JP6261109B2 - Extraction and purification method of silkworm sericin - Google Patents

Description

  The present invention relates to a method for extracting and purifying sericin from silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm silkworm.

  Conventionally, silk protein is extracted from silk thread produced by silk insects including silkworms (rabbits) as well as wild silkworms and applied to, for example, food and drink, supplements, cosmetics, and pharmaceutical materials for health promotion. In particular, in recent years, interest in the functionality of sericin, a silk protein of silkworm (rabbit), has increased.

  It is known that sericin is present in the state of a colloid covered with fibrous hard protein fibroin, and its acquisition and use is not always easy compared to fibroin, but as a method for extracting sericin from silkworm (rabbit) For example, the methods of Patent Documents 1 and 2 have been proposed.

  According to Patent Document 1, a silkworm (rabbit) cocoon is scoured at a temperature of 95 ° C. or more for 3 to 4 hours with a strong alkaline aqueous solution (1.5% silicon soda) containing a surfactant such as Marcel soap and having a pH of 11.5 or more. A method for extracting sericin by treatment is described.

  In Patent Document 2, a method of sericin extraction from silkworm insects and the like in a urea solution with a concentration of 2 to 8 mol / l and extraction at a temperature of 100 ° C. to 130 ° C. for several minutes to several hours. Have been described.

JP-A-4-202435

  However, when silkworm cocoons are treated with a high-temperature alkaline aqueous solution for a long time as in the methods of Patent Documents 1 and 2, the influence of alteration, decomposition, etc. due to hydrolysis into silk protein is large. For this reason, in the case of the conventional method, the composition (amino acid composition) and molecular weight become unstable due to loss of sericin-derived components to be obtained or fibroin-derived components, etc. There is a problem that it is not always easy to specify a functional function.

  The present invention has been made in view of the circumstances as described above, suppresses the loss of sericin-derived components and the incorporation of fibroin-derived components, and extracts sericin stably and efficiently from silkworm-derived silk thread or cocoon layer. It is an object to provide a method for purification.

In order to solve the above-mentioned problems, the silkworm sericin extraction / purification method of the present invention comprises the following steps: (1) a silkworm-derived silk thread or silkworm layer is heated at a temperature of 90 ° C to 98 ° C and 0.05 to 0.07M NaOH 1 to 3 minutes to obtain a scouring solution containing silkworm sericin, and (2) dialysis of the scouring solution to obtain a silkworm sericin solution.

In this silkworm sericin extraction / purification method, the bath ratio of the silk thread or cocoon layer to the NaOH solution in the scouring step is more preferably 1:25 to 1: 100.

  According to the method of the present invention, loss of sericin-derived components and mixing of fibroin-derived components can be suppressed, and sericin is extracted and purified from silkworm-derived silk fibers stably and efficiently.

It is the flowchart which illustrated one Embodiment of the sericin extraction / purification method of this invention. 2 is a diagram showing the results of SDS-PAGE electrophoresis (silver staining) of sericin obtained in Example 1. FIG. It is a figure which shows the light absorbency of a sericin powder solution.

  As the silk thread or silkworm layer used as a raw material for the sericin extraction / purification method of the present invention, a silkworm silkworm (B. mori) belonging to the silkworm family (Bombycidae) spun is used. The silkworm may be any cultivated mulberry leaf or artificial feed. Further, as the silk thread spun by the silkworm, for example, a silkworm-shaped and oval-shaped silkworm layer formed by the silkworm before hatching may be incised, and the silkworm layer appropriately cut into pieces may be used.

  Hereinafter, an embodiment of the sericin extraction / purification method of the present invention will be described.

  The method for extracting and purifying sericin according to the present invention includes (1) a scouring step of immersing silkworm-derived silk thread or cocoon layer in an alkaline solution of 90 to 98 ° C. and 0.05 to 0.07 M to obtain a sericin containing sericin, And (2) dialyzing the scouring solution.

  In the sericin extraction / purification method of the present invention, preferably, a silkworm-derived silk thread or silkworm layer can be washed before the scouring process.

  Washing of the silk thread or the silk thread layer can be performed, for example, by immersing the silkworm-derived silk thread or silk thread layer obtained in hot water (pure water) at about 40 ° C. to 80 ° C. for about 2 to 10 minutes. In this case, the bath ratio of the kite string or cocoon layer and warm water can preferably be exemplified in the range of about 1:50 to 1: 200. In the present invention, the “bath ratio” refers to the ratio between the weight of the kite string or cocoon layer and the weight (liquid amount) of the liquid (water, alkaline solution).

  The washing process can remove foreign matters and bacteria attached to the silk thread or the silk layer. By removing impurities and the like in advance by the washing step, it is possible to prevent impurities from being mixed into the sericin extract.

  Next, in the scouring step, silk fiber that has been washed appropriately is immersed in an alkaline solution of 90 to 98 ° C. and 0.05 to 0.07 M to extract sericin.

  As the alkaline solution, for example, strong alkaline NaOH, KOH, LiOH and the like can be preferably exemplified, but among them, NaOH and KOH are preferable, and NaOH is particularly preferable. Since it is not necessary to use an expensive solvent, the cost can be kept low.

  When the concentration of the alkaline solution is in the range of 0.05 to 0.07 M, loss of sericin-derived components and mixing of fibroin-derived components can be suppressed, and sericin can be extracted stably and efficiently. When the concentration of the alkaline solution is smaller than 0.05M, extraction of sericin is insufficient or sericin is not dissolved in the solution. On the other hand, for example, when the concentration of the alkaline solution is higher than 0.07M, sericin is excessively decomposed, making it difficult to analyze the specific function, and there is a possibility that components derived from fibroin are mixed in the scouring solution.

  Furthermore, it is difficult to extract sericin efficiently when the temperature of the alkaline solution is 90 ° C or lower, whereas when the temperature of the alkaline solution is 98 ° C or higher, the sericin is excessively decomposed and has a specific function. Analysis becomes difficult, and components derived from fibroin may be mixed in the scouring solution.

  The preferred bath ratio of the silk thread or cocoon layer to the alkaline solution is in the range of about 1:25 to 1: 100, more preferably about 1:50. When the bath ratio is within this range, sericin can be extracted effectively.

  In the scouring step, the time for immersing the silk fiber in the alkaline solution can be about 1 to 10 minutes. If scouring time is less than 1 minute, extraction of sericin may be insufficient, and if scouring exceeds 10 minutes, sericin is excessively decomposed and it is difficult to analyze the specific function of sericin. At the same time, components derived from fibroin may be mixed into the scouring liquid.

  In the method of the present invention, a low-concentration (0.05 to 0.07M) alkaline solution using a strong alkali agent that has not been studied at all in the scouring process is used, thereby greatly reducing the scouring time at high temperatures. Therefore, there is no fear that the sericin-derived component is damaged.

  Furthermore, the time for immersing the silk fiber in the alkaline solution is more preferably 1 to 3 minutes. Although it depends on the bath ratio and the concentration of the alkaline solution, the yield of sericin usually does not increase even when scouring for more than 3 minutes, the sericin is decomposed and fibroin-derived components are mixed into the scouring solution. Increase the likelihood of doing so.

  Next, the scouring liquid in which the extraction residue remains is filtered using, for example, a glass filter, and separated into the extraction residue and the scouring liquid. This scouring solution contains high molecular weight sericin, while the extraction residue contains fibroin.

  Then, this scouring solution is dialyzed to obtain a sericin solution (dialysis step). In addition, you may perform the process of filtering a scouring liquid with a filter paper before this dialysis process.

  The method of dialysis of the scouring liquid is not particularly limited, but as a simple and reliable method, for example, dialysis with water can be exemplified. Specifically, for example, a commercially available cellulose tube for dialysis is filled with a scouring solution, and dialysis with running water or pure water at about 5 to 25 ° C. can be performed for a predetermined time. Dialysis can also be performed by adjusting the pH with various organic acids. Furthermore, electrodialysis using a commercially available apparatus can be employed. In addition, performing a filtration process suitably after dialysis is also considered. The sericin-derived component does not flow out by dialysis, and for example, a sericin solution from which low molecules such as pigments, fatty acids and inorganic components contained in the silk thread or cocoon layer are removed can be obtained. Sericin contained in this sericin solution has a molecular weight of about 32 to 44 kDa.

  In the method of the present invention, particularly in the scouring step, the cocoon string or cocoon layer is scoured in an extremely short time with an alkaline solution of 90 to 98 ° C. and 0.05 to 0.07 M. For this reason, the sericin solution dialyzed from the scouring solution does not contain fibroin-derived components, and sericin is not decomposed too much, and its amino acid composition and molecular weight are stable. For this reason, the extracted sericin is excellent as a sample for analyzing a specific function.

  The sericin solution can be freeze-dried to obtain a purified soluble powder of sericin, which can be used in foods, drinks, supplements, cosmetics, pharmaceutical materials, and the like. In addition, for example, soluble powder from a sericin solution can be suitably used for various functional analyzes of sericin.

  Specifically, for example, in the case of a cosmetic containing sericin (sericin powder) obtained by the method of the present invention, water (purified water, hot spring water, deep water, etc.), oil agent, surfactant, metal soap , Moisturizer, gelling agent, alcohol, water-soluble polymer, powder, pH adjuster, film-forming agent, resin, UV protection agent, inclusion compound, antibacterial agent, perfume, deodorant, salt, refreshing agent , Animal / microbe-derived extracts, plant extracts, blood circulation promoters, astringents, antiseborrheic agents, active oxygen scavengers, cell activators, keratolytic agents, enzymes, hormones, vitamins, etc. Can do.

  The sericin extraction / purification method of the present invention is not limited to the above form.

  Hereinafter, the method for extracting and purifying sericin according to the present invention will be described in more detail with reference to examples. However, the method for extracting and purifying sericin according to the present invention is not limited to the following examples.

<Example 1> Extraction and purification of silkworm-derived sericin The steps of silkworm-derived sericin extraction and purification will be described with reference to FIG. FIG. 1 is a flowchart illustrating an embodiment of the sericin extraction / purification method of the present invention.

  The material used was a cocoon layer obtained by cutting a silkworm ginger. This soot layer was washed with 65 ° C. pure water (bath ratio 1: 100) for 3 minutes to remove impurities and bacteria.

  Next, the washed soot layer was immersed in 98 ° C., 0.06 M NaOH (bath ratio 1:50) for 3 minutes to extract sericin to obtain a scouring solution.

  Further, this scouring liquid is filtered with a 17G glass filter so that no precipitate is mixed, and is separated into an extraction residue (fibroin) and a scouring liquid. Further, this scouring liquid is filtered with a filter paper at room temperature (25 ° C.). It was.

  Then, the obtained scouring solution is filled into a dialysis cellulose tube (SPECTRUM Laboratories) and subjected to running water dialysis for 2 to 3 days and pure water dialysis for 1 day at 20 ° C., followed by filtration with filter paper again. A sericin solution was obtained.

  This sericin solution was freeze-dried to obtain a soluble sericin powder (sericin powder), which was subjected to the following analysis.

<Example 2> Analysis of silkworm-derived sericin (1) SDS-PAGE electrophoresis analysis Using the soluble powder of sericin obtained in Example 1 as a sample, tricine-SDS polyacrylamide electrophoresis was performed. Specifically, the soluble powder of sericin was separated using a vertical slab gel in which a concentrated gel was layered on a 16% (T), 3% (C) separation gel. The cathode chamber is 0.2M Tris, 0.1M Tricine, 0.1% (W / V) SDS, the anode chamber is 0.2M Tris-HCl buffer (pH 8.9), and the sample for electrophoresis (10 μl) is the same amount. Tricine SDS-PAGE sample buffer [50 mM trice HCl (pH 6.8), 4% (w / v) SDS, 12% (w / v) glycerol, 2% (v / v) β-mercaptethanol, 0.01% (w / v) Serva blue G] and heat-treated for 3 minutes were used. Proctin Marker Kit (MWRange 97,000-14,400 GE Healthcare Bioscience) was used as the molecular weight marker. Electrophoresis was energized at a low voltage of 30 V until entering the separation gel and then reaching the lower end of the separation gel at 80 V. The gel after electrophoresis was subjected to protein detection by staining with silver staining. Silver staining was performed according to a silver staining method modified from Oakley's method (Inoue et al. 1986).

The results are shown in FIG. The left lane is a molecular weight marker, and the right lane is a sericin band obtained in Example 1. In the sericin obtained in Example 1, a plurality of polymer components of 3 to 4 components were confirmed, and it was found that the loss of the sericin-derived component was suppressed without the sericin being excessively decomposed. Specifically, component bands can be observed at 44 kDa, 37 kDa and 32 kDa. Therefore, the soluble powder of sericin obtained in Example 1 can be identified as a sericin powder mainly having a molecular weight of 32 kDa to 44 kDa.
(2) Amino acid composition analysis The soluble powder of sericin obtained in Example 1 was dissolved in 1 ml of 6N hydrochloric acid and sealed under reduced pressure. The sealed tube is hydrolyzed (110 ° C / 24 hours), vacuum-dried, dissolved in Lithium citrate sample buffer (pH 2.2), and filtered through a 0.45 µm pore size filter (diameter 4 mm, Millipore). An analysis sample was obtained. The analysis sample was injected into an amino acid analyzer (JLC-500 / v, JEOL Ltd.) and the amino acid composition was measured.

  The results are shown in Table 1.

  As shown in Table 1, the amino acid composition of sericin obtained in Example 1 is characterized by the fact that serine is the largest at 30.8%, then aspartic acid is 17.7%, and glycine is 17.2%, which is silkworm (rabbit) sericin. It shows well. It is generally known that the most common amino acid in the amino acid composition of sericin is 30 to 35% of serine, then 13 to 18% of aspartic acid, and further 12 to 17% of glycine (for example, Komatsu 1 (1980) Hojo Yasumasa, Continuation Silk Structure, Chemistry and Structure of Sericin, pp379-415, Faculty of Textile Science, Shinshu University), the composition tendency is maintained even with the composition of sericin obtained by the method of the present invention (Example 1) Has been. Strictly speaking, the composition differs depending on the cultivar, production area, year and cocoon season, and naturally, the reduction rate of the silk thread and the cocoon layer is different, but the numerical trend with many compositional amino acids is the same or close. It can be determined that the measured amino acid is derived from sericin.

  Therefore, according to the method of the present invention (Example 1), loss of sericin-derived components and mixing of fibroin-derived components can be suppressed, and sericin can be extracted and purified stably and efficiently from silkworm-derived silk cocoons or silkworm layers. confirmed.

<Example 3> Examination of scouring time The influence on the sericin solution when the scouring time was changed was examined.

  Specifically, in the same extraction / purification method as in Example 1, the scouring time (98 ° C., time of immersion in 0.06M NaOH) is 1 minute, 3 minutes (same as Example 1), 5 minutes The change in the yield of sericin and the reduction rate was examined when the time was changed to 10 minutes.

  The kneading rate was calculated by the following formula: kneading rate (%) = (weight of cocoon layer−residue weight after scouring) / weight of cocoon layer × 100. Moreover, the yield of sericin was calculated by the following formula, yield (%) = recovered sericin amount / reduction amount × 100.

  The results are shown in Table 2.

  As shown in Table 2, as the scouring time of the treatment progresses, the reduction rate increases, but the yield decreases. About this, as the processing time advances, sericin is sufficiently refined and dispersed in the solution, but at the same time hydrolysis and lowering of the molecule progress, so that the dialysis membrane shed during dialysis and the yield decreased. It is guessed.

  Therefore, for example, when analyzing the specific function of sericin, it was confirmed that the scouring time is preferably about 1 to 3 minutes, more preferably about 3 minutes.

<Example 4> Measurement of absorbance 0.1 mg of sericin powder obtained in Example 1 was dissolved in 1 ml of pure water, and the absorption amount of this aqueous solution was determined as a wavelength spectrum with an absorptiometer (JASCO Corporation).

  The results are shown in FIG.

  As shown in FIG. 3, the presence of tyrosine and phenylalanine, which are constituent amino acids of sericin, gives an absorption peak at a wavelength of 280 nm, and it decreases sharply to around 310 nm, but gradually decreases to around 500 nm thereafter. It was confirmed that That is, the sericin powder obtained by the extraction / purification method of Example 1 has a high absorption in the short wavelength region (UV C and UV B) in the ultraviolet region and a low absorption in the UV A region. Has reached about 500 nm, and it was confirmed that its absorption wavelength region is quite wide.

Therefore, the sericin powder obtained by the extraction / purification method of Example 1 can be used as a cosmetic ingredient, for example, to impart UV absorption to the cosmetic.

Claims (2)

The following steps:
(1) A step of immersing a silkworm-derived silk thread or silkworm layer in a NaOH solution of 90 to 98 ° C. and 0.05 to 0.07 M for 1 to 3 minutes to obtain a scouring solution containing silkworm sericin; Dialysis the liquid to obtain a silkworm sericin solution,
A method for extracting and purifying silkworm sericin, comprising:   The method for extracting and purifying silkworm sericin according to claim 1, wherein the bath ratio of the silk thread or cocoon layer and the NaOH solution in the scouring step is 1:25 to 1: 100.