Growth promoters for Staphylococcus epidermidis and selective growth promoters for microorganisms

Abstract

To provide techniques for promoting the proliferation of Staphylococcus epidermis, or for promoting the proliferation of Staphylococcus epidermis without promoting the proliferation of one or more microorganisms selected from Staphylococcus aureus, Malassezia globosa, Cutibacterium acnes, and Candida albicans.SOLUTION: Disclosed is a proliferation promoting agent for Staphylococcus epidermis comprising 1-kestose as an effective ingredient. According to the invention, it is possible to promote the proliferation of useful microorganisms without promoting the proliferation of useless or harmful microorganisms for skin health. Thus, the invention contributes to acquisition or maintenance of a bacterial balance preferred for skin health.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a growth promoter for Staphylococcus epidermidis and a selective growth promoter for microorganisms, which contain 1-kestose as an active ingredient. [Background technology]

[0002] A wide variety of microorganisms exist on the skin of humans and animals, and a healthy state is maintained by maintaining a balance among them. Examples of such microorganisms that normally reside on the skin include Staphylococcus epidermidis, Staphylococcus aureus, Malassezia globosa, Cutibacterium acnes, Propionibacterium acnes, and Candida albicans.

[0003] Staphylococcus epidermidis is a facultative anaerobic bacterium that is present on the skin surface and in pores. This bacterium feeds on sweat and sebum, producing glycerin and fatty acids. Fatty acids keep the skin slightly acidic and inhibit the growth of other harmful microorganisms. Glycerin also plays a role in maintaining the barrier function of the skin. For these reasons, Staphylococcus epidermidis is generally considered a beneficial bacterium, and it is preferable to prevent its numbers from decreasing in order to maintain healthy skin (Non-Patent Document 1).

[0004] Staphylococcus aureus is also a facultative anaerobic bacterium that is present on the skin surface and in pores. Although the presence of a small number of Staphylococcus aureus is not problematic, it is highly pathogenic among Staphylococcus aureus bacteria, and if it grows too much, it can cause dermatitis or suppuration of wounds (Non-Patent Document 1).

[0005] Malassezia is a peanut-shaped yeast-like fungus with a diameter of 3 to 5 μm, and is classified as a basidiomycete. Although the presence of a small number of this fungus is not a problem, an excess of this fungus is undesirable because it is involved in the onset or worsening of skin diseases such as tinea versicolor, dandruff, Malassezia folliculitis, Malassezia intertrigo, and seborrheic dermatitis (Non-Patent Document 2).

[0006] In addition, acne bacteria are anaerobic bacteria that hate oxygen and are present in pores and sebaceous glands. When the amount of sebum secreted increases or the pores become clogged due to some abnormality, this bacteria proliferates excessively, causing inflammation and developing acne (Non-Patent Document 1).

[0007] Candida is a dimorphic fungus that can take either a yeast or filamentous form. It is always present on the surface of the skin and is not highly pathogenic, but it causes endogenous opportunistic infections when the immune system is weakened by the use of immunosuppressants or antitumor therapy. Such infections range from mild ones such as oral candidiasis to severe or fatal ones such as candidemia and disseminated candidiasis.

[0008] From the above, it can be said that it is preferable to maintain or increase the number of beneficial bacteria such as Staphylococcus epidermidis and not increase harmful or opportunistic bacteria such as Staphylococcus aureus, Malassezia fungus, Propionibacterium acnes, and Candida fungus in order to maintain healthy skin. Therefore, a technology for obtaining or maintaining a favorable balance of bacterial numbers for the skin is required. For example, Patent Document 1 discloses a selective antibacterial composition that has an antibacterial effect only on Staphylococcus aureus without affecting the growth of Staphylococcus epidermidis. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent No. 4011787 [Non-patent literature]

[0010] [Non-Patent Document 1] Yoshida Rika, About the resident bacteria of the skin, September 1, 2016, Healthcare column, Research activities, public lectures, and adult lectures, Tokyo Health Care University, [online], [Retrieved April 8, 2021], Internet <url: https: www.thcu.ac.jp research column detail.html?id="1101"> [Non-Patent Document 2] Kanto Chemical Co., Ltd., Malassezia, fungus, bacterial test reagent, clinical test drug, product information, [online], [searched on April 9, 2021], Internet <url: https: products.kanto.co.jp web index.cgi?c="t_product_table&pk=826"> Summary of the Invention [Problem to be solved by the invention]

[0011] However, the technology described in Patent Document 1 has a concern that suppressing the number of Staphylococcus aureus bacteria may result in the proliferation of other harmful bacteria, and is not sufficient as a technology for obtaining or maintaining a favorable balance in the number of bacteria. The present invention has been made to solve such problems, and aims to provide a technology for promoting the proliferation of Staphylococcus epidermidis, or for promoting the proliferation of Staphylococcus epidermidis without promoting the proliferation of one or more microorganisms selected from Staphylococcus aureus, Malassezia fungi, Propionibacterium acnes, and Candida fungi. [Means for solving the problem]

[0012] As a result of intensive research, the present inventors have found that 1-kestose can promote the growth of Staphylococcus epidermidis, but does not promote the growth of Staphylococcus aureus, Malassezia fungi, Propionibacterium acnes, or Candida fungi. Based on this finding, the present inventors have completed the following inventions.

[0013] (1) The growth promoter for Staphylococcus epidermidis according to the present invention contains 1-kestose as an active ingredient.

[0014] (2) The selective microbial growth promoter of the present invention has a selective microbial growth-promoting effect of promoting the growth of Staphylococcus epidermidis, but not promoting the growth of one or more microorganisms selected from Staphylococcus aureus, Malassezia fungi, Propionibacterium acnes, and Candida fungi, and contains 1-kestose as an active ingredient.

[0015] (3) The agent according to the present invention may be used as an external preparation. Effect of the Invention

[0016] According to the present invention, it is possible to promote the growth of Staphylococcus epidermidis. Furthermore, according to the present invention, it is possible to promote the growth of Staphylococcus epidermidis without promoting the growth of one or more microorganisms selected from Staphylococcus aureus, Malassezia, Propionibacterium acnes, and Candida. In other words, according to the present invention, it is possible to promote the growth of beneficial microorganisms without promoting the growth of microorganisms that are useless or harmful to skin health. Therefore, according to the present invention, it is possible to contribute to obtaining or maintaining a bacterial count balance that is favorable for skin health.

[0017] In addition, 1-kestose, which is the active ingredient of the present invention, is a type of oligosaccharide that is also contained in vegetables and grains, and has been ingested as a food or food component since ancient times. In addition, since no toxicity was found in any of mutagenicity tests, acute toxicity tests, subchronic toxicity tests, and chronic toxicity tests, it is extremely safe (Food and Development, Vol. 49, No. 12, p. 9, 2014). Therefore, according to the present invention, it is possible to promote the growth of Staphylococcus epidermidis without concerns about safety or side effects. [Brief description of the drawings]

[0018] [Figure 1] 1 is a table showing the composition of the medium used in the growth test of Example 1. [Diagram 2] The top figure is a table showing the viable counts of S. epidermidis cultured in the presence of various concentrations of the test substance, while the bottom figure is a bar graph showing the viable counts. [Diagram 3] The top figure is a table showing the viable counts of Staphylococcus aureus cultured in the presence of various concentrations of the test substance, and the bottom figure is a bar graph showing the viable counts. [Figure 4] The top figure is a table showing the viable counts of Malassezia fungi cultured in the presence of various concentrations of the test substance, while the bottom figure is a bar graph showing the viable counts. [Diagram 5] The top figure is a table showing the viable cell counts of P. acnes cultured in the presence of various concentrations of the test substance, while the bottom figure is a bar graph showing the viable cell counts. [Figure 6] The top figure is a table showing the viable counts of Candida fungi cultured in the presence of various concentrations of the test substance, and the bottom figure is a bar graph showing the viable counts. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention is described in detail below. The present invention provides a growth promoter for Staphylococcus epidermidis and a selective growth promoter for microorganisms. In this specification, these agents may be collectively referred to as "the agent of the present invention" or "the agent" or any one of the agents may be referred to as "the agent of the present invention".

[0020] In the present invention, the term "microbe" is used as a concept that comprehensively includes organisms that are too small to be seen with the naked eye, such as bacteria and fungi. That is, in the present invention, the term "microbe" is synonymous with "microorganism."

[0021] "Promoting growth" refers to increasing the degree of increase in the number of microorganisms (bacterial count). Similarly, "growth-promoting action" refers to the action of increasing the degree of increase in the bacterial count. Furthermore, "growth promoter" refers to an agent used to promote the growth of microorganisms.

[0022] In the present invention, whether or not the agent has a growth-promoting effect can be determined by a culture test, as shown in the examples described later. In the culture test, a sample containing glycerin, which is widely used as a moisturizing agent and lubricant in cosmetics and pharmaceuticals, and a sample containing neither the agent nor glycerin are used as controls for comparison. That is, in the same type of medium, (a) one containing the agent, (b) one containing glycerin, and (c) one containing neither the agent nor glycerin are prepared. After inoculating the same amount of microorganisms into these media and culturing them for a predetermined period of time, the amount of bacteria in the medium is measured. The amount of bacteria can be measured by any of known methods such as colony counting, turbidity, dry cell weight, wet cell weight, and real-time PCR. As a result, if the amount of bacteria in (a) is greater than that in either (b) or (c), it can be determined that the agent has promoted the growth of the microorganism.

[0023] On the other hand, if the amount of bacteria in (A) is smaller than that in either (B) or (C) in the above culture test, it can be determined that the agent does not promote the growth of the microorganism (does not have a growth-promoting effect). In other words, in the present invention, "does not promote growth" includes cases where the increase in the number of bacteria is greater than that when neither the agent nor glycerin is added, but is smaller than that when glycerin is added.

[0024] The term "selective microbial growth promoter" refers to an agent that has a selective growth-promoting effect on microorganisms. Here, the selective growth-promoting effect on microorganisms refers to an effect of promoting the growth of Staphylococcus epidermidis, but not promoting the growth of one or more microorganisms selected from Staphylococcus aureus, Malassezia fungus, Propionibacterium acnes, and Candida fungus.

[0025] As described above, Staphylococcus epidermidis is a microorganism that is beneficial to skin health. When confirming the effects of the present invention, strains (e.g., ATCC12228, NBRC12993) stored in depositories such as the National Institute of Technology and Evaluation (2-49-10 Nishihara, Shibuya-ku, Tokyo) (NITE) Biotechnology Center can be used.

[0026] Staphylococcus aureus, Malassezia, Propionibacterium acnes and Candida are all microorganisms that may be detrimental to skin health, as mentioned above. In order to confirm the effectiveness of the present invention, the following strains stored in preservation institutions such as the NITE Biotechnology Center and the American Type Culture Collection can be used: Staphylococcus aureus: e.g. ATCC6538, NBRC13276, ATCC6538P, NBRC12732, NBRC14462 Malassezia fungi: e.g. NBRC101597 Propionibacterium acnes: e.g. NBRC107605 Candida: e.g. NBRC1594, ATCC10231

[0027] 1-kestose is a trisaccharide oligosaccharide consisting of one glucose molecule and two fructose molecules. 1-kestose may be produced according to a method known to those skilled in the art, or commercially available 1-kestose may be used for convenience. Commercially available products include purified products containing 1-kestose at high purity (mass % of 1-kestose when the total amount of sugar is taken as 100%) and mixtures of fructooligosaccharides containing 1-kestose at a relatively low purity of about 30 to 70 mass%, and any of these may be used.

[0028] The agent may be used as it is as a cosmetic, pharmaceutical, quasi-drug, etc., or may be mixed with other ingredients to be used as a raw material for cosmetics, pharmaceuticals, quasi-drugs, etc.

[0029] This agent exerts its effect by promoting the proliferation of Staphylococcus epidermidis, which is beneficial to skin health among the normal skin bacteria, or by promoting the proliferation of Staphylococcus epidermidis without promoting the proliferation of Staphylococcus aureus, Malassezia fungus, Propionibacterium acnes, and Candida fungus, which are harmful to skin health, thereby creating a favorable skin environment. Therefore, this agent can be used as a preparation for use on the skin, i.e., as an external preparation.

[0030] Examples of the topical agent of the present invention include those that are directly applied, attached, sprayed, etc. to the skin (cosmetics, quasi-drugs, pharmaceuticals), as well as hygiene products such as skin cleansers, bath additives, disinfectants, and bactericides. Examples of the product dosage form include creams, sheets, lotions, emulsions, gels, aerosols, roll-ons, sticks, powders, tablets, etc. Products containing the agent can be produced by adding 1-kestose to raw materials that are normally used in the product (for example, ingredients such as oils, surfactants, alcohols, preservatives, chelating agents, antioxidants, thickeners, fragrances, and bactericides).

[0031] The content of 1-kestose can be set appropriately depending on the form and use of the product, and examples of the content include 0.001 to 40 mass%, 0.01 to 40 mass%, 0.1 to 35 mass%, 0.1 to 30 mass%, 0.1 to 10 mass%, and 0.1 to 5 mass%.

[0032] The present invention will be described below based on examples. It should be noted that the technical scope of the present invention is not limited to the characteristics shown by these examples. EXAMPLES

[0033] Example 1: Growth activity against various microorganisms (1) Test materials The test substances were glycerin and 1-kestose. For glycerin, an aqueous solution containing glycerin at a concentration of 95% by mass or more (commercial product) was used, and for 1-kestose, a composition containing 1-kestose at a purity of 95% by mass or more (powder; commercial product) was used. The concentrations of the test substances in the medium were the final concentrations of each of glycerin and 1-kestose.

[0034] The test bacteria and medium used were those shown in Table 1 below. The composition of the medium is shown in Figure 1. The test bacteria were obtained from the NITE Biotechnology Center. Of the test bacteria, only P. acnes was cultured under anaerobic conditions in an anaerobic jar. [Table 1]

[0035] (2) Growth test The test bacteria were cultured on an agar medium for 22 to 48 hours. The colonies that appeared were picked up and suspended in sterile saline to prepare a bacterial suspension. A liquid medium was placed in a test tube in advance, and the bacterial suspension was added thereto to prepare a bacterial solution. Meanwhile, the test substance was dissolved in sterile saline to prepare a test substance solution of a dilution concentration series. The dissolved agar medium, bacterial solution, and test substance solution were combined and placed in a petri dish to prepare a culture solution. The test substance solution was added using a dilution concentration series so that the final concentrations of the test substance in the culture solution were 0.005 mass%, 0.010 mass%, 0.050 mass%, 0.100 mass%, 0.500 mass%, and 1.000 mass%. As a control sample, a culture solution without the test substance solution (final concentration of the test substance: 0 mass%) was also set. After the culture solution was solidified, it was cultured at 30°C for 24 hours. The number of colonies was then counted to calculate the concentration per culture solution (cfu / mL), which was taken as the viable bacterial count. The results for Staphylococcus epidermidis are shown in Figure 2, those for Staphylococcus aureus in Figure 3, those for Malassezia fungi in Figure 4, those for Propionibacterium acnes in Figure 5, and those for Candida fungi in Figure 6.

[0036] As shown in Figure 2, the higher the concentration of 1-kestose added, the higher the viable cell count of Staphylococcus epidermidis. Furthermore, at all concentrations of the test substance of 0.005 mass%, 0.010 mass%, 0.05 mass%, 0.1 mass%, 0.5 mass%, and 1 mass%, the viable cell count was significantly higher with 1-kestose than with glycerin. These results demonstrate that 1-kestose can promote the growth of Staphylococcus epidermidis.

[0037] On the other hand, as shown in Figure 3, the viable cell count of Staphylococcus aureus was almost constant regardless of the concentration of 1-kestose added. Furthermore, the viable cell count was greater in glycerin than in 1-kestose at all concentrations of the test substance of 0.005 mass%, 0.010 mass%, 0.05 mass%, 0.1 mass%, 0.5 mass%, and 1 mass%. This result demonstrated that 1-kestose hardly promotes the growth of Staphylococcus aureus.

[0038] Furthermore, as shown in Figure 4, no clear positive correlation was observed between the concentration of 1-kestose added and the viable cell count for Malassezia. Furthermore, at test substance concentrations of 0.005 mass%, 0.010 mass%, 0.05 mass%, 0.1 mass%, 0.5 mass%, and 1 mass%, glycerin produced a greater viable cell count than 1-kestose. These results demonstrate that 1-kestose does little to promote the growth of Malassezia.

[0039] As shown in Figure 5, the viable cell count of Propionibacterium acnes was almost constant regardless of the concentration of 1-kestose added. Moreover, the viable cell count was greater in glycerin than in 1-kestose at all concentrations of the test substance of 0.005 mass%, 0.010 mass%, 0.05 mass%, 0.1 mass%, 0.5 mass%, and 1 mass%. This result demonstrated that 1-kestose hardly promotes the proliferation of Propionibacterium acnes.

[0040] As shown in Figure 6, the viable cell count of Candida was almost constant regardless of the concentration of 1-kestose added. Moreover, the viable cell count was greater in glycerin than in 1-kestose at all concentrations of the test substance of 0.005 mass%, 0.010 mass%, 0.05 mass%, 0.1 mass%, 0.5 mass%, and 1 mass%. This result demonstrated that 1-kestose does little to promote the growth of Candida.< / url:> < / url:>

Claims

1. A growth promoter for Staphylococcus epidermidis, containing 1-kestose as an active ingredient.

2. A selective microbial growth-promoting agent containing 1-kestose as an active ingredient, the selective microbial growth-promoting effect being such that the agent has physical properties of promoting the growth of Staphylococcus epidermidis, but not promoting the growth of one or more microorganisms selected from Staphylococcus aureus, Malassezia globosa, Cutibacterium acnes, and Candida albicans.

3. The agent according to claim 1 or 2, which is used as an external preparation.

Images