WO2016076647A1 - Method for preparing collagenase and method for preparing collagen tripeptide using same - Google Patents

Abstract

A method for preparing collagenase of the present invention comprises: a first step for centrifuging a Bacillus subtilis strain; a second step for concentrating the centrifuged supernatant; and a third step for purifying the supernatant using ion exchange chromatography. A method for preparing a collagen tripeptide comprises: a first step for mixing pre-treated fish scales and water at a weight ratio of 2:8; a second step for heat-treating the mixture at 90°C for 5 hours; a third step for adding the collagenase prepared by the above-described method, followed by degradation at 35°C for 12 hours; a fourth step for removing foreign materials from the component in the third step through centrifugation; a fifth step for purifying the component by ion exchange chromatography; a sixth step for concentrating the purified component; a seventh step for purifying the concentrated component using activated carbon; and an eighth step for removing microorganisms from the component using a filter.

Description

Method for producing collagen degrading enzyme and method for producing collagen tripeptide using same

The present invention relates to a method for producing collagen tripeptides, and more particularly, to a method for producing collagen tripeptides having a high content of collagen tripeptides using collagen degrading enzymes that specifically produce collagen tripeptides.

Collagen is an animal's fibrous protein consisting of about 18 amino acids such as proline, oxyproline, glycine, and glutamic acid, and is a special structural protein that accounts for 30% of the 5,000 kinds of proteins that make up the human body. to be. In particular, the skin, bones, tendons are present in a lot, especially the dermal layer 70% of the skin is composed of collagen collagen plays a very important role as a skin component. Collagen is a three-stranded form of amino acids are combined in the form of a polypeptide, the molecular weight is very large, about 300,000.

Low-molecular collagen is broken down by collagen degrading enzymes and lowered to molecular weight 5,000 or less (usually 3,000 to 5,000). It is also called a collagen peptide. As it is absorbed into the human body in amino acid form. Since most proteins have molecular weight of 12,000 ~ 70,000, collagen has a molecular weight of 300,000, which is very difficult to absorb. Therefore, low molecular weight collagen is extracted, separated and purified to facilitate digestion and absorption in the human body. It is made in the form of a peptide having a molecular weight of 5,000 or less.

Collagen tripeptide is a small collagen (molecular weight of 200 ~ 500) that is linked to three amino acids (glycine-x-y), and is known to easily penetrate the skin due to its small molecular weight. On the other hand, if more than four amino acids are linked, the molecular structure is large and cannot penetrate the skin. Collagen tripeptides contribute to shortening the time to repair damaged collagen tissue by reconnecting each other immediately after skin penetration. For example, AMOREPACIFIC has applied for a patent for a composition containing collagen tripeptides to promote skin regeneration after laser treatment (Republic of Korea Publication No. 10-2013-0122569). In addition, even when ingested has a high absorption rate in the body than low molecular collagen has the advantage of maximizing the bioefficiency of collagen.

Generally, collagen is mainly made of cows, pigs, fish, squids, etc., and can be hydrolyzed by enzymes to obtain collagen peptide compositions. However, by using a protease that is generally used, the content of tripeptide is very low, and thus it is difficult to obtain a high content of the peptide peptide. Therefore, there is a steady need for an efficient manufacturing method for producing a high content of tripeptide collagen hydrolyzate.

In order to solve the above problems, it is an object of the present invention to provide a method for producing a collagen degrading enzyme having a high yield of collagen tripeptides.

Another object of the present invention is to provide a method for producing collagen tripeptides using high yield collagen degrading enzymes.

In order to achieve the above object, the present invention provides a method for producing collagen degrading enzyme, the first step of centrifuging the Bacillus subtilis strain, the second step of concentrating the centrifuged supernatant, and the ion exchange chromatography method It characterized in that it comprises a third step of purification using.

The ion exchange chromatography method is used in cation exchange chromatography, characterized in that using sodium chloride (NaCl) at a concentration of 0.1 to 0.3 M.

The ion exchange chromatography is characterized by using anion exchange chromatography and using sodium chloride (NaCl) at a concentration of 0.09 to 0.155 M.

In another embodiment of the present invention, the method for preparing collagen tripeptide is characterized by preparing collagen tripeptides using collagen degrading enzymes prepared by the method described above.

In another embodiment of the present invention, a method for preparing collagen tripeptide includes a first step of mixing pretreated young with water at a weight ratio of 2: 8, and a second step of heat-treating the mixture for 90 to 5 hours. , A third step of decomposing the collagen degrading enzyme prepared by the method described above for 35 to 12 hours, a fourth step of removing foreign matter through centrifugation from the components in the third step, and A fifth step of purifying by ion exchange chromatography, a sixth step of concentrating the purified component, a seventh step of purifying the concentrated component using activated carbon, and disinfecting the component using a filter It is characterized by including the eighth step.

In the method for producing collagen degrading enzyme according to the present invention and the method for producing collagen tripeptides using the same, the following effects are obtained.

By using a new enzyme having high tripeptide productivity in the collagen hydrolysis process through an enzyme without changing the existing manufacturing process, it is possible to simply prepare a collagen hydrolyzate having a high content of tripeptide.

1 is a separation and purification using a cationic ion resin of collagen degrading enzyme prepared according to a preferred embodiment of the present invention. A is the result of SDS-PAGE analysis of purified collagenase (M: protein size marker, conc: concentrated sample, wash: wash, E1 ~ E5: elution sample 1-5). B shows the peak of the collagenase purification according to the concentration change of the buffer in the AKTA prime (arrow is an indication for each elution samples).

Figure 2 is a separation and purification using the anion ion resin of the collagen degrading enzyme prepared by the preferred embodiment of the present invention. A is the result of SDS-PAGE analysis of purified collagenase (M: protein size marker, P1: peak 1). B shows the peak of collagen degrading enzyme purification according to the concentration change of the buffer in the AKTA prime (arrow is an indication for each elution sample).

3 is a graph showing the results of enzyme activity by temperature of collagen degrading enzyme prepared by a preferred embodiment of the present invention.

Figure 4 is a graph showing the temperature stability results over time of collagen degrading enzyme prepared by the preferred embodiment of the present invention.

5 is a graph showing the results of enzyme activity by pH of collagen degrading enzyme prepared by a preferred embodiment of the present invention.

6 is HPLC data comparing collagen tripeptide productivity of collagenase and general protease prepared by the preferred embodiment of the present invention.

Figure 7 is a production process for producing a collagen hydrolyzate having high collagen tripeptide content by using a collagen degrading enzyme prepared according to a preferred embodiment of the present invention.

8 is HPLC data analyzing collagen tripeptide (CTP) content of a product produced using collagen degrading enzyme prepared according to a preferred embodiment of the present invention.

9 is HPLC data analyzing glycine-proline-hydroxyproline content of a product produced using collagen degrading enzyme prepared according to a preferred embodiment of the present invention.

Hereinafter, with reference to the accompanying drawings, a preferred embodiment of the method for producing collagen degrading enzyme according to the present invention and a method for producing collagen tripeptides using the same will be described in detail.

The method for producing collagen degrading enzyme according to the present invention includes a first step of centrifuging a Bacillus subtilis strain, a second step of concentrating the centrifuged supernatant, and a third step of purification using ion exchange chromatography. It can be configured to include.

In another embodiment of the present invention, a method for preparing collagen tripeptides using collagen degrading enzymes includes a first step (S10) of mixing pretreated young with water at a weight ratio of 2: 8, and mixing the mixture at 90 ° C. A second step (S20) of heat treatment for a time, a third step (S30) of decomposing the collagen degrading enzyme prepared according to claim 1 for 12 hours at 35 ° C, and centrifugation at the components in the third step. A fourth step (S40) of removing foreign matter through separation, a fifth step (S50) of purifying the component by ion exchange chromatography, a sixth step (S60) of concentrating the purified component, and And a seventh step (S70) of purifying the concentrated component using activated carbon, and an eighth step (S80) of disinfecting the component using a filter.

First, specific examples for the process of preparing the collagen degrading enzyme of the present invention will be described.

<Example 1> Selection of new strains possessing collagen degrading activity

Bacillus subtilis, a GRAS (generally recognized as safe) strain, is known to be harmless to the human body and can produce a variety of proteases, lipolytic enzymes and sugar converting enzymes for food production and feed addition. It has been used a lot. Some Bacillus subtilis have also been reported to have collagen degrading activity (Nagano H et al., Biosci Biotehnol Biochem, 2000, 64 (1): 181-3; Tran LH et al., J Food Sci, 2002) Years 67 (3): 1184-7). In the present invention, a collagen degrading enzyme derived from a new Bacillus subtilis having collagen degrading activity was isolated and attempted to be applied to the collagen degrading process.

Soil samples were collected from Jinju, Gyeongsangnam-do, 30 g of soil was suspended in 270 ml of sterile PBS buffer and allowed to stand at room temperature for about 30 minutes to remove coarse soil particles and impurities. 10 ml of the supernatant was sufficiently mixed in 90 ml of sterile PBS buffer to prepare 100 ml, and the same operation was repeated again to prepare 100 ml of the third, fourth and fifth dilutions.

200 ml of the prepared 3rd, 4th, and 5th dilutions were added to a solid medium containing 1% bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl containing 1% skim milk and 1.5% agar. After culturing at 30 ° C. for 24 hours, colonies with clear zones were selected. Selected strains were incubated for 24 hours at 30 rpm at 30 ℃ in LB liquid medium 25 ℃ and the supernatant was taken to measure the titer. The titer is measured as follows. Purified pork skin was placed in a buffer (50 mM Tris-HCl (pH 7.4), 5 mM CaCl ₂ ) and dissolved at 56 ° C. to form a final 2% substrate solution. After 150 μl of substrate solution was pre-heated at 30 to 15 minutes, 100 μl of enzyme was added thereto and reacted at 30 ° C. for 30 minutes. 500 μl of 0.01 N HCl was added to stop the enzymatic reaction. 50 μl of 2% ninhydrin solution was added thereto, and the mixture was boiled for 4 minutes. The absorbance was measured at 570 nm. In the definition of enzyme unit value, 1 μmol L-leucine, which is released when reacted with substrate in pH 7.4 solution with calcium ion at 30 ° C for 30 minutes, was defined as 1 unit of enzyme.

As a result of the measurement, five strains (# 8, # 9, # 15, # 60, # 86) showed strong collagen degradation activity (Table 1). The highest five strains were selected as one of the Bacillus strains (# 86) by analyzing the physiological characteristics of the strain through the API50CHB kit (bioMerieux). In addition, strains were identified through analysis of the nucleotide sequence of the 16S rRNA gene. As a result of 16S rRNA sequencing, the selected strain showed more than 99% homology with Bacillus subtilis. This strain was named Bacillus subtilis BP and was deposited on July 13, 2015 by the Biotechnology Research Institute Gene Bank (Accession No. KCTC 12866BP).

Enzyme Activity of Selected Strains (U / ml)

Sample	U / ml	Sample	U / ml	Sample	U / ml	Sample	U / ml	Sample	U / ml
#One	0.51	# 64	0.16	# 156	1.24	# 232	0.57	# 465	0.76
# 3	0.12	# 71	0.68	# 158	0.98	# 240	0.67	# 474	1.08
#8	4.65	# 86	3.41	# 161	0.21	# 261	0.43	# 568	0.60
# 9	2.90	# 103	0.57	# 166	0.43	# 263	0.38	# 577	0.71
# 10	0.56	# 110	0.69	# 169	1.42	# 265	0.28	# 592	0.54
# 15	2.60	# 115	0.45	# 173	0.67	# 274	1.21	# 603	0.31
# 36	0.55	# 117	0.32	# 176	0.53	# 394	0.89	# 615	0.37
# 38	0.1	# 133	0.31	# 201	0.45	# 439	0.74	# 702	0.29
# 51	0.21	# 140	0.86	# 204	0.31	# 450	0.12	# 716	0.51
# 60	2.92	# 142	0.12	# 209	0.13	# 463	0.36	# 773	0.43

Example 2 Isolation of New Collagen Degrading Enzyme (BP)

Isolation and purification of Bacillus-derived collagen degrading enzyme (BP) was attempted. The strains were incubated in 1000 ml of mTB medium (yeast extract 2.4%, tryptone 1.2%, glycerol 1%, KH2PO4 2.31%, K2HPO4 12.54%) for 24 hours at 30 ° C. at 180 rpm, and then centrifuged at 6500 rpm for 15 minutes. . The resulting supernatant was carefully transferred to a new tube and concentrated 5 times using a 30 kDa filter. The concentrated supernatant was purified by ion exchange chromatography using an AKTA prime apparatus.

Purification conditions used were A buffer (50 mM Tris-HCl, pH 7.5) as a binding buffer and gradient using B buffer (50 mM Tris-HCl, pH 7.5, 0.5 M NaCl). The flow rate was flowed at 5 ml / min. The purification was performed using cation exchange chromatography and anion exchange chromatography. As the ion resin, SP sepharose resin (GE Healthcare, new-Jersey, USA), which is a cation resin, and Q sepharose resin (GE Healthcare, new-Jersey, USA) respectively.

Initial cation exchange chromatography gave 0.5 M NaCl gradient and received purified protein for each fraction. As shown in FIG. 1B, the protein purification of the fractions (E1 to E5) showing the protein pits in the purification profile was confirmed on the SDS-PAGE, and the proteins expected as target enzymes in E1 and E2 were included. , E1 was confirmed to have the most collagen degrading enzyme (arrow of Figure 1A).

NaCl concentrations of E1 to E5 are E1: 0.1-0.2 M, E2: 0.2-0.3 M, E3: 0.3-0.4 M. E4: 0.4-0.5 M, E5: 0.5 M. The NaCl concentration that can be separated was found to be 0.1-0.3 M, and the recovery rate of the target enzyme was the best at the NaCl concentration of 0.1-0.2 M, which is the E1 part. Next, E1 was purified by anion exchange chromatography.

The buffer used was 0.25 M NaCl and gave the same gradient as cation exchange chromatography. As shown in FIG. 2B, the anion exchange resin in the purification profile showed that a protein peak appeared in 0.09 to 0.155 M of NaCl (arrow of FIG. 2B).

As a result of confirming the purification degree of this fraction on SDS-PAGE, it was confirmed that the target enzyme was mostly purified (P1 of FIG. 2A). Therefore, in anion exchange chromatography, it was found that the recovery rate of the target enzyme was best in 0.09 to 0.155 M NaCl. Thereafter, the purified new collagen degrading enzyme was named BP.

Example 3 Enzyme Reaction Condition Experiment of BP

The titer was measured by performing an enzymatic reaction at a temperature of 20 ~ 70 ℃ in the optimum temperature experiment. As shown in FIG. 3, the experimental results have enzymatic activity at 30-55 ° C., and show the highest specific activity and relative activity at 50 ° C., and activity drops sharply from 60 ° C. Confirmed.

Temperature stabilization experiments were conducted for actual mass production. The temperature was adjusted to 30 ° C, 35 ° C, 40 ° C, and 50 ° C, and samples of each temperature were sampled for each hour from 0 to 12 hours to confirm their remaining activity.

As shown in FIG. 4, as a result, the lower the temperature, the more stable the enzyme was observed, and it was confirmed that the activity of the enzyme remained about 59.3% after 12 hours at 35 ° C and 40 ° C. From these results, it was desirable to set the production process of the collagen tripeptide to 35.

Next, the optimum pH was examined. Donpi gelatin substrates were prepared for each pH (50 mM citrate-Na ₂ HPO ₄ (pH 5.0-6.0), 50 mM Tris-HCl (pH 6.0-9.0), 50 mM Na ₂ CO ₃ -Na ₂ HCO ₃ ( pH 9.0 ~ 10.0)) and then determine the optimum pH by measuring the activity of the enzyme. BP showed the highest activity at pH 7.4 and high activity in the neutral zone up to pH 6 ~ 10. However, as shown in Figure 5, it was confirmed that the activity of the enzyme is similarly reduced toward the acidic or basic.

Hereinafter, a process of preparing collagen tripeptides using the collagen degrading enzyme (BP) will be described.

Example 4 Confirmation of Collagen Tripeptide Productivity of BP

Each of the four samples was prepared by uniformly mixing the weight ratio of young and water in a reactor equipped with a stirrer at a ratio of 20:80 in a pre-treated fish (fish scales) at a ratio of 20:80 for 5 hours. BP (manufacturer: Amicogen), Alcalase 2.4L FG (manufacturer: Novozyme, vendor: Biosis), Flavorzyme 1,000L (manufacturer: Novozyme, vendor: Biosis), Collupμlin MG (manufacturer: Novozyme) (Commercially available from Daejong Corp.) was used to compare collagen tripeptide productivity.

Reaction conditions (temperature, pH, enzyme use, reaction time) of each enzyme are as follows.

    BP: 35 ° C., pH 7.4, 30 unit / g (young), 12 h

    Alcalase 2.4L FG: 60 ° C, pH 7.0, 30 unit / g (young), 12 hours

    -Flavorzyme 1,000L: 55 ℃, pH 6.0, 30 unit / g (young), 12 hours

    Collupμlin MG: 60 ° C, pH 7.0, 30 unit / g (child), 12 hours

After the enzyme treatment, heat treatment at 80 ℃ 30 minutes to inactivate the enzyme. Next, comparative analysis of the four enzymes confirmed the collagen tripeptide production of BP.

Collagen peptide analysis was performed using HPLC (gilson), Superdex ^TM Peptide 10 / 300GL column, mobile phase (10 mM Tris-Cl (pH 7.4), 0.15 M NaCl, 5 mM CaCl ₂ ), flow rate: 0.5 ml / Collagen tripeptide (CTP) was analyzed under min conditions. As a standard of CTP, glycine-proline-hydroxyproline (GPH) consisting of three amino acids was used.

As shown in FIG. 6, in the case of the CTP standard, peaks were formed at about 55 minutes. As a result of comparing the degradation activity of the four enzymes, BP showed that most of the peaks were late and collagen became low molecular weight, and large peaks appeared at the same time in the standard material, indicating that the CTP content was high. Could. Alcalase 2.4L FG had higher molecular weight than other Flavorzyme 1000L and collupin MG, but showed little CPT content. As a result, it was found that BP is very effective for collagen tripeptide production.

Example 5 Establishment of high content collagen tripeptide manufacturing process using BP

A manufacturing method using BP has been established to produce products containing high collagen tripeptides. The pretreated broiler (fish scales) was uniformly mixed with the weight ratio of the broth and water in a reactor equipped with a stirrer at a ratio of 20:80 and heat-treated at about 90 ° C. for 5 hours.

The heat-treated solution was corrected to pH 7.4 in solution by adding 10% NaOH at 35 ° C. BP was added to the calibrated solution and reacted at 35 ° C. for 12 hours after adding about 30 units / g (young). After enzymatic reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 30 minutes. Enzyme-treated collagen peptide solution was removed using an ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion column purification.

The liquid was concentrated to Brix 35% using a vacuum pressure concentrator, and then decolorized and deodorized through activated carbon tablets. Activated charcoal tablets were filtered by filter press filtration and microfiltration, and then powdered with a spray dryer to perform a quality test, and a high content tripeptide was prepared through a packaging step (FIG. 7).

<Comparative Example>: Preparation of high content collagen tripeptide using general protease

Collagen tripeptide productivity was compared using Alcalase 2.4L FG (manufacturer: Novozyme, vendor: Biosis), which is typically used for low molecular weight collagen digestion for comparison with the new BP. The pretreated broiler (fish scales) was uniformly mixed with the weight ratio of the broth and water in a reactor equipped with a stirrer at a ratio of 20:80 and heat-treated at about 90 ° C. for 5 hours.

The heat-treated solution was corrected to pH 7.5 in solution by adding 10% NaOH at 55 ° C. Alcalase enzyme was added to the calibrated solution, and about 30 units / g (young) were treated at 35 ° C. for 12 hours. After enzymatic reaction, heat treatment was performed at 80 to 30 minutes to inactivate the enzyme.

Enzyme-treated collagen peptide solution was removed using an ultra-fast serial centrifuge to remove foreign substances such as metal ions through ion column purification. The liquid was concentrated to Brix 35% using a vacuum pressure concentrator, and then decolorized and deodorized through activated carbon tablets. Activated charcoal refining solution was sterilized by filter press filtration and membrane filtration, and then powdered with a spray dryer.

CTP content was carried out in the same manner as in Example 2, GPH content analysis was carried out in the following manner. HPLC (gilson) was used and the content of GPH was analyzed under the conditions of Jupiter 4u Proteo 90A column, mobile phase 10 mM Tris-Cl (pH 7.4), 5 mM CaCl ₂ , flow rate: 0.5 ml / min. Samples were analyzed for collagen hydrolysates prepared using BP and Alcalase and other companies purchased from Jellice, Japan.

As shown in Figure 8, in the case of the CTP content, it was confirmed that the peak appears at the same position as the CTP standard in the collagen hydrolyzate prepared by using BP, although the peak occurs at the same position in other products, BP sample It can be seen that the content is small because the size of the peak is small compared to.

As shown in FIG. 9, in the case of the GPH content, the pits of GPH did not appear in Alcalase as in the result of CTP, and it was found that the content of the BP sample was very high compared to other products.

As a result of quantitative comparison of collagen tripeptide (CTP) content of products produced using BP and Alcalase, BP is 56%, Alcalase is 0%, and other products have 18.1%. In the case of glycine-proline-hydroxyproline (GPH), a standard of CTP, BP contained 13.1%, Alcalase 0%, and other products 2.6%. These results show that BP can be used to prepare collagen hydrolyzate with higher content of collagen tripeptide than existing products.

Comparison of Collagen Tripeptide (CTP) and Standard (GFP) Contents

Enzyme Type	CTP content (%)	GPH content (%)
BP	56.0	13.1
Alcalase	0	0
Other company's product (Japan)	18.1	2.6

The rights of the present invention are not limited to the embodiments described above, but are defined by the claims, and those skilled in the art can make various modifications and adaptations within the scope of the claims. It is self-evident.

Collagen tripeptide is a small collagen with three amino acids linked to it, and its small molecular weight makes it easy to penetrate the skin and reconnects to each other immediately after skin penetration, thereby reducing the time for repairing damaged collagen tissue. It can be used in various ways as a cosmetic or skin treatment.

Claims (5)

1. Centrifuging the Bacillus subtilis strain;

   A second step of concentrating the centrifuged supernatant and

   A method for producing collagen degrading enzyme comprising a third step of purifying by ion exchange chromatography.
2. The method of claim 1,

   The ion exchange chromatography method,

   A method of producing collagen degrading enzyme, characterized in that it is used in cation exchange chromatography and sodium chloride (NaCl) is used at a concentration of 0.1 to 0.3 M.
3. The method of claim 1,

   The ion exchange chromatography method,

   Sodium chloride (NaCl) using anion exchange chromatography using a concentration of 0.09 to 0.155 M method of producing a collagen degrading enzyme.
4. Method for producing a collagen tripeptide using the collagen degrading enzyme prepared by claim 1 to prepare a collagen tripeptide.
5. A first step of mixing the pretreated young with water at a weight ratio of 2: 8;

   A second step of heat-treating the mixture at 90 ° C. for 5 hours;

   A third step of dissolving the collagen degrading enzyme prepared by claim 1 for 12 hours at 35 ° C .;

   A fourth step of removing foreign matter from the components in the third step by centrifugation;

   A fifth step of purifying the component by ion exchange chromatography;

   A sixth step of concentrating the purified component;

   A seventh step of purifying the concentrated component using activated carbon and

   Method of producing a collagen tripeptide comprising the eighth step of disinfecting the component using a filter.

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