Composition capable of promoting osteogenic activity and preparation method thereof

Abstract

The invention relates to the technical field of nutritional supplement preparation, in particular to a composition with osteogenesis promoting activity and a preparation method thereof.The preparation method comprises the steps that chondroitin sulfate, bone collagen peptide and casein are taken according to the proportion and added into a container, then 1 L of deionized water is added, the pH value of the solution is adjusted to 7.0-9.0, and a solution A is obtained; dissolving the solution A in a water bath kettle; adding alkaline protease into the solution A, and slowly stirring for enzymolysis to obtain enzymatic hydrolysate; and performing enzyme deactivation on the enzymatic hydrolysate, adjusting the pH value to 7, adding flavourzyme for enzymolysis, then adding beta-cyclodextrin, performing uniform stirring, performing vacuum concentration until the solid content is 25%, and performing spray drying to obtain powder. In this way, the technical problems that in the prior art, a fracture injury health-care composition is indefinite in action mechanism and effect, high in production cost, difficult in raw material obtaining and poor in user experience are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of nutritional supplements, and in particular to a composition with osteogenic activity and a preparation method thereof. Background Art

[0002] Fractures are common diseases, generally caused by the destruction of the mechanical integrity of bones, accompanied by local soft tissue and blood vessel damage. The healing process is the repair of bone tissue, a continuous process of clearing damage and regenerating repair, which is mainly regulated by bone formation led by osteoblasts and bone resorption led by osteoclasts. Both play an important role in the bone reconstruction process. The reconstruction process of human bones is generally considered to consist of several stages: the absorption phase in which osteoclast progenitor cells are recruited to the surface of damaged bones, osteoclasts absorb damaged bones, osteoclast apoptosis and osteoblast progenitor cells are recruited, mature osteoblasts produce new bone matrix, and mineralize the formation phase. Under normal physiological conditions, the homeostasis of bones is maintained by the appropriate ratio between bone tissue formation and resorption. In the process of fracture and other injuries, promoting the proliferation of osteoblasts is of vital significance for fracture healing, osteoporosis treatment and bone defect repair.

[0003] At present, the health care products for fracture injuries on the market are mostly focused on the combination of ingredients such as calcium supplementation, relief of tissue swelling and inflammation, blood circulation and blood stasis, lubrication and relief of tissue pain and somatosensory sensation. For example, patent CN119185447A uses a group of Chinese medicine powder paste composition to promote bone recovery. Patent CN118557635A uses a group of strong medicine composition to relieve pain, promote callus formation, and increase bone density. There are few active ingredients with a clear mechanism of action for the healing of bone tissue after fracture injury.

[0004] In summary, most of the fracture injury health care compositions in the prior art follow the formulation and compatibility of traditional Chinese medicine, which have problems such as unclear mechanism of action and effect, high production cost, difficulty in obtaining raw materials and poor user experience. Summary of the invention

[0005] The purpose of the present invention is to provide a composition with osteogenic activity and a preparation method thereof, aiming to solve the technical problems of the fracture injury health care composition in the prior art, such as unclear mechanism of action and effect, high production cost, difficulty in obtaining raw materials and poor user experience.

[0006] To achieve the above object, the present invention adopts a composition with osteogenic activity, including 25g-35g chondroitin sulfate, 25g collagen peptide and 40g-45g casein, 0.5g-0.8g alkaline protease, 0.3g flavor protease and 10g β-cyclodextrin.

[0007] The present invention also provides a method for preparing a composition having osteogenic activity, which is used to prepare the composition having osteogenic activity as described above. The steps include: Step 1: firstly, add chondroitin sulfate, collagen peptide and casein in proportion to a container, then add 1 L of deionized water and adjust the pH value of the solution to 7.0-9.0 to obtain solution A; Step 2: Dissolve solution A in a water bath; Step 3, adding alkaline protease to solution A, and slowly stirring to hydrolyze to obtain an enzymolysis solution; Step 4: inactivate the enzyme in the hydrolyzate and adjust the pH value to 7, add flavor protease for hydrolysis, then add β-cyclodextrin and stir evenly, concentrate under reduced pressure to a solid content of 25%, and spray dry into powder.

[0008] Wherein, in step 1, 4 mol / L NaOH is used to adjust the pH value of the solution to 7.0-9.0.

[0009] Wherein, in step 2, when solution A is dissolved in a water bath, the temperature is 45-60°C.

[0010] Among them, in step three, before adding alkaline protease, it is necessary to ensure that the solution has no obvious particles and the pH value has no obvious change.

[0011] Wherein, in step three, alkaline protease is added, and during the enzymatic hydrolysis process, NaOH with a concentration of 2 mol / L is used to maintain the pH value of the solution at 7.0-9.0.

[0012] Among them, in step 4, the specific method of inactivating the enzyme in the enzymatic solution and adjusting the pH value to 7 is: The enzymatic solution obtained by adding alkaline protease was inactivated in a boiling water bath at 100°C for 10 minutes, and the pH value of the system was adjusted to 7 with citric acid.

[0013] Wherein, in step 4, when flavor protease is added for enzymatic hydrolysis, the enzymatic hydrolysis process is reacted in a water bath at 45° C. for 3 hours.

[0014] The invention discloses a composition with osteogenic activity and a preparation method thereof. Firstly, chondroitin sulfate, collagen peptide and casein are added to a container in proportion, and then 1L of deionized water is added and the pH value of the solution is adjusted to 7.0-9.0 to obtain solution A; solution A is dissolved in a water bath; alkaline protease is added to solution A and slowly stirred for enzymolysis to obtain enzymolysis solution; enzyme is inactivated in the enzymolysis solution and the pH value is adjusted to 7, flavor protease is added for enzymolysis, and then beta-cyclodextrin is added for uniform stirring, reduced pressure concentration is performed to a solid content of 25%, and spray drying is performed to obtain powder.

[0015] The enzymatic treatment method adopted by the present invention makes the chondroitin sulfate, collagen peptide and casein raw materials that originally have no osteogenic activity have significant osteogenic activity, osteogenic differentiation activity and osteocyte mineralization activity after treatment. These functions are closely related to bone formation, have the potential to prevent osteoporosis and promote injury repair, and expand the function; The process of the composition treatment of the present invention uses the most simplified steps and simple enzyme combination to reduce production costs and facilitate scale-up production and implementation. The bitterness removal treatment is carried out simultaneously in the process, which not only achieves the efficacy of osteogenic activity, but also avoids the negative experience of the bitter taste of the material. At the same time, the addition of β-cyclodextrin makes it easier for the material to be dried and formed, and the product formula is applied; The raw materials used in the present invention are commonly used raw materials for bone and joint health, which are easy to obtain and do not require special raw material sources.

[0016] In this way, the technical problems of the fracture injury health care composition in the prior art, such as unclear mechanism of action and effect, high production cost, difficulty in obtaining raw materials and poor user experience, are solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The present invention is a flow chart of the preparation method of the composition having osteogenic activity.

[0019] Figure 2 It is a schematic diagram of the osteoblast toxicity test results of the present invention.

[0020] Figure 3 It is a schematic diagram of the osteoblast proliferation experimental results of the present invention.

[0021] Figure 4 It is a schematic diagram of the osteoblast differentiation activity detection results of the present invention.

[0022] Figure 5 It is a schematic diagram of the osteoblast mineralization activity detection results of the present invention. DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0024] The invention provides a composition with osteogenic activity, comprising 25g-35g of chondroitin sulfate, 25g of collagen peptide, 40g-45g of casein, 0.5g-0.8g of alkaline protease, 0.3g of flavor protease and 10g of beta-cyclodextrin.

[0025] See also Figure 1 , Figure 1 is a flow chart of a method for preparing a composition having osteogenic activity of the present invention. The present invention also provides a method for preparing a composition having osteogenic activity, which is used to prepare the composition having osteogenic activity as described above. The steps include: Step 1: firstly, add chondroitin sulfate, collagen peptide and casein in proportion to a container, then add 1 L of deionized water and adjust the pH value of the solution to 7.0-9.0 to obtain solution A; Step 2: Dissolve solution A in a water bath; Step 3, adding alkaline protease to solution A, and slowly stirring to hydrolyze to obtain an enzymolysis solution; Step 4: inactivate the enzyme in the hydrolyzate and adjust the pH value to 7, add flavor protease for hydrolysis, then add β-cyclodextrin and stir evenly, concentrate under reduced pressure to a solid content of 25%, and spray dry into powder.

[0026] In step 1, 4 mol / L NaOH is used to adjust the pH value of the solution to 7.0-9.0.

[0027] In step 2, solution A is dissolved in a water bath at a temperature of 45-60°C.

[0028] In step 3, before adding alkaline protease, it is necessary to ensure that the solution has no obvious particles and the pH value has no obvious change.

[0029] In step three, alkaline protease is added, and during the enzymatic hydrolysis process, 2 mol / L NaOH is used to maintain the pH value of the solution at 7.0-9.0.

[0030] In step 4, the specific method of inactivating the enzyme in the enzymatic solution and adjusting the pH value to 7 is as follows: The enzymatic solution obtained by adding alkaline protease was inactivated in a boiling water bath at 100°C for 10 minutes, and the pH value of the system was adjusted to 7 with citric acid.

[0031] In step 4, flavor protease is added for enzymatic hydrolysis, and the enzymatic hydrolysis process is carried out in a water bath at 45° C. for 3 hours. Example

[0032] In this embodiment, the composition having osteogenic activity is prepared as follows: Take 35g chondroitin sulfate, 25g collagen and 40g casein into a container, add 1L deionized water, adjust the pH value of the solution to 7.0-9.0 with 4mol / L NaOH, and dissolve in a 45-60℃ water bath; When the solution has no obvious particles and the pH value has no obvious change, add 0.5g of alkaline protease, maintain the pH value of the mixture hydrolyzate to 7.0-9.0 with 2mol / L NaOH in a constant temperature water bath and slowly stir for 4h; The hydrolyzate was inactivated in a 100°C boiling water bath for 10 min, the pH of the system was adjusted to 7 with citric acid, 0.3 g of flavor protease was added, the mixture was reacted in a 45°C water bath for 3 h, 10 g of β-cyclodextrin was added and stirred evenly, the mixture was concentrated under reduced pressure to a solid content of about 25%, and spray-dried into powder. Example

[0033] In this embodiment, the composition having osteogenic activity is prepared as follows: Take 20g chondroitin sulfate, 25g collagen and 55g casein into a container, add 1L deionized water, adjust the pH value of the solution to 7.0-9.0 with 4mol / L NaOH, and dissolve in a 45-60℃ water bath; When the solution has no obvious particles and the pH value has no obvious change, add 0.8g of alkaline protease, maintain the pH value of the mixture hydrolyzate to 7.0-9.0 with 2mol / L NaOH in a constant temperature water bath and slowly stir for 4h; The hydrolyzate was inactivated in a 100°C boiling water bath for 10 min, the pH of the system was adjusted to 7 with citric acid, 0.3 g of flavor protease was added, the mixture was reacted in a 45°C water bath for 3 h, 10 g of β-cyclodextrin was added and stirred evenly, the mixture was concentrated under reduced pressure to a solid content of about 25%, and spray-dried into powder. Comparative Example 1 (adjusting alkaline protease): Take 35g chondroitin sulfate, 25g collagen and 40g casein into a container, add 1L deionized water, adjust the pH value of the solution to 7.0-9.0 with 4mol / L NaOH, and dissolve in a 45-60℃ water bath; When the solution has no obvious particles and the pH value has no obvious change, the pH value of the mixture hydrolyzate is maintained at 7.0-9.0 with 2 mol / L NaOH in a constant temperature water bath and stirred slowly for 4 h; The hydrolyzate was placed in a boiling water bath at 100°C for 10 min, the pH of the system was adjusted to 7 with citric acid, 0.3 g of flavor protease was added, and the mixture was reacted in a water bath at 45°C for 3 h. 10 g of β-cyclodextrin was added and stirred evenly, the mixture was concentrated under reduced pressure to a solid content of about 25%, and spray-dried into powder. Comparative Example 2 (adjusting flavor protein and β-cyclodextrin): Take 35g chondroitin sulfate, 25g collagen and 40g casein into a container, add 1L deionized water, adjust the pH value of the solution to 7.0-9.0 with 4mol / L NaOH, and dissolve in a 45-60℃ water bath; When the solution has no obvious particles and the pH value has no obvious change, add 0.5g of alkaline protease, maintain the pH value of the mixture hydrolyzate to 7.0-9.0 with 2mol / L NaOH in a constant temperature water bath and slowly stir for 4h; The hydrolyzate was inactivated in a 100°C boiling water bath for 10 min, the pH of the system was adjusted to 7 with citric acid, 0.1 g of flavor protease was added, the mixture was reacted in a 45°C water bath for 3 h, 5 g of β-cyclodextrin was added and stirred evenly, the mixture was concentrated under reduced pressure to a solid content of about 25%, and spray-dried into powder.

[0034] A composition having osteogenic activity and a preparation method thereof according to the present embodiment are used. First, chondroitin sulfate, collagen peptide and casein are added to a container in proportion, and then 1 L of deionized water is added and the pH value of the solution is adjusted to 7.0-9.0 to obtain solution A; solution A is dissolved in a water bath; alkaline protease is added to solution A and slowly stirred for enzymolysis to obtain an enzymolysis solution; the enzymolysis solution is inactivated and the pH value is adjusted to 7, flavor protease is added for enzymolysis, and then β-cyclodextrin is added and stirred evenly, and the solution is concentrated under reduced pressure to a solid content of 25%, and spray-dried into powder.

[0035] The enzymatic treatment method adopted by the present invention makes the chondroitin sulfate, collagen peptide and casein raw materials that originally have no osteogenic activity have significant osteogenic activity, osteogenic differentiation activity and osteocyte mineralization activity after treatment. These functions are closely related to bone formation, have the potential to prevent osteoporosis and promote injury repair, and expand the function; The process of the composition treatment of the present invention uses the most simplified steps and simple enzyme combination to reduce production costs and facilitate scale-up production and implementation. The bitterness removal treatment is carried out simultaneously in the process, which not only achieves the efficacy of osteogenic activity, but also avoids the negative experience of the bitter taste of the material. At the same time, the addition of β-cyclodextrin makes it easier for the material to be dried and formed, and the product formula is applied; The raw materials used in the present invention are commonly used raw materials for bone and joint health, which are easy to obtain and do not require special raw material sources.

[0036] In this way, the technical problems of the fracture injury health care composition in the prior art, such as unclear mechanism of action and effect, high production cost, difficulty in obtaining raw materials and poor user experience, are solved.

[0037] The present invention achieves the purpose of promoting osteoblast proliferation activity by hydrolyzing the mixture of collagen peptide and casein in coordination with alkaline protease; After proteolysis, a debittering treatment process is carried out in series, and the bitterness of the product is greatly reduced by combining flavor protease treatment and β-cyclodextrin adsorption. The osteogenic activity in which osteoblasts participate is a very complex and delicate process, which is acted upon by a variety of regulatory factors. The osteogenic process mainly includes four stages, osteoblast proliferation, extracellular matrix maturation, extracellular matrix mineralization and osteoblast apoptosis. Among them, osteoblast mineralization refers to the orderly deposition of inorganic matter on organic matter, that is, calcium phosphate forms hydroxyapatite on collagen. In addition, osteoblast differentiation refers to that osteoblasts undergo a series of differentiation into pre-osteoblasts, and then continue to differentiate into functional osteoblasts, and this process is a prerequisite for bone occurrence and formation. There is no obvious boundary between these four stages, and normal bone formation is finally completed. Therefore, the present application also investigates and evaluates the beneficial effects of the composition from several key points of osteogenic activity, osteogenic differentiation activity, and osteogenic mineralization. 1. Osteogenic activity: please refer to Figure 2 and Figure 3 , Figure 2 is a schematic diagram of the osteoblast toxicity test results of the present invention, Figure 3 It is a schematic diagram of the osteoblast proliferation experimental results of the present invention.

[0038] Osteoblast proliferation assay (CCK-8 method): MC3T3-E1 cells, add 100ul cell suspension of 1*104 in a 96-well culture plate, culture in a 37℃, 5% carbon dioxide incubator for 24h, add basal culture medium after the cells adhere to the wall and continue to culture for 24h, discard the culture medium, add 100ul complete culture medium or complete culture medium containing different test samples to the culture plate, culture in the incubator for 24h, discard the culture medium, add 100ul culture medium and 10ulCCK-8 solution to each well, incubate the culture plate in the incubator for 4h, and measure the absorbance at a wavelength of 450nm with an enzyme marker. Set up a blank group (blank culture medium without cells + CCK-8 reagent), a control group (control cells without samples + CCK-8 reagent), and an experimental group (sample-treated cells + CCK-8 reagent); Based on the experimental results, the cytotoxic effects of each sample on cells were first verified. When the sample loading concentration was 4 mg / ml, each sample showed low cytotoxicity ( Figure 2 ), and the cell proliferation activity test can be performed below this concentration later. The results of the osteoblast proliferation experiment showed that the enzymatically treated Examples 1 and 2 were significantly higher than the control group, indicating that the modified samples can effectively promote the proliferation of osteoblasts. Further data analysis showed that the sample concentration was positively correlated with the cell proliferation rate, verifying the positive role of the tested samples in promoting osteoblast proliferation. 2. Osteoblastic differentiation activity: please refer to Figure 4 , Figure 4 It is a schematic diagram of the osteoblast differentiation activity detection results of the present invention.

[0039] Osteoblast differentiation activity assay (ALP alkaline phosphatase): Alkaline phosphatase (ALP) is a product of the early differentiation of MC3T3-E1 cells and an important indicator for determining its osteogenic activity. The increase in ALP activity indicates the promotion of osteoblast differentiation. MC3T3-E1 cells were inoculated in a 24-well culture plate at a density of 8*104 / ml and cultured in a 5% carbon dioxide incubator at 37°C for 24 hours. The culture medium was discarded, and 500ul differentiation medium (10% fetal bovine serum (α-MEM), 5mmol / L β-glycerophosphate, 1% penicillin and streptomycin (w / v), 50ug / ml ascorbic acid and 10-7mol / L dexamethasone) and samples of different concentrations were added to the culture plate for bone differentiation detection. Untreated cells were used as blank controls. After 3 days of differentiation, the expression of ALP was detected using an alkaline phosphatase (ALP) staining kit.

[0040] Depend on Figure 4 It can be seen that the ALP activity of the composition components after enzymatic hydrolysis was significantly improved, indicating that the sample has significant osteogenic differentiation activity, which further verifies the sample's promoting effect on the osteogenic differentiation of MC3T3-E1 cells. 3. Osteocyte mineralization activity: please refer to Figure 5 , Figure 5 It is a schematic diagram of the osteoblast mineralization activity detection results of the present invention.

[0041] Characterization of osteoblast mineralization activity (Alizarin red staining) MC3T3-E1 cells were seeded in 6-well plates at 2*104 cells / well and cultured to 80% confluence. All groups were cultured with osteogenic agents (50ug / ml ascorbic acid, 100nM dexamethasone, 10mM β-glycerophosphate in α-MEM). The experimental groups were given samples of different concentrations at the same time and cultured for 21 days. Subsequently, they were fixed with 4% paraformaldehyde solution for 15 minutes, and alizarin red staining solution was added and incubated for 30 minutes. PBS was used to wash the excess dye from the cells, and the stained cells were dissolved with 10% hexadecylpyridine, and the absorbance was measured at 540nm by a microplate reader.

[0042] based on Figure 5 Compared with the control group, both Example 1 and Example 2 can significantly improve the absorbance value of the sample at 540 nm, indicating that more mineralized nodules are produced after being treated by the process of the present invention. The results show that the composition treated by the method of the present invention has a stronger effect of promoting osteocyte mineralization. 4. Evaluation of debittering effect: Bitterness evaluation method: After rinsing the mouth with distilled water, the assessor takes about 2 mL of the product solution and puts it in the mouth, spits it out after 5-10 seconds, and then tastes the next sample after rinsing the mouth. The tasting interval of each sample is 3-5 minutes. The solutions with the mass concentration of quinine sulfate of 0, 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% are selected as reference solutions. The bitterness degree is not bitter, slightly bitter, weakly bitter, generally bitter, bitter, and extremely bitter, and the corresponding bitterness values ​​are 1, 2, 3, 4, 5, and 6 respectively.

[0043] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A composition having osteogenic activity, characterized in that: The invention comprises 25g-35g chondroitin sulfate, 25g collagen peptide, 40g-45g casein, 0.5g-0.8g alkaline protease, 0.3g flavor protease and 10g beta-cyclodextrin.

2. A method for preparing a composition having osteogenic activity, for preparing the composition having osteogenic activity as claimed in claim 1, characterized in that: The steps include: Step 1: firstly, add chondroitin sulfate, collagen peptide and casein in proportion to a container, then add 1 L of deionized water and adjust the pH value of the solution to 7.0-9.0 to obtain solution A; Step 2: Dissolve solution A in a water bath; Step 3, adding alkaline protease to solution A, and slowly stirring to hydrolyze to obtain an enzymolysis solution; Step 4: inactivate the enzyme in the hydrolyzate and adjust the pH value to 7, add flavor protease for hydrolysis, then add β-cyclodextrin and stir evenly, concentrate under reduced pressure to a solid content of 25%, and spray dry into powder.

3. The method for preparing the composition having osteogenic activity according to claim 2, characterized in that: In step 1, 4 mol / L NaOH is used to adjust the pH value of the solution to 7.0-9.

0.

4. The method for preparing the composition having osteogenic activity according to claim 3, characterized in that: In step 2, solution A is dissolved in a water bath at a temperature of 45-60°C.

5. The method for preparing the composition having osteogenic activity according to claim 4, characterized in that: In step 3, before adding alkaline protease, it is necessary to ensure that the solution has no obvious particles and the pH value has no obvious change.

6. The method for preparing the composition having osteogenic activity according to claim 5, characterized in that: In step three, alkaline protease is added, and during the enzymatic hydrolysis process, 2 mol / L NaOH is used to maintain the pH value of the solution at 7.0-9.

0.

7. The method for preparing the composition having osteogenic activity according to claim 6, characterized in that: In step 4, the specific method of inactivating the enzyme in the enzymatic solution and adjusting the pH value to 7 is as follows: The enzymatic solution obtained by adding alkaline protease was inactivated in a boiling water bath at 100°C for 10 minutes, and the pH value of the system was adjusted to 7 with citric acid.

8. The method for preparing the composition having osteogenic activity according to claim 7, characterized in that: In step 4, flavor protease is added for enzymatic hydrolysis, and the enzymatic hydrolysis process is carried out in a water bath at 45° C. for 3 hours.

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