Sericin-containing powder and method for producing sericin-containing powder

Abstract

To provide a sericin-containing powder with good dispersibility even though it contains high molecular weight sericin, and to provide a method for producing the same.SOLUTION: Provided is a sericin-containing powder that contains sericin and acid, the sericin being a sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE method, and the pH being 5.0 or less when made into a 1.0 mass% aqueous dispersion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a sericin-containing powder and a method for producing the same. More specifically, the present invention relates to a sericin-containing powder that contains high-molecular-weight sericin and has excellent dispersibility, and a method for producing the same. [Background technology]

[0002] The cocoon threads spun by silkworms and other insects are mainly composed of two proteins: sericin and fibroin. Of these, fibroin is the protein that makes up the fibers of the cocoon, and sericin is the protein that coats the outer layer of the fibers formed by fibroin and is involved in the adhesion of the fibers.

[0003] The sericin contained in cocoons is solubilized when the cocoons are boiled in alkaline hot water to extract fibroin during the silk thread degumming process. Therefore, sericin is usually obtained from the wastewater of this degumming process, but its molecular weight has been reduced by hydrolysis.

[0004] On the other hand, it is known that high-molecular-weight sericin has superior properties to low-molecular-weight sericin (e.g., Reference 1). A method for obtaining high-molecular-weight sericin is known to be extraction under mild conditions using an aqueous solution of lithium bromide as an extraction solvent (Reference 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-40905 [Patent Document 2] Japanese Patent Application Laid-Open No. 2006-111667 Summary of the Invention [Problem to be solved by the invention]

[0006] However, high-molecular-weight sericin generally has poor solubility and dispersibility, making it difficult to incorporate into cosmetic formulations, especially when it is in powder form. Furthermore, even when high-molecular-weight sericin is obtained in an aqueous solution, it gels over time, making it difficult to obtain in a stable state or to incorporate into cosmetic formulations.

[0007] The present invention has been made in light of the above-mentioned circumstances, and its object is to provide a sericin-containing powder that can achieve both high molecular weight and good dispersibility, and a method for producing the same. [Means for solving the problem]

[0008] The gist of the present invention is as follows. [1] A sericin-containing powder containing sericin and acid, The sericin is sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE, A sericin-containing powder characterized by having a pH of 5.0 or less when dispersed in 1.0% by mass water. [2] The sericin-containing powder according to [1], having a degree of dispersion of 33% or more as measured by the following method. [Dispersion measurement method] 10.0 mg of sericin-containing powder was added to 1.00 mL of ion-exchanged water, accurately measured with a measuring pipette, and heated at 60°C for 30 minutes in a 2.0 mL screw-cap tube (Watson, No. 1392-200-SS-CS). After heating, the mixture was centrifuged at 9,500 x g for 5 minutes, and 0.20 mL of the supernatant was removed. The mixture was then mixed in a vortex mixer (approximately 2700 rpm for 5 seconds) and sonicated at room temperature for 5 minutes in an ultrasonicator (Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water. After sonication, the tube was placed vertically and the height (H) of the liquid portion and the height (h) of the high-concentration portion were measured. The degree of dispersion (%) was calculated using the following formula: Dispersion degree (%)=h / H×100 [3] The sericin-containing powder according to [1] or [2], wherein the acid is at least one selected from the group consisting of an aliphatic monocarboxylic acid, an aliphatic polycarboxylic acid, and an inorganic acid. [4] An extraction step of obtaining sericin from the cocoons using an organic solvent; a step of obtaining a sericin-containing gel by mixing the sericin extract with an acidic aqueous solution having a pH of 5.0 or less; and a drying step of powdering the gel obtained as described above. [5] The method according to [4], wherein the organic solvent is at least one selected from the group consisting of sulfoxide solvents and amide solvents. [6] The method according to [4] or [5], wherein the organic solvent contains at least one selected from the group consisting of alkali metal halides and alkaline earth metal halides. [7] The method according to any one of [4] to [6], wherein the acidic aqueous solution contains at least one acid selected from the group consisting of an aliphatic monocarboxylic acid, an aliphatic polycarboxylic acid, and an inorganic acid. [8] The method according to any one of [4] to [7], further comprising a washing step of washing the obtained gel with an acidic aqueous solution after the sericin-containing gel obtaining step and before the drying step. [9] The method according to [8], wherein the acidic aqueous solution used in the washing step contains at least one acid selected from the group consisting of aliphatic monocarboxylic acids, aliphatic polycarboxylic acids, and inorganic acids.

[10] A manufacturing method described in any of [4] to [9], wherein the sericin contained in the sericin-containing powder is sericin in which at least one band is detected in the range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a sericin-containing powder that contains high-molecular-weight sericin yet has good dispersibility (particularly dispersibility in water), and a method for producing the same. Because the sericin-containing powder of the present invention has good dispersibility, it is easy to incorporate into cosmetic formulations and is easy to handle. Furthermore, if sericin is stored in solution, hydrolysis of the sericin may occur during storage, and undesired gelation may occur, particularly in the case of sericin with a high molecular weight. However, because the sericin of the present invention is in powder form, it is highly stable during storage. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram relating to the measurement of the dispersity. [Figure 2] FIG. 2 is a photograph showing the results of measuring the dispersibility of the sericin-containing powder obtained in Example 3. [Figure 3] FIG. 3 is a photograph showing the results of measuring the dispersibility of the sericin-containing powder obtained in Comparative Example 1. [Figure 4] FIG. 4 is a photograph showing the results of SDS-PAGE of sericin in the sericin-containing powder obtained in Example 13 after storage for a certain period of time. [Figure 5] FIG. 5 is a photograph showing the results of confirming the film-forming ability of the sericin-containing powder obtained in Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Sericin-containing powder The sericin-containing powder of the present invention is characterized by containing high-molecular-weight sericin and an acid, and having a pH of 5.0 or less when dispersed in 1.0% by mass in water. The sericin-containing powder of the present invention having such a constitution has excellent dispersibility (particularly dispersibility in water) despite containing high-molecular-weight sericin.

[0012] 1.1 Sericin The sericin contained in the sericin-containing powder of the present invention is one in which clear bands are detected by SDS-polyacrylamide gel electrophoresis (PAGE), rather than bands that are not visible or smeared. Specifically, at least one band is detected in the range corresponding to a molecular weight of 150 kDa or higher. The band may be detected in the range corresponding to a molecular weight of 150 kDa or higher, preferably in the range corresponding to a molecular weight of 160 kDa or higher. The number of bands detected in this range may be one or more, and there is no upper limit. However, the number of bands detected in this range is preferably one to five, and more preferably two to five. Furthermore, a sericin-containing powder containing sericin in which one or more clear bands are detected by SDS-PAGE in the range corresponding to a molecular weight of 250 kDa or higher is also preferred. A sericin-containing powder containing sericin in which such bands are detected indicates that the sericin has not been degraded and exhibits the properties characteristic of high-molecular-weight sericin. The molecular weights corresponding to the bands detected in SDS-PAGE were determined by the marker proteins (e.g., Precision Plus Protein TM The molecular weight of the sericin can be determined by comparing it with the bands of the molecular weight fractions of a two-color standard (e.g., manufactured by BIO-RAD). The constituent ratio of the bands can be calculated from the density of the bands. However, the sericin contained in the sericin-containing powder of the present invention is not particularly limited as long as it is a high-molecular-weight sericin in which at least one band is detected within the range corresponding to a molecular weight of 150 kDa or more.

[0013] The content of sericin in the sericin-containing powder of the present invention is preferably 50% by mass or more, more preferably 60% by mass or more, even more preferably 70% by mass or more, and even more preferably 80% by mass or more, from the viewpoint of the dispersibility of the sericin-containing powder, in order to fully exert the functions of sericin, such as antioxidant activity, tyrosinase inhibitory activity, and film-forming ability. From the viewpoint of the dispersibility of the sericin-containing powder, the content of sericin is preferably 99.9% by mass or less, and more preferably 99.5% by mass or less.

[0014] 1.2 Acid The acid contained in the sericin-containing powder of the present invention may be an organic acid or an inorganic acid. The organic acid is preferably a carboxylic acid compound, with aliphatic monocarboxylic acids and aliphatic polycarboxylic acids being more preferred. Examples of aliphatic monocarboxylic acids include saturated aliphatic monocarboxylic acids such as acetic acid, propionic acid, butyric acid, and valeric acid, and unsaturated aliphatic monocarboxylic acids such as sorbic acid. Aliphatic saturated monocarboxylic acids are preferred, with saturated aliphatic monocarboxylic acids having 1 to 8 carbon atoms being more preferred. Examples of aliphatic polycarboxylic acids include saturated aliphatic polycarboxylic acids such as oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, adipic acid, and citric acid, and unsaturated aliphatic polycarboxylic acids such as maleic acid, fumaric acid, citraconic acid, and mesaconic acid. Examples of aliphatic polycarboxylic acids include saturated aliphatic polycarboxylic acids having 2 to 10 carbon atoms, more preferably saturated aliphatic polycarboxylic acids having 2 to 8 carbon atoms, and even more preferably saturated aliphatic oxypolycarboxylic acids having 2 to 8 carbon atoms, with citric acid being particularly preferred. Examples of inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and bromic acid, with hydrochloric acid being preferred. The acid is preferably an aliphatic monocarboxylic acid, an aliphatic polycarboxylic acid, or an inorganic acid, more preferably an aliphatic polycarboxylic acid or an inorganic acid, and even more preferably citric acid or hydrochloric acid. These acids may be used alone or in combination of two or more.

[0015] The acid content in the sericin-containing powder is, for example, preferably 0.1% by mass or more, more preferably 0.3% by mass or more, even more preferably 0.5% by mass or more, and preferably 35% by mass or less, more preferably 25% by mass or less, and even more preferably 20% by mass or less. When the acid content in the sericin-containing powder is within the above range, the dispersibility is better.

[0016] The acid content in the sericin-containing powder can be determined by a known compound analysis method such as HPLC.

[0017] The acid may be used in combination with a salt thereof (alkali metal salt, alkaline earth metal salt, amine salt, ammonium salt, etc.) as needed, but the content of the acid does not include the content of the salt.

[0018] 1.3 Characteristics The sericin-containing powder of the present invention has a pH of 5.0 or less when dispersed in a 1.0% by mass aqueous solution. The pH is preferably 4.0 or less, more preferably 3.0 or less, and even more preferably 2.7 or less, and the lower limit may be, for example, 1.5. The sericin-containing powder of the present invention contains an acid, and the pH of the aqueous dispersion is within the above range, resulting in excellent dispersibility. The pH of the aqueous dispersion is measured after sufficient dispersion treatment. For example, it is preferable to heat the dispersion at 60°C for 30 minutes or more, and then disperse the dispersion at room temperature for 5 minutes or more using an ultrasonic device. Alternatively, the dispersion may be centrifuged, and the resulting supernatant may be used to measure the pH.

[0019] The sericin-containing powder of the present invention preferably has a dispersity of 33% or more, more preferably 45% or more, even more preferably 50% or more, even more preferably 60% or more, even more preferably 70% or more, particularly preferably 75% or more, or may even be 100%, as measured by the following method.

[0020] [Dispersion measurement method] 10.0 mg of sericin-containing powder was added to 1.00 mL of ion-exchanged water, accurately measured with a measuring pipette, and heated at 60°C for 30 minutes in a 2.0 mL screw-cap tube (Watson, No. 1392-200-SS-CS). After heating, the mixture was centrifuged at 9,500 x g for 5 minutes, and 0.20 mL of the supernatant was removed. The mixture was then mixed in a vortex mixer (approximately 2700 rpm for 5 seconds) and sonicated at room temperature for 5 minutes in an ultrasonicator (Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water. After sonication, the tube was placed vertically and the height (H) of the liquid portion and the height (h) of the high-concentration portion were measured. The degree of dispersion (%) was calculated using the following formula: Dispersion degree (%)=h / H×100 The high-concentration part is a part that is thicker and cloudier than the supernatant part, and an interface is formed between the supernatant part and the high-concentration part, so its height can be determined. When the liquid part is uniform overall and no interface is observed, the degree of dispersion is 100%. If the interface is not horizontal, for example, if it is uneven due to partial swelling, determine h at the lowest point.

[0021] The sericin-containing powder of the present invention does not necessarily have good solubility, but it is preferable that it has better solubility than ordinary polymeric sericin. Specifically, "good solubility" preferably means high solubility in water, for example, a solubility of 0.020 g / 100 g of aqueous solution or greater, as measured by the following method. The solubility is preferably 0.035 g / 100 g of aqueous solution or greater, more preferably 0.040 g / 100 g of aqueous solution or greater, even more preferably 0.050 g / 100 g of aqueous solution or greater, even more preferably 0.070 g / 100 g of aqueous solution or greater, and particularly preferably 0.080 g / 100 g of aqueous solution or greater, and may be 0.3 g / 100 g of aqueous solution or less. Furthermore, the solubility may be 0.001 g / 100 g of aqueous solution or greater.

[0022] [Solubility measurement method] Sericin-containing powder was added to ion-exchange water to a sericin concentration of 1.0% by mass, and dissolved at 60°C for 30 minutes. After dissolution, the solution was centrifuged at an acceleration of 9,500 × g for 5 minutes, and the supernatant was collected. The sericin concentration in the resulting aqueous solution was calculated from the absorbance at 280 nm. The mass of dissolved sericin was determined from the calculated sericin concentration. The sericin concentration was calculated by creating a calibration curve. The dissolution rate was then calculated using the following formula from the mass of dissolved sericin and the mass of sericin initially added, and the solubility was calculated using this dissolution rate. Dissolution rate of sericin (wt%) = (mass of dissolved sericin / mass of added sericin) x 100 Solubility of sericin (g / 100g of aqueous solution) = 100 (g) × 1 (wt%) × solubility of sericin (wt%)

[0023] 1.4 Other ingredients The sericin-containing powder of the present invention may contain other components in addition to sericin and acid as long as the above-mentioned properties are not impaired. The content of the other components is preferably 30% by mass or less, more preferably 10% by mass or less, and may be 0.1% by mass or more of the total amount of the sericin-containing powder. However, it is particularly preferred that the sericin-containing powder does not contain any other components.

[0024] 2. Method for producing sericin-containing powder Conventionally, sericin has been obtained by hydrolyzing and eluting cocoons, cocoon filaments, and raw silk using hydrolysis methods such as acid hydrolysis, alkaline hydrolysis, and enzymatic degradation. However, in the method for producing a sericin-containing powder of the present invention, sericin is extracted from cocoons, cocoon filaments, raw silk, etc. using an organic solvent without hydrolysis. An acidic aqueous solution of pH 5.0 or less is then added to the extract to form a sericin-containing gel, and the resulting gel is dried to obtain a sericin-containing powder. Therefore, the sericin-containing powder obtained by the production method of the present invention contains unhydrolyzed sericin. However, as long as the sericin-containing powder contains high-molecular-weight sericin (sericin having a band in the range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE), it may also contain sericin hydrolyzed during the extraction. Furthermore, the sericin-containing powder may also contain fibroin. The sericin-containing powder obtained by the production method of the present invention contains an acid and has excellent dispersibility.

[0025] 2.1 Extraction process The production method of the present invention includes a step of extracting sericin from cocoons, cocoon filaments, or raw silk using an organic solvent as an extraction solvent.

[0026] The cocoons, cocoon filaments, and raw silk used for sericin extraction are not particularly limited as long as they are produced by silkworms or other silkworms, and silk produced by wild silkworms of the Bombycidae family, such as the mulberry silkworm and the mulberry silkworm, wild silkworms of the Saturniidae family, such as the wild silkworm, the tussah silkworm, and the tasar silkworm, or insects such as honeybees, hornets, spiders, and caddisflies may also be used, and there are no particular limitations on the place of origin. Furthermore, silkworms may be natural or may be those that have been subjected to mutation, breeding, or genetic engineering. For example, since high molecular weight sericin tends to be extracted efficiently, silkworms with poor fibroin production ability can be used. Specifically, silkworms with reduced fibroin production ability due to mutation or breeding (e.g., Sericin Hope (sericin C), sericin N, Nd silkworms, Nd-s silkworms, MNS300, MCS300, etc.) can be used.

[0027] Silkworm cocoons are structures created by silkworm larvae secreting silk proteins from their silk glands during pupation, and are obtained by separating the pupae from the cocoons. Raw silk is obtained by combining several cocoon threads (silk fibers) extracted from the cocoons to form a thread.

[0028] The cocoons may be used as they are, or may be processed by cutting, crushing, drying, or the like.

[0029] Examples of the organic solvent include ether-based solvents, ester-based solvents, hydrocarbon-based solvents, ketone-based solvents, nitrile-based solvents, halogen-based solvents, amide-based solvents, and sulfoxide-based solvents. Amide-based solvents such as dimethylformamide and dimethylacetamide, and sulfoxide-based solvents such as dimethyl sulfoxide and diethyl sulfoxide are preferred, sulfoxide-based solvents are more preferred, and dimethyl sulfoxide is even more preferred. These organic solvents may be used alone or in combination of two or more.

[0030] The amount of organic solvent used is preferably 5 to 50 times by mass, more preferably 10 to 40 times by mass, relative to the amount of cocoons, cocoon filaments, raw silk, etc.

[0031] The organic solvent preferably contains an inorganic salt, more preferably an alkali metal halide and / or an alkaline earth metal halide. Examples of alkali metal halides include lithium halide, sodium halide, and potassium halide, and examples of alkaline earth metal halides include calcium halide, strontium halide, and barium halide. Examples of inorganic salts include lithium chloride, lithium bromide, sodium chloride, sodium bromide, potassium chloride, potassium bromide, calcium chloride, and calcium bromide, and calcium chloride is more preferred. These inorganic salts may be used alone or in combination of two or more.

[0032] The total concentration of inorganic salts in the organic solvent is preferably 0.3 mol / L or more and 3.0 mol / L or less, more preferably 0.5 mol / L or more and 2.0 mol / L or less, and even more preferably 0.8 mol / L or more and 1.5 mol / L or less.

[0033] The extraction temperature is preferably 100°C or lower, more preferably 80°C or lower, from the viewpoint of suppressing hydrolysis of sericin, and is preferably 0°C or higher from the viewpoint of extraction efficiency.

[0034] The extraction time can be set appropriately depending on the amount of raw materials used and the equipment used for extraction, but is preferably 48 hours or less, more preferably 24 hours or less, even more preferably 12 hours or less, and even more preferably 6 hours or less, and from the viewpoint of extraction efficiency, preferably 0.5 hours or more.

[0035] The concentration of sericin in the extract can be set appropriately depending on the extraction conditions, but is preferably 0.1% by mass or more and 20% by mass or less, more preferably 0.5% by mass or more and 18% by mass or less, and even more preferably 1.0% by mass or more and 15% by mass or less.

[0036] 2.2 Gel acquisition process The production method of the present invention includes a step of mixing the sericin extract obtained in the extraction step with an acidic aqueous solution having a pH of 5.0 or less to form a sericin-containing gel.

[0037] By setting the pH of the acidic aqueous solution to 5.0 or less, a sericin-containing powder with excellent dispersibility can be produced. The pH of the acidic aqueous solution is preferably 4.0 or less, more preferably 3.0 or less, even more preferably 2.8 or less, and even more preferably 2.5 or less, with no particular lower limit, and may be 1.0 or more.

[0038] The acidic aqueous solution contains an acid, which may be an organic acid or an inorganic acid. The organic acid is preferably a carboxylic acid compound, with aliphatic monocarboxylic acids and aliphatic polycarboxylic acids being more preferred. Examples of aliphatic monocarboxylic acids include saturated aliphatic monocarboxylic acids such as acetic acid, propionic acid, butyric acid, and valeric acid, and unsaturated aliphatic monocarboxylic acids such as sorbic acid. Aliphatic saturated monocarboxylic acids are preferred, with saturated aliphatic monocarboxylic acids having 1 to 8 carbon atoms being more preferred. Examples of aliphatic polycarboxylic acids include saturated aliphatic polycarboxylic acids such as oxalic acid, malonic acid, succinic acid, malic acid, tartaric acid, glutaric acid, adipic acid, and citric acid, and unsaturated aliphatic polycarboxylic acids such as maleic acid, fumaric acid, citraconic acid, and mesaconic acid. Examples of aliphatic polycarboxylic acids include saturated aliphatic polycarboxylic acids having 2 to 10 carbon atoms, more preferably saturated aliphatic polycarboxylic acids having 2 to 8 carbon atoms, and even more preferably saturated aliphatic oxypolycarboxylic acids having 2 to 8 carbon atoms, with citric acid being particularly preferred. Examples of inorganic acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, and bromic acid, with hydrochloric acid being preferred. The acidic aqueous solution preferably contains at least one acid selected from the group consisting of aliphatic monocarboxylic acids, aliphatic polycarboxylic acids, and inorganic acids, more preferably contains an aliphatic polycarboxylic acid and / or an inorganic acid, and even more preferably contains citric acid and / or hydrochloric acid. These acids may be contained alone or in combination of two or more.

[0039] The acid concentration of the acidic aqueous solution is preferably 2.0 mmol / L or more, more preferably 5.0 mmol / L or more, and although there is no particular upper limit, it is preferably 1 mol / L or less. Note that the acid concentration does not include the concentrations of acid salts (alkali metal salts, alkaline earth metal salts, amine salts, ammonium salts, etc.).

[0040] The volume of the acidic aqueous solution used is preferably 0.5 to 50 times the volume of the sericin extract, more preferably 1.0 to 20 times, and even more preferably 2.0 to 10 times. When the volume of the acidic aqueous solution is within this range, a sericin-containing gel with good properties can be obtained without excessive aggregation.

[0041] After adding the acidic aqueous solution to the sericin extract, the mixture is mixed by a known method such as using a vortex mixer to obtain a mixture, which is then allowed to stand at room temperature or under cooling (e.g., 1 to 10°C) for, for example, 1 hour to 3 days to allow gel formation to proceed.

[0042] The resulting sericin-containing gel is preferably one in which the sericin does not aggregate excessively and has good softness.

[0043] The sericin-containing gel may be extracted from the mixture by filtration or centrifugation. The conditions for centrifugation are not particularly limited as long as the gel precipitates and the gel can be separated from the liquid by decantation. However, preferred conditions are, for example, 5 to 40°C, 2,000 to 20,000 × g, and 1 to 30 minutes.

[0044] 2.3 Cleaning process The production method of the present invention preferably includes a step of washing the obtained gel with an acidic aqueous solution (hereinafter also referred to as a washing solution) after the gel obtaining step and before the drying step. By washing the sericin-containing gel, the organic solvent used in the extraction step and, if necessary, the inorganic salt used can be removed.

[0045] The washing liquid contains an acid, and examples of the acid include the same acids as those contained in the acidic aqueous solution used in the gel obtaining step, and the preferred embodiments thereof are also the same.

[0046] The pH of the cleaning solution is preferably 5.0 or less, more preferably 4.0 or less, even more preferably 3.0 or less, even more preferably 2.8 or less, and particularly preferably 2.5 or less, with no particular lower limit and may be 1.0 or more.

[0047] The acid concentration of the cleaning solution is preferably 1.0 mmol / L or more, more preferably 2.0 mmol / L or more, and even more preferably 5.0 mmol / L or more. There is no particular upper limit, but it is preferably 1 mol / L or less. Note that the acid concentration does not include the concentration of acid salts (alkali metal salts, alkaline earth metal salts, amine salts, ammonium salts, etc.).

[0048] The washing liquid may contain the same acid as the acidic aqueous solution used in the gel obtaining step, and may also have the same pH and acid concentration as the acidic aqueous solution used in the gel obtaining step. Preferably, the washing liquid contains the same acid as the acidic aqueous solution used in the gel obtaining step.

[0049] The amount of washing solution used is preferably 0.5 to 50 times by volume, more preferably 1.0 to 20 times by volume, and even more preferably 2.0 to 10 times by volume, relative to the sericin extract used in the gel obtaining step.

[0050] The washing treatment is carried out by adding a washing solution to the sericin-containing gel, mixing by a known method such as using a vortex mixer, centrifuging, and decanting. The conditions for centrifugation are not particularly limited as long as the gel is precipitated by centrifugation and the gel can be separated from the liquid by decantation. However, for example, conditions within the range of 5 to 40°C, 2,000 to 20,000 × g, and 1 to 30 minutes are preferred.

[0051] The number of washings is not particularly limited as long as unnecessary substances (particularly inorganic salts used as needed) can be removed, but is preferably 1 to 10 times, more preferably 3 to 6 times. When washing is performed multiple times, washing may be performed with one type of washing solution, or two or more types of washing solutions may be combined.

[0052] 2.4 Drying process The production method of the present invention includes a step of drying a sericin-containing gel to powder it.

[0053] The drying method is not particularly limited as long as it can produce a powder by drying, but examples thereof include freeze-drying, reduced pressure (vacuum, low vacuum) drying, spray-drying, etc., with freeze-drying and spray-drying being preferred, and freeze-drying being more preferred. All of these drying methods may be performed using a known drying apparatus, and drying conditions such as drying temperature and drying time may be appropriately set depending on the drying method.

[0054] The sericin-containing powder obtained after the drying treatment has excellent dispersibility (particularly dispersibility in water) while containing high-molecular-weight sericin, and preferably has properties similar to those of the sericin-containing powder described above.

[0055] The yield of sericin is preferably 20% by mass or more, more preferably 25% by mass or more, and even more preferably 30% by mass or more, and the upper limit is not particularly limited and may be 100% by mass or 85% by mass or less. The yield of sericin can be determined by the following method.

[0056] [Sericin yield measurement method] First, the sericin concentration in the sericin extract is calculated from the absorbance at 280 nm, and the mass of sericin in the sericin-containing powder (the mass of sericin obtained by extraction) is determined from the calculated sericin concentration. The sericin concentration is calculated by creating a calibration curve. The yield is then calculated using the following formula from the mass of sericin obtained by extraction and the mass of the cocoons (or cocoon filaments or raw silk) used to extract the sericin. Yield (wt%) = (mass of sericin obtained by extraction / mass of cocoons used for sericin extraction) × 100

[0057] The total content of inorganic salts in the sericin-containing powder is preferably 500 ppm or less, more preferably 300 ppm or less, even more preferably 100 ppm or less, even more preferably 50 ppm or less, or even 0 ppm. Even if inorganic salts are used in the extraction process, the total content of inorganic salts can be kept within the above range by including a washing process. If the content is within the above range, the storage stability of the sericin-containing powder is further improved. Furthermore, the safety of the sericin-containing powder when applied to skin preparations, etc. is also excellent. In this specification, unless otherwise specified, "ppm" means a value calculated by mass conversion.

[0058] The production method of the present invention preferably does not include a step of using a protein denaturant such as a surfactant, so the resulting sericin-containing powder does not contain such a protein denaturant and there is no risk of the higher-order structure of sericin being changed.

[0059] The sericin-containing powder obtained by the present invention is a dry powder and therefore has excellent storage stability. Specifically, even after storage for one month under various temperature conditions (e.g., room temperature, 50°C), sericin maintains its high molecular weight without undergoing hydrolysis. Furthermore, while storage in a solution state can cause undesired gelation and make handling difficult, the dry powder eliminates the risk of gelation during storage.

[0060] The sericin-containing powder of the present invention contains high-molecular-weight sericin and has good dispersibility, making it possible to obtain a dispersion (mixture) with excellent film-forming ability. Therefore, the sericin-containing powder of the present invention can be suitably used as an active ingredient in moisturizers that promote skin protection and protective agents that supplement the barrier function. [Example]

[0061] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples, and it is possible to carry out the invention by making appropriate modifications within the scope of the above and below-described aims, and all such modifications are included in the technical scope of the present invention.

[0062] Measurements and evaluations in the examples and comparative examples were carried out by the following methods.

[0063] (1) Properties of sericin-containing gel Regarding the properties of the sericin-containing gel, if it was soft it was judged as having good properties (◯), and if it was hard due to aggregation it was judged as having poor properties (×).

[0064] (2) Sericin yield First, the sericin concentration in the sericin extract is calculated from the absorbance at 280 nm, and the mass of sericin in the sericin-containing powder (the mass of sericin obtained by extraction) is determined from the calculated sericin concentration. The sericin concentration is calculated by creating a calibration curve. The yield is then calculated using the following formula from the mass of sericin obtained by extraction and the mass of the cocoons (or cocoon filaments or raw silk) used to extract the sericin. Three measurements are taken, and the average value is the sericin yield of the sample. Yield (wt%) = (mass of sericin obtained by extraction / mass of cocoons used for sericin extraction) × 100

[0065] (3) Solubility of sericin-containing powder 10.0 mg of sericin-containing powder was added to 1.00 mL of ion-exchanged water accurately measured with a measuring pipette and heated at 60°C for 30 minutes in a 2.0 mL screw-cap tube (Watson, No. 1392-200-SS-CS). After heating, the mixture was centrifuged at 9,500 × g for 5 minutes, and 0.20 mL of the supernatant was collected. The sericin concentration in the resulting aqueous solution was calculated from the absorbance at 280 nm. The mass of dissolved sericin was calculated from the calculated sericin concentration. The sericin concentration was calculated by creating a calibration curve. The dissolution rate was then calculated using the following formula from the mass of dissolved sericin and the mass of sericin initially added, and the solubility was calculated using this dissolution rate. Three measurements were performed, and the average value was used as the solubility of the sample. Dissolution rate of sericin (wt%) = (mass of dissolved sericin / mass of added sericin) x 100 Solubility of sericin (g / 100g of aqueous solution) = 100 (g) × 1 (wt%) × solubility of sericin (wt%)

[0066] (4) Dispersibility and dispersibility of sericin-containing powder After collecting 0.20 mL of the supernatant for the solubility measurement, the remainder was mixed in a vortex mixer (approximately 2700 rpm, 5 seconds), and then ultrasonicated for 5 minutes at room temperature in an ultrasonic treatment device (Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water. After ultrasonic treatment, the tube was held upright and the height H of the liquid portion and the height h of the high-concentration portion in the tube were measured, and the dispersity (%) was calculated using the following formula. Two measurements were performed, and the average value was used as the dispersity of the sample. H and h in dispersity measurement are shown in Figure 1. Dispersion degree (%)=h / H×100 The high-concentration part is a part that is thicker and cloudier than the supernatant part, and an interface is formed between the supernatant part and the high-concentration part, so its height can be determined. When the liquid part is uniform overall and no interface is observed, the degree of dispersion is 100%. If the interface is not horizontal, for example, if it is uneven due to partial swelling, determine h at the lowest point.

[0067] Regarding the dispersibility of the sericin-containing powder, a degree of dispersion of 50% or more and 100% or less was judged as very good (◎), 33% or more and less than 50% was judged as good (○), and less than 33% was judged as poor (×).

[0068] (5) pH of 1.0% by mass aqueous dispersion of sericin-containing powder 10.0 mg of sericin-containing powder was added to 1.00 mL of ion-exchanged water accurately measured with a measuring pipette and heated at 60°C for 30 minutes. The mixture was sonicated for 5 minutes at room temperature using an ultrasonicator (Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water, followed by centrifugation at 9,500 × g for 5 minutes. The pH of the supernatant (300 μL) was measured using a pH meter (HORIBA, Ltd., LAQUAtwin B-712). Two measurements were taken, and the average was used as the pH of the sample.

[0069] (6) Molecular weight of sericin Weigh out 2-3 mg of sericin-containing powder, add 100 times the weight of sericin in the powder with 8M LiBr aqueous solution, and dissolve at 35°C for 2 hours. The resulting solution is used to measure the molecular weight by SDS-PAGE. The gel used is a Multigel II Mini Gradient Gel 2-15% 17-well (Cosmo Bio Co., Ltd.), and the molecular weight marker is Precision Plus Protein TM A two-color standard (BIO-RAD, product number 1610374) was used.

[0070] Regarding the molecular weight of sericin, if at least one band was detected in the range corresponding to a molecular weight of 150 kDa or more, it was judged as good (○), and if no band was detected in the range corresponding to a molecular weight of 150 kDa or more, it was judged as poor (×).

[0071] (7) Calcium ion concentration The calcium ion concentration in the cleaning solution was measured by measuring the supernatant of the cleaning solution using a compact calcium ion meter (LAQUAtwin Ca-11, manufactured by Horiba, Ltd.). The calcium ion concentration in the sericin-containing powder was measured using a compact calcium ion meter (LAQUAtwin Ca-11, manufactured by Horiba, Ltd.) for a dispersion prepared by adding 1.00 mL of ion-exchanged water to 10.0 mg of sericin-containing powder and heating the mixture at 60°C for 30 minutes. The calcium ion concentration was calculated from the measured value. Three measurements were taken, and the average value was used as the calcium ion concentration of the sample.

[0072] (8) Storage stability The sericin-containing powder is stored at room temperature or 50°C for two weeks or one month. The molecular weight of the sericin is measured using the stored sericin-containing powder by the method described above. Furthermore, 10 mg of the stored sericin-containing powder is mixed with 1 mL of ion-exchanged water in a 2.0 mL screw-cap tube and dispersed at 60°C for 30 minutes. The dispersed sample is freeze-dried to form a sericin-containing powder, and the molecular weight of the sericin is measured using the sericin-containing powder by the method described above.

[0073] If high molecular weight sericin was maintained even after storage (especially if at least one band was detected in the range corresponding to a molecular weight of 150 kDa or more), the storage stability was determined to be good.

[0074] (9) Film forming ability 20 mg of sericin-containing powder was added to 2 mL of ion-exchanged water and mixed at 60°C for 30 minutes to obtain a mixture. The entire mixture was spread evenly on a dialysis membrane (Spectra / Por, No. 4, MWCO: 12,000-14,000) and left overnight on a melamine sponge soaked in ion-exchanged water to remove electrolytes from the mixture. The dialysis membrane was dried at 50°C for 2 hours and then re-swelled in ion-exchanged water to immerse the entire membrane. The resulting membrane was then peeled off from the membrane.

[0075] If a uniform film without cracks or defects could be formed, the film forming ability was judged to be good.

[0076] Example 1 A flask was charged with 1.5 g of sericin "Hope" cocoons obtained from selectively bred silkworms and 30 mL of 1 M CaCl2-DMSO solution, which was then stirred and dissolved at 70°C for 1 hour. A 50 mL centrifuge tube was charged with 2 mL of the resulting solution (extract) and 10 mL of 10 mM hydrochloric acid (pH 2.0), vortex-mixed (approximately 2700 rpm, 5 seconds x 2), and then refrigerated (4°C) overnight to form a sericin-containing gel. The results of observing the shape of the resulting gel are shown in Table 1. The gel-containing solution was centrifuged at 25°C and 9,500 x g for 5 minutes, and the supernatant was discarded. To this solution, 10 mL of the same acidic aqueous solution used to obtain the sericin-containing gel was added, and the mixture was mixed using a vortex mixer (approximately 2700 rpm, 5 seconds x 2 times), followed by centrifugation at 25°C and 9,500 x g for 5 minutes, and the supernatant was discarded. This washing process was repeated five times. The washed gel was freeze-dried to obtain a sericin-containing powder.

[0077] (Examples 2 to 11, Comparative Example 1) A sericin-containing powder was obtained in the same manner as in Example 1, except that the acidic aqueous solution used was changed to the acidic aqueous solution shown in Table 1.

[0078] The sericin yield and molecular weight, the solubility and dispersibility of the sericin-containing powder, and the pH of a 1.0% by mass aqueous dispersion of the sericin-containing powder were measured using the sericin-containing powders obtained in Examples 1 to 11 and Comparative Example 1. The measurement results are shown in Table 1. Photographs of the solution after dispersion treatment in the measurement of dispersibility are shown in Figures 2 and 3. Figure 2 corresponds to Example 3, and Figure 3 corresponds to Comparative Example 1.

[0079] Furthermore, the sericin-containing powders obtained in Examples 1 to 11 and Comparative Example 1 were freeze-dried to obtain sericin-containing powders. The molecular weight of sericin in each sericin-containing powder was confirmed by SDS-PAGE. The results of confirming the molecular weight of sericin in the sericin-containing powders after dispersion treatment are shown in Table 1.

[0080] [Table 1]

[0081] In Table 1, the citrate buffer is specifically a citric acid-trisodium citrate buffer.

[0082] As shown in Table 1, the sericin-containing powders obtained in Examples 1 to 11 contained high-molecular-weight sericin and had high dispersibility. Furthermore, the high-molecular-weight sericin was maintained even after the dispersion treatment.

[0083] Example 12 A flask was charged with 1.5 g of sericin-containing cocoons obtained from selectively bred silkworms and 30 mL of 1M CaCl2-DMSO solution. The mixture was stirred and dissolved at 70°C for 1 hour. A 500 mL centrifuge tube was charged with 20 mL of the resulting solution (extract) and 100 mL of 10 mM citric acid solution (pH 2.7). The mixture was then mixed at 50-60°C and then refrigerated (4°C) overnight to form a sericin-containing gel. The gel-containing solution was centrifuged at 9,500 × g for 10 minutes at 4°C, and the supernatant was discarded. A washing process was performed five times: adding 100 mL of the same acidic aqueous solution used to obtain the sericin-containing gel, mixing, centrifuging at 9,500 × g for 10 minutes at 4°C, and discarding the supernatant. The washed gel was then freeze-dried to obtain a sericin-containing powder. The sericin-containing powder thus obtained was used to measure the sericin yield and molecular weight, the solubility and dispersibility of the sericin-containing powder, and the pH of a 1.0% by mass aqueous dispersion of the sericin-containing powder. The results are shown in Table 2. The calcium ion concentration in the sericin-containing powder thus obtained and in the supernatant after washing were also measured. The results are shown in Table 3.

[0084] [Table 2]

[0085] [Table 3]

[0086] As shown in Table 2, the sericin-containing powder obtained in Example 12 contained high-molecular-weight sericin and had a high degree of dispersion and excellent dispersibility. Furthermore, as shown in Table 3, in the examples that included a washing step, the concentration of alkaline earth metals derived from inorganic salts in the resulting sericin-containing powder was low. In other words, it was found that the content of inorganic salts can be reduced by the washing step.

[0087] Example 13 A sericin-containing powder was obtained in the same manner as in Example 5, except that the amount of dissolving solution (extraction solution) used in the sericin-containing gel preparation step was 4 mL, the amount of acidic aqueous solution used was 20 mL, and the amount of acidic aqueous solution used in the washing step was 20 mL. The pH of a 1.0% by mass aqueous dispersion of the resulting sericin-containing powder was measured and found to be 2.8. The storage stability and film-forming ability of the resulting sericin-containing powder were also evaluated. The results of the storage stability evaluation are shown in Figure 4, and the results of the film-forming ability evaluation are shown in Figure 5. In Figure 4, from left to right, the molecular weight marker corresponds to the sericin-containing powder after storage at room temperature for one month (left: before dispersion treatment, right: after dispersion treatment), the sericin-containing powder after storage at 50°C for two weeks (left: before dispersion treatment, right: after dispersion treatment), the sericin-containing powder after storage at 50°C for one month (left: before dispersion treatment, right: after dispersion treatment), and the molecular weight marker.

[0088] As shown in Figure 4, the sericin-containing powder obtained in Example 13 did not undergo hydrolysis of the sericin even after long-term storage, and the high molecular weight sericin was maintained, demonstrating high storage stability. Furthermore, the sericin-containing powder after long-term storage maintained the high molecular weight sericin even after dispersion treatment. As shown in Figure 5, a uniform, crack-free, and semi-transparent film with sufficient strength for easy handling was formed from the sericin-containing powder obtained in Example 13. Therefore, it can be said that the sericin-containing powder of the present invention has good film-forming ability. [Explanation of symbols]

[0089] 1 screw cap tube 2 Liquid part 3 High concentration area H Liquid height h Height of high-density area

Claims

1. A sericin-containing powder containing sericin, The sericin-containing powder is a powder obtained by adding an acid to the sericin and drying it, The sericin is a sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE, the acid is citric acid and / or hydrochloric acid; The pH of a 1.0 mass% aqueous dispersion is 5.0 or less, A sericin-containing powder characterized by having a degree of dispersion of 33% or more as measured by the following method. [Dispersion measurement method] 10.0 mg of the sericin-containing powder was added to 1.00 mL of ion-exchanged water accurately measured with a measuring pipette, and heated at 60°C for 30 minutes in a 2.0 mL screw-cap tube (manufactured by Watson, No. 1392-200-SS-CS). After the heat treatment, the mixture was centrifuged at an acceleration rate of 9,500 × g for 5 minutes, and 0.20 mL of the supernatant was removed. After mixing with a vortex mixer (approximately 2700 rpm for 5 seconds), the mixture was subjected to ultrasonic irradiation for 5 minutes at room temperature using an ultrasonicator (manufactured by Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water. After the ultrasonic treatment, the tube is placed vertically, and the height H of the liquid portion and the height h of the high concentration portion in the tube are measured, and the degree of dispersion (%) is calculated according to the following formula. Dispersity (%) = h / H x 100

2. an extraction step of obtaining sericin from the cocoons using an organic solvent; a step of obtaining a sericin-containing gel by mixing the sericin extract with an acidic aqueous solution having a pH of 5.0 or less; a drying step of powdering the gel obtained as described above, The method further includes a washing step of washing the obtained gel with an acidic aqueous solution after the sericin-containing gel obtaining step and before the drying step, the organic solvent is dimethyl sulfoxide, the organic solvent contains an inorganic salt, the inorganic salt is calcium chloride, the acidic aqueous solutions in the sericin-containing gel obtaining step and the washing step each contain citric acid and / or hydrochloric acid; the sericin contained in the sericin-containing powder is sericin in which at least one band is detected in a range corresponding to a molecular weight of 150 kDa or more by SDS-PAGE; A method for producing a sericin-containing powder, wherein the sericin-containing powder has a degree of dispersion of 33% or more as measured by the following method. [Dispersion measurement method] 10.0 mg of the sericin-containing powder was added to 1.00 mL of ion-exchanged water accurately measured with a measuring pipette, and heated at 60°C for 30 minutes in a 2.0 mL screw-cap tube (manufactured by Watson, No. 1392-200-SS-CS). After the heat treatment, the mixture was centrifuged at an acceleration rate of 9,500 × g for 5 minutes, and 0.20 mL of the supernatant was removed. After mixing with a vortex mixer (approximately 2700 rpm for 5 seconds), the mixture was subjected to ultrasonic irradiation for 5 minutes at room temperature using an ultrasonicator (manufactured by Sonic Tech, ST805, 42 kHz, 35 W) containing 0.5 L of water. After the ultrasonic treatment, the tube is placed vertically, and the height H of the liquid portion and the height h of the high concentration portion in the tube are measured, and the degree of dispersion (%) is calculated according to the following formula. Dispersity (%) = h / H x 100

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