KR20160057294A

KR20160057294A - The Collagen Hydrolysate Manufacturing Method and the Collagen Tripeptides Manufacturing Method Using the Collagen Hydrolysate

Info

Publication number: KR20160057294A
Application number: KR1020150124453A
Authority: KR
Inventors: 신용철; 박철; 최수림; 왕은선; 김이수; 임재명; 정진희; 원주은; 윤지훈; 김태영
Original assignee: 아미코젠주식회사
Priority date: 2014-11-13
Filing date: 2015-09-02
Publication date: 2016-05-23
Also published as: JP6457652B2; KR101831431B1; CN107532157B; CN107532157A; JP2017537978A

Abstract

The method for producing collagenase according to the present invention comprises a first step of centrifuging the strain of Bacillus subtilis, a second step of concentrating the supernatant separated by centrifugation, and a third step of purifying by using ion exchange chromatography Wherein the step of preparing collagen tripeptide comprises a first step of mixing the pretreated child with water in a weight ratio of 2: 8, a second step of heat-treating the mixture at 90 ° C for 5 hours, , A third step of adding collagenolytic enzyme prepared by the above method and decomposing at 35 DEG C for 12 hours, a fourth step of removing foreign matters through centrifugation in the components in the third step, A sixth step of concentrating the purified component; a fifth step of purifying the concentrated component by using ion exchange chromatography; And an eighth step of sterilizing the component using a filter.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing collagen hydrolyzate and a method for producing collagen tripeptide using the same,

The present invention relates to a method for producing a collagen tripeptide, and more particularly, to a method for producing a collagen tripeptide having a high content of collagen tripeptide using a collagenase which specifically produces a collagen tripeptide.

Collagen is an animal fibrotic protein composed of about 18 amino acids such as proline, oxyproline, glycine and glutamic acid. In the case of humans, collagen is a special structural protein that accounts for 30% of the 5,000 proteins constituting the human body to be. Especially, it exists in skin, bones, tendons and especially 70% of dermis in skin is composed of collagen, and collagen plays a very important role as a constituent of skin. Collagen is a polypeptide in which various amino acids are bound to three strands and has a molecular weight of about 300,000.

Low-molecular collagen is decomposed by collagenase, etc. and is low-molecular-weighted with molecular weight less than 5,000 (usually 3,000 ~ 5,000). It is also called collagen peptide. When this low-molecular collagen enters the human body, it is further degraded by proteinase in human body. In the form of amino acids. Since the molecular weight of most proteins is 12,000 ~ 70,000, the molecular weight of collagen is very high as 300,000 and it is more difficult to be absorbed. Therefore, after the extraction, separation and purification process for easy digestion and absorption by human body with low molecular weight collagen, It is made in the form of a peptide having a molecular weight of 5,000 or less.

Collagen tripeptide refers to a small collagen (molecular weight of 200-500) with three amino acids linked (glycine-x-y) and is known to easily penetrate the skin due to its small molecular weight. On the other hand, when four or more amino acids are connected, the molecular structure is large and the skin can not penetrate. The collagen tripeptide contributes to shortening the time to repair damaged collagen tissue by being directly linked back to each other after permeation of the skin. For example, AMOREPACIFIC applied for a patent on a composition containing collagen tripeptide to promote skin regeneration after laser treatment (Korea Publication No. 10-2013-0122569). In addition, when taken, the absorption rate of the body is higher than that of the low molecular weight collagen, thereby maximizing the bioactivity of the collagen.

In general, collagen is mainly used as a raw material such as beef, pig, fish, squid and the like, and can be hydrolyzed by an enzyme to obtain a collagen peptide composition. However, the content of the tripeptide is very low due to the proteolytic enzyme generally used, which makes it difficult to obtain a high-content peptidase composition. Therefore, there is a continuing need for an efficient preparation method for producing a high-content tripeptide collagen hydrolyzate.

[Patent Document 1] Korean Patent Laid-Open Publication No. 2013-0122569 (published on July 31, 2013)

[Publication 1] Nagano H et al., "Purification of collagenase and specificity of its related enzyme from Bacillus subtilis FS-2." Biosci Biotehnol Biochem, 2000, 64 (1): 181-3 [Literature 2] Tran LH et al., "Isolation and Characteristics of Bacillus subtilis CN2 and its Collagenase Production" J Food Sci, 2002 67 (3): 1184-7

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

Another object of the present invention is to provide a method for producing a collagen tripeptide using a collagenase having a high yield.

In order to accomplish the above object, the present invention provides a method for producing collagenase, comprising a first step of centrifuging the strain of Bacillus subtilis, a second step of concentrating the supernatant separated by centrifugation, an ion exchange chromatography method And a third step of purifying by using the catalyst.

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

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

According to another embodiment of the present invention, a collagen tripeptide is produced by using the collagenase produced by the method described above.

In another aspect of the present invention, there is provided a method for preparing collagen tripeptide comprising the steps of: mixing pre-treated young children with water at a weight ratio of 2: 8; , A third step of adding collagenolytic enzyme prepared by the above method to decompose for 35 to 12 hours, a fourth step of removing foreign matters through centrifugation in the components in the third step, A sixth step of purifying the purified component by ion exchange chromatography, a sixth step of concentrating the purified component, a seventh step of purifying the concentrated component using activated charcoal, And an eighth step.

The method for producing collagenase according to the present invention and the method for producing collagen tripeptide using the same have the following effects.

It is possible to produce a collagen hydrolyzate having a high content of tripeptide in a simple manner by using a novel enzyme having high tripeptide productivity in the collagen hydrolysis process using the enzyme without changing the conventional manufacturing process.

1 is a separation and purification using a cationic ion generator of collagenolytic enzyme prepared according to a preferred embodiment of the present invention. A is the result of SDS-PAGE analysis of the purified collagenase (M: protein size marker, conc: concentrated sample, wash: wash, E1 to E5: elution sample 1 to 5). B shows the peak of collagenase degradation according to the change of buffer concentration in AKTA prime (the arrows indicate the respective elution samples).
FIG. 2 is a separation and purification using an anion ion resin of collagenolytic enzyme prepared according to a preferred embodiment of the present invention. A is the result of SDS-PAGE analysis of the purified collagenase (M: protein size marker, P1: peak 1). B shows the peak of collagenase degradation according to the change of buffer concentration in AKTA prime (arrows are for each elution sample).
FIG. 3 is a graph showing the results of enzymatic activity of the collagenase according to the present invention produced by a preferred embodiment of the present invention. FIG.
4 is a graph showing the temperature stability results of the collagenolytic enzyme prepared according to the preferred embodiment of the present invention with time.
FIG. 5 is a graph showing the results of enzymatic activity of collagenase according to pH, which is produced according to a preferred embodiment of the present invention.
FIG. 6 shows HPLC data comparing collagen tripeptide productivity of collagenase and general protease prepared by the preferred embodiment of the present invention.
FIG. 7 is a process for producing a collagen hydrolyzate having a high content of collagen tripeptide using the collagenase produced by the preferred embodiment of the present invention.
FIG. 8 is HPLC data of collagen tripeptide (CTP) content of a product produced using the collagenase produced by the preferred embodiment of the present invention.
FIG. 9 shows HPLC data of the glycine-proline-hydroxyproline content of a product produced using the collagenase produced by the preferred embodiment of the present invention.

Hereinafter, preferred embodiments of a method for producing collagenase according to the present invention and a method for producing collagen tripeptide using the same will be described in detail with reference to the accompanying drawings.

The method for producing collagenase according to the present invention comprises a first step of centrifuging the strain of Bacillus subtilis, a second step of concentrating the supernatant separated by centrifugation, and a third step of purifying by using ion exchange chromatography And the like.

In another aspect of the present invention, there is provided a method for preparing a collagen tripeptide using collagenase, comprising the steps of: (a) mixing a pretreated child with water at a weight ratio of 2: 8 (S10) A third step (S30) of decomposing for 12 hours at 35 DEG C by the addition of the collagenolytic enzyme produced by the first step; and a third step (S30) of decomposing the components in the third step A fifth step S50 of purifying the components by ion exchange chromatography, a sixth step S60 of concentrating the purified components, and a fourth step S60 of removing the foreign substances through separation, A seventh step (S70) of purifying the concentrated component using activated charcoal, and an eighth step (S80) of filtering the component using a filter.

First, a specific example of a process for producing collagenase, which is the present invention, will be described.

&Lt; Example 1 > Selection of new strains possessing collagen decomposing activity

Bacillus subtilis, a GRAS (generally recognized as safe) strain, is known to be harmless to humans and can produce a variety of proteolytic enzymes, lipolytic enzymes, and glycosyltransferases. It has been widely used. In addition, some Bacillus subtilis have also been reported to have collagenolytic activity (Nagano H et al., Biosci Biotehnol Biochem, 2000, 64 (1): 181-3; Tran LH et al., J Food Sci, 2002 67 (3): 1184-7). In the present invention, a collagen degrading enzyme derived from a novel Bacillus subtilis having collagen decomposing activity was isolated and tried to be applied to a collagen degradation process.

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

200 ml of the prepared 3rd, 4th and 5th dilutions were added to LB (1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl) solid medium containing 1% skim milk and 1.5% agar After culturing at 30 ° C for 24 hours, colonies having a clear zone were selected. The selected strains were cultured in LB liquid medium at 25 캜 for 24 hours at 30 캜 and 180 rpm, and the supernatant was taken to measure the activity. The method of measuring the activity is as follows. The purified pork was placed in a buffer (50 mM Tris-HCl (pH 7.4), 5 mM CaCl ₂ ) and dissolved at 56 ° C to make a final 2% substrate solution. 150 μl of the substrate solution was pre-heated at 30 ° C for 15 minutes, and then 100 μl of the enzyme was added and reacted at 30 ° C for 30 minutes. 500 μl of 0.01 N HCl was added to stop the enzyme reaction, and 50 μl of 2% ninhydrin solution was added. After boiling for 4 minutes, absorbance was measured at 570 nm. The enzyme unit value is defined as 1 unit of L-leucine, which is liberated when reacted with substrate in pH 7.4 solution containing calcium ions at 30 ° C for 30 minutes, as 1 unit of enzyme.

As a result, five strains (# 8, # 9, # 15, # 60, and # 86) showed strong collagenolytic activity (Table 1). One of five strains (# 86) was selected by analyzing the physiological characteristics of the strains through API50 CHB kit (bioMerieux). In addition, strains were identified by analyzing the base sequence of the 16S rRNA gene. Sequence analysis of 16S rRNA revealed that the selected strains were 99% or more homologous with Bacillus subtilis. This strain was named Bacillus subtilis BP and deposited on July 13, 2015 (Accession No. KCTC 12866 BP) at the Genetics Research Institute of Biotechnology.

The enzyme activity of the selected strain (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 Collagenase (BP)

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

The 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 5 ml / min. Purification was carried out by cation exchange chromatography and anion exchange chromatography. The cation resin, SP sepharose resin (GE Healthcare, New Jersey, USA) and Q sepharose resin (GE Healthcare, new Jersey, USA), respectively.

For the first cation exchange chromatography, a gradient was applied with 0.5 M NaCl and the purified protein was obtained for each fraction. As shown in FIG. 1B, protein purification of the fractions (E1 to E5) in which the protein pits appeared in the purification profile was confirmed by SDS-PAGE. As a result, proteins expected to be the target enzymes were contained in E1 and E2 , It was confirmed that E1 contained the greatest amount of collagenase (arrow mark in Fig. 1A).

Since the NaCl concentrations of E1 to E5 are 0.1 to 0.2 M in E1, 0.2 to 0.3 M in E2, 0.3 to 0.4 in M. E4, 0.4 to 0.5 M in E5 and 0.5 M in E5, The separable NaCl concentration was found to be 0.1 ~ 0.3 M, and it was found that the recovery of the target enzyme was the best at the NaCl concentration of 0.1 ~ 0.2 M which is the E1 fraction. Next, E1 was purified by anion exchange chromatography.

At this time, the buffer used was 0.25 M NaCl, and the gradient was the same as that of the cation exchange chromatography. As shown in FIG. 2B, the anion exchange resin in the purification profile showed protein peaks at 0.09-0.155 M NaCl (arrows in FIG. 2B).

The purification of this fraction was confirmed by SDS-PAGE, and it was confirmed that most of the target enzyme was purified (P1 in FIG. 2A). Therefore, in the anion exchange chromatography, the recovery of the target enzyme was the best at 0.09 to 0.155 M NaCl. Thereafter, the purified new collagenase was named BP.

&Lt; Example 3 > Experiments of enzyme reaction conditions of BP

The enzyme activity was measured at 20 ~ 70 ℃ for optimal temperature. As shown in FIG. 3, the enzyme activity is shown at 30 to 55 ° C., and the specific activity and relative activity at 50 ° C. are highest at 60 ° C., .

A temperature stabilization experiment was applied to actual mass production. The temperature was adjusted to 30 ℃, 35 ℃, 40 ℃, and 50 ℃. Sampling of each temperature sample from 0 to 12 hours was sampled to confirm residual activity.

As shown in FIG. 4, it was shown that the enzyme stabilized at a lower temperature as a result of the experiment, and 59.3% of enzyme activity remained after 12 hours at 35 ° C and 40 ° C. As a result, it was desirable to set the production process of the collagen tripeptide to 35 at present.

Next, the optimum pH was examined. (50 mM citrate-Na ₂ HPO ₄ (pH 5.0 to 6.0), 50 mM Tris-HCl (pH 6.0 to 9.0) and 50 mM Na ₂ CO ₃ -Na ₂ HCO ₃ pH 9.0 ~ 10.0)) and the enzyme activity was measured to confirm the optimum pH. BP showed the highest activity at pH 7.4 and high activity in the neutral zone from pH 6 to 10. However, as shown in Fig. 5, it has been confirmed that the activity of the enzyme is similarly lowered toward acidity or basicity.

Hereinafter, the process for producing the collagen tripeptide using the above collagenase (BP) will be described.

Example 4 Confirmation of Collagen Tripeptide Productivity of BP

The pretreated juvenile (fish scales) were uniformly mixed in a reactor equipped with a stirrer at a weight ratio of 20:80 to the water and heat-treated at about 90 ° C for 5 hours to prepare four samples. Four samples were loaded with BP (manufacturer: Amicon), Alcalase 2.4L FG (manufacturer: Novozymes, purchased from Biosys), Flavourzyme 1,000L (manufacturer: Novozymes, purchased from Biosys), Collupulin MG , Purchased from Dae Jong Sang Company) were used to compare the productivity of collagen tripeptide.

The reaction conditions (temperature, pH, enzyme usage, reaction time) of each enzyme are as follows.

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

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

- Flavourzyme 1,000 L: 55 ° C, pH 6.0, 30 unit / g (young), 12 hours

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

After the enzyme treatment, the enzyme was inactivated by heat treatment at 80 ° C for 30 minutes. Next, comparative analysis of the four enzymes was performed to confirm the collagen tripeptide producibility of BP.

The prepared collagen peptide analysis was performed using a HPLC (gilson Inc.), 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 / min, the collagen tripeptide (CTP) was analyzed. As a standard substance of CTP, glycine-proline-hydroxyproline (GPH) consisting of three amino acids was used.

As shown in FIG. 6, when the CTP standard material was analyzed, it was confirmed that a peak was formed at about 55 minutes. As a result of comparing the degradation activities of four enzymes, it was found that collagen became low molecular mass in the peak of BP mostly at the late time, and a large peak was observed in the standard substance at the same time, and the content of CTP was high I could. Alcalase 2.4L FG showed higher content of low molecular weight than other Flavourzyme 1000L and collpin MG but showed almost no CPT content. As a result, it was found that BP is very effective for production of collagen tripeptide.

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

A production method using BP was established to produce a product containing a high content collagen tripeptide. The pretreated juveniles (fish scales) were uniformly mixed in a ratio of 20:80 by weight in a reactor equipped with a stirrer and heat-treated at about 90 ° C for 5 hours.

The heat-treated solution was adjusted to pH 7.4 in solution by adding 10% NaOH at 35 캜. About 30 units / g of BP was added to the calibrated solution and reacted at 35 ° C for 12 hours. After the enzymatic reaction, the enzyme was inactivated by heat treatment at 80 ° C for 30 minutes. The enzyme-treated collagen peptide solution was removed by using an ultra-high speed continuous centrifuge and foreign substances such as metal ions were removed through ion column purification.

The solution was concentrated to 35% Brix using a vacuum decompression concentrator, and decolorized and deodorized through activated carbon purification. The purified purified activated carbon was sterilized by filter press filtration and membrane filtration, powdered by a spray dryer, quality tested, and packed to produce a high-content tripeptide (FIG. 7).

&Lt; Comparative Example >: Production of high-content collagen tripeptide using general protease

For comparison with the manufacturing process using new BP, the productivity of collagen tripeptide was compared using Alcalase 2.4L FG (manufactured by Novozymes, purchased from Biosys), which is generally used for low molecular weight collagen degradation process. The pretreated juveniles (fish scales) were uniformly mixed in a ratio of 20:80 by weight in a reactor equipped with a stirrer and heat-treated at about 90 ° C for 5 hours.

The heat-treated solution was adjusted to pH 7.5 in a solution by adding 10% NaOH at 55 캜. About 30 units / g of Alcalase enzyme was added to the calibrated solution and treated at 35 ° C for 12 hours. After the enzymatic reaction, the enzyme was inactivated by heat treatment for 80 to 30 minutes.

The enzyme-treated collagen peptide solution was removed by using an ultra-high speed continuous centrifuge and foreign substances such as metal ions were removed through ion column purification. The solution was concentrated to 35% Brix using a vacuum decompression concentrator, and decolorized and deodorized through activated carbon purification. The purified activated carbon filtration was filtered by filter press and membrane filtration, and then pulverized with a spray drier to compare the CTP and GPH contents.

The CTP content was measured in the same manner as in Example 2, and the GPH content analysis was performed as follows. The content of GPH was analyzed on a Jupiter 4u Proteo 90A column, mobile phase 10 mM Tris-Cl (pH 7.4), 5 mM CaCl ₂ , and a flow rate of 0.5 ml / min using HPLC (gilson). The samples were analyzed for collagen hydrolyzate prepared using BP and Alcalase and other products purchased from Jellice Japan.

As shown in FIG. 8, in the case of the CTP content, it was confirmed that peaks appeared at the same position as the CTP standard material in the collagen hydrolyzate prepared using BP, The peak size was small and the content was small.

As shown in FIG. 9, GPH content was not significantly different from that of other products, and the peak of the BP sample was larger than that of the other products.

As shown in Table 1, the content of collagen tripeptide (CTP) in products produced using BP and Alcalase was 56%, the content of Alcalase was 0%, and the content of other companies was 18.1% In the case of glycine-proline-hydroxyproline (GPH), which is a standard substance of CTP, 13.1% of BP, 0% of Alcalase and 2.6% of other products were contained. These results show that BP can produce collagen hydrolysates with high content of collagen tripeptide compared to existing products.

Comparison of content of collagen tripeptide (CTP) and standard substance (GFP) Enzyme type CTP content (%) GPH content (%) BP 56.0 13.1 Alcalase 0 0 Third-party products (Japan) 18.1 2.6

It is to be understood that the invention is not limited to the disclosed embodiment, but is capable of many modifications and variations within the scope of the appended claims. It is self-evident.

Claims

A first step of centrifuging the strain of Bacillus subtilis;
A second step of concentrating the centrifuged supernatant and
And a third step of purifying by using an ion exchange chromatography method.

The method according to claim 1,
In the ion exchange chromatography method,
Characterized in that sodium chloride (NaCl) is used in a concentration of 0.1 to 0.3 M in cation exchange chromatography.

The method according to claim 1,
In the ion exchange chromatography method,
Characterized in that anion exchange chromatography is used and sodium chloride (NaCl) is used at a concentration of 0.09 to 0.155 M.

A process for producing a collagen tripeptide, wherein the collagen tripeptide is produced using the collagenase produced according to claim 1.

A first step of mixing pretreated children with water at a weight ratio of 2: 8;
A second step of heat-treating the mixture at 90 DEG C for 5 hours;
A third step of adding the collagenolytic enzyme prepared according to claim 1 and decomposing at 35 DEG C for 12 hours;
A fourth step of removing foreign matters through centrifugal separation in the third step;
A fifth step of purifying the above components by ion exchange chromatography;
A sixth step of concentrating the purified component;
A seventh step of purifying the concentrated component using activated carbon, and
And a step of sterilizing the above components with a filter to produce a collagen tripeptide.