JP2004269478A - Method for production of collagen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a collagen production method by which collagen can efficiently be mass-produced from the skin of fishes. <P>SOLUTION: This method for producing collagen comprises the processes of: insolubilizing the epidermal layer of the fishes skin and a cutis layer portion of the epidermal layer side including pigmentary cells; swelling at least a part of the cutis layer which is not insolubilized; and recovering the swollen cutis layer. In the method, it is possible to include a filtering process and also a centrifuging process. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing collagen, and more particularly to a method for producing collagen that can efficiently produce collagen from fish skin in large quantities.
[0002]
[Prior art]
Collagen has been applied to biomaterials such as artificial skin because of its excellent applicability to the human body. Collagen is also used in cosmetics because it gives tension to the skin and keeps the skin moist.
[0003]
Collagen is manufactured industrially primarily by extraction from mammalian or fish skin. In particular, fish such as salmon and trout become industrial waste after being processed. Therefore, research on obtaining collagen from fish has been actively conducted in recent years in order to effectively utilize this industrial waste.
[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, fish skin from which scales, excess body, and the like have been removed is cut into squares each having a side of about 3 cm. Then, this is degreased and washed by immersing it in a solution such as chloroform or methanol, and then immersed in a buffer solution or the like to remove neutral proteins. Next, collagen is extracted by immersing the treated skin in acetic acid or the like, and further pepsin is added to the solution to perform atherosclerosis treatment of the collagen. After that, the solution is centrifuged to collect a supernatant. . The salting out and centrifugation of the supernatant are repeated several times to remove impurities such as pepsin, and then ultracentrifugation is performed by centrifugal force equivalent to 100,000 × G (gravity) to remove impurities such as pigment cells. Remove. Thereafter, the liquid after the above treatment is sterile-filtered, and the filtrate is dialyzed and lyophilized to obtain collagen.
[0005]
Here, for example, as shown in FIG. 3, the thickness of the subcutaneous layer 3 including the epidermis layer 1 and the dermis layer 2 is about 5 to 7 mm and the mammalian skin has an appropriate thickness. It is possible to easily remove hair roots and pigment cells 4 together with the epidermis layer 1 by a method such as cutting. However, fish skin has a layer 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. 4, and is considerably thinner than mammalian skin. It is very difficult to remove the pigment cells 14 after removing the scale 16. Therefore, in a conventional collagen production method using fish skin, collagen is extracted by immersion in acetic acid or the like while containing pigment cells 14. Therefore, in order to obtain high-quality, high-purity collagen, it was necessary to remove impurities such as pigment cells 14 by performing ultracentrifugation with 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 since the ultracentrifuge is expensive, the number of ultracentrifuges that can be mass-produced must be uniform. The production cost was very inefficient.
[0007]
In order to solve such a problem, a method of extracting natural collagen from pigment-free fish skin such as flat fish has been disclosed (for example, see Patent Document 1).
[0008]
[Patent Document 1]
Japanese Patent Publication No. 2722014/0009
[Patent Document 2]
Japanese Patent Application Laid-Open No. 2001-200000
[Patent Document 3]
Japanese Patent Application Laid-Open No. 9-278639
[Problems to be solved by the invention]
However, in the method according to Patent Literature 1, fish that can be used are limited to certain non-pigmented fish such as flat fish, and it is necessary to select 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 that can efficiently produce collagen from fish skin in large quantities.
[0013]
[Means for Solving the Problems]
The present invention is a step of insolubilizing the epidermis layer of fish skin and a portion of the dermis layer containing pigment cells on the epidermis layer side, and a step of swelling at least a part of the dermis layer that has not been insolubilized, Recovering the swollen dermis layer. Here, the method for producing collagen of the present invention can include a step of filtering. In addition, the method for producing collagen of the present invention can include a step of centrifugation.
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, an embodiment of the present invention will be described with reference to FIG.
[0015]
(Pretreatment step)
FIG. 1 shows a flowchart of an example of the method for producing collagen according to the present invention. As shown in FIG. 1, in the method for producing collagen according to the present invention, first, fish skin is prepared, and scales, fins, and unnecessary bodies below the dermis layer are removed from the prepared fish skin. Then, the skin of the fish is formed, and the portion not used for the production of collagen is removed.
[0016]
Here, fish generally refers to animals that breathe in gills, have fins, and live in water. 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, or different types of fish may be mixed and used, regardless of the type of fish used. You don't have to. Therefore, in the present invention, collagen can be efficiently produced in large quantities.
[0017]
Next, the fish skin after the above-mentioned shaping can be washed using 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 adhering 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 liquid and stirring the same. Note that the washing may be performed one or more times.
[0018]
Next, the skin layer of fish skin and the portion of the dermis layer containing pigment cells on the side of the skin layer are insolubilized. Here, the “insolubilization treatment” refers to denaturation of a protein constituting a part of a dermis layer including a pigment cell on the side of the epidermis layer and the epidermis layer of fish skin, thereby converting these parts to an organic acid or the like. It refers to a treatment for preventing these parts from swelling and / or dissolving even when immersed in an acid.
[0019]
Due to this insolubilization treatment, even when the dermis layer is swollen by immersing fish skin in an organic acid or the like as described later, the insolubilized dermis layer containing pigment cells does not swell. Therefore, the pigment cells contained in the dermis layer of the fish that has been insolubilized can be easily separated by collecting only the dermis layer which has increased in thickness by swelling.
[0020]
The insolubilization treatment can be performed, for example, by immersing a portion of the fish skin to be insolubilized in a salad powder aqueous solution, sodium hydroxide aqueous solution, calcium hydroxide aqueous solution, or the like. Above all, in the insolubilization treatment, the portion of the fish skin to be insolubilized is preferably immersed in an aqueous solution of ash powder for 10 to 60 minutes, more preferably for 15 to 45 minutes. In these cases, the insolubilization treatment of the epidermis layer and the like and the sterilization treatment of fish skin can be performed simultaneously and efficiently.
[0021]
It is considered that the protein on the skin portion of the fish immersed in the aqueous solution of the ground bean powder 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 layer thickness of the epidermis and dermis, which is only about 1-5 mm, can be about 5-15 mm. Therefore, the swollen portion of the dermis layer and the portion of the insolubilized dermis layer containing the pigment cells can be easily separated, so that purification such as ultracentrifugation to remove the pigment cells can be performed. The steps can be largely omitted. Therefore, in the present invention, collagen can be efficiently obtained in large quantities.
[0023]
Here, the swelling of the dermis layer is preferably such that the fish skin after the insolubilization treatment is immersed in an acid such as an organic acid (for example, acetic acid or citric acid) for 1 to 4 days, and immersed for 2 to 3 days. Is more preferred. In these cases, the dermis layer tends to be sufficiently swollen in a shorter time.
[0024]
The swelling of the dermis layer is preferably performed so that the thickness of the dermis layer after swelling is 110 to 130% of the thickness of the dermis layer before swelling, and is performed so as to be 115 to 125%. Is more preferable. In these cases, the swollen dermis layer is more easily collected, and therefore, there is a tendency that collagen can 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 cut off from the portion containing the pigment cells of the epidermis layer and the dermis layer and collected. As a method for this recovery, for example, a method in which a skin layer or the like hardened by the above insolubilization treatment is fixed and a blade or the like is used and physically cut off by hand, a method in which only the recovered portion of the dermis layer is squeezed, or a method in which the dermis layer is used There may be a method of sucking only the collected portion.
[0026]
In the present invention, as described above, the epidermis layer of fish skin and the portion of the dermis layer containing pigment cells on the epidermis layer side are insolubilized, and at least a part of the dermis layer that has not been insolubilized is swollen. Then, the swollen dermis layer is collected, and the portion of the dermis layer containing the insolubilized pigment cells is not collected. Therefore, since pigment cells cannot be contained in the collected dermis layer, even if the purification step is largely omitted for the collected dermis layer, the purity of the obtained collagen tends not to deteriorate.
[0027]
(Purification step)
Next, salting out of the recovered dermis layer may be performed. When salting out is performed, the purity of the collagen obtained by the present invention can be further increased. Here, the salting out is performed, for example, by immersing the collected dermis layer in an aqueous solution of an electrolyte such as sodium chloride or the like, and then collecting the precipitated salting out substance by stirring it. The salted-out product is collected, for example, using a filtration means. In addition, salting out may be performed one or more times.
[0028]
Then, the collected salted-out material is immersed in an alcohol such as isopropanol, for example, to extract lipids contained in the salted-out material, thereby performing defatting. The degreasing can be performed one or more times.
[0029]
(Drying process)
The collagen is obtained by drying the defatted salted-out product. Drying can be performed, for example, by a method such as drying under reduced pressure, 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 compounding agents 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 of fish skin.
[0031]
Further, collagen can be extracted after the dermis layer swollen in the above production method is collected. The collagen is extracted by, for example, immersing the recovered swelled dermis layer in an acid solution and stirring the solution.
[0032]
Moreover, in the above-mentioned production method, atherosclerosis of collagen can also be performed. Atherosclerosis of collagen is performed, for example, by dissolving the collected dermis layer, adding pepsin or the like to the dispersed acid solution, and stirring. In addition, the atherolation can be performed after the extraction of collagen or simultaneously with the extraction of collagen.
[0033]
After the atherosclerosis of collagen, for example, the extract of the atherosclerotic collagen is filtered using a pre-filter to remove extraction residues, and further filtered using an ultrafiltration filter to extract the collagen. Can be concentrated. Next, the concentrated extract of the collagen is filtered using a sterile filter, and the obtained supernatant is dialyzed against deionized water and then freeze-dried.
[0034]
In addition, the above-described production method may include a step of centrifuging as appropriate. In this case, since the collagen and the like that cannot be completely solubilized contained in the acid solution can be removed, the purity of the collagen obtained by the present invention can be further increased. In addition, centrifugation may be performed once or more.
[0035]
Further, in the above-described production method, a step of appropriately filtering may be included. 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. In addition, filtration may be performed once or more.
[0036]
【Example】
Hereinafter, the present invention will be described using examples, but the present invention is not limited thereto.
[0037]
(Example 1)
First, salmon skin was prepared, and scales, fins and unnecessary body were removed from the salmon skin. The mass of the salmon skin after this treatment was 3270 g in a state containing water. Next, unnecessary portions of the salmon skin were removed to form salmon skin. At this time, the mass of the salmon skin was 2500 g in a state containing water. Thereafter, the salmon skin was sufficiently washed with purified water to remove scales and the like adhering 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% aqueous solution of salami powder for 20 minutes to insolubilize the epidermal layer of the salmon skin and the dermis layer containing pigment cells on the epidermal layer side. Processed.
[0039]
Next, the salmon skin was immersed in 25 liters of a 0.075 M citric acid aqueous 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, an aqueous sodium chloride solution in which 367 g of sodium chloride was dissolved in 2 liters of purified water was gradually added to the collected dermis layer. Then, the total amount of the added aqueous sodium chloride solution became 9 liters. Thereafter, salting out was performed by stirring for 24 hours. Next, the salted out product was recovered using a filtration means.
[0041]
Next, 15 liters of isopropanol was added to the collected salted out product, and the mixture was stirred for 24 hours to extract lipid. Thereafter, the suspension after the treatment was filtered to collect all the residues. 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)
The collected dermis layer after swelling was further immersed in 54 liters of a 0.5 M acetic acid aqueous solution for 2 days, further added with 2.6 g of pepsin to the collagen extract, and left for 24 hours to perform atherogenization. Then, 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]
Next, the collagen extract after concentration was filtered using a sterile 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 collected.
[0044]
(Comparative Example 1)
First, as in Example 1, unnecessary parts such as scales, fins, and body were removed to prepare a salmon skin whose shape was adjusted. The mass of the salmon skin was 2500 g with water. Next, the salmon skin was chopped into a square having a side of about 3 cm. The shredded salmon skin was immersed in 24 liters of a mixture of chloroform and methanol at a volume ratio of 1: 1 and stirred for 24 hours to extract lipids. Then, the defatted salmon skin 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 immersed for 24 hours in 24 liters of 2-amino-2-hydroxymethyl-1,3-propanediol-hydrochloric acid buffer solution containing 20% of sodium chloride and having a pH of 7.5 for 24 hours. The protein was removed. Then, it was sufficiently 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, further added with 2.6 g of pepsin, and left to stand for 24 hours to perform atherolation. Then, the collagen extract was filtered using an ultrafiltration filter to remove the extraction residue containing pigment cells, and the collagen extract was concentrated by filtering using an ultrafiltration filter. Next, the concentrated extract of collagen 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 the description of the production methods of Examples 1 and 2 and Comparative Example 1, “M” indicates a molar concentration (moles of solute (mol) / volume of solvent (L)).
[0047]
As described above, in the methods of Examples 1 and 2, collagen was obtained in a higher yield than in the method of Comparative Example 1. This is because in the methods of Examples 1 and 2, almost all of the pigment cells could be removed before the dermis layer of the salmon skin was recovered, so that an ultrafiltration filter was used as compared with the method of Comparative Example 1. It is considered that the step of removing the pigment cells by filtration was omitted.
[0048]
The embodiments and examples disclosed this time are to be considered in all respects as illustrative and not restrictive. 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, since pigment cells can be almost completely removed in the pretreatment step of fish skin, the production step can be largely omitted. Therefore, according to the present invention, collagen can be efficiently produced in large quantities 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 epidermis layer of fish skin and a portion of the dermis layer containing the pigment cells on the epidermis layer side, a step of swelling at least a part of the dermis layer that has not been insolubilized, Collecting a layer. 2. The method for producing collagen according to claim 1, comprising a step of filtering. The method for producing collagen according to claim 1, comprising a step of centrifuging.

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