Palmon cynaroides bone collagen peptide capable of promoting longitudinal growth of bones as well as preparation method and application thereof

Abstract

The invention provides king salmon bone collagen peptide for promoting longitudinal growth of bones as well as a preparation method and application thereof, and relates to the technical field of collagen peptide. The preparation method comprises the following steps: (1) carrying out steam explosion treatment on a king salmon bone raw material to obtain pretreated king salmon bones; then mixing with pepsin, and carrying out primary enzymolysis under a pressurizing condition to obtain a primary enzymolysis mixture; and mixing the first enzymolysis mixture with collagenase and ficin, carrying out second enzymolysis under a pressurization condition, collecting an enzymatic hydrolysate, and carrying out ultrafiltration on the enzymatic hydrolysate to obtain the collagen peptide. Under the pressurizing condition, pepsase is used for enzymolysis for the first time, then the pepsase is compounded with collagenase and ficin protease, collagen is subjected to sufficient enzymolysis through the synergistic effect of the three proteases, the diversity of cleavage sites is improved, the molecular weight of polypeptide is reduced, the activity of the polypeptide is improved, longitudinal growth of bones is promoted, and the effect of strengthening the bone is achieved. The bone density is improved.

Description

Technical Field

[0001] The present invention belongs to the field of collagen peptides, and in particular relates to a salmon collagen peptide for promoting vertical bone growth, a preparation method and an application thereof. Background Art

[0002] Collagen peptides are hydrolyzed products of collagen. Their smaller molecular weight makes them more readily absorbed by the body than collagen itself. After protein digestion, they are primarily absorbed directly as peptides. Collagen peptides have antioxidant, anti-tumor, blood pressure-lowering, liver-protecting, calcium-boosting, hormone-regulating, and skin-beautifying properties.

[0003] Compared to terrestrial collagen, marine collagen is less polluting and has a lower molecular weight, making it easier for the human body to absorb. Extensive research is currently underway on the extraction of marine collagen. Chinese invention patent CN105925649A discloses a method for preparing low-molecular-weight, defatted squid protein functional peptides, which includes pretreatment, decolorization, and enzymatic hydrolysis and pressurization. The pretreatment step involves removing the squid's internal organs to obtain squid plates; the plates are then scored and crushed to produce minced squid fragments. The decolorization and fixation step involves ultrasonic decolorization and fixation of the squid fragments, followed by extraction to produce a squid protein extract. The enzymatic hydrolysis and pressurization step involves sequentially enzymatic hydrolysis and pressurization of the squid protein extract to produce the active peptides. The enzymatic hydrolysis process involves first adjusting the squid protein extract to an alkaline state, then adding alkaline protease for hydrolysis for 1-3 hours at 55-65°C. The enzyme is then inactivated to produce a squid protein hydrolyzate. The molecular weight of the active peptides obtained by this method is mostly below 3KD, and the oligopeptide content is around 80%-85%.

[0004] Chinese invention patent CN112062834A discloses a method for extracting and preparing deep-sea fish skin collagen peptides, comprising the following steps: S1, pre-treating the deep-sea fish skin to remove the fishy smell; S2, beating 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, significantly reducing the amount of protein in the fish skin to produce peptides, thereby increasing the final protein peptide extraction rate.

[0005] Currently, most methods for preparing marine collagen peptides focus on improving the extraction yield or reducing the molecular weight of the peptides, while little attention has been paid to the effects of marine collagen peptides on bone growth and development. After absorption by the human body, collagen is broken down into small amino acids, which serve as the raw materials for the synthesis of the body's own collagen. During bone growth, osteoblasts use these raw materials to synthesize new collagen, forming bone matrix fibers. These fibers then combine with minerals such as calcium and phosphorus to form bone, promoting bone growth and development. Alternatively, collagen and its breakdown products (such as peptides) can stimulate the proliferation and differentiation of osteoblasts through signaling pathways, enhancing their ability to synthesize bone matrix (such as collagen and osteocalcin), thereby promoting new bone formation. Collagen peptides can also inhibit the differentiation and activity of osteoclasts, reducing bone resorption and maintaining balanced bone metabolism.

[0006] The organic matter in bones is primarily composed of proteins (such as collagen), which are important raw materials for bone growth and repair. Peptide chains with specific endpoints or small peptides are more likely to promote bone growth. Furthermore, the vertebrae of marine organisms are often considered low-value raw materials. Even if enzymatic hydrolysis is used, the degree of enzymatic degradation is limited due to the dense structure of the raw materials.

[0007] Therefore, it is necessary to find a marine biological bone spine raw material rich in high-quality protein, perform specific extraction and enzymatic hydrolysis on its protein, and obtain a collagen peptide product rich in low molecular weight protein peptides, so that the protein peptide product can be easily absorbed and utilized, provide support for the synthesis of organic matter in bones, enhance the toughness and strength of bones, promote the longitudinal growth of bones, and improve bone density. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides a salmon bone collagen peptide for promoting longitudinal bone growth, as well as a preparation method and application. The salmon bones are subjected to steam explosion treatment to break the dense physical structure of the salmon bones, which is beneficial to the denaturation of proteins and the dissolution of sugars. Under pressurized conditions, pepsin is used for the first enzymatic hydrolysis, and then the peptide is compounded with collagenase and ficin. The synergistic effect of the three proteases ensures that the collagen is fully enzymatically hydrolyzed, and the diversity of cleavage sites is increased, the molecular weight of the polypeptide is reduced, the activity of the polypeptide is increased, more active sites are exposed, the longitudinal growth of bones is promoted, and bone density is significantly improved.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: First, the present invention provides a method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the steps of: (1) steam explosion-treating the king salmon bone raw material and crushing it to obtain pretreated king salmon bone; (2) mixing the pretreated king salmon bones with pepsin, and performing a first enzymatic hydrolysis under pressure to obtain a first enzymatic hydrolysis mixture; (3) The first enzymatic hydrolysis mixture is mixed with collagenase and ficin, and a second enzymatic hydrolysis is performed under pressure, and the enzymatic hydrolysis solution is collected and ultrafiltered to obtain collagen peptides; Pepsin, collagenase and ficin constitute a complex protease.

[0010] Preferably, in step (1), the king salmon bone raw material is king salmon bone after removing foreign proteins and defatting; the treatment of removing foreign proteins and defatting is a conventional treatment method in the art: after steaming the fish bones, add them to a sodium chloride solution, stir at 2-5°C overnight, wash with clean water, and drain; mix the king salmon bones after removing foreign proteins with an ethanol solution, stir at 2-5°C for 12-24 hours, wash with clean water, and drain.

[0011] Preferably, in step (1), the steam explosion treatment is specifically: 180-210°C, 1.2-1.8 MPa, maintaining pressure for 5-15 min, and instantaneous pressure release.

[0012] Further preferably, in step (1), the steam explosion treatment is specifically: 190° C., 1.6 MPa pressure maintenance for 10 min, and instantaneous pressure release.

[0013] Preferably, in step (1), the particle size of the crushed material is 100-300 mesh.

[0014] Further preferably, in step (1), the particle size of the crushed product is 200 mesh.

[0015] Preferably, in step (2) and step (3), the pressurization condition is: 5-10 MPa.

[0016] Further preferably, in step (2) and step (3), the pressurization condition is: 6-8 MPa.

[0017] Preferably, in step (2), the enzymatic hydrolysis conditions for the first time are as follows: the enzymatic hydrolysis temperature is 35-43°C, the enzymatic hydrolysis time is 3-6 hours, the amount of pepsin is 30-40 U / g (pretreated king salmon bones), the enzymatic hydrolysis solid-liquid ratio is 1 g:15-30 mL (water), and the enzymatic hydrolysis pH is 2-3.5.

[0018] Further preferably, in step (2), the enzymatic hydrolysis conditions for the first time are as follows: the enzymatic hydrolysis temperature is 40° C., the enzymatic hydrolysis time is 5 h, the amount of pepsin is 35 U / g, the enzymatic hydrolysis solid-liquid ratio is 1 g:20 mL, and the enzymatic hydrolysis pH is 2.4.

[0019] Preferably, in step (2), during the first enzymatic hydrolysis, acid needs to be 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.

[0020] Preferably, in step (3), the second enzymatic hydrolysis is carried out under the following conditions: enzymatic hydrolysis temperature is 40-50°C, enzymatic hydrolysis time is 4-10h, the amount of collagenase is 30-50U / g (pretreated king salmon bones), the enzymatic hydrolysis solid-liquid ratio is 1g:15-30mL (water), and the enzymatic hydrolysis pH is 6.0-7.7.

[0021] Further preferably, in step (3), the second enzymatic hydrolysis is carried out under the following conditions: enzymatic hydrolysis temperature is 42° C., enzymatic hydrolysis time is 6 h, the amount of collagenase is 40 U / g, the enzymatic hydrolysis solid-liquid ratio is 1 g:20 mL, and the enzymatic hydrolysis pH is 6.8.

[0022] Preferably, in step (3), the amount of ficin used is 10-25 U / g (mass of pretreated king salmon bones).

[0023] Further preferably, in step (3), the amount of ficin used is 20 U / g.

[0024] Preferably, in the composite protease, the usage ratio of pepsin, collagenase and ficin is 30-40:30-50:10-25, with the unit being U:U:U.

[0025] Further preferably, in the composite protease, the usage ratio of pepsin, collagenase and ficin is 35:40:20, with the unit being U:U:U.

[0026] Preferably, in step (3), during the second enzymatic hydrolysis, a base needs to be added to adjust the pH. The base is a base commonly used in the art, and is not limited to sodium carbonate, sodium bicarbonate, or sodium hydroxide.

[0027] Preferably, in step (3), the ultrafiltration uses an ultrafiltration membrane to collect collagen peptides below 10 kDa.

[0028] In the present invention, the king salmon bones are bones obtained by removing skin, meat and viscera from king salmon.

[0029] Then, the present invention provides a collagen peptide prepared by the above preparation method.

[0030] Finally, the present invention provides the use of the above collagen peptide in the preparation of food and / or medicine that promotes bone growth, enhances bone toughness and strength, and improves bone density.

[0031] Preferably, the food is ordinary food or health food.

[0032] In the present invention, the "instantaneous pressure release" refers to the instantaneous release of pressure through high-temperature and high-pressure steam to destroy the cell structure.

[0033] Compared with the prior art, the present invention has the following beneficial effects: 1. The present 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 the human body's composition pattern. As a small molecule, collagen peptides are easily absorbed and utilized, and can provide support for the synthesis of organic matter in bones, enhance the toughness and strength of bones, promote the longitudinal growth of bones, and improve bone density.

[0034] 2. The present invention adopts steam explosion instantaneous pressure release treatment, which is conducive to softening the bone tissue structure and enlarging the pores, thereby making the denatured collagen easy to mix with the protease, thereby effectively exerting the enzymatic effect of the protease; in addition, during the enzymatic hydrolysis process, the triple helix structure of the collagen is further compressed and broken due to the pressure, and converted into a random coil structure that is easier to hydrolyze; at the same time, high pressure can change the spatial conformation of the enzyme, enhance its catalytic activity, thereby improving the enzymatic hydrolysis efficiency, reducing the molecular weight of the collagen peptide, completing the enzymatic hydrolysis at a lower temperature, and ensuring the activity of the collagen peptide product.

[0035] 3. The present invention uses three specific enzymes to perform two enzymatic hydrolysis treatments. Pepsin, collagenase and ficin work synergistically with each other to improve the enzymatic hydrolysis efficiency under mild conditions, ensuring a high yield of collagen peptides while reducing the molecular weight of collagen peptides, increasing the activity of collagen peptides, exposing more active sites, and promoting the growth of osteoblasts and the longitudinal growth of bones. DETAILED DESCRIPTION

[0036] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0037] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified herein, 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 herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.

[0038] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels. The products of different manufacturers did not significantly affect the effects.

[0039] In the following embodiments of the present invention, the king salmon bone raw material is the bones of king salmon after the skin, meat, and internal organs are removed; the bones are subjected to a process of removing foreign proteins and defatting: the fish bones are steamed, added to a sodium chloride solution, stirred at 2-5°C overnight, washed with clean water, and drained; the king salmon bones from which foreign proteins have been removed are mixed with an ethanol solution, stirred at 2-5°C for 12-24 hours, washed with clean water, and drained.

[0040] Example 1 A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the following steps: (1) The king salmon bone raw material was subjected to steam explosion treatment: 190°C, 1.6 MPa pressure maintenance for 10 min, and instantaneous pressure release; then the raw material was crushed and passed through a 200-mesh sieve to obtain the pretreated king salmon bone; (2) The pretreated salmon bones were mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis was 5 h, the amount of pepsin was 35 U / g (mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (mass of the pretreated salmon bones: volume of water), and malic acid was used to adjust the enzymatic hydrolysis pH to 2.4; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 42°C, the enzymatic hydrolysis time was 6 h, the amount of collagenase was 40 U / g (the mass of the pretreated king salmon bone), the amount of ficin was 20 U / g (the mass of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (the mass of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH was 6.8 using sodium hydroxide solution; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0041] Example 2 The difference from Example 1 is that the amount of the composite protease is different.

[0042] A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the following steps: (1) The king salmon bone raw material was subjected to steam explosion treatment: 190°C, 1.6 MPa pressure maintenance for 10 min, and instantaneous pressure release; then the raw material was crushed and passed through a 200-mesh sieve to obtain the pretreated king salmon bone; (2) The pretreated salmon bones were mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis was carried out for 5 h, the amount of pepsin was 30 U / g (mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (mass of the pretreated salmon bones: volume of water), and malic acid was used to adjust the enzymatic hydrolysis pH to 2.4; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 42°C, the enzymatic hydrolysis time was 6 h, the amount of collagenase was 50 U / g (the mass of the pretreated king salmon bone), the amount of ficin was 10 U / g (the mass of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (the mass of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH of the sodium hydroxide solution was 6.8; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0043] Example 3 The difference from Example 1 is that the amount of the composite protease is different.

[0044] A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the following steps: (1) The king salmon bone raw material was subjected to steam explosion treatment: 190°C, 1.6 MPa pressure maintenance for 10 min, and instantaneous pressure release; then the raw material was crushed and passed through a 200-mesh sieve to obtain the pretreated king salmon bone; (2) The pretreated salmon bones were mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis was 5 h, the amount of pepsin was 40 U / g (mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (mass of the pretreated salmon bones: volume of water), and malic acid was used to adjust the enzymatic hydrolysis pH to 2.4; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under a pressure of 7±1 MPa: the enzymatic hydrolysis temperature was 42°C, the enzymatic hydrolysis time was 6 h, the amount of collagenase was 30 U / g (the mass of the pretreated king salmon bone), the amount of ficin was 25 U / g (the mass of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (the mass of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH was 6.8 using sodium hydroxide solution; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0045] Example 4 A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the following steps: (1) The king salmon bone raw material was subjected to steam explosion treatment: 180°C, 1.2MP pressure for 15 minutes, and instantaneous pressure release; then the raw material was crushed and passed through a 300-mesh sieve to obtain the pretreated king salmon bone; (2) The pretreated salmon bones were mixed with pepsin and the first enzymatic hydrolysis was carried out under a pressure of 6±1 MPa: the enzymatic hydrolysis temperature was 35°C, the enzymatic hydrolysis was carried out for 6 h, the amount of pepsin was 35 U / g (the mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:30 mL (the mass of the pretreated salmon bones: the volume of water), and citric acid was used to adjust the enzymatic hydrolysis pH to 2; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under a pressure of 6±1 MPa: the enzymatic hydrolysis temperature was 50°C, the enzymatic hydrolysis time was 4 h, the amount of collagenase was 40 U / g (the mass of the pretreated king salmon bone), the amount of ficin was 20 U / g (the mass of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1 g:30 mL (the mass of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH was 7.7 using sodium hydroxide solution; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0046] Example 5 A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, comprising the following steps: (1) The king salmon bone raw material was subjected to steam explosion treatment: 210°C, 1.8 MPa pressure maintenance for 5 min, and instantaneous pressure release; then the raw material was crushed and passed through a 100-mesh sieve to obtain the pretreated king salmon bone; (2) The pretreated salmon bones were mixed with pepsin and subjected to the first enzymatic hydrolysis under a pressure of 8±1 MPa: the enzymatic hydrolysis temperature was 43°C, the enzymatic hydrolysis was 3 h, the amount of pepsin was 35 U / g (the mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:15 mL (the mass of the pretreated salmon bones: the volume of water), and acetic acid was used to adjust the enzymatic hydrolysis pH to 3.5; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under a pressure of 8±1 MPa: the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis time was 10 h, the amount of collagenase was 40 U / g (the mass of the pretreated king salmon bone), the amount of ficin was 20 U / g (the mass of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1 g:15 mL (the mass of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH of the sodium carbonate solution was 6.0; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0047] Comparative Example 1 The difference from Example 1 is that the steam explosion treatment in step (1) is replaced by ordinary heating steam treatment, specifically: The king salmon bone raw material was placed in boiling water steam and steamed for 1 hour, then crushed and passed through a 200-mesh sieve to obtain pre-treated king salmon bones. Steps (2) to (3) were the same as in Example 1.

[0048] Comparative Example 2 Different from Example 1, the pressurization conditions in step (2) and step (3) are replaced by ultrasonic treatment.

[0049] (1) Same as Example 1; (2) The pretreated salmon bones were mixed with pepsin and subjected to the first enzymatic hydrolysis under ultrasonic conditions (ultrasonic treatment was performed for 1 minute every 4 minutes): the enzymatic hydrolysis temperature was 40°C, the enzymatic hydrolysis time was 5 hours, the amount of pepsin was 35 U / g (mass of the pretreated salmon bones), the enzymatic hydrolysis solid-liquid ratio was 1 g:20 mL (mass of the pretreated salmon bones: volume of water), and malic acid was used to adjust the enzymatic hydrolysis pH to 2.4; the first enzymatic hydrolysis mixture was obtained; (3) The first enzymatic hydrolysis mixture was mixed with collagenase and ficin, and the second enzymatic hydrolysis was carried out under ultrasonic conditions (ultrasonic treatment was performed for 1 minute every 4 minutes): the enzymatic hydrolysis temperature was 42°C, the enzymatic hydrolysis time was 6 hours, the amount of collagenase was 40 U / g (the weight of the pretreated king salmon bone), the amount of ficin was 20 U / g (the weight of the pretreated king salmon bone), the enzymatic hydrolysis solid-liquid ratio was 1g:20mL (the weight of the pretreated king salmon bone: the volume of water), and the enzymatic hydrolysis pH of sodium hydroxide solution was 6.8; The enzymatic hydrolysate was collected and the collagen peptides below 10 kDa were collected using an ultrafiltration membrane.

[0050] Comparative Example 3 The difference from Example 1 is that collagenase is replaced by trypsin. The rest is the same as Example 1.

[0051] Comparative Example 4 The difference from Example 1 is that ficin is replaced by papain. The rest is the same as Example 1.

[0052] Comparative Example 5 The difference from Example 1 is that the amount of the composite protease is different.

[0053] The dosage of pepsin was 50 U / g (weight of king salmon bones after pretreatment); The dosage of collagenase was 15 U / g (weight of king salmon bones after pretreatment); The dosage of ficin was 30 U / g (weight of king salmon bones after pretreatment).

[0054] The rest are the same as in Example 1.

[0055] Test 1 Collagen peptide molecular weight The molecular weight of the collagen peptides in each example and comparative example was tested according to the high performance size exclusion chromatography (HPSEC) method specified in Appendix A of GB 31645-2018. The relative molecular mass distribution results are shown in Table 1.

[0056] Table 1

[0057] As can be seen from Table 1, the preparation method of the present invention, which uses a specific steam explosion treatment, pressurized enzymatic hydrolysis treatment, and the selection of three components, pepsin, collagenase, and ficin, for enzymatic hydrolysis, can thoroughly enzymatically hydrolyze king salmon bones, obtaining collagen peptides with a high proportion of small molecular weight, thereby increasing the activity of the polypeptide components and facilitating absorption. Compared with Comparative Examples 1 and 2, which use conventional steam treatment and ultrasonic enzymatic hydrolysis, the preparation method of the present invention can promote the close catalytic binding of proteases and proteins, thereby facilitating enzymatic hydrolysis to obtain small-molecule polypeptides. The three proteases, pepsin, collagenase, and ficin, of the present invention cooperate with each other to improve the enzymatic hydrolysis effect, further breaking down large-molecule proteins and polypeptides into small-molecule polypeptides.

[0058] Experiment 2 Effects on the Growth of Osteoblasts Experimental methods: Step 1: Osteoblasts (Procell CL-0202) were cultured in a cell culture incubator for 3 hours, then the culture medium was changed and cultured for another 24 hours. The culture medium was then removed and digested with 0.25% trypsin for 5 minutes. The digestion was terminated by adding an equal volume of cell culture medium. The cells were harvested into a 10 mL centrifuge tube and centrifuged at 1200 rpm for 3 minutes. The supernatant was removed and fresh cell culture medium was added, pipetting evenly. The cells were evenly distributed among three culture dishes and then topped up with culture medium. When the cells reached 80% confluence, the above procedure was repeated for passage. The osteoblast culture medium used was a commercially available Saos-2 cell-specific culture medium.

[0059] Step 2: Effect of collagen peptide on cell activity: After cells are fully plated, collect the cells into a 10 mL centrifuge tube, centrifuge at 1200 rpm for 3 min, remove the supernatant, add new cell culture medium, pipette evenly and count with a hemocytometer. 4cells in a total volume of 200 μL and cultured overnight at 37°C and 5% CO2. The collagen peptides of each embodiment and comparative example were then added to a final concentration of 800 μg / mL, 4 replicates were set for each concentration, and the polypeptide treatment time was set to 24 hours. 400 μM hydrogen peroxide was then used for treatment for 3 hours, and then a culture medium containing 10% CCK-8 was added to each well for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and then the cell activity was calculated. No hydrogen peroxide was added to the blank control group; the control group was treated with 400 μM hydrogen peroxide and no protein peptide was added for treatment.

[0060] The results of osteoblast activity in each group are shown in Table 2.

[0061] Table 2

[0062] In Table 2, there are significant differences compared with the control group. △ P<0.05, △△ P < 0.01; there is a significant difference between the comparative example group and the example 1 group. # P<0.05, ## P<0.01.

[0063] As can be seen from Table 2, the collagen peptide obtained by the preparation method of the present invention can significantly improve damaged osteoblasts and promote the growth of osteoblasts due to the high content of small molecule active ingredients and the small molecule peptides exposing more active sites. The cell survival rate is significantly better than that of the control example.

[0064] Experiment 3: Bone growth promotion test 1. Experimental Animals: Sixty healthy male mice of the FVB / N inbred strain, aged 2-3 months, were fed a normal diet in an SPF-grade enclosure for three days before establishing an osteoporosis model. The mice were randomly divided into 12 groups: a normal group, a model group, groups treated with Examples 1-5, and groups treated with Comparative Examples 1-5. Except for the normal group, the remaining 11 groups were gavaged daily with 150 mg / kg of retinoic acid for two weeks to establish an osteoporosis model.

[0065] After the retinoic acid-induced osteoporosis model was established, the mice in the normal group and the model group were given free drinking water every day in addition to the normal diet; the Example 1-Example 5 groups and the Comparative Example 1-Comparative Example 5 groups were fed with 0.6g / 20g of collagen peptide every day in addition to the normal diet and drinking water, and the feeding time lasted for 6 weeks.

[0066] 2. Measurement of mouse femur length After 3 weeks of feeding, the mice were killed, and their femurs were excised and dried in an oven. The femurs were measured with a vernier caliper and the lengths were recorded. The femur length results are shown in Table 3.

[0067] Table 3

[0068] In Table 3, there are significant differences compared with the model group. △ P<0.05, △△ P < 0.01; there is a significant difference between the comparative example group and the example 1 group. # P<0.05.

[0069] As can be seen from Table 3, the preparation method of the present invention is easier to enzymatically hydrolyze the raw materials; the obtained collagen peptides, compared with the collagen peptides prepared by the preparation method of the comparative example, expose more active sites and are more conducive to promoting the growth of bones and related cells.

[0070] Experiment 4: Detection of bone density improvement effect 1.1 Experimental animals and environment Healthy SPF female SD rats, weighing 300 ± 20 g, 3 months old.

[0071] 1.2 Animal grouping and drug administration The basic formula of estrogen-free feed refers to AIN-93G feed.

[0072] After one week of adaptive feeding, the experimental animals were randomly divided into groups based on body weight and then underwent ovariectomy to establish an osteoporosis model. Five days after surgery, blood was collected from the tail tip to measure serum estradiol levels to determine model success. Successful model rats were randomly divided into 12 groups of 5 rats each: sham-operated group with saline solution; ovariectomized group with saline solution; ovariectomized group with collagen peptides from the example (5 groups in total); and ovariectomized group with collagen peptides from the comparative example (5 groups in total). Collagen peptides were administered orally daily at a dose of 500 mg / kg for a total of 90 days.

[0073] After the gavage period, the rats were killed by cervical dislocation, and their femurs were removed. The proximal femoral bone density (BMD) of the rat left femurs was measured using a Perkin Elmer Quantum GX micro-CT. BMD results for each group are shown in Table 4.

[0074] Table 4

[0075] In Table 4, there are significant differences compared with the model group. △△ P < 0.01; there is a significant difference between the comparative example group and the example 1 group. ##P<0.01.

[0076] As can be seen in Table 4, the bone density of the rats in the model group was significantly lower than that in the sham-operated group; the improvement effect in the Example group was significant, and was even better than that in the Control Group, significantly increasing the bone density of the rats. This indicates that the collagen peptide prepared by the preparation method of the present invention has a significant effect on improving bone density.

[0077] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing king salmon collagen peptide for promoting longitudinal bone growth, characterized in that: Including steps: (1) steam explosion-treating the king salmon bone raw material and crushing it to obtain pretreated king salmon bone; (2) mixing the pretreated king salmon bones with pepsin, and performing a first enzymatic hydrolysis under pressure to obtain a first enzymatic hydrolysis mixture; (3) The first enzymatic hydrolysis mixture is mixed with collagenase and ficin, and a second enzymatic hydrolysis is performed under pressure, and the enzymatic hydrolysis solution is collected and ultrafiltered to obtain collagen peptides; Pepsin, collagenase and ficin form a composite protease, and the dosage ratio of pepsin, collagenase and ficin is 30-40:30-50:10-25, and the unit is U:U:U.

2. The preparation method according to claim 1, characterized in that In step (1), the steam explosion treatment is specifically as follows: 180-210°C, 1.2-1.8 MPa, maintaining pressure for 5-15 min, and instantaneous pressure release; the crushed particle size is 100-300 mesh.

3. The preparation method according to claim 2, characterized in that In step (1), the steam explosion treatment is specifically as follows: 190° C., 1.6 MPa pressure maintenance for 10 min, and instantaneous pressure release; the crushed particle size is 200 mesh.

4. The preparation method according to claim 1, characterized in that In step (2) and step (3), the pressurization condition is: 5-10 MPa.

5. The preparation method according to claim 1, characterized in that In step (2), the first enzymatic hydrolysis is carried out under the following conditions: enzymatic hydrolysis temperature is 35-43°C, enzymatic hydrolysis time is 3-6h, the amount of pepsin is 30-40U / g, the enzymatic hydrolysis solid-liquid ratio is 1g:15-30mL, and the enzymatic hydrolysis pH is 2-3.

5.

6. The preparation method according to claim 1, characterized in that In step (3), the second enzymatic hydrolysis is carried out under the following conditions: enzymatic hydrolysis temperature is 40-50°C, enzymatic hydrolysis time is 4-10h, the amount of collagenase is 30-50U / g, the enzymatic hydrolysis solid-liquid ratio is 1g:15-30mL, and the enzymatic hydrolysis pH is 6.0-7.

7.

7. The preparation method according to claim 1, characterized in that In step (3), the dosage of ficin is 10-25 U / g.

8. The preparation method according to claim 1, characterized in that In the composite protease, the usage ratio of pepsin, collagenase and ficin is 35:40:20, and the unit is U:U:U.

9. The collagen peptide prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the collagen peptide according to claim 9 in the preparation of food and / or medicine having the effects of promoting bone growth, enhancing bone toughness and strength, and improving bone density.

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