JP2001200000A - Method for producing collagen derived from marine organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable the removal of finely divided particles such as coloring matter, the concentration of collagen and the removal of added pepsin or the like and to realize mass production in an inexpensive equipment with pollution of outside excluded by employing and adding new purification and concentration processes because it is clifficult to put the present method for producing collagen from salmon, etc., into practical use due to the thinness of salmon skin leading to the impossibility of physically removing impurity-containing epidermis alone and therefore to both of the impossibility of mass production and the expensiveness of equipment because of finally performing the removal of impurities by an ultracentrifuge. SOLUTION: It is the same way as a conventional one that a preprocessing process for degreasing and cleansing skins of marine organisms is followed by an extracting process for extracting collagen with an organic acid and also making it atheromatous using pepsin, etc. Then, a purified collagen solution is obtained by a purifying process for removing impurities from the crude collagen solution obtained there and for making the solution pass through a purification membrane. The collagen solution is applied with a concentrating process for making it pass through a concentration membrane and is finally applied with a conventional filtration process for sterilization to obtain a product of soluble collagen in enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for mass-producing acid-soluble collagen useful for various medical biomaterials, cosmetic materials, food materials, and the like, using the skin of marine organisms such as salmon as a raw material.

[0002]

2. Description of the Related Art As is known, the above collagen is mainly extracted from the skin of mammals such as cows and pigs as a raw material. In recent years, not only the above mammals but also the skin of marine organisms that become industrial waste after processing. Studies have been conducted to extract and produce collagen from corn. First, in the former method of producing collagen using mammals, as shown in FIG. 6, the skin layer is composed of an epidermis layer a, a dermis layer b, and a subcutaneous layer c, and the length from the epidermis layer a to the dermis layer b is about 5 to 7 mm. Collagen is contained in the dermis layer b, and impurities such as pigment are contained in the epidermal layer a, respectively.

[0003] For this reason, in mammals, as a pretreatment step, relatively thick skin is preceded by the next extraction step.
Since the epidermis layer a containing the pigment and the like is physically removed by means such as separation, it is possible to obtain collagen with little contamination of the pigment and the like as compared with marine organisms described below. . Then, the collagen solution obtained in the above-mentioned extraction step is subjected to a centrifugal separator, and after removing impurities, a collagen is produced through a sterile filtration step. In this case, the centrifugal separator is also large. As a result, a large amount of collagen can be purified at a time, and its mass production is possible.

However, as compared with the above-mentioned mammals, the means for producing collagen from marine organisms is still in the course of research, so that the following methods are at a stage of trial on a small scale. First, the skin layers of marine organisms such as salmon are formed of three layers as in FIG.
The thickness between the epidermis layer a and the dermis layer b excluding the subcutaneous layer c is about 1 to 2 mm, which is extremely thin compared to mammals, and collagen is of course contained in the dermis layer b. In addition, the pigment cells d are present in the upper layer of the dermis layer b, and the amount of the impurities is considerably larger than that of mammals.

Therefore, in the case of marine organisms, as described above, since the skin layer is thin, it is practically impossible to remove only a portion containing a pigment or the like, such as a mammal.
In the pre-treatment step, salmon skin is obtained to the extent that scale e, body, and fins in FIG. 7 are removed, and as shown in FIG. 8, lipids are extracted and washed, as shown in FIG. The treated sample obtained by the above is treated with an organic acid as is known to extract collagen, and at the same time, pepsin or the like is added to the treated sample, or the extract is subjected to centrifugation after the extraction treatment as shown in the above figure. The second extraction step is performed by selecting one of the methods to be performed. As a result, impurities such as pigments are naturally present in the solution after collagen extraction in the form of fine particles in large numbers.Moreover, since these fine particles are very light in weight, ordinary centrifugation in the above-mentioned extraction step is performed. The separation treatment does not precipitate and cannot be sufficiently removed.

[0006] Therefore, the crude collagen obtained in the above extraction step is subjected to salting out and centrifugation in the next third purification step, whereby the precipitate obtained by salting out is recovered by centrifugation. Pepsin is removed and impurities are removed, and acetic acid is added thereto to obtain an acid-soluble collagen, which is further subjected to an ultracentrifugal separator to collect the supernatant. Then, as shown in FIG. 8 described above, the process proceeds to the final fourth sterilization filtration step. Here, the concentrated solution obtained by the recovered supernatant in the previous process is sterilized and filtered through a filtration sterilization membrane, and the permeated solution is deionized. Either dialysis with water or sterilization filtration is performed before this dialysis, and then lyophilization is performed to obtain enzyme-soluble collagen.

[0007]

According to the above-described method for producing marine collagen derived from marine organisms, salting out and centrifugation are performed in the third purification step, but the centrifugation is repeated many times. Then, light fine particles as impurities which existed in large numbers could not be completely removed even after a considerable time, and thereafter, equivalent to ultracentrifugation (100,000 × G).
The microparticles must be settled and removed by applying centrifugal force. However, as an ultracentrifugal separator, it is not possible to obtain a material that can be purified in large quantities at one time, and the amount of collagen that can be purified at one time is about 400 cc. Because it is very expensive, it is considered irrational in terms of capital investment.
In view of the difficulty in removing impurities such as pigments from fish skin as a solution to this problem, in the production of the collagen, collagen is extracted from fish skin of colorless pigments, particularly from flat fish skin. (Japanese Patent No. 2722014) has also been proposed.

The present invention has been studied in view of the above-mentioned disadvantages of the prior art, and the first and second pretreatment steps according to claim 1 are different from the method for producing collagen according to FIG. There is no essential difference between the extraction step and the final sterilization filtration step. However, instead of performing centrifugation, salting out and ultracentrifugation as in the third purification step, a purification step by purification filtration treatment is performed. And by setting the concentration step by the subsequent concentration treatment, it is possible to remove fine particles such as dyes without using a conventional ultracentrifugal separator by the former purification and filtration treatment, in the latter concentration step Attempts are being made to concentrate collagen and to remove impurities such as pepsin, which have conventionally been removed by centrifugation.
According to the first aspect of the present invention, the purification and filtration treatment in the above-mentioned purification step makes it possible to continuously remove impurities such as pigments from the crude collagen solution after the extraction step without using expensive equipment, and the collagen according to the required production scale The first object is to realize a large-scale purification of. Moreover, the production schedule can be significantly shortened compared to the conventional production method described above,
The second purpose is to make it possible to adopt a closed production line unlike centrifugal separation, and to make it possible to manufacture with confidence from the aspect of sanitation without worrying about external contamination. From the skin of marine organisms, which are regarded as waste, we are trying to make collagen, which is desired to be provided at low cost, available to medical, cosmetic, and food industries.

In a second aspect, the present invention relates to a purification membrane for removing unnecessary substances from the crude collagen solution from the extraction step in the purification step in the above-mentioned claim 1, wherein the membrane size is less than 1.0 μm. The first object of the present invention is to make it possible to exhibit a more desirable purification effect by further reducing the collagen recovery rate and the pigment fine particle removal efficiency by setting the thickness to 0.1 μm. is there.
According to a second aspect of the present invention, there is provided a concentration membrane for removing impurities having a molecular weight fraction or less of a purified collagen solution from the purification and filtration step in the concentration step of the first aspect of the present invention. It is a second object of the present invention to achieve a more desirable concentration effect by making the ultrafiltration membrane of (1) compatible with a reduction in the removal rate of impurities such as pepsin and a reduction in the recovery rate of collagen.

In the case of claim 3, the purifying membrane has a size of 0.2 μm in order to improve the reliability of the purifying effect and the concentrating effect. ~ 0.6 μm, membrane size of concentration membrane is 10K
It specifies that it is more desirable to specify ~ 100K.

According to a fourth aspect of the present invention, in the first to third aspects, the crude collagen solution, the purified collagen solution, and the concentrated collagen solution are provided with respect to the purified membrane and the concentration membrane, respectively. Permeate perpendicular to the flow direction, eliminate the deposition of impurities on the membrane surface to prevent clogging, and further improve the purity of the finally obtained enzyme-soluble collagen. And

Next, in claim 5 or later, while the above-mentioned one is in the extraction step, the extraction and the atherolation are simultaneously advanced to increase the amount of extracted collagen, 8 in that crude impurities were removed from the crude collagen solution obtained and passed through a purification membrane to carry out purification and filtration, thereby removing impurities and performing atherosylation. Instead of centrifugation as in the conventional example, a purification and filtration treatment is adopted here to make it easier to remove impurities in a subsequent purification step.

According to a sixth aspect of the present invention, as in the second aspect of the first aspect, the extraction step and the purification step of the fifth aspect have a membrane size of less than 1.0 μm to 0.1 μm, and a concentration step. In the film size is less than 1000K10
A membrane for concentration of K is adopted to promote the purification effect and the concentration effect, respectively. In claim 7, 0.2 μm to 0.6 μm for each membrane size as in claim 3 described above,
By limiting the selection range to 10K to 100K, more desirable results of the above-mentioned purification effect and concentration effect are intended to be obtained. In the case of claim 8,
In contrast to Claims 5 to 7, a crude collagen solution, first and second purified collagen solutions, and a concentrated collagen solution are provided in a purification membrane in an extraction step or a purification step, and further in a concentration membrane in a concentration step. An attempt is made to improve the impurity removal efficiency by transmitting light perpendicular to the flow direction.

[0014]

According to the present invention, in order to achieve the above object, there is provided a pretreatment step for obtaining a treated sample by degreased and then washed marine skin.
An extraction step of extracting a crude collagen solution by adding an organic acid to the treated sample to extract collagen and adding a proteolytic enzyme to perform atherolation, and purifying by removing insolubles from the crude collagen solution A purification step of passing through a membrane, and a concentration step of concentrating the purified collagen solution obtained by passing the purified collagen solution through a concentration membrane to remove impurities having a molecular weight fraction or less. The concentrated collagen solution is characterized by comprising a sterilization filtration step of recovering a sample after sterilization filtration by preceding any of the sterilization filtration treatment of passing through a filtration sterilization membrane by dialysis treatment and pressurization with sterilization gas. It is an object of the present invention to provide a method for producing marine organism-derived collagen.

According to a second aspect, in addition to the contents of the first aspect, an organic acid is added to the treated sample obtained in the pretreatment step to extract collagen,
An extraction step of extracting a crude collagen solution by adding at least a protease and performing atherolation, and removing an insoluble substance from the crude collagen solution to reduce the membrane size to less than 1.0 μm.
A purification step of passing through a purification membrane using a microfiltration membrane of 0.1 μm, and concentration by passing the purified collagen solution obtained through a purification membrane having a membrane size of less than 1000K to 10K as an ultrafiltration membrane. Then, any of a concentration step for removing impurities having a molecular weight fraction or less, and a sterilization filtration treatment in which the concentrated collagen solution thus obtained is passed through a filtration sterilization membrane by pressurizing with a dialysis treatment and a sterilization gas. By preceding the method, the contents of the method include a sterilizing filtration step of collecting a sample after the sterilizing filtration.

Next, in claim 3, according to claim 1 or claim 2, the purification membrane in the purification step and the concentration membrane in the concentration step are each 0.2 μm to 0.65 μm.
m, and a film size of 10K to 100K.

According to a fourth aspect of the present invention, in the first to third aspects, a crude collagen solution, a purified collagen solution, and a concentrated collagen solution are respectively provided in the purification membrane in the purification step and the concentration membrane in the concentration step. That is, the light is transmitted in a direction orthogonal to the flow direction.

Next, according to the fifth aspect, only the extraction step is different from the first to fourth aspects, and collagen is extracted by adding an organic acid to the treated sample obtained in the pretreatment step. Insoluble matter is removed from the obtained crude collagen solution, and the crude collagen solution is purified by filtration through a purification membrane, and then a proteolytic enzyme is added to perform atherolation.

According to a sixth aspect of the present invention, in the extraction step, the film size is less than 1.0 μm.
Purification and filtration through a purification membrane with a microfiltration membrane of 0.1 μm and a membrane size of less than 1000K to 10
The content added is such that it is concentrated by passing through a concentration membrane which is an ultrafiltration membrane of K.

In claim 7, the range of both membrane sizes in claim 6 is further reduced to 0.2 μm to 0.65 μm and 10 K to 1 μm respectively for the purified membrane and the filter sterilized membrane.
00K.

According to the present invention, the crude membrane in the extraction step permeates the crude collagen solution in a direction parallel to the flow direction, and the purified membrane in the extraction step is subjected to purification. The crude collagen solution, the first and second purified collagen solutions, and the concentrated collagen solution pass through the purification membrane in the step and the concentration membrane in the concentration step, respectively, in a direction perpendicular to the flow direction. That is what it does.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Claims 1 to 4 of the present invention
The method for producing collagen according to the present invention will be described below with reference to FIGS. 1, 2, 4, and 5. In claim 1, a first pretreatment step, a second extraction step, a third purification step, and a 4 and a fifth sterilization filtration step. In the first pretreatment step, as in the previous practice, fish skin such as salmon skin is cut into approximately 3 cm squares under running water by removing unnecessary parts such as body as described above, for example, mixing chloroform and methanol. The minced fish skin is put into the solution and stirred, the solvent is exchanged, and the mixture is repeatedly stirred, then replaced with methanol, replaced with deionized water, washed, and further, a buffer solution containing 20% NaCl, etc. Washing with deionized water and washing with deionized water are repeated by exchanging the washing solution to remove the neutral protein, thereby obtaining the treated sample shown in FIG.

The contents of the second extraction step are the same as those of the extraction tank 1 in the manufacturing apparatus shown in FIG.
Athelation is carried out by adding 0.5M acetic acid and 1% of pepsin or other proteolytic enzyme to the weight of the substrate relative to a and 1b and stirring the mixture as is known. If a crude collagen solution is obtained by performing atherosimilation simultaneously with the extraction of collagen by the organic acid as described above, there is an advantage that the amount of extracted collagen is increased, but at the same time, impurities such as pigments are also large. It is also known that they will be mixed into the solution.

The thus obtained crude collagen S1
After removing insolubles using a nylon mesh or the like, the pumps 2a and 2b in FIG. 2 are operated to transfer the sample to the sample tank 3, where a new third purification step is performed. That is, after the crude collagen S1 in the sample tank 3 is passed through the purification membrane 4 while controlling the flow rate using the pumps 3a and 3b, the purified collagen solution S2 obtained here is transferred to the purification / concentration tank 5. . In the illustrated example, the residue and the like that have been blocked by the purification membrane 4 return to the sump tank 3 through the sample circulation channel 6 and increase the amount of collagen recovered. The solvent such as acetic acid described above is removed from the tank 7 by the pump 9 through the solvent addition flow path 8,
By flowing the sample to the sample tank 3, the collagen molecules contained in the residue and the like are to be further recovered.

In the fourth concentration step to be carried out next, the purified collagen solution S2 contained in the purification / concentration tank 5 shown in FIG. After being passed through the sample circulation channel 12 to the purification / concentration tank 5 in the same manner as above, the insoluble matter generated at this time is discharged from the drainage channel 13 to the waste liquid tank 14 together with the solvent. Thus, in the concentration step, the purified collagen solution S2 having an increased liquid volume is concentrated and impurities having a molecular weight fraction or less are removed to obtain a concentrated collagen solution S3.

The fifth sterilization filtration step to be further performed is:
As shown in FIG. 1, the concentrated collagen solution S3 obtained in the purification / concentration tank 5 is filtered from a concentrated sample flow path 16 by a pump 15 for filtration and sterilization. After the solution is sent to the tank 17 and the entire amount of the solution has been sent, the membrane size of the filtration sterilization membrane 20 is reduced to, for example, 0. 0 by the pressurized gas 19 using sterilized air or sterilized nitrogen gas from the pressurized gas flow path 18. 2
This is passed through using a material such as 2 μm, whereby the sample S4 is collected after filtration and sterilization as a product,
This is sent to the lyophilizer 21 to produce a product, and thus an enzyme-soluble collagen is obtained. Of course, during this time, it is preferable to control the temperature throughout the entire process and maintain the temperature at 10 ° C. or lower, preferably at about 4 ° C. to 5 ° C., in order to prevent denaturation of the protein.

In this connection, in the purification step, the purification membrane 4 is washed, flushed, and the initial fresh water permeation flow rate is checked in advance. After the flushing, if necessary, the system is optimized with a buffer or the like. Is good. Further, it is desirable to add a solvent or the like to the stock solution as appropriate. The pressure and temperature are set over time, and when a sufficient amount of permeate is obtained, the pump 3b is stopped, and the clarified solution in the permeate side system is recovered as much as possible. I do.
As a concentration procedure, it is preferable to optimize the inside of the system for the concentration membrane 11 with the same washing, flushing, confirmation of the initial fresh water permeation flow rate, and necessary buffers and the like. It is preferable to carry out the concentration at a higher concentration.

Next, claim 2 will be described in detail. Here, a purification membrane 4 as a microfiltration (MF) membrane used in a purification step performed after the extraction step described in claim 1, Subsequently, with respect to the concentration membrane 11 used in the enrichment step, the respective membrane sizes are selected within a specific range. First, the membrane size of the purification membrane 4 is preferably less than 1.0 μm to 0.1 μm. That is, if the membrane size is 1.0 μm or more, it becomes difficult to remove the fine particles of the dye, while if the membrane size is smaller than 0.1 μm, it is confirmed that the collagen recovery rate decreases. Was completed. For the concentration membrane 11, it is preferable to use an ultrafiltration (UF) membrane having a molecular weight fraction of less than 1000K to 10K. That is, when the temperature is higher than 1000K, the recovery of collagen is reduced. On the other hand, when the temperature is lower than 10K, the removal rate of impurities such as pepsin decreases.

According to the third aspect, in the first or second aspect, a more desirable dimensional range is selected for the membrane size of the purification membrane 4 and the concentration membrane 11. About 0.2 μm
When the thickness is 0.65 μm, by setting the thickness to 0.65 μm or less, the fine particle removal rate of the dye is not reduced.
By setting it to be not less than μm, it was possible to guarantee that the recovery rate of collagen was not reduced. In addition, in the case of the concentration membrane 11, by setting the membrane size to 10K to 100K, it was possible to guarantee that the rate of removing impurities such as pepsin and the like and the rate of collecting collagen were not reduced.

In claim 4, the above-mentioned claim 1 is described.
And a purification membrane 4 used in the purification filtration step used in claim 3.
And the concentration membrane 11 in the concentration step, both of which are tangential flow filtration (TF
F) is used. In general, the membrane type which is frequently used is such that the membrane F1 and the flow direction D1 of the permeate solution are orthogonal to each other as shown in FIG. However, in the above-mentioned TFF, as shown in FIG. 5, the permeated solution of the crude collagen solution S1 or the purified collagen solution S2 is passed through the purification membrane 4 or the concentration membrane 11 in a direction orthogonal to the flow direction D2. The light passes through a certain pair of films F2, and flows out to the respective transmission sides P2 and P3. Therefore T
By using the FF, accumulation of impurities and the like on the film surface of the film F2 is prevented by the flow of the permeation solution, and a permeation state without clogging can be maintained. As a result, according to claim 4, the purification filtration treatment and the concentration treatment can be performed within a very short period of time, and the recovery rate of collagen and the removal rate of impurities such as pigment fine particles and pepsin can be improved. Becomes

Next, a detailed description will be given of claim 5 and thereafter. First, claim 5 has the following differences from claim 1 described above. That is, as shown in FIG. 1, regarding the extraction step, the extraction processing and the atherification processing are not performed simultaneously as described above, but are performed separately. In this case, the manufacturing apparatus as shown in FIG. Can be implemented. Here, the difference between the manufacturing apparatus of FIG. 3 and that of FIG. 2 is that the sample tank 3 and the purification / concentration tank 5 are communicated with each other by a first purified sample flow path 23 connected with a pump 22. Of course, the same members as those in FIG. 2 are denoted by the same reference numerals.

In carrying out claim 5 using the same manufacturing apparatus, in order to carry out the extraction step different from claim 1, the processed sample S1 obtained by the pretreatment step in the same manner as before is used. The crude collagen solution S1 obtained by extruding collagen by adding an organic acid in the extraction tank 1a is
It is put into the sample tank 3 by the pump 2a. The first purified collagen S5 is passed through the purification membrane 4 and flows into the purified / concentrated collagen 5 while controlling the flow rate and the like by the pumps 3a and 3b in the same manner as in FIG. The residue returns to the sample tank 3 via the sample circulation channel 6 in the same manner as before. In order to increase the recovery amount, a certain amount of solvent is added from the solvent addition tank 7 to the sample tank 3 through the solvent addition channel 8. Next, in claim 5, the first purified collagen solution S5 in the purification / concentration tank 5 is returned to the sample tank 3 by the pump 22 via the first purified sample channel 23 described above, and the pepsin By adding the above, the atheromatization process is performed. The atherosylated product is subjected to a purification and filtration treatment in the next purification step. In this embodiment, the purification is repeated again through the purification membrane 4 as described above, and the second purified collagen thus obtained is obtained. Solution S
6 flows into the purification / concentration tank 5 described above. Thereafter, the sample S7 after the second filtration sterilization is recovered in the same manner as in the above-mentioned claim 1 through the concentration step and the sterilization filtration step.

Next, in claim 6, as in claim 2 with respect to claim 1, the membrane size of the purification membrane in the extraction step in claim 5 is less than 1.0 μm to 0.1 μm, and the purification step is carried out. By specifying the membrane size of the membrane and the membrane for concentration in the concentration step to be less than 1000 K to 10 K, it is intended to ensure both the collection of collagen and the removal of impurities such as dyes.

Further, in the seventh aspect, as in the third aspect of the invention, the specific value of the film size is limited to 0.2 μm to 0.65 μm and 10 K to 100 K, respectively.
By doing so, more desirable results are obtained.

According to the eighth aspect, a normal filtration membrane is used for the purification membrane in the extraction step in the same manner as in the fourth aspect of the invention, but the purification membrane in the purification step and the membrane for concentration in the concentration step are used. Is the tangential flow
By using filtration (TFF), the reliability of the action of removing impurities is to be maintained.

[0036]

Since the present invention can be carried out as described above, according to the first aspect, in the conventional production method, impurities such as pigment cells are removed in the purification step after the extraction step. In addition, centrifugal separation and salting-out cannot achieve the purpose and further rely on ultracentrifugation. Since the concentration step is performed after the purification step, large-scale purification of collagen according to the production scale can be realized without expensive equipment for removing impurities such as dyes, thereby satisfying social demands for collagen. It becomes possible. Further, according to the first aspect, it has been confirmed that the number of manufacturing days is significantly reduced as compared with the conventional manufacturing method. That is, when a specific comparative example is shown, centrifugation and salting out in a conventional purification step are repeatedly performed two to three times, which requires 10 to 20 days. A new purification filtration step and a subsequent concentration step can be completed in 1 to 2 days, and both steps are different from the conventional centrifugation or ultracentrifugation in a closed production line. Therefore, there is no need to worry about external contamination, and it is possible to manufacture the apparatus safely from a sanitary point of view.

According to the second aspect of the present invention, the size of each of the purification membrane and the membrane for concentration in each of the above two steps is specified, thereby achieving both the recovery rate of collagen and the efficiency of removing pigment. Desirable compatibility between the recovery rate of collagen and the removal rate of impurities such as pepsin can be achieved. Further, in claim 3, by limiting the range of the membrane size in claim 2, it is possible to further promote the effect of claim 2, and in claim 4, the purification membrane and the concentration membrane are: Instead of using a normal filtration membrane, the T
By adopting FF, it becomes possible to further improve the purity of the final product relating to collagen.

Next, in the fifth aspect, in the extraction step which is also performed in the conventional production method, centrifugal separation is performed between each of the extraction and the atherolation. Since the first purified collagen solution was obtained by abolishing the separation process and performing the purification and filtration process, the impurities were removed from the collagen in the purification process and the purification and filtration process before the concentration process. Therefore, purification is easier and purification of collagen with high purity is greatly beneficial. And claim 6,
As for the items 7 and 8, the same additional effects as those of the above-mentioned claims 1 to 3 and 4 can be exerted, and the details described in the above description are used.

[Brief description of the drawings]

FIG. 1 is a process explanatory diagram showing a method for producing marine organism-derived collagen according to claims 1 and 5 of the present invention.

FIG. 2 is an apparatus system diagram showing an example of a manufacturing apparatus capable of implementing the manufacturing method according to claim 1;

FIG. 3 is an apparatus system diagram showing an example of a manufacturing apparatus that can be used to carry out the manufacturing method according to claim 5;

FIG. 4 is an operation explanatory view showing a general-purpose purification film used for removing impurities.

FIG. 5 is an operation explanatory diagram showing a tangential flow filtration (TFF) used for removing impurities.

FIG. 6 is a longitudinal sectional view showing a mammalian skin layer.

FIG. 7 is a longitudinal sectional view showing a skin layer of a marine organism.

FIG. 8 is a process explanatory view showing a conventional method for producing collagen derived from marine organisms.

[Explanation of symbols]

 4 Purification membrane 11 Concentration membrane 20 Filtration sterilization membrane S1 Crude collagen solution S2 Purified collagen solution S3 Concentrated collagen solution S4 Sample after sterile filtration S5 First purified collagen solution S6 Second purified collagen solution S7 Sample after sterile filtration

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mina Okamura 1007 Izumizawa, Chitose-shi, Hokkaido Daido Hoku Chitose Research Center Co., Ltd. F-term (reference) 4H045 AA20 CA52 EA01 EA15 EA34 FA16 FA70 GA01 GA10

Claims (8)

[Claims] 1. A pretreatment step of obtaining a treated sample by delipidating and washing the skin of a marine organism to obtain a treated sample, adding an organic acid to the treated sample to extract collagen, and adding a proteolytic enzyme to atherotrate. Performing an extraction step of extracting a crude collagen solution, a purification step of removing insolubles from the crude collagen solution and passing through a purification membrane, and passing the purified collagen solution obtained thereby through a concentration membrane. A concentration step of removing the impurities having a molecular weight fraction or less, and a sterilization filtration treatment in which the concentrated collagen solution thus obtained is passed through a filtration sterilization membrane by dialysis treatment and pressurization with a sterilization gas. A method for producing a collagen derived from a marine organism, which comprises: Law. 2. A pre-treatment step of obtaining a treated sample by degreased and washed skin of marine organisms, adding an organic acid to the treated sample to extract collagen, and adding a proteolytic enzyme to atherogenate. Performing an extraction step to extract a crude collagen solution, and removing insolubles from the crude collagen solution to reduce the membrane size to less than 1.0 μm to 0.1 μm.
a purification step of passing through a purification membrane with a microfiltration membrane of μm, and concentrating the purified collagen solution obtained by passing the purified collagen solution through a concentration membrane which is an ultrafiltration membrane having a membrane size of less than 1000K to 10K. A concentration step for removing impurities having a molecular weight fraction or less, and any of the sterilized filtration treatment in which the concentrated collagen solution thus obtained is passed through a filtration sterilization membrane by pressurizing with a dialysis treatment and a sterilization gas. A method for producing marine organism-derived collagen, which comprises a sterile filtration step of collecting a sample after sterile filtration. 3. The purification membrane in the purification step and the concentration membrane in the concentration step are each 0.2 μm to 0.65 μm,
The method for producing marine organism-derived collagen according to claim 1 or 2, wherein the membrane has a membrane size of 0K to 100K. 4. The method according to claim 1, wherein the crude collagen solution and the purified collagen solution permeate through the purification membrane in the purification step and the concentration membrane in the concentration step, respectively, in a direction perpendicular to the flow direction. The method for producing marine organism-derived collagen according to any one of claims 1 to 3. 5. A pre-treatment step for obtaining a treated sample by degreased skin of a marine organism and then washing it, and removing an insoluble matter from a crude collagen solution obtained by adding an organic acid to the treated sample to extract collagen. An extraction step of removing and passing through a purification membrane for purification and filtration, and then adding a protease to carry out atherolation, and a purification step of passing the first purified collagen solution obtained through the purification membrane. And a concentration step of concentrating the second purified collagen solution thus obtained by passing it through a membrane for concentration to remove impurities having a molecular weight fraction or less. A sterilization filtration step of collecting a sample after sterilization filtration by preceding any of a dialysis treatment and a sterilization filtration treatment of passing through a filtration sterilization membrane by pressurizing with a sterilizing gas. A method for producing collagen derived from marine organisms. 6. A pretreatment step of obtaining a treated sample by degreased and washed skin of a marine organism, and removing an insoluble substance from a crude collagen solution obtained by adding an organic acid to the treated sample to extract collagen. An extraction step of removing and filtering by passing through a purification membrane with a microfiltration membrane having a membrane size of less than 1.0 μm to 0.1 μm, and then adding at least a protease to carry out atherolation; A purification step in which the first purified collagen solution obtained by the above is passed through a purification membrane using a microfiltration membrane having the same membrane size as described above, and an ultrafiltration membrane having a membrane size of less than 1000 K to 10 K through the second purification collagen solution obtained thereby. A concentration step of removing the impurities having a molecular weight fraction or less by passing through a concentration membrane which is a concentrated collagen solution obtained by the dialysis treatment. And a sterilizing filtration step of collecting a sample after the sterilizing filtration by preceding any of the sterilizing filtration processes of passing through a filtration sterilizing membrane by pressurizing with a sterilizing gas. Method. 7. The purification membrane in the extraction step and the purification filtration step and the concentration membrane in the concentration step each have a size of 0.2 μm.
The method for producing marine organism-derived collagen according to claim 5 or 6, wherein the membrane size is from about 0.65 µm to about 10K to 100K. 8. The purified membrane in the extraction step allows the crude collagen solution to permeate in a direction parallel to the flow direction, and the purified membrane in the purification step and the membrane for concentration in the concentration step each contain crude collagen solution. 8. The method for producing marine organism-derived collagen according to claim 5, wherein the solution and the first and second purified collagen solutions pass through in a direction orthogonal to the flow direction.