Method for producing monounsaturated fatty acids with 16 carbon atoms

Abstract

To provide a method for producing a monounsaturated fatty acid having 16 carbon atoms by a method other than chemical synthesis.SOLUTION: A method for producing a monounsaturated fatty acid having 16 carbon atoms comprises a step of culturing bacteria of the genus Bifidobacterium capable of producing a monounsaturated fatty acid having 16 carbon atoms in a liquid medium.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing C16 monounsaturated fatty acids using Bifidobacterium. Background Art

[0002] Atopic dermatitis is a disease characterized by itchy eczema as the main lesion that repeats exacerbation and remission. It is known that most cases of atopic dermatitis onset in childhood, and after temporary cure, it relapses with severe symptoms in adolescence. The number of atopic dermatitis patients in Japan has been increasing year by year, reaching approximately 350,000 in 2008 and approximately 510,000 in 2017 (Ministry of Health, Labour and Welfare, 2017), and the disease is developing into one of the major social issues.

[0003] In recent years, it has been clarified that one of the causative factors of atopic dermatitis is inflammation induced by *Staphylococcus aureus*. In addition to atopic dermatitis, inflammation induced by *Staphylococcus aureus* is also considered to be a causative factor of chronic dermatitis and rough skin. Therefore, in order to treat, improve or prevent this type of dermatitis, there is a demand for suppressing the proliferation and activity of *Staphylococcus aureus*. Antibiotics are generally used to suppress microorganisms such as *Staphylococcus aureus*, but since antibiotics have strong antibacterial activity, there has been a problem that beneficial microorganisms such as skin resident bacteria are also suppressed.

[0004] On the other hand, sapenoic acid (6-cis-C16:1), a monounsaturated fatty acid with 16 carbon atoms, is known to have antibacterial activity against Staphylococcus aureus. It has been reported that sapenoic acid exhibits selective antibacterial activity, showing strong antibacterial activity against Staphylococcus aureus, while showing no antibacterial activity against Staphylococcus epidermidis, a closely related species that is a beneficial bacterium that contributes to skin moisturizing (Non-Patent Literature 1). Furthermore, Non-Patent Literature 1 reports that palmitoleic acid (9-cis-C16:1) and 7-cis-hexadecenoic acid (7-cis-C16:1), which are also monounsaturated fatty acids with 16 carbon atoms, exhibit similar selective antibacterial activity to sapenoic acid. Therefore, in order to treat, improve, or prevent dermatitis such as atopic dermatitis while maintaining a healthy skin microbiome, it is expected that monounsaturated fatty acids with 16 carbon atoms that exhibit selective antibacterial activity, showing antibacterial activity against harmful Staphylococcus aureus but not against beneficial Staphylococcus epidermidis, will be used. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Toshihiro Nagao, Ayaka Uyama, Shigemitsu Tanaka, and Tetsuzo Sugino, "Fatty Acids that Control Skin Microbiota," Biotechnology, The Japan Society for Biotechnology, October 2020, Vol. 98, No. 10, pp. 525-528. [Overview of the project] [Problems that the invention aims to solve]

[0006] When using monounsaturated fatty acids with 16 carbon atoms for the treatment, improvement, or prevention of atopic dermatitis, topical application of these fatty acids through lotions, creams, or other skin preparations, or oral administration through foods and beverages containing these fatty acids, is considered. Therefore, it is desirable to obtain monounsaturated fatty acids with 16 carbon atoms as natural or naturally derived ingredients that are easily usable in topical skin preparations and foods and beverages. However, the natural resources from which these monounsaturated fatty acids with 16 carbon atoms can be extracted are limited, making it difficult to obtain them as natural or naturally derived ingredients. Consequently, monounsaturated fatty acids with 16 carbon atoms are generally produced as synthetic materials through chemical synthesis, which presents a problem in that they are difficult to use in topical skin preparations and foods and beverages.

[0007] Therefore, the present invention has been made in view of the above-mentioned points, and its object is to provide a method for producing monounsaturated fatty acids having 16 carbon atoms by a method other than chemical synthesis. [Means for solving the problem]

[0008] Inspired by fermentation production by microorganisms, the inventors diligently investigated the search for microorganisms capable of producing 16-carbon monounsaturated fatty acids and analyzed their characteristics to determine efficient production methods. As a result, they discovered that Bifidobacterium bacteria, commonly known as Bifidobacteria, which are yogurt-producing bacteria and generally well-known probiotics, can produce 16-carbon monounsaturated fatty acids. Based on this finding, the present invention was completed.

[0009] To solve the above problems, the present invention provides a method for producing a C16 monounsaturated fatty acid, which includes a step of culturing Bifidobacterium bacteria having the ability to produce C16 monounsaturated fatty acids in a liquid medium. This makes it possible to produce a C16 monounsaturated fatty acid by a method other than chemical synthesis. Specifically, by culturing Bifidobacterium bacteria having the ability to produce C16 monounsaturated fatty acids in a liquid medium, the target fatty acid is accumulated in the cells of the Bifidobacterium bacteria that have grown efficiently in the liquid medium.

[0010] Furthermore, the Bifidobacterium bacteria used in the production method of the present invention may preferably be Bifidobacterium adolescentis, Bifidobacterium boum, Bifidobacterium sp. JCM7042 strain, or mutants thereof. This allows for the selection of Bifidobacterium bacteria that have high productivity of 16-carbon monounsaturated fatty acids and are suitable for the production of the target fatty acid.

[0011] Furthermore, it is preferable that the mutant strain of Bifidobacterium bacteria used in the production method of the present invention be Bifidobacterium sp. AD2 strain (NITE BP-03576). This allows for the selection of a novel Bifidobacterium bacterium that exhibits particularly high productivity of monounsaturated fatty acids with 16 carbon atoms and is suitable for the production of the target fatty acid.

[0012] Furthermore, in the method for producing a C16 monounsaturated fatty acid of the present invention, it is also preferable that the C16 monounsaturated fatty acid is 7-cis-hexadecenoic acid. This allows for the selection of a C16 monounsaturated fatty acid that is produced by Bifidobacterium bacteria, exhibits antibacterial activity against harmful Staphylococcus aureus, but not against beneficial Staphylococcus epidermidis, thus exhibiting selective antibacterial activity.

[0013] Furthermore, it is preferable that the liquid culture medium in the production method of the present invention is a liquid culture medium further containing 0.001 to 1 g / L of L-cysteine ​​or a salt thereof in addition to MRS medium. This makes it possible to improve the productivity of Bifidobacterium bacteria in producing 16-carbon monounsaturated fatty acids, particularly 7-cis-hexadecenoic acid.

[0014] Furthermore, it is preferable that the culture in the production method of the present invention be carried out by passing carbon dioxide through the liquid culture medium. This can improve the productivity of Bifidobacterium bacteria in producing 16-carbon monounsaturated fatty acids, particularly 7-cis-hexadecenoic acid, and furthermore, enable the efficient production of 16-carbon monounsaturated fatty acids even under large-scale culture conditions.

[0015] Furthermore, the cultivation in the production method of the present invention is preferably carried out under temperature conditions of 32 to 35°C. This makes it possible to improve the productivity of Bifidobacterium bacteria in producing 16-carbon monounsaturated fatty acids, particularly 7-cis-hexadecenoic acid.

[0016] Furthermore, the method for producing the Staphylococcus aureus-selective antimicrobial agent of the present invention includes a step of culturing Bifidobacterium bacteria, which have the ability to produce a monounsaturated fatty acid with 16 carbon atoms, in a liquid medium, and this monounsaturated fatty acid with 16 carbon atoms is 7-cis-hexadecenoic acid. Because Bifidobacterium bacteria produce 7-cis-hexadecenoic acid, a Staphylococcus aureus-selective antimicrobial agent is obtained that does not exhibit antimicrobial activity against beneficial Staphylococcus epidermidis but exhibits antimicrobial activity against harmful Staphylococcus aureus. This Staphylococcus aureus-selective antimicrobial agent includes not only the crude extract of total fatty acids obtained from the bacterial cells of Bifidobacterium bacteria after the culturing step, but also 7-cis-hexadecenoic acid isolated and purified from the crude extract of total fatty acids.

[0017] Furthermore, the present invention provides a method for producing food and beverages for improving atopic dermatitis, which includes a step of culturing Bifidobacterium bacteria capable of producing a monounsaturated fatty acid with 16 carbon atoms in a liquid medium. This monounsaturated fatty acid with 16 carbon atoms is 7-cis-hexadecenoic acid. As a result, 7-cis-hexadecenoic acid, which does not exhibit antibacterial activity against beneficial Staphylococcus epidermidis but does exhibit antibacterial activity against Staphylococcus aureus, one of the causes of atopic dermatitis, is produced by Bifidobacterium bacteria, thus providing a 7-cis-hexadecenoic acid-containing material that is easily usable in food and beverages. Food and beverages containing this 7-cis-hexadecenoic acid-containing material can be used as food and beverages for improving atopic dermatitis.

[0018] Furthermore, the present invention provides a method for producing a cosmetic composition for improving atopic dermatitis, which includes a step of culturing Bifidobacterium bacteria capable of producing a monounsaturated fatty acid with 16 carbon atoms in a liquid medium. This monounsaturated fatty acid with 16 carbon atoms is 7-cis-hexadecenoic acid. As a result, 7-cis-hexadecenoic acid, which does not exhibit antibacterial activity against beneficial Staphylococcus epidermidis but does exhibit antibacterial activity against Staphylococcus aureus, one of the causes of atopic dermatitis, is produced by Bifidobacterium bacteria, thus providing a 7-cis-hexadecenoic acid-containing material that is easily usable in cosmetics. Cosmetics containing this 7-cis-hexadecenoic acid-containing material can be used as a cosmetic composition for improving atopic dermatitis.

[0019] Furthermore, the novel Bifidobacterium strain of the present invention is Bifidobacterium sp. AD2 strain (NITE BP-03576). By culturing this novel Bifidobacterium strain AD2, monounsaturated fatty acids with 16 carbon atoms, particularly 7-cis-hexadecenoic acid, can be produced with high efficiency. [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a method for producing a monounsaturated fatty acid having 16 carbon atoms that has the following excellent effects, as well as a method for producing a selective antibacterial agent for Staphylococcus aureus and a food or cosmetic product for improving atopic dermatitis. (1) By culturing Bifidobacterium, monounsaturated fatty acids with 16 carbon atoms, particularly 7-cis-hexadecenoic acid, can be produced. (2) By optimizing the culture conditions such as the culture medium composition, culture temperature, and gas aeration, the production of monounsaturated fatty acids with 16 carbon atoms can be improved. (3) Because it is produced using Bifidobacteria, which are used in the production of fermented foods and probiotics, it is highly safe and the product is easily usable in the fields of food and beverages and cosmetics. [Brief explanation of the drawing]

[0021] [Figure 1]It is a graph showing the intracellular localization of 7-cis-hexadecenoic acid produced by bifidobacteria in Example 2. The vertical axis represents the amount of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in 1 L of the culture solution. [Figure 2] It is a graph showing the antibacterial activity of a fatty acid extract extracted from bifidobacteria against Staphylococcus aureus and Staphylococcus epidermidis in Example 3. [Figure 3] It is a graph showing the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture solution and the proportion (%) of 7-cis-hexadecenoic acid in total fatty acids when bifidobacteria are cultured in MRS liquid medium, CSL liquid medium and TOS liquid medium in Example 4, wherein Figure 3(a) is the graph obtained when culturing Bifidobacterium sp. JCM7042 strain, and Figure 3(b) is the graph obtained when culturing Bifidobacterium boum JCM1211 strain. [Figure 4] It is a graph showing the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture solution and the proportion (%) of 7-cis-hexadecenoic acid in total fatty acids when bifidobacteria are cultured in MRS liquid medium and MRS+L-Cys liquid medium in Example 6, wherein Figure 4(a) is the graph obtained when culturing Bifidobacterium sp. JCM7042 strain, and Figure 4(b) is the graph obtained when culturing Bifidobacterium boum JCM1211 strain. [Figure 5] It is a graph showing the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture solution and the culture time (h) when bifidobacteria are subjected to scale-up culture in TOS liquid medium, MRS liquid medium and MRS+L-Cys liquid medium by aeration culture with carbon dioxide gas or nitrogen gas in Example 7. [Figure 6] It is a graph showing the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture solution when bifidobacteria are cultured in liquid media with varying amounts of L-cysteine added to MRS liquid medium in Example 8. [Figure 7] This graph shows the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture medium when the culture temperature of Bifidobacterium was changed in Example 9. [Figure 8] This graph shows the amount (mg / L) of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in the culture medium and the culture time (h) when Bifidobacterium was cultured at a temperature of 34°C and scaled up by carbon dioxide aeration culture in Example 9. [Figure 9] In Example 10, for the wild-type Bifidobacterium strain (JCM7042 strain) and its mutant strains (AD1 strain, AD2 strain), Figure 9(a) is a graph showing the amount of 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture medium (mg / L), and Figure 9(b) is a graph showing the accumulation of 7-cis-hexadecenoic acid per unit weight of dry cells (mg / gDCW). [Modes for carrying out the invention]

[0022] The following describes in detail a method for producing a 16-carbon monounsaturated fatty acid and a method for producing food, beverages, and cosmetics for improving atopic dermatitis according to one embodiment of the present invention. The method for producing a 16-carbon monounsaturated fatty acid according to this embodiment includes a step of culturing Bifidobacterium bacteria having the ability to produce a 16-carbon monounsaturated fatty acid in a liquid medium.

[0023] (Bacteria of the genus Bifidobacterium) First, the Bifidobacterium bacteria used in this embodiment will be described. The Bifidobacterium bacteria in this embodiment are Bifidobacterium bacteria that have the ability to produce monounsaturated fatty acids with 16 carbon atoms. Specifically, for example, Bifidobacterium adolescentis, Bifidobacterium thermophilum, Bifidobacterium boum, Bifidobacterium ruminantium, Bifidobacterium gallinarum, Bifidobacterium dentium, Bifidobacterium breve, and Bifidobacterium sp. JCM7042 strain, as well as their mutant strains, are preferred. Of these, Bifidobacterium adolescentis, Bifidobacterium boum, and Bifidobacterium sp. JCM7042 strain, as well as their mutant strains, are more preferably used from the viewpoint of excellent production of monounsaturated fatty acids with 16 carbon atoms.

[0024] The mutant strains are not particularly limited as long as they have the ability to produce monounsaturated fatty acids with 16 carbon atoms. However, as shown in Example 10 described later, in the present invention, mutant strains AD1 and AD2, derived from Bifidobacterium sp. JCM7042, have been obtained as mutant strains with excellent productivity of monounsaturated fatty acids with 16 carbon atoms. These strains exhibit superior productivity of monounsaturated fatty acids with 16 carbon atoms compared to the parent strain JCM7042. Of these, strain AD2, which shows particularly excellent productivity, i.e., Bifidobacterium sp. AD2, has been internationally deposited with a patent microorganism depositary as follows. (1) Accession number: NITE BP-03576 (2) Original deposit date: December 22, 2021 (3) Depository institution: National Institute of Technology and Evaluation, Patent Microorganism Depository Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan)

[0025] (Monounsaturated fatty acids with 16 carbon atoms) In this embodiment, the C16 monounsaturated fatty acid is a C16 monounsaturated fatty acid that exhibits selective antibacterial activity, showing antibacterial activity against harmful Staphylococcus aureus but not against beneficial Staphylococcus epidermidis. Specifically, this includes at least one fatty acid selected from the group consisting of sapienic acid (6-cis-C16:1), palmitoleic acid (9-cis-C16:1), and 7-cis-hexadecenoic acid (7-cis-C16:1). Of these, 7-cis-hexadecenoic acid (7-cis-C16:1), which was found to be produced by Bifidobacterium and whose productivity was improved in the examples described later, is particularly preferred. Since 7-cis-hexadecenoic acid (7-cis-C16:1) is a fatty acid with the selective antibacterial activity described above, it can be used for the treatment, improvement, or prevention of dermatitis such as atopic dermatitis while maintaining a healthy skin microbiome.

[0026] (Liquid culture medium) In this embodiment, any liquid medium capable of culturing and growing so-called Bifidobacterium bacteria can be used as the liquid medium for culturing Bifidobacterium. Specifically, although not particularly limited, examples include MRS medium, TOS medium, TOS propionic acid medium, CSL medium, BL medium, and GAM broth. The composition of MRS liquid medium is as follows per liter of liquid medium: 10 g of proteospeptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 1 g of polysorbate 80, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 0.1 g of magnesium sulfate (heptahydrate), 0.05 g of manganese sulfate, and 2 g of dipotassium monohydrogen phosphate. However, the amounts of each component may differ by about ±20%, some components may be replaced with components having similar functions, and other components may be added. Furthermore, the composition of TOS liquid medium may include 10g of tryptone, 1g of yeast extract, 3g of potassium dihydrogen phosphate, 4.8g of dipotassium monohydrogen phosphate, 3g of ammonium sulfate, 0.2g of magnesium sulfate (heptahydrate), 0.5g of L-cysteine ​​hydrochloride (monohydrate), and 10g of galactooligosaccharide per liter of liquid medium. However, the amounts of each component may differ by approximately ±20%, some components may be substituted with components having similar functions, and other components may be added. Furthermore, the composition of TOS propionic acid liquid medium may include 10g of peptone, 1g of yeast extract, 3g of potassium dihydrogen phosphate, 4.8g of dipotassium monohydrogen phosphate, 3g of ammonium sulfate, 0.2g of magnesium sulfate (heptahydrate), 0.5g of L-cysteine ​​hydrochloride (monohydrate), 15g of sodium propionate, and 10g of galactooligosaccharide per liter of liquid medium. However, the amounts of each component may differ by approximately ±20%, some components may be substituted with components having similar functions, and other components may be added.Furthermore, the composition of the CSL liquid medium may include 55g of corn steep liquor, 10g of glucose, 1mL of polysorbate 80, 1g of dipotassium monohydrogen phosphate, and 1g of potassium dihydrogen phosphate per liter of liquid medium. However, the amounts of each component may differ by approximately ±20%, some components may be substituted with components having similar functions, and other components may be added. Furthermore, the composition of BL liquid medium may include 2.4g of meat extract, 10g of proteose peptone, 5g of peptone, 3g of soybean peptone, 5g of yeast extract, 3.2g of liver extract, 10g of glucose, 0.5g of soluble starch, 1g of dipotassium monohydrogen phosphate, 1g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate (heptahydrate), 0.01g of ferrous sulfate (heptahydrate), 0.01g of sodium chloride, 0.007g of manganese sulfate, 0.2g of antifoaming agent (silicone), 1g of polysorbate 80, and 0.5g of L-cysteine ​​hydrochloride (monohydrate) per liter of liquid medium. However, the amounts of each component may differ by approximately ±20%, some components may be substituted with components having similar functions, and other components may be added. Furthermore, the composition of GAM broth may include 10g of peptone, 3g of soybean peptone, 10g of proteose peptone, 13.5g of digested serum powder, 5g of yeast extract, 2.2g of meat extract, 1.2g of liver extract, 3g of glucose, 2.5g of potassium dihydrogen phosphate, 3g of sodium chloride, 5g of soluble starch, 0.3g of L-cysteine ​​hydrochloride (monohydrate), and 0.3g of sodium thioglycolate per liter of liquid medium. However, the amounts of each component may differ by approximately ±20%, some components may be substituted with components having similar functions, and other components may be added.

[0027] Of the liquid media described above, MRS liquid medium is preferred as the liquid medium used for cultivation from the viewpoint of improving the productivity of monounsaturated fatty acids with 16 carbon atoms, particularly 7-cis-hexadecenoic acid (7-cis-C16:1). By culturing in MRS liquid medium, the concentration of 7-cis-hexadecenoic acid in the culture medium can be increased by 2 to 5 times compared to CSL liquid medium or TOS liquid medium. Furthermore, from the viewpoint of further improving the productivity of 7-cis-hexadecenoic acid, it is particularly preferable to use a medium to which L-cysteine ​​or a salt thereof is added to MRS liquid medium. By culturing in MRS liquid medium to which L-cysteine ​​is added, the concentration of 7-cis-hexadecenoic acid in the culture medium can be increased by 2 times compared to culturing in MRS liquid medium without L-cysteine. The amount of L-cysteine ​​or its salt added to the MRS liquid medium is preferably in the range of 0.001 to 1.0 g / L, more preferably in the range of 0.01 to 0.7 g / L, and particularly preferably in the range of 0.1 to 0.6 g / L, from the viewpoint of the productivity of 7-cis-hexadecenoic acid in the culture medium.

[0028] (Culture conditions) In this embodiment, when culturing Bifidobacteria, it is preferable to cultivate them under microaerophilic or anaerobic conditions that facilitate their growth, and specifically, liquid static culture or aerated culture is preferred. Here, in aerated culture, it is particularly preferable to culture with carbon dioxide aerated, from the viewpoint of improving the productivity of monounsaturated fatty acids with 16 carbon atoms, especially 7-cis-hexadecenoic acid (7-cis-C16:1). By culturing with carbon dioxide aerated in the culture medium, the production rate and amount of 7-cis-hexadecenoic acid can be significantly improved. Among these, by using a medium in which L-cysteines are added to MRS liquid medium as the liquid medium for culturing with carbon dioxide aerated, the productivity of 7-cis-hexadecenoic acid is further improved, so that 7-cis-hexadecenoic acid can be efficiently produced in scale-up culture.

[0029] In this embodiment, the culture temperature for Bifidobacteria is preferably a temperature suitable for the growth of Bifidobacteria. However, from the viewpoint of improving the productivity of monounsaturated fatty acids with 16 carbon atoms, particularly the productivity of 7-cis-hexadecenoic acid (7-cis-C16:1), the culture temperature is preferably in the range of 30°C to 37°C, more preferably in the range of 32°C to 35°C, and even more preferably 34°C, as shown in the examples described later.

[0030] Furthermore, the culture time for Bifidobacteria in this embodiment can be set as appropriate, and the culture can be performed until the desired concentration of monounsaturated fatty acids with 16 carbon atoms is reached. For example, it is preferable to culture for about 12 hours to 5 days, and more preferably for about 24 hours to 72 hours.

[0031] (Use of culture medium) Monounsaturated fatty acids with 16 carbon atoms, such as 7-cis-hexadecenoic acid (7-cis-C16:1), produced by Bifidobacterium, are accumulated as polar lipids within the Bifidobacterium cells. Therefore, when using the culture medium obtained through the cultivation process, the culture medium itself can be used depending on the purpose, or the bacterial cells can be recovered from the culture medium by centrifugation or other means and used in their individual form. Furthermore, within the limits that do not impair the effects of the present invention, various additional treatments such as washing, drying (freeze-drying, L-drying, spray-drying, etc.) or heat treatment can be applied to the recovered bacterial cells and culture medium. The recovered bacterial cells can be used as live or dead cells, or a mixture of live and dead cells may be used. Furthermore, it is also possible to extract and use total fatty acids, including 16-carbon monounsaturated fatty acids such as 7-cis-hexadecenoic acid, accumulated within the recovered bacterial cells, or to further separate and purify the 16-carbon monounsaturated fatty acids such as 7-cis-hexadecenoic acid from the extracted total fatty acids and use them.

[0032] (Method for producing a selective antibacterial agent for Staphylococcus aureus) The method for producing a Staphylococcus aureus-selective antimicrobial agent according to this embodiment includes a step of culturing Bifidobacterium bacteria having the ability to produce 7-cis-hexadecenoic acid in a liquid medium. The Bifidobacterium bacteria having the ability to produce 7-cis-hexadecenoic acid, the liquid medium used for cultivation, the cultivation conditions, and the manner of use of the culture solution are the same as described above, and the effects are also the same. This Staphylococcus aureus-selective antimicrobial agent includes not only the crude extract of total fatty acids obtained from the bacterial cells of Bifidobacterium bacteria after the cultivation step, but also 7-cis-hexadecenoic acid separated and purified from the crude extract of total fatty acids. Furthermore, specific methods for producing a Staphylococcus aureus-selective antimicrobial agent include a step of recovering the bacterial cells of Bifidobacterium bacteria after the cultivation step and an extraction step of obtaining a crude extract of total fatty acids from the bacterial cells after the recovery step, but it is sufficient to obtain an agent having antimicrobial activity selective for Staphylococcus aureus, and is not particularly limited. It is preferable that the total fatty acids are extracted as free fatty acids from the total lipids of the bacterial cells. Furthermore, it is possible to include a purification step after the extraction process in which 7-cis-hexadecenoic acid is separated and purified from the crude extract of total fatty acids. This allows for the acquisition of a Bifidobacterium-derived antibacterial agent that does not exhibit antibacterial activity against beneficial Staphylococcus epidermidis but does exhibit antibacterial activity against Staphylococcus aureus.

[0033] (Method of manufacturing food, beverages, and cosmetics for improving atopic dermatitis) The method for producing a food or beverage or cosmetic for improving atopic dermatitis according to this embodiment includes a step of culturing Bifidobacterium bacteria capable of producing 7-cis-hexadecenoic acid in a liquid medium. The Bifidobacterium bacteria capable of producing 7-cis-hexadecenoic acid, the liquid medium used for cultivation, the cultivation conditions, and the manner of use of the culture solution are the same as described above, and the effects are also the same. The 7-cis-hexadecenoic acid produced by Bifidobacterium in this way is not a chemically synthesized product, but a fermentation product of Bifidobacterium. Furthermore, Bifidobacterium has long been used for maintaining human health as a fermented food such as yogurt and as a probiotic, and is highly safe, so 7-cis-hexadecenoic acid can be produced as an easily usable material in food and beverages such as supplements and beverages, and cosmetics such as lotions and creams.

[0034] Culture solutions containing cultures produced by Bifidobacterium, recovered bacterial cells, or processed products thereof, as well as 7-cis-hexadecenoic acid extracted from recovered bacterial cells, can be used in all forms of food and beverages, including supplements such as tablets, capsules, powders, granules, and gels; beverages such as fermented milk, lactic acid bacteria drinks, soft drinks, and sports drinks; dairy products such as yogurt and ice cream; confectionery such as candy, gum, and chocolate; bread, porridge, cereal, noodles, jelly, soup, and seasonings; and can be used for the improvement, treatment, or prevention of atopic dermatitis. Furthermore, these food and beverages can be combined with various other functional ingredients, other microorganisms, sugars, vitamins, minerals, amino acids, or proteins.

[0035] Furthermore, the culture medium containing the culture produced by Bifidobacterium, the recovered bacterial cells, or processed products thereof, as well as 7-cis-hexadecenoic acid extracted from the recovered bacterial cells, can be applied to various cosmetic formulations by conventional methods as dosage forms typically applied to the skin. For example, they can be used in liquid formulations such as lotions, emulsions, gels, powders or creams, patches, packs, cleansing agents, soaps or hair care products, or makeup products, and can be used for the improvement, treatment, or prevention of atopic dermatitis. In addition, various ingredients commonly used in cosmetics can be incorporated, to the extent that they do not impair the effects of this invention. Examples include moisturizers, emollients, plant extracts, plant oils, pH adjusters, surfactants, thickeners, vitamins, amino acids, preservatives, fragrances, and pigments. Note that the term "cosmetics" in this specification includes not only cosmetics but also quasi-drugs.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way by these examples. [Examples]

[0037] [Example 1] 1. Examination of Bifidobacterium capable of producing monounsaturated fatty acids with 16 carbon atoms. In this example, we searched for microorganisms that produce monounsaturated fatty acids with 16 carbon atoms. In particular, we selected and tested microorganisms that have a long history of use in fermented foods and are highly safe, so that they can be used as raw materials for food and beverages and topical skin preparations.

[0038] Specifically, the experiment was conducted as follows: MRS liquid medium, consisting of the composition shown in Table 1, was dispensed into 12 mL screw-cap test tubes, various preserved bacterial strains were inoculated, and the screw-cap test tubes were sealed and left to stand to perform liquid static culture. The culture temperature was 37°C, and the culture was performed until the turbidity reached OD660=1 to obtain the pre-culture solution. Subsequently, for the main culture, 12 mL of MRS liquid medium was dispensed into screw-cap test tubes, 120 μL of the pre-culture solution was added, and the screw-cap test tubes were sealed and left to stand to perform liquid static culture. The culture temperature was 37°C, and the culture period for the main culture was 2 days.

[0039] [Table 1]

[0040] The culture medium from this culture was centrifuged at 3260 × g for 10 minutes to collect the bacterial cells, which were then dried in a 95°C oven for 2 hours. The dried bacterial weight was calculated by subtracting the tare weight from the weight of the test tube after drying the bacterial cells. Tricosanoic acid (C23:0) was used as the internal standard and dissolved in dichloromethane to a concentration of 0.2 mg / mL. 1 mL of dichloromethane containing the internal standard and 2 mL of 10% hydrochloric acid-methanol were added to the dried bacterial cells, the test tube was sealed, and incubated at 55°C for 2 hours. This simultaneously extracted fatty acids from the bacterial cells and methylated the extracted fatty acids. 1 mL of pure water and 3 mL of n-hexane were added and mixed vigorously, then centrifuged at 3260 × g for 10 minutes, and the upper hexane layer was transferred to a new test tube. After removing the hexane using a centrifugal evaporator, the obtained fatty acid methyl ester fraction was dissolved in 100 μL of chloroform and subjected to gas chromatography (GC) analysis under the following conditions. Furthermore, the content of each fatty acid component was quantified relatively based on the content of the internal standard and the area value of each fatty acid peak. • Column: GC capillary column for fatty acid separation (Model number: TC-70, product of GL Sciences Co., Ltd.) • Column size: Inner diameter 0.25 mm x length 60 m, film thickness 0.25 μm Column temperature: 160℃ • Heating rate: 2°C / min to 230°C • Holding time: 10 minutes at 230°C • Evaporation chamber temperature: 250℃ Detector temperature: 250℃ ·Injection volume: 1μL

[0041] As a result, we discovered that Bifidobacterium bacteria (Bifidobacteria), which are well-known as probiotics and are used in yogurt production, produce a monounsaturated fatty acid with 16 carbon atoms. The results are shown in Table 2 below. Total fatty acids (mg / L) indicate the total amount of fatty acids contained in the culture medium, and 7-cis-C16:1 (%) indicates the proportion of 7-cis-hexadecenoic acid in the total fatty acids. We determined the structure of the monounsaturated fatty acid with 16 carbon atoms produced by these Bifidobacteria, and it was revealed to be 7-cis-hexadecenoic acid (7-cis-C16:1). Among the Bifidobacteria, in particular, Bifidobacterium sp. JCM7042 strain, Bifidobacterium adolescentis, and Bifidobacterium boum were found to accumulate nearly 2% of 7-cis-C16:1 in their total fatty acids.

[0042] [Table 2]

[0043] [Example 2] 2. Examination of the localization of monounsaturated fatty acids with 16 carbon atoms produced by Bifidobacterium. In this example, we analyzed the lipid form in which 7-cis-hexadecenoic acid accumulates within the cells of Bifidobacterium sp. JCM7042, which produces 7-cis-hexadecenoic acid.

[0044] Specifically, the experiment was conducted as follows: 12 mL of TOS liquid medium, consisting of the composition shown in Table 3 below, was dispensed into a screw-top test tube, and Bifidobacterium sp. JCM7042 strain (hereinafter referred to as "JCM7042 strain") was inoculated. Liquid static culture was performed by sealing the screw-top test tube and allowing it to stand. The culture temperature was 37°C, and the culture was performed until the turbidity reached OD660=1 to obtain the pre-culture medium. Next, for the main culture, 10 mL of the pre-culture medium was added to 1 L of TOS liquid medium, and liquid static culture was performed with the tube tightly sealed. The culture temperature was 37°C, and the culture period for the main culture was 3 days.

[0045] [Table 3]

[0046] A large quantity of bacterial cells was recovered by centrifugation of 1 L of the culture medium from the main culture. Total lipids were extracted from the wet bacterial cells using the Bligh-Dyer method, with water / chloroform / methanol (final ratio 2 / 2.5 / 2.5, v / v / v) used for extraction. The extracted total lipids were developed on a thin-layer chromatography using a hexane:diethyl ether:acetic acid = 80:20:1 developing solvent, and then an 80% acetone solution containing 0.01% (w / v) primuline was sprayed, and the lipid spots were detected with 365 nm ultraviolet light. In this way, lipids from the bacterial cells were extracted and fractionated, and the fatty acid composition and composition amount of each lipid species were analyzed. Many fatty acids exist as constituent fatty acids of polar lipids (mainly phospholipids), and as shown in the graph in Figure 1, it was confirmed that 7-cis-hexadecenoic acid (7-cis-C16:1) is also included in polar lipids. UK1 and UK2 in the graph in Figure 1 represent unknown substances.

[0047] [Example 3] 3. Examination of the antibacterial activity of fatty acids produced by Bifidobacterium. In this example, total fatty acids were extracted from cultured Bifidobacterium sp. JCM7042 strain, which was confirmed to effectively produce 7-cis-hexadecenoic acid in Examples 1 and 2, and the antibacterial activity of the crude extract was investigated.

[0048] Specifically, the experiment was conducted as follows. Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the Bifidobacterium. Pre-culture was performed using the TOS liquid medium shown in Table 3, as in Example 2. The culture temperature was set to 37°C, and the culture was performed until the turbidity reached OD660 = 1.0 to obtain the pre-culture solution. Subsequently, for the main culture, 0.14 mL of the pre-culture solution was added to 14 mL of TOS liquid medium, and liquid static culture was performed at 37°C with the container tightly sealed. After culturing until the turbidity reached OD660 = 1.0, the entire volume was transferred to a 15 mL tube. The tubes were centrifuged at 4000 × g for 15 minutes at 4°C to collect the bacterial cells. The bacterial cells were washed once with PBS and then suspended in 100 μL of PBS. 50 μL of 5N HCl and 400 μL of acetonitrile were added, vortexed for 1 minute, incubated at 100°C for 1 hour, and cooled to room temperature. Subsequently, 100 μL of methanol, 800 μL of t-butyl methyl ether, and 400 μL of ultrapure water were added, and vortexed for 1 minute. The mixture was centrifuged at 300 × g for 5 minutes, and the upper layer was collected in a new 15 mL tube. 800 μL of ultrapure water was added to the collected upper layer, vortexed for 1 minute, and then centrifuged at 300 × g for 5 minutes, and the upper layer was collected in a new 15 mL tube. The upper layer was collected in a dried 1.5 mL tube and dried using a centrifugal evaporator. At this time, the weight of the 1.5 mL tube was measured to determine the amount of fatty acid extract collected. In this way, total fatty acids derived from Bifidobacterium cells were obtained as a crude extract of free fatty acids separated from total lipids.

[0049] The antibacterial activity was measured using Staphylococcus aureus and Staphylococcus epidermidis. For S. aureus, strains JCM20624 (reference strain) and S. aureus 15R2 were used, and for S. epidermidis, strain 15R5 was used. Strains S. aureus 15R2 and S. epidermidis 15R5 were isolated from the same individual, a patient with severe atopic dermatitis (strain held by the Laboratory of Environmental Microbial Engineering, Gifu University).

[0050] Two 96-well round-bottom plates were used; the first was used for diluting the Bifidobacterium fatty acid extract, and the second for culture. First, the fatty acid extract was serially diluted in the first plate. 11 μL of DMSO and 99 μL of NB medium were added to rows 3-10 and 12. 22 μL of 20,000 ppm fatty acid extract dissolved in DMSO and 198 μL of NB medium were added to row 2. Row 2 was mixed by pipetting, and 110 μL was added to row 3. Row 3 was mixed by pipetting, and 110 μL was added to row 4. This process was repeated, and 110 μL from row 10 was added to row 11. Row 12 was left untouched. Next, the bacterial suspension was prepared in the second plate. The pre-culture solution of the above bacterial strain, cultured to OD=1.0, was diluted 1000-fold, and 1.0 × 10⁶ was added. 5 The bacterial suspension concentration was adjusted to achieve a CFU / mL. 100 μL of this suspension was added to each well in rows 2-10 and row 12. Row 11 was used as a negative control, and 100 μL of NB medium alone was added. 100 μL of the diluted fatty acid extract prepared in the first 96-well plate was added to the corresponding wells in the second 96-well plate and mixed by pipetting. In this way, the fatty acid extract in the medium was prepared at 10 different concentrations: 1000 ppm, 500 ppm, 250 ppm, 125 ppm, 63 ppm, 31 ppm, 16 ppm, 8 ppm, 4 ppm, and 0 ppm. The second plate was covered with a gas-exchangeable plate seal (BreezeEasy, Diversified Biotech product) and incubated at 37°C. After incubation, the OD of each well was measured. 660Turbidity was measured, and the growth rate (%) was calculated by determining the percentage of turbidity in each well relative to the turbidity at a fatty acid extract concentration of 0 ppm, which was set to 100%.

[0051] Figure 2 shows the antibacterial activity of fatty acid extracts obtained from cultured Bifidobacterium sp. JCM7042 strain. According to these results, when the fatty acid extract was present at concentrations of 60 ppm or higher, the growth rate of Staphylococcus aureus (S. aureus) decreased to less than 50%, while Staphylococcus epidermidis (S. epidermidis) maintained a growth rate of over 60% across fatty acid extract concentrations from 0 to 1000 ppm. This indicates that the fatty acid extract containing 7-cis-hexadecenoic acid exhibits selective antibacterial activity against Staphylococcus aureus (S. aureus), a causative agent of skin inflammation, even in such a crude state. Furthermore, since the proportion of 7-cis-hexadecenoic acid in the crude fatty acid extract obtained in this example was approximately 2-4%, the concentration of 7-cis-hexadecenoic acid at the concentration (approximately 60 ppm) where a decrease in growth rate was observed was calculated to be approximately 1.2-2.4 μg / mL. The MIC of 7-cis-hexadecenoic acid itself against Staphylococcus aureus is approximately 3 μg / mL.

[0052] [Example 4] 4. Improving the productivity of 7-cis-hexadecenoic acid by Bifidobacterium (1) We cultured Bifidobacterium that produce 7-cis-hexadecenoic acid using different types of culture media and investigated the relationship between these media and 7-cis-hexadecenoic acid productivity.

[0053] Using Bifidobacterium sp. JCM7042 strain (JCM7042 strain) and Bifidobacterium boum JCM1211 strain (boum1211 strain) as Bifidobacteria, cultivation was carried out in the same manner as in Example 1, except that the MRS liquid medium in Example 1 was replaced with the TOS liquid medium shown in Table 3 or the CSL liquid medium having the composition shown in Table 4 below. 7-cis-hexadecenoic acid contained in each culture medium was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0054] [Table 4]

[0055] Figure 3(a) shows the results for strain JCM7042, and Figure 3(b) shows the results for strain boum1211. 7-cis-C16:1 (mg / L) indicates the amount of 7-cis-hexadecenoic acid contained in the culture medium, and 7-cis-C16:1 (%) indicates the percentage of 7-cis-hexadecenoic acid in the total fatty acids. The data for the MRS liquid medium is the same as that obtained in Example 1.

[0056] These results indicate that using MRS liquid medium as the culture medium promotes the production of 7-cis-hexadecenoic acid by Bifidobacterium, resulting in a concentration of 7-cis-hexadecenoic acid in the culture medium of 2 mg / L or higher. On the other hand, when using TOS liquid medium, although the concentration of 7-cis-hexadecenoic acid in the culture medium was lower than that of MRS liquid medium, it was found that the proportion of 7-cis-hexadecenoic acid in the total fatty acids improved.

[0057] [Example 5] 5. Improving the productivity of 7-cis-hexadecenoic acid by Bifidobacterium (2) The effects of adding or substituting components found in MRS medium or components thought to be involved in fatty acid production on the growth of Bifidobacterium and the production of 7-cis-hexadecenoic acid (7-cis-C16:1) were investigated in TOS liquid medium with the composition shown in Table 3.

[0058] Specifically, the tests were conducted as follows: Liquid media were prepared by adding the additive components shown in Table 5 to TOS liquid media consisting of the compositions shown in Table 3. In the case of liquid media with "glucose" as the additive component, glucose was added in place of galactooligosaccharides in the composition of TOS liquid media, with the aim of substituting them. In the case of liquid media with "peptone" and "casamino acid" as the additive components, peptone or casamino acid was added in place of tryptone in the composition of TOS liquid media, with the aim of substituting them. Furthermore, for liquid media with "tryptone" as the additive component, indicated by an asterisk (*) in Table 5, since TOS liquid media originally contains "tryptone," a control test was conducted by preparing a liquid media with a composition obtained by removing tryptone from the composition of TOS liquid media for comparison. Similarly, for the liquid media marked with an asterisk (*) in Table 5, which contain "L-cysteine ​​hydrochloride" as an additive, since TOS liquid medium originally contains "L-cysteine ​​hydrochloride," a control test was conducted by preparing a liquid medium with a composition that omits L-cysteine ​​hydrochloride from the TOS liquid medium composition for comparison.

[0059] Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the Bifidobacterium, and cultivation was carried out in the same manner as in Example 1, except that the MRS liquid medium in Example 1 was replaced with the various liquid media prepared as described above. 7-cis-hexadecenoic acid (7-cis-C16:1) contained in each culture medium was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1. Regarding the growth of Bifidobacterium, compared to the control group without added components, an increase in cell weight was evaluated as "◎", maintaining the cell weight as "○", decreasing as "△", and a significant decrease as "×". Furthermore, regarding the production of 7-cis-hexadecenoic acid (7-cis-C16:1), an increase in production was evaluated as "◎", maintaining production as "○", decreasing as "△", and a significant decrease as "×". For additives such as "tryptone" or "L-cysteine ​​hydrochloride," the evaluation was conducted by comparing the results with control tests using liquid media without these components. The results are shown in Table 5 below.

[0060] [Table 5]

[0061] These results show that L-cysteine ​​is an essential amino acid in the culture of Bifidobacterium and has the effect of improving the growth of Bifidobacterium. In addition, it has been revealed that it also has the effect of improving the productivity of 7-cis-hexadecenoic acid. Furthermore, it was shown that in liquid culture media supplemented with calcium carbonate or methyl oleate, the growth of Bifidobacterium improved, but the production of 7-cis-hexadecenoic acid was either unaffected or decreased.

[0062] [Example 6] 6. Improving the productivity of 7-cis-hexadecenoic acid by Bifidobacterium (3) We investigated whether further improvements in the productivity of 7-cis-hexadecenoic acid could be achieved by combining the liquid culture medium and additive components that demonstrated high productivity of 7-cis-hexadecenoic acid in Examples 4 and 5.

[0063] Specifically, the experiment was conducted as follows: Bifidobacterium sp. JCM7042 strain (JCM7042 strain) and Bifidobacterium boum JCM1211 strain (boum1211 strain) were used as Bifidobacteria. MRS liquid medium shown in Table 1 and MRS+L-Cys liquid medium were prepared by adding L-cysteine ​​hydrochloride monohydrate to the MRS liquid medium shown in Table 1 to a concentration of 0.5 g / L. Pre-culture and main culture were performed in these two liquid media in the same manner as in Example 1. 7-cis-hexadecenoic acid contained in each main culture solution was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0064] Figure 4(a) shows the results for strain JCM7042, and Figure 4(b) shows the results for strain boum1211. 7-cis-C16:1 (mg / L) indicates the amount of 7-cis-hexadecenoic acid contained in the culture medium, and 7-cis-C16:1 (%) indicates the percentage of 7-cis-hexadecenoic acid in the total fatty acids. The data for the MRS liquid medium is the same as that obtained in Example 1.

[0065] These results indicate that using a liquid medium prepared by adding L-cysteine ​​to MRS liquid medium as the culture medium promotes the production of 7-cis-hexadecenoic acid by Bifidobacterium, resulting in a concentration of 7-cis-hexadecenoic acid in the culture medium exceeding 4 mg / L. Furthermore, it was found that the proportion of 7-cis-hexadecenoic acid in the total fatty acids also improved.

[0066] [Example 7] 7. Improving the productivity of 7-cis-hexadecenoic acid by Bifidobacterium (4) In order to scale up the culture of Bifidobacterium, we investigated culture conditions that would increase the productivity of 7-cis-hexadecenoic acid.

[0067] Specifically, the experiment was conducted as follows: Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the Bifidobacterium. MRS liquid medium shown in Table 1, MRS+L-Cys liquid medium prepared by adding 0.5 g / L of L-cysteine ​​hydrochloride monohydrate to the MRS liquid medium shown in Table 1, and TOS liquid medium shown in Table 3 were prepared, and pre-culture was performed using each liquid medium. The culture temperature was set to 37°C, and the culture was performed until the turbidity reached OD=1 to obtain the pre-culture solution. Subsequently, for the main culture, 30 mL of the pre-culture solution was added to a 3.5 L capacity medium bottle containing 3 L of the same liquid medium as the pre-culture solution. Pure carbon dioxide or pure nitrogen gas was passed through the medium bottle, and the culture was performed at 37°C while stirring with a stirrer at 700 rpm. At the predetermined sampling time, each culture solution was collected with a syringe, and 5 mL of the culture solution was centrifuged to recover the bacterial cells. The amount of 7-cis-hexadecenoic acid contained in each culture medium was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0068] The results are shown in Figure 5. In the graph in Figure 5, the solid line and black circle markers represent the results of culturing in MRS+L-Cys liquid medium with carbon dioxide aeration, the solid line and star markers represent the results of culturing in MRS liquid medium with carbon dioxide aeration, the dashed line and triangle markers represent the results of culturing in MRS liquid medium with nitrogen gas aeration, the solid line and square markers represent the results of culturing in TOS liquid medium with carbon dioxide aeration, and the dashed line and diamond markers represent the results of culturing in TOS liquid medium with nitrogen gas aeration. The vertical axis shows the amount of 7-cis-hexadecenoic acid contained in the culture medium, and the horizontal axis shows the time (h) during which the culture was performed.

[0069] The results of this scale-up culture test revealed that, compared to the test group aerated with nitrogen gas, aerating with carbon dioxide increased the maximum accumulation of 7-cis-hexadecenoic acid by approximately double, significantly improving the production of 7-cis-hexadecenoic acid. Furthermore, the addition of L-cysteine ​​to the MRS liquid medium increased the production rate of 7-cis-hexadecenoic acid, and it was found that the time to reach the maximum accumulation was approximately halved compared to the MRS medium without L-cysteine.

[0070] [Example 8] 8. Examination of L-cysteine ​​concentration in liquid culture medium We prepared MRS media with varying amounts of L-cysteine ​​and investigated the effect on 7-cis-hexadecenoic acid production by Bifidobacterium. Specifically, the experiment was conducted as follows: Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the Bifidobacterium. To MRS liquid media with the composition shown in Table 1, L-cysteine ​​hydrochloride monohydrate was added at concentrations of 0.001 g / L, 0.01 g / L, 0.1 g / L, 0.5 g / L, 1.0 g / L, and 5.0 g / L to prepare L-cysteine-containing MRS liquid media. These L-cysteine-containing MRS liquid media and a control MRS liquid medium (without L-cysteine) were cultured in the same manner as in Example 1. The amount of 7-cis-hexadecenoic acid contained in each culture medium was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0071] The results are shown in Figure 6. These results indicate that a high concentration of L-cysteine ​​hydrochloride in the liquid medium (5.0 g / L) significantly inhibits the production of 7-cis-hexadecenoic acid. Therefore, it was inferred that the concentration of L-cysteine ​​hydrochloride in the liquid medium is preferably in the range of 0.001 to 1.0 g / L, and more preferably in the range of 0.01 to 0.7 g / L.

[0072] [Example 9] 9. Examination of culture temperature The effects of varying the culture temperature on the production of 7-cis-hexadecenoic acid by Bifidobacterium were investigated. Specifically, the experiment was conducted as follows: Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the Bifidobacterium. Pre-culture was performed at 37°C using MRS liquid medium with the composition shown in Table 1, in the same manner as in Example 1. Subsequently, for the main culture, the culture temperature was set in the range of 28°C to 37°C, and the main culture was performed in the same manner as in Example 1, except that culture was performed at each culture temperature. The amount of 7-cis-hexadecenoic acid contained in each culture solution was quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0073] The results are shown in Figure 7. These results indicate that the optimal culture temperature for improving the productivity of 7-cis-hexadecenoic acid is 30°C to 37°C. In particular, the 32°C to 35°C range showed excellent productivity, with the concentration of 7-cis-hexadecenoic acid in the culture medium exceeding 2.5 mg / L.

[0074] Therefore, in the scale-up culture performed in Example 7 described above, a test was conducted to confirm whether the productivity of 7-cis-hexadecenoic acid would be further improved by performing a scale-up culture with carbon dioxide aeration using the same procedure and method as in Example 7, except that the culture temperature of the main culture was changed to 34 degrees Celsius. Bifidobacterium sp. JCM7042 strain (JCM7042 strain) was used as the bifidobacteria, and the liquid medium used was MRS+L-Cys liquid medium, which is MRS liquid medium shown in Table 1 to which L-cysteine ​​hydrochloride monohydrate was added to a concentration of 0.5 g / L.

[0075] The results are shown in Figure 8. The vertical axis represents the amount of 7-cis-hexadecenoic acid contained in the culture medium, and the horizontal axis represents the time (h) during the culture. These results show that culturing at 34°C further improves the productivity of 7-cis-hexadecenoic acid, with the amount of 7-cis-hexadecenoic acid in the culture medium reaching 4 mg / L.

[0076] [Example 10] 10. Creation of 7-cis-hexadecenoic acid production-enhanced strains through mutation introduction. In this example, we attempted to obtain a strain that enhances the production of 7-cis-hexadecenoic acid by mutagenesis. Specifically, the experiment was conducted as follows: Bifidobacterium sp. JCM7042 strain was used as the parent strain of Bifidobacterium. 12 mL of TOS liquid medium, consisting of the composition shown in Table 3, was dispensed into a screw-top test tube, the JCM7042 strain was inoculated, and liquid static culture was performed by sealing the screw-top test tube and allowing it to stand. The culture temperature was 37°C, and the culture was continued until the turbidity reached OD=1. 10 μL of ethyl methanesulfonate (final concentration 1% (v / v)) was added to 1 mL of this culture solution, mixed, and incubated at 37°C for 1 hour to induce mutagenesis.

[0077] Nile Red was selected as the indicator reagent for this study. Nile Red is incorporated into the lipid fraction of the bacterial cells, causing it to turn red. Therefore, by picking colonies that turned red more quickly and more rapidly on the agar medium, mutant strains with improved fatty acid production capacity can be obtained. 100 μL of the bacterial suspension after mutation introduction treatment was spread onto TOS agar medium coated with Nile Red, and anaerobic culture was performed at 37°C using an oxygen scavenger and carbon dioxide generator (Mitsubishi Gas Chemical Co., Ltd. product, Anaeropack® Kenki). In this study, colonies with a strong red color were selected as priority. Liquid culture was performed on each of the picked strains in the same manner as in Example 1, except that they were cultured in TOS liquid medium. The components of each fatty acid contained in each culture medium were quantified by gas chromatography (GC) analysis using the same method and conditions as in Example 1.

[0078] As a result, after primary and secondary screening, we were able to obtain two mutant strains that showed higher 7-cis-hexadecenoic acid (7-hexadecenoic acid) production than the wild-type strain JCM7042. Figure 9(a) shows the amount of 7-cis-hexadecenoic acid (mg / L) contained in the culture medium of the wild-type strain (JCM7042) and the mutant strains AD1 and AD2, cultured in TOS liquid medium. Figure 9(b) shows the accumulation of 7-cis-hexadecenoic acid (mg / gDCW) per dry cell weight. Of these, strain AD2, which had the highest 7-cis-hexadecenoic acid production, was deposited with the Patent Microorganism Depository Center as NITE BP-03576.

[0079] The present invention is not limited to the embodiments or examples described above, and its technical scope also includes various design modifications that do not depart from the gist of the invention as described in the claims. [Industrial applicability]

[0080] This invention provides a monounsaturated fatty acid with 16 carbon atoms that exhibits selective antibacterial activity against harmful Staphylococcus aureus but not against beneficial Staphylococcus epidermidis, as well as foods and cosmetics containing the same. Therefore, it will be widely useful in industries such as the food and cosmetics sectors, quasi-drugs, and pharmaceuticals. [Accession Number]

[0081] Accession number: NITE BP-03576, Bifidobacterium sp. AD2 strain, Original deposit date: December 22, 2021, Depositing institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818, Japan)

Claims

1. The process includes culturing Bifidobacterium bacteria, which have the ability to produce monounsaturated fatty acids with 16 carbon atoms, in a liquid medium. The aforementioned monounsaturated fatty acid having 16 carbon atoms is 7-cis-hexadecenoic acid. The Bifidobacterium species mentioned above may be Bifidobacterium breeve, Bifidobacterium sp. JCM7042 strain, Bifidobacterium animalis subspp. lactis JCM10602 strain, Bifidobacterium boum JCM1211 strain, Bifidobacterium ruminantium JCM8222 strain, Bifidobacterium thermophyllum JCM1207 strain, or Bifidobacterium sp. A method for producing a monounsaturated fatty acid having 16 carbon atoms, characterized by being the AD2 strain (NITE BP-03576).

2. The method for producing a C16 monounsaturated fatty acid according to claim 1, characterized in that the liquid medium is a liquid medium further containing 0.001 to 1 g / L of L-cysteine ​​or a salt thereof in addition to MRS medium.

3. The method for producing a 16-carbon monounsaturated fatty acid according to claim 1 or 2, characterized in that the culture is carried out by passing carbon dioxide through the liquid culture medium.

4. The method for producing a 16-carbon monounsaturated fatty acid according to claim 1 or 2, characterized in that the culture is carried out under temperature conditions of 32 to 35°C.

5. A step of culturing Bifidobacterium bacteria, which have the ability to produce monounsaturated fatty acids with 16 carbon atoms, in a liquid medium. A step of obtaining a composition containing a 16-carbon monounsaturated fatty acid produced by the Bifidobacterium bacteria from the bacterial cells of the Bifidobacterium bacteria, and The process includes a step of preparing an agent containing a composition comprising the aforementioned monounsaturated fatty acid having 16 carbon atoms, or an agent containing the aforementioned monounsaturated fatty acid having 16 carbon atoms obtained therefrom. The aforementioned monounsaturated fatty acid having 16 carbon atoms is 7-cis-hexadecenoic acid. The aforementioned Bifidobacterium genus bacteria, Bifidobacterium breeve, This refers to Bifidobacterium sp. JCM7042 strain, Bifidobacterium animalis subspp. lactis JCM10602 strain, Bifidobacterium boum JCM1211 strain, Bifidobacterium ruminantium JCM8222 strain, Bifidobacterium thermophyllum JCM1207 strain, or Bifidobacterium sp. AD2 strain (NITE BP-03576). A method for producing a selective antimicrobial agent for Staphylococcus aureus, characterized by the following:

6. A step of culturing Bifidobacterium bacteria, which have the ability to produce monounsaturated fatty acids with 16 carbon atoms, in a liquid medium. A step of obtaining a composition containing a 16-carbon monounsaturated fatty acid produced by the Bifidobacterium bacteria from the bacterial cells of the Bifidobacterium bacteria, and The process includes a step of preparing a food or beverage containing a composition comprising the aforementioned monounsaturated fatty acid having 16 carbon atoms, or a food or beverage containing the aforementioned monounsaturated fatty acid having 16 carbon atoms obtained therefrom. The aforementioned monounsaturated fatty acid having 16 carbon atoms is 7-cis-hexadecenoic acid. The Bifidobacterium species mentioned above may be Bifidobacterium breeve, Bifidobacterium sp. JCM7042 strain, Bifidobacterium animalis subspp. lactis JCM10602 strain, Bifidobacterium boum JCM1211 strain, Bifidobacterium ruminantium JCM8222 strain, Bifidobacterium thermophyllum JCM1207 strain, or Bifidobacterium sp. This is strain AD2 (NITE BP-03576). A method for producing food and beverages for improving atopic dermatitis, characterized by the above.

7. A step of culturing Bifidobacterium bacteria, which have the ability to produce monounsaturated fatty acids with 16 carbon atoms, in a liquid medium. A step of obtaining a composition containing a 16-carbon monounsaturated fatty acid produced by the Bifidobacterium bacteria from the bacterial cells of the Bifidobacterium bacteria, and The process includes a step of preparing a cosmetic composition containing the aforementioned monounsaturated fatty acid having 16 carbon atoms, or a cosmetic composition containing the aforementioned monounsaturated fatty acid having 16 carbon atoms obtained therefrom. The aforementioned monounsaturated fatty acid having 16 carbon atoms is 7-cis-hexadecenoic acid. The Bifidobacterium species mentioned above may be Bifidobacterium breeve, Bifidobacterium sp. JCM7042 strain, Bifidobacterium animalis subspp. lactis JCM10602 strain, Bifidobacterium boum JCM1211 strain, Bifidobacterium ruminantium JCM8222 strain, Bifidobacterium thermophyllum JCM1207 strain, or Bifidobacterium sp. This is strain AD2 (NITE BP-03576). A method for producing a cosmetic for improving atopic dermatitis, characterized by the above.

8. Bifidobacterium sp. AD2 strain (NITE BP-03576).

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