CN111116703A - Method for preparing animal-derived extracellular matrix polypeptide by physical method - Google Patents
Method for preparing animal-derived extracellular matrix polypeptide by physical method Download PDFInfo
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- CN111116703A CN111116703A CN202010008547.1A CN202010008547A CN111116703A CN 111116703 A CN111116703 A CN 111116703A CN 202010008547 A CN202010008547 A CN 202010008547A CN 111116703 A CN111116703 A CN 111116703A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K1/14—Extraction; Separation; Purification
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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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, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to the technical field of preparation methods of bioactive substances, in particular to a method for preparing animal-derived extracellular matrix polypeptides by a physical method. The method comprises the following steps: 1, cleaning, cutting and carrying out high-temperature treatment on animal-derived tissues to obtain tissue samples; 2, carrying out deep cooling treatment on the tissue sample, then unfreezing the tissue sample by using water at the temperature of 25-35 ℃, and circulating for 3-5 times to obtain the unfrozen tissue sample; 3, physically crushing the thawed tissue sample to obtain powder particles; 4, dissolving the powder particles in water, and stirring and emulsifying at a high speed to obtain slurry; 5 heating and dissolving the slurry, carrying out suction filtration, collecting filtrate, and drying to obtain a powder semi-finished product; and 6, carrying out irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide. The invention adopts a set of combined physical preparation method to prepare the ultra-low molecular weight sterile composite polypeptide rich in the type III collagen sequence, and provides high bioactive components for skin whitening, nursing, repairing and the like.
Description
Technical Field
The invention belongs to the technical field of preparation methods of bioactive substances, and particularly relates to a method for preparing animal-derived extracellular matrix polypeptides by a physical method.
Background
Peptides are short chains consisting of amino acids, are precise protein fragments, have nanometer-sized molecules, and are easy to absorb by intestines, stomach, blood vessels and skin. The small molecular polypeptide has higher skin permeability and is easier to be absorbed by human skin, and simultaneously, due to the fact that the molecular weight is small to a certain degree, qualitative leap of biological activity occurs. The smaller the molecular weight of the peptide, the shorter the "amino acid chain" and the easier it is to be absorbed and utilized by the human body.
Due to the unique biological personality and outstanding functional performance of the small molecular polypeptide, the small molecular polypeptide has huge application space in the fields of daily cosmetics, hair washing and protecting, food health products, biomedicine, even textile and the like. The micromolecule polypeptide has wide application in the field of beauty, and leads to a new trend of healthy skin care in the high-end beauty market in China. However, most of the existing small molecular polypeptides are chemically synthesized, and have the defects of single component, high unit price, multiple chemical additives, insufficient utilization of the compatibility of various natural compound polypeptides and the like.
Disclosure of Invention
In order to solve the above problems, the present invention provides, in a first aspect, a method for physically preparing an extracellular matrix polypeptide of animal origin, comprising the steps of:
(1) cleaning, cutting and high-temperature treating animal-derived tissues to obtain tissue samples;
(2) carrying out deep cooling treatment on the tissue sample, then unfreezing the tissue sample by using water at the temperature of 25-35 ℃, and circulating for 3-5 times to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample to obtain powder particles;
(4) dissolving the powder particles in water, and stirring and emulsifying at a high speed to obtain slurry;
(5) heating and dissolving the slurry, performing suction filtration, collecting filtrate, and drying to obtain a powder semi-finished product;
(6) and (3) carrying out irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
In a preferred embodiment, the animal-derived tissue includes one or more of urinary bladder submucosa matrix, small intestine submucosa matrix, peritoneal matrix, and basement membrane matrix of terrestrial mammals such as pig, cow, sheep, etc.
As a preferred technical proposal, the high temperature in the step (1) is 100-140 ℃.
As a preferable technical scheme, the cryogenic treatment in the step (2) is specifically carried out at-100 to-60 ℃ for 20 to 28 hours.
As a preferable technical scheme, the physical pulverization in the step (3) is specifically pulverization at a low temperature of-200 to-40 ℃.
As a preferable technical solution, the mass ratio of the powder particles to the water in the step (4) is (1-5): 100, respectively; the high-speed stirring speed is 18000-24000 rpm.
As a preferable technical scheme, the heating dissolution in the step (5) is carried out for one or more times at 80-120 ℃.
As a preferable technical solution, the drying in the step (5) is selected from one of freeze drying, spray drying and forced air drying.
According to a second aspect of the invention, the animal-derived extracellular matrix polypeptide prepared by the method for preparing the animal-derived extracellular matrix polypeptide by using the physical method is provided.
The third aspect of the invention provides application of the animal-derived extracellular matrix polypeptide prepared by the method for preparing the animal-derived extracellular matrix polypeptide by a physical method in beautifying and protecting skin, functional beverages and nutritional supplementary food additives.
Has the advantages that: the invention takes natural animal-derived tissues as polypeptide sources, adopts a set of combined physical preparation technology, comprises high-temperature treatment, repeated ultralow-temperature circulating freezing, physical crushing, high-temperature dissolution, freeze drying and ray irradiation methods, reduces the use of chemical reagents, and prepares the ultralow molecular weight sterile composite polypeptide rich in type III collagen sequences. The molecular weight is continuously reduced, and the effect of percutaneous absorption is finally achieved, so that the bioactive components are improved for skin whitening, nursing and repairing. In addition, the selected raw materials of the natural animal-derived tissue comprise type I collagen, type III collagen, type IV collagen and the like, and the low molecular weight polypeptide sequence prepared by the natural animal-derived tissue has the advantages of rich active ingredients, long metabolic cycle, long activity maintaining time and low cost, and can be used as additives of beauty treatment, skin care, functional beverages, nutritional supplementary foods and the like.
Drawings
FIG. 1 is a graph showing the molecular weight distribution of the extracellular matrix polypeptides of animal origin prepared in examples 1 to 5.
FIG. 2 is a graph showing the molecular weight distribution of the extracellular matrix polypeptides of animal origin prepared in comparative examples 1 to 4.
FIG. 3 is a scanning electron micrograph (20KX) of the extracellular matrix polypeptide derived from an animal obtained in example 1.
Reference numerals: m-standard protein MARKER; 1# -example 1; 2# -example 2; 3# -example 3; 4# -example 4; 5# -example 5; 1-comparative example 1; 2-comparative example 2; 3-comparative example 3; 4-comparative example 4.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "including". As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase will render the claim closed except for the materials described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Approximating language, as used herein in the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes portions that are literally received by modifying or otherwise modifying such quantity without substantially changing the basic function to which it is related. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.
In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.
In view of the above technical problems, the present invention provides, in a first aspect, a method for physically preparing an extracellular matrix polypeptide of animal origin, comprising the steps of:
(1) cleaning, cutting and high-temperature treating animal-derived tissues to obtain tissue samples;
(2) carrying out cryogenic treatment on the tissue sample, unfreezing the tissue sample by using warm water at the temperature of 25-35 ℃, and circulating for 3-5 times to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample to obtain powder particles;
(4) dissolving the powder particles in water, and stirring and emulsifying at a high speed to obtain slurry;
(5) heating and dissolving the slurry, performing suction filtration, collecting filtrate, and drying to obtain a powder semi-finished product;
(6) and (3) carrying out irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
In a preferred embodiment, the animal-derived tissue comprises a combination of one or more of urinary bladder submucosa matrix, small intestine submucosa matrix, peritoneal matrix, and basement membrane matrix of a terrestrial mammal such as pig, cow, sheep, and the like.
In a most preferred embodiment, the tissue of animal origin is a porcine small intestine submucosa matrix.
In a preferred embodiment, the high temperature in step (1) is 100-140 ℃.
In a most preferred embodiment, the elevated temperature in step (1) is 120 ℃.
The inventor finds that the treatment of the animal-derived matrix at the temperature of 100-140 ℃ can eliminate bacteria and viruses on the surface of the animal-derived matrix, can achieve the degreasing effect and improve the sterilization efficiency through long-time research. The possible reason is that if the temperature is lower than 100 ℃, the bacillus cannot be killed, and if the temperature is higher than 140 ℃, the structure of the protein can be damaged to influence the activity of the final product, so that the bacillus can be killed without influencing the product quality by treating the animal-derived substrate at the temperature of 100 ℃ and 140 ℃, the sterilization time is shortened, and the sterilization efficiency is improved.
In a preferred embodiment, the cryogenic treatment in the step (2) is carried out at-100 to-60 ℃ for 20 to 28 hours.
In a most preferred embodiment, the cryogenic treatment in step (2) is specifically a treatment at-80 ℃ for 24 h.
In a preferred embodiment, the mass ratio of the warm water and the tissue sample in the step (2) is (8-12): 1.
in a most preferred embodiment, the mass ratio of the warm water and the tissue sample in the step (2) is 10: 1.
in a preferred embodiment, the physical pulverization in the step (3) is specifically pulverization at a low temperature of-200 to-40 ℃.
In a most preferred embodiment, the physical pulverization in the step (3) is specifically pulverization at-196 ℃.
In a preferred embodiment, the particle size of the powder particles in the step (3) is 200 mesh or less.
The inventors of the present invention have found, through long-term studies, that powder particles having a uniform particle diameter can be obtained by the steps (2) and (3). The possible reason is that when the tissue sample is placed at-100 to-60 ℃ for treatment for 20-28h, then the tissue sample is unfrozen by water at 25-35 ℃, and tissue fibers and structures are loosened after repeating for 2-3 times, so that the tissue sample is deeply crushed; then the mixture is immediately placed at the temperature of-196 ℃ for crushing, can be crushed into particles with certain particle size, and can be hydrolyzed into micromolecular polypeptide to the maximum extent without influencing the biological activity of the micromolecular polypeptide.
In a preferred embodiment, the water in step (4) is purified water.
The purified water refers to water for medicine prepared by pure water through a distillation method, an ion exchange method, a reverse osmosis method or other suitable methods, and does not contain any additive.
In a preferred embodiment, the mass ratio of the powder particles to the water in the step (4) is (1-5): 100, respectively; the high-speed stirring speed is 18000-24000 rpm.
In a most preferred embodiment, the mass ratio of the powder particles to the water in the step (4) is 3: 100, respectively; the rotation speed of the high-speed stirring is 21000 rpm.
In a preferred embodiment, the heating dissolution in the step (5) is carried out by treating for 20-40min at 80-120 ℃ for one or more times.
In a most preferred embodiment, the heating dissolution in step (5) is carried out by one or more treatments at 100 ℃ for 30 min.
In a preferred embodiment, the drying in step (5) is selected from one of freeze drying, spray drying, and forced air drying.
In a most preferred embodiment, the drying is freeze drying.
In a preferred embodiment, the suction filtration in the step (5) is specifically nylon gauze suction filtration.
In a preferred embodiment, the nylon gauze has a pore size of 30 to 50 μm.
In a most preferred embodiment, the nylon scrim has a pore size of 37.5 μm.
In a preferred embodiment, the radiation dose in the step (6) is 15 to 35 KGy.
In a preferred embodiment, the radiation dose in the step (6) is 30 KGy.
The inventor finds that after long-term research, the filtrate obtained by filtering the soluble slurry through nylon gauze with the aperture of 30-50 mu m is freeze-dried and then is subjected to irradiation treatment, so that the micromolecular polypeptide with good quality can be obtained. The possible reason is that the aperture of the nylon gauze is 30-50 μm, only water and small molecular substances are allowed to pass through, and the obtained filtrate is freeze-dried, so that the loss of the activity of the components is less; the composite material has excellent color and form, complete dehydration, good rehydration property and easy storage and transportation, and finally has a sterilization effect by adopting irradiation treatment, thereby enhancing the quality of the small molecular polypeptide composite.
In a most preferred embodiment, a method of physically preparing an extracellular matrix polypeptide of animal origin comprises the steps of:
(1) cleaning animal derived tissue, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) immediately carrying out subzero treatment on the tissue sample at-80 ℃ for 24h, and then adding 30 ℃ warm water according to a mass ratio of 10: 1, unfreezing the tissue sample, and circulating for 3 times in such a way to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample at-196 ℃ to prepare powder particles with the particle size of less than 200 meshes;
(4) mixing the powder particles according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying for 5min at 21000rpm to obtain slurry;
(5) heating the slurry to 100 deg.C, treating for 30min, vacuum filtering with 37.5 μm nylon gauze, collecting filtrate, and freeze drying to obtain semi-finished powder;
(6) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
According to a second aspect of the invention, the animal-derived extracellular matrix polypeptide prepared by the method for preparing the animal-derived extracellular matrix polypeptide by using the physical method is provided.
In a preferred embodiment, the animal-derived extracellular matrix polypeptide is a sterile, complex polypeptide enriched in a type III collagen sequence.
The type III collagen is in a sparse network shape and is scattered around the type I collagen, and the higher the content of the type III collagen is, the finer the fiber bundle is. Is a main structural protein of human body and other vertebrates, plays a main role in supporting tissues and connective tissues, and is called as bones in bones and meat in meat, and type III collagen determines the elasticity and tenderness of skin.
In a preferred embodiment, the extracellular matrix polypeptide of animal origin has a molecular weight of 4000-10000 Da.
In a more preferred embodiment, the extracellular matrix polypeptide of animal origin has a molecular weight of 6000-8000 Da.
The third aspect of the invention provides application of the animal-derived extracellular matrix polypeptide prepared by the method for preparing the animal-derived extracellular matrix polypeptide by a physical method in beautifying and protecting skin, functional beverages and nutritional supplementary food additives.
Examples
In order to better understand the above technical solutions, the following detailed descriptions will be provided with reference to specific embodiments. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention. In addition, the starting materials and plant extracts used are commercially available, unless otherwise specified.
Example 1
Example 1 provides a method for physically preparing an extracellular matrix polypeptide of animal origin, comprising the steps of:
(1) cleaning porcine small intestine submucosa matrix, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) immediately carrying out subzero treatment on the tissue sample at-80 ℃ for 24h, and then adding 30 ℃ warm water according to a mass ratio of 10: 1, unfreezing the tissue sample, and circulating for 3 times in such a way to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample at-196 ℃ to prepare powder particles with the particle size of 200 meshes;
(4) mixing the powder particles according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying for 5min at 21000rpm to obtain slurry;
(5) heating the slurry to 100 ℃ for 30 min; then, nylon gauze with the aperture of 37.5 mu m is used for suction filtration, filtrate is collected, and powder semi-finished products are obtained by freeze drying;
(6) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
Example 2
Example 2 is substantially the same as example 1 except that the step (1) is to wash the porcine peritoneal matrix, divide it into strips having a length of 10cm and a width of 2cm, and treat them at 120 ℃ for 30min to obtain tissue samples.
Example 3
Example 3 is essentially the same as example 1 except that the elevated temperature in step (1) is 140 ℃.
Example 4
Example 4 is substantially the same as example 1 except that the cryogenic treatment in the step (2) is specifically carried out at-100 ℃ for 20 hours.
Example 5
Example 5 is substantially the same as example 1, except that the heating dissolution in the step (5) is carried out for 20min at 120 ℃, and after cooling, a proper amount of purified water is supplemented and the solution is heated to 120 ℃ for 20 min.
Comparative example 1
Comparative example 1 is essentially the same as example 1 except that it comprises the following steps:
(1) cleaning porcine small intestine submucosa matrix, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) physically crushing the tissue sample at-196 ℃ to prepare powder particles with the particle size of 200 meshes;
(3) mixing the powder particles according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying for 5min at 18000rpm to obtain slurry;
(4) heating the slurry to 100 ℃ for 30 min; then, nylon gauze with the aperture of 37.5 mu m is used for suction filtration, filtrate is collected, and powder semi-finished products are obtained by freeze drying;
(5) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
Comparative example 2
Comparative example 2 is substantially the same as example 1 except that it comprises the steps of:
(1) cleaning porcine small intestine submucosa matrix, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) immediately carrying out subzero treatment on the tissue sample at-80 ℃ for 24h, and then adding 30 ℃ warm water according to a mass ratio of 10: 1, unfreezing the tissue sample, and circulating for 3 times in such a way to obtain the unfrozen tissue sample;
(3) and (3) mixing the unfrozen tissue sample according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying for 5min at 23000rpm to obtain slurry;
(4) heating the slurry to 100 deg.C, treating for 30min, vacuum filtering with 37.5 μm nylon gauze, collecting filtrate, and freeze drying to obtain semi-finished powder;
(5) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
Comparative example 3
Comparative example 3 is substantially the same as example 1 except that it comprises the steps of:
(1) cleaning porcine small intestine submucosa matrix, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) immediately carrying out subzero treatment on the tissue sample at-80 ℃ for 24h, and then adding 30 ℃ warm water according to a mass ratio of 10: 1, unfreezing the tissue sample, and circulating for 3 times in such a way to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample at-196 ℃ to prepare powder particles with the particle size of 200 meshes;
(4) mixing the powder particles according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying for 5min at 21000rpm to obtain slurry;
(5) filtering the serous fluid with nylon gauze with the aperture of 37.5 mu m, collecting filtrate, and freeze-drying to obtain a powdery semi-finished product;
(6) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
Comparative example 4
Comparative example 4 is substantially the same as example 1 except that it comprises the steps of:
(1) cleaning porcine small intestine submucosa matrix, cutting into strips with length of 10cm and width of 2cm, and treating at 120 deg.C for 30min to obtain tissue sample;
(2) immediately carrying out subzero treatment on the tissue sample at-80 ℃ for 24h, and then adding 30 ℃ warm water according to a mass ratio of 10: 1, unfreezing the tissue sample, and circulating for 3 times in such a way to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample at-196 ℃ to prepare powder particles with the particle size of 200 meshes;
(4) mixing the powder particles according to a mass ratio of 3: 100, mixing the mixture in pure water, and emulsifying the mixture for 30min at 8000rpm to obtain slurry;
(5) heating the slurry to 100 ℃ for 30 min; then, nylon gauze with the aperture of 37.5 mu m is used for suction filtration, filtrate is collected, and powder semi-finished products are obtained by freeze drying;
(6) and (3) carrying out 30KGy irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
Evaluation of Performance
The molecular weight distribution of the polypeptide is measured by an agarose gel electrophoresis method; wherein M is a standard protein MARKER; # 1 is the animal-derived extracellular matrix polypeptide prepared in example 1; # 2 is the animal-derived extracellular matrix polypeptide prepared in example 2; # 3 is the animal-derived extracellular matrix polypeptide prepared in example 3; # 4 is the animal-derived extracellular matrix polypeptide prepared in example 4; # 5 is the extracellular matrix polypeptide of animal origin prepared in example 5. 1 is the animal-derived extracellular matrix polypeptide prepared in comparative example 1; 2 is the animal-derived extracellular matrix polypeptide prepared in comparative example 2; 3 is the animal-derived extracellular matrix polypeptide prepared in comparative example 3; and 4 is the extracellular matrix polypeptide of animal origin prepared in comparative example 4.
The results of the molecular weight tests are shown in the following figure:
FIG. 1 is a graph showing the molecular weight distribution of the extracellular matrix polypeptides of animal origin prepared in examples 1 to 5.
FIG. 2 is a graph showing the molecular weight distribution of the extracellular matrix polypeptides of animal origin prepared in comparative examples 1 to 4.
FIG. 3 is a scanning electron micrograph (20KX) of the extracellular matrix polypeptide of animal origin prepared in example 1.
As can be seen from FIG. 1 and FIG. 2, the molecular weight of the extracellular matrix polypeptide of animal origin prepared by the method of the present invention is 4000-10000Da, and is intensively distributed at 6000-8000 Da; the molecular weight of the extracellular matrix polypeptide of animal origin prepared by the comparative example is 4600-40000Da and is intensively distributed at 10000-26000 Da. As can be seen from FIG. 3, the physical size of the extracellular matrix polypeptide molecule of animal origin prepared by the method of the present invention reaches the nanometer level.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. The use of some numerical ranges in the claims also includes sub-ranges, processes, features and principles within them, and equivalent variations or modifications in these ranges should also be construed as being covered by the appended claims where possible.
Claims (10)
1. A method for preparing an extracellular matrix polypeptide of animal origin by a physical method, comprising the steps of:
(1) cleaning, cutting and high-temperature treating animal-derived tissues to obtain tissue samples;
(2) carrying out cryogenic treatment on the tissue sample, unfreezing the tissue sample by using warm water at the temperature of 25-35 ℃, and circulating for 3-5 times to obtain the unfrozen tissue sample;
(3) physically crushing the thawed tissue sample to obtain powder particles;
(4) dissolving the powder particles in water, and stirring and emulsifying at a high speed to obtain slurry;
(5) heating and dissolving the slurry, performing suction filtration, collecting filtrate, and drying to obtain a powder semi-finished product;
(6) and (3) carrying out irradiation treatment on the powder semi-finished product to obtain the animal source extracellular matrix polypeptide.
2. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the tissue derived from an animal comprises a combination of one or more of a urinary bladder submucosa matrix, a small intestine submucosa matrix, a peritoneal matrix, and a basement membrane matrix of a terrestrial mammal such as a pig, a cow, a sheep, and the like.
3. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the elevated temperature in the step (1) is 100-140 ℃.
4. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the cryogenic treatment in the step (2) is carried out at-100 to-60 ℃ for 20 to 28 hours.
5. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the physical pulverization in the step (3) is specifically pulverization at a low temperature of-200 to-40 ℃.
6. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the mass ratio of the powder particles to water in the step (4) is (1-5): 100, respectively; the high-speed stirring speed is 18000-24000 rpm.
7. The method for physically preparing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the heat solubilization in the step (5) is carried out by one or more treatments at 80-120 ℃.
8. The method for physically producing an extracellular matrix polypeptide derived from an animal according to claim 1, wherein the drying in the step (5) is one selected from the group consisting of freeze drying, spray drying and forced air drying.
9. An animal-derived extracellular matrix polypeptide produced by the method for producing an animal-derived extracellular matrix polypeptide according to any one of claims 1 to 8.
10. The animal-derived extracellular matrix polypeptide prepared by the method for preparing an animal-derived extracellular matrix polypeptide according to claim 9, wherein the molecular weight of the animal-derived extracellular matrix polypeptide is 4000-10000 Da.
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