JP5253760B2 - Method for producing sericin aqueous solution - Google Patents

Description

  The present invention relates to a method for producing a sericin aqueous solution, more specifically, a method for preparing a low molecular weight sericin aqueous solution by hydrolyzing a high molecular weight sericin aqueous solution, and a sericin aqueous solution obtained by the hydrolysis, Relates to a cosmetic composition containing the sericin aqueous solution.

  Sericin is a protein that constitutes silk fiber together with fibroin, and is extracted from silk fiber or the like using alkali, acid, surfactant, proteolytic enzyme, ionic water, hot water or the like. Such sericin was conventionally discarded as a refining waste liquid in the refining process of silk fibers, but in recent years, its use as a functional material in fields such as application fields related to biological functions, cosmetics, and foods has been studied. .

  As a method for separating and recovering sericin, the following Patent Document 1 discloses a technique of treating silk fabric with high-temperature and high-pressure water, and freezing and thawing the obtained sericin aqueous solution to extract sericin. It is described that the sericin obtained by this method has an average molecular weight of 100,000 or more, and the aqueous solution gels at 0 to 40 ° C.

  As described in Patent Document 1, an aqueous solution of high molecular weight sericin having an average molecular weight of 50,000 or more is cloudy and gelled because the solubility of high molecular weight sericin in water is poor. . That is, in a high molecular weight sericin aqueous solution, sericin is not completely dissolved, the solution becomes cloudy, has a high viscosity, and forms a colloidal gel over time or aggregates and precipitates. Therefore, it is difficult to handle due to the high viscosity, resulting in inconveniences such as poor appearance and water separation.

  In such a state, it is difficult to use in applications that require high uniformity and transparency. In addition, when a high-molecular weight sericin aqueous solution is used as a raw material for cosmetics such as skin care and hair care, a phenomenon such that most sericin permeates and adsorbs inside the skin and hair cannot be expected, and its effect is difficult to be confirmed. Therefore, there is a need for a method for preparing a low molecular weight sericin aqueous solution that can efficiently reduce the molecular weight of high molecular weight sericin obtained by extraction with high-temperature and high-pressure water and has high transparency and uniformity.

  As a method for obtaining a low molecular weight sericin aqueous solution, the following Patent Document 2 proposes a method of hydrolyzing the sericin of the silk thread using ionic water generated by electrolyzing water or an electrolyte aqueous solution with an ionic water generator. Yes.

Further, in Patent Document 3 below, a silk protein raw material derived from rabbit or tengu is dissolved in a neutral salt aqueous solution, and a peptide bond between specific amino acid residues of the silk protein using a peptide bond cleaving substance such as a proteolytic enzyme. A method for producing a silk protein aqueous solution having an average molecular weight of 10,000 or more and less than 200,000 is disclosed. Patent Document 4 below relates to a method for producing fibroin peptide instead of sericin, and discloses a technique for reducing the molecular weight by degrading silk fibroin with a neutral salt and hydrolyzing with a proteolytic enzyme. Has been. In these methods, since high molecular weight sericin or fibroin is eluted from silk thread into an aqueous solution containing a high concentration of neutral salt, a desalting step such as dialysis is required after hydrolysis with a peptide bond cleaving substance.
JP-A-11-131318 Japanese Patent Application Laid-Open No. 10-029909 JP 2004-300142 A Japanese Patent Application Laid-Open No. 07-0667686

  As described above, a high-molecular weight sericin aqueous solution having a weight average molecular weight of 50,000 or more obtained by treating silk fabric with high-temperature and high-pressure water is poorly soluble and cloudy, and is gel-like at room temperature. Therefore, an attempt was made to obtain a transparent low molecular weight sericin aqueous solution by hydrolyzing the high molecular weight sericin aqueous solution with a proteolytic enzyme, an alkali, an acid or a combination thereof.

  However, although it was confirmed that the molecular weight was reduced by gel filtration chromatography, the solubility was not improved. That is, the decomposition with alkali did not improve the appearance while remaining cloudy despite the low molecular weight. Moreover, in the decomposition | disassembly by an acid or a proteolytic enzyme, aggregation precipitation produced and it was inferior to an external appearance and performance. Such low molecular weight sericin is hardly useful for industrial use, and it is necessary to establish a method for decomposing high molecular weight sericin that does not cause cloudiness or aggregation precipitation.

  This invention is made | formed in view of the above point, and it aims at providing the method which can prepare the low molecular weight sericin aqueous solution which has high transparency and uniformity.

  The inventors of the present invention have intensively studied in view of the above problems, and have thought that it is effective to solubilize high molecular weight sericin with an anionic surfactant before hydrolysis. As previously proposed by the present applicant, the aqueous solution of high molecular weight sericin can be improved in solubility by an anionic surfactant to suppress colloidal gel formation and aggregation precipitation (Japanese Patent Application No. 2006-2006). 248198). For this reason, it was considered that the above problem could be solved if hydrolysis was performed using the high-molecular weight sericin aqueous solution obtained in this way and having excellent uniformity.

  However, even when using a uniform high molecular weight aqueous sericin solution solubilized with an anionic surfactant, hydrolysis with acid causes aggregation to precipitate sericin that has been solubilized due to a decrease in solubility. Turned out to be. In addition, the hydrolysis with alkali did not cause aggregation and precipitation, but the hydrolysis required a long time and was inefficient.

  Therefore, the present inventors paid attention to hydrolysis by a proteolytic enzyme in which the reaction proceeds in a short time, and caused white turbidity and aggregation precipitation by combining solubilization by an anionic surfactant and hydrolysis by a proteolytic enzyme. Thus, the present inventors have found that an aqueous sericin solution having a low molecular weight can be obtained in a short time, and completed the present invention.

That is, the method for producing an aqueous sericin solution according to the present invention comprises adding an anionic surfactant to a stock solution of an aqueous high molecular weight sericin solution and mixing by heating , solubilized with an anionic surfactant and having a weight average molecular weight of 50,000 or more. An aqueous solution is prepared, and the resulting high molecular weight sericin aqueous solution is prepared by hydrolyzing sericin with a proteolytic enzyme while maintaining the pH at 7 to 11 to prepare a low molecular weight sericin aqueous solution having a weight average molecular weight of less than 50,000. It is.

  In the present invention, when cysteine protease is used as the proteolytic enzyme, the proteolytic enzyme is inactivated by the anionic surfactant and hydrolysis does not proceed easily. Therefore, in this case, it is preferable to add a reducing agent and hydrolyze sericin in the presence of the reducing agent as an improvement measure for suppressing the inactivation of cysteine protease by the anionic surfactant. Thereby, the activity of cysteine protease is maintained, and sericin can be reduced in a short time without causing cloudiness or aggregation precipitation.

  In the present invention, an alkaline protease can be used as the proteolytic enzyme. In this case, the activity can be maintained without adding a reducing agent, and sericin can be produced in a short time without causing cloudiness or aggregation precipitation. The molecular weight can be reduced.

  According to the present invention, when preparing a low molecular weight sericin aqueous solution from a high molecular weight sericin aqueous solution, it is possible to suppress the occurrence of aggregation precipitation and white turbidity, and thus excellent transparency and uniformity and suitable for a wide range of applications. A low molecular weight sericin aqueous solution can be obtained. In addition, since a proteolytic enzyme is used, the molecular weight can be reduced in a short time, and the molecular weight of the sericin peptide can be controlled. Moreover, when it mix | blends for cosmetics because of low molecular weight, the permeability to the inside of skin or hair can be improved.

  Hereinafter, matters related to the implementation of the present invention will be described.

  In the present invention, high molecular weight sericin refers to sericin having a weight average molecular weight (Mw) of 50,000 or more. The low molecular weight sericin is a sericin or sericin peptide having a weight average molecular weight of less than 50,000, and the weight average molecular weight is more preferably 20,000 or less, and still more preferably 10,000 or less. By using such low molecular weight sericin, for example, when blended for cosmetics, the penetration into the skin and hair can be improved.

  The upper limit of the weight average molecular weight of the high molecular weight sericin is not particularly limited, but it is preferably 200,000 or less from the viewpoint of uniformity and transparency in a state solubilized with a surfactant. The lower limit of the weight average molecular weight of the low molecular weight sericin is not particularly limited, but is preferably 500 or more.

  In the present invention, the weight average molecular weight is measured by gel filtration chromatography.

A method for producing an aqueous sericin solution according to an embodiment of the present invention includes:
(1) preparing a high molecular weight sericin aqueous solution solubilized with a surfactant;
(2) a step of hydrolyzing sericin with a proteolytic enzyme to the prepared high molecular weight sericin aqueous solution;
Is included.

  In the step (1), the solubilized high molecular weight sericin aqueous solution can be prepared by adding an anionic surfactant to the stock solution of the high molecular weight sericin aqueous solution that has become cloudy and coagulated and heated and mixed.

  As the stock solution of the high molecular weight sericin aqueous solution, one obtained by subjecting silk fibers (cocoon yarn) to extraction treatment with high-temperature water (for example, 100 to 140 ° C.) is preferable. If extracted with such high-temperature water, an aqueous solution containing sericin having a higher molecular weight than that obtained by alkali extraction can be obtained. Such a method for extracting sericin with high-temperature water can be carried out using a known method as described in Patent Document 1, for example, and is not particularly limited. The stock solution of the high molecular weight sericin aqueous solution obtained by high-temperature water extraction usually does not completely dissolve sericin at room temperature (hence, it can also be referred to as a sericin aqueous dispersion), becomes cloudy and aggregates. The liquid is thickened by precipitation.

  After adding a predetermined amount of an anionic surfactant to the stock solution of this high molecular weight sericin aqueous solution, the mixture is stirred for a predetermined time (for example, about 10 minutes) under heating conditions (preferably 80 to 95 ° C.), and then rapidly cooled. . Thereby, the high molecular weight sericin aqueous solution excellent in uniformity is obtained.

  In addition, when high transparency is required, you may add a pressure process to the obtained high molecular weight sericin aqueous solution. The pressure treatment can be performed using a known high-pressure processor such as a microfluidizer, a homogenizer, or an optimizer. Transparency is improved by applying pressure to the high molecular weight sericin aqueous solution to form fine particles. Although it does not specifically limit as a processing pressure, It is suitable that it is 50-100 Mpa. Further, the number of times of processing is preferably 2 passes or more.

  An anionic surfactant is used for the solubilization. By using an anionic surfactant, the solubility of sericin can be improved. On the other hand, other surfactants such as a cationic surfactant and an amphoteric surfactant do not improve the solubility of high molecular weight sericin. In some cases, the solubility may rather decrease. The reason why the anionic surfactant improves the solubility of sericin is not clear, but when the lipophilic group of the anionic surfactant is adsorbed on the lipophilic region of sericin, the hydrophilic group is an anion. It is presumed that an electrostatic repulsive force is generated within or between sericin molecules, promoting the unfolding state (random structure) of sericin and preventing aggregation between sericin molecules.

  The anionic surfactant can be used without particular limitation as long as it can solubilize sericin. For example, amino acid-based activator, polyoxyethylene alkyl ether acetate, alkyl sulfate (for example, sodium dodecyl sulfate) Etc. Preferably, amino acid activators such as N-acyl-N-methyl-β-alanine salt, N-acyl-D, L-alanine salt, N-acyl-sarcosine salt, polyoxyethylene alkyl ether acetate, These are highly effective in improving the solubility of high molecular weight sericin among anionic surfactants, and can contribute not only to uniformity but also to improvement of transparency.

  The acyl group of the amino acid activator is not particularly limited, but is preferably derived from a fatty acid having 8 to 20 carbon atoms, and more preferably derived from a fatty acid having 12 to 18 carbon atoms. These fatty acid compositions may be single or a mixture of two or more. Moreover, the alkyl group of the polyoxyethylene alkyl ether acetate is not particularly limited, but preferably has 8 to 20 carbon atoms, and more preferably has 12 to 18 carbon atoms.

  The salt of the anionic surfactant is not particularly limited, but metal salts such as sodium salt, potassium salt and magnesium salt, and triethanolamine salt are preferable.

  The above-mentioned solubilization means that the solubility of high molecular weight sericin in water is improved to obtain a uniform sericin aqueous solution, and all high molecular weight sericin is required to be completely dissolved in water. Not what you want. The concentration of the surfactant required for such solubilization is not particularly limited, but it is preferably 0.1 times or more by weight with respect to the total concentration of sericin. In addition, if the concentration is too high, the activity of the proteolytic enzyme tends to decrease with an anionic surfactant in the step (2), so it is preferable to keep the upper limit as low as possible. Is preferred.

  The total concentration of sericin in the high molecular weight sericin aqueous solution is preferably less than 3% by weight. If the sericin concentration is too high, it will be difficult to obtain a high-molecular-weight sericin aqueous solution with good solubility even if the surfactant concentration is increased, and it will be difficult to prepare a low-molecular weight sericin aqueous solution having high transparency and uniformity. . The sericin concentration is more preferably 0.5 to 2.5% by weight.

  The proteolytic enzyme used in the step (2) is not particularly limited as long as it can hydrolyze sericin. For example, cysteine proteases such as papain, bromelain, chymopapain and ficin, metalloproteases such as thermolysin, trypsin and chymotrypsin. And serine proteases such as subtilisin, and proteases derived from microorganisms such as Aspergillus, Rhizopus and Bacillus. These can be used alone or in combination of two or more.

  As the proteolytic enzyme, one having activity in the neutral region to the alkaline region is used. Specifically, an alkaline protease having an enzyme activity in the alkaline region (particularly, alkaline proteinase), for example, derived from the genus Bacillus Alkaline protease (subtilisin etc.) etc. are used suitably. In addition, cysteine proteases such as papain and bromelain (particularly cysteine proteinase (SH enzyme)) are also preferably used in cosmetic applications because they are derived from plants.

  The concentration of the proteolytic enzyme is not particularly limited, but is preferably 0.001 to 0.5 times by weight with respect to the total concentration of sericin.

  In the above step (2), when cysteine protease is used as a proteolytic enzyme, sericin is hydrolyzed in the presence of a reducing agent. The reducing agent is not particularly limited as long as it can suppress inactivation of cysteine protease by an anionic surfactant. That is, the reducing agent can suppress the deactivation due to oxidation of the SH group at the active center such as papain or bromelain, and can maintain the active SH group by reduction. Specific examples include cysteine or a salt thereof (for example, cysteine hydrochloride), cysteamine or a salt thereof, dithiothreitol, 2-mercaptoethanol, glutathione, and the like. These may be used alone or in combination of two or more. it can. Particularly preferred is cysteine or cysteamine or a salt thereof.

  Although the density | concentration of a reducing agent is not specifically limited, It is preferable that it is 0.1 to 1 time by weight ratio with respect to the usage-amount of a proteolytic enzyme.

  In the step (2), more specifically, a proteolytic enzyme is added to the high molecular weight sericin aqueous solution prepared in the step (1), and the enzyme is subjected to a predetermined time (for example, 15 to 15) in an optimum temperature range where the enzyme exhibits a catalytic function. Sericin is hydrolyzed by stirring (for 30 minutes). Here, when the enzyme is a cysteine protease, it is preferable to add the enzyme after adding a reducing agent to the high molecular weight sericin aqueous solution prepared in step (1) and stirring and mixing.

  At that time, if the pH of the aqueous solution is smaller than 7, even when solubilized, the solubility of sericin is lowered and becomes cloudy again, aggregates and precipitates, or is markedly colored. Therefore, the pH is always adjusted to 7 or more. It is preferable. In addition, when the pH of the aqueous solution exceeds 11, it affects the activity of the proteolytic enzyme, aggregates and precipitates, or is markedly colored. Therefore, the pH is preferably adjusted to 11 or less. As mentioned above, in the said hydrolysis, pH is maintained at 7-11, More preferably, pH is maintained at 8-10. In addition, pH adjustment is carried out using inorganic alkalis such as sodium hydroxide and potassium hydroxide, various alkalis such as organic alkalis such as ethanolamine, diethanolamine and triethanolamine, inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, citric acid, malic acid, Various acids such as organic acids such as succinic acid and lactic acid can be used. By adjusting the pH, salt is generated in the aqueous solution, but since the amount thereof is small, the desalting step as in Patent Documents 3 and 4 is not necessary.

  As described above, after sericin is reduced in molecular weight by hydrolysis with a proteolytic enzyme, the enzyme can be inactivated by heating and stirring to a predetermined temperature. Then, the low molecular weight sericin aqueous solution is obtained by cooling. The inactivated proteolytic enzyme may be left in the low molecular weight sericin aqueous solution as it is.

  Thus, according to the present embodiment, hydrolyzing sericin with a proteolytic enzyme while maintaining the pH at 7 to 11 with respect to the high-molecular weight sericin aqueous solution improved in solubility by adding an anionic surfactant. Thus, a low molecular weight sericin aqueous solution that does not cause aggregation and has high transparency, uniformity, and penetration ability can be prepared in a very short time.

  The resulting low molecular weight sericin aqueous solution comprises a low molecular weight sericin, an anionic surfactant, optionally a reducing agent, a deactivated proteolytic enzyme, an acid and / or alkali for adjusting pH and / or a salt thereof. In the range which does not impair the effect of invention, you may contain other components, such as antiseptic | preservative.

  A cosmetic composition according to one embodiment of the present invention is obtained by blending the low molecular weight sericin aqueous solution. The blending amount is not particularly limited, but is usually blended in the cosmetic composition within a range of 0.001 to 0.1% by weight as the sericin concentration.

  Various components that are generally blended in cosmetic compositions can be blended in the cosmetic composition. For example, alcohols such as ethanol, glycerin, sorbitol, sorbitan fatty acid esters such as sorbitan monolaurate, sorbitan monoisostearate, sorbitan tristearate, glycerol fatty acid esters such as glycerol monostearate, glycerol monooleate, Surfactant such as polyoxyethylene hydrogenated castor oil, polyether-modified silicone surfactant, dimethicone polyol, plasticizer, antioxidant, UV absorber, moisturizer, fragrance, dye, pigment, pigment, preservative, vitamin Agents, hormone agents, deodorants, pH adjusters, fixing agents and the like.

  The use of the cosmetic composition is not particularly limited, and hair care cosmetics such as hair gel, hair shampoo, hair treatment, hair rinse, styling lotion, hair mist, styling mousse, hair spray, skin lotion, milky lotion, moisture cream, moisture lotion, etc. Skin care cosmetics, cleaning agents such as body shampoos, makeup cosmetics such as mascara and foundation.

  Hereinafter, the present invention will be specifically described by way of examples, but the scope of the present invention is not limited thereto. The weight average molecular weight and appearance measurement / evaluation method are as follows.

(Weight average molecular weight)
・ Sample preparation method:
1. 1. Diluted with an aqueous solution having the same composition as the mobile phase so that the concentration of sericin is 0.1% by weight. Above 1. 2. Boiling for 5 minutes 2. Was passed through a syringe filter (“DISMIC-13HP” manufactured by ADAVANTEC, PTFE 0.45 μm) to obtain a sample.
Reagents: disodium hydrogen phosphate (manufactured by Nacalai Tesque), sodium dihydrogen phosphate (manufactured by Nacalai Tesque), sodium chloride (manufactured by Nacalai Tesque), molecular weight marker ("MW-Marker" manufactured by Oriental Yeast), molecular weight 12,400-290,000)
·Analysis conditions:
UV detector: “SPD-6A” manufactured by Shimadzu Corporation, measuring wavelength 210 nm
Column: “Asahipak GS-520HG” manufactured by Showa Denko KK
Column temperature: 40 ° C
Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.3 M sodium chloride
Flow rate ... 0.6ml / min Injection volume ... 10μl
Molecular weight calculation method: A calibration curve was created by measuring a molecular weight marker, and a weight average molecular weight was measured.

(Appearance evaluation)
The appearance of the aqueous solution of sericin was evaluated by visual inspection, and “○” indicates that the uniformity and transparency are good, “Δ” indicates that cloudiness is generated, and “×” indicates that aggregation precipitation has occurred. .

Example 1
(1) High-molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) was added to 99.5 parts by weight of an anionic surfactant (polyoxy Ethylene lauryl ether acetate sodium salt, “Neohaitenol ECL-30S, manufactured by Daiichi Kogyo Seiyaku Co., Ltd., aqueous solution having a surfactant concentration of 30% by weight) 0.5 part by weight is added, and 10 parts in a 95 ° C. water bath are added. After heating and stirring for a minute, the mixture was rapidly cooled in an ice bath to obtain a solubilized high molecular weight sericin aqueous solution.

(2) L-cysteine hydrochloride monohydrate (Wako Pure Chemical Industries, Ltd.) was prepared by appropriately adding 10% by weight sodium hydroxide to 99.76 parts by weight of the solubilized high molecular weight sericin aqueous solution to adjust the pH to about 9. (0.04 parts by weight), and stirred until uniform.

(3) Further, 10% by weight sodium hydroxide was added as appropriate to adjust the pH to about 9, and 0.2 parts by weight of papain (“Papain W-40” manufactured by Amano Enzyme Co., Ltd., 50% by weight of papain) was added uniformly. The solution was stirred until the pH was about 8.

(4) After stirring at 50 ° C. for 30 minutes, the enzyme was deactivated by heating and stirring at 95 ° C. for 30 minutes, and quenched in a water bath to obtain a low molecular weight sericin aqueous solution.

(5) The prepared low molecular weight sericin aqueous solution was allowed to stand at 5 ° C. for 24 hours, and the appearance of the aqueous solution was visually evaluated, and then the weight average molecular weight was measured.

(Example 2)
In (3) of Example 1 above, 0.2 parts by weight of bromelain (“Bromelain F” manufactured by Amano Enzyme) was added instead of papain, and the rest was the same as in Example 1 to obtain a low molecular weight sericin aqueous solution. The appearance was evaluated and the weight average molecular weight was measured.

(Example 3)
In (2) of Example 1 above, the amount of the solubilized high molecular weight sericin aqueous solution was 99.86 parts by weight, the amount of L-cysteine hydrochloride monohydrate added was 0.04 parts by weight, In 3), the amount of papain added was 0.1 parts by weight, and the others were the same as in Example 1, to obtain a low molecular weight sericin aqueous solution, and the appearance evaluation and the weight average molecular weight were measured.

Example 4
(1) High-molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) was added to 98.3 parts by weight of an anionic surfactant (N— Lauroyl-N-methyl-β-alanine sodium salt, “Enazicol L-30ANT” manufactured by Lion, an aqueous solution having a surfactant concentration of 30% by weight) and 1.7 parts by weight were added in a 95 ° C. water bath. After heating and stirring for a minute, the mixture was rapidly cooled in an ice bath to obtain a solubilized high molecular weight sericin aqueous solution. Thereafter, pressure treatment was performed with a microfluidizer (“Ultra High Pressure Homogenizer M-110-E / H” manufactured by Mizuho Kogyo Co., Ltd.) (treatment pressure = 100 MPa, number of treatments = 2 passes).

(2) L-cysteine hydrochloride monohydrate (Wako Pure Chemical Industries, Ltd.) was prepared by appropriately adding 10% by weight sodium hydroxide to 99.76 parts by weight of the solubilized high molecular weight sericin aqueous solution to adjust the pH to about 9. (0.04 parts by weight), and stirred until uniform.

(3) Further, 10% by weight sodium hydroxide is added as appropriate to adjust the pH to about 9, and 0.2 parts by weight of bromelain (“Bromelain F” manufactured by Amano Enzyme, Inc., 85% by weight of bromelain) is added to make uniform. Until the pH was about 8.

(4) After stirring at 50 ° C. for 30 minutes, the enzyme was deactivated by heating and stirring at 95 ° C. for 30 minutes, and quenched in a water bath to obtain a low molecular weight sericin aqueous solution.

(5) The prepared low molecular weight sericin aqueous solution was allowed to stand at 5 ° C. for 24 hours, and the appearance of the aqueous solution was visually evaluated, and then the weight average molecular weight was measured.

(Example 5)
In (1) of Example 4 above, as an anionic surfactant, N-coconut oil fatty acid-D, L-alanine triethanolamine salt (“Amlite ACT-12” manufactured by Ajinomoto Co., Inc., surfactant concentration of 30% by weight) In the same manner as in Example 4, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Example 6)
In (1) of Example 4 above, as an anionic surfactant, N-coconut oil fatty acid-sarcosine triethanolamine salt (“Sarcosineate CT-30” manufactured by Nikko Chemicals Co., Ltd., aqueous solution having a surfactant concentration of 30% by weight) In the same manner as in Example 4, a low molecular weight sericin aqueous solution was obtained, and the appearance evaluation and the weight average molecular weight were measured.

(Example 7)
(1) High-molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) was added to 99.5 parts by weight of an anionic surfactant (polyoxy Ethylene lauryl ether acetate sodium salt, “Neohaitenol ECL-30S, manufactured by Daiichi Kogyo Seiyaku Co., Ltd., aqueous solution having a surfactant concentration of 30% by weight) 0.5 part by weight is added, and 10 parts in a 95 ° C. water bath are added. After heating and stirring for a minute, the mixture was rapidly cooled in an ice bath to obtain a solubilized high molecular weight sericin aqueous solution.

(2) 10 wt% sodium hydroxide was appropriately added to 99.85 parts by weight of a solubilized high molecular weight sericin aqueous solution to adjust the pH to about 9, and an alkaline protease ("Protin SD-AY10" manufactured by Daiwa Kasei Co., Ltd.) 15% by weight, 0.15 parts by weight of an endo-type alkaline protease derived from Bacillus licheniformis (Bacillus subtilis), EC 3.4.21.14) was added, and the mixture was stirred until uniform, and the pH was confirmed to be about 9.

(4) After stirring at 50 ° C. for 30 minutes, the enzyme was deactivated by heating and stirring at 95 ° C. for 30 minutes, and quenched in a water bath to obtain a low molecular weight sericin aqueous solution.

(5) The prepared low molecular weight sericin aqueous solution was allowed to stand at 5 ° C. for 24 hours, and the appearance of the aqueous solution was visually evaluated, and then the weight average molecular weight was measured.

(Example 8)
In (1) of Example 7 above, as an anionic surfactant, N-lauroyl-N-methyl-β-alanine sodium salt (“Enazicol L-30ANT” manufactured by Lion Corporation, the surfactant concentration is 30% by weight. A low molecular weight sericin aqueous solution was obtained in the same manner as in Example 7 except that the amount added was 98.3 parts by weight with respect to 98.3 parts by weight of the high molecular weight sericin aqueous solution. Measurements were made.

Example 9
In (1) of Example 7 above, as an anionic surfactant, N-coconut oil fatty acid-D, L-alanine triethanolamine salt (“Amilite ACT-12” manufactured by Ajinomoto Co., Inc., surfactant concentration of 30% by weight) The amount of addition to 98.3 parts by weight of the high molecular weight sericin aqueous solution was 1.7 parts by weight, and the others were the same as in Example 7 to obtain a low molecular weight sericin aqueous solution. The molecular weight was measured.

(Example 10)
In (1) of Example 7 above, as an anionic surfactant, N-coconut oil fatty acid-sarcosine triethanolamine salt (“Sarcosineate CT-30” manufactured by Nikko Chemicals Co., Ltd., aqueous solution having a surfactant concentration of 30% by weight) ), And the addition amount with respect to 98.3 parts by weight of the high-molecular weight sericin aqueous solution was 1.7 parts by weight. Otherwise, the low-molecular weight sericin aqueous solution was obtained in the same manner as in Example 7, and the appearance evaluation and measurement of the weight average molecular weight were performed. Went.

(Example 11)
(1) High-molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) was added to 99.5 parts by weight of an anionic surfactant (polyoxy Ethylene lauryl ether acetate sodium salt, “Neohaitenol ECL-30S, manufactured by Daiichi Kogyo Seiyaku Co., Ltd., aqueous solution having a surfactant concentration of 30% by weight) 0.5 part by weight is added, and 10 parts in a 95 ° C. water bath are added. After heating and stirring for a minute, the mixture was rapidly cooled in an ice bath to obtain a solubilized high molecular weight sericin aqueous solution.

(2) 10 wt% sodium hydroxide was appropriately added to 99.85 parts by weight of the solubilized high molecular weight sericin aqueous solution to adjust the pH to 11, and alkaline protease ("Buroreza FG-F" manufactured by Amano Enzyme, Protease 10) 0.15 part by weight of an endo-type alkaline protease derived from Bacillus subtilis, EC 3.4.21.62) was added and stirred until it was uniform, and it was confirmed that the pH was about 10.

(4) After stirring at 60 ° C. for 30 minutes, the mixture was heated and stirred at 95 ° C. for 30 minutes to deactivate the enzyme, and quenched in a water bath to obtain a low molecular weight sericin aqueous solution.

(5) The prepared low molecular weight sericin aqueous solution was allowed to stand at 5 ° C. for 24 hours, and the appearance of the aqueous solution was visually evaluated, and then the weight average molecular weight was measured.

(Comparative Example 1: No solubilization)
(1) High-molecular-weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) 99.72 parts by weight with 10% by weight sodium hydroxide The pH was appropriately adjusted to about 9, and 0.08 part by weight of L-cysteine hydrochloride monohydrate (manufactured by Wako Pure Chemical Industries, Ltd., special grade) was added and stirred until uniform.

(2) Next, 10 wt% sodium hydroxide was added as appropriate to adjust the pH to about 9, and 0.2 parts by weight of papain ("Papain W-40" manufactured by Amano Enzyme Inc., papain 50 wt%) was added, It stirred until it became uniform and it confirmed that pH was about 8.

(3) After stirring at 50 ° C. for 30 minutes, the enzyme was inactivated by heating and stirring at 95 ° C. for 30 minutes, and then rapidly cooled in a water bath to obtain a low molecular weight sericin aqueous solution.

(4) The prepared low molecular weight sericin aqueous solution was allowed to stand at 5 ° C. for 24 hours, and the appearance of the aqueous solution was visually evaluated, and then the weight average molecular weight was measured.

(Comparative Example 2: No solubilization and reducing agent)
10 wt% sodium hydroxide was appropriately added to 99.8 parts by weight of a high molecular weight sericin aqueous solution ("DK-sericin MT" manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000). PH is adjusted to about 9, and 0.2 parts by weight of papain (“Papain W-40” manufactured by Amano Enzyme, 50% by weight of papain) is added and stirred until it is uniform, and the pH is about 8. confirmed. Thereafter, as in (3) and (4) of Comparative Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative example 3: no reducing agent)
(1) High molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) 99.85 parts by weight with an anionic surfactant (polyoxy 0.15 parts by weight of sodium lauryl ether acetate sodium salt, “Neohaitenol ECL-30S” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., an aqueous solution having a surfactant concentration of 30% by weight) and added in a 95 ° C. water bath. After heating and stirring for a minute, the mixture was rapidly cooled in an ice bath to obtain a solubilized high molecular weight sericin aqueous solution.

(2) 10 wt% sodium hydroxide was appropriately added to 99.8 parts by weight of the solubilized high molecular weight sericin aqueous solution to adjust the pH to about 9, and papain ("Papain W-40", Papain 50 manufactured by Amano Enzyme Inc.) 0.2% by weight) was added and stirred until uniform, confirming that the pH was about 8.

(3) Thereafter, as in (3) and (4) of Comparative Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 4: No solubilization, reducing agent and pH adjustment)
High-molecular-weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) was added to 99.8 parts by weight of papain (“Papain W-” manufactured by Amano Enzyme). 40 ", 50% by weight of papain) was added and stirred until uniform. Thereafter, as in (3) and (4) of Comparative Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 5: No pH adjustment)
After obtaining a high-molecular weight sericin aqueous solution solubilized in the same manner as in (1) of Comparative Example 3, L-cysteine hydrochloride monohydrate (Wako Pure Chemical Industries, Ltd.) was added to 99.72 parts by weight of the obtained high-molecular weight sericin aqueous solution. 0.08 parts by weight of a special grade) was added and stirred until uniform. Thereafter, 0.2 part by weight of papain (“Papain W-40” manufactured by Amano Enzyme, 50% by weight of papain) was added and stirred until uniform. The pH of the solution at this time was about 6. Thereafter, as in (3) and (4) of Comparative Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 6: pH adjusted to 12)
After obtaining a solubilized high molecular weight sericin aqueous solution in the same manner as in (1) of Comparative Example 3, 10 wt% sodium hydroxide was appropriately added to 99.72 parts by weight of the obtained high molecular weight sericin aqueous solution to adjust the pH to about The mixture was adjusted to 9, 0.08 parts by weight of L-cysteine hydrochloride monohydrate (manufactured by Wako Pure Chemical Industries, Ltd., special grade) was added, and the mixture was stirred until uniform. Thereafter, 0.2 part by weight of papain (“Papain W-40” manufactured by Amano Enzyme, 50% by weight of papain) is added, stirred until uniform, and 10% by weight of sodium hydroxide is added as appropriate to adjust the pH to about 12. Adjusted. Thereafter, as in (3) and (4) of Comparative Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 7: No solubilization and alkaline hydrolysis)
(1) High molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1% by weight, weight average molecular weight 61,000) in 99.5 parts by weight, sodium hydroxide (Wako Pure Chemical Industries, Ltd.) 0.5 parts by weight of Kogyo Co., Ltd., special grade) was added and stirred at 50 ° C. for 16 hours.

(2) The appearance of the aqueous solution after standing at 5 ° C. for 24 hours was evaluated, and the weight average molecular weight was measured.

(Comparative Example 8: Solubilization and alkaline hydrolysis)
After obtaining a solubilized high molecular weight sericin aqueous solution in the same manner as (1) of Comparative Example 3, sodium hydroxide (made by Wako Pure Chemical Industries, Ltd., special grade) was added to 99.5 parts by weight of the obtained high molecular weight sericin aqueous solution. 0.5 part by weight was added and stirred at 50 ° C. for 16 hours. Then, it neutralized and rapidly cooled in the water bath, the low molecular weight sericin aqueous solution was obtained, and the external appearance evaluation after standing still at 5 degreeC for 24 hours and the measurement of the weight average molecular weight were performed.

(Comparative Example 9: No solubilization and acid hydrolysis)
(1) High molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) 99.5 parts by weight, hydrochloric acid (manufactured by Sasaki Chemical Co., Ltd.) , Hydrochloric acid concentration 35%, primary) was added, and the mixture was stirred at 50 ° C. for 16 hours, neutralized and quenched in a water bath to obtain a low molecular weight sericin aqueous solution.

(2) The appearance of the aqueous solution after standing at 5 ° C. for 24 hours was evaluated and the weight average molecular weight was measured.

(Comparative Example 10: with solubilization and acid hydrolysis)
After obtaining a solubilized high molecular weight sericin aqueous solution in the same manner as in (1) of Comparative Example 3, 99.5 parts by weight of the obtained high molecular weight sericin aqueous solution was mixed with hydrochloric acid (manufactured by Sasaki Chemical Co., Ltd., hydrochloric acid concentration 35%, first grade). 0.5 parts by weight) was added and stirred at 50 ° C. for 16 hours. Then, it neutralized and rapidly cooled in the water bath, the low molecular weight sericin aqueous solution was obtained, and the external appearance evaluation after standing still at 5 degreeC for 24 hours and the measurement of the weight average molecular weight were performed.

(Comparative Example 11: Amphoteric surfactant)
(1) High-molecular weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) 99.5 parts by weight, amphoteric surfactant (coconut oil Fatty acid amidopropyl betaine, “Amorgen CB-H” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., an aqueous solution having a surfactant concentration of 30% by weight) and 0.5 parts by weight are added and heated and stirred in a 95 ° C. water bath for 10 minutes. Quenched in an ice bath.

(2) Thereafter, as in (2) to (5) of Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 12: Cationic surfactant)
(1) High-molecular-weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) 99.5 parts by weight, cationic surfactant (lauroyl chloride) 0.5 parts by weight of trimethylammonium, “Daiichi Kogyo Seiyaku Co., Ltd.“ Cathogen TML ”, aqueous solution having a surfactant concentration of 30% by weight) was added and stirred in a water bath at 95 ° C. for 10 minutes, and then in an ice bath. It was cooled quickly.

(2) Thereafter, as in (2) to (5) of Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

(Comparative Example 13: Nonionic surfactant)
(1) High-molecular-weight sericin aqueous solution (“DK-sericin MT” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., sericin concentration 1.1 wt%, weight average molecular weight 61,000) 0.5 parts by weight of oxyethylene tridecyl ether, “Neugen TDS-500” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., an aqueous solution having a surfactant concentration of 30% by weight), and heated and stirred for 10 minutes in a 95 ° C. water bath After that, it was rapidly cooled in an ice bath.

(2) Thereafter, as in (2) to (5) of Example 1, a low molecular weight sericin aqueous solution was obtained, and appearance evaluation and weight average molecular weight were measured.

The results of the above Examples and Comparative Examples are as shown in Table 1. According to Examples 1 to 11, high molecular weight sericin could be reduced in a short reaction time of about 30 minutes. Moreover, the obtained low molecular weight sericin aqueous solution was excellent in transparency and uniformity.

(Example 12: Moisture lotion)
Concentrated glycerin (manufactured by Sakamoto Yakuhin Kogyo) 3 parts by weight, dipropylene glycol (manufactured by BASF Japan) 7.5 parts by weight, citric acid (manufactured by Nacalai Tesque) 0.02 parts by weight, sodium citrate (manufactured by Nacalai Tesque) ) 0.1 parts by weight, PCA isostearic acid glyceres-25 ("Pirotel GPI-25" manufactured by BASF Japan) 0.15 parts by weight, ethanol (manufactured by Japan Ethanol Co., Ltd.) 4 parts by weight, quince seed extract (Koei Kogyo Co., Ltd.) “Malmero BG”) 7 parts by weight, jojoba leaf extract (manufactured by Koei Kogyo Co., Ltd.) 1 part by weight, low molecular weight sericin aqueous solution (1% by weight) obtained in Example 1, 5 parts by weight, preservative and perfume And, the purified water was mixed with the remainder (however, in total 100 parts by weight) to prepare a moisturizing lotion.

  The preparation method is as follows. (1) Thoroughly mix concentrated glycerin, dipropylene glycol, citric acid, sodium citrate, and purified water. (2) Thoroughly mix the preservative, PCA glyceres-25 isostearate, ethanol, and perfume. (3) The quince BG, jojoba leaf extract, and the low molecular weight sericin aqueous solution are sufficiently mixed. (4) While stirring the liquid of the above (1), the liquid of the above (2) is added and made uniform, and then the liquid of the above (3) is added and made uniform.

(Example 13: Hair shampoo)
POE sodium lauryl ether sulfate (Daiichi Kogyo Seiyaku "Haitenol 325L", 25% solution) 24 parts by weight, lauric acid amidopropyl betaine (Daiichi Kogyo Seiyaku "Amogen LB-C", 30% solution) 30 Parts by weight, 5 parts by weight of polyglyceryl-10 laurate ("Sunsoft M-12J" manufactured by Taiyo Kagaku), 2 parts by weight of polyglyceryl-6 caprylate ("Sunsoft Q-81F" manufactured by Taiyo Kagaku), glycerin (new 5 parts by weight of Nippon Rika Co., Ltd., 2 parts by weight of ethylene glycol distearate (“NIKKOL EGDS” manufactured by Nikko Chemicals), 0.5 part by weight of the low molecular weight sericin aqueous solution (1% by weight) obtained in Example 1, antiseptic A hair shampoo was prepared by blending an appropriate amount of the agent, an appropriate amount of citric acid, and purified water with the remainder (however, a total of 100 parts by weight).
The preparation method is as follows. (1) All raw materials other than citric acid are mixed, heated to about 70 ° C. and uniformly dissolved. (2) Cool to 30 ° C. with stirring and adjust the pH to 6.8 with citric acid.

(Example 14: Hair treatment)
24 parts by weight of behentrimonium chloride (“NIKKOL CA-2580” manufactured by Nikko Chemicals, 80% solution), 30 parts by weight of distearyldimonium chloride (“NIKKOL CA-3475” manufactured by Nikko Chemicals, 75% solution), stearin 5 parts by weight of glyceryl acid ("NIKKOL MGS-BV" manufactured by Nikko Chemicals), 2 parts by weight of cetanol ("Cetanol # 2500" manufactured by Kyowa Technos), 5 parts by weight of oleyl alcohol ("Oleil # 1500HS" manufactured by Kyowa Technos) , 2 parts by weight of low melting point wax (“NIKKOL Nico Wax LM” manufactured by Nikko Chemicals), 0.2 part by weight of olive oil (“NIKKOL olive oil” manufactured by Nikko Chemicals), appropriate amount of DL-α-tocopherol (manufactured by Nacalai Tesque), dimethicone (“KF-96 manufactured by Shin-Etsu Chemical Co., Ltd. -100cs ", kinematic viscosity 100 mm ² / s) 1 part by weight, dimethicone (Shin-Etsu Chemical Co., Ltd." KF-96-1000cs "kinematic viscosity 1000mm ² /s)0.5 parts, low molecular weight obtained in Example 1 A hair treatment was prepared by blending 0.5 parts by weight of an aqueous sericin solution (1% by weight), an appropriate amount of preservatives, and the remaining amount of purified water (however, a total of 100 parts by weight). Street. (1) Uniformity while heating behentrimonium chloride, distearyldimonium chloride, glyceryl stearate, cetanol, oleyl alcohol, low melting wax, olive oil, tocopherol, dimethicone, low molecular weight sericin aqueous solution, preservative to 80 ° C To. (2) The above-mentioned liquid (1) is added and emulsified while heating and stirring purified water up to 80 ° C. (3) Cool to 35 ° C. with stirring.

  The low molecular weight sericin aqueous solution of the present invention can be used mainly as a cosmetic material, and in addition to various fields such as pharmaceuticals, quasi drugs, foods, biotechnology and other fields related to ecology, and fiber treatment agents. Can be used.

Claims (4)

To prepare a weight average molecular weight of 50,000 or more high molecular weight aqueous sericin solution solubilized with anionic surfactant by heating mixed with an anionic surfactant to the stock solution of high molecular weight aqueous sericin solution, the high molecular weight aqueous sericin solution obtained On the other hand, a method for producing a sericin aqueous solution, in which a sericin aqueous solution having a weight average molecular weight of less than 50,000 is prepared by hydrolyzing sericin with a proteolytic enzyme while maintaining the pH at 7-11.   The method for producing an aqueous sericin solution according to claim 1, wherein the anionic surfactant is at least one anionic surfactant selected from the group consisting of an amino acid-based surfactant and a polyoxyethylene alkyl ether acetate.   The method for producing an aqueous sericin solution according to claim 1 or 2, wherein the proteolytic enzyme is a cysteine protease and hydrolyzes sericin in the presence of a reducing agent.   The method for producing an aqueous sericin solution according to claim 1 or 2, wherein the proteolytic enzyme is an alkaline protease.