Preparation method and application of yak bone marrow protein peptide for increasing bone mineral density

Abstract

The invention relates to the field of small molecule peptide extraction, in particular to a preparation method and application of yak bone marrow protein peptide for increasing bone mineral density, and solves the problems that in the cryopreservation process, the activity of protein is reduced, enzymolysis is not thorough after a polyphenol compound is combined with protein, and the activity of small molecule peptide is low. Comprising the following steps: embedding a polyphenol compound by using a special embedding material to form a protein protection body, mixing yak bone marrow and the protein protection body, performing ultrasonic dispersion, freeze-drying, putting into an extracting solution, adding petroleum ether into a lower-layer extracting solution for extracting, removing grease and the special embedding material in one step, then adding saturated ammonium sulfate, filtering, and dissolving by using clear water. The method comprises the following steps: firstly, preparing a macromolecular protein active solution, carrying out ultrafiltration enzymolysis on the macromolecular protein active solution to obtain a small molecular peptide solution, and carrying out post-treatment to obtain the high-activity protein peptide. According to the method, the protein is convenient to transport after being refrigerated at low temperature, and the activity of small molecule peptide after enzymolysis is higher.

Description

Technical Field

[0001] The invention relates to the technical field of small molecule peptide extraction, and in particular to a preparation method and application of yak bone marrow protein peptide for increasing bone density. Background Art

[0002] Yak bone marrow protein has extremely high nutritional and medicinal value. The bioactive peptides contained in it have higher biological activity than ordinary bone marrow protein. It not only plays a role in the treatment of hypertension and tumor inhibition, but can also be used as a drug carrier, making it easier to deliver drugs directly to the lesion site for treatment. In order to meet higher drug delivery performance and pursue a smaller amount of drug to achieve the maximum therapeutic effect, there are extremely high requirements for the activity of yak bone marrow protein peptides. Therefore, the protein extracted from bovine bone marrow has different degrees of loss of activity from extraction to storage, transportation and use. It is easily affected by temperature, humidity, physical and chemical environment, etc., resulting in the inactivation of the small molecule polypeptides obtained by enzymatic hydrolysis and difficult to use.

[0003] In the process of purifying protein, the protein must first be released from the tissue and maintained in its natural state so that it does not lose its activity. The protein is then freeze-dried for easy transportation, and then separated and purified from oils for use. Therefore, the proteins purchased on the market are freeze-dried products, which are purified and separated when used. Freeze-drying first will fill the space of the protein molecular chain with lipids and other substances, which has sufficient steric hindrance to prevent the protein from deforming and inactivating. However, this method has a destructive effect on the activity of the protein, mainly due to the formation of ice crystals during freezing and deterioration. The existing solution is to add sugars and complex phosphates to indirectly act on the protein, but this method reduces the purity of the protein, and more purification steps are required later, resulting in a decrease in the activity of the protein peptide.

[0004] In order to reduce the impact of low-temperature freezing on proteins, polyphenol compounds are used to contact and combine with proteins, thereby bonding with the peptide bonds of the proteins to achieve hydrogen bonds or ionic bonds and better protect the activity of the proteins. However, this method makes it difficult to decompose the proteins into small molecular peptides during subsequent enzymatic hydrolysis. Polarized enzymatic hydrolysis conditions can separate polyphenols and achieve enzymatic hydrolysis, but this method is more unfavorable for retaining protein activity, so this problem needs to be solved. Summary of the invention

[0005] In order to solve the problems that the activity of protein decreases due to freezing deterioration during cryopreservation, and that polyphenol compounds combine with proteins to cause incomplete enzymatic hydrolysis and low activity of small molecule peptides, the present invention provides a method for preparing yak bone marrow protein peptides for increasing bone density.

[0006] The technical solution adopted by the present invention is as follows:

[0007] The protocol includes the following preparation steps:

[0008] S1. Using a special embedding material to embed the polyphenol compound to form a protein protection body, pre-treating the yak bone marrow, mixing the pre-treated yak bone marrow with the protein protection body, performing ultrasonic dispersion, and freeze-drying to prepare a protein powder;

[0009] The special embedding material is collodion;

[0010] S2, placing the protein powder in the extraction solution, the solid-liquid ratio is 1:10-20g / mL, the extraction temperature is 40-50°C, the time is 2-3h, the extraction number is 2-3 times, the oil and water layer is separated, petroleum ether is added to the lower layer of the extract for extraction, the oil and special embedding material are removed in one step, and the protein solution is separated again, and then saturated ammonium sulfate is added to the protein solution, filtered and dissolved with clean water to obtain a macromolecular protein active solution;

[0011] S3. Ultrafiltration is performed on the macromolecular protein active solution to obtain an enzymatic hydrolysis preparatory solution, complex enzymes are added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis to obtain a small molecule peptide solution, and then post-processing is performed to obtain a highly active protein peptide.

[0012] The present application mainly improves and stabilizes the activity of protein peptides through two steps. First, collodion is used as an embedding component of polyphenol compounds. The embedding material will not connect or react with any active sites of proteins, and the stability of the environment is maintained to the maximum extent, so that the active sites of proteins can be preserved. In addition, the use of collodion reduces the bonding connection between polyphenol compounds and proteins, weakens the difficulty of separating proteins from polyphenol compounds, and does not make subsequent enzymatic hydrolysis difficult. After mixing with yak bone marrow, ultrasonic dispersion is performed. 70% of bovine bone marrow is fat, which facilitates the maximum dispersion of collodion between proteins and is used for bonding a small amount of key active sites. The remaining sites of the protein are occupied by collodion, thereby protecting the protein from the influence of freeze-drying temperature and preventing freezing and deterioration.

[0013] Secondly, the present application uses collodion as the embedding material, which is soluble in petroleum ether and can be removed at the same time as the grease removal step. The related embedding agents in the field, such as gelatin, sodium alginate, chitosan, carrageenan and instant agar, do not have the effect of synchronously removing grease and grease, and are prone to cause residues. The residual substances mentioned above will improve the tolerance and stability of the protein, but on the contrary will inhibit the enzymatic hydrolysis of the protein, making it difficult to form small molecule peptides. The collodion is easy to remove and can also remove a large amount of polyphenol compounds, ensuring the smooth enzymatic hydrolysis of the protein. After the removal with ether, the protein is further purified 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 affecting the activity of the protein.

[0014] Finally, enzymatic hydrolysis is performed to hydrolyze the highly active protein into highly active protein peptides.

[0015] Preferably, the polyphenol compound is one of catechins, anthocyanins or theaflavins, and the weight ratio of the polyphenol compound to the special embedding material is 2-4:10.

[0016] The polyphenols used in the present application serve as excellent hydrogen donors, forming hydrogen bonds with the carbonyl groups of proteins, and some hydrophobic amino acid residues of proteins can also produce hydrophobic interactions with the non-polar aromatic rings of polyphenols, thereby enhancing the antioxidant and anti-degradation capabilities of proteins. However, excessive interactions will lead to the formation of covalent bonds between polyphenol compounds and proteins, so encapsulation materials are used to limit this hydrophobic interaction to prevent excessive linkage.

[0017] Preferably, the mass ratio of the pretreated yak bone marrow to the protein protector is 3-5:1, the ultrasonic treatment power is between 200-500W, each working time is 5s and the interval is 5s, and it is repeated 30-60 times.

[0018] Preferably, the pretreatment steps are as follows:

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

[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 comprises freezing a mixture of yak bone marrow and the protein protector to -20°C and freeze-drying the mixture, and then adding liquid nitrogen to crush the mixture, wherein the mass ratio of liquid nitrogen to the protein protector is 5:1.

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

[0023] Preferably, the ultrafiltration membrane used in the ultrafiltration has a molecular weight cutoff of 10KDa-50KDa.

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

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

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

[0027] Bone protease: It is mainly composed of endoenzymes, exoenzymes and flavor enzymes, which can avoid the formation of bitter peptides and achieve thorough enzymatic hydrolysis.

[0028] Elastase: It can digest and decompose elastin in connective tissue protein, including enzymatic hydrolysis of peptide bond-bound, amide-bound and ester-bound ones.

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

[0030] The obtained small molecule peptide liquid is centrifuged for 8-10 minutes at a centrifugal speed of 2000-3000 rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1-5 KDa to obtain a highly active protein peptide.

[0031] The small molecule peptide prepared by the preparation method of the yak bone marrow protein peptide for increasing bone density in the present application has high activity, can be used as a drug carrier, improves the drug delivery efficiency, and has stronger activity and stability.

[0032] The beneficial effects of the present invention are:

[0033] 1. The present application uses collodion as an embedding material for polyphenol compounds, which does not affect the activity of proteins and is easy to remove, does not reduce the efficiency of enzymatic hydrolysis, does not affect the activity of enzymatic hydrolysis, and the obtained small molecule peptides have high activity.

[0034] 2. The present application uses ultrasonic treatment of the collodion embedding material and the protein liquid, which has good dispersibility and bonds to the exposed active sites without over-bonding to the unexposed active sites, thus facilitating the subsequent removal of polyphenol compounds and not affecting the enzymatic hydrolysis. The enzymatic hydrolysis environment is more mild and stable, and the obtained small molecule peptides are less inactivated. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a line graph of the protein peptide activity of the examples of the present invention and the comparative examples. DETAILED DESCRIPTION

[0036] The following will refer to the attached Figure 1 The embodiments of the present invention are described in detail. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0037] Preparation Example 1

[0038] Preparation of protein powder

[0039] Weigh 40 g of nitrocellulose cotton, 200 g of 70 wt% ethanol and 560 g of ether, dissolve the nitrocellulose cotton in the ether, then mix and stir 240 g of catechins and ethanol and drop them into the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0040] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1600 g was weighed, and then 160 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0041] The pretreated yak bone marrow and the protein protector were mixed in a mass ratio of 4:1, and stirred under ultrasonic treatment. The ultrasonic treatment power was between 300 W, and the working time was 5 s and the interval was 5 s. This was repeated 50 times until the protein protector was dispersed to an invisible state to obtain a mixture.

[0042] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[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 and stir 160g of anthocyanin with ethanol and add dropwise to the ether solution containing nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0046] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1200 g was weighed, and then 120 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0047] The pretreated yak bone marrow and the protein protector were mixed in a mass ratio of 3:1, and stirred under ultrasonic treatment of an ultrasonic processor. The ultrasonic treatment power was between 200 W, and the working time was 5 s and the interval was 5 s, and the process was repeated 30 times until the protein protector was dispersed to an invisible state to obtain a mixture.

[0048] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[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, stir, and drop into the ether solution containing nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0052] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 2000 g was weighed, and then 200 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0053] The pretreated yak bone marrow and the protein protector were mixed in a mass ratio of 5:1, and stirred under ultrasonic treatment of an ultrasonic processor. The ultrasonic treatment power was between 500 W, and the working time was 5 s and the interval was 5 s, and the process was repeated 60 times until the protein protector was dispersed to an invisible state to obtain a mixture.

[0054] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[0055] Preparation Example 4 - Carrageenan was used to encapsulate polyphenol compounds instead of collodion

[0056] Preparation of protein powder

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

[0058] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1600 g was weighed, and then 160 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0059] The pretreated yak bone marrow and carrageenan-embedded emulsion were mixed in a mass ratio of 1.2:1, and stirred under ultrasonic treatment of an ultrasonic processor. The ultrasonic treatment power was between 300 W, and the working time was 5 s and the interval was 5 s, and the process was repeated 50 times until the protein protected body was dispersed to an invisible state to obtain a mixture.

[0060] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[0061] Preparation Example 5 - Dispersion of protein protectants 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 and stir 240g of catechins and ethanol and drop them into the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0064] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1600 g was weighed, and then 160 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

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

[0066] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[0067] Preparation Example 6—Pretreatment of Yak Bone Marrow without 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 and stir 240g of catechins and ethanol and drop them into the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0070] Separate the yak bone marrow from the yak bones, wash them with deionized water, remove the broken bones and flesh, weigh 1600g, mix the yak bone marrow with the protein protector in a mass ratio of 3-5:1, and stir under ultrasonic treatment of an ultrasonic processor with an ultrasonic treatment power between 200-500W. Work for 5s and rest for 5s, repeat 30-60 times, until the protein protector is dispersed to an invisible state to obtain a mixture.

[0071] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

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

[0073] Preparation of protein powder

[0074] Weigh 40 g of nitrocellulose cotton, 200 g of 70% ethanol and 560 g of ether, dissolve the nitrocellulose cotton in the ether, then mix and stir 400 g of catechins and ethanol and drop them into the ether solution containing the nitrocellulose cotton. After stirring and homogenizing, slowly evaporate the solvent to obtain a protein protected body in a viscous colloid state.

[0075] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1600 g was weighed, and then 160 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0076] The pretreated yak bone marrow and the protein protector were mixed in a mass ratio of 4:1, and stirred under ultrasonic treatment of an ultrasonic processor. The ultrasonic treatment power was between 300 W, and the working time was 5 s and the interval was 5 s, and the process was repeated 50 times until the protein protector was dispersed to an invisible state to obtain a mixture.

[0077] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[0078] Preparation Example 8 - The amount of collodion used exceeds the maximum amount

[0079] Preparation of protein powder

[0080] Weigh 40 g of nitrocellulose cotton, 200 g of 70% ethanol and 560 g of ether, dissolve the nitrocellulose cotton in the ether, then mix 80 g of catechins and ethanol, stir, and dropwise add to the ether solution containing the nitrocellulose cotton, stir and homogenize, slowly evaporate the solvent to obtain a protein protector in a viscous colloid state.

[0081] The yak bone marrow was separated from the yak bones and washed with deionized water to remove the broken bones and flesh and blood. 1600 g was weighed, and then 160 g of 10 wt% trehalose solution was mixed with the yak bone marrow and stirred for 10 min. The pretreated yak bone marrow was filtered through a filter and was in a jelly-like state.

[0082] The pretreated yak bone marrow and the protein protector were mixed in a mass ratio of 4:1, and stirred under ultrasonic treatment of an ultrasonic processor. The ultrasonic treatment power was between 300 W, and the working time was 5 s and the interval was 5 s, and the process was repeated 50 times until the protein protector was dispersed to an invisible state to obtain a mixture.

[0083] The dispersed mixture was frozen to -20°C and freeze-dried, and then liquid nitrogen was added to pulverize it. The mass ratio of liquid nitrogen to protein protection body was 5:1, and protein powder was obtained.

[0084] Example 1

[0085] A method for preparing yak bone marrow protein peptide for increasing bone density

[0086] The protein powder prepared in Preparation Example 1 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15g / mL, an extraction temperature of 45°C, an extraction time of 2.5h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution.

[0087] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0088] Example 2

[0089] A method for preparing yak bone marrow protein peptide for increasing bone density

[0090] The protein powder prepared in Preparation Example 2 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:10 g / mL, an extraction temperature of 40°C, an extraction time of 2 h, and two extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution.

[0091] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 10KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 1:2:2:1; the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis is performed for 4 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 8 minutes at a centrifugal speed of 2000rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1KDa to obtain a highly active protein peptide.

[0092] Example 3

[0093] A method for preparing yak bone marrow protein peptide for increasing bone density

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

[0095] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 50KDa to obtain an enzymatic hydrolysis preparatory solution, a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 3:3:2:2; the enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis is performed for 5h, a small molecule peptide solution is obtained, and the obtained small molecule peptide solution is centrifuged for 10min at a centrifugal speed of 3000rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 5KDa to obtain a highly active protein peptide.

[0096] Comparative Example 1

[0097] A method for preparing yak bone marrow protein peptide for increasing bone density

[0098] The protein powder prepared in Preparation Example 4 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15 g / mL, an extraction temperature of 45°C, an extraction time of 2.5 h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution.

[0099] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0100] Comparative Example 2

[0101] A method for preparing yak bone marrow protein peptide for increasing bone density

[0102] The protein powder prepared in Preparation Example 5 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15g / mL, an extraction temperature of 45°C, an extraction time of 2.5h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution;

[0103] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0104] Comparative Example 3

[0105] A method for preparing yak bone marrow protein peptide for increasing bone density

[0106] The protein powder prepared in Preparation Example 6 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15g / mL, an extraction temperature of 45°C, an extraction time of 2.5h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution.

[0107] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0108] Comparative Example 4

[0109] A method for preparing yak bone marrow protein peptide for increasing bone density

[0110] The protein powder prepared in Preparation Example 7 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15g / mL, an extraction temperature of 45°C, an extraction time of 2.5h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and then saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution;

[0111] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0112] Comparative Example 5

[0113] A method for preparing yak bone marrow protein peptide for increasing bone density

[0114] The protein powder prepared in Preparation Example 8 was placed in a 0.5wt% sodium chloride solution to extract protein, with a solid-liquid ratio of 1:15g / mL, an extraction temperature of 45°C, an extraction time of 2.5h, and three extractions. The oil-water layer was separated, and petroleum ether was added to the lower extract to extract, and the oil and special embedding material were removed in one step. The protein solution was separated again, and saturated ammonium sulfate was added to the protein solution, filtered, and dissolved in clean water to obtain a macromolecular protein active solution;

[0115] The macromolecular protein active solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 20KDa to obtain an enzymatic hydrolysis preparatory solution, and a composite enzyme is added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis, wherein the amount of the composite enzyme is 2% of the mass of the enzymatic hydrolysis preparatory solution, and the composite enzyme includes trypsin, papain, bone protein hydrolase and elastase, and the mass addition ratio of the above four enzymes is 2:2:2:1; the enzymatic hydrolysis temperature is 53°C, the enzymatic hydrolysis is performed for 4.5 hours, a small molecule peptide solution is obtained, and then the obtained small molecule peptide solution is centrifuged for 9 minutes at a centrifugal speed of 2600rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 3KDa to obtain a highly active protein peptide.

[0116] Comparative Example 6 - No step of saturated ammonium sulfate and clean water

[0117] Comparative Example 7: No ultrafiltration of macromolecular protein active solution

[0118] Experiments and data

[0119] The highly active protein peptides prepared in all the above examples and comparative examples were tested for biological activity and the protein content after protein extraction was tested.

[0120] The detection method is as follows:

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

[0122] Control group: boric acid + ACE + hippurylhistidylleucine + Tris-HCl buffer

[0123] Example group: protein peptide + ACE + hippuryl histidyl leucine + Tris-HCl buffer protein peptide activity: control group - example group / control group * 100%

[0124] The protein content was determined using the BCA method, with the unit being 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 activity of the above protein peptides is plotted as follows: Figure 1 shown.

[0128] analyze

[0129] According to the experimental data of Example 1, Example 2 and Example 3, the present application can effectively reduce the binding of polyphenols with proteins by using collodion to embed polyphenol compounds, while also retaining a large amount of protein activity.

[0130] According to the data of comparative example 1, the protein peptide activity of comparative example 1 is low and the content of small molecules is low, which proves that the embedding of collodion does not affect the effect of protease on the molecular chain in the subsequent enzymatic hydrolysis process, while carrageenan reduces the enzymatic hydrolysis efficiency, resulting in a decrease in the content of small molecule proteins.

[0131] According to the data of Comparative Example 2, the protein peptide activity of Comparative Example 2 is lower and the small molecule content is normal, which proves that ultrasonic treatment of dispersed protein protectors can effectively increase the protection of polyphenol compounds to proteins and will not aggregate in large quantities, which is a factor that has a greater impact on the activity of protein peptides.

[0132] According to the data of Comparative Example 3, the protein peptide activity of Comparative Example 3 is the lowest, and the content of small molecule protein is normal, indicating that the pretreatment of trehalose can effectively form a layer of ink on the protein surface, thereby protecting the protein from losing activity in subsequent reactions.

[0133] According to the data of Comparative Example 4, the protein peptide activity and protein content of Comparative Example 4 decreased slightly, proving that the amount of collodion used was small, resulting in incomplete embedding of polyphenol compounds, thereby inhibiting the enzymatic hydrolysis process of macromolecular proteins.

[0134] According to the data of Comparative Example 5, excessive use of collodion will lead to a significant decrease in protein peptide activity and protein content, making it difficult for polyphenol compounds to effectively contact proteins, significantly reducing activity, and incomplete enzymatic hydrolysis.

[0135] According to the experimental data of Comparative Examples 6 and 7, the presence of ammonium sulfate inhibits the enzymatic hydrolysis process, but has little effect on the activity of the protein peptide.

[0136] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle 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 these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A method for preparing yak bone marrow protein peptide for increasing bone density, characterized in that: The method comprises the following preparation steps: S1. Using a special embedding material to embed the polyphenol compound to form a protein protection body, pre-treating the yak bone marrow, mixing the pre-treated yak bone marrow with the protein protection body, performing ultrasonic dispersion, and freeze-drying to prepare a protein powder; The special embedding material is collodion; S2, placing the protein powder in the extraction solution, the solid-liquid ratio is 1:10-20g / mL, the extraction temperature is 40-50°C, the time is 2-3h, the extraction number is 2-3 times, the oil and water layer is separated, petroleum ether is added to the lower layer of the extract for extraction, the oil and special embedding material are removed in one step, and the protein solution is separated again, and then saturated ammonium sulfate is added to the protein solution, filtered and dissolved with clean water to obtain a macromolecular protein active solution; S3. Ultrafiltration is performed on the macromolecular protein active solution to obtain an enzymatic hydrolysis preparatory solution, complex enzymes are added to the enzymatic hydrolysis preparatory solution for enzymatic hydrolysis to obtain a small molecule peptide solution, and then post-processing is performed to obtain a highly active protein peptide.

2. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The polyphenol compound is one of catechins, anthocyanins or theaflavins, and the weight ratio of the polyphenol compound to the special embedding material is 2-4:

10.

3. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The mass ratio of the pretreated yak bone marrow to the protein protector is 3-5:1, the ultrasonic treatment power is between 200-500W, each working time is 5s and the interval is 5s, and it is repeated 30-60 times.

4. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The steps of the pretreatment are as follows: The yak bone marrow is separated from the yak bone and washed with deionized water to remove broken bones and flesh and blood. The yak bone marrow is then soaked in a trehalose solution, wherein the mass ratio of the trehalose solution to the yak bone marrow is 1-2:

10. The pretreated yak bone marrow is obtained after filtration.

5. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The freeze-drying step of the protein powder includes freezing a mixture of yak bone marrow and the protein protector to -20°C for freeze-drying, and then adding liquid nitrogen for pulverization, wherein the mass ratio of liquid nitrogen to the protein protector is 5:

1.

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

7. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The ultrafiltration membrane used in the ultrafiltration has a molecular weight cutoff of 10KDa-50KDa.

8. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The complex enzyme comprises trypsin, papain, osteoprotease and elastase, and the mass addition ratio of the four enzymes is 1-3:2-3:2:1-2.

9. The method for preparing a yak bone marrow protein peptide for increasing bone density according to claim 1, characterized in that: The post-processing comprises the following steps: The obtained small molecule peptide liquid is centrifuged for 8-10 minutes at a centrifugal speed of 2000-3000 rpm / min, the precipitate is discarded and the supernatant is taken, and the supernatant is ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 1-5 KDa to obtain a highly active protein peptide.

10. Use of the preparation method of the yak bone marrow protein peptide for increasing bone density according to any one of claims 1 to 9 in drug carriers and health products.

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