Fresh bovine bone collagen peptide for supplementing joint nutrition and increasing bone density and its preparation method

Abstract

The present invention belongs to the technical field of bioactive peptide processing, and particularly relates to fresh bovine bone collagen peptide for supplementing joint nutrition and increasing bone density and a preparation method thereof. The preparation method includes enzymatic hydrolysis, and the enzymatic hydrolysis uses a compound protease including carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin; the mass ratio of the carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin is 0.5-1:0.5-1:2-4:1-3:1-3. The molecular weight of the collagen peptide prepared by the present invention is less than 5000 daltons, and this collagen peptide can effectively improve bone density, bone mineral content, bone biomechanical properties and bone metabolism indexes, and has good health care and therapeutic effects on elderly osteoporotic fracture patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive peptide processing, and particularly relates to fresh bovine bone collagen peptide for supplementing joint nutrition and increasing bone density and a preparation method thereof. Background Art

[0002] Fresh bovine bone collagen peptide is a small molecule peptide obtained by subjecting collagen extracted from bovine bone to a process treatment. It is easily absorbed by the human body and has various health effects, including beauty care, calcium supplementation and bone health, and improvement of immunity.

[0003] The extraction method of fresh bovine bone collagen peptide has a significant impact on its final efficacy. Different extraction methods not only affect the purity, molecular weight distribution and biological activity of collagen peptide, but also further affect its absorption and utilization efficiency in the human body, thus affecting its final health efficacy. The extraction of fresh bovine bone collagen peptide usually involves multiple steps such as raw material pretreatment, steaming, enzymatic hydrolysis, filtration, purification, concentration and drying. Each of these steps may affect the quality and efficacy of the final product. Specifically, it is reflected in (1) purity and molecular weight distribution: during the extraction process, through fine filtration and purification steps, impurities can be removed to improve the purity of collagen peptide. High-purity collagen peptide is more easily absorbed by the human body, thus more effectively exerting its health efficacy. Molecular weight distribution is also an important factor affecting efficacy. Smaller molecular weights mean higher biological activity because they are more easily able to cross cell membranes and enter cells to play their roles. Therefore, by controlling the enzymatic hydrolysis conditions, collagen peptide with a specific molecular weight distribution can be obtained to optimize its efficacy. (2) Retention of biological activity: During the extraction process, conditions such as high temperature, strong acid and strong base should be avoided as much as possible to prevent the destruction of the biological activity of collagen peptide. Adopting mild extraction conditions can maximize the retention of the original biological activity of collagen peptide, thus ensuring the exertion of its health efficacy. (3) Absorption and utilization: The extraction method also affects the absorption and utilization efficiency of collagen peptide in the human body. For example, collagen peptide powder obtained by methods such as spray drying has better solubility and dispersibility, and is thus more easily absorbed and utilized by the human body. (4) Selection of raw materials: In the preparation of ordinary collagen peptide, gelatin is mostly used as the raw material. Currently, the top-grade edible gelatin is made from the scraps of animal skins and bones such as cows, sheep and pigs. The raw material source is unstable and the safety is not guaranteed. Moreover, there is also the use of industrial gelatin adulterated with edible gelatin (made from the leftovers cut from tanned leather in leather factories) as the raw material, which poses a great safety hazard in the food processing field. And currently, the alkali method is generally used to produce gelatin at home and abroad. The alkali method process is specifically as follows: raw material sorting (sorting different types of raw materials), lime water pre-soaking (immersing the raw materials in lime water of a certain concentration for 7-15 days), washing and neutralization (after washing, using acid to neutralize the remaining lime, which generally takes 3-5 days to complete), glue boiling and concentration (heating the raw materials after acid-base treatment in an extraction tank to boil the glue), gel drying (the concentrated glue solution is dried and crushed to obtain the finished product). The process of preparing gelatin from animal skins or bones involves high-temperature steaming or acid-base treatment, etc. At present, the degradation law of collagen during this process is not very clear, but it can be determined that the polypeptide sequences in gelatin caused by thermal degradation or acid-base treatment processes are not completely the same; furthermore, the preparation process of gelatin also affects the amino acid composition in collagen. Commonly, phenomena such as deamidation of glutamine or asparagine occur during thermal degradation or acid treatment processes, affecting the amino acid composition of gelatin and the sequences of polypeptides therein.Finally, the collagen peptide processed with gelatin loses the smell of the substance itself, greatly reducing its nutritional value and thus significantly reducing the product efficacy.

[0004] The fresh bovine bone collagen peptide powder is prepared from fresh bovine bones through various processes such as bone cleaning, crushing, water extraction, and enzymatic hydrolysis. No chemical reagents are added during the production process, which is green and healthy, ensuring that the original nutrients of the materials are not damaged.

[0005] In summary, the extraction method of fresh bovine bone collagen peptide has an important impact on its final efficacy. At present, the extraction technology of collagen peptide is still in continuous development, but there are still problems of insufficient technological innovation. How to develop high-quality and highly active collagen peptide products to better exert their health effects is one of the urgent problems to be solved currently. Summary of the Invention

[0006] To solve the above problems, the present invention provides an extraction method of fresh bovine bone collagen peptide, and the obtained bovine bone collagen peptide has better health care effects.

[0007] On the one hand, the present invention provides a preparation method of bovine bone collagen peptide, comprising the following steps:

[0008] S1. Pretreatment: Clean and crush bovine bones to obtain bone blocks;

[0009] S2. Cooking: Cook the bone blocks obtained in step S1 to obtain bone soup;

[0010] S3. Defatting: Perform three-phase separation of bone oil, liquid, and bone residue on the bone soup obtained in step S2 to obtain a defatted crude collagen solution.

[0011] S4. Add a compound protease to the crude collagen solution obtained in step S3 and perform enzymatic hydrolysis to obtain a collagen peptide solution;

[0012] S5. Filtration: Filter the collagen peptide solution obtained in step S4 to obtain a purified collagen peptide solution;

[0013] S6. Concentration: Concentrate the purified collagen peptide solution obtained in step S5 under reduced pressure to obtain a concentrated solution;

[0014] S7. Drying: Spray-dry the concentrated solution obtained in step S6 to obtain bovine bone collagen peptide powder; The compound protease is carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin. Specifically, the bovine bones can be selected from fresh, safe, and pollution-free bovine leg bones.

[0015] Specifically, the steaming conditions described in step S2 can be: 100 - 120°C, 1.5 - 2.0 kPa.

[0016] Preferably, the steaming conditions described in step S2 can be: 100 - 110°C, 1.8 - 2.0 kPa.

[0017] More preferably, the steaming conditions described in step S2 can be: 110°C, 2.0 kPa.

[0018] Specifically, the addition amount of the compound protease described in step S4 can be 2‰ - 5‰ of the crude collagen content, such as 2‰, 3‰, 4‰, 5‰, and any value within this range.

[0019] Preferably, in some embodiments, the addition amount of the compound protease described in step S4 can be 2‰ of the weight of the crude collagen solution;

[0020] In some other embodiments, the addition amount of the compound protease described in step S4 can be 5‰ of the weight of the crude collagen solution.

[0021] Specifically, the conditions for enzymatic hydrolysis in step S4 can be 40 - 50°C, 2 - 4 h.

[0022] Preferably, the conditions for enzymatic hydrolysis in step S4 can be 40 - 45°C, 2 - 3 h.

[0023] More preferably, the enzymatic hydrolysis conditions in step S5 can be 45°C, 3 h.

[0024] Specifically, the mass ratio of the carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin can be 0.5 - 1:0.5 - 1:2 - 4:1 - 3:1 - 3.

[0025] More specifically, in some embodiments, the mass ratio of the carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin can be 0.5:0.5:4:1:1;

[0026] In some other embodiments, the mass ratio of the carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin can be 1:1:2:3:3.

[0027] Specifically, the enzyme activities of the carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin are respectively: 10,000 - 50,000 U / g, 10,000 - 100,000 U / g, 100,000 - 300,000 U / g, 50,000 - 150,000 U / g, and 2,000 - 6,000 U / g. More specifically, the enzyme activities of the carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin are respectively: 10,000 - 30,000 U / g, 10,000 - 60,000 U / g, 200,000 - 300,000 U / g, 100,000 - 150,000 U / g and 2,000 - 5,000 U / g.

[0028] Preferably, the enzyme activities of the carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin are respectively: 30,000 U / g, 60,000 U / g, 200,000 U / g, 100,000 U / g and 4,000 U / g.

[0029] Specifically, the filtration in step S5 is successively ceramic membrane filtration, nanofiltration and ultrafiltration; the molecular weight of the collagen peptide in the collagen peptide purification solution is less than 8,000 Daltons.

[0030] More specifically, the ultrafiltration can be a three-step ultrafiltration method.

[0031] Preferably, in the first step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 20,000 - 30,000 Daltons; in the second step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 12,000 - 18,000 Daltons; in the third step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 5,000 - 8,000 Daltons. More preferably, in the first step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 20,000 Daltons; in the second step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 15,000 Daltons; in the third step, ultrafiltration is carried out through an ultrafiltration membrane with a molecular weight cut-off of 5,000 Daltons.

[0032] More specifically, the molecular weight of the collagen peptide is less than 5,000 Daltons.

[0033] Specifically, the conditions for vacuum concentration in step S6 can be: 0.1 - 0.3 MPa.

[0034] More specifically, the conditions for vacuum concentration in step S6 can be: 0.1 - 0.2 MPa.

[0035] Preferably, the conditions for vacuum concentration in step S6 can be: 0.1 MPa.

[0036] Specifically, the conditions for spray drying in step S7 can be: the inlet air temperature can be 180 - 200 °C, the outlet air temperature can be 85 - 100 °C, and the pressure of the high-pressure homogenizing pump can be 60 - 80 kg.

[0037] More specifically, the conditions for spray drying in step S7 can be: the inlet air temperature can be 190 - 200 °C, the outlet air temperature can be 95 - 100 °C, and the pressure of the high-pressure homogenizing pump can be 70 - 80 kg.

[0038] Preferably, the conditions for spray drying in step S7 can be: the inlet air temperature can be 190 °C, the outlet air temperature can be 95 °C, and the pressure of the high-pressure homogenizing pump can be 70 kg.

[0039] On the other hand, the present invention provides bovine bone collagen peptides obtained by using the aforementioned preparation method.

[0040] On the other hand, the present invention provides the application of the aforementioned bovine bone collagen peptides in the preparation of drugs for supplementing joint nutrition and increasing bone density.

[0041] On the other hand, the present invention provides the application of the aforementioned bovine bone collagen peptides in the preparation of foods for supplementing joint nutrition and increasing bone density.

[0042] On the other hand, the present invention provides the application of the aforementioned bovine bone collagen peptides in the preparation of health products for supplementing joint nutrition and increasing bone density.

[0043] Technical effects achieved by the present invention: (1) The fresh bovine bone collagen peptides provided by the present invention can effectively increase the bone density and bone mineral content of ovariectomized mice.

[0044] (2) The fresh bovine bone collagen peptides provided by the present invention can effectively improve the bone biomechanical properties of ovariectomized mice.

[0045] (3) The fresh bovine bone collagen peptides provided by the present invention can effectively improve the bone metabolism indexes of elderly osteoporotic fracture patients.

[0046] (4) The fresh bovine bone collagen peptides provided by the present invention can effectively increase the bone density of elderly osteoporotic fracture patients. Detailed implementation manners

[0047] The following combines specific embodiments to further elaborate on the present invention in detail. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0048] Purchase sources of the enzymes of the present invention:

[0049] Carboxypeptidase, Nanning Dongheng Huadao Biotechnology Co., Ltd., 30,000 U / g;

[0050] Aminopeptidase, Shandong Longkete Enzyme Preparation Co., Ltd., 60,000 U / g;

[0051] Neutral protease, Nanning Dongheng Huadao Biotechnology Co., Ltd., 200,000 U / g;

[0052] Phytase, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., 100,000 U / g;

[0053] Trypsin, Nanning Dongheng Huadao Biotechnology Co., Ltd., 4000U / g.

[0054] Example 1

[0055] A method for preparing fresh bovine bone collagen peptide, comprising the following steps:

[0056] S1. Raw material selection: Select fresh, safe and pollution-free fresh bovine leg bones as raw materials.

[0057] S2. Pretreatment: Wash the selected bovine leg bones to remove surface impurities and blood stains. Then, crush the bovine bones into bone blocks of 2 cm by a bone crusher.

[0058] S3. Cooking: Put the treated bovine bones into a cooking tank for cooking, the cooking temperature is 110 °C and the pressure is 2.0 kPa.

[0059] S4. Use a three-phase horizontal screw centrifuge to separate the bone oil, the liquid material and the bone residue to ensure the purity of the separated liquid. Then, filter the liquid material to remove the fat in the bone broth to obtain a crude collagen solution.

[0060] S5. Enzymolysis: Add compound protease at 2‰ w / w of the crude collagen content (carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin with a mass ratio of 0.5:0.5:4:1:1) to the crude collagen solution, carry out an enzymolysis reaction at 45 °C for 3 h, inactivate the enzyme at high temperature after enzymolysis is completed, and centrifuge to take the supernatant to obtain a collagen peptide solution.

[0061] S6. Filtration: Filter the collagen peptide solution through a ceramic membrane to collect the filtrate; the clarified filtrate after filtration is further subjected to nanofiltration to collect the retentate; the collected retentate is processed by a three-step ultrafiltration method: first ultrafilter through an ultrafiltration membrane with a pore size of 20,000 Daltons to collect the filtrate; then ultrafilter the filtrate through an ultrafiltration membrane with a pore size of 15,000 Daltons to collect the filtrate; then separate the protein peptides with a molecular weight less than 5000 Daltons from the filtrate through a membrane with a pore size of 5000 Daltons to obtain a purified collagen peptide solution.

[0062] S7. Concentration: Concentrate the purified collagen peptide solution under reduced pressure to obtain a concentrated solution after concentration. When concentrating under reduced pressure, control the pressure at 0.1 MPa.

[0063] S8. Drying: Spray-dry the concentrated solution prepared in step S8 through an atomized granulation device to obtain bovine bone collagen peptide powder. The conditions for spray-drying by the atomized granulation device are: the inlet air temperature is 190 °C, the outlet air temperature is 95 °C, and the pressure of the high-pressure homogenizing pump is 70 kg.

[0064] The enzyme activity of the carboxypeptidase is 10,000 - 50,000 U / g, the enzyme activity of the aminopeptidase is 10,000 - 100,000 U / g, the enzyme activity of the neutral protease is 100,000 - 300,000 U / g, the enzyme activity of the phytase is 50,000 - 150,000 U / g, and the enzyme activity of the trypsin is 2,000 - 6,000 U / g.

[0065] The enzyme activities in this example are as follows: the enzyme activity of the carboxypeptidase is 30,000 U / g, the enzyme activity of the aminopeptidase is 60,000 U / g, the neutral protease is 200,000 U / g, the enzyme activity of the phytase is 100,000 U / g, and the enzyme activity of the trypsin is 4,000 U / g.

[0066] Example 2

[0067] A method for preparing fresh bovine bone collagen peptide, which is different from Example 1 in step S5: S5. Enzymatic hydrolysis: Add a compound protease (carboxypeptidase, aminopeptidase, neutral protease, phytase, and trypsin with a mass ratio of 1:1:2:3:3) accounting for 5‰ w / w of the crude collagen content to the crude collagen solution, carry out an enzymatic hydrolysis reaction at 45°C for 3 h, inactivate the enzyme at high temperature after the enzymatic hydrolysis is completed, centrifuge to obtain the supernatant, and obtain a collagen peptide solution.

[0068] Comparative Example 1

[0069] A method for preparing fresh bovine bone collagen peptide, which is different from Example 1 in that the phytase in step S5 is replaced with trypsin, that is, add a compound protease (carboxypeptidase, aminopeptidase, neutral protease, and trypsin with a mass ratio of 0.5:0.5:4:2) accounting for 2‰ w / w of the crude collagen content to the crude collagen solution.

[0070] Comparative Example 2

[0071] A method for preparing fresh bovine bone collagen peptide, which is different from Example 1 in that the trypsin in step S5 is replaced with phytase, that is, add a compound protease (carboxypeptidase, aminopeptidase, neutral protease, and phytase with a mass ratio of 0.5:0.5:4:2) accounting for 2‰ w / w of the crude collagen content to the crude collagen solution.

[0072] Comparative Example 3

[0073] A method for preparing fresh bovine bone collagen peptide, which is different from Example 1 in that no trypsin and phytase are added in step S5, that is, add a compound protease (carboxypeptidase, aminopeptidase, and neutral protease with a mass ratio of 0.5:0.5:4) accounting for 2‰ w / w of the crude collagen content to the crude collagen solution.

[0074] Test Example 1 Influence of Fresh Bovine Bone Collagen Peptide on Osteoporosis

[0075] 1.1 Animal Modeling

[0076] SPF - level Balb / c mice, healthy, 12 - week - old female and non - pregnant.

[0077] Modeling method:

[0078] (1) After intraperitoneal anesthesia of the mice with 40 mg / kg of 3% sodium pentobarbital, place them supine and depilate the abdomen.

[0079] (2) Under sterile conditions, cut the skin and muscle layer along the mid - abdominal line at the inguinal level to expose the abdominal cavity.

[0080] (3) Isolate the uterus and remove the ovaries. After cleaning the wound, suture the skin and the underlying layer in two layers. After the mice wake up, place them back in a clean cage and return them to the breeding room for breeding. Regularly observe the status and death of the mice and record them.

[0081] (4) Three days after the operation, inject 40,000 units of penicillin into each mouse every day to prevent infection.

[0082] (5) In the control group, only about 1 g of fat around the ovaries is removed, while the ovaries are retained, and the other treatments are the same.

[0083] Standard for successful modeling: The body weight of the rats in the ovariectomized group increased rapidly, their activity ability decreased, their hair color lacked luster and was slightly dull, while the body weight of the mice in the control group increased slowly, their hair color was bright, and their activities were normal. The estradiol level and femoral BMD in the ovariectomized group decreased significantly.

[0084] 1.2 Animal grouping and drug administration

[0085] The ovariectomized mice are divided into a model group, a positive drug group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group, with 15 mice in each group. Take 15 mice from the above - mentioned control group as the control group. The control group and the model group are respectively given an equal amount of saline, and the positive drug group is given 12 μg / kg by gavage. The Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group are respectively given 20 mg / kg of the corresponding bovine bone collagen peptide powder of the example and the comparative example by gavage. All animals are administered once a day for 10 weeks.

[0086] 1.3 Detection indexes

[0087] 1.3.1 Bone mineral density (BMD) detection

[0088] Two hours after the last administration of the BMD drug, place the anesthetized rats supine on the BMD tester table, fully extend the femoral bones of the rats to the maximum extent to expose the femoral bones fully under X - rays, turn on the BMD tester to scan both femoral bones, and measure the BMD values.

[0089] 1.3.2 Determination of bone mineral content (BMC)

[0090] After detecting BMD, the bilateral femurs of the rats were excised, the surrounding muscle tissues were cleaned, dried in an oven (at a temperature of 90 °C) for 1 h, and weighed on a balance (accuracy: 0.1 mg). After carbonization in an electric furnace, it was placed in a muffle furnace (800 °C) for 2 h. After the bone ash cooled down, it was weighed on an electronic balance. 10 L of 3% nitric acid solution was added with 0.05 g and 0.1 g of gray matter respectively, transferred to a 25 L colorimetric tube, and left at room temperature for 48 h. The solution was colorless and transparent, and the bone mineral content was measured.

[0091] 1.3.3 Bone biomechanics determination

[0092] The CTM2050S universal tensile testing machine was used in the experiment. The entire right femur was taken, immersed in normal saline at room temperature for 3 h, taken out and restored to room temperature. After casting with adhesive at both ends, it was placed on a three-point compression test bench with a spacing of 22 mm. The direction of the short axis of the specimen cross-section was consistent with the acceleration direction, and the maximum loading speed was 2 mm / min. The pressure frame switch was started, and after the femur fractured, the computer obtained data on the maximum load, bone stress, elastic modulus, energy absorption, and structural strength.

[0093] The experimental results were expressed as mean ± standard deviation. Analyzed with SPSS version 20.0 software, one-way ANOVA was used for comparison among multiple groups. For homogeneous variance, t-test was used; for heterogeneous variance, non-parametric test was used.

[0094] 1.4 Results and analysis

[0095] 14.1 Bone density detection results

[0096] The results are shown in Table 1:

[0097] Table 1 Bone density detection data

[0098]

[0099]

[0100] Note: Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group, *P < 0.05, **P < 0.01.

[0101] The above results indicate that compared with the model group, the positive drug and Examples 1-2 can effectively increase the bone density of mice, and the effects of Examples 1-2 are better than those of the positive drug. Comparative Examples 1-2 also have a certain effect on increasing bone density, but the effect is significantly inferior to that of Examples 1-2.

[0102] 14.2 Bone mineral content detection results

[0103] The results are shown in Table 2:

[0104] Table 2 Bone mineral content detection data

[0105] Group Bone mineral content / g Control group 0.40±0.02** Model group 0.36±0.01## Positive drug group 0.41±0.03** Example 1 group 0.41±0.02** Example 2 group 0.40±0.02** Comparative example 1 group 0.39±0.02** Comparative example 2 group <![CDATA[0.38±0.01* # > Comparative example 3 group 0.37±0.01##

[0106] Note: Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group, *P < 0.05, **P < 0.01.

[0107] The above results show that, compared with the model group, the positive drug, Examples 1-2 and Comparative Example 1 can all effectively increase the bone mineral content of mice, and there is no significant difference from the control group. The effect of Comparative Example 2 is worse than that of Examples 1-2 and Comparative Example 1, and Comparative Example 3 has no effect on increasing the bone mineral content of mice.

[0108] 1.3.3 Results of bone biomechanics measurement

[0109] The results are shown in Table 3:

[0110] Table 3 Bone biomechanics test data

[0111]

[0112]

[0113] Note: Compared with the control group, #P < 0.05, ##P < 0.01; compared with the model group, *P < 0.05, **P < 0.01.

[0114] The above results show that there are no significant differences in the data of maximum load, energy absorption and structural strength among the groups in the bone biomechanics test, and there is no statistical significance. In terms of bone stress, compared with the model group, the positive drug and Examples 1-2 can both significantly increase the bone stress of mice, and the effect is comparable to that of the control group; the effects of Comparative Examples 1-3 are significantly inferior to those of Examples 1-2. In terms of elastic modulus, compared with the model group, the positive drug, Examples 1-2 and Comparative Examples 1-3 all have an increasing effect, among which Examples 1-2 are comparable to the positive drug, and the effect is significantly better than that of Comparative Examples 1-3.

[0115] Test Example 2 Effect of fresh bovine bone collagen peptide on fractures in elderly osteoporosis

[0116] 2.1 Population screening criteria

[0117] Inclusion criteria: aged 60 - 80 years old; in line with the diagnosis of fractures in elderly osteoporosis according to the diagnostic criteria; without other serious infectious diseases, tumors, etc.; the patients voluntarily participate and sign the informed consent form.

[0118] The diagnostic criteria for osteoporotic fractures in the elderly refer to the "Diagnosis and Treatment Guidelines for Primary Osteoporosis". The diagnostic criteria for osteoporosis are as follows: fragility fractures of the hip or vertebra; the T-value of axial bone mineral density or the bone mineral density of the distal 1 / 3 of the radius measured by dual-energy X-ray absorptiometry ≤ -2.5; bone mineral density measurement meeting low bone mass (-2.5 < T-value < -1.0) + fragility fractures of the proximal humerus, pelvis, or distal forearm; meeting one of the three criteria.

[0119] Exclusion criteria: age < 60 years or > 80 years; patients with fractures caused by other reasons; patients with allergic constitution or allergic to the drugs related to this study; patients with combined tumors, severe hepatic and renal insufficiency; those who are not voluntary or have not signed the informed consent form.

[0120] 2.2 Grouping and drug administration

[0121] 120 eligible elderly patients with osteoporotic fractures were randomly divided into 6 groups. The positive drug group was given calcitriol soft capsules at a dose of 0.25 μg / time, 3 times a day. The implementation group 1, implementation group 2, control group 1, control group 2, and control group 3 were given 150 mg / time, 2 times a day. The curative effects were observed after 7 weeks of treatment in all 6 groups.

[0122] 2.3 Detection indicators

[0123] 2.3.1 Detection of bone metabolism indicators

[0124] 5 mL of venous blood was drawn from the patients before and after treatment and centrifuged

[0125] The supernatant was taken and stored at low temperature. The PINP, β-CTX, and OC in the serum of the patients were measured using an elisa kit.

[0126] 2.3.2 Bone mineral density detection

[0127] The changes in bone mineral density of the two groups of patients before and after treatment were detected using the American Prodigy dual-energy X-ray bone densitometer.

[0128] 2.4 Detection results

[0129] 2.4.1 Detection results of bone metabolism indicators of patients before and after treatment

[0130] The detection results are shown in Table 4:

[0131] Table 4 Detection data of bone metabolism indicators of patients before and after treatment

[0132]

[0133] Note: Compared with the positive drug group, #P < 0.05, ##P < 0.01; compared with before treatment, *P < 0.05, **P < 0.01.

[0134] The above results show that the positive drug and Examples 1-2 can significantly reduce PINP and significantly increase OC compared with before treatment, and Examples 1-2 are superior to the positive drug in terms of efficacy. There is no significant difference in reducing β-CTX between the positive drug group and before treatment, and there are significant differences between Examples 1-2 and before treatment as well as the positive drug.

[0135] 2.4.1 Bone density test results of patients before and after treatment

[0136] The test results are shown in Table 5:

[0137] Table 5 Bone density test data of patients before and after treatment

[0138]

[0139] Note: Compared with the positive drug group, # P < 0.05, ## P < 0.01; compared with before treatment, *P < 0.05, **P < 0.01.

[0140] The above results show that the positive drug, Examples 1-2 and Comparative Example 1 all have the effect of enhancing bone density compared with before treatment. Among them, the effects of Examples 1-2 are significantly superior to those of the positive drug and Comparative Example 1.

Claims

1. A method for preparing bovine collagen peptide, characterized in that: The following steps are involved: S1. Pretreatment: washing and crushing the cattle bones to obtain bone blocks; S2, steaming: steaming the bone blocks in step S1 to obtain bone broth; S3, fat removal: the bone broth of step S2 is subjected to three-phase separation of bone oil, liquid and bone residue to obtain a crude collagen solution after degreasing; S4, adding a composite protease to the crude collagen solution obtained in step S3 for enzymatic hydrolysis to obtain a collagen peptide solution; S5, filtration: filtering the collagen peptide solution of step S4 to obtain a purified collagen peptide solution; S6, concentration: concentrating the purified collagen peptide solution in step S5 under reduced pressure to obtain a concentrated solution; S7, drying: spray drying the concentrated solution of step S6 to obtain bovine collagen peptide powder; The composite protease is carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin; The amount of the composite protease added in step S4 is 2‰-5‰ w / w of the crude collagen content; The mass ratio of the carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin is 0.5-1:0.5-1:2-4:1-3:1-3; The enzyme activities of the carboxypeptidase, aminopeptidase, neutral protease, phytase and trypsin are respectively 10,000-50,000 U / g, 10,000-100,000 U / g, 100,000-300,000 U / g, 50,000-150,000 U / g and 2,000-6,000 U / g.

2. The preparation method according to claim 1, characterized in that: The cooking conditions in step S2 are: 100-120° C., 1.5-2.0 kPa.

3. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis conditions in step S4 are 40-50° C. for 2-4 h.

4. The preparation method according to claim 1, characterized in that: The filtration in step S5 is ceramic membrane, nanofiltration and ultrafiltration in sequence; the molecular weight of the collagen peptide in the collagen peptide purification solution is less than 8000 Daltons.

5. The preparation method according to claim 4, characterized in that: The molecular weight of collagen peptides is less than 5000 Daltons.

6. Use of the preparation method according to any one of claims 1 to 5 in preparing a health product for increasing bone density.

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