CN111893156A - Preparation method of refined yak bone collagen peptide - Google Patents
Preparation method of refined yak bone collagen peptide Download PDFInfo
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- CN111893156A CN111893156A CN202010765751.8A CN202010765751A CN111893156A CN 111893156 A CN111893156 A CN 111893156A CN 202010765751 A CN202010765751 A CN 202010765751A CN 111893156 A CN111893156 A CN 111893156A
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- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 110
- 102000008186 Collagen Human genes 0.000 title claims abstract description 85
- 108010035532 Collagen Proteins 0.000 title claims abstract description 85
- 229920001436 collagen Polymers 0.000 title claims abstract description 85
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 71
- 238000002360 preparation method Methods 0.000 title description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000001179 sorption measurement Methods 0.000 claims abstract description 18
- 239000000706 filtrate Substances 0.000 claims abstract description 16
- 239000011347 resin Substances 0.000 claims abstract description 16
- 229920005989 resin Polymers 0.000 claims abstract description 16
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000001914 filtration Methods 0.000 claims abstract description 12
- 238000000227 grinding Methods 0.000 claims abstract description 11
- 230000001954 sterilising effect Effects 0.000 claims abstract description 11
- 238000007670 refining Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 238000011068 loading method Methods 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 5
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 239000002002 slurry Substances 0.000 claims description 15
- 108091005658 Basic proteases Proteins 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 235000013372 meat Nutrition 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 239000000287 crude extract Substances 0.000 claims description 5
- 238000005520 cutting process Methods 0.000 claims description 5
- 238000005238 degreasing Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 4
- 238000001694 spray drying Methods 0.000 claims description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 238000000926 separation method Methods 0.000 abstract description 6
- 238000011033 desalting Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 30
- 241000283690 Bos taurus Species 0.000 description 7
- 238000000746 purification Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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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- 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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Abstract
The invention discloses a method for refining yak bone collagen peptide, which is characterized by comprising the following steps: (1) pre-treating; (2) crushing and grinding; (3) heating and extracting; (4) carrying out enzymolysis; (5) centrifuging and filtering; (6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 1-4 column volumes by using clear water to remove impurities after sample loading is finished, eluting by using 55-80% ethanol water solution, and collecting eluent to obtain yak bone collagen peptide purified liquid; (7) concentrating under reduced pressure; (8) and (5) drying and sterilizing. The invention purifies the yak bone collagen peptide by utilizing a macroporous adsorption resin separation technology, has the functions of enriching small molecular peptides, decoloring, desalting and removing peculiar smell, and is a production method capable of obviously improving the quality of the yak bone collagen peptide.
Description
Technical Field
The invention belongs to the technical field of collagen extraction, and particularly relates to a preparation method of refined yak bone collagen peptide.
Background
With the continuous improvement of living standard of people, the demand of cattle and mutton at home and abroad is continuously increased, and meanwhile, the breeding scale of cattle and sheep at home is rapidly enlarged under the support of the national government, but the low utilization rate of cattle and sheep bones leads to the serious waste of resources. Yak is a rare half-wild special population in China and even the world, mainly grows in blue sky and regions such as Qinghai-Tibet plateau and Qinghai where pasture is luxurious, the regions are high in cold and lack of oxygen and the freezing period is as long as about half a year, and the yak is also mixed with various rare Chinese herbal medicines in the pasture eaten in summer, so that the yak in the environment is delicious in meat quality and is a treasure throughout the world, wherein the yak bone is an absolutely-owned high-quality collagen extraction treasure house.
However, the quality gap between bovine bone collagen peptide and imported bone collagen peptide in domestic market at present mainly lies in: 1) the small molecular peptide concentration in the bovine bone collagen peptide in China is not enough; 2) the bovine bone collagen peptide products in China are yellow in color; 3) the peculiar smell of the domestic bovine bone collagen peptide product cannot be completely and effectively removed. The main technical reason for these problems is that the separation and purification process is too complicated or too crude, the traditional process mainly includes methods such as simple filtration, membrane filtration, ion exchange, and activated carbon adsorption, and in actual production, the adoption of the too simple separation and purification process can cause the above problems in the product, and the adoption of the too complicated separation and purification process can cause the production cost to be too high and the yield to be seriously reduced. Therefore, in order to solve the above problems, it is necessary to develop an efficient purification process for yak bone collagen peptide.
Disclosure of Invention
Aiming at the problems, the invention provides a method for refining yak bone collagen peptide, which purifies the yak bone collagen peptide by using a macroporous adsorption resin separation technology, has the effects of enriching small molecular peptides, decoloring, desalting and removing peculiar smell, and is a production method capable of obviously improving the quality of the yak bone collagen peptide.
The invention is realized by the following technical scheme.
A method for refining yak bone collagen peptide is characterized by comprising the following steps:
(1) pretreatment: removing fat, meat residue and the like on the surface of fresh yak bones, cleaning, cutting into blocks, degreasing, removing foreign proteins, and finally washing with clear water until the pH value is neutral;
(2) crushing and grinding: crushing the yak bones obtained in the step (1), adding a small amount of water, and grinding into slurry to obtain yak bone slurry;
(3) heating and extracting: adding water into the yak bone slurry for heating and extracting to obtain a yak bone collagen extracting solution;
(4) enzymolysis: adjusting the pH of the yak bone collagen extracting solution to a proper range, and adding enzyme for enzymolysis to obtain a yak bone collagen peptide crude extracting solution;
(5) centrifugal filtration: carrying out centrifugal filtration on the crude extract of the yak bone collagen peptide, and collecting filtrate to obtain yak bone collagen peptide filtrate;
(6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 1-4 column volumes by using clear water to remove impurities after sample loading is finished, eluting by using 55-80% ethanol water solution, and collecting eluent to obtain yak bone collagen peptide purified liquid;
(7) and (3) concentrating under reduced pressure: concentrating the purified yak bone collagen peptide solution under reduced pressure until the solution has no alcohol taste, and recovering ethanol to obtain concentrated yak bone collagen peptide solution;
(8) drying and sterilizing: and (3) carrying out freeze drying or spray drying on the yak bone collagen concentrated solution, and sterilizing to obtain the yak bone collagen peptide.
As a specific technical scheme, the heating and extracting conditions in the step (3) are as follows: the material-liquid ratio is 1: 5-1: 10kg/L, the extraction temperature is 75-90 ℃, and the extraction time is 4-10 h.
As a specific technical scheme, the enzyme adopted in the enzymolysis step in the step (4) is alkaline protease.
As a specific technical scheme, the enzymolysis conditions in the step (4) are as follows: the addition amount of the alkaline protease is 1-5 g/100g based on the mass of the substrate, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 3-8 h.
The invention has the beneficial effects that:
1) the invention selects high-quality fresh yak bones as raw materials, firstly, the raw materials are ensured to have good and stable quality from the source, and the problem of uneven raw materials of the yak bones on the market at present is solved.
2) The invention applies the macroporous adsorption resin separation technology to the production process for separating and purifying the yak bone collagen peptide, the process fully utilizes the adsorption performance and the molecular sieve effect of the macroporous adsorption resin, has the functions of enriching small molecular peptides, decoloring, desalting and removing peculiar smell in the purification process of the yak bone collagen peptide, and is a production method capable of obviously improving the quality of the yak bone collagen peptide.
Detailed Description
The present invention will be further described with reference to the following detailed description, which should be construed as illustrative only, and not limiting the scope of the invention, which is to be given the full breadth of the appended claims, and all changes that can be made by those skilled in the art and which are, therefore, intended to be embraced therein.
Example 1
A method for refining yak bone collagen peptide comprises the following steps:
(1) pretreatment: weighing 1.03kg of fresh yak bones, removing fat, meat residues and the like on the surfaces of the fresh yak bones, cleaning, cutting into blocks, degreasing, removing foreign proteins, and finally washing with clear water until the pH value is neutral;
(2) crushing and grinding: crushing the yak bones obtained in the step (1), adding a small amount of water, and grinding into slurry to obtain yak bone slurry;
(3) heating and extracting: adding 5L of water into the yak bone slurry, and heating and extracting to obtain yak bone collagen extract;
(4) enzymolysis: adjusting the pH of the yak bone collagen extracting solution to 9.11, adding enzyme for enzymolysis to obtain a yak bone collagen peptide crude extracting solution;
(5) centrifugal filtration: centrifuging the crude extract of yak bone collagen peptide (5000rpm for 10min), filtering, and collecting filtrate to obtain yak bone collagen peptide filtrate;
(6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 2 column volumes by using clear water to remove impurities after sample loading is finished, eluting by using 55% ethanol water solution, and collecting eluent to obtain yak bone collagen peptide purified liquid;
(7) and (3) concentrating under reduced pressure: concentrating the purified yak bone collagen peptide solution under reduced pressure until the solution has no alcohol smell, and recovering ethanol to obtain yak bone collagen concentrated solution 0.88L;
(8) drying and sterilizing: and (3) carrying out freeze drying and ultraviolet sterilization on the yak bone collagen concentrated solution to obtain 121.26g of yak bone collagen peptide.
Example 2
A method for refining yak bone collagen peptide comprises the following steps:
(1) pretreatment: weighing 1.01kg of fresh yak bones, removing fat, meat residues and the like on the surfaces of the fresh yak bones, cleaning, cutting into blocks, degreasing, removing foreign proteins, and finally washing with clear water until the pH value is neutral;
(2) crushing and grinding: crushing the yak bones obtained in the step (1), adding a small amount of water, and grinding into slurry to obtain yak bone slurry;
(3) heating and extracting: adding 10L of water into the yak bone slurry, heating and extracting at 80 deg.C for 6 hr to obtain yak bone collagen extract;
(4) enzymolysis: adjusting the pH value of the yak bone collagen extracting solution to 8.9, adding alkaline protease for enzymolysis for 5 hours at the enzymolysis temperature of 55 ℃ to obtain a yak bone collagen peptide crude extracting solution; wherein the addition amount of the alkaline protease based on the mass of the substrate is 4g/100 g;
(5) centrifugal filtration: carrying out centrifugal filtration on the crude extract of the yak bone collagen peptide, and collecting filtrate to obtain yak bone collagen peptide filtrate;
(6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 3 column volumes by using clear water to remove impurities after sample loading is finished, eluting 3 column volumes by using 55% and 70% ethanol aqueous solutions respectively, collecting and combining eluates, and thus obtaining a yak bone collagen peptide purified liquid;
(7) and (3) concentrating under reduced pressure: concentrating the purified yak bone collagen peptide solution under reduced pressure until the solution has no alcohol taste, and recovering ethanol to obtain concentrated yak bone collagen peptide solution;
(8) drying and sterilizing: and (3) freeze-drying and sterilizing the yak bone collagen concentrated solution to obtain 114.32g of yak bone collagen peptide.
Example 3
A method for refining yak bone collagen peptide comprises the following steps:
(1) pretreatment: weighing 1.05kg of fresh yak bones, removing fat, meat residues and the like on the surfaces of the fresh yak bones, cleaning, cutting into blocks, degreasing by adopting an alkaline reagent, cleaning by using clear water, soaking by using a salt solution with a certain concentration to remove foreign proteins, and finally washing by using clear water until the pH value is neutral;
(2) crushing and grinding: crushing the yak bones obtained in the step (1) into small blocks of about 5 multiplied by 5mm by using a crusher, then adding a small amount of water and grinding into slurry to obtain yak bone slurry;
(3) heating and extracting: adding water into the yak bone slurry according to the material-liquid ratio of 1:8kg/L, heating and extracting at 85 ℃ for 8h to obtain a yak bone collagen extracting solution;
(4) enzymolysis: adjusting the pH value of the yak bone collagen extracting solution to 8.7, adding alkaline protease for enzymolysis for 6h at the enzymolysis temperature of 50 ℃, and naturally cooling the enzymolysis solution to below 50 ℃ to obtain a yak bone collagen peptide crude extracting solution; wherein the addition amount of the alkaline protease based on the mass of the substrate is 3g/100 g;
(5) centrifugal filtration: carrying out centrifugal filtration on the crude extract of the yak bone collagen peptide, and collecting filtrate to obtain yak bone collagen peptide filtrate;
(6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 2 column volumes by using clear water to remove impurities after sample loading is finished, eluting 4 column volumes by using 80% ethanol water solution, and collecting eluent to obtain yak bone collagen peptide purified liquid;
(7) and (3) concentrating under reduced pressure: concentrating the purified yak bone collagen peptide solution under reduced pressure until the solution has no alcohol taste, and recovering ethanol to obtain concentrated yak bone collagen peptide solution;
(8) drying and sterilizing: and (3) carrying out freeze drying and ultraviolet sterilization on the yak bone collagen concentrated solution to obtain 116.29g of yak bone collagen peptide.
Claims (4)
1. A method for refining yak bone collagen peptide is characterized by comprising the following steps:
(1) pretreatment: removing fat, meat residue and the like on the surface of fresh yak bones, cleaning, cutting into blocks, degreasing, removing foreign proteins, and finally washing with clear water until the pH value is neutral;
(2) crushing and grinding: crushing the yak bones obtained in the step (1), adding a small amount of water, and grinding into slurry to obtain yak bone slurry;
(3) heating and extracting: adding water into the yak bone slurry for heating and extracting to obtain a yak bone collagen extracting solution;
(4) enzymolysis: adjusting the pH of the yak bone collagen extracting solution to a proper range, and adding enzyme for enzymolysis to obtain a yak bone collagen peptide crude extracting solution;
(5) centrifugal filtration: carrying out centrifugal filtration on the crude extract of the yak bone collagen peptide, and collecting filtrate to obtain yak bone collagen peptide filtrate;
(6) purifying with macroporous adsorption resin: passing the yak bone collagen peptide filtrate through a pre-filled macroporous adsorption resin chromatographic column at a proper flow rate, eluting 1-4 column volumes by using clear water to remove impurities after sample loading is finished, eluting by using 55-80% ethanol water solution, and collecting eluent to obtain yak bone collagen peptide purified liquid;
(7) and (3) concentrating under reduced pressure: concentrating the purified yak bone collagen peptide solution under reduced pressure until the solution has no alcohol taste, and recovering ethanol to obtain concentrated yak bone collagen peptide solution;
(8) drying and sterilizing: and (3) carrying out freeze drying or spray drying on the yak bone collagen concentrated solution, and sterilizing to obtain the yak bone collagen peptide.
2. The method for refining yak bone collagen peptide as claimed in claim 1, wherein the heating and extracting conditions of step (3) are as follows: the material-liquid ratio is 1: 5-1: 10kg/L, the extraction temperature is 75-90 ℃, and the extraction time is 4-10 h.
3. The method for refining yak bone collagen peptide according to claim 1, wherein the enzyme used in the enzymatic hydrolysis step in step (4) is alkaline protease.
4. The method for refining yak bone collagen peptide as claimed in claim 3, wherein the enzymolysis condition of step (4) is: the addition amount of the alkaline protease is 1-5 g/100g based on the mass of the substrate, the enzymolysis temperature is 40-55 ℃, and the enzymolysis time is 3-8 h.
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Cited By (3)
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| CN113151391A (en) * | 2021-05-18 | 2021-07-23 | 青海国肽生物科技有限公司 | Yak bone collagen peptide with function of improving abundance of intestinal microorganisms and preparation method thereof |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN115969043A (en) * | 2022-12-31 | 2023-04-18 | 无锡正大生物股份有限公司 | Production method of bovine bone collagen immunoregulation peptide solid beverage |
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| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN115969043A (en) * | 2022-12-31 | 2023-04-18 | 无锡正大生物股份有限公司 | Production method of bovine bone collagen immunoregulation peptide solid beverage |
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Application publication date: 20201106 |