KR20150078870A - Collagen method of preparing - Google Patents

Abstract

The present invention relates to a method for producing collagen.
The present invention is characterized in that liquid collagen is concentrated to a high concentration by using a tangential flow filtration (TFF) and a centrifuge to use as a medical material.
The present invention having the above-described structure allows the collagen to be prepared at various concentrations for use as a medical material. Particularly, collagen which is concentrated at a high concentration has advantages of excellent performance for the purpose and easy storage This greatly improves the quality and reliability of the product, so that it can provide a good image by satisfying the various needs of users who are users.

Description

Collagen method of preparing < RTI ID = 0.0 >

The present invention relates to a method for producing high-concentration collagen suitable for medical materials, and more particularly to a method for producing high-concentration collagen suitable for medical use, It has the advantages of excellent performance and easy storage compared to low concentration, which greatly improves the quality and reliability of the product, so that it can provide a good image by satisfying the various needs of users as consumers.

As you know, collagen is a structural protein that is distributed in various tissues of animals and is also called collagen, accounting for about 30% of the total weight.

Collagen is now known to be about 20 kinds, collagen type 1 being the most, morphologically being in the form of fibrous solid, and the three strands of the polypeptide being twisted by hydrogen bonds, the length of which is about 300 kda.

Collagen is dissolved in dilute acid or dilute alkali and can be prepared in liquid form. The higher the concentration, the higher the viscosity.

Collagen is suitable for living tissues and is a biodegradable material and widely used as one of medical materials such as tissue restorative material, skin graft material, bone graft material, and cell culture material.

On the other hand, in order to remove foreign materials and microorganisms from collagen produced from various raw materials and made into a liquid form, they are generally subjected to conventional filtration to produce medical materials.

As shown in FIG. 1, the conventional filtration method forms a pressure and a fluid flow in a direction perpendicular to the filtration membrane to filter out substances larger than the pore size of the filtration membrane and to pass small substances through the filtration membrane. The filtration method of this type permeates the object to be filtrated at once, and the filtration efficiency is lowered as the amount of filtration accumulated by accumulating the unpassed substance in the filtration membrane, and finally, the filtration membrane is clogged. Therefore, the conventional filtration method is dependent on the capacity and the membrane area of the filtration object, and has a low filtration concentration.

In order to filter the collagen of the polymer protein according to the restriction requirements, it should be prepared in a low concentration liquid phase. In particular, in case of microbial filtration for removing microorganisms, about 5 mg / mL of collagen to permeate the filtration membrane having a small pore size of 0.2 to 0.45 micrometer Or less.

In a method of concentrating a collagen solution prepared at a low concentration at a high concentration, general evaporation condensation is difficult to apply because of the possibility of thermal deformation of collagen, and a method of concentrating using a volatile solvent is also required to remove the residual solvent separately. Method is not suitable.

In order to solve the above problems, the following prior art documents have been filed. In other words, In the prior art 1, Patent Registration No. 1105603 (Jan. 06, 2012) (Application No. 2011-0089802) (name: method of producing collagen solution by salt precipitation compression concentration method). The above-mentioned prior art can shorten the drying time for moisture removal by compressing and concentrating the collagen by salt precipitation. In addition, since the collagen solution can be produced at a high concentration, the collagen in the living body The present invention relates to a method for producing a collagen solution by using a salt precipitation compressive concentration method so that an effect on tissue repair can be maintained for a long time. In the prior art 2, Patent Application No. 2009-0087716 (2009-08-18) (Application No. 2008-0013126) (entitled "Method for Producing Concentrated Vinegar Beverage Containing High-Concentration Collagen)". In the above-mentioned prior art, a collagen peptide derived from cattle, swine or fish is added with vinegar vinegar, acidulant, saccharides, sweeteners, sugar alcohols, fruit juice, purified water and proteolytic enzyme as a processing aid, And a method of manufacturing a beverage. However, the above-mentioned prior arts also have a great problem that collagen can not be manufactured at various concentrations in order to use it as a medical material. In addition, the above-mentioned prior arts have not been able to concentrate collagen at a high concentration, resulting in a significant problem of poor performance and inability to store. In addition, in the case of the prior art 1, there is a problem that salt concentration can not be suitably removed during salt precipitation compression concentration, and osmotic pressure is changed when applied to a human body, so that it can not be used as a medical material.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve all the problems of the prior art as described above. In order to use liquid collagen as a medical material, it is concentrated at a high concentration using Tangential Flow Filtration (TFF) and a centrifuge The second object of the present invention based on the above technical construction is to prepare collagen at various concentrations in order to use it as a medical material. Particularly, a third object of the present invention is to provide a collagen- The fourth purpose is to improve the quality and reliability of the product. Therefore, it is possible to satisfy the various needs of users and to provide good images The present invention provides a method for producing high-concentration collagen.

In order to achieve the above object, the present invention provides a method for producing collagen, comprising the steps of: (1) concentrating using collagen filtration; And concentrating the concentrated collagen using a centrifugal separator (step 2).

In the present invention, the step of concentrating using a tangential flow filtration (TFF) is carried out by feeding a collagen solution into a storage tank and then transferring the collagen solution to a contact line filtration net. The solution flows along a tangent line, The solution that has not passed through the filtration membrane is recovered to the storage tank, and the solution having passed through the filtration membrane is discharged to the waste water tank.

Further, the present invention provides a method for producing collagen, wherein the pore opening of the filtration device of the contact line filtration device is 1/6 to 1/2 of the size of the substance to be concentrated.

In addition, the present invention is characterized in that the filtration net area of the contact line filtration apparatus of the present invention has a pore size of 100 kda molecular weight cutoff (MWCO), and a polyethersulfone tangential flow filtration membrane is formed in a membrane of 0.02 to 0.1 square meters per liter of collagen solution Wherein the area of the collagen is in the range of 1: 1 to 1: 1.

In the present invention, the collagen concentrated by using the contact line filtration apparatus is adjusted to PH (neutral pH 6.0 to 8.0), maintained at 25 to 35 ° C for 4 to 24 hours, Water, and collagen), and the recovered collagen is placed in a container of a centrifuge and centrifuged to concentrate the collagen.

In addition, the present invention provides a method for producing collagen, wherein the collagen in a semi-solid state is put into a mixing container (FIG. 5) through the centrifuge and stirred uniformly with a mixed solvent to prepare a liquid.

As described above in detail, the present invention can reduce energy and energy by concentrating a collagen solution at a concentration of 5 to 10 mg / ml using a centrifuge at a concentration of 2 to 3 mg / ml, And the concentration, concentration, and dilution are repeated so that the enzyme, salt, and other low-molecular substances used in the previous step are removed through the contact-filtration membrane, thereby increasing the purity so as to be suitable for medical materials.

The present invention based on the technical structure described above enables the production of collagen at various concentrations in order to use collagen as a medical material.

In particular, the present invention is advantageous in that the collagen concentrated at a high concentration has excellent performance for the purpose and is easy to store as compared with a low concentration.

The present invention greatly improves the quality and reliability of a product due to the above-mentioned effects, and thus is a very useful invention that can provide a good image by satisfying various needs of consumers who are users.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic view illustrating a method of filtering collagen according to a conventional method.
FIG. 2 is a view showing a case where a tangential flow filtration device (TFF) applied to the present invention filters a collagen
A diagram illustrating the law.
FIG. 3 is a cross-sectional view of a tangential flow filtration apparatus (TFF)
Explanation of the expression.
Fig. 4 is a schematic configuration diagram of a pH tank applied to the present invention. Fig.
FIG. 5 is a schematic configuration diagram of a mixer vessel applied to the present invention. FIG.

The high-concentration collagen and the preparation method thereof applied to the present invention are constructed as shown in Figs. 2 to 5.

In the following description of the present invention, detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

First, the present invention is characterized in that liquid collagen is concentrated at a high concentration for use as a medical material.

At this time, in order to use liquid collagen as a medical material, collagen is produced at a high concentration using a tangential flow filtration (TFF) (FIGS. 2 and 3) and a centrifuge (FIGS. 4 and 5) .

In particular, it is preferable that the highly concentrated collagen applied to the present invention is concentrated to a concentration of 10 mg / mL to 400 mg / mL.

When the collagen is concentrated to 10 mg / mL or less, the collagen is not easy to store, and it is difficult to apply it to various products as a medical material. When the collagen is concentrated to 400 mg / mL or more, It is preferable to concentrate the high-concentration collagen to a concentration of 10 mg / mL to 400 mg / mL.

The present invention provides high-concentration collagen prepared by the above-described method.

Hereinafter, embodiments of the present invention will be described in detail.

(Embodiment 1)

A method for concentrating collagen at a high concentration using the tangential flow filtration (TFF) applied to the present invention is as follows.

First, the pig skin is sectioned to obtain a collagen solution having a concentration of 2 to 3 mg / mL, which is treated with pepsin enzyme in an acidic solution.

Thereafter, the collagen solution is subjected to a step of removing foreign matter using a filtration membrane having a void of 0.5 to 2.0 micrometers.

Then, the collagen solution is put into the storage tank, and then the solution is transferred to the tangential flow filtration net through the pump. Then, the solution is caused to flow along the tangent line so that some force of the solution flows in the direction of the filtration membrane, (FIG. 3). ≪ / RTI >

Finally, the solution which has not passed through the tangential flow filtration membrane is returned to the storage tank, and the permeate is discharged to a separate wastewater tank (FIG. 3) to produce high-concentration collagen.

Particularly, the pore of the filtration membrane applied to the present invention is preferably 1/6 to 1/2 of the size of the substance to be concentrated.

If the pore size of the filtration membrane used for the tangential flow filtration is smaller than 1/6 of the size of the substance to be concentrated, it is difficult to concentrate the filtration membrane because the size and amount of the permeate material is small. 2, there is a risk of loss of concentrate. Therefore, it is preferable that the pore size of the filtration membrane is 1/6 to 1/2 of that of the concentrated material, and that the filtration membrane of the collagen solution is preferably a MWCO filtration membrane having a molecular weight of 50-150 kda.

Also, the pore of the filtration membrane applied to the present invention has a pore size of 100 kda molecular weight cutoff (MWCO), and a tether flow filtration membrane made of polyethersulfone is used to have a membrane area of 0.02 to 0.1 m 2 per liter of collagen solution .

The tangential flow filtration of the present invention differs from the conventional filtration method in that the solution flows along the tangent line between the filtration membrane and the solution so that a partial force of the solution flows in the direction of the filtration membrane. At this time, a substance smaller than the pore of the filtration membrane is discharged as a permeate, and the non-permeated substance is collected so as to be filtered again. This tangential flow filtration prevents the clogging of the filtration membrane by the flow direction of the solution, and can be performed several times to concentrate the material larger than the pore of the filtration membrane.

The material of the filtration membrane is preferably polyethersulfone, which is suitable for collagen solution with diluted acid or diluted alkali, washing solution, and storage solution.

The pump for transferring the collagen solution to the filtration membrane of the tangential flow filtration device uses a pump capable of transferring highly viscous solution, easy to clean, and preventing contamination of the collagen solution. For this purpose, it is suitable to use a rotary lobe pump or a peristaltic pump.

When the collagen solution is concentrated using the tangential flow filtration apparatus, liquid collagen of about 10 mg / mL can be prepared

The comparison chart of the concentration before and after tangential flow filtration (TFF) of the first embodiment of the present invention is as follows.

case Concentration before concentration (mg / mL) Concentration after concentration (mg / mL) Concentration ratio One 3 11 3.6 times 2 2 7 3.5 times 3 3 9 3.0 times 4 2 7 3.5 times 5 3 7 2.3 times

As described above, it can be seen that the present invention increases the concentration ratio after concentration.

(Second Embodiment)

A method for concentrating collagen at a high concentration using the centrifugal separator according to the present invention is as follows.

First, the collagen solution at a concentration of 2 to 5 mg / mL extracted from pig skin was concentrated using a contact line filtration apparatus (Example 1), and then filtered stepwise using a filtration membrane having a pore size of 2.0 to 0.2 micrometer, Of the collagen solution.

Thereafter, the aseptic state collagen solution is concentrated using a contact line filtration apparatus (Example 1), and then put into a pH titration tank sterilized with steam.

Then, the temperature of the collagen solution in the titration tank is adjusted to be 25 to 35 DEG C, and the mixture is allowed to stand for 4 hours to 1 day so that the collagen coagulates and separates the water and the layer.

Thereafter, the collagen and separated water in the pH titration tank are separately removed, and the collagen layer is aseptically dispensed into the sterilized centrifuge vessel in the aseptic operation space and the centrifuge vessel is sealed.

Then, the centrifuge is put into a centrifuge and centrifugation is performed by setting the number of revolutions at which gravity acceleration of 4,000 to 6,000 g is applied.

Thereafter, the centrifuged centrifuge vessel is brought into the aseptic operation space and aseptically operated to remove the separated water by centrifugation.

In addition, sterilization of the mixer vessel takes place in the aseptic operation space where the centrifuged collagen and the acid solution are introduced.

Finally, the hopper container is sealed, and then homogenously stirred using an agitator to concentrate at a concentration of 100 mg / mL to produce a high concentration of collagen.

Particularly, the step of adding the aseptic state collagen solution into a pH titration tank sterilized with steam is followed by a step of titrating the pH close to neutrality (pH 6.0 to 8.0) using hydrochloric acid (HCl) and sodium hydroxide solution (NaOH) It is of course possible to produce collagen at a high concentration.

In the present invention, collagen contained in a dilute acid or a dilute alkali solution is aggregated when it is titrated to neutral or neutral pH (pH 6.0 to 8.0) at room temperature, and the collagen layer having a different specific gravity from water is separated . Using this method, the collagen and water are firstly separated and centrifuged at a gravitational acceleration of about 4,000 ~ 6,000 g using a centrifuge to separate the collagen and water from each other. At this time, the water in the centrifuge container is removed and the concentrated collagen is recovered. The recovered collagen is further mixed with hydrochloric acid (HCl) to form a liquid, and the mixture is stirred with a mixer to produce a high concentration collagen solution of about 100 mg / mL.

In the above method, the container for titrating pH, the container used for centrifugal separation, and the container for the mixer should be properly sterilized and sealed to prevent contamination. The mixer should also be equipped with a stirring device capable of stirring a high viscosity collagen solution.

A comparison table of concentration before and after concentration using the centrifugal separator of the second embodiment of the present invention is as follows.

case Concentration before concentration (mg / mL) Concentration after concentration (mg / mL) Concentration ratio 34 3 120 40.0 times 35 2 95 47.5 times 36 3 100 33.3 times 41 4 96 24.0 times 48 3 103 34.3 times

As described above, the present invention can confirm that the concentration ratio after concentration is greatly increased compared with before the concentration.

In the meantime, it is needless to say that, in the present invention, the semi-solid collagen is put into a mixing container through the centrifuge and stirred uniformly with a mixed solvent to prepare a liquid.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.

It is to be understood that the invention is not to be limited to the specific forms thereof which are to be described in the foregoing description, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. .

The method for producing high-concentration collagen according to the present invention comprises concentrating using high-concentration collagen by using a centrifugal separator, producing a high concentration of collagen by using a centrifugal separator, thereby reducing treatment capacity and energy (energy) And it is advantageous in that purity can be increased to suit medical collagen by eliminating low molecular substances such as enzymes and salts consumed in the previous process through the filtration membrane by repeatedly concentrating or concentrating and diluting. Therefore, by implementing the invention of the present invention, it is possible to contribute to industrial development by promoting technological development, which is worth protecting.

Claims (6)

A method for producing collagen, comprising the steps of: (1) concentrating the collagen using a contact line filtration apparatus; And
And concentrating the concentrated collagen using a centrifuge (step 2).
The method according to claim 1,
In the step of concentrating using Tangential Flow Filtration (TFF), the collagen solution is put into a storage tank and transferred to a contact line filtration net. The solution flows along the tangent line, Wherein the solution is returned to the storage tank, and the solution having passed through the contact-sintering filtration membrane is discharged to the waste water tank.
The method according to claim 1,
Wherein the filtration mesh pore of the contact line filtration apparatus is 1/6 to 1/2 of the size of the substance to be concentrated.
The method according to claim 1,
The filtration net area of the filtration apparatus of the present invention has a pore size of 100 kda molecular weight cutoff (MWCO), and a polyethersulfone tangential flow filtration membrane has a membrane area of 0.02 to 0.1 m 2 per liter of collagen solution ≪ / RTI >
The method according to claim 1,
(Collagen, water, and water) was prepared by keeping the pH of the concentrated collagen (pH 6.0 to 8.0) close to neutrality and maintaining the temperature at 25 to 35 ° C for 4 to 24 hours, And collecting the recovered collagen in a container of a centrifuge, followed by centrifuging to concentrate the collagen.
6. The method of claim 5,
The collagen in a semi-solid state is put into a mixing container (FIG. 5) through the centrifuge and stirred uniformly with a mixed solvent to prepare a liquid phase again.

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