WO2015034007A1 - 低毒性ソホロリピッド含有組成物及びその用途 - Google Patents
低毒性ソホロリピッド含有組成物及びその用途 Download PDFInfo
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Definitions
- the present invention relates to a low toxicity sophorolipid-containing composition and its use.
- amino acid surfactants such as N-acyl-L-glutamate triethanolamine and polyglycerin fatty acid ester surfactants such as polyglyceryl laurate are relatively safe surfactants. It has come to be used frequently. Moreover, since there are few safety problems, soap (fatty acid salt) is reviewed again. However, it is known that medium chain fatty acids having a carbon chain length of C6 to 10 such as soap are highly irritating. Further, among the above, amino acid surfactants are known to have relatively low irritation (hypoallergenicity), but even that is not satisfactory.
- biosurfactant which is a biologically derived surfactant
- sophorolipid which is one of glycolipid type biosurfactants
- sophorolipid is a fermentation product obtained from yeast fermentation and has been known to have high safety.
- Patent Document 1 describes that sophorolipid is hypoallergenic.
- the degree of low irritation is equivalent to that of amino acid surfactants, and further irritation (reduction in toxicity) is required.
- lactone type sophorolipid which is one embodiment of sophorolipid, has relatively high toxicity (Non-patent Document 1).
- An object of the present invention is to provide a low-toxic sophorolipid-containing composition. Moreover, an object of this invention is to provide the use of a low toxicity sophorolipid containing composition.
- the “low toxicity sophorolipid-containing composition” means a low toxicity composition containing a low toxicity sophorolipid.
- the present inventors have made extensive studies, and in addition to the lactone type sophorolipid contained in the sophorolipid-containing composition produced by yeast fermentation, in addition to fatty acid, hydroxy fatty acid, and acid type sophorolipid. It was found that the acetyl group contained in each of the sophorolipid-containing compositions had a considerable adverse effect on the cytotoxicity of the sophorolipid-containing composition, and by removing these, the less toxic sophorolipid-containing composition, particularly the irritation to the eyes and mucous membranes, was extremely high. It was confirmed that a low sophorolipid-containing composition was obtained.
- sophorolipid may be abbreviated as “SL”.
- SL sophorolipid-containing composition
- low-toxic SL-containing composition the “low-toxic sophorolipid-containing composition” of the present invention
- Low-toxic SL-containing composition (I-1) Contains at least a coloring component derived from an SL-producing yeast culture, acid type SL, fatty acid and hydroxy fatty acid, acid type SL, lactone type SL, fatty acid and hydroxy A low-toxic SL-containing composition, characterized in that when the total amount of fatty acids is 100% by mass, the respective proportions are as follows in terms of dry weight: (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.
- (I-2) Acid type SL, lactone type Sl, and 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 the low-toxic SL-containing composition according to (I-1), which satisfies at least one of (ii): 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.
- the ratio of acid type SL, lactone type SL, and 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 is as follows:
- 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 to 100% by mass, and the proportion of lactone type SL is 0 to 2% by mass, preferably 0 to 1.5% by mass, more preferably 0 to 1% by mass, and (I-1) to The low-toxic SL-containing composition according to any one of (I-3).
- 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 to 1, preferably 0.005 Of (I-1) to (I-4), preferably 0.8 to 0.8, more preferably 0.01 to 0.6, particularly preferably 0.01 to 0.5, and still more preferably 0.4 or less.
- (I-6) Ester equivalent of 1 g of ethanol soluble content is 0.01-2 mgKOH / g, preferably 0.01-1.5 mgKOH / g, more preferably 0.1-1.5 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.
- the equivalent of 1 g of ethanol soluble component has a hydroxyl value of 460 to 630 mg KOH / g, preferably 545 to 630 mg KOH / g, more preferably 560 to 630 mg KOH / g, particularly preferably 575 to 630 mg KOH / g, and more particularly
- the low-toxic SL-containing composition according to any one of (I-1) to (I-6), preferably 575 to 585 mg KOH / g.
- the low lethal concentration (IC 50 ) against HeLa cells is 2000 ppm or more, preferably 3000 to 60000 ppm.
- Toxic SL-containing composition is 2000 ppm or more, preferably 3000 to 60000 ppm.
- 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
- (II-2) A cosmetic, food and drink, quasi-drug, pharmaceutical product comprising the low-toxic SL-containing composition according to any one of (I-1) to (I-13); or Additives for cosmetics, foods and drinks, quasi drugs or pharmaceuticals.
- the present invention also includes a method for producing the low-toxicity sophorolipid-containing composition and a method for reducing the toxicity of the sophorolipid-containing composition.
- 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, (III-1) or (III- The production method described in 2).
- 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-3), which is reverse phase column chromatography using an ODS resin as a mobile phase and an aqueous ethanol solution as a mobile phase.
- the step of eliminating the acetyl group bonded to SL is at least one treatment selected from hydrolysis treatment and enzyme treatment (III-1) to (III-4) The manufacturing method described in any one of.
- 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-6) Manufacturing method to be described.
- a method for detoxifying an SL-containing composition comprising preparing a detoxified SL-containing composition: (A) Acid type SL: 94 to 99.99% by mass, (B) Lactone type SL: 0 to 2% by mass (C) Total amount of fatty acid and hydroxy fatty acid: 0.01 to 4% by mass.
- (IV-2) Acid type SL, lactone type SL, and 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 as follows (i) And a method for detoxifying as described in (IV-1), which comprises preparing a detoxified SL-containing composition in the range of at least one of (ii): 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) The ratio of the acid type SL, the lactone type SL, and the acid type SL, the lactone type SL, and the fatty acid and the hydroxy fatty acid (total amount) included per 100% by mass of the total amount of the fatty acid and the hydroxy fatty acid are within the following ranges.
- (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
- 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 content of the lactone-type SL is 0 to 2% by mass, preferably 0 to 1.5% by mass, more preferably 0 to 1% by mass.
- the ester value of 1 g equivalent of ethanol soluble component is 0.01-2 mgKOH / g, preferably 0.01-1.5 mgKOH / g, more preferably 0.1-1.5 mgKOH / g, especially A method for reducing poisoning according to any one of (IV-1) to (IV-4), characterized in that a composition containing SL with reduced poisoning, preferably 0.8 to 1.5 mg KOH / g, is prepared. .
- the hydroxyl value of the equivalent of 1 g of ethanol soluble component is 460 to 630 mg KOH / g, preferably 545 to 630 mg KOH / g, more preferably 560 to 630 mg KOH / g, particularly preferably 575 to 630 mg KOH / g, and more particularly
- the SL-containing culture obtained by culturing the SL-producing microorganism or a treated product thereof is further (2) a step of eliminating the acetyl group bonded to SL, and (3) removal of the lactone-type SL
- the step of removing fatty acids and / or hydroxy fatty acids is at least one treatment selected from a solvent extraction method, an adsorption method, and chromatography, (IV-1) to (IV- 7) The method for reducing toxicity described in any one of the above.
- 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-8), which is reverse phase column chromatography using an ODS resin as a mobile phase and an aqueous ethanol solution as a mobile phase.
- step of eliminating the acetyl group bonded to SL is at least one treatment selected from hydrolysis treatment and enzyme treatment, (IV-7) to (IV-9) The method for reducing poisoning according to any one of the above.
- step (IV-12) any one of (IV-7) to (IV-11), wherein (3) the step of removing lactone-type SL is at least one treatment selected from hydrolysis treatment and chromatography Detoxification method to be described.
- an SL-containing composition having low cytotoxicity can be provided.
- the low-toxic SL-containing composition has a surface-active effect and has extremely little cytotoxicity and irritation to the eyes and mucous membranes. Therefore, cosmetics, pharmaceuticals, quasi-drugs and the like applied to the skin, wounds and inflamed areas
- eye care products such as eyewashes and contact lens mounting liquids, oral drugs applied to the oral mucosa, and points applied to the nasal mucosa It can be suitably blended in nasal drugs and the like.
- 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.
- 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.)
- Sophorolipid 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 ⁇ 1 ⁇ 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 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.
- 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 carbon number of the hydrocarbon chain of R 3 is usually 11 to 20, preferably 13 to 17, and more preferably 14 to 16.
- 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.
- 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 be 45:55 to 10:90 (dry weight ratio).
- SL-producing yeast include 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 it (Candida bombicola ATCC22214, etc.).
- 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 solution of these yeasts (R. Hommel, Biodegradation, 1, 107 (1991)).
- Candida floricola strain ZM-1502 (FERM P-21133) and Candida floricola NBRC10700T are SL-producing yeast that selectively produce only acid form SL.
- Japanese Patent Laid-Open No. 2008-247845 Japanese Patent Laid-Open No. 2008-247845. Therefore, when the acid form SL is selectively produced, the SL-producing yeast can be preferably used.
- 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.
- 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 composition at least in terms of cytotoxicity and has the following characteristics. .
- acid type SL When the coloring component derived from the SL-producing yeast culture, acid type SL, fatty acid and hydroxy fatty acid is contained at least, and the total amount of acid type SL, lactone type SL, fatty acid and hydroxy fatty acid is 100% by mass, The proportion is converted to dry weight as follows: (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.
- the acid type SL is contained in a ratio of 94 to 99.99% by mass in terms of dry matter. This is a total amount of the acid type SL, the lactone type SL, the fatty acid and the hydroxy fatty acid in the low-toxic SL-containing composition of 100% by mass. % Of acid type SL contained in% (dry matter weight). 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 the acid type SL contained in 100% by mass of the ethanol-soluble component of the low-toxic SL-containing composition is 94 to 99.99% by 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 ratio of the acid type SL can be calculated from the ester value and the ether extract content of the low-toxic SL-containing composition.
- 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.
- 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 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).
- 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.
- 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.
- mold SL as SL production yeast 0.01 mass% can be mentioned.
- 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.
- the low-toxic SL-containing composition of the present invention includes low-toxic SL-containing compositions containing lactone-type SL in a proportion of 0.04 to 2.0% by mass (Examples 1 to 10). However, these are one aspect
- the ratio of the lactone type SL can be determined from the ester value (mg KOH / g) of the low-toxic SL-containing composition.
- 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 ratio 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.
- 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 mgKOH / g. From the viewpoint of reduction in toxicity, 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.
- the surfactant 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, particularly preferably 0.8 to 1.5 mgKOH / g.
- 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.
- 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.
- Fatty acid and hydroxy fatty acid are contained in a 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, and total amount of fatty acid and hydroxy fatty acid of 100. It is the ratio (dry matter weight) of fatty acids and hydroxy fatty acids contained in 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 is a fatty acid contained in a medium used for culture of SL-producing yeast, and is a fatty acid calculated as the ether extract by the method for measuring the ether extract content described in Examples. Specifically, it is a saturated or unsaturated fatty acid having 6 to 24 carbon atoms.
- Saturated fatty acids such as C15), palmitic acid
- examples of the hydroxy fatty acid used herein include fatty acids obtained by substituting at least one hydrogen atom with a hydroxy group in the above fatty acid.
- the ratio of the fatty acid and hydroxy fatty acid 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 an upper limit. Moreover, as a minimum, 0.01 mass% can be mentioned suitably.
- 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 acid form is contained to some extent in the range of 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.
- the low-toxic SL-containing composition of the present invention includes low-toxic SL-containing compositions containing fatty acids and hydroxy fatty acids in a total amount of 0.1 to 4% by mass (Examples 1 to 10). However, these are one aspect
- the mainstream are saturated and unsaturated fatty acids having 16 and 18 carbon atoms.
- 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). .
- 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-toxic SL-containing composition.
- 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 (
- 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.
- 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 “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.
- the low-toxic SL-containing composition targeted by the present invention includes those having at least one of the following properties (4) and (5) in addition to the properties (1) to (3) described above. .
- the hydroxyl value of the equivalent of 1 g of ethanol soluble component is 460 to 630 mgKOH / g.
- the absorbance (OD 440 ) of the aqueous solution in which the low-toxic SL-containing composition is dissolved so that the ethanol-soluble content is 10% by mass is 0.001-1.
- “Hydroxyl number (mg KOH / g)” in the present invention in which the hydroxyl value of 1 g of ethanol soluble component is 460 to 630 mg KOH / g, is a sample (low toxicity) corresponding to 1 g of ethanol soluble component. This is the number of mg of potassium hydroxide required to completely acetylate the OH group of the compound contained in the SL-containing composition), thereby free of the SL in the SL contained in the sample (low-toxic SL-containing composition). The proportion of hydroxyl groups can be measured.
- the “hydroxyl value (mg KOH / g)” can be measured according to the standard oil analysis test method (2.3.6.2-1996) established by the Japan Oil Chemical Association (Japan). The details are as described in Test Example 1.
- the “hydroxyl value (mg KOH / g)” of the low toxicity SL-containing composition of the present invention is preferably 460 to 630 mg KOH / g for a sample equivalent to 1 g of ethanol-soluble matter.
- the amount is preferably 545 to 630 mg KOH / g, more preferably 560 to 630 mg KOH / g, and particularly preferably 575 to 630 mg KOH / g.
- the low-toxic SL-containing composition of the present invention includes low-toxic SL-containing compositions having a hydroxyl value of 575 to 585 mg KOH / g (Examples 1 to 10), which are one aspect of the present invention.
- the present invention is not limited to these.
- coloring of the low-toxic SL-containing composition includes coloring components other than acid type SL, lactone type SL, fatty acid and hydroxy fatty acid. It means to include.
- a coloring component originates in the culture of SL production yeast, and it will not restrict
- 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.
- the hue (OD 440 ) of the aqueous solution is in the range of 0.001 to 1.
- it is 0.005 to 0.8, more preferably 0.01 to 0.6, and particularly preferably 0.01 to 0.5.
- the low-toxic SL-containing composition of the present invention includes low-toxic SL-containing compositions having a hue (OD 440 ) of 0.1 to 0.4 (Examples 1 to 7). However, these are one aspect
- the low-toxic SL-containing composition targeted by the present invention is characterized by having at least one of the following physical properties (a) to (c) in addition to the above properties: (A) Evaporation residue: 1 to 100% (B) Loss on drying: 0-99% (C) Ethanol soluble content: 1 to 100%.
- the “evaporation residue (%)” is, as described in Test Example 1, the residue when the sample is evaporated is shown in mass percentage (mass%).
- 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%, and still more preferably 60%. To 100%, even more preferably 70 to 100%, particularly preferably 80 to 100%, more particularly preferably 90 to 100%.
- the “loss on drying (%)” means the weight loss (% by mass) when the sample is dried as described in Test Example 1, and in this way, the present invention has low toxicity in the present invention.
- the content of moisture and other volatile substances (low boiling point compounds) in the SL-containing 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.
- the weight loss on drying (%) of the low toxicity SL-containing composition of the present invention is 0 to 99%, preferably 0 to 95%, more preferably 0 to 90%, still more preferably 0 to 90%. It is 30%, still more preferably 0 to 20%, particularly preferably 0 to 20%, more particularly preferably 0 to 10%.
- the “ethanol-soluble content (%)” is the content (mass%) of the substance dissolved in ethanol contained in the sample, as described in Test Example 1, and this makes it possible to dissolve ethanol mixed in the sample. It is possible to ascertain the content of polar substances such as surfactants.
- the “ethanol-soluble content (%)” can be measured according to JIS K3362-2008. The details are as described in Test Example 1.
- the ethanol-soluble content (%) of the low-toxic SL-containing composition of the present invention is 1 to 100%, preferably 5 to 100%, more preferably 10 to 100%, and still more preferably It is 85 to 100%, still 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.
- the low-toxic SL-containing composition of the present invention has an infrared absorption spectrum of at least a wave number of about 1024 cm ⁇ 1 , 1706 to 1730 cm ⁇ 1 , 2854 cm ⁇ 1 , 2924 cm ⁇ 1 , and 3000 to 3500 cm ⁇ 1. It has an infrared absorption band (absorption peak) in the vicinity.
- 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.
- 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).
- IC 50 cell lethal concentration
- 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 ) for Hela cells of the low-toxic SL-containing composition of the present invention include 2000 to 60000 ppm. It is preferably 3000 to 60000 ppm, more preferably 5000 to 60000 ppm, and particularly preferably 10,000 to 60000 ppm.
- CMC critical micelle concentration
- 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 To 200, more preferably 17 to 200, particularly preferably 33 to 200.
- the low-toxic SL-containing composition of the present invention includes low-toxic SL-containing compositions whose “IC 50 / CMC” is 106.7 to 200 (Examples 1 to 10). However, these are one aspect
- 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 low-toxic SL-containing composition of the present invention has a surfactant activity, but has extremely little cytotoxicity and irritation to eyes and mucous membranes. Since it can be said to be substantially non-toxic and non-irritating at practical concentrations, it is used as a low-toxic and low-irritant anionic surfactant, as well as food and drink that requires safety and low irritation (non-irritant) It can be suitably used for products, pharmaceuticals, quasi drugs, cosmetics and the like. It can also be used as an additive for foods and drinks, pharmaceuticals, quasi drugs or cosmetics.
- foods and drinks In addition to foods and beverages, foods and drinks here have specific functions such as health supplements, health functional foods, foods for specified health use, or supplements, and are consumed for the purpose of maintaining health. Food and drink.
- the term “cosmetics” is used in a concept including “cosmetics” and “fragrance products” such as perfume, eau de cologne and perfume.
- 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.), for example, makeup cosmetics (foundation, lipstick, etc.), basic cosmetics (skin lotion, emulsion, etc.), hair cosmetics (hair tonic, hair liquid, hair cream, etc.) And toiletry products (toothpaste, mouthwash, shampoo, rinse, soap, facial cleanser, bath preparation, etc.).
- the low-toxic SL-containing composition of the present invention can be suitably used for a composition for external use in which low irritation (nonirritation) is particularly required.
- the composition for external use include cosmetics for sensitive skin (cosmetics, fragrance products), external medicines or quasi-drugs applied to wounds and inflamed skin, eyes, nasal cavity, oral cavity, etc.
- Pharmaceuticals or quasi drugs for example, eye care products such as nasal drops, eye drops, eye ointments, eye washing liquids, nasal liquids, contact lens mounting liquids) and the like that are applied to the mucous membranes of the above.
- the low-toxic SL-containing composition of the present invention When 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.
- 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 is converted to the amount of acid type SL. 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 When used as an additive (food additive, pharmaceutical additive, quasi-drug additive) for foods, beverages, pharmaceuticals, quasi drugs, or cosmetics, it contains low-toxic SL
- the composition may be used as it is as an additive, and other components may be used as long as the desired surface-active effect is obtained and the characteristics of the present invention such as low toxicity / hypoallergenicity are not impaired. You may mix
- Such other components can be appropriately set according to the target product such as food and drink, pharmaceuticals, quasi drugs, and cosmetics.
- 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 is converted into the amount of acid type SL.
- Examples are 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 When the low-toxic SL-containing composition of the present invention is used by adding to a food / beverage product, pharmaceutical, quasi-drug or cosmetic, that is, the low-toxic SL-containing composition of the present invention is used for food / beverage product, pharmaceutical or quasi-drug.
- the amount of the low-toxic SL-containing composition to be blended in these various products is appropriately set within a range in which a desired surfactant effect is exhibited according to the purpose and properties of each product.
- the low-toxic SL-containing composition of the present invention is added to foods and drinks, pharmaceuticals, quasi drugs or cosmetics, so that the CMC of these foods and drinks is 300 ppm or more.
- a percentage can be mentioned. For example, it is 0.005 to 99.9% by mass, preferably 0.01 to 50% by mass, more preferably 0.02 to 10% by mass in terms of the amount of acid type SL blended in these foods and beverages. Particularly preferred is 0.1 to 5% by mass.
- SL-producing yeast belonging to the genus Candida, which is known to produce SL (acid type, lactone type)
- 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.
- SL produced by Rhodotorula (Candida) Bogoriensis NRCC9862 is 13-[(2′-O- ⁇ -D-glucopyranosyl- ⁇ -D-glucopyranosyl) oxycosic] ', 6 "-diacetate, in which the hydroxyl group at the center of the alkyl group and a sophorose are glycosidically bonded.
- This SL is different from the general formulas (1) and (2), but is composed of sophorose and a hydroxy fatty acid. This is the same, and it is included in the SL targeted by the present invention.
- Candida floricola strain ZM-1502 (FERM P-21133) and Candida floricola strain NBRC10700T are SL-producing yeasts that selectively produce only acid type SL in the present invention. It can be preferably used.
- SL-producing yeast 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.
- 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.
- 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 is preferable.
- the present invention is not limited to this, and widely known methods can be applied as long as the effects of the present invention are not hindered.
- the known method may be 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, it is known that the fatty acid part of SL depends 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.
- 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.
- a mixture of hydrophilic sugar and hydrophobic fat as a carbon source is preferable. Glucose is frequently used as the hydrophilic substrate.
- 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).
- the content rate of acid type SL is less than 45 mass% (solid conversion) in SL total amount.
- all SL contained in the SL-containing fraction (SL-containing culture) obtained by culturing acid-type SL-producing yeast is acid-type SL.
- 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 the 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 the pH to about 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
- the low-toxic SL-containing composition of the present invention can be prepared by subjecting the SL-containing culture or a treated product thereof to at least (1) a step of removing fatty acids and / or hydroxy fatty acids (fatty acid removal step).
- fatty acid removal methods include (a) solvent extraction method, (b) adsorption method, and (c) chromatography.
- 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.
- the treatment order is not particularly limited and is not particularly limited.
- solvent extraction or adsorption is performed first as in (a) ⁇ (c) or (b) ⁇ (c). Preferably it is done.
- the solvent extraction method is a separation method utilizing the fact that fatty acids and hydroxy fatty acids are more hydrophobic than SL.
- 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
- the pH of the SL-containing liquid is adjusted in advance to an acidic region in order to increase the removal rate of fatty acids and hydroxy fatty acids.
- the carboxyl group of fatty acid and hydroxy fatty acid is protonated and more easily recovered in a solvent.
- 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, and more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4.
- a pH adjuster is usually used for adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region.
- pH adjuster examples 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 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.
- 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. Further, alumina oxide described in JP 2008-64489 A can also be used. Particularly preferred for adsorbing a highly hydrophobic compound is activated carbon.
- 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.
- the pH of the SL-containing liquid is adjusted in advance to an acidic region in order to increase the adsorption and removal rate of fatty acids and hydroxy fatty acids.
- the carboxyl group of fatty acid and hydroxy fatty acid is protonated and more easily adsorbed by the adsorbent.
- 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, and more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4.
- 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 an adsorbent and sulfuric acid is used as a pH adjuster.
- an SL-containing composition equivalent to an ethanol-soluble content of 30% by mass (SL-containing culture)
- sulfuric acid is added to the treated product) to adjust to pH 3
- activated carbon is added thereto and mixed so as to be 5 to 15% by mass of the total amount.
- 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 50 to less than 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.
- 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.
- ODS resin or the like is preferably used as the filler.
- 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.
- 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.
- 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).
- 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)
- the top of the separation tower for example, an eluent having an ethanol concentration of less than about 70 to 80% (ethanol aqueous solution)
- Equilibrate the column (2)
- the eluent having a concentration of about 70 to less than 80% is supplied from the top of the separation column, and the acid-type SL-containing fraction is selectively eluted and recovered.
- 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 the latter stepwise elution method is used.
- 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)].
- the ethanol aqueous solution of less than 70% can be used for the purpose of removing the odor component, pigment
- 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.
- 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.
- 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 to 10 atmospheres, preferably about 1 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.
- the time which performs an alkali hydrolysis can be suitably set with the number of acetyl groups couple
- 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.
- the enzyme generally used is acetylesterase, and examples thereof include acetylesterase isolated from Aspergillus niger, Rhodococcus sp., Meyerozyma guilliermondii.
- the conditions suitable for the reaction of acetylesterase are pH 5 to 8, preferably 6 to 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.
- the reaction temperature is preferably 20 to 40 ° C, particularly preferably 20 to 35 ° C.
- 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.
- a 0.1M phosphoric acid aqueous solution pH 7.0
- An example is a method of adding acetylesterase to 50 U and stirring at room temperature (25 ° C.) for 1 day.
- 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).
- 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.
- 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.
- hydrolysis treatment a widely known method can be used as long as the effect of the present invention is not hindered.
- 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.
- 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.
- 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 to 10 atmospheres, preferably about 1 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.
- the time which performs alkali hydrolysis can be suitably set with the ratio of lactone type SL contained in SL containing composition to process.
- 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.
- ODS octadecyl silica gel
- the chromatography employed in the present invention is preferably partition chromatography, particularly reverse phase chromatography.
- ODS resin or the like is preferably used as the filler.
- 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.
- 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).
- the pH of the SL-containing liquid to be subjected to chromatography is preferably less than about pH 6 because the pKa value of the acid type SL is pH 6.1 to 6.4. More specifically, the pH is about 1 to less than 6, preferably about pH 1 to 5, and more preferably about pH 1 to 4.5. The pH is particularly preferably about 2 to 4.
- a pH adjuster is usually used for adjusting the SL-containing liquid (SL-containing culture or its treated product) to the acidic region. As the pH adjuster, those described in the above (a) can be used similarly.
- SL-containing composition SL-containing culture or treated product thereof
- 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.
- 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)
- the top of the separation tower for example, an eluent having an ethanol concentration of less than about 70 to 80% (ethanol aqueous solution)
- Equilibrate the column (2)
- the eluent having a concentration of about 70 to less than 80% is supplied from the top of the separation column, and the acid-type SL-containing fraction is selectively eluted and recovered.
- 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 the latter stepwise elution method is used.
- 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)].
- the ethanol aqueous solution of less than 70% can be used for the purpose of removing the odor component, pigment
- the ethanol concentration to be equilibrated in [(1) step] is made to coincide with the ethanol concentration used in [(3 ′) step].
- the (IV-2) fatty acid removal step described above may be carried out independently on the SL-containing culture or its treated product.
- the fatty acid removal step may be a combination of (IV-3) deacetylation step and / or (IV-4) lactone type SL removal step. In this case, the order of these steps is There is no particular limitation as long as the object of the present invention can be achieved.
- step (IV-2) fatty acid removal
- step (eg, (a) to (c)) may be followed by the step (IV-3) deacetylation (eg, (d) to (e)), or conversely (IV-3)
- acetylation step for example, (d) to (e)
- step (IV-2) fatty acid removal step for example, (a) to (c)
- (IV-2) fatty acid removal step after (IV-3) deacetylation step specifically, at least one of (d) hydrolysis treatment and (e) enzyme treatment
- 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.
- (IV-4) a lactone type SL removal step ( For example, after (f) to (g)), (IV-2) fatty acid removal step (eg (a) to (c)) and (IV-3) deacetylation step (eg (d) to (e) )) May be carried out in any combination.
- 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.
- (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.
- a lactone-type SL removal step for example, (f) to (g)
- (IV-2) fatty acid removal steps for example, (a) to (c)
- Preferable combinations include a method in which at least one of (a) solvent extraction method and (c) chromatography is performed after the hydrolysis treatment (f).
- (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.
- 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.
- Reference Production Example 1 Production of SL (Preparation of crudely purified SL-containing composition-1)
- 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, 48% by mass of 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.
- a tabletop centrifuge Westphalia: manufactured by Westphalia Separator AG
- Reference Production Example 2 Preparation of crude purified SL-containing composition-2
- the crude purified SL-containing composition- 1 fractionated in the above-mentioned Reference Production Example 1 was adjusted to pH 14 by adding an aqueous sodium hydroxide solution, 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 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).
- 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).
- 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).
- 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).
- Examples 8 to 10 Production of low-toxic SL-containing composition
- an aqueous sodium hydroxide solution was added to adjust the pH to 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.
- 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.
- 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.
- the hydroxyl value of 1 g of the sample is calculated based on the following formula.
- 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].
- JIS K 3331 JIS K 3331
- 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.
- 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.
- 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.
- the weight was measured after returning to room temperature, 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.
- Each peak detected at a retention time of 20-55 minutes is compared with the retention time of known hydroxy fatty acids and fatty acids (standard) to identify hydroxy fatty acids and fatty acids, and the area of each peak is 20- 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.
- the notation method of the obtained fatty acid describes the number of double bonds with a carbon number after C and then a semicolon. That is, in the case of oleic acid having 18 carbon atoms and one double bond, C18: 1.
- (OH) is added at the end.
- C18: 1 (OH) is obtained.
- 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.
- Equation 7 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.
- 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 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.
- Example 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.
- Test results Test results obtained for the SL-containing compositions (crudely purified SL-containing composition-1, roughly purified SL-containing composition-2, example products 1 to 10) prepared in the reference production examples and examples. Is shown in Table 3.
- the component of the commercially available anionic surfactant A is N-acyl-L-glutamate triethanolamine, the component B is Surfactin Na, and the component C is ⁇ -olefin sulfonate Na.
- HeLa cells (Kurabo) were seeded in a 96-well plate at a concentration of 2 ⁇ 10 4 cells / well, 10% NCS (Newborn Calf Serum: manufactured by Invitrogen), non-essential amino acids, 58 ⁇ g / ml L-glutamic acid, The cells were cultured in Dulbecco's Modified EAGLE MEDIUM medium (manufactured by Nissui Pharmaceutical) containing 60 ⁇ g / ml kanamycin at 37 ° C. and 5% CO 2 for 72 hours. The medium was replaced with the medium containing the test sample.
- Dulbecco's Modified EAGLE MEDIUM medium manufactured by Nissui Pharmaceutical
- FIG. 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.
- the IC 50 of these Example products is 1.3 times or more of the IC 50 (1500 ppm) of the roughly purified SL-containing composition-2 obtained during the production thereof, and this is performed on the roughly purified SL-containing composition-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).
- 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).
- the IC 50 values of the low-toxic SL-containing compositions of the present invention shown in Examples 1 to 10 are much larger than the IC 50 of Tween 20, they are “non-irritating” substances. It is judged that there is.
- 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.
- the IC 50 value of the low-toxic SL-containing composition of the present invention has a ratio (IC 50 / CMC) to CMC of 6.7 to 200 times, 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 higher fatty acid, and higher fatty acids and hydroxy fatty acids are used. Cytotoxicity was measured.
- oleic acid and 12-hydroxystearic acid were each dissolved in 99.5% ethanol so that the concentration was 100,000 ppm, and passed through a GHP membrane filter (0.45 ⁇ m) (manufactured by Nippon Pole). These were diluted with 99.5% ethanol to a concentration of 1,000 ppm to 50,000 ppm. 990 ⁇ l of 10% NCS medium was added to 10 ⁇ l of each of these diluted solutions to prepare a test solution for cytotoxicity (prepared 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.
- IC 50 cell lethal concentration
- the cell lethal concentrations (IC 50 ) of oleic acid and 12-hydroxystearic acid were 300 ppm and 160 ppm, respectively.
- 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).
- the cytotoxicity of the SL-containing composition increases when it is mixed into the low-toxic SL-containing composition of the present invention.
- 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 the equivalent of 1 g of ethanol soluble component: 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.
- SL-containing compositions were measured for critical micelle concentration (CMC) and surface tension lowering ability, and performance as a surfactant (wetting property, solubilizing power, detergency, foaming property) was evaluated.
- CMC critical micelle concentration
- 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 surface tension may increase or decrease. I can't guess.
- 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.
- a low-toxic and low-toxic SL-containing composition can be prepared safely and with a simple method. Since the low-toxic SL-containing composition of the present invention has low toxicity to cells and is hypoallergenic, it can be applied not only to foods and drinks, but also to external products (including wounds) and mucous membranes. It can be suitably applied to medicines or quasi drugs.
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Abstract
Description
(I-1)SL産生酵母培養物に由来する着色成分、酸型SL、脂肪酸及びヒドロキシ脂肪酸を少なくとも含有し、酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量を100質量%とした場合に、それぞれの割合が乾燥重量に換算して下記であることを特徴とする、低毒性SL含有組成物;
(1)酸型SL:94~99.99質量%、
(2)ラクトン型SL:0~2質量%、
(3)脂肪酸及びヒドロキシ脂肪酸の総量:0.01~4質量%。
ラクトン型SL:好ましくは0より多く2質量%以下、より好ましくは0.1~2質量%、さらに好ましくは0.1~1.5質量%、特に好ましくは0.8~1.5質量%、
脂肪酸及びヒドロキシ脂肪酸(総量):好ましくは0.01~2.4質量%、より好ましくは0.01~1.2質量%、さらに好ましくは0.01~0.24質量%。
酸型SL:好ましくは96.1質量%以上、より好ましくは97.9質量%以上、さらに好ましくは99.31質量%以上、
ラクトン型SL:好ましくは0より多く2質量%以下、より好ましくは0.1~2質量%、さらに好ましくは0.1~1.5質量%以下、特に好ましくは0.8~1.5質量%、
脂肪酸及びヒドロキシ脂肪酸(総量):好ましくは0.01~2.4質量%、より好ましくは0.01~1.2質量%、さらに好ましくは0.01~0.24質量%。
(a)蒸発残分:1~100%、
(b)乾燥減量:0~99%、
(c)エタノール可溶分:1~100%。
(II-1)(I-1)~(I-13)のいずれかに記載する低毒性SL含有組成物を有効成分とするアニオン性界面活性剤。
(III-1)SL産生酵母を培養することによって得られるSL含有培養物またはその処理物を、(1)脂肪酸及び/又はヒドロキシ脂肪酸を除去する工程に供することを特徴とする、(I-1)~(I-12)のいずれかに記載する低毒性SL含有組成物の製造方法。
(IV-1)SL産生酵母を培養することによって得られるSL含有培養物またはその処理物を(1)脂肪酸及び/又はヒドロキシ脂肪酸を除去する工程を有する方法に供し、SL含有組成物に含まれる酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量を100質量%とした場合に、それぞれの割合が乾燥重量に換算して下記の範囲にある低毒化SL含有組成物を調製することを特徴とする、SL含有組成物の低毒化方法:
(a)酸型SL:94~99.99質量%、
(b)ラクトン型SL:0~2質量%、
(c)脂肪酸及びヒドロキシ脂肪酸の総量:0.01~4質量%。
ラクトン型SL:好ましくは0より多く2質量%以下、より好ましくは0.1~2質量%、さらに好ましくは0.1~1.5質量%、特に好ましくは0.8~1.5質量%、
脂肪酸及びヒドロキシ脂肪酸(総量):好ましくは0.01~2.4質量%、より好ましくは0.01~1.2質量%、さらに好ましくは0.01~0.24質量%。
(a)酸型SL:好ましくは96.1質量%以上、より好ましくは97.9質量%以上、さらに好ましくは99.31質量%以上、
(b)ラクトン型SL:好ましくは0より多く2質量%以下、より好ましくは0.1~2質量%、さらに好ましくは0.1~1.5質量%、特に好ましくは0.8~1.5質量%、
(c)脂肪酸及びヒドロキシ脂肪酸:好ましくは0.01~2.4質量%、より好ましくは0.01~1.2質量%、さらに好ましくは0.01~0.24質量%。
ソホロリピッド(SL)は、一般的にソホロース又はヒドロキシル基が一部アセチル化したソホロースと、ヒドロキシ脂肪酸とからなる糖脂質である。なお、ソホロースとは、β1→2結合した2分子のブドウ糖からなる糖である。ヒドロキシ脂肪酸とは、ヒドロキシル基を有する脂肪酸である。また、SLは、ヒドロキシ脂肪酸のカルボキシル基が遊離した酸型(下記一般式(1))と、分子内のソホロースが結合したラクトン型(下記一般式(2))とに大別される。ある種の酵母(SL産生酵母)の発酵によって得られるSLは、通常、下記一般式(1)で示されるSLと一般式(2)で示されるSLの混合物であり、脂肪酸鎖長(R3)が異なるもの、ソホロースの6’(R2)及び6”位(R1)がアセチル化あるいはプロトン化されたものなど、30種以上の構造同族体の集合体として得られる。
本発明が対象とする低毒性SL含有組成物は、前述する従来公知のSL組成物とは、少なくとも細胞毒性の点で相違し、下記の特徴を備えている。
(1)酸型SL:94~99.99質量%、
(2)ラクトン型SL:0~2質量%、
(3)脂肪酸及びヒドロキシ脂肪酸の総量:0.01~4質量%。
(1)酸型SLを乾燥物換算で94~99.99質量%の割合で含有する
これは低毒性SL含有組成物中の酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%中に含まれる酸型SLの割合(乾燥物重量)である。これは、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれる酸型SLの割合に相当する。従って、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれる酸型SLの割合は94~99.99質量%であるということができる。当該酸型SLの割合として、好ましくは96.1質量%以上、より好ましくは97.9質量%以上、特に好ましくは99.31質量%以上である。
これは低毒性SL含有組成物に含まれる酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%中に含まれるラクトン型SLの割合(乾燥物重量)である。これは、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれるラクトン型SLの割合に相当する。従って、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれるラクトン型SLの割合は0~2質量%であるということができる。当該ラクトン型SLの割合は、少ないほうが毒性の低いSL含有組成物を取得するうえで好ましいが、0より多く2質量%以下の範囲で或る程度含まれているほうが表面張力低下能が良好であり、界面活性剤としての性能(濡れ性、可溶化力、洗浄力、起泡性)に優れる(試験例5参照)。
上限:好ましくは1.5質量%以下、より好ましくは0.9質量%以下、さらに好ましくは0.45質量%以下、特に好ましくは0.1質量%以下、
下限:好ましくは0質量%。なお、SL産生酵母としてラクトン型SLを産生する酵母を使用する場合には0.01質量%を挙げることができる。
上限:好ましくは2質量%以下、より好ましくは1.5質量%以下、
下限:好ましくは0質量%より多く、より好ましくは0.1質量%以上、さらに好ましくは0.8質量%以上。
これは低毒性SL含有組成物の酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%中に含まれる脂肪酸及びヒドロキシ脂肪酸の割合(乾燥物重量)である。これは、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれる脂肪酸及びヒドロキシ脂肪酸の合計の割合に相当する。従って、低毒性SL含有組成物のエタノール可溶分100質量%中に含まれる脂肪酸及びヒドロキシ脂肪酸の合計の割合は0.01~4質量%であるということができる。
(4)エタノール可溶分1g相当物の水酸基価が460~630mgKOH/gである。
(5)エタノール可溶分が10質量%になるように低毒性SL含有組成物を溶解した水溶液の吸光度(OD440)が0.001~1である。
本発明でいう「水酸基価(mg KOH/g)」とは、エタノール可溶分1gに相当する試料(低毒性SL含有組成物)に含まれる化合物のOH基を完全にアセチル化するために要する水酸化カリウムのmg数であり、これにより当該試料(低毒性SL含有組成物)に含まれるSL中のフリーの水酸基の割合を測定することができる。水酸基価(mg KOH/g)が高ければSL中に含まれるフリーの水酸基の割合が多く(つまりアセチル化されている水酸基が少ない=アセチル基が少ない)、水酸基価(mg KOH/g)が低ければSL中に含まれるフリーの水酸基の割合が低い(つまりアセチル化されている水酸基が多い=アセチル基が多い)という関係にある。
本発明が対象とする低毒性SL含有組成物は白色以外の色に着色している。
(a)蒸発残分:1~100%、
(b)乾燥減量:0~99%、
(c)エタノール可溶分:1~100%。
上記に説明するように本発明の低毒性SL含有組成物は、界面活性作用を有しながらも、細胞毒性及び眼や粘膜に対する刺激性が極めて少なく、実用濃度では実質的に無毒性及び無刺激性といえることから、低毒性且つ低刺激性のアニオン性界面活性剤として使用される他、安全性や低刺激性(無刺激性)が求められる飲食品、医薬品、医薬部外品、香粧品等に好適に用いることができる。また飲食品用、医薬品用、医薬部外品用または香粧品用の添加剤として用いることもできる。
(IV-1)原料(SL含有培養物またはその処理物)
低毒性SL含有組成物の製造の原料として用いるSL含有培養物またはその処理物としては、SL産生酵母の培養物またはその処理物であってSLを含有する粗精製物を広く挙げることができる。SL産生酵母としては公知のものを用いることができ、例えば、前述するキャンディダ(スタメレラ)・ボンビコーラ(Candida bombicola)を好適に挙げることができる。なお、キャンディダ(スタメレラ)・ボンビコーラは生物資源バンクであるATCCに登録されており、そこから入手することができる(Candida bombicola ATCC22214など)。また、本発明の低毒性SL含有組成物の製造には、SL(酸型、ラクトン型)を産生することが知られているキャンディダ属に属する他のSL産生酵母を使用することもできる。かかるSL産生酵母として、例えばキャンディダ・マグノリエ(Candida magnoliae)、キャンディダ・グロペンギッセリ(Candida gropengisseri)、及びキャンディダ・アピコーラ(Candida apicola)、キャンディダ・ペトロフィラム(Candida petrophilum)、キャンディダ・ボゴリエンシス(Candida bogoriensis)、キャンディダ・バチスタエ(Candida batistae)を挙げることができる。これらの酵母は、保存機関から分譲された菌株又はその継代培養によって得られた菌株であってもよい。ここで、ロドトルラ(キャンディダ)・ボゴリエンシス NRCC9862(Rhodotorula(Candida)bogoriensis NRCC9862)が生産するSLは、13-[(2’-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] docosanoic acid6’, 6”-diacetateであり、アルキル基の中央のヒドロキシル基とソホロースがグリコシド結合している。このSLは前記一般式(1)及び(2)とは異なるが、ソホロースとヒドロキシ脂肪酸から構成される点では同じであり、本発明が対象とするSLに含まれる。
本発明の低毒性SL含有組成物は、SL含有培養物またはその処理物を少なくとも(1)脂肪酸及び/又はヒドロキシ脂肪酸を除去する工程(脂肪酸除去工程)に供することで調製することができる。
溶剤抽出法は、脂肪酸及びヒドロキシ脂肪酸がSLよりも疎水性が高いことを利用した分離方法である。一般的にSLを含有する溶液(通常、水)と相溶性のない溶剤を使用して、疎水性化合物である脂肪酸及びヒドロキシ脂肪酸をSL含有液から抽出除去する方法である。溶剤として使用されるものは、SL含有液と相溶性のない溶剤であればよく、特に制限されないものの、例えば酢酸エチル、ジエチルエーテル(エーテル)、及びヘキサンなどが挙げられる。特に酸型SLの回収率と脂肪酸およびヒドロキシ脂肪酸の除去率の高さからジエチルエーテル(エーテル)が好ましい。
吸着法は、使用する吸着剤に対するSL並びに脂肪酸及びヒドロキシ脂肪酸の親和性の差を利用した分離方法である。吸着剤としては、一般的に疎水性化合物を選択的に吸着することができるものを使用することができ、例えば活性炭、シリカゲル、ゼオライト、及びイオン交換樹脂などが挙げられる。また、特開2008-64489号公報に記載されている酸化アルミナも用いることができる。疎水性の高い化合物を吸着するうえで特に好ましいのは活性炭である。なお、イオン交換樹脂としては、強酸性カチオン交換樹脂、弱酸性カチオン交換樹脂、強塩基性アニオン交換樹脂、及び弱塩基性アニオン交換樹脂を挙げることができる。好ましくは強塩基性アニオン交換樹脂、及び弱塩基性アニオン交換樹脂である。
クロマトグラフィーは、両親媒性であるSLの構造を利用した分離方法である。一般的に、固定相として用いられる充填剤(吸着剤)には、当該分野で公知の任意のシリカゲル、オクタデシルシリカゲル(ODS)樹脂、イオン交換樹脂、または合成吸着剤などが用いられる。本発明で採用するクロマトグラフィーは、分配クロマトグラフィー、特に逆相クロマトグラフィーであることが好ましい。当該逆相クロマトグラフィーによると、環境及び人体に対して安全性の高い溶離液(移動相)を使用することができる。
(1)カラム塔最上部(以下、分離塔塔頂)から約70~80%未満濃度の溶離液(例えば、エタノール濃度が約70~80%未満の溶離液(エタノール水溶液))を供給し、カラムを平衡化する。
(2)分離塔塔頂からSL含有組成物(SL含有培養物の処理物)を添加する。
(3)分離塔塔頂から約70~80%未満濃度の前記溶離液を供給し、酸型SL含有画分を選択的に溶出させて回収する。
脱アセチル化方法としては、(d)加水分解処理、及び(e)酵素処理を例示することができる。なお、これらの脱アセチル化方法は、一種単独で行ってもよいし、2種以上を任意に組み合わせて使用することもできる。2以上の処理を併用する場合、処理の順序は順不同であり特に制限はされない。なお、脱アセチル化工程は、SL含有培養物またはその処理物が、アセチル化SLを産生するSL産生酵母によって調製されたものである場合に好適に用いられる処理工程であり、SL含有培養物またはその処理物が、非アセチル化SLを選択的に産生するSL産生酵母(アセチルトランスフェラーゼ欠失SL産生酵母)によって調製されたものである場合に適用されない。
加水分解処理には、本発明の効果を妨げない限り、広く公知の方法を用いることができる。例えば、水酸化物の金属塩(ナトリウム、カリウム、カルシウム及びマグネシウムなど)、炭酸塩、リン酸塩、またはアルカノールアミン等の塩基を用いたアルカリ加水分解を好適に挙げることができる。さらに、加水分解処理には、各種の触媒、例えば、アルコール等を用いることも可能である。前記アルカリ加水分解を行う場合の温度、圧力及び時間は、SL含有培養物またはその処理物に含まれるSLのアセチル基を脱離するという目的及び効果が達成できるものである限り特に制限されないが、目的産物である酸型SLの分解や化学修飾等の副反応を抑制しながら、効率的に脱アセチル化を進行させることのできる温度、圧力及び時間を採用することが好ましい。この点から、反応温度は通常約30℃~120℃の範囲であり、好ましくは約50℃~90℃である。圧力は通常約1気圧~10気圧の範囲であり、好ましくは約1気圧~2気圧である。反応時間は通常約10分~5時間の範囲であり、好ましくは約1時間~3時間である。また、アルカリ加水分解を行う時間は、処理するSL含有組成物中のSLに結合しているアセチル基の数によって適宜設定できる。
酵素を用いてSLに結合しているアセチル基を解離する方法は、アセチルエステルからアルコールと酢酸を生成する酵素を利用した方法である。酵素として利用されるのは、一般的にアセチルエステラーゼであり、例えばAspergillus niger、Rhodococcus sp.、Meyerozyma guilliermondiiから単離されたアセチルエステラーゼが挙げられる。
ラクトン型SL除去方法としては、(f)加水分解処理、及び(g)クロマトグラフィーを例示することができる。これらのラクトン型SL除去方法は、(f)及び(g)のいずれかひとつを単独で行ってもよいし、2つを任意に組み合わせて使用することもできる。2つの処理を併用する場合、処理の順序は順不同であり特に制限はされないが、好ましくは(f)→(g)である。なお、ラクトン型SL除去処理工程は、SL含有培養物またはその処理物が、酸型SLとラクトン型SLの両方を産生するSL産生酵母(ラクトン型/酸型SL産生酵母)によって調製されたものである場合に好適に用いられる処理工程であり、SL含有培養物またはその処理物が、酸型SLを選択的に産生するSL産生酵母(酸型SL産生酵母)によって調製されたものである場合に適用されない。
ここで用いられる加水分解処理は、SL含有培養物またはその処理物に含まれるラクトン型SLのラクトン環を開環して酸型SLに変換する処理である。
ここで用いられるクロマトグラフィーは、SL含有培養物またはその処理物に含まれるラクトン型SLを選択的に除去する処理である。
(1)カラム塔最上部(以下、分離塔塔頂)から約70~80%未満濃度の溶離液(例えば、エタノール濃度が約70~80%未満の溶離液(エタノール水溶液))を供給し、カラムを平衡化する。
(2)分離塔塔頂からSL含有組成物(SL含有培養物の処理物)を添加する。
(3)分離塔塔頂から約70~80%未満濃度の前記溶離液を供給し、酸型SL含有画分を選択的に溶出させて回収する。
前述する製造方法によれば、SL産生酵母から調製とされるSL含有組成物を低毒化及び低刺激性化し、本発明の低毒性SL含有組成物を取得することができる。従って、上記の製造方法は、SL産生酵母の培養により製造されるSL含有組成物の低毒化方法または低刺激性化方法と言い換えることができる。
培養培地として、1L当たり、含水グルコース10g(日本食品化工社製、製品名:日食含水結晶ブドウ糖)、ペプトン10g(オリエンタル酵母社製、製品名:ペプトンCB90M)、酵母エキス5g(アサヒフードアンドヘルスケア社製、製品名:ミーストパウダーN)を含有する液体培地を使用し、30℃で2日間、Candidabombicola ATCC22214を振盪培養し、これを前培養液とした。
前記参考製造例1で分取した粗精製SL含有組成物-1に水酸化ナトリウム水溶液を加えてpH14に調整し、80℃で2時間処理して加水分解(アルカリ加水分解)を行った。次いで、室温に戻してから硫酸(9.8M水溶液)を用いてpH7.5に調整し、発生した不溶物をろ過除去して、ろ液を「粗精製SL含有組成物-2」(参考製造例品2)として得た。
前記参考製造例2で得た粗精製SL含有組成物-2を、硫酸(9.8M水溶液)を用いてpH3.0に調整した。
固定相:C18カラム(コスモシル40C18―PREP、ナカライテスク、15kg)移動相:50%及び70% エタノール水溶液。
前記参考製造例2で得た粗精製SL含有組成物-2を、硫酸(9.8M水溶液)を用いてpH3.0に調整した。
前記参考製造例2で得た粗精製SL含有組成物-2を、硫酸(9.8M水溶液)を用いてpH3.0に調整した。これを、ジエチルエーテル(エーテル)を用いて下記の方法で抽出した(溶剤抽出法)。
実施例4:7mlエーテルを用いた抽出作業を1回実施(→実施例品4)
実施例5:15mlエーテルを用いた抽出作業を1回実施(→実施例品5)
実施例6:15mlエーテルを用いた抽出作業を2回実施(→実施例品6)
実施例7:15mlエーテルを用いた抽出作業を3回実施(→実施例品7)。
前記参考製造例1で分取した粗精製SL含有組成物-1に水酸化ナトリウム水溶液を加えてpH14に調整し、80℃で15分間(実施例8)、80℃で30分間(実施例9)、及び80℃で45分間(実施例10)加熱することで、加水分解(アルカリ加水分解)を行った。次いで、これらを室温に戻してから硫酸(9.8M水溶液)を用いてpH7.5に調整し、発生した不溶物をろ過除去した。さらに、硫酸(9.8M水溶液)を用いてpH3.0に調整し、50mlのスクリューキャップ付きガラス遠沈管にエタノール可溶分が3gになるように加えて、これに蒸留水を添加し、全量が15mlになるように調整した。これにヘキサンを加えて激しく混合したあと、200×gで2分間遠心を行って2層に分離させ、上層のヘキサン層を除去した、(抽出作業)。この抽出作業を5回行った。その後、80℃条件下で放置してヘキサンを除去し、SL含有組成物を得た(実施例品8~10)。
上記の参考製造例1及び2、並びに実施例1~10で調製したSL含有組成物(粗精製SL含有組成物-1、粗精製SL含有組成物-2、実施例品1~10)について、下記の方法に従って、エステル価(mg KOH/g)、水酸基価(mg KOH/g)、エーテル抽出物含量(%)、色相(OD440)、蒸発残分(%)、乾燥減量(%)、エタノール可溶分(%)、及び赤外吸収スペクトル(cm-1)を測定した。また、HPLC分析を行った。
(A)エステル価
けん化価(エタノール可溶分1g相当の試料中の遊離酸の中和及びエステルのけん化に要する水酸化カリウムのmg数:JIS K 3331、日本油化学協会規定の基準油脂分析試験法[2.3.2.1-1996])と酸価(エタノール可溶分1g相当の試料中に含有する遊離酸を中和するのに要する水酸化カリウムのmg数:JIS K 3331、日本油化学協会法の基準油脂分析試験法[2.3.1-1996])との差として求めることができる他、直接測定する方法として、下記の方法を用いることができる。
水酸基価(エタノール可溶分1g相当の試料に含まれる遊離のヒドロキシル基をアセチル化するために必要な酢酸を中和するのに要する水酸化カリウムのmg数:日本油化学協会規定の基準油脂分析試験法[2.3.6.2-1996])から求めることができる。
アセチル化試薬:12.5 gの無水酢酸を100 mlの全量フラスコに入れ、ピリジンを標線まで加え、注意しながら十分に混ぜる。このようにして調製した液は、湿気、二酸化炭素、および酸の蒸気にふれないようにし、褐色ビンに保存する。
試料1gを正確に首長丸底フラスコに投入し、適当量のアセトンを加えて105℃で加温し、水分を除去する。その後、アセチル化試薬を5 ml正しく加え、95~100 ℃に加熱する。1時間加熱した後、フラスコを加熱浴から取って空冷させ、1 mlの蒸留水を加えて混合したあと、フラスコを再度加熱浴に入れ、10分間加熱する。再び引き上げて空冷させ、漏斗の壁に凝縮した液を5 mlの中性エタノールで洗い流しながら加える。このフラスコの内容物にフェノールフタレイン溶液を加え、0.5 N KOH/EtOHで滴定し、下式に基づいて試料1gの水酸基価を算出する。また、下記式3中、酸価は、試料1g中に含有する遊離酸を中和するのに要する水酸化カリウムのmg数であり、日本油化学協会法の基準油脂分析試験法(JIS K 3331)[2.3.1-1996]に従って求めることができる。エタノール可溶分1g相当の試料の水酸基価は、当該試料のエタノール可溶分(%)から、下式4に従って求めることができる。
エーテル抽出物含量は、エタノール可溶分1g相当の試料からエーテルを用いて抽出される物質の量を質量百分率で示したものである。
試料1gを50ml容量のナス型フラスコに投入し、10%水酸化ナトリウム水溶液を10ml加えて冷却管を接続し、80℃で2時間加温を行った。10%塩酸水溶液を10ml加えて中和させ、99.5%エタノールを加えながらエバポレーターで水分を留去した。その後、99.5%エタノールを10ml加えて超音波処理を行いながら分散させ、分散液をガラス漏斗を用いてろ過し、ろ液を50ml容ナス型フラスコに移した。99.5%エタノールでさらに洗いこみを行ったあと、エタノールをエバポレーターで留去させ、 残留物を15mlスクリューキャップ付きガラス遠沈管に移し、硫酸(関東化学製)を用いてpHを3に調整し、全量を蒸留水で5mlに合わせた。5mlのエーテルを加えて激しく混合し、卓上遠心機H-108M2(コクサン製)を用いて1000 rpmで2分間遠心し、2層に分離させた。上層を重量既知の100mlビーカーに移し、新たに5mlのエーテルを加え、合計で3回エーテル抽出作業を行った。エーテル抽出液の入った100mlビーカーを50℃のインキュベーターに投入してエーテルを除去し、さらに105℃のインキュベーターで30分間置き、エーテルを完全に除去した。
(C)エーテル抽出物含量で得られたエーテル抽出物を99.5%エタノールに1%溶液となるように溶解し、下表の条件でHPLC分析を行った。このHPLC分析条件により、酸型SLは8~20分、ヒドロキシ脂肪酸は20~45分、脂肪酸は45~55分のリテンションタイムにそれぞれ検出される。20~55分のリテンションタイムに検出される各ピークを、既知のヒドロキシ脂肪酸および脂肪酸(標準品)のリテンションタイムと対比することでヒドロキシ脂肪酸及び脂肪酸を同定し、且つ個々のピークの面積を20~55分のリテンションタイムに検出されるピークの面積の総和で除することで、エタノール可溶分1gに対する脂肪酸及びヒドロキシ脂肪酸の割合を算出した。つまり、下式10によりそれぞれ個別の含量を算出した。
色相(OD440nm)は、エタノール可溶分が10質量%になるように、被験試料をアルカリ水溶液(2% Na2CO3in 0.1N NaOH)に溶解して調製した水溶液の、波長440nmにおける吸光度を測定することで求めることができる。
蒸発残分(%)は、試料の重量を精密に秤量した後、JIS K0067-1992規定の第2法(熱板上で加熱蒸発する方法)に従って蒸発乾固し、その残分を量り、下式7から求めることができる。
乾燥減量(%)は、試料の重量を精密に秤量した後、JIS K0067-1992規定の第1法(大気圧下で加熱乾燥する方法)に従って加熱乾燥し(105±2℃、2時間)、乾燥後の減量を量り、下式8から求めることができる。
エタノール可溶分は、試料をエタノールで溶解し、エタノールに溶ける物質の量を示したものである。
三角フラスコ及びガラスろ過器の重量を正確に測定する。これらの重量は105℃で2時間以上乾燥後、デシケーター内で放冷してから測定する。三角フラスコに試料約5gを1mg単位まで正確に量り取り、エタノールを試料の100mL添加して、ガラス管を付けて水浴上で30分間加熱し、時々振り混ぜながら溶解する。なお、粉状または粒状試料には95vol%エタノールを使用し、液状又はペースト状試料には99.5vol%のエタノールを使用する。温溶液のままガラスろ過器を用いてろ過し、三角フラスコの残量に再びエタノール50mLを加えて溶解する。温溶液をガラスろ過器を用いてろ過し、熱エタノールで三角フラスコ及びガラスろ過器をよく洗浄する。室温まで放冷し、全量フラスコ250mLにろ液および洗液を移し、エタノールを標線まで加え、この中から、全量ピペットを用いて、100mLずつ質量既知の2個のビーカー200mLに分取する。そのうちの1個を、水浴上で加熱してエタノールを除いた後、105±2℃に調節した乾燥器で1時間乾燥し、デシケーターで放冷後、重量を正確に測定する(乾燥残量)。
強熱残分試験は、被験試料を下記の方法[第1法]で強熱した後に残留する物質の量を測定する方法である。通常、有機物中に不純物として含まれる無機物の含量を知る目的で行われるが、場合によっては、有機物中に構成成分として含まれる無機物又は揮発性無機物中に含まれる不純物の量を測定するために行なわれる。例えば、本発明において「強熱残分0.1%以下(第1法、1g)」と規定したものは、被験試料約1gを精密に量り、下記第1法の操作法によって強熱したとき、その残分が被験試料の採取量の0.10%以下であることを示す。
[第1法]るつぼの上で試料を硫酸少量で潤し、徐々に加熱してなるべく低温でほとんど灰化又は揮散させた後、硫酸で潤し、完全に灰化し、恒量になるまで強熱(450~550℃)する。これをデシケーター(シリカゲル)中で放冷した後、質量を精密に量る。得られた測定値(残分)とあらかじめ測定しておいた採取量から、下式9により強熱残分(%)を算出する。
赤外吸収スペクトルの測定には、液体試料は105±2℃で3時間加熱乾燥固化したものを使用し、固体試料はそのまま使用した。赤外吸収スペクトルは、フーリエ変換赤外分光分析装置SpectrumTM100(パーキンエルマージャパン製)を使用し、ATR法で分析した。
参考製造例及び実施例で調製したSL含有組成物(粗精製SL含有組成物-1、粗精製SL含有組成物-2、実施例品1~10)について得られた試験結果を表3に示す。
上記の参考製造例2、及び実施例1~10で得られたSL含有組成物(粗精製SL含有組成物-2、実施例品1~10)について、臨界ミセル濃度(CMC)及び表面張力低下能を測定した。また比較対照として、市販のアニオン界面活性剤(A:「アミノソフトLT-12」(30%):味の素(株)製、B:「サーファクチンNa」(100%):和光純薬工業(株)製、C:「リポランLJ-441」(37%):ライオン(株)製)、Tween 20(Polyoxyethylene Sorbitan Monolaurate (20 E.O.))及びSLS(Sodium Lauryl Sulfate)についても同様に臨界ミセル濃度(CMC)及び表面張力低下能を測定した。なお、市販のアニオン界面活性剤Aの成分はN-アシル-L-グルタミン酸トリエタノールアミン、Bの成分はサーファクチンNa、Cの成分はα-オレフィンスルホン酸Naである。
臨界ミセル濃度(CMC)及び表面張力低下能の測定はWilhelmy法に準拠して測定した。自動表面張力計 CBVP-Z型一式(協和界面科学株式会社製)を使用し、20℃、pH7の条件で測定を行った。なお、pH調整には、水酸化ナトリウム水溶液または塩酸水溶液を使用した。
各被験試料の臨界ミセル濃度(CMC)及び表面張力低下能の測定結果を、表4に示す。
上記の参考製造例2、及び実施例1~10で得られたSL含有組成物(粗精製SL含有組成物-2、実施例品1~10)について、Hela細胞を用いて細胞毒性試験を行った。また比較対照として、前述する市販アニオン界面活性剤A~C、並びにTween 20及びSLSについても同様に細胞毒性試験を行った。
HeLa細胞(クラボウ)を96ウェルプレートに2×104cells/wellの濃度で播種し、10%NCS(Newborn Calf Serum:invitrogen製)、非必須アミノ酸、58μg/mlL-グルタミン酸、60μg/mlカナマイシンを含んだDulbecco’s Modified EAGLE MEDIUM培地(日水製薬製)で37℃、5%CO2下で72時間培養した。被験試料を含んだ培地に交換し、48時間後、1mg/ml MTT(3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)入り培地に交換した。2時間処理し、イソプロパノールで色素であるホルマザンを抽出し、波長570nmの吸光度を測定した。細胞生存率%は下式から求めた。
上記のMTTアッセイから細胞生存率を算出し、得られた細胞生存率から細胞致死濃度(IC50)を求めた。
SL含有組成物に多く含まれている高級脂肪酸としてオレイン酸を使用し、またヒドロキシ脂肪酸として12-ヒドロキシステアリン酸を使用して、高級脂肪酸及びヒドロキシ脂肪酸の細胞毒性を測定した。
下記の構成を有する高純度SL含有組成物に、ラクトン型SLを添加して、酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%とした場合のラクトン型SLの割合が0~2質量%(エタノール可溶分1g相当物のエステル価:0~2mgKOH/g)の範囲になるように調製した(実施例品11~15)。
(1)酸型SL、ラクトン型SL、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%あたり、酸型SL99.95質量%、ラクトン型0質量%、脂肪酸及びヒドロキシ脂肪酸(総量)0.05質量%
(2)エタノール可溶分が10質量%になるように溶解した水溶液の波長440nmにおける吸光度(OD440):0.08
(3)エタノール可溶分1g相当物の水酸基価:596mgKOH
(4)Hela細胞に対する細胞致死濃度(IC50):63000ppm。
以下に本願発明の低毒性SL含有組成物を含む医薬品及び医薬部外品の処方を示す。
1-メントール 0.02
d-カンフル 0.001
d-ボルネオール 0.005
ユーカリ油 0.01
ミント油 0.002
塩酸ナファゾリン 0.0015
メチル硫酸ネオスチグミン 0.004
マレイン酸クロルフェニラミン 0.03
塩酸ビリドキシン 0.08
酢酸トコフェロール 0.04
L-アスパラギン酸マグネシウム・カリウム 1.4
アミノエチルスルホン酸 0.8
ホウ酸 0.5
ホウ砂 0.1
濃塩化ベンザルコニウム液50(日本薬局方) 0.015
クロロブタノール 0.2
低毒性SL含有組成物(実施例1~10) 0.3
塩酸/水酸化ナトリウム 適量
精製水 残量
合 計 100.00
処方例2 洗眼薬 (pH5.8)
1-メントール 0.01
イプシロンアミノカプロン酸 0.1
グリチルリチン酸二カリウム 0.1
硫酸亜鉛 0.05
マレイン酸クロルフェニラミン 0.003
L-アスパラギン酸カリウム 0.1
塩化カルシウム 0.05
ホウ酸 1.5
ホウ砂 0.015
濃塩化ベンザルコニウム液50(日本薬局方) 0.004
クロロブタノール 0.2
低毒性SL含有組成物(実施例1~10) 0.2
エタノール 0.1
塩酸/水酸化ナトリウム 0.01
精製水 残量
合 計 100.00
処方例3 コンタクトレンズ装着液(pH7.0)
1-メントール 0.015
アミノエチルスルホン酸 0.5
塩化カリウム 0.08
塩化ナトリウム 0.15
ホウ酸 0.9
ホウ砂 0.2
ソルビン酸カリウム 0.1
エデト酸ナトリウム(日本薬局方) 0.05
低毒性SL含有組成物(実施例1~10) 0.5
塩酸/水酸化ナトリウム 適量
精製水 残量
合計 100.0
処方例4 コンタクトレンズ装着液(pH7.5)
1-メントール 0.01
塩化カリウム 0.05
塩化ナトリウム 1
リン酸水素ナトリウム 0.15
リン酸二水素ナトリウム 0.01
低毒性SL含有組成物(実施例1~10) 0.02
20%ポリヘキサメチレンビグアニド液 0.0005
(商品名:コスモシルCQ)
塩酸/水酸化ナトリウム 適量
精製水 残量
合 計 100.0
処方例5 創傷洗浄剤(pH6.5)
塩化ナトリウム 0.9
低毒性SL含有組成物(実施例1~10) 0.05
塩酸/水酸化ナトリウム 適量
精製水 残量
合 計 100.0
Claims (15)
- SL産生酵母培養物に由来する着色成分、酸型ソホロリピッド、脂肪酸及びヒドロキシ脂肪酸を少なくとも含有し、酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量を100質量%とした場合に、それぞれの割合が乾燥重量に換算して下記であることを特徴とする、低毒性ソホロリピッド含有組成物;
(1)酸型ソホロリピッド:94~99.99質量%、
(2)ラクトン型ソホロリピッド:0~2質量%、
(3)脂肪酸及びヒドロキシ脂肪酸の総量:0.01~4質量%。 - 酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%あたりに含まれるラクトン型ソホロリピッドの割合、並びに脂肪酸及びヒドロキシ脂肪酸(総量)の割合が、それぞれ下記(i)並びに(ii)のいずれか少なくとも一方を充足する、請求項1記載の低毒性ソホロリピッド含有組成物:
(i)ラクトン型ソホロリピッドの割合:0より多く2質量%以下、
(ii)脂肪酸及びヒドロキシ脂肪酸(総量)の割合:0.01~2.4質量%。 - 酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%あたりに含まれるラクトン型ソホロリピッドの割合が0.1~1.5質量%である、請求項1または2に記載する低毒性SL含有組成物。
- エタノール可溶分が10質量%になるように低毒性SL含有組成物を溶解した水溶液の波長440nmにおける吸光度(OD440)が0.001~1である、請求項1~3のいずれかに記載する低毒性SL含有組成物。
- エタノール可溶分1g相当物の水酸基価が460~630mgKOH/gである、請求項1~4のいずれかに記載する低毒性SL含有組成物。
- HeLa細胞に対する細胞致死濃度(IC50)が2000~60000ppmであることを特徴とする、請求項1~5のいずれかに記載する低毒性SL含有組成物。
- HeLa細胞に対する細胞致死濃度(IC50)と臨界ミセル濃度(CMC)との比(IC50/CMC)が6.7~200であることを特徴とする、請求項1~6のいずれかに記載する低毒性SL含有組成物。
- 請求項1~7のいずれかに記載する低毒性ソホロリピッド含有組成物を有効成分とするアニオン性界面活性剤。
- 請求項1~7のいずれかに記載する低毒性ソホロリピッド含有組成物を含有することを特徴とする香粧品、飲食品、医薬部外品、医薬品またはこれらの添加物。
- 上記香粧品、飲食品、医薬部外品または医薬品が、粘膜、傷口または炎症部に適用されるものである、請求項9に記載する香粧品、飲食品、医薬部外品、医薬品またはこれらの添加物。
- ソホロリピッド産生酵母を培養することによって得られるソホロリピッド含有培養物またはその処理物を、(1)脂肪酸及び/又はヒドロキシ脂肪酸を除去する工程に供することを特徴とする、請求項1~7のいずれかに記載する低毒性SL含有組成物の製造方法。
- ソホロリピッド産生微生物を培養することによって得られるソホロリピッド含有培養物またはその処理物を、さらに(2)ソホロリピッドに結合したアセチル基を脱離する工程、及び(3)ラクトン型ソホロリピッドを除去する工程の少なくとも1方の工程に供することを特徴とする、請求項11に記載する製造方法。
- ソホロリピッド産生酵母を培養することによって得られるソホロリピッド含有培養物またはその処理物を(1)脂肪酸及び/又はヒドロキシ脂肪酸を除去する工程を有する方法に供し、ソホロリピッド含有組成物に含まれる酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量を100質量%とした場合に、それぞれの割合が乾燥重量に換算して下記の範囲にある低毒化ソホロリピッド含有組成物を調製することを特徴とする、ソホロリピッド含有組成物の低毒化方法:
(a)酸型ソホロリピッド:94~99.99質量%、
(b)ラクトン型ソホロリピッド:0~2質量%、
(c)脂肪酸及びヒドロキシ脂肪酸の総量:0.01~4質量%。 - 酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%あたりに含まれるラクトン型ソホロリピッドの割合、並びに脂肪酸及びヒドロキシ脂肪酸(総量)の割合が、それぞれ下記(i)並びに(ii)のいずれか少なくとも一方の範囲にある低毒化SL含有組成物を調製することを特徴とする、請求項13に記載する低毒化方法:
ラクトン型ソホロリピッドの割合:0より多く2質量%以下、
脂肪酸及びヒドロキシ脂肪酸(総量)の割合:0.01~2.4質量%。 - 酸型ソホロリピッド、ラクトン型ソホロリピッド、並びに脂肪酸及びヒドロキシ脂肪酸の総量100質量%あたりに含まれるラクトン型ソホロリピッドの割合が0.1~1.5質量%である低毒化ソホロリピッド含有組成物を調製することを特徴とする、請求項13または14に記載する低毒化方法。
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JPWO2015034007A1 (ja) | 2017-03-02 |
CN105683329B (zh) | 2018-04-06 |
EP3042940A1 (en) | 2016-07-13 |
EP3042940A4 (en) | 2017-03-29 |
US20160324747A1 (en) | 2016-11-10 |
CN105683329A (zh) | 2016-06-15 |
US10688031B2 (en) | 2020-06-23 |
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JP2020015917A (ja) | 2020-01-30 |
JP6644548B2 (ja) | 2020-02-12 |
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