CN111662376B - Small molecule bovine bone marrow peptide and extraction method thereof - Google Patents
Small molecule bovine bone marrow peptide and extraction method thereof Download PDFInfo
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 78
- 241000283690 Bos taurus Species 0.000 title claims abstract description 58
- 210000001185 bone marrow Anatomy 0.000 title claims abstract description 56
- 238000000605 extraction Methods 0.000 title claims abstract description 36
- 150000003384 small molecules Chemical class 0.000 title claims description 16
- 210000000988 bone and bone Anatomy 0.000 claims abstract description 56
- 150000001875 compounds Chemical class 0.000 claims abstract description 37
- 102000008186 Collagen Human genes 0.000 claims abstract description 31
- 108010035532 Collagen Proteins 0.000 claims abstract description 31
- 229920001436 collagen Polymers 0.000 claims abstract description 30
- 108091005804 Peptidases Proteins 0.000 claims abstract description 29
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 29
- 102000035195 Peptidases Human genes 0.000 claims abstract description 24
- 102000004190 Enzymes Human genes 0.000 claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 claims abstract description 23
- 239000004365 Protease Substances 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 238000001914 filtration Methods 0.000 claims abstract description 15
- 239000000796 flavoring agent Substances 0.000 claims abstract description 15
- 235000019634 flavors Nutrition 0.000 claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 238000004140 cleaning Methods 0.000 claims abstract description 11
- 239000002131 composite material Substances 0.000 claims abstract description 8
- 238000001035 drying Methods 0.000 claims abstract description 7
- 230000000415 inactivating effect Effects 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- 239000007787 solid Substances 0.000 claims abstract description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 5
- 238000009835 boiling Methods 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 230000000694 effects Effects 0.000 claims description 18
- 235000019419 proteases Nutrition 0.000 claims description 14
- 208000010392 Bone Fractures Diseases 0.000 claims description 11
- 208000006670 Multiple fractures Diseases 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 102000004142 Trypsin Human genes 0.000 claims description 7
- 108090000631 Trypsin Proteins 0.000 claims description 7
- 239000012588 trypsin Substances 0.000 claims description 7
- 108090000145 Bacillolysin Proteins 0.000 claims description 6
- 108010004032 Bromelains Proteins 0.000 claims description 6
- 102000035092 Neutral proteases Human genes 0.000 claims description 6
- 108091005507 Neutral proteases Proteins 0.000 claims description 6
- 235000019835 bromelain Nutrition 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 5
- 238000013329 compounding Methods 0.000 claims description 3
- 108010007119 flavourzyme Proteins 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 24
- 229920001184 polypeptide Polymers 0.000 abstract description 19
- 238000002360 preparation method Methods 0.000 abstract description 6
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 230000007062 hydrolysis Effects 0.000 abstract description 4
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 4
- 230000002209 hydrophobic effect Effects 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 description 17
- 238000004364 calculation method Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 238000009928 pasteurization Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a small molecular bovine bone marrow peptide and an extraction method thereof, wherein the extraction method comprises the following steps: crushing and cleaning fresh bones; decocting and filtering the cleaned fresh bones, and collecting decoction; centrifuging the decoction to remove fat to obtain bone collagen solution; regulating the concentration of the collagen solution, and adding a compound enzymolysis agent for enzymolysis to obtain an enzymolysis solution, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme; and (3) sterilizing and inactivating enzyme, separating solid from liquid, concentrating and drying the enzymolysis liquid. According to the preparation method, the specific composite enzymolysis agent is selected, so that bone collagen can be fully hydrolyzed into micromolecular polypeptide through one-time hydrolysis, the extraction rate of bovine bone marrow peptide is improved, meanwhile, hydrophobic amino acid contained in the polypeptide can be hydrolyzed, the taste of bitter peptide can be improved, and the extracted bovine bone marrow peptide is ensured to have excellent taste and flavor.
Description
Technical Field
The invention relates to the technical field of polypeptide preparation, in particular to a small molecular bovine bone marrow peptide and an extraction method thereof.
Background
Niu Gusui peptide is a protein fragment obtained by enzymolysis of bovine bone marrow protein, and can be directly absorbed by human body to directly participate in vital activity; bovine bone marrow peptide has the beneficial effects of repairing damaged cells, stimulating immunoglobulin to produce antibodies, activating the activity of an intracellular lysosome system and the like.
At present, the extraction method of the bovine bone marrow peptide mainly comprises the steps of crushing, steaming, enzymolysis for multiple times, enzyme deactivation, filtration, concentration and drying to obtain the bovine bone marrow peptide; however, the bovine bone marrow peptides prepared by the existing method have the problems of low extraction rate, low content of small molecular weight bovine bone marrow peptides and long production period caused by multiple enzymolysis.
In view of this, the invention is proposed inter alia!
Disclosure of Invention
The first aim of the invention is to provide an extraction method of small molecular bovine bone marrow peptide, which can significantly improve the extraction rate of extracting bovine bone marrow peptide from bovine bone, and only needs to adopt a complex enzyme preparation for enzymolysis once, so that the production period is shorter.
The second object of the present invention is to provide a small-molecule bovine bone marrow peptide which has a high small-molecule peptide content and can be better absorbed by human body.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
an embodiment of the present invention provides a method for extracting small molecule bovine bone marrow peptide, which includes the following steps:
crushing and cleaning fresh bones;
decocting and filtering the cleaned fresh bones, and collecting decoction;
centrifuging the decoction to remove fat to obtain bone collagen solution;
regulating the concentration of the collagen solution, and adding a compound enzymolysis agent for enzymolysis to obtain an enzymolysis solution, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme;
sterilizing and inactivating enzyme, separating solid from liquid, concentrating with ultrafiltration membrane, and drying.
According to the preparation method, the specific composite enzymolysis agent is selected, and the collagen protein can be fully hydrolyzed into the micromolecular polypeptide through one-time hydrolysis, so that the extraction rate of the bovine bone marrow peptide is improved, meanwhile, the hydrophobic amino acid contained in the polypeptide can be hydrolyzed, the taste of the bitter peptide can be improved, and the extracted bovine bone marrow peptide is ensured to have excellent taste and flavor.
Research shows that the molecular weight of the bovine bone marrow peptide is below 180 and is mainly amino acid instead of polypeptide, so that the higher the content of the polypeptide with the molecular weight larger than 180 and close to the molecular weight 180 in the bovine bone marrow peptide is, the better the content of the polypeptide with the molecular weight between 180 and 500 in the extracted bovine bone marrow peptide can be obviously improved by selecting a specific composite enzymolysis agent, and the content of the polypeptide with the molecular weight between 180 and 500 can be particularly up to 41 percent, so that the bovine bone marrow peptide extracted by the application is easier to digest and absorb, and has better utilization rate.
Preferably, the crushing is to crush the fresh bones to 2-4 cm 3 The method comprises the steps of carrying out a first treatment on the surface of the The cleaning is to put broken bones into a cleaning machine to clean for 20-25 min, and then put into clear water to soak for 4-5 h.
Preferably, the mass ratio of the compound protease to the bone proteolytic enzyme to the flavor enzyme in the compound enzymolysis agent is (1-3) to 1; more preferably, the mass ratio of the compound protease, the bone proteolytic enzyme and the flavor enzyme in the compound enzymolysis agent is 2:2:1.
Preferably, the bone proteolytic enzyme activity is (30-35). Times.10 4 u/g, the activity of the flavor enzyme is (3-6) multiplied by 10 4 u/g。
Preferably, each part of the compound protease is prepared by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin; more preferably, the bromelain activity is (20 to 30). Times.10 4 u/g, neutral protease activity is (5-10). Times.10 4 u/g, trypsin activity is 2000-4000 u/g.
Preferably, the enzymatic hydrolysis comprises:
regulating the pH value of the collagen solution to 6.0-6.2, and adding a composite enzymolysis agent at 54-56 ℃ for enzymolysis for 3-4 h.
Through the specific limitation of enzymolysis conditions, the activity of each enzyme is regulated, the bone collagen is hydrolyzed into polypeptide by one-time hydrolysis under the combined action of different enzymes, and the amount and the size of hydrolyzed polypeptide are ensured.
Preferably, the concentration of the collagen solution is regulated to 5-8 Baume, and the mass ratio of the composite enzymolysis agent to the collagen solution is (1-2) to 100.
Preferably, the boiling and filtering the cleaned fresh bone, and collecting the boiling liquid comprises:
placing the cleaned fresh bones into clear water for first decoction and filtration to obtain first decoction and decoction broken bones;
then the boiled crushed bones and clear water are boiled and filtered for the second time to obtain second boiling liquid;
combining the first decoction and the second decoction to obtain decoction;
wherein: in the first time of boiling, the mass ratio of the fresh bone to the clear water is 1:5-6, the boiling temperature is 110-120 ℃, and the boiling time is 2-3 hours;
in the second boiling, the mass ratio of the boiled broken bones to the clean water is 1: (3-4), the boiling temperature is 110-120 ℃ and the boiling time is 1-2 h.
Through the specific boiling process, bovine bone marrow protein can be better extracted, and the extraction rate of bovine bone marrow peptide can be better ensured.
Preferably, the filtration employs a filter media pore size of 150 mesh.
Preferably, the pore size of the filter medium used for the solid-liquid separation is 0.2-0.5 μm.
The second aspect of the invention provides the small-molecule bovine bone marrow peptide prepared by the extraction method.
Compared with the prior art, the invention has the beneficial effects that at least:
according to the preparation method, the specific composite enzymolysis agent is selected, so that bone collagen can be fully hydrolyzed into micromolecular polypeptide through one-time hydrolysis, the extraction rate of bovine bone marrow peptide is improved, meanwhile, hydrophobic amino acid contained in the polypeptide can be hydrolyzed, the taste of bitter peptide can be improved, and the extracted bovine bone marrow peptide is ensured to have excellent taste and flavor.
The bovine bone marrow peptide prepared by the invention has the advantages that the molecular weight is more than 180, the content of the polypeptide which is close to the molecular weight of 180 is higher, the content of the polypeptide with the molecular weight of 180-500 in the bovine bone marrow peptide can be up to 41%, so that the bovine bone marrow peptide extracted by the application is easier to digest and absorb, and the application has better utilization rate.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.
It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
The following examples used the following raw materials:
bone proteolytic enzyme: the activity is (30-35) x 10 4 u/g, purchased from Toyowa biotechnology limited;
flavor enzyme: the activity is (3-6) x 10 4 u/g; purchased from eastern Henghua road biotechnology limited;
each part of compound protease is prepared by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin, wherein:
bromelain: the activity is (20-30) x 10 4 u/g, purchased from Shanghai Yuan Yes Biotechnology Co., ltd;
neutral protease: the activity is (5-10) x 10 4 u/g, purchased from Toyowa biotechnology limited;
trypsin: the activity is 2000-4000 u/g, purchased from Toyowa Biotechnology Co., ltd.
Example 1
The embodiment is an extraction method of small molecule bovine bone marrow peptide, which comprises the following steps:
crushing fresh bone to 2-4 cm 3 Placing the crushed bones into a cleaning machine for cleaning for 20min, and then placing into clear water for soaking for 5h;
placing the cleaned fresh bones into clear water for first decoction and filtering by adopting a filter bag with 150 meshes to obtain first decoction and decoction broken bones;
then the boiled crushed bones are boiled in clear water for the second time and filtered by a filter bag with 150 meshes to obtain second boiling liquid;
combining the first decoction and the second decoction to obtain decoction, wherein: in the first decoction, the mass ratio of the fresh bone to the clear water is 1:5, the decoction temperature is 110 ℃, and the decoction time is 3 hours;
in the second boiling, the mass ratio of the boiled broken bones to the clean water is 1:4, the boiling temperature is 110 ℃, and the boiling time is 1h;
centrifuging the decoction at a rotation speed of 15000r/min to remove fat and obtain bone collagen solution;
regulating the concentration of the collagen solution to 5 Baume, regulating the pH of the collagen solution to 6.0-6.2, and adding a compound enzymolysis agent at 54 ℃ according to the mass ratio of the compound enzymolysis agent to the collagen solution of 1.5:100 for enzymolysis for 4 hours, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme according to the mass ratio of 3:1:1;
and (3) sterilizing and inactivating enzyme by using a pasteurization method, and then performing solid-liquid separation, concentration and drying by using a filter medium with the pore diameter of 0.5 mu m to obtain the micromolecular bovine bone marrow peptide. The weight of the small molecular bovine bone marrow peptide obtained in example 1 was weighed, and the extraction rate was calculated from the extraction rate=weight of small molecular bovine bone marrow peptide/weight of fresh bone by 100%, and the calculated result was 12.3%.
Example 2
The embodiment is an extraction method of small molecule bovine bone marrow peptide, which comprises the following steps:
crushing fresh bone to 2-4 cm 3 Placing the crushed bones into a cleaning machine for cleaning for 25min, and then placing the crushed bones into clear water for soaking for 4h;
placing the cleaned fresh bones into clear water for first decoction and filtering by adopting a filter bag with 150 meshes to obtain first decoction and decoction broken bones;
then the boiled crushed bones are boiled in clear water for the second time and filtered by a filter bag with 150 meshes to obtain second boiling liquid;
combining the first decoction and the second decoction to obtain decoction, wherein: in the first decoction, the mass ratio of the fresh bone to the clear water is 1:6, the decoction temperature is 120 ℃, and the decoction time is 2 hours;
in the second boiling, the mass ratio of the boiled broken bones to the clean water is 1:3, the boiling temperature is 120 ℃, and the boiling time is 2 hours;
centrifuging the decoction at a rotation speed of 15000r/min to remove fat and obtain bone collagen solution;
regulating the concentration of the collagen solution to 8 Baume, regulating the pH of the collagen solution to 6.0-6.2, and adding a compound enzymolysis agent at 56 ℃ according to the mass ratio of the compound enzymolysis agent to the collagen solution of 1.5:100 for enzymolysis for 3 hours, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme according to the mass ratio of 1:3:1;
and (3) sterilizing and inactivating enzyme by using a pasteurization method, and then performing solid-liquid separation, concentration and drying by using a filter medium with the pore diameter of 0.5 mu m to obtain the micromolecular bovine bone marrow peptide.
The extraction rate of the small molecule bovine bone marrow peptide in this example was calculated according to the calculation method of the extraction rate in example 1, and the calculation result was 12.8%.
Example 3
The embodiment is an extraction method of small molecule bovine bone marrow peptide, which comprises the following steps:
crushing fresh bone to 2-4 cm 3 Placing the crushed bones into a cleaning machine for cleaning for 25min, and then placing into clear water for soaking for 5h;
placing the cleaned fresh bones into clear water for first decoction and filtering by adopting a filter bag with 150 meshes to obtain first decoction and decoction broken bones;
then the boiled crushed bones are boiled in clear water for the second time and filtered by a filter bag with 150 meshes to obtain second boiling liquid;
combining the first decoction and the second decoction to obtain decoction, wherein: in the first decoction, the mass ratio of the fresh bone to the clear water is 1:5, the decoction temperature is 115 ℃, and the decoction time is 3 hours;
in the second boiling, the mass ratio of the boiled broken bones to the clean water is 1:3.5, the boiling temperature is 115 ℃ and the boiling time is 1.5h;
centrifuging the decoction at a rotation speed of 15000r/min to remove fat and obtain bone collagen solution;
regulating the concentration of the collagen solution to 6 Baume, regulating the pH of the collagen solution to 6.0-6.2, and adding a compound enzymolysis agent at 55 ℃ according to the mass ratio of the compound enzymolysis agent to the collagen solution of 1.5:100 for enzymolysis for 3 hours, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme according to the mass ratio of 2:2:1;
and (3) sterilizing and inactivating enzyme by using a pasteurization method, and then performing solid-liquid separation, concentration and drying by using a filter medium with the pore diameter of 0.5 mu m to obtain the micromolecular bovine bone marrow peptide.
The extraction rate of the small molecule bovine bone marrow peptide in this example was calculated according to the calculation method of the extraction rate in example 1, and the calculation result was 13.6%.
Comparative example 1
The comparative example is an extraction method of small molecule bovine bone marrow peptide, which is basically the same as that of example 3, except that each part of compound protease is compounded by 3 parts of trypsin, 2 parts of papain and 3 parts of pepsin.
The extraction rate of the small molecule bovine bone marrow peptide in this example was calculated according to the calculation method of the extraction rate in example 1, and the calculation result was 10.4%.
Comparative example 2
The present comparative example is a method of extracting small molecule bovine bone marrow peptide, which is substantially the same as that of example 3, except that the bone proteolytic enzyme is replaced with the compound protease of the same fraction.
According to the calculation method of example 1 of the present application, the extraction content of bovine bone marrow peptide in the present comparative example was calculated, and the calculation result was 11.1%.
Comparative example 3
The comparative example is an extraction method of small molecule bovine bone marrow peptide, which is basically the same as the extraction method in example 3, except that the method is modified by adjusting the pH of the collagen solution to 6.0-6.2, adding a compound enzymolysis agent at 50 ℃ for enzymolysis for 3h, adjusting the pH of the collagen solution to 6.4-6.6, and adding a compound enzymolysis agent at 50 ℃ for enzymolysis for 3 h.
According to the calculation method of example 1 of the present application, the extraction content of bovine bone marrow peptide in the present comparative example was calculated, and the calculation result was 9.2%.
Experimental example
Selecting the same batch and appearance qualityThe cattle backbone is crushed to 2 cm to 4cm 3 The method comprises the steps of carrying out a first treatment on the surface of the Then dividing into 6 parts randomly, extracting according to the extraction methods of the bovine bone marrow peptides in examples 1-3 and comparative examples 1-3, and detecting the content of the extracted bovine bone marrow peptides in different molecular weight ranges; the detection unit is a Jiangnan university analysis test center, and the detection results are shown in table 1:
TABLE 1 content distribution of bovine bone marrow peptides extracted by different extraction methods
As can be seen from table 1:
according to the embodiment of the invention, the collagen can be fully hydrolyzed by selecting a specific compound enzyme preparation and hydrolyzing in one step, so that the polypeptide content of the prepared bovine bone marrow peptide with the molecular weight of 180-500 is the highest, and the polypeptide content of the bovine bone marrow peptide with the molecular weight of below 1000 can reach more than 80%.
Therefore, the bovine bone marrow peptide prepared by the invention has higher polypeptide content with the molecular weight larger than 180 and close to the molecular weight of 180, and the polypeptide content with the molecular weight between 180 and 500 in the bovine bone marrow peptide can reach 41 percent, so that the bovine bone marrow peptide extracted by the application is easier to digest and absorb, and has better utilization rate.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.
Claims (5)
1. The extraction method of the small molecular bovine bone marrow peptide is characterized by comprising the following steps:
crushing and cleaning fresh bones;
decocting and filtering the cleaned fresh bones, and collecting decoction;
centrifuging the decoction to remove fat to obtain bone collagen solution;
regulating the concentration of the collagen solution, and adding a compound enzymolysis agent for enzymolysis to obtain an enzymolysis solution, wherein the compound enzymolysis agent consists of compound protease, bone proteolytic enzyme and flavor enzyme;
sterilizing and inactivating enzyme, separating solid from liquid, concentrating and drying the enzymolysis liquid;
the mass ratio of the compound protease to the bone proteolytic enzyme to the flavor enzyme in the compound enzymolysis agent is (1-3) to 1;
the activity of the bone proteolytic enzyme is (30-35) x 10 4 u/g, the activity of the flavor enzyme is (3-6) multiplied by 10 4 u/g；
Each part of compound protease is prepared by compounding 3 parts of bromelain, 2 parts of neutral protease and 3 parts of trypsin;
the bromelain activity is (20-30) x 10 4 u/g, neutral protease activity is (5-10). Times.10 4 u/g, trypsin activity is 2000-4000 u/g;
regulating the concentration of the collagen solution to 5-8 Baume, wherein the mass ratio of the composite enzymolysis agent to the collagen solution is 1.5:100;
the enzymolysis comprises the following steps: regulating the pH value of the collagen solution to 6.0-6.2, and adding a composite enzymolysis agent at 54-56 ℃ for enzymolysis for 3-4 h.
2. The extraction method according to claim 1, wherein the mass ratio of the compound protease, the bone proteolytic enzyme and the flavourzyme in the compound enzymolysis agent is 2:2:1.
3. The method according to claim 1, wherein the steps of decocting and filtering the washed fresh bone and collecting the decoction include:
placing the cleaned fresh bones in clear water for first decoction and filtration to obtain first decoction and decoction broken bones;
placing the boiled crushed bones in clear water for second boiling and filtering to obtain second boiling liquid;
combining the first decoction and the second decoction to obtain decoction;
wherein: in the first time of boiling, the mass ratio of the fresh bone to the clear water is 1:5-6, the boiling temperature is 110-120 ℃, and the boiling time is 2-3 hours;
in the second boiling, the mass ratio of the boiled broken bones to the clean water is 1: (3-4), the boiling temperature is 110-120 ℃ and the boiling time is 1-2 h.
4. The extraction method according to claim 1, wherein the filtration medium used for the filtration has a pore size of 150 mesh;
the aperture of the filter medium adopted by the solid-liquid separation is 0.2-0.5 mu m.
5. The small molecule bovine bone marrow peptide prepared by the extraction method of any one of claims 1-4.
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