KR101837118B1 - A Method for Extracting Collagen using Bacterial Fermentation - Google Patents

Abstract

The present invention relates to a method for extracting collagen using a bacterial culture product, and more particularly, to a method for extracting collagen using a cultured product of bacteria without using an acid method commonly used in collagen extraction, And a method for extracting collagen which can be used as a medical raw material.

Description

A method for extracting collagen using bacterial fermentation products,

The present invention relates to a method for extracting collagen using a bacterial culture product.

Collagen is a major protein in the connective tissue of the body, accounting for a very large portion of 25 to 35% of body composition proteins. The collagen composition ratio of the human body was 18% in the dentin, 70% in the dermis under the epidermis, 50% in the articular cartilage, 80% in the bone organisms, 80% in the tendons connecting the bones and muscles, .

The collagen found in animals is mostly Type I collagen, consisting of a monomer of 300 kDa, and has a covalent bond at a specific site. Therefore, collagen found in mature tissues has low solubility. The amino acids constituting the collagen are glutamic acid, hydroxyproline, glycine, proline and alanine, and the content of hydroxyproline, which is specifically present only in collagen, is high.

As collagen becomes older, it loses its ability to synthesize in the body. At about age 18, the rate of collagen production starts to drop rapidly and is known to be less than half of that at age 40 at age 18. In addition, when older, metabolism becomes dull, old collagen does not decompose and accumulates continuously, and collagen synthesis material becomes short, and aging is promoted.

 For this reason, in the field of food, cosmetics, and medicine, collagen development and cosmetics using it, and development of edible materials and biological materials are continuously studied. Collagen obtained from cattle and pigs has been mainly used until now, but after the outbreak of mad cow disease in cattle, collagen derived from fish and other livestock products other than cattle and pigs attracted attention and was used as a raw material for cosmetics, health foods and medicines Is under review. This is because infectious diseases common to humans in fish and other livestock products, which are evolutionarily far away from humans, are relatively uncommon, and they are more safe than cattle or pig collagen.

In addition to conventional livestock such as collagen pork, there has been known a method of extracting collagen mainly by adding a chemical solvent, especially an acid, to a specific region of an animal such as a squid, a deer antler, a chicken leg, and the like.

Korean Patent Registration No. 10-892605 proposes a method for producing an extract for seasoning having high collagen content using chicken legs and a technique for utilizing the extract for seasoning. Here, techniques for bleaching and sterilizing chicken feet with a chlorine-based disinfectant, extracting proteins containing collagen by hydrothermal or proteolytic enzymes, and then filtering, separating, and concentrating them to prepare an extract for seasoning having a high collagen content is proposed.

Korean Patent Laid-Open Publication No. 2012-120571 discloses a method in which squid endothelial parts are separated and collected, washed with alkaline electrolytic water, sterilized and disinfected with acidic electrolytic water, and then squid endothelium is dried and pulverized at a moisture content of 10 to 20% And the collagen peptide is extracted by hydrolysis to concentrate and spray-dry. In Korean Patent Laid-Open Publication No. 2012-134935, an aqueous solution of sodium hydroxide is added to a dried and crushed squid skin powder, and non-collagen There has been proposed a method of extracting a collagen composition by removing proteins, neutralizing and rinsing, adding water and a Nutraceutical enzyme to hydrolyze them, and secondly hydrolyzing the proteins with a flavonoid enzyme, followed by filtration and drying.

Korean Patent Registration No. 10-1105603 discloses a method of treating collagen with high purity through acid dissolution, pepsin treatment, salt precipitation, filtration, secondary acid dissolution, secondary salt precipitation, phase separation and concentration, and acid dissolution in animal tissues Has been proposed. Here, the animal tissue is softened with a hydrochloric acid solution, and the collagen is separated from the tissue by using a pepsin enzyme, and the precipitated collagen is precipitated with sodium chloride to recover the enzyme-separated collagen, which is then dissolved in a phosphoric acid solution and diluted After removing the collagen by filtration and salt precipitation, the collagen precipitate is pressurized, concentrated, redissolved in a phosphoric acid solution and neutralized with a sodium hydroxide solution to propose a method of producing a collagen solution. However, such a collagen extraction method is a typical extraction method using an acid, a base treatment and an enzyme treatment, and has a problem in that it is difficult to commercialize it because the process is complicated and the purity is poor.

Korean Patent Laid-Open Publication No. 2012-76901 discloses a method for preparing duck collagen for preparing a collagen extract having high collagen content which can be used as cosmetic raw materials and food raw materials from duck wings and flippers, A raw material pre-treatment step of quenching each of the small pieces together with the husks at a temperature of less than -20 ° C, a step of removing bleached and excessive fats by using sodium hydroxide, and a step of removing soluble collagen extract , A desalting step of removing the salt used for extraction, and a drying step of obtaining a dried collagen-containing dried product by concentrating and drying it. However, this method of extracting collagen is a method of extracting collagen using an acid and a base at the tissue part of a duck, and it focuses on improvement of extraction efficiency of collagen through removal of fat and drying concentration. However, it does not escape from the conventional methods and collagen content It is difficult to expect high purity in terms of its purity and also it is necessary to use a chemical reagent which is harmful to various human bodies when extracting collagen and it is necessary to improve the pollution when scaling up.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

The present inventors have studied a method for efficiently extracting high purity collagen from an animal or plant tissue. As a result, when the bacterial culture product is treated with collagen-containing tissues and the collagen is extracted at a temperature of 15 to 25 ° C., high-yield and high-purity collagen The present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for extracting collagen using a bacterial culture product.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for extracting collagen using a bacterial culture product.

The present inventors have studied a method for effectively extracting high-purity collagen from an animal or plant tissue. As a result, in the case of extracting collagen at a temperature of 15 to 25 ° C by treating the bacterial culture product with collagen-containing tissues, it is possible to extract collagen with high yield and high purity I can see that I can get it.

As used herein, the term "bacteria comprising collagen or proteolytic enzyme" is used generically to refer to bacteria that secrete an enzyme involved in collagen or proteolysis when cultured. The present inventors analyzed the bacteria through experiments and found that they secrete the enzyme involved in collagen or protein degradation in Bacillus sp. Microorganism when cultured.

According to a preferred embodiment of the present invention, the bacteria comprising the collagen or protease are Bacillus < RTI ID = 0.0 > Cereus (Bacillus cereus) or keurep Silas pneumoniae (Klebsiella pneumoniae ), but are not limited thereto.

Hereinafter, a method for extracting high-purity collagen using the cultured product of the bacteria including the collagen or protease will be described step by step.

(a) culturing a bacteria comprising a collagen or proteolytic enzyme;

First, the bacteria containing the collagen or proteolytic enzyme are cultured in a culture medium. It is preferable that the culture solution is carried out under conditions in which the bacteria can sufficiently secrete the collagen or proteolytic enzyme. Preferable conditions are, for example, to float 3 to 7 ml of bacteria per 150 to 350 ml of the liquid culture broth and incubate at 25 to 40 캜 while rotating at 100 to 200 rpm, but the optimum temperature varies depending on the type of bacteria .

In addition, the culture may contain components of the host family sufficient for the bacteria to secrete collagen or proteolytic enzymes. Components of the family, for _{example, Peptone, NH 4 H 2 PO} 4, NaCl, MgSO 4 · 7H 2 O and CaCl _{2 ·} may include the 2H ₂ O, more preferably Peptone 0.5 to 1.5%, NH ₄ H ₂ PO ₄ 0.1 to 0.5%, NaCl 0.2 to 0.8%, MgSO _{4 ·} 7H ₂ O may include 0.01 to _{0.05%, CaCl 2 · 2H 2} O 0.01 % to 0.03% tween 20, and 0.005 to 0.02%.

(b) culturing the bacteria in a culture medium The culture product  Collecting steps:

Then, the bacterial culture product containing the collagen or proteolytic enzyme is collected from the culture solution.

According to a preferred embodiment of the present invention, the collection of the bacterial culture products is carried out by centrifuging the culture medium in which the bacteria have been cultured and collecting it from the supernatant. The conditions of the centrifugation are not limited, but centrifugation is preferably performed at a speed of 5,000 to 20,000 rpm at 2 to 8 ° C for 10 to 20 minutes. A syringe filter may be used to prevent further contamination by the bacteria, preferably a 0.1 to 0.22 micron syringe filter, and more preferably a 0.1 micron syringe filter for reliable microwave plasma removal.

(c) contacting the collagen-containing animal or plant tissue with the bacteria The culture product  And extracting the collagen at a temperature of 15 to 25 DEG C:

One of the great features of the present invention is that high purity collagen is extracted at a temperature of 15 to 25 DEG C, preferably at a temperature of 20 to 25 DEG C. In the conventional extraction method, it has been common to use a large amount of an acidic solvent or to perform extraction at a low temperature of about 4 DEG C, but the present invention is characterized in that a large amount of collagen can be obtained at a high temperature of 15 to 25 DEG C.

According to one embodiment of the present invention, when extracting collagen at a high temperature of pH 5 to 8 and 15 to 25 ° C, the collagen is extracted by about 3 to 5 times, preferably 4 It was possible to extract high purity collagen at a high yield of about 5 times (FIG. 3).

According to a preferred embodiment of the present invention, the animal or plant tissue used in the present invention is minced before the bacterial culture product treatment.

The animal or plant tissue that can be used in the present invention is not limited as long as it includes a collagen component and preferably is an animal tissue, more preferably a bird, a mammal or a fish tissue, most preferably a duck's foot, May be used.

The tissue may be used as it is, but the tissue may be cut to maximize the contact between the tissue containing collagen and the culture product of bacteria, preferably a unit of about 1 mm to about 10 mm in size It is good to deviate three.

According to one embodiment of the present invention, the cleavage tissue is washed with distilled water and then added to the bacterial product at 5 to 13% (w / v) and stirred for 24 to 72 hours at a temperature of 20 to 25 ° C at 150 rpm And allowed to degrade into tissue through bacterial culture products. Thereafter, the viscous solution containing the collagen dissolved in the tissue was centrifuged at 12,000 rpm for 30 minutes at 4 ° C.

For effective collection of the extracted collagen, the present invention may further comprise the following steps.

(d) The collagen solution obtained in the collagen extraction step is collected With buffer  Termination of enzymatic reaction to stop the enzyme reaction by dialysis:

To stop the enzyme reaction, use a buffer, preferably sodium phosphate buffer. According to one embodiment of the present invention, the supernatant obtained above is collected, and 0.02 M sodium phosphate buffer (pH 7.0) is dialyzed with external dialysis solution for 12 to 48 hours to stop the enzyme reaction.

(e) dialysis step of dissolving the solution obtained by stopping the enzyme reaction in an acid, precipitating with a salt, and dialyzing with distilled water:

If the enzyme reaction is stopped through the above step, it may be further dissolved in an acid, followed by precipitation with a salt and dialysis with distilled water. According to one embodiment of the present invention, the solution in which the enzyme reaction is stopped is collected and centrifuged to obtain pellets. The pellet is dissolved in 0.5 M of acetic acid, and 1 M of salt is added thereto. After 12 to 24 hours of precipitation, (3500 Mw), dialyzing the distilled water for 24 to 48 hours or more with dialysis external fluid, collecting the collagen, and lyophilizing it to finally obtain high-purity collagen.

The collagen extracted by the process according to the present invention has a protein content of 30% or more lower than that of the existing collagen and a hydroxyproline content of 5 to 10 times higher than that of the conventional collagen. In addition, most of collagen type 1 and type 3 collagen are contained and their viscosity is also excellent, and thus they are very suitable for cosmetics, edible and medical biomaterials.

The high purity collagen extracted according to the present invention can be used in a gel or solution state, and can be used as a film type or a support made of a tissue engineering material.

The features and advantages of the present invention are summarized as follows:

(i) culturing the bacteria comprising the collagen or protease in a culture medium; Collecting the bacterial culture product from the culture medium in which the bacteria are cultured; And an animal or plant tissue containing collagen, and extracting the collagen at a temperature of 15 to 25 캜.

(Ii) Using the extraction method according to the present invention, collagen can be obtained at a high yield of 4 to 5 times or more as compared with the conventional collagen extraction method, and the use of an acidic solvent can be remarkably reduced, thereby being environmentally friendly. In addition, the extraction method according to the present invention can obtain stable high purity collagen at a temperature of 15 to 25 ° C, which is more economical than the prior art.

FIG. 1 is a result of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis showing the composition of collagen extracted and extracted according to an embodiment of the present invention.
FIG. 2 is a graph showing the amount of collagen extracted from the collagen extracted and extracted as a comparative example and the amount of hydroxyproline extracted from the collagen extracted by the method of the present invention.
Fig. 3 shows the amount and yield of collagen obtained by modifying the extraction temperature according to an embodiment of the present invention.
FIG. 4 shows a collagen sheet prepared by dissolving collagen powder obtained by freeze-drying of collagen extracted in accordance with an embodiment of the present invention in an acidic solution, followed by lyophilization and EDC / NHS cross-linking.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

1. Bacterial culture step

Bacteria were suspended in Nutrient Broth (NB) culture and incubated at 37 ° C for 24, 48 and 72 hours.

2. Bacterial product production step

_{Peptone, NH 4 H 2 PO 4} , NaCl, MgSO 4 · 7H 2 O, CaCl 2 · 2H 2 O, Tween 20 2~3 drops, after manufacturing the generation culture medium of pH 7.0 conditions, the bacteria 5 per 300 ㎖ liquid culture medium Ml was suspended and incubated in a 37 ° C CO ₂ incubator while being rotated at 150 rpm.

3. Bacterial production product collection stage

After the bacteria were cultured in the production medium for 24 hours, the culture medium in which the bacteria were suspended was collected and centrifuged at 12,000 rpm, 15 minutes, and 4 ° C. The supernatant was then filtered using a 0.1-0.22 micron syringe filter and stored frozen at -80 ° C until use.

4. Cutting tissue preparation step

The flippers were soaked in distilled water for one day to remove the blood and washed several times with distilled water. The flippers were finely cut into a size of 1 to 10 mm, and this cut tissue was used as a raw material. The cut material was stored in a refrigerator of -20 ° C or less and rapidly frozen.

5. Preparation of animal tissue containing collagen

The cut tissue is prepared by cutting and cleaving at the preparation stage of the cut tissue of an animal containing collagen. Washed with distilled water and cut to about 1 to 10 mm.

6. Collagen extraction step

The cut tissues were washed with cold distilled water, and the tissues were placed in a 10% (w / v) solution of the bacterial culture preparation prepared above, and the mixture was incubated for 48 hours at 25 ° C with stirring at 150 rpm. Respectively. The viscous solution containing the degraded collagen in the tissue was then centrifuged at 12,000 rpm.

7. Dialysis step

After the supernatant was collected, 0.02 M sodium phosphate buffer (pH 7.0) was dialyzed for 24 hours in an external dialysis solution to facilitate stable collagen removal. After the dialysis was completed, the solution was collected and centrifuged to obtain pellets. The pellet obtained above was dissolved in 0.5 M acetic acid, and a 1 M salt was added thereto. The pellet was precipitated for 12 to 24 hours, then charged into a cellulose ultrafiltration membrane (3500 Mw), dialyzed for 24 hours or longer with dialysis external solution, Were collected and lyophilized.

Comparative Example

For the same samples, the bone was removed from the flippers by the existing collagen extraction method, and the flour was cut into small pieces each with a small size, followed by quenching, followed by removal of the bleached and excessive fats by using sodium hydroxide, Soluble collagen extract is prepared and the salt is removed and concentrated and dried. As a result of the pretreatment steps, the fat removal step, the collagen extraction step, and the collagen extraction step, which are shown in Examples 1 to 5 described in Korean Patent Publication No. 10-2012-0076901, Desalting and concentration steps, etc., to obtain a collagen extract.

Experimental Example  1: Analysis of collagen composition

In order to examine the composition of the collagen extracted in the above example, SDS-PAGE analysis was performed using 5 to 20% gradient gel, and it was shown in FIG. S in Fig. 1 was selected as a standard material in the present invention as commercially available collagen purchased from Sigma. A is collagen extracted using the acid-soluble method of the comparative example, B is collagen extracted with Bacillus cereus, and C is collagen extracted with Bacillus cereus CNA1. As shown in FIG. 1, it was confirmed that type I collagen having a molecular weight of 200, 140, or 120 kDa was mostly present, and that it contained a large amount of type III collagen. Usually, animal collagen has a type 1 collagen 116kDa, type 1 collagen α2 chain 97.4 kDa, and type 2 collagen has a β chain 205 kDa.

Experimental Example  2: Analysis of collagen and protein content

In order to measure the collagen content of the collagen extracted in the above examples, the content of hydroxyproline specifically decomposed when the collagen was decomposed was measured to analyze the content of collagen. As a result of the measurement of hydroxyproline content, it was found that the content of hydroxyproline in the collagen extracted by the extraction method of the present invention was two times or more higher than that of the conventional method. The measurement results are shown in Fig. In FIG. 2, A is high-purity collagen extracted by the method of Example by culturing Bacillus cereus, Bacillus cereus CNA1, and B and C, respectively.

Experimental Example  3: Analysis of collagen yield by extraction temperature

The yield of collagen was determined by extracting collagen at different extraction temperatures according to the above examples. The extraction temperature is usually 4 ° C due to the denaturation of collagen, but in the present invention, the bacterial culture product is stably extracted at a high temperature of 15 to 25 ° C to increase the yield. When the bacterial culture product was added to 1 kg of animal tissue and extracted at 20 ° C, collagen was obtained 5 times higher than when extracted at 4 ° C (FIG. 3).

Experimental Example  4: Production of collagen gel sheet

The collagen extracted in the present invention was dissolved in MES buffer to a concentration of 10 wt% and crosslinked using EDC / NHS to prepare a collagen gel sheet which can be used for tissue engineering (FIG. 4).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

A method for extracting collagen using a bacterial culture product comprising the steps of:
(a) culturing bacteria from the Bacillus celebrity the mouse (Bacillus cereus) culture medium containing collagen, or protease;
(b) collecting the bacterial culture product from the culture medium in which the bacteria have been cultured;
(c) preparing an amputated tissue by cutting an animal or plant tissue containing collagen to a size of 1 to 10 mm; And
(d) treating the cultivated tissue without acid treatment with the bacterial culture product and extracting collagen at a temperature of 20 to 25 캜.
delete delete The method of claim 1, wherein the culture medium is _{Peptone, NH 4 H 2 PO 4} , NaCl, MgSO 4 · 7H 2 O and CaCl _{2 ·} extraction of collagen comprising the 2H ₂ O.
The method of claim 1, wherein the collection of bacterial culture products is performed by centrifuging the culture medium in which the bacteria have been cultured, and then collecting the culture from the supernatant.
6. The method of claim 5, wherein the collection from the supernatant is performed by a 0.1 to 0.22 micron syringe filter.
delete delete The method of extracting collagen according to claim 1, wherein the extraction is carried out at a pH of 5 to 8.
The method of extracting collagen according to claim 1, wherein the extraction method further comprises: (e) collecting the collagen solution obtained in the collagen extraction step and dialyzing with a buffer to stop the enzyme reaction.
The method of extracting collagen according to claim 10, wherein the buffer is sodium phosphate buffer.
The method for extracting collagen according to claim 10, wherein the extraction method further comprises: (f) a step of dialysis with distilled water by dissolving the solution obtained through the step of stopping the enzyme reaction in an acid, .
The method of extracting collagen according to claim 1, wherein the animal is a bird, a mammal, or a fish.

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