WO2023029161A1 - 天然胶原材料及天然胶原材料的制备方法、用途 - Google Patents

天然胶原材料及天然胶原材料的制备方法、用途 Download PDF

Info

Publication number
WO2023029161A1
WO2023029161A1 PCT/CN2021/124986 CN2021124986W WO2023029161A1 WO 2023029161 A1 WO2023029161 A1 WO 2023029161A1 CN 2021124986 W CN2021124986 W CN 2021124986W WO 2023029161 A1 WO2023029161 A1 WO 2023029161A1
Authority
WO
WIPO (PCT)
Prior art keywords
collagen
solution
treatment
natural collagen
small intestinal
Prior art date
Application number
PCT/CN2021/124986
Other languages
English (en)
French (fr)
Inventor
景伟
王华生
赵博
魏鹏飞
李红阳
胡苗苗
Original Assignee
北京博辉瑞进生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京博辉瑞进生物科技有限公司 filed Critical 北京博辉瑞进生物科技有限公司
Publication of WO2023029161A1 publication Critical patent/WO2023029161A1/zh

Links

Images

Classifications

    • 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]
    • 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
    • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • 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
    • A61Q19/08—Anti-ageing preparations
    • 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

Definitions

  • the disclosure belongs to the field of biomedical materials, in particular, the disclosure relates to a natural collagen material, a preparation method and application of the natural collagen material, and a dermal filler.
  • Collagen is an important structural protein of animal extracellular matrix, accounting for about 25-30% of its total protein content. Collagen supports the integrity, elasticity and strength of connective tissue, thereby maintaining the form and function of skin, cartilage and bones. Affected by natural aging and external environmental factors, the collagen in the skin tissue is lost, and various skin problems such as skin sagging, wrinkles, and pigmentation appear. How to repair skin damage and delay skin aging is an important issue that people generally pay attention to.
  • Type I collagen fibers are thick in diameter, tightly arranged, in the form of dense bundles, criss-crossing, very suitable for forming a strong fiber network, and are relatively hard collagen.
  • Type III collagen fibers are fine fibers, in a sparse network shape, and the higher the content, the thinner the fiber bundles, and the higher the elasticity.
  • the content ratio of type I/III collagen in infant skin is about 1. With age, type III collagen decreases and type I collagen increases. The content ratio of type I/III collagen in adult skin is about 2.5, and the content of type III collagen decreases precipitously.
  • Type III collagen With the loss of type III collagen, the dermal tissue will collapse in disguise, fiber breakage, and fat atrophy, resulting in skin loss of elasticity, luster, pigmentation, and wrinkles.
  • Type III collagen has excellent elasticity and repair function. Therefore, supplementing exogenous type III collagen is an important way to delay aging and repair skin damage.
  • the sources of collagen mainly include natural collagen extracted from tissues, and artificial collagen obtained through techniques such as genetic engineering.
  • natural collagen which is generally extracted from the tendons, dermis and tails of cattle, pigs, rats, fish and other animals.
  • the natural collagen extracted by traditional methods is dominated by type I collagen, and the content of type III collagen is low.
  • the impurity components (nucleic acid fragments, cell fragments) in collagen extracted by acid method, alkali method, salt method, enzymatic method and other methods require complex purification processes to remove impurity components.
  • High-purity type III collagen can be prepared by genetic engineering methods, but there are problems such as complicated preparation process, high cost, and long cycle.
  • the prokaryotic expression system of Escherichia coli is mostly used in the preparation of collagen at present, and certain endotoxins may be produced in bacterial cells such as Escherichia coli and mixed into collagen, which limits its application in the field of biomedicine.
  • the content of type III collagen in natural collagen extracted by traditional methods is low and the content of impurities is high; however, the preparation of type III collagen by genetic engineering has problems of high cost, complicated preparation process, and certain biological safety hazards.
  • the present disclosure provides a natural collagen material, which uses the submucosa of the small intestine as a raw material to obtain a high content of type III collagen, has low immunogenicity, high biological safety, and has excellent skin repair and skin elasticity restoration. It is suitable as a subcutaneous filling material for damaged and aging skin.
  • the present disclosure provides a natural collagen material, wherein the natural collagen material includes type I collagen and type III collagen; based on the total mass of the natural collagen material, the amount of the type I collagen The content is 30%-35%, and the content of the type III collagen is 65%-70%;
  • the natural collagen material is derived from small intestinal submucosa, preferably porcine small intestinal submucosa.
  • the residual amount of DNA in the natural collagen material is lower than 4ng/mg, and the residual amount of ⁇ -Gal is lower than 1.0U/L.
  • the present disclosure provides a method for preparing a natural collagen material, which includes the following steps:
  • the small intestine submucosa material was taken, and the virus inactivation treatment, degreasing treatment, decellularization treatment and enzymatic treatment were carried out in sequence to obtain a crude collagen extraction solution;
  • the natural collagen material is the natural collagen material described in the first aspect.
  • the step of virus inactivation treatment includes: washing the small intestinal submucosa material and soaking it in a virus inactivation solution, and performing virus inactivation under shaking conditions Treatment; preferably, the volume ratio of the small intestinal submucosa material to the virus inactivation solution is (0.5-5):30; preferably, the virus inactivation solution contains (0.5-5)% (v/v) peroxygen An aqueous solution of acetic acid and (20-25)% (v/v) ethanol; preferably, the time for the virus inactivation treatment is (1-5) h.
  • the step of degreasing treatment includes: soaking the small intestinal submucosa material after virus inactivation treatment in a degreasing solution, and performing degreasing treatment under ultrasonic conditions;
  • the volume ratio of the small intestinal submucosa material to the degreasing solution is (0.1-1):2; preferably, the degreasing solution is an alkaline lipase solution of 1-2wt%; preferably, the degreasing solution
  • the pH is 7-10; preferably, the small intestine submucosa material is soaked in the degreasing solution, and the degreasing solution is replaced after ultrasonic treatment for 30-60 min, and the operation is repeated more than 3 times.
  • the step of decellularization includes: soaking the defatted small intestinal submucosa material in a decellularization solution, and performing decellularization under ultrasonic conditions
  • the volume ratio of the small intestinal submucosa material to the decellularization solution is (0.5-5):30;
  • the decellularization solution contains 0.02-0.05wt% trypsin and 0.1-0.4wt% EDTA phosphate buffered saline; preferably, the time of the decellularization treatment is 30 min, and then washed and dried to obtain decellularized small intestinal submucosa material.
  • step of enzymatic hydrolysis treatment includes:
  • the soaked small intestinal submucosa material is pulverized and stirred by crushing equipment, added with pepsin, centrifuged after enzymatic hydrolysis treatment, and the precipitate is removed to obtain a crude collagen extraction solution;
  • the volume ratio of the small intestinal submucosa material to the acidic solution is (0.5-5):30; preferably, the acidic solution is (0.1-1) mol/L acetic acid solution, and the small intestinal submucosa material is in the acidic solution
  • the soaking time is 12h, and the temperature equipment is 4-10°C; preferably, the mass ratio of small intestinal submucosa material to pepsin is (8-12):1; preferably, the enzymolysis treatment is performed under stirring conditions 48h-96h.
  • step of purifying includes:
  • the crude collagen extraction solution is filtered once and subjected to salting-out treatment to obtain precipitated particles; the precipitated particles are dissolved in an acidic solution, washed and dissolved for a second time, separated, filtered and concentrated through membrane separation to obtain a collagen concentrate containing natural collagen materials.
  • the present disclosure provides the natural collagen material according to the first aspect, or the use of the natural collagen material prepared according to the method described in the second aspect in the preparation of medical cosmetic materials or skin care products; preferably, the The medical cosmetic material is a dermal filler; preferably, the dermal filler is an injection.
  • the present disclosure provides a dermal filler, wherein the dermal filler includes the natural collagen material according to the first aspect, or the natural collagen material prepared according to the method of the second aspect.
  • the natural collagen material provided by the present disclosure is derived from the submucosa of the small intestine. Compared with the collagen extracted from the Achilles tendon, dermis, tail and other parts, the content of type III collagen in the natural collagen material in the present disclosure Significantly improved, with excellent elasticity and skin repair function, can effectively restore skin elasticity, firm skin, increase skin moisture, achieve damaged skin repair, anti-aging and other effects.
  • the preparation method of the natural collagen material provided by the present invention can remove intracellular components (such as DNA fragments, cell membrane and nucleus fragments, etc.) Residues of immune source substances improve the biological safety of natural collagen materials, simplify the purification process of natural collagen materials, and reduce the difficulty and cost of preparation.
  • intracellular components such as DNA fragments, cell membrane and nucleus fragments, etc.
  • the dermal filler provided by the present disclosure includes the natural collagen material provided by the present disclosure, which is suitable for subcutaneous tissue filling, effectively tightens the skin, improves skin wrinkles and damage, and restores the elasticity, luster and moisture of the skin Spend.
  • the natural collagen material compared with fillers such as hyaluronic acid, the natural collagen material has a longer retention time and better durability of the effect.
  • Figure 1 shows the SEM image of the natural collagen material
  • A, B, C, and D in Figure 1 are scanning electron microscope images of 1.00 mm, 500 ⁇ m, 500 ⁇ m, and 100 ⁇ m in order.
  • Figure 2 shows the Fourier transform infrared spectrum of natural collagen material.
  • Figure 3 shows the UV-Vis absorption spectrum of natural collagen material.
  • Figure 4 shows the circular dichroism spectrum of native collagen material.
  • references to “some specific/preferred embodiments”, “other specific/preferred embodiments”, “embodiments” and the like refer to specific elements described in relation to the embodiments (for example, A feature, structure, property and/or characteristic) is included in at least one embodiment described herein and may or may not be present in other embodiments.
  • references to “some specific/preferred embodiments”, “other specific/preferred embodiments”, “embodiments” and the like refer to specific elements described in relation to the embodiments (for example, A feature, structure, property and/or characteristic) is included in at least one embodiment described herein and may or may not be present in other embodiments.
  • the described elements may be combined in any suitable manner in the various embodiments.
  • numerical range represented by "numerical value A - numerical value B" means the range which includes numerical value A and B of an end point.
  • v/v is used to represent volume percentage content
  • wt% is used to represent mass percentage content
  • the "water” includes any feasible water that can be used such as deionized water, distilled water, ion exchanged water, double distilled water, high-purity water, and purified water.
  • the temperature when “normal temperature” and “room temperature” are used, the temperature may be 10-40°C.
  • a first aspect of the present disclosure provides a natural collagen material, the natural collagen material includes type I collagen and type III collagen; based on the total mass of the natural collagen material, the content of the type I collagen is 30-35%, the content of the type III collagen is 65-70%;
  • the natural collagen material is derived from small intestinal submucosa, preferably porcine small intestinal submucosa.
  • the natural collagen material of the present disclosure is obtained from Small Intestinal Submucosa (SIS) as a raw material, and the extracted collagen has a relatively high content of type III collagen, which provides a natural, safe and low immunogenicity for supplementing collagen loss.
  • a source of collagen suitable as a skin collagen supplement. After the natural collagen material is applied to damaged or aging skin, it can tighten the skin and restore skin elasticity, making the skin delicate and shiny, effectively improving the condition of damaged skin and delaying skin aging.
  • the natural collagen material is derived from the porcine small intestine submucosa, which contains a relatively high content of type III collagen, and is suitable as a subcutaneous filling material for restoring skin elasticity.
  • the natural collagen material provided by the present disclosure has a low content of immunogenic substances, wherein the residual DNA is less than 4ng/mg, and the enzyme-linked immunosorbent assay method is used to detect ⁇ -galactosidase ( ⁇ -Gal) , the residual amount is lower than the detection limit of ⁇ -galactosidase detection kit 1.0U/L.
  • the natural collagen material has low content of immune source substances and high biological safety. It is suitable for implantation into subcutaneous tissue, and it can be used as a filling material to repair damaged and aging skin and restore skin elasticity.
  • a second aspect of the present disclosure provides a method for preparing a natural collagen material, comprising the steps of:
  • the small intestine submucosa material was taken, and the virus inactivation treatment, degreasing treatment, decellularization treatment and enzymatic treatment were carried out in sequence to obtain a crude collagen extraction solution;
  • the preparation method of the present disclosure can effectively remove intracellular components such as nucleic acid fragments, cell membranes, and nuclear fragments through the degreasing and decellularization of the small intestinal submucosa material, and reduce the residue of immunogenic substances.
  • the preparation method of the present disclosure can effectively simplify the purification treatment steps of collagen, and can produce natural collagen materials with high content of type III collagen and high biological safety.
  • the preparation method of the present disclosure has the advantages of low cost, simple steps, and easy operation.
  • a natural collagen material with a content of type I collagen of 30-35% and a content of type III collagen of 65-70% based on the total mass can be prepared, which is a skin Repairing and anti-aging provides a natural collagen material with a reasonable ratio of type III collagen and type I collagen.
  • the small intestine submucosa material is the middle small intestine submucosa material that is retained after the mucosal layer and fascia layer of the small intestine are removed.
  • the small intestinal submucosa material was washed with water and used for subsequent processing.
  • the washed material When measuring the volume of small intestinal submucosa material, the washed material should be placed on a stainless steel net for 1-5 minutes and then put into a graduated cylinder for measurement.
  • the step of virus inactivation treatment includes: washing the submucosa material of the small intestine, soaking it in a virus inactivation solution, and performing virus inactivation treatment under shaking conditions.
  • the virus inactivation solution it is an aqueous solution comprising (0.5-5)% (v/v) peracetic acid and (20-25)% (v/v) ethanol.
  • the content of peracetic acid in the virus inactivation solution is 0.8% (v/v), 1% (v/v), 1.5% (v/v), 2% (v/v), 2.5% ( v/v), 3% (v/v), 3.5% (v/v), 4% (v/v), 4.5% (v/v) and so on;
  • the content of ethanol in the virus inactivation solution is 21% (v/v), 22% (v/v), 23% (v/v), 24% (v/v), etc.
  • the virus inactivation solution is an aqueous solution comprising 1% (v/v) peracetic acid and 24% (v/v) ethanol.
  • the volume ratio of small intestinal submucosa material to virus inactivation solution is 1:30. Soak the submucosa material of the small intestine in the virus inactivation solution and soak under shaking conditions. The submucosa material of the small intestine is immersed in the virus inactivation solution for 1-5 hours, preferably 2 hours, and the shaking speed is 40 r/min. The soaked small intestinal submucosa material was washed with water. Oscillation can be achieved by means of a shaker, a magnetic stirrer, and the like.
  • the virus components in the small intestinal submucosa material can be effectively inactivated, and the content of bacterial endotoxin can be further reduced to ensure the biological safety of the natural collagen material.
  • the step of degreasing treatment includes: immersing the small intestinal submucosa material after virus inactivation treatment in a degreasing solution, and performing degreasing treatment under ultrasonic conditions.
  • the degreasing solution is 1-2wt% alkaline lipase solution.
  • the concentration of the alkaline lipase solution is 1wt%, 1.2wt%, 1.4wt%, 1.5wt%, 1.7wt%, 2wt%, etc.
  • the pH of the degreasing solution is 7-10, for example, the pH is 7, 8, 9, 10, etc.
  • the volume ratio of the small intestinal submucosa material to the defatting solution (0.1-1):2, exemplary, the volume ratio of the small intestinal submucosa material to the defatting solution is 0.1:2, 0.2:2, 0.5:2, 0.8:2 , 1:2 and so on.
  • the volume ratio of the small intestinal submucosa material to the degreasing solution is 1:10, the small intestinal submucosa material is soaked in the degreasing solution, and the degreasing solution is replaced after degreasing under ultrasonic conditions for 30-60 minutes; repeat the operation more than 3 times, and then Wash small intestinal submucosa material.
  • the degreasing treatment can effectively reduce the non-collagen impurity components mixed in the crude collagen extract, thereby simplifying the subsequent purification process and avoiding the loss of collagen.
  • the selection of 2wt% alkaline lipase solution in this disclosure can more effectively remove unnecessary impurities such as lipids and reduce the loss of collagen components during the extraction process .
  • the decellularization step includes: soaking the defatted small intestinal submucosa material in a decellularization solution, and performing decellularization under ultrasonic conditions.
  • the decellularization solution it is phosphate buffered saline containing 0.02-0.05 wt% trypsin and 0.1-0.4 wt% EDTA.
  • the content of trypsin in the phosphate buffer is 0.02wt%, 0.03wt%, 0.04wt%, 0.05wt% etc.
  • the content of EDTA is 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt% %etc.
  • the content of trypsin in the phosphate buffer solution is 0.02 wt%
  • the content of EDTA is 0.1 wt%.
  • the small intestinal submucosa material was soaked in the decellularization solution for 30 minutes under ultrasonic conditions. Further, the small intestinal submucosa material and the decellularized solution were soaked in an ultrasonic cleaning machine for 30 minutes at a volume ratio of 1:30.
  • the drying treatment condition is to dry the small intestine submucosa material at a temperature of 37° C. for 24 hours.
  • the conditions of the decellularization treatment in the present disclosure are mild, can fully remove immune source components such as nucleic acid, cell membrane, and nuclear fragments, reduce the immunogenicity of natural collagen materials, make them suitable for implantation into subcutaneous tissue, and avoid loss of collagen components.
  • the present disclosure can reduce fat, nucleic acid, ⁇ -Gal and other immunogenic substances to the minimum by combining degreasing and decellularization treatment, so the purification process of the crude collagen extraction solution only needs one salting out and microporous membrane filtration. Finish.
  • the step of enzymatic treatment includes: taking the decellularized small intestinal submucosa material, crushing it, and immersing it in an acidic solution; crushing and stirring the soaked small intestinal submucosa material with a crushing device, adding pepsin, Centrifuge after enzymatic treatment to remove the precipitate to obtain crude collagen extract solution.
  • the acidic solution is 0.1-1 mol/L acetic acid solution, for example, the concentration of acetic acid solution is 0.2 mol/L, 0.3 mol/L, 0.5 mol/L, 0.7 mol/L, 0.8 mol/L, etc. As preferably, the concentration of the acetic acid solution is 0.5mol/L.
  • the volume ratio of the small intestinal submucosa material to the acidic solution is (0.5-5):30, for example, 0.8:30, 1:30, 2:30, 3:30, 4:30 and so on.
  • the small intestine submucosa material is soaked in the acidic solution at a volume ratio of 1:30.
  • the small intestine submucosa material is soaked in an acidic solution at a low temperature of 4-10° C. for 12 hours, so as to promote the dissolution of collagen.
  • the soaked small intestine submucosa material was pulverized and stirred for 5 minutes with a pulverizer at low temperature, and then added to the acidic solution according to the mass ratio of pepsin and small intestine submucosa material at a ratio of 1: (8-12).
  • Pepsin mechanical stirring at low temperature (4-10°C) for 48-96 hours to fully dissolve the collagen in the small intestinal submucosa material, so as to improve the extraction efficiency of type III collagen and type I collagen.
  • the mass ratio of pepsin to small intestinal submucosa material is 1:10; after pepsin is added to the acidic solution, the time for mechanical stirring at low temperature is 72 hours.
  • the mixed solution is centrifuged, and the obtained supernatant is the collagen crude extraction solution in which the collagen is dissolved.
  • the purification step includes: filtering the crude collagen extraction solution once and then performing salting-out treatment to obtain precipitated particles; dissolving the precipitated particles in an acidic solution, washing and dissolving them for the second time, and separating, filtering, and concentrating , to obtain a collagen concentrate containing natural collagen material.
  • the acidic solution is 0.5 mol/L acetic acid solution to fully dissolve the natural collagen material in the precipitated particles.
  • a microfiltration membrane is used to filter the solution to filter out particles in the solution.
  • the pore size of the microfiltration membrane for primary filtration is 0.2-0.4 ⁇ m, and the insoluble particles in the crude collagen extraction solution can be removed by primary filtration.
  • the pore size of the filter membrane filtered after the second cleaning is 0.05-0.1 ⁇ m. After the second cleaning, the filter can remove excess small molecular impurities and improve the purity of the collagen concentrate.
  • the purification step in the present disclosure only needs to remove pepsin and residual non-collagen components in the material. Therefore, only one salting-out and microporous membrane filtration is required to complete the purification of the crude collagen extraction solution, which has simple steps and The advantage of less collagen loss.
  • the natural collagen material is obtained by freeze-drying the collagen concentrate.
  • the preparation method provided by the present disclosure through the interaction of each step and optimization of the treatment conditions of each step, can simplify the purification process, realize the efficient extraction of collagen in the small intestine submucosa material, and obtain the collagen containing 65%-70% Type III collagen and 30%-35% type I collagen of natural collagen material.
  • the proportion of type III collagen in natural collagen materials is high, the content of immune source substances is low, and the biological safety is high, and it has a loose and porous structure, which is suitable for cell adhesion and growth; natural collagen materials maintain the triple helix conformation of collagen, and have With high collagen activity, it is suitable as a subcutaneous filler material for repairing damaged and aging skin.
  • the third aspect of the present disclosure provides a dermal filler, comprising the natural collagen material according to the first aspect, or the natural collagen material prepared according to the method of the second aspect.
  • the fourth aspect of the present disclosure provides the use of natural collagen materials.
  • the natural collagen materials are used as or prepared as medical cosmetic materials.
  • the cosmetic medical material is a dermal filler.
  • the natural collagen material can be mixed with one or more auxiliary materials to be made into any applicable dosage form.
  • auxiliary materials include solvents, thickeners, co-solvents, emulsifiers, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, surfactants, etc.
  • the dermal filler is an injection.
  • natural collagen materials are used in the preparation of skin care products.
  • Skin care products can be in the form of patches, lotions, gels, lotions or creams, etc.
  • the natural collagen material in this disclosure has a ratio of type III collagen and type I collagen close to that of infant skin, has a high content of type III collagen, is suitable for subcutaneous filling, can effectively restore skin elasticity, tighten skin, Increases skin hydration and radiance.
  • the present embodiment provides the preparation method of natural collagen material, comprises the steps:
  • Virus inactivation prepare a virus inactivation aqueous solution (1% (v/v) peracetic acid+24% (v/v) ethanol), soak the cleaned small intestinal submucosa material in the virus inactivation solution, The ratio of small intestinal submucosa material to solution is 1:30, placed on a shaker, soaked at room temperature for 2 hours, and the shaker speed is 40r/min. Wash with purified water after soaking.
  • Decellularization prepare decellularized phosphate buffer solution (0.025wt% trypsin+0.19wt% EDTA), transfer the small intestinal submucosa material to the decellularized solution, and the ratio of the small intestinal submucosa material to the decellularized solution is 1: 30 (v:v), ultrasonically soaked in an ultrasonic cleaner for 30 minutes, washed with purified water after ultrasonication, and dried in an oven at 37°C for 24 hours.
  • Acid immersion cut the dried small intestinal submucosa material into pieces of about 1 cm 2 , soak the decellularized small intestinal submucosa material in 0.5mol/L acetic acid solution at 4°C for 12 hours, and mix the decellularized small intestinal submucosa material with the decellularized material.
  • the cell solution ratio was 1:30 (v:v).
  • the crude collagen solution is treated with a microfiltration membrane, and the insoluble particles are first filtered out with a filter membrane with a pore size of 0.2-0.4 microns.
  • Use neutral salt sodium chloride or ammonium sulfate for salting out after salting out, centrifuge to collect precipitated particles, wash with distilled water, dissolve with 0.5mol/L acetic acid, and then use ultrafiltration membrane with a pore size of 0.05-0.1 micron for concentration treatment , to remove excess small molecule impurities and obtain high-purity collagen concentrate.
  • Drying freeze-dry the high-purity collagen concentrate to obtain natural collagen materials.
  • After pre-cooling turn on the vacuum pump for vacuum drying, and set the temperature of the cold trap It is -35°C to -45°C, such as -37°C, -38°C, -40°C, -42°C, -44°C and so on.
  • the freeze-drying temperature is -40°C.
  • the native collagen material is obtained by a lyophilization step.
  • the residual amount of DNA in the natural collagen material is less than 4ng/mg, and the enzyme-linked immunosorbent immunoassay method is used to detect ⁇ -galactosidase ( ⁇ -Gal), and the residual amount is lower than the detection limit of ⁇ -galactosidase detection kit 1.0 U/L.
  • the natural collagen material prepared in Example 1 is tested as follows:
  • SEM The microstructure and morphology of lyophilized native collagen materials were studied by SEM. As shown in Figure 1, SEM shows a highly interconnected porous structure. The natural collagen material generally presents a loose and porous structure. It can also be seen that some fibrous structures exist with a pore size range of 150-500 ⁇ m, which is suitable for cell adhesion and growth.
  • Figure 2 shows the Fourier transform infrared spectrum of natural collagen material.
  • in the infrared spectrum of collagen near 3293.53cm -1 is the NH stretching vibration absorption peak
  • near 1545.88cm -1 is the NH bending of the amide II band Vibration absorption peak.
  • the ratio of the absorption peaks around 1450 and 1230cm -1 was greater than 0.5, indicating the integrity of the collagen triple helix structure.
  • the ratio of the peak intensities of collagen at 1455.99 and 1236.05 cm -1 is 1.02, which is the characteristic value of collagen.
  • Freeze-dried collagen was dissolved in 0.5mol/L acetic acid solution to make the concentration of collagen solution reach 0.5mg/mL.
  • a UV spectrophotometer to scan the ultraviolet light of the collagen solution in the wavelength range of 200-400nm. absorption spectrum.
  • Figure 3 shows the UV-Vis absorption spectrum of natural collagen material. Larger light absorption occurs near the wavelength of 235nm.
  • the ultraviolet spectrum of collagen is actually the result of the addition of various ultraviolet chromogenic groups of protein molecules. Since type I collagen has hydroxyproline and proline with benzene ring structure, it has the maximum ultraviolet absorption The peak is at 235nm, which is consistent with the characteristic peak of ultraviolet absorption of type I collagen reported in the literature. The appearance of this absorption peak is mainly due to the n ⁇ * transition of the carbonyl group in the peptide chain.
  • a collagen solution with a concentration of 20 ⁇ g/mL was prepared, and a circular dichroism spectrometer was used to scan in the range of 180-260 nm. Setting parameters: the thickness of the liquid pool is 1mm, the scanning speed is 10nm/min, the scanning temperature is 19°C, and the slit width is 1.5nm.
  • the Rpn of collagen with triple helix structure ranges from 0.09 to 0.15.
  • Figure 4 shows the circular dichroism chromatogram of the natural collagen material, the chromatogram conforms to the characteristics of collagen, and its Rpn value is 0.112, indicating the integrity of the collagen triple helix structure. The results showed that the purification process did not damage the triple helical conformation of collagen.
  • HPLC-MS high-performance liquid chromatography-mass spectrometry
  • the natural collagen material contains 65%-70% of type III collagen and 30%-35% of type I collagen by mass.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Birds (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Materials For Medical Uses (AREA)
  • Peptides Or Proteins (AREA)

Abstract

天然胶原材料，天然胶原材料的制备方法、应用，和皮下填充剂，属于生物医用材料领域。天然胶原材料中III型胶原蛋白的含量显著提高，具有优异的弹性和皮肤修复功能，可有效恢复皮肤弹性、紧致肌肤，增加皮肤水润度，实现受损皮肤修复、抗衰老等功效，适合作为受损、衰老皮肤的皮下填充材料。

Description

天然胶原材料及天然胶原材料的制备方法、用途 技术领域
本公开属于生物医用材料领域，具体来说，本公开涉及一种天然胶原材料，天然胶原材料的制备方法、应用，和皮下填充剂。
背景技术
胶原蛋白(collagen)是动物细胞外基质的重要结构蛋白，约占其蛋白总含量的25～30％。胶原蛋白支持结缔组织的完整性、弹性和强度，从而保持皮肤、软骨和骨骼的形态和功能。受自然衰老和外界环境因素的影响，皮肤组织的胶原蛋白流失，出现皮肤松弛、皱纹、色斑等各种困扰人们的皮肤问题。如何修复皮肤损伤，延缓皮肤衰老，是当前人们普遍关注的重要问题。
人体所含的胶原蛋白有28种之多，而分布于皮肤中的主要是I型胶原和III型胶原。I型胶原纤维直径粗大，排列紧密，呈致密的条束状，纵横交错，非常适合形成坚固的纤维网络，是相对坚硬的胶原蛋白。III型胶原纤维为细纤维，呈疏网状，其含量越高纤维束越细，富有较高的弹性。婴儿皮肤中I/III型胶原的含量比约为1。随着年龄的增长，III型胶原不断减少，I型胶原不断增加。成人皮肤中的I/III型胶原的含量比约为2.5，III型胶原的含量断崖式降低。而伴随III型胶原的流逝，真皮组织会变相坍塌、纤维断裂、脂肪萎缩，导致皮肤失去弹性、光泽，出现色斑、皱纹等。III型胶原具有优异的弹性和修复功能，因此，补充外源性的III型胶原，是延缓衰老、修复皮肤损伤的重要途径。
目前，胶原蛋白的来源主要包括从组织中提取的天然胶原，以及通过基因工程等技术获得的人工胶原。目前广泛应用的是以天然获取的胶原为主，一般从牛、猪、鼠、鱼类等动物的肌腱、真皮和尾巴中提取。但是，传统方法提取的天然胶原中以I型胶原为主，其III型胶原的含量占比低。并且，通过酸法、碱法、盐法、酶法等方法提取的胶原中杂质成分(核酸片段、细 胞碎片)，需要复杂的提纯工艺进行杂质成分的脱除。
采用基因工程手段可制备高纯度的III型胶原，存在制备过程复杂、成本高、周期长等问题。另一方面，目前在胶原蛋白的制备多采用大肠杆菌的原核表达系统，大肠杆菌等细菌细胞内可能产生一定的内毒素混入胶原蛋白中，限制了其在生物医疗领域中的应用。
发明内容
发明要解决的问题
鉴于现有技术中存在的问题。例如，传统方法提取的天然胶原中III型胶原蛋白的含量低，且杂质含量高；而基因工程的方法制备III型胶原蛋白存在成本高、制备过程复杂、并存在一定生物安全隐患的问题。为此，本公开提供了一种天然胶原材料，其以小肠粘膜下层为原料获取III型胶原蛋白的含量占比高，免疫原性低、生物安全性高，具有优异的修复皮肤、恢复皮肤弹性的功能，适合作为受损、衰老皮肤的皮下填充材料。
用于解决问题的方案
第一方面，本公开提供了一种天然胶原材料，其中，所述天然胶原材料包括I型胶原蛋白和III型胶原蛋白；以所述天然胶原材料的总质量计，所述I型胶原蛋白的含量为30％-35％，所述III型胶原蛋白的含量为65％-70％；
所述天然胶原材料来源于小肠粘膜下层，优选为猪小肠粘膜下层。
在一些实施方式中，根据本公开所述的天然胶原材料，其中，所述天然胶原材料中DNA残留量低于4ng/mg，α-Gal残留量低于1.0U/L。
第二方面，本公开提供了一种天然胶原材料的制备方法，其中，包括如下步骤：
取小肠粘膜下层材料，依次进行病毒灭活处理、脱脂处理、脱细胞处理和酶解处理，得到胶原粗提溶液；
将胶原粗提溶液提纯、干燥，得到天然胶原材料；
优选地，所述天然胶原材料为第一方面所述的天然胶原材料。
在一些实施方式中，根据本公开所述的制备方法，其中，所述病毒灭活处理的步骤包括：将小肠粘膜下层材料清洗后浸泡于病毒灭活溶液中，在振荡条件下进行病毒灭活处理；优选地，小肠粘膜下层材料与病毒灭活溶液的体积比为(0.5-5):30；优选地，所述病毒灭活溶液是包含(0.5-5)％(v/v)过氧乙酸和(20-25)％(v/v)乙醇的水溶液；优选地，所述病毒灭活处理的时间为(1-5)h。
在一些实施方式中，根据本公开所述的制备方法，其中，所述脱脂处理的步骤包括：将病毒灭活处理后的小肠粘膜下层材料浸泡于脱脂溶液中，在超声条件下进行脱脂处理；优选地，所述小肠粘膜下层材料与脱脂溶液的体积比为(0.1-1):2；优选地，所述脱脂溶液为1-2wt％的碱性脂肪酶溶液；优选地，所述脱脂溶液pH为7-10；优选地，所述小肠粘膜下层材料浸泡于脱脂液中，超声处理30-60min后置换脱脂液，重复操作3次以上。
在一些实施方式中，根据本公开所述的制备方法，其中，所述脱细胞处理的步骤包括：将脱脂处理后的小肠粘膜下层材料浸泡于脱细胞溶液中，在超声条件下进行脱细胞处理；优选地，所述小肠粘膜下层材料与脱细胞溶液的体积比为(0.5-5):30；优选地，所述脱细胞溶液为包含0.02-0.05wt％胰蛋白酶和0.1-0.4wt％EDTA的磷酸盐缓冲液；优选地，所述脱细胞处理的时间为30min，然后清洗、干燥，得到脱细胞的小肠粘膜下层材料。
在一些实施方式中，根据本公开所述的制备方法，其中，所述酶解处理的步骤包括：
取脱细胞的小肠粘膜下层材料，破碎后，浸泡于酸性溶液中；
将浸泡后的小肠粘膜下层材料采用粉碎设备进行粉碎搅拌，加入胃蛋白酶，酶解处理后离心，去除沉淀，得到胶原粗提溶液；
优选地，小肠粘膜下层材料与酸性溶液的体积比为(0.5-5):30；优选地，所述酸性溶液为(0.1-1)mol/L的醋酸溶液，小肠粘膜下层材料在酸性溶液中浸泡的时间为12h，温度设备为4-10℃；优选地，小肠粘膜下层材料与胃蛋白 酶的质量比为(8-12):1；优选地，所述酶解处理是在搅拌条件下处理48h-96h。
在一些实施方式中，根据本公开所述的制备方法，其中，所述提纯的步骤包括：
将胶原粗提溶液一次过滤后进行盐析处理，得到沉淀颗粒；将沉淀颗粒溶解于酸性溶液中，二次清洗、溶解，并通过膜分离过滤、浓缩，得到包含天然胶原材料的胶原浓缩液。
第三方面，本公开提供了根据第一方面所述的天然胶原材料，或根据第二方面所述的方法制备的天然胶原材料在制备医疗美容材料或护肤品中的用途；优选地，所述医疗美容材料为皮下填充剂；优选地，所述皮下填充剂为注射剂。
第四方面，本公开提供了一种皮下填充剂，其中，所述皮下填充剂包括根据第一方面所述的天然胶原材料，或根据第二方面所述的方法制备的天然胶原材料。
发明的效果
在一些实施方式中，本公开提供的天然胶原材料，来源于小肠粘膜下层，与跟腱、真皮、尾巴等部位提取的胶原蛋白相比，本公开中的天然胶原材料中III型胶原蛋白的含量显著提高，具有优异的弹性和皮肤修复功能，可有效恢复皮肤弹性、紧致肌肤，增加皮肤水润度，实现受损皮肤修复、抗衰老等功效。
在一些实施方式中，本发明提供的天然胶原材料的制备方法，通过脱脂、脱细胞处理，可去除小肠粘膜上层组织中的细胞内成分(如DNA片段、细胞膜和细胞核碎片等)，减少核酸等免疫源物质的残留，提高天然胶原材料的生物安全性，并且能够简化对天然胶原材料的纯化工艺，降低制备难度和制备成本。
在一些实施方式中，本公开提供的皮下填充剂，包含本公开提供的天然胶原材料，适合进行皮下组织填充，有效紧致皮肤，改善皮肤的皱纹和损伤， 恢复皮肤的弹性、光泽、水润度。并且，与玻尿酸等填充剂相比，天然胶原材料的保持时间长，作用效果的持久性好。
附图说明
图1示出了天然胶原材料的SEM图，图1中A、B、C、D依次为1.00mm，500μm、500μm、100μm视野下的扫描电镜图片。
图2示出了天然胶原材料的傅里叶变换红外光谱图。
图3示出了天然胶原材料的紫外-可见吸收光谱图。
图4示出了天然胶原材料的圆二色谱图。
具体实施方式
以下将详细说明本发明的各种示例性实施例、特征和方面。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好地说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段、器材和步骤未作详细描述，以便于凸显本发明的主旨。
如无特殊声明，本说明书中所使用的单位均为国际标准单位，并且本发明中出现的数值，数值范围，均应当理解为包含了工业生产中所不可避免的系统性误差。
本说明书中，使用“可以”表示的含义包括了进行某种处理以及不进行某种处理两方面的含义。
本说明书中，所提及的“一些具体/优选的实施方案”、“另一些具体/优选的实施方案”、“实施方案”等是指所描述的与该实施方案有关的特定要素(例如，特征、结构、性质和/或特性)包括在此处所述的至少一种实施方案中，并且可存在于其它实施方案中或者可不存在于其它实施方案中。另外，应理 解，所述要素可以任何合适的方式组合在各种实施方案中。
本说明书中，使用“数值A～数值B”表示的数值范围是指包含端点数值A、B的范围。
本说明书中，使用“v/v”表示体积百分比含量，使用“wt％”为质量百分比含量。
另外，本说明书中，所述“水”包含去离子水、蒸馏水、离子交换水、双蒸水、高纯水、纯净水等能够使用的任何可行的水。
本说明书中，使用“常温”、“室温”时，其温度可以是10-40℃。
天然胶原材料
本公开的第一方面提供了一种天然胶原材料，所述天然胶原材料包括I型胶原蛋白和III型胶原蛋白；以所述天然胶原材料的总质量计，所述I型胶原蛋白的含量为30-35％，所述III型胶原蛋白的含量为65-70％；
所述天然胶原材料来源于小肠粘膜下层，优选为猪小肠粘膜下层。
本公开的天然胶原材料以小肠粘膜下层(Small Intestinal Submucosa，SIS)为原料获得，提取的胶原中III型胶原含量较高，为补充胶原蛋白流失提供了一种天然、安全、免疫原性低的胶原蛋白来源，适合作为皮肤胶原蛋白的补充材料。天然胶原材料在施用于受损或衰老皮肤后，能够发挥紧致皮肤、恢复皮肤弹性的功效，使皮肤细致、有光泽，有效改善受损皮肤状态、延缓皮肤衰老。
在一些优选实施方式中，天然胶原材料来源于猪小肠粘膜下层，该组织中含有较高含量的III型胶原蛋白，适合作为修复皮肤弹性的皮下填充材料。
在一些实施方式中，本公开提供的天然胶原材料，免疫源物质的含量低，其中DNA残留量低于4ng/mg，采用酶联免疫竞争法方法检测α-半乳糖苷酶(α-Gal)，其残留量低于α-半乳糖苷酶检测试剂盒检测限1.0U/L。天然胶原材料免疫源物质的含量低，生物安全性高，适合向皮下组织植入，作为填充材料修复受损、衰老皮肤，恢复皮肤弹性。
天然胶原材料的制备方法
本公开的第二方面提供了天然胶原材料的制备方法，包括如下步骤：
取小肠粘膜下层材料，依次进行病毒灭活处理、脱脂处理、脱细胞处理和酶解处理，得到胶原粗提溶液；
将胶原粗提溶液提纯、干燥，得到天然胶原材料。
本公开的制备方法通过对小肠粘膜下层材料的脱脂、脱细胞处理，能够有效去除核酸片段、细胞膜、细胞核碎片等细胞内成分，减少免疫源物质的残留。与传统的组织胶原提取方法相比，本公开的制备方法可有效简化对胶原的纯化处理步骤，能够制得III型胶原含量高、生物安全性高的天然胶原材料。与基因工程制备III型胶原的方法相比，本公开的制备方法具有成本低、步骤简单、易于操作等优势。
在一些实施方式中，应用本公开的制备方法，能够制得以总质量计，I型胶原蛋白的含量为30-35％，III型胶原蛋白的含量为65-70％的天然胶原材料，为皮肤修复、抗衰老提供了一种III型胶原蛋白与I型胶原蛋白合理配比的天然胶原材料。
在一些实施方式中，对于小肠粘膜下层材料，是去除了小肠的粘膜层和筋膜层后，留取的中间的小肠粘膜下层材料。小肠粘膜下层材料用清水洗净后用于后续处理。
当计量小肠粘膜下层材料体积时，为将清洗后的材料在不锈钢网上静置1-5min后放入量筒计量。
在一些实施方式中，所述病毒灭活处理的步骤包括：将小肠粘膜下层材料清洗后浸泡于病毒灭活溶液中，在振荡条件下进行病毒灭活处理。
对于病毒灭活溶液，是包含(0.5-5)％(v/v)过氧乙酸和(20-25)％(v/v)乙醇的水溶液。示例性的，病毒灭活溶液中过氧乙酸的含量为0.8％(v/v)、1％(v/v)、1.5％(v/v)、2％(v/v)、2.5％(v/v)、3％(v/v)、3.5％(v/v)、4％(v/v)、4.5％(v/v)等等；病毒灭活溶液中乙醇的含量为21％(v/v)、22％(v/v)、23％(v/v)、24％(v/v)等等。在一些优选的实施方式中，病毒灭活溶液是包含1％(v/v)过氧乙酸和24％(v/v)乙醇的水溶液。
按照小肠粘膜下层材料与病毒灭活溶液为(0.5-5):30的体积比，例如，0.8:30、1:30、2:30、3:30、4:30等等。在一些优选的实施方式中，小肠粘膜下层材料与病毒灭活溶液的体积比为1:30。将小肠粘膜下层材料浸泡于病毒灭活溶液，振荡条件下浸泡。小肠粘膜下层材料在病毒灭活溶液中浸泡的时间为1-5h，优选浸泡2h，振荡的速率为40r/min。浸泡后的小肠粘膜下层材料用水进行清洗。振荡可以通过摇床、磁搅拌子等方式实现。
通过上述的病毒灭活处理，可有效灭活小肠粘膜下层材料中的病毒成分，并能进一步降低细菌内毒素含量，保证天然胶原材料的生物安全性。
在一些实施方式中，脱脂处理的步骤包括：将病毒灭活处理后的小肠粘膜下层材料浸泡于脱脂溶液中，在超声条件下进行脱脂处理。
进一步的，脱脂溶液为1-2wt％的碱性脂肪酶溶液。示例性的，碱性脂肪酶溶液的浓度为1wt％、1.2wt％、1.4wt％、1.5wt％、1.7wt％、2wt％等。在一些优选的实施方式中，脱脂溶液的pH为7-10，例如，pH为7、8、9、10等。按照小肠粘膜下层材料与脱脂溶液为(0.1-1):2的体积比，示例性的，小肠粘膜下层材料与脱脂溶液的体积比为0.1:2、0.2:2、0.5:2、0.8:2、1:2等等。优选地，小肠粘膜下层材料与脱脂溶液的体积比1:10，将小肠粘膜下层材料浸泡于脱脂溶液中，在超声条件下脱脂处理30-60min后更换脱脂液；重复操作3次以上，然后用水清洗小肠粘膜下层材料。
通过脱脂处理，可有效减少胶原粗提液中混入的非胶原杂质成分，从而简化后续的纯化处理工艺，避免胶原的损失。与现有将组织至于水中超声清洗的处理方式相比，本公开中选择2wt％的碱性脂肪酶溶液，能够更加有效地去除脂质等不需要的杂质成分，减少提取过程中胶原成分的损耗。
在一些实施方式中，脱细胞处理的步骤包括：将脱脂处理后的小肠粘膜下层材料浸泡于脱细胞溶液中，在超声条件下进行脱细胞处理。
对于脱细胞溶液，是包含0.02-0.05wt％胰蛋白酶和0.1-0.4wt％EDTA的磷酸盐缓冲液。示例性的，磷酸盐缓冲液中胰蛋白酶的含量为0.02wt％、0.03wt％、0.04wt％、0.05wt％等等，EDTA的含量为0.1wt％、0.2wt％、0.3 wt％、0.4wt％等等。在一些优选的实施方式中，磷酸盐缓冲液中胰蛋白酶的含量为0.02wt％，EDTA的含量为0.1wt％。按照小肠粘膜下层材料与脱细胞溶液为(0.5-5):30的体积比，将小肠粘膜下层材料浸泡于脱细胞溶液，超声条件下浸泡30min。进一步的，是将小肠粘膜下层材料与脱细胞溶液按照1:30的体积比，在超声清洗机中浸泡30min。
超声后，将小肠粘膜下层材料清洗、干燥。在一些更为具体的实施方式中，干燥的处理条件是将小肠粘膜下层材料在37℃的温度条件下烘干24h。
本公开中脱细胞处理的条件温和，可以充分去除核酸、细胞膜、细胞核碎片等免疫源成分，降低天然胶原材料的免疫原性，使其适合植入皮下组织，并且避免胶原成分的损失。
现有的胶原提取工艺中，在得到胶原粗提液后，需要反复进行盐析-溶解的纯化操作，去除胶原中混有的核酸等杂质成分。这个过程会导致胶原蛋白的损失，并且由于使用了大量的中性盐，后期还需要进行清洗或透析，耗时长且操作过程复杂。而本公开通过结合脱脂、脱细胞处理，可将脂肪、核酸、α-Gal等免疫源物质降到最低，因此对胶原粗提溶液的纯化过程仅需要一次盐析和微孔滤膜过滤即可完成。
在一些实施方式中，酶解处理的步骤包括：取脱细胞的小肠粘膜下层材料，破碎后，浸泡于酸性溶液中；将浸泡后的小肠粘膜下层材料采用粉碎设备进行粉碎搅拌，加入胃蛋白酶，酶解处理后离心，去除沉淀，得到胶原粗提溶液。
进一步地，酸性溶液为0.1-1mol/L的醋酸溶液，示例性的，醋酸溶液的浓度为0.2mol/L、0.3mol/L、0.5mol/L、0.7mol/L、0.8mol/L等。作为优选，醋酸溶液的浓度为0.5mol/L。将烘干的小肠粘膜下层材料剪碎后，按照小肠粘膜下层材料与酸性溶液的体积比为(0.5-5):30，例如，0.8:30、1:30、2:30、3:30、4:30等等。在一些优选的实施方式中，按照1:30的体积比将小肠粘膜下层材料浸泡于酸性溶液中。在一些优选地实施方式中，小肠粘膜下层材料在4-10℃的低温下浸泡于酸性溶液中12h，以促进胶原蛋白的溶解。
对于酶解处理，是将浸泡后的小肠粘膜下层材料用粉碎机低温粉碎搅拌5min，然后按照胃蛋白酶与小肠粘膜下层材料的质量比为1:(8-12)的比例，向酸性溶液中加入胃蛋白酶，低温(4-10℃)机械搅拌48-96h，使小肠粘膜下层材料中的胶原蛋白充分溶解，以提高对III型胶原蛋白及I型胶原蛋白的提取效率。在一些优选的实施方式中，胃蛋白酶与小肠粘膜下层材料的质量比为1:10；向酸性溶液中加入胃蛋白酶后，低温机械搅拌的时间为72h。
酶解处理后，对混合溶液进行离心，所得上清液即为溶解有胶原蛋白的胶原粗提溶液。
在一些实施方式中，提纯的步骤包括：将胶原粗提溶液一次过滤后进行盐析处理，得到沉淀颗粒；将沉淀颗粒溶解于酸性溶液中，二次清洗、溶解，并通过膜分离过滤、浓缩，得到包含天然胶原材料的胶原浓缩液。
进一步地，酸性溶液为0.5mol/L的醋酸溶液，以充分溶解沉淀颗粒中的天然胶原材料。
对于过滤，是采用微滤膜过滤溶液，以过滤掉溶液中的颗粒。其中，一次过滤的微滤膜孔径为0.2-0.4μm，通过一次过滤可以去除胶原粗提溶液中的不溶性的颗粒。二次清洗后过滤的滤膜孔径为0.05-0.1μm，通过二次清洗后过滤可以去除多余的小分子杂质，提高胶原浓缩液的纯度。本公开中的提纯步骤，只需去除胃蛋白酶和材料中残余的非胶原成分，因此，仅需一次盐析和微孔滤膜过滤即可完成对胶原粗提溶液的提纯，具有步骤简单、对胶原损耗少的优势。
在一些实施方式中，通过对胶原浓缩液进行冷冻干燥处理，得到天然胶原材料。
本公开提供的制备方法，通过各步骤的相互作用，以及对各步骤的处理条件进行优化，可简化纯化工艺，实现对小肠粘膜下层材料中胶原蛋白的高效提取，得到包含65％-70％的III型胶原蛋白以及30％-35％的I型胶原蛋白的天然胶原材料。天然胶原材料中III型胶原蛋白的比例高，免疫源物质的含量低、生物安全心高，并呈疏松、多孔结构，适合细胞的黏附、生长；天 然胶原材料保持胶原蛋白的三螺旋构象，具有高的胶原蛋白活性，适合作为皮下填充材料，用于修复受损、衰老的皮肤。
皮下填充剂
本公开的第三方面提供了一种皮下填充剂，包括根据第一方面所述的天然胶原材料，或根据第二方面的方法制备的天然胶原材料。
天然胶原材料的用途
本公开的第四方面提供了天然胶原材料的用途，在一些实施方式中，天然胶原材料作为或制备医疗美容材料。在一些具体的实施方式，医疗美容材料为皮下填充剂。天然胶原材料可以与一种或多种辅料混合，被制作成任何可施用的剂型。示例性的，辅料包括溶剂、增稠剂、助溶剂、乳化剂、粘合剂、崩解剂、润滑剂、润湿剂、渗透压调节剂、pH调节剂、稳定剂、表面活性剂等等。在一些优选的实施方式中，皮下填充剂为注射剂。
在另外一些实施方式中，天然胶原材料用于制备护肤品。护肤品可以是贴片型、水剂型、凝胶型、乳液型或霜剂型等。
本公开中的天然胶原材料，其III型胶原蛋白和I型胶原蛋白的配比接近婴儿期皮肤，具有高含量的III型胶原蛋白，适用于皮下填充，能够有效恢复皮肤弹性，紧致肌肤，增加皮肤水润、光泽度。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
本实施例提供天然胶原材料的制备方法，包括如下步骤：
(1)清洗预处理：在室温下，刮除小肠的粘膜层和筋膜层，留取中间小肠粘膜下层，将材料使用纯化水清洗备用。
(2)病毒灭活：配制病毒灭活水溶液(1％(v/v)过氧乙酸+24％(v/v)乙醇)，将清洗后的小肠粘膜下层材料浸泡在病毒灭活溶液中，小肠粘膜下层材料与溶液比例为1:30，放置在摇床上，室温下浸泡2h，摇床速率为40r/min。浸泡完后采用纯化水清洗。
(3)脱脂：配置浓度为2wt％的碱性脂肪酶溶液，将病毒灭后的小肠粘膜下层材料浸泡在脱脂溶液中，小肠粘膜下层材料与脱脂液的比例为1:10(v:v)，在超声清洗机中超声浸泡30min，置换脱脂溶液，反复操作3次。纯化水清洗小肠粘膜下层材料。
(4)脱细胞：配制脱细胞磷酸盐缓冲液(0.025wt％胰蛋白酶+0.19wt％EDTA)，将小肠粘膜下层材料转移至脱细胞溶液中，小肠粘膜下层材料与脱细胞溶液比例为1:30(v:v)，在超声清洗机中超声浸泡30min，超声后采用纯化水清洗，将小肠粘膜下层材料放置在37℃烘箱中干燥24h。
(5)酸浸泡：将烘干后的小肠粘膜下层材料裁剪成大约1cm 2的碎片，采用0.5mol/L的醋酸溶液4℃浸泡脱细胞后的小肠粘膜下层材料12h，小肠粘膜下层材料与脱细胞溶液比例为1:30(v:v)。
(6)酶解：将浸泡后的小肠粘膜下层材料用粉碎机低温粉碎搅拌5min，在溶液中加入胃蛋白酶(胃蛋白酶与小肠粘膜下层材料质量比为1:10)，低温机械搅拌72h。酶解后将混合液离心处理，8000r/min，离心10min，取上清液即为粗提胶原溶液。
(7)纯化：将粗提胶原溶液采用微滤膜处理，先用孔径为0.2-0.4微米的滤膜过滤掉不溶颗粒。采用中性盐氯化钠或者硫酸铵进行盐析，盐析后离心取沉淀颗粒，蒸馏水清洗后采用0.5mol/L的醋酸进行溶解，再采用孔径为0.05-0.1微米的超滤膜进行浓缩处理，去除多余的小分子杂质，得到高纯度的胶原浓缩液。
(8)干燥：将高纯胶原浓缩液冷冻干燥，得到天然胶原材料。将胶原浓缩液放入冷冻干燥机(北京博医康实验仪器有限公司，FD-1A-50)冷阱中进行预冷60-120min，预冷后开启真空泵，进行真空干燥，冷阱温度设定为 -35℃至-45℃，例如-37℃、-38℃、-40℃、-42℃、-44℃等等。优选地，冷冻干燥的温度为-40℃。通过冻干步骤得到天然胶原材料。
天然胶原材料中DNA残留量低于4ng/mg，采用酶联免疫竞争法方法检测α-半乳糖苷酶(α-Gal)，其残留量低于α-半乳糖苷酶检测试剂盒检测限1.0U/L。
性能测试
对实施例1制备的天然胶原材料进行如下测试：
1、SEM分析
剪取适量大小合适的天然胶原材料，粘贴于样品台上，使用扫描电镜观察胶原海绵表面形貌，加速电压设定为10kV。
用SEM研究了冻干的天然胶原材料的微观结构和形态。如图1所示，SEM显示高度互连的多孔结构，天然胶原材料总体呈现疏松、多孔的结构,还可见部分纤维化结构的存在孔径范围为150-500μm，适合细胞的黏附、生长。
2、傅里叶变换红外光谱检测
称取约2mg天然胶原材料，液氮冷冻研磨粉碎，与200mg溴化钾混合均匀，研磨完全后在15MPa下压片制样，使用红外光谱仪在4000～400cm -1进行红外光谱扫描，扫描64次，分辨率为4cm -1。
图2示出了天然胶原材料的傅里叶变换红外光谱图。胶原蛋白红外谱图中在3293.53cm -1附近是N-H的伸缩振动吸收峰，1634.48cm -1附近是酰胺Ⅰ带的C＝O伸缩振动吸收峰，1545.88cm -1附近是酰胺Ⅱ带的N-H弯曲振动吸收峰。1450和1230cm -1附近吸收峰比值大于0.5，表示胶原蛋白三螺旋结构的完整性。胶原蛋白在1455.99和1236.05cm -1处峰强度的比值为1.02，是胶原蛋白的特征值。
3、紫外-可见吸收光谱法
冻干胶原溶解于0.5mol/L醋酸溶液中，使胶原溶液浓度达到 0.5mg/mL，以0.5mol/L HAC溶液作对比，使用紫外分光光度计扫描胶原溶液在200～400nm波长区间内的紫外吸收光谱。
图3示出了天然胶原材料的紫外-可见吸收光谱图。在波长235nm附近产生较大的光吸收。胶原蛋白的紫外光谱图实际上是蛋白质分子的各种紫外生色基团加和的结果，由于Ⅰ型胶原蛋白中具有含苯环结构的羟脯氨酸和脯氨酸，使其最大紫外吸收峰在235nm处，这与文献报道的Ⅰ型胶原蛋白的紫外吸收特征峰相符。该吸收峰的出现主要是由于肽链中羰基的n→π*跃迁产生的。
4、圆二色谱分析(CD)
配制浓度为20μg/mL的胶原溶液，使用圆二色谱仪在180～260nm范围内进行扫描。设置参数：液池厚度1mm，扫描速度10nm/min，扫描温度19℃，狭缝宽度1.5nm。
在远紫外区域，胶原蛋白的CD光谱中，在222nm处具有最大正峰值，在197nm处具有最大负峰值，并且交叉点在约213nm处。正峰与负峰的比率(Rpn)是三螺旋结构构象所独有的，可用于鉴定三螺旋构象。
具有三螺旋结构的胶原蛋白的Rpn范围为0.09～0.15。图4示出了天然胶原材料的圆二色谱图，色谱图符合胶原蛋白的特征，其Rpn值为0.112，表明胶原蛋白三螺旋结构的完整性。结果显示，纯化过程不会损害胶原蛋白的三螺旋构象。
5、胶原成分分析
本实验采用高效液相色谱-质谱联用技术，将样品中胶原用胰蛋白酶特异性降解，再通过高效液相色谱-质谱(HPLC-MS)联用技术检索和识别I、III型胶原的特征多肽，对其进行定量分析，可识别材料中不同类型胶原并准确定量。将标准品和样品分别变性并酶解，采用HPLC-MS检测酶解物。其中，取冻干后的天然胶原材料，粉碎后称取10mg，加入NH 4HCO 3缓冲液(50mM，pH 8.0)配置成1mg/mL的溶液。在100℃下变性10min，冷却至室温后按照胶原与胰蛋白酶为50:1的质量比例加入胰蛋白酶，37℃酶解 16h，酶解产物直接通过HPLC-MS检测其中胶原成分。通过针对检测结果的分析，天然胶原材料包含质量百分含量65％-70％的III型胶原蛋白以及质量百分含量30％-35％的I型胶原蛋白。
本公开的上述实施例仅是为清楚地说明本公开所作的举例，而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本公开权利要求的保护范围之内。

Claims (10)

  1. 一种天然胶原材料，其特征在于，所述天然胶原材料包括I型胶原蛋白和III型胶原蛋白；以所述天然胶原材料的总质量计，所述I型胶原蛋白的含量为30％-35％，所述III型胶原蛋白的含量为65％-70％；
    所述天然胶原材料来源于小肠粘膜下层，优选为猪小肠粘膜下层。
  2. 根据权利要求1所述的天然胶原材料，其特征在于，所述天然胶原材料中DNA残留量低于4ng/mg，α-Gal残留量低于1.0U/L。
  3. 一种天然胶原材料的制备方法，其特征在于，包括如下步骤：
    取小肠粘膜下层材料，依次进行病毒灭活处理、脱脂处理、脱细胞处理和酶解处理，得到胶原粗提溶液；
    将胶原粗提溶液提纯、干燥，得到天然胶原材料；
    优选地，所述天然胶原材料为权利要求1-2任一项所述的天然胶原材料。
  4. 根据权利要求3所述的制备方法，其特征在于，所述病毒灭活处理的步骤包括：将小肠粘膜下层材料清洗后浸泡于病毒灭活溶液中，在振荡条件下进行病毒灭活处理；优选地，小肠粘膜下层材料与病毒灭活溶液的体积比为(0.5-5):30；优选地，所述病毒灭活溶液是包含(0.5-5)％(v/v)过氧乙酸和(20-25)％(v/v)乙醇的水溶液；优选地，所述病毒灭活处理的时间为(1-5)h。
  5. 根据权利要求3或4所述的制备方法，其特征在于，所述脱脂处理的步骤包括：将病毒灭活处理后的小肠粘膜下层材料浸泡于脱脂溶液中，在超声条件下进行脱脂处理；优选地，所述小肠粘膜下层材料与脱脂溶液的体积比为(0.1-1):2；优选地，所述脱脂溶液为1-2wt％的碱性脂肪酶溶液；优选地，所述脱脂溶液pH为7-10；优选地，所述小肠粘膜下层材料浸泡于脱脂液中，超声处理30-60min后置换脱脂液，重复操作3次以上。
  6. 根据权利要求3-5任一项所述的制备方法，其特征在于，所述脱细胞处理的步骤包括：将脱脂处理后的小肠粘膜下层材料浸泡于脱细胞溶液中，在超声条件下进行脱细胞处理；优选地，所述小肠粘膜下层材料与脱细胞溶液的体积比为(0.5-5):30；优选地，所述脱细胞溶液为包含0.02-0.05wt％胰蛋白酶和0.1-0.4wt％EDTA的磷酸盐缓冲液；优选地，所述脱细胞处理的时间 为30min，然后清洗、干燥，得到脱细胞的小肠粘膜下层材料。
  7. 根据权利要求3-6任一项所述的制备方法，其特征在于，所述酶解处理的步骤包括：
    取脱细胞的小肠粘膜下层材料，破碎后，浸泡于酸性溶液中；
    将浸泡后的小肠粘膜下层材料采用粉碎设备进行粉碎搅拌，加入胃蛋白酶，酶解处理后离心，去除沉淀，得到胶原粗提溶液；
    优选地，小肠粘膜下层材料与酸性溶液的体积比为(0.5-5):30；优选地，所述酸性溶液为(0.1-1)mol/L的醋酸溶液，小肠粘膜下层材料在酸性溶液中浸泡的时间为12h，温度设备为4-10℃；优选地，小肠粘膜下层材料与胃蛋白酶的质量比为(8-12):1；优选地，所述酶解处理是在搅拌条件下处理48h-96h。
  8. 根据权利要求3-7任一项所述的制备方法，其特征在于，所述提纯的步骤包括：
    将胶原粗提溶液一次过滤后进行盐析处理，得到沉淀颗粒；将沉淀颗粒溶解于酸性溶液中，二次清洗、溶解，并通过膜分离过滤、浓缩，得到包含天然胶原材料的胶原浓缩液。
  9. 根据权利要求1-2任一项所述的天然胶原材料，或根据权利要求3-8任一项所述的方法制备的天然胶原材料在制备医疗美容材料或护肤品中的用途；优选地，所述医疗美容材料为皮下填充剂；优选地，所述皮下填充剂为注射剂。
  10. 一种皮下填充剂，其中，所述皮下填充剂包括根据权利要求1-2任一项所述的天然胶原材料，或根据权利要求3-8任一项所述的方法制备的天然胶原材料。
PCT/CN2021/124986 2021-09-06 2021-10-20 天然胶原材料及天然胶原材料的制备方法、用途 WO2023029161A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111037549.4 2021-09-06
CN202111037549.4A CN113717273A (zh) 2021-09-06 2021-09-06 天然胶原材料及天然胶原材料的制备方法、用途

Publications (1)

Publication Number Publication Date
WO2023029161A1 true WO2023029161A1 (zh) 2023-03-09

Family

ID=78681841

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/124986 WO2023029161A1 (zh) 2021-09-06 2021-10-20 天然胶原材料及天然胶原材料的制备方法、用途

Country Status (2)

Country Link
CN (1) CN113717273A (zh)
WO (1) WO2023029161A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116179634A (zh) * 2023-04-27 2023-05-30 北京盛美诺生物技术有限公司 一种iii型胶原蛋白肽及其制备方法

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113694254A (zh) * 2021-09-06 2021-11-26 北京博辉瑞进生物科技有限公司 骨修复材料及骨修复材料的制备方法、应用
CN114163518A (zh) * 2022-02-14 2022-03-11 北京思尔根生物科技有限公司 一种从猪小肠粘膜下层中提取活性胶原蛋白的方法
CN115536742B (zh) * 2022-10-14 2023-09-08 斐缦(长春)医药生物科技有限责任公司 一种医美真皮填充用胶原蛋白及其制备方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060246033A1 (en) * 2005-03-02 2006-11-02 Cook Biotech Incorporated Injectable bulking agent compositions
CN101361989A (zh) * 2008-09-03 2009-02-11 陕西瑞盛生物科技有限公司 双层膜状组织修补材料及其制备方法
CN101361990A (zh) * 2008-09-03 2009-02-11 陕西瑞盛生物科技有限公司 一种双层人工皮肤及其制备方法
CN101366975A (zh) * 2008-09-03 2009-02-18 陕西瑞盛生物科技有限公司 脱细胞小肠粘膜下层生物材料的制备方法
CN101433735A (zh) * 2007-11-13 2009-05-20 北京大清生物技术有限公司 一种sis组织修复材料的制备方法
CN109331228A (zh) * 2018-11-26 2019-02-15 陈德夫 一种抗感染的小肠粘膜下层生物材料的制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101126104B (zh) * 2007-05-22 2011-01-19 上海市食品研究所 采用酸酶复合制备天然活性胶原蛋白的方法
CN101366977B (zh) * 2008-09-03 2013-07-24 陕西瑞盛生物科技有限公司 具有生物活性的组织修补材料及其制备方法
CN102462561A (zh) * 2010-11-19 2012-05-23 北京迈迪顶峰医疗科技有限公司 一种sis软组织修复补片及其制备方法
CN104312140B (zh) * 2014-10-22 2017-02-15 四川大学华西医院 一种聚氨酯/小肠粘膜下层复合材料及用途
CN108785746B (zh) * 2018-07-05 2021-05-04 承功（厦门）生物科技有限公司 一种脱细胞猪小肠粘膜下层快速制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060246033A1 (en) * 2005-03-02 2006-11-02 Cook Biotech Incorporated Injectable bulking agent compositions
CN101433735A (zh) * 2007-11-13 2009-05-20 北京大清生物技术有限公司 一种sis组织修复材料的制备方法
CN101361989A (zh) * 2008-09-03 2009-02-11 陕西瑞盛生物科技有限公司 双层膜状组织修补材料及其制备方法
CN101361990A (zh) * 2008-09-03 2009-02-11 陕西瑞盛生物科技有限公司 一种双层人工皮肤及其制备方法
CN101366975A (zh) * 2008-09-03 2009-02-18 陕西瑞盛生物科技有限公司 脱细胞小肠粘膜下层生物材料的制备方法
CN109331228A (zh) * 2018-11-26 2019-02-15 陈德夫 一种抗感染的小肠粘膜下层生物材料的制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116179634A (zh) * 2023-04-27 2023-05-30 北京盛美诺生物技术有限公司 一种iii型胶原蛋白肽及其制备方法
CN116179634B (zh) * 2023-04-27 2023-08-18 北京盛美诺生物技术有限公司 一种iii型胶原蛋白肽及其制备方法

Also Published As

Publication number Publication date
CN113717273A (zh) 2021-11-30

Similar Documents

Publication Publication Date Title
WO2023029161A1 (zh) 天然胶原材料及天然胶原材料的制备方法、用途
Li et al. Plant protein amyloid fibrils for multifunctional sustainable materials
US8197853B2 (en) Process for solubilizing protein from a proteinaceous material and compositions thereof
Santos et al. Extraction and characterization of highly purified collagen from bovine pericardium for potential bioengineering applications
US20190359650A1 (en) Methods for extracting keratin proteins
CN110655568A (zh) 一种酸性酶解法提取胶原蛋白的方法
US20070017447A1 (en) Avian eggshell membrane polypeptide extraction via fermentation process
US20170281783A1 (en) Low protein percentage gelling compositions
CN113527466A (zh) 一种植入级ⅱ型胶原蛋白的制备方法
JP4863433B2 (ja) 魚鱗由来コラーゲンの取得方法
US10792239B2 (en) White keratin compositions
CN108977488B (zh) 皮肤密集修复用弹性蛋白及其制备方法
Earland et al. Studies on the structure of keratin IV. The molecular structure of some morphological components of keratins
CN114774501B (zh) 鱼皮胶原蛋白海绵材料及其制备方法
CN113603768B (zh) 一种鱼源胶原蛋白的制备方法
CN109575119A (zh) 兔毛角蛋白的新型提取方法及其在化妆品中的应用
CN108277252A (zh) 一种从鱼骨中提取胶原蛋白制备方法
CN115232203A (zh) 一种提取胶原蛋白的方法
AU2018204128B2 (en) White Keratin Compositions
US20190194297A1 (en) Process to extract and recover keratin and keratin associated protein from animal body parts
CN114106148B (zh) 一种鱼皮源医用级非变性胶原及其制备方法
KR102563616B1 (ko) 줄기세포 유래 수용성 세포외기질 및 이의 제조방법
JP7138873B2 (ja) コラーゲン含有組成物の製造方法及びコラーゲン単離物
KR20130031194A (ko) 누에 실샘 유래 가수분해물의 수득방법
KR101106706B1 (ko) 실고기 유래 콜라겐 및 그 제조 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21955690

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21955690

Country of ref document: EP

Kind code of ref document: A1