JP2022190142A - Methods for producing lipid and fatty acid composition, and fatty acid composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fatty acid composition useful as a selective antibacterial agent that contains a high proportion of palmitoleic acid, inhibits Staphylococcus aureus but does not inhibit Staphylococcus epidermidis, namely an antibacterial agent that keeps the balance of skin indigenous flora.

SOLUTION: A novel transformant of yeast is prepared. From lipids produced by the transformant, a fatty acid composition is obtained that contains a high proportion of palmitoleic acid, with a low content of fatty acids having 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid).

SELECTED DRAWING: None

COPYRIGHT: (C)2023,JPO&INPIT

Description

The present invention relates to novel methods for producing lipid and fatty acid compositions and fatty acid compositions, and also novel transformed strains of Saccharomyces cerevisiae useful for producing the lipid and fatty acid compositions, and the The present invention relates to external skin preparations, quasi-drugs and cosmetics containing a 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 (mutations in the filaggrin gene, etc., family history, history of allergic diseases, tendency to produce IgE antibodies, etc.), imbalance of the epidermal flora, nutritional factors (high linoleic It develops due to multiple factors such as acid edible oil and foods made from it) 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, etc. produced by Staphylococcus aureus stimulate the immune system and are involved in exacerbation of inflammation. Furthermore, it has been elucidated that V8 protease produced by Staphylococcus aureus destroys the barrier function of the epidermis, allowing this bacterium to enter the skin and aggravates atopic dermatitis (non Patent document 3).

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. The Staphylococcus epidermidis is a beneficial bacterium, and in recent years, it has been attracting attention as a skin-beautifying bacterium in the cosmetics industry and the like.

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.
Macadamia nut oil contains about 20% by weight of palmitoleic acid, but also contains 50% by weight or more of oleic acid. The present inventors have found that oleic acid inhibits the antibacterial activity of palmitoleic acid against Staphylococcus aureus (Japanese Patent Application 2017-034097), and macadamia nut oil is not practical.
Sardine oil contains about 10% by weight of palmitoleic acid, and it is possible to purify it as palmitoleic acid ethyl ester from the by-product of refining eicosapentaenoic acid (EPA) ethyl ester by precision distillation or the like. It is not suitable for cosmetic applications, as it is a raw material of and is not favored by plant-oriented consumers.
In addition, seaberry pulp oil and other vegetable oils, such as linoleic acid and linoleic acid, have two or more double bonds and have poor oxidation stability, and 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.

On the other hand, a diacylglyceroltransferase gene-disrupted strain of budding yeast (Saccharomyces cerevisiae) was transformed with a gene encoding an N-terminal deleted diacylglyceroltransferase to enhance the ability to produce lipids. Furthermore, a technique for producing a lipid with a high palmitoleic acid content has also been disclosed (Patent Document 2).
However, the lipid produced by the transformed strain of Saccharomyces cerevisiae disclosed in Patent Document 2 contains oleic acid in an amount similar to palmitoleic acid, and for the reasons described above, a selective antibacterial agent against Staphylococcus aureus Not suitable for use as

The present inventors have previously found that lipids derived from microorganisms belonging to the genus Pseudozyma or the genus Moesziomyces were produced using the transformed strain of budding yeast described in Patent Document 2 above. (Japanese Patent Application No. 2017-034097) disclosed a technique for selectively liberating fatty acids at the sn-1,3 positions to increase the content of palmitoleic acid using lipase or the like.
However, in the above technique, the fatty acid at the sn-1,3 positions is liberated and recovered, so the theoretical yield is only 67%.
In addition, the present inventors utilize the fatty acid specificity of lipase derived from microorganisms belonging to the genus Candida to terminate the hydrolysis reaction at a stage where the hydrolysis rate is low, thereby increasing the content of palmitoleic acid. also disclosed, but this method gave low yields of palmitoleic acid.
In terms of yield, the method using lipase or the like derived from microorganisms belonging to the genus Pseudozyma or Moesziomyces is preferable. It required a process.

In addition, 41.2% to 52.9% of workers working in intensive care units (ICUs) of hospitals who repeatedly wash their hands and use alcohol disinfection have Staphylococcus aureus on their hands. has been reported (Non-Patent Documents 4 and 5).
Therefore, workers in food factories, etc., and those engaged in housework at home who frequently wash their hands and disinfect their hands frequently and have rough skin, such as rough hands, may have a yellowish color on their skin, similar to patients with atopic dermatitis. Suggests the possibility of staphylococcal growth.
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.

International Publication No. 2011/011486 JP 2015-146778 A

S. Higaki et al.; International J. Dermatology 38 265 (1999) H. H. Kong et al.; Genome Research 22 8505 (2012) Teruaki Nakatsuji et al.; Journal of Investigative Dermatology 136, 2192-2200 (2016) 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

In the process of examining the use of palmitoleic acid for external skin preparations, the present inventors found that fatty acids with 18 carbon atoms and one double bond, such as oleic acid and vaccenic acid, were found to have antibacterial activity of palmitoleic acid. was found to inhibit As a result of further studies to solve the above problems, the content of fatty acids (oleic acid and vaccenic acid) containing a high content of palmitoleic acid and having 18 carbon atoms and one double bond (oleic acid and vaccenic acid) We have found that a low fatty acid composition is effective as a selective antibacterial agent against Staphylococcus aureus, and have succeeded in obtaining a novel yeast transformant that can be suitably used for the production of the fatty acid composition. As a result, the present invention has been completed.

That is, the present invention relates to the following.
[1] A transformant strain of Saccharomyces cerevisiae having lipogenic ability, comprising a diacylglycerol acyltransferase (DGA1) gene and a Δ9 desaturase (OLE1) gene on the chromosome, or a diacylglycerol acyltransferase (DGA1) gene, protein phosphatase methyltransferase 1 (PPM1) gene, and Δ9 desaturase (OLE1) gene are disrupted or impaired, and the diacylglycerol acyltransferase gene, palmitoleate synthesis-specific Δ9 desaturation A transformed strain in which the enzyme gene and cytochrome _b5 gene are overexpressed.
[2] The overexpressed diacylglycerol acyltransferase gene is a gene encoding a protein lacking the N-terminus of Saccharomyces cerevisiae diacylglycerol acyltransferase. Transformed strain.
[3] The overexpressed palmitoleic acid synthesis-specific Δ9 desaturase gene is the Caenorhabditis elegans Δ9 desaturase (cFAT5) gene or mouse Δ9 desaturase (mSCD3) gene, the transformant of [1] or [2].
[4] The transformed strain according to any one of [1] to [3], wherein the overexpressed cytochrome b5 gene is the cytochrome b5 _{( CYB5} ) gene of Saccharomyces cerevisiae.
[5] Furthermore, the leucine synthase (LEU2) gene is overexpressed, [1]-[4
], the transformed strain according to any one of the above.
[6] A fatty acid (C18:1) (oleic acid and vaccenic acid) containing 55% by weight or more of palmitoleic acid and having 18 carbon atoms and one unsaturated bond based on the total amount of fatty acid residues in the lipid The transformant strain according to any one of [1] to [5], which is a transformant strain capable of producing a lipid containing 10% by weight or less of these in total.
[7] Culturing the transformed strain of Saccharomyces cerevisiae according to any one of [1] to [6] in the presence of methionine, and collecting lipids from the cultured cells , a method for the production of lipids.
[8] The lipid contains 55% by weight or more of palmitoleic acid and has 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaxene The production method according to [7], wherein the lipid contains a total of 10% by weight or less of acid).
[9] A method for producing a fatty acid composition, which comprises converting the lipid produced by the production method according to [7] or [8] into a free fatty acid.
[10] The fatty acid composition contains 55% by weight or more of palmitoleic acid and a fatty acid (C18:1) having 18 carbon atoms and one unsaturated bond (oleic acid and vaccenic acid) in total The production method according to [9], which is a fatty acid composition containing 10% by weight or less.
[11] The production method according to [10], wherein the fatty acid composition contains 60% by weight or more of palmitoleic acid.
[12] Containing 55% by weight or more of palmitoleic acid and containing 10% by weight or less in total of fatty acids having 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid) , the fatty acid composition.
[13] The composition according to [12], wherein the content of palmitoleic acid is 60% by weight or more. [14] The composition according to [12] or [13], containing 1% by weight or less of polyunsaturated fatty acids.
[15] A selective antibacterial agent against Staphylococcus aureus, containing the composition according to any one of [12] to [14].
[16] An external skin preparation containing the composition according to any one of [12] to [14].
[17] A quasi-drug containing the composition according to any one of [12] to [14].
[18] Cosmetics containing the composition according to any one of [12] to [14].

According to the present invention, the content of palmitoleic acid is high and the content of fatty acids (oleic acid and vaccenic acid) having 18 carbon atoms and one double bond that reduces the antibacterial activity of palmitoleic acid is low. A new transformant strain of yeast can be obtained that is useful for producing a fatty acid composition.
By using the transformant strain, a lipid suitable for producing the fatty acid composition can be obtained, and the fatty acid composition can be obtained efficiently and easily.
The fatty acid composition of the present invention is a selective antibacterial agent that has selective antibacterial activity against Staphylococcus aureus, inhibits Staphylococcus aureus, and does not inhibit Staphylococcus epidermidis, namely As an antibacterial agent that maintains the balance of resident epidermal flora, as an external skin preparation, quasi-drug or cosmetic for preventing aggravation of symptoms of atopic dermatitis, or improving symptoms of atopic dermatitis, It can be preferably used.
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. 1 is a diagram showing the inhibitory effect of oleic acid on the antibacterial activity of palmitoleic acid in Test Example 4. FIG. FIG. 2 is a diagram showing the inhibitory effect of vaccenic acid on the antibacterial activity of palmitoleic acid in Test Example 4. FIG.

The present invention provides a novel transformant strain of Saccharomyces cerevisiae having lipogenic ability (hereinafter sometimes referred to as "the transformant strain of the present invention").
The transformant strain of the present invention is a transformant strain of Saccharomyces cerevisiae having the ability to produce lipids, comprising a diacylglycerol acyltransferase (DGA1) gene and a Δ9 desaturase (OLE1) gene on the chromosome, or the diacylglycerol acyltransferase (DGA1) gene, protein phosphatase methyltransferase 1 (PPM1) gene and Δ9 desaturase (OLE1) gene are disrupted or impaired, and the diacylglycerol acyltransferase gene, palmitoleic acid synthesis A specific Δ9 desaturase gene, the cytochrome _b5 gene, is overexpressed.

As Saccharomyces cerevisiae having lipid-producing ability, any yeast including natural baker's yeast and wine yeast may be used as long as it belongs to Saccharomyces cerevisiae. Considering the high efficiency of transformation and ease of handling, laboratory yeast standard strains with selectable markers such as auxotrophs, which are established culture strains and are widely used as parent strains for transformation, or the strains It is preferable to use the transformed strain used. Typical auxotrophic standard strains include BY4741 strain, S288C strain, W303 strain, etc. It is particularly preferable to use transformant strains genetically engineered to enhance lipid-producing ability using these standard strains as hosts. .
As used herein, the term "lipid" refers to a compound that is insoluble in water and soluble in organic solvents and to which fatty acids are covalently bonded.

Disruption or decreased function of the diacylglycerol acyltransferase (DGA1) gene, Δ9 desaturase (OLE1) gene, and protein phosphatase methyltransferase 1 (PPM1) gene on the chromosome means that the expression of these genes is constantly inhibited, Alternatively, it means that the expression is suppressed.
In the above-described standard strain or its transformant, the diacylglycerol acyltransferase (DGA1) gene and Δ9 desaturase (OLE1) gene on the chromosome, or the diacylglycerol acyltransferase (DGA1) gene, protein phosphatase methyltransferase 1 (PPM1) ) gene and the Δ9 desaturase (OLE1) gene can be used as a method for disrupting or lowering the function of the gene, a general method for disrupting or lowering the function of the gene in Saccharomyces cerevisiae , Preferably, it can be performed by the method described in Kanzaka et al. (JP 2014-054239).

For example, DNA is prepared by adding the sequences of the upstream region and the downstream region of the target gene to be disrupted to both ends of the marker gene, and the host is transformed with the DNA, whereby the introduced DNA can be disrupted. Homologous recombination can occur in the upstream and downstream regions of the target gene to disrupt the target gene. Mutants in which the target gene has been disrupted can be identified by phenotypic changes due to the expression of the marker gene.
In addition, after isolating the gene to be disrupted, it is cleaved with an appropriate restriction enzyme, and a marker gene is inserted into the cleaved site to transform using a fragment obtained. A method of amplifying by polymerase chain reaction (PCR) using a primer containing a portion and transforming, a method of generating random mutations with a mutagen or the like, and a method of screening for mutations in the target gene, etc. can be used. .
Examples of marker genes include genes that complement auxotrophic mutations such as amino acids and antibiotic drug resistance genes. It preferably contains a vector with the LEU2 gene encoding isopropylmalate dehydrogenase or a gene of other organisms homologous thereto as a marker. When several types of plasmids coexist, any marker gene other than LEU2 can be used as long as the transformed strain of the microorganism to be expressed can be selected. and the URA3 gene, etc. When LEU2 is not used as a marker gene, lipid production can be achieved by expressing LEU2 or other enzyme genes of the leucine synthesis pathway and genes homologous to these in other organisms in vectors having other marker genes. It can contribute to the improvement of sexuality.

Overexpression of the diacylglycerol acyltransferase gene, palmitoleic acid synthesis _- specific Δ9 desaturase gene, and cytochrome b5 gene means that the expression of these genes is higher than that in Saccharomyces cerevisiae before transformation. is enhanced and the expression of proteins encoded by these genes is increased.
For the purposes of the present invention, the diacylglycerol acyltransferase gene is preferably a protein lacking the N-terminus of Saccharomyces cerevisiae diacylglycerol acyltransferase (DGA1p), more preferably the N-terminal side. A protein lacking any amino acid sequence from the 2nd to the 37th, more preferably, a protein lacking the sequence from the 2nd to any of the 29th to 37th amino acids on the N-terminal side and genes encoding

The palmitoleic acid synthesis-specific Δ9 desaturase gene is a gene encoding a Δ9 desaturase capable of specifically synthesizing palmitoleic acid, and is a type of nematode, Caenorhabditis elegans. elegans) palmitoleate synthesis-specific Δ9 desaturase (cFAT5) or mouse palmitoleate synthesis-specific Δ9 desaturase (mSCD3).

As the cytochrome b5 gene, a gene encoding cytochrome b5 _{( CYB5} ) of Saccharomyces cerevisiae and the like are preferably used.

In order to overexpress the diacylglycerol acyltransferase gene, the palmitoleic acid synthesis _- specific Δ9 desaturase gene, and the cytochrome b5 gene, according to the usual transformation method of Saccharomyces cerevisiae, on the chromosome Disruption of the diacylglycerol acyltransferase (DGA1) and Δ9 desaturase (OLE1) genes, or the diacylglycerol acyltransferase (DGA1), protein phosphatase methyltransferase 1 (PPM1) and Δ9 desaturase (OLE1) genes Transformation of a standard strain of Saccharomyces cerevisiae or a transformed strain thereof that has been modified or reduced in function is carried out.
The above transformation may be performed by incorporating the diacylglycerol acyltransferase gene, the palmitoleic acid synthesis-specific Δ9 desaturase gene, and the cytochrome _b5 gene into a vector that replicates extrachromosomally, or a vector that integrates into the chromosome. However, it is preferable to use a plasmid containing the gene.

From the viewpoint of lipid productivity, it is preferable that the LEU2 gene, which encodes leucine synthase, is overexpressed as described above.

The transformant strain of Saccharomyces cerevisiae described above, that is, the diacylglycerol acyltransferase (DGA1) gene and Δ9 desaturase (OLE1) gene on the chromosome, or the diacylglycerol acyltransferase (DGA1) gene, protein phosphatase The methyltransferase 1 (PPM1) gene and the Δ9 desaturase ( _OLE1 ) gene are disrupted or functionally impaired, and the diacylglycerol acyltransferase gene, palmitoleic acid synthesis-specific Δ9 desaturase gene, cytochrome b5 A novel transformant strain of Saccharomyces cerevisiae in which the gene is overexpressed contains a high content of palmitoleic acid described later and a fatty acid having 18 carbon atoms and one unsaturated bond ( C18:1) (oleic acid and vaccenic acid) with the ability to produce lipids substantially free.

Therefore, the present invention provides a method for producing a lipid using the novel transformant strain of Saccharomyces cerevisiae described above (hereinafter also referred to as a "method for producing a lipid of the present invention"). offer.
The method for producing lipids of the present invention comprises culturing the novel transformant described above in the presence of methionine and collecting lipids from the cultured cells.

Cultivation of novel transformant strains of Saccharomyces cerevisiae can be performed according to the method described in Kanzaka et al. (JP 2014-054239 A).
For example, it can be cultured at 15° C. to 30° C., preferably 18° C. to 27° C., for 7 to 14 days, preferably 7 to 10 days, using SD medium or the like.
As the transformant strain, it is preferable to transform using the methionine-auxotrophic strain BY4741 or the like as a standard strain. It is more preferable to carry out in the presence of L concentration of methionine.
By culturing the novel transformant strain of Saccharomyces cerevisiae under the medium and culture conditions described above, lipids are accumulated in the cells. In the lipid production method of the present invention, the cultured cells are collected by centrifugation, ultrafiltration, etc., crushed in an organic solvent such as chloroform, methanol, etc. The organic solvent can be used to extract, separate and purify the produced lipids.

The lipid produced by the production method of the present invention contains a high content of palmitoleic acid and has 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid). is 10% by weight or less.
Here, "contains a high content of palmitoleic acid" means that a large amount of palmitoleic acid is contained as a fatty acid residue contained as a constituent of the lipid. The content of palmitoleic acid is 55% by weight or more, preferably 60% by weight or more, based on the total amount of fatty acid residues.
Here, palmitoleic acid is a C16 unsaturated fatty acid ((Z)-9-hexadecenoic acid) having one double bond at the 9-position.
Considering the lipid-producing ability of the transformed strain of Saccharomyces cerevisiae used in the production method of the present invention, the content of palmitoleic acid in the lipid produced by the production method of the present invention is usually 80%. % by weight or less.
In addition, the lipid produced by the production method of the present invention has a reduced content of fatty acids (C18:1) having 18 carbon atoms and one unsaturated bond (oleic acid and vaccenic acid). The total content is 10% by weight or less, preferably 6% by weight or less, more preferably 3.5% by weight or less, based on the total amount of fatty acid residues in the lipid.
Here, oleic acid is an unsaturated fatty acid having 18 carbon atoms and having one double bond at the 9-position ((Z
)-9-octadecenoic acid), 18:1(n-9)), and vaccenic acid is an unsaturated fatty acid having 18 carbon atoms and having one double bond at the 11-position ((Z)-11-octadecenoic acid ), 18:1(n-7)).
Considering the function of the Δ9 desaturase gene introduced into the transformant strain of Saccharomyces cerevisiae used in the production method of the present invention, the lipid produced by the production method of the present invention has , A fatty acid having 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid) is contained in a slight amount, for example, at least about 0.4% by weight based on the total amount of fatty acid residues in the lipid. can be

The present invention also provides a method for producing a fatty acid composition, which comprises decomposing a lipid produced by the above-described method for producing a lipid of the present invention into free fatty acids (hereinafter referred to as "fatty acid composition of the present invention (also referred to as "method of manufacturing").
The method for decomposing the lipid produced by the lipid production method of the present invention into free fatty acids is not particularly limited as long as it is a method capable of liberating fatty acids mainly from triacylglycerol. and hydrolysis by.
Saponification can be carried out by a method and conditions known per se. For example, using a base such as sodium hydroxide or potassium hydroxide, the saponification is usually performed at 40°C to 90°C for 5 minutes to 60 minutes, preferably 50°C. React for 10-30 minutes at ~75°C.

In the lipid produced by the lipid production method of the present invention, as will be described later, since there is no particular position specificity in the binding site of the fatty acid, lipase can cleave the ester bond between glycerol and free fatty acid. As long as it is a lipase, its origin etc. are not limited, but pancreatic lipase and Candida genus, Diutina genus, Pseudozyma genus, Moesziomyces genus, Alcaligenes are preferred. ), Pseudomonas, Burkholderia, Rhizopus, Rhizomucor, Thermomyces, etc. can be used.
In the present invention, the lipase 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 amount of lipase to be added is usually 0.01 to 10 parts by weight, preferably 0.03 to 1.5 parts by weight, per 100 parts by weight 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 weight, more preferably 5% to 95% by weight. . For immobilized enzymes, water may or may not be added.
Further, hydrolysis with lipase is usually carried out at 10° C. to 60° C. for 5 minutes to 24 hours, preferably at 30° C. to 40° C. for 10 minutes to 120 minutes, usually pH=4 to 9, preferably pH=5 to 7.
In addition, it is not necessary to adjust the pH with a buffer solution or the like, and water, lipid and lipase may be simply mixed.
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 an aqueous potassium hydroxide solution to the reaction solution to saponify the free fatty acids and transferring them to the aqueous layer, followed by extraction with a non-polar organic solvent such as n-hexane. can be removed.

The fatty acids liberated by the above saponification, hydrolysis with lipase, etc. are (1) separated by column chromatography using a silica gel column or the like, and (2) an alkaline agent such as an aqueous potassium hydroxide solution is added to the reaction solution to saponify the free fatty acids. After transferring to the aqueous layer, unreacted triglycerides are removed by extraction with a low-polarity organic solvent such as n-hexane, etc., acid such as hydrochloric acid is added to the aqueous layer to make it acidic, and then n-hexane. A method of extracting with a low-polarity organic solvent such as , and removing the solvent by evaporation; can.

In each step of the method for producing fatty acids of the present invention, purification means such as recrystallization and various types of chromatography, solid-liquid separation means such as filtration and centrifugation, and the like, as appropriate and necessary, within a range that does not impair the characteristics of the present invention. etc. can be used.

Fatty acid (C18:1) (oleic acid and vaccenic acid) containing 55% by weight or more of palmitoleic acid and having 18 carbon atoms and one unsaturated bond (oleic acid and vaccenic acid) is produced by the above-described method for producing fatty acids of the present invention. Fatty acid compositions containing up to 10% by weight in total can be produced.

Therefore, the present invention contains 55% by weight or more of palmitoleic acid, a fatty acid (C18: 1) having 18 carbon atoms and one unsaturated bond (oleic acid and vaccenic acid), and 10 weights in total of these % or less (hereinafter also referred to as the "fatty acid composition of the present invention" in this specification).

The fatty acid composition of the present invention preferably contains 60% by weight or more, more preferably 65% by weight or more of palmitoleic acid.
Considering the efficiency of conversion to free fatty acids in the production method of the present invention, the content of palmitoleic acid in the fatty acid composition of the present invention is usually 80% by weight 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 has 18 carbon atoms and one unsaturated bond (C18: 1) (oleic acid and vaccenic acid), which suppresses the antibacterial activity of palmitoleic acid. The total content of fatty acids (C18:1) (oleic acid and vaccenic acid) having one is 10% by weight or less, preferably 6% by weight or less, and 3.5% by weight or less. It is more preferable to have
In addition, considering the function of the Δ9 desaturase gene introduced into the transformant strain of Saccharomyces cerevisiae used in the production method of the present invention, the fatty acid composition of the present invention has a carbon number of Fatty acids having one unsaturated bond at 18 (C18:1) (oleic acid and vaccenic acid) are contained in small amounts, for example, at least about 0.4% by weight in total.

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% by weight).

As described above, the fatty acid composition of the present invention contains 55% by weight 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 has a high content of palmitoleic acid having selective antibacterial activity against Staphylococcus aureus (55% by weight or more based on the total amount of the fatty acid composition), and the above selection of palmitoleic acid The content of fatty acids with 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid) that inhibits the antibacterial activity is low (the total of these is 10% by weight or less), so it is atopic. It suppresses the growth of Staphylococcus aureus, which is involved in exacerbation of dermatitis, but does not suppress the growth of Staphylococcus epidermidis, which is a resident bacterium of healthy skin, and Staphylococcus epidermidis. 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 (disodium calcium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, 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% to 10% by weight as the content of palmitoleic acid with respect to the total amount of the antibacterial agent of the present invention, and 0.005 It is preferably from 0.01% to 0.5% by weight, more preferably from 0.01% to 0.5% by weight.

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. , preferably 0.1 μg/cm ² to 50 μg/cm ² , more preferably 0.2 μg/cm ² to 10 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.

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

[Examples 1 to 3, Comparative Examples 1 to 3] Preparation of transformants of Saccharomyces cerevisiae having lipid production ability (1) DGA1 gene and OLE1 gene disrupted strains, or DGA1 gene, PPM1 gene and OLE1 Generation of gene-disrupted strains

In the Saccharomyces cerevisiae strain BY4741Δdga1 (Mat a leu2Δ0 his3 Δ1 ura3 Δ0 met15 Δ0 dga1::kanMX4) (Invitrogen Life Technologies), the protein phosphatase methyltransferase (PPM1) gene and the Δ9 deficiency were identified. Disruption of the saturase (OLE1) gene was performed by replacing the entire open reading frame (ORF) with the auxotrophic marker gene URA3 gene and/or LEU2 gene according to the method of Kanzaka et al. went.

A URA3 cassette that disrupts the PPM1 gene was constructed as follows.
Using a plasmid (pT-URA3) cloned from the promoter region to the terminator region of the URA3 gene as a template, 5'-TCCTTGTGACTCCGCATAAACTAGATGATAAAGAGTACAAACAAGTCGCCCGGTAATCTCCGAGCAG-3' (SEQ ID NO: 1) and 5'-ATAAACGGTAAGCATATTAAGATCAAATTAGTTGAGGCTGTAAATAAAAAACGACCGAGATTCCCGG-3' (SEQ ID NO: 2) were used as primers. As
Amplification was performed by PCR using KOD plus (Toyobo Co., Ltd.), which is a DNA polymerase with high replication fidelity. PCR was carried out in a buffer solution containing 0.25 μM primer concentration and 1.2 mM magnesium chloride, followed by reaction at 94° C. for 2 minutes, followed by 94° C. (15 seconds)/55° C. (30 seconds)/68° C. (90 seconds) as one cycle, and was repeated 30 times. The amplified construct is PCR purification kit (Qiagen Co., Ltd.)
and introduced into Saccharomyces cerevisiae BY4741Δdga1 strain using a yeast transformation kit (Invitrogen Life Technologies) to prepare a Δdga1Δppm1 disruption strain.
Disruption of the PPM1 gene was performed using two primers outside the ORF of the PPM1 gene (5'-AAAGAAGATGGGTCAGGG-3' (SEQ ID NO: 3) and 5'-GTAGTGTAAGTACAGGTA-3' (SEQ ID NO: 4)), PCR (“GeneTaq”, Nippon Gene Co., Ltd.) was performed using genomic DNA extracted with an extraction reagent (“ISOPLANT”, Nippon Gene Co., Ltd.) as a template for confirmation.
PCR was carried out with a primer concentration of 0.5 μM, in the attached buffer, after reacting at 95 ° C. for 2 minutes, 95 ° C. (30 seconds) / 40 ° C. (30 seconds) / 72 ° C. (90 seconds) The cycle was repeated 25 times. The resulting product was separated by 0.7 (w/v) agarose gel electrophoresis, but it was difficult to distinguish the amplified product from the presence or absence of disruption of the PPM1 gene. Nippon Gene), separated on a 2 (w/v) % agarose gel, and confirmed by low-molecular-weight degradation products of the wild-type strain and the disrupted strain.
A strain resistant to 5-fluoroorotic acid (5-FOA, Wako Pure Chemical Industries, Ltd.) was further obtained from the resulting Δppm1-disrupted strain. The resulting resistant strain was confirmed to be auxotrophic for uracil, and transformation with pL1091-5 (a plasmid complementing uracil auxotrophy) was made possible.

Also, the LEU2 cassette that disrupts the OLE1 gene was constructed as follows. A plasmid cloned from the promoter region to the terminator region of the LEU2 gene (pT-LEU2
) as a template, 5′-GATAGTTGTGGTGATCATATTATAAACAGCACTAAAACATTACAACAAAGCAGGTATCGTAAGATGC-3′ (SEQ ID NO: 5) and 5′-TTTCAATTTTTTTTTATGGTAGTTGCAGTTTTGTTATTGTAATGTGATACGTTGAGCCATTAGTATC-3′ (SEQ ID NO: 6) as primers, KODplu
It was amplified by PCR using s (Toyobo Co., Ltd.).
PCR was carried out in a buffer solution containing 0.25 μM primer concentration and 1.2 mM magnesium chloride, followed by reaction at 94° C. for 2 minutes, followed by 94° C. (15 seconds)/55° C. (30 seconds)/68° C. (120 seconds) as one cycle, and was repeated 30 times. The amplified construct was purified with a PCR purification kit (Qiagen Co., Ltd.) and transformed into Saccharomyces cerevisiae BY4741Δdga1 strain or the above using a yeast transformation kit (Invitrogen Life Technologies). was introduced into the Δdga1Δppm1-disrupted strain of , and Δdga1Δole1 double-disrupted strains or Δdga1Δppm1Δole1 triple-disrupted strains were generated.
Disruption of the OLE1 gene was extracted from the disrupted strain using two primers outside the ORF of OLE1 (5'-AATAGATAGTTGTGGTGATC-3' (SEQ ID NO: 7) and 5'-GGTAGTTGCAGTTTTGTTAT-3' (SEQ ID NO: 8)). It was confirmed by PCR (“GeneTaq”, Nippon Gene Co., Ltd.) using the obtained genomic DNA as a template. PCR was performed by adding 5 (v/v)% DMSO to the attached buffer solution with a primer concentration of 0.25 μM, reacting at 95° C. for 2 minutes, and then heating at 95° C. (30 seconds)/45° C. (30 seconds). /72°C (120 seconds) was set as one cycle, and was repeated 30 times. The resulting product was separated by 0.7 (w/v) % agarose gel electrophoresis, and a larger band (1865 bp) than the wild strain (1621 bp) was obtained, confirming that it was a disrupted strain.

The obtained Δdga1Δole1 and Δdga1Δppm1Δole1 strains do not require leucine for growth because the OLE1 gene is disrupted using the LEU2 gene as a selection marker, but they cannot synthesize unsaturated fatty acids, so unsaturated fatty acids are added to the medium. You can't grow without it. Therefore, SD medium containing 10 (w / v) % glucose (100 g / L glucose, 1.7 g / L yeast nitrogen base, no amino acid / ammonium sulfate, 5 g / L ammonium sulfate, 20 mg / L uracil, 60 mg / L Leucine, 20 mg/L histidine, 20 mg/L methionine) was added with 0.25 (w/v) % polyoxyethylene nonylphenyl ether (Tergitol NP-40) and 0.05 (w/v) % oleic acid Screening of these disruptants was performed using the culture medium.

(2) Preparation of a transformant overexpressing a gene encoding diacylglycerol acyltransferase lacking the N-terminus, a palmitoleic acid synthesis _- specific Δ9 desaturase gene and a cytochrome b5 gene

(I) According to the method of Kansaka et al. (JP 2014-054239 A), the gene encoding the protein (Dga1ΔNp) in which the N-terminal 29 residues of diacylglycerol acyltransferase are deleted is incorporated into the pL1091-5 vector pL1091- 5/DGA1ΔN was produced.

(ii) Δ9 desaturase (cFAT5) gene of Caenorhabditis elegans as the palmitoleic acid synthesis-specific Δ9 desaturase gene,
and the mouse Δ9 desaturase (mSCD3) gene were codon-optimized for expression in Saccharomyces cerevisiae by the OptimumGene™ algorithm (Genscript) for gene synthesis. and obtained as pUC57/cFAT5 and pUC57/mSCD3.
A DNA fragment obtained by digesting pUC57/cFAT5 with BamHI and SacI was subcloned into pL1177-2 to obtain pL1177-2/cFAT5.
For pUC57/mSCD3, 5'-GCAAGCTTATGCCAGGTCACTTATTA-3' (
SEQ ID NO: 9) and 5'-ATGCGGCCGCTTAACCTGATTTATGGGA-3' (SEQ ID NO: 10) as primers were amplified by PCR using KOD plus (Toyobo Co., Ltd.), and the resulting product was cleaved with HindIII and NotI. The DNA fragment was subcloned into pL1177-2 to give pL1177-2/mSCD3.

(iii) The CYB5 gene encoding cytochrome b ₅ is composed of 5′-ATCTCGAGCATGCCTAAAGTTTACAG-3′ (SEQ ID NO: 11) and 5′-ATGCGGCCGCTGGGCTATTCTGGAAGAA-3′ (SEQ ID NO: 12) using Saccharomyces cerevisiae genomic DNA as a template. ) as primers, PCR was performed using KOD plus (Toyobo Co., Ltd.), the resulting product was cleaved with XhoI and NotI, and subcloned into pL2137-26 to obtain pL2137-26/CYB5.

(iv) To each of the Δdga1Δole1 and Δdga1Δppm1Δole1 strains prepared above, according to the method of Kanzaka et al. Transformation was performed using CYB5.
Since it is inefficient to obtain a transformant strain by culturing strains with three kinds of plasmids at once, strains transformed with two kinds of plasmids are obtained first, and then the resulting transformant strains are used as the rest. , to obtain a transformant containing the three plasmids.
The resulting transformants (Δdga1Δole1 + DGA1ΔN, cFAT5, CYB5, Δdga1Δole1 + DGA1ΔN
, mSCD3, CYB5, Δdga1Δppm1Δole1+DGA1ΔN, cFAT5, CYB5) were used as Examples 1-3.

On the other hand, transformants (Δdga1Δole1+DGA1ΔN, OLE1, Δdga1Δole1+DGA1ΔN, cFAT5,
Δdga1Δole1+DGA1ΔN, OLE1, CYB5) were prepared and designated as Comparative Examples 1 to 3, respectively.
The OLE1 gene was introduced by transformation using pL1172-2/OLE1 prepared according to the method of Kanzaka et al. (Patent Document 2).
In addition, transformant strains (Δdga1+DGA1ΔN, Δdga1+DGA1ΔN, cFAT5, Δppm1+DGA1ΔN, Δdga1+DGA1ΔN, PPM1, Δdga1Δppm1+DGA1ΔN) were prepared by introducing the DGA1ΔN gene, cFAT5 gene, and PPM1 gene into the Δdga1 strain, Δppm1 strain, and Δdga1Δppm1 strain, respectively. The cells were cultured in the same manner as in the test example, and the produced lipids were analyzed.
Introduction of the PPM1 gene was carried out using Saccharomyces cerevisiae genomic DNA as a template, 5'-ATGGATCCCCATGGAGAGAATCATAC-3' (SEQ ID NO: 13) and 5'-ATGCGGCCGCTCACCACTGAGCCTTCAT-3' (SEQ ID NO: 14) as primers, using KOD plus. (Toyobo Co., Ltd.), the resulting product was cleaved with BamHI and NotI, and subcloned into pL1177-2 to obtain pL1177-2/PPM1.

[Test Example 1] Analysis of lipids produced by a transformant strain of Saccharomyces cerevisiae 100 g/L glucose, 1.7 g/L yeast nitrogen base, no amino acid/ammonium sulfate, 5 g/L ammonium sulfate, 0.02 g/L or 2 g/L methionine) at 30°C or 20°C, 120 rpm rotary Cultured for 7 days in a shaker.
After culturing, the cells were concentrated by centrifugation (3000 rpm, 5 minutes), and the wet cells collected by filtration were added to the solution in chloroform/methanol (2:1) and the same amount of glass beads (No .06, Toshin Riko Co., Ltd.), crushed for 10 minutes with a homogenizer (Nippon Seiki Co., Ltd.), filtered to remove glass beads, and added saturated saline to the collected extract. Layer distribution was performed, and the lower lipid layer was dehydrated with magnesium sulfate and concentrated to obtain a lipid extract.
Moreover, the analysis of the fatty acid composition was performed as follows. That is, the collected cells were washed with distilled water, the obtained pellets were heated at 105° C. for 3 hours, and the dry weight was measured. Then, 1 mL of 10 (w/v)% hydrochloric acid methanol (prepared by dropping acetyl chloride to 10 (w/v)% in chilled methanol) and 0.5 mL of dichloromethane were added to the dried cells. , 60° C. for 3 hours to produce a fatty acid methyl ester, 250 nmol of heptadecanoic acid methyl ester was added as an internal standard, and 1 mL of hexane and 1 mL of saturated saline were further added to partition into two layers. Fatty acid methyl esters distributed in the hexane layer were measured with a gas chromatograph (“GC-17A”, column; “TC-70”) (Shimadzu Corporation), and the total fatty acid content was calculated from the ratio to the internal standard. and determined the fatty acid composition. Also, the lipid content was determined by dividing the total fatty acid content by the dry weight of the cells.
Table 1 shows the analysis results of the lipids produced by each of the transformed strains of Examples 1-3 and Comparative Examples 1-3.

As shown in Table 1, the transformant of Example 1 in which the DGA1ΔN gene, the cFAT5 gene and the CYB5 gene were introduced into the Δdga1Δole1 strain, and the transformant of Example 2 in which the DGA1ΔN gene, the mSCD3 gene and the CYB5 gene were introduced into the Δdga1Δole1 strain. The transformant strain and the transformant strain of Example 3 in which the DGA1ΔN gene, the cFAT5 gene and the CYB5 gene were introduced into the Δdga1Δppm1Δole1 strain contain a high content of palmitoleic acid and have 18 carbon atoms and one unsaturated bond. It was confirmed that lipids containing almost no fatty acids (C18:1) (oleic acid and vaccenic acid) were produced.
On the other hand, each of the transformed strains of Comparative Examples 1 and 3, in which the OLE1 gene was introduced into the Δdga1Δole1 strain, produced lipids containing a high content of C18:1 (oleic acid and vaccenic acid). In addition, the transformant strain of Comparative Example 2, into which the cFAT5 gene was introduced but the CYB5 gene was not introduced, contained a high content of palmitoleic acid and almost all of C18:1 (oleic acid and vaccenic acid). However, inhibition of yeast growth was observed, and a decrease in lipid production was observed.
In addition, although data are not shown, each of the Δdga1 strain, Δppm1 strain, and Δdga1Δppm1 strain was transformed with the DGA1ΔN gene, cFAT5 gene, and PPM1 gene (Δdga1 + DGA1ΔN, Δdga1 + DGA1ΔN, cFAT5, Δppm1 + DGA1ΔN, Δdga1 + DGA1ΔN, PPM1, Δdga1Δppm1
+DGA1ΔN) contained significant amounts of C18:1 (oleic acid and vaccenic acid) as well as palmitoleic acid.

[Test Example 2] Examination of culture conditions for a transformant strain of Saccharomyces cerevisiae having lipid-producing ability The transformant strain (Δdga1Δppm1Δole1+DGA1ΔN, cFAT5, CYB5) of Example 3 was cultured under the following culture conditions and grown. and effects on lipid production. In addition, except for the culture conditions shown below, culture was performed in the same manner as in Test Example 1, and the dry weight of the cells was measured in the same manner as in Test Example 1, and the total fatty acid content, lipid content, and fatty acid composition were measured. was analyzed.
(1) Medium Nitrogen source-deficient SD medium containing 10 (w/v)% glucose (100 g/L glucose, 1.7 g/L yeast nitrogen base, no amino acid/ammonium sulfate, 5 g/L ammonium sulfate, 0.2 g/L) 02 g/L methionine), nitrogen source-deficient methionine-rich SD medium containing 10 (w/v)% glucose (100 g/L glucose, 1.7 g/L yeast nitrogen base, no amino acid/ammonium sulfate, 5 g /L ammonium sulfate, 2 g/L methionine), nitrogen source-deficient natural medium (100 g/L glucose, 4 g/L yeast extract), nitrogen source-deficient natural medium containing high concentration of methionine (100 g/L glucose, 4 g/L yeast extract, 2 g/L methionine) were used, respectively.
(2) Culture temperature and culture period Culture was performed at 20°C for 4 days, 7 days and 10 days, respectively.

The results are shown in Table 2.

As shown in Table 2, when cultured in SD medium containing 10 (w/v)% glucose deficient in nitrogen sources, both lipid content and palmitoleic acid content increased up to 10 days of culture. It was suggested that 10 days of culture is suitable for extraction. It was also confirmed that the methionine concentration in the medium had little effect.
Natural medium containing yeast extract had lower lipid content than SD medium, but lipids containing high content of palmitoleic acid and little C18:1 (oleic acid and vaccenic acid) The results suggested that culture using a natural medium is also possible.

[Examples 4 and 5] Lipid production using a transformant of Saccharomyces cerevisiae Using the transformant of Saccharomyces cerevisiae of Example 3 (Δdga1Δppm1Δole1+DGA1ΔN, cFAT5, CYB5) , lipids were prepared as follows.
(1) Preculture 50 mL of SD medium (20 g/L glucose, 1.7 g/L yeast nitrogen base, no amino acid/ammonium sulfate, 5 g/L ammonium sulfate, 0.02 g/L methionine) was added to a 300 mL conical flask with baffles. Then, the above transformant was inoculated and cultured with a rotary shaker at 160 rpm at 30° C. for 2 days.
(2) Main culture (i) 10 (w/v)% GSD medium (100 g/L glucose, 1.7 g/L yeast nitrogen base, no amino acid/ammonium sulfate, 5 g/L ammonium sulfate, 0.02 g/L methionine) was added to 3 L, the preculture solution was inoculated, and culture was performed for 12 days at 23° C., 250 rpm, and an air flow rate of 1.5 L/min (Example 4).
(ii) Put 6 L of natural medium (100 g/L glucose, 4 g/L yeast extract) in a 10 L capacity culture apparatus, inoculate the pre-culture solution, and culture for 10 days at 23 ° C., 250 rpm, and aeration rate of 3 L / min. (Example 5).
(3) Lipid extraction and analysis In the main culture of Example 4 above, on the 4th, 7th and 12th days of culture, in the main culture of Example 5, on the 4th day, 7th day and On the 10th day, the cells were collected from each culture medium and lipids were extracted (Examples 4 and 5). Methyl esterification was carried out at 70 ° C. for 5 hours, and "GC-2025AF"(column;"HR-SF-10") (manufactured by Shimadzu Corporation) was used as a gas chromatograph. The dry weight, total fatty acid content, lipid content and fatty acid composition of the cells were determined in the same manner as in .
Table 3 shows the results.

As shown in Table 3, in Example 4 in which the main culture was performed using a 10 (w / v) % GSD medium, the culture was performed for 12 days, and in Example 5 in which the main culture was performed using a natural medium. After 10 days of culture, sufficient lipid production was observed, and a lipid containing palmitoleic acid with a high content exceeding 65% by weight could be obtained. Furthermore, in any of the examples, the content of the fatty acid (18:1) (oleic acid and vaccenic acid) having 18 carbon atoms and one unsaturated bond in the lipid is 6.0% by weight or less, was very low.
Also in Example 5 using the natural medium, although a slight decrease in fatty acid production was observed compared to Example 4 using the GSD medium, favorable production of palmitoleic acid was observed. It was confirmed that by using the transformant strain of the present invention, the target lipid can be produced even when using a natural medium.

[Examples 6 and 7] Production of fatty acid composition (1) Preparation of crude yeast oil The transformed strain of Saccharomyces cerevisiae of Example 3 above was cultured as described in Example 4 above. recovered the body.
200 mL of 0.5 mm to 0.7 mm glass beads and 200 mL of chloroform/methanol (2:1, v/v) were added to 10 g of the collected wet cells and homogenized at 10,000 rpm for 10 minutes ("AM-3 ”, Nippon Seiki Co., Ltd.). After collecting the solvent layer by filtering with filter paper, the precipitate was washed with 100 mL of chloroform/methanol (2:1, v/v) to collect the solvent layer. The two solvent layers were mixed and the solvent was removed to approximately 30 mL using an evaporator. This was extracted three times with 50 mL of hexane, the hexane layer was desolvated, and 1.7 g of crude yeast oil was recovered.

(2) Purification of yeast-derived triglycerides and analysis of fatty acid composition at positions sn-1, 3 and sn-2 of the purified yeast triglycerides Loaded onto silica gel chromatography (2.6 cm × 26 cm) equilibrated with n-hexane:ethyl acetate = 98:2 (volume ratio), 40 mL × 15 bottles of the solvent and hexane:ethyl acetate = 95:5 ( Volume ratio) Elution was performed with 40 mL×15 tubes. 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 triglycerides.

Regarding the purified yeast triglyceride obtained, the fatty acid composition at the sn-1, 3 and sn-2 positions was measured according to the method of Y. Watanabe et al. (J. Oleo Sci., 64, 1193 (2015)) as follows. did.
To 100 mg of the purified yeast triglyceride, 1 g of ethanol and immobilized lipase derived from Pseudozyma antarctica ("Novozyme (No
vozym) 435)” (Novozymes)) 0.044 g was 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 ("WAT051900" (Waters) (Company)), 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 purified triglyceride and sn-2 monoglyceride derived from the triglyceride, and a methanol solution of 28% by weight of sodium methoxide were added and 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 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:
(i) Hold at 150°C for 0.5 minutes (ii) 150°C to 170°C; temperature rise at 4°C/min (iii) 170°C to 195°C; temperature rise at 5°C/min (iv) 195°C Up to 215°C; heated at 10°C/min (v) held at 215°C for 11 minutes

The fatty acid composition (% by weight) at positions sn-1 and 3 was calculated from the fatty acid composition (% by weight) of purified yeast triglycerides (composition of all fatty acids at positions sn-1, 2, and 3) and the fatty acid composition (% by weight) of sn-2 monoglyceride. The results are shown in Table 4.

As shown in Table 4, the purified yeast triglycerides derived from the transformed strain of Saccharomyces cerevisiae of Example 3 contained more palmitoleic acid than conventional vegetable oils and fats, and oleic acid ( C18:1, n-9) and vaccenic acid (C18:1, n-7) were found to be almost absent. Furthermore, 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.
Also, as shown in Table 4, Saccharomyces cerevisiae of Example 3
In purified yeast triglycerides from transformants of S. cerevisiae, there is a large difference between the fatty acid composition of total triglycerides (total fatty acid composition at sn-1,2,3 positions) and that at sn-1,3 positions. was not accepted.
In the lipid produced using the budding yeast transformant described in Patent Document 2, the palmitoleic acid (C16: 1) content at the sn-1 and 3 positions is about twice as high as that at the sn-2 position. , on the other hand, the 3.6-fold higher content of vaccenic acid and oleic acid (C18:1) at the sn-2 position than at the sn-1,3 position indicates that the fatty acid at the sn-1,3 position is selectively liberated. It was necessary to selectively hydrolyze or ethanolyze at the sn-1,3 positions using a lipase that causes the reaction to occur (theoretical yield = 67%) (Japanese Patent Application No. 2017-034097).
However, when a fatty acid composition is produced from purified yeast triglycerides derived from the transformant strain of the present invention, fatty acids can be liberated by saponification decomposition without worrying about the bonding position of the fatty acid residue in the triglyceride. (Theoretical yield = 100%), suggesting that the purified yeast triglycerides are more useful as starting materials for producing the fatty acid compositions of the present invention.

(3) Preparation of fatty acid composition After dissolving 0.9 g of sodium hydroxide in 2.5 mL of water, add 50 mL of ethanol, add 1.7 g of crude yeast oil prepared in (1) above, Saponification was performed at 65° C. for 60 minutes with occasional stirring.
After cooling, 75 mL of water was added and extracted twice with 50 mL of hexane to remove contaminants (squalene, cholesterol, etc.). After adding concentrated hydrochloric acid to the water/ethanol layer until it became acidic, it was extracted twice with 50 mL of hexane, and the solvent was removed by an evaporator to recover 1.24 g of free fatty acids (sn-1, 2, 3 positions). (Example 6).
The fatty acid composition of the resulting fatty acid composition was analyzed by the same method as for the purified yeast triglycerides. Table 5 shows the results.

As shown in Table 5, the fatty acid composition of Example 6 contains palmitoleic acid at a content close to 70% by weight, and the content of oleic acid and vaccenic acid is very small (3.5% in total). 4% by weight).

[Test Example 3] Evaluation of antibacterial activity of fatty acid composition The following sample containing the fatty acid composition of Example 6 was subjected to the following minimum inhibitory concentration (minimum inhibitory concentration) according to the standard method of the Japanese Society of Chemotherapy, the liquid dilution method. concentration, MIC) was measured to evaluate the antibacterial activity.

(1) Samples to be tested The samples evaluated for antibacterial activity are as follows.
(i) Fatty acid composition of Example 6 (ii) Fatty acid composition obtained by liberating fatty acids at sn-1 and 3 positions with lipase from the purified triglyceride derived from the transformant strain of Example 3 described above (Example 7 fatty acid composition)
(iii) by the method of Kanzaka et al. (Patent Document 2), sn-1, sn-1, Fatty acid composition obtained by liberating fatty acid at position 3 (referred to as fatty acid composition of Comparative Example 4)
Specifically, 2 g of ethanol and 0.088 g of Novozym 435 (Novozymes) were added to 200 mg of the purified yeast triglyceride, reacted at 30° C. for 2.5 hours with reciprocating shaking, filtered, and filtered. Clear was collected. Then, the solvent was removed by an evaporator, and the Sep-Pack silica cartridge ("WAT051900" (Waters) equilibrated with n-hexane: diethyl ether = 8: 2 (volume ratio) in halves. (Company)), and the fatty acid ethyl ester was eluted with 10 mL of the solvent.
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 collected, the solvent was removed by an evaporator, and the free fatty acid was collected and obtained.
(iv) A fatty acid composition (the fatty acid composition of Comparative Example 5 and do)
That is, 3 g of ethanol, 0.1 mL of water and 0.1 g of 5M sodium hydroxide were added to 50 mg of the purified yeast triglyceride, and saponified by heating at 60° C. for 15 minutes. After cooling to room temperature, 4 mL of water was added, 2M hydrochloric acid was added until acidified, and the mixture was extracted twice with 3 mL of n-hexane. The hexane layer was collected, the solvent was removed by an evaporator, and the free fatty acid was collected and obtained.
(v) Reagent palmitoleic acid (9-cis-C16:1, Tokyo Chemical Industry Co., Ltd.)
(vi) reagent sapienic acid (6-cis-C16:1, Kanto Kagaku Co., Ltd.)
(vii) dimethyl sulfoxide (DMSO) used as solvent

(2) Preparation of Test Sample Solution Each of the above fatty acid compositions (i) to (vi) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10,000 μg/mL.

(3) Test strains The test strains evaluated for antibacterial activity are as follows.
(i) Staphylococcus aureus subsp. aureus NBRC100910 strain (Type strain)
aureus NBRC13276 strain (iii) Staphylococcus aureus subsp. aureus NBRC14462 strain (iv) Staphylococcus aureus subsp. aureus NBRC12732 strain (v) Staphylococcus aureus subsp.
(vi) Staphylococcus aureus subsp. aureus JCM8702 strain (MRSA strain)
(vii) Staphylococcus aureus subsp. aureus JCM16544 strain (MRSA strain)
(viii) Staphylococcus aureus subsp. aureus JCM16545 strain (MRSA strain)
(ix) Staphylococcus aureus subsp. aureus JCM16546 strain (MRSA strain)
(x) Staphylococcus epidermidis NBRC100911 strain (Type strain)
(xi) Staphylococcus epidermidis NBRC12933 strain (xii) Staphylococcus epidermidis ATCC35984 strain

(4) Preparation of test strain suspension B. Medium (0.5% by weight bonito extract (Wako Pure Chemical Industries, Ltd.), 1% by weight polypeptone (Nippon Pharmaceutical Co., Ltd.), 0.5% by weight 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 was added to fresh N. cerevisiae so that the inoculum amount was 10% by weight. B. 3.6 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. culture medium, 2.0
^N.O. B. A test strain suspension was prepared by dilution with medium (pH=6.0).

(5) Measurement of Minimum Inhibitory Concentration (MIC) Dispense 234 μL of the test strain suspension (2.0×10 ⁴ cfu/mL) into the 2nd row of a 96-well round-bottom microplate, then into the 3rd to 12th rows. was dispensed in 130 μL portions. 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 MIC was taken as the lowest concentration of the test sample at which the
Table 6 shows the measurement results of MIC.

As shown in Table 6, sn-1,
The fatty acid composition obtained by selectively liberating the fatty acid at the 3-position (fatty acid composition of Comparative Example 4) was obtained by liberating the fatty acids at the sn-1, 2, and 3-positions by saponifying and decomposing the fat. Compared to the fatty acid composition (fatty acid composition of Comparative Example 5), it has a high antibacterial activity against Staphylococcus aureus, and the MIC against S. aureus NBRC13276 strain, IID1677 strain, JCM16546 strain, etc. is at least one digit lower. there were.
However, the fatty acid composition of Comparative Example 4 contains a high content of palmitoleic acid, but also contains a fatty acid (C18:1) having 18 carbon atoms and one unsaturated bond (oleic acid and vaccenic acid). Therefore, for the S. aureus NBRC14462 strain, which exhibits a strong inhibitory effect of contaminating C18:1 (oleic acid and vaccenic acid) on the antibacterial activity of palmitoleic acid,
No antibacterial activity was observed.
On the other hand, since the fatty acid composition of Example 6 of the present invention hardly contains C18:1 (oleic acid and vaccenic acid), good antibacterial activity was also observed against S. aureus NBRC14462 strain. The MIC of the fatty acid composition of Example 6 of the present invention was lower than that of the fatty acid composition of Comparative Example 4 by one digit or more. Against S. aureus IID1677 strain and JCM8702 strain, the fatty acid composition of Example 6 of the present invention exhibited 4 to 8 times higher antibacterial activity in MIC value than the fatty acid composition of Comparative Example 4 above. Indicated.

Further, a fatty acid composition (fatty acid composition of Example 7) obtained by selectively liberating fatty acids at the sn-1,3 positions with lipase from the oil produced by the transformant strain of Example 3 of the present invention. showed almost the same MIC as the fatty acid composition of Example 6 obtained by liberating the fatty acids at the sn-1,2,3 positions by saponifying and decomposing the fats and oils.
That is, in the lipid (triglyceride) produced by the transformant strain of Saccharomyces cerevisiae of the present invention, the binding site of palmitoleic acid has no positional specificity, and the sn-1 and 3 positions are converted using lipase. It was shown that there is no need for selective release, suggesting that the use of the lipid produced by the transformant strain of the present invention can omit the hydrolysis step with lipase and improve the yield. was done.

Furthermore, as shown in Table 6, against the three strains of S. epidermidis, the MICs of each fatty acid composition of Examples 6 and 7 of the present invention were as high as 500 μg/mL or 1000 μg/mL, with most It was found not to exhibit antibacterial activity.

From the above results of Test Example 3, each of the fatty acid compositions of Examples 6 and 7 of the present invention suppresses the growth of a wide range of strains of S. aureus, which is a bad bacterium, and suppresses the growth of a wide range of strains of S. epidermidis, which is a good bacterium. It was shown that it does not inhibit growth, suggesting that it has an excellent function of restoring healthy skin flora.

[Test Example 4] Examination of the inhibitory effect of oleic acid and vaccenic acid on the antibacterial activity of palmitoleic acid The inhibitory effects of oleic acid and vaccenic acid on the antibacterial activity of palmitoleic acid against Staphylococcus aureus are described below. investigated.

(1) Test sample (i) Palmitoleic acid and oleic acid (9-cis-C18:1, Tokyo Chemical Industry Co., Ltd.), palmitoleic acid: oleic acid (weight ratio) = 100: 0, 90: 10, Samples containing palmitoleic acid and oleic acid were prepared by mixing at respective mixing ratios of 80:20, 67:33, 50:50, 33:67 and 20:80.
(ii) palmitoleic acid and vaccenic acid (11-cis-C18:1), palmitoleic acid: vaccenic acid (weight ratio) = 100:0, 90:10, 80:20, 67:33, 50:50; Samples containing palmitoleic acid and vaccenic acid were prepared by mixing at respective mixing ratios of 33:67 and 20:80.
In addition, vaccenic acid was prepared by saponifying and decomposing vaccenic acid ethyl ester (Tokyo Kasei Kogyo Co., Ltd.) in the same manner as in Example 6 above to convert it into free fatty acids.

(2) Preparation of test sample solution The samples of each mixing ratio prepared in (i) and (ii) above were prepared by dissolving them in DMSO so that the total fatty acid concentration was 40,000 μg/mL.

(3) Test strains The following strains were used as Staphylococcus aureus.
(i) Staphylococcus aureus subsp. aureus NBRC100910 strain (Type strain)
aureus NBRC13276 strain (iii) Staphylococcus aureus subsp. aureus NBRC14462 strain (iv) Staphylococcus aureus subsp. aureus NBRC12732 strain (v) Staphylococcus aureus subsp.

(4) Measurement of MIC A test strain suspension was prepared in the same manner as in Test Example 3, and the MIC was measured. However, 234 μL of the test bacterial suspension was dispensed into the first well of the microplate, and 130 μL of each was dispensed into the wells of the second to twelfth rows. After 26 μL of the test sample solution was added to the test bacteria suspension in the first row and suspended, 130 μL of this suspension was sequentially added to the wells in the 2nd to 12th rows for dilution.
Visually confirm the turbidity of the culture solution or the presence or absence of precipitated bacteria, and the minimum concentration of the test sample at which the growth of the test strain is no longer observed (total concentration of palmitoleic acid and oleic acid, or total concentration of palmitoleic acid and vaccenic acid MIC of palmitoleic acid was converted from the mixing ratio of fatty acids in each test sample and shown in FIGS.
In addition, for example, when the mixed weight ratio of palmitoleic acid and oleic acid is 20:80 and growth of the test strain is observed in the wells of the first row, the MIC of the total concentration of palmitoleic acid and oleic acid is >4. ,000 μg/mL, so the MIC for palmitoleic acid is >800 μg/mL. Since MICs with concentrations higher than this cannot be evaluated, they are indicated as detection limit values in FIGS.

As shown in FIGS. 1 and 2, the higher the content of oleic acid or vaccenic acid in the test sample, the higher the MIC value of palmitoleic acid and the lower the antibacterial activity of palmitoleic acid against each test strain. Admitted.
The above results shown in Test Example 4 indicate that oleic acid and vaccenic acid each have an inhibitory effect on the antibacterial activity of palmitoleic acid against Staphylococcus aureus. In addition, the degree of inhibitory effect on the antibacterial activity of palmitoleic acid varied depending on the test strain.

As detailed above, according to the present invention, palmitoleic acid, which has selective antibacterial activity against Staphylococcus aureus, is contained at a high concentration, and the number of carbon atoms that inhibits the antibacterial activity of palmitoleic acid is 18 and has one unsaturated bond (C18:1) (oleic acid and vaccenic acid) with a low content of fatty acid compositions can be obtained.
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 (6)

containing 55% by weight or more of palmitoleic acid and containing 10% by weight or less in total of fatty acids having 18 carbon atoms and one unsaturated bond (C18:1) (oleic acid and vaccenic acid); A fatty acid composition containing 0.1% by weight or less of unsaturated fatty acids. 2. The composition according to claim 1, wherein the content of palmitoleic acid is 60% by weight or more. A selective antibacterial agent against Staphylococcus aureus, comprising a composition according to claim 1 or 2. An external skin preparation comprising the composition according to claim 1 or 2. A quasi-drug containing the composition according to claim 1 or 2. Cosmetics containing the composition according to claim 1 or 2.

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