JP2007124901A - Method for extracting collagen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a human-derived I or III type collagen that has no possibility of cross infection, a high take rate and a long-lasting effect and is useful in the field of plastic surgery and cosmetic surgery. <P>SOLUTION: In the example of extraction of I type collagen, the collagen is extracted through a cleaning process for firstly homogenizing a human fat tissue and approximately removing blood and cleaning the homogenized fat tissue, a cleaning process for cleaning the fat tissue with an acidic solution containing a proteolytic enzyme inhibitor, a defatting treatment process for removing a fat component, a pepsin digestion process for digesting pepsin, a centrifugal separation process for centrifuging a soluble supernatant, a dialysis process for dialyzing the supernatant, a centrifugal separation process for centrifuging the supernatant, a salting out process for adding a buffer solution and an alkali to the supernatant and carrying out salting-out at pH 7-8, a cleaning process for cleaning a precipitate, centrifuging the precipitate and cleaning the obtained precipitate with a buffer solution, a centrifugal separation process for carrying out a centrifugal separation operation after the cleaning process and a dialysis process for dialyzing the supernatant. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for extracting type I and type III collagen used in areas such as plastic surgery and cosmetic surgery. In particular, the present invention relates to a method for extracting type I and type III collagen from adipose tissue collected from the human body so that the human body can use it with peace of mind.

Collagens constituting human skin are mainly type I collagen and type III collagen.
Currently, in the fields of plastic surgery and cosmetic surgery, subcutaneous injection type collagen preparations for skin correction such as Zyderm (collagen injection agent manufactured by Collagen USA) are widely used for correction of facial wrinkles and scars.

  The subcutaneous injection type collagen preparation for skin correction (Zyderm) is a weakly antigenic atelocollagen obtained by treating bovine skin-derived collagen with pepsin and removing most of the antigenic telopeptide portion of collagen. is there. This collagen is a milky white gel substance having a viscosity in which the collagen is dispersed in phosphate buffered saline and lidocaine is added at a rate of 0.3% as a soothing agent.

  By the way, since the above-mentioned collagen preparation is derived from animals such as cattle, it has immunogenicity and risk of cross infection.

  Thus, it has been proposed to collect a part of human-derived skin, proliferate the skin-derived cells by a culture operation, and use it as an injection.

  However, this proposed technique involves the invasion of the skin of the healthy part of the patient and requires a culturing operation, so that there is a problem that it takes time and cost.

  In the fields of plastic surgery and cosmetic surgery, free fat transplantation and aspiration fat injection have been proposed as techniques for tissue augmentation.

  This is an autograft and there is no risk of cross infection. However, it has been pointed out that the survival rate is low and the effects do not last long.

  Therefore, the problem to be solved by the present invention is to solve the above problems. That is, it is to provide human-derived type I or type III collagen that is used in areas such as plastic surgery and cosmetic surgery where there is no risk of cross-infection, high survival rate, and long-lasting effects.

The subject is a digestion step C for digesting human adipose tissue with a proteolytic enzyme,
Centrifugation step D for subjecting the supernatant after the digestion step C to a centrifugation operation,
Dialysis step E for dialysis operation on the supernatant after centrifugation step D,
Centrifugation step F, in which an acid is added to the precipitate obtained by centrifuging after the dialysis step E, and the centrifuging operation is performed.
Centrifugation step G in which the precipitate obtained by subjecting the supernatant after centrifugation step F to salting-out operation is subjected to centrifugation operation after washing,
Dialysis step H in which the supernatant after the centrifugation step G is dialyzed
It is solved by the type I collagen extraction method characterized by comprising.

In particular, in the type I collagen extraction method, the centrifugation step G includes:
A salting-out step Ga for adding a buffer solution to the supernatant after the centrifugal separation step F and adding an alkali to perform a salting-out operation in a weakly alkaline state;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
Centrifugation step Gc for performing a centrifugal separation operation after the washing step Gb
It is solved by the type I collagen extraction method characterized by comprising.

In addition, the type I collagen extraction method,
The method is solved by a type I collagen extraction method, further comprising a blood removal step A for performing blood removal treatment before the digestion step C.

In addition, the type I collagen extraction method,
A blood removal step A for performing blood removal treatment before the digestion step C;
A defatting process B for performing a defatting process after the blood removal process A and before the digestion process C
It is solved by the type I collagen extraction method characterized by further comprising.

Among them, a washing step Aa for homogenizing human adipose tissue and substantially removing and washing the contained blood,
After the washing step Aa, a washing step Ab for washing with an acidic solution containing a protease inhibitor;
After the washing step Ab, a defatting treatment step B for removing fat contained using an organic solvent,
Pepsin digestion step C, in which pepsin is added to the residue from which fat has been removed and pepsin digestion is performed after the fat removal treatment step B;
A centrifugation step D for subjecting the soluble supernatant after the pepsin digestion step C to a centrifugation operation;
A dialysis step E in which a dialysis operation is performed on the supernatant after the centrifugation step D;
Centrifugation step F in which an acid is added to a precipitate obtained by performing a centrifugation operation after the dialysis step E to perform a centrifugation operation;
A salting-out step Ga in which a buffer solution is added to the supernatant after the centrifugation step F and an alkali is added to perform a salting-out operation at a pH of 7-8;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
A centrifugal separation step Gc for performing a centrifugal separation operation after the washing step Gb;
Dialysis step H in which the supernatant after the centrifugation step Gc is dialyzed
It is solved by the type I collagen extraction method characterized by comprising.

Further, the above-mentioned problem is a digestion step C for digesting human adipose tissue with a proteolytic enzyme,
Centrifugation step D for subjecting the supernatant after the digestion step C to a centrifugation operation,
Dialysis step E for dialysis operation on the supernatant after centrifugation step D,
Centrifugation step F, in which an acid is added to the precipitate obtained by centrifuging after the dialysis step E, and the centrifuging operation is performed.
Centrifugation step G in which the precipitate obtained by subjecting the supernatant after centrifugation step F to salting-out operation is subjected to centrifugation operation after washing,
After washing the precipitate after the centrifugation step G, the centrifugation operation is performed, and the supernatant is dialyzed.
It is solved by the type III collagen extraction method characterized by comprising.

In particular, in the method for extracting type III collagen, the centrifugation step G includes:
A salting-out step Ga for adding a buffer solution to the supernatant after the centrifugal separation step F and adding an alkali to perform a salting-out operation in a weakly alkaline state;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
Centrifugation step Gc for performing a centrifugal separation operation after the washing step Gb
It is solved by the type III collagen extraction method characterized by comprising.

The type III collagen extraction method,
This is solved by a type III collagen extraction method further comprising a blood removal step A in which blood removal treatment is performed before the digestion step C.

The type III collagen extraction method,
A blood removal step A for performing blood removal treatment before the digestion step C;
A defatting process B for performing a defatting process after the blood removal process A and before the digestion process C
It is solved by the type III collagen extraction method characterized by further comprising.

Among them, a washing step Aa for homogenizing human adipose tissue and substantially removing and washing the contained blood,
After the washing step Aa, a washing step Ab for washing with an acidic solution containing a protease inhibitor;
After the washing step Ab, a defatting treatment step B for removing fat contained using an organic solvent,
Pepsin digestion step C, in which pepsin is added to the residue from which fat has been removed and pepsin digestion is performed after the fat removal treatment step B;
A centrifugation step D for subjecting the soluble supernatant after the pepsin digestion step C to a centrifugation operation;
A dialysis step E in which a dialysis operation is performed on the supernatant after the centrifugation step D;
Centrifugation step F in which an acid is added to a precipitate obtained by performing a centrifugation operation after the dialysis step E to perform a centrifugation operation;
A salting-out step Ga in which a buffer solution is added to the supernatant after the centrifugation step F and an alkali is added to perform a salting-out operation at a pH of 7-8;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
A centrifugal separation step Gc for performing a centrifugal separation operation after the washing step Gb;
Dialysis step I in which the precipitate after the centrifugation step Gc is washed, then subjected to a centrifugal separation operation, and the supernatant is subjected to a dialysis operation.
It is solved by the type III collagen extraction method characterized by comprising.

  According to the present invention, human-derived type I collagen or type III collagen can be obtained at low cost.

  Moreover, when the adipose tissue collected from the body by means such as liposuction is used as a raw material, there is no risk of cross-infection and it is considered that the safety is high.

  Furthermore, since it is type I collagen or type III collagen derived from human beings, especially because it is type I collagen or type III collagen from its own living body, the engraftment rate when injected is high and the effect is thought to last long. It is done.

The type I collagen extraction method according to the present invention includes a digestion step C for digesting human adipose tissue with a proteolytic enzyme, a centrifugation step D for subjecting the supernatant after the digestion step C to centrifugation, and a post-centrifugation step D Dialysis step E for dialysis operation on the supernatant in
Obtained by subjecting the precipitate obtained by centrifuging operation after dialysis step E to acid by adding acid to the centrifuging step F and subjecting the supernatant after centrifuging step F to salting out. A centrifugal separation step G in which a centrifugal separation operation is performed after washing the deposited precipitate, and a dialysis step H in which a dialysis operation is performed on the supernatant after the centrifugal separation step G. The centrifugation step G includes, in particular, a salting-out step Ga in which a buffer solution is added to the supernatant after the centrifugation step F and an alkali is added to perform a salting-out operation in a weakly alkaline state, and a salting-out step A washing step Gb for washing the precipitate after Ga and washing the precipitate obtained by performing a centrifugation operation with a buffer, and a centrifugation step Gc for carrying out a centrifugation operation after the washing step Gb are provided. . Prior to the digestion step C, a blood removal step A for performing blood removal treatment is further provided. Further, after the blood removal step A and before the digestion step C, a defatting treatment step B for performing a defatting treatment is further provided. In particular, a washing step Aa for homogenizing human adipose tissue and substantially removing and washing the contained blood, and a washing step Ab for washing with an acidic solution containing a protease inhibitor after the washing step Aa, After the washing step Ab, a fat removal treatment step B for removing the contained fat using an organic solvent, and after the fat removal treatment step B, pepsin is added to the residue from which the fat content has been removed. Pepsin digestion step C for performing pepsin digestion, centrifugation step D for subjecting the soluble supernatant after the pepsin digestion step C to centrifugation, and dialysis step for subjecting the supernatant after the centrifugation step D to dialysis E, a centrifugation step F in which acid is added to a precipitate obtained by performing a centrifugation operation after the dialysis step E, and a centrifugation operation is performed; and a buffer is added to the supernatant after the centrifugation step F A salting-out process Ga in which a salting-out operation is performed at a pH of 7 to 8 by adding an alkali, and a precipitate obtained after the salting-out process Ga is washed and centrifuged. A washing step Gb for washing with a buffer solution, a centrifugation step Gc for performing a centrifugation operation after the washing step Gb, and a dialysis step H for performing a dialysis operation on the supernatant after the centrifugation step Gc are provided.

  The type III collagen extraction method according to the present invention shares most of the steps of the type I collagen extraction method according to the present invention. That is, the same process can be performed up to the process close to the final process. That is, in the type I collagen extraction method, the supernatant after the centrifugation step G is subjected to a dialysis operation. The precipitate after the centrifugation step G is washed, then subjected to the centrifugation operation, and the supernatant. A type III collagen is obtained by subjecting to a dialysis operation.

  Hereinafter, specific examples will be described.

  First, 300 g of human thigh adipose tissue collected by a liposuction technique was homogenized, and blood was roughly removed with purified water. Thereafter, it was washed with purified water for 24 hours.

  Next, it was washed with 0.05 M Tris-HCl (pH 7.5) containing 1 M NaCl for 4 days. Thereafter, it was further washed with purified water for 24 hours.

  Thereafter, in order to remove serum components, a 0.5 M acetic acid solution containing 4 mM EDTA, 2 mM α-toluenesulfonyl fluoride, 10 mM N-ethylmaleimide, and 1 μg / ml Pepstatin A as a protease inhibitor is removed. Washed for 3 days. Thereafter, it was washed with purified water for 24 hours.

  Next, the washed adipose tissue (about 108 mL including tissue and water) was placed in a separatory funnel, and 270 mL of methanol and 135 mL of chloroform were added and mixed. Thereafter, the liquid in the funnel was separated into two layers. Therefore, 135 mL of chloroform was further added, and then 135 mL of purified water was added and mixed. Thereafter, the liquid in the funnel was separated into three layers. After separating into three layers, the lower layer was removed. And only the fluff layer was fractionated. The solvent was removed from the collected fluff layer, 500 mL of 0.5 M acetic acid solution containing 250 mg of pepsin was added, and pepsin digestion was performed over 24 hours.

  The soluble supernatant after pepsin digestion was subjected to ultracentrifugation at 100,000 g × 1 hour. Thereafter, the supernatant was dialyzed against 0.5 M acetic acid (containing 0.7 M NaCl), and then subjected to ultracentrifugation at 100000 g × 1 hour.

  After the ultracentrifugation operation, the precipitate (containing type I and type III collagen) was dissolved in 0.5 M acetic acid, and thereafter, ultracentrifugation operation was performed again at 100,000 g × 1 hour.

  Then, add 0.5 M Tris-HCl buffer (pH 7.4) so that the obtained supernatant becomes 0.05 M, and add 5 M NaOH while measuring the pH after providing buffer capacity. The pH was 7.4.

  The precipitate obtained by salting out (stirring for 24 hours) to 4.4 M with respect to the liquid thus obtained was added to a 0.05 M Tris-HCl buffer (pH 7.5: 4. Washed for 24 hours with 4M NaCl).

  After this washing, an ultracentrifugation operation of 100,000 g × 1 hour was performed. The resulting precipitate was washed with 0.05 M Tris-HCl buffer (pH 7.5: containing 4.4 M NaCl) for 48 hours.

  After this washing, an ultracentrifugation operation of 100,000 g × 1 hour was performed. The obtained precipitate was washed with 0.05M Tris-HCl buffer (pH 7.5: containing 2.4M NaCl) for 48 hours.

  After this washing, an ultracentrifugation operation of 100,000 g × 1 hour was performed. The resulting precipitate was washed with 0.05 M Tris-HCl buffer (pH 7.5: containing 1.7 M NaCl) for 48 hours. And after washing | cleaning, the ultracentrifugation operation of 100000 g x 1 hour was performed. The resulting supernatant was dialyzed against 0.1M acetic acid. Subsequently, freeze-drying was performed to obtain 25.5 mg of a sample (type I collagen).

  The precipitate obtained by the ultracentrifugation operation was washed with 0.05 M Tris-HCl buffer (pH 7.5: containing 1.0 M NaCl) for 48 hours.

  After this washing, an ultracentrifugation operation of 100,000 g × 1 hour was performed. The resulting supernatant was dialyzed against 0.1M acetic acid. Subsequently, freeze-drying was performed to obtain 30.4 mg of a sample (type III collagen).

  The outline of the above process is shown in FIG.

  The collagen obtained as described above was analyzed for amino acids using an amino acid analyzer (Hitachi Model 835). That is, 300 μL of 6N hydrochloric acid was added to 0.83 mg of the sample, hydrolyzed at 110 ° C. for 22 hours, and then dried under reduced pressure. The residue was dissolved in 500 μL of pure water, filtered through a 0.22 μm filter, and the filtrate was diluted 10 times and subjected to amino acid analysis. The results are shown in Table-1.

この表−１におけるハイドロキシプロリンはコラーゲンのみに存在するものであるから、抽出された試料はコラーゲンであることが判る。又、ハイドロキシプロリンは、コラーゲンに存在するアミノ酸の約１０％であり、表−１も約１０％である。 Table-1

Since hydroxyproline in Table-1 is present only in collagen, it can be seen that the extracted sample is collagen. Hydroxyproline is about 10% of amino acids present in collagen, and Table 1 is also about 10%.

  Next, the type I collagen and type III collagen obtained as described above were diluted to a concentration of 10 mg / mL with an electrophoresis sample buffer solution (non-reducing) and an electrophoresis sample buffer solution (reduction), respectively. In order to convert to SDS, the sample was heated at 95 ° C. for 5 minutes to prepare non-reduced and reduced electrophoresis samples. Then, the electrophoresis gel plate was opened, the electrophoresis apparatus was assembled, a sample of 15 μL and a molecular weight marker of 5 μL were spotted on a 10% SDS gel lane, and electrophoresis was performed at 20 mA per gel for about 2 hours.

  After the completion of electrophoresis, the gel was taken out from the electrophoresis apparatus, and the transfer apparatus was assembled from the bottom in the order of filter paper, PVDF membrane, gel, and filter paper, and transferred at 15 V for 30 minutes. After the transfer, the PVDF membrane was rinsed with purified water and then immersed in a blocking solution, followed by shaking at room temperature for 1 hour for blocking. Then, shake with TBS-Tween for 10 minutes to wash, cut into each lane, and soak a PVDF membrane into which each type of primary antibody was cut into a 2500-fold diluted solution with TBS-Tween, and overnight at 4 ° C. Shaking.

The next day, the PVDF membrane was washed twice for 10 minutes while shaking with TBS-Tween. Thereafter, the secondary antibody was immersed in a solution diluted 300 times with TBS-Tween and shaken at room temperature for 2 hours. Thereafter, the PVDF membrane was washed twice for 10 minutes while being again shaken with TBS-Tween. This washed PVDF membrane was diluted 3000 times with STBS-AP complex with TBS-Tween (streptavidin 10 μL and biotinylated Alkaline Phosphatase
10 μL was added to 30 mL of TBS-Tween) and shaken at room temperature for 2 hours. Thereafter, the plate was washed twice for 10 minutes while being shaken with TBS-Tween. After washing, it was immersed in a coloring solution and shaken until it developed color. After the color development, the color developing solution was discarded, and the mixture was washed 3 times for 10 minutes while being shaken with purified water.

  And what reacted the primary antibody with respect to type I collagen and type III collagen is shown in FIG.

As a result, four bands were confirmed in the case where the primary antibody against type I collagen was reacted. When collagen is migrated in a denatured state (a state in which the three-stranded helix structure of the molecule is broken), β and γ chains with crosslinks between α chains have low mobility, and α chains without crosslinks between chains move. Since it is large, both can be easily identified. Chain composition of type I collagen molecules, alpha ₁ chain (I type) 2 and alpha ₂ chain (I-type) is formed by one, it is determined from the intensity ratio of the electrophoretic band patterns and band, most mobile The band appearing at the bottom with the highest degree was identified as α ₂ chain (type I), and the second strongest band from the bottom was identified as α ₁ chain (type I).

In the case of reacting the primary antibody against type III collagen, one band was confirmed for the reduced type and three bands were observed for the non-reduced type. In type III collagen molecule {α ₁ chain (type III)} ₃ , since there is a disulfide bridge between α chains, the denaturation alone does not migrate much, but type III has an SS bond in the molecule, When the disulfide bond is blocked with a reducing agent, α ₁ chain (type III) shows the same mobility as α ₁ chain (type I), and it is considered that α chain and β chain are generated. Also from this result, it can be seen that those extracted as described above are type I collagen and type III collagen.

  Furthermore, it investigated by HPLC method. That is, standard type I collagen and type III collagen were prepared and subjected to a liquid chromatogram. The type I collagen and type III collagen extracted as described above were applied to a liquid chromatogram. As a result, it was found that those extracted as described above were type I collagen and type III collagen.

Flow diagram of the extraction method according to the present invention HPLC and electrophoretic diagram of collagen springed up by the method of the present invention Katsumi Utaka

Claims (10)

Digestion process C for digesting human adipose tissue with proteolytic enzymes;
Centrifugation step D for subjecting the supernatant after the digestion step C to a centrifugation operation,
Dialysis step E for dialysis operation on the supernatant after centrifugation step D,
Centrifugation step F, in which an acid is added to the precipitate obtained by centrifuging after the dialysis step E, and the centrifuging operation is performed.
Centrifugation step G in which the precipitate obtained by subjecting the supernatant after centrifugation step F to salting-out operation is subjected to centrifugation operation after washing,
Dialysis step H in which the supernatant after the centrifugation step G is dialyzed
And a method for extracting type I collagen. Centrifugation step G
A salting-out step Ga for adding a buffer solution to the supernatant after the centrifugal separation step F and adding an alkali to perform a salting-out operation in a weakly alkaline state;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
Centrifugation step Gc for performing a centrifugal separation operation after the washing step Gb
The method for extracting type I collagen according to claim 1, comprising: The method for extracting type I collagen according to claim 1 or 2, further comprising a blood removal step A for performing blood removal treatment before the digestion step C. A blood removal step A for performing blood removal treatment before the digestion step C;
A defatting process B for performing a defatting process after the blood removal process A and before the digestion process C
The type I collagen extraction method according to any one of claims 1 to 3, further comprising: A washing step Aa for homogenizing human adipose tissue and substantially removing and washing the contained blood;
After the washing step Aa, a washing step Ab for washing with an acidic solution containing a protease inhibitor;
After the washing step Ab, a defatting treatment step B for removing fat contained using an organic solvent,
Pepsin digestion step C, in which pepsin is added to the residue from which fat has been removed and pepsin digestion is performed after the fat removal treatment step B;
A centrifugation step D for subjecting the soluble supernatant after the pepsin digestion step C to a centrifugation operation;
A dialysis step E in which a dialysis operation is performed on the supernatant after the centrifugation step D;
Centrifugation step F in which an acid is added to a precipitate obtained by performing a centrifugation operation after the dialysis step E to perform a centrifugation operation;
A salting-out step Ga in which a buffer solution is added to the supernatant after the centrifugation step F and an alkali is added to perform a salting-out operation at a pH of 7-8;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
A centrifugal separation step Gc for performing a centrifugal separation operation after the washing step Gb;
Dialysis step H in which the supernatant after the centrifugation step Gc is dialyzed
And a method for extracting type I collagen. Digestion process C for digesting human adipose tissue with proteolytic enzymes;
Centrifugation step D for subjecting the supernatant after the digestion step C to a centrifugation operation,
Dialysis step E for dialysis operation on the supernatant after centrifugation step D,
Centrifugation step F, in which an acid is added to the precipitate obtained by centrifuging after the dialysis step E, and the centrifuging operation is performed.
Centrifugation step G in which the precipitate obtained by subjecting the supernatant after centrifugation step F to salting-out operation is subjected to centrifugation operation after washing,
After washing the precipitate after the centrifugation step G, the centrifugation operation is performed, and the supernatant is dialyzed.
And a method of extracting type III collagen. Centrifugation step G
A salting-out step Ga for adding a buffer solution to the supernatant after the centrifugal separation step F and adding an alkali to perform a salting-out operation in a weakly alkaline state;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
Centrifugation step Gc for performing a centrifugal separation operation after the washing step Gb
The method for extracting type III collagen according to claim 6, comprising: The method for extracting type III collagen according to claim 6 or 7, further comprising a blood removal step A for performing blood removal treatment before the digestion step C. A blood removal step A for performing blood removal treatment before the digestion step C;
A defatting process B for performing a defatting process after the blood removal process A and before the digestion process C
The type III collagen extraction method according to any one of claims 6 to 8, further comprising: A washing step Aa for homogenizing human adipose tissue and substantially removing and washing the contained blood;
After the washing step Aa, a washing step Ab for washing with an acidic solution containing a protease inhibitor;
After the washing step Ab, a defatting treatment step B for removing fat contained using an organic solvent,
Pepsin digestion step C, in which pepsin is added to the residue from which fat has been removed and pepsin digestion is performed after the fat removal treatment step B;
A centrifugation step D for subjecting the soluble supernatant after the pepsin digestion step C to a centrifugation operation;
A dialysis step E in which a dialysis operation is performed on the supernatant after the centrifugation step D;
Centrifugation step F in which an acid is added to a precipitate obtained by performing a centrifugation operation after the dialysis step E to perform a centrifugation operation;
A salting-out step Ga in which a buffer solution is added to the supernatant after the centrifugation step F and an alkali is added to perform a salting-out operation at a pH of 7-8;
A washing step Gb for washing the precipitate after the salting-out step Ga, and washing the precipitate obtained by centrifuging with a buffer;
A centrifugal separation step Gc for performing a centrifugal separation operation after the washing step Gb;
Dialysis step I in which the precipitate after the centrifugation step Gc is washed, then subjected to a centrifugal separation operation, and the supernatant is subjected to a dialysis operation.
And a method of extracting type III collagen.

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