the same as in Example 1.

[0080] Comparative Example 2

[0081] Unlike Example 1, the pressurization conditions in steps (2) and (3) are replaced with ultrasonic treatment.

[0082] (1) Same as Example 1;

[0083] (2) The pretreated king salmon bone was mixed with pepsin and subjected to the first enzymatic hydrolysis under ultrasonic conditions (ultrasonic treatment for 1 minute every 4 minutes): the hydrolysis temperature was 40℃, the hydrolysis time was 5h, the amount of pepsin was 35U / g (mass of pretreated king salmon bone), the solid-liquid ratio of hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and the pH of hydrolysis was adjusted to 2.4 with malic acid; the first hydrolysis mixture was obtained.

[0084] (3) The first enzymatic hydrolysis mixture was mixed with collagenase and figase, and a second enzymatic hydrolysis was carried out under ultrasonic conditions (ultrasonic treatment for 1 minute every 4 minutes): the enzymatic hydrolysis temperature was 42℃, the enzymatic hydrolysis time was 6h, the amount of collagenase was 40U / g (mass of pretreated king salmon bone), the amount of figase was 20U / g (mass of pretreated king salmon bone), the solid-liquid ratio of enzymatic hydrolysis was 1g:20mL (mass of pretreated king salmon bone: volume of water), and sodium hydroxide solution was used for enzymatic hydrolysis at pH 6.8;

[0085] Collect the enzymatic hydrolysate and use an ultrafiltration membrane to collect the collagen peptides below 10 kDa.

[0086] Comparative Example 3

[0087] The difference from Example 1 is that collagenase is replaced with trypsin. Everything else is the same as in Example 1.

[0088] Comparative Example 4

[0089] The difference from Example 1 is that fig protease is replaced with papain. Everything else is the same as in Example 1.

[0090] Comparative Example 5

[0091] Unlike Example 1, the amount of complex protease used is different.

[0092] The amount of pepsin used was 50 U / g (weight of pretreated king salmon bone).

[0093] The amount of collagenase used was 15 U / g (weight of pretreated king salmon bone).

[0094] The amount of fig protease used was 30 U / g (weight of pretreated king salmon bone).

[0095] Everything else is the same as in Example 1.

[0096] Experiment 1: Molecular weight of collagen peptides

[0097] The molecular weight of collagen peptides in each example and comparative example was determined by high performance size exclusion chromatography (HPSEC) as specified in Appendix A of GB 31645-2018. The relative molecular mass distribution results are shown in Table 1.

[0098] Table 1

[0099]

[0100] As shown in Table 1, the preparation method of this invention, employing specific steam explosion treatment, pressurized enzymatic hydrolysis, and selecting three components—pepsin, collagenase, and figase—for enzymatic hydrolysis, can thoroughly hydrolyze king salmon bones, yielding collagen peptides with a high proportion of small molecular weights, enhancing the activity of multiple polypeptide components, and making them easily absorbed. Compared to Comparative Examples 1-2, which used ordinary steam treatment and ultrasonic enzymatic hydrolysis, the preparation method of this invention can promote the tight binding and catalysis of proteases and proteins, thus facilitating enzymatic hydrolysis and yielding small-molecule polypeptides. The pepsin, collagenase, and figase of this invention work synergistically to improve the enzymatic hydrolysis effect, further breaking down large-molecule proteins and polypeptides into smaller-molecule polypeptides.

[0101] Experiment 2 Effects on osteoblast growth

[0102] Experimental methods:

[0103] Step 1: Osteoblasts (ProcellCL-0202) were cultured in a cell culture incubator for 3 hours, then the medium was changed, and the cells were cultured for another 24 hours. Afterwards, the culture medium was removed, and 0.25% trypsin was added for digestion for 5 minutes. The digestion was stopped by adding the same volume of cell culture medium. The cells were collected into 10mL centrifuge tubes, centrifuged at 1200rpm for 3 minutes, the supernatant was discarded, and fresh cell culture medium was added and mixed thoroughly. The mixture was then evenly distributed into 3 culture dishes, and the culture medium was replenished to the minimum. When the cells reached 80% coverage of the plates, the above steps were repeated for passage. The culture medium used for osteoblasts was purchased Saos-2 cell culture medium.

[0104] Step 2, Effect of collagen peptides on cell viability: After the cells were plated, they were collected into 10 mL centrifuge tubes, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, fresh cell culture medium was added, the cells were mixed thoroughly, and the cells were counted using a hemocytometer. 10 μL of collagen peptides were seeded into each well of a 96-well plate. 4 Cells were cultured overnight at 37°C with 5% CO2, with a total volume of 200 μL. Collagen peptides from each example and comparative example were then added to a final concentration of 800 μg / mL, with four replicates per concentration. The peptide treatment time was set to 24 h. Afterward, each well was treated with 400 μM hydrogen peroxide for 3 h, followed by incubation in culture medium containing 10% CCK-8 for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The blank control group received no hydrogen peroxide; the control group received 400 μM hydrogen peroxide treatment without the addition of protein peptides.

[0105] The osteoblast activity results for each group are shown in Table 2.

[0106] Table 2

[0107]

[0108] Table 2 shows significant differences compared to the control group. △ P < 0.05 △△ P < 0.01; the comparative example group showed a significant difference compared to the Example 1 group. # P < 0.05 ## P < 0.01.

[0109] As can be seen from Table 2, the collagen peptides obtained by the preparation method of the present invention have a high content of small molecule active ingredients and more active sites exposed by the small molecule peptides, which can significantly improve damaged osteoblasts, promote osteoblast growth, and have a cell survival rate that is significantly better than that of the comparative example.

[0110] Experiment 3: Detection of bone growth promotion

[0111] 1. Experimental Animals: Sixty healthy male FVB / N strain mice aged 2-3 months were used to establish an osteoporosis model after being fed normally and stably in an SPF-grade housing for three days. The 60 mice were randomly divided into 12 groups: normal group, model group, Examples 1-5, and Comparative Examples 1-5. Except for the normal group, the other 11 groups were administered 150 mg / kg of retinoic acid daily by gavage to establish the osteoporosis model for two weeks.

[0112] After the retinoic acid-induced osteoporosis model was established, mice in the normal group and the model group were given free access to water in addition to their normal diet. In addition to their normal diet and water, mice in Examples 1-5 and Comparative Examples 1-5 were fed with 0.6g / 20g of collagen peptides daily for 6 weeks.

[0113] 2. Measurement of mouse femur length

[0114] Mice were sacrificed after 3 weeks of feeding, and their femurs were dissected and dried in an oven. The length of the removed femurs was measured using calipers, and the results were recorded. The femur length results are shown in Table 3.

[0115] Table 3

[0116]

[0117] Table 3 shows significant differences compared to the model group. △ P < 0.05 △△ P < 0.01; the comparative example group showed a significant difference compared to the Example 1 group. # P < 0.05.

[0118] As can be seen from Table 3, the preparation method of the present invention makes it easier to enzymatically hydrolyze the raw materials; the collagen peptides obtained, compared with the collagen peptides prepared by the comparative method, expose more active sites, which is more conducive to promoting the growth of bones and related cells.

[0119] Experiment 4: Detection of bone mineral density improvement

[0120] 1.1 Laboratory Animals and Environment

[0121] Healthy SPF-grade female SD rats, weighing 300±20g, 3 months old.

[0122] 1.2 Animal grouping and drug administration

[0123] The basic formula for estrogen-free feed is based on AIN-93G feed.

[0124] After one week of acclimatization feeding, experimental animals were randomly divided into groups according to body weight, followed by ovariectomy to establish an osteoporosis model. Five days after surgery, blood was collected from the tail tip to determine serum estradiol levels, thus assessing model success. Successfully modeled rats were randomly divided into 12 groups of 5 rats each: sham-operated group + saline; ovariectomized model group + saline; ovariectomized model group + collagen peptides (5 groups total); and ovariectomized model group + collagen peptides (5 groups total). The daily gavage dose of collagen peptides was 500 mg / kg, and the entire gavage period was 90 days.

[0125] Rats were euthanized by cervical dislocation after the gavage cycle, and their femurs were harvested. The bone mineral density (BMD) of the left femur near the femur was measured using Perkin Elmer Quantum GX micro-CT after extraction. The BMD results for each group are shown in Table 4.

[0126] Table 4

[0127]

[0128] Table 4 shows significant differences compared to the model group. △△ P < 0.01; the comparative example group showed a significant difference compared to the Example 1 group. ## P < 0.01.

[0129] As shown in Table 4, the bone mineral density of the model group rats was significantly lower than that of the sham-operated group; the improvement effect of the example group was significant, and the improvement effect was better than that of the control group, significantly increasing the bone mineral density of the rats. This indicates that the collagen peptides prepared by the method of the present invention have a significant effect on improving bone mineral density.

[0130] 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 a king salmon collagen peptide that promotes longitudinal bone growth, characterized in that, Including the following steps: (1) The raw material of king salmon bone is subjected to steam explosion treatment and crushed to obtain pretreated king salmon bone; the steam explosion treatment is specifically: 180-210℃, 1.2-1.8MP pressure maintained for 5-15min, and instantaneous pressure release; (2) The pretreated king salmon bone is mixed with pepsin and subjected to the first enzymatic hydrolysis for 3-6 hours under a pressure of 5-10 MPa to obtain the first enzymatic hydrolysis mixture; the amount of pepsin used is 30-40 U / g. (3) Mix the first enzymatic hydrolysis mixture with collagenase and figase, and carry out a second enzymatic hydrolysis for 4-10 hours under a pressure of 5-10 MPa. Collect the enzymatic hydrolysate, and ultrafilter the enzymatic hydrolysate to obtain collagen peptides. The amount of collagenase used is 30-50 U / g, and the amount of figase used is 10-25 U / g. The complex protease consists of pepsin, collagenase, and figase, with a ratio of 30-40:30-50:10-25, expressed in U:U:U.

2. The preparation method according to claim 1, characterized in that, In step (1), the particle size of the pulverized material is 100-300 mesh.

3. The preparation method according to claim 1, characterized in that, In step (1), the steam explosion treatment specifically involves maintaining a pressure of 190°C and 1.6 MPa for 10 minutes, followed by instantaneous pressure release; the particle size of the pulverized material is 200 mesh.

4. The preparation method according to claim 1, characterized in that, In step (2), the first enzymatic hydrolysis is performed under the following conditions: the hydrolysis temperature is 35-43℃, the solid-liquid ratio is 1g:15-30mL, and the hydrolysis pH is 2-3.

5.

5. The preparation method according to claim 1, characterized in that, In step (3), the second enzymatic hydrolysis is carried out under the following conditions: the hydrolysis temperature is 40-50℃, the solid-liquid ratio is 1g:15-30mL, and the hydrolysis pH is 6.0-7.

7.

6. The preparation method according to claim 1, characterized in that, In the complex protease, the ratio of pepsin, collagenase and fig protease is 35:40:20, in units of U:U:U.

7. Collagen peptides prepared by the preparation method according to any one of claims 1-6.

8. The use of the collagen peptide according to claim 7 in the preparation of health food products that promote bone growth, enhance bone toughness and strength, and improve bone density.

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