Method for evaluating efficacy of composition using skin resident bacteria

Abstract

The present invention provides a method for evaluating the efficacy of a composition. The method for evaluating the efficacy of a composition comprises the steps of: selecting a medium for creating a dominant state of a target strain among a plurality of skin resident bacteria; selecting an inoculation order of the plurality of skin resident bacteria so as to create the dominant state of the target strain; inoculating the plurality of skin resident bacteria into the selected medium according to the selected inoculation order; creating the dominant state of the target strain; applying a composition for evaluation in the created dominant state; and evaluating an effect on the skin.

Description

Method for evaluating the efficacy of a composition using skin flora

[0001] The present application relates to a method for evaluating the efficacy of a composition using skin flora.

[0002] The microbiome, a portmanteau of "microbe" and "biome," refers to the collective of microorganisms that exist within and influence the human body. These microorganisms tend to dominate when two or more species are exposed to an equal environment. Most strains are known to create a dominant environment through exocrine substances and other metabolites.

[0003] The skin is home to resident microbes, primarily found in the outermost layer of the epidermis and the upper part of hair follicles (supraderial layer). Beyond directly applying a composition to the target strain on the skin to evaluate its effects, a simpler and more efficient method is needed to assess its effects on the skin.

[0004] Accordingly, the present invention aims to provide an evaluation method capable of evaluating the efficacy of a composition by forming a microbiome.

[0005] The purpose of this application is to provide a method for evaluating the efficacy of a composition using skin flora.

[0006] The technical problems of the present application are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0007] According to an embodiment of the present application, a method for evaluating the efficacy of a composition is provided. The method for evaluating the efficacy may include: selecting a medium to form a dominant state of a target strain among a plurality of skin resident bacteria; selecting an inoculation order of the plurality of skin resident bacteria to form a dominant state of the target strain; inoculating the plurality of skin resident bacteria into the selected medium according to the selected inoculation order; creating a dominant state of the target strain; applying a composition for evaluation in the created dominant state; and evaluating the effect on the skin.

[0008] Additionally, the inoculation of the above multiple skin flora may be performed within 48 hours from the first inoculation to the last inoculation.

[0009] Additionally, the above inoculation may be performed with three or more strains with a time difference of at least three hours.

[0010] Additionally, the inoculation may include inoculating the second strain at least 3 hours after inoculating the first strain, and inoculating the third strain at least 6 hours after inoculating the second strain.

[0011] Additionally, the dominant state may be a state in which the plurality of skin flora are co-dominant or at least one strain among the plurality of strains is dominant.

[0012] Additionally, the dominant state may include at least one of a first section in which the first strain is dominant, a second section in which the second strain is dominant, a third section in which the third strain is dominant, or a fourth section in which the first strain, the second strain, and the third strain are co-dominant.

[0013] In addition, the skin flora may include three or more species selected from among Staphylococcus strains, Streptococcus strains, Malassezia strains, Propionibacterium strains, and Candida strains.

[0014] In addition, the skin flora includes at least three species selected from among Staphylococcus aureus, Propionibacterium acnes, Corynebacterium minutissimum, Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus warneri, Streptococcus mitis, Micrococcus luteus or Acinetobacter johnsonii, Streptococcus pyogens, Malassezia furfur, or Candida albicans. Can be.

[0015] Additionally, the step of selecting the badge may include selecting the type and mixing ratio of the badge.

[0016] Additionally, the medium may include a method for evaluating the efficacy of a composition, wherein the medium is a single medium or a mixed medium comprising a first medium and a second medium.

[0017] In addition, the mixed medium may include a method for evaluating the efficacy of a composition in which the first medium and the second medium are mixed in a weight ratio of 1:0.001 to 20.

[0018] Additionally, the first medium may include peptone, beef extract, yeast extract, dextrose, sodium chloride, soluble starch, cysteine, sodium acetate, and agar.

[0019] Additionally, the first medium may be a Reinforced Clostridial Medium (RCM) medium.

[0020] Additionally, the second medium may include tryptone, soytone, dextrose, sodium chloride and dipotassium phosphate.

[0021] Additionally, the second medium may be a TSB (Tryptic Soy Broth) medium.

[0022] According to an embodiment of the present application, a co-culture model of a microbiome can be provided for evaluating the skin influence of a composition for evaluation.

[0023] Additionally, to evaluate the efficacy of the composition, multiple strains can be sequentially inoculated to create a microbiome environment in which the desired strain is dominant.

[0024] In addition, by appropriately adjusting the type and mixing ratio of the medium, a dominant state between strains can be artificially created, thereby simulating the environment in which the desired strain is dominant, thereby creating the desired evaluation environment.

[0025] In addition, by creating a similar evaluation environment using a co-culture model without directly collecting microbiome samples, the efficacy of a composition can be efficiently evaluated without directly targeting the human body.

[0026] To facilitate a more thorough understanding of the drawings cited in the detailed description of this application, a brief description of each drawing is provided.

[0027] Figures 1a to 1c are graphs showing the growth results of three strains in RCM medium.

[0028] Figure 2 is a graph showing the absorbance measured in Manufacturing Examples 11 to 13.

[0029] Figure 3 shows the results of counting the number of S. epidermidis and S. aureus in RCM medium, expressed in 3-hour units.

[0030] Figures 4a to 4c show the results of counting each strain at 3-hour intervals from the start of culture at different inoculation times.

[0031] Figure 5 shows the results of counting the number of P. acnes bacteria according to the ratio of the medium in the mixed medium.

[0032] Figure 6 shows the results of counting the number of P. acnes, S. epidermidis, and S. aureus in a mixed medium.

[0033] The structural or functional descriptions of the embodiments disclosed in this specification are merely illustrative for the purpose of explaining embodiments according to the technical concept of the present application. Furthermore, embodiments according to the technical concept of the present application may be implemented in various forms other than those disclosed herein. Furthermore, the technical concept of the present application is not limited to the embodiments described herein.

[0034] Among the properties mentioned in this specification, if the measurement temperature affects the properties, the properties are properties measured at room temperature and pressure, unless otherwise specified.

[0035] The terms a to b used in this specification mean a and b inclusive and within the range between a and b. For example, including a to b by weight means including within the range of a to b by weight.

[0036] In this specification, terms including ordinal numbers, such as "first," "second," etc., may be used to describe various components, but the components are not limited by the terms. The terms are used solely to distinguish one component from another. For example, without departing from the scope of the present application, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component. The term "and / or" includes a combination of a plurality of related items or any one of a plurality of related items.

[0037] The terminology used in this specification is for the purpose of describing embodiments and is not intended to limit and / or restrict the present application. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprises" or "has" are intended to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0038]

[0039] Hereinafter, exemplary embodiments according to the present application will be described in detail with reference to the contents described in the attached drawings.

[0040]

[0041] According to an embodiment of the present application, a method for evaluating the efficacy of a composition is provided. The method may include: selecting a medium to form a dominant state of a target strain among a plurality of skin resident bacteria; selecting an inoculation order of the plurality of skin resident bacteria to form a dominant state of the target strain; inoculating the plurality of skin resident bacteria into the selected medium according to the selected inoculation order; creating a dominant state of the target strain; applying a composition for evaluation in the created dominant state; and evaluating the effect on the skin.

[0042]

[0043] skin flora

[0044] The skin resident bacteria of the present application may be three or more species selected from among Staphylococcus strains, Streptococcus strains, Malassezia strains, Propionibacterium strains, and Candida strains.

[0045] Specifically, it can be a harmful bacteria or a beneficial bacteria, and a harmful bacteria can refer to a strain that has a harmful effect on the skin or scalp, such as an inflammatory response, and a beneficial bacteria can refer to a strain among the resident bacteria of the skin or scalp that helps the skin or scalp, such as by alleviating inflammation or inhibiting the growth of harmful bacteria.

[0046] The above skin harmful bacteria may be at least one of Staphylococcus aureus, Propionibacterium acnes, Corynebacterium minutissimum, or Cutibacterium acnes.

[0047] The above skin beneficial bacteria may be at least one of Staphylococcus epidermidis, Staphylococcus warneri, Streptococcus mitis, Micrococcus luteus, or Acinetobacter johnsonii.

[0048] In one embodiment of the present application, the skin flora may include Staphylococcus aureus, Propionibacterium acnes, and Staphylococcus epidermidis.

[0049] The above strains are skin resident bacteria that inhabit the skin, and by culturing them together to form a microbiome, the strains can be used to evaluate the effect of an evaluation composition containing an effective ingredient on the skin.

[0050]

[0051] Dominant state

[0052] In this specification, the dominant state means a state determined based on the size of the clusters between strains in each cluster, the number of strains, etc. when multiple strains are co-cultured, and specifically, it means whether the expression and proliferation of a specific strain is quantitatively superior, inferior, or equal to that of other strains.

[0053] A dominant state may mean a state in which, in a culture of multiple strains, one specific strain has an advantage in development or proliferation over other specific strains.

[0054] A co-dominant state may mean a state in which multiple strains are cultured at similar rates without equal or substantial superiority in occurrence or proliferation. A co-dominant state may be a state in which the ratio of the numbers of multiple strains or the difference in the number of strains is below a threshold value. For example, the threshold for the ratio of the numbers of strains is 1.0 (log 10 ) or less, preferably 0.5 (log 10 ) may be less than or equal to:

[0055] In an embodiment of the present application, the dominant state may be a co-dominant state in which all of the plurality of skin resident bacteria are dominant, or a state in which at least one strain among the plurality of strains is dominant.

[0056] In an embodiment of the present application, the dominant state may include at least one of a first section in which a first strain is dominant, a second section in which a second strain is dominant, a third section in which a third strain is dominant, or a fourth section in which the first strain, the second strain, and the third strain are co-dominant during culture by inoculating multiple strains into a medium.

[0057]

[0058] Badge selection step

[0059] In this specification, a medium is a mixture containing nutrients that enable strains, microorganisms, or cells to be cultured or proliferated, and may include at least one of water, a carbon source, a nitrogen source, minerals, and growth factors. In the medium, carbon sources, nitrogen sources, minerals, growth factors, etc., excluding water, may be referred to as effective ingredients that cause changes in the state of strains (e.g., changes in the number of strains).

[0060] For example, the carbon source may include glucose, sucrose, dextrin, glycerol, or starch, etc.; the nitrogen source may include peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate, other organic or inorganic nitrogen-containing compounds, etc.; the inorganic salt may include magnesium, manganese, calcium, iron, or potassium, etc.; and the growth factor may include, but is not limited to, vitamins such as nicotinic acid, pantothenic acid, folic acid, and biotin, amino acids such as tryptophan, and nucleic acid bases such as adenine.

[0061] In an embodiment of the present application, the medium may be a single medium; or a mixed medium comprising a first medium and a second medium.

[0062] Specifically, the single badge may be a first badge or a second badge.

[0063] The first medium may comprise peptone, beef extract, yeast extract, dextrose, sodium chloride, soluble starch, cysteine, sodium acetate and agar, and may be, for example, RCM medium.

[0064] The second medium may contain tryptone, soytone, dextrose, sodium chloride and dipotassium phosphate, and may be, for example, TSB medium.

[0065] In an embodiment of the present application, the mixed medium may be a mixture of the first medium and the second medium in a weight ratio of 1:0.001 to 20, specifically, 1:0.05 to 10, 1:0.1 to 5.

[0066] As the weight ratio of the second medium to the first medium increases, the dominant state of the strain can be reversed to form the desired microbiome.

[0067] In the method for evaluating the efficacy of the composition of the present invention, a medium may be selected to simulate an environment in which skin harmful and beneficial bacteria coexist to evaluate the efficacy of the composition for evaluation. In an embodiment of the present application, the step of selecting the medium may include selecting the type and mixing ratio of the medium.

[0068] Specifically, a medium can be selected to form a dominant state of a desired strain among multiple skin flora, thereby artificially creating a dominant state of a desired strain.

[0069] In one embodiment of the present application, when it is desired to establish P. acnes as a dominant state for evaluating the efficacy of the composition, a medium containing RCM may be selected, or a medium containing RCM alone or a mixed medium containing RCM and TSB in a weight ratio of 1:0.001 to 20 may be selected.

[0070] In another embodiment of the present application, when it is desired to establish a co-dominant state of S. aureus and S. epidermidis (or not to establish a dominant state of P. acnes) for evaluating the efficacy of the composition, RCM may be selected as a single medium or a mixed medium in which RCM and TSB are mixed at a weight ratio of 1:0.001 to 20 may be selected.

[0071] For example, a medium with an increased proportion of TSB in a mixed medium can be selected to create a co-dominant condition of S. aureus and S. epidermidis.

[0072]

[0073] Vaccination order selection step and vaccination step

[0074] Microorganisms go through the stages of lag phase, log phase, stationary phase, and death phase depending on the culture time after inoculation. Each period is different for each strain, and even for the same strain, it can vary depending on the medium.

[0075] In an embodiment of the present application, a step of selecting an inoculation order of the plurality of skin resident bacteria is included to form a dominant state of the desired strain.

[0076] In an embodiment of the present application, inoculation of multiple skin resident bacteria may be performed within 48 hours from the first to the last inoculation to establish a desired dominant state, taking into account the growth patterns of multiple strains. Specifically, the inoculation may be performed within 36 hours, for example, within 24 hours, from the first to the last inoculation.

[0077] By setting the inoculation interval within the above range, it is possible to establish a superiority / inferiority relationship between strains before each strain reaches the death phase.

[0078] In an embodiment of the present application, the inoculation may be performed with three or more strains, each with a time difference of at least three hours.

[0079] Specifically, the inoculation may include inoculating the second strain at least 3 hours after inoculating the first strain, and inoculating the third strain at least 6 hours after inoculating the second strain. For example, the inoculation may include inoculating the second strain at least 6 hours after inoculating the first strain, and inoculating the third strain at least 6 hours after inoculating the second strain.

[0080] In one embodiment of the present application, S. epidermids may be inoculated 6 hours after P. acnes inoculation, and S. aureus may be inoculated 3 hours after the S. epidermidis inoculation. In another embodiment of the present application, S. epidermids may be inoculated 14 hours after P. acnes inoculation, and S. aureus may be inoculated 3 hours after the S. epidermidis inoculation.

[0081] By setting the inoculation order and time difference of multiple strains, it is possible to induce a change in dominance for each strain and create the desired microbiome environment.

[0082] The above multiple skin resident bacteria can be inoculated at a concentration of 0.1 to 5% of the medium, specifically, at a concentration of 0.1 to 2%, but is not limited thereto. For example, the multiple strains can be inoculated at a concentration of 1.0 × 10 5 1.0 × 10 7 It can be inoculated at a number of CFU / mL, but is not limited to this.

[0083]

[0084] Stage of establishing a dominant state and stage of approving a composition for evaluation

[0085] The above multiple skin resident bacteria can be mixed and cultured (i.e., co-cultured) under the same conditions to create a dominant state. Mixed culture refers to culturing different types of strains simultaneously in a single system, and can be performed in various ways. For example, it can be performed in a stationary culture method in which the medium is fixed and not stirred and cultured, a shaking culture method in which the medium is continuously shaken on a shaker and cultured, and an aerated stirring culture method in which sterile air or a mixed gas of a certain composition is supplied to the medium and stirred and cultured. In addition, for example, when using a shaking culture method or a stirring culture method, the culture can be performed at a stirring speed of 80 to 180 rpm. In addition, the culture can be performed under temperature conditions of, for example, 30 to 37°C and can be a stationary culture. However, the present invention is not limited thereto.

[0086] In the embodiments of the present application, once the desired dominant state is reached, the desired microbiome environment can be created by maintaining the dominant state. The desired dominant state can be maintained by controlling temperature, medium, and rpm.

[0087] When multiple strains are mixed and cultured, multiple dominant states may be exhibited until the culture is completed. Specifically, when three strains are mixed and cultured, a state in which the first strain is dominant, a state in which the second strain is dominant, and a state in which the third strain is dominant may be exhibited. In this case, a state in which two strains are dominant or a co-dominant state in which all three strains are dominant may also be exhibited.

[0088] Depending on the inoculation order, inoculation time difference, type of medium, and mixing ratio of medium, the dominant state may be the same or include multiple dominant states during the culture period.

[0089] According to an embodiment of the present application, a method for evaluating the efficacy of the composition includes the steps of creating a desired dominant state and applying the composition for evaluation in the created dominant state.

[0090] In an embodiment of the present application, the culture may be performed until a critical culture time after application of the composition for evaluation. Here, the critical culture time may refer to an appropriate time for evaluating the effect of the composition, such as, for example, the time at which a change in the state of the strain occurs effectively and clearly, the time at which high reactivity is observed when the composition is applied, etc.

[0091] For example, the culture critical time may refer to the time period during which a strain proliferates through culture and reaches a target dominant state, and before a change in the dominant state occurs due to the death of the strain, etc. In addition, for example, the culture critical time may be the time when a strain proliferates through culture and is closest to the target dominant state, or the time period after a predetermined period of time has elapsed therefrom.

[0092] For example, when the target dominant state is the dominant state of a specific strain, the culture critical time may be when the specific strain reaches the dominant state but the difference in number between other strains is the greatest or when a predetermined time has elapsed therefrom. Furthermore, for example, when the target dominant state is a co-dominant state between strains, the culture critical time may be when the strains reach the co-dominant state but the difference in number between the strains is the smallest or when a predetermined time has elapsed therefrom. For example, the culture critical time may be 24 to 48 hours, specifically 24 to 30 hours, and more specifically 24 to 26 hours. However, the present invention is not limited thereto.

[0093] In an embodiment, the evaluation composition may be applied once or multiple times to a strain culture that has reached the established dominant state, and at this time, the evaluation composition may be applied after a predetermined period of time has elapsed. The predetermined period of time may be determined by the strain to be cultured, the type and concentration of the medium, the target dominant state, etc. For example, when culturing Staphylococcus aureus (harmful bacteria) as the first strain and Staphylococcus epidermidis (beneficial bacteria) as the second strain, the evaluation composition may be applied after 12 to 25 hours, specifically 18 to 24 hours, have elapsed since the start of the culture.

[0094]

[0095] Steps to evaluate the impact on the skin

[0096] Additionally, a step of evaluating the effect on the skin after applying the composition may be included. Specifically, the evaluation of the effect may include a step of observing changes in status between the strains.

[0097] The step of observing the change in status among the strains may be performed after the dominant status among the plurality of strains in the medium reaches the target dominant status. The change in status among the plurality of strains may be observed after the application of the evaluation composition. For example, the change in status among the strains may include at least one of a change in the dominant status among the plurality of strains and a change in the number of strains among the plurality of strains.

[0098] The above state change can be observed using, but is not limited to, the absorbance method, and can be observed using various methods and / or instruments such as the surface plate method, the injection plate method, a hemocytometer, and a spectrophotometer. For example, the first absorbance of the medium before the target substance is applied is measured, and the second absorbance of the medium is measured after the target substance is applied to the medium, and the state change can be observed from the difference between the first absorbance and the second absorbance.

[0099] Depending on the embodiment, the status change between multiple strains can be expressed in various ways, such as numbers, images, and graphs. Multiple graphs can be generated for each medium and / or each strain to easily understand the status change of the overall strains in the medium and the status change of each strain. At this time, the graph can be expressed on a planar coordinate system including a horizontal axis and a vertical axis, wherein the horizontal axis represents the time since the start of the culture and / or the time the target substance was applied, and the vertical axis can represent the number of strains, etc. However, the present invention is not limited thereto.

[0100] In an embodiment, the skin impact assessment method may further include a step of assessing the impact of the composition for assessment on the formed microbiome.

[0101] Specifically, if the co-dominance of the first strain and the second strain is maintained even after the evaluation composition is approved in a co-dominance state of the first strain and the second strain, the evaluation composition can be evaluated as being able to help maintain a healthy skin condition while appropriately maintaining the balance of the skin microbiome's flora.

[0102] For example, if the number of harmful bacteria decreases after the evaluation composition is approved in a state where the first strain is dominant or in a state where harmful bacteria and beneficial bacteria co-dominant, the evaluation composition can be evaluated as being able to inhibit the growth of harmful bacteria and prevent skin inflammation, etc.

[0103] In addition, the above skin impact evaluation may be an impact that the composition for evaluation may have on skin flora, such as hair loss, anti-inflammation, antibacterial, and skin safety, but is not limited thereto.

[0104]

[0105] Hereinafter, the present invention will be described through manufacturing examples, working examples, and experimental examples, but the scope of the present invention is not limited by the contents presented below.

[0106]

[0107] Manufacturing example

[0108]

[0109] (1) Manufacturing examples 1 to 4: Cultivation according to medium type

[0110]

[0111] As shown in [Table 1] below, Propionibacterium acnes (P. acnes) was inoculated into each medium and cultured in an aerobic environment for 24 hours.

[0112] The medium of Manufacturing Example 1 used a medium containing 38 g of RCM in 1 L of purified water, and the medium of Manufacturing Examples 2 to 4 used NB No. 2 medium (25 g in 1 L of purified water), BHI medium (37 g in 1 L of purified water), and TSB medium instead of the RCM.

[0113] Each badge contains the following components:

[0114] - RCM medium: 1 wt% peptone, 1 wt% beef extract, 1 wt% yeast extract, 0.5 wt% dextrose, 0.5 wt% sodium chloride, 0.1 wt% water-soluble protease, 0.05 wt% cysteine-HCl, 0.3 wt% sodium acetate, 0.05 wt% agar

[0115] - NB No.2 medium: 1 wt% special meat extract, 1 wt% peptone, 0.5 wt% sodium chloride

[0116] - BHI medium: calf brain 0.77 wt%, beef heart 0.98 wt%, proteose peptone 1 wt%, dextrose 0.2 wt%, sodium chloride 0.5 wt%, disodium phosphate 0.25 wt%

[0117] - TSB medium: tryptone 1.7 wt%, soytone 0.3 wt%, dextrose 0.25 wt%, sodium chloride 0.5 wt%, dipotassium phosphate 0.25 wt%

[0118] Dispense 14 mL of the concentration-adjusted medium into each 15 mL vial and add each strain at a concentration of 1% (140 μL, 10 6 CFU / mL) and cultured at 37°C.

[0119]

[0120]

[0121]

[0122] After culturing the above-mentioned manufacturing examples 1 to 4, 24 hours after the start of culture, the absorbance of the medium was measured at a wavelength of 600 nm to confirm the growth of the strain. The results are shown in Table 2 below.

[0123]

[0124]

[0125]

[0126] As shown in Table 2 above, for P. acnes, RCM medium showed an absorbance that was at least 4.1 times and at most 75.9 times higher than other media, confirming that RCM medium was the optimal medium for the strain.

[0127]

[0128] (2) Manufacturing Examples 5 to 7: Cultivation according to strain

[0129]

[0130] As shown in [Table 3] below, Propionibacterium acnes (P. acnes), Staphylococcus epidermidis (S. epidermidis), and Staphylococcus aureus (S. aureus) were inoculated into RCM medium and cultured in an aerobic environment for 24 hours.

[0131] A medium containing 30 g of RCM in 1 L of purified water was used, 14 mL of the medium was dispensed into each 15 mL vial, and each strain was cultured at a concentration of 1% (140 μL, 10 6 CFU / mL) and each strain was cultured at 37°C.

[0132]

[0133]

[0134]

[0135] The results of counting the strains 24 hours after the start of culture of the above manufacturing examples 5 to 7 are shown in Table 4. The strain count was performed by counting the strains using physiological saline solution (0.85% NaCl) at a rate of 10 for each strain. 6 , 10 7 , 10 8The diluted solution was spread in 3 batches and cultured at 37℃ for 24 hours. The number of strains was measured and the average value was calculated.

[0136]

[0137]

[0138]

[0139]

[0140] As shown in [Table 4] above, in the case of P. acnes in the RCM medium, 9.2 log 10 For S. epidermidis, 8.1 log 10 For S. aureus, 8.3 log 10 was counted as .

[0141]

[0142] (3) Manufacturing examples 8 to 10: Mixed culture of two strains

[0143]

[0144] As shown in [Table 5] below, two species each of Propionibacterium acnes (P. acnes), Staphylococcus epidermidis (S. epidermidis), and Staphylococcus aureus (S. aureus) were inoculated onto RCM medium and cultured in an aerobic environment for 24 hours.

[0145] A medium containing 30 g of RCM in 1 L of purified water was used, and 14 mL of the medium was dispensed into each 15 mL vial, and each strain was added at a concentration of 1% (104 μL, 10 6 CFU / mL) and the strains were cultured at 37°C.

[0146]

[0147]

[0148]

[0149] The results of counting the strains 24 hours after the start of culture in Manufacturing Examples 8 to 10 are shown in Table 6. The counting method is the same as that used in Manufacturing Examples 5 to 7.

[0150]

[0151]

[0152]

[0153] As shown in Table 6 above, unlike when cultured alone in RCM medium, in the case of P. acnes, the bacterial population was measured to be reduced compared to Preparation Example 5, in the case of S. epidermidis, the bacterial population was measured to be equivalent to or reduced compared to Preparation Example 6, and in the case of S. aureus, the bacterial population was measured to be increased compared to Preparation Example 7.

[0154] When the two types were co-cultured for 24 hours, the dominant state was S. aureus, followed by P. acnes and S. epidermidis, which was different from the expected dominance results in Manufacturing Examples 5 to 7.

[0155]

[0156] (3) Manufacturing examples 11 to 13: Cultivation of three strains in RCM medium

[0157]

[0158] As shown in [Table 7] below, Propionibacterium acnes (P. acnes), Staphylococcus epidermidis (S. epidermidis), and Staphylococcus aureus (S. aureus) were inoculated into RCM medium and cultured in an aerobic environment for 72 hours.

[0159] Dispense 14 mL of medium into each 20 mL vial, and add each strain at a concentration of 1% (140 μL, 10 6 CFU / mL) and the strains were cultured at 37°C.

[0160]

[0161]

[0162]

[0163] The growth of the strain was confirmed by measuring the absorbance of the medium at a wavelength of 600 nm at 3-hour intervals for up to 12 hours from the start of culture in Manufacturing Examples 11 to 13, at 12-hour intervals until 2548 hours, and at 24-hour intervals thereafter. The results are shown in Figures 1a to 1c.

[0164] Referring to Figures 1a to 1c, in the case of P. acnes, the log phase entry time was confirmed to be 12 hours, in the case of S. epidermidis, the log phase entry time was confirmed to be 9 hours, and in the case of S. aureus, the log phase entry time was confirmed to be 3 hours.

[0165] Figure 2 is a graph that merges Figures 1a to 1c, and confirms the logarithmic phase entry time and the dominant state of each strain. The absorbance under TSB single culture growth conditions of S. epidermidis and S. aureus was also plotted to compare the logarithmic phase and strain growth patterns.

[0166] Specifically, for S. epidermidis and S. aureus, it was confirmed that when cultured in RCM medium compared to TSB medium, the logarithmic phase entry was delayed and the absorbance was 15 to 30% lower than when cultured in TSB medium.

[0167] In addition, the results of measuring the number of S. epidermidis and S. aureus in RCM medium every 3 hours (see Fig. 3) showed that the number of bacteria was approximately 1 log higher than that of culture in TSB medium. 10 It decreased.

[0168] At the same time, for these two strains, they enter the death phase after 48 hours, so the strains of S. epidermidis and S. aureus were inoculated within 48 hours, but the inoculation time of the three strains was set to be adjusted to within 48 hours from the first inoculation to the last inoculation.

[0169] In summary of the above results, in order to similarly create the logarithmic phase entry time in subsequent examples and form the desired dominant state, P. acnes, S. epidermidis, and S. aureus were sequentially inoculated within 48 hours.

[0170]

[0171] Examples and Comparative Examples

[0172]

[0173] (1) Examples 1 to 3: Mixed culture of three strains according to inoculation time

[0174]

[0175] As shown in [Table 8] below, three strains were cultured in RCM medium. The RCM medium used was a medium containing 30 g of RCM in 1 L of purified water, and 14 mL of the medium was dispensed into each 15 mL vial, and each strain was cultured at a concentration of 1% (140 μL, 10 6 CFU / mL) and the strains were cultured at 37°C.

[0176] At this time, the inoculation time of the strains in [Table 8] means the time difference from the previous inoculation, for example, in the case of Example 1, P. acnes, S. epidermidis, and S. aureus were inoculated at the same time. In the case of Example 2, S. epidermidis was inoculated 6 hours after P. acnes inoculation, and S. aureus was inoculated 3 hours after S. epidermidis inoculation. In the case of Example 3, S. epidermidis was inoculated 14 hours after P. acnes inoculation, and S. aureus was inoculated 3 hours after S. epidermidis inoculation.

[0177]

[0178]

[0179]

[0180] As shown in [Table 8] above, the results of counting each strain at 3-hour intervals from the start of culture at different inoculation times are shown in Figures 4a to 4c.

[0181] The graphs in Figures 4a to 4c are growth graphs of the three strains with different culture times, merged with the same starting point.

[0182] Referring to Fig. 4a, when 3 strains are cultured simultaneously as in Example 1, the coefficient value between strains is 1.1 to 2.2 log around 24 hours after the first inoculation of the strains after culturing. 10 It was calculated that S. aureus was dominant.

[0183] In Example 1, 48 hours after the first inoculation, as S. epidermidis and S. aureus entered the death phase, P. acenes became dominant.

[0184] In Example 2, when S. epidermids was inoculated 6 hours after P. acnes inoculation and S. aureus was inoculated 3 hours after S. epidermidis inoculation, the dominant state changed from Example 1, and S. epidermids became dominant 18 hours after inoculation, and P. acenes became dominant after 48 hours (see Figure 4b).

[0185] When other strains were inoculated after the P. acnes phase (Example 3), P. acnes was established as a dominant strain, as shown in Fig. 4c (see Fig. 4b).

[0186]

[0187] (2) Examples 4 to 8: Cultivation of P. acnes strains in mixed media

[0188]

[0189] RCM medium was prepared by adding 30 g of RCM to 1 L of purified water, and TSB medium was prepared by adding 30 g of RCM to 1 L of purified water.

[0190] A total of 14 mL of medium was mixed in the ratio of Table 9, and P. acnes strain was added at a concentration of 1% (140 μL, 10 6 CFU / mL) and the strains were cultured at 37°C.

[0191]

[0192]

[0193]

[0194] As shown in [Table 9] above, each medium was mixed at the above ratio, and the results of counting the number of P. acnes after culturing for 72 hours are shown in Figure 5. The measurement of the number of P. acnes was the same as the measurement method in other manufacturing examples and examples.

[0195] Referring to Figure 5, it was found that there was no difference in the total number of P. acnes bacteria after 72 hours of culture.

[0196]

[0197] (3) Example 9: Cultivation of three strains in a mixed medium

[0198]

[0199] In Example 3, three strains were cultured in the same manner as in Example 3, except that the medium was changed to the same manner as in Example 6. The mixing ratio of the medium was set to 1:1, and the results of the strain count measurement over time are as shown in Figure 6.

[0200] The graph in Figure 6 is a merger of growth graphs of the three strains with different culture times starting from the same starting point.

[0201] Referring to Figure 6, when the inoculation time of each strain was set to be similar to the logarithmic phase entry time of each strain and sequentially inoculated, and a mixed medium was used for the composition of the medium unlike Example 3, the number of S. aureus cells was approximately 8.4 log when about 18 hours had passed after the first strain inoculation. 10 was measured as , whereas S. epidermidis was about 7.9 log 10As for P. acnes, it is about 6.1 log 10 It was measured that S. aureus was dominant.

[0202] Meanwhile, it was confirmed that the number of P. acnes bacteria increased after 36 hours, and that P. acnes became dominant after 48 hours.

[0203]

[0204] Experimental Example: Antibacterial activity evaluation using the evaluation composition

[0205]

[0206] The effects of the evaluation composition on harmful or beneficial bacteria in an artificially formed skin microbiome were evaluated using the culture models of Examples 1 to 3 and 9.

[0207] In each example, three strains were added to 20 mL of medium at a concentration of 1% (200 μL, 10 6 CFU / mL) and cultured the strain at 37°C and a stirring speed of 100 rpm.

[0208] At 24 hours after the start of the culture, 1 mg / mL of the evaluation composition was applied to each medium. At this time, the evaluation composition included ylang-ylang oil, lavender oil, and lemongrass oil. After applying the evaluation composition, the strains for each medium were counted at 1 hour, 3 hours, and 6 hours (i.e., 19 hours, 21 hours, and 24 hours from the start of the culture). The strain counting was performed in the same manner as in other manufacturing examples and examples.

[0209] According to the results of the above experimental example, when the evaluation composition was applied for 24 hours in a state where P. acnes was dominant, it was confirmed that the number of P. acnes bacteria decreased, the strains restored balance, and the microbiome could be stabilized.

[0210]

[0211] While specific portions of the contents of this application have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and do not limit the scope of this application. Therefore, the substantial scope of this application is defined by the appended claims and their equivalents.

Claims

1. A step of selecting a medium to form a dominant state of a desired strain among multiple skin resident bacteria; A step of selecting the inoculation order of the plurality of skin-resident bacteria to form a dominant state of the desired strain; A step of inoculating the above-mentioned plurality of skin resident bacteria into the selected medium according to the above-mentioned selected inoculation order; A step for creating a dominant state of the desired strain; A step of applying a composition for evaluation in the above-mentioned dominant state; and A method for evaluating the efficacy of a composition, comprising: a step of evaluating the effect on the skin; 2. A method for evaluating the efficacy of a composition, wherein inoculation of the plurality of skin resident bacteria in paragraph 1 is performed within 48 hours from the first inoculation to the last inoculation.

3. A method for evaluating the efficacy of a composition, wherein in the first paragraph, the inoculation is performed with three or more strains at a time interval of at least three hours.

4. In paragraph 1, the inoculation is performed at least 3 hours after the inoculation of the first strain, and the second strain is inoculated. A method for evaluating the efficacy of a composition, comprising inoculating a third strain at least 6 hours after inoculating the second strain.

5. A method for evaluating the efficacy of a composition in claim 1, wherein the dominant state is a state in which the plurality of skin resident bacteria are co-dominant or at least one strain among the plurality of strains is dominant.

6. A method for evaluating the efficacy of a composition in the first paragraph, wherein the dominant state includes at least one of a first section in which the first strain is dominant, a second section in which the second strain is dominant, a third section in which the third strain is dominant, or a fourth section in which the first strain and the third strain are co-dominant.

7. A method for evaluating the efficacy of a composition in claim 1, wherein the skin resident bacteria are at least three species selected from among Staphylococcus strains, Streptococcus strains, Malassezia strains, Propionibacterium strains, and Candida strains.

8. A method for evaluating the efficacy of a composition, wherein the step of selecting a medium in the first paragraph includes selecting the type and mixing ratio of the medium.

9. A method for evaluating the efficacy of a composition in the first paragraph, wherein the medium is a single medium or a mixed medium including a first medium and a second medium.

10. A method for evaluating the efficacy of a composition in claim 9, wherein the mixed medium is a mixture of the first medium and the second medium in a weight ratio of 1:0.001 to 20.

11. A method for evaluating the efficacy of a composition according to claim 9, wherein the first medium comprises peptone, beef extract, yeast extract, dextrose, sodium chloride, soluble starch, cysteine, sodium acetate and agar.

12. A method for evaluating the efficacy of a composition according to claim 9, wherein the second medium comprises tryptone, soytone, dextrose, sodium chloride and dipotassium phosphate.

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