CN111808185A - Method for extracting elastin peptide from bovine cartilage - Google Patents
Method for extracting elastin peptide from bovine cartilage Download PDFInfo
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- CN111808185A CN111808185A CN202010652449.1A CN202010652449A CN111808185A CN 111808185 A CN111808185 A CN 111808185A CN 202010652449 A CN202010652449 A CN 202010652449A CN 111808185 A CN111808185 A CN 111808185A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 34
- 210000000845 cartilage Anatomy 0.000 title claims abstract description 27
- 241000283690 Bos taurus Species 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 20
- 102000016942 Elastin Human genes 0.000 title claims abstract description 18
- 108010014258 Elastin Proteins 0.000 title claims abstract description 18
- 229920002549 elastin Polymers 0.000 title claims abstract description 18
- 239000012528 membrane Substances 0.000 claims abstract description 40
- 239000000843 powder Substances 0.000 claims abstract description 22
- 102000004190 Enzymes Human genes 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 12
- 239000004365 Protease Substances 0.000 claims abstract description 12
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 238000001816 cooling Methods 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 238000001728 nano-filtration Methods 0.000 claims abstract description 8
- 238000001694 spray drying Methods 0.000 claims abstract description 8
- 238000000605 extraction Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000010008 shearing Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 230000001877 deodorizing effect Effects 0.000 claims abstract description 5
- 238000000227 grinding Methods 0.000 claims abstract description 5
- 238000010438 heat treatment Methods 0.000 claims abstract description 5
- 238000003756 stirring Methods 0.000 claims abstract description 5
- 230000000415 inactivating effect Effects 0.000 claims abstract description 4
- 239000008213 purified water Substances 0.000 claims abstract description 4
- 239000002904 solvent Substances 0.000 claims abstract description 4
- 238000000926 separation method Methods 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- 239000002131 composite material Substances 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims 2
- 239000005909 Kieselgur Substances 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 4
- 238000010521 absorption reaction Methods 0.000 abstract description 3
- 239000000047 product Substances 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- 210000004177 elastic tissue Anatomy 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- VEVRNHHLCPGNDU-MUGJNUQGSA-O desmosine Chemical compound OC(=O)[C@@H](N)CCCC[N+]1=CC(CC[C@H](N)C(O)=O)=C(CCC[C@H](N)C(O)=O)C(CC[C@H](N)C(O)=O)=C1 VEVRNHHLCPGNDU-MUGJNUQGSA-O 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000009295 crossflow filtration Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000006467 substitution reaction 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]
-
- 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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- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Gastroenterology & Hepatology (AREA)
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Abstract
The invention discloses a method for extracting elastin peptide from bovine cartilage, comprising the following steps: grinding bovine cartilage into powder; transferring the powder to an extraction tank, adding purified water 3-4 times the weight of the powder, heating to 95 ℃, and stirring for 2 hours; cooling to 50-55 ℃, adding compound protease, and inactivating enzyme after reaction to obtain enzyme shearing liquid; cooling to 50-60 deg.C, and decolorizing and deodorizing; separating solute and solvent by using a permeable membrane; a nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules; concentrating, and spray drying. The method utilizes different heat stability of protein peptide and non-peptide substances to extract bovine cartilage powder at high temperature, the extract is sheared by compound protease and then concentrated and filtered by different permeable membranes to obtain small peptide with the molecular weight within the range of 150-1000Dal, and the peptide with the molecular weight range accounts for more than 85 percent of the peptide, so that the absorption efficiency can be greatly increased.
Description
Technical Field
The invention relates to the technical field of biological extraction, in particular to a method for extracting elastin peptide from bovine cartilage.
Background
Two forms of elastin have been found: elastin I is present in the ligamentum nuchae, aorta and skin, and elastin II is available from cartilage.
Elastin is the main component of elastic fibres (elastic fibers) in the rawhide tissue. The peptide chain of elastin contains 713 or more amino acid residues. Unlike collagen and keratin, elastin has an amino acid sequence in which there is no continuous repetitive periodic structure throughout the peptide chain, but there are alternating hydrophobic and hydrophilic skin segments. Desmosine and open-chain desmosine formed from oxidized lysyl are unique cross-linked structures of elastin. These cross-linked structures are combined in more than two pairs. Elastic fibers are found primarily in ligaments and vessel walls. The presence of elastic fibers in combination with collagen fibers imparts elasticity and tensile strength to the tissue.
The existing method for extracting protein peptide from bovine cartilage adopts a preparation method of bovine cartilage collagen peptide with application number of 201710562191.4, and the method sequentially comprises the steps of pretreatment, desugarization treatment, online enzymolysis, hydrolysis, refining and filtering by adopting a precision filter, spray drying by adopting a high-pressure pump and the like.
Disclosure of Invention
The present invention provides a method for extracting elastin peptide from bovine cartilage.
The invention is realized by the following technical scheme:
a method for extracting elastin peptide from cattle cartilage comprises the following steps:
A. grinding bovine cartilage into powder;
B. transferring the powder to an extraction tank, adding purified water 3-4 times the weight of the powder, heating to 95 ℃, and stirring for 2 hours;
C. cooling to 50-55 ℃, adding compound protease, and inactivating enzyme after reaction to obtain enzyme shearing liquid;
D. cooling to 50-60 deg.C, and decolorizing and deodorizing;
E. separating solute and solvent by using a permeable membrane;
F. a nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules;
G. concentrating, and spray drying.
According to the scheme, the protein peptide and the non-peptide substance have different heat stability, the bovine cartilage powder is extracted at high temperature, the extract is sheared by the compound protease and then concentrated and filtered by different permeable membranes, the small peptide with the molecular weight within the range of 150 plus 1000Dal is obtained, the peptide with the molecular weight within the range accounts for more than 85% of the peptide, the absorption efficiency can be greatly increased, and a good foundation is provided for the research on the efficacy of peptide products with different molecular weight ranges.
Preferably, the permeable membrane in the step E is a composite tubular membrane, the membrane inlet pressure is 1.6-1.7/Bar, the membrane outlet pressure is 0.8-1.0 Bar, the temperature is 55-60 ℃, the flow rate of the concentrated solution is 300-400LPM, and the flow rate of the clear solution is 70-90 LPM.
Preferably, when the nanofiltration membrane is used for separation, the membrane inlet pressure is 10-20Bar, the membrane outlet pressure is 10-20Bar, the flow rate of the concentrated solution is 20-40LPM in the first 5 minutes, the flow rate of the clear solution is more than 100LPM, and the pressure of the filter is 0.35 MPA.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. according to the invention, the high-temperature extraction is carried out on the bovine cartilage powder by utilizing different heat stability of protein peptide and non-peptide substances, the extract is sheared by compound protease and then concentrated and filtered by different permeable membranes to obtain the small peptide with the molecular weight within the range of 150 plus 1000Dal, and the peptide with the molecular weight within the range accounts for more than 85% of the peptide, so that the absorption efficiency can be greatly increased, and a good basis is provided for the research on the efficacy of peptide products with different molecular weight ranges.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention.
FIG. 1 is a flow chart of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1
A method for extracting elastin peptide from cattle cartilage as shown in figure 1 comprises the following steps:
A. grinding bovine cartilage into powder;
B. transferring the powder to an extraction tank, adding purified water 3-4 times the weight of the powder, heating to 95 ℃, and stirring for 2 hours;
C. cooling to 50-55 ℃, adjusting to the optimal reaction condition of the enzyme, adding compound protease, and inactivating the enzyme after the reaction is finished to obtain enzyme shearing liquid; specifically, the enzyme deactivation can be carried out by heating. The addition amount of the compound protease is 0.2 percent of the weight of the bovine cartilage powder, so that the saturation of the compound protease is ensured, and the compound protease is not wasted excessively.
D. Cooling to 50-60 deg.C, and decolorizing and deodorizing; the method preferably adopts a plate-and-frame filter, the plate-and-frame filter is precoated with diatomite, activated carbon accounting for 0.5 percent of the weight of the bovine cartilage powder is added into the enzyme shearing liquid, the mixture is mixed and stirred for 0.5 hour, and the pressure is applied during the filtration of the plate-and-frame filter until the filtrate is yellow clear transparent liquid.
E. Separating solute and solvent by using a permeable membrane; the permeable membrane can adopt a composite tubular membrane, and in the separation operation process, the membrane inlet pressure is controlled to be 1.6-1.7/Bar, the membrane outlet pressure is controlled to be 0.8-1.0 Bar, the temperature is 55-60 ℃, the flow rate of concentrated solution is 300-400LPM, and the flow rate of clear solution is 70-90 LPM. In the separation process, the product has no phase change, the effective components are completely retained, and the membrane and the pipeline system meet the food sanitation level requirement, thereby greatly improving the product quality. Removing mycelium, pigment and small molecular impurities.
F. A nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules; because the operation mode of the membrane adopts cross-flow filtration, the interception substances retained on the surface of the membrane pores can be automatically cleaned during working, thereby achieving the purpose of predetermined purification and separation. In the separation process, the membrane inlet pressure is controlled to be 10-20Bar, the membrane outlet pressure is controlled to be 10-20Bar, the flow rate of the concentrated solution is 20-40LPM in the first 5 minutes, the flow rate of the clear solution is more than 100LPM, and the pressure of the filter is 0.35 MPA.
G. Concentrating, and spray drying.
Example 2
Based on the principle of the foregoing embodiment, the present embodiment discloses a specific embodiment mode, specifically:
pretreatment: cleaning ox cartilage, grinding, and pulverizing.
Adding 1kg of ox cartilage powder and 4kg of hot water into an extraction tank, keeping the water temperature at 95 ℃, and extracting for 2 hours.
Cooling to 55 ℃, adding compound protease accounting for 0.2 percent of the weight of the bovine cartilage powder, and controlling the water temperature to be 50-55 ℃ for enzymolysis. After the enzymolysis is finished, the temperature is raised to 95 ℃, and the temperature is kept for 30 min.
Cooling to 50-60 deg.C, and removing odor and decolorizing; precoating the plate-and-frame filter with diatomite, adding 30g of active carbon, mixing and stirring for 0.5h, and pressurizing the mixture through the plate-and-frame filter until the filtrate is yellow clear transparent liquid.
The method adopts a composite tubular membrane equal permeable membrane for separation, wherein the membrane inlet pressure is 1.6Bar, the membrane outlet pressure is 0.8Bar, the temperature is 55 ℃, the concentrated solution flow is 300LPM, and the clear solution flow is 79 LPM. A nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules: the membrane inlet pressure is 12Bar, the membrane outlet pressure is 10Bar, the concentrated solution flow is 23LPM at the first 5 minutes, the clear solution flow is more than 100LPM, and the filter pressure is 0.35 MPA.
Concentrating the enzymolysis liquid by a double-effect concentrator until the solid content is 40-60 ℃, and then carrying out centrifugal spray drying. And during centrifugal spray drying, controlling the temperature of a feed inlet at 172 ℃, the temperature of air outlet at 105 ℃ and the feeding speed at 200r/min, and spray drying the concentrated filtrate to obtain 200g of elastin peptide powder.
By adopting the method of the embodiment, the protein peptide is extracted and detected, and the content of the detected protein is more than 90%. Due to the separation by the nanofiltration membrane, the molecular mass of the peptide reaches 85 percent at 150-1000 Dal.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for extracting elastin peptide from bovine cartilage, comprising the following steps:
A. grinding bovine cartilage into powder;
B. transferring the powder to an extraction tank, adding purified water 3-4 times the weight of the powder, heating to 95 ℃, and stirring for 2 hours;
C. cooling to 50-55 ℃, adding compound protease, and inactivating enzyme after reaction to obtain enzyme shearing liquid;
D. cooling to 50-60 deg.C, and decolorizing and deodorizing;
E. separating solute and solvent by using a permeable membrane;
F. a nanofiltration membrane is adopted to separate 150-1000Dal organic micromolecules;
G. concentrating, and spray drying.
2. The method as claimed in claim 1, wherein the permeable membrane in step E is a composite tubular membrane with an inlet pressure of 1.6-1.7/Bar, an outlet pressure of 0.8-1.0 Bar, a temperature of 55-60 ℃, a concentrate flow rate of 300-400LPM and a clear flow rate of 70-90 LPM.
3. The method for extracting elastin peptide from ox cartilage as claimed in claim 1, wherein decolorizing and deodorizing treatment is performed by plate and frame filter, diatomaceous earth is precoated on the plate and frame filter, activated carbon with 0.5% weight of ox cartilage powder is added into the enzyme shearing liquid, and the mixture is mixed and stirred for 0.5h, and pressurized during filtering by plate and frame filter.
4. The method of claim 1, wherein the separation is performed with a nanofiltration membrane at a membrane inlet pressure of 10-20Bar and a membrane outlet pressure of 10-20Bar, the flow rate of the concentrate is 20-40LPM 5 minutes before the start, the flow rate of the clear solution is greater than 100LPM, and the pressure of the filter is 0.35 MPA.
5. The method of claim 1, wherein the enzyme-cleaved solution is concentrated to a soluble solids content of 40-60 ° and then spray-dried.
6. The method for extracting elastin peptide from bovine cartilage as claimed in claim 1, wherein the amount of compound protease is 0.2% of the weight of bovine cartilage powder.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112176017A (en) * | 2020-10-27 | 2021-01-05 | 威海市宇王集团海洋生物工程有限公司 | Method for efficiently extracting protein peptide from sea cucumber intestines |
CN114794489A (en) * | 2022-04-20 | 2022-07-29 | 河南磐康健康管理股份有限公司 | Composition for preventing and improving joint diseases and preparation method thereof |
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CN109943616A (en) * | 2019-04-18 | 2019-06-28 | 威海市宇王集团海洋生物工程有限公司 | A method of the abstraction function albumen from giant salamander |
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CN101913583A (en) * | 2010-08-20 | 2010-12-15 | 曹荣军 | Method for extracting chondroitin, collagen and high-calcium powder from animal cartilage |
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CN112176017A (en) * | 2020-10-27 | 2021-01-05 | 威海市宇王集团海洋生物工程有限公司 | Method for efficiently extracting protein peptide from sea cucumber intestines |
CN114794489A (en) * | 2022-04-20 | 2022-07-29 | 河南磐康健康管理股份有限公司 | Composition for preventing and improving joint diseases and preparation method thereof |
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