KR20070010813A - Process for preparing collagen - Google Patents

Abstract

A method for preparing a collagen is provided to be able to obtain the highly pure collagen having low molecular weight, thereby being usefully used as raw materials or additives in various fields including beauty, health drink, health food, medicine, and cosmetic. The method comprises the steps of: (a) pre-treating pig skin by base-treating and acid-treating it in sequence so as to remove foreign materials therefrom; (b) treating the pre-treated pig skin with an enzyme to obtain hydrolysate; (c) after adding a dispersion solution to the hydrolysate, adding a basic solution thereto so as to crush the hydrolysate having high molecular weight into the hydrolysate having low molecular weight; (d) separating the obtained hydrolysate having low molecular weight substances to remove foreign materials therefrom; (e) after heat-treating the purified fraction obtained from the step(d), filtering it; and (f) concentrating the filtrate, and purifying a collagen having low molecular weight. The solution obtained by the step(f) is dried and pulverized to obtain the powdery collagen.

Description

Process for preparing collagen {Process for preparing collagen}

The present invention relates to a method for producing collagen, and more particularly, to a production method capable of producing high purity low molecular weight collagen with high efficiency.

Generally, collagen is a fibrous protein that is one of the proteins that make up the skin, bones, and tendons. They are especially distributed in the connective tissues of the body, especially in our body contains about 20% protein, of which collagen accounts for about 30%. The molecular structure of the collagen has a collagen type structure in which two polypeptide chains are wound and bonded to each other by hydrogen bonds. Collagen with this structure has functions such as adhesion of cells, support of body and organs, activation of cell function, cell proliferation, hemostasis, immunity, etc. It is a protein.

Collagen having such characteristics is currently used as a raw material or additives in various fields, including beauty, health drinks, health foods, medicines and cosmetics, and its application field is gradually expanding due to its functional characteristics.

Such collagen generally collects bones and skins of cows or pigs, and in order to separate collagen from tissues, organic solvent extraction, acid / base treatment, trypsin and hyaluronidase can be used to obtain collagen as an insoluble substance. Because of their high molecular weight, digestion and absorption in the human body are difficult, and the purity is reduced due to various impurities included in raw materials, thereby limiting their use.

The technical problem to be achieved by the present invention is to provide a production method capable of obtaining high purity low molecular weight collagen with high efficiency.

The present invention to achieve the above technical problem,

(a) a pretreatment step of removing the foreign matter by sequentially performing base treatment and acid treatment of pig skin;

(b) a hydrolysis step of enzymatically treating the pretreated pig skin to obtain a hydrolyzate;

(c) pretreatment by adding the dispersion solution to the obtained hydrolyzate, followed by adding a basic solution to break down the high molecular weight hydrolyzate to a low molecular weight;

(d) a purification step of removing the foreign matter by separating the obtained low molecular weight hydrolyzate by material;

(e) heat treating the purified fractions followed by filtration; And

(F) provides a method for producing collagen comprising the step of purifying the low molecular weight collagen after concentrating the filtrate.

The manufacturing method may further obtain a powdery collagen by further comprising the step of drying and powdering the solution obtained by purifying the low molecular weight collagen.

The enzyme treatment of step (b) means a step of obtaining a hydrolyzate by adding pretreated pork skin to an acidic solution together with a hydrolase, wherein the available hydrolase may be pepsin, trypsin, papain, reline, collagenase. Agent or cathepsin can be used.

The basic solution of step (c) is a basic substance containing an alkali metal, it is preferable to use an aqueous solution such as sodium hydroxide, calcium hydroxide, potassium hydroxide or magnesium hydroxide.

The separation and debris removal process for each material of step (d) comprises suspending low molecular weight hydrolyzate using volcanic stone, diatomaceous earth, talc, perlite, cellulose powder, or fine powder ion exchange resin and filtering the same;

Dissolving and homogenizing the hydrolyzate by adding sodium chloride under acidic conditions, and then filtering and recovering the obtained supernatant; And

Adding the separated supernatant to a column filled with Sephadex-G gel and separating with buffer to remove desalting and acetic acid.

Purification of the low molecular weight collagen of step (f) comprises the step of adding the concentrated low molecular weight collagen to the column filled with Sephadex-G gel, and then fractionating using a buffer.

Hereinafter, the present invention will be described in more detail.

As described above, the present invention enables high-efficiency, low-molecular-weight collagen to be produced at high efficiency using a suitable sterilization, extraction, and purification filtration process from pig skin containing a relatively high content of collagen.

Such collagen production process of the present invention, (a) a pretreatment step of removing the foreign matter by sequentially performing a base treatment and an acid treatment of the donpi; (b) a hydrolysis step of enzymatically treating the pretreated pig skin to obtain a hydrolyzate; (c) pretreatment by adding the dispersion solution to the obtained hydrolyzate, followed by adding a basic solution to break down the high molecular weight hydrolyzate to a low molecular weight; (d) a purification step of removing the foreign matter by separating the obtained low molecular weight hydrolyzate by material; (e) heat treating the purified fractions followed by filtration; And (f) purifying the low molecular weight collagen after concentrating the filtrate.

Step (a) is a pretreatment step to remove and disinfect bacteria from pig skin and to perform a base treatment and an acid treatment sequentially as a pretreatment process for removing the remaining amount of hair, color and keratin. Such a process is preferably carried out in a clean environment and refrigerated state in order to prevent corruption of pork skin and maintain freshness.

For this pretreatment, the excess lipid layer is removed from the pig skin kept in the refrigerated state in a clean vinyl cloth and cut into a size convenient for work. Subsequently, the base material is sufficiently submerged in a basic solution of 0.01 M to 2.0 M concentration, for example, sodium hydroxide, calcium hydroxide, potassium hydroxide, or sodium hypochlorite refrigeration solution for base treatment, and it is immersed for 1 to 48 hours to deposit bacteria and the like. Bacteria were removed and disinfected. Next, the basic solution is removed in order to remove fine hairs and the like present on the surface of the pig skin and soaked in an acid solution of 0.01M to 2.0M, for example, a formic acid refrigeration solution, and soaked for 1 to 48 hours. The lipid layer which can be generated in this process can be removed by washing several times with water or the like.

The acid treatment, which is part of the pretreatment process, is immersed for 12 to 72 hours in a refrigerated state by immersing the washed pork skin sufficiently in an EDTA solution containing about 0.01 to 1 M hydrochloric acid solution, for example Tris-HCl. Can exfoliate. The deposition solution is then removed, washed several times with water and then drained. When the extraction of the pig skin is delayed, it is preferable to keep the freshness for later processing by freezing it in a plastic bag.

In the pretreatment it is possible to further remove the lipid layer through additional acid treatment, for which the pig skin is refrigerated in an acid solution of 0.01 to 2 M concentration, for example acetic acid solution, having a capacity of about 1 to 10 times the weight of the pig skin. After being deposited for 1 to 72 hours in the state, the separated lipids are primarily removed. At this time, it is preferable to remove the separated lipids through a sieve while stirring every 3 hours in the deposited state.

If the primary lipid extraction process is not sufficient, the secondary lipid extraction process may be further performed. Homogenized by homogenizer (for example, knife homogenizer), homogenized, and acid of 0.01M to 2.0M concentration. The solution, for example acetic acid solution, may be further added in an amount of about 1 to 10 times the weight of the pig skin, and then the solution may be stirred under refrigerated conditions for 12 to 72 hours and then the separated lipids may be removed. At this time, it is more preferable to remove the separated lipids through a sieve while stirring every 3 hours in the deposited state.

The acid treatment or base treatment described above is preferably carried out by heating in order to further increase the treatment efficiency, but preferably at about 50 ° C. or less without fear of protein denaturation.

By removing such bacteria, foreign substances, and lipid layers, it is possible to increase the product purity of the desired collagen, and to remove various causes of odor and rot, thereby providing a product having improved stability.

As described above, the base treatment and the acid treatment are sequentially carried out to remove the bacteria and various foreign substances, and the enzyme treatment of the following step (b) is performed on the pretreated pig skin obtained by extracting the lipid layer. do.

Such enzyme treatment is adjusted to pH 2.0 to 3.0 by adding an acid, for example acetic acid, to the extract from which the lipid layer is removed in the pretreatment process, and then the hydrolytic enzyme of the total extract is dissolved and added to acetic acid, for example. . Such enzyme treatment is stirred for 1 to 48 hours in a constant temperature water bath, it is preferable to remove the separated lipids through a sieve in this process. The temperature of the constant temperature bath is preferably high, but more preferably maintained at 30 to 70 ° C. to prevent protein denaturation.

As the hydrolase that can be used for the enzyme treatment, pepsin, trypsin, papain, reline, collagenase, cathepsin and the like can be used. They hydrolyze and decompose the muscle matrix protein contained in the donpi, and the hydrolysis products produce not only low molecular weight collagen but also high molecular weight collagen.

The content of the hydrolase used in the enzyme treatment can be appropriately selected in consideration of the decomposition time and processing efficiency, in the present invention, it is more preferable to add the content of 0.0001 to 0.01 (W / V)% of the pig skin extract Do. When the content is insufficient, it is difficult to achieve sufficient hydrolysis, and when it is exceeded, the additional hydrolysis rate due to the excess content is insignificant, which is not economical, which is not preferable.

The hydrolyzate obtained by such an enzyme treatment contains not only low molecular weight but also high molecular weight hydrolyzate, and it is necessary to make them low molecular weight. To this end, the dispersion solution addition and molecular weight crushing process of step (c) are performed.

The addition of the dispersion solution to the hydrolyzate is for the pretreatment of the high molecular weight hydrolyzate, and the pH is adjusted to 5.0 to 5.5 by adding a basic solution such as sodium hydroxide solution, and then about 1 to 10.0 (V / V)% alcohols such as isopropyl alcohol are added and stirred at room temperature for 0.5 to 10 hours. The separated lipids are filtered off and left to stand for at least 24 hours in a refrigerated state to perform a pretreatment step.

Next, after standing, the lipid layer of the supernatant solution is removed and the solution is homogenized by stirring in a water bath. The pH is first adjusted to neutral by further addition of a basic solution, for example an aqueous sodium hydroxide solution, stirred and homogenized, and the pH is then adjusted to 11.5 to 12.5 by further addition of the basic solution described above. After stirring for 1 to 10 hours while increasing the temperature of the internal solution in the water bath to about 40 to 70 ℃, the solution is filtered to remove foreign matter and insoluble matter. The pH is neutralized with an acid solution, for example acetic acid, and the molecular weight crushing process is performed by standing for 1 to 72 hours in a refrigerated state. The high molecular weight hydrolyzate is converted to a low molecular weight by the molecular weight crushing process, so that digestion and absorption by the human body are further improved, thereby making it possible to manufacture collagen with improved quality.

In this way, the hydrolyzate that has undergone molecular weight crushing process has a higher purity quality through the separation and purification of each substance in the step (d) and the removal of foreign substances. Such purification may be performed only by a general filtration process using a simple micro filter, an ultra filter, or the like, but in the present invention, it is more preferable to undergo a tertiary purification process to obtain a target having a higher purity quality which is harmless to the human body.

First, as the primary tablet, after standing in the molecular weight crushing step, the lipid layer of the supernatant solution is removed, and then the solution is homogenized in a water bath at 30 to 50 ° C. Low-molecular-weight hydrolyzate is suspended using volcanic stone, diatomaceous earth, talc, perlite, cellulose powder, or fine powder ion exchange resin, and then filtered to remove foreign matter and insoluble material, thereby performing primary purification. The volcanic stone is preferably washed several times with water before use to remove contaminants, and after completely removing water, is suspended at a concentration of 0.1 to 5.0 (W / W)% of the extraction solution.

Next is a secondary purification step, the acid solution, for example acetic acid solution is added to the filtrate after the first step to adjust the pH to 5.0 to 5.5, and then 0.1 to 5.0 M sodium hydroxide is added to complete the solid content Dissolve and homogenize. It is left for 1 to 72 hours in the refrigerated state and the supernatant is collected by filtration. This process is repeated several times to perform a secondary purification process, preferably, the acid solution addition, sodium hydroxide addition, refrigeration and filtering process is preferably performed two to five times more.

After the second purification step is to perform a third purification step, this process is carried out for the desalination and removal of acetic acid. First, a gel, for example Sephadex-G gel, is sufficiently inflated with water to remove fine particles, and then the gel is filled into the prepared column and washed with sufficient flow of water. After sufficient washing with a buffer such as 0.01 M sodium phosphate buffer (pH 7.4), the separated supernatant is added to the column, preferably no more than half of the fill. Separation with 0.01 M sodium phosphate pH 7.4 buffer is used to recover fractions free of desalination and acetic acid.

The fractional water may be additionally subjected to a heat treatment process to inactivate viruses, etc., which may be included in the solution. For this purpose, a water bath having a temperature maintained at 40 to 80 ° C. may be prepared and then subjected to the purification process. After distilling the purified liquid into a glass container in a water bath, and maintaining the internal temperature of the purified liquid at about 60 ℃ and incubated for 1 to 48 hours to inactivate the virus contained in the solution to further improve the safety of the product Done. After the heat treatment is completed, the solution is quenched to room temperature, and the foreign substances generated during the heat treatment are filtered out.

Next, the purity of the final product is improved through the concentration and further purification in step (f). To this end, a concentrator is installed to prevent minimal contamination, followed by filling and rapidly concentrating the purification liquid.

When the concentration is completed, the final fourth purification process is carried out. To this end, a gel capable of molecular weight separation is filled into the column, and the gel is sufficiently washed with a buffer such as sodium phosphate buffer. Subsequently, the purification liquid is filled in an appropriate amount, and the flow rate is appropriately fractionated. Begin the fractionation to identify the initial protein and collect the fraction solution. Collection of fractions is desirable in terms of efficiency in recovering all of the proteins identified.

After finishing the fourth purification process, the sterilization filtration process may be further performed for aseptic treatment of the final product. In this case, install a 0.2 micrometer cartridge filter sterilization filter set, perform a leak test to check for abnormalities, and then treat the sterilization filter set with a high-pressure steam sterilizer to aseptically. The fractions are filtered using positive pressure. After filtration, check for abnormalities in the sterile filtration set. If anomalies occur, refiltration is essential to aseptic products.

Collagen according to the invention may be used in the form of a liquid by performing the purification process, it is also possible to use it after drying and powdering it. When powdered, it is possible to further suppress decay or deterioration of the product, which is advantageous in terms of long-term storage.

The material obtained through such a process is a pure collagen material, and can be usefully used as a raw material or an additive in various fields including beauty, health beverage, health food, medicine and cosmetics. In particular, the collagen obtained according to the production method of the present invention will have a low molecular weight and high purity, and thus its usefulness is greater.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(a) preprocessing stage

It is cut into pieces that are convenient for work by removing excess lipid layer from pig skin kept in refrigerated state in clean plastic bag. The pig skin was sufficiently submerged in 0.1 (W / V)% sodium hypochlorite refrigerated solution, soaked for about 24 hours to remove bacteria and disinfect. The sodium hypochlorite solution was removed and soaked in 0.1 (W / V)% formic acid refrigeration solution for 1 day to allow the remaining amount of hair to be removed. It was then washed several times with water to remove the lipid layer. Washed pork skin was sufficiently immersed in 0.1mMol EDTA solution containing about 50mMol Tris-HCl (pH 7.6) to remove color and dead skin in a refrigerated state for more than 12 hours. The deposition solution was then removed, washed several times with water and then drained.

The pig skin was immersed in a refrigerated state for 2 days in a 0.5 molar acetic acid solution having a capacity about twice the weight of the pig skin. At this time, while immersing every 3 hours while immersed, the separated lipids were removed by sieving to perform a primary extraction process.

Honkpi was ground and homogenized using a homogenizer (Knife Homogenizer), and 0.5 mol acetic acid solution was further added in an amount of about 2 times the total weight (total 6 times the weight). The solution was then stirred for 24 hours or more under refrigerated conditions. Lipids separated during stirring were removed by sieving to carry out a second extraction process.

(b) enzyme treatment step

After adding acetic acid to the extract to adjust the pH to 2.0, pepsin corresponding to about 0.001 (W / V)% of the total extract was dissolved in acetic acid and added. Subsequently, the mixture was stirred at a constant temperature of about 37 ° C. for 3 hours, and the separated lipids were sieved off.

(c) Dispersion solution addition and molecular weight crushing step

After adjusting to pH 5.0 by adding 10N NaOH to the enzyme-treated solution, 2.0 (V / V)% of isopropyl alcohol was added and stirred at room temperature for 1.5 hours. The separated lipids were filtered off and left to stand for at least 24 hours in a chilled state.

After standing, the lipid layer of the supernatant solution was removed and the solution was homogenized by stirring in a water bath of about 37 ° C. Further 10N NaOH was added, the pH was first adjusted to neutral, stirred and homogenized. The pH was further adjusted to 11.5-12.5 by further adding 10N NaOH. After the temperature of the internal solution reached 60 ° C in a 60 ° C water bath and stirred for 3 hours, the solution was filtered to remove foreign substances and insoluble matters. The pH was adjusted to neutral with acetic acid solution and left for 24 hours in a cold state.

(d) Separation and Removal of Foreign Materials

Primary refining

After standing, the lipid layer of the supernatant solution was removed and the solution was homogenized in a 37 ° C. water bath. Volcanic stone was washed three times with water to completely remove water and suspended at a concentration of 2.0 (W / W)% of the extract solution. The solution was then filtered to remove debris and insoluble matters.

Secondary purification

The pH was adjusted to 5.0 by adding acetic acid, and then homogenized after complete dissolution by addition of 1.0 M sodium hydroxide. It was left to stand for 24 hours or more in a refrigerated state, and the supernatant was collected by filtration. Acetic acid was added again to adjust pH to 4.0-4.5. 2.0M sodium hydroxide was further added to complete dissolution and then homogenized. After standing for 24 hours or more in a refrigerated state, the supernatant was collected by filtration. Acetic acid was added thereto to adjust the pH to 3.0 to 3.5. The solution was then homogenized by adding and stirring 1.0 M sodium hydroxide. The solution was allowed to stand for 24 hours or more in a refrigerated state, and then the supernatant was collected by filtration.

Tertiary tablets

After the Sephadex-G gel was sufficiently inflated with water to remove fine particles, the gel was filled into the prepared column and washed with sufficient flow. After being sufficiently washed with 0.01 M sodium phosphate pH 7.4 buffer, the separated supernatant was added to the column so that no more than half of the fill water. Separation with 0.01 M sodium phosphate pH 7.4 buffer recovered the desalted and acetic acid-free fractions.

(e) heat treatment and filtration steps

The constant temperature water tank was filled with water, and a thermometer was installed to maintain the temperature of the water bath at 60 ° C. Subsequently, the purified liquid was placed in a glass container and deposited in a water bath, and then the internal temperature of the purified liquid reached 60 ° C. and incubated for 10 hours to inactivate viruses and the like. The solution after the heat treatment was quenched to room temperature, and the foreign substances generated during the heat treatment were filtered out using a filtration set.

(f) concentration and fourth purification step

After the concentrator was installed to prevent minimal contamination, the purified liquid was filled and concentrated quickly. Then, a gel capable of molecular weight separation was filled into the column, and the gel was sufficiently washed with 0.01 M sodium phosphate pH 7.4 buffer. Subsequently, the purified liquid was filled in an appropriate amount and fractionated with an appropriate flow rate. Fractions were started to identify the first protein and fractions were collected. Collection of fractions recovered all of the proteins identified to produce collagen derived from pork skin.

Subsequently, a bactericidal filtration set is installed, a leak test is performed to check for abnormalities, and then the bactericidal filtration set is treated to be aseptic. Fractions were removed using positive pressure. After filtration, check for abnormalities in the sterile filtration set. If abnormality occurred, it was refiltered.

The molecular weight and the purity of the finally obtained pure collagen were measured using HPLC, and the molecular weight range was 20,000 or less and the purity was about 92%.

The method for producing collagen according to the present invention can produce high-purity low molecular weight collagen from pig skin with high efficiency, and thus obtained collagen can be used in various fields, including beauty, health beverage, health food, medicine and cosmetics. It can be usefully used as an additive.

Claims (7)

(a) a pretreatment step of removing the foreign matter by sequentially performing base treatment and acid treatment of pig skin; (b) a hydrolysis step of enzymatically treating the pretreated pig skin to obtain a hydrolyzate; (c) pretreatment by adding the dispersion solution to the obtained hydrolyzate, followed by adding a basic solution to break down the high molecular weight hydrolyzate to a low molecular weight; (d) a purification step of removing the foreign matter by separating the obtained low molecular weight hydrolyzate by material; (e) heat treating the purified fractions followed by filtration; And (F) a method for producing collagen comprising the step of purifying the low molecular weight collagen after concentrating the filtrate. The method of claim 1, further comprising drying and powdering a solution obtained by purifying the low molecular weight collagen of step (f). The method of claim 1, wherein the enzyme treatment of step (b) is a step of obtaining hydrolyzate by adding the pretreated pig skin to the acidic solution together with the hydrolase. The method for producing collagen according to claim 3, wherein the hydrolase is pepsin, trypsin, papain, relin, collagenase or cathepsin. The method of claim 1, wherein the basic solution of step (c) is an aqueous solution of sodium hydroxide, calcium hydroxide, potassium hydroxide or magnesium hydroxide. The method of claim 1, wherein the step of separating and removing the foreign matter by the material of step (d), Suspending low molecular weight hydrolyzate using volcanic stone, diatomaceous earth, talc, perlite, cellulose powder, or fine powder ion exchange resin and filtering it; Dissolving and homogenizing the hydrolyzate by adding sodium hydroxide under acidic conditions, and then filtering and recovering the supernatant obtained by standing. And A method for producing collagen, comprising the steps of adding a separated supernatant to a column packed with Sephadex-G gel and separating it with a buffer to remove desalination and acetic acid. The method of claim 1, wherein the purification of the low molecular weight collagen of step (f) comprises adding concentrated low molecular weight collagen to the column filled with Sephadex-G gel, and then fractionating it using a buffer. Method for producing collagen.