JP3532817B2 - Method for producing marine organism-derived collagen - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to a variety of medical biomaterials, cosmetics, materials and food material to the skin of marine life in the raw material BACKGROUND OF THE INVENTION
Such as to mass-produce useful acid-soluble collagen thereto
A method for manufacturing.

[0002]

BACKGROUND OF THE INVENTION Collagen used in the above fields is mainly
The mammalian skin such as cattle and pigs have been extracted as a raw material in
It In recent years, as well as mammals, where it is industrial waste
The collagen extraction production Then Some from marine organisms of the skin of the
Cormorant research has been carried out.

[0003] The mammal to be used in the production method of collagen
Skin is shown in FIG. Referring to FIG. 6, the skin layer of mammals is composed of an epidermis layer a , a dermis layer b, and a subcutaneous layer c. this
The skin layer is about 5 to 7 mm from the epidermis layer a to the dermis layer b.
Rather not thickness. Among them, collagen is contained in the dermal layer b
Is, impurities such as dyes are contained in the skin layer a. did
Therefore, the pigment cell d is also on the epidermal layer a side. In the this Yonaho milk <br/> such skin, in the pretreatment step preceding the extraction step
The separating means, relatively thick have <br/> skin or al physically removing the skin layer a containing and color elements. This is the original marine life below
When the comparison that the charge is that the collagen solution mixed absence etc. N photo <br/> such as color element is obtained.
Collagen solution obtained in the extraction process after an applied and impurities are further removed in the centrifugal separator, Ru through sterile filtration step. Thus collagen is produced . Since a large size this sometimes as a <br/> centrifugal separator are available can be purified a large amount of collagen at a time, it leads to mass production of Koller <br/> Gen.

On the other hand, means for producing collagen from marine organisms
Is in the process of being researched . The method described below for this is that has been tried on a small scale.

Used in the production of collagen from marine organisms
The salmon etc. are as follows. The skin layer is the above
It is the same three-layer with. But the thickness of the skin layer a except subcutaneous layer c to between the dermal layer b are as about 1~2mm and thin <br/> rather in Figure 7, contains a collagen in its thin dermal layer b. Moreover, pigment cells d and the like existing in the upper layer portion of the dermis layer b
Neat, the greater the amount if mammals and specific solid. Therefore salmon
For marine life such as the reason that the skin layer is thin, it is difficult to remove only the part containing such dyes.

Therefore, collagen from marine organisms such as salmon
The manufacturing means performs the following in the pretreatment process .
For salmon skin, scale (e) , body, and fin ( fin) of Fig. 7
The extent to which and the like are removed. Washing the salmon skin from or extracted lipids or shredded as in FIG. For this Ushite <br/> obtained treated samples, as is known, either Atero of it like is added simultaneously pepsin when extracting the collagen is treated with an organic acid, or extraction as in FIG. 8 < After the extraction treatment, the second extraction step is carried out by selecting one of the methods such as applying a centrifuge and then performing an atherogenic treatment. Impurities <br/> such crude collagen extraction solution in the dye are present many become fine particles. Moreover, since these fine particles are very light and do not precipitate by the usual centrifugal separation treatment in the above extraction step , they cannot be sufficiently removed. So
As a measure against the crude collagen obtained in the above extraction process
Do not salt out, centrifuge, or ultracentrifuge in the next purification step .
Hang on. At the beginning of the purification process, salting out and centrifugation are repeated . That precipitate obtained in salting was collected by centrifugation
Perform the removal and removal of impurities pepsin Te, to which Ru to give the acid soluble collagen with acetic acid. This was then recovering the supernatant subjected ultracentrifuge. Figure 8 after this
In moves to final sterile filtration step. Of this sterile filtration process
Time, before the collected supernatant concentrate sterile filtered through sterile filtration membranes in step, either dialysis permeate with deionized water therefrom, or to carry out the sterilization filtration by prior dialysis, Thereafter, Ru give enzyme soluble collagen by lyophilization.

[0007]

As described above when due to manufacturing method of marine biological collagen [0005], intends row salting and centrifuged at purification step. But just repeat the centrifugation
When not completely significant impurities present many as over time (light particle) is removed, precipitated particulates then giving centrifugal force ultracentrifugation equivalent (10 00 00 × G)
It must be removed by Shingarijo. On the other hand large amount purified at a time
Since there is no possible ultracentrifugation apparatus, the amount of collagen can be purified per operation is suppressed to about 400 cc. Rice cake
Of course , a huge amount of capital investment will be required to match the number of units to the mass production scale . Difficulty because of the removal of impurities such as dye from this fish skin, the manufacturing method of the content to extract collagen from fish skin, especially flat fish skin colorless dye (Japanese Patent No. 2722014) is or is proposed .

[0008] The present invention has been name of <br/> is view of the problems of the prior art, and an object thereof as follows. (1) Centrifuge / salt as in the existing process for the purification process
Purification and filtration instead of precipitation and ultracentrifugation, and then
The concentration process by the concentration process is set. Super by doing this
Enables fine particle removal without using a centrifuge
Or removing impurities such as pepsin along with collagen concentration
To enable. (2) Obtained in the extraction process by the purification filtration process in the purification process.
Continuous removal of impurities from the crude collagen solution
Enables large-scale purification of collagen according to production scale. This
This avoids huge capital investment in the mass purification of collagen
Understand. (3) The manufacturing schedule is significantly shortened, while the manufacturing aspect
To allow the adoption of a closed manufacturing line and external pollution
To eliminate the worry of and to be relieved in hygiene. Produced
Cheap Coller from the skin of marine life that was considered industrial waste
To be able to provide Gen to medical-related, makeup-related, and food-related
To (4) Remove insoluble matter from crude collagen solution from extraction process
In the purification step to
Collagen of less than 0 μm to 0.1 μm
Efficiency of dye removal without lowering the recovery rate of dyes
It also exerts a more desirable purification effect without lowering. (5) Molecular weight fractionation of purified collagen solution from purification filtration step
In the concentration process to remove the following impurities,
Ultrafiltration membrane with a membrane size of less than 1000K-10K
This reduces the removal rate of impurities such as pepsin and
More desirable concentration by avoiding a decrease in collagen recovery rate
Increase the effect. (6) The membrane size of the purified membrane was 0.2 μm to 0.6 μm
The size of the concentrating membrane to 10K-100K
This further enhances the purification effect and the concentration effect. (7) Each solution (crude collagen solution, purified collagen solution,
Concentrated collagen solution, etc. is used for each membrane (purified membrane / concentrated
Permeation membrane (for example, a compressive membrane) so that the permeation is perpendicular to the flow direction . By doing so, impurities were prevented from accumulating on the film surface and clogging did not occur .
Ri, the purity of the finally obtained enzyme soluble collagen or further improved. (8) Remove insoluble matter from crude collagen solution obtained by extraction
Then, purify it through a purification membrane to remove impurities.
Remove and atheroize it. Thus by centrifugation
By performing the purification filtration process without
It is easier to remove impurities.

[0009]

According to a first aspect of the present invention, in order to solve the problems]
A method for producing collagen derived from marine organisms achieves the intended purpose
In order to achieve this, the following means for solving the problems are featured. Ie
The process according to claim 1 includes a pretreatment step of obtaining a washed and treated samples this was degreased skin marine organisms, to together when extracting collagen with an organic acid added to the treated sample an extraction step of extracting the crude collagen solution by Atero by addition of proteolytic enzymes thereto, obtained by removing insolubles from the crude collagen solution
When the crude collagen solution is purified with a purification membrane ,
Permeation through the purification membrane in the direction orthogonal to
A purification step that, to perpendicular to the purified collagen solution is its flow direction when the thus obtained purified collagen solution was concentrated concentrating membrane to remove molecular weight fraction or less impurities
A concentration step to go through the concentrating layer in the direction that, the sterilizing gas concentrated collagen solution thereby obtained
Sterile filtration or in pressurized passing sterile filtration membranes
It is characterized by comprising a sterilizing filtration step of collecting a sample after sterilizing filtration by performing these treatments in advance of any one of dialysis treatments for dialysis of the concentrated collagen solution .

Coral derived from marine organisms according to claim 2 of the present invention
In order to achieve the intended purpose,
It is characterized by a problem solving means. That is, according to claim 2.
The manufacturing method includes a pretreatment step of degreasing the skin of marine organisms and then washing the same to obtain a treated sample, and a crude collagen solution in which collagen is extracted by adding an organic acid to the treated sample. it or to remove insoluble material was passed in purified membrane from
An extraction step or to Atero by addition of further proteolytic enzyme to it or purified filtered Te, the first time the purification of the first purified collagen solution thereby obtained in purified membrane
The purified collagen solution is refined in a direction orthogonal to its flow direction.
A purification step to make go through the film, the second purified Koller when removing the second purified collagen solution concentrated membrane with concentrated by <br/> molecular weight fractionation following impurities thereby obtained
The gen solution passes through the concentration membrane in a direction orthogonal to its flow direction.
A concentration step to go spent, sterile filtration or the concentrate collagen solution is passed through a sterile filtration membrane thus obtained was concentrated collagen solution is pressurized with sterile gas
These are preceded by any of the dialysis treatments
It is characterized by comprising a sterilization filtration step of collecting a sample after sterilization filtration by performing a treatment .

[0011] According to claim 3 of the present invention, the marine organism-derived cola
The method for producing a gene is the method according to claim 1 or 2.
The purification membrane in the purification step has a membrane size of less than 1.0 μm to 0.1 μm , and the concentration membrane in the concentration step is an ultrafiltration membrane having a membrane size of less than 1000K to 10K.
It is characterized by

[0012] A marine organism-derived cola according to claim 4 of the present invention.
The method for producing a gene according to any one of claims 1 to 3.
In law, purified membrane film size in the purification step 0.2
μm~0.65μm are those of the concentrated <br/> reduced membrane in the concentration step is of film size 10K~100K
Characterize.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Marine organism-derived coller according to the present invention
Illustrated in Figure 1, Figure 2 and FIGS. 4 and 5 in Gen manufacturing method
The product comprises a pretreatment step , an extraction step , a purification step, a concentration step, and a sterile filtration step.

Referring to FIG . 1, the pretreatment process is as follows.
It is a thing. For fish skins such as salmon skin, remove unnecessary parts such as the body in running water and shred into 3 cm squares . This minced
If example Tato the already fish skin and stirred was put in a mixed solution of chloroform and methanol. Further to repeat the stirring and replace the solvent speaking. As after the original by or replaced with deionized water or substituted in methanol after washing the minced already fish skins, further cleaning liquid exchange washing with cleaning and deionized water by a buffer solution such as a solution containing 20% NaCl to remove the neutral protein repeatedly with. Thus the processed sample shown in Figure 1
It is obtained.

[0015] When the extraction step with reference to Figure 1, Figure 2
The processed tanks are added to the extraction tanks 1a and 1b of the exemplified manufacturing equipment.
Sample was charged, this to 0. And 5M acetic acid, and 1% pepsin or other proteolytic enzymes to substrate weight was added, perform Atero reduction by stirring them. like this
To, and Atero simultaneously with extraction of collagen with an organic acid
To obtain a crude collagen solution Te, while the amount of collagen that is extracted is the advantage of increasing, so that the coming mixed into simultaneously impurities mass in solution, such as a dye.

[0016] Thus crude collagen S that obtained in the above
1, after removal of the insoluble matter such as nylon mesh, transferred to a sample tank 3 by operating the pump 2a · 2b in FIG. 2, subjected then to a novel purification process. You
The crude collagen S1 of ie sample tank 3 through the purified membrane 4 while controlling the flow rate of a pump 3a · 3b,
Purified is that purified collagen solution S2 obtained in the film passage of this -
Transfer to the concentration tank 5 . In the case of the example shown in the drawing, the sample circulation channel 6 is used to remove the residue and the like blocked by the purification membrane 4.
Ru return to the sump tank 3 via the. Further, since the measure recovery amount increasing collagen, the solvent with acetic acid or the like of the pump 9
Solvent addition tank 7 by operation → solvent addition flow path 8 → sample
It is made to flow like the tank 3 and added to the tank 3 .
This collagen molecules contained in such the residue is recovered in further <br/>.

[0017] With respect to the next concentration step, purified collagen solution S2, which enters the purification and concentration tank 5 in FIG. 2,
It is concentrated by passing through the concentration membrane 11 while controlling the flow rate and the like by the pump 10, and then returning to the purification / concentration tank 5 via the sample circulation channel 12. On this occasion
Insoluble matter occurs in the Ru is discharged from the drain passage 13 together with the solvent into the waste tank 14. With such a concentration step, the concentration of purified collagen solution S2, which increases the liquid volume, removal of the molecular weight fraction below impurities and is made, it is Ru obtained concentrated collagen solution S3.

The sterilization filtration step is shown in Figure 1 the far
Ri, Ru conventional substantially the same as the contents Der. This is Figure 2
Concentrated collagen solution S3, in the purification and concentration tank 5
By operating the pump 15 so that the purification / concentration tank 5 → concentrated sample flow path 16 → filtration sterilization tank 17 flow
It Concentrated collagen solution S in the purification / concentration tank 5
When all 3 has been sent out , the sterilized air or sterilized nitrogen gas, which is a pressurized gas 19, is sterilized by filtration from the gas passage 18 for pressurization.
It is sent into the tank 17 for use and flows out from the inside of the tank 17.
A concentrated collagen solution S3 with a membrane size of 0.22
It is passed through a filter sterilization membrane 20 of μm . Filter sterilization after passing through this membrane
The post sample S4 is collected as a product or is sent to the freeze dryer 21 to be a product . Thus by the enzyme soluble collagen Ru obtained. During this time, temperature control is performed throughout the entire process, and it is preferable to maintain the temperature at 10 ° C. or lower, preferably at 4 ° C. to 5 ° C. This is in order to prevent denaturation of proteins
To do.

Regarding the purification step, it is necessary to confirm the washing / flushing / initial fresh water permeation flow rate of the purification membrane 4 in advance, and after flushing, optimize the system with a buffer etc. if necessary. Good. In addition, suitable for the stock solution
Yichun, is to hydrolysis and solvent not desirable. In addition , the pressure and temperature were set over time, and a sufficient amount of permeate was obtained .
Or stop can pump 3b, or the clarified liquid in the permeation side system as much as possible collected. Like the above for enrichment procedure, it is to optimize the system, such as the confirmation-required buffer wash flushing initial Shimizu permeation rate of the concentration membrane 11
Yes. It may be carried out in the concentration ratio as needed for enrichment.

The extract or purified membrane 4 as microfiltration (MF) membrane used in the purification step after the step out, to the concentrating film 11 used Succeeding concentration step which selects a film size within a specific range The time is as follows. The membrane size of the purified membrane 4 is preferably less than 1.0 μm to 0.1 μm. The reason is that when the film size is 1.0 μm or more, it becomes difficult to remove the fine particles of the dye, and the film size is less than 0.
It becomes smaller when the collagen recovery than 1μm is lowered. As the concentration membrane 11, it is preferable to use an ultrafiltration (UF) membrane having a molecular weight fraction of less than 1000K to 10K. The reason is because decreases the recovery of co <br/> collagen in the case of more than 1000 K, the removal rate of impurities such as small comes to pepsin than 10K is decreased.

[0021] attached to the membrane size of the refining film 4 and concentrated for the film 11
Te, the Hatsugi when selecting a more desirable size range
Growls 0.2 μm to 0.65 μm for purified membrane
You to. The reason is that the fine particle removal rate of the dye does not decrease when the particle size is 0.65 μm or less, and the particle size is 0.2 μm or more.
This is because the recovery rate of collagen does not decrease . It does not decrease the removal rate of impurities such as pepsin by a film size of 10K~100K for concentrating film 11, the recovery of collagen nor decrease.

[0022] - tangential Both the concentrating layer 11 of purified membrane 4 and concentration step of refining filtration step Flow
Filtration (TFF) is used . Incidentally usually frequently used as film type ones, since the the film F1 and the permeate solution flow direction D1 as shown in FIG. 4 orthogonal, the solution in the direction parallel to the flow direction D1 flows out after passing through the permeate side P1 .
On the other hand, in the case of the TFF shown in FIG. 5, the permeation solution such as the crude collagen solution S1 and the purified collagen solution S2 is
Purification in a direction perpendicular to the those of the flow direction D2 film 4 and concentrating film 11 (the pair of film F2) passes, each of the permeate side P
It flows out to the 2 · P3. So when using TFF
It may be deposited, such as impurity with respect to the film surface of the film F2 is prevented by the flow of the permeate solution, holding the free transmission state of clogging. This means that the purification filtration process and the concentration process can be performed within an extremely short period of time, and the recovery rate of collagen and the removal rate of impurities such as pigment fine particles and pepsin are improved .

[0023] In regard Extraction process as <br/> listed in Figure 1, the extraction process and Atero treatment is to be performed independently in not simultaneous. Is an example shown in Figure 3 for this
Kill in the implementation in the manufacturing equipment. The manufacturing apparatus of FIG.
Sample via the first purified sample flow path 23 with 2
The tank 3 and the purification and concentration tank 5 is communicated, this point is different from that of FIG. But the others
It is substantially the same as that of FIG. Therefore, in the case of FIG.
In this case, the same members as those in FIG. 2 are designated by the same reference numerals.

[0024] Extraction process using the manufacturing apparatus of Figure 3 is the following
Is like. Since the treated samples S1 obtained in the previous SL the same way the pre-process is Ru extraction tank 1a near, which
Adding an organic acid extruded City, collagen, crude collagen soluble
Liquid S1 is obtained. Crude collagen solution S1 thus obtained
Is charged into the sample tank 3 by the pump 2a. Further, as in FIG. 2, the first purified collagen S5 is passed through the purification membrane 4 while controlling the flow rate with the pumps 3a and 3b.
To the purification / concentration tank 5. For even the remaining Tomebutsu
Similar to the above, it is returned to the sample tank 3 via the sample circulation flow path 6 . A certain amount of solvent in order to increase the recovery amount
The or added to San <br/> Purutanku 3 through solvent addition channel 8 from the solvent added for the tank 7. A pump 22 is provided for the first purified collagen solution S5 in the purification / concentration tank 5.
Back through the first purified sample flow path 23 was example to support Npurutanku 3, wherein intends rows Atero process by adding such as pepsin. While you purified filtration in the next stage of the purification process of those Atero reduction in write, perform repeated purification via purified membrane 4 again in the embodiment, thereby the second purified collagen solution S6 was obtained Pour into the purification / concentration tank 5 . Thereafter recovering the second filter sterilized after sample S7 like the <br/> said at Rukoto through the concentration step with a sterile filtration step.

Also in this embodiment, the purified membrane used in the extraction step is specified to have a membrane size of less than 1.0 μm to 0.1 μm, and the purified membrane used in the purification step or the concentration membrane used in the concentration step has a membrane size of 100.
It is specified to be less than 0K to 10K . Both the co <br/> Lage down recovery of ensuring the removal of impurities such as dyes That way
Let 0.2μm~ each particular value of said film size
When the thickness is 0.65 μm and 10K to 100K , more desirable results are obtained .

In this embodiment , an ordinary filtration membrane is used as the purification membrane in the extraction step, but tangential flow filtration (TFF) is used as the purification membrane in the purification step and the concentration membrane in the concentration step. As a result, the reliability of the impurity removing action is maintained.

[0027]

The following effects can be obtained by the method for producing collagen derived from marine organisms according to the present invention. (1) Since the existing purification process is abolished and the concentration process is performed after the purification process by the purification filtration process, expensive equipment is not required for removing impurities such as pigments, and mass purification of collagen according to the production scale is realized. can do. This also means that it can meet the social demands for collagen . (2) base ratio to the conventional method collagen production dates can be greatly shortened. By the way, centrifugation at traditional purification process
And salting out is repeated 2-3 times, and 10-20
It took a day . In contrast to the purification filtration step in the present invention and this
The concentrating process that follows can be completed in 1-2 days.
it can. (3) for the purified filtration step and concentration steps, unlike the conventional centrifugation or ultracentrifugation, to adopt a closed production line
Worry of external contamination in the can Rukoto is not name. Therefore also it is possible to safely producing collagen from <br/> sanitation surface
It (4) By identifying each film size for purified membrane and concentrating film on the both steps, or both and the collagen recovery and color containing <br/> removal efficiency, respectively, the desired collagen recovery It is compatible with the removal rate of impurities such as pepsin . this
The effect is further enhanced by limiting the range of membrane size .
It (5) for the purified membrane and concentrating film that has been adopted the TFF
As a result, the purity of the final collagen product can be further improved. (6) abolished the far <br/> heart separation process have been made between the extraction and Atero of the extraction process, it is so arranged to obtain a first purified collagen solution with purified filtration, purification steps subsequent thereto and in the concentration step prior the purification filtration, Ru is performed the removal of impurities to collagen. Therefore, purification is easy
Ri, large door be Ekisuru in the production of high-purity collagen
It

[Brief description of drawings]

[1] process explanatory view showing one embodiment of the present onset bright Way Method

2 is a system diagram of a manufacturing apparatus for carrying out the method of FIG.
Explanatory diagram outlined in

FIG. 3 is an explanatory view schematically showing another embodiment of the method of the present invention together with a system diagram of its manufacturing apparatus.

[4] created <br/> for illustration of purified membrane which is generally to remove impurities

[5] The present invention is used for removing impurities in the process TFF (tangential flow fill trays
Of action)

FIG. 6 is a longitudinal sectional view schematically showing the skin layer of mammals.

FIG. 7 is a vertical cross sectional explanatory view schematically showing the skin layer of marine organisms.

[8] of a conventional marine-derived collagen Manufacturing how shown schematically
The process illustration

[Explanation of symbols]

4 Purification membrane 11 Concentrating 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 Sterile filtered sample

Continuation of the front page (56) Reference JP-A-9-278639 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 14/78 C07K 1/14 C07K 1/34 BIOSIS / CA / MEDLINE / W PIDS (STN)

Claims (4)

(57) [Claims] 1. A This preprocessing step to obtain a washed and treated samples this was degreased skin marine organisms, to together when extracting collagen with an organic acid added to the treated sample
An extraction step of extracting the crude collagen solution by Atero by addition of proteolytic enzymes Les, purified that is obtained by removing the insoluble matter from the crude collagen solution with purified membranes
The direction in which the crude collagen solution is perpendicular to the flow direction
So that go through the purification membrane purification step and, the purified Koller when removing this by purified collagen solution concentrated membrane with concentrated by <br/> molecular weight fractionation following impurities obtained
The gen solution passes through the concentration membrane in a direction orthogonal to its flow direction.
A concentration step to go spent, sterile filtration or the concentrate collagen solution is passed through a sterile filtration membrane thus obtained was concentrated collagen solution is pressurized with sterile gas
These are preceded by any of the dialysis treatments
A method for producing collagen derived from marine organisms, which comprises a sterilization filtration step of recovering a sample after sterilization filtration by performing a treatment . 2. A pretreatment step of degreasing the skin of marine organisms and then washing this to obtain a treated sample, and removing insoluble matter from a crude collagen solution obtained by adding collagen to the treated sample to extract collagen. in or the extraction step or to Atero by addition of the passing to fine <br/> made filtered or more proteolytic enzyme to it in purified membrane thereto, purified membrane first purified collagen solution thereby obtained When refining the first purification
Purification membrane with collagen solution in a direction orthogonal to its flow direction
A purification step to make go through the second purification collagen When this second purification collagen solution obtained by the concentrated in concentrating film removing molecular <br/> weight fraction following impurities
The solution permeates through the concentrating membrane in a direction orthogonal to its flow direction.
A concentration step in going way, thereby sterilizing filtration process or the concentrate collagen solution to the concentrated collagen solution is passed through a pressurized filter sterilized membrane sterile gas obtained translucency
These treatments are preceded by any of the dialysis treatments
The method for producing collagen derived from marine organisms, which comprises performing a sterilizing filtration to recover a sample by performing a sterilizing filtration. 3. The purified membrane in the purification step has a membrane size of 1.0.
The method for producing collagen derived from marine organisms according to claim 1 or 2 , wherein the concentration membrane in the concentration step is an ultrafiltration membrane having a membrane size of less than 1000K-10K. 4. The purified membrane used in the purification step has a membrane size of 0.2.
μm~0.65μm are of Claim concentrated <br/> reduced membrane in the concentration step is of film size 10K~100K
The method for producing collagen derived from marine organisms according to any one of 1 to 3 .