JP2018140940A - Fatty acid composition and production method thereof, as well as skin external preparations, quasi drugs, and cosmetics containing fatty acid composition concerned - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide fatty acid compositions which contain palmitoleic acid at high content and are useful as a selective antimicrobial agent against Staphylococcus aureus.SOLUTION: The present invention relates to a fatty acid composition which contains 55 mol% or more of palmitoleic acid by isolating fatty acid at positions sn-1 and sn-3 with lipase from oils and fats containing triglyceride having palmitoleic acid at positions sn-1 position and sn-3 and subsequently by recovering the isolated fatty acid. The content ratio of palmitoleic acid and oleic acid (palmitoleic acid/oleic acid) is 2.4 or more, preferably 3 or more in a molar ratio, and preferably polyunsaturated fatty acids are not contained substantially.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a novel fatty acid composition and a method for producing the same, and further to a skin external preparation, a quasi-drug and a cosmetic containing the fatty acid composition.

  According to the Japanese Dermatological Association guidelines, atopic dermatitis is accompanied by various non-specific irritation reactions and idiosyncratic symptoms, including skin abnormalities such as skin dryness and barrier function abnormalities caused by abnormalities in the epidermis, especially stratum corneum. It is said to be a disease of the eczema / dermatitis group, which is caused by chronic allergic reaction and pruritus caused by chronic allergic reaction. Particularly in developed countries, the incidence is high, 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 disease, nature of easily producing IgE antibody, etc.), imbalance in the normal bacterial flora of the epidermis, nutritional factors (high linoleic acid edible oil and it) It develops due to multiple factors such as foods used as raw materials) 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 patients with atopic dermatitis, and that Staphylococcus aureus dramatically increases in the inflamed part of patients with atopic dermatitis (Non-patent Document 1, 2) It has been suggested that abnormal growth of Staphylococcus aureus in the epidermis may be a cause of exacerbation of atopic dermatitis. This is because protein A, δ toxin, V8 protease and the like produced by Staphylococcus aureus are involved in exacerbation of inflammation.

Therefore, atopic dermatitis patients are also treated with antibiotics, such as topical use of mupirocin, fusidic acid, or internal use of dicloxacillin, cephalexin, erythromycin and the like.
However, antibiotics such as those mentioned above are resident bacteria of the skin that inhibit the growth of Staphylococcus aureus and suppress the exacerbation of inflammation caused by the secretion of Staphylococcus aureus. It is highly possible that even the staphylococcus epidermidis shown will be sterilized and the balance of the epidermal resident flora will not be improved.

On the other hand, it has been reported that the content of sapienoic acid ((Z) -6-hexadecenoic acid) in the sebum of patients with atopic dermatitis is reduced to about 1/10 compared to healthy subjects (non-patent literature). 3) It is reported that palmitoleic acid ((Z) -9-hexadecenoic acid), which is an isomer of sapienoic acid and has experience in eating, has antibacterial activity against Staphylococcus aureus (Patent Document 1). Thus, the use of palmitoleic acid in a topical skin preparation has been studied.
However, there are few vegetable oils that have been eaten and have been confirmed to be safe and contain a large amount of palmitoleic acid. Although it is contained in the fruit pulp of sea berries (Hippophae rhamnoides), about 40% by mass, The cultivation area is limited, and it cannot be said that it is suitable as a practical source of palmitoleic acid.
In addition, seaberry pulp oil and vegetable oils, for example, linoleic acid, linolenic acid, etc. have two or more double bonds and have poor oxidative stability. Since the fatty acid composition obtained from vegetable oil has a problem in oxidation stability, it is not preferable for use in a skin external preparation or the like.

Furthermore, among workers working in hospital intensive care units (ICU) who repeat hand washing and alcohol disinfection, 41.2% to 52.9% have been reported to detect S. aureus in their hands. (Non-Patent Documents 4 and 5).
Therefore, Staphylococcus aureus is also proliferating in the skin of workers in food factories and those who are engaged in household chores at home, who frequently repeat hand washing and disinfection and have rough skin such as rough hands. The possibility is suggested.
Therefore, not only to prevent or improve the symptoms of atopic dermatitis, but also to prevent or improve rough skin caused by washing and disinfection of the skin, There is a high need to prevent or suppress, maintain and improve the balance of the normal epidermis flora.

International Publication No. 2011/011486

S. Higaki et al .; International J. Dermatology 38 265 (1999) H. H. Kong et al .; Genome Research 22 8505 (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 is a selective antibacterial agent that contains palmitoleic acid in a high content, suppresses Staphylococcus aureus, and does not suppress Staphylococcus epidermidis, that is, an antibacterial agent that maintains the balance of the resident epidermis. It was aimed to provide a useful fatty acid composition.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained a lipase from the fats and oils containing triglycerides having palmitoleic acid at the sn-1 position and the sn-3 position. The present invention was completed by successfully obtaining a fatty acid composition containing 55 mol% or more of palmitoleic acid by liberating the fatty acid and then collecting the liberated fatty acid.

That is, the present invention relates to the following.
[1] A fatty acid composition containing 55 mol% or more palmitoleic acid.
[2] The composition according to [1], wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid / oleic acid) is 2.4 or more in terms of molar ratio.
[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 molar ratio.
[4] The composition according to any one of [1] to [3], which does not substantially contain a polyunsaturated fatty acid.
[5] A selective antibacterial agent against Staphylococcus aureus, comprising the composition according to any one of [1] to [4].
[6] A skin external 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] A cosmetic 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 by lipase from an oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions, and (2) The manufacturing method of the fatty acid composition which contains the process of collect | recovering the fatty acid liberated at the process, and contains 55 mol% or more palmitoleic acid.
[10] The fat containing the triglyceride having palmitoleic acid at the sn-1 position and the sn-3 position is more than the sn-1 position and the content ratio of palmitoleic acid and oleic acid in the whole triglyceride (palmitoleic acid / oleic acid) The production method according to [9], wherein the content ratio of palmitoleic acid and oleic acid at the sn-3 position (palmitoleic acid / oleic acid) is larger.
[11] The production method according to [9] or [10], wherein the fat containing a triglyceride having palmitoleic acid at the sn-1 position and the sn-3 position is an oil produced by Saccharomyces cerevisiae .
[12] The production method according to [11], wherein Saccharomyces cerevisiae is a transformant 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 the fatty acid at the sn-1 and sn-3 positions of the triglyceride.
[14] The lipase that preferentially liberates the sn-1 and sn-3 fatty acids of triglyceride is a lipase derived from a microorganism belonging to the genus Pseudozyma or a lipase derived from a microorganism belonging to the genus Alcaligenes. [13] The production method according to [13].
[15] The production according to [14], wherein the step of liberating the fatty acids at the sn-1 and sn-3 positions with a lipase derived from a microorganism belonging to the genus Pseudozyma is performed 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 the fatty acid at the sn-1 and sn-3 positions of the triglyceride.
[17] The production method according to [16], wherein the lipase that specifically releases the fatty acid at the sn-1 position and the sn-3 position of the triglyceride 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 liberates palmitoleic acid preferentially over oleic acid.
[19] The production method according to [18], wherein the lipase that releases palmitoleic acid preferentially over oleic acid is a lipase derived from a microorganism belonging to the genus Candida.

According to the present invention, it is possible to obtain a fatty acid composition containing palmitoleic acid having a selective antibacterial activity at a high concentration unprecedented against Staphylococcus aureus.
The fatty acid composition of the present invention is a selective antibacterial agent that suppresses the growth of Staphylococcus aureus and does not suppress the growth of Staphylococcus epidermidis, that is, as an antibacterial agent that maintains the balance of the resident epidermis. It can be suitably used as a skin external preparation, quasi-drug or cosmetic for preventing the deterioration of symptoms of atopic dermatitis or improving the symptoms of atopic dermatitis.
Further, the fatty acid composition of the present invention is not only atopic dermatitis, but also a skin external preparation or quasi-drug for preventing or improving rough skin such as rough hands caused by washing, disinfection, etc. of skin for housework or work. Or it can be used suitably also as cosmetics.

It is a figure which shows the examination result of Test Example 3 about the effective amount of the fatty-acid composition in a skin external preparation.

  The present invention provides a fatty acid composition containing palmitoleic acid in an amount of 55 mol% or more based on the total amount of the composition (hereinafter also referred to as “the fatty acid composition of the present invention”).

Palmitoleic acid contained in the fatty acid composition of the present invention is an unsaturated fatty acid having 16 carbon atoms ((Z) -9-hexadecenoic acid) having one double bond at the 9-position.
The fatty acid composition of the present invention preferably contains palmitoleic acid in an amount of 58 mol% or more, more preferably 60 mol% or more, based on the total amount of the composition.
Moreover, when the efficiency etc. in the manufacturing method using the lipase mentioned later are considered, content of the palmitoleic acid in the fatty acid composition of this invention is 70 mol% or less normally.

The fatty acid composition of the present invention may contain a saturated or unsaturated fatty acid having about 10 to 20 carbon atoms in addition to palmitoleic acid, 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 ( (Palmitoleic acid / oleic acid) is preferably 2.4 or more, more preferably 3 or more in terms of molar ratio.
Furthermore, the fatty acid composition of the present invention is unstable to oxygen, and when formulated as a skin external preparation or the like, 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, It is preferable that substantially no polyunsaturated fatty acid such as 12-octadecatrienoic acid) is contained.
Here, “substantially free of polyunsaturated fatty acid” means that the polyunsaturated fatty acid is not detected by a usual analysis method of the polyunsaturated fatty acid, for example, a quantification method by 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 usually has a liquid state at 20 ° C. because the content of long-chain saturated fatty acids is small.

  The fatty acid composition of the present invention releases fatty acids at the sn-1 and sn-3 positions by lipase from fats and oils containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions. By collecting, it can be suitably obtained. In addition, the fats and oils have a content ratio of palmitoleic acid and oleic acid at the sn-1 and sn-3 positions (palmitoleic acid) based on the content ratio of palmitoleic acid and oleic acid in the whole triglyceride (palmitoleic acid / oleic acid). / Oleic acid is more preferably a larger fat.

The fats and oils containing the above-mentioned triglycerides are fats and oils obtained from plants, microorganisms such as yeast, etc., preferably fats and oils having a food experience, and are fats and oils produced by Saccharomyces cerevisiae It is more preferable.
As Saccharomyces cerevisiae, it is preferable to use a transformant having a high lipid-producing ability. Such a transformant is a transformant overexpressing an N-terminal deletion gene of diacylglycerol acyltransferase (DGAT) as described in JP-A-2015-146778, and includes sn-1 Those having the ability to produce lipids with a high content of triglycerides having palmitoleic acid at the position and sn-3 position can be preferably used. Furthermore, a transformed strain in which the N-terminal deletion gene of DGAT is overexpressed with a DGAT gene (DGA1) -disrupted strain of Saccharomyces cerevisiae is more preferable. In particular, TGAT lacking 29 residues at the N-terminus was overexpressed in Saccharomyces cerevisiae with reduced function of ESA1 gene encoding histone acetyltransferase in cells such as DGA1 gene disrupted strains. Converted strains are preferably used.
In addition, Kansaka et al. (Appl. Microbiol. Biotechnol. 99 201-210 (2015)), Japanese Patent Application Laid-Open No. 2015-146778, for producing and culturing a transformant overexpressing the DGAT N-terminal deletion gene. Can be carried out according to the method described in the Gazette.

In order to obtain the fatty acid composition of the present invention, as lipase, a lipase that preferentially releases the fatty acid at the sn-1 position and the sn-3 position of the triglyceride, or the fatty acid at the sn-1 position and the sn-3 position of the triglyceride It is preferable to use a lipase that specifically releases.
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, microorganisms belonging to the genus Alcaligenes Lipases derived from microorganisms belonging to the genus Psudomonas and microorganisms belonging to the genus Burkholderia, and the like. Among them, lipases derived from microorganisms belonging to the genus Pseudozyma and alkaligenes ( A lipase derived from a microorganism belonging to the genus Alcaligenes) is more preferable.
Examples of 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, Rhizopus Mihei ( Preferable examples include microorganisms belonging to the genus Rhizomucor such as Rhizomucor miehei, lipases derived from microorganisms belonging to the genus Thermomyces such as Thermomyces lanuginosus, and pancreatic lipase. Of these, lipases derived from microorganisms belonging to the genus Rhizopus are more preferable.

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

The lipase described above may be a free lipase, or may be immobilized by binding to a carrier such as a resin or encapsulating in a microcapsule or a liposome.
In the present invention, commercially available lipases provided by various companies can be used.

The reaction of liberating the fatty acids at the sn-1 and sn-3 positions by lipase from the oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions depends on the type of lipase used. It can be set appropriately.
The amount of lipase added is usually 0.01 to 10 parts by mass, preferably 0.03 to 1.5 parts by mass, with respect to 100 parts by mass of triglyceride as a substrate. In the case of a free enzyme, the reaction is carried out in an aqueous system, and the amount of water added to the triglyceride is preferably 0.01% by mass to 99% by mass, more preferably 5% by mass to 95% by mass. . In the case of an immobilized enzyme, water may or may not be added.
The enzyme 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 = 3-9, preferably pH = 5-7, usually 0. Let it run for 5 to 18 hours. When the reaction time becomes long, the liberation of fatty acids proceeds at all of the sn-1 to sn-3 positions, or acyl group transfer occurs. Therefore, the reaction time is preferably about 2 to 3 hours. In order to stop the hydrolysis reaction by lipase, it is preferable to carry out deactivation by heating, deactivation by short-chain alcohols such as ethanol and methanol, and oil-water separation by standing or centrifugation. In the case of an immobilized enzyme, the immobilized enzyme need only be removed by standing or filtration. Unreacted triglyceride is removed by adding an alkaline agent such as potassium hydroxide to the reaction solution, saponifying the free fatty acid and transferring it to the aqueous layer, and then extracting it with a nonpolar 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, when the lipase is reacted in the presence of an excess amount of ethanol of about 500 parts by mass with respect to 100 parts by mass of the substrate triglyceride, , Sn-1 and sn-3 positions are almost specific, which is preferable. 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 an ordinary method.

  The fatty acid liberated by the lipase is separated by (1) 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. Then, unreacted triglycerides are removed by extraction with a low polar organic solvent such as n-hexane, and acidified by adding an acid such as hydrochloric acid to the aqueous layer, and then with a low polar organic solvent such as n-hexane. It can be recovered by ordinary methods such as extraction and evaporation of the solvent, (3) removal of water by oil-water separation, etc., and recovery of free fatty acids 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 Low oleic acid, which inhibits the antibacterial activity, suppresses the growth of Staphylococcus aureus, which is involved in the exacerbation of atopic dermatitis, but is an epidermis grape that is a normal skin resident bacterium The growth of Staphylococcus epidermidis is not suppressed, 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 and maintains or improves the normal bacterial flora of the skin to improve atopic dermatitis. Caused by topical skin preparations, quasi-drugs, cosmetics, etc. that are effective in preventing the worsening of symptoms or improving the symptoms of atopic dermatitis, or by cleaning or disinfecting skin for housework or work It can be preferably used as an external preparation for skin, quasi-drug, cosmetics and the like effective in preventing or improving rough skin.

Therefore, the present invention provides a selective antibacterial agent (hereinafter also referred to as “the antibacterial agent of the present invention”) against Staphylococcus aureus comprising the fatty acid composition of the present invention.
The antibacterial agent of the present invention is added to the fatty acid composition of the present invention, if necessary, by adding a general additive used in the field of pharmaceutical preparations, and a formulation means well known in the field of pharmaceutical preparations, for example, the 17th revision Japanese Pharmacopoeia General Formulation [3] Can be prepared by the methods described in each section of the formulation, oily; liquid, such as suspension, emulsion, etc .; semi-solid, such as gel, paste, cream A solid form such as powder, granule, tablet, capsule or the like.
For the purpose 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 additives include excipients, binders, disintegrants, lubricants, coating agents, bases, solvents, emulsifiers, dispersants, suspending agents, stabilizers, thickeners, pH adjusters, Antioxidants, preservatives, preservatives, flavoring agents, sweeteners, fragrances, colorants and the like can be mentioned.

  Excipients include anhydrous silicic acid, calcium silicate, magnesium aluminate, starch (corn starch, potato starch, wheat starch, etc.), sugar (glucose, lactose, sucrose, etc.), sugar alcohol (sorbitol, maltitol) , Mannitol, etc.).

  Examples of the binder include gelatin, sodium caseinate, starch (such as hydroxypropyl starch, pregelatinized starch, and partially pregelatinized starch), cellulose and derivatives thereof (crystalline cellulose, hydroxypropylcellulose, carboxymethylcellulose, and the like).

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

  Examples of the lubricant include talc, synthetic aluminum silicate, magnesium silicate, calcium stearate, magnesium stearate and the like.

  Coatings 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 the base include hydrocarbons (such as liquid paraffin) and polyethylene glycol (such as Macrogol 400 and Macrogol 1500).

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

  As an emulsifier, nonionic surfactant (sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, sucrose fatty acid ester, etc.), anionic surfactant (sodium alkyl sulfate, N-acyl glutamate, etc.) ), Refined soybean lecithin and the like.

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

  Examples of the suspending agent include sodium alginate, nonionic surfactant (polyoxyethylene lauryl ether, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, propylene glycol fatty acid ester, and the like).

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

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

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

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

Examples of the preservative include sodium benzoate, sorbic acid, potassium sorbate, dehydroacetic acid, sodium dehydroacetate, paraoxybenzoic acid ester (such as methyl parahydroxybenzoate) and the like.
Examples of the preservative include benzoic acid, sodium benzoate, sorbitol, paraoxybenzoic acid esters (such as methyl paraoxybenzoate), and propylene glycol.

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

  Examples of the fragrances include orange essence, l-menthol, d-borneol, vanillin, linalool and the like.

  Colorants include tar dyes (food red 2, food blue 1, food yellow 4, etc.), inorganic pigments (bengala, yellow iron sesquioxide, titanium oxide, etc.), natural dyes (anato dyes, turmeric dyes, carotenoids) Etc.).

  One or two or more of the above-mentioned additives can be used as necessary.

  As content of the fatty acid composition of this invention in the antibacterial agent of this invention, it is 0.001 mass%-10 mass% normally as content of the palmitoleic acid with respect to the whole quantity of the antibacterial agent of this invention, 0.005 It is preferable that it is mass%-5 mass%.

The application amount of the antibacterial agent of the present invention is 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 target”), the antibacterial agent of the present invention. dosage form, is appropriately determined by the application route and the like, application target is a human adult, when applied by external application as an application amount of palmitoleic acid, per normal 0.02μg ^/ cm ² ~200μg ^/ cm 2 by weight, ^{preferably, 0.1μg / cm 2 ~100μg / cm} 2.
The above amounts can be applied once to several times a day.
The application period of the antibacterial agent of the present invention is appropriately determined depending on the state of the skin observed in the application target (the state of the balance of the skin resident bacterial flora) and the like, but is usually 1 day to 30 days, preferably 3 Days to 15 days.
In addition, since the antibacterial agent of this invention uses the palmitoleic acid obtained from lipids, such as yeast confirmed safety | security, as an active ingredient, it is highly safe and suitable for continuous application.

  Examples of animals to which the antibacterial agent of the present invention is applied (hereinafter also referred to as “target animals”) 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 application amount of the antibacterial agent of the present invention may be appropriately set according to the type, sex, body weight, skin condition, etc.

The antibacterial agent of the present invention improves the balance of skin resident bacterial flora to a normal state, or maintains the balance of skin resident bacterial flora to a normal state, thereby aggravating the symptoms of atopic dermatitis. It is effective for prevention or improvement 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 frequently used not only for patients with atopic dermatitis, but also for those who are likely to have rough skin symptoms such as rough hands, rough skin, and other purposes for housework and duties. Even in those who present, it can be suitably applied to prevent or ameliorate rough skin symptoms.

The present invention also provides a skin external preparation containing the fatty acid composition of the present invention (hereinafter also referred to as “the skin external preparation of the present invention” in the present specification).
Here, “external preparation for skin” refers to a pharmaceutical applied to a skin lesion site for external use, but also includes a transdermal preparation for the purpose of delivering an active ingredient to the circulating bloodstream through the skin. It is.
The external preparation for skin of the present invention is prepared by adding, to the fatty acid composition of the present invention, general additives used in the manufacture of external preparations for skin, if necessary, solid preparations for external use such as external powders; liniments, lotions Liquids for external use such as liquids (emulsions, emulsions, suspensions, etc.); sprays such as aerosols for external use, pump sprays; ointments such as oily and water-soluble ointments; oil-in-water or in oil It can be provided in a dosage form such as a water-type cream agent; a gel agent such as an aqueous gel agent or an oily gel agent; a patch such as a tape agent or a patch.
The external preparation for skin of the present invention is described in the general method for producing an external preparation for skin, for example, “11. Preparations for application to the skin” in the 17th revised Japanese Pharmacopoeia Preparation General Rules [3] Can be produced according to the methods described.

  The skin external preparation of the present invention includes the above-mentioned excipients, binders, disintegrants, lubricants, bases, solvents, emulsifiers, dispersants, suspending agents, stabilizers, thickeners, pH adjustments. 1 type (s) or 2 or more types, such as an agent, an antioxidant, antiseptic | preservative, a preservative, a fragrance | flavor, and a coloring agent, 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, glycyrrhetinic acid; adrenal glands such as clobetasone butyrate, etc. Cortical steroids; antibiotics such as sodium fusidate and mupirocin; and immunomodulating drugs such as tacrolimus hydrate can be included.

  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 in order to prevent deterioration of inflammation or improve symptoms.
In addition, the topical skin preparation of the present invention is used to prevent or ameliorate rough skin symptoms even in those who are likely to have rough skin symptoms or those who have rough skin symptoms such as rough skin caused by washing, disinfection, etc. It can be suitably applied.
The application amount and application period of the external preparation for skin of the present invention per day depend on the type, sex, age, degree of skin symptom, the dosage form of the external preparation for skin of the present invention, etc. It is determined appropriately. For example, in the case of a human adult, the application amount of palmitoleic acid can be applied so as to be the same as the application amount described above for the antibacterial agent of the present invention. Can be applied.

Furthermore, the present invention relates to a quasi-drug containing the fatty acid composition of the present invention, particularly a skin quasi-drug (hereinafter also referred to as “the quasi-drug of the present invention”) or a cosmetic (hereinafter referred to as “the quasi-drug of the present invention”). In the present specification, it is also referred to as “the cosmetic of the present invention”).
Here, the “quasi-drug” refers to those that have a milder effect on the human body than pharmaceuticals but have some improvement effect. It is called “foreign goods” So-called medicinal cosmetics are included in quasi-drugs for external use on the skin.
“Cosmetics” is applied to the skin or the like for the purpose of cleansing the body or making it look beautiful, and means a product with a relaxed action.
The quasi-drug or cosmetic of the present invention can be produced in accordance with the above-mentioned skin external preparation of the present invention, and forms such as skin lotion, cosmetic liquid, milky lotion, cream, face wash, pack, body wash, etc. Can be provided at.

  In the quasi-drug or cosmetic of the present invention, a polyhydric alcohol (glycerin, 1,3-butylene glycol, dipropylene glycol, etc.), an amino acid or a salt thereof (alanine, serine, Proline, DL-pyrrolidone carboxylate, etc.), proteins (whey, water-soluble collagen, hydrolyzed elastin, etc.), nucleic acids (deoxyribonucleic acid sodium, etc.), mucopolysaccharides (sodium chondroitin sulfate, sodium hyaluronate, etc.), heparin analogues, Moisturizers such as plant extracts (Ashitaba extract, cucumber extract, birch extract, etc.); azulenes (azurene, guaiazulene, ethyl guaiazulene sulfonate, sodium guaiazulene sulfonate, etc.), allantoin and its derivatives (allantoin, ascorb Acid allantoin, allantoin glycyrrhetinic acid, etc.), vitamins ((ascorbyl / tocopheryl) potassium phosphate, panthenol, etc.), plant extracts (such as chamomile extract, licorice extract, leafy aloe extract, etc.), glycyrrhizic acid and its salts ( Glycyrrhizic acid, ammonium glycyrrhizinate, dipotassium glycyrrhizinate), glycyrrhetinic acid and its derivatives (glycyrrhetinic acid, glyceryl glycyrrhetinate, pyridoxine glycyrrhetinate, disodium succinylglycyrrhetinate), etc. be able to.

  The content of the fatty acid composition of the present invention in the quasi-drug or cosmetic of the present invention can be appropriately determined according to the skin external preparation of the present invention, but the amount of palmitoleic acid applied per day is It is preferable that the fatty acid composition of the present invention is set to be applied so as to achieve the above-mentioned daily application amount of the antibacterial agent of the present invention.

The quasi-drug or cosmetic of the present invention prevents the deterioration of the skin condition of an atopic dermatitis patient, improves the skin condition of an atopic dermatitis patient, or causes rough skin symptoms such as rough hands. It can be suitably used to prevent or ameliorate rough skin symptoms in those who are likely to present or who have rough skin symptoms.
In addition, the quasi-drug or cosmetic of the present invention is mainly composed of human skin, particularly humans having a predisposition for atopy, or particularly the normal bacterial flora of human skin that exhibits rough skin symptoms caused by washing, disinfection, etc. of the skin. It is preferably used to maintain the balance in a normal state.
In particular, the quasi-drug or cosmetic product of the present invention is highly safe because it contains palmitoleic acid obtained from lipids such as yeast whose safety has been confirmed as an active ingredient, for the purpose of daily skin care, It can be applied continuously over a long 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 by lipase from an oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions; and (2) A step of recovering the fatty acid liberated in the above step is included.

  In order to liberate the fatty acids at the sn-1 and sn-3 positions in the present invention, such as oils and fats containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions and the type of lipase that can be used in the present invention The conditions for the enzymatic reaction, 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, within the range that does not impair the characteristics of the present invention, if necessary, purification means such as recrystallization and various chromatography, solid-liquid separation means such as filtration and centrifugation, etc. Can be used.

  Further, the present invention will be described in detail by examples.

[Reference Example 1] Preparation of Purified Yeast Triglyceride The fat and oil produced by the transformant of Saccharomyces cerevisiae was recovered by the method described in Kansaka et al. (Japanese Patent Laid-Open No. 2015-146778). The solvent remaining in the oil (5 g) was removed by an evaporator, and 3.57 g of the oil after removal of the solvent was equilibrated with n-hexane: ethyl acetate = 98: 2 (volume ratio) (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 triacylglyceride 15 to 25 fractions were collected, and the solvent was removed by an evaporator to obtain 2.74 g of purified yeast triglyceride of Reference Example 1.

For the purified yeast triglyceride of Reference Example 1, the fatty acid composition of the sn-1, 3 and sn-2 positions is as follows according to the method of Y. Watanabe et al. (J. Oleo Sci., 64, 1193 (2015)). 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) 30) The mixture was reacted for 3 hours with reciprocal shaking at 0 ° C. and filtered to recover the supernatant. Next, the solvent was removed by an evaporator, and the whole amount was equilibrated with n-hexane: diethyl ether = 8: 2 (volume ratio). A Sep-Pack silica cartridge (“WAT051900” (Waters) And the fatty acid ethyl ester and diglyceride were eluted and removed with 30 mL of the solvent. Next, 10 mL of diethyl ether was used to elute monoglyceride having one acyl group at the sn-2 position (sn-2 monoglyceride), 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 above-derived sn-2 monoglyceride and a methanol solution of 28% by mass of sodium methoxide were added and heated at 75 ° C. for 15 minutes to methylate the fatty acid. went. After allowing to cool, 0.5 mL of n-hexane was added and mixed, then 3 mL of water was added and mixed, and the fatty acid methyl ester was extracted with n-hexane, and the n-hexane layer was recovered.
The collected n-hexane layer was analyzed under the following conditions with a capillary gas chromatograph (“Agilent 6890N” (Agilent Technologies)) to quantify fatty acids.

<Analysis conditions>
Capillary column: DB-23 (0.25 mm × 30 m) (Agilent Technologies)
Inlet temperature: 245 ° C
Injection volume: 3 μL
Detector: Hydrogen 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 sn-1, 2, 3 positions) and the fatty acid composition (mol%) of the sn-2 monoglyceride of Reference Example 1, Calculated. The results are shown in Table 1.

As shown in Table 1, it was confirmed that the purified yeast triglyceride of Reference Example 1 derived from a transformant of Saccharomyces cerevisiae contained more palmitoleic acid than conventional vegetable oils and fats. On the other hand, it was observed that polyunsaturated fatty acids which are poor in oxidative stability and are undesirable from the viewpoint of formulation stability are hardly contained.
Moreover, as shown in Table 1, in the purified yeast triglyceride of Reference Example 1, the palmitolein at the sn-1,3 position compared to the fatty acid composition in the whole triglyceride (total fatty acid composition at the sn-1,2,3 position). It can be seen that the acid content is high and the oleic acid content at the sn-1,3-position is low, that is, the content ratio of palmitoleic acid to oleic acid (palmitoleic acid / oleic acid) is high.
That is, the purified yeast triglyceride of Reference Example 1 contains 20 mol% or more of palmitoleic acid, but sea berry oil containing about 10 mol% of linoleic acid, or macadamia containing 2 mol% to 3 mol% of linoleic acid and linolenic acid, respectively. Compared to nut oil, it is useful as a starting material for producing the fatty acid composition of the present invention.

[Example 1] Preparation of fatty acid composition using immobilized lipase derived from Pseudozyma antarctica To 200 mg of the purified yeast triglyceride of Reference Example 1, 2 g of ethanol and Novozyme 435 (Novozumes) 0.088 g was added, reacted for 2.5 hours with reciprocal shaking at 30 ° C., and filtered to recover the supernatant. Next, the solvent was removed by an evaporator, and a Sep-Pack silica cartridge (“WAT051900” (Waters) equilibrated with n-hexane: diethyl ether = 8: 2 (volume ratio) by half. And the fatty acid ethyl ester was eluted with 10 mL of the solvent. Furthermore, 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 ester obtained by two column fractions was mixed, and after solvent removal (estimated 130 mg), 3 g of ethanol and 0.2 g of 5M sodium hydroxide were added, and the mixture was heated at 60 ° C. for 15 minutes to saponify. Then, after cooling to room temperature, 4 mL of water was added, 2 M hydrochloric acid was added until it became acidic, and then extracted twice with 3 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and the free fatty acid was recovered (the fatty acid composition of Example 1).
On the other hand, sn-2 monoglyceride obtained by two column fractions was mixed, and after solvent removal (estimated 50 mg), ethanol 3 g, water 0.1 mL and 5 M sodium hydroxide 0.1 g were added, Heated for minutes and saponified. Then, after cooling to room temperature, 4 mL of water was added, 2 M hydrochloric acid was added until it became acidic, and then extracted twice with 3 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and free fatty acids were recovered to obtain the fatty acid composition of Comparative Example 1.
In addition, when the above reaction using Novozume 435 is performed all day and night, all of the fatty acids at positions sn-1, 2, and 3 of the triglyceride are converted into ethyl esters. The composition can be considered to be the same as the fatty acid composition in the purified yeast triglyceride of Reference Example 1 used as a starting material. Therefore, for comparison, a fatty acid composition was prepared in the case of reacting day and night as follows.
To 200 mg of the purified yeast triglyceride of Reference Example 1, 2 g of ethanol and 0.088 g of Novozymes 435 (Novozymes) are added, reacted for 24 hours while reciprocating at 30 ° C., filtered, and the supernatant Was recovered and the solvent was removed with an evaporator. Next, 6 g of ethanol and 0.4 g of 5M sodium hydroxide were added to the obtained ethyl ester (about 200 mg), and the mixture was heated at 60 ° C. for 15 minutes for saponification. Subsequently, it cooled to room temperature, 10 mL of water was added, 2 M hydrochloric acid was added until it became acidic, and it extracted twice with 10 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and free fatty acids were recovered (reaction product for 24 hours).

[Example 2] Preparation of fatty acid composition using lipase derived from Rhizopus japonicus In 200 mg of purified yeast triglyceride of Reference Example 1, 1.8 mL of water and a lipase derived from Rhizopus japonicus ("Lipase A" −10D ”(Nagase ChemteX Corporation)) (25 mg / mL) 0.053 mL was added and reacted at 35 ° C. with stirring at 350 rpm for 2.5 hours. Then, 10 mL of ethanol was added to stop the reaction. The hydrolysis rate was determined by 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 extracted three times with 15 mL of n-hexane to remove unreacted triglycerides. Next, 2M hydrochloric acid was added to the aqueous layer until acidic, and extracted twice with 15 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and free fatty acid was recovered (fatty acid composition of Example 2).
For comparison, a fatty acid composition was prepared by allowing the reaction with the above lipase to be performed overnight.
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) are added and reacted at 35 ° C. with stirring at 350 rpm for 24 hours. Thereafter, the same operation as described above was performed to recover free fatty acids (reaction product for 24 hours).

[Example 3] Preparation of fatty acid composition using lipase derived from Candida rugosa To 100 mg of purified yeast triglyceride of Reference Example 1, 0.9 mL of water and lipase derived from Candida rugosa ("lipase OF" “(Nagoya Sangyo Co., Ltd.)” (10 mg / mL) 0.011 mL was added and reacted at 35 ° C. with stirring at 350 rpm for 2.5 hours, followed by addition of 10 mL of ethanol to stop the reaction, and 50 mM. The hydrolysis rate was determined by titrating 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 extracted three times with 15 mL of n-hexane to remove unreacted triglycerides. Next, 2M hydrochloric acid was added to the aqueous layer until acidic, and extracted twice with 15 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and free fatty acids were recovered (fatty acid composition of Example 3).
For comparison, a fatty acid composition was prepared by allowing the reaction with the above lipase to be performed overnight.
After adding 0.84 mL of water and 0.06 mL of lipase OF (Nagoya Sangyo Co., Ltd.) (10 mg / mL) to 100 mg of the purified yeast triglyceride of Reference Example 1, the mixture was reacted at 35 ° C. with stirring at 350 rpm for 24 hours. The same operation as above was performed to recover free fatty acids (reaction product for 24 hours).

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

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. . Subsequently, it cooled to room temperature, 4 mL of water was added, 2M hydrochloric acid was added until it became acidic, and it extracted twice with 3 mL of n-hexane. The hexane layer was recovered, the solvent was removed with an evaporator, and free fatty acid was recovered to obtain the fatty acid composition of Comparative Example 2.

[Test Example 1] Analysis of fatty acid composition About each fatty acid composition of Examples 1 to 4 and 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 sample and a methanol solution of 14% by mass of boron trifluoride methanol complex were added, and heated at 75 ° C. for 15 minutes to perform fatty acid methyl esterification. After allowing to cool, 0.5 mL of n-hexane was added and mixed, then 3 mL of water was added and mixed, and the fatty acid methyl ester was extracted with n-hexane, and the n-hexane layer was recovered.
The collected n-hexane layer was analyzed with a capillary gas chromatograph (“Agilent 6890N” (Agilent Technologies)) under the same conditions as in the analysis of the fatty acid composition of the purified yeast triglyceride of Reference Example 1 above. Was quantified.
The analysis results of the fatty acid composition are shown in Table 2.

  Table 3 shows the hydrolysis rates during preparation of the fatty acid compositions of Examples 2 to 4. In addition, 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. The estimated hydrolysis rate is listed.

As shown in Table 2, in the fatty acid composition of Example 1, the palmitoleic acid content was improved and the oleic acid content was decreased compared to the purified yeast triglyceride of Reference Example 1, and palmitoleic acid and oleic acid It was observed that the content ratio (palmitoleic acid / oleic acid) increased from 1.62 to 3.52 in molar ratio. Moreover, the fatty acid composition of the fatty acid composition of Example 1 is close to the fatty acid composition at the sn-1,3 position of the purified yeast triglyceride of Reference Example 1, and in the presence of an excess amount of ethanol, Pseudozyma antarctica ) -Derived immobilized lipase was confirmed to preferentially release 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 reaction for 2.5 hours is estimated to be about 67%. That is, it was suggested that the method using an immobilized lipase derived from Pseudozyma antarctica requires a step of saponifying the produced fatty acid ethyl ester, but has a high yield.

Since the fatty acid composition of Example 2 was obtained by a lipase derived from Rhizopus japonicus that acts specifically on the sn-1,3 position of triglyceride, the fatty acid of Example 1 was obtained as shown in Table 2. The palmitoleic acid content is higher than the composition and the oleic acid content is low, and the content ratio of palmitoleic acid and oleic acid (palmitoleic acid / oleic acid) is 3.99 in terms of molar ratio. It was.
However, as shown in Table 3, in the preparation of the fatty acid composition of Example 2, the hydrolysis rate after reaction for 2.5 hours was slightly low, and a tendency for the yield to be slightly low was observed. This is because in the reaction with lipase derived from Rhizopus japonicus, both hydrolysis and esterification occur, and triacylglycerol, diacylglycerol, monoacylglycerol and free fatty acids are equilibrated at a certain ratio. This is because there is a tendency to become.
In addition, lipase derived from Rhizopus japonicus is specific to the sn-1,3 position, but when the reaction time is increased, the di-2 or monoglyceride sn-2 position and sn-1 position or sn-3 position. Since non-enzymatic acyl transfer occurs between the two, and the fatty acid at the sn-2 position is released slowly, the content ratio of palmitoleic acid and oleic acid after reaction for 24 hours (palmitoleic acid / oleic acid) is , Decreased after 2.5 hours of reaction.

The fatty acid composition of Example 3 uses a lipase derived from Candida rugosa that acts equally on the sn-1,2,3 positions of triglycerides but acts better on palmitoleic acid than oleic acid. Since it was prepared and the lipase hardly acts on saturated fatty acids, Table 2 shows a higher palmitoleic acid content than the compositions of other examples.
Further, from Table 3, it was confirmed that the hydrolysis of the triglyceride proceeded almost completely when the reaction time was 24 hours during the preparation of Example 3.

The fatty acid composition of Example 4 acts equally on the sn-1,2,3 positions of triglycerides or acts preferentially on the sn-1,3 positions, but does not show fatty acid specificity ( Since it was prepared using a lipase derived from a microorganism belonging to the genus (Alcaligenes), the palmitoleic acid content was slightly improved as compared with the purified yeast triglyceride of Reference Example 1 as shown in Table 2, but the extent was as in Example 1. It was a little low compared with each of the fatty acid composition of ~ 3.
From Table 3, it was confirmed that the hydrolysis of the triglyceride proceeded almost completely when the reaction time was set to 24 hours during the preparation of the fatty acid composition of Example 4.

[Test Example 2] Evaluation of antibacterial activity Each fatty acid composition of Examples 1 to 4, a fatty acid composition obtained by reacting in each Example for 24 hours for comparison (24-hour reaction product), and comparison For each fatty acid composition of Examples 1 and 2, the minimum inhibitory concentration (MIC) was measured according to a micro liquid dilution method which is a standard method of the Japanese Society of Chemotherapy, and antibacterial activity was evaluated.
(1) Test strain suspension
Staphylococcus aureus NBRC13276 strain and Staphylococcus epidermidis NBRC100911 strain, respectively. B. 1 platinum ear implant in 3 mL of medium (0.5% by weight salmon meat extract (Wako Pure Chemical Industries, Ltd.), 1% by weight polypeptone (Nippon Pharmaceutical Co., Ltd.), 0.5% by weight sodium chloride, pH = 7.0) Bacteria and cultured overnight at 37 ° C. with shaking. This pre-culture solution was reconstituted with new N. cerevisiae so that the inoculum was 20% by mass. B. Inoculated into 4 mL of the medium and pre-cultured at 37 ° C. for 3 hours with shaking. From the measured value of turbidity (OD ₆₆₀ ) at ₆₆₀ nm, the viable cell count was calculated as 6.4 × 10 ⁸ colony forming unit (cfu) / mL at OD ₆₆₀ = 1. The culture solution is N. so as to be 2.0 × 10 ⁴ cfu / mL. B. A test strain suspension was prepared by dilution with medium (pH = 6.0).
(2) Test sample Each fatty acid composition of Examples 1 to 4, a fatty acid composition (24-hour reaction product) obtained by reacting in each Example for 24 hours, and Comparative Example 1, Each fatty acid composition of No. 2 was dissolved in dimethyl sulfoxide (DMSO) so as to be 10,000 μg / mL, and used as each test sample.
Further, as a control, antibacterial activity was similarly evaluated for the reagents palmitoleic acid and oleic acid (Tokyo Chemical Industry Co., Ltd.).
(3) Measurement of MIC 234 μL of the test strain suspension (2.0 × 10 ⁴ cfu / mL) was dispensed into the second row of the 96-well round bottom microplate, and 130 μL was dispensed into the 3rd to 11th rows. did. Next, 26 μL of the test sample (10,000 μg / mL) was added to the second row of the microplate, and was sufficiently suspended by pipetting and diluted 10-fold. Next, 130 μL of this suspension was added to and suspended in the third row of the microplate, and diluted stepwise by two times.
After culturing the microplate by standing at 37 ° C. for 2 days, the growth of the test strain is judged by visually confirming the turbidity of the culture solution or the presence or absence of precipitated cells. The minimum concentration of the test sample that could not be obtained was defined as MIC.

  The evaluation results of antibacterial activity are shown in Table 4.

As shown in Table 4, palmitoleic acid selectively suppressed the growth of Staphylococcus aureus NBRC13276 at low concentrations, but oleic acid did not show effective antibacterial activity against Staphylococcus aureus NBRC13276.
The content of palmitoleic acid was high, the content of oleic acid was low, and the content ratio of palmitoleic acid and oleic acid (palmitoleic acid / oleic acid) was improved as compared with the purified yeast triglyceride of Reference Example 1. Regarding the fatty acid composition, good antibacterial activity was observed against Staphylococcus aureus NBRC13276 strain. On the other hand, it was confirmed that the growth of Staphylococcus epidermidis NBRC100911 strain, which is a resident skin bacterium, was not affected.
In contrast, the fatty acid composition of Comparative Example 1 derived from sn-2 monoglyceride and having a high oleic acid content does not exhibit antibacterial activity against Staphylococcus aureus NBRC13276 strain, and is a saponified purified triglyceride of Reference Example 1, In the fatty acid composition of Comparative Example 2 containing palmitoleic acid and oleic acid, a decrease in antibacterial activity was observed as compared with the fatty acid composition of each example, and the presence of oleic acid caused the antibacterial activity of palmitoleic acid against Staphylococcus aureus NBRC13276. Was suggested to be inhibited.
In addition, the fatty acid composition of Example 4 having a slightly lower palmitoleic acid content than other examples also shows a selective antibacterial activity against Staphylococcus aureus NBRC13276 strain, although it is lower than the other examples. It was.
The 24-hour reaction product compositions of Examples 1, 3, and 4 in which hydrolysis of triglycerides almost completely proceeded had the same content of palmitoleic acid and oleic acid as the fatty acid composition of Comparative Example 2, As shown in FIG. 4, antibacterial activity against Staphylococcus aureus NBRC13276 was not observed.

[Test Example 3] Examination of effective amount of fatty acid composition in topical skin preparation by co-culture method (1) Test bacteria B. Agar medium (0.1% by weight salmon meat extract (Wako Pure Chemical Industries, Ltd.), 1% by weight polypeptone (Nippon Pharmaceutical Co., Ltd.), 7.5% by weight sodium chloride, 2% by weight agar, pH = 6.0) The same plate was coated with Staphylococcus aureus NBRC13276 strain and Staphylococcus epidermidis NBRC100911 strain at 3,000 cfu / cm ² , respectively.
(2) Sample As a model of the external preparation for skin, N. B. Fatty acid of Example 1 in liquid medium (0.5% by weight salmon meat extract, 1% by weight polypeptone, 0.5% by weight sodium chloride, 0.4% by weight thickener (guar gum), pH = 6.0) The composition was suspended to 0.5% by mass, and then diluted stepwise by 2 times until it became 0.001% by mass.
In addition, the thing which does not add the fatty acid composition of Example 1 was made into the control | contrast (control).
(3) Calculation of effective amount of fatty acid composition The above-mentioned sample and control were coated with the above-mentioned two kinds of test bacteria so as to be ² mg / cm ² . B. It apply | coated to the agar medium. After culturing at 37 ° C. for 2 days, the grown bacteria were collected with a cotton swab, suspended in 7.5% by mass saline, and diluted appropriately, and then applied to mannitol-salt agar medium supplemented with egg yolk (As One Co., Ltd.). After culturing at 37 ° C. for 1 day, Staphylococcus aureus NBRC13276 and Staphylococcus epidermidis NBRC100911 were discriminated based on the shape and color of the grown colonies and whether the yolk reaction was positive or negative.
(3) Results The above examination results are shown in FIG. When the content of the fatty acid composition of Example 1 in the sample which is a model of the external preparation for skin is 0.0313% by mass or more, the growth of Staphylococcus aureus NBRC13276 is completely suppressed, and only the Staphylococcus epidermidis NBRC100911 grows. Was recognized.
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 amount of the fatty acid composition applied to the skin was 0.625 μg / cm ² , and palmitolein The amount of acid applied is 0.37 μg / cm ² .
Therefore, when applying the skin external preparation of this invention, possibility that the growth of Staphylococcus aureus could be suppressed by 0.37 microgram / cm < ² > as an application amount of a palmitoleic acid was suggested.

As described above in detail, according to the present invention, a fatty acid composition containing palmitoleic acid having selective antibacterial activity against Staphylococcus aureus at an unprecedented high concentration can be obtained.
The fatty acid composition of the present invention is a selective antibacterial agent against Staphylococcus aureus, and is particularly effective for preventing exacerbation of symptoms of atopic dermatitis or improving symptoms of atopic dermatitis. It can be used as an quasi-drug or cosmetic.
Furthermore, the fatty acid composition of the present invention is a topical skin preparation, quasi-drug, or cosmetic that is effective in preventing or improving rough skin caused by washing, disinfection, etc., of which the detection of Staphylococcus aureus has been reported. As can be used.

Claims (19)

  A fatty acid composition containing 55 mol% or more of palmitoleic acid.   The composition according to claim 1, wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid / oleic acid) is 2.4 or more in molar ratio.   The composition according to claim 1, wherein the content ratio of palmitoleic acid and oleic acid (palmitoleic acid / oleic acid) is 3 or more in molar ratio.   The composition of any one of Claims 1-3 which does not contain a polyunsaturated fatty acid substantially.   The selective antibacterial agent with respect to Staphylococcus aureus containing the composition of any one of Claims 1-4.   The skin external preparation containing the composition of any one of Claims 1-4.   A quasi-drug containing the composition according to any one of claims 1 to 4.   Cosmetics containing the composition of any one of Claims 1-4.   (1) a step of releasing fatty acids at the sn-1 and sn-3 positions by lipase from an oil containing triglycerides having palmitoleic acid at the sn-1 and sn-3 positions; and (2) free in the above step. The manufacturing method of the fatty acid composition which contains the process of collect | recovering the made fatty acid and contains 55 mol% or more of palmitoleic acid.   The fat and oil containing triglyceride having palmitoleic acid at the sn-1 position and the sn-3 position shows the sn-1 position and the sn-3 from the content ratio of palmitoleic acid and oleic acid in the whole triglyceride (palmitoleic acid / oleic acid). The production method according to claim 9, wherein the content ratio of palmitoleic acid and oleic acid in the position (palmitoleic acid / oleic acid) is larger.   The manufacturing method according to claim 9 or 10, wherein the oil containing a triglyceride having palmitoleic acid at the sn-1 position and the sn-3 position is an oil produced by Saccharomyces cerevisiae.   The production method according to claim 11, wherein Saccharomyces cerevisiae is a transformant overexpressing a N-terminal deletion gene of diacylglycerol acyltransferase.   The production method according to any one of claims 9 to 12, wherein the lipase is a lipase that preferentially liberates the fatty acid at the sn-1 position and the sn-3 position of the triglyceride.   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. 14. The production method according to 13.   The production method according to claim 14, wherein the step of liberating the sn-1 and sn-3 fatty acids with a lipase derived from a microorganism belonging to the genus Pseudozyma is performed in the presence of an excess amount of ethanol.   The manufacturing method according to any one of claims 9 to 12, wherein the lipase is a lipase that specifically releases fatty acids at the sn-1 and sn-3 positions of triglycerides.   The production method according to claim 16, wherein the lipase that specifically releases the fatty acid at the sn-1 position and the sn-3 position of the triglyceride is a lipase derived from a microorganism belonging to the genus Rhizopus.   The production method according to any one of claims 9 to 12, wherein the lipase is a lipase that releases palmitoleic acid preferentially over oleic acid.   The production method according to claim 18, wherein the lipase that releases palmitoleic acid preferentially over oleic acid is a lipase derived from a microorganism belonging to the genus Candida.