JP3516059B2 - How to extract and obtain high molecular weight sericin - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for obtaining sericin, and more specifically, sericin is extracted from a fiber (cocoon thread) spit by silkworms in a high molecular weight while suppressing its decomposition. , How to get efficiently.

[0002]

2. Description of the Related Art Fibers spun into silkworms, that is, cocoon threads, are composed of proteins of fibroin and sericin, and sericin is present in a fibroin-coated state. Traditionally, after the cocoon thread is reeled into raw silk,
It has been used by being scoured and processed into silk fabrics. Sericin itself, which constitutes the cocoon thread, is hardly soluble in hot water due to association and crystallization of sericin molecules, but it can be dissolved in alkaline hot water. Therefore, as the scouring of raw silk, usually, a method of adding a surfactant such as Marcel soap to an alkaline aqueous solution and treating at 95 ° C. or higher for several hours, a method of using a proteolytic enzyme such as papain or chymotrypsin, etc. are adopted. Has been done.

[0003]

When the conventional method of scouring with an alkaline aqueous solution is adopted, sericin tends to have a low molecular weight due to the cleavage of the molecular chain due to hydrolysis. Therefore, sericin obtained by extracting from the waste liquid from the scouring process has almost an average molecular weight of 50,000.
The following (see JP-A-1-168906 and JP-A-4-202435). Originally, sericin, which constitutes cocoon or raw silk, has an average molecular weight of 6
Despite being set at 30,000 to 300,000 (measured by SDS electrophoresis), sericin obtained by extraction from the waste liquid of the scouring process which is actually used has an average molecular weight of 50 as described above. No more than 1,000. Such sericin is mainly used as a fiber modifier.

However, in recent years, it has been noted that sericin has bioprotective properties, and new materials utilizing the unique properties of sericin have been actively developed. According to the findings of the present inventors, the average molecular weight is 50,0.
It was found that low molecular weight sericin of 00 or less is easily soluble in water, has low strength and is fragile, has no film forming property, and is not easily compatible with the human body. Furthermore, conversely, in the case of high molecular weight sericin having an average molecular weight of more than 50,000, the physical strength and the like gradually increase as it exceeds it, resulting in a water-insoluble substance having excellent film-forming properties. Therefore, it was found that the applicability to the human body is improved.

From the above points, in order to effectively utilize the bioprotective properties of sericin, it is necessary to extract and obtain high molecular weight sericin having an average molecular weight of more than 50,000. The present invention has been made based on the above technical background. That is, the present invention is a new material, for example,
It is an object of the present invention to provide a method for obtaining a high molecular weight sericin which can be expected to be used as a wound dressing material, a cosmetic additive and the like. More specifically, it is an object of the present invention to provide a method for efficiently extracting sericin from cocoon thread, raw thread, or the like while keeping its high molecular weight as much as possible while suppressing its decomposition.

[0006]

Means for Solving the Problems The present inventors have established a method for obtaining high molecular weight sericin by carefully examining the relationship between the extraction conditions (extractant and temperature) of sericin and the molecular weight of the obtained sericin. .

That is, the present invention provides (1), cocoons spun from silkworms, cocoons obtained from the cocoons, raw silk, raw silk that has not been refined or semi-refined, cloth prepared from the raw silk, and those The residual yarn is heated at a temperature over 100 ° C. for several minutes to
A method of obtaining sericin having an average molecular weight of 50,000 or more comprises extracting with an aqueous urea solution for 10 minutes .

Further, (2), the temperature exceeds 100 ° C. 13
1. Extraction with urea aqueous solution at a temperature of 0 ° C. or lower
Exists in the method of obtaining sericin. and again,
(3), cocoon spun from silkworm, cocoon yarn obtained from the cocoon, raw silk, unrefined or semi-refined raw silk, cloth prepared from the raw silk, and residual threads thereof at a temperature of more than 100 ° C. In 10 minutes or less, it exists in the method of acquiring sericin with an average molecular weight of 50,000 or more which consists of extracting with urea aqueous solution. And again (4), 100 ℃
A method of obtaining sericin to be extracted with an aqueous urea solution at a temperature of over 130 ° C and below 130 ° C.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a raw material for obtaining sericin in the present invention, basically, all protein fibers spun by silkworms of domestic silkworms and wild silkworms are targeted, and fibroin derived from these protein fibers is targeted. The raw material is a substance containing and sericin. Further, the cocoon of the sericin silkworm, which does not contain fibroin and spits only sericin, can be used as a raw material. These raw materials may be in the form of cocoon, cocoon yarn, raw silk, unwoven product of silk knitted fabric or incompletely scoured product, sericin-containing fiber, powder, film and the like, and all of them can be used as raw materials.

The extraction of sericin is 2 to 8 per 1 g of the raw material.
Immersion in 20-60 ml of an aqueous urea solution having a concentration of mol / l,
It takes a few minutes at a temperature above 100 ℃ and below 130 ℃.
U When the extraction temperature is 100 ° C. or lower, the extraction efficiency of sericin is poor, and when the extraction temperature exceeds 130 ° C., urea is easily decomposed, which is not preferable. Here, in order to obtain high-molecular-weight sericin in higher yield, it is preferable to perform extraction at a temperature as high as possible and in a short time. As extraction time
Is preferably 10 minutes or less, and longer extraction time
If so, the decomposition of sericin will occur, so be careful.
There is a point.

After completion of the extraction, separation and recovery of sericin from the extract are carried out, for example, by the following method. 1) Method using molecular sieve chromatography; Fractionation The column chromatography method using Sephadex or the like having a molecular weight of about 5,000 to 600,000 is used to collect fractions having a molecular weight of more than 50,000.

2) Method using ultrafiltration membrane; filtration is carried out using an ultrafiltration membrane having a molecular weight cutoff of 50,000 or more, and the polymer fractions are collected and concentrated.

3) A method of performing fractional precipitation with an organic solvent;
Depending on the molecular weight of the high molecular weight sericin to be extracted, a hydrophilic organic solvent such as methyl alcohol, ethyl alcohol, propyl alcohol, or acetone is added at 50 to 80% (weight ratio) to selectively precipitate the high molecular weight sericin. Let

The high-molecular-weight sericin obtained by the present invention (usually, the molecular weight of these high-molecular-weight sericin is analyzed by liquid chromatography and / or SDS electrophoresis) has a high molecular weight, so that In addition to being molded and used as a new raw material, it can be naturally used as a powder or a solution. The high molecular weight sericin obtained by the present invention is highly hygroscopic, tough, and hardly soluble in hot water.
Therefore, it is also useful as a medical material such as a wound dressing and a cosmetic additive, and also as a fiber improving material, a treating agent, a coating agent, etc. for improving the comfort of wearing a textile product. is there. Hereinafter, the present invention will be described with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

[0015]

【Example】

[Example 1] 5 g of silkworm cocoon thread was dipped in a 30 times amount of aqueous urea solution (2 mol / l concentration) and subjected to extraction treatment at 120 ° C (2 atm) for 5 minutes. This extract was applied to a column of High load Q manufactured by Pharmacia (inner diameter 2.6 cm x length 10
cm) to adsorb sericin. Then, the column was eluted with 0.2 mol sodium sulfate and the protein fractions were collected, desalted by filtration with Centriflow 50 ultrafiltration membrane manufactured by Amicon, and freeze-dried to obtain 1 g of powdered sericin. The average molecular weight of sericin obtained by this method was about 180 as measured by a chromatographic method (Sephacryl S-500).
It was 000.

Example 2 The extraction temperature was 110 ° C. (1.4
(Atmospheric pressure), the silkworm cocoon thread was extracted under the same conditions as in Example 1 above to obtain 0.75 g of sericin having an average molecular weight of about 200,000.

[Embodiment 3] The extraction temperature is 105 ° C. (1.2
(Atmospheric pressure) except that the silkworm cocoon thread was extracted under the same conditions as in Example 1 above to obtain 0.65 g of sericin having an average molecular weight of about 220,000.

[0018]

[Comparative Example 1] [Comparative Example 1] A cocoon yarn of a silkworm was extracted under the same conditions as in Example 1 except that the extraction temperature was 100 ° C (1 atm), and sericin 0. 25 g was obtained.

Comparative Example 2 The cocoon thread of domestic silkworm was extracted under the same conditions as in Example 1 except that the extraction temperature was 80 ° C. (1 atm), but sericin was hardly extracted.

[Comparative Example 3 ] 5 g of silkworm cocoon thread was dipped in 30 times the amount of water and heated to 120 ° C.
Extraction treatment was performed at (2 atm) for 5 minutes. After filtering this extract, 4 times the volume of ethyl alcohol was added and the resulting precipitate was collected by centrifugation. This was washed with ethyl alcohol for dehydration, washed with ethyl ether, dried, and then dried.
55 g of powdered sericin was obtained. The average molecular weight of sericin obtained by this method was measured by a chromatographic method (Sephacryl S-50
It was about 180,000 as measured by 0).

[Comparative Example 4 ] A cocoon thread of a silkworm was extracted under the same conditions as in Comparative Example 3 except that the extraction temperature was 110 ° C (1.4 atm), and sericin 0 having an average molecular weight of about 180,000 was prepared. 0.45 g was obtained.

[Comparative Example 5 ] A cocoon thread of a silkworm was extracted under the same conditions as in Comparative Example 3 except that the extraction temperature was 105 ° C (1.2 atm), and sericin 0 having an average molecular weight of about 200,000 was prepared. 0.25 g was obtained.

[Comparative Example 6 ] A cocoon thread of a silkworm was extracted under the same conditions as in Comparative Example 3 except that the extraction temperature was 100 ° C. (1 atm), and 0.15 g of sericin having an average molecular weight of about 200,000 was obtained. Got

Comparative Example 7 The above Comparative Example except that the extraction temperature was 80 ° C. (1 atm).
The silkworm silkworm silkworm silkworms were extracted under the same conditions as in Example 3 , but sericin was hardly extracted.

The results of the above Examples and Comparative Examples are summarized in Table 1. As is clear from Table 1, when the aqueous urea solution is used, the extraction rate is almost doubled as compared with the case where hot water containing no urea is used. Particularly, in Example 1, the raw material cocoon yarn is used. It will be understood that more than 80% of the sericin contained therein is extracted. In addition, when the aqueous urea solution is used, the extraction temperature is 1
It can be seen that when the temperature exceeds 00 ° C, the extraction rate is greatly increased.

[0026]

[Reference example]

Reference Example 1 Average molecular weight of about 220 obtained in Example 3,
000 sericin was used as an aqueous solution (Sample 1). Further, sericin having an average molecular weight of about 220,000 obtained in Example 3 was made into a 0.1 wt% sodium carbonate solution, boiled for 5 minutes, and dialyzed to obtain an aqueous sericin solution (Sample 2). ). Similarly, after boiling for 30 minutes, it was dialyzed to obtain an aqueous solution of sericin from which sodium carbonate was removed (Sample 3). Similarly, after boiling for 2 hours, it was dialyzed to obtain an aqueous solution of sericin from which sodium carbonate was removed (Sample 4). The above four samples were allowed to stand at room temperature and the time until gelation was investigated. The results and the chromatographic method (Sephacryl S-50
The molecular weight determined in 0) is shown in [Table 2].

Reference Example 2 Films (thickness 50 μm ± 5 μm) were prepared from the aqueous solutions of Sample 1 to Sample 4 of Reference Example 1 above, and the strength of each film is shown in Table 3. Strength is 2
Tensile strength of film at 0 ° C. and 65% RH (holding width 7 mm, holding interval 2 cm, pulling speed about 100 / mi
n). Each of these films was treated with steam at 110 ° C. ± 5 ° C. for 1 hour to crystallize sericin. Hot water of sericin (100 ℃ ± 3 ℃,
When the solubility was measured for 1 hour), the results shown in [Table 4] were obtained.

Here, the solubility is the original weight (A) and the precipitate in hot water after 1 hour is recovered by centrifugation (2000 G), and the weight (B) after drying is calculated by the following equation (1) ) Was calculated. (1) Hot water solubility (%) = (A−B / A) × 100

Reference Example 3 A Cg nylon knitted fabric was dipped in the aqueous sericin solution of Sample 1 to Sample 4 of Reference Example 1 for 1 hour, and the measured weight after drying was taken as Dg. Next, these were steam-treated at 110 ° C. ± 5 ° C. for 1 hour, dried, and then subjected to hot water treatment (100 ° C. ± 3 ° C., 1 hour) and dried again, and the weight measurement value was taken as Eg. The results are shown in [Table 5].

Here, the "adhesion amount of sericin" and the "adhesion amount of sericin after hot water treatment" were calculated by the following equations (1) and (2). (1) Adhesion amount of sericin (%) = (D−C) / C × 10
0 (2) Adhesion amount (%) of sericin after hot water treatment = (E-
C) / C x 100

[0031]

From the above results, the sericin obtained by the extraction method of the present invention has a large molecular weight, unlike the conventional low-molecular-weight (hydrolyzed) sericin obtained by extraction from the scouring liquid. In addition to increasing strength and film-forming ability, it also has hot water insolubility. In addition, there is an advantage that it also has the characteristics of having good hygroscopicity and touch. It is extremely useful for medical materials such as wound dressing and cosmetic additives that take advantage of the biological protection characteristic of sericin.

[0032]

[Table 1]

[0033]

[Table 2]

[0034]

[Table 3]

[0035]

[Table 4]

[0036]

[Table 5]

Continuation of front page (56) Reference JP-A-58-26809 (JP, A) JP-A-1-168906 (JP, A) JP-A-4-202435 (JP, A) JP-A-51-41401 (JP) , A) Gamo Takumi, Extraction by cleavage of disulfide bond of cocoon layer protein and fractionation by gel electrophoresis, Japan Silkworm Journal, Japan, 1973, Vol. 41, No. 1, p. 17-23 (58) Fields investigated (Int.Cl. ⁷ , DB name) C08H 1/00-1/06 D01C 3/00 C07K 16/00-16/46

Claims (4)

(57) [Claims] 1. A cocoon spun from a silkworm, a cocoon thread obtained from the cocoon, a raw silk, an unrefined or semi-refined raw silk, a cloth prepared from the raw silk, and a residual thread thereof are higher than 100 ° C. A method for obtaining sericin having an average molecular weight of 50,000 or more, which comprises extracting with an aqueous urea solution at a temperature for several minutes to 10 minutes . 2. The method for obtaining sericin according to claim 1, which comprises extracting with an aqueous urea solution at a temperature of more than 100 ° C. and not more than 130 ° C. 3. A cocoon spun from a silkworm, a cocoon thread obtained from the cocoon, a raw silk, an unrefined or semi-refined raw silk, a cloth prepared from the raw silk, and a residual thread thereof are higher than 100 ° C. A method for obtaining sericin having an average molecular weight of 50,000 or more, which comprises extracting with an aqueous urea solution at a temperature of 10 minutes or less . 4. The method for obtaining sericin according to claim 3, which comprises extracting with an aqueous urea solution at a temperature of more than 100 ° C. and not more than 130 ° C.