Staphylococcus aureus biofilm formation control agent and topical skin agent

Abstract

To provide an agent for controlling the formation of a biofilm of Staphylococcus that can suitably control the formation of each biofilm of Staphylococcus epidermidis and Staphylococcus aureus, and an external preparation for skin.SOLUTION: The present invention provides an agent for controlling the formation of a biofilm of Staphylococcus, and an external preparation for skin, each containing at least one of a chitosan derivative chemically modified with an amino group moiety, and β chitin fibers.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an agent for controlling staphylococcal biofilm formation, which is used by being applied to the skin, and an external preparation for skin. [Background technology]

[0002] Various microorganisms exist on human skin as normal skin flora. Examples of normal skin flora include Staphylococcus epidermidis, which is beneficial to the skin, and Staphylococcus aureus, which is harmful to the skin. Both Staphylococcus epidermidis and Staphylococcus aureus are known to form biofilms on the skin. It is known that Staphylococcus epidermidis is more prevalent than Staphylococcus aureus on healthy skin, while Staphylococcus aureus is more prevalent on diseased skin. Therefore, in order to improve skin diseases, it is believed that Staphylococcus epidermidis should be more prevalent than Staphylococcus aureus. In other words, it is believed to be preferable to activate the formation of biofilms by Staphylococcus epidermidis while suppressing the formation of biofilms by Staphylococcus aureus.

[0003] However, because the above two types of staphylococci are taxonomically close, it is relatively difficult to make only the biofilm formation of S. epidermidis predominant. For example, even when a conventional biofilm control agent is applied to the skin, the biofilm formation of S. epidermidis and S. aureus is simultaneously suppressed or promoted. Therefore, there is a demand for a staphylococcal biofilm control agent that can either promote only the biofilm formation of S. epidermidis or suppress only the biofilm formation of S. aureus.

[0004] In response to this, an application agent for controlling normal skin bacteria is known that can more significantly inhibit the growth of harmful Staphylococcus aureus than beneficial Staphylococcus epidermidis (for example, Patent Document 1). The topical agent for controlling resident skin bacteria described in Patent Document 1 contains magnesium lactate and / or calcium lactate as active ingredients. The topical agent for controlling resident skin bacteria described in Patent Document 1 inhibits the growth of both Staphylococcus epidermidis and Staphylococcus aureus when applied to the skin, but is more effective at inhibiting the growth of Staphylococcus aureus than Staphylococcus epidermidis.

[0005] However, the liniment for controlling skin resident bacteria described in Patent Document 1 inhibits the growth of both Staphylococcus epidermidis and Staphylococcus aureus, and therefore has the problem that it is not necessarily sufficient in terms of its ability to inhibit biofilm formation by Staphylococcus epidermidis without significantly inhibiting biofilm formation by Staphylococcus epidermidis, or to promote biofilm formation by Staphylococcus epidermidis without substantially promoting biofilm formation by Staphylococcus aureus. In other words, it does not have the ability to suitably control biofilm formation by two types of staphylococcus. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 2018-052891 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above-mentioned problems, an object of the present invention is to provide a staphylococcal biofilm formation control agent and an external skin preparation that can suitably control biofilm formation by two types of staphylococcus. [Means for solving the problem]

[0008] The agent for controlling staphylococcal biofilm formation according to the present invention is characterized by containing at least one of a chitosan derivative chemically modified at an amino group moiety and β-chitin fiber. The agent for controlling staphylococcal biofilm formation can suitably control the formation of biofilms by Staphylococcus epidermidis and Staphylococcus aureus.

[0009] The agent for controlling staphylococcal biofilm formation according to the present invention preferably contains the chitosan derivative and the β-chitin fiber. In the agent for controlling staphylococcal biofilm formation according to the present invention, the chitosan derivative is preferably partially myristoylated carboxymethylchitosan.

[0010] The topical skin preparation according to the present invention is characterized by containing the above-mentioned agent for controlling staphylococcal biofilm formation. By applying the above-mentioned topical skin preparation to the skin, biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus can be suitably controlled. [Effects of the Invention]

[0011] The agent for controlling staphylococcal biofilm formation and the topical skin preparation of the present invention have the effect of being able to suitably control biofilm formation by two types of staphylococcus. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the effect of a composition of the present embodiment containing a chitosan derivative on the formation of a staphylococcal biofilm. [Figure 2A] Graph showing the effect of the composition of the present embodiment containing β-chitin fibers on staphylococcal biofilm formation. [Figure 2B] Graph showing the effect of a composition containing cellulose fibers on Staphylococcus aureus biofilm formation. [Figure 3] Graph showing the effect of the composition of the present embodiment containing a chitosan derivative and β-chitin fibers on staphylococcal biofilm formation. [Figure 4A] 1 is a graph showing the effect of a composition of the present embodiment containing a chitosan derivative on the formation and proliferation of Staphylococcus aureus biofilms. [Figure 4B]A graph showing the effect of the composition of this embodiment containing β-chitin fibers on staphylococcal biofilm formation and proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0013] An embodiment of the agent for controlling staphylococcal biofilm formation and the topical skin preparation (topical skin composition) according to the present invention will be described below. Hereinafter, both of these may be simply referred to as the composition.

[0014] The composition of the present embodiment contains at least one of a chitosan derivative chemically modified with an amino group moiety and β-chitin fiber.

[0015] The chitosan derivative is a compound in which the amino group attached to the carbon atom at position 2 in the D-glucosamine structure constituting chitosan is chemically modified. The chitosan derivative is soluble in water. Examples of the chitosan derivatives include acylated chitosan derivatives in which a monovalent organic acid is partially amide-bonded to the amino groups. In other words, examples of the chitosan derivatives include acylated chitosan derivatives in which a monovalent organic acid is amide-bonded to some of the amino groups in chitosan. Examples of the chitosan derivatives include acylated chitosan derivatives in which one H of the amino group is substituted with an acyl group. In the chitosan derivatives described above, the H of the hydroxy group bonded to the carbon atom constituting the pyranose ring (for example, the carbon atom at the 3rd position) may also be substituted with an acyl group.

[0016] Examples of organic acids that partially form amide bonds as described above include fatty acids having 12 to 22 carbon atoms. Specific examples include lauric acid, myristic acid, palmitic acid, stearic acid, and behenic acid. In other words, examples of acyl groups as substituents as described above include aliphatic acyl groups having 12 to 22 carbon atoms. Specific examples of aliphatic acyl groups include lauroyl, myristoyl, palmitoyl, stearoyl, and behenoyl groups. In other words, the chitosan derivatives include aliphatic acylated chitosan derivatives such as partially lauroylated chitosan derivatives, partially myristoylated chitosan derivatives, partially palmitoylated chitosan derivatives, and partially stearoylated chitosan derivatives.

[0017] In the chitosan derivatives described above, some of the amino groups are usually chemically modified. As the chitosan derivatives, partially acylated chitosan derivatives in which some amino groups have been chemically modified with organic groups are preferred, and partially aliphatic acylated chitosan derivatives in which some amino groups have been chemically modified with aliphatic organic groups are more preferred, as they can more fully promote biofilm formation by Staphylococcus epidermidis (so-called beneficial bacteria). The partially aliphatic acylated chitosan derivative is preferably at least one selected from the group consisting of partially lauroylated chitosan derivatives, partially myristoylated chitosan derivatives, partially palmitoylated chitosan derivatives, partially stearoylated chitosan derivatives, and partially behenoylated chitosan derivatives, and more preferably a partially myristoylated chitosan derivative.

[0018] The chitosan derivatives may be further chemically modified at the oxygen atom adjacent to the carbon at position 6 in the D-glucosamine structure. Examples of functional groups bonded to the oxygen atoms by such chemical modification include carboxymethyl groups, hydroxyethyl groups, hydroxypropyl groups, methyl groups, etc. The functional group is preferably a carboxymethyl group, in that it can favorably control biofilm formation by two types of staphylococci.

[0019] The average molecular weight of the partially aliphatic acylated carboxymethyl chitosan is preferably 500,000 to 1,000,000. The introduction rate of aliphatic groups (e.g., myristoyl groups) in the partially aliphatic acylated carboxymethyl chitosan is preferably 0.10% to 50.0%.

[0020] The introduction rate (%) of aliphatic groups in partially aliphatic acylated carboxymethyl chitosan refers to the introduction rate per 100 hexosamine residues, which are the constituent monosaccharides of carboxymethyl chitosan. For example, partially aliphatic acylated carboxymethyl chitosan with an introduction rate of 10.0% of aliphatic groups means that 10 myristoyl groups are introduced into 100 carboxymethyl glucosamine residues, which are the constituent monosaccharides. As the partially myristoylated carboxymethylchitosan, one in which myristoyl groups have been introduced into carboxymethylchitosan having an average molecular weight of 500,000 to 1,000,000 at an introduction rate of 0.10% to 50.0% is preferred.

[0021] Commercially available products can be used as the chitosan derivatives. For example, a partially aliphatic acylated carboxymethyl chitosan (partially myristoylated carboxymethyl chitosan) available under the product name "MC Chitosan" (manufactured by Pierce) can be used.

[0022] The chitosan derivative is contained in the composition in an amount of, for example, 0.001% by mass to 1.000% by mass (solid content equivalent).

[0023] The concentration of the chitosan derivative in the composition of this embodiment (solid content equivalent) is preferably 0.002% by mass or more and 0.500% by mass or less, and more preferably 0.003% by mass or more and 0.100% by mass or less. The above-mentioned content has the advantage of further improving epidermal keratinization.

[0024] The composition of the present embodiment, containing the chitosan derivative but not β-chitin fiber, does not significantly affect the growth inhibition of Staphylococcus aureus (so-called bad bacteria), but can inhibit biofilm formation by S. aureus, which is prone to biofilm formation. In contrast, a composition containing the chitosan derivative does not significantly affect the growth inhibition of Staphylococcus epidermidis (so-called good bacteria), but promotes biofilm formation by S. epidermidis, which does not naturally form biofilms. Therefore, after application to the skin, the composition of the present embodiment containing the chitosan derivative can preferentially promote the colonization of S. epidermidis (so-called good bacteria) over S. aureus (so-called bad bacteria) on the skin. In other words, it can inhibit the formation of biofilms by S. aureus (so-called bad bacteria) and promote the formation of biofilms by S. epidermidis (so-called good bacteria) and colonization on the skin. In this way, the ability to effectively control biofilm formation by two types of staphylococcus can improve the condition of the skin.

[0025] The β-chitin fibers that may be contained in the composition of this embodiment include fibrous materials formed from poly-N-acetylglucosamine. Such fibrous materials are generally water-insoluble and therefore can be dispersed in a solvent containing water. β-chitin fibers are formed from multiple fibrous materials, and in each fibrous material, multiple sugar chains are bundled and extend in the fiber length direction. The thickness of each fibrous substance is usually 1 nm or more and 100 nm or less.

[0026] Beta-chitin fibers are composed of multiple fibrous materials, and in each fibrous material, multiple sugar chains of poly-beta-1,4-N-acetyl-D-glucosamine extend in the fiber length direction and are adjacent to each other in the fiber diameter direction.

[0027] β-chitin fibers are prepared from β-chitin derived from, for example, squid midbone (cartilage). Compared to α-chitin, β-chitin has fewer hydrogen bonds in its molecule and a weaker crystalline structure, resulting in improved swelling in water, uniform dispersibility, affinity for living organisms and cells, and enzymatic degradability. β-chitin fibers are composed of multiple fibrous materials, and in each fibrous material, multiple poly-β-1,4-N-acetyl-D-glucosamine sugar chains extend along the fiber length and are adjacent in the fiber diameter direction. In the β-chitin of β-chitin fibers, the β-1,4 bonds of adjacent sugar chains are oriented in the same direction along the chain length. On the other hand, in α-chitin fibers prepared from α-chitin obtained from the exoskeleton (shell) of crustaceans such as crabs and shrimp, the β-1,4 bonds of adjacent poly-β-1,4-N-acetyl-D-glucosamine sugar chains are oriented in opposite directions along the chain length.

[0028] In α-chitin fibers, the β-1,4 bonds of adjacent poly-β-1,4-N-acetyl-D-glucosamine sugar chains are oriented in opposite directions along the chain length, whereas in β-chitin fibers, the β-1,4 bonds of adjacent sugar chains are oriented in the same direction along the chain length.

[0029] As the β-chitin fiber, for example, a raw material named "β-chitin nanofiber liquid (β-chitin NF)" (manufactured by Yaegaki Hakko Giken Co., Ltd.) can be used.

[0030] The β-chitin fiber is contained in the composition in an amount of, for example, 0.001% by mass or more and 1.000% by mass or less (solid content equivalent).

[0031] The concentration of β-chitin fiber (solid content equivalent) in the composition of this embodiment is preferably 0.002% by mass or more and 0.500% by mass or less, and more preferably 0.003% by mass or more and 0.100% by mass or less. The above-mentioned content has the advantage of further improving epidermal keratinization.

[0032] The composition of this embodiment, containing β-chitin fiber but not chitosan derivative, has little effect on the growth inhibition of Staphylococcus epidermidis (so-called beneficial bacteria) and little effect on biofilm formation. In contrast, the composition of this embodiment containing β-chitin fiber has little effect on the growth inhibition of Staphylococcus aureus (so-called harmful bacteria), but can suppress biofilm formation by Staphylococcus aureus, which is naturally prone to biofilm formation. Therefore, after application to the skin, the composition of this embodiment containing β-chitin fiber can preferentially promote the colonization of Staphylococcus epidermidis (so-called beneficial bacteria) on the skin over Staphylococcus aureus (so-called harmful bacteria). In other words, by inhibiting the formation of biofilms and colonization of Staphylococcus aureus (so-called harmful bacteria) on the skin, it can promote the colonization of Staphylococcus epidermidis (so-called beneficial bacteria) on the skin. In this way, the ability to effectively control biofilm formation by two types of staphylococcus can improve the condition of the skin.

[0033] The composition of this embodiment preferably contains both the chitosan derivative and β-chitin fibers, and particularly preferably contains the partially aliphatic acylated carboxymethylchitosan as the chitosan derivative. When the composition of this embodiment contains both the chitosan derivative and β-chitin fibers, the β-chitin fibers inhibit biofilm formation by Staphylococcus aureus (bad bacteria), while the chitosan derivative promotes biofilm formation by Staphylococcus epidermidis (good bacteria). This allows the composition applied to the skin, etc., to more favorably colonize Staphylococcus epidermidis (good bacteria) on the skin than Staphylococcus aureus (bad bacteria). This more favorable control of biofilm formation by the two types of staphylococcus can improve the condition of the skin.

[0034] In the composition of this embodiment, the mass ratio (X / Y) of the chitosan derivative (X) to the β-chitin fiber (Y) is preferably 0.01 or more, more preferably 0.05 or more, and even more preferably 0.1 or more, in terms of solid matter. The mass ratio (X / Y) is preferably 200 or less, more preferably 150 or less, and even more preferably 100 or less. When the mass ratio (X / Y) is within the above range, biofilm formation by Staphylococcus epidermidis can be promoted while biofilm formation by Staphylococcus aureus can be further suppressed, thereby improving the condition of the skin when the composition is applied to the skin.

[0035] The composition of this embodiment typically contains water. In addition to the above-mentioned components, the composition of this embodiment may also contain components commonly incorporated into cosmetics, topical skin preparations, and the like. Examples of components that the composition of this embodiment may contain include polyhydric alcohols such as dipropylene glycol, glycerin, pentylene glycol, propylene glycol, 1,3-butylene glycol, and diglycerin. Other examples include preservatives, antioxidants, anti-inflammatory agents, UV absorbers, UV scattering agents, vitamins, and enzymes. The composition of this embodiment is composed of components specified in the Standards for Quasi-Drug Ingredients, Standards for Ingredients by Cosmetic Type, Standards for Cosmetic Ingredients, Japanese Pharmacopoeia, and Official Standards for Food Additives, among others.

[0036] The state of the composition of the present embodiment is not particularly limited, but is usually liquid.

[0037] The composition of this embodiment can be produced by mixing and stirring the above-mentioned chitosan derivative or β-chitin fiber with other components by a general method.

[0038] The composition of this embodiment is used by being applied to the skin, for example. The composition is an external preparation for the human body that can be applied to, for example, the skin of the face, neck, limbs, scalp, hair, and mucous membranes of the nostrils, lips, ears, genitals, anus, etc. The composition is not particularly restricted by classification as a cosmetic, quasi-drug, pharmaceutical, etc. under the Pharmaceutical and Medical Device Act, and is applicable to a variety of uses.

[0039] The staphylococcal biofilm formation control agent and topical skin preparation of the present invention are as exemplified above, but the present invention is not limited to the above-exemplified embodiments. Furthermore, in the present invention, various forms employed in general topical skin preparations and the like can be employed within a range that does not impair the effects of the present invention. [Example]

[0040] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0041] <Substances used in the test> [Liquid containing chitosan derivatives chemically modified with amino groups] Partially myristoylated carboxymethyl chitosan (carboxymethyl chitosan myristamide) Product name: "MC Chitosan" (Pierce Co.) Solid content: 1.0% by mass [β-chitin fiber-containing liquid] Raw material name: "β-chitin nanofiber liquid (β-chitin NF)" (manufactured by Yaegaki Fermentation Engineering Co., Ltd.) The beta-chitin powder obtained by alkali and acid treatment of squid bones was dispersed in water. This dispersion was then subjected to high-pressure collisions to obtain a dispersion containing fibrous materials with diameters of 2 to 8 nm. The solid content of the liquid was 1.6% by mass. [Reference: Cellulose fiber-containing liquid] (Product name: "LeoCrysta" manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) Solid content: 2% by mass A solution diluted to 20% concentration (5 times diluted) (solid content 0.4% by mass) was used as the test solution.

[0042] <Test 1> Biofilm formation of the above chitosan derivatives [Preparation of test solution A] Using TSB (Tryptone Soy Broth) medium, the chitosan derivative-containing solution was adjusted in concentration from 10% by mass to 0.02% by mass in a two-fold dilution series (10 concentrations). [Preparation of bacteria-containing solution B] Staphylococcus aureus (a) and Staphylococcus epidermidis (b), which are normal skin bacteria, were purchased from the National Biotechnology Center of the National Institute of Technology and Evaluation (NBRC). (A) Staphylococcus aureus (bad bacteria) Staphylococcus aureus : NBRC 13276 (a) Staphylococcus epidermidis (good bacteria) Staphylococcus epidermidis : NBRC 12993 The above test bacteria were pre-cultured in TSB (Tryptone Soy Broth) medium at 37°C under aerobic conditions until they reached a steady state. Equal amounts of the pre-cultured bacteria were mixed and used to prepare 1 x 10 8 The concentration was adjusted with TSB medium to cfu / mL to prepare a bacteria-containing solution. [Biofilm formation / quantification] 1. Test solution A (containing the above chitosan derivative) prepared using TSB medium was dispensed into each well of a 96-well microplate (manufactured by IWAKI Polystyrene) at 100 μL (11 concentrations ranging from 10% by mass to 0.01% by mass in a 2-fold dilution series (n=4 for each concentration)). 2. 10 μL of the above bacteria-containing solution B was inoculated into each well. 3. TSB medium without the chitosan derivative was used as a control. The wells were cultured at 4.37°C under aerobic conditions for 24 hours to form a biofilm (hereinafter referred to as BF) of normal skin bacteria on the bottom of the well. 5. After the formation of BF was completed, the well was washed with sterile water and dried. 6. 100 μL of 0.1% (w / v) crystal violet solution was added to each well, and staining was carried out for 30 minutes. 7. After staining, the plate was washed three times with sterilized water. 8. 100 μL of ethanol was added to each well to extract the crystal violet in the biofilm, and the absorbance (OD570) was measured.

[0043] The results of Test 1 are shown in Figure 1. As can be seen from Figure 1, the chitosan derivative (partially myristoylated carboxymethyl chitosan) was able to inhibit BF formation by Staphylococcus aureus, while promoting BF formation by Staphylococcus epidermidis.

[0044] <Test 2> Biofilm formation of β-chitin fibers [Preparation of test solution C and test solution D] Using TSB (Tryptone Soy Broth) medium, the concentrations of each solution containing β-chitin fiber and cellulose fiber were adjusted in a two-fold dilution series from 10% by mass to 0.01% by mass (11 concentrations). - Test solution C (containing β-chitin fiber) - Test solution D (containing cellulose fiber) [Preparation of bacteria-containing solution B] The above-mentioned skin resident bacteria (a) and skin resident bacteria (b) were used. Each test bacterium was pre-cultured in TSB (Tryptone Soy Broth) medium at 37°C under aerobic conditions until it reached a steady state. Equal amounts of these pre-cultured bacteria were mixed and used to make 1 x 10 8 The concentration was adjusted with TSB medium to cfu / mL to prepare bacteria-containing solution B. [Biofilm formation / quantification] 1. 100 μL of test solutions C and D containing β-chitin fibers or cellulose fibers prepared using TSB medium were dispensed into each well of a 96-well microplate (IWAKI polystyrene). (11 concentrations ranging from 10% by mass to 0.01% by mass in a 2-fold dilution series (n=4 for each concentration)) 2. 10 μL of the above bacteria-containing solution B was inoculated into each well. 3. The TSB medium containing neither β-chitin fiber nor cellulose fiber was used as a control. The wells were cultured at 4.37°C under aerobic conditions for 24 hours to form a biofilm (hereinafter referred to as BF) of normal skin bacteria on the bottom of the well. 5. After the formation of BF was completed, the well was washed with sterile water and dried. 6. 100 μL of 0.1% (w / v) crystal violet solution was added to each well, and staining was carried out for 30 minutes. 7. After staining, the plate was washed three times with sterilized water. 8. 100 μL of ethanol was added to each well to extract the crystal violet in the biofilm, and the absorbance (OD570) was measured.

[0045] The results of Test 2 are shown in Figure 2A (β-chitin fiber) and Figure 2B (cellulose fiber). As can be seen from Figure 2A, at a certain concentration, β-chitin fiber was able to suppress BF formation by Staphylococcus aureus while promoting BF formation by Staphylococcus epidermidis. Furthermore, as can be seen from Figure 2B, even when cellulose fiber was added to the medium, BF formation by the beneficial bacterium Staphylococcus epidermidis was more strongly suppressed than that by the harmful bacterium Staphylococcus aureus.

[0046] <Test 3> Biofilm formation when the chitosan derivative and the β-chitin fiber are combined [Preparation of test solution E] Each sample of the chitosan derivative-containing solution and the β-chitin fiber-containing solution was diluted with TSB medium to the following final concentrations.

[0047] [Table 1] [Preparation of bacteria-containing solution B] The above-mentioned skin resident bacteria (a) and skin resident bacteria (b) were used. Each test bacterium was pre-cultured in TSB (Tryptone Soy Broth) medium at 37°C under aerobic conditions until it reached a steady state. Equal amounts of these pre-cultured bacteria were mixed and used to make 1 x 10 8 The concentration was adjusted with TSB medium to cfu / mL to prepare bacteria-containing solution B. [Biofilm formation / quantification] 1. Test solution E prepared using TSB medium was dispensed into each well of a 96-well microplate (IWAKI polystyrene) at 100 μL (each concentration, n=4). 2. 10 μL of the above bacteria-containing solution B was inoculated into each well. 3. A TSB medium containing 5% by mass of the chitosan derivative but not containing the β-chitin fiber was used as a control. The wells were cultured at 4.37°C under aerobic conditions for 24 hours to form a biofilm (hereinafter referred to as BF) of normal skin bacteria on the bottom of the well. 5. After the formation of BF was completed, the well was washed with sterile water and dried. 6. 100 μL of 0.1% (w / v) crystal violet solution was added to each well, and staining was carried out for 30 minutes. 7. After staining, the plate was washed three times with sterilized water. 8. 100 μL of ethanol was added to each well to extract the crystal violet in the biofilm, and the absorbance (OD570) was measured.

[0048] The results of Test 3 are shown in Figure 3. As can be seen from Figure 3, by combining the above chitosan derivative with β-chitin fiber, it was possible to suppress BF formation by Staphylococcus aureus while promoting BF formation by Staphylococcus epidermidis.

[0049] <Test 4> Measurement of biofilm formation amount and number of growing viable bacteria The relationship between the biofilm formation-promoting effect of chitosan derivatives and the growth-promoting effect of viable bacteria The relationship between the inhibitory effect of β-chitin fiber on biofilm formation and its promoting effect on viable bacterial growth [Preparation of Test Solution F and Test Solution G] Using TSB (Tryptone Soy Broth) medium, the chitosan derivative-containing solution was diluted two-fold from a 10% by mass concentration (5 concentrations) to prepare test solution F. Using TSB (Tryptone Soy Broth) medium, the concentration of the β-chitin fiber-containing solution was adjusted by 2-fold dilution from 10% by mass (5 concentrations) to prepare test solution G. [Preparation of bacteria-containing solution B] The above-mentioned skin resident bacteria (a) and (b) were pre-cultured in TSB (Tryptone Soy Broth) medium at 37°C under aerobic conditions until they reached a steady state. Equal amounts of these pre-cultured bacteria were mixed and used to prepare 1 x 10 8 The concentration was adjusted with TSB medium to cfu / mL to prepare bacteria-containing solution B. [Biofilm formation] (same procedure for two microplates) 1. 100 μL of test solutions F and G containing β-chitin fibers prepared using TSB medium were dispensed into each well of a 96-well microplate (IWAKI Polystyrene). 2. 10 μL of the above bacteria-containing solution B was inoculated into each well. 3. TSB medium containing no active ingredient was used as a control. The wells were cultured at 4.37°C under aerobic conditions for 24 hours to form a biofilm (hereinafter referred to as BF) of normal skin bacteria on the bottom of the well. [Quantification of biofilm] (First microplate) 5. After the formation of BF was completed, the well was washed with sterile water and dried. 6. 100 μL of 0.1% (w / v) crystal violet solution was added to each well, and staining was carried out for 30 minutes. 7. After staining, the plate was washed three times with sterilized water. 8. 100 μL of ethanol was added to each well to extract the crystal violet in the biofilm, and the absorbance (OD570) was measured. [Measurement of the amount of biofilm formed in the wells and the total viable airborne bacteria] (Second microplate) 9. An extracellular ATP elimination reagent was added to each well of the second microplate. 10. After the extracellular ATP elimination treatment was completed, the ATP extraction reagent was added and stirred with a pipette to detach the biofilm at the bottom of each well. 11. The sample in the well was transferred to a measurement tube. 12. After adding an ATP luminescent reagent and stirring, the amount of ATP was measured. 13. This amount of ATP luminescence corresponds to the amount of biofilm formed in the well and the total viable planktonic bacteria. [ATP measurement] Measuring device: Microtech Nichion Luminometer Measurement reagent: Kikkoman Biochemifa

[0050] The results of Test 4 are shown in Figures 4A and 4B. As shown in Figure 4A, the chitosan derivatives did not necessarily strongly inhibit the growth of normal skin bacteria, but they promoted BF formation by Staphylococcus epidermidis and inhibited BF formation by Staphylococcus aureus. Furthermore, as shown in Figure 4B, the same was true for β-chitin fiber, and relatively high concentrations of β-chitin fiber tended to inhibit the growth of Staphylococcus aureus. In other words, chitosan derivatives and chitin fibers can promote or suppress biofilm formation, despite not significantly inhibiting the growth of resident skin bacteria. Therefore, the mechanism of action of chitosan derivatives and chitin fibers in inhibiting or promoting biofilm formation is thought to be different from the mechanism of action of promoting or suppressing the growth of resident skin bacteria.

[0051] As a reference test, a similar test was conducted using alpha-chitin fiber. As with the cellulose fiber mentioned above, alpha-chitin fiber more strongly inhibited BF formation by the beneficial bacterium Staphylococcus epidermidis than by the harmful bacterium Staphylococcus aureus.

[0052] From the above results, it is believed that applying a test sample (composition) corresponding to the example to the skin can promote biofilm formation by Staphylococcus epidermidis while suppressing biofilm formation by Staphylococcus aureus. Separate tests confirmed that the transepidermal water loss (TEWL) decreases as the number of Staphylococcus epidermidis on the skin increases. The lower the transepidermal water loss (TEWL) value, the less water is evaporated from the skin, which can be said to improve the skin's condition. Therefore, the skin can be made healthier. [Industrial Applicability]

[0053] The staphylococcal biofilm formation control agent and topical skin preparation of the present invention are used by being applied to the skin, etc., for example, to maintain healthy skin, etc. Furthermore, the staphylococcal biofilm formation control agent and topical skin preparation of the present invention are used by being applied to the skin, etc., for example, to improve the condition of skin, etc., which has deteriorated due to inflammation, etc.

Claims

1. A staphylococcal biofilm formation control agent comprising 0.001% by mass or more and 1.000% by mass or less of partially myristoylated carboxymethyl chitosan.

2. the partially myristoylated carboxymethyl chitosan; A staphylococcal biofilm formation control agent as described in claim 1, which contains beta-chitin fibers containing multiple fibrous objects with a thickness of 1 nm or more and 100 nm or less, and 0.001 mass% or more and 1.000 mass% or less of beta-chitin fibers.

3. A staphylococcal biofilm formation control agent as described in claim 2, wherein the mass ratio (X / Y) of the partially myristoylated carboxymethyl chitosan (X) to the β-chitin fiber (Y) is 0.01 or more and 200 or less.

4. A β-chitin fiber containing a partially myristoylated carboxymethyl chitosan and a plurality of fibrous substances having a thickness of 1 nm or more and 100 nm or less, A topical skin preparation containing the partially myristoylated carboxymethyl chitosan and the β-chitin fiber in an amount of 0.001% by mass or more and 1.000% by mass or less, respectively.

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