JP2012082174A - Method of separating fibroin and sericin in cricula cocoon layer, and fibroin and sericin obtained by the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of separating and obtaining fibroin and sericin from a cocoon layer of Cricula trifenestrata.SOLUTION: The method includes: a washing step (1) of washing a Cricula cocoon layer; a refining step (2) of dissolving the sericin by immersing the Cricula cocoon layer obtained through the step (1) in a solution having 0.1-1.0% of NaCOat 80-120°C in a bath ratio of 1:70 to 1:150; and a separating step (3) of leaching the refined solution obtained through the step (2) to separate the refined solution into solid fibroin and sericin solution.

Description

  The present invention relates to a method for separating fibroin and sericin in a cricula soot layer. The present invention also relates to fibroin and sericin obtained by this method.

  Conventionally, various methods have been proposed for extracting and purifying silkworm fibroin and sericin from Bombyx mori (for example, Patent Document 1), and studies on the functions of silkworm fibroin and sericin from rabbits have been proposed. Is underway. Specifically, for example, attempts have been made to use silk protein obtained by a process such as extraction from a rabbit silkworm layer as a food for health promotion or as a cosmetic ingredient.

On the other hand, the present inventors have advanced research on silk protein of Antheraea yamamai, and have established its purification method and practical use (Patent Document 2). Tengu's cocoon layer contained a lot of calcium oxalate Ca (COO) ₂ and it was difficult to obtain pure sericin.However, the development of this purification method enabled the removal of Ca (COO) ₂ and the high yield of purified sericin. Acquisition is realized.

  On the other hand, Cricula trifenestrata is a kind of wild boar that belongs to the Solanumaceae family inhabiting Southeast Asia, and its cocoon has a rough mesh-like golden color. It is expensive and traded as a raw material. Further, Patent Document 3 proposes an antitumor agent, an antioxidant, and a prolyl endopeptidase inhibitor containing an extract of coconut trifenestrata.

  However, in the extraction method of Patent Document 3, fibroin and sericin have not been extracted and purified from the cocoon of Cricula trifenestrata. If technology to extract and purify fibroin and sericin from Cricula trifenestrata straw is established, it is expected to be used more effectively in the fields of cosmetics and health supplements.

  Then, the present inventors examined applying the purification method (Patent Document 2) of silk protein from the Antheraea yamamai cocoon layer to Cricula trifenestrata.

  However, wild silk silk proteins (fibroin, sericin) are notably different in constituent amino acid composition and higher order structure depending on the species, and contain complex inorganic salts and pigments. For this reason, when the method for purifying silk protein from Antheraea yamamai cocoon layer (Patent Document 2) is applied to Cricula trifenestrata, high molecular weight fibroin and sericin are obtained separately. It was difficult.

Japanese Patent Laid-Open No. 2004-238312 JP 2008-208123 A JP 2009-280503 A

  The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a method for separating and obtaining fibroin and sericin from the cocoon layer of Cricula trifenestrata. Yes.

The method of separating the fibroin and sericin of the cruciferous soot layer of the present invention includes the following steps: (1) a washing step for washing the clicular soot layer; (2) ° C., bath ratio 1 0.1% 1.0% Na ₂ CO ₃ solution: 70-1: scouring process to dipped to dissolve sericin 150; obtained and (3) the step (2) And a separation step of filtering the scoured liquid into solid fibroin and sericin solution.

  In the separation method of the present invention, the washing treatment in the step (1) is preferably carried out by washing the crucible soot layer with water at 50 ° C to 80 ° C.

  Furthermore, the fibroin of the present invention can be obtained by the separation method.

  The sericin powder of the present invention is obtained by dialysis and lyophilization of the sericin solution obtained by the separation method.

  The sericin powder of the present invention preferably has a molecular weight of 47 kDa.

  The method for producing sericin powder of the present invention comprises a step of dialysis and lyophilization of the sericin solution obtained by the separation method.

  According to the present invention, fibroin and sericin can be separated and obtained from the cocoon layer of Cricula trifenestrata.

It is the flowchart which illustrated the separation and refinement | purification method of fibroin and sericin and the powdering acquisition method of sericin from a cricula soot layer. It is a Blue native-PAGE electrophoresis diagram of sericin powder obtained by the method of the present invention. It is a figure which shows the ultraviolet part and visible part wavelength absorption spectrum of the sericin powder obtained by the method of this invention. It is a figure which shows the ultraviolet part and visible part wavelength absorbance spectrum of the extract A in a reference example.

The method for separating fibroin and sericin in the cricula soot layer of the present invention includes the following steps (1) to (3):
(1) A cleaning process for cleaning the crucible soot layer;
(2) The crucible soot layer that has undergone the step (1) is immersed in a Na ₂ CO ₃ solution at 80 ° C. to 120 ° C. and 0.1% to 1.0% at a bath ratio of 1:70 to 1: 150. A scouring step in which sericin is dissolved; and (3) a separation step in which the scouring solution obtained in the step (2) is filtered and separated into a solid fibroin and a sericin solution.

  The cricula cocoon layer used in the present invention is a silk fiber obtained by incising a ginger of Cricula trifenestrata, and refers to one formed mainly by fibroin and sericin. The crucible wrinkle layer can be processed in an incised state, but can be cut, pulverized, or the like into an appropriate size by a known means, for example.

  The first step in the method of the present invention is a step of cleaning the crucible soot layer. Specifically, the crucible soot layer is washed with water at 50 ° C. to 80 ° C., preferably with pure water at about 65 ° C. The washing time can be about 3 to 5 minutes. In addition, at this time, when the bath ratio of the crucible soot layer to water (pure water) is about 1:80 to 1: 120, the crucible soot layer can be more efficiently washed. By this washing step, water-soluble pigments and contaminants contained in the crucible soot layer can be removed. Moreover, in this washing | cleaning process, a soot layer can also be stirred suitably. And after washing | cleaning, it solid-liquid-separates into a soot layer and a filtrate with a glass filter, for example.

The second step is a step of refining the soot layer that has been solid-liquid separated through the first step. Specifically, the soot layer washed and separated in the first step is immersed in a Na ₂ CO ₃ solution at 80 ° C. to 120 ° C. and 0.1% to 1.0% in a bath ratio of 1:70 to 1: 150. Can be done. Since sericin is soluble in water, high-molecular weight sericin can be separated and eluted from the fibroin constituting the crucible soot layer by this scouring treatment. By setting to such scouring conditions, sericin can be efficiently extracted from the soot layer.

In particular, when the ratio of the Na ₂ CO ₃ solution bath in the soot layer is 1:70 or less, the scouring effect is low, and when the ratio of the Na ₂ CO ₃ solution bath is 1: 150 or more, the subsequent steps are processed. This is not preferable because it takes time and effort. Thus, Na ₂ CO ₃ solution temperature, associated with conditions such as concentration, Na ₂ CO ₃ solution bath ratio range is 1: 70 to 1: 150, more preferably, a bath ratio of 1: 75 to 1: It can be about 85. Further, the immersion time can be appropriately determined according to conditions such as the degree of chopping of the cocoon layer, the temperature, concentration, and bath ratio of the Na ₂ CO ₃ solution, but about 40 minutes to 90 minutes as a guide. can do.

  The third step is a step of filtering the scouring liquid (including the solid cocoon layer) obtained in the second step and separating it into a fibrous solid fibroin and a sericin solution. For filtration, for example, a glass filter, filter paper, or the like can be used as appropriate. Further, in this separation step, a physical action can be given to the fibrous solid content, whereby fibroin and sericin can be efficiently separated. Examples of physical actions include compression and squeezing. More specifically, for example, as the compression, a method of compressing a fibrous solid content by a press device, a method of applying a centrifugal force, or the like can be employed. Moreover, an automatic or manual mortar etc. can also be utilized as milking.

  Further, sericin powder can be obtained by dialysis treatment of the sericin solution obtained in the third step and lyophilization. Specifically, the sericin solution obtained in the third step can be filled in, for example, a cellulose tube and dialyzed with tap water or pure water at about 20 ° C to 25 ° C. And the powdered sericin extract can be obtained by freeze-drying the solution after this dialysis by a well-known means.

  This powdery sericin extract (sericin powder) is usually confirmed to have a high molecular weight of 45 to 50 kDa, depending on the extraction conditions. Therefore, by the method of the present invention, fibroin and sericin can be separated from the cricula soot layer, and the resulting sericin powder has high stability and excellent workability, and has a new functionality derived from the cricula soot layer. As a material, it can be effectively used in the fields of food, cosmetics and the like.

  Hereinafter, the method for separating fibroin and sericin of the cricula soot layer of the present invention will be described, but the present invention is not limited to the following examples.

  FIG. 1 is a flow chart illustrating a method for separating and purifying fibroin and sericin from a cricula soot layer of the present invention and a method for obtaining powdered sericin. Hereinafter, the method of the present invention will be described in detail with reference to the flowchart of FIG.

<1> Separation of fibroin and sericin from cricula cocoon layer (1) Material A cuticle of cricula trifenestrata (US Corporation, Indonesia) was cut open to obtain a cricula cocoon layer.

(2) Washing step 50 g of the crucible soot layer was immersed in pure water of 65 L and 5 L for 3 minutes (bath ratio 1: 100) and washed. Then, it filtered with a 17G glass filter, and isolate | separated solid content (a soot layer) and a solution.

(3) Scouring Step Scouring was carried out by immersing the solid content (salted layer) after washing in a 0.5% Na ₂ CO ₃ solution at 98 ° C. at a bath ratio of 1:75 for 1.5 hours.

In the conventional method for obtaining sericin sericin (Patent Document 2), sericin was obtained by scouring the swordfish layer by immersing it in a Na ₂ CO ₃ solution at a bath ratio of 1:50. However, it is thought that the solubility of the sericin is completely different between the cricula cocoon layer and the tengu layer, because the cocoon layer morphological structure, amino acid composition, molecular higher order structure of fibroin and sericin, and binding (aggregation) are different. In the case where the cricula soot layer was immersed in a Na ₂ CO ₃ solution at a bath ratio of 1:50, it was difficult to obtain sericin from the cricula soot layer. That is, when the Na ₂ CO ₃ solution bath ratio was 1:50, fibroin and sericin could not be sufficiently separated, and the processing conditions of the scouring step in the present invention were specific to the crucible soot layer.

In addition, in the conventional method for obtaining sericin sericin (Patent Document 2), after scouring the cocoon layer, it is left at room temperature (25 ° C.) for 3 days or more to precipitate impurities such as calcium oxalate Ca (COO) _2. Although it was necessary, the cricula soot layer had a small amount of these contaminants, and the precipitation step by standing was unnecessary.

(4) Separation process The scouring solution after the scouring treatment was filtered with a 17G glass filter, so that the fibroin and the sericin solution having a solid content (salted layer portion) were separated.

  Furthermore, about a sericin solution, the solution obtained by filtering with a filter paper at normal temperature (25 degreeC) is filled into a cellulose tube, and it dialyzes against running water with 20 degreeC tap water for 3 days, Then, pure water is used for 2-3 hours. The sericin powder is extracted from the cricula cocoon layer by freeze-drying with a freeze-dryer (Tokyo Science Instruments Co., Ltd., EYELA FDU-810). The rate was 13.86%.

  In addition, it is well known that the silk protein of the cocoon layer is composed of two types of fibroin, which is oriented along the fiber axis and has strong intermolecular bonding and cohesion, and sericin that is non-oriented and has almost no intermolecular bonding. . That is, although there is a difference in solubility depending on the species, sericin is characterized by being dissolved in water, and the scouring step (3) is a step for actively removing sericin using this feature. . Therefore, it can be judged that the solid content separated after the scouring treatment is fibroin. Further, it is confirmed that the filtrate from which fibroin has been removed contains a high molecular weight protein (molecular weight measurement and amino acid composition analysis described later), and it can be judged that the solution contains sericin (sericin solution). it can.

<2> Molecular weight of sericin powder The molecular weight of the sericin powder obtained by the method of the present invention was measured by Blue native-PAGE electrophoresis as follows.
For Blue Native-PAGE electrophoresis, a 4-16% gradient gel was used (Invitrogen). An electrophoresis buffer (Invitrogen) was placed in the electrophoresis tank, and an electrophoresis sample (20 μl) was prepared by adding an equal amount of sample buffer (Invitrogen). A protein marker kit (MWRange 97,000-14,400, GE Healthcare Bioscience) was used as the molecular weight marker. Electrophoresis was performed at a constant voltage of 150 V, and the gel was decolorized after electrophoresis according to Invitrogen's BN-PAGE Manual. Specifically, the gel after electrophoresis was placed in 100 ml of fixative (40% methanol, 10% acetic acid), heated in a microwave for 45 seconds, shaken in a shaker at room temperature for 5 minutes, and the fixative was discarded. . Subsequently, 100 ml of decolorizing solution (8% acetic acid) was added, heated in a microwave for 45 seconds, and shaken with a shaker until the background became clear. The protein used for electrophoresis was quantified according to the measurement procedure of BCA protein assay reagent kit (Pierce).

  As a result, as shown in FIG. 2, it was confirmed that the sericin powder obtained by the method of the present invention was 47 kDa. Since this sericin powder has a high molecular weight, it is considered to have high stability and excellent workability, and can be effectively used in various fields.

<3> Amino acid composition of sericin powder The amino acid composition of the sericin powder obtained by the method of the present invention was analyzed by an amino acid analyzer (JEOL Ltd., JLC-500 / V) and compared with the amino acid composition of rabbit sericin and tengu sericin. .

  In addition, about the tengu sericin, what was acquired by the method of patent document 2 was used, and the data described in patent document 2 were quoted about the rabbit sericin.

  The results are shown in Table 1.

  As shown in Table 1, the amino acid composition of sericin powder (T. Cricula column in the table) from the Cricula trifenestrata cocoon layer obtained by the method of the present invention is as follows. It was confirmed that the amino acid composition of sericin is different. Specifically, sericin powder (T. Cricula) obtained by the method of the present invention was relatively rich in serine and glycine. Furthermore, the sericin powder (T. Cricula) obtained by the method of the present invention contained a large amount of histidine as compared with rabbit sericin and tengu sericin.

<4> Spectral analysis of sericin powder With respect to a solution obtained by dissolving sericin powder obtained by the method of the present invention in water, ultraviolet and visible wavelength absorbance spectra were obtained.

  As shown in FIG. 3, the absorbance decreases while drawing a parabola along the long wavelength side from the maximum value at the short wavelength of 225 nm, but has small convex peaks at wavelengths of 240 nm, 280 nm, and 335 nm, and further the wavelength of 370 nm , A spectrum showing a gentle peak at 490 nm was obtained.

<5> Reference Example (1) After the washing step in the method of the present invention, the filtrate separated from the solid content (soot layer) was further filtered through a filter paper to obtain an extract A (solution).
When this extract is freeze-dried in the same manner as the drying process in order to obtain the present purified sericin powder, an extract A powder can be obtained. The yield of Extract A was 0.32% (dry powder).

  The UV and visible wavelength absorbance spectra of this extract A (solution) were obtained, and decreased while drawing a parabola along the long wavelength side from the maximum value at the short wavelength of 220 nm, but small at wavelengths of 275 nm, 340 nm, and 370 nm. A spectrum showing a gentle peak was obtained from the convex peak to wavelengths of 390 nm and 490 nm (FIG. 4). This extract A has a golden color and is considered to contain sericin (some amount) of the outermost layer of the cricula cocoon layer, a water-soluble pigment, impurities, and the like.

(2) The solid content (the soot layer) after the washing step in the method of the present invention was immersed in 98 ° C. water at a bath ratio of 1:50 for 20 minutes to 30 minutes. Thereafter, the mixture was filtered through a 17G glass filter, and the filtrate was further filtered through filter paper to obtain an extract B (solution).
This extract was freeze-dried in the same manner as in the case of the purified sericin powder obtained in the Example, whereby Extract B powder could be obtained. The yield of Extract B was 1.23% (dry powder). This extract B is considered to contain sericin in the outermost layer of the soot layer and a dye that binds to the sericin. This extract B can be expected to have a physiological activity different from the purified sericin obtained in the examples.

Claims (6)

A method for separating fibroin and sericin in a cricula soot layer, comprising the following steps:
(1) A cleaning process for cleaning the crucible soot layer;
(2) The crucible soot layer that has undergone the step (1) is immersed in a Na ₂ CO ₃ solution at 80 ° C. to 120 ° C. and 0.1% to 1.0% at a bath ratio of 1:70 to 1: 150. A scouring step for dissolving sericin; and (3) a separation method comprising filtering the scouring liquid obtained in the step (2) and separating the sericin into a solid fibroin and a sericin solution.   The separation method according to claim 1, wherein the washing step (1) comprises washing the crucible soot layer with water at 50 ° C. to 80 ° C.   Fibroin obtained by the separation method according to claim 1 or 2.   A sericin powder obtained by dialysis and lyophilization of a sericin solution obtained by the separation method according to claim 1 or 2.   The sericin powder according to claim 4, which has a molecular weight of 47 kDa. A method for producing sericin powder, comprising a step of dialysis and lyophilization of a sericin solution obtained by the separation method according to claim 1 or 2.