JP2017095400A - Collagen, collagen sheet, method for producing collagen and method for producing collagen sheet - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a collagen with high viscoelasticity capable of maintaining its shape only with collagen, to provide a sheet thereof, and to provide a production method thereof.SOLUTION: This collagen has viscoelasticity higher than conventional collagens that are derived from the skin. Thereby, the collagen sheet using this collagen is processable into a sheet shape with a collagen solely. Therefore, there is no need of adding an artificial crosslinking agent component or highly viscous substance which may cause damage to a human body, and the collagen sheet with a 100% collagen component can be obtained. It also has a particularly excellent effect for skin cares and medical applications because of having a fibrogenesis ability higher than conventional collagens. Furthermore, the collagen is absorbed entirely by the human body and disappears because of containing no additives, so that residues and the like never remain.SELECTED DRAWING: Figure 1

Description

  The present invention relates to collagen used for cosmetics or medical use, a collagen sheet, and a method for producing the same.

  Collagen is a natural polymer material that has been used together with cellulose for a long time. In recent years, collagen has been used not only in cosmetics but also in medical fields such as artificial skin and artificial bone, and is expected to be widely applied in the future. These collagens are generally extracted from the skin and bones of pigs, cattle or fish. However, conventional collagen extracted from skin and bone has low viscosity and elasticity, and the shape cannot be maintained with collagen alone. For this reason, in order to maintain these conventional collagens in the shape of a sheet or the like, it is necessary to add an artificial crosslinking agent or mix a highly viscous substance as described in, for example, [Patent Document 1] below. is there.

JP 2002-220319 A

  However, these additives such as cross-linking agents are harmful to chemical allergy carriers and cannot be said to be highly safe, and there is a problem that users are limited. Further, these additives are not preferred because they may not be absorbed by the human body and remain as a residue on the skin surface.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a highly viscoelastic collagen, a collagen sheet, and a method for producing the same that can maintain the shape only with collagen.

The present invention
(1) Collagen containing no artificial crosslinking agent component,
Viscosity of 1.0 Pa or more and 3.0 Pa when shear deformation is applied to an aqueous solution of collagen adjusted to a concentration of 0.25 wt%, pH of 2.0 to 3.5, and liquid temperature of 10 ° C. at a shear rate of 1 s ^−1. The storage elastic modulus after 30 minutes of heating the collagen aqueous solution adjusted to a concentration of 0.25 wt% and pH 6.8 to 7.4 to 37 ° C. is 60 Pa to 120 Pa. The above-mentioned problems are solved by providing the collagen.
(2) Collagen containing both or one of type I collagen and type III collagen derived from pig ears that does not contain an artificial crosslinking agent component,
Viscosity of 1.0 Pa or more and 3.0 Pa when shear deformation is applied to an aqueous solution of collagen adjusted to a concentration of 0.25 wt%, pH of 2.0 to 3.5, and liquid temperature of 10 ° C. at a shear rate of 1 s ^−1. The storage elastic modulus after 30 minutes of heating the collagen aqueous solution adjusted to a concentration of 0.25 wt% and pH 6.8 to 7.4 to 37 ° C. is 60 Pa to 120 Pa. The above-mentioned problems are solved by providing the collagen.
(3) The problem is solved by providing the collagen according to (2) above, wherein the pig ears are 3 to 7 months old and have a body weight of 50 to 130 kg.
(4) The collagen sheet according to any one of (1) to (3) above,
Contains no artificial crosslinker component,
Hold the shape without tearing in a single state without a holding body,
The problem is solved by providing a collagen sheet characterized by being absorbed and extinguished after being attached to a human body.
(5) The above (4), wherein the solution is dissolved by contact with reducing alkaline ionized water having a pH of 10.5 to 12.5, and the solution exhibits weak acidity of pH 4.5 to pH 6.0. The above-mentioned problem is solved by providing the collagen sheet described above.
(6) After removing the epidermis of the pig ears to the dermis, a step of cutting to a predetermined size (pig ear pretreatment step: step S100);
A process of degreasing the cut pig ears with an organic degreasing liquid (degreasing process: step S104);
A process of extracting collagen from a degreased pig ear with a solution containing an acid and a digestive enzyme (extraction process: step S106);
A process of purifying the extracted collagen (purification process: step S110);
The above-mentioned problem is solved by providing a method for producing collagen, comprising a step of drying purified collagen (drying step: step S112).
(7) Solving the above-mentioned problem by providing a method for producing collagen according to (6) above, wherein pig ears having an age of 3 months or more and 7 months or less and a body weight of 50 kg or more and 130 kg or less are used. To do.
(8) After removing the epidermis of the pig ears to the dermis, the step of cutting into a predetermined size (pig ear pretreatment step: step S100);
A process of degreasing the cut pig ears with an organic degreasing liquid (degreasing process: step S104);
A process of extracting collagen from a degreased pig ear with a solution containing an acid and a digestive enzyme (extraction process: step S106);
A process of purifying the extracted collagen (purification process: step S110);
A step of dissolving the purified collagen in a predetermined acidic solution (dissolution step: step S200);
A process of casting the dissolved collagen into a predetermined mold and drying it into a sheet (molding process: step S202 to de-frame process: step S206);
The above-mentioned problems are solved by providing a method for producing a collagen sheet characterized by comprising:
(9) By providing a method for producing a collagen sheet as described in (8) above, wherein the above-mentioned problem is achieved by using a pig ear whose age is 3 months or more and 7 months or less and whose weight is 50 kg or more and 130 kg or less. Solve.

  The collagen and collagen sheet according to the present invention have high viscoelasticity. Thereby, a sheet | seat shape can be maintained only with collagen. In addition, the method for producing collagen and the method for producing a collagen sheet according to the present invention can produce collagen and a collagen sheet capable of maintaining the shape only with collagen.

It is a flowchart of the manufacturing method of the collagen which concerns on this invention. It is the graph which compared the storage elastic modulus G 'of the collagen aqueous solution under neutral conditions. 2 is a graph comparing viscosity η of collagen aqueous solutions under acidic conditions. It is the graph which compared the fibrosis degree (turbidity) of the collagen aqueous solution under neutral conditions. It is the figure which compared the cell growth rate by collagen. It is a flowchart of the manufacturing method of the collagen sheet which concerns on this invention. It is the graph which compared the moisture retention of collagen.

  DESCRIPTION OF EMBODIMENTS Embodiments of collagen, a collagen sheet, and a manufacturing method thereof according to the present invention will be described with reference to the drawings. Note that the collagen and collagen sheet according to the present invention are purified and produced by extracting collagen from pig ears, focusing on the fact that type I collagen contained in pig ears is higher in viscoelasticity than type I collagen at other sites. To do.

  FIG. 1 is a flowchart showing a method for producing collagen according to the present invention. First, a pig ear (hereinafter referred to as a pig ear) is prepared. This pig ear is preferably a pig whose age is 3 months or more and 7 months or less and whose weight is 50 kg or more and 130 kg or less. Moreover, it is preferable to use a pig that is healthy and not sick. In addition, when the age of a pig exceeds 7 months, the collagen of a pig ear will become hard and a quality collagen cannot be acquired. Moreover, the pigs of this age and weight are generally pigs that are processed into meat, and according to this configuration, pig ears that are disposed of at the time of meat processing can be stably obtained at low cost. Moreover, the pig ears to be disposed of can be effectively used.

  Next, the epidermis is removed until the pig dermis is exposed. Then, the pig ear from which the epidermis has been deleted is cut into a predetermined size (pig ear pretreatment process: step S100). The cutting dimension at this time is better as it is finer, and preferably about several millimeters square to 1 cm square. Next, the cut pig ear is degreased with a known organic degreasing solution (degreasing step: step S104). As this organic degreasing liquid, methanol, ethanol, chloroform, acetone, xylene, or the like can be used alone or in combination. Thereby, the lipid in a pig ear elutes in this organic type degreasing liquid. Next, the degreased pig ears are thoroughly washed to remove the organic degreasing solution together with the lipids.

  Next, collagen is extracted from the defatted pig ear with a solution containing an acid and a digestive enzyme. As the acid used at this time, it is preferable to use acetic acid having a predetermined concentration. Moreover, it is preferable to use pepsin as a digestive enzyme used at this time. Then, the degreased pig ears are immersed for a predetermined time while maintaining this extraction solution at a low temperature of several degrees C. (extraction step: step S106). Thereby, the collagen in the pig ear is eluted in the extraction solution.

Next, the extracted solution is separated by, for example, a centrifugal separator to obtain a supernatant. The remaining pig ears after extraction of collagen are discarded. Next, the digestive enzyme is removed from the obtained supernatant. The digestive enzyme is preferably removed by dialysis using a predetermined dialysis membrane and a dialysate having a predetermined concentration (for example, a disodium hydrogen phosphate (Na ₂ HPO ₄ ) aqueous solution) (separation step: Step S108).

  Next, the obtained collagen is purified (purification step: step S110). Any method may be used for the purification of collagen. For example, the following method is used. First, the collagen gelled by the dialysis fluid in the separation step is centrifuged to remove the supernatant. Next, the centrifuged collagen is redissolved with an acid solution such as an acetic acid aqueous solution or a neutral solution such as water. Next, a desired concentration of saline is added to salt out the desired collagen. Next, the salted out collagen is centrifuged to remove the supernatant.

  Next, the centrifuged collagen is redissolved with an acid solution such as an acetic acid aqueous solution or a neutral solution such as water, and then the salt is removed by dialysis or the like (salt removal step: step S111). This completes the purification of the desired collagen.

  Next, the purified collagen is dried (drying step: step S112). Any method may be used as a method for drying the collagen, but lyophilization (freeze drying) is particularly preferable. Thereby, the highly viscoelastic collagen obtained from pig ears is obtained.

  In addition, the collagen obtained from the pig ear as described above is analyzed by CBB staining by SDS-PAGE (SDS-polyacrylamide gel electrophoresis) and Western blocking using anti-type I and anti-type III collagen antibodies. As a result, it was suggested that this collagen is type I collagen contained in a large amount in animal skin and the like. In addition, type III collagen often coexists in type I collagen, and it was confirmed that type III collagen was also contained in the pig ear collagen.

  Here, a comparison result between collagen obtained from pig ears (hereinafter referred to as pig ear collagen) and conventional collagen obtained from pig skin (hereinafter referred to as pig skin collagen) as described above. Indicates.

  First, a known pH buffer solution was added to a collagen aqueous solution in which pork ear collagen and porcine skin collagen were dissolved to prepare a collagen aqueous solution having a collagen concentration of 0.25 wt% and a pH of 6.8 to 7.4 (neutral). And the storage elastic modulus G '(Pa) of this collagen aqueous solution at 37 degreeC was measured using the rotary rheometer (Thermo Scientific: HAKKE MARS). The result is shown in FIG. The solid line in FIG. 2 indicates the storage elastic modulus G ′ of pig ear collagen, and the broken line indicates the storage elastic modulus G ′ of pig skin collagen.

  From FIG. 2, the storage elastic modulus G ′ of pig ear collagen showed about 90 Pa after 30 minutes (1800 sec) after heating to 37 ° C. On the other hand, the storage elastic modulus G ′ of pig skin collagen is about 10 Pa 30 minutes after heating, and it can be seen that pig ear collagen has a storage elastic modulus G ′ that is extremely higher than that of porcine skin collagen. Here, 10 mM of genipin as an artificial cross-linking agent was added to a skin collagen aqueous solution having a concentration of 0.25 wt% and adjusted to pH 7.0 to 7.4, and the storage modulus G of conventional collagen 20 minutes after heating at 37 ° C. 'Was about 100 Pa. From this, the storage elastic modulus G ′ of pig ear collagen is slightly inferior to conventional collagen with a cross-linking agent added, but it has a storage elastic modulus G ′ capable of maintaining the shape sufficiently by itself. I understand. In addition, the storage modulus G 'of pig ear collagen slightly fluctuates depending on the age, growth state, individual difference, type, etc. of the pig used, and the range is generally 60 Pa or more and 120 Pa or less. Within this range, the shape of the collagen can be maintained without adding an artificial crosslinking agent component or the like.

  In addition, the viscosity η (Pa) at 10 ° C. of a pig ear collagen aqueous solution and a pig skin collagen aqueous solution having a collagen concentration of 0.25 wt% and pH 2.0 to 3.5 (acidic) prepared in the same manner was measured using a rotary rheometer (Thermo Scientific). FIG. 3 shows the result of measurement using a company (HAKKE MARS). The black square mark in FIG. 3 indicates the viscosity η of pig ear collagen, and the white square mark indicates the viscosity η of pig skin collagen.

From FIG. 3, the viscosity η of the swine ear collagen aqueous solution when shear deformation was applied at a shear rate of 1 s ⁻¹ (1 / s) showed 2.3 Pa, whereas that of porcine skin collagen was about 1.05 Pa. It can be seen that porcine ear collagen has a higher viscosity η than porcine skin collagen. The pig ear collagen has a viscosity η that slightly increases and decreases depending on the pig used as described above, and the range is approximately 1.0 Pa to 3.0 Pa under the above conditions (shear rate 1 s ⁻¹ ).

  Next, while maintaining a pig ear collagen aqueous solution and a pig skin collagen aqueous solution having a collagen concentration of 0.1 wt% and pH 7.4 (neutral) at 37 ° C., the change in turbidity OD was measured by measuring the absorbance at a wavelength of 400 nm. evaluated. The result is shown in FIG. In addition, the continuous line in FIG. 4 shows the turbidity of pig ear collagen, and a broken line shows the turbidity of pig skin collagen. From FIG. 4, it was recognized that the pig ear collagen had an increase in turbidity due to the association of collagen molecules and fiber formation from the initial stage of the measurement compared to the pig skin collagen. From this, it can be seen that porcine ear collagen is superior to porcine skin collagen in terms of fiber forming ability which is important for material creation.

Next, a pig ear collagen and a pig skin collagen having a collagen concentration of 0.1 wt% and pH 7.4 (neutral) were dissolved and applied to a glass slide chamber, and NIH3T3 cells were added to 5.0 × 10 ⁵ thereon. After seeding one by one, the cell proliferation rate after 12 hours was measured. The cell growth rate was measured by MTT assay. The measurement results of the cell growth rate of pig ear collagen and pig skin collagen after the test are shown in FIG. FIG. 5 shows the ratio when the cell growth rate is 1 when NIH3T3 cells are seeded in a medium without collagen. FIG. 5 shows that pig ear collagen has the same cell growth rate as porcine skin collagen or higher than porcine skin collagen.

  These results suggest that the pig ear collagen according to the present invention has higher viscoelasticity, fiber-forming ability, and cell proliferation ability than conventional pig skin collagen.

  Next, the manufacturing method of the collagen sheet which concerns on this invention is demonstrated using the flowchart of FIG. First, collagen is obtained from pig ears in the same manner as in steps S100 to S112. In addition, when extracting, refine | purifying, and sheet-izing collagen from a pig ear continuously, you may abbreviate | omit the drying process of step S112. Next, this collagen (pig ear collagen) is dissolved in a predetermined acidic solution (dissolution step: step S200). Although there is no limitation in particular in the acidic solution used at this time, a citric acid aqueous solution, malic acid aqueous solution, ascorbic acid aqueous solution, lactic acid aqueous solution, hydrochloric acid aqueous solution, and acetic acid aqueous solution are preferable, and it is preferable to use especially 0.5 wt% malic acid aqueous solution.

  Next, the aqueous collagen solution is poured into a mold having a predetermined shape (molding step: step S202). As the shape of this formwork, it can be any shape depending on the application, such as circular, oval, square, rectangular, etc. Also good. In particular, when preparing a collagen sheet for medical use, neutrality of pH 6.8 to pH 7.4 is obtained by adding distilled water or a buffer solution having a predetermined pH to the collagen aqueous solution before being poured into the mold. It is preferable to keep it.

  Next, the poured collagen aqueous solution is dried together with the mold (sheet drying step: step S204). Any method may be used as the drying method, but the following two drying methods are particularly preferable. First, when obtaining an acidic collagen sheet to which no buffer solution is added, the entire mold is dried by freeze drying (freeze drying). According to this drying method, a relatively low density and thick collagen sheet can be obtained in a relatively short time. The collagen sheet obtained by this drying method is particularly suitable for skin care and cosmetic applications. In addition, the collagen sheet by this technique has pH 4.5 to pH 6.0 suitable for human skin by using electrolysis-reducing alkaline ionized water having a pH of 10.5 to 12.5 as water used for dissolution. Shows weak acidity.

  When a neutral collagen sheet is obtained by adjusting the pH by adding a buffer solution, the collagen aqueous solution poured into the mold is heated to about 37 ° C. and air-dried for 1 to 12 hours to gel. Next, the gelled collagen is washed with distilled water to remove the buffer component. Then, this air drying and washing are repeated a plurality of times to sufficiently remove the buffer component, and then completely air dried under the same conditions. Thereby, a neutral (physiological condition) collagen sheet is obtained. By this drying method, a relatively dense and thin neutral collagen sheet (collagen film) can be obtained. And since the collagen sheet (collagen film) obtained by this drying method is neutral, thin and high density, it is particularly suitable for medical use.

  Next, the dried collagen sheet is deframed to obtain a collagen sheet (collagen film) having a predetermined shape and thickness (deframement step: step S206).

  Since the collagen sheet (collagen film) according to the present invention produced in this way is produced from pork ear collagen having high viscoelasticity as described above, collagen can be added without adding an artificial crosslinking agent component or the like. The shape can be maintained without tearing in the state of a single sheet. Needless to say, it is not necessary to attach and handle a holding body such as a holding film. Furthermore, the collagen sheet (collagen film) according to the present invention dissolves quickly in 30 seconds to 2 minutes with normal water such as various distilled water, lotion, alkaline reduction water, etc., as in the case of conventional collagen sheets. In addition, since the collagen sheet according to the present invention is composed only of collagen, it is dissolved by being stuck to a target position of the human body and containing water so that it is completely absorbed by the human body and disappears. At this time, no residue remains on the skin surface.

  Here, a collagen sheet (hereinafter referred to as a pig ear collagen sheet) having a diameter of about 2 cm prepared from pork ear collagen as described above and a collagen sheet (hereinafter referred to as pig) similarly prepared using pig skin collagen. It is a skin collagen sheet.) And 300 μL of distilled water are dropped on a normal filter paper not containing collagen for comparison, and dried under conditions of a humidity of 25% to 30% and a temperature of 23 ° C. to 25 ° C. The amount of water evaporation (g) was measured. The result is shown in FIG. 7 indicates the water evaporation amount of the pig ear collagen sheet, the triangle mark indicates the water evaporation amount of the porcine skin collagen sheet, and the circle mark indicates the water evaporation amount of the filter paper. It can be seen from FIG. 7 that the amount of water evaporation of the pig ear collagen sheet and the pig skin collagen sheet is less than the amount of water evaporation of the filter paper after 2 hours (120 min), and both have moisture retention. In addition, the amount of water evaporated between the pig ear collagen sheet and the pig skin collagen sheet is the same, and the pig ear collagen (pig ear collagen sheet) has inferior moisture retention compared to the collagen sheet using the existing pig skin. I understand that.

  As described above, the collagen according to the present invention has higher viscoelasticity than conventional collagen derived from skin. Thereby, the collagen sheet using the collagen according to the present invention can be processed into a sheet shape with collagen alone. For this reason, it is not necessary to add an artificial crosslinking agent component or a highly viscous substance that may cause harm to the human body, and a collagen sheet with 100% collagen component can be obtained. As a result, the collagen according to the present invention can be used with peace of mind by all persons regardless of the presence or absence of allergies. Moreover, since it has higher fiber-forming ability than conventional collagen, it has particularly excellent effects for skin care and medical use.

  Further, since the collagen according to the present invention does not contain an additive, it is completely absorbed by the human body and disappears without any residue remaining. Therefore, it is not necessary to wash the residue particularly when used for a medical sheet.

  And, the collagen according to the present invention may be used by directly applying to the skin or affected part of the human body as a paste, and it is blended in a cosmetic liquid, lotion, milky lotion, cream, gel, foundation, base cream, BB cream, etc. May be used. In addition, the collagen sheet according to the present invention is molded to an appropriate thickness, so that it can be used not only for cosmetics such as skin care sheets, but also for medical sheets such as bandages, oblates, periodontal ligaments, pressure ulcer sheets, corneas and tympanic membranes Is possible.

  And the manufacturing method of the collagen which concerns on this invention, and the manufacturing method of a collagen sheet can manufacture said high-viscoelasticity collagen and a collagen sheet from a pig ear.

  The collagen, collagen sheet, collagen production method and collagen sheet production method shown above are examples, and the production conditions, production techniques, steps, procedures, usage methods, etc. are not limited to the above examples. The present invention can be modified and implemented without departing from the scope of the present invention.

S100 Pig ear pretreatment process
S104 Degreasing process
S106 Extraction process
S110 Purification process
S200 dissolution process
S202 Molding process (sheeting process)
S204 Sheet drying process (sheet forming process)
S206 De-frame process (sheet process)

Claims (9)

Collagen that does not contain an artificial crosslinker component,
Viscosity of 1.0 Pa or more and 3.0 Pa when shear deformation is applied to an aqueous solution of collagen adjusted to a concentration of 0.25 wt%, pH of 2.0 to 3.5, and liquid temperature of 10 ° C. at a shear rate of 1 s ^−1. The storage elastic modulus after 30 minutes of heating the collagen aqueous solution adjusted to a concentration of 0.25 wt% and pH 6.8 to 7.4 to 37 ° C. is 60 Pa to 120 Pa. And collagen. A collagen containing both or one of type I collagen and type III collagen derived from pig ears, which does not contain an artificial crosslinking agent component,
Viscosity of 1.0 Pa or more and 3.0 Pa when shear deformation is applied to an aqueous solution of collagen adjusted to a concentration of 0.25 wt%, pH of 2.0 to 3.5, and liquid temperature of 10 ° C. at a shear rate of 1 s ^−1. The storage elastic modulus after 30 minutes of heating the collagen aqueous solution adjusted to a concentration of 0.25 wt% and pH 6.8 to 7.4 to 37 ° C. is 60 Pa to 120 Pa. And collagen. 3. The collagen according to claim 2, wherein pig ears having a age of 3 months or more and 7 months or less and a body weight of 50 kg to 130 kg are used. A collagen sheet of collagen according to any one of claims 1 to 3,
Contains no artificial crosslinker component,
Hold the shape without tearing in a single state without a holding body,
A collagen sheet that is absorbed and disappears after being attached to the human body. The collagen according to claim 4, wherein the collagen dissolves by contact with reducing alkaline ionized water having a pH of 10.5 to 12.5, and the solution exhibits weak acidity of pH 4.5 to pH 6.0. Sheet. After removing the epidermis of the pig ears to the dermis, cutting to a predetermined size,
Degreasing the cut pig ears with an organic degreasing solution;
Extracting collagen from a degreased pig ear with a solution containing an acid and a digestive enzyme;
A step of purifying the extracted collagen;
And a step of drying the purified collagen. The method for producing collagen according to claim 6, wherein pig ears having a age of 3 months to 7 months and a weight of 50 kg to 130 kg are used. After removing the epidermis of the pig ears to the dermis, cutting to a predetermined size,
Degreasing the cut pig ears with an organic degreasing solution;
Extracting collagen from a degreased pig ear with a solution containing an acid and a digestive enzyme;
A step of purifying the extracted collagen;
Dissolving purified collagen in a predetermined acidic solution;
A process of pouring the dissolved collagen into a predetermined form and drying to form a sheet;
A method for producing a collagen sheet, comprising: 9. The method for producing a collagen sheet according to claim 8, wherein pig ears having a age of 3 months or more and 7 months or less and a weight of 50 kg or more and 130 kg or less are used.

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