KR20110062001A - Method of isolation of collagen and collagen isolated by thereof - Google Patents

Abstract

PURPOSE: A method for isolating collagen is provided to ensure environmentally-friendly property and cheaply isolate marine collagen. CONSTITUTION: A method for isolating collagen comprises: a step of adding an acidic solvent and performing sonication at 20 kHz of frequency and 0-10°C for 0.1-24 hours; a step of centrifuging the extract to obtain supernatant; a step of adding sodium chloride(NaCl) to the supernatant; and a step of isolating precipitate. The acidic solvent is acetic acid.

Description

Method of isolation and collagen isolated by {Method of isolation of collagen and collagen isolated by flowering}

The present invention relates to a method for separating collagen and collagen separated thereby, specifically, high yield of collagen separation and separation in a short time, and environmentally friendly marine collagen is separated at a low cost because a small amount of acid is used. The method for isolating collagen and thus for the collagen isolated.

Collagen is a fibrous protein contained in almost all tissues such as skin, blood vessels and teeth, and occupies about 30% of the protein constituting the body. About 40% of the collagen in the human body is present in the skin, 20% is contained in the bones and cartilage, and is widely distributed anywhere in the body such as blood vessels and intestines. Such collagen has been used for food in the form of gelatin, and the ingested collagen is digested by digestive enzymes in the digestive tract and absorbed in the form of most amino acids, which enhances immune function and promotes cell regeneration to strengthen joints. It is known to have an excellent effect on skin beauty by activating skin metabolism and maintaining moisturizing power.

Collagen is mainly extracted from animal skins, bones, and knee joints of cattle, or from human bodies or placental tissues when human collagen is needed for special purposes. However, recently, the recognition of animal collagen, such as the occurrence of BSE is deteriorating, and the value of marine collagen extracted from fish skin, scales, etc. is increasing.

Marine collagen is a type I collagen having a low melting point compared to skin collagen of livestock, and is mainly extracted with acid, causing environmental pollution due to acid.

An object of the present invention to solve the above problems is high separation yield of collagen and can be separated in a short time, because the use of a small amount of acid is environmentally friendly and low cost collagen separation method that can be separated by this separation To provide collagen.

The present invention to achieve the above object,

It provides a method for separating collagen comprising a collagen extraction step of adding an acidic solvent and sonication to extract collagen.

The acidic solvent may be acetic acid.

The acidic solvent may be 0.01 to 0.5 M concentration.

The acidic solvent may be added at 100 to 300 parts by weight based on the sample to be treated.

The sonication may be performed for 0.1 to 24 hours.

The ultrasonic wave may use a frequency of 20 kHz.

The ultrasound may have an amplitude of 20 to 80%.

The ultrasonic treatment may be performed at 0 to 10 ℃.

The collagen separation method may include a centrifugation step of obtaining a supernatant by centrifuging the solution obtained after the collagen extraction step; And a separation step of precipitating by adding sodium chloride (NaCl) to the supernatant and dialysis the precipitate.

In order to achieve the other object of the present invention,

It provides collagen obtained by the method for separating collagen.

The collagen separation method of the present invention not only has high separation yield of collagen, but also can separate collagen with high yield within a short time. In addition, since the collagen can be separated in a high yield even with a small amount of acid when the collagen is separated, the amount of acid used can be reduced, so it can be said to be environmentally friendly.

According to the collagen separation method of the present invention, high-molecular-weight collagen, which is not a hydrolyzate form of collagen, may be extracted while maintaining the basic structure.

According to the collagen separation method of the present invention, marine collagen, which has recently increased in value, can be separated.

Collagen isolated by the method of the present invention can be usefully used in cosmetics, external application for skin or health food.

Hereinafter, the present invention will be described in detail so that those skilled in the art can easily practice.

Collagen (collagen) separation method of the present invention includes a collagen extraction step of extracting collagen by adding an acidic solvent and sonication. The collagen separation method is capable of separating collagen in a high yield while using less acid by sonication in the presence of an acid solvent, it is possible to separate the collagen in a desired yield in a shorter time than the conventional acid treatment method.

By this method, marine collagen derived from marine organisms such as fish skin and scales can be separated. The sample can be used while stored at a temperature below -20 ℃ after the pretreatment process to remove and degreasing salt-soluble protein. Specifically, after washing the skin and scales of marine organisms and cutting them to a certain size, 10 to 30 parts by weight of sodium chloride (NaCl) solution is added to the sample and stirred, followed by centrifugation at 5,000 to 10,000 rpm to remove the supernatant. Salt-soluble proteins can be removed. This process can be repeated several times at a temperature of less than 4 ℃. The degreasing process may be carried out by washing the precipitate obtained after the centrifugation with purified water and stirring for at least 24 hours by adding ethanol.

The collagen extraction step is a process of adding an acidic solvent to the marine organism sample subjected to the pretreatment. Collagen is dissolved and extracted by the acidic solvent.

The acidic solvent may be hydrochloric acid or acetic acid, but is not limited thereto. Preferably, acetic acid can be used.

The acidic solvent may be added at a concentration of 0.01 to 0.5 M. Collagen is known to be insoluble in water, dilute acid and dilute alkali, but in the present invention, since the sonication is performed in parallel, the collagen can be extracted even when treated with the above low concentration of acid. The extraction yield of collagen is significantly lower when the acidic solvent is contained at a concentration of less than 0.01 M. When the acidic solvent is included at a concentration of more than 0.5 M, the extraction yield of the collagen is insignificant, but the acid concentration is increased. Costs are increased and are not environmentally friendly and collagen can be broken down to produce by-products in the form of hydrolysates.

The acidic solvent may be added at 100 to 300 parts by weight based on the sample to be treated. If it is added less than 100 parts by weight, the extraction rate of collagen is lowered and the extraction yield of collagen is significantly lowered, and if it exceeds 300 parts by weight, the increase in extraction rate and extraction yield is inefficient.

The collagen extraction step is an ultrasonic treatment after adding an acidic solvent to the sample subjected to the pretreatment process. Due to the nature of marine collagen derived from an acid-soluble marine organism, it is difficult to cause structural changes of collagen fibers when ultrasonication is not performed without adding an acidic solvent. Therefore, an acidic solvent is necessary for separation of collagen. In the present invention, sonication is performed in the presence of an acidic solvent so that collagen can be eluted with a high yield even in an acidic solvent having a minimum concentration. Thus, the amount of acid used for separation of collagen can be significantly reduced.

The sonication may be performed at 0 ° C. to 10 ° C., preferably at 4 ° C. In the above temperature range, the collagen may be extracted as it is, instead of the hydrolyzate, in the form of collagen, which functions in a living body having a molecular weight of about 300,000. Collagen has three polypeptide chains with molecular weights of about 100,000, forming a triple helix structure. These chains are stabilized by hydrogen bonds with each other, and when heated, these hydrophobic bonds are cleaved to form gelatin on a random coil. When treated under a temperature of less than 0 ℃, the separation of collagen may be insignificant, and if it exceeds 10 ℃ there is a fear that the hydrogen bond of the collagen is cleaved and transformed into gelatin or extracted as a hydrolyzate of collagen other than collagen.

The ultrasonic wave may have an amplitude of 20 to 80% at a frequency of 20 kHz. The person skilled in the art can arbitrarily modify the frequency and amplitude as long as it corresponds to the ultrasonic wave that can produce the amplitude at the frequency. Within the frequency and amplitude range, the collagen separation yield is excellent and the collagen functioning in vivo, rather than the hydrolyzate of collagen, can be obtained. If the range is exceeded, the increase in the separation yield of collagen is insignificant and inefficient.

The sonication may be performed for 0.1 to 24 hours. When the sonication is performed in less than 0.1 hour, the separation yield of collagen may be low, and if it exceeds 24 hours, the separation yield of collagen does not increase, but only the treatment time is long.

Collagen separation method of the present invention after the collagen extraction step centrifugation step; And a separating step.

The centrifugation step is a step of obtaining a supernatant by centrifuging the solution obtained after the collagen extraction step. By extracting the supernatant by centrifugation of the extraction solution, collagen dissolved in an acidic solution can be obtained at a higher concentration.

Specifically, the solution in which collagen is dissolved after extraction may be centrifuged at 5,000 to 10,000 rpm for 5 to 20 minutes to take only the supernatant thereof.

The separation step is a step of precipitating by adding sodium chloride (NaCl) to the supernatant obtained after the centrifugation and dialysis the precipitate. Sodium chloride precipitates the collagen dissolved in the supernatant and is purified to obtain pure collagen.

Specifically, by adding sodium chloride to about 5% by weight to the supernatant obtained after the centrifugation, the collagen can be precipitated and dialyzed with purified water to obtain collagen, and the obtained collagen can be used by lyophilization.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. This is for the purpose of illustrating the invention only, and therefore, the scope of the invention is not limited.

< Preparation > Sample Preparation

The sample was used by receiving frozen fish and sea bream (scales) of perch from Ohsung Fish Farm. Fish skin was removed from the remaining perch muscles and scales, washed with ice water to remove impurities, and cut into 1.0 cm × 1.0 cm in size. In order to completely remove the salt-soluble protein attached to the skin, 20 times of 0.5 M NaCl solution was added to the weight of the skin, stirred well, and centrifuged at 6,000 rpm for 10 minutes to remove the supernatant. This operation was repeated three times, and all operations were performed at 4 degrees C or less. The precipitate obtained by centrifugation was washed again with purified water stored at 4 ° C. or lower, and about 20 times the weight of cold ethanol was added thereto, followed by degreasing with stirring at 4 ° C. for 24 hours to obtain a purified sample. Purified samples were taken out if necessary while being stored below -20 ° C. On the other hand, young children were used while removing impurities and washing with water at -20 ℃.

< Example  1-4> Separation of Collagen by Ultrasonication

After adding acetic acid in an amount of about 200-fold 0.01 to 0.5 M to the sample prepared in the preparation example, it was subjected to ultrasonic treatment at 4 ℃ under the conditions of Table 1 below. Pulse on / off was set to 20 sec / 20 sec. The viscous solution obtained after sonication was centrifuged at 6,000 rpm for 10 minutes, and then the supernatant was separated and NaCl was added to the supernatant to 5 wt% to obtain a white precipitate. The white precipitate was centrifuged, dialyzed with purified water, and lyophilized to extract collagen.

division Amplitude
(%) Acetic acid concentration
(M) Example 1 Example 1-1 20 0.01 Example 1-2 0.1 Example 1-3 0.5 Example 2 Example 2-1 40 0.01 Example 2-2 0.1 Example 2-3 0.5 Example 3 Example 3-1 60 0.01 Example 3-2 0.1 Example 3-3 0.5 Example 4 Example 4-1 80 0.01 Example 4-2 0.1 Example 4-3 0.5

< Comparative example  1-4> Isolation of Acid Soluble Collagen

200-fold amount of 0-0.5 M acetic acid was added on the conditions of the following Table 2 with respect to the sample weight, and it extracted with stirring at 4 degreeC for 24 hours. The obtained viscous solution was centrifuged at 6,000 rpm for 10 minutes, the supernatant was separated, and NaCl was added to the supernatant to 5 wt% to obtain a white precipitate. The white precipitate was centrifuged, dialyzed with purified water, and lyophilized to extract collagen.

division Acetic acid concentration
(M) Comparative Example 1 0 Comparative Example 2 0.01 Comparative Example 3 0.1 Comparative Example 4 0.5

< Experimental Example  1> Measurement of collagen yield

The supernatant obtained by centrifuging the samples obtained in the above Examples and Comparative Examples for 20 minutes at 15,000 × g was measured for protein content using the Biuret method (Gornall, A, G., 1949). Collagen yield was calculated by the following formula as the ratio of protein content after sonication to total protein content.

Collagen yield (%) = (protein concentration in total supernatant / total protein concentration) x 100

Collagen yield with treatment time is shown in FIGS. 1 to 5. 1 to 4 show the yield of collagen according to the acetic acid concentration of Examples 1 to 4, Figure 5 shows the yield of collagen according to the acid concentration of Comparative Examples 1 to 5.

According to FIGS. 1 to 4, the yield of collagen was increased rapidly as compared with FIG. 5 without the sonication. Moreover, as the amplitude increases, the rate of increase increases rapidly. According to FIG. 5, collagen was hardly separated when treated with only acetic acid having a low concentration of 0.01 M without ultrasonication. According to FIG. 1, when treated with ultrasonic waves having 20% of amplitude at 20 kHz, Even at low concentrations of acetic acid, the amount of collagen separation began to increase. 2 to 4, the higher the concentration, the greater the increase in collagen, and the higher the amplitude under the same conditions, the faster the increase rate appeared. Therefore, the resolution of collagen seems to increase depending on the concentration of acetic acid and the ultrasonic amplitude.

< Experimental Example  2> Maximum yield measurement of collagen

The yield of collagen was measured in the same manner as in Experimental Example 1, and when the yield increased, the speed k _i was calculated by the following equation.

K _i = (n _t -n _o )

Where n _t : solubility after sonication t time

n _o : Solubility before ultrasonic treatment

t: ultrasonic processing time

The results are shown in FIGS. 6 and 7. FIG. 6 shows the maximum yield of collagen when treated with ultrasonic waves under acidic solvents of Examples 1 to 4 and when treated only with acidic solvents of Comparative Examples 1 to 4. FIG. In Fig. 6, each symbol represents 0% (?), 20% (?), 40% (?), 60% (?), And 80% (?) Amplitude. 7 shows the rate of increase in collagen yield.

According to FIG. 6, as the concentration of acetic acid is increased, as the amplitude of ultrasonic waves is increased, the maximum yield of collagen is also high, and the maximum yield when acetic acid concentration is 0.1 M and the maximum yield when 0.5 M are similar to each other. It can be assumed that even if the concentration increases above 0.5 M, the increase in maximum yield will be minimal. In addition, even when ultrasonication was performed without adding acetic acid, collagen separation hardly occurred. In the comparative example treated only with an acidic solvent, the maximum yield also increased as the concentration of acetic acid increased, but the rate of increase was slower than when the ultrasonic treatment was performed in parallel. In addition, when the concentration of acetic acid is 0 M, collagen separation does not occur, it can be seen that the acid is required for collagen separation.

According to FIG. 7, the relationship between the amplitude of the ultrasonic wave and the acetic acid concentration on the increase rate of collagen yield was examined in detail, and the linear relationship was shown under any conditions, and their respective relational expressions were as follows. In other words, the relation between acetic acid and Amplitude at 0.01 M was y = 0.0198x + 0.2296, y = 0.0418x + 0.6832 at 0.1 M, and y = 0.044x + 1.2633 at 0.5 M. According to this equation, the slope was different according to the concentration of acetic acid, the slope was increased at 0.1 M or more than 0.01 M acetic acid, and the collagen separation occurred rapidly in the presence of 0.1 M or more acetic acid. In addition, when the 0.1 M and 0.5 M acetic acid was compared, the slopes were almost similar, and the acetic acid concentration did not significantly affect the yield increase rate of collagen at an acetic acid concentration of 0.5 M or more.

< Experimental Example  3> of isolated collagen SDS - PAGE pattern

The subunit composition of the collagen obtained by the above Examples and Comparative Examples was examined by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was performed using a 7.5% slab gel by Lammli method (Lammli, V.K. et al., 1970). To the collagen samples isolated in Examples and Comparative Examples, 8 M urea, 2% mercaptoethanol, 2% SDS and 20 mM Tris-HCl (pH 8.0) were added and dissolved, and heated at 100 ° C. for 2 minutes. Fixing and staining were performed using Coomassie brillant blue by Neuhoff (Neuhoff V. et al. 1988). The results of Example 1 treated with an ultrasonic wave of 20% of amplitude and Example 3 obtained by treating with an amplitude of 60% are shown in FIG. 8, and the results of Comparative Examples 2 to 4 treated with different concentrations of acetic acid are shown in FIG. 9. Indicated.

According to FIG. 8, when treated with 20% amplitude ultrasonic waves in the presence of 0.01 M acetic acid, components corresponding to collagen began to be observed after 3 hours, and α1, α2, β, and chain were clearly observed as the ultrasonic treatment time increased. . This tendency became more pronounced as the concentration of acetic acid increased, and the alleged multimer of collagen was observed at the top of the gel. In the case of treatment with 60% of amplitude, the overall tendency was the same as that of treatment with 20% of amplitude. However, as the acidic solvent concentration increased and the sonication time increased, it was observed that components that are presumed to be decomposition products of collagen were observed. In particular, when sonication under 0.5 M acetic acid was performed for 24 hours, a decrease in α1 and β chains, which are major subunits of collagen, occurred, and an unspecific peptide was observed to stain the background of the gel.

According to FIG. 9, the subunit composition of the collagen separated and treated only with an acidic solvent was examined. As a result, α1 and β chains corresponding to collagen were observed after 6 hours of reaction under 0.01 M acetic acid. After 24 hours, α2 and chains were observed. These phenomena began to be observed more significantly as the acetic acid concentration increased, and quantitatively increased α1, α2, β and chains as the reaction time increased.

As a result, the sonication under acidic solvent increased α1, α2, β and chain equivalent to collagen more rapidly, and the sonication under acidic solvent resulted in the separation of collagen in a short time.

< Experimental Example  4> Determination of collagen separation time

According to FIG. 5, when the collagen is separated by adding 0.5 M acetic acid, about 20% of the total collagen content is separated when treated for about 24 hours. Therefore, in order to compare the treatment time, the time taken when ultrasonic treatment in the presence of acetic acid until the same yield as the yield when extracting collagen for 24 hours in the presence of 0.5 M acetic acid was measured and the results are shown in FIG. Indicated.

According to FIG. 10, it took 24 hours in 0.01 M acetic acid-40% Amplitude, but shortened to 12 hours in 0.01 M acetic acid-80% Amplitude. It also took 12 hours at 0.1 M acetic acid-40% Amplitude, 8 hours at 0.1 M acetic acid-80% Amplitude, 8 hours at 0.5 M acetic acid-40% Amplitude, and 1.5 hours at 0.5 M acetic acid-80% Amplitude. Did.

As a result, even when a small amount of acid of 0.01 M was used, the same yield as that of 0.5 M acetic acid was obtained when the sonication was performed in parallel, and when the sonication was performed under the same acidic solvent concentration (0.5 M) (80 M). % amplitude) processing time was only 1.5 hours, resulting in a 16-times reduction.

From the above results, when the acid and the ultrasonic wave are separated when separating collagen from the skin, the same yield can be obtained even with low concentration of acid, which is expected to be environmentally friendly and speed up the separation process.

< Experimental Example  5> Confirmation of Collagen

In the examples, pepsin (1: 10,000. Yakuri pure chem., Co .. ltd., In order to confirm whether collagen separated by sonication under acidic solvent was separated in the form of collagen or gelatin was isolated. Digestive power of collagen by Japan) was examined. Collagen has a very strong structure and is known to be degraded by collagenase. However, when collagen is gelatinized by heat, digestion occurs with digestive enzymes. Therefore, pepsin-treated collagen isolated by ultrasound using this property can be determined to be separated in the form of gelatin when digestion occurs, and can be determined to be separated in the form of collagen if digestion does not occur. The collagen concentration was adjusted to 1 mg / ml and gelatinized by heating at 100 ° C. for 5 minutes. 0.5% pepsin was added to the collagen and gelatin for 0-30 minutes at 10 ° C, and then 8 M SDS, 2% mercaptoethanol and 20 mM Tris-HCl (pH 8.0) were added to each sample at 100 ° C. The enzyme activity was stopped by heating for 2 minutes. Then digestion pattern was analyzed by SDS-PAGE (Lammli method). At this time, the collagen standard used Acid soluble Collagen (Type I, from white rabbit skin., Sigma, USA.). The result is shown in FIG. In Figure 11, No 1 is a reference type TYPE I collagen, No 2 is collagen separated by sonication at 80% amplitude for 12 hours in 0.01 M acetic acid, No 3, 4, 5 was treated with pepsin The result is. No6 is gelatinized by heat-treating collagen of No2 at 100 degreeC, and No7, 8, and 9 are the result of having analyzed the gelatin by pepsin. In Fig. 11, each No. Means:

S: molecular weight marker

No. 1: I type collagen (acid soluble)

No. 2: Collagen of fish skin Isolated by sonication with acetic acid.

No. 3,4,5: Collagen treated with pepsin for 10, 20 and 30 mim.

No. 6: Gelatin obtained from collagen (No. 2) by heating at 100 ° C.

No. 7,8,9: Gelatin (No. 6) treated with pepsin for 10, 20 and 30 mim.

According to FIG. 11, collagen (No. 2) isolated by ultrasonic waves in an acidic solvent did not change α and β chains, which are major components of collagen, even when pepsin treatment (No. 3, 4, and 5). However, the collagen was heat treated and gelatinized (No. 6), followed by Pepsin treatment to completely lose α and β chains, which are major components of collagen, and to increase the low molecular weight (No. 7, 8, and 9). Therefore, it was confirmed from the above results that the component separated by ultrasonic waves was collagen, not gelatin or hydrolyzate of collagen.

As described above, the collagen separation method of the present invention is environmentally friendly because it can separate collagen in the same yield obtained in the presence of a high concentration of acidic solvent even in the presence of a low concentration of acidic solvent, and it is environmentally friendly. It is separable and efficient.

In addition, it can be seen that by the separation method of the present invention, the collagen is extracted with a molecular weight of 300,000 or more, rather than a hydrolyzate of collagen.

1 to 4 show the separation yield of collagen when the collagen is separated by the method of Examples 1 to 4.

Figure 5 shows the separation yield of collagen when separated by the method of Comparative Examples 1 to 4.

Figure 6 shows the maximum yield of collagen when separated by the method of Examples 1 to 4.

Figure 7 shows the maximum yield of collagen when separated by the method of Comparative Examples 1 to 4.

Figure 8 shows the SDS-PAGE pattern of the collagen when the collagen is separated by the method of Examples 1 and 3.

Figure 9 shows the SDS-PAGE pattern of the collagen separated when the collagen is separated by the method of Comparative Examples 2 to 4.

10 shows the same yields as 24 hours treatment with 0.5M acetic acid.

The time taken for sonication in the presence of an acidic solvent is shown.

Figure 11 shows the evaluation of digestibility when gelatinized collagen and collagen isolated.

Claims (10)

And a collagen extraction step of extracting collagen by adding an acidic solvent and sonicating the collagen. The method of claim 1, Separation method of collagen, characterized in that the acidic solvent is acetic acid. The method of claim 1, Separation method of the collagen, characterized in that the acidic solvent is 0.01 to 0.5M concentration. The method of claim 1, Separation method of the collagen, characterized in that the acid solvent is added to 100 to 300 parts by weight based on the sample to be treated. The method of claim 1, Said sonication is performed for 0.1 to 24 hours. The method of claim 1, The method of separating collagen, characterized in that the frequency of the ultrasonic wave is 20 kHz. The method of claim 1, Separation method of the collagen, characterized in that the amplitude of the ultrasound is 20 to 80%. The method of claim 1, Separation method of collagen, characterized in that the sonication is carried out at 0 to 10 ℃. The method of claim 1, Centrifuging the supernatant by centrifuging the solution obtained after the collagen extraction step; And separating the collagen by adding a sodium chloride (NaCl) to the supernatant and precipitating the precipitate. Collagen, characterized in that separated by the method of any one of claims 1 to 9.

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