Preparation method of polypeptide of hu sheep bone
By enzymatically hydrolyzing and purifying Hu sheep bone powder using silica gel column chromatography, Hu sheep bone polypeptides were prepared. This solved the problem of limited utilization of bone protein through enzymatic hydrolysis, achieved efficient hydroxyl radical scavenging and calcium chelation, and increased the added value of Hu sheep bone.
Patent Information
- Application Number
- CN202610227379.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-26
- Publication Date
- 2026-06-26
AI Technical Summary
In existing technologies, there are few methods for utilizing bone protein through enzymatic hydrolysis, resulting in low added value of animal bones. Furthermore, chemical degradation methods can lead to the loss of amino acids and the generation of toxic and harmful substances.
The bone powder of Hu sheep was enzymatically hydrolyzed using a complex protease (trypsin and papain), and then purified by silica gel column chromatography to prepare Hu sheep bone polypeptides. Modified silica gel was used to improve the separation effect.
It improved the hydroxyl radical scavenging rate and calcium chelation rate of Hu sheep bone polypeptides. After calcium chelation, it had a good proliferative effect on cells, thus enhancing the utilization value of Hu sheep bone.
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Figure CN122278980A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of polypeptide preparation technology, specifically relating to a method for preparing a polypeptide from sheep bone. Background Technology
[0002] Currently, the production methods for protein hydrolysates are divided into chemical degradation and enzymatic degradation. Chemical methods utilize acids and alkalis to hydrolyze proteins, but the reaction conditions are harsh, and amino acids are severely damaged during the process. Experiments have shown that alkaline hydrolysis of proteins can cause L-amino acids to form D-amino acids, accompanied by the production of toxic and harmful substances. Enzyme engineering is a modern biotechnology, and there are many processes for enzymatically hydrolyzing plant and animal proteins, but methods for enzymatically hydrolyzing bone protein are less common. In my country, most animal bones are used to produce bone glue, bone oil, and bone meal; for example, a large amount of bone meal is used in animal feed and fertilizer, with low added value. Enzymatic hydrolysis is a way to optimize the utilization of bone protein, hydrolyzing collagen protein, which is not easily digested and absorbed by the human body, into collagen peptides, thereby improving its nutritional value and functional properties. Summary of the Invention
[0003] The purpose of this invention is to provide a method for preparing a sheep bone polypeptide with high hydroxyl radical scavenging rate, high calcium chelation rate, and good cell proliferation effect after calcium chelation.
[0004] The technical solution adopted by the present invention to achieve the above objectives is as follows: A method for preparing Huzhou sheep bone polypeptides includes: pretreating fresh Huzhou sheep bones to obtain Huzhou sheep bone powder, and then enzymatically hydrolyzing the Huzhou sheep bone powder to obtain Huzhou sheep bone polypeptides; the enzymatic hydrolysis uses a complex protease, which includes trypsin and papain, and the mass ratio of trypsin to papain in the complex protease is 1:0.1-10; the enzymatic hydrolysis is purified by silica gel column separation. This invention improves the utilization of Huzhou sheep bones, optimizes the utilization of Huzhou sheep bone protein, and increases the added value of Huzhou sheep bones by preparing Huzhou sheep bone polypeptides from fresh Huzhou sheep bones.
[0005] Preferably, the pretreatment includes removing the meat scraps from the fresh sheep bones, washing them, and then chopping them into sheep bone pieces.
[0006] More preferably, the sheep bone blocks are soaked in hexane, washed, and then pressure-cooked before being dried and pulverized to obtain sheep bone powder.
[0007] Preferably, the relationship between the amount of trypsin used and the amount of sheep bone powder used is 6000-10000 U / g.
[0008] Preferably, in the enzymatic hydrolysis treatment, the enzymatic hydrolysis reaction is carried out at 30-37℃ for 0.5-6 hours.
[0009] Preferably, the filler in the silica column is silica gel or modified silica gel.
[0010] Preferably, in the preparation of modified silica gel, silica gel is first activated with acid, then functionalized with 3-aminopropyltriethoxysilane, and then reacted with modified monomers to prepare modified silica gel.
[0011] More preferably, the modified monomer is cyclohexyl vinyl ether. In this invention, after activating and functionalizing silica gel, it is reacted with cyclohexyl vinyl ether to prepare modified silica gel with a cyclohexyl vinyl ether group structure. This modified silica gel has a good separation effect on sheep bone polypeptides. The obtained sheep bone polypeptides can improve the scavenging effect of hydroxyl radicals and the calcium chelation effect. The sheep bone polypeptide calcium chelate has a good cell proliferation effect.
[0012] Preferably, in the preparation of Hu sheep bone powder, the scraps of meat on the fresh Hu sheep bones are removed, the bones are washed with deionized water, chopped into Hu sheep bone pieces, the Hu sheep bone pieces are soaked in n-hexane for 12-48 hours, washed with deionized water, and then pressure-cooked at 110-125℃ for 0.5-2 hours. After cooling to room temperature, the bones are dried, pulverized at 25000-30000 r / min, and passed through a 60-100 mesh sieve to obtain Hu sheep bone powder.
[0013] More preferably, in the preparation of Hu sheep bone powder, the amount of Hu sheep bone blocks used is 20-60 wt% of hexane. Deionized water is used in appropriate amounts during washing and during high-pressure cooking.
[0014] Preferably, in the preparation of Hu sheep bone polypeptide, Hu sheep bone powder is added to deionized water and mixed, the pH is adjusted to 5-8, a complex protease is added, and the enzymatic hydrolysis reaction is carried out at 30-37℃ for 0.5-6 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated by boiling water bath for 10-30 minutes, cooled to room temperature, and the supernatant is collected by centrifugation to prepare Hu sheep bone enzymatic hydrolysate. The Hu sheep bone enzymatic hydrolysate is separated and purified by silica gel column chromatography and freeze-dried to prepare Hu sheep bone polypeptide.
[0015] More preferably, in the preparation of Hu sheep bone polypeptide, the amount of Hu sheep bone powder used is 2-10 wt% of deionized water.
[0016] More preferably, in the preparation of sheep bone polypeptide, the complex protease includes trypsin and papain, and the mass ratio of trypsin to papain in the complex protease is 1:0.1-10.
[0017] More preferably, in the preparation of Hu sheep bone polypeptide, the relationship between the amount of trypsin used and the amount of Hu sheep bone powder used is 6000-10000 U / g.
[0018] More preferably, in the preparation of sheep bone polypeptide, the filler in the silica column is silica gel.
[0019] More preferably, in the preparation of the sheep bone polypeptide, the packing material in the silica column is modified silica. The preparation of modified silica includes the preparation of activated silica and the preparation of functionalized silica.
[0020] Preferably, in the preparation of activated silica gel, silica gel is added to hydrochloric acid solution, stirred at 60-90°C for 4-12 hours, then washed until neutral, and dried to obtain activated silica gel.
[0021] More preferably, in the preparation of activated silica gel, the hydrochloric acid content in the hydrochloric acid solution is 4-8M, and the amount of silica gel used is 2-10wt% of the hydrochloric acid solution.
[0022] Preferably, in the preparation of functionalized silicone, activated silicone and 3-aminopropyltriethoxysilane are added to an organic reagent and mixed thoroughly. Then, the mixture is stirred at 50-70°C for 3-12 hours. After the reaction is completed, the mixture is cooled, washed, and dried to obtain functionalized silicone.
[0023] More preferably, in the preparation of functionalized silicone, the organic reagent is anhydrous ethanol, the amount of activated silicone used is 5-20 wt% of anhydrous ethanol, and the amount of 3-aminopropyltriethoxysilane used is 50-200 wt% of activated silicone.
[0024] Preferably, in the preparation of modified silica gel, functionalized silica gel is added to acetonitrile, then modified monomers and initiators are added, and the mixture is stirred and reacted at a temperature of 50-70°C for 6-24 hours. After the reaction is completed, the mixture is washed and dried to obtain modified silica gel.
[0025] More preferably, in the preparation of modified silica gel, the amount of functionalized silica gel used is 1-10 wt% of acetonitrile, the amount of modified monomer is cyclohexyl vinyl ether used is 5-30 wt% of functionalized silica gel, the amount of initiator is AIBN used is 0.5-2 wt% of functionalized silica gel, and the inert gas is nitrogen.
[0026] More preferably, in the preparation of modified silica gel, the modifying monomer also includes diethyl allyl malonate, and the amount of diethyl allyl malonate used is 1-10 wt% of the functionalized silica gel. In this invention, in addition to using cyclohexyl vinyl ether as the modifying monomer, diethyl allyl malonate can also be added to obtain a group structure containing cyclohexyl vinyl ether and diethyl allyl malonate. Under the action of the functional group structure formed by 3-aminopropyltriethoxysilane and the group structure formed by cyclohexyl vinyl ether and diethyl allyl malonate, the separation effect of sheep bone polypeptide is good, the chelation effect of the obtained sheep bone polypeptide on calcium is better, and the sheep bone polypeptide calcium chelate has a good cell proliferation effect.
[0027] This invention discloses the sheep bone polypeptide prepared by the above method.
[0028] This invention discloses a method for preparing a calcium chelate of sheep bone polypeptide, including the preparation method described above.
[0029] Preferably, in the preparation of the lake sheep bone polypeptide calcium chelate, the lake sheep bone polypeptide is mixed with calcium chloride solution, the pH of the solution is adjusted to 5-9, and the mixture is chelated by shaking in a constant temperature water bath at 20-40℃ for 0.5-2h. Then anhydrous ethanol is added, and the mixture is centrifuged at 0-10℃. The precipitate is freeze-dried to obtain the lake sheep bone polypeptide calcium chelate.
[0030] More preferably, in the preparation of the sheep bone polypeptide calcium chelate, the calcium chloride solution is made by mixing calcium chloride and deionized water, and the calcium chloride content in the calcium chloride solution is 0.5-2 wt%.
[0031] More preferably, in the preparation of the sheep bone polypeptide calcium chelate, the amount of sheep bone polypeptide used is 0.5-2 wt% of the calcium chloride solution.
[0032] More preferably, in the preparation of the sheep bone polypeptide calcium chelate, the volume of anhydrous ethanol used is 4-16 times the volume of calcium chloride solution used.
[0033] This invention employs a method of pretreating fresh Hu sheep bones to obtain Hu sheep bone powder, followed by enzymatic hydrolysis to prepare Hu sheep bone polypeptides. The enzymatic hydrolysis utilizes a complex protease comprising trypsin and papain, with a mass ratio of trypsin to papain of 1:0.1-10. The hydrolysis is further purified using silica gel column separation. The silica gel column is filled with silica gel or modified silica gel. The modified silica gel is prepared by first activating the silica gel with acid, then functionalizing it with 3-aminopropyltriethoxysilane, and finally reacting it with a modified monomer. Therefore, this invention provides the following beneficial effects: the Hu sheep bone polypeptides exhibit high hydroxyl radical scavenging rate and good calcium chelation rate; the calcium chelated Hu sheep bone polypeptide calcium chelate has a good cell proliferation effect. Thus, this invention provides a method for preparing Hu sheep bone polypeptides with high hydroxyl radical scavenging rate, high calcium chelation rate, and good cell proliferation effect after calcium chelation. Attached Figure Description
[0034] Figure 1 This is a graph showing the scavenging rate of hydroxyl radicals.
[0035] Figure 2 This is a graph showing the calcium chelation rate.
[0036] Figure 3 This is a graph showing cell proliferation rate. Detailed Implementation
[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0038] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.
[0039] Example 1: A method for preparing a bone polypeptide from Hu sheep Preparation of Huzhou sheep bone meal: Remove meat scraps from fresh Huzhou sheep bones, wash with deionized water, chop into bone pieces, soak the bone pieces in n-hexane for 24 hours, wash with deionized water, autoclave at 120℃ for 1 hour, cool to room temperature, dry, pulverize at 28000 r / min, and pass through an 80-mesh sieve to obtain Huzhou sheep bone meal. The amount of Huzhou sheep bone pieces used is 50 wt% of n-hexane. Deionized water is used appropriately during washing and autoclaving.
[0040] Preparation of Hu sheep bone polypeptides: Hu sheep bone powder was mixed with deionized water, the pH was adjusted to 6, a complex protease was added, and the enzymatic hydrolysis reaction was carried out at 37℃ for 3 hours. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 15 minutes, cooled to room temperature, and the supernatant was collected by centrifugation to prepare Hu sheep bone enzymatic hydrolysate. The Hu sheep bone enzymatic hydrolysate was purified by silica gel column chromatography and freeze-dried to obtain Hu sheep bone polypeptides. The amount of Hu sheep bone powder used was 5 wt% of deionized water. The complex protease included trypsin and papain, with a mass ratio of trypsin to papain of 1:1. The ratio of trypsin to Hu sheep bone powder used was 8000 U / g. The silica gel column was filled with silica gel.
[0041] Example 2: A method for preparing a bone polypeptide from Hu sheep The difference between this embodiment and Example 1 lies in the preparation of the sheep bone polypeptide; the filler in the silica column is replaced with modified silica.
[0042] Preparation of activated silica gel: Silica gel was added to a hydrochloric acid solution and stirred at 70°C for 8 hours. The mixture was then washed until neutral and dried to obtain activated silica gel. The hydrochloric acid content in the hydrochloric acid solution was 5 M, and the amount of silica gel used was 5 wt% of the hydrochloric acid solution.
[0043] Preparation of functionalized silica gel: Activated silica gel and 3-aminopropyltriethoxysilane were added to an organic reagent and mixed thoroughly. The mixture was then stirred at 60°C for 6 hours. After the reaction was completed, the mixture was cooled, washed, and dried to obtain the functionalized silica gel. The organic reagent was anhydrous ethanol. The amount of activated silica gel used was 10 wt% of anhydrous ethanol, and the amount of 3-aminopropyltriethoxysilane used was 100 wt% of activated silica gel.
[0044] Preparation of modified silica gel: Functionalized silica gel was added to acetonitrile, followed by the addition of a modified monomer and an initiator. The mixture was stirred at 60°C for 12 hours. After the reaction was complete, the silica gel was washed and dried to obtain the modified silica gel. The amount of functionalized silica gel used was 5 wt% of acetonitrile, the amount of the modified monomer was cyclohexyl vinyl ether (25 wt% of functionalized silica gel), and the amount of the initiator was AIBN (1 wt% of functionalized silica gel). Nitrogen was used as the inert gas.
[0045] Preparation of Hu sheep bone polypeptides: Hu sheep bone powder was mixed with deionized water, the pH was adjusted to 6, a complex protease was added, and the enzymatic hydrolysis reaction was carried out at 37℃ for 3 hours. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 15 minutes, cooled to room temperature, and the supernatant was collected by centrifugation to prepare Hu sheep bone enzymatic hydrolysate. The Hu sheep bone enzymatic hydrolysate was purified by silica gel column chromatography and freeze-dried to obtain Hu sheep bone polypeptides. The amount of Hu sheep bone powder used was 5 wt% of deionized water. The complex protease included trypsin and papain, and the mass ratio of trypsin to papain in the complex protease was 1:1. The relationship between the amount of trypsin used and the amount of Hu sheep bone powder used was 8000 U / g. The packing material in the silica gel column was modified silica gel.
[0046] Example 3: A method for preparing a bone polypeptide from Hu sheep The difference between this embodiment and Example 2 lies in the preparation of the modified silicone.
[0047] Preparation of modified silica gel: Functionalized silica gel was added to acetonitrile, followed by the addition of a modified monomer and an initiator. The mixture was stirred at 60°C for 12 hours. After the reaction was complete, the silica gel was washed and dried to obtain the modified silica gel. The amount of functionalized silica gel used was 5 wt% of acetonitrile, the amount of the modified monomer was cyclohexyl vinyl ether (10 wt% of functionalized silica gel), and the amount of the initiator was AIBN (1 wt% of functionalized silica gel). Nitrogen was used as the inert gas.
[0048] Example 4: A method for preparing a bone polypeptide from Hu sheep The difference between this embodiment and Example 2 lies in the preparation of the modified silicone.
[0049] Preparation of modified silica gel: Functionalized silica gel was added to acetonitrile, followed by the addition of modified monomers and an initiator. The mixture was stirred at 60°C for 12 hours. After the reaction was complete, the silica gel was washed and dried to obtain the modified silica gel. The amount of functionalized silica gel used was 5 wt% of acetonitrile. The modified monomers were cyclohexyl vinyl ether and diethyl allyl malonate. The amount of cyclohexyl vinyl ether used was 25 wt% of the functionalized silica gel, and the amount of diethyl allyl malonate used was 8 wt%. The initiator was AIBN, and the amount of AIBN used was 1 wt% of the functionalized silica gel. Nitrogen was used as the inert gas.
[0050] Example 5: A method for preparing a bone polypeptide from a lake sheep The difference between this embodiment and Example 2 lies in the preparation of the modified silicone.
[0051] Preparation of modified silica gel: Functionalized silica gel was added to acetonitrile, followed by the addition of modified monomers and an initiator. The mixture was stirred at 60°C for 12 hours. After the reaction was complete, the silica gel was washed and dried to obtain the modified silica gel. The amount of functionalized silica gel used was 5 wt% of acetonitrile. The modified monomers were cyclohexyl vinyl ether and diethyl allyl malonate. The amount of cyclohexyl vinyl ether used was 25 wt% of the functionalized silica gel, and the amount of diethyl allyl malonate used was 2 wt%. The initiator was AIBN, and the amount of AIBN used was 1 wt% of the functionalized silica gel. Nitrogen was used as the inert gas.
[0052] Comparative Example 1: A method for preparing a bone polypeptide from a lake sheep The difference between this comparative example and Example 2 lies in the preparation of the modified silicone.
[0053] Preparation of modified silica gel: Functionalized silica gel was added to acetonitrile, followed by the addition of a modified monomer and an initiator. The mixture was stirred at 60°C for 12 hours. After the reaction was complete, the silica gel was washed and dried to obtain the modified silica gel. The amount of functionalized silica gel used was 5 wt% of acetonitrile, the amount of the modified monomer was cyclohexyl vinyl ether (2 wt% of functionalized silica gel), and the amount of the initiator was AIBN (1 wt% of functionalized silica gel). Nitrogen was used as the inert gas.
[0054] Example 6: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of Hu sheep bone polypeptide calcium chelate: Hu sheep bone polypeptide was mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25°C with shaking for 1 hour. Then anhydrous ethanol was added, and the mixture was centrifuged at 5°C. The precipitate was freeze-dried to obtain Hu sheep bone polypeptide calcium chelate. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of Hu sheep bone polypeptide used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The Hu sheep bone polypeptide was derived from Example 1.
[0055] Example 7: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of Hu sheep bone polypeptide calcium chelate: Hu sheep bone polypeptide was mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25°C with shaking for 1 hour. Then anhydrous ethanol was added, and the mixture was centrifuged at 5°C. The precipitate was freeze-dried to obtain Hu sheep bone polypeptide calcium chelate. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of Hu sheep bone polypeptide used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The Hu sheep bone polypeptide was derived from Example 2.
[0056] Example 8: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of Hu sheep bone polypeptide calcium chelate: Hu sheep bone polypeptide was mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25°C with shaking for 1 hour. Then anhydrous ethanol was added, and the mixture was centrifuged at 5°C. The precipitate was freeze-dried to obtain Hu sheep bone polypeptide calcium chelate. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of Hu sheep bone polypeptide used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The Hu sheep bone polypeptide was derived from Example 3.
[0057] Example 9: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of Hu sheep bone polypeptide calcium chelate: Hu sheep bone polypeptide was mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25°C with shaking for 1 hour. Then anhydrous ethanol was added, and the mixture was centrifuged at 5°C. The precipitate was freeze-dried to obtain Hu sheep bone polypeptide calcium chelate. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of Hu sheep bone polypeptide used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The Hu sheep bone polypeptide was derived from Example 4.
[0058] Example 10: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of Hu sheep bone polypeptide calcium chelate: Hu sheep bone polypeptide was mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25°C with shaking for 1 hour. Then anhydrous ethanol was added, and the mixture was centrifuged at 5°C. The precipitate was freeze-dried to obtain Hu sheep bone polypeptide calcium chelate. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of Hu sheep bone polypeptide used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The Hu sheep bone polypeptide was derived from Example 5.
[0059] Comparative Example 2: A method for preparing a calcium chelate of sheep bone polypeptides Preparation of calcium chelate of sheep bone peptides: Sheep bone peptides were mixed with calcium chloride solution, the pH of the solution was adjusted to 7, and chelation was carried out in a constant temperature water bath at 25℃ with shaking for 1 h. Then anhydrous ethanol was added, and the mixture was centrifuged at 5℃. The precipitate was freeze-dried to obtain the calcium chelate of sheep bone peptides. The calcium chloride solution was prepared by mixing calcium chloride and deionized water, with a calcium chloride content of 1 wt%. The amount of sheep bone peptides used was 1 wt% of the calcium chloride solution, and the volume of anhydrous ethanol used was 8 times the volume of the calcium chloride solution. The sheep bone peptides were derived from Comparative Example 1.
[0060] Experimental example: The antioxidant capacity of the sheep bone polypeptides prepared in Examples 1-5 and Comparative Example 1 was tested in this invention. Using the scavenging of hydroxyl radicals as a representative measure, the salicylic acid method was used to determine the hydroxyl radical scavenging rate of the sheep bone polypeptides. In the test, 1 mL of 8 mg / L sheep bone polypeptide solution was used. The sheep bone polypeptide solution was prepared by mixing sheep bone polypeptides with deionized water. The results are as follows: Figure 1As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, and D1 is Comparative Example 1. In this invention, sheep bone is first processed to obtain sheep bone powder, then enzymatically hydrolyzed with a compound enzyme, and finally purified by silica gel column chromatography to obtain sheep bone polypeptide. The sheep bone polypeptide prepared by this invention has a good scavenging effect on hydroxyl radicals. In the silica gel column purification process of this invention, modified silica gel can also be used to purify the sheep bone polypeptide. In the preparation of modified silica gel, the silica gel is first activated with hydrochloric acid to obtain activated silica gel, then 3-aminopropyltriethoxysilane is reacted with the activated silica gel in anhydrous ethanol to obtain functionalized silica gel. Finally, the functionalized silica gel... Modified silica gel is prepared by reacting with a modifying monomer, including cyclohexyl vinyl ether. When the modified silica gel prepared by cyclohexyl vinyl ether is used in the purification of sheep bone polypeptide, the resulting sheep bone polypeptide exhibits a better scavenging effect against hydroxyl radicals. This indicates that the modified silica gel of the present invention can better prepare sheep bone polypeptide with a good scavenging effect against hydroxyl radicals. When the amount of cyclohexyl vinyl ether used is too low, it cannot effectively increase the amount of sheep bone polypeptide with a good scavenging effect against hydroxyl radicals. In the present invention, diethyl allyl malonate can also be added as a modifying monomer to modify the functionalized silica gel. The resulting modified silica gel can further increase the amount of sheep bone polypeptide with a good scavenging effect against hydroxyl radicals.
[0061] This invention calculated the calcium chelation rate of the sheep bone polypeptides prepared into calcium chelates in Examples 6-10 and Comparative Example 2. The calcium chelation rate was defined as the ratio of the reduced calcium content in the supernatant after centrifugation to the calcium content before chelation. The calcium content was determined by flame atomic absorption spectrometry. The results are as follows: Figure 2As shown, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, and D2 is Comparative Example 2. In this invention, sheep bone is first processed to obtain sheep bone powder, then enzymatically hydrolyzed with a compound enzyme, and finally purified by silica gel column chromatography to obtain sheep bone polypeptide. The sheep bone polypeptide prepared by this invention has a good chelating effect on calcium. In the silica gel column purification process of this invention, modified silica gel can also be used to purify the sheep bone polypeptide. In preparing modified silica gel, the silica gel is first activated with hydrochloric acid to obtain activated silica gel, and then 3-aminopropyltriethoxysilane is reacted with the activated silica gel in anhydrous ethanol to obtain functionalized silica gel. Finally, modified silica gel was prepared by reacting functionalized silica gel with modified monomers, including cyclohexyl vinyl ether. In the purification of sheep bone polypeptides, the modified silica gel prepared by cyclohexyl vinyl ether showed improved calcium chelation of the resulting sheep bone polypeptides, indicating that the modified silica gel in this invention can better prepare sheep bone polypeptides with good calcium chelation. When the amount of cyclohexyl vinyl ether used is too low, it cannot effectively increase the amount of sheep bone polypeptides with good calcium chelation. In this invention, diethyl allyl malonate can also be added as a modifying monomer to modify the functionalized silica gel, and the resulting modified silica gel can increase the amount of sheep bone polypeptides with good calcium chelation only.
[0062] In this invention, the effects of the sheep bone polypeptide calcium chelate in Examples 6-10 and Comparative Example 2 on cells were tested. MC3T3-E1 cells were subjected to a treatment of 5 × 10⁻⁶ cells / year. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates, with 100 μL of LMEM medium added to each well. The plates were cultured overnight, and the supernatant was discarded. Then, 100 μL of sheep bone polypeptide calcium chelate solution was added. A control group with the same concentration of calcium chloride solution was included. Cells were cultured at 37°C and 5% CO2 for 48 hours. Cell proliferation was detected using CCK-8 assay. The cell proliferation rate in the control group was 100%. Results are as follows: Figure 3As shown, S6 is Example 6, S7 is Example 7, S8 is Example 8, S9 is Example 9, S10 is Example 10, D2 is Comparative Example 2, and B is the control group. In this invention, sheep bone is first processed to obtain sheep bone powder, then enzymatically hydrolyzed with a compound enzyme, and finally purified by silica gel column chromatography to obtain sheep bone polypeptide. After preparing the sheep bone polypeptide calcium chelate, the sheep bone polypeptide calcium chelate has a good cell proliferation effect. In the silica gel column purification process of this invention, modified silica gel can also be used to purify the sheep bone polypeptide. In the preparation of modified silica gel, the silica gel is first activated with hydrochloric acid to obtain activated silica gel, then 3-aminopropyltriethoxysilane is reacted with the activated silica gel in anhydrous ethanol to obtain functionalized silica gel. Finally, the functionalized silica gel is reacted with modified monomers to prepare modified silica gel. The modified monomers include cyclohexylvinyl In the purification of sheep bone polypeptides, modified silica gel prepared by cyclohexyl vinyl ether modification, after preparing sheep bone polypeptide calcium chelates, improved cell proliferation. This indicates that the modified silica gel in this invention can better prepare sheep bone polypeptides with good calcium chelation effects. Preparing sheep bone polypeptides into calcium chelates improves cell proliferation. However, when the amount of cyclohexyl vinyl ether used is too low, it cannot effectively increase the amount of sheep bone polypeptides with good calcium chelation effects, and the improvement in cell proliferation is not significant. In this invention, diethyl allyl malonate can also be added as a modifying monomer to modify the functionalized silica gel. The resulting modified silica gel can increase the amount of sheep bone polypeptides with good calcium chelation effects only. Preparing sheep bone polypeptides into calcium chelates further improves cell proliferation.
[0063] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.
[0064] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.
Claims
1. A method for preparing a bone polypeptide from Hu sheep, comprising: Fresh Hu sheep bones were pretreated to prepare Hu sheep bone powder, and then the Hu sheep bone powder was enzymatically hydrolyzed to prepare Hu sheep bone polypeptides. The enzymatic hydrolysis used a complex protease, which included trypsin and papain, with a mass ratio of trypsin to papain of 1:0.1-10. The enzymatic hydrolysis was purified by silica gel column separation.
2. The preparation method according to claim 1, characterized in that, The pretreatment includes removing meat scraps from fresh Hu sheep bones, washing them, and then chopping them into Hu sheep bone pieces.
3. The preparation method according to claim 2, characterized in that, The sheep bone blocks were soaked in hexane, washed, and then pressure-cooked before being dried and pulverized to obtain sheep bone powder.
4. The preparation method according to claim 1, characterized in that, The relationship between the amount of trypsin used and the amount of sheep bone powder used is 6000-10000 U / g.
5. The preparation method according to claim 1, characterized in that, In the enzymatic hydrolysis treatment, the enzymatic hydrolysis reaction is carried out at 30-37℃ for 0.5-6 hours.
6. The preparation method according to claim 1, characterized in that, The filler in the silica column is silica gel or modified silica gel.
7. The preparation method according to claim 1, characterized in that, In the preparation of the modified silica gel, the silica gel is first activated with acid, then functionalized with 3-aminopropyltriethoxysilane, and then reacted with modified monomers to prepare the modified silica gel.
8. The preparation method according to claim 7, characterized in that, The modified monomer is cyclohexyl vinyl ether.
9. The sheep bone polypeptide prepared by any one of claims 1-8.
10. A method for preparing a calcium chelate of sheep bone polypeptide, comprising the preparation method described in any one of claims 1-8.