JP6157524B2 - Low toxicity sophorolipid-containing composition and use thereof - Google Patents

Description

  The present invention relates to a low toxicity sophorolipid-containing composition and use thereof.

  Surfactants are frequently used in various cosmetics, pharmaceuticals, and foods and drinks, and consumers who suffer from rough hands and dermatitis caused by heavy use of surfactants are also increasing. In fact, according to the survey of the number of patients by the Ministry of Health, Labor and Welfare (by classification of injury and disease), the number of patients with atopic dermatitis increased from 224,000 to 399,000 from 1987 to 1999, as of 2011. But it is said that there are 369,000 patients. From this, it is considered that there are potentially many consumers who regularly feel a disorder due to a surfactant (for example, rough skin, rough skin, cracks, eczema, hair rustling, etc.).

  In addition to the skin, it is known that discomfort, inflammation, or suppuration occurs due to foreign matter adhering to mucous membrane sites such as the pharynx, nasal cavity, and vagina, and wound sites. In order to prevent and ameliorate such symptoms, it is considered effective to remove foreign substances by washing these parts with a cleaning material or the like. In this case, an excellent cleaning effect can be obtained by using a cleaning material containing a surfactant or the like. On the other hand, surfactants are usually applied to the skin and hair, and have strong irritation to the mucous membrane sites such as the eyes, pharynx, nasal cavity, vagina and oral cavity, and wound sites, so the concentration should be low. For example, the application site may be red and swollen (erythema), and may inhibit wound healing. Therefore, the use of a surfactant or a cleaning material containing the same on a mucosal site or a wound site is avoided.

  Biosurfactants, which are biologically derived surfactants, are substances that are expected to be industrially used as next-generation surfactants because of their high biodegradability and safety. Among them, sophorolipid, which is one of glycolipid type biosurfactants, is a fermentation product obtained from yeast fermentation, and has been known to have high safety (Patent Document 1). However, the degree of mildness is the same as that of amino acid surfactants, and according to Non-Patent Document 1, an extract obtained by extracting a culture of Candida bombicola ATCC 22214 with ethyl acetate In addition to natural sophorolipid mixtures that have been washed with fatty acids to remove lactone sophorolipids, and ester sophorolipids such as methyl esters, ethyl esters (including monoacetyl and diacetyl types), and hexyl esters, It is known that toxicity is relatively high among high sophorolipids. For this reason, further reduction in stimulation (reduction in toxicity) is required.

JP 2009-275145 A

Shah V, Doncel GF, Seyoum T, Eaton KM, Zalenskaya I, Hagver R, Azim A, Gross R (2005), Sophorolipids, Microbial Glycolipids with Anti-HumanImmunodeficiency Virus and Sperm-Immobilizing Activities. Antimicrob. Agents Chemother. 49 (10 ), 4093-4100 Karen M.J.Saerens, Lien Saey, Wim Soetaert (2011) One-Step Production of Unacetylated Sophorolipids by an Acetyltransferase Negative Candida bombicola. Biotechnol. Bioeng. 108 (12). 2923-2931

  It is an object of the present invention to provide a low toxicity sophorolipid-containing composition. Moreover, an object of this invention is to provide the use of a low toxicity sophorolipid containing composition. In the present invention, the “low toxicity sophorolipid-containing composition” means a low toxicity composition containing a low toxicity sophorolipid.

  The inventors of the present invention have made extensive studies to solve the above problems. In addition to the lactone type sophorolipid contained in the sophorolipid-containing composition produced by yeast fermentation, fatty acids, hydroxy fatty acids, and conventional safety It has been found that the acetyl groups contained in acid-type sophorolipids, which are considered to have high properties, have a significant adverse effect on the cytotoxicity of the sophorolipid-containing compositions, respectively, and by removing these, the lower-toxic sophorolipid-containing composition Sophorolipid-containing composition (hypoallergenic sophorolipid-containing composition) that is extremely irritating to products, particularly the eyes, oral cavity, and other mucosal sites (throat, nasal cavity, ear cavity, genital organs, anus, etc.) and wound sites It was confirmed that

  In addition, the present inventors have found that the low-toxic sophorolipid-containing composition does not have a problem such as a feeling of tension or squeak when applied to the skin or hair, but also has a moisturizing effect on itself, against the skin and hair. It has been found that it has a protective action (skin protective action, hair protective action) and an action of repairing hair, and as a result, it can moisturize the skin and prevent damage to the skin and hair.

  The present invention has been completed based on such findings, and includes the following embodiments. In the following description, “Sophorolipid” may be abbreviated as “SL”. Specifically, the “low-toxic sophorolipid-containing composition” of the present invention is also referred to as “low-toxic SL-containing composition”.

(I) Low-toxic SL-containing composition (I-1) A low-toxic SL-containing composition having the following characteristics (a) to (c):
(A) It contains at least acid type SL, fatty acid, and hydroxy fatty acid derived from SL-producing yeast culture, and the total amount of acid type SL, lactone type SL, fatty acid, and hydroxy fatty acid is 100% by mass. (1) Acid type SL: 94 to 99.99% by mass,
(2) Lactone type SL: 0 to 2% by mass,
(3) Total amount of fatty acid and hydroxy fatty acid: 0.01 to 4% by mass;
(B) The ratio of lactone-type SL with respect to 100 mass parts of acid-type SL contained in a low toxicity SL containing composition, or the ratio of a fatty acid and a hydroxy fatty acid (total amount) is 0-2.12 mass parts or 0.01-4, respectively. .25 parts by weight;
(C) Neither the acid type SL nor the lactone type SL contained in the low-toxic SL-containing composition has an acetyl group.

(I-2) Acid type SL, lactone type SL, the ratio of lactone type SL contained per 100% by mass of the total amount of fatty acid and hydroxy fatty acid, and the ratio of fatty acid and hydroxy fatty acid (total amount) are the following (i) and The low-toxic SL-containing composition according to (I-1), which satisfies at least one of (ii):
(I) Lactone type SL: preferably more than 0 and 2% by mass or less, more preferably 0.1 to 2% by mass, still more preferably 0.1 to 1.5% by mass, particularly preferably 0.8 to 1.%. 5% by mass,
(Ii) Fatty acid and hydroxy fatty acid (total amount): preferably 0.01 to 2.4% by mass, more preferably 0.01 to 1.2% by mass, and still more preferably 0.01 to 0.24% by mass.

(I-3) Acid type SL, lactone type SL, the ratio of acid type SL, lactone type SL, and fatty acid and hydroxy fatty acid (total amount) contained per 100% by mass of the total amount of fatty acid and hydroxy fatty acid are as follows: The composition containing low toxicity SL described in I-1):
(I) Acid type SL: Preferably it is 96.1 mass% or more, More preferably, it is 97.9 mass% or more, More preferably, it is 99.31 mass% or more,
(Ii) Lactone type SL: preferably more than 0 and 2% by mass or less, more preferably 0.1 to 2% by mass, still more preferably 0.1 to 1.5% by mass, and particularly preferably 0.8 to 1%. .5% by mass,
(Iii) Fatty acid and hydroxy fatty acid (total amount): preferably 0.01 to 2.4% by mass, more preferably 0.01 to 1.2% by mass, and still more preferably 0.01 to 0.24% by mass.

  (I-4) The ratio of the acid type SL is 98 to 100% by mass, preferably 98.5 to 100% by mass, more preferably 99 to 100% by mass, per 100% by mass of the total amount of the acid type SL and the lactone type SL. More preferably, it is 99.5-100 mass%, The ratio of lactone type SL is 0-2 mass%, Preferably it is 0-1.5 mass%, More preferably, it is 0-1 mass%, (I-1)- The low-toxic SL-containing composition according to any one of (I-3).

(I-5) The absorbance (hue: OD ₄₄₀ ) at a wavelength of 440 nm of an aqueous solution in which a low-toxic SL-containing composition is dissolved so that the ethanol-soluble content is 10% by mass is 0.001-1, preferably 0.005. -0.8, more preferably 0.01-0.6, particularly preferably 0.01-0.5, even more preferably 0.4 or less, of (I-1) to (I-4) The low-toxic SL-containing composition described in any one of the above.

  (I-6) Ester value of 1 g equivalent of ethanol soluble component is 0.01 to 2 mgKOH / g, preferably 0.01 to 1.5 mgKOH / g, more preferably 0.1 to 1.5 mgKOH / g, especially The low-toxic SL-containing composition according to any one of (I-1) to (I-5), preferably 0.8 to 1.5 mgKOH / g.

  (I-7) The hydroxyl value of the equivalent of 1 g of ethanol soluble component is 545 to 630 mg KOH / g, preferably 560 to 630 mg KOH / g, more preferably 575 to 630 mg KOH / g, and particularly preferably 575 to 585 mg KOH / g. A low-toxic SL-containing composition according to any one of (I-1) to (I-6).

(I-8) The low lethal concentration (IC ₅₀ ) against HeLa cells is 2000 ppm or more, preferably 3000 to 60000 ppm. Toxic SL-containing composition.

(I-9) Ratio (IC ₅₀ / CMC) of cell lethal concentration (IC ₅₀ ) to critical micelle concentration (CMC) for HeLa cells is 6.7 to 200, preferably 10 to 200, more preferably 17 to 200 The low-toxic SL-containing composition according to any one of (I-1) to (I-8), particularly preferably 33 to 200.

(I-10) The low-toxic SL-containing composition according to any one of (I-1) to (I-9), wherein the composition has at least one physical property of any of the following (a) to (c): :
(A) Evaporation residue: 1 to 100%
(B) Loss on drying: 0 to 99%,
(C) Ethanol soluble content: 1 to 100%.

In (I-11) Infrared absorption spectrum having at least wavenumber 1024cm around ^-1, 1706～1730Cm around ^-1, 2854Cm around ^-1, 2924 cm around ^-1, and 3000~3500cm infrared absorption band around ^-1, ( The low-toxic SL-containing composition described in any one of (I-1) to (I-10).

  (I-12) The low-toxic SL-containing composition according to any one of (I-1) to (I-11), which has a solid form.

  (I-13) The low-toxic SL-containing composition according to (I-12), wherein the solid form is a powder or a granule.

(II) Use of low-toxic SL-containing composition (II-1) An anionic surfactant comprising the low-toxic SL-containing composition described in any one of (I-1) to (I-13) as an active ingredient.

  (II-2) Skin moisturizing agent, rough skin improving agent, skin protecting agent, or hair protecting agent comprising the low-toxic SL-containing composition according to any one of (I-1) to (I-13) as an active ingredient .

  (II-3) Cosmetics, quasi-drugs, pharmaceuticals, medical devices or daily life characterized by containing the low-toxic SL-containing composition according to any one of (I-1) to (I-13) Miscellaneous goods; or additives for cosmetics, quasi-drugs, pharmaceuticals, medical devices or daily miscellaneous goods.

  (II-4) Cosmetics, quasi-drugs, pharmaceuticals, medical devices or the like described in (II-3) that are applied to skin, hair, mucous membranes, or wounds (such as wounds or inflamed areas) Daily miscellaneous goods; or additives for cosmetics, quasi-drugs, pharmaceuticals, medical equipment or daily miscellaneous goods.

  (II-5) The above cosmetics, quasi-drugs, pharmaceuticals, medical equipment or daily miscellaneous goods are body wash, hair cleanser, eye wash, eye drop, cosmetic, oral cleanser, mucous membrane or wound cleanser. Cosmetics, quasi-drugs, pharmaceuticals, medical devices, or daily miscellaneous goods described in (II-3) or (II-4), which are at least one selected from the group consisting of cosmetics and wound dressings Or these additives.

(II-6) A cosmetic or pharmaceutical product according to (II-5), which comprises the low-toxic SL-containing composition according to any one of (I-1) to (I-13) in the following ratio: Quasi-drugs, pharmaceuticals, medical equipment or daily miscellaneous goods or their additives:
Body wash: 0.01-100% by mass,
Hair cleaning agent: 0.01 to 30% by mass,
Eye wash: 0.001-10% by mass,
Eye drops: 0.001 to 10% by mass,
Cosmetics: 0.01-20% by mass,
Oral cleansing agent: 0.001 to 10% by mass,
Mucosal or wound cleaning material: 0.001 to 30% by mass,
Wound dressing: 0.001 to 30% by mass.

  The present invention also includes a method for producing the above-mentioned low-toxic SL-containing composition and a method for reducing the toxicity of the SL-containing composition.

(III) Method for producing low-toxic SL-containing composition (III-1) SL-containing culture obtained by culturing SL-producing yeast or treated product thereof (1) Step of removing fatty acid and / or hydroxy fatty acid And, if necessary, (2) a step of removing an acetyl group bonded to SL, and / or (3) a step of removing lactone-type SL, (I-1) to The manufacturing method of the low toxicity SL containing composition in any one of (I-13).

  (III-2) (1) The production according to (III-1), wherein the step of removing fatty acid and / or hydroxy fatty acid is at least one treatment selected from a solvent extraction method, an adsorption method, and chromatography Method.

  (III-3) Solvent extraction method is extraction method using diethyl ether as solvent; adsorption method is a method using activated carbon, silica gel, zeolite, ion exchange resin and alumina oxide as adsorbent; chromatography is stationary phase The production method described in (III-2), which is reverse phase column chromatography using an ODS resin as a mobile phase and an aqueous ethanol solution as a mobile phase.

  (III-4) (2) The step of removing the acetyl group bonded to SL is at least one treatment selected from hydrolysis treatment and enzyme treatment (III-1) to (III-3) The manufacturing method described in any one of.

  (III-5) The production method according to (III-4), wherein the enzyme treatment is a treatment using acetylesterase.

  (III-6) (3) The step of removing lactone-type SL is at least one treatment selected from hydrolysis treatment and chromatography, and any of (III-1) to (III-5) Manufacturing method to be described.

  (III-7) The production method according to (III-6), wherein the chromatography is reverse phase column chromatography using an ODS resin as a stationary phase and an aqueous ethanol solution as a mobile phase.

(IV) Method for reducing toxicity of SL-containing composition (IV-1) (1) Step of removing fatty acid and / or hydroxy fatty acid from SL-containing culture obtained by culturing SL-producing yeast or treated product thereof, And (2) removing the acetyl group bonded to SL, and / or (3) removing the lactone-type SL, if necessary, and having the following characteristics (a) to (c) A method for detoxifying a SL-containing composition comprising preparing a detoxified SL-containing composition having:
(A) When the total amount of acid-type SL, lactone-type SL, fatty acid and hydroxy fatty acid contained in the attenuated SL-containing composition is 100% by mass, the respective ratios are converted into dry weights within the following ranges. (1) Acid type SL: 94 to 99.99% by mass,
(2) Lactone type SL: 0 to 2% by mass,
(3) Total amount of fatty acid and hydroxy fatty acid: 0.01-4% by mass;
(B) The ratio of lactone type SL or fatty acid and hydroxy fatty acid (total amount) to 100 parts by mass of acid type SL contained in the low-toxic SL-containing composition is 0 to 0.212 or 0.01 to 4.25 parts by mass, respectively.
(C) Neither the acid type SL nor the lactone type SL contained in the low-toxic SL-containing composition has an acetyl group.

(IV-2) Acid type SL, lactone type SL, the proportion of lactone type SL contained per 100% by mass of the total amount of fatty acid and hydroxy fatty acid, and the proportion of fatty acid and hydroxy fatty acid (total amount) are the following (i) and (Ii) A method for reducing poisoning according to (IV-1), characterized in that a composition containing SL with reduced poisoning in at least one of the ranges is prepared:
Lactone type SL: preferably more than 0 and 2% by mass or less, more preferably 0.1 to 2% by mass, further preferably 0.1 to 1.5% by mass, particularly preferably 0.8 to 1.5% by mass ,
Fatty acid and hydroxy fatty acid (total amount): preferably 0.01 to 2.4% by mass, more preferably 0.01 to 1.2% by mass, and still more preferably 0.01 to 0.24% by mass.

(IV-3) Acid type SL, lactone type SL, the ratio of acid type SL, lactone type SL, and fatty acid and hydroxy fatty acid (total amount) contained per 100% by mass of the total amount of fatty acid and hydroxy fatty acid are in the following ranges. A method for detoxification as described in (IV-1), which comprises preparing a composition with reduced SL content:
(A) Acid type SL: Preferably it is 96.1 mass% or more, More preferably, it is 97.9 mass% or more, More preferably, it is 99.31 mass% or more,
(B) Lactone type SL: preferably more than 0 and 2% by mass or less, more preferably 0.1 to 2% by mass, still more preferably 0.1 to 1.5% by mass, and particularly preferably 0.8 to 1.%. 5% by mass,
(C) Fatty acid and hydroxy fatty acid: preferably 0.01 to 2.4% by mass, more preferably 0.01 to 1.2% by mass, and still more preferably 0.01 to 0.24% by mass.

  (IV-4) The ratio of the acid type SL is 98 to 100% by mass, preferably 98.5 to 100% by mass, more preferably 99 to 100% by mass, per 100% by mass of the total amount of the acid type SL and the lactone type SL. Particularly preferably, it is 99.5 to 100% by mass, and the lactone-type SL content is 0 to 2% by mass, preferably 0 to 1.5% by mass, and more preferably 0 to 1% by mass. A method for reducing toxicity according to any one of (IV-1) to (IV-3), which comprises preparing a composition.

  (IV-5) The ester value of the equivalent of 1 g of ethanol soluble component is 0.01 to 2 mgKOH / g, preferably 0.01 to 1.5 mgKOH / g, more preferably 0.1 to 1.5 mgKOH / g, particularly Preferably, the attenuated SL-containing composition according to any one of (IV-1) to (IV-4), wherein a reduced attenuated SL-containing composition of 0.8 to 1.5 mg KOH / g is prepared. .

  (IV-6) Low hydroxyl group value of ethanol equivalent 1g equivalent is 545-630 mgKOH / g, preferably 560-630 mgKOH / g, more preferably 575-630 mgKOH / g, particularly preferably 575-585 mgKOH / g A method for reducing poisoning according to any one of (IV-1) to (IV-5), wherein a composition containing a poisoned SL is prepared.

  (IV-7) (1) The step of removing fatty acid and / or hydroxy fatty acid is at least one treatment selected from a solvent extraction method, an adsorption method, and chromatography, (IV-1) to (IV- 6) The method for reducing toxicity described in any one of the above.

  (IV-8) Solvent extraction method is extraction method using diethyl ether as solvent; adsorption method is a method using activated carbon, silica gel, zeolite, ion exchange resin and alumina oxide as adsorbent; chromatography is stationary phase As described in (IV-7), which is reverse phase column chromatography using an ODS resin as a mobile phase and an aqueous ethanol solution as a mobile phase.

  (IV-9) (2) The step of removing the acetyl group bonded to SL is at least one treatment selected from hydrolysis treatment and enzyme treatment, (IV-7) to (IV-8) The method for reducing poisoning according to any one of the above.

  (IV-10) The method for reducing poisoning according to (IV-9), wherein the enzyme treatment is treatment using acetylesterase.

  (IV-11) (3) The step of removing lactone-type SL is at least one treatment selected from hydrolysis treatment and chromatography, and any of (IV-7) to (IV-10) Detoxification method to be described.

  (IV-12) The method for reducing toxicity described in (IV-11), wherein the chromatography is reverse phase column chromatography using an ODS resin as a stationary phase and an aqueous ethanol solution as a mobile phase.

  According to the present invention, an SL-containing composition having low cytotoxicity can be provided. The low-toxic SL-containing composition has a surface-active effect, and has extremely low cytotoxicity and irritation to mucous membranes and wound sites (low toxicity and low irritation), so that the skin, ocular mucosa, oral mucosa, and the Suitable for cosmetics, pharmaceuticals, quasi-drugs, medical devices, etc. applied to other mucosal sites (throat, nasal cavity, ear cavity, genital organs, anus, etc.) and wound sites (such as wounds and inflamed areas) be able to. Specifically, eye care products such as eye drops applied to the ocular mucosa, eye wash and contact lens mounting liquids, oral preparations applied to the oral mucosa, and nasal drops such as nasal drops applied to the nasal mucosa It can mix | blend suitably for a formulation etc.

  For this reason, cosmetics, pharmaceuticals and quasi-drugs containing the low-toxicity (hypoallergenic) SL-containing composition of the present invention include not only the skin but also the ocular mucosa, oral mucosa, and other mucosal sites (throats). Nasal cavity, ear cavity, genital organs, anus, etc.) and wound sites (such as wounds and inflamed areas).

  In addition, the low toxicity (low irritation) SL-containing composition of the present invention has a moisturizing action, a protective action for skin and hair, and an action for improving the skin barrier function. It can be preferably used to protect the hair and moisturize it.

SL-containing compositions obtained in Reference Production Example 2 and Examples 1 to 10 (crudely purified SL-containing composition-2, Example products 1 to 10), Tween 20, and commercially available anionic surfactants A to C (A : “Aminosoft LT-12” (30%): Ajinomoto Co., Inc., B: “Surfactin Na” (100%): Wako Pure Chemical Industries, Ltd., C: “Lipolane LJ-441” (37 %): Manufactured by Lion Co., Ltd.)) and SLS, the cell lethality concentration (IC ₅₀ ) calculated from the cytotoxicity test and the CMC calculated in Test Example 2 are plotted. It is the figure which showed the recovery effect of the rough skin created by processing the upper arm part of human by SDS. (A) It is the figure which showed the hair protection effect with respect to healthy hair. (B) It is the figure which showed the hair repair effect with respect to damaged hair.

(I) General sophorolipid (known SL)
Sophorolipid (SL) is a glycolipid generally composed of sophorose or sophorose having a partially acetylated hydroxyl group and a hydroxy fatty acid. Sophorose is a sugar composed of two molecules of glucose linked by β1 → 2. Hydroxy fatty acid is a fatty acid having a hydroxyl group. SL is broadly classified into an acid type in which the carboxyl group of hydroxy fatty acid is liberated (the following general formula (1)) and a lactone type in which sophorose in the molecule is bound (the following general formula (2)). SL obtained by fermentation of a certain kind of yeast (SL-producing yeast) is usually a mixture of SL represented by the following general formula (1) and SL represented by the general formula (2), and the fatty acid chain length (R ³ ) Are different, and the 6 ′ (R ² ) and 6 ″ positions (R ¹ ) of sophorose are acetylated or protonated, and are obtained as an aggregate of 30 or more structural homologues.

In the general formula (1) or (2), R ⁰ is either a hydrogen atom or a methyl group. R ¹ and R ² are each independently a hydrogen atom or an acetyl group. R ³ is a saturated aliphatic hydrocarbon chain or an unsaturated aliphatic hydrocarbon chain having at least one double bond, and may have one or more substituents. Examples of the substituent include a halogen atom, a hydroxyl group, a lower (C _1-6 ) alkyl group, a halo lower (C _1-6 ) alkyl group, a hydroxy lower (C _1-6 ) alkyl group, and a halo lower (C ₁ -C) _{. 6} ) An alkoxy group and the like can be mentioned. The number of carbon atoms of the hydrocarbon chain of ^{R 3} is usually 11 to 20, preferably 13 to 17, more preferably 14 to 16. Here, examples of the halogen atom bonded to the halogen atom or the alkyl group or alkoxy group include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

  In a culture solution obtained by fermentation of SL-producing yeast, SL is usually present as a mixture of acid type SL represented by general formula (1) and lactone type SL represented by general formula (2). Yes. The ratio of the acid type SL and the lactone type SL contained in the culture solution can usually include 45:55 to 10:90 (dry weight ratio).

  A preferred example of the SL-producing yeast is Candida bombicola. The genus Candida has been changed to the name of the genus Starmerella. The yeast is a known SL-producing yeast known to produce a significant amount of SL (acid type, lactone type) [Canadian Journal of Chemistry, 39,846 (1961) (Note: described in the literature) The genus Torlopsis falls under the genus Candida, but as described above, it is currently classified into the genus Starmerella.), Applied and Environmental Microbiology, 47, 173 (1984), etc.]. Candida (Starmelera) and Bonbicola are registered in the biological resource bank ATCC (American Type Culture Collection), and can be obtained from them (Candida bombicola ATCC22214, etc.). For the production of the low-toxic SL-containing composition of the present invention, another SL-producing yeast belonging to the genus Candida (Starmelera), which is known to produce SL (acid type, lactone type), is used. You can also Examples of such SL-producing yeast include Candida magnoliae, Candida gropengisseri, Candida apicola, Candida petrophilum, and Candida bogoriensis. bogoriensis) and Candida batistae. It has already been reported that SL is produced in a relatively large amount in the culture medium of these yeasts (R. Hommel, Biodegradation, 1, 107 (1991)).

  Candida floricola (Candida floricola) strain ZM-1502 (FERM P-21133) and Candida floricola (Candida floricola) NBRC10700T strain are SL-producing yeasts that selectively produce only acid type SL having a diacetyl group. It is known (Japanese Patent Laid-Open No. 2008-247845). Therefore, when the acid form SL is selectively produced, the SL-producing yeast can be preferably used. However, since the acid type SL has a diacetyl group as described above, a deacetylation treatment is required to obtain the low-toxic SL-containing composition of the present invention described later.

  For the cultivation of these SL-producing yeasts, sugars such as glucose (hydrophilic substrate) as a carbon source, fatty acids such as fatty acids and fatty acid triglycerides, or fats and oils such as vegetable oils containing fatty acids as constituents (hydrophobic substrates) ) Is used. Other components of the medium are not particularly limited and can be appropriately selected from medium components generally used for yeast.

(II) Low-toxic SL-containing composition The low-toxic SL-containing composition targeted by the present invention is different from the above-described conventionally known SL compositions in at least cytotoxicity (cell irritation) and skin moisturizing action. However, it has the following features.

(A) It contains at least acid type SL, fatty acid, and hydroxy fatty acid derived from SL-producing yeast culture, and the total amount of acid type SL, lactone type SL, fatty acid, and hydroxy fatty acid is 100% by mass. Is converted to dry weight as follows:
(1) Acid type SL: 94-99.99 mass%,
(2) Lactone type SL: 0 to 2% by mass,
(3) Total amount of fatty acid and hydroxy fatty acid: 0.01-4% by mass,
(B) The ratio of the lactone type SL to the 100 parts by mass of the acid type SL contained in the low-toxic SL-producing yeast culture, or the ratio of the fatty acid and the hydroxy fatty acid (total amount) is 0 to 2.12 or 0.01 to 4.25, respectively. Part by mass,
(C) Neither acid-type SL nor lactone-type SL contained in a low-toxic SL-producing yeast culture has an acetyl group.

Hereinafter, these will be described.
(A) (1) Containing acid type SL at a ratio of 94 to 99.99% by mass in terms of dry matter, this is the total amount of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid in the composition containing low toxicity SL It is a ratio (dry matter weight) of acid type SL contained in 100 mass%. This corresponds to the ratio of the acid form SL contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition. Therefore, it can be said that the ratio of acid-type SL contained in 100 mass% of ethanol soluble parts of a low toxicity SL containing composition is 94-99.99 mass%. The ratio of the acid type SL is preferably 96.1% by mass or more, more preferably 97.9% by mass or more, and particularly preferably 99.31% by mass or more.

  The low-toxic SL-containing composition of the present invention includes a low-toxic SL-containing composition containing acid SL at a ratio of 95 to 99.86% by mass (Examples 1 to 10). It is one aspect of the invention and is not limited thereto.

  The proportion of the acid type SL can be calculated from the ester value and the ether extract content of the low-toxic SL-containing composition. As will be described later, in the present invention, “ester value” and “ether extract content” are the acid type SL, lactone type SL, and the total amount of fatty acid and hydroxy fatty acid contained in the low-toxic SL-containing composition, respectively, of 100% by mass. Corresponds to the ratio of “lactone type SL” and the ratio of “fatty acid and hydroxy fatty acid”. Therefore, a value obtained by subtracting the sum of these “ester value” and “ether extract content” from 100 corresponds to the ratio (mass%) of the acid type SL contained in the low-toxic SL-containing composition.

(A) (2) Containing 0 to 2 mass% of lactone type SL in terms of dry matter, this is a total amount of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid contained in the low toxicity SL-containing composition 100 It is the ratio (dry matter weight) of the lactone type SL contained in mass%. This corresponds to the proportion of lactone-type SL contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition. Therefore, it can be said that the ratio of lactone-type SL contained in 100 mass% of ethanol soluble parts of a low toxicity SL containing composition is 0-2 mass%. A smaller lactone-type SL ratio is preferable in obtaining an SL-containing composition having low toxicity, but a surface tension reducing ability is better if it is contained to some extent within a range of more than 0 and 2% by mass or less. Yes, it is excellent in performance as a surfactant (wetting property, solubilizing power, detergency, foaming property) (see Test Example 5).

For this reason, the ratio (upper limit, lower limit) of the lactone type SL contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition when focusing on low toxicity is as follows:
Upper limit: preferably 1.5% by mass or less, more preferably 0.9% by mass or less, further preferably 0.45% by mass or less, particularly preferably 0.1% by mass or less,
Lower limit: Preferably 0% by mass. In addition, when using yeast which produces lactone type | mold SL as SL production yeast, 0.01 mass% can be mentioned.

On the other hand, it possesses excellent performance as a surfactant (wetability, solubilizing power, detergency, foaming property) (hereinafter collectively referred to as “surfactant activity”) and is excellent in low toxicity. The ratio (upper limit, lower limit) of the lactone type SL contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition is as follows:
Upper limit: preferably 2% by mass or less, more preferably 1.5% by mass or less,
Lower limit: preferably more than 0% by mass, more preferably 0.1% by mass or more, still more preferably 0.8% by mass or more.

  In addition, the low toxicity SL containing composition of this invention contains the low toxicity SL containing composition which contains lactone type SL in the ratio of 0.04-2.0 mass% (Examples 1-10). However, these are one aspect | mode of this invention, and are not limited to these.

  The ratio of the lactone type SL can be determined from the ester value (mg KOH / g) of the low-toxic SL-containing composition.

  Specifically, the “ester value (mg KOH / g)” as used in the present invention is required to completely saponify the ester contained in a sample (low-toxic SL-containing composition) corresponding to 1 g of ethanol-soluble matter. This is the number of mg of potassium hydroxide, whereby the ratio of the ester bond of the lactone ring contained in the sample (low-toxic SL-containing composition) can be grasped. The ester value (mg KOH / g) correlates with the proportion of lactone type SL in the total amount (100% by mass) of SL contained in the SL-containing composition. % Of the lactone type SL in% can be calculated.

  Here, the ethanol-soluble component substantially corresponds to the total amount of the acid type SL, lactone type SL, fatty acid and hydroxy fatty acid contained in the SL-containing composition, so that the acid value SL, The lactone type SL and the acid type SL contained in 100% by mass of the total amount of fatty acid and hydroxy fatty acid can be calculated.

  The “ester value (mg KOH / g)” can be measured according to the standard method for analyzing fats and oils (2.3.3-1996) established by the Japan Oil Chemical Association (Japan). The details are as described in Test Example 1. The ester value (mg KOH / g) of the low toxicity SL-containing composition of the present invention is usually 0 to 2 mg KOH / g. From the viewpoint of reducing the poisoning, it is preferably 0 to 1.5 mgKOH / g, more preferably 0.01 to 0.9 mgKOH / g, and particularly preferably 0 to 0.45 mgKOH / g. On the other hand, for the purpose of achieving low toxicity while maintaining good performance (wetting property, solubilizing power, detergency, foaming property) as a surfactant, it is preferably more than 0 (for example, 0.01 mg KOH). / G) 2 mgKOH / g or less, more preferably 0.1 to 2 mgKOH / g, still more preferably 0.1 to 1.5 mgKOH / g, and particularly preferably 0.8 to 1.5 mgKOH / g.

  In addition, when the total amount of acid type SL and lactone type SL contained in the low-toxic SL-containing composition is 100% by mass, the ratio of acid type SL and lactone type SL contained therein (acid type SL: lactone type SL, The weight ratio is 98: 2 to 100: 0, preferably 98.5: 1.5 to 100: 0, and more preferably 99: 1 to 100: 0. In addition, when using yeast which produces lactone type SL as SL production yeast, acid type SL may not become 100 mass%, and in this case, the ratio (weight ratio) of acid type SL and lactone type SL is 99. 99: 0.01 may be sufficient.

(A) (3) Fatty acid and hydroxy fatty acid are contained in a ratio of the total amount of 0.01 to 4% by mass in terms of dry matter. This is a low-toxic SL-containing composition of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid. It is the ratio (dry matter weight) of fatty acids and hydroxy fatty acids contained in a total amount of 100% by mass. This corresponds to the total proportion of fatty acid and hydroxy fatty acid contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition. Therefore, it can be said that the total ratio of the fatty acid and the hydroxy fatty acid contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition is 0.01 to 4% by mass.

  The target fatty acid here is a fatty acid contained in a medium used for culturing SL-producing yeast, and is a fatty acid calculated as the ether extract by the method for measuring an ether extract content described in Examples. Specifically, it is a saturated or unsaturated fatty acid having 6 to 24 carbon atoms. More specifically, caproic acid (C6), enanthic acid (C7), caprylic acid (C8), pelargonic acid (C9), capric acid (C10), lauric acid (C12), myristic acid (C14), pentadecylic acid ( Saturated fatty acids such as C15), palmitic acid (C16), margaric acid (C17), stearic acid (C18), arachidic acid (C20), docosanoic acid (C22), lignoceric acid (C24); palmitoleic acid (C16: 1 ), Oleic acid (C18: 1), linoleic acid (C18: 2), and linolenic acid (C18: 3), arachidonic acid (C20: 4), eicosapentaenoic acid (C20: 5), docosahexynoic acid (C22: 6) ) And other unsaturated fatty acids.

  Moreover, as a hydroxy fatty acid made into object here, the fatty acid by which at least 1 hydrogen atom is substituted by the hydroxy group in said fatty acid can be mentioned.

  The ratio of the fatty acid and hydroxy fatty acid (total amount) is preferably 2.4% by mass or less, more preferably 1.2% by mass or less, and particularly preferably 0.24% by mass or less as the upper limit. Moreover, as a minimum, 0.01 mass% can be mentioned suitably. Although the amount of fatty acid and hydroxy fatty acid is preferably as small as possible in order to obtain a SL-containing composition having low toxicity, in the present invention, it is more preferable that the amount of the fatty acid and hydroxy fatty acid is contained in a range from 0.01 to 4% by mass. There is an advantage that the surface activity of SL is maintained. Although not restrained, the reason is that fatty acids and hydroxy fatty acids exhibit a kind of chelating effect in the test sample. Specifically, the chelating action of fatty acid and hydroxy fatty acid supplements metal ions such as K, Na, Ca and Mg contained in the test sample, thereby suppressing salt formation between the metal ion and acid type SL. Thus, it is considered that the effect of the acid type SL as a surfactant is maintained. In addition, the low toxicity SL containing composition of this invention contains the low toxicity SL containing composition which contains a fatty acid and a hydroxy fatty acid in the ratio of 0.1-4 mass% in total (Examples 1-10). However, these are one aspect | mode of this invention, and are not limited to these.

  Among the fatty acids and hydroxy fatty acids contained in the low-toxic SL-containing composition of the present invention, the mainstream are saturated and unsaturated fatty acids having 16 and 18 carbon atoms. Specifically, the main fatty acids contained in the low-toxic SL-containing composition are a saturated fatty acid having 16 carbon atoms and an unsaturated fatty acid having 1 or 2 double bonds having 18 carbon atoms. The main hydroxy fatty acid contained in the low-toxic SL-containing composition is a saturated fatty acid having 16 carbon atoms and an unsaturated fatty acid having 1 or 2 double bonds having 18 carbon atoms (see Test Example 1 and Table 2). . For this reason, it can also be said that the ratio of the fatty acid and the hydroxy fatty acid (total amount) is substantially the ratio of the fatty acid having 16 and 18 carbon atoms and the hydroxy fatty acid (total amount).

  The ratio of the fatty acid and hydroxy fatty acid can be determined from the ether extract content (%) of the low toxicity SL-containing composition.

  Specifically, the “ether extract content (%)” referred to in the present invention is the ratio of a substance extracted with ether from a sample (low-toxic SL-containing composition) corresponding to 1 g of ethanol-soluble matter ( Thus, the ratio of fatty acids and hydroxy fatty acids contained in the sample (low-toxic SL-containing composition) can be grasped. That is, since the “ether extract content (%)” correlates with the proportion of fatty acid and hydroxy fatty acid in 100% by mass of the ethanol-soluble component, the ethanol-soluble component is determined from the “ether extract content (%)”. The ratio of the fatty acid and hydroxy fatty acid in 100% by mass can be calculated. As described above, the ethanol-soluble component substantially corresponds to the total amount of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid contained in the SL-containing composition. The amount of fatty acid and hydroxy fatty acid contained in 100% by mass of the total amount of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid can be calculated.

  The details of the method for measuring the “ether extract content (%)” are as described in Test Example 1. The “ether extract content (%)” of the low toxicity SL-containing composition of the present invention is usually 0.01 to 4% by mass, preferably 0.01 to 2.4% by mass, more preferably 0.01 to 1%. .2% by mass, particularly preferably 0.01 to 0.24% by mass. In particular, a range of 0.1 to 4% by mass can be exemplified.

(B-1) The ratio of the lactone type SL to 100 parts by mass of the acid type SL contained in the low toxicity SL-containing composition is 0 to 2.12 parts by mass ,
This ratio can be easily calculated from the contents of acid-type SL and lactone-type SL contained in the low-toxic SL-containing composition. The said ratio becomes like this. Preferably it is 0.01-1.5 mass parts, More preferably, it is 0.01-1.0 mass part.

(B-2) The ratio of fatty acid and hydroxy fatty acid (total amount) to 100 parts by mass of acid type SL contained in the low-toxic SL-containing composition is 0.01 to 4.25 parts by mass ;
This ratio can be easily calculated from the contents of acid type SL, fatty acid and hydroxy fatty acid contained in the low-toxic SL-containing composition. The said ratio becomes like this. Preferably it is 0.05-2.5 mass parts, More preferably, it is 0.1-1.0 mass part.

(C) The acid-type SL and lactone-type SL contained in the low-toxic SL-containing composition have higher cytotoxicity than SL having no acetyl group, as compared to SL having no acetyl group. From this, it is considered that the acetyl group of SL is related to cytotoxicity in both acid type SL and lactone type SL. On the other hand, both the acid type SL and the lactone type SL contained in the low toxicity SL-containing composition of the present invention are characterized by not having an acetyl group.

  The ratio of the acetyl group contained in the SL-containing composition can be determined from the hydroxyl value of the equivalent of 1 g ethanol-soluble component of the SL-containing composition. The low toxicity SL-containing composition of the present invention does not have an acetyl group. For this reason, the hydroxyl value should just reflect this. Although the hydroxyl value (for 7) of the low-toxic SL-containing composition is theoretically 630 mgKOH / L, in reality, 545 to 630 mgKOH / L can be usually mentioned in consideration of the reaction efficiency (about 90%). That is, when the hydroxyl value of the 1 g equivalent of the ethanol-soluble component of the SL-containing composition is 545 to 630 mgKOH / g, it can be determined that SL contained in the SL-containing composition does not have an acetyl group.

  The “hydroxyl value (mg KOH / g)” as used in the present invention is necessary for completely acetylating the OH group of a compound contained in a sample (low-toxic SL-containing composition) corresponding to 1 g of ethanol-soluble matter. This is the number of mg of potassium hydroxide, whereby the proportion of free hydroxyl groups in SL contained in the sample (low-toxic SL-containing composition) can be measured. If the hydroxyl value (mg KOH / g) is high, the percentage of free hydroxyl groups contained in the SL is large (that is, the number of acetylated hydroxyl groups is small = the acetyl group is small), and the hydroxyl value (mg KOH / g) is low. For example, the ratio of free hydroxyl groups contained in SL is low (that is, there are many acetylated hydroxyl groups = many acetyl groups).

The “hydroxyl value (mg KOH / g)” can be measured according to the standard method for analyzing fats and oils (2.3.6.2-1996) established by the Japan Oil Chemical Association (Japan). The details are as described in Test Example 1. As described above, the “hydroxyl value (mg KOH / g)” of the low-toxic SL-containing composition of the present invention is preferably 545 to 630 mg KOH / g for a sample equivalent to 1 g of ethanol-soluble matter. Preferably it is 560-630 mgKOH / g, More preferably, it is 575-630 mgKOH / g. In addition, although the low toxicity SL containing composition of this invention contains the low toxicity SL containing composition whose hydroxyl value is 575-585 mgKOH / g (Examples 1-10), these are 1 aspect of this invention. Yes, the present invention is not limited to the low-toxic SL-containing composition targeted by the present invention, in addition to the characteristics of (a) (1) to (3), (b) and (c) described above. Further, those having at least one of the following characteristics (d) to (h) are included.

(D) Low toxic SL-containing composition targeted by the present invention in which the absorbance (OD ₄₄₀ ) of an aqueous solution in which a low-toxic SL-containing composition is dissolved so that the ethanol-soluble content is 10% by mass is 0.001-1 The thing contains what is colored in colors other than white.

  Since acid type SL, lactone type SL, fatty acid, and hydroxy fatty acid are all colorless or white, when the low-toxic SL-containing composition is colored, the coloration includes acid type SL, lactone type SL, fatty acid and It means that coloring components are included in addition to hydroxy fatty acids. In addition, a coloring component originates in the culture of SL production yeast, and it will not restrict | limit in particular in this limit. As an example of the possibility, melanoidin can be exemplified, but is not restricted thereto.

The coloring degree of the low-toxic SL-containing composition of the present invention is specifically determined by dissolving the low-toxic SL-containing composition in an alkaline solution (2% Na ₂ CO ₃ in 0.1N NaOH) to obtain an ethanol-soluble component. This can be evaluated by measuring the absorbance (hue: OD ₄₄₀ ) at a wavelength of 440 nm of an aqueous solution prepared so that the total concentration of the solution becomes 10% by mass. If low toxicity SL-containing composition of the present invention was prepared as described above, the hue of the aqueous solution (OD ₄₄₀₎ is in the range of 0.001 to 1. Preferably it is 0.005-0.8, More preferably, it is 0.01-0.6, Most preferably, it is 0.01-0.5. Incidentally, the low toxicity SL-containing composition of the present invention, the hue (OD ₄₄₀₎ is included low toxicity SL-containing composition is 0.1 to 0.4 (Example 1-7). However, these are one aspect | mode of this invention, and are not limited to these.

(E) Evaporation residue: 1 to 100%
Here, the “evaporation residue (%)” is, as described in Test Example 1, the residue when the sample is evaporated is shown in mass percentage (mass%). In the present invention, it is possible to ascertain the content of substances mixed in the low-toxic SL-containing composition, in particular, high-boiling substances. The “evaporation residue (%)” can be measured according to the second method of JIS K0067-1992. The details are as described in Test Example 1. The evaporation residue (%) of the low toxicity SL-containing composition of the present invention is 1 to 100%, preferably 5 to 100%, more preferably 10 to 100%, still more preferably 60. -100%, even more preferably 70-100%, particularly preferably 80-100%, more particularly preferably 90-100%.

(F) Loss on drying: 0 to 99%
The “loss on drying (%)” is the weight loss when the sample is dried as described in Test Example 1 in mass percentage (% by mass). The content of moisture and other volatile substances (low boiling point compounds) in the contained composition can be ascertained. The “loss on drying (%)” can be measured according to the first method of JIS K0067-1992. The details are as described in Test Example 1. Loss on drying (%) of the low-toxic SL-containing composition of the present invention is 0 to 99%, preferably 0 to 95%, more preferably 0 to 90%, and still more preferably 0 to 90%. 30%, even more preferably 0 to 20%, particularly preferably 0 to 20%, and even more preferably 0 to 10%.

(G) Ethanol soluble content: 1 to 100%
As described in Test Example 1, the “ethanol-soluble content (%)” is the content (mass%) of a substance dissolved in ethanol contained in a sample, and thereby the ethanol solubility mixed in the sample. The content of polar substances such as surfactants can be ascertained. The “ethanol soluble content (%)” can be measured according to JIS K3362-2008. The details are as described in Test Example 1. The ethanol-soluble component (%) of the low-toxic SL-containing composition of the present invention is 1 to 100%, preferably 5 to 100%, more preferably 10 to 100%, more preferably It is 85 to 100%, more preferably 90 to 100%, particularly preferably 95 to 100%, more particularly preferably 98 to 100%. The ethanol-soluble content (%) is the content ratio (mass%) of the target sample, acid-type SL and lactone-type SL, fatty acid, hydroxy fatty acid when the low-toxic SL-containing composition is 100 mass% in the present invention. Indicates.

(H) Infrared absorption spectrum The low-toxic SL-containing composition of the present invention more preferably has an infrared absorption spectrum of at least about 1024 cm ⁻¹ , about 1706 to 1730 cm ⁻¹ , about 2854 cm ⁻¹ , and about 2924 cm ^−1. And an infrared absorption band (absorption peak) in the vicinity of 3000 to 3500 cm ⁻¹ .

  The shape of the low-toxic SL-containing composition of the present invention is not particularly limited, and may be liquid, emulsion, or solid. The solid form is preferred, and examples of the solid form include tablet form, pill form, powder form, granule form, and capsule form. Preferably it is a powder form or a granular form, More preferably, it is a powder form.

  The low-toxic SL-containing composition of the present invention has a surfactant action, but among the SL-producing yeast fermentation by-products produced in the production process, the components showing cytotoxicity are selectively removed, and their contamination is low. It is characterized by that. Examples of the cytotoxic component include, but are not limited to, lactone type SL; fatty acids and hydroxy fatty acids having 16 and 18 carbon atoms; and SL having an acetyl group.

The toxicity of the low-toxic SL-containing composition of the present invention can be evaluated by, for example, a cell lethal concentration (IC ₅₀ ) calculated from a cytotoxicity test using Hela cells (human cervical epithelial cancer-derived cells). However, in evaluating the action and effect of the low-toxic SL-containing composition in terms of both surface-active action, the “cell lethal concentration (IC ₅₀ )” of the low-toxic SL-containing composition is set to the “criticality of the low-toxic SL-containing composition. It is preferable to obtain a value (IC ₅₀ / CMC) divided by “micelle concentration (CMC)”.

The cytotoxicity test using Hela cells, the method for calculating the cell lethal concentration (IC ₅₀ ) therefrom, and the method for measuring the critical micelle concentration (CMC) are as described in detail in Test Examples 3 and 2, respectively.

Examples of the cell lethal concentration (IC ₅₀ ) with respect to Hela cells of the low toxicity SL-containing composition of the present invention include 2000 to 60000 ppm. Preferably it is 3000-60000 ppm, More preferably, it is 5000-60000 ppm, Most preferably, it is 10000-60000 ppm.

  Examples of the critical micelle concentration (CMC) of the low toxicity SL-containing composition of the present invention include 50 to 500 ppm. Preferably it is 50-400 ppm, More preferably, it is 50-300 ppm, Most preferably, it is 100-300 ppm.

The “IC ₅₀ / CMC” of the low toxicity SL-containing composition of the present invention calculated from these cell lethal concentration (IC ₅₀ ) and critical micelle concentration (CMC) is in the range of 6.7 to 200, preferably 10 It is -200, More preferably, it is 17-200, Most preferably, it is 33-200. Incidentally, the low toxicity SL-containing composition of the present invention, "IC ₅₀ / CMC" are included low toxicity SL-containing composition is from 106.7 to 200 (Examples 1-10). However, these are one aspect | mode of this invention, and are not limited to these.

In addition, as described in Test Example 3, the low toxicity SL-containing composition of the present invention has extremely low irritation to the eyes and mucous membranes from its cell lethal concentration (IC ₅₀ ), and is low in toxicity and at the same time to the eyes and mucosa And hypoallergenic (non-irritating). The mucosa herein includes mucous membranes other than ocular mucosa, specifically, mucous membranes such as oral mucosa, throat mucosa, nasal mucosa, ear cavity mucosa, genital mucosa, and anus.

(III) Use of low-toxic SL-containing composition As described above, the low-toxic SL-containing composition of the present invention has a surface-active action, specifically a good cleaning action and foaming property, while being cytotoxic. In addition, there is very little irritation to eyes and mucous membranes, and it can be said to be substantially non-toxic and non-irritating at practical concentrations. Therefore, it can be used as an anionic surfactant having low toxicity and low irritation. Further, it can be suitably used for pharmaceuticals, quasi-drugs, medical devices, cosmetics and daily miscellaneous goods that require safety and hypoallergenicity (non-irritant). It can also be used as an additive for pharmaceuticals, quasi-drugs, medical devices, cosmetics or daily miscellaneous goods.

  Here, the term “cosmetics” is used in a concept including “fragrance products” such as perfume, eau de cologne and perfume, “cosmetics” and “quasi-drugs”. Cosmetics are used to clean, beautify, enhance attractiveness, change appearance, or keep skin or hair healthy, such as by rubbing, spraying or other similar methods on the body (for example, applying Etc.). Cosmetics include makeup cosmetics (foundation, lipstick, etc.), basic cosmetics (skin lotion, emulsion, etc.), hair cosmetics (hair tonic, hair liquid, hair cream, etc.), body cosmetics (body lotion, hand cream, tanning). Stopper preparations, etc.), toiletry products (toothpaste, mouthwash, shampoo, rinse, soap, body wash, facial cleanser, bath preparation, eye wash, etc.) Quasi-drugs (medicinal cosmetics, antiperspirants, hair color, insecticide) Agents), aromatic products (perfume, eau de toilette, eau de cologne, body powder, odor bags, etc.). Examples of the drug include external preparations (skin external preparations, eye drops, oral preparations, nasal drops, ear drops, suppositories, etc.), internal medicines and injections. Examples of medical devices include wound dressings. Examples of daily miscellaneous goods include dish vegetable detergents, laundry detergents, softeners, bleaches, residential detergents, bath detergents, toilet detergents, and fragrances.

  The low-toxic SL-containing composition of the present invention can be suitably used for an external composition that requires low irritation (nonirritation). Specifically, the composition for external use includes cosmetics for sensitive skin (cosmetics, fragrance products), external medicines applied to wounds and inflamed skin, quasi-drugs for external use, cosmetics (for example, wound parts) Pharmaceuticals, quasi-drugs, and cosmetics (eg, eye drops) that are applied to mucous membranes such as eye care, nasal cavity and oral cavity, etc. Eye care products such as eye ointments, eyewashes, contact lens mounting liquids, nasal rinses, nasal drops, and stomatitis therapeutic agents). In addition, the form of the said wound part coating | covering material is not ask | required in particular, A gel form, powder form, a sheet form, and other shapes can be mentioned widely.

  When the low-toxic SL-containing composition of the present invention is used as an anionic surfactant, the low-toxic SL-containing composition may be used as an anionic surfactant as it is, or has a surfactant effect and has low toxicity. / Other ingredients may be blended as long as the feature of the present invention, i.e. hypoallergenicity, is not impaired. Such other components include distilled water as a solvent, ion-exchanged water, and ethanol; sodium chloride and potassium chloride as additives; glycerin, propylene glycol, and hexylene glycol as solubilizers; Xanthan gum, alginic acid, dextran, etc. as agents; citric acid, lactic acid, malic acid, hydrochloric acid, sulfuric acid, boric acid, sodium hydroxide, potassium hydroxide, etc. as pH adjusters; phosphoric acid compounds, nitrilotrimethyl as chelating agents Acetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and the like; other examples include, but are not limited to, dyes and enzymes. When blending other components, the amount of the low-toxic SL-containing composition of the present invention contained in the anionic surfactant is not limited as long as it exhibits a surfactant effect, but the amount of deacetylated SL is not limited. In terms of conversion, 0.005 to 99.9% by mass, preferably 0.01 to 50% by mass, more preferably 0.02 to 10% by mass, and particularly preferably 0.1 to 5% by mass. .

  The low-toxic SL-containing composition of the present invention is added to pharmaceuticals, quasi-drugs, medical devices, cosmetics or daily miscellaneous goods additives (pharmaceutical additives, quasi-drug additives, medical device additives, cosmetic additives) In addition, when used as a daily miscellaneous goods additive), the low-toxic SL-containing composition may be used as such as it is, or has a desired surface active effect and has low toxicity / low irritation. You may mix | blend another component as long as the characteristic of this invention is not impaired. Such other components can be appropriately set according to the target product such as pharmaceuticals, quasi-drugs, medical devices, cosmetics or daily miscellaneous goods. When blending other components, the amount of the low-toxic SL-containing composition of the present invention contained in the additive is not limited as long as the desired surface-active effect is exhibited, but the amount of deacetylated acid SL is not limited. In terms of conversion, 0.005 to 99.9% by mass, preferably 0.01 to 50% by mass, more preferably 0.02 to 10% by mass, and particularly preferably 0.1 to 5% by mass. .

When the low-toxic SL-containing composition of the present invention is used by adding to a pharmaceutical, quasi-drug, medical device, cosmetic or daily miscellaneous product, that is, using the low-toxic SL-containing composition of the present invention, When preparing external products, medical devices, cosmetics, or daily miscellaneous goods, the amount of the low-toxic SL-containing composition contained in these various products exhibits a desired surface-active effect depending on the purpose and properties of each product. It is set as appropriate within the range. Although not limited, the low-toxic SL-containing composition of the present invention can be added to pharmaceuticals, quasi-drugs, medical devices, cosmetics or daily miscellaneous goods to increase the CMC of these pharmaceuticals to 300 ppm or more. Can be mentioned. For example, the amount of the low-toxic SL-containing composition of the present invention blended in these pharmaceuticals and the like is usually 0.001 to 99.9% by mass, preferably 0.00, in terms of the amount of deacetylated SL. It can select suitably from the range of 001-50 mass% according to a product (for example, 0.02-10 mass%, 0.1-5 mass%, etc.). Specifically, the following ratios can be given according to each product.
Body cleansing agent: 0.01 to 100% by mass, preferably 1 to 90% by mass, more preferably 5 to 800% by mass
Hair cleaning agent: 0.01 to 30% by mass, preferably 1 to 25% by mass, more preferably 5 to 20% by mass
Eye wash: 0.001 to 10% by mass, preferably 0.01 to 8% by mass, more preferably 0.05 to 5% by mass
Eye drops: 0.001 to 10% by mass, preferably 0.01 to 8% by mass, more preferably 0.05 to 5% by mass
Cosmetic: 0.01 to 20% by mass, preferably 0.05 to 15% by mass, more preferably 0.1 to 10% by mass
Oral cleansing agent: 0.001 to 10% by mass, preferably 0.05 to 8% by mass, more preferably 0.1 to 5% by mass
Mucosal or wound cleaning agent: 0.001 to 30% by mass, preferably 0.01 to 25% by mass, more preferably 0.1 to 20% by mass
Wound dressing: 0.001 to 30% by mass, preferably 0.005 to 25% by mass, more preferably 0.01 to 20% by mass.

  When the low-toxic SL-containing composition of the present invention is used by adding it to a pharmaceutical product, quasi-drug, cosmetic product or daily miscellaneous product, these pharmaceutical products are used for the purpose of use as long as the effects of the present invention are not impaired. Accordingly, whitening agents, anti-aging agents, moisturizers, ultraviolet absorbers, antibacterial agents, and other components can be further blended.

  For example, whitening agents include L-ascorbic acid or a derivative thereof, tranexamic acid or a derivative thereof, hydroquinone and a derivative thereof, lucinol, edetic acid or a derivative thereof, arbutin, placental extract, t-AMCHA, acerola extract, agetsu extract, Ellagic acid or its derivatives, flame extract, chamomile extract, chamomile flower extract, kiwi extract, glutathione, tocotrienols, ferulic acid, raspberry ketone, lucinol, walnut extract, pyridoxine dipalmitate, sulfur, kojic acid or its derivatives, glucosamine or its derivatives Derivatives, hydroxycinnamic acid or its derivatives, glutathione, arnica extract, urgonum extract, Sakuhakuhi extract, Psycho extract, bowfish extract, garlic mycelium culture or extract thereof, shiitake Examples include nanoki extract, peach leaf extract, ages extract, cucumber extract, jellyfish extract, touki extract, yokuinin extract, oyster leaf extract, daiou extract, button pipi extract, hamamelis extract, maroni extract, hypericum extract, oil-soluble licorice extract and the like.

  For example, examples of the anti-inflammatory agent include glycyrrhizic acid or a salt thereof, glycyrrhetinic acid or a salt thereof, isopropylaminocaproic acid or a salt thereof, allantoin, lysozyme chloride, guaiazulene, methyl salicylate, and γ-oryzanol.

  Antioxidants include α-carotene, β-carotene, γ-carotene, lycopene, cryptoxanthine, lutein, zeaxanthin, isozeaxanthin, rhodoxanthine, capsanthin, crocetin and other carotenoids; 1,4-diazacyclooctane, 2, 5-dimethylfuran, 2-methylfuran, 2,5-diphenylfuran, 1,3-diphenylisobenzofuran, α-tocopherol, β-tocopherol, γ-tocopherol, d-tocopherol, histidine, tryptophan, methionine, alanine or its alkyl ester, dibutyl Flavono such as tannins such as hydroxytoluene, butylhydroxyanisole, ascorbic acid, tannic acid, epicatechin, epicacatechin, epicatechin gallate, epicacatechin gallate, etc. , Propyl gallate, lacanca extract, astaxanthin, carotene, tocopherol, ascorbic acid derivative, tetrasodium edetate, erythorbic acid, tocopherol acetate, retinol acetate, dibitylhydroxytoluene, ascorbyl stearate, ascorbyl palmitate, methylsilanoldiol Reyltocopherol / silicic acid mixture, benzyl nicotinate, photosensitizer 401, aspartic acid, adenosine triphosphate 2Na, aminobutyric acid, fennel extract, Dutch mustard extract, caffeine, chlorella extract, saffron extract, ginger extract, soybean extract , Tisoiso extract, folic acid, retinol, retinol palmitate, inositol, turmeric extract, oryzanol, carotene, carrot extract, wheat embryo Kiss, Senkyu Extract, Chimp Extract, Toki Extract, Toki Root Extract, Dokudami Extract, Tocopherol, Tocopherol Nicotinate, Button Extract, Ergocalciferol, Pyridoxine Dicaprylate, Batyl Alcohol, Glycyrrhetinic Acid Stearate, Glycyrrhizic Acid, Glycyrrhetinic Acid, Diphenhydramine Hydrochloride, Lysozyme chloride, aminocaproic acid, litchi extract, yokoinin, merirot extract, button extract, toki extract, toki root extract, senkyu extract, geno-chocolate extract, allantoin, arnica extract, arnica flower extract, oxon extract, lauren extract, licorice extract, Cattail extract, chamomile extract, calamine, kawara mugwort extract, licorice extract, guaiazulene, gardenia extract, ku Zasa extract, glycyrrhizic acid 2K, stearyl glycyrrhetinate, gentian extract, black tea extract, Comfrey extract, Comfrey leaf extract, tocopherol acetate, methyl salicylate, zinc oxide, citrus extract, purple root extract, perilla extract, perilla leaf extract, periwinkle extract, peonies Extract, Honeysuckle extract, Sage extract, Horseshoe extract, Horseshoe extract, Achillea millefolium extract, Prunus extract, Sorocera extract, Periwinkle extract, Pyridoxine HCl, Biwa leaf extract, Fuyubodaiju extract, Peach leaf / fruit extract, Cornflower extract, Examples are cypress extract, mugwort extract, lettuce extract, roman chamomile extract, bitumen extract, kakarol, polyamine and the like.

  As moisturizers, sodium pyridonecarboxylate, glycol, glycerin, glucose, maltose, maltitol, sucrose, fructose, xylitol, sorbitol, maltotriose, threitol, erythritol, amylolytic sugar-reducing alcohol, sorbitol, polyhydric alcohols ( Ethylene glycol, 1,4-butylene glycol, diglycerin, triglycerin, tetraglycerin, 1,3-butylene glycol, propylene glycol, dipropylene glycol, polyethylene glycol, 1,3-propanediol, pentylene glycol, hexanediol, Octanediol), serine, glycine, threonine, alanine, collagen, hydrolyzed collagen, hydronectin, fibronectin, keratin, ella Chin, royal jelly, chondroitin sulfate heparin, glycerophospholipid, glyceroglycolipid, sphingophospholipid, sphingoglycolipid, linoleic acid or its esters, eicosapentaenoic acid or its esters, pectin, bifidobacteria fermentation product, lactic acid fermentation product, yeast Extract, Ganoderma mycelium culture or extract thereof, wheat germ oil, avocado oil, rice germ oil, jojoba oil, soybean phospholipid, γ-oryzanol, belude oyster extract, juniper extract, dianthus extract, typhoid extract, diatomaceous earth extract Extract, burdock extract, mannen wax extract, arnica extract, wheat bran, pyrrolidone carboxylic acid, tomato extract, camellia oil, soybean phospholipid, hyaluronic acid, threonine, ammonium glycolate, methyl silano alginate , Yokuinin, Toki Extract, Toki Root Extract, Soybean Extract, Asparagus Extract, DNA-Na, PCA-Na, RNA-Na, Ashitaba Extract, Aspartic Acid, Amatya Extract, Rahan Fruit Extract, Lactoferrin, Alanine, Arginine, Na Alginic Acid, Altea extract, aloe vera extract, oyster extract, barley buckthorn extract, oyster leaf, hydrolyzed keratin, hydrolyzed collagen, hydrolyzed conchiolin, hydrolyzed egg shell membrane, hydrolyzed egg white, hydrolyzed silk, hydrolyzed soy protein, brown alga extract, Karin extract, raspberry extract, xylitol, chitosan, cucumber extract, cucumber fruit extract, guava leaf extract, guava fruit extract, quince seed extract, glycine, glycerin, glucose, grapefruit extract, grapefruit fruit ex , Clematis extract, burdock extract, rice fermented liquor, chondroitin sulfate Na, fish collagena, hawthorn extract, shiitake extract, diou extract, glycerin, cystine, cysteine, horsetail extract, mallow extract, serine, sorbitol, soy protein, tomato extract, Sodium lactate, lactobacilli, fermented soybean extract, urea, wild rose extract, almond extract, almond fruit extract, corn oil, honey, sodium hyaluronate, fuchno extract, betaine, loofah extract, maltitol, maltose, mannitol, lily extract, lysine, apple extract , Lotus root extract, royal jelly, lanolin fatty acid polyethylene glycol 1000, lanolin fatty acid isopropyl, isopropyl myristate, isobutyl myristate, hexyldecanol, milk Myristyl, lanolin fatty acid, tricaprylglyceryl, oleyl alcohol, octyldodecyl oleate, decyl oleate, reduced lanolin, octyldodecanol, almond oil, avocado oil, olive oil, oleic acid, orange rough oil, cocoa butter, carrot extract, Sesame oil, sasanqua oil, safflower oil, dihydrocholesterol, squalane, cholesteryl stearate, ceramide 2, evening primrose oil, sunflower oil, sunflower oil, cefnide 3, sunflower seed oil hybrid sunflower oil, phytosphingosine, grape seed oil, jojoba oil, Examples are jojoba seed oil, mineral oil, mink oil, macadamia nut oil, meadow foam oil, yuri oil, eucalyptus leaf oil, lanolin, linoleic acid, rosehip oil, petrolatum and polyglutamic acid. .

  Ultraviolet absorbers include amyl benzoate, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomenthyl salicylate, benzyl cinnamate, 2-ethoxyethyl paramethoxycinnamate, para Octyl methoxycinnamate, glyceryl mono-2-ethylhexanoate diparamethoxycinnamate, isopropyl paramethoxycinnamate, diisopropyl diisopropylcinnamate ester mixture, urocanic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenone Sulfonic acid or its salt, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenone sodium disulfonate, dihydro Xylbenzophenone, tetrahydroxybenzophenone, 4-tert-butyl-4′-methoxy-dibenzoylmethane, 2,4,6-trianilino-p- (carbo-2′-ethylhexyl-1′-oxy) -1,3, 5-triazine, 2- (2-hydroxy-5-methylphenyl) benzotriazole, 2-ethylhexyl paradimethylaminobenzoate, ethyl paraaminobenzoate, toptylmethoxydipenzoylmethane, oxybenzone-1, ethyl guaiazulenesulfonate, synoxate And anthranilic acid ultraviolet absorbers (for example, homomenthyl-N-acetylanthranilate), ethyl hexyl dimethoxybenzylidenedioxoimidazolidinepropionate, and the like. Examples of the ultraviolet scattering agent include titanium oxide and zinc oxide.

  As antibacterial agents, alum extract, halocarban, chlorophenesin, lysozyme chloride, alkyldiaminoethylglycine hydrochloride, isopropylmethylphenol, thymol, hexachlorophene, berberine, thioxolone, salicylic acid or derivatives thereof, benzoic acid, sodium benzoate, Paraoxybenzoic acid ester, parachlorometacresol, benzalkonium chloride, phenoxyethanol, isopropylmethylphenol, carboxylic acid, sorbic acid, potassium sorbate, hexachlorophene, chlorhexidine chloride, trichlorocarbanilide, photosensitizer, bis (2-pyridylthio- 1-oxide) zinc, thianthol, hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenol Butoxyethanol, resorcinol, azulene, salicylic acid, zinc pyrithione, sodium mononitroguayacol, fennel extract, pepper extract, cetylpyridinium chloride, etc. benzethonium chloride and undecylenic acid derivatives.

  The low-toxic SL-containing composition of the present invention, as shown in test examples to be described later, itself has a skin moisturizing action, an action for improving skin and hair damage (rough skin, hair cuticle damage), skin and hair. There is a protective effect. Therefore, the low toxicity SL-containing composition of the present invention can be used as a moisturizer (skin, hair), a rough skin improving agent, a skin protecting agent, a hair protecting agent and the like. In this case, the ratio of the low-toxic SL-containing composition of the present invention contained in 100% by mass of each product exhibits its effect according to the purpose of the product (whitening, improvement of rough skin, skin / hair protection, etc.). The amount may be any amount, and can be appropriately selected from a range of 0.01 to 10% by mass in terms of the amount of deacetylated SL contained in the low-toxic SL-containing composition.

(IV) Method for producing low-toxic SL-containing composition
(IV-1) Raw material (SL-containing culture or processed product thereof)
Examples of the SL-containing culture used as a raw material for the production of the low-toxic SL-containing composition or a processed product thereof include a wide variety of SL-producing yeast cultures or processed products thereof containing SL. Known SL-producing yeasts can be used, and for example, the above-mentioned Candida bombicola can be preferably mentioned. Candida (Starmelera) and Bonbicola are registered with the biological resource bank ATCC and can be obtained from them (Candida bombicola ATCC22214, etc.). In addition, for the production of the low-toxic SL-containing composition of the present invention, other SL-producing yeast belonging to the genus Candida, which is known to produce SL (acid type, lactone type), can also be used. Examples of such SL-producing yeast include Candida magnoliae, Candida gropengisseri, Candida apicola, Candida petrophilum, and Candida bogoriensis. bogoriensis) and Candida batistae. These yeasts may be strains distributed from preservation institutions or strains obtained by subculture thereof. Here, SL produced by Rhodotorula (Candida) Bogoriensis NRCC9862 (Rhodotorula (Candida) bogoriensis NRCC9862) is 13-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxycosic] 6 ', 6 "-diacetate, where the hydroxyl group at the center of the alkyl group and the sophorose are glycoside-bonded. This SL is different from the general formulas (1) and (2), but is composed of sophorose and hydroxy fatty acid. This is the same, and it is included in the SL targeted by the present invention.

  Candida floricola ZM-1502 strain (FERM P-21133) and Candida floricola NBRC10700T strain are SL-producing yeasts that selectively produce only acid type SL having a diacetyl group. is there. In the production of the low-toxic SL-containing composition of the present invention, deacetylation is essential, but it can be suitably used for obtaining a low-toxic SL-containing composition that does not contain lactone-type SL. Hereinafter, in order to distinguish the SL-producing yeast from the SL-producing yeast that produces both the acid-type SL and the lactone-type SL (lactone-type / acid-type SL-producing yeast), it is also referred to as “acid-type SL-producing yeast”. Called.

  Furthermore, acetyltransferase-deficient Candida bombicola described in Non-Patent Document 2 can also be used, and according to such acetyltransferase-deficient SL-producing yeast, non-acetylated SL is produced in one stage. be able to.

  As a method for culturing SL-producing yeast, for example, a method of culturing by simultaneously giving a high concentration of sugar and a hydrophobic oily substrate can be mentioned. Alternatively, not limited to this, a widely known method can be applied as long as the effect of the present invention is not hindered. The known method may be one described in JP-A-2002-045195 (Patent Document 2). Specifically, a method of culturing SL-producing yeast using glucose as a sugar and a carbon source composed of a fatty acid and vegetable oil as a hydrophobic oily substrate can be used.

  The medium composition is not particularly limited, but the fatty acid portion of SL is known to depend on the fatty acid chain length of the hydrophobic substrate added as a medium component and its ratio, and can be controlled to some extent. For example, as the hydrophobic substrate, oleic acid or a lipid containing a high proportion of oleic acid is suitable. Examples thereof include vegetable oils such as palm oil, rice bran oil, rapeseed oil, olive oil and safflower oil, and animal oils such as pork fat and beef tallow. In the case where SL is stably fermented and produced at a high yield and yield, a mixture of hydrophilic sugar and hydrophobic fat as a carbon source is preferable. Glucose is frequently used as the hydrophilic substrate.

  From the obtained culture solution, for example, liquid components are separated and removed by a conventional solid-liquid separation method such as centrifugation or decantation, and then the solid content is washed with water to obtain an SL-containing fraction (SL-containing culture). be able to. The SL-containing fraction (SL-containing culture) obtained by culturing lactone-type / acid-type SL-producing yeast is a mixture of lactone-type SL and acid-type SL (lactone-type / acid-type SL-containing composition). Usually, the content rate of acid type SL is less than 45 mass% (solid conversion) in SL total amount. On the other hand, SL contained in the SL-containing fraction (SL-containing culture) obtained by culturing acid-type SL-producing yeast is all acid-type SL having a diacetyl group.

  The method for recovering the lactone type / acid type SL-containing composition (or acid type SL-containing composition) from the culture solution of SL-producing yeast may be a known method as long as the effects of the present invention are not hindered. And a method described in JP-A-2003-9896 (Patent Document 3). Such a method is a method of controlling the solubility of SL in water by adjusting the pH of the SL-producing yeast culture solution or the SL-containing fraction prepared therefrom. Specifically, for example, the pH of the SL-producing yeast culture solution is adjusted to about 6 to 7 with an aqueous NaOH solution or the like to solubilize SL, and this is centrifuged and recovered, followed by an aqueous sulfuric acid solution or the like. SL is insolubilized by adding and adjusting to pH 2-3. The lactone type / acid type SL-containing composition (or acid type SL-containing composition) can be prepared as a hydrated product of about 50% by decanting after standing.

(IV-2) Step of removing fatty acid and / or hydroxy fatty acid (fatty acid removal step)
The method for producing a low-toxic SL-containing composition of the present invention includes (1) a step of removing fatty acids and / or hydroxy fatty acids from the SL-containing culture or a processed product thereof (fatty acid removal step).

  Examples of the fatty acid removal method include (a) solvent extraction method, (b) adsorption method, and (c) chromatography. In addition, these fatty acid removal methods may be performed individually by 1 type, and can also be used in combination of 2 or more types arbitrarily. When two or more treatments are used in combination, the treatment order is not particularly limited and is not particularly limited. Preferably, however, solvent extraction or adsorption is performed first as in (a) → (c) or (b) → (c). Preferably it is done.

(A) Solvent extraction method The solvent extraction method is a separation method utilizing the fact that fatty acids and hydroxy fatty acids are more hydrophobic than SL. In general, this is a method in which a fatty acid and a hydroxy fatty acid, which are hydrophobic compounds, are extracted and removed from a SL-containing liquid using a solvent that is not compatible with a solution containing SL (usually water). What is used as a solvent should just be a solvent incompatible with SL containing liquid, and although it does not restrict | limit in particular, For example, ethyl acetate, diethyl ether (ether), hexane, etc. are mentioned. In particular, diethyl ether (ether) is preferred because of the high recovery rate of acid SL and the high removal rate of fatty acids and hydroxy fatty acids.

  In order to increase the removal rate of fatty acids and hydroxy fatty acids, it is preferable to adjust the pH of the SL-containing liquid to be treated to an acidic region in advance. By making it into an acidic region, the carboxyl group of fatty acid and hydroxy fatty acid is protonated and more easily recovered in a solvent. Although not particularly limited, the acidic region can include about pH 6 or less. More specifically, the pH is about 1 to less than 6, preferably about pH 1 to 5, more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4. For adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region, a pH adjuster is usually used. Examples of the pH adjuster include inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, boric acid and hydrofluoric acid; organic acids such as formic acid, acetic acid, malic acid, citric acid, oxalic acid, glutamic acid and aspartic acid. Is used.

  The specific operation of the solvent extraction method will be described by taking, for example, the case of using diethyl ether (ether) as a solvent and sulfuric acid as a pH adjuster, as an example. First, an SL-containing composition (SL-containing culture or treated product thereof) Is added to a separatory funnel so that the ethanol-soluble content is 20% by mass, the pH is adjusted to 3, and the total volume is made up to 100 ml with distilled water. Next, add 1/2 to 2 times volume of ether to this, mix vigorously and let stand, and transfer the lower layer to another separatory funnel. Further, add 1/2 to 2 times volume of ether to the aqueous phase component transferred to another separatory funnel, and mix vigorously in the same manner and let stand. Such extraction operation is performed at least twice, preferably three times or more. After the extraction operation, it is preferable to remove ether by heating or decompressing.

(B) Adsorption method The adsorption method is a separation method that utilizes the difference in the affinity of SL and fatty acids and hydroxy fatty acids for the adsorbent used. As the adsorbent, those that can selectively adsorb hydrophobic compounds can be used, and examples thereof include activated carbon, silica gel, zeolite, and ion exchange resin. Moreover, the alumina oxide described in Unexamined-Japanese-Patent No. 2008-64489 can also be used. Particularly preferred for adsorbing a highly hydrophobic compound is activated carbon. Examples of the ion exchange resin include strong acid cation exchange resins, weak acid cation exchange resins, strong basic anion exchange resins, and weak basic anion exchange resins. A strong basic anion exchange resin and a weak basic anion exchange resin are preferred.

  Moreover, in order to increase the adsorption removal rate of fatty acids and hydroxy fatty acids, it is preferable to adjust the pH of the SL-containing liquid to be treated to an acidic region in advance. By setting it as an acidic region, the carboxyl group of fatty acid and hydroxy fatty acid is protonated and more easily adsorbed by the adsorbent. Although not particularly limited, the acidic region can include about pH 6 or less. More specifically, the pH is about 1 to less than 6, preferably about pH 1 to 5, more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4. For adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region, a pH adjuster is usually used. As the pH adjuster, those described in the above (a) can be used similarly.

  The specific operation of the adsorption method will be described by taking, for example, the case where activated carbon is used as the adsorbent and sulfuric acid is used as the pH adjuster. Alternatively, sulfuric acid is added to the treated product) to adjust to pH 3, and activated carbon is added thereto and mixed so as to be 5 to 15% by mass of the total amount. From the viewpoint of improving the adsorption efficiency of fatty acids and hydroxy fatty acids by activated carbon, and filtering and removing activated carbon, the above mixed solution can be heated. The heating temperature is not particularly limited, and examples thereof include 40 to less than 100 ° C, preferably less than 50 to 100 ° C, more preferably less than 55 to 100 ° C, and particularly preferably less than 60 to 100 ° C.

(C) Chromatography Chromatography is a separation method that utilizes the amphiphilic SL structure. In general, any silica gel, octadecyl silica gel (ODS) resin, ion exchange resin, or synthetic adsorbent known in the art is used as a filler (adsorbent) used as a stationary phase. The chromatography employed in the present invention is preferably partition chromatography, particularly reverse phase chromatography. According to the reverse phase chromatography, it is possible to use an eluent (mobile phase) that is highly safe for the environment and the human body.

  When reverse phase chromatography is used as chromatography, ODS resin or the like is preferably used as the filler. Using an ODS resin in which a hydrophobic octadecyl group or the like is chemically modified on a silica gel carrier, utilizing a hydrophobic interaction with an alkyl side chain of SL, an SL-containing composition (SL-containing culture or treated product thereof) Can efficiently remove fatty acids and hydroxy fatty acids. As the eluent (mobile phase) for reverse phase chromatography, it is preferable to use a solvent having a polarity stronger than that of the filler used as the stationary phase from the viewpoint of separation efficiency and the like. As such an eluent, for example, a mixed solution of lower alcohol such as methanol and ethanol and water can be mentioned. From the viewpoint of safety and environment, a mixed solution of ethanol and water is preferable. The eluent preferably contains a volatile acid component in a proportion of about 0.01 to 0.2% by volume, preferably about 0.05 to 0.1% by volume. Examples thereof include formic acid, acetic acid, and trifluoroacetic acid (TFA).

  Moreover, in order to increase the adsorption amount of the acid type SL to the stationary phase and increase the recovery rate of the acid type SL, it is preferable to previously adjust the pH of the SL-containing liquid to be subjected to chromatography to an acidic region. As an acidic region, since the pKa value of acid type SL is pH 6.1-6.4, it is preferably less than about pH 6. More specifically, the pH is about 1 to less than 6, preferably about pH 1 to 5, more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4. For adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region, a pH adjuster is usually used. As the pH adjuster, those described in the above (a) can be used similarly.

The specific operation of the chromatography will be described by taking, for example, the case where an ODS resin is used as a stationary phase and an aqueous ethanol solution is used as a mobile phase. An SL-containing composition (SL-containing culture or treated product) is used as the stationary phase. In the state where impurities including fatty acids and hydroxy fatty acids are adsorbed and retained by flowing an eluent (ethanol aqueous solution) having an ethanol concentration of less than about 70 to 80% (meaning volume%; the same applies hereinafter). A fraction containing acid type SL can be collected. Specifically, for example, the following method can be exemplified.
(1) An eluent having a concentration of less than about 70 to 80% (for example, an eluent having an ethanol concentration of less than about 70 to 80% (ethanol aqueous solution)) is supplied from the top of the column tower (hereinafter, the top of the separation tower). Equilibrate the column.
(2) Add SL-containing composition (processed product of SL-containing culture) from the top of the separation tower.
(3) The eluent having a concentration of less than about 70 to 80% is supplied from the top of the separation column, and the acid-type SL-containing fraction is selectively eluted and recovered.

  In each step of (1) and (3), the ethanol concentration in the eluent may be increased over time within the above concentration range (gradient elution method), or within the above concentration range. (Stepwise elution method). Preferably, the latter stepwise elution method is used. For example, an SL-containing composition is added to a stationary phase (column filler) equilibrated with an aqueous ethanol solution having an ethanol concentration of 70% [step (2)]. Then, an ethanol aqueous solution having an ethanol concentration of 70% is flowed to elute and collect the target acid-type SL-containing fraction [step (3)]. Moreover, before [(3) process], the ethanol aqueous solution of less than 70% can be used for the purpose of removing the odor component, pigment | dye component, and salts in SL containing culture from an acid type SL containing fraction [ (3 ′) Step]. In this case, the ethanol concentration to be equilibrated in [(1) step] is made to coincide with the ethanol concentration used in [(3 ′) step].

  The low-toxic SL-containing composition of the present invention comprises an SL-containing culture or a processed product thereof in addition to the above-described (1) step of removing fatty acids and / or hydroxy fatty acids (fatty acid removal step), It can also be prepared by subjecting it to at least one of a step of removing an acetyl group (deacetylation step) and a step of removing (3) lactone-type SL (lactone-type SL removal treatment step). These processing steps will be described below.

(IV-3) Step of removing acetyl group of SL (deacetylation step)
Examples of the deacetylation method include (d) hydrolysis treatment and (e) enzyme treatment. In addition, these deacetylation methods may be performed individually by 1 type, and can also be used in combination of 2 or more types arbitrarily. When two or more processes are used in combination, the order of the processes is not limited and is not particularly limited. The deacetylation step is a treatment step that is preferably used when the SL-containing culture or its treated product is prepared by an SL-producing yeast that produces acetylated SL. The treatment product is not applied when it is prepared by an SL-producing yeast that selectively produces non-acetylated SL (acetyltransferase-deficient SL-producing yeast).

(D) Hydrolysis treatment As long as the effects of the present invention are not hindered, widely known methods can be used for the hydrolysis treatment. For example, alkaline hydrolysis using a base such as a metal salt of hydroxide (such as sodium, potassium, calcium and magnesium), carbonate, phosphate, or alkanolamine can be preferably mentioned. Further, various catalysts such as alcohol can be used for the hydrolysis treatment. The temperature, pressure and time for performing the alkaline hydrolysis are not particularly limited as long as the purpose and effect of eliminating the acetyl group of SL contained in the SL-containing culture or its treated product can be achieved. It is preferable to employ a temperature, pressure, and time at which deacetylation can proceed efficiently while suppressing side reactions such as decomposition and chemical modification of the target product acid type SL. In this respect, the reaction temperature is usually in the range of about 30 ° C to 120 ° C, preferably about 50 ° C to 90 ° C. The pressure is usually in the range of about 1 atmosphere to 10 atmospheres, preferably about 1 atmosphere to 2 atmospheres. The reaction time is usually in the range of about 10 minutes to 5 hours, preferably about 1 hour to 3 hours. Moreover, the time which performs an alkali hydrolysis can be suitably set with the number of acetyl groups couple | bonded with SL in the SL containing composition to process.

(E) Enzyme treatment A method of dissociating an acetyl group bonded to SL using an enzyme is a method using an enzyme that generates alcohol and acetic acid from an acetyl ester. As the enzyme, generally used is acetylesterase, and examples thereof include acetylesterase isolated from Aspergillus niger, Rhodococcus sp., Meyerozyma guilliermondii.

  Suitable conditions for the reaction of acetylesterase are pH 5-8, preferably 6-7.5. As long as it falls within this range as a pH adjusting agent, a commonly used pH adjusting agent can be used. For example, sodium hydroxide, potassium hydroxide, sulfuric acid, hydrochloric acid and the like can be used. As reaction temperature, 20-40 degreeC is preferable and 20-35 degreeC is especially preferable. The reaction time is usually 6 hours or longer, preferably 12 hours or longer, particularly preferably 1 day or longer.

  The deacetylation treatment using the enzyme is not particularly limited as long as the objective effect of the present invention is not hindered. For example, a 0.1M phosphoric acid aqueous solution (pH 7.0) is used so that the SL-containing composition becomes 10% by mass. An example is a method of adding acetylesterase to 50 U and stirring at room temperature (25 ° C.) for 1 day.

(IV-4) Step of removing lactone-type SL (Lactone-type SL removal step)
Examples of the lactone-type SL removal method include (f) hydrolysis treatment and (g) chromatography. In these lactone-type SL removal methods, either one of (f) and (g) may be performed alone, or two may be used in any combination. When two processes are used in combination, the order of the processes is not particularly limited and is not particularly limited, but is preferably (f) → (g). In addition, the lactone type SL removal treatment step is prepared by SL-producing yeast (lactone type / acid type SL producing yeast) in which the SL-containing culture or the treated product produces both acid type SL and lactone type SL. The SL-containing culture or the processed product thereof is prepared by SL-producing yeast that selectively produces acid-type SL (acid-type SL-producing yeast). Not applicable.

(F) Hydrolysis treatment The hydrolysis treatment used here is a treatment for opening the lactone ring of the lactone type SL contained in the SL-containing culture or the treated product to convert it into the acid type SL.

  A widely known method can be used for the hydrolysis treatment as long as the effects of the present invention are not hindered. For example, alkaline hydrolysis using a base such as a metal salt of hydroxide (such as sodium, potassium, calcium and magnesium), carbonate, phosphate, or alkanolamine can be preferably mentioned. Further, various catalysts such as alcohol can be used for the hydrolysis treatment. The temperature, pressure, and time for performing the alkaline hydrolysis are not particularly limited as long as the purpose and effect of opening the lactone ring of the lactone type SL contained in the SL-containing culture or its treated product can be achieved. However, it is preferable to employ a temperature, pressure, and time at which the lactone ring can be efficiently opened while suppressing side reactions such as decomposition and chemical modification of the acid type SL that is the target product. In this respect, the reaction temperature is usually in the range of about 30 ° C to 120 ° C, preferably about 50 ° C to 90 ° C. The pressure is usually in the range of about 1 atmosphere to 10 atmospheres, preferably about 1 atmosphere to 2 atmospheres. The reaction time is usually in the range of about 10 minutes to 5 hours, preferably about 1 hour to 3 hours. Moreover, the time which performs alkali hydrolysis can be suitably set with the ratio of lactone type SL contained in SL containing composition to process.

(G) Chromatography The chromatography used here is a treatment for selectively removing lactone-type SL contained in the SL-containing culture or its treated product.

  In general, any silica gel, octadecyl silica gel (ODS) resin, ion exchange resin, synthetic adsorbent and the like known in the art are used as a filler (adsorbent) used as a stationary phase. The chromatography employed in the present invention is preferably partition chromatography, particularly reverse phase chromatography.

  When reverse phase chromatography is used as chromatography, ODS resin or the like is preferably used as the filler. As the eluent (mobile phase) for reverse phase chromatography, it is preferable to use a solvent having a polarity stronger than that of the filler used as the stationary phase in view of separation efficiency and the like. As such an eluent, for example, a mixed solution of lower alcohol such as methanol and ethanol and water can be mentioned. From the viewpoint of safety and environment, a mixed solution of ethanol and water is preferable. The eluent preferably contains a volatile acid component in a proportion of about 0.01 to 0.2% by volume, preferably about 0.05 to 0.1% by volume. Examples thereof include formic acid, acetic acid, and trifluoroacetic acid (TFA).

  Moreover, in order to increase the adsorption amount of the acid type SL to the stationary phase and increase the recovery rate of the acid type SL, it is preferable to previously adjust the pH of the SL-containing liquid to be subjected to chromatography to an acidic region. As an acidic region, since the pKa value of acid type SL is pH 6.1-6.4, it is preferably less than about pH 6. More specifically, the pH is about 1 to less than 6, preferably about pH 1 to 5, more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4. For adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region, a pH adjuster is usually used. As the pH adjuster, those described in the above (a) can be used similarly.

The specific operation of the chromatography will be described by taking, for example, the case where an ODS resin is used as a stationary phase and an aqueous ethanol solution is used as a mobile phase. An SL-containing composition (SL-containing culture or treated product) is used as the stationary phase. In the state in which impurities including lactone type SL are adsorbed and retained by flowing an eluent (ethanol aqueous solution) having an ethanol concentration of less than about 70 to 80% (meaning volume%; the same shall apply hereinafter). The SL-containing fraction can be collected. Specifically, for example, the following method can be exemplified.
(1) An eluent having a concentration of less than about 70 to 80% (for example, an eluent having an ethanol concentration of less than about 70 to 80% (ethanol aqueous solution)) is supplied from the top of the column tower (hereinafter, the top of the separation tower). Equilibrate the column.
(2) Add SL-containing composition (processed product of SL-containing culture) from the top of the separation tower.
(3) The eluent having a concentration of less than about 70 to 80% is supplied from the top of the separation column, and the acid-type SL-containing fraction is selectively eluted and recovered.

  In each step of (1) and (3), the ethanol concentration in the eluent may be increased over time within the above concentration range (gradient elution method), or within the above concentration range. (Stepwise elution method). Preferably, the latter stepwise elution method is used. For example, an SL-containing composition is added to a stationary phase (column filler) equilibrated with an aqueous ethanol solution having an ethanol concentration of 70% [step (2)]. Then, an ethanol aqueous solution having an ethanol concentration of 70% is flowed to elute and collect the target acid-type SL-containing fraction [step (3)]. Moreover, before [(3) process], the ethanol aqueous solution of less than 70% can be used for the purpose of removing the odor component, pigment | dye component, and salts in SL containing culture from an acid type SL containing fraction [ (3 ′) Step]. In this case, the ethanol concentration to be equilibrated in [(1) step] is made to coincide with the ethanol concentration used in [(3 ′) step].

  The manufacturing method of the low toxicity SL containing composition of this invention can be selected with the kind of SL which SL production yeast to use produces. Specifically, when the SL-containing culture or its treated product has only deacetylated acid type SL (non-acetylated acid type SL), (IV-2) fatty acid described above The removal step may be performed alone. On the other hand, when the SL-containing culture or its treated product contains SL having an acetyl group and / or contains lactone type SL in addition to acid type SL, (IV-2) in the fatty acid removal step (IV-3) A combination of a deacetylation step and / or (IV-4) a lactone-type SL removal step may be used. In this case, the order of these steps is not particularly limited as long as the object of the present invention can be achieved.

  As a combination aspect of two or more steps, for example, when an SL-containing culture or a processed product thereof is prepared from acid-type SL-producing yeast (for example, see Patent Document 1), (IV-2) fatty acid removal The step (eg, (a) to (c)) may be followed by (IV-3) deacetylation step (eg, (d) to (e)) or vice versa. After the acetylation step (for example, (d) to (e)), (IV-2) fatty acid removal step (for example, (a) to (c)) may be performed. Although not limited, it is preferable to perform (IV-2) fatty acid removal step after (IV-3) deacetylation step, specifically, at least one of (d) hydrolysis treatment and (e) enzyme treatment After the treatment, there can be mentioned a method of performing at least one of the treatment consisting of (a) solvent extraction method, (b) adsorption method and (c) chromatography. A combination of (d) hydrolysis treatment and (a) solvent extraction method and / or (c) chromatography is preferred.

  Further, for example, when the SL-containing culture or the treated product thereof is prepared from a lactone type / acid type SL producing yeast (for example, see Reference Production Example 1), (IV-4) a lactone type SL removal step ( For example, after (f) to (g)), (IV-2) fatty acid removal step (for example, (a) to (c)) and (IV-3) deacetylation step (for example, (d) to (e) )) May be carried out in any combination. (IV-4) The lactone-type SL removal step (f) and (g) are (IV-3) the deacetylation step (d) and (IV-2) the fatty acid removal step (c), respectively. Therefore, (IV-3) deacetylation or (IV-2) fatty acid removal can be performed simultaneously by performing (IV-4) lactone-type SL removal treatment. A preferable combination is a method in which (d) (or (f)) hydrolysis treatment is followed by at least one of (a) solvent extraction method or (c) (or (g)) chromatography. be able to.

  Furthermore, for example, when the SL-containing culture or a processed product thereof is prepared from an acetyltransferase-deficient SL-producing yeast (see, for example, Non-Patent Document 2), (IV-4) a lactone-type SL removal step ( For example, (f) to (g)) and (IV-2) fatty acid removal steps (for example, (a) to (c)) may be combined in any order. Preferable combinations include (f) a method of performing at least one of (a) solvent extraction method and (c) chromatography after the hydrolysis treatment.

(V) Method for reducing the toxicity of an SL-containing composition According to the production method described above, the SL-containing composition prepared from SL-producing yeast is reduced in toxicity and hypoallergenic, and the low-toxic SL-containing composition of the present invention is used. You can get things. Therefore, the above production method can be paraphrased as a method for reducing the toxicity or the method for reducing the irritancy of the SL-containing composition produced by culturing SL-producing yeast.

  The method for reducing toxicity (or the method for reducing irritation) can be carried out according to the method described in (IV) above, and all the descriptions in the column (IV) described above are incorporated in this column. Moreover, the low toxicity SL containing composition of this invention demonstrated in the (III) column can be prepared and acquired by the said reduction method (or reduction method). Therefore, all the descriptions in the column (III) are incorporated for the low-toxic SL-containing composition obtained by reducing and reducing the SL-containing composition prepared from the SL-producing yeast.

  Hereinafter, the present invention will be described more specifically based on examples and test examples. However, the present invention is not limited to these examples and test examples, and many modifications are possible by those having ordinary knowledge in the art within the technical idea of the present invention.

Reference Production Example 1: Production of SL (Preparation of crudely purified SL-containing composition-1)
As a culture medium, 10 g of hydrous glucose per liter (manufactured by Nippon Shokuhin Kako Co., Ltd., product name: eclipse hydrous crystal glucose), 10 g of peptone (manufactured by Oriental Yeast Co., Ltd., product name: Peptone CB90M), 5 g of yeast extract (Asahi Food and Health) Candida mbicocola ATCC 22214 was shake-cultured at 30 ° C. for 2 days using a liquid medium containing a product of Care Co., product name: Mist Powder N), and this was used as a preculture solution.

  This preculture was inoculated into a main culture medium (3 L) charged in a 5 L fermentor at a rate of 4% of the charged amount, and cultured at 30 ° C. for 6 days under conditions of aeration 0.6 vvm. Fermented. As the main culture medium, 100 g of hydrous glucose per liter, 50 g of palm olein (manufactured by NOF Corporation, product name: Permaly 2000), 50 g of oleic acid (manufactured by ACID CHEM, product name: Palmac 760), 1 g of sodium chloride, phosphoric acid Medium containing 10 g of monopotassium, 10 g of magnesium sulfate heptahydrate, 2.5 g of yeast extract (manufactured by Asahi Food and Healthcare, product name: Mist Powder N), and 1 g of urea (pH 4.5 to 4 before sterilization) .8) was used.

  The fermentation was stopped on the 6th day from the start of the culture, the culture solution taken out from the fermenter was heated, returned to room temperature, and allowed to stand for 2 to 3 days, so that a liquid brown precipitate layer, It was separated into three layers, a milky white solid layer and a supernatant. After removing the supernatant, industrial water or groundwater was added in the same amount as the removed supernatant. While stirring this, a 48 mass% sodium hydroxide solution was gradually added to adjust the pH to 6.5 to 6.9 to solubilize SL contained in the culture solution. This was centrifuged with a tabletop centrifuge (Westphalia: manufactured by Westphalia Separator AG) to precipitate a milky white solid, and the supernatant was collected. While the collected supernatant was stirred, a 62.5% by mass sulfuric acid aqueous solution was gradually added to adjust the pH to 2.5 to 3.0, thereby resolubilizing SL. After leaving this to stand for 2 days, the supernatant was removed as much as possible by decantation, and the residue was obtained as “crudely refined SL-containing composition-1” (about 50% hydrated product, reference production example product 1).

Reference Production Example 2: Preparation of Crude Purified SL-Containing Composition-2 To the crude purified SL-containing composition- 1 fractionated in Reference Production Example 1, an aqueous sodium hydroxide solution was added to adjust the pH to 14, and then at 80 ° C. for 2 hours It processed and hydrolyzed (alkali hydrolysis). Next, after returning to room temperature, the pH is adjusted to 7.5 using sulfuric acid (9.8 M aqueous solution), the generated insoluble matter is removed by filtration, and the filtrate is “crudely purified SL-containing composition-2” (reference production). Obtained as Example 2).

Example 1: Production of low-toxic SL-containing composition The crude purified SL-containing composition-2 obtained in Reference Production Example 2 was adjusted to pH 3.0 using sulfuric acid (9.8 M aqueous solution).

This was subjected to reverse phase column chromatography under the following conditions.
Stationary phase: C18 column (Cosmosil 40C18-PREP, Nacalai Tesque, 15 kg) Mobile phase: 50% and 70% aqueous ethanol.

  Specifically, 1.2 kg of crude purified SL-containing composition-2 adjusted to pH 3.0 (approximately 3% of crude purified SL-containing composition-2 as an ethanol-soluble component with respect to 15 kg of stationary phase filling amount) First, 35 L of 50% ethanol aqueous solution was added to the C18 column, and water-soluble impurities (odor and salts, some pigment substances) were eluted and removed. Subsequently, 30 L of 70% ethanol aqueous solution was supplied to the column, and after the 70% ethanol solution started to elute, the first 15 L was recovered from the C18 column as a fraction containing SL.

  The obtained SL-containing liquid was subjected to an evaporator (Toyo Chemical Food Plant), and the solvent (ethanol) was distilled off and concentrated. The concentrate was subjected to a spray dryer (dry powder pulverizer) (SUS304 R-3 type, water evaporation capacity MAX 5 kg / h, manufactured by Sakamoto Giken Co., Ltd.) to form a dry powder. The spray drying conditions were an atomizer of 12,000 rpm and a bath temperature of 105 ° C. As a result, a fine powder was obtained (Example product 1).

Example 2: Production of low-toxic SL-containing composition Crude purified SL-containing composition-2 obtained in Reference Production Example 2 was adjusted to pH 3.0 using sulfuric acid (9.8 M aqueous solution).

  1.2 kg of the crude purified SL-containing composition-2 adjusted to pH 3.0 is put into a separatory funnel, 400 ml of diethyl ether is added and mixed, separated into two layers, and the lower layer is put into a new separatory funnel. It was collected. 400 ml of diethyl ether was added again, and extraction with diethyl ether was performed three times. The recovered liquid extracted from the lower layer was allowed to stand until the odor of ether was not felt under 50 ° C. conditions, and then subjected to reverse phase column chromatography in the same manner as in Example 1. Specifically, the recovered liquid in which diethyl ether was volatilized was added to a C18 column, and first, 35 L of a 50% ethanol aqueous solution was applied thereto to elute and remove water-soluble impurities (odor and salts, some pigment substances). Subsequently, 30 L of 70% ethanol aqueous solution was supplied, and after the 70% ethanol solution started to elute, the first 15 L was recovered as a SL-containing fraction from the C18 column.

  The obtained SL-containing liquid was subjected to an evaporator (Toyo Chemical Food Plant), and the solvent (ethanol) was distilled off and concentrated. The concentrate was subjected to a spray dryer (dry powder pulverizer) (SUS304 R-3 type, water evaporation capacity MAX 5 kg / h, manufactured by Sakamoto Giken Co., Ltd.) to form a dry powder. The spray drying conditions were an atomizer of 12,000 rpm and a bath temperature of 105 ° C. As a result, a fine powder was obtained (Example product 2).

Examples 3 to 7: Production of low-toxic SL-containing composition The crude purified SL-containing composition-2 obtained in Reference Production Example 2 was adjusted to pH 3.0 using sulfuric acid (9.8 M aqueous solution). This was extracted by the following method using diethyl ether (ether) (solvent extraction method).

  Specifically, the roughly purified SL-containing composition-2 adjusted to pH 3.0 was added to a 50 ml glass centrifuge tube with a screw cap so that the ethanol-soluble content was 3 g, and distilled water was added thereto, The total volume was adjusted to 15 ml. The following amount of ether was added to this and mixed vigorously, followed by centrifugation at 200 × g for 2 minutes to separate into two layers, and the upper ether layer was removed (extraction operation). This extraction operation was performed as follows.

Example 3: An extraction operation using 3 ml ether was performed once (→ Example product 3).
Example 4: An extraction operation using 7 ml ether was performed once (→ Example product 4).
Example 5: The extraction operation using 15 ml ether was performed once (→ Example product 5).
Example 6: Extraction operation using 15 ml ether was performed twice (→ Example product 6).
Example 7: Extraction with 15 ml ether was carried out 3 times (→ Example product 7).

  Then, it was left to stand under 50 ° C. to remove ether, and SL-containing compositions were obtained (Example products 3 to 7).

Examples 8 to 10: Production of low-toxic SL-containing composition Sodium hydroxide aqueous solution was added to the crude purified SL-containing composition-1 fractionated in Reference Production Example 1 to adjust to pH 14, and the mixture was adjusted to 80 ° C for 15 minutes ( Example 8) Hydrolysis (alkali hydrolysis) was performed by heating at 80 ° C. for 30 minutes (Example 9) and at 80 ° C. for 45 minutes (Example 10). Subsequently, these were returned to room temperature, adjusted to pH 7.5 using sulfuric acid (9.8 M aqueous solution), and the generated insoluble matter was removed by filtration. Further, adjust the pH to 3.0 using sulfuric acid (9.8M aqueous solution), add ethanol-soluble component to 3ml in a 50ml glass centrifuge tube with screw cap, and add distilled water to this. Was adjusted to 15 ml. Hexane was added thereto and mixed vigorously, and then centrifuged at 200 × g for 2 minutes to separate into two layers, and the upper hexane layer was removed (extraction operation). This extraction operation was performed five times. Then, it was left to stand at 80 ° C. to remove hexane, and SL-containing compositions were obtained (Example products 8 to 10).

Test Example 1 Method for Measuring Physical Properties of Each Sample SL-containing composition prepared in Reference Production Examples 1 and 2 and Examples 1 to 10 (crudely purified SL-containing composition-1, roughly purified SL-containing composition-2, For Example products 1 to 10), the ester value (mg KOH / g), hydroxyl value (mg KOH / g), ether extract content (%), hue (OD ₄₄₀ ), evaporation residue ( %), Loss on drying (%), ethanol-soluble content (%), and infrared absorption spectrum (cm ⁻¹ ). In addition, HPLC analysis was performed.

  (1) Outline of the test

(2) Test method
(A) Saponification value of ester value (mg of potassium hydroxide required for neutralization of free acid and saponification of ester in a sample equivalent to 1 g of ethanol-soluble component: JIS K 3331, standard oil analysis test specified by Japan Oil Chemical Association Method [2.3.2.1-1996]) and acid number (mg of potassium hydroxide required to neutralize free acid contained in a sample equivalent to 1 g of ethanol-soluble matter: JIS K 3331, Japan Oil Chemistry Association method In addition, the following method can be used as a direct measurement method.

  [Direct method] Weigh approximately 3 g of sample correctly in a saponification flask, add 50 mL of 95vol% ethanol, shake well with a phenolphthalein indicator, and neutralize with a 0.1 mol / L potassium hydroxide standard solution. Acid value is required). Next, add 25 mL of 0.5 mol / L potassium hydroxide-ethanol standard solution correctly, attach a condenser to the flask, and adjust the heating temperature so that the refluxing ethanol does not reach the top of the condenser while stirring occasionally. Heat gently. Allow the flask contents to boil for 30 minutes and then immediately cool.Remove the cooler and add a few drops of phenolphthalein indicator while the contents do not solidify in agar. Add 0.5 mol / L hydrochloric acid standard solution. Titration was performed, and when the indicator disappeared slightly for 30 seconds, the end point was determined, and the amount of 0.5 mol / L hydrochloric acid standard solution used was determined as “the amount of 0.5 mol / L hydrochloric acid standard solution used in this test ( mL) ”. In parallel, take 50 mL of 95vol% ethanol, add 25 mol of 0.5 mol / L potassium hydroxide-ethanol standard solution correctly, perform a blank test, and use 0.5 mol / L hydrochloric acid standard solution required for the blank test. The amount is defined as “Amount used of 0.5 mol / L hydrochloric acid standard solution for blank test (mL)”, and the ester value of 1 g of the sample is calculated from the following formula 1. The ester value of a sample corresponding to 1 g of ethanol-soluble component can be determined from the ethanol-soluble component (%) of the sample according to the following formula 2.

(B) Hydroxyl value Hydroxyl value (mg of potassium hydroxide required to neutralize acetic acid necessary for acetylating free hydroxyl group contained in a sample equivalent to 1 g of ethanol-soluble component: Japan Oil Chemical Association) It can be obtained from the standard oil and fat analysis test method [2.3.6.2-1996]).

[reagent]
Acetylation reagent: Add 12.5 g of acetic anhydride to a 100 ml volumetric flask, add pyridine to the marked line, and mix well with caution. The solution thus prepared is kept away from moisture, carbon dioxide and acid vapors and stored in brown bottles.

[Test method]
Put 1 g of the sample into the round neck flask accurately, add an appropriate amount of acetone and warm at 105 ° C. to remove the water. Then add 5 ml of acetylating reagent correctly and heat to 95-100 ° C. After heating for 1 hour, the flask is removed from the heating bath, allowed to cool, and 1 ml of distilled water is added and mixed, and then the flask is placed in the heating bath again and heated for 10 minutes. Pull it up again to cool it down, add the condensed liquid on the wall of the funnel while washing with 5 ml of neutral ethanol. A phenolphthalein solution is added to the contents of the flask, titrated with 0.5 N KOH / EtOH, and the hydroxyl value of 1 g of the sample is calculated based on the following formula. Further, in the following formula 3, the acid value is the number of mg of potassium hydroxide required to neutralize the free acid contained in 1 g of the sample, and is a standard oil analysis test method (JIS K 3331 ) [2.3.1-1996]. The hydroxyl value of a sample corresponding to 1 g of ethanol-soluble matter can be determined from the ethanol-soluble content (%) of the sample according to the following formula 4.

(C) Ether extract content (%)
The ether extract content indicates the amount of a substance extracted with ether from a sample corresponding to 1 g of ethanol-soluble matter in terms of mass percentage.

[Measuring method]
1 g of a sample was put into a 50 ml capacity eggplant type flask, 10 ml of 10% aqueous sodium hydroxide solution was added, a condenser tube was connected, and the mixture was heated at 80 ° C. for 2 hours. 10 ml of 10% hydrochloric acid aqueous solution was added for neutralization, and water was distilled off with an evaporator while adding 99.5% ethanol. Thereafter, 10 ml of 99.5% ethanol was added and dispersed while performing ultrasonic treatment, the dispersion was filtered using a glass funnel, and the filtrate was transferred to a 50 ml eggplant type flask. After further washing with 99.5% ethanol, the ethanol was distilled off with an evaporator, the residue was transferred to a glass centrifuge tube with a 15 ml screw cap, and the pH was adjusted to 3 using sulfuric acid (manufactured by Kanto Chemical). The total amount was adjusted to 5 ml with distilled water. 5 ml of ether was added and mixed vigorously, followed by centrifugation at 1000 rpm for 2 minutes using a tabletop centrifuge H-108M2 (manufactured by Kokusan) to separate into two layers. The upper layer was transferred to a 100 ml beaker with a known weight, 5 ml of ether was newly added, and ether extraction was performed three times in total. A 100 ml beaker containing the ether extract was put into a 50 ° C. incubator to remove the ether, and further placed in a 105 ° C. incubator for 30 minutes to completely remove the ether.

  After returning to room temperature, the weight was measured, and the ether extract content in 1 g of the sample was calculated based on the following formula 5. The ether extract content of a sample corresponding to 1 g of ethanol-soluble component can be determined from the ethanol-soluble component (%) of the sample according to the following formula 6.

(D) Composition analysis of ether extract by HPLC analysis (C) Ether extract obtained with ether extract content was dissolved in 99.5% ethanol to form a 1% solution, and HPLC analysis was performed under the conditions shown in the table below. Went. Under this HPLC analysis condition, acid type SL is detected at a retention time of 8 to 20 minutes, hydroxy fatty acid is detected at a retention time of 20 to 45 minutes, and fatty acid is detected at a retention time of 45 to 55 minutes. Each peak detected at a retention time of 20 to 55 minutes is compared with the retention time of a known hydroxy fatty acid and fatty acid (standard) to identify the hydroxy fatty acid and the fatty acid, and the area of each peak is 20 to By dividing by the sum of the peak areas detected at the retention time of 55 minutes, the ratio of fatty acids and hydroxy fatty acids to 1 g of ethanol-soluble fraction was calculated. That is, the individual contents were calculated by the following formula 10.

  In the notation method of the obtained fatty acid, the number of carbon atoms is described after C, and then the number of double bonds is represented with a semicolon in between. That is, in the case of oleic acid having 18 carbon atoms and one double bond, C18: 1. When a hydroxy fatty acid is represented, (OH) is added at the end. That is, in the case of hydroxyoleic acid having 18 carbon atoms, C18: 1 (OH) is obtained.

(E) Hue (OD ₄₄₀ )
The hue (OD _{440 nm} ) is the absorbance at 440 nm of an aqueous solution prepared by dissolving a test sample in an alkaline aqueous solution (2% Na ₂ CO ₃ in 0.1N NaOH) so that the ethanol-soluble component is 10% by mass. It can be obtained by measuring.

(F) Evaporation residue (%)
The evaporation residue (%) is precisely weighed and then evaporated to dryness according to the second method (method of heating and evaporating on a hot plate) specified in JIS K0067-1992. It can be obtained from Equation 7.

(G) Loss on drying (%)
Loss on drying (%) is obtained by accurately weighing the weight of the sample, followed by heat drying according to the first method (method of heat drying under atmospheric pressure) specified in JIS K0067-1992 (105 ± 2 ° C, 2 hours) The weight loss after drying can be measured and calculated from the following formula 7.

(H) Ethanol soluble content (%)
The ethanol-soluble component indicates the amount of a substance dissolved in ethanol after dissolving the sample with ethanol.

[Measuring method]
Weigh accurately the Erlenmeyer flask and glass filter. These weights are measured after drying at 105 ° C. for 2 hours or longer and then allowing to cool in a desiccator. About 5 g of the sample is accurately weighed to the 1 mg unit in an Erlenmeyer flask, add 100 mL of the sample, attach a glass tube, heat on a water bath for 30 minutes, and dissolve while occasionally shaking. In addition, 95 vol% ethanol is used for powdery or granular samples, and 99.5 vol% ethanol is used for liquid or pasty samples. It filters using a glass filter with a warm solution, and 50 mL of ethanol is added again to the remaining amount of the Erlenmeyer flask and dissolved. The warm solution is filtered using a glass filter, and the Erlenmeyer flask and glass filter are thoroughly washed with hot ethanol. The mixture is allowed to cool to room temperature, the filtrate and washings are transferred to a 250-mL volumetric flask, ethanol is added up to the marked line, and 100 mL of each volume is dispensed into two 200-mL beakers of known mass using a pipette. One of them is heated on a water bath to remove ethanol, dried in a dryer adjusted to 105 ± 2 ° C for 1 hour, allowed to cool in a desiccator, and accurately weighed (residual amount) .

  The ethanol soluble content (%) is calculated from the following formula 8.

(I) Ignition residue test The ignition residue test is a method for measuring the amount of a substance remaining after ignition of a test sample by the following method [first method]. Usually, it is performed for the purpose of knowing the content of inorganic substances contained as impurities in organic substances, but in some cases, it is carried out to measure the amount of impurities contained in inorganic substances or volatile inorganic substances contained as constituents in organic substances. It is. For example, in the present invention, what is defined as “ignition residue 0.1% or less (first method, 1 g)” is obtained when about 1 g of a test sample is accurately weighed and ignited by the following first method. This indicates that the remainder is 0.10% or less of the collected amount of the test sample.

[Sample Collection Method] A platinum, quartz or magnetic crucible is heated to a constant weight, allowed to cool in a desiccator (silica gel), and then its mass is accurately measured (collected amount). A sample in the range of ± 10% of the specified amount is precisely measured, and the following operation is performed.
[First Method] Moisten the sample with a small amount of sulfuric acid on a crucible, gradually incinerate or volatilize it as low as possible by gradually heating, then moisten with sulfuric acid, completely incinerate, and ignite until constant weight (450 ~ 550 ° C). This is allowed to cool in a desiccator (silica gel) and then weighed accurately. From the obtained measured value (residue) and the collected amount measured in advance, the ignition residue (%) is calculated by the following formula 9.

(J) Infrared absorption spectrum For the measurement of infrared absorption spectrum, a liquid sample was used which was solidified by heating and drying at 105 ± 2 ° C. for 3 hours, and the solid sample was used as it was. The infrared absorption spectrum was analyzed by an ATR method using a Fourier transform infrared spectroscopic analyzer Spectrum ^™ 100 (manufactured by PerkinElmer Japan).

(3) Test results Test results obtained for the SL-containing compositions (crudely purified SL-containing composition-1, roughly purified SL-containing composition-2, and example products 1 to 10) prepared in the reference production examples and examples. Is shown in Table 3.

Test Example 2 Performance Evaluation as Surfactant About the SL-containing compositions (crudely purified SL-containing composition-2, example products 1 to 10) obtained in Reference Production Example 2 and Examples 1 to 10 above, The critical micelle concentration (CMC) and surface tension reducing ability were measured. As a comparative control, a commercially available anionic surfactant (A: “Aminosoft LT-12” (30%): manufactured by Ajinomoto Co., Inc., B: “Surfactin Na” (100%): Wako Pure Chemical Industries, Ltd. ), C: “Lipolane LJ-441” (37%): Lion Co., Ltd.), Tween 20 (Polyoxyethylene Sorbitan Monolaurate (20 EO)) and SLS (Sodium Lauryl Sulfate) in the same manner. ) And surface tension reducing ability. The component of the commercially available anionic surfactant A is N-acyl-L-glutamic acid triethanolamine, the component B is surfactin Na, and the component C is α-olefin sulfonic acid Na.

(1) Experimental method The critical micelle concentration (CMC) and surface tension reducing ability were measured according to the Wilhelmy method. An automatic surface tension meter CBVP-Z type set (manufactured by Kyowa Interface Science Co., Ltd.) was used, and measurement was performed under the conditions of 20 ° C. and pH 7. In addition, sodium hydroxide aqueous solution or hydrochloric acid aqueous solution was used for pH adjustment.

(2) Experimental results Table 4 shows the measurement results of the critical micelle concentration (CMC) and the surface tension reducing ability of each test sample.

Test Example 3 Cytotoxicity Test Using Hela Cells About the SL-containing composition (crudely purified SL-containing composition-2, Example products 1-10) obtained in Reference Production Example 2 and Examples 1-10 above Cytotoxicity tests were performed using Hela cells. As a comparative control, cytotoxicity tests were similarly conducted on the above-described commercially available anionic surfactants A to C, Tween 20 and SLS.

(1) Experimental method
HeLa cells (Kurabo) are seeded in a 96-well plate at a concentration of 2 × 10 ⁴ cells / well, and 10% NCS (Newborn Calf Serum: manufactured by invitrogen), non-essential amino acids, 58 μg / ml L-glutamic acid, 60 μg / ml kanamycin The cells were cultured for 72 hours at 37 ° C. under 5% CO _{2 in the} contained Dulbecco's Modified EAGLE MEDIUM medium (manufactured by Nissui Pharmaceutical). The medium was replaced with the medium containing the test sample. After 48 hours, the medium was replaced with a medium containing 1 mg / ml MTT (3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide). After treatment for 2 hours, formazan, which is a pigment, was extracted with isopropanol, and absorbance at a wavelength of 570 nm was measured. The cell viability% was obtained from the following formula.

(2) Experimental results The cell viability was calculated from the above MTT assay, and the cell lethal concentration (IC ₅₀ ) was determined from the obtained cell viability.

A graph plotting the cell lethal concentration (IC ₅₀ ) calculated from the cell viability and the CMC calculated in Test Example 1 is shown in FIG. Table 5 shows the results of summarizing the CMC (ppm), cell lethal concentration (IC ₅₀ ), and division value (IC ₅₀ / CMC) of these two values calculated in Test Example 1 for each test sample.

As a result, the IC ₅₀ of Example Product 10 is 2000Ppm～60000ppm, remarkably as compared with the commercially available surfactant and Tween 20 and SLS of IC ₅₀ (10~700ppm) increased significantly toxic Turned out to be low. The IC ₅₀ of these examples products are in the crude obtained in the course of manufacturing SL-containing composition-2 IC ₅₀ (1500 ppm) of 1.3 times or more, performed on crude SL-containing compositions -2 It was confirmed that toxic components were remarkably removed by the treatment (solvent extraction treatment, hydrophobic column chromatography).

The cytotoxicity test conducted in this study has been proposed as an alternative to the Draize test (an alternative to the eye irritation test) that is widely used as an eye irritation test for evaluating irritation to the eyes and mucous membranes ( Kenji Okamoto “Trends in alternative methods for eye irritation testing in Japan”, FRAGRANCE JOURNAL 2005-2, P.67-71; “Guidelines for evaluating eye irritation of cosmetic ingredients using alternative methods (Research Group for Health and Welfare) (Altern. Animal Test. Experiment, 5 (Supplement), 1988). According to the proposal of the Ministry of Health and Welfare described in the latter paper, cytotoxicity tests were similarly conducted on Tween 20 as a non-irritating standard substance and SLS as a positive control substance together with the test substance, and the IC ₅₀ was larger than Tween 20 It is described that a test substance with a value is “substantially non-irritating”, and a test substance with an IC ₅₀ smaller than Tween 20 and larger than SLS can be evaluated as “mildly irritating” (the latter See Note 8 of the paper).

As shown in the above table, since the IC ₅₀ values of the low-toxic SL-containing compositions of the present invention shown in Examples 1 to 10 are much higher than the IC _{50 of} Tween 20, they are “non-irritating” substances. It is judged that there is. In addition, since the IC ₅₀ value of the low-toxic SL-containing composition of the present invention is much larger than the IC ₅₀ value of commercially available surfactants, it is particularly low toxic and less irritating among the conventionally known surfactants. It is sex.

Further, the IC ₅₀ value of the low-toxic SL-containing composition of the present invention is 6.7 to 200 times the ratio to CMC (IC ₅₀ / CMC), which is far from CMC. It was confirmed that the function as a surfactant can be sufficiently exhibited while ensuring (stimulation).

Test Example 4 Cytotoxicity of Higher Fatty Acids and Hydroxy Fatty Acids Oleic acid is used as a higher fatty acid and 12-hydroxystearic acid is used as a hydroxy fatty acid. Cytotoxicity was measured.

Specifically, oleic acid and 12-hydroxystearic acid were each dissolved in 99.5% ethanol so as to be 100,000 ppm, and passed through a GHP membrane filter (0.45 μm) (manufactured by Nippon Pole). These were diluted with 99.5% ethanol to a concentration of 1,000 ppm to 50,000 ppm. 990 μl of 10% NCS medium was added to 10 μl of each of these dilutions to prepare a test solution for cytotoxicity (preparing a test solution having a concentration range of 10 ppm to 1,000 ppm). Using this as a test solution, the survival rate (%) of Hela cells was measured according to the method described in Test Example 3, and the cell lethal concentration (IC ₅₀ ) was calculated for each of oleic acid and 12-hydroxystearic acid.

As a result, the cell lethal concentrations (IC ₅₀ ) of oleic acid and 12-hydroxystearic acid were 300 ppm and 160 ppm, respectively. These IC ₅₀ values are as small as 1/6 to 1/375 times the IC ₅₀ values (2000 to 60000 ppm, see Table 4) of the low-toxic SL-containing compositions of the present invention (Example products 1 to 10). However, there is a concern that the cytotoxicity of the SL-containing composition increases when it is mixed into the low-toxic SL-containing composition of the present invention. In other words, by preparing an SL-containing composition so that it does not contain higher fatty acids such as 16 to 18 carbon atoms and hydroxy fatty acids, or contains such fatty acids, the SL-containing composition contains low toxicity. It can be seen that the composition can be obtained.

Test Example 5 Influence on Surface Activity by Lactone-type SL A lactone-type SL is added to a high-purity SL-containing composition having the following constitution, and the total amount of acid-type SL, lactone-type SL, fatty acid and hydroxy fatty acid is 100 mass. %, The ratio of the lactone-type SL was adjusted to be in the range of 0 to 2% by mass (the ester value of 1 g of the ethanol soluble content: 0 to 2 mg KOH / g) (Example products 11 to 15). .

[High purity SL-containing composition]
(1) Acid type SL 99.95% by mass, lactone type 0% by mass, fatty acid and hydroxy fatty acid (total amount) 0.05% by mass per 100% by mass of acid type SL, lactone type SL, and fatty acid and hydroxy fatty acid
(2) Absorbance (OD ₄₄₀ ) at a wavelength of 440 nm of an aqueous solution dissolved so that the ethanol-soluble content is 10% by mass: 0.08
(3) Hydroxyl value of 1 g equivalent of ethanol soluble content: 596 mg KOH
(4) Cell lethal concentration (IC ₅₀ ) for Hela cells: 63000 ppm.

  For these SL-containing compositions, the critical micelle concentration (CMC) and surface tension reducing ability were measured, and the performance as a surfactant (wetting, solubilizing power, detergency, foaming property) was evaluated. The critical micelle concentration (CMC) and the surface tension reducing ability were measured according to the Wilhelmy method in the same manner as in Test Example 2. Specifically, an automatic surface tension meter CBVP-Z type set (manufactured by Kyowa Interface Science Co., Ltd.) was used, and measurement was performed under the conditions of 20 ° C. and pH 7.

  The results are shown in Table 6.

  In general, when a nonionic surfactant (an example of a lactone type SL) is added to an anionic surfactant (an example of an acid type SL), the surface tension may increase or decrease. I can't guess. As shown in Table 6, the SL-containing composition of the present invention decreases in surface tension up to a predetermined amount when a nonionic surfactant (lactone type SL) is added to an anionic surfactant (acid type SL). Confirmed to do. Specifically, the surface tension (minimum surface tension value) decreased as the proportion of lactone type SL increased from 0% to 1.5%. However, it reached a peak at 1.5%, and the surface tension did not change even when more lactone type SL was added.

  The decrease in surface tension is one of the advantages required for a surfactant, and it is predicted that the present composition will improve the performance as a surfactant by containing lactone-type SL at a concentration of up to 2%. The effect of improving the cleaning power, wettability, solubilizing power, and foamability is expected.

Test Example 6 Preparation of body wash and its evaluation (foaming property, rinsing property, skin irritation)
Using the low-toxic SL-containing composition obtained in Example 1 above, according to the formulation described in Table 7, body wash (the present body wash 1-4 (liquid) and 5-6 (solid)) And the characteristics (foaming property, rinsing property, and skin irritation) of each body cleansing agent were evaluated. In addition, as comparative tests, using the roughly purified SL-containing product-2 obtained in Reference Production Example 2 above, comparative body cleansing materials 1 to 5 (liquid) and 6 to 7 (solid) described in Table 8 Similarly, each of the above characteristics was evaluated.

(1) Experimental method (1-1) Foaming and rinsing properties Each of the body cleansing materials described in Tables 7 and 8 (invention body cleaning materials 1 to 6, comparative body cleaning materials 1 to 1) was given to five subjects with self-named sensitive skin. 7) was put out on the wet palm, and both palms were rubbed together for 30 seconds to evaluate foaming. The liquid detergent was used in an amount of 1 ml, and the solid detergent was used by rubbing an amount corresponding to 1 ml of the liquid detergent with the palm. After that, they were rinsed by rubbing their palms with running water at 40 ° C. for 30 seconds, and the rinse property of the cleaning material was evaluated. There are 4 stages of foaming (very foaming: ◎, foaming: ◯, slightly foaming: △, no foaming: x), and rinsing properties are 2 stages (not both slimy and squeaky: ◯, slimy or squeaky Feel: evaluated with x).

(1-2) Skin irritation Each test body wash diluted to 1% with purified water is applied to the filter paper part of a tape for skin sensitization test (patch test) (Fin Chamber [registered trademark] manufactured by Smart Practice Japan). 20 μl was added and affixed around the middle of the forearm bent side of the left hand and wrist of subjects (self-proposed sensitive skin 20-50s, 10 men and women). After acting for 20 minutes, the tape was removed and left unwashed. The determination was made 40 minutes after the tape was removed. For subjects with erythema, observation was continued until the erythema disappeared. In addition, at the time of determination, the presence or absence of stimuli such as tingling and tingling was also examined.

  The application site was observed with the naked eye 40 minutes after removing the tape. The determination was in accordance with the standards of the Japanese Contact Dermatitis Society in the table below.

  The judgment was “−: 0 points, ±: 1 point, +: 2 points, ++: 3 points, +++: 4 points”, and the scores of each subject were averaged. The evaluation of skin irritation was evaluated in four stages: “average score 0 to 0.30 points: ◎, 0.31 to 0.80 points: ○, 0.81 to 1.20: Δ, 1.21 or more: ×”.

(2) Experimental results The results are shown in Tables 8 and 9.

  As can be seen from the results of the body cleansing material 6 of the present invention, the low-toxic SL-containing composition targeted by the present invention itself has very good foaming properties and very good rinsing properties. Furthermore, the low-toxic SL-containing composition has extremely low irritation to the skin, and it did not change even when a surfactant with high skin irritation (Na Laureth sulfate) was used in combination (invention body wash 3).

  Further, the body cleansing agent of the present invention has good foaming properties and good rinsing properties even when it contains a low-toxic SL-containing composition at an extremely low concentration of 0.01% by mass (the present invention body cleaning material 1). Met. Furthermore, even when a low-toxic SL-containing composition is contained at a high concentration of 10% by mass, or when a surfactant having a low foaming property and rinsing property (Na Laureth sulfate) is used in combination, the foaming property is extremely good, and further rinsing is performed. The property was also good.

  Based on these facts, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, a body cleansing agent having good foaming and rinsing properties and less skin irritation is prepared and provided. You can see that you can.

Test Example 7 Preparation and evaluation of hair cleanser (foaming property, rinsing property, skin irritation)
Using the low toxicity SL-containing composition obtained in Example 1, according to the formulation described in the following table, liquid hair washes (pH 4 to 7) were produced (invention hair washes 1 to 4), and each The properties (foaming properties, rinsing properties, and skin irritation) of the hair cleanser were evaluated. Moreover, said each characteristic was similarly evaluated about the comparative hair washing material (pH 4-10) described in the following table as a comparative test.

(1) Experimental method (1-1) Foaming and rinsing properties Each of the hair washing materials described in the following table (the hair washing material of the present invention) was given to 5 female subjects (hair length: degree to be applied to the shoulder) of self-proposed sensitive skin. (1 to 4, comparative hair washing agent) 6 ml was used, and the hair was washed with hot water of 40 ° C., and the rinsing property was evaluated from the foaming property and the squeaking condition of the hair at the time of rinsing. There are 4 stages of foaming (very foaming: ◎, foaming: ○, slightly foaming: △, no foaming: ×), and rinsing properties are 3 stages (not feeling squeaking: ○, feeling squeaking slightly: △, feeling squeaking :).

(1-2) Skin irritation Evaluation was performed in the same manner as in Test Example 6.

(2) Experimental results The results are shown in the table below.

  As can be seen from the results, the hair cleanser of the present invention prepared using the low-toxic SL-containing composition is extremely irritating to the skin even when the low-toxic SL-containing composition is contained at a high concentration of 30% by mass. It was low (Invention Hair Wash 3), and it did not change even when a surfactant (Na laureth sulfate, cocamidopropyl betaine) with high skin irritation was used in combination (Invention Hair Wash 4). Moreover, even if the hair cleansing material of the present invention contains a low-toxic SL-containing composition at a low concentration of 0.01% by mass (the present hair cleaning material 1), the foaming property is good and the rinsing property is also good. Met. Furthermore, when a low-toxic SL-containing composition is contained at a high concentration of 30% by mass (inventive hair cleansing material 3), a surfactant having poor foaming properties and rinsing properties (sodium laureth sulfate or cocamidopropyl betaine) was used in combination. Even in the case (Invention Hair Wash 4), the foaming property was very good and the rinsing property was also good. Furthermore, it is generally known that a hair cleanser formulated with soap is not easy to foam, but it contains a low-toxic SL content, resulting in a cleanser with good foam ease. (Invention hair cleanser 5).

  Based on these facts, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, a hair cleanser having good foaming and rinsing properties and less skin irritation is prepared and provided. You can see that you can.

Test Example 8 Preparation of eye wash and its evaluation (eye mucous membrane irritation)
Using the SL-containing composition obtained in Example 2 above, a liquid eye wash (pH 6) was produced according to the formulation described in Table 11 (the present eye wash 1 to 3), and the eye of each eye wash The irritation to the mucous membrane was evaluated. Further, as a comparative test, the irritation to the ocular mucosa was similarly evaluated for Comparative Eyewashes 1 and 2 (pH 6) shown in Table 11.

(1) Experimental method:
(Evaluation of eye / mucosal irritation)
Cytotoxicity tests have been proposed as an alternative to eye / mucosal irritation tests that evaluate irritation of eyes and mucous membranes. Therefore, a cytotoxicity test was performed using rabbit corneal epithelium-derived cells (SIRC) cells. First, 100 μL of each eyewash diluted in MEM medium stepwise was added to a 96-well plate, and SIRC cells were seeded there at a concentration of 1 × 10 ⁴ cells / well, at 37 ° C. and 5% CO ₂ . Cultured for 72 hours. After the culture, the medium was replaced with a medium containing 0.5 mg / mL MTT, and further cultured for 2 hours. Formazan (dye) was extracted from the obtained culture using isopropanol, and the absorbance at 570 nm was measured. Cell viability (%) was determined by the following formula.

  The same test was conducted using the comparative control substance triethanolamine (TEA) (comparative test), and the cell viability (%) obtained above was higher than the cell viability obtained in the comparative test. Cytotoxicity is lower than TEA), the case of the same degree was judged as ◯ (cytotoxicity is equivalent to TEA), and the case of low was judged as x (cytotoxicity higher than TEA).

(2) Experimental results The results are shown in Table 11 together.

  As can be seen from the results, the eye wash of the present invention prepared using the low-toxic SL-containing composition is extremely irritating to the eye mucosa even when the low-toxic SL-containing composition is contained at a high concentration of 10% by mass. It was low (invention eyewash 3). In addition, it was confirmed that the eye wash of the present invention exhibits a good detergency even when it contains a low-toxic SL-containing composition at an extremely low concentration of 0.001% by mass (the present invention eye wash 1). .

  From these facts, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, it is possible to prepare and provide an eye wash with good detergency and low irritation to the eye mucosa. I understand.

Test Example 9 Preparation of eye drops and their evaluation (eye irritation)
Using the SL-containing composition obtained in Example 5, a liquid eye drop (pH 6) was produced according to the formulation described in Table 12 (the present eye drops 1 to 3), and the eye irritation of each eye drop Evaluated. In addition, as a comparative test, the ophthalmic mucous membrane irritation was similarly evaluated for comparative eye drops 1 and 2 (pH 6) shown in Table 12.

(1) Experimental method (1-1) Ocular mucosal irritation Rabbit corneal epithelium-derived cells (Test Example 8) using eye drops 1 to 3 and comparative eye drops 1 and 2 of the present invention as test samples ( Cytotoxicity tests using SIRC cells were performed to evaluate eye mucosal irritation.

(2) Experimental results The results are shown in Table 12 together.

  As can be seen from the results, the eye drop of the present invention prepared using the low-toxic SL-containing composition was similar to the eye wash evaluated in Test Example 8, and the low-toxic SL-containing composition was as high as 10% by mass. Even though it was contained, the ocular mucosal irritation was extremely low (the eye drop 3 of the present invention). From this, it can be seen that by using the low-toxic SL-containing composition provided in the present invention as a surfactant, it is possible to prepare and provide an eye drop having low eye mucosal irritation.

Test Example 10 Preparation of mouthwash and its evaluation (mucosal irritation)
Using the SL-containing composition obtained in Example 9 above, a liquid mouth washes (pH 6.5) was produced according to the formulation described in Table 13 (invention mouthwash 1 to 3), and each wash The oral mucosal irritation of the oral fluid was evaluated. In addition, as a comparative test, oral mucosal irritation was similarly evaluated for comparative mouthwashes 1 to 3 (pH 6.5) shown in Table 13.

(1) Experimental method (oral mucosal irritation)
As test samples, using the mouthwashes 1 to 3 and comparative mouthwashes 1 to 3 of the present invention, a cytotoxicity test using rabbit corneal epithelium-derived cells (SIRC) cells was performed in the same manner as in Test Example 8, Oral mucosal irritation was evaluated.

(2) Experimental results The results are shown in Table 13 together.

  As can be seen from the results, the oral cleansing liquid of the present invention prepared using the low-toxic SL-containing composition is extremely irritating to the oral mucosa even when the low-toxic SL-containing composition is contained at a high concentration of 10% by mass. It was low (invention mouthwash 3). In addition, it was confirmed that the oral cleansing liquid of the present invention exhibits good detergency even when it contains a low-toxic SL-containing composition at an extremely low concentration of 0.001% by mass (inventive mouthwash 1). It was.

  Therefore, it is possible to prepare and provide a mouthwash with good detergency and low oral mucosal irritation by using the low-toxic SL-containing composition provided in the present invention as a surfactant. I understand that I can do it.

Test Example 11 Preparation and evaluation of mucosal cleaning material (mucosal irritation)
Using the SL-containing composition obtained in Example 1 above, a liquid mucosal cleaning material (pH 6) was produced according to the formulation described in Table 14 (invention mucosal cleaning liquids 1 to 4), and each cleaning liquid was rinsed. And mucosal irritation were evaluated. In addition, as a comparative test, the rinsing properties and mucous membrane irritation properties were similarly evaluated for the comparative mucosal cleaning solutions 1 to 2 (pH 6) shown in Table 14.

(1) Experimental method mucosal irritation Rabbit corneal epithelium-derived cells (SIRC) cells were used as test samples in the same manner as in Test Example 8 using the mucosal lavage fluids 1 to 4 and the comparative mucosal lavage fluids 1 and 2 of the present invention. Cytotoxicity tests were performed to evaluate mucosal irritation.

(2) Experimental results The results are shown in Table 14.

  As can be seen from the results, the mucosal lavage fluid of the present invention prepared using the low-toxic SL-containing composition has extremely low mucosal irritation even when the low-toxic SL-containing composition is contained at a high concentration of 30% by mass. (Invention mucosa washing solution 3). Further, even when the mucosal cleaning liquid of the present invention and the low-toxic SL-containing composition were contained at an extremely low concentration of 0.001% by mass (the mucosal cleaning liquid 1 of the present invention), it was confirmed that good detergency was exhibited.

  From these facts, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, it is possible to prepare and provide a mucosal cleaning solution with good detergency and low mucosal irritation. Recognize.

Test Example 12 Preparation of wound cleaning agent and its evaluation (detergency and wound part irritation)
Using the SL-containing composition obtained in Example 4 above, a liquid wound cleanser (pH 5.5) was produced according to the formulation described in Table 15 (the present wound cleanser 1-4). The cleaning power and wound part irritation of each cleaning agent were evaluated. Moreover, as a comparative test, the cleaning power and wound part irritation were similarly evaluated for the cleaning agents for comparative wounds 1-2 (pH 5.5) shown in Table 15.

(1) Experimental method (1-1) Detergency The detergency was evaluated by the following method. A test piece was prepared by uniformly spreading “model dirt” (see below) on a stainless steel piece whose weight had been measured in advance. The model soil was dried and weighed. Each wound cleaner (the present invention wound cleaners 1 to 4 and comparative wound cleaners 1 and 2) was applied to the surface of the soil, allowed to stand for 5 minutes, and then rinsed with running water for 10 seconds. After drying, the weight of the test piece was measured, and the washing rate was calculated from the weight ratio of the model dirt before and after washing.

Detergency was evaluated according to the following criteria from the obtained cleaning rate.
[Detergency]
◎: Cleaning rate 75-100%, ○: Cleaning rate 50-74%, Δ: Cleaning rate 25-49%, ×: Cleaning rate 0-24%.

[Model dirt]
A mixture of model sebum stain and model blood stain at 5: 1 was used as “model stain”.
(Composition of model sebum stain)
Macadamia nut oil 40%, Isopropyl myristate 20%, Palmitic acid 15%, Squalene 10%, Oleic acid 10%, Cholesterol 5%;
(Model blood stain composition)
Sheep sterile defibrinated blood.

(1-2) Wound site irritation Cytotoxicity evaluation was performed using fibroblasts. First, fibroblasts were seeded in a 96-well plate at a concentration of 1 × 10 ⁴ cells / well and cultured in DMEM medium at 37 ° C. and 5% CO ₂ for 72 hours. Each of the wound cleansing agents (the present invention wound cleansing agents 1-4, comparative wound cleansing agents 1-2) was replaced with a DMEM medium added at an arbitrary concentration, and cultured at 37 ° C. under 5% CO ₂ for 48 hours. . After the culture, the medium was replaced with a medium containing 0.5 mg / mL MTT, and further cultured for 2 hours. Formazan (dye) was extracted from the obtained culture using isopropanol, and the absorbance at 570 nm was measured. The cell viability was calculated by the following formula.

  The same test was performed using the comparative control substance triethanolamine (TEA) (comparative test), and the cell viability (%) obtained above was higher than the cell viability obtained in the comparative test. Toxicity is lower than TEA), the case of the same degree is judged as ○ (cytotoxicity is equivalent to TEA), and the case of low is judged as × (cytotoxicity is higher than TEA).

(2) Experimental results The results are shown in Table 15.

  As can be seen from these results, the wound cleansing agent of the present invention prepared using the low-toxic SL-containing composition is irritating to the wound even if it contains the low-toxic SL-containing composition at a high concentration of 30% by mass. Was very low (Inventive wound cleaning agent 3). Moreover, even if the wound cleaning agent of the present invention contains a low-toxic SL-containing composition at an extremely low concentration of 0.001% by mass (present invention wound cleaning agent 1), it exhibits good cleaning power. Was confirmed.

  Based on these facts, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, preparing and providing a wound cleanser with good detergency and low wound area irritation You can see that

Test Example 13 Preparation of wound dressing and its evaluation (irritability, dispersibility)
Using the SL-containing composition obtained in Example 6 above, a gel wound dressing (pH 7) was produced according to the formulation described in Table 16 (the present wound dressing 1 to 8), and each coating The wound irritation and dispersibility of the material was evaluated. Moreover, as a comparative test, the wound part irritation and dispersibility were similarly evaluated for the comparative wound dressing (pH 7) described in Table 17.

(1) Experimental method (1-1) Wound part irritation As a test sample, the present invention wound dressing materials 1 to 8 and comparative wound dressing materials 1 to 4 were used, and fibroblasts were used as in Test Example 12. Cytotoxicity tests were performed to assess wound irritation.

(1-2) Dispersibility When preparing the present wound dressings 1-8 and comparative wound dressings 1-4, it was evaluated whether each compounding component was uniformly dissolved. Evaluation was performed in two stages (uniformly dissolved: ◯, aggregated or insoluble: x).

(2) Experimental results The results are shown in Tables 16 and 17.

  As can be seen from the results, the wound dressing of the present invention prepared by blending the low-toxic SL-containing composition was much less irritating than the wound dressing not blended with the low-toxic SL-containing composition. (Invention wound dressing 1-8). The dispersibility of each component was also good.

  From this, by using the low-toxic SL-containing composition provided in the present invention, the dispersibility of each compounding component is enhanced, and the irritation caused by additives such as surfactants and antibacterial agents is alleviated and suppressed. It can be seen that, as a result, a less irritating wound dressing can be prepared and provided.

Test Example 14 Evaluation of Moisturizing Action and Skin Barrier Function Improving Action of Low Toxicity SL-Containing Composition Using the low toxicity SL-containing composition obtained in Example 1 above, a test sample was prepared according to the formulation described in Table 18. Manufactured (invention lotions 1 to 3) and evaluated the moisturizing action and skin barrier function improving action of each lotion. Further, as comparative tests, Comparative Lotions 1 to 3 shown in Table 18 were similarly evaluated for the moisturizing action and the skin barrier function improving action.

(1) Experimental method (1-1) Moisturizing effect The inside of the human upper arm was marked, and the stratum corneum moisture content was measured using a stratum corneum moisture meter (Corneometer CM825 (Courage + Khazaka)) (initial value). Each lotion (invention lotions 1 to 3 and comparative lotions 1 to 3) was applied to the site where the initial value was measured at a rate of 20 μL / cm ² . After exposing the inside of the upper arm for 4 hours in a room with a relative humidity of 50% and a temperature of 20 ° C., the moisture content of the stratum corneum was measured. The rate of change before and after application of each lotion was determined, and the moisturizing effect of each lotion was evaluated based on the following criteria.

[Moisturizing effect]
◎: Change rate of stratum corneum water content 120% or more ○: Change rate of stratum corneum water content 100% to less than 120% Δ: Change rate of stratum corneum water content 90% to less than 100% ×: Change rate of stratum corneum water content 90% or less.

(1-2) Skin Improvement Action A 5% SDS solution was brought into contact with the human upper arm for 1 hour to artificially cause rough skin. The texture of the rough skin was observed with a microscope (microscope VHX-1000 / VH-220R (Keyence)). In addition, transdermal moisture transpiration was measured using Tewameter TM300 (Courage + Khazaka). Each lotion (invention lotions 1 to 3 and comparative lotions 1 to 3) was applied to this site twice a day at a rate of 20 μL / cm ² . After performing this for 4 days, the skin texture was observed. In addition, transdermal moisture transpiration was measured using Tewameter TM300 (Courage + Khazaka). The rate of change before and after using each lotion was determined, and the skin barrier function improving action of each lotion was evaluated based on the following criteria.

[Skin barrier function improving effect]
◎: Moisture transpiration rate change rate is less than 80% ○: Moisture transpiration rate is less than 80-90% △: Moisture transpiration amount is less than 90-100% ×: Moisture transpiration amount is 100% or more.

[Skin texture]
A: Skin texture returned to normal. B: Skin texture slightly improved.
(Triangle | delta): The texture of skin has hardly changed.
X: The texture of the skin deteriorated.

(2) Experimental results The results are shown in Table 18 together. Further, FIG. 1 shows skin images obtained by observing the skin after treatment with 5% SDS solution and after applying for 4 days with the lotion 1 of the present invention with a microscope.

  From this result, it can be seen that the low-toxic SL-containing composition has not only low irritation but also skin moisturizing action and skin barrier function improving action. In particular, it has been found that a lotion containing a low-toxic SL-containing composition at a ratio of 10% by mass is excellent in both the moisturizing action and the action of improving the barrier function of the skin. Therefore, by using the low-toxic SL-containing composition provided in the present invention, a hypoallergenic external preparation (cosmetics, dermopharmaceutical, dermatological quasi-drug) having a moisturizing action and a skin barrier function improving action. It can be seen that can be prepared and provided.

Test Example 16 Evaluation of moisturizing action and irritation of lotion Using the composition containing low toxicity SL obtained in Example 1, lotion (pH 5.0) was produced according to the formulation described in Table 19 (this Invention lotion 1-3), the moisturizing action and irritation of each lotion were evaluated. Moreover, as a comparative test, the moisturizing action and irritation were similarly evaluated for comparative lotions 1 to 3 (pH 5.0) shown in Table 19.

(1) Experimental Method (1-1) Evaluation of Moisturizing Action and Stinging Applying each lotion (invention lotion 1 to 3 and comparative lotion 1 to 3) to the face after washing the face to 5 specialist panelists. I was asked to evaluate moistness and sting. The evaluation of moist feeling is “moist: ◎, slightly moist: ○, not very moist: △, not moist: x”, and the evaluation of sting (irritation) is “no irritation: “Slightly stimulated: Δ, very stimulating: x”.

(1-2) Irritation
(A) Skin irritation Using the skin lotions of the present invention 1 to 3 and comparative skin lotions 1 to 3, a patch test was conducted on 10 subjects in the same manner as in Test Example 6, and the skin irritation of each skin lotion. Evaluated.

(2) Experimental results The results are shown in Table 19 together.

  As can be seen from the results, the skin lotion of the present invention prepared using the low-toxic SL-containing composition is irritating to the skin even when the low-toxic SL-containing composition is contained at a high concentration of 10% by mass and 20% by mass. It was confirmed that the properties were extremely low and increased without impeding the moisture retention of glycerin and sodium hyaluronate (invention lotions 2 and 3).

  Therefore, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, it is possible to prepare and provide a cosmetic having good moisturizing properties and low skin irritation. I understand that I can do it.

Test Example 16 Evaluation of Irritation and Storage Stability of Whitening Cream Using the low toxicity SL-containing composition obtained in Example 1, a whitening cream (pH 5) was produced according to the formulation described in Table 20 (the present invention). Cream 1-4), the storage stability, skin irritation and feeling of use of each cream were evaluated. Moreover, as a comparative test, the storage stability, skin irritation, and feeling of use were similarly evaluated for comparative creams 1 to 2 (pH 5) shown in Table 20.

Each cream was produced as follows.
First, each component of the A phase and each component of the B phase are mixed and heated to 70 ° C. After heating and dissolving, phase B is gradually added to phase A with stirring, and emulsified with a homomixer. It is then cooled to room temperature with stirring.

(1) Experimental method (1-1) Evaluation of storage stability Conditions of each whitening cream (present creams 1 to 4 and comparative creams 1 to 2) at room temperature (25 ± 5 ° C), 50 ° C, and -5 ° C It was stored for one month below, and the change in state was observed after one month. Evaluation was performed in three stages (no change: ◯, slightly separated: Δ, clearly separated: x).

(1-2) Skin irritation Using the creams 1 to 4 of the present invention and the comparative creams 1 to 2, a patch test was conducted on 10 subjects as in Test Example 6, and the skin irritation of each cream was determined. evaluated.

(1-3) Usability 5 professional panelists applied each whitening cream (Invention creams 1-4, Comparative creams 1-2) to the face after face washing, feeling of use (extensibility, moist feeling, Skin familiarity) was evaluated. Each evaluation item was evaluated in four stages (very good: ◎, good: ◯, somewhat bad: △, bad: ×).

(2) Experimental results The results are shown in Table 20 together.

  As can be seen from the results, the whitening cream of the present invention prepared using the low-toxic SL-containing composition is irritating to the skin even when the low-toxic SL-containing composition is contained at a high concentration of 10% by mass and 20% by mass. It was confirmed that the properties were extremely low and improved without impairing the storage stability of the cream (inventive creams 3 and 4). Moreover, all of the whitening creams of the present invention had good usability.

  From these, by using the low-toxic SL-containing composition provided in the present invention as a surfactant, a cosmetic having good storage stability and feeling of use and having low skin irritation is prepared. It can be seen that it can be provided.

Test Example 18 Hair Protection and Restoration Effect (1) Test Method (1-1) Evaluation Preparation of Test Hair (30 cm Length, 10 g Hair Bundle) (i) Healthy Hair 10% healthy black human hair (30 cm) After immersing in an aqueous solution of alkyl ether sulfate sodium (AES) (pH 7, room temperature) for 1 minute, washed with running water and dried with a dryer, healthy hair (10 g of hair bundle) was obtained.

(Ii) Damaged hair Dilute healthy black human hair (30cm) in bleach (6% hydrogen peroxide and 1% (2 / w) mixture of 2% ammonia) (30 ° C) for 30 minutes (bath ratio) : Hair / bleaching agent = 1/10 (w / w)), and then washed with running water. Next, it is immersed in a buffer solution (pH 4.0) of 0.1 M citric acid and 0.2 M disodium hydrogen phosphate for 5 minutes, washed with running water, immersed in distilled water (room temperature) for 5 minutes, then taken out and dried with a dryer. did. A hair bundle of 10 g obtained by performing this treatment three times was used as damaged hair.

(1-2) Test Method Fluorescein sodium salt (SIGMA Batch116K0094 EC208-253) in an aqueous solution containing the low-toxic SL-containing composition of the present invention (Example 1) at a concentration of 1% by mass to a concentration of 0.08% by weight. -0) was added to prepare a test solution. For comparison and control tests, instead of the low-toxic SL-containing composition (Example 1), an aqueous solution containing 2% by mass of “Aqulio TXF-875” (manufactured by Nippon Seika Co., Ltd.) and water In the same manner as above, a fluorescent substance was added to prepare a test solution. “Aqulio TXF-875” is known to have moisture retention and to promote penetration of active ingredients. Each evaluation test hair (normal hair, damaged hair) was immersed in these for 30 minutes, and then the cross section of each evaluation test hair was observed with a fluorescence microscope.

(2) Test result The result of healthy hair is shown in FIG. 3 (A), and the result of damaged hair is shown in FIG. 3 (B). As is clear from this, the test hair treated with the low-toxic SL-containing composition is confirmed to have the test solution penetrating into the hair while protecting the hair surface of both healthy and damaged hair. It was done. From this, it is expected that the use of the low-toxic SL-containing composition of the present invention not only protects the hair surface by coating, but also penetrates into the hair and can recover damaged hair.

[Prescription example]
The formulation of various products containing the low toxicity SL containing composition of this invention is shown below.

Formulation Example 1 Oil cleansing agent polyglyceryl-6 myristate 12.0 (mass%)
Polyglyceryl oleate-6 2.0
Polyglyceryl laurate-10 2.0
Polyglyceryl pentaisostearate-10 4.0
Lauric acid 2.0
Ethyl hexyl palmitate 10.0
Triethylhexanoin 45.0
Low-toxic SL-containing composition (Examples 1 to 10) 5.0
Butylene glycol 10.0
Purified water balance
Total amount 100.0% by mass.

Formulation example 2 Out bath treatment <br/> Ethanol 5 (mass%)
Hydroxypropyltrimonium chloride starch 0.3
Low-toxic SL-containing composition (Examples 1 to 10) 1
Purified water remaining
Total amount 100.0% by mass.

Formulation Example 3 Eye drops (pH 6.6)
1-Menthol 0.02 (mass%)
d-Camphor 0.001
d-borneol 0.005
Eucalyptus oil 0.01
Mint oil 0.002
Naphazoline hydrochloride 0.0015
Neostigmine methylsulfate 0.004
Chlorpheniramine maleate 0.03
Viridoxine hydrochloride 0.08
Tocopherol acetate 0.04
Magnesium and potassium L-aspartate 1.4
Aminoethylsulfonic acid 0.8
Boric acid 0.5
Borax 0.1
Concentrated benzalkonium chloride solution 50 (Japanese Pharmacopoeia) 0.015
Chlorobutanol 0.2
Low-toxic SL-containing composition (Examples 1 to 10) 0.3
Hydrochloric acid / sodium hydroxide
Purified water remaining
Total 100.00% by mass.

Formulation Example 4 Eyewash (pH 5.8)
1-Menthol 0.01 (mass%)
Epsilon aminocaproic acid 0.1
Dipotassium glycyrrhizinate 0.1
Zinc sulfate 0.05
Chlorpheniramine maleate 0.003
Potassium L-aspartate 0.1
Calcium chloride 0.05
Boric acid 1.5
Borax 0.015
Concentrated benzalkonium chloride solution 50 (Japanese Pharmacopoeia) 0.004
Chlorobutanol 0.2
Low-toxic SL-containing composition (Examples 1 to 10) 0.2
Ethanol 0.1
Hydrochloric acid / sodium hydroxide 0.01
Purified water remaining
Total 100.00% by mass.

Formulation Example 5 Contact Lens Wearing Solution (pH 7.0)
1-Menthol 0.015 (mass%)
Aminoethylsulfonic acid 0.5
Potassium chloride 0.08
Sodium chloride 0.15
Boric acid 0.9
Borax 0.2
Potassium sorbate 0.1
Sodium edetate (Japanese Pharmacopoeia) 0.05
Low-toxic SL-containing composition (Examples 1 to 10) 0.5
Hydrochloric acid / sodium hydroxide
Purified water remaining
Total 100.0% by mass.

Formulation Example 6 Contact Lens Wearing Solution (pH 7.5)
1-Menthol 0.01 (mass%)
Potassium chloride 0.05
Sodium chloride 1
Sodium hydrogen phosphate 0.15
Sodium dihydrogen phosphate 0.01
Low-toxic SL-containing composition (Examples 1 to 10) 0.02
20% polyhexamethylene biguanide solution 0.0005
(Product name: Cosmosil CQ)
Hydrochloric acid / sodium hydroxide
Purified water remaining
Total 100.0% by mass.

Formulation Example 7 Wound cleansing agent (pH 6.5)
Sodium chloride 0.9 (mass%)
Low-toxic SL-containing composition (Examples 1 to 10) 0.05
Hydrochloric acid / sodium hydroxide
Purified water remaining
Total 100.0% by mass.

Claims (4)

Skin moisturizer, rough skin improving agent, skin protective agent, or hair protective agent comprising as an active ingredient a low toxicity sophorolipid-containing composition having the following characteristics (a) to (c) and (b) and (b):
(A) It contains at least acid type sophorolipid, fatty acid and hydroxy fatty acid derived from SL-producing yeast culture, and when the total amount of acid type sophorolipid, lactone type sophorolipid, fatty acid and hydroxy fatty acid is 100% by mass, The proportion is converted to dry weight as follows:
(1) Acid-type sophorolipid: 94-99.99 mass%,
(2) Lactone type sophorolipid: 0 to 2% by mass,
(3) Total amount of fatty acid and hydroxy fatty acid: 0.01 to 0.24% by mass ,
(B) The ratio of the lactone type sophorolipid to the 100 parts by mass of the acid type sophorolipid contained in the low toxicity sophorolipid-containing composition, or the ratio of the fatty acid and the hydroxy fatty acid (total amount) is 0 to 2.12 parts by mass or 0.01 to 1 part by mass (c) Neither the acid-type sophorolipid nor the lactone-type sophorolipid contained in the low-toxic sophorolipid-containing composition has an acetyl group,
(A) The cell lethal concentration (IC ₅₀ ) for HeLa cells is 10,000 to 60000 ppm.
(B) the ratio of a cell lethal concentration on HeLa cells and (IC ₅₀₎ and the critical micelle concentration (CMC) (IC ₅₀ / CMC) is 33 to 200. A hypersensitive skin cosmetic comprising the following (a) to (c) and a low-toxic sophorolipid-containing composition having the characteristics (a) and (b); applied to a wound or an inflamed area Externally used quasi-drugs, externally used pharmaceuticals, cosmetics or external medical devices; pharmaceuticals, quasi-drugs or cosmetics applied to the mucous membrane in the eye or nostril:
(A) When containing at least acid sophorolipid derived from a sophorolipid-producing yeast culture, fatty acid and hydroxy fatty acid, and the total amount of acid sophorolipid, lactone sophorolipid, fatty acid and hydroxy fatty acid is 100% by mass, The proportion is converted to dry weight as follows:
(1) Acid-type sophorolipid: 94-99.99 mass%,
(2) Lactone type sophorolipid: 0 to 2% by mass,
(3) Total amount of fatty acid and hydroxy fatty acid: 0.01 to 0.24% by mass ,
(B) The ratio of lactone type sophorolipid to fatty acid and hydroxy fatty acid (total amount) with respect to 100 parts by mass of acid type sophorolipid contained in the low-toxic sophorolipid-containing composition is 0 to 2.12 parts by mass or 0.01 to 1 part by mass (c) Neither the acid-type sophorolipid nor the lactone-type sophorolipid contained in the low-toxic sophorolipid-containing composition has an acetyl group,
(A) The cell lethal concentration (IC ₅₀ ) for HeLa cells is 10,000 to 60000 ppm.
(B) the ratio of a cell lethal concentration on HeLa cells and (IC ₅₀₎ and the critical micelle concentration (CMC) (IC ₅₀ / CMC) is 33 to 200. "   The above sensitive skin cosmetics, quasi-drugs, pharmaceuticals, cosmetics, or external medical devices are body wash, hair cleansers, eye wash, eye drops, cosmetics, mucosa or wound cleansers, and wound dressings. The hypersensitive skin cosmetic, quasi-drug, pharmaceutical, cosmetic or external medical device according to claim 2, which is at least one selected from the group consisting of: The sensitive skin cosmetic, quasi-drug, pharmaceutical, cosmetic or external medical device according to claim 3, wherein the composition contains a low-toxic sophorolipid-containing composition in the following ratio:
Body wash: 0.01-100% by mass,
Hair cleaning agent: 0.01 to 30% by mass,
Eye wash: 0.001-10% by mass,
Eye drops: 0.001 to 10% by mass,
Cosmetics: 0.01-20% by mass,
Mucosal or wound cleaning material: 0.001 to 30% by mass,
Wound dressing: 0.001 to 30% by mass.

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