A method for preparing yak bone marrow protein peptides that increase bone density and their application.

Abstract

This invention relates to the field of small molecule peptide extraction, specifically a method for preparing and applying yak bone marrow protein peptides that increase bone density. It solves the problems of decreased protein activity during cryopreservation, incomplete enzymatic hydrolysis after polyphenol compounds bind to proteins, and low activity of small molecule peptides. The method includes using a special embedding material to embed polyphenol compounds, forming a protein protectant; ultrasonically dispersing the mixture of yak bone marrow and the protein protectant; freeze-drying and placing the mixture in an extraction solution; adding petroleum ether to the lower layer of the extraction solution for extraction, removing oil and the special embedding material in one step; then adding saturated ammonium sulfate; filtering and dissolving in water to obtain a high-molecular-weight protein active solution; ultrafiltration and enzymatic hydrolysis of the high-molecular-weight protein active solution to obtain a small molecule peptide solution; and further post-processing to obtain highly active protein peptides. This invention achieves convenient transportation of proteins after low-temperature refrigeration while maintaining higher activity of the enzymatically hydrolyzed small molecule peptides.

Description

Technical Field

[0001] This invention relates to the field of small molecule peptide extraction technology, specifically to a method for preparing and applying yak bone marrow protein peptides that increase bone density. Background Technology

[0002] Yak bone marrow protein has extremely high nutritional and medicinal value. Compared with ordinary bone marrow protein, the bioactive peptides it contains have higher bioactivity, which not only plays a role in the treatment of hypertension and tumor inhibition, but also can serve as a drug carrier, making it easier to deliver drugs directly to the lesion site for treatment. In order to meet the requirements of higher drug delivery performance and pursue the goal of achieving the maximum therapeutic effect with less drug, there are extremely high requirements for the activity of yak bone marrow protein peptides. Therefore, the activity of proteins extracted from bovine bone marrow is lost to varying degrees from extraction to preservation, transportation and use. It is easily affected by temperature, humidity and physicochemical environment, which causes the small molecule peptides obtained by enzymatic hydrolysis to become inactive and difficult to use.

[0003] The process of purifying proteins begins with releasing them from tissues while maintaining their native state to preserve their activity. Next, the proteins are freeze-dried for easy transport, followed by lipid separation and purification before use. Therefore, commercially available proteins are freeze-dried products, requiring further purification before use. Freeze-drying fills the spaces between protein molecules with lipids and other substances, providing sufficient steric hindrance to prevent denaturation and inactivation. However, this method has a destructive impact on protein activity, primarily due to ice crystal formation during freezing. Current solutions involve adding sugars and complex phosphates to indirectly affect protein activity, but this reduces purity, requiring further purification steps and ultimately decreasing peptide activity.

[0004] To reduce the impact of cryogenic freezing on proteins, polyphenolic compounds are used to contact and bind with proteins, thereby forming hydrogen or ionic bonds and better protecting protein activity. However, this method makes it difficult to break down proteins into small peptides during subsequent enzymatic digestion. Polarized enzymatic digestion conditions are required to separate multiple compounds and achieve digestion, but this method is even less conducive to preserving protein activity. Therefore, this problem needs to be addressed. Summary of the Invention

[0005] To address the issues of decreased protein activity due to freezing degradation during cryopreservation, and incomplete enzymatic hydrolysis and low activity of small peptides caused by the binding of polyphenolic compounds to proteins, this invention provides a method for preparing yak bone marrow protein peptides that increase bone density.

[0006] The technical solution adopted in this invention is as follows:

[0007] The method includes the following preparation steps:

[0008] S1. Polyphenolic compounds are encapsulated using special encapsulating materials to form protein protectants. Yak bone marrow is pretreated, and the pretreated yak bone marrow is mixed with the protein protectants and dispersed by ultrasonication. The mixture is then freeze-dried to produce protein powder.

[0009] The special embedding material is collodion;

[0010] S2. Place the protein powder in the extraction solution with a material-to-liquid ratio of 1:10-20 g / mL, an extraction temperature of 40-50℃, an extraction time of 2-3 hours, and 2-3 extractions. Separate the oil and water layers, add petroleum ether to the lower layer of the extract for extraction, remove the oil and special embedding material in one step, separate again to obtain a protein solution, add saturated ammonium sulfate to the protein solution, filter and dissolve in water to obtain a macromolecular protein active solution.

[0011] S3. Ultrafiltration is performed on the active solution of macromolecular proteins to obtain an enzymatic hydrolysis preparation solution. A complex enzyme is added to the enzymatic hydrolysis preparation solution for enzymatic hydrolysis to obtain a small molecule peptide solution. Then, post-processing is performed to obtain highly active protein peptides.

[0012] This application primarily enhances and stabilizes the activity of protein peptides through two steps. First, collodion is used as an embedding component for polyphenolic compounds. This embedding material does not bind or react with any active sites of the protein, maximizing environmental stability and preserving the protein's active sites. Furthermore, the use of collodion reduces the bonding between polyphenolic compounds and proteins, making separation between them easier and preventing difficulties in subsequent enzymatic hydrolysis. After mixing with yak bone marrow, the mixture is ultrasonically dispersed. Since 70% of yak bone marrow is fat, collodion can disperse maximally among the proteins, allowing for a small amount of bonding to key active sites. The remaining protein sites are occupied by collodion, thus protecting the proteins from the effects of freeze-drying temperatures and preventing freeze-drying degradation.

[0013] Secondly, this application uses kerosene as an embedding material, which is soluble in petroleum ether and can remove grease simultaneously. In contrast, related embedding agents in the field, such as gelatin, sodium alginate, chitosan, carrageenan, and instant agar, do not have the effect of removing grease at the same time and are prone to leaving residues. The residues of the above substances can improve the tolerance and stability of proteins, but conversely, they can inhibit the enzymatic hydrolysis of proteins and make it difficult to form small peptides. Keratin is easy to remove and can also remove a large number of polyphenolic compounds, ensuring the smooth progress of protein enzymatic hydrolysis. After removal with ether, the protein is further purified by using saturated ammonium sulfate and water. This step only involves salting out, which is a physical change and will not affect the activity of the protein, thereby further removing the components that affect the activity of the protein.

[0014] Finally, enzymatic hydrolysis is performed, which breaks down the highly active protein into highly active protein peptides.

[0015] Preferably, the polyphenolic compound is one of catechin, anthocyanin or theaflavin, and the ratio of the amount of the polyphenolic compound to the amount of the special encapsulating material by weight is 2-4:10.

[0016] The polyphenols used in this application serve as excellent hydrogen donors, forming hydrogen bonds with the carbonyl groups of proteins. Furthermore, some hydrophobic amino acid residues of proteins can also generate hydrophobic interactions with the nonpolar aromatic rings of polyphenols, enhancing the antioxidant capacity and resistance to denaturation of proteins. However, excessive interactions can lead to the formation of covalent bonds between polyphenol compounds and proteins. Therefore, embedding materials are used to limit this hydrophobic interaction to prevent over-linking.

[0017] Preferably, the mass ratio of pretreated yak bone marrow to the protein protectant is 3-5:1, the ultrasonic treatment power is between 200-500W, the treatment is repeated 30-60 times with a 5-second interval between treatments.

[0018] Preferably, the preprocessing steps are as follows;

[0019] The yak bone marrow is separated from the yak bones and washed with deionized water to remove bone fragments and flesh. Then, the yak bone marrow is soaked in a trehalose solution with a mass ratio of 1-2:10 to the yak bone marrow. After filtration, the pretreated yak bone marrow is obtained.

[0020] Trehalose forms non-covalent bonds with the amino acid residues of proteins, preventing exposed protein residues from denaturing at low temperatures.

[0021] Preferably, the freeze-drying step of the protein powder includes freezing the mixture of yak bone marrow and protein protectant to -20°C for freeze-drying, and then adding liquid nitrogen for pulverization, wherein the mass ratio of liquid nitrogen to protein protectant is 5:1.

[0022] Preferably, the extract is a 0.5 wt% sodium chloride solution.

[0023] Preferably, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 10 kDa to 50 kDa.

[0024] Preferably, the complex enzyme includes trypsin, papain, osteoprotease and elastase, and the mass ratio of the above four enzymes is 1-3:2-3:2:1-2.

[0025] Trypsin: It can cleave the carboxyl side of lysine and arginine residues in polypeptide chains.

[0026] Papain: A thiol protease that hydrolyzes the carboxyl terminus of arginine and lysine in proteins and polypeptides, and preferentially hydrolyzes peptide bonds of amino acids with two carboxyl groups at the N-terminus or aromatic L-amino acids.

[0027] Bone proteinase: mainly composed of endopeptidase, exopeptidase and flavor enzyme, which can avoid the generation of bitter peptide flavor and is completely enzymatically hydrolyzed.

[0028] Elastase: It can digest and break down elastin in connective tissue proteins, including peptide-bound, amide-bound, and ester-bound proteins.

[0029] Preferably, the post-processing includes the following steps:

[0030] The obtained small molecule peptide solution was centrifuged for 8-10 minutes at a speed of 2000-3000 rpm / min. The precipitate was discarded and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1-5 kDa to obtain highly active protein peptides.

[0031] The method for preparing yak bone marrow protein peptides that increases bone density, as described in this application, produces small molecule peptides with high activity, which can serve as drug carriers, improving drug delivery efficiency and exhibiting stronger activity and stability.

[0032] The beneficial effects of this invention are as follows:

[0033] 1. This application uses collodion as an embedding material for polyphenolic compounds, which does not affect protein activity, is easy to remove, does not reduce enzymatic hydrolysis efficiency, does not affect enzymatic hydrolysis activity, and the resulting small molecule peptides have high activity.

[0034] 2. This application uses ultrasonic treatment to treat the collodion-embedded material and protein solution, which has good dispersibility and binds to the exposed active sites without over-binding the unexposed active sites. This facilitates the subsequent removal of polyphenolic compounds and does not affect the enzymatic hydrolysis process. The enzymatic hydrolysis environment is more mild and stable, and the resulting small molecule peptides are less inactivated. Attached Figure Description

[0035] Figure 1 This is a line graph showing the protein peptide activity of the embodiments and comparative examples of the present invention. Detailed Implementation

[0036] The following will refer to the attached reference. Figure 1 The various embodiments of the present invention will be described in detail below. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0037] Preparation Example 1

[0038] Preparation of protein powder

[0039] Weigh 40g of nitrocellulose cotton, 200g of 70wt% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 240g of catechin with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0040] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1600g of the marrow was weighed and then 160g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0041] The pretreated yak bone marrow and protein protectants were mixed at a mass ratio of 4:1 and stirred under ultrasonic treatment. The ultrasonic treatment power was between 300W, with a 5-second interval between each 5-second operation, and the mixture was repeated 50 times until the protein protectants were dispersed to an invisible state, thus obtaining the mixture.

[0042] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0043] Preparation Example 2

[0044] Preparation of protein powder

[0045] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 160g of anthocyanins with ethanol and add the mixture dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0046] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1200g of the marrow was weighed and then 120g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0047] Pretreated yak bone marrow and protein protectants were mixed at a mass ratio of 3:1 and stirred under ultrasonic treatment by an ultrasonic processor with an ultrasonic power of 200W. The mixture was repeated 30 times, with a 5-second interval between each 5-second operation, until the protein protectants were dispersed to an invisible state, thus obtaining the mixture.

[0048] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0049] Preparation Example 3

[0050] Preparation of protein powder

[0051] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 320g of theaflavins with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0052] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 2000g of the marrow was weighed and then mixed with 200g of 10wt% trehalose solution. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0053] The pretreated yak bone marrow and protein protectants were mixed at a mass ratio of 5:1 and stirred under ultrasonic treatment by an ultrasonic processor with an ultrasonic power of 500W. The mixture was repeated 60 times, with a 5-second interval between each 5-second operation, until the protein protectants were dispersed to an invisible state, thus obtaining the mixture.

[0054] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0055] Preparation Example 4 – Encapsulation of polyphenol compounds using carrageenan instead of collodion

[0056] Preparation of protein powder

[0057] 240g of tea polyphenols were mixed with 1000g of water to prepare a clear solution. Then, 200g of 5wt% sodium carbonate ethanol suspension was added to the clear solution. 180g of 1wt% carrageenan solution was then prepared. The carrageenan solution was added dropwise to the above mixture and the pH was adjusted to 7.6 to obtain a carrageenan-encapsulated emulsion of tea polyphenols, which was in a relatively thin colloidal state.

[0058] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1600g of the marrow was weighed and then 160g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0059] Pretreated yak bone marrow was mixed with carrageenan embedding emulsion at a mass ratio of 1.2:1 and stirred under ultrasonic treatment with an ultrasonic processor at a power of 300W. The mixture was repeated 50 times with a 5-second interval between each 5-second interval until the protein protectant was dispersed to an invisible state, thus obtaining the mixture.

[0060] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0061] Preparation Example 5 – Protein Protectors Dispersed Without Ultrasonic Treatment

[0062] Preparation of protein powder

[0063] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 240g of catechin with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0064] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1600g of the marrow was weighed and then 160g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0065] The pretreated yak bone marrow and protein protectant were mixed at a mass ratio of 4:1 and stirred for 10 minutes to obtain the mixture.

[0066] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0067] Preparation Example 6 – Pretreatment of yak bone marrow without the use of trehalose

[0068] Preparation of protein powder

[0069] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 240g of catechin with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0070] Separate the yak bone marrow from the yak bones and wash them with deionized water to remove bone fragments and flesh. Weigh 1600g of the mixture and mix the yak bone marrow with the protein protectant at a mass ratio of 3-5:1. Stir the mixture under ultrasonic treatment with an ultrasonic processor at a power between 200-500W, working for 5 seconds and then resting for 5 seconds, repeating the process 30-60 times until the protein protectant is dispersed to an invisible state, thus obtaining the mixture.

[0071] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0072] Preparation Example 7 – The amount of collodion used is less than the minimum amount.

[0073] Preparation of protein powder

[0074] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 400g of catechin with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0075] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1600g of the marrow was weighed and then 160g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0076] The pretreated yak bone marrow and protein protectants were mixed at a mass ratio of 4:1 and stirred under ultrasonic treatment by an ultrasonic processor with an ultrasonic power of 300W. The mixture was repeated 50 times with a 5-second interval between each 5-second interval until the protein protectants were dispersed to an invisible state, thus obtaining the mixture.

[0077] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0078] Preparation Example 8 – The amount of collodion used exceeded the maximum amount.

[0079] Preparation of protein powder

[0080] Weigh 40g of nitrocellulose cotton, 200g of 70% ethanol and 560g of ether. Dissolve the nitrocellulose cotton in the ether. Then, mix 80g of catechin with ethanol and add it dropwise to the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protectant, which is a viscous colloidal substance.

[0081] The yak bone marrow was separated from the yak bones and washed with deionized water to remove bone fragments and flesh. 1600g of the marrow was weighed and then 160g of 10wt% trehalose solution was mixed with the yak bone marrow. The mixture was stirred for 10 minutes and filtered through a sieve to obtain the pre-treated yak bone marrow, which at this point had a jelly-like consistency.

[0082] The pretreated yak bone marrow and protein protectants were mixed at a mass ratio of 4:1 and stirred under ultrasonic treatment by an ultrasonic processor with an ultrasonic power of 300W. The mixture was repeated 50 times with a 5-second interval between each 5-second interval until the protein protectants were dispersed to an invisible state, thus obtaining the mixture.

[0083] The dispersed mixture was freeze-dried at -20°C, and then liquid nitrogen was added for pulverization. The mass ratio of liquid nitrogen to protein protectant was 5:1 to obtain protein powder.

[0084] Example 1

[0085] A method for preparing yak bone marrow protein peptides to increase bone density

[0086] The protein powder prepared in Preparation Example 1 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0087] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0088] Example 2

[0089] A method for preparing yak bone marrow protein peptides to increase bone density

[0090] The protein powder prepared in Preparation Example 2 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:10 g / mL, the extraction temperature was 40℃, the extraction time was 2 h, and the extraction was performed twice. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0091] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 1:2:2:1. The hydrolysis was carried out at 50°C for 4 hours to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 8 minutes at a speed of 2000 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1 kDa to obtain highly active protein peptides.

[0092] Example 3

[0093] A method for preparing yak bone marrow protein peptides to increase bone density

[0094] The protein powder prepared in Preparation Example 3 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:20 g / mL, the extraction temperature was 50℃, the extraction time was 3 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0095] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 3:3:2:2. The hydrolysis was carried out at 55°C for 5 hours to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 10 minutes at a speed of 3000 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa to obtain highly active protein peptides.

[0096] Comparative Example 1

[0097] A method for preparing yak bone marrow protein peptides to increase bone density

[0098] The protein powder prepared in Preparation Example 4 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0099] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0100] Comparative Example 2

[0101] A method for preparing yak bone marrow protein peptides to increase bone density

[0102] The protein powder prepared in Preparation Example 5 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0103] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0104] Comparative Example 3

[0105] A method for preparing yak bone marrow protein peptides to increase bone density

[0106] The protein powder prepared in Example 6 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0107] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0108] Comparative Example 4

[0109] A method for preparing yak bone marrow protein peptides to increase bone density

[0110] The protein powder prepared in Preparation Example 7 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0111] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0112] Comparative Example 5

[0113] A method for preparing yak bone marrow protein peptides to increase bone density

[0114] The protein powder prepared in Preparation Example 8 was placed in a 0.5 wt% sodium chloride solution to extract the protein. The material-to-liquid ratio was 1:15 g / mL, the extraction temperature was 45℃, the extraction time was 2.5 h, and the extraction was performed 3 times. The oil and water layers were separated, and petroleum ether was added to the lower extract for extraction to remove the oil and special embedding material in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution. The solution was filtered and dissolved in water to obtain a macromolecular protein active solution.

[0115] The active solution of large molecular weight proteins was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20 kDa to obtain an enzymatic hydrolysis preparation solution. A complex enzyme, comprising trypsin, papain, osteoprotease, and elastase, was added to the preparation solution for enzymatic hydrolysis at a mass ratio of 2:2:2:1. The hydrolysis was carried out at 53°C for 4.5 h to obtain a small molecular weight peptide solution. The obtained small molecular weight peptide solution was then centrifuged for 9 min at a speed of 2600 rpm. The precipitate was discarded, and the supernatant was collected. The supernatant was then ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa to obtain highly active protein peptides.

[0116] Comparative Example 6 – without the step of adding saturated ammonium sulfate and water

[0117] Comparative Example 7 – Solution of macromolecular protein without ultrafiltration

[0118] Experiments and Data

[0119] The bioactivity of the highly active protein peptides prepared in all the above examples and comparative examples was tested, and the protein content after protein extraction was also tested.

[0120] The detection method is as follows:

[0121] The highly active protein peptide was reacted with ACE and hippurylhistylleucine in Tris-HCl buffer. The reaction was started and timed. After the reaction was completed, the amount of hippuric acid produced was measured. Enzyme activity = change in product concentration / time.

[0122] Control group: Boric acid + ACE + hippuryl histidine leucine + Tris-HCl buffer

[0123] Example group: Protein peptide + ACE + hippuryl histamine leucine + Tris-HCl buffer. Protein peptide activity: Control group - Example group / Control group * 100%.

[0124] Protein content was determined using the BCA method, with units of mg / mL.

[0125] The experimental data are shown in Table 1:

[0126] Protein peptide activity Small molecule protein content Example 1 92.1％ 21.44 Example 2 90.4％ 19.68 Example 3 88.7％ 21.32 Comparative Example 1 84.5％ 14.21 Comparative Example 2 71.4％ 20.82 Comparative Example 3 62.7％ 20.43 Comparative Example 4 73.2％ 18.64 Comparative Example 5 68.9％ 12.61 Comparative Example 6 80.4％ 14.71 Comparative Example 7 77.2％ 9.74

[0127] The activities of the above protein peptides were plotted as curves, such as... Figure 1 As shown.

[0128] analyze

[0129] According to the experimental data of Examples 1, 2 and 3, this application can effectively reduce the binding of polyphenolic substances with proteins by using collodion to encapsulate polyphenolic compounds, while also preserving a large amount of protein activity.

[0130] According to the data from Comparative Example 1, the protein peptides in Comparative Example 1 have lower activity and lower content of small molecules, proving that the encapsulation of collodion does not affect the action of protease on molecular chains during enzymatic hydrolysis, while carrageenan reduces the efficiency of enzymatic hydrolysis, resulting in a decrease in the content of small molecule proteins.

[0131] According to the data from Comparative Example 2, the protein peptide activity of Comparative Example 2 is lower, while the content of small molecules is normal. This proves that ultrasonic treatment of the dispersed protein protectant can effectively increase the protection of proteins by polyphenolic compounds, preventing them from accumulating in large quantities. This is a factor that has a significant impact on the activity of protein peptides.

[0132] According to the data from Comparative Example 3, the protein peptide activity of Comparative Example 3 was the lowest, while the content of small molecule proteins was normal. This indicates that the pretreatment with trehalose can effectively form a layer of ink on the protein surface, thereby protecting the protein from losing its activity in subsequent reactions.

[0133] According to the data from Comparative Example 4, the protein peptide activity and protein content of Comparative Example 4 both decreased slightly, indicating that the amount of collodion used was relatively small, resulting in incomplete encapsulation of polyphenol compounds, which inhibited the enzymatic hydrolysis process of macromolecular proteins.

[0134] According to the data from Comparative Example 5, excessive use of collodion leads to a significant decrease in protein peptide activity and protein content, making it difficult for polyphenolic compounds to effectively contact proteins, resulting in a substantial reduction in activity and incomplete enzymatic hydrolysis.

[0135] Based on the experimental data from Comparative Examples 6 and 7, it can be seen that the presence of ammonium sulfate inhibits the enzymatic hydrolysis process, but has a relatively low impact on the activity of protein peptides.

[0136] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A method for preparing yak bone marrow protein peptides that increase bone density, characterized in that, The preparation process includes the following steps: S1. Encapsulating polyphenolic compounds with a special embedding material to form a protein protectant, pretreating yak bone marrow, mixing the pretreated yak bone marrow with the protein protectant, ultrasonically dispersing the mixture, and freeze-drying it to obtain protein powder; the special embedding material is collodion; S2. Placing the protein powder in an extraction solution with a material-to-liquid ratio of 1:10-20 g / mL, an extraction temperature of 40-50℃, an extraction time of 2-3 h, and 2-3 extractions; separating the oil and water layers, adding petroleum ether to the lower layer of the extract for extraction to remove the oil and special embedding material in one step, separating again to obtain a protein solution, then adding saturated ammonium sulfate to the protein solution, filtering and dissolving in water to obtain a macromolecular protein active solution; S3. Ultrafiltration is performed on the macromolecular protein active solution to obtain an enzymatic hydrolysis preparative solution. A complex enzyme is added to the preparative solution for enzymatic hydrolysis to obtain a small molecule peptide solution. Further post-treatment yields highly active protein peptides. The polyphenolic compound is one of catechin, anthocyanin, or theaflavins, and the weight ratio of the polyphenolic compound to the special encapsulating material is 2-4:

10. The mass ratio of the pretreated yak bone marrow to the protein protectant is 3-5:

1. The ultrasonic treatment power is between 200-500W, with a 5-second interval between treatments, repeated 30-60 times. The freeze-drying step of the protein powder includes freezing the mixture of yak bone marrow and the protein protectant to -20℃ for freeze-drying, followed by pulverization with liquid nitrogen. The mass ratio of liquid nitrogen to the protein protectant is 5:

1.

2. The method for preparing yak bone marrow protein peptides for increasing bone density according to claim 1, characterized in that, The pretreatment steps are as follows: Separate yak bone marrow from yak bones, wash with deionized water to remove bone fragments and flesh, then soak the yak bone marrow in trehalose solution, wherein the mass ratio of trehalose solution to yak bone marrow is 1-2:10, and filter to obtain pretreated yak bone marrow.

3. The method for preparing yak bone marrow protein peptides for increasing bone density according to claim 1, characterized in that, The extract is a 0.5 wt% sodium chloride solution.

4. The method for preparing yak bone marrow protein peptides for increasing bone density according to claim 1, characterized in that, The ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 10 kDa to 50 kDa.

5. The method for preparing yak bone marrow protein peptides for increasing bone density according to claim 1, characterized in that, The complex enzyme includes trypsin, papain, osteoprotein hydrolase, and elastase, with the mass ratio of the four enzymes being 1-3:2-3:2:1-2.

6. A method for preparing yak bone marrow protein peptides for increasing bone density according to claim 1, characterized in that, The post-processing includes the following steps: Centrifuge the obtained small molecule peptide solution for 8-10 minutes at a speed of 2000-3000 rpm / min, discard the precipitate and take the supernatant, and use an ultrafiltration membrane with a molecular weight cutoff of 1-5 kDa to ultrafilter the supernatant to obtain highly active protein peptides.

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