JP7168921B2 - Fatty acid composition, method for producing the same, external skin preparation, quasi-drug, and cosmetic containing the fatty acid composition - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel fatty acid composition and a method for producing the same, and further to external skin preparations, quasi-drugs and cosmetics containing the fatty acid composition.

According to the guidelines of the Japanese Dermatological Association, atopic dermatitis is accompanied by physiological skin abnormalities such as skin dryness and barrier dysfunction caused by abnormalities of the epidermis, especially the stratum corneum, and various non-specific irritation reactions and specific skin irritations. It is considered to be one of the diseases of the eczema/dermatitis group whose pathology is chronic inflammation and pruritus associated with allergic reactions. The prevalence is particularly high in developed countries, and it is said that 15-30% of children and 5% of adults are affected.

Atopic dermatitis is caused by atopic predisposition (family history, history of allergic diseases, tendency to produce IgE antibodies, etc.), imbalance of epidermal flora, nutritional factors (high linoleic acid edible oil and It develops due to multiple factors such as food used as material, etc.) and environmental factors (dust, stress, irregular life, etc.).
In recent years, it has been reported that Staphylococcus aureus is frequently detected in the skin of atopic dermatitis patients, and that Staphylococcus aureus dramatically increases in the inflamed areas of atopic dermatitis patients (Non-Patent Document 1, 2) It has been suggested that overgrowth of Staphylococcus aureus on the epidermis may be the cause of exacerbation of atopic dermatitis. It is believed that this is because protein A, delta toxin, V8 protease, etc. produced by Staphylococcus aureus are involved in aggravation of inflammation.

Therefore, patients with atopic dermatitis are also treated with antibiotics, such as external application of mupirocin, fusidic acid, etc., and oral administration of dicloxacillin, cephalexin, erythromycin, etc.
However, antibiotics such as those mentioned above inhibit the growth of Staphylococcus aureus, which is a resident skin bacterium, and have an inhibitory effect on exacerbation of inflammation caused by secretions of Staphylococcus aureus. It is highly likely that even the staphylococcus epidermidis (Staphylococcus epidermidis) is killed, and the imbalance of the indigenous epidermal flora is not improved.

On the other hand, it has been reported that the content of sapienic acid ((Z)-6-hexadecenoic acid) in the sebum of atopic dermatitis patients is reduced to about 1/10 compared to healthy subjects (Non-Patent Document 3) It is reported that palmitoleic acid ((Z)-9-hexadecenoic acid), which is an isomer of sapienic acid and has been eaten, has antibacterial activity against Staphylococcus aureus (Patent Document 1). Thus, the use of palmitoleic acid in external preparations for skin has been investigated.
However, among vegetable oils that have been eaten and have been confirmed to be safe, there are few that contain a large amount of palmitoleic acid. Its cultivation area is limited, and it cannot be said that it is suitable as a practical supply source of palmitoleic acid.
In addition, including seaberry pulp oil, vegetable oils such as linoleic acid, linoleic acid, etc., have two or more double bonds and have poor oxidation stability, polyunsaturated fatty acids (two double bonds Since the fatty acid composition obtained from the vegetable oil contains a considerable amount of the unsaturated fatty acids described above, the fatty acid composition obtained from the vegetable oil has a problem of oxidation stability, and is not preferable for use in external skin preparations and the like.

In addition, it was reported that 41.2% to 52.9% of workers working in intensive care units (ICUs) of hospitals who repeatedly washed their hands and disinfected with alcohol had Staphylococcus aureus on their hands. (Non-Patent Documents 4 and 5).
Therefore, Staphylococcus aureus also proliferates on the skin of workers in food factories, etc., and those who are engaged in housework at home, who frequently wash their hands and disinfect their hands repeatedly, and have rough skin such as rough hands. A possibility is suggested.
Therefore, not only to prevent aggravation of symptoms of atopic dermatitis or to improve symptoms, but also to prevent or improve rough skin caused by skin cleaning and disinfection, abnormal growth of Staphylococcus aureus There is a great need to prevent or suppress, maintain and improve the normal balance of the normal flora of the epidermis.

WO2011/011486

S. Higaki et al.; International J. Dermatology 38 265 (1999) H. H. Kong et al.; Genome Research 22 850-859 (2012) H. Takigawa et al.; Dermatology 211 240 (2005) M. Rosenthal et al.; Pathogens 3 1-13 (2014) P. Horn et al.; Scand. J. Clin. Lab. Invest. 67 165-177 (2007)

Therefore, the present invention provides a selective antibacterial agent that contains a high content of palmitoleic acid and inhibits Staphylococcus aureus but does not inhibit Staphylococcus epidermidis, that is, an antibacterial agent that maintains the balance of the epidermal resident flora. The purpose was to provide a fatty acid composition useful as

The present inventors, as a result of intensive studies to solve the above problems, found that from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions, lipase at the sn-1 and sn-3 positions By liberating the fatty acid and then recovering the liberated fatty acid, the inventors succeeded in obtaining a fatty acid composition containing 55 mol % or more of palmitoleic acid, thereby completing the present invention.

That is, the present invention relates to the following.
[1] A fatty acid composition containing 55 mol% or more of palmitoleic acid.
[2] The composition according to [1], wherein the molar ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) is 2.4 or more.
[3] The composition according to [1], wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) is 3 or more in terms of molar ratio.
[4] The composition according to any one of [1] to [3], which does not substantially contain polyunsaturated fatty acids.
[5] A selective antibacterial agent against Staphylococcus aureus, containing the composition according to any one of [1] to [4].
[6] An external skin preparation containing the composition according to any one of [1] to [4].
[7] A quasi-drug containing the composition according to any one of [1] to [4].
[8] Cosmetics containing the composition according to any one of [1] to [4].
[9] (1) a step of liberating fatty acids at the sn-1 and sn-3 positions with a lipase from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions, and (2) the above A method for producing a fatty acid composition containing 55 mol% or more of palmitoleic acid, comprising a step of recovering fatty acids liberated in the step.
[10] Fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions have a ratio of palmitoleic acid to oleic acid in the entire triglyceride (palmitoleic acid / oleic acid), and The production method according to [9], wherein the oil has a higher content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) at the sn-3 position.
[11] The production method according to [9] or [10], wherein the oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions is produced by Saccharomyces cerevisiae. .
[12] The production method of [11], wherein Saccharomyces cerevisiae is a transformant strain overexpressing an N-terminal deletion gene of diacylglycerol acyltransferase.
[13] The production method according to any one of [9] to [12], wherein the lipase is a lipase that preferentially liberates fatty acids at the sn-1 and sn-3 positions of triglycerides.
[14] The lipase that preferentially releases fatty acids at the sn-1 and sn-3 positions of triglycerides is a lipase derived from a microorganism belonging to the genus Pseudozyma or a lipase derived from a microorganism belonging to the genus Alcaligenes. , the manufacturing method according to [13].
[15] The production of [14], wherein the step of liberating fatty acids at the sn-1 and sn-3 positions with a lipase derived from a microorganism belonging to the genus Pseudozyma is carried out in the presence of an excess amount of ethanol. Method.
[16] The production method according to any one of [9] to [12], wherein the lipase is a lipase that specifically releases fatty acids at the sn-1 and sn-3 positions of triglycerides.
[17] The production method of [16], wherein the lipase that specifically releases fatty acids at the sn-1 and sn-3 positions of triglycerides is a lipase derived from a microorganism belonging to the genus Rhizopus.
[18] The production method according to any one of [9] to [12], wherein the lipase is a lipase that preferentially releases palmitoleic acid over oleic acid.
[19] The production method of [18], wherein the lipase that preferentially releases palmitoleic acid over oleic acid is a lipase derived from a microorganism belonging to the genus Candida.

INDUSTRIAL APPLICABILITY According to the present invention, a fatty acid composition containing palmitoleic acid having selective antibacterial activity against Staphylococcus aureus at an unprecedentedly high concentration can be obtained.
The fatty acid composition of the present invention is a selective antibacterial agent that inhibits the growth of Staphylococcus aureus but does not inhibit the growth of Staphylococcus epidermidis. It can be suitably used as an external skin preparation, quasi-drug or cosmetic for preventing exacerbation of symptoms of dermatitis or improving symptoms of atopic dermatitis.
In addition, the fatty acid composition of the present invention can be used not only for atopic dermatitis, but also for external skin preparations and quasi-drugs for preventing or improving rough skin such as rough hands caused by washing, disinfecting, etc. of the skin during housework and work. Alternatively, it can be suitably used as cosmetics.

FIG. 2 is a diagram showing the examination results of Test Example 3 on the effective amount of a fatty acid composition in an external preparation for skin.

The present invention provides a fatty acid composition containing 55 mol% or more of palmitoleic acid relative to the total amount of the composition (hereinafter also referred to as "fatty acid composition of the present invention").

Palmitoleic acid contained in the fatty acid composition of the present invention is a C16 unsaturated fatty acid ((Z)-9-hexadecenoic acid) having one double bond at the 9-position.
The fatty acid composition of the present invention preferably contains 58 mol% or more, more preferably 60 mol% or more, of palmitoleic acid relative to the total amount of the composition.
Considering the efficiency of the production method using lipase, which will be described later, the content of palmitoleic acid in the fatty acid composition of the present invention is usually 70 mol % or less.

In addition to palmitoleic acid, the fatty acid composition of the present invention may contain saturated or unsaturated fatty acids having about 10 to 20 carbon atoms, but from the viewpoint of selective antibacterial activity against Staphylococcus aureus Since oleic acid suppresses the antibacterial activity of palmitoleic acid, it is desirable that the content of oleic acid ((Z)-9-octadecenoic acid) is small, and the content ratio of palmitoleic acid and oleic acid ( The molar ratio of palmitoleic acid/oleic acid) is preferably 2.4 or more, more preferably 3 or more.
Furthermore, the fatty acid composition of the present invention is unstable against oxygen, and linoleic acid ((9Z, 12Z)- 9,12-octadecadienoic acid), α-linolenic acid (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid), γ-linolenic acid (6Z,9Z,12Z)-6,9, 12-octadecatrienoic acid) and other polyunsaturated fatty acids are preferably not substantially contained.
Here, "substantially free of polyunsaturated fatty acids" means that the polyunsaturated fatty acids are not detected by a normal analysis method for the polyunsaturated fatty acids, such as a quantification method using gas chromatography. That is, it is below the detection limit (0.1 mol %).

As described above, the fatty acid composition of the present invention contains 55 mol% or more of palmitoleic acid having a melting point of -1°C, and has a low content of long-chain saturated fatty acids, so that it usually exhibits a liquid state at 20°C.

The fatty acid composition of the present invention liberates the fatty acids at the sn-1 and sn-3 positions by lipase from oils and fats containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions, and the released fatty acids are It can be suitably obtained by collection. In addition, from the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) in the entire triglyceride, the content ratio of palmitoleic acid and oleic acid at the sn-1 and sn-3 positions (palmitoleic acid /oleic acid) is more preferable.

The above-mentioned triglyceride-containing fats and oils are fats and oils obtained from plants, microorganisms such as yeast, etc., and are preferably fats and oils that have been eaten, and are fats and oils produced by Saccharomyces cerevisiae. is more preferable.
As Saccharomyces cerevisiae, it is preferable to use a transformant strain with high lipid-producing ability. Examples of such a transformant include a transformant overexpressing the N-terminal deletion gene of diacylglycerol acyltransferase (DGAT) as described in JP-A-2015-146778, wherein sn-1 Those having the ability to produce lipids with high triglyceride content having palmitoleic acid at the and sn-3 positions can be preferably used. Furthermore, a transformant in which the DGAT N-terminal deleted gene is overexpressed in a DGAT gene (DGA1)-disrupted strain of Saccharomyces cerevisiae is more preferable. In particular, the N-terminal 2
DGAT lacking 9 residues is preferably used as a transformant overexpressed in Saccharomyces cerevisiae in which the function of the ESA1 gene encoding intracellular histone acetyltransferase is reduced, such as a DGA1 gene disruption strain. be done.
In addition, the preparation, culture, etc. of the transformant overexpressing the N-terminal deleted gene of DGAT are described in Kanzaka et al. It can be carried out according to the method described in the publication.

In order to obtain the fatty acid composition of the present invention, a lipase that preferentially releases fatty acids at the sn-1 and sn-3 positions of triglycerides, or fatty acids at the sn-1 and sn-3 positions of triglycerides It is preferred to use a lipase that specifically releases the
Examples of lipases that preferentially release fatty acids at the sn-1 and sn-3 positions of triglycerides include microorganisms belonging to the genus Pseudozyma such as Pseudozyma antarctica, and microorganisms belonging to the genus Alcaligenes. , Psudomonas, and Burkholderia, among others. A lipase derived from a microorganism belonging to the genus Alcaligenes is more preferred.
Lipases that specifically release fatty acids at the sn-1 and sn-3 positions of triglycerides include microorganisms belonging to the genus Rhizopus such as Rhizopus japonicus and Rhizopus oryzae, and Rhizomucor mihei ( Preferred examples include lipases derived from microorganisms belonging to the genus Rhizomucor such as Rhizomucor miehei, microorganisms belonging to the genus Thermomyces such as Thermomyces lanuginosus, and pancreatic lipase. Among them, lipases derived from microorganisms belonging to the genus Rhizopus are more preferable.

In the present invention, a lipase that preferentially releases palmitoleic acid over oleic acid can also be used.
Preferred examples of such lipases include lipases derived from microorganisms belonging to the genus Candida, such as Candida rugosa, and microorganisms belonging to the genus Geotrichum, such as Geotrichum candidum. , lipases derived from microorganisms belonging to the genus Candida are more preferred.

The lipase described above may be a free lipase, or may be immobilized by being bound to a carrier such as a resin or encapsulated in microcapsules or liposomes.
In the present invention, commercially available lipases provided by various companies can be used.

The reaction in which fatty acids at the sn-1 and sn-3 positions are liberated by lipase from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions varies depending on the type of lipase used. It can be set appropriately.
The amount of lipase to be added is usually 0.01 to 10 parts by mass, preferably 0.03 to 1.5 parts by mass, per 100 parts by mass of triglyceride as a substrate. In the case of a free enzyme, the reaction is an aqueous system, and the amount of water added to the triglyceride is preferably 0.01% to 99% by mass, more preferably 5% to 95% by mass. . For immobilized enzymes, water may or may not be added.
The enzymatic reaction is carried out using a solvent such as water or hexane as necessary, usually at 5° C. to 60° C., preferably 20° C. to 40° C., pH=3 to 9, preferably pH=5 to 7. .5 to 18 hours. If the reaction time is long, liberation of fatty acids may proceed at all of the sn-1 to sn-3 positions, or transacylation may occur. Therefore, the reaction time is preferably about 2 to 3 hours. In order to stop the hydrolysis reaction by lipase, it is preferable to perform deactivation by heating, deactivation by short-chain alcohol such as ethanol or methanol, oil-water separation by standing or centrifugation, or the like. In the case of immobilized enzymes, it is only necessary to remove the immobilized enzymes by standing or filtering. Unreacted triglycerides are removed by adding an alkaline agent such as potassium hydroxide to the reaction solution to saponify free fatty acids and transferring them to the aqueous layer, followed by extraction with a non-polar organic solvent such as n-hexane. can do.
When a lipase derived from a microorganism belonging to the genus Pseudozyma is used as the lipase, reaction is performed in the presence of an excess amount of about 500 parts by mass of ethanol with respect to 100 parts by mass of triglyceride as a substrate. , sn-1 and sn-3 positions, and thus are preferred. In such a case, since the fatty acids at the sn-1 and sn-3 positions are liberated as ethyl esters, the free fatty acids are recovered after saponification by a conventional method.

The fatty acid liberated by the lipase is (1) separated by column chromatography using a silica gel column or the like, and (2) an alkaline agent such as potassium hydroxide is added to the reaction solution to saponify the free fatty acid and transfer it to the aqueous layer. After that, unreacted triglycerides are removed by extraction with a low-polarity organic solvent such as n-hexane, and the aqueous layer is acidified by adding an acid such as hydrochloric acid, and then with a low-polarity organic solvent such as n-hexane. It can be recovered by a usual method such as a method of extracting and evaporating the solvent to remove it, and (3) a method of removing water by oil-water separation or the like and then recovering the free fatty acid by distillation.

The fatty acid composition of the present invention has a high content of palmitoleic acid having selective antibacterial activity against Staphylococcus aureus (55 mol% or more based on the total amount of the fatty acid composition), and the above selection of palmitoleic acid Staphylococcus aureus, which is involved in the exacerbation of atopic dermatitis due to its low content of oleic acid, which inhibits its antibacterial activity
However, it does not suppress the growth of Staphylococcus epidermidis, which is a resident bacterium of healthy skin, and there is little risk of reducing Staphylococcus epidermidis.
Therefore, the fatty acid composition of the present invention is useful as a selective antibacterial agent against Staphylococcus aureus, maintains or improves the normal flora of the skin in a healthy state, and is effective in treating atopic dermatitis. or as a topical skin preparation, quasi-drugs, or cosmetics that are effective in preventing aggravation of the symptoms of atopic dermatitis, or as a result of cleaning or disinfecting the skin during housework or work. It can be preferably used as an external skin preparation, a quasi-drug, a cosmetic, etc. that are effective in preventing or improving rough skin.

Accordingly, the present invention provides a selective antibacterial agent against Staphylococcus aureus (hereinafter also referred to as "antibacterial agent of the present invention") containing the fatty acid composition of the present invention.
The antibacterial agent of the present invention can be obtained by adding general additives used in the field of formulation, if necessary, to the fatty acid composition of the present invention, and by formulating means well known in the field of formulation, such as the 17th revision. It can be prepared by the method described in the Japanese Pharmacopoeia General Rules for Preparations [3] Preparations, etc., and is oily; liquids such as suspensions and emulsions; it can be in the form of solids such as powder, granules, tablets, and capsules.
For purposes of the present invention, the antibacterial agent of the present invention is preferably in a form that can be externally applied to the skin.

Examples of the above additives include excipients, binders, disintegrants, lubricants, coating agents, bases, solvents, emulsifiers, dispersants, suspending agents, stabilizers, thickeners, pH adjusters, Antioxidants, preservatives, preservatives, corrigents, sweeteners, flavoring agents, coloring agents and the like can be mentioned.

Excipients include anhydrous silicic acid, calcium silicate, magnesium aluminosilicate, starch (corn starch, potato starch, wheat starch, etc.), sugars (glucose, lactose, sucrose, etc.), sugar alcohols (sorbitol, maltitol). , mannitol, etc.).

Binders include gelatin, sodium caseinate, starch (hydroxypropyl starch, pregelatinized starch, partially pregelatinized starch, etc.), cellulose and derivatives thereof (crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, etc.).

Examples of disintegrants include povidone, crospovidone, cellulose and derivatives thereof (crystalline cellulose, methylcellulose, etc.).

Lubricants include talc, synthetic aluminum silicate, magnesium silicate, calcium stearate, magnesium stearate and the like.

Coating agents include methacrylic acid copolymers (methacrylic acid/ethyl acrylate copolymer, etc.), methacrylate copolymers (ethyl acrylate/methyl methacrylate copolymer, ethyl acrylate/methyl methacrylate/methacrylate trimethylammonium ethyl copolymer, etc.).

Examples of bases include hydrocarbons (liquid paraffin, etc.), polyethylene glycols (Macrogol 400, Macrogol 1500, etc.) and the like.

Examples of solvents include purified water, monohydric alcohols (ethanol etc.), polyhydric alcohols (propylene glycol, glycerin etc.) and the like.

Emulsifiers include nonionic surfactants (sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid ester, etc.), anionic surfactants (sodium alkyl sulfate, N-acylglutamate, etc.). ), refined soybean lecithin, and the like.

Dispersants include gum arabic, propylene glycol alginate, nonionic surfactants (polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene polyoxypropylene glycol, etc.), anionic surfactants ( sodium alkyl sulfate, etc.).

Suspending agents include sodium alginate, nonionic surfactants (polyoxyethylene lauryl ether, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, propylene glycol fatty acid ester, etc.).

Stabilizers include adipic acid, ethylenediaminetetraacetate (calcium disodium salt, disodium salt, etc.), α-cyclodextrin, β-cyclodextrin and the like.

Examples of thickening agents include water-soluble polymers (sodium polyacrylate, carboxyvinyl polymer, etc.), polysaccharides (sodium alginate, xanthan gum, tragacanth, etc.), and the like.

Examples of pH adjusters include hydrochloric acid, phosphoric acid, acetic acid, citric acid, lactic acid, sodium hydroxide, sodium hydrogen phosphate and the like.

Antioxidants include dibutylhydroxytoluene (BHT), butylhydroxyanisole (BHA), dl-α-tocopherol, erythorbic acid, propyl gallate and the like.

Examples of antiseptics include sodium benzoate, sorbic acid, potassium sorbate, dehydroacetic acid, sodium dehydroacetate, p-hydroxybenzoic acid esters (methyl p-hydroxybenzoate, etc.) and the like.
Examples of preservatives include benzoic acid, sodium benzoate, sorbitol, p-hydroxybenzoic acid esters (methyl p-hydroxybenzoate, etc.), propylene glycol, and the like.

Examples of flavoring agents include ascorbic acid, erythritol, disodium 5'-guanylate, citric acid, sodium L-glutamate, tartaric acid, and DL-malic acid.
Sweeteners include aspartame, licorice extract, saccharin and the like.

Fragrances include orange essence, l-menthol, d-borneol, vanillin, linalool and the like.

Coloring agents include tar pigments (food red No. 2, food blue No. 1, food yellow No. 4, etc.), inorganic pigments (red iron oxide, yellow ferric oxide, titanium oxide, etc.), natural pigments (annatto pigment, turmeric pigment, carotenoid etc.).

One or more of the above additives may be used as necessary.

The content of the fatty acid composition of the present invention in the antibacterial agent of the present invention is usually 0.001% by mass to 10% by mass, and 0.005 as the content of palmitoleic acid with respect to the total amount of the antibacterial agent of the present invention. It is preferably from 5% by mass to 5% by mass.

The application amount of the antibacterial agent of the present invention depends on the type, sex, age, skin condition of the subject to which the antibacterial agent of the present invention is applied (hereinafter also referred to as "application subject" in the present specification), the antibacterial agent of the present invention However, when the subject of application is an adult human and external application is applied, the dose of palmitoleic acid is usually 0.02 μg/cm ² to 200 μg/cm ² per application. and preferably 0.1 μg/cm ² to 100 μg/cm ² .
The above amounts can be applied once to several times a day.
The period of application of the antibacterial agent of the present invention is appropriately determined depending on the skin condition observed in the subject (the state of balance of the resident skin flora) and the like, but it is usually 1 to 30 days, preferably 3 days. days to 15 days.
Since the antibacterial agent of the present invention contains palmitoleic acid obtained from lipids such as yeast whose safety has been confirmed as an active ingredient, it is highly safe and suitable for continuous application.

Animals to which the antibacterial agent of the present invention is applied (hereinafter also referred to as "target animals" in this specification) include mammals (humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, cows, , horses, donkeys, pigs, sheep, etc.). When the antibacterial agent of the present invention is applied to target animals other than humans, the amount of application of the antibacterial agent of the present invention may be appropriately set according to the type, sex, weight, skin condition, etc. of the target animal.

The antibacterial agent of the present invention improves the imbalance of the skin flora to a normal state, or maintains the balance of the skin flora to a normal state, thereby preventing the symptoms of atopic dermatitis from worsening. It is effective for prevention or amelioration of symptoms of atopic dermatitis, and can be suitably applied to patients with atopic dermatitis or animals exhibiting symptoms of atopic dermatitis.
In addition, the antibacterial agent of the present invention is used not only for patients with atopic dermatitis, but also for those who frequently wash, disinfect, etc. their skin for housework or work, and who may exhibit rough skin symptoms such as rough hands, or who have rough skin symptoms. It can also be suitably applied to prevent or improve symptoms of rough skin in those who exhibit symptoms.

The present invention also provides an external preparation for skin containing the fatty acid composition of the present invention (hereinafter also referred to as "the external preparation for skin of the present invention").
Here, “external preparations for skin” refer to pharmaceuticals that are externally applied to lesions on the skin, but also include transdermal absorption-type preparations that aim to deliver active ingredients to the circulating bloodstream through the skin. be
The external preparation for skin of the present invention is prepared by adding general additives used in the production of external preparations for skin to the fatty acid composition of the present invention, if necessary, and solid preparations for external use such as powders for external use; liniments, lotions; Liquid preparations for external use such as agents (solutions, emulsions, suspensions, etc.); Sprays such as external aerosols and pump sprays; Ointments such as oleaginous ointments and water-soluble ointments; Oil-in-water or in-oil It can be provided in dosage forms such as water-type creams; gels such as aqueous gels and oily gels; patches such as tapes and poultices.
The topical preparation for skin of the present invention is described in general methods for manufacturing topical preparations for skin, for example, in the 17th revision of the Japanese Pharmacopoeia General Rules for Formulations [3] Articles for preparations, Section "11. It can be manufactured according to the method described.

The skin preparation for external use of the present invention contains the above excipients, binders, disintegrants, lubricants, bases, solvents, emulsifiers, dispersants, suspending agents, stabilizers, thickening agents, and pH adjusters. One or more of agents, antioxidants, preservatives, preservatives, fragrances, colorants, etc. can be used.

In addition, the topical skin preparation of the present invention includes non-steroidal anti-inflammatory drugs such as ibuprofen piconal, suprofen, bufexamac, bendazac, ufenamate and glycyrrhetinic acid; Cortical steroids; antibiotics such as sodium fusidate and mupirocin; immunoregulatory topical agents such as tacrolimus hydrate and the like can be contained.

The content of the fatty acid composition of the present invention in the external preparation for skin of the present invention is the same as the content described above for the antibacterial agent of the present invention.

The external preparation for skin of the present invention can be suitably applied to patients with atopic dermatitis or animals exhibiting symptoms of atopic dermatitis to prevent exacerbation of inflammation or improve symptoms.
In addition, the topical preparation for skin of the present invention can be applied to people who are likely to develop symptoms of rough skin such as rough skin caused by washing, disinfecting, etc. of the skin, or to those who exhibit rough skin symptoms, in order to prevent or improve rough skin symptoms. It can be suitably applied.
The daily application amount and application period of the topical skin preparation of the present invention depend on the type, sex, age, degree of skin symptoms, dosage form of the topical skin preparation of the present invention, etc. to which the topical skin preparation of the present invention is applied. Determined as appropriate. For example, in the case of human adults, the amount of palmitoleic acid can be applied in an amount similar to the amount described above for the antibacterial agent of the present invention, and the number and period of times described above for the antibacterial agent of the present invention. can be applied in

Furthermore, the present invention provides a quasi-drug containing the fatty acid composition of the present invention, especially a quasi-drug for skin external use (hereinafter also referred to as "the quasi-drug of the present invention" in this specification) or a cosmetic (hereinafter referred to as Also referred to herein as "cosmetics of the present invention").
Here, "quasi-drugs" refer to drugs that have a milder effect on the human body than drugs, but have some improvement effect. It is called "foreign product". So-called medicinal cosmetics are included in skin external quasi-drugs.
The term "cosmetics" refers to those applied to the skin, etc., for the purpose of cleaning the body and making the appearance beautiful, and having a mild action.
The quasi-drugs or cosmetics of the present invention can be produced according to the external preparation for skin of the present invention described above, and can be in the form of lotions, beauty essences, emulsions, creams, facial cleansers, packs, body cleansers, and the like. can be provided in

The quasi-drugs or cosmetics of the present invention may contain polyhydric alcohols (glycerin, 1,3-butylene glycol, dipropylene glycol, etc.), amino acids or salts thereof (alanine, serine, proline, sodium DL-pyrrolidonecarboxylate, etc.), proteins (whey, water-soluble collagen, hydrolyzed elastin, etc.), nucleic acids (sodium deoxyribonucleic acid, etc.), mucopolysaccharides (sodium chondroitin sulfate, sodium hyaluronate, etc.), heparin-like substances, Moisturizers such as plant extracts (Angelica keiskei koidz. extract, cucumber extract, white birch extract, etc.); allantoin, allantoing glycyrrhetinic acid, etc.), vitamins ((ascorbyl/tocopheryl) potassium phosphate, panthenol, etc.), plant extracts (chamomile extract, licorice extract, aloe vera extract, etc.), glycyrrhizic acid and its salts (glycyrrhizic acid) , ammonium glycyrrhizinate, dipotassium glycyrrhizinate, etc.), glycyrrhetinic acid and its derivatives (glycyrrhetinic acid, glyceryl glycyrrhetinate, pyridoxine glycyrrhetinate, disodium succinyl glycyrrhetinate, etc.). can.

The content of the fatty acid composition of the present invention in the quasi-drugs or cosmetics of the present invention can be appropriately determined according to the skin preparation for external use of the present invention. It is preferable to apply the fatty acid composition of the present invention so as to achieve the above daily dose for the antibacterial agent of the present invention.

The quasi-drugs or cosmetics of the present invention are used to prevent deterioration of the skin condition of patients with atopic dermatitis, to improve the skin condition of patients with atopic dermatitis, or to relieve rough skin symptoms such as rough hands. It can be preferably used to prevent or improve rough skin symptoms in those who are likely to develop symptoms or who exhibit rough skin symptoms.
In addition, the quasi-drugs or cosmetics of the present invention are mainly used for human skin, especially for humans with atopic predisposition, or for humans with rough skin symptoms caused by washing, disinfecting, etc. of the skin. It is preferably used to maintain balance in a normal state.
In particular, the quasi-drugs or cosmetics of the present invention are highly safe because they contain as an active ingredient palmitoleic acid obtained from lipids such as yeast whose safety has been confirmed. It can be applied continuously over an extended period of time.

Furthermore, the present invention provides a method for producing the above-described fatty acid composition of the present invention (hereinafter also referred to as "the production method of the present invention" in the present specification).
The production method of the present invention includes (1) a step of liberating fatty acids at the sn-1 and sn-3 positions with lipase from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions, and (2) including a step of recovering the fatty acid liberated in the above step;

Fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions that can be used in the present invention, and types of lipases, etc. In the present invention, for liberating fatty acids at the sn-1 and sn-3 positions The conditions for the enzymatic reaction of (1), the method for recovering the liberated fatty acid, and the like are as described above for the fatty acid composition of the present invention.

In each step of the production method of the present invention, purification means such as recrystallization and various types of chromatography, solid-liquid separation means such as filtration and centrifugation, etc., are appropriately and optionally used within the range not impairing the characteristics of the present invention. can be used.

Further, the present invention will be described in detail with reference to examples.

[Reference Example 1] Preparation of Purified Yeast Triglycerides Oils and fats produced by transformants of Saccharomyces cerevisiae were collected by the method described in Kanzaka et al. (JP-A-2015-146778). The solvent remaining in the fat (5 g) was removed by an evaporator, and 3.57 g of the desolvated fat was subjected to silica gel chromatography (2. 6 cm×26 cm) and eluted with 40 mL×15 of the solvent and 40 mL×15 of hexane:ethyl acetate=95:5 (volume ratio). Fraction No. containing triacylglycerides. Fractions 15 to 25 were collected and the solvent was removed by an evaporator to obtain 2.74 g of purified yeast triglyceride of Reference Example 1.

Regarding the purified yeast triglyceride of Reference Example 1, the fatty acid composition at the sn-1, 3 and sn-2 positions was determined according to the method of Y. Watanabe et al. (J. Oleo Sci., 64, 1193 (2015)) as follows. It was measured.
To 100 mg of the purified yeast triglyceride of Reference Example 1, 1 g of ethanol and 0.044 g of immobilized lipase derived from Pseudozyma antarctica ("Novozyme 435" (Novozumes)) were added, and 30
The mixture was allowed to react for 3 hours with reciprocating shaking at ℃, filtered, and the supernatant was recovered. Next, the solvent was removed with an evaporator, and the total amount was equilibrated with n-hexane: diethyl ether = 8: 2 (volume ratio) Sep-Pack silica cartridge ("WAT05190
0" (Waters)), and fatty acid ethyl esters and diglycerides were eluted and removed with 30 mL of the solvent. Then, the monoglyceride having one acyl group at the sn-2 position (sn-2 monoglyceride) was eluted with 10 mL of diethyl ether, and the solvent was removed with a rotary evaporator.

To 3 mL of methanol, 5 μL each of the triglyceride of Reference Example 1 and the sn-2 monoglyceride derived above, and a methanol solution of 28% by mass of sodium methoxide were added and heated at 75° C. for 15 minutes to effect methyl esterification of the fatty acid. gone. After allowing to cool, 0.5 mL of n-hexane was added and mixed, then 3 mL of water was added and mixed, fatty acid methyl ester was extracted with n-hexane, and the n-hexane layer was recovered.
The recovered n-hexane layer was analyzed with a capillary gas chromatograph (“Agilent 6890N” (Agilent Technologies) under the following conditions to quantify fatty acids.

<Analysis conditions>
Capillary column: DB-23 (0.25 mm x 30 m) (Agilent Technologies)
Inlet temperature: 245°C
Injection volume: 3 μL
Detector: Flame ionization detector (FID) (250°C)
Column temperature:
(1) Hold at 150°C for 0.5 minutes (2) 150°C to 170°C; temperature rise at 4°C/min (3) 170°C to 195°C; temperature rise at 5°C/min (4) 195°C Up to 215°C; temperature rise at 10°C/min (5) Hold at 215°C for 11 minutes

From the fatty acid composition (mol%) of the purified yeast triglyceride (composition of all fatty acids at the sn-1,2,3 positions) and sn-2 monoglyceride of Reference Example 1, the fatty acid composition (mol%) at the sn-1,3 positions was determined. Calculated. The results are shown in Table 1.

As shown in Table 1, it was found that the purified yeast triglycerides of Reference Example 1 derived from the transformant of Saccharomyces cerevisiae contained more palmitoleic acid than conventional vegetable oils and fats. On the other hand, it was found that the polyunsaturated fatty acid, which is poor in oxidation stability and undesirable from the viewpoint of formulation stability, is hardly contained.
In addition, as shown in Table 1, in the purified yeast triglyceride of Reference Example 1, palmitolein It can be seen that the acid content is high and the oleic acid content at the sn-1,3 positions is low, that is, the content ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) is high.
That is, the purified yeast triglycerides of Reference Example 1 contain 20 mol% or more of palmitoleic acid, but seaberry oil containing about 10 mol% of linoleic acid, and macadamia containing 2 mol% to 3 mol% of linoleic acid and linoleic acid, respectively. Compared to nut oils, it is useful as a starting material for making the fatty acid compositions of the present invention.

[Example 1] Preparation of fatty acid composition using immobilized lipase derived from Pseudozyma antarctica 0.088 g was added, and the reaction was allowed to proceed for 2.5 hours with reciprocating shaking at 30° C., followed by filtration to recover the supernatant. Next, the solvent was removed with an evaporator, and Sep-Pack silica cartridges ("WAT0519
00" (Waters), and the fatty acid ethyl ester was eluted with 10 mL of the solvent. Furthermore, the diglyceride was eluted and removed with 30 mL of the solvent. Next, sn-2 monoglyceride was eluted with 10 mL of diethyl ether, and the solvent was removed with an evaporator.
The fatty acid ethyl esters obtained by column fractionation twice were mixed, and after desolvation (estimated 130 mg), 3 g of ethanol and 0.2 g of 5M sodium hydroxide were added and saponified by heating at 60° C. for 15 minutes. After cooling to room temperature, 4 mL of water was added, and 2M hydrochloric acid was added until the mixture became acidic, followed by extraction with 3 mL of n-hexane twice. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered (fatty acid composition of Example 1).
On the other hand, the sn-2 monoglycerides obtained from two column fractionations were mixed, and after desolvation (estimated 50 mg), 3 g of ethanol, 0.1 mL of water and 0.1 g of 5 M sodium hydroxide were added, and the mixture was stirred at 60° C. for 15 minutes. Heated for 1 minute to saponify. After cooling to room temperature, 4 mL of water was added, and 2M hydrochloric acid was added until the mixture became acidic, followed by extraction with 3 mL of n-hexane twice. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered to obtain a fatty acid composition of Comparative Example 1.
In addition, when the above reaction using Novozume 435 is carried out overnight, all the fatty acids at the sn-1, 2, and 3 positions of the triglyceride are converted into ethyl esters. The composition can be considered to be similar to the fatty acid composition in the purified yeast triglycerides of Reference Example 1 used as the starting material. Therefore, for comparison, a fatty acid composition was prepared after reacting overnight as follows.
To 200 mg of the purified yeast triglyceride of Reference Example 1, 2 g of ethanol and 0.088 g of Novozyme 435 (Novozumes) were added, reacted at 30° C. for 24 hours with reciprocating shaking, filtered, and filtered. was collected and the solvent was removed by an evaporator. Next, 6 g of ethanol and 0.4 g of 5M sodium hydroxide were added to the obtained ethyl ester (approximately 200 mg), and the mixture was saponified by heating at 60° C. for 15 minutes. After cooling to room temperature, 10 mL of water was added, and 2M hydrochloric acid was added until the mixture became acidic, followed by extraction with 10 mL of n-hexane twice. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered (24-hour reaction product).

[Example 2] Preparation of fatty acid composition using lipase derived from Rhizopus japonicus To 200 mg of purified yeast triglyceride of Reference Example 1, 1.8 mL of water and lipase derived from Rhizopus japonicus ("Rhizopus japonicus) -10D" (Nagase ChemteX Corporation)) (25 mg/mL) (0.053 mL) was added, and the reaction was allowed to proceed for 2.5 hours while stirring at 350 rpm at 35°C, and then 10 mL of ethanol was added to stop the reaction. , 50 mM potassium hydroxide to determine the percent hydrolysis.
Next, 10 mL of water and 1 mL of 5M potassium hydroxide were added to the solution after titration, and the solution was extracted three times with 15 mL of n-hexane to remove unreacted triglycerides. Then, 2M hydrochloric acid was added to the aqueous layer until it became acidic, and the aqueous layer was extracted twice with 15 mL of n-hexane. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered (fatty acid composition of Example 2).
For comparison, a fatty acid composition was prepared by allowing the above lipase to react overnight as follows.
To 200 mg of the purified yeast triglyceride of Reference Example 1, 1.53 mL of water and 0.27 mL of lipase A-10D (Nagase ChemteX Corporation) (25 mg/mL) were added.
After reacting for 24 hours while stirring at 350 rpm at 0 C, the same operation as above was performed to recover free fatty acid (24-hour reaction product).

[Example 3] Preparation of fatty acid composition using Candida rugosa-derived lipase ” (Meito Sangyo Co., Ltd.)) (10 mg / mL) 0.011 mL was added and reacted for 2.5 hours while stirring at 350 rpm at 35 ° C., and then 10 mL of ethanol was added to stop the reaction. Percent hydrolysis was determined by titration of free fatty acids with potassium hydroxide.
Next, 10 mL of water and 1 mL of 5M potassium hydroxide were added to the solution after titration, and the solution was extracted three times with 15 mL of n-hexane to remove unreacted triglycerides. Then, 2M hydrochloric acid was added to the aqueous layer until it became acidic, and the aqueous layer was extracted twice with 15 mL of n-hexane. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered (fatty acid composition of Example 3).
For comparison, a fatty acid composition was prepared by allowing the above lipase to react overnight as follows.
To 100 mg of the purified yeast triglyceride of Reference Example 1, 0.84 mL of water and 0.06 mL of Lipase OF (Meito Sangyo Co., Ltd.) (10 mg/mL) were added and reacted at 35°C for 24 hours while stirring at 350 rpm. , and the same operation as above was performed to recover the free fatty acid (24-hour reaction product).

[Example 4] Preparation of a fatty acid composition using a lipase derived from a microorganism belonging to the genus Alcaligenes To 200 mg of the purified yeast triglyceride of Reference Example 1, 1.97 mL of water and a lipase derived from a microorganism belonging to the genus Alcaligenes ("Lipase QLM" (Meito Sangyo Co., Ltd.)) (50 mg / mL) 0.03 mL was added and reacted for 2.5 hours while stirring at 350 rpm at 35 ° C., and then 10 mL of ethanol was added to react. Percent hydrolysis was determined by stopping and titrating free fatty acids with 50 mM potassium hydroxide.
Next, 10 mL of water and 1 mL of 5M potassium hydroxide were added to the solution after titration, and the solution was extracted three times with 15 mL of n-hexane to remove unreacted triglycerides. Then, 2M hydrochloric acid was added to the aqueous layer until it became acidic, and the aqueous layer was extracted twice with 15 mL of n-hexane. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered (fatty acid composition of Example 4).
For comparison, a fatty acid composition was prepared by allowing the above lipase to react overnight as follows.
After adding 0.6 mL of water and 0.4 mL of Lipase QLM (Meito Sangyo Co., Ltd.) (50 mg/mL) to 100 mg of the purified yeast triglyceride of Reference Example 1, and reacting for 24 hours while stirring at 350 rpm at 35°C. , and the same operation as above was performed to recover the free fatty acid (24-hour reaction product).

[Comparative Example 2] Preparation of Fatty Acid Composition by Saponification To 50 mg of the purified yeast triglyceride of Reference Example 1, 3 g of ethanol, 0.1 mL of water and 0.1 g of 5M sodium hydroxide were added and saponified by heating at 60° C. for 15 minutes. . Then cool to room temperature, add 4 mL of water, add 2M hydrochloric acid until acidic, and add 3 mL of n-hexane.
Extracted twice with L. The hexane layer was recovered, the solvent was removed by an evaporator, and the free fatty acid was recovered to obtain a fatty acid composition of Comparative Example 2.

[Test Example 1] Analysis of fatty acid composition For each fatty acid composition of Examples 1 to 4 and a fatty acid composition (24-hour reaction product) obtained by reacting for 24 hours in each example for comparison, fatty acid Compositional analysis was performed.
To 3 mL of methanol, 5 μL of each of the above samples and a methanol solution of 14% by mass of boron trifluoride-methanol complex were added, and the mixture was heated at 75° C. for 15 minutes to carry out methyl esterification of fatty acids. After allowing to cool, 0.5 mL of n-hexane was added and mixed, then 3 mL of water was added and mixed, fatty acid methyl ester was extracted with n-hexane, and the n-hexane layer was recovered.
The recovered n-hexane layer was analyzed with a capillary gas chromatograph ("Agilent 6890N" (Agilent Technologies) under the same conditions as the analysis of the fatty acid composition of the purified yeast triglycerides in Reference Example 1 above. was quantified.
Table 2 shows the analysis results of the fatty acid composition.

Table 3 shows the hydrolysis rates during the preparation of the fatty acid compositions of Examples 2-4. When the fatty acid composition of Example 1 was prepared (when immobilized lipase derived from Pseudozyma antarctica was used), the hydrolysis rate could not be determined by titration. Estimated hydrolysis rates are given.

As shown in Table 2, in the fatty acid composition of Example 1, compared to the purified yeast triglyceride of Reference Example 1, the palmitoleic acid content was improved and the oleic acid content was decreased, and palmitoleic acid and oleic acid content ratio (palmitoleic acid/oleic acid) increased from 1.62 to 3.52 in terms of molar ratio. In addition, the fatty acid composition of the fatty acid composition of Example 1 is close to the fatty acid composition of the sn-1,3 positions of the refined yeast triglyceride of Reference Example 1, and in the presence of an excess amount of ethanol, Pseudozyma antarctica )-derived immobilized lipase preferentially released palmitoleic acid bound to the sn-1,3 positions of triglycerides.
Moreover, as shown in Table 3, in the preparation of the fatty acid composition of Example 1, the hydrolysis rate after 2.5 hours of reaction is estimated to be about 67%. That is, it was suggested that the method using immobilized lipase derived from Pseudozyma antarctica requires a step of saponifying the produced fatty acid ethyl ester, but is a method with high yield.

The fatty acid composition of Example 2 was obtained by a lipase derived from Rhizopus japonicus that specifically acts on the sn-1,3 positions of triglycerides, so as shown in Table 2, the fatty acid of Example 1 The palmitoleic acid content is higher and the oleic acid content is lower than the composition, and the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) is also as high as 3.99 in terms of molar ratio. was taken.
However, as shown in Table 3, in the preparation of the fatty acid composition of Example 2, the hydrolysis rate after 2.5 hours of reaction was slightly low, and the yield tended to be slightly low. This is because in the reaction by lipase derived from Rhizopus japonicus, both hydrolysis and esterification reactions occur, and triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids are equilibrated in a fixed ratio. This is because there is a tendency to
In addition, although the lipase derived from Rhizopus japonicus is specific for the sn-1 and 3 positions, the longer the reaction time, the more the sn-2 and sn-1 or sn-3 positions of the diglyceride or monoglyceride. Since non-enzymatic acyl group transfer occurs between them and the fatty acid at the sn-2 position is slowly released, the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) after reacting for 24 hours is , decreased compared to after 2.5 hours of reaction.

The fatty acid composition of Example 3 acts evenly on the sn-1,2,3 positions of triglycerides, but with a lipase from Candida rugosa that acts better on palmitoleic acid than on oleic acid. In Table 2, the palmitoleic acid content was shown to be higher than the compositions of other examples, since the lipase was prepared and the lipase is less sensitive to saturated fatty acids.
Moreover, from Table 3, it was confirmed that hydrolysis of triglyceride proceeded almost completely when the reaction time was set to 24 hours in the preparation of Example 3.

The fatty acid composition of Example 4 acts evenly on the sn-1,2,3 positions of triglycerides, or acts preferentially on the sn-1,3 positions, but does not exhibit fatty acid specificity. Alcaligenes) was prepared using a lipase derived from microorganisms belonging to the genus, Table 2
As shown in , the palmitoleic acid content is slightly improved from the purified yeast triglyceride of Reference Example 1, but the degree is slightly lower than each of the fatty acid compositions of Examples 1-3.
From Table 3, when the reaction time was 24 hours in the preparation of the fatty acid composition of Example 4, it was found that the hydrolysis of triglyceride proceeded almost completely.

[Test Example 2] Evaluation of antibacterial activity Each fatty acid composition of Examples 1 to 4 and a fatty acid composition (24-hour reaction product) obtained by reacting for 24 hours in each example for comparison, and comparison For each of the fatty acid compositions of Examples 1 and 2, the minimum inhibitory concentration (MIC) was measured according to the standard method of the Japanese Society of Chemotherapy, the microdilution method, to evaluate the antibacterial activity.
(1) test strain suspension
Staphylococcus aureus NBRC13276 strain and Staphylococcus epidermidis NBRC100911 strain were each isolated from N. B. Medium (0.5% by mass bonito extract (Wako Pure Chemical Industries, Ltd.), 1% by mass polypeptone (Nihon Pharmaceutical Co., Ltd.), 0.5% by mass sodium chloride, pH = 7.0) 1 platinum loop planted in 3 mL Inoculated and pre-incubated overnight at 37°C with shaking. This pre-preculture solution was added to fresh N.C. B. 4 mL of medium was inoculated and pre-incubated for 3 hours at 37° C. with shaking. From the measured turbidity (OD ₆₆₀ ) at 660 nm, the number of viable bacteria at OD ₆₆₀ = 1 was calculated as 6.4 × 10 ⁸ colony forming units (cfu)/mL. The culture medium was 2.0×10 ⁴ cf
N.u/mL. B. A test strain suspension was prepared by dilution with medium (pH=6.0).
(2) Test samples Each fatty acid composition of Examples 1 to 4 and a fatty acid composition (24-hour reaction product) obtained by reacting for 24 hours in each example for comparison, and Comparative Example 1, 2 was dissolved in dimethyl sulfoxide (DMSO) to 10,000 μg/mL to prepare each test sample.
As controls, palmitoleic acid and oleic acid (Tokyo Kasei Kogyo Co., Ltd.) as reagents were similarly evaluated for antibacterial activity.
(3) Measurement of MIC Dispense 234 μL of the test strain suspension (2.0×10 ⁴ cfu/mL) into the 2nd column of a 96-well round-bottom microplate, and 130 μL each into the 3rd to 11th columns. did. Then, 26 μL of the test sample (10,000 μg/mL) was added to the second row of the microplate, well suspended by pipetting, and diluted 10-fold. Next, 130 μL of this suspension was added to the third column of the microplate, suspended, and serially diluted by 2-fold.
After standing and culturing the microplate at 37 ° C. for 2 days, the growth of the test strain was determined by visually confirming the turbidity of the culture solution or the presence or absence of precipitated cells, and the growth of the test strain was observed. The lowest concentration of the sample tested at which no scavenging occurred was taken as the MIC.

Table 4 shows the evaluation results of the antibacterial activity.

As shown in Table 4, palmitoleic acid selectively inhibited the growth of Staphylococcus aureus NBRC13276 strain at low concentrations, but oleic acid did not exhibit effective antibacterial activity against Staphylococcus aureus NBRC13276 strain.
The palmitoleic acid content is high, the oleic acid content is low, and the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) is improved compared to the refined yeast triglyceride of Reference Example 1. The fatty acid composition was found to have good antibacterial activity against Staphylococcus aureus NBRC13276 strain. On the other hand, it was found to have no effect on the growth of the Staphylococcus epidermidis NBRC100911 strain, which is a resident skin bacterium.
On the other hand, the fatty acid composition of Comparative Example 1, which is derived from sn-2 monoglyceride and has a high oleic acid content, does not exhibit antibacterial activity against the Staphylococcus aureus NBRC13276 strain.
In the fatty acid composition of Comparative Example 2, which is a saponified product of purified triglycerides and contains palmitoleic acid and oleic acid, a decrease in antibacterial activity was observed compared to the fatty acid composition of each example. antibacterial activity against Staphylococcus aureus NBRC13276 strain.
In addition, the fatty acid composition of Example 4, which has a palmitoleic acid content slightly lower than that of the other examples, has a selective antibacterial activity against the Staphylococcus aureus NBRC13276 strain, although it is lower than that of the other examples. rice field.
The 24-hour reaction product compositions of Examples 1, 3, and 4, in which triglyceride hydrolysis proceeded almost completely, had palmitoleic acid content and oleic acid content comparable to those of the fatty acid composition of Comparative Example 2. 4, no antibacterial activity against Staphylococcus aureus NBRC13276 strain was observed.

[Test Example 3] Examination of the effective amount of the fatty acid composition in the external skin preparation by the co-cultivation method (1) Test bacteria N. B. Agar medium (0.1% by mass bonito extract (Wako Pure Chemical Industries, Ltd.), 1% by mass polypeptone (Nihon Pharmaceutical Co., Ltd.), 7.5% by mass sodium chloride, 2% by mass agar, pH = 6.0) Staphylococcus aureus NBRC13276 strain and Staphylococcus epidermidis NBRC100911 strain were each applied to the same plate at 3,000 cfu/cm ² .
(2) Samples As a model of external preparations for skin, N.O. B. Liquid medium (0.5% by mass bonito extract, 1% by mass polypeptone, 0.5% by mass sodium chloride, 0.4% by mass thickening agent (guar gum), pH = 6.0), the fatty acid of Example 1 The composition was suspended to 0.5% by weight and then serially diluted in 2-fold increments to 0.001% by weight.
In addition, what did not add the fatty acid composition of Example 1 was made into the control (control).
(3) Calculation of the Effective Amount of Fatty Acid Composition N. spp. to which the above two types of test bacteria were applied so that the above sample and control were 2 mg/cm ² . B. It was applied on an agar medium. After culturing at 37° C. for 2 days, grown bacteria were collected with a cotton swab, suspended in 7.5% by mass saline, diluted appropriately, and applied to a mannitol salt agar medium (AS ONE Corporation) containing egg yolk. After culturing at 37°C for 1 day, the Staphylococcus aureus NBRC13276 strain and the Staphylococcus epidermidis NBRC100911 strain were discriminated based on the shape and color of the grown colonies and whether the egg yolk reaction was positive or negative.
(3) Results The results of the above study are shown in FIG. The content of the fatty acid composition of Example 1 in the sample that is the model of the external preparation for skin is 0.0313% by mass or more, and Staphylococcus aureus NBRC13276
The growth of the strains was completely suppressed, and only the Staphylococcus epidermidis NBRC100911 strain was observed to grow.
When a skin external preparation model sample containing 0.0313% by mass of a fatty acid composition was applied to the skin at 2 mg/cm ² , the applied amount of the fatty acid composition on the skin was 0.625 μg/cm ² , and palmitolein The amount of acid applied is 0.37 μg/cm ² .
Therefore, it was suggested that the growth of Staphylococcus aureus could be inhibited at a dose of 0.37 μg/cm ² of palmitoleic acid when the external preparation for skin of the present invention was applied.

As detailed above, according to the present invention, it is possible to obtain a fatty acid composition containing palmitoleic acid having selective antibacterial activity against Staphylococcus aureus at an unprecedentedly high concentration.
The fatty acid composition of the present invention is effective as a selective antibacterial agent against Staphylococcus aureus, especially for prevention of exacerbation of symptoms of atopic dermatitis or improvement of symptoms of atopic dermatitis. It can be used as a quasi-product or cosmetic.
Furthermore, the fatty acid composition of the present invention is an external skin preparation, quasi-drug, or cosmetic that is effective in preventing or improving rough skin caused by washing, disinfecting, etc. of the skin reported to contain Staphylococcus aureus. can be used as

Claims (16)

Staphylococcus aureus containing a fatty acid composition containing 55 mol% or more of palmitoleic acid and having a content ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) of 2.4 or more in molar ratio ) selective antibacterial agents. 2. The antibacterial agent according to claim 1, wherein the molar ratio of palmitoleic acid to oleic acid in the fatty acid composition (palmitoleic acid/oleic acid) is 3 or more. 3. The antimicrobial agent according to claim 1 or 2, wherein the fatty acid composition is substantially free of polyunsaturated fatty acids. An external skin preparation comprising a fatty acid composition containing 55 mol % or more of palmitoleic acid and having a molar ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) of 2.4 or more. 5. The external preparation for skin according to claim 4, wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) in the fatty acid composition is 3 or more in terms of molar ratio. 6. The external preparation for skin according to claim 4 or 5, wherein the fatty acid composition does not substantially contain polyunsaturated fatty acids. A quasi-drug containing a fatty acid composition containing 55 mol % or more of palmitoleic acid and having a molar ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) of 2.4 or more. 8. The quasi-drug according to claim 7, wherein the molar ratio of palmitoleic acid to oleic acid in the fatty acid composition (palmitoleic acid/oleic acid) is 3 or more. The quasi-drug according to claim 7 or 8, wherein the fatty acid composition does not substantially contain polyunsaturated fatty acids. A cosmetic comprising a fatty acid composition containing 55 mol % or more of palmitoleic acid and having a molar ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) of 2.4 or more. 11. The cosmetic product according to claim 10, wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) in the fatty acid composition is 3 or more in terms of molar ratio. 12. Cosmetic product according to claim 10 or 11, wherein the fatty acid composition is substantially free of polyunsaturated fatty acids. A method for producing a fatty acid composition containing 55 mol% or more of palmitoleic acid and having a molar ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) of 2.4 or more,
(1) a step of liberating fatty acids at the sn-1 and sn-3 positions from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions with lipase; A production method, including a step of recovering the fatty acid that has been allowed to react. Fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions have a content ratio of palmitoleic acid and oleic acid in the entire triglyceride (palmitoleic acid / oleic acid), the sn-1 position and sn-3 14. The production method according to claim 13, wherein the oil or fat has a higher content ratio of palmitoleic acid to oleic acid (palmitoleic acid/oleic acid) at each position. The production method according to claim 13 or 14, wherein the oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions is produced by Saccharomyces cerevisiae. 16. The production method according to claim 15, wherein Saccharomyces cerevisiae is a transformant strain overexpressing an N-terminal deletion gene of diacylglycerol acyltransferase.

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