JP4204883B2 - Method for producing collagen - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing collagen, and more particularly to a method for producing collagen, which can efficiently produce large amounts of collagen from fish skin.
[0002]
[Prior art]
Collagen is applied to biomaterials such as artificial skin because of its excellent applicability to the human body. Collagen is also added to cosmetics because it gives skin tension and keeps the skin moist.
[0003]
Collagen is industrially produced mainly by extraction from mammalian or fish skin. In particular, fish such as salmon and trout become industrial waste after such processing. Therefore, in recent years, in order to effectively use this industrial waste, research for obtaining collagen from fish has been actively conducted.
[0004]
FIG. 2 shows a flowchart of an example of a conventional method for producing collagen using fish skin. In the conventional method for producing collagen using fish skin, first, as shown in FIG. 2, the fish skin from which scales, excess body and the like are removed is shredded into a square shape having a side of about 3 cm. And after degreasing and washing | cleaning by immersing this in solutions, such as chloroform and methanol, it immerses in a buffer solution etc. and removes neutral protein. Next, collagen is extracted by immersing the treated skin in acetic acid or the like, and further, pepsin is added to the solution to subject the collagen to an atherogenic treatment, and then the solution is centrifuged to collect the supernatant. . The supernatant is salted out and centrifuged several times to remove impurities such as pepsin and then ultracentrifugated with a centrifugal force equivalent to 100,000 × G (gravity) to remove impurities such as pigment cells. Remove. Thereafter, the treated liquid is sterile filtered, and the filtrate is dialyzed and freeze-dried to obtain collagen.
[0005]
Here, since the skin of mammals has a moderate thickness on the subcutaneous layer 3 that combines the epidermis layer 1 and the dermis layer 2 as shown in FIG. It is possible to easily remove the hair roots and pigment cells 4 together with the epidermal layer 1 by a method such as cutting. However, the fish skin has a thickness of about 1 to 5 mm on the subcutaneous layer 13 including the epidermis layer 11 and the dermis layer 12 as shown in FIG. It is very difficult to remove the pigment cells 14 after removing the scales 16. Therefore, in the conventional method for producing collagen using fish skin, collagen is extracted by immersing it in acetic acid or the like while containing the pigment cells 14. Accordingly, in order to obtain high-quality collagen with high purity, it was necessary to remove impurities such as pigment cells 14 by performing ultracentrifugation with a centrifugal force equivalent to 100,000 × G (gravity) as described above. .
[0006]
However, the amount of collagen that can be processed by one ultracentrifugation is as small as about 400 cc, and the ultracentrifugation device is expensive. The production cost was very inefficient.
[0007]
In order to solve such a problem, a method for extracting natural collagen from the skin of a pigmentless fish such as flat fish has been disclosed (for example, see Patent Document 1).
[0008]
[Patent Document 1]
Japanese Patent No. 2722014 [0009]
[Patent Document 2]
Japanese Patent Laid-Open No. 2001-200000
[Patent Document 3]
Japanese Patent Laid-Open No. 9-278639
[Problems to be solved by the invention]
However, in the method according to Patent Document 1, the fish that can be used is limited to certain non-pigmented fish such as flat fish, and it is necessary to sort out the non-pigmented fish and the like, so that collagen can be efficiently used. It could not be manufactured in large quantities.
[0012]
In view of the above circumstances, an object of the present invention is to provide a method for producing collagen, which can efficiently produce a large amount of collagen from fish skin.
[0013]
[Means for Solving the Problems]
The present invention comprises a step of insolubilizing the skin layer of fish skin and a portion of the dermis layer containing pigment cells on the epidermis layer side, a step of swelling at least a portion of the dermis layer that has not been insolubilized, And a step of recovering a swollen dermis layer. Here, the manufacturing method of the collagen of this invention can include the process of filtering. Moreover, the manufacturing method of the collagen of this invention can include the process of centrifuging.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The embodiment of the present invention will be described below with reference to FIG.
[0015]
(Pretreatment process)
FIG. 1 shows a flowchart of an example of a method for producing collagen according to the present invention. As shown in FIG. 1, the method for producing collagen according to the present invention first prepares fish skin, and removes unnecessary bodies and the like under the scales, folds and dermis layer from the prepared fish skin. Then, after that, fish skin is molded to remove portions not used for collagen production.
[0016]
Here, fish refers to animals that generally breathe with a shark and have a shark and live underwater. Examples of fish that can be used in the present invention include salmon, trout, and cod. Here, as the fish used in the present invention, only the same type of fish may be used, and different types of fish can be mixed and used. There is no need to do. Therefore, in the present invention, collagen can be efficiently produced in large quantities.
[0017]
Next, the fish skin after the molding can be washed with a washing solution such as purified water or an organic solvent (for example, chloroform, methanol or a mixture thereof). When the fish skin is washed, scales, lipids and the like attached to the fish skin surface can be removed. The washing can be performed, for example, by immersing the fish skin after the treatment in the washing solution and then stirring it. The washing can be performed once or more.
[0018]
Next, insolubilization treatment is performed on the skin layer of the skin of fish and the portion of the dermis layer containing pigment cells on the skin layer side. Here, the “insolubilization treatment” means that the protein constituting the epidermis layer of the skin of fish and the dermis layer containing the pigment cells on the epidermis layer side is denatured, etc. It refers to a treatment that prevents these parts from swelling and / or dissolving even when immersed in an acid.
[0019]
Even when the skin of fish is immersed in an organic acid or the like to swell the dermis layer as described later, the insolubilized portion of the dermis layer containing pigment cells does not swell. Therefore, by collecting only the dermis layer that has been swollen and increased in thickness, pigment cells contained in the dermis layer of fish that have been insolubilized can be easily separated.
[0020]
The insolubilization treatment can be performed, for example, by immersing a portion of fish skin to be insolubilized in a salty powder aqueous solution, a sodium hydroxide aqueous solution, a calcium hydroxide aqueous solution or the like. Especially, insolubilization treatment, it is preferable to immerse the part to be insolubilized in fish skin for 10 to 60 minutes, and more preferably for 15 to 45 minutes. In these cases, the insolubilization treatment of the skin layer and the like and the sterilization treatment of fish skin can be efficiently performed simultaneously.
[0021]
It is thought that the protein of the fish skin part immersed in the salty-powder aqueous solution can be denatured, for example.
[0022]
Next, at least a part of the dermis layer that has not been insolubilized is swelled. By swelling the dermis layer, the combined thickness of the epidermis layer and the dermis layer, which is only about 1-5 mm, can be about 5-15 mm. Therefore, since the swollen dermis layer part and the insolubilized dermis layer part containing the pigment cells can be easily separated, purification such as ultracentrifugation to remove the pigment cells is possible. The process can be omitted significantly. Therefore, in the present invention, a large amount of collagen can be obtained efficiently.
[0023]
Here, the swelling of the dermis layer is preferably performed by immersing the fish skin after the insolubilization treatment in an acid such as an organic acid (for example, acetic acid or citric acid) for 1 to 4 days, and for 2 to 3 days. Is more preferable. In these cases, the dermis layer tends to swell sufficiently in a shorter time.
[0024]
The swelling of the dermis layer is preferably performed such that the thickness of the dermis layer after swelling is 110 to 130% of the thickness of the dermis layer before swelling, and is 115 to 125%. It is more preferable. In these cases, since it becomes easier to collect the swollen dermis layer, collagen tends to be efficiently produced in large quantities. Here, the “thickness of the dermis layer” refers to the thickness of the dermis layer in a portion that has not been insolubilized.
[0025]
Thereafter, the swollen dermis layer is separated and recovered from the epidermis layer and the portion of the dermis layer containing pigment cells. As this recovery method, for example, the skin layer cured by the above insolubilization process is fixed and a blade is used, and a method of physically cutting by a human hand, a method of squeezing only a recovery part of the dermis layer, or a dermis layer There may be a method of sucking only the collection portion.
[0026]
In the present invention, as described above, the skin layer of fish skin and the part of the dermis layer containing pigment cells on the skin layer side are insolubilized, and at least a part of the dermis layer that has not been insolubilized is swollen. Thus, the swollen dermis layer is recovered, and the part of the dermis layer containing the insolubilized pigment cells is not recovered. Therefore, pigment cells cannot be contained in the collected dermis layer, and therefore the purity of the obtained collagen tends not to deteriorate even if the purification step is largely omitted for the collected dermis layer.
[0027]
(Purification process)
Next, the recovered dermis layer can be salted out. When salting out is performed, the purity of the collagen obtained by the present invention can be further increased. Here, salting out is performed, for example, by immersing the recovered dermis layer in an aqueous electrolyte solution such as sodium chloride, and then recovering the precipitated salting out by stirring it. The salted-out matter is collected using, for example, a filtering means. The salting out can be performed once or more.
[0028]
The recovered salted-out product is degreased by, for example, immersing the salted-out product in an alcohol such as isopropanol to extract the lipid contained in the salted-out product. In addition, degreasing can be performed once or more.
[0029]
(Drying process)
Collagen is obtained by drying the defatted salted-out product. Drying may be performed by a method such as vacuum drying, freeze drying, or spray drying. The collagen obtained by the present invention is suitably used as a raw material for biomaterials such as artificial skin, cosmetic ingredients or health food additives.
[0030]
(Other)
In the above production method, degreasing is performed after salting out, but it can also be performed after shaping fish skin.
[0031]
Moreover, collagen can also be extracted after collection | recovery of the dermis layer swollen in the said manufacturing method. Collagen extraction is performed, for example, by immersing the recovered swollen dermis layer in an acid solution and stirring the solution.
[0032]
In the above production method, collagen can be atelolated. The collagen is atelolated by, for example, dissolving the recovered dermis layer and then adding pepsin or the like to the dispersed acid solution and stirring. In addition, atelolation can be performed after collagen extraction or simultaneously with collagen extraction.
[0033]
After the collagen is atelolated, for example, the extract of the collagen is removed by filtering the extracted solution of atelocollagen using a pre-filter and further filtering using an ultrafiltration filter. Can be concentrated. Next, the collagen extract concentrated using a sterilizing filter is filtered, and the resulting supernatant is dialyzed against deionized water and then freeze-dried or the like.
[0034]
Moreover, in the said manufacturing method, the process of centrifuging can be included suitably. In this case, collagen and the like that are not completely solubilized contained in the acid solution can be removed, so that the purity of the collagen obtained by the present invention can be further increased. In addition, centrifugation can be performed once or more.
[0035]
Moreover, in the said manufacturing method, the process of filtering can be included suitably. Filtration is preferably performed after salting out. Also in this case, the purity of the collagen obtained by the present invention can be further increased. Filtration can be performed using, for example, a prefilter or an ultrafiltration filter. The filtration can be performed once or more.
[0036]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated using an Example, this invention is not limited to this.
[0037]
Example 1
First, salmon skin was prepared, and scales, straw and unnecessary bodies were removed from the salmon skin. The mass of the salmon skin after this treatment was 3270 g in a state containing moisture. Next, unnecessary portions of the salmon skin were removed to form the salmon skin. The mass of the salmon skin at this time was 2500 g in a state containing moisture. Thereafter, the salmon skin was sufficiently washed with purified water to remove scales and the like attached to the surface of the salmon skin.
[0038]
Then, the surface side of the salmon skin is immersed in 12.5 liters of a 1% concentration of salicy powder aqueous solution for 20 minutes to insolubilize the skin layer of the salmon skin and the dermis layer containing pigment cells on the side of the skin layer. Processed.
[0039]
Next, this salmon skin was immersed in 25 liters of a 0.075 M aqueous citric acid solution for 2 days to swell the dermis layer of the salmon skin that had not been insolubilized. The thickness of the dermis layer after swelling was 125% of the thickness of the dermis layer before swelling. Then, the swollen dermis layer was squeezed out and 7 liters were collected.
[0040]
Next, a sodium chloride aqueous solution in which 367 g of sodium chloride was dissolved in 2 liters of purified water was gradually added to the collected dermis layer. And the total amount of the added sodium chloride aqueous solution became 9 liters. Then, salting out was performed by stirring for 24 hours. Next, the salted-out product was recovered using a filtering means.
[0041]
Next, 15 liters of isopropanol was added to the recovered salted-out product, and the mixture was stirred for 24 hours to extract lipids. Then, all the residues were collected by filtering the suspension after the treatment. The residue was dried under reduced pressure to remove isopropanol to obtain collagen. The mass of the obtained collagen was 256 g in a dry state.
[0042]
(Example 2)
2.6 g of pepsin was further added to a collagen extract obtained by immersing the recovered dermis layer after swelling in 54 liters of a 0.5 M aqueous acetic acid solution for 2 days, and the mixture was allowed to stand for 24 hours to effect atherogenesis. Thereafter, the extraction residue was removed from the collagen extract by filtration using a prefilter, and the collagen extract was concentrated by filtration using an ultrafiltration filter.
[0043]
Subsequently, the concentrated collagen extract was filtered using a sterilizing filter, and the supernatant was dialyzed against deionized water for 5 days and then freeze-dried to obtain collagen. The mass of the obtained collagen was 182 g in a dry state. In Example 2, the same operation as in Example 1 was performed before the swollen dermis layer was recovered.
[0044]
(Comparative Example 1)
First, the salmon skin which prepared the shape by removing unnecessary parts, such as a scale, a salmon, and a body like Example 1, was prepared. The mass of this salmon skin was 2500 g with moisture. Next, the salmon skin was cut into a square shape with a side of about 3 cm. The salmon skin after shredding was immersed in 24 liters of a mixture of chloroform and methanol in a volume ratio of 1: 1, and lipid extraction was performed by stirring for 24 hours. The salmon skin after degreasing was immersed in 24 liters of methanol and washed by stirring for 24 hours. Thereafter, the salmon skin was thoroughly washed with deionized water. Then, the washed salmon skin is neutralized by immersing it in 24 liters of 2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloric acid buffer solution of pH 7.5 containing 20% sodium chloride for 24 hours. Protein was removed. Thereafter, it was thoroughly washed with deionized water.
[0045]
Next, the salmon skin after the above treatment was immersed in 25 liters of a 0.5 M acetic acid aqueous solution for 2 days to extract collagen, and 2.6 g of pepsin was further added and allowed to stand for 24 hours for atherogenesis. Then, the collagen extract was filtered using an ultrafiltration filter to remove an extraction residue containing pigment cells, and further filtered using an ultrafiltration filter to concentrate the collagen extract. Next, the concentrated collagen extract was filtered using a sterile filter, and the supernatant was dialyzed against deionized water for 5 days. Then, collagen was obtained by freeze-drying. The mass of the obtained collagen was 127 g in a dry state.
[0046]
In addition, in description of the manufacturing method of the said Examples 1-2 and the comparative example 1, "M" shows molar concentration (the number of moles of solute (mol) / volume of a solvent (L)).
[0047]
As described above, in the methods of Examples 1 and 2, collagen was able to be obtained in a high yield as compared with the method of Comparative Example 1. This is because the method of Examples 1 and 2 was able to remove most of the pigment cells before recovering the dermis layer of salmon skin, and therefore used an ultrafiltration filter compared to the method of Comparative Example 1. This is probably because the process of removing the pigment cells by filtration was omitted.
[0048]
It should be understood that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.
[0049]
【The invention's effect】
As described above, in the present invention, most of the pigment cells can be removed in the pretreatment process of fish skin, so that the manufacturing process can be largely omitted. Therefore, according to the present invention, a large amount of collagen can be efficiently produced from fish skin.
[Brief description of the drawings]
FIG. 1 is a flowchart of an example of a method for producing collagen according to the present invention.
FIG. 2 is a flowchart of an example of a conventional method for producing collagen using fish skin.
FIG. 3 is a schematic enlarged cross-sectional view of mammalian skin.
FIG. 4 is a schematic enlarged sectional view of fish skin.
[Explanation of symbols]
1,11 epidermis layer, 2,12 dermis layer, 3,13 subcutaneous layer, 4,14 pigment cells, 5 hairs, 16 scales.

Claims (3)

A step of insolubilizing the skin layer of fish skin and a portion of the dermis layer containing pigment cells on the epidermis layer side, a step of swelling at least a part of the dermis layer that has not been insolubilized, and a swollen dermis And a step of recovering the layer. The method for producing collagen according to claim 1, further comprising a step of filtering. The method for producing collagen according to claim 1, further comprising a step of centrifuging.

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