Topical Compositions and Uses Thereof

Abstract

Described herein are methods of improving the appearance of skin or at least one sign of skin aging using Micrococcus luteus compositions. The invention also relates to topical compositions, kits, and methods of making topical compositions useful in such methods.

Description

[Technical field]

[0001] The present invention relates to methods for improving the appearance of skin or at least one sign of skin aging using Micrococcus luteus compositions, and also to topical compositions and kits useful in such methods. [Background technology]

[0002] Skin care products containing probiotic microorganisms are becoming more and more well known.The microorganisms or related products that have been used in skin care products to date are generally Bifidobacterium spp., Lactobacillus spp. (now known as Limosilactobacillus spp., Lacticaseibacillus spp., Lactiplantibacillus spp., Ligilactobacillus spp.), Lactococcus spp. and Streptococcus spp., or filtrates or lysates made from said bacteria.However, the role they play in interacting with skin microflora is not fully understood.

[0003] WO2006104403 (Blis Technologies Limited) describes Micrococcus luteus (M. luteus) compositions and their therapeutic use for controlling skin diseases or disorders. Probiotic strain Q24, deposited at Deutsche Sammlung von Mikro organisms Und Zellkulturen GmbH, Braunschweig, Germany under accession number DSM 17172, is also provided. This document is incorporated herein by reference in its entirety.

[0004] ANZCTR, Probiotics for Eczema Treatment (Registration Number: ACTRN12616000022460) describes a clinical trial testing the use of a lysate of Micrococcus luteus Q24 for the treatment of eczema. No results are provided.

[0005] The present applicants have unexpectedly identified a new role for M. luteus in cosmetic applications, including improving the appearance of skin or at least one sign of skin aging. Based on this unexpected discovery, there is a need to develop new cosmetic treatment methods and compositions suitable for cosmetic applications.

[0006] Working with probiotic organisms is difficult: probiotics are sensitive and often react unpredictably to the various freeze-drying processes, the drugs, processing conditions and formulation agents used in such processes.

[0007] In the harsh freeze-drying process, microorganisms are generally protected using various lyoprotectants and cryoprotectants. The applicants have also surprisingly found that M. luteus can be formulated in the absence of lyoprotectants or cryoprotectants without significant loss of viability of the probiotic microorganism.

[0008] Preparation of formulations containing Micrococcus luteus can be difficult because M. luteus has been found to be unstable in aqueous and polar solvents, is chemically sensitive, and is heat sensitive.

[0009] It is an object of the present invention to provide methods of improving the appearance of skin or at least one sign of skin aging using Micrococcus luteus compositions; and / or to at least provide the public with a useful choice.

[0010] Other objects of the invention will become apparent from the following description, given by way of example only.

[0011] Any discussion of documents, acts, materials, devices, articles or the like included in this specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of those matters form part of the prior art base or were common general knowledge in the art relevant to the present invention prior to the priority date. Summary of the Invention

[0012] In a first aspect, the present invention relates to a method of improving the appearance of skin or at least one sign of aging comprising applying to the skin a topical composition comprising Micrococcus luteus Q24.

[0013] In a second aspect, the present invention provides a topical composition comprising Micrococcus luteus Q24, a viscosity modifier, a dispersant, and an oil vehicle, the composition being in an amount of about 1×10 4 ~Approx. 1×10 10 Contains Micrococcus luteus Q24 in the amount of cfu / g.

[0014] In a third aspect, the present invention provides a topical composition comprising Micrococcus luteus Q24, hydrophobic silica, polysorbate 80 and an oil vehicle.

[0015] In a fourth aspect, the present invention provides a method for producing a medicament for the treatment of a cancer, comprising: Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, About 2 to about 10% w / w of hydrophobic silica about 0.5 to about 2% w / w of polysorbate 80, and A sufficient amount of oil vehicle, A topical composition comprising:

[0016] In a fifth aspect, the present invention provides a topical composition as defined in any one of the first to fourth aspects for improving the appearance of the skin or at least one sign of aging.

[0017] In a sixth aspect, the present invention relates to the use of Micrococcus luteus Q24 in the manufacture of a medicament for improving the appearance of the skin or at least one sign of ageing.

[0018] In a seventh aspect, the present invention provides a two-phase composition comprising an oil phase and an aqueous phase, said oil phase comprising a topical composition according to any one of the second to fourth aspects.

[0019] In an eighth aspect, the present invention provides a kit comprising a topical composition comprising Micrococcus luteus Q24 and an aqueous composition.

[0020] In a ninth aspect, the present invention relates to a method for producing a pharmaceutical composition comprising the steps of: a) mixing an oil vehicle and a dispersant; b) adding Micrococcus luteus Q24 and a viscosity modifier to the mixture from step a); c) homogenizing the mixture from step b) to provide a composition; The present invention relates to a method for producing a topical composition comprising Micrococcus luteus Q24, comprising:

[0021] The following embodiments and preferences may relate to any of the above aspects, either alone or in any combination of any two or more.

[0022] In various embodiments, the composition comprises about 1×10 3 ~Approx. 1×10 12 Contains Micrococcus luteus Q24 in the amount of cfu / g.

[0023] In various embodiments, the composition comprises a viscosity modifier.

[0024] In various embodiments, the composition comprises a viscosity modifier in an amount of about 3 to about 15% w / w.

[0025] In various embodiments, the viscosity modifier is selected from the group consisting of hydrophobic silica, hydrophilic silica, white beeswax, yellow beeswax, paraffin wax, jojoba wax, microcrystalline wax, ethyl cellulose, stearic acid, xanthan gum, tapioca starch, Carbopol polymer, cocoa butter, shea butter, and combinations of any two or more thereof.

[0026] In various embodiments, the composition comprises a dispersant.

[0027] In various embodiments, the composition comprises a dispersant in an amount of about 0.1 to about 5% w / w.

[0028] In various embodiments, the dispersing agent is selected from the group consisting of polysorbate 80, polysorbate 20, sorbitan oleate, egg lecithin, soy lecithin, polyoxyl 35 castor oil, and combinations of any two or more thereof.

[0029] In various embodiments, the composition comprises an oil vehicle.

[0030] In various embodiments, the oil vehicle is selected from the group consisting of a medium chain triglyceride, a vegetable oil, or a combination thereof.

[0031] In various embodiments, the medium chain triglyceride is a caprylic / capric triglyceride.

[0032] In various embodiments, the vegetable oil is selected from the group consisting of sunflower oil, rapeseed oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grape seed oil, and combinations of two or more thereof.

[0033] In various embodiments, the Micrococcus luteus Q24 is lyoprotectant-free.

[0034] In various embodiments, the composition further comprises one or more additional probiotics.

[0035] In various embodiments, the one or more additional probiotics are selected from the group consisting of Streptococcus spp., Lactobacillus spp., Rimosilactobacillus spp., Lacticaseibacillus spp., Ligylactobacillus spp., Lactipranchibacillus spp., Bifidobacterium spp., Saccharomyces spp., and combinations of any two or more thereof.

[0036] In various embodiments, the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus salivarius24SMB, Streptococcus oralis (e.g., S. oralis 89a), and combinations of any two or more thereof. In various embodiments, the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus salivarius 24SMB, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius DB-B5, and combinations of any two or more thereof.

[0037] In various embodiments, the composition comprises about 1×10 3 ~Approx. 1×10 12 Each contains additional probiotics in the amount of cfu / g.

[0038] In various embodiments, the composition further comprises an inhibitory activity enhancer, a buffering agent, an antibacterial agent, a prebiotic, a fragrance, an antioxidant, a colorant, a skin protectant, an antimicrobial agent, an aluminum salt, a mineral pigment, an odor absorber or neutralizer, a sunscreen, and combinations of any two or more thereof.

[0039] In various embodiments, the inhibitory activity enhancer is selected from the group consisting of sodium chloride, ethylenediaminetetraacetic acid, arginine, calcium carbonate, and combinations of any two or more thereof.

[0040] In various embodiments, the buffering agent is selected from the group consisting of calcium carbonate, magnesium carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, magnesium carbonate, urea, hydrated aluminum oxides, hydrated aluminum oxide, bentonite clay, kaolin clay and combinations thereof.

[0041] In various embodiments, the antibacterial agent is selected from the group consisting of xylitol, erythritol, epidermin, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, and combinations thereof. In various embodiments, the antibacterial agent is selected from the group consisting of xylitol, erythritol, epidermin, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, salivaricin 9, salivaricin MPS, and combinations thereof.

[0042] In various embodiments, the prebiotic is selected from the group consisting of manuka honey powder, olive squalene, pomegranate seed oil, flax seed oil, coconut oil, colloidal oatmeal, vitamin E, vitamin C, retinol (vitamin A), olive oil, hyaluronic acid, calendula oil, almond oil, tomato oil, allantoin, aloe vera powder, colloidal oatmeal, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide liquid, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, niacinamide, sunscreen, and combinations of any two or more thereof.

[0043] In various embodiments, the prebiotic is selected from the group consisting of yeast extract, cysteine, maltodextrin, inulin, licorice root, licorice extract, honey, and combinations of any two or more thereof.

[0044] In various embodiments, the fragrance is selected from the group consisting of rose water, orange blossom, rose gardenia, peony, white jasmine, ylang ylang oil, geranium oil, rose oil, and combinations of any two or more thereof.

[0045] In various embodiments, the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalene, vitamin C, beta-carotene, retinyl acetate, retinyl palmitate, retinol, niacinamide, green tea, green tea extract, caffeine, and combinations of any two or more thereof.

[0046] In various embodiments, the antioxidant is selected from olive squalane, pomegranate seed oil, flaxseed oil, coconut oil, colloidal oatmeal, vitamin E, vitamin C, retinol (vitamin A), olive oil, hyaluronic acid, calendula oil, almond oil, tomato oil, and combinations of any two or more thereof.

[0047] In various embodiments, the skin protectant is selected from the group consisting of ceramides, collagen, elastin, coenzyme Q10, hydrolyzed collagen, hyaluronic acid, sodium hyaluronate, retinol, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-38, acetyl hexapeptide-8, heptapeptide-14, heptapeptide-15-palmitate, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, alpha hydroxy acids, beta hydroxy acids, vitamin B5, seaweed, seaweed extract, silicone, xylitol, oat extract, oatmeal powder, colloidal oatmeal, aloe vera, and combinations of any two or more thereof. In various embodiments, the skin protectant is selected from the group consisting of ceramides, collagen, elastin, coenzyme Q10, hydrolyzed collagen, hyaluronic acid, sodium hyaluronate, retinol, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-38, acetyl hexapeptide-8, heptapeptide-14, heptapeptide-15-palmitate, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, alpha hydroxy acids, beta hydroxy acids, vitamin B5, seaweed, seaweed extracts, silicone, xylitol, oat extract, oatmeal powder, colloidal oatmeal, aloe vera, sunscreen, and combinations of any two or more thereof.

[0048] In various embodiments, the antimicrobial agent is selected from the group consisting of zinc, salicylic acid, azelaic acid, benzoyl peroxide, and combinations thereof.

[0049] In various embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane and about 0.1 to about 10% w / w pomegranate seed oil.

[0050] In various embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane and about 0.1 to about 3% w / w vitamin E.

[0051] In various embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane and about 0.1 to about 10% w / w pomegranate seed oil and about 0.1 to about 3% w / w vitamin E.

[0052] In various embodiments, improving the appearance of the skin or at least one sign of aging includes skin looking more radiant, skin looking healthier, skin feeling more hydrated, smaller pores, skin feeling softer, skin appearing clearer, reduced wrinkles, reduced dryness, reduced blemishes, reduced impurities, increased moisture, and reduced sebum production.

[0053] In various embodiments, the composition is non-aqueous.

[0054] In various embodiments, when applied, the composition is applied in combination with an aqueous phase.

[0055] In various embodiments, the composition has a viscosity of about 20,000 to about 500,000 cp at 25° C. In various embodiments, the composition has a viscosity of about 20,000 to about 2,000,000 cp at 25° C.

[0056] In various embodiments, the composition has a shelf life of at least 6 months at 25° C. and 60% RH.

[0057] In various embodiments, the oil vehicle is a medium chain triglyceride.

[0058] In various embodiments, the composition further comprises 0.1-10% w / w prebiotic(s).In various embodiments, the composition further comprises 0.1-35% w / w prebiotic(s).

[0059] In various embodiments, the prebiotic(s) is selected from olive squalane, pomegranate seed oil, vitamin E, or combinations thereof.

[0060] In various embodiments, the prebiotic is selected from combinations of olive squalane and pomegranate seed oil; olive squalane and Vitamin E; and olive squalane, pomegranate seed oil and Vitamin E.

[0061] In various embodiments, the kit includes a dispensing system having a first container and a second container, the first container containing an oil phase including Micrococcus luteus Q24 and the second container containing an aqueous phase.

[0062] In various embodiments, the particle size (Dv90) of Micrococcus luteus Q24 is less than about 300 μm.

[0063] In various embodiments, the particle size of Micrococcus luteus Q24 is less than about 250 μm or less than about 100 μm.

[0064] In various embodiments, the method is carried out without heating.

[0065] The present invention may also be broadly described as being present in any or all combinations of two or more of the parts, elements and features referred to or shown in the specification of this application, either individually or collectively, of said parts, elements or features, and where specific integers having known equivalents in the art to which this invention pertains are referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0066] Reference to a numerical range disclosed herein (e.g., 1-10) is also intended to incorporate reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and also any rational number range within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and therefore all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are to be considered as being expressly set forth in this application in a similar manner.

[0067] References herein to patent specifications, other external documents or other sources of information are generally for the purpose of providing a context for discussing features of the present invention, and unless specifically stated otherwise, references to such external documents should not be construed as an admission in any jurisdiction that such documents or such sources are prior art or form part of the common general knowledge in the art.

[0068] To those skilled in the art to which this invention pertains, numerous modifications in structure and widely differing embodiments and applications of the invention will be suggested without departing from the scope of the invention as set forth in the appended claims. The disclosures and descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[0069] Although the present invention is broadly as defined above, those skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description provides examples. [Brief description of the drawings]

[0070] The invention will now be described with reference to the accompanying drawings in which:

[0071] [Figure 1]Figure 1 shows the shelf life stability of freeze-dried raw ingredients of Micrococcus luteus Q24 under refrigerated storage conditions at 4°C. P1: trehalose as freeze-dried protection agent. P2: Blis Technologies Ltd, trimix blend of trehalose, maltodextrin and lactitol. P3: No freeze-dried protection agent.

[0072] [Diagram 2] FIG. 2 displays the percentage of participants showing changes in skin parameters after use of the compositions of the present invention compared to baseline using data obtained from a Skin analyzer device (Dermo Prime(dp) / viso, CHOWIS, Korea).

[0073] [Diagram 3] Figure 3 shows images captured from participants using the composition of the present invention from baseline to day 25. Parameters included pores, age spots, impurities, and wrinkles, and parameters were measured using a skin analyzer (dP / viso, CHOWIS, Korea).

[0074] [Figure 4] FIG. 4 shows examples of changes in a range of parameters in participants before and after application of a composition of the invention over a 25 day period.

[0075] [Diagram 5] FIG. 5 shows the stability of Q24 in formulations 14, 17, 19, 21, 28, 30 and 32 (see Table 1) and Cetomacrogol cream at 25° C. / 60% RH.

[0076] [Figure 6]Figure 6 displays a scatter plot showing the viability (compared to the PBS negative control treatment) of individual tissues given each dose level of Blis Q24. Treatment groups 5-9 received Blis Q24 at doses of 105-109 cfu / mL, respectively. Thick horizontal bars indicate group means, error bars indicate significant differences at 95% confidence intervals*, P<0.05.

[0077] [Figure 7] Figure 7 shows scatter plots of individual IL-6, IL8 and IL-18 levels measured in conditioned medium of tissues on days 0 and 4. Horizontal bars are group means, error bars indicate significant differences at 95% confidence intervals**, P<0.01.

[0078] [Figure 8] FIG. 8 shows histology of EpiDerm treated with Blis Q24 for 5 days (and PBS and Vitamin C - data not shown). Sections were stained with hematoxylin and eosin (H&E) and imaged at 40x magnification. Arrow "B" indicates the permeable membrane through which the tissue grew. Tissue layers shown are "C" stratum basale, "D" stratum granularis, and "E" stratum corneum. Arrow labeled "A" indicates nuclear remunants trapped in the stratum corneum (presence may indicate enhanced stratum corneum production).

[0079] [Figure 9] Figure 9 shows scatter plots of individual changes in IL-8 levels measured in conditioned medium of tissues receiving combinations of main treatment and ±0.5% SDS on days 4 and 5. Horizontal bars are group means and error bars indicate significant differences with 95% confidence intervals***, P<0.001.

[0080] [Figure 10] FIG. 10 shows skin quality parameters of the Blis Q24 "active" composition versus placebo compared to baseline.

[0081] [Figure 11] FIG. 11 shows skin quality parameters for the Blis Q24 "Live" versus Blis Q24 "Dead" compositions compared to baseline.

[0082] [Figure 12] FIG. 12 shows the growth curve of Q24, a potential prebiotic.

[0083] [Figure 13] FIG. 13 shows the growth curves of prebiotic combinations-olive squalane and pomegranate seed oil; olive squalane and oatmeal flour (colloidal oatmeal); and olive squalane and vitamin E.

[0084] [Figure 14] FIG. 14 shows the growth of commensal species in the presence of prebiotics.

[0085] Detailed Description of the Invention definition The term "comprising" as used in this specification and claims means "consisting at least in part of." When interpreting each statement in this specification and claims containing the term "comprising," features other than the term or the term preceded by the term may also be present. Related terms such as "comprise," "comprising," and "comprises" are to be interpreted in the same manner.

[0086] As used herein, the term "and / or" means "and" or "or," or both.

[0087] As used herein, "(s)" following a noun refers to the plural and / or singular form of the noun.

[0088] Common chemical and biological terms used herein, for example in formulas, have their ordinary meanings.

[0089] As used herein, the term "subject" refers to mammals, including humans, dogs, cats, horses, sheep, cattle and other domestic and farm animals.

[0090] The unit "cfu / g" means colony forming units per gram. Colony forming units (CFU) are units used to estimate the number of viable bacteria in a sample. Determination of colony forming units requires the culture of the microorganism and counts only viable cells, i.e. cells that are able to grow and form visible colonies.

[0091] The unit "% w / w" means the percentage weight based on the total weight of the composition.

[0092] As used herein, the terms "lyoprotectant-free" or "cryoprotectant-free" mean that M. luteus is produced in the absence of a lyoprotectant or cryoprotectant or both.

[0093] As used herein, the term "improving the appearance of skin or at least one sign of aging in a subject" means improving at least one parameter commonly used for skin analysis, including skin radiance, skin health, skin hydration, pore size, skin softness, skin clarity, moisture level, sebum level, wrinkles, dryness, roughness, appearance of dullness, appearance of spots including age spots, and impurities.

[0094] Topical Compositions and Methods Described herein is a method for improving the appearance of skin or at least one sign of aging, comprising applying to the skin a topical composition comprising Micrococcus luteus Q24. Also described herein is a topical composition comprising Micrococcus luteus Q24 for improving the appearance of skin or at least one sign of aging. Also described herein is the use of Micrococcus luteus Q24 in the manufacture of a medicament for improving the appearance of skin or at least one sign of aging.

[0095] Described herein is a topical composition comprising Micrococcus luteus Q24, a viscosity modifier, a dispersant, and an oil vehicle, the composition being administered in an amount of about 1×10 4 ~Approx. 1×10 10 Contains Micrococcus luteus Q24 in the amount of cfu / g.

[0096] Also described herein is a topical composition comprising Micrococcus luteus Q24, hydrophobic silica, polysorbate 80 and an oil vehicle.

[0097] In addition, what is described in this specification is Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, about 2 to about 10% w / w of hydrophobic silica, about 0.5 to about 2% w / w of polysorbate 80, and A sufficient amount of oil vehicle, A topical composition comprising:

[0098] In various embodiments, the oil vehicle is a medium chain triglyceride.

[0099] The topical compositions described herein can be used to improve the appearance of skin or at least one sign of aging.

[0100] Described herein is the use of Micrococcus luteus Q24 in the manufacture of a medicament for improving the appearance of skin or at least one sign of aging.

[0101] Q24 useful in the present invention has been found in tissue culture models to be well tolerated, not induce anti-inflammatory responses, have anti-inflammatory effects, and promote the growth of the stratum corneum, which can help rejuvenate and moisturize the skin and reduce skin pores and wrinkles (see Example 9).

[0102] Micrococcus luteus Q24 Micrococcus luteus is a normal bacterium (commensal organism) on human skin and is important in maintaining the balance between the various microbiota of the skin.

[0103] M. luteus Q24 was deposited at the Deutsche Sammlung von Mikro organisms Und Zellkulturen GmbH, Braunschweig, Germany on March 10, 2005 and was given the accession number DSM 17172. M. luteus strain Q24 is described in WO2006104403, which is incorporated herein by reference.

[0104] In various embodiments, M. luteus is a live probiotic.

[0105] In various embodiments, the compositions of the invention that are useful in the methods of the invention comprise about 1×10 3 ~Approx. 1×10 12 cfu / g of Micrococcus luteus Q24. In various embodiments, the composition comprises about 1×10 4 ~Approx. 1×10 12 , about 1×10 5 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 7 ~Approx. 1×10 12 , about 1×108 ~Approx. 1×10 12 , about 1×10 4 ~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 7 ~Approx. 1×10 9 cfu / g of Micrococcus luteus Q24. In various embodiments, the composition comprises about 1×10 9 Contains Micrococcus luteus Q24 in the amount of cfu / g.

[0106] In various embodiments, Micrococcus luteus Q24 is freeze-dried or lyophilized. In various embodiments, Micrococcus luteus Q24 is provided in a lyoprotectant or cryoprotectant. Lyoprotectants and cryoprotectants are commonly used in the manufacture of products, including probiotics, to protect and maintain cell viability. The terms "lyoprotectant" and "cryoprotectant" refer to a composition that protects an active ingredient, in this case Micrococcus luteus Q24. A lyoprotectant protects when dried, whereas a cryoprotectant protects when frozen. Similar compositions can have both functions, and the terms are used interchangeably herein, unless otherwise specified.

[0107] Suitable lyoprotectants or cryoprotectants will be known to those of skill in the art. In various embodiments, the lyoprotectant is trehalose or a trimix comprising trehalose, maltodextrin and lactitol.

[0108] Surprisingly, applicants have identified that Micrococcus luteus Q24 can also be freeze-dried without significant loss of cell viability in the absence of a lyoprotectant or cryoprotectant, as shown in Figure 1. This result is counterintuitive given that microorganisms routinely need to be protected during the freeze-drying process. In various embodiments, Micrococcus luteus Q24 does not include a lyoprotectant in the composition.

[0109] As can be seen from the above, this discovery translates into important manufacturing advantages where the lyophilization step is simplified and costs are reduced where no lyoprotectant or cryoprotectants are required.

[0110] Viscosity modifier In various embodiments, the composition includes a viscosity modifier, which can be advantageously used to adjust the release profile of the probiotics and also to modify the viscosity of the composition.

[0111] In various embodiments, the composition comprises a viscosity modifier in an amount of about 3 to about 15% w / w. For example, the composition may comprise a viscosity modifier in an amount of about 3 to about 10%, or about 3 to about 9%, or about 3 to about 8%, or about 4 to about 15%, or about 4 to about 10%, or about 4 to about 9%, or about 4 to about 8%, or about 5 to about 15%, or about 5 to about 10%, or about 5 to about 9%, or about 5 to about 8% w / w. For example, the composition may comprise a viscosity modifier in an amount of about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% w / w, based on the total weight of the composition. In various embodiments, the composition comprises a viscosity modifier (e.g., hydrophobic silica) in an amount of about 7% w / w.

[0112] Suitable viscosity modifiers include, but are not limited to, hydrophobic silica, hydrophilic silica, white wax, yellow wax, paraffin wax, jojoba wax, microcrystalline wax, ethylcellulose, stearic acid, xanthan gum, tapioca starch, carbopol polymers (e.g., 971p, 974p), cocoa butter, shea butter, and combinations of any two or more thereof.Preferably, the viscosity modifier is hydrophobic silica (e.g., Aerosil R972)®.

[0113] Dispersants In various embodiments, the composition includes a dispersing agent. Advantageously, the dispersing agent can facilitate the dispersion of solid particles in the composition. For example, the dispersing agent can facilitate the dispersion of probiotics in the composition. Additionally, the dispersing agent can aid in the emulsification of the oil phase with the aqueous phase when mixed together before application. The dispersing agent can be a non-ionic dispersing agent or an amphoteric dispersing agent. Examples of non-ionic dispersing agents include, but are not limited to, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), sorbitan oleate (Span 80), polyoxyl 35 castor oil (Cremaphor EL). Examples of amphoteric dispersing agents include, but are not limited to, lecithins, such as egg lecithin and soy lecithin.

[0114] In various embodiments, the composition comprises a dispersant in an amount of about 0.1 to about 5% w / w. For example, the composition may comprise a dispersant in an amount of about 0.5 to about 4%, about 0.5 to about 3%, about 0.5 to about 2.5% w / w, about 0.5 to about 2% w / w, or about 1 to about 2% w / w. In various embodiments, the composition comprises a dispersant (e.g., Tween 80) in an amount of about 1% w / w or about 2% w / w.

[0115] Oil Vehicle In various embodiments, the composition comprises an oil vehicle. Suitable oil vehicles include, but are not limited to, medium-chain triglycerides and vegetable oils. Preferably, the medium-chain triglycerides are tri(caprylic / capric) glycerides, such as Miglyol 812N (triglyceride esters of caprylic and capric fatty acids and vegetable-derived glycerol derived from saturated coconut oil / palm kernel oil). In various embodiments, the vegetable oil is selected from the group consisting of sunflower oil, rapeseed oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grape seed oil, and any two or more combinations thereof.

[0116] In various embodiments, the composition comprises a quantity sufficient (qs) amount of the non-aqueous carrier, i.e., an amount that brings the total % w / w of the composition to 100%. In various embodiments, the composition comprises a quantity of the non-aqueous carrier of about 55 to about 95% w / w. For example, the composition may comprise about 60 to about 95% or about 60 to about 90% or about 65 to about 90% or about 65 to about 90% or about 70 to about 95% or about 75 to about 95% or about 75 to about 90% or about 80 to about 90% or about 80 to about 90% w / w or about 85 to about 95% or about 85 to about 90% about 88 to about 93% w / w. In various embodiments, the composition comprises a quantity of the non-aqueous carrier of 88, 89, 90, 91, 92 or 93% w / w.

[0117] In various embodiments, the composition is non-aqueous. In various embodiments, the composition is substantially anhydrous. In various embodiments, the composition contains less than 7% water, less than 5% water, less than 3% water, less than 2% water, less than 1% water, less than 0.5% water, less than 0.1% water, or less than 0.01% water. In the present composition, water includes moisture absorbed from the surroundings.

[0118] Additional Probiotics In various embodiments, the composition further comprises one or more additional probiotics. Suitable additional probiotics include Lactobacillus species (e.g., L. acidophilus), Rimosilactobacillus species (e.g., L. reuteri, formerly Lactobacillus reuteri), Lacticaseibacillus species (e.g., L. rhamnosus, formerly Lactobacillus rhamnosus), Rigilactobacillus species (e.g., L. salivarius, formerly Lactobacillus salivarius), Lactiplantibacillus species (e.g., L. plantarum, formerly Lactobacillus plantarum), Bifidobacterium species (e.g., B. bifidum, B. longum, or B. lactis BB12 ... BB12), Streptococcus species (e.g., S. oralis, S. oralis 89a, S. uberis, S. salivarius 24SMB, S. salivarius M18, S. salivarius K12, or S. salivarius DB-B5), and Saccharomyces species (e.g., S. boulardii or S. cerevisiae).

[0119] S. salivarius K12 was deposited on October 8, 1999 at the Deutsche Sammlung von Mikro organismen Und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124, Braunschweig, Germany, and was assigned the accession number DSM 13084. S. salivarius M18 was deposited on December 12, 2001 at the Deutsche Sammlung von Mikro organismen Und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124, Braunschweig, Germany, and was assigned the accession number DSM 14685.

[0120] In various embodiments, the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius 24SMB, and any combination of two or more thereof. In various embodiments, the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus salivarius 24SMB, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius DB-B5, and any combination of two or more thereof.

[0121] In various embodiments, the composition comprises about 1×10 3 ~Approx. 1×10 12 cfu / g of each additional probiotic. For example, the composition may contain about 1×10 4 ~Approx. 1×10 12 , about 1×10 5 ~Approx. 1×10 12 , about 1×10 6 ~Approx. 1×10 12 , about 1×10 7 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 12 , about 1×10 4~Approx. 1×10 10 , about 1×10 5 ~Approx. 1×10 10 , about 1×10 6 ~Approx. 1×10 10 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 10 , about 1×10 4 ~Approx. 1×10 9 , about 1×10 5 ~Approx. 1×10 9 , about 1×10 6 ~Approx. 1×10 9 , about 1×10 7 ~Approx. 1×10 9 cfu / g of each additional probiotic. In various embodiments, the composition comprises about 1×10 9 Each contains additional probiotics in the amount of cfu / g.

[0122] Additional Additives Those skilled in the art will appreciate that the topical composition may contain other additives conventionally used in topical compositions, such as moisturizers. Those skilled in the art will further appreciate that the additives must be compatible with the viability and efficacy of the probiotics. Such additives may provide or improve the therapeutic, cosmetic, stability, and / or appearance properties of the composition. Examples of suitable additives include, but are not limited to, inhibitory activity enhancers, buffers, antibacterial agents, prebiotics, fragrances, antioxidants, colorants, skin protectants, antimicrobial agents, aluminum salts, mineral pigments, odor absorbers or neutralizers, or sunscreens. Such additives may be included in the compositions of the present invention in amounts typical of topical formulations. A variety of pharmaceutically acceptable additives suitable for topical application of live or lyophilized bacteria are well known in the art. Those skilled in the art will appreciate that any additional additives must not be inhibitory to Micrococcus luteus Q24.

[0123] Those skilled in the art will also appreciate that some additives have dual function, such as, for example, an antioxidant or skin protectant that also functions as a prebiotic.

[0124] In various embodiments, the composition further comprises inhibitory activity enhancers, buffers, antibacterial agents, prebiotics, fragrances, antioxidants, colorants, skin protectants, antimicrobial agents, aluminum salts, mineral pigments, odor absorbers or neutralizers, sunscreens, and combinations of any two or more thereof.

[0125] In various embodiments, the inhibitory activity enhancer is selected from the group consisting of sodium chloride, ethylenediaminetetraacetic acid, arginine, calcium carbonate, and combinations of any two or more thereof.

[0126] In various embodiments, the buffering agent is selected from the group consisting of calcium carbonate, magnesium carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium carbonate, urea, hydrated aluminum oxide, bentonite clay, kaolin clay, and combinations thereof.

[0127] In various embodiments, the antibacterial agent is selected from the group consisting of xylitol, erythritol, epidermin, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, and combinations thereof. In various embodiments, the antibacterial agent is selected from the group consisting of xylitol, erythritol, epidermin, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, salivaricin 9, salivaricin MPS, and combinations thereof.

[0128] In various embodiments, the composition includes a prebiotic that can enhance the growth of Q24, enhance antibacterial activity, or enhance the production of compounds to improve skin quality parameters.

[0129] In various embodiments, the prebiotic is selected from the group consisting of manuka honey, olive squalane, pomegranate seed oil, flaxseed oil, coconut oil, colloidal oatmeal, vitamin E, vitamin C, olive oil, hyaluronic acid, calendula oil, almond oil, tomato oil, allantoin, aloe vera powder, colloidal oatmeal, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide liquid, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and combinations of any two or more thereof.

[0130] In various embodiments, the prebiotic is selected from the group consisting of yeast extract, cysteine, maltodextrin, inulin, licorice root, licorice extract, honey, and combinations of any two or more thereof.In various embodiments, the prebiotic is selected from the group consisting of yeast extract, cysteine, maltodextrin, inulin, licorice root, licorice extract, and combinations of any two or more thereof.

[0131] In various embodiments, the prebiotic may be an oil prebiotic or a powdered probiotic. In various embodiments, the composition comprises an oil prebiotic and / or a powdered prebiotic. In various embodiments, the oil prebiotic is selected from the group consisting of olive squalane, pomegranate seed oil, flaxseed oil, coconut oil, vitamin E, olive oil, calendula oil, almond oil, tomato oil, fructooligosaccharide liquid, and any combination of two or more thereof. In various embodiments, the powdered prebiotic is selected from the group consisting of manuka honey, colloidal oatmeal, vitamin C, hyaluronic acid, allantoin, aloe vera powder, colloidal oatmeal, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, and any combination of two or more thereof.

[0132] In various embodiments, the prebiotic is a combination of olive squalane and pomegranate seed oil; olive squalane and vitamin E; or olive squalane, pomegranate seed oil and vitamin E. The inventors have surprisingly identified that the combination of olive squalane and pomegranate seed oil or olive squalane and vitamin E has a synergistic effect on the growth of Micrococcus luteus Q24.

[0133] In various embodiments, the composition comprises from about 0.1% to about 10% w / w of prebiotic, for example, from about 0.1% to about 8%, or from about 0.1% to about 6%, or from about 0.1% to about 5%, or from about 0.3% to about 8%, or from about 0.3% to about 6%, or from about 0.3% to about 5%, or from about 0.5% to about 8%, or from about 0.5% to about 6%, or from about 0.5% to about 5%, or from about 1% to about 8%, or from about 1% to about 6%, or from about 1% to about 5% w / w of prebiotic.

[0134] In various embodiments, the composition comprises from about 0.1% to about 35% w / w prebiotics, e.g., from about 0.1% to about 30%, from about 0.1% to about 25%, or from about 0.1% to about 20%, or from about 0.1% to about 18%, or from about 0.1% to about 16%, or from about 0.1% to about 15%, or from about 0.1% to about 12%, or from about 0.1% to about 10%, or from about 0.1% to about 8%, or from about 0.1% to about 6%, or from about 0.1% to about 5%, or from about 0.1% to about 2%, or from about 0.3% to about 35%, or from about 0.3% to about 30%, or about 0.3% ~ about 25%, or about 0.3% to about 20%, or about 0.3% to about 18%, or about 0.3% to about 16%, or about 0.3% to about 15%, or about 0.3% to about 12%, or about 0.3% to about 10%, or about 0.3% to about 8%, or about 0.3% to about 6%, or about 0.3% to about 5%, or about 0.3% to about 2%, or about 0.5% to about 35%, or about 0.5% to about 30%, or about 0.5% to about 25%, or about 0.5% to about 20%, or about 0.5% to about 18%, or about 0.5% to about 16%, or about 0.5% to about 15%, or about 0.5% to about about 12%, or about 0.5% to about 10%, or about 0.5% to about 8%, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 2%, or about 0.5% to about 35%, or about 1% to about 30%, or about 1% to about 25%, or about 1% to about 20%, or about 1% to about 18%, or about 1% to about 16%, or about 1% to about 15%, or about 1% to about 12%, or about 1% to about 10%, or about 1% to about 8%, or about 1% to about 6%, or about 1% to about 5%, or about 0.5% to about 35%, or about 3% to about 30%, or about 3% to about 2 5% or about 3% to about 20% or about 3% to about 18% or about 3% to about 16% or about 3% to about 15% or about 3% to about 12% or about 3% to about 10% or about 3% to about 8% or about 3% to about 6% or about 3% to about 5% or about 4% to about 30% or about 4% to about 25% or about 4% to about 20% or about 4% to about 18% or about 4% to about 16% or about 4% to about 15% or about 4% to about 12% or about 4% to about 10% or about 4% to about 8% or about 4% to about 6% or about 4% to about 5% w / w of prebiotics.

[0135] In various embodiments, the composition comprises an oil prebiotic in an amount of about 35% w / w prebiotic, e.g., from about 0.1% to about 30%, from about 0.1% to about 25%, or from about 0.1% to about 20%, or from about 0.1% to about 18%, or from about 0.1% to about 16%, or from about 0.1% to about 15%, or from about 0.1% to about 12%, or from about 0.1% to about 10%, or from about 0.1% to about 8%, or from about 0.1% to about 6%, or from about 0.1% to about 5%, or from about 0.1% to about 2%, or from about 0.3% to about 35%, or from about 0.3% to about 30%, or is about 0.3% to about 25%, or about 0.3% to about 20%, or about 0.3% to about 18%, or about 0.3% to about 16%, or about 0.3% to about 15%, or about 0.3% to about 12%, or about 0.3% to about 10%, or about 0.3% to about 8%, or about 0.3% to about 6%, or about 0.3% to about 5%, or about 0.3% to about 2%, or about 0.5% to about 35%, or about 0.5% to about 30%, or about 0.5% to about 25%, or about 0.5% to about 20%, or about 0.5% to about 18%, or about 0.5% to about 16%, or about 0.5% to about 15%, or about 0.5% to about 12%, or about 0.5% to about 10%, or about 0.5% to about 8%, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 2%, or about 0.5% to about 35%, or about 1% to about 30%, or about 1% to about 25%, or about 1% to about 20%, or about 1% to about 18%, or about 1% to about 16%, or about 1% to about 15%, or about 1% to about 12%, or about 1% to about 10%, or about 1% to about 8%, or about 1% to about 6%, or about 1% to about 5%, or about 0.5% to about 35%, or about 3% to about 30%, or about 3% or about 25% or about 3% to about 20% or about 3% to about 18% or about 3% to about 16% or about 3% to about 15% or about 3% to about 12% or about 3% to about 10% or about 3% to about 8% or about 3% to about 6% or about 3% to about 5% or about 4% to about 30% or about 4% to about 25% or about 4% to about 20% or about 4% to about 18% or about 4% to about 16% or about 4% to about 15% or about 4% to about 12% or about 4% to about 10% or about 4% to about 8% or about 4% to about 6% or about 4% to about 5% oil prebiotics.

[0136] In various embodiments, the composition comprises from about 0.1% to about 20%, or from about 0.1% to about 18%, or from about 0.1% to about 16%, or from about 0.1% to about 15%, or from about 0.1% to about 12%, or from about 0.1% to about 10%, or from about 0.1% to about 8%, or from about 0.1% to about 6%, or from about 0.1% to about 5%, or from about 0.1% to about 2%, or from about 0.3% to about 20%, or from about 0.3% to about 18%, or from about 0.3% to about 16%, or about 0.3% to about 15%, or about 0.3% to about 12%, or about 0.3% to about 10%, or about 0.3% to about 8%, or about 0.3% to about 6%, or about 0.3% to about 5%, or about 0.3% to about 2%, or about 0.5% to about 20%, or about 0.5% to about 18%, or about 0.5% to about 16%, or about 0.5% to about 15%, or about 0.5% to about 12%, or about 0.5% to about 10%, or about 0.5% to about 8% %, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 2%, or about 1% to about 20%, or about 1% to about 18%, or about 1% to about 16%, or about 1% to about 15%, or about 1% to about 12%, or about 1% to about 10%, or about 1% to about 8%, or about 1% to about 6%, or about 1% to about 5%, or about 3% to about 20%, or about 3% to about 18%, or about 3% to about 16%, or about 3% to about 15%, or The powdered prebiotic comprises an amount of about 3% to about 12%, or about 3% to about 10%, or about 3% to about 8%, or about 3% to about 6%, or about 3% to about 5%, or about 4% to about 20%, or about 4% to about 18%, or about 4% to about 16%, or about 4% to about 15%, or about 4% to about 12%, or about 4% to about 10%, or about 4% to about 8%, or about 4% to about 6%, or about 4% to about 5% w / w of powdered prebiotic.

[0137] In certain embodiments, the composition comprises about 0.1 to about 30% w / w, or about 3% to about 8% w / w, or about 4% to about 6% w / w, or about 5% w / w olive squalane. In certain embodiments, the composition comprises about 0.1 to about 30% w / w, or about 3% to about 8% w / w, or about 4% to about 6% w / w, or about 5% w / w olive squalane. In certain embodiments, the composition comprises about 5% w / w flaxseed oil. In certain embodiments, the composition comprises about 1% w / w colloidal oatmeal. In certain embodiments, the composition comprises about 0.1 to about 2% w / w, or about 0.2 to about 1.5%, or about 0.2 to about 0.6%, or about 0.3%, or about 0.5 to about 1.3%, or about 0.8 to about 1.2%, or about 1% w / w vitamin E.

[0138] The present inventors have unexpectedly identified that the combination of the prebiotics olive squalane and pomegranate seed oil; and the combination of the prebiotics olive squalane and vitamin E are synergistic. As a result, in certain embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane, about 3% to about 8% w / w or about 4 to about 7% w / w or about 5% w / w or about 0.5% to about 3% w / w or about 0.5 to about 2% w / w or about 1% w / w olive squalane, and about 0.1 to about 10% w / w pomegranate seed oil or about 2 to about 8% w / w or about 3 to about 7% w / w or about 4 to about 6% w / w or about 3 to about 5% w / w or about 5% w / w or about 0.5 to about 3% w / w or about 0.5 to about 2% w / w or about 1% w / w pomegranate seed oil. In certain embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane, about 3% to about 8% w / w olive squalane or about 4 to about 7% w / w olive squalane, for example about 5% w / w olive squalane, and about 0.1 to about 3% w / w or about 0.1 to about 2% w / w or about 0.2 to about 1.5% or about 0.2 to about 0.6% or about 0.3% or about 0.5 to about 1.3% or about 0.8 to about 1.2% or about 1% w / w vitamin E.

[0139] In certain embodiments, the composition comprises about 0.1% to about 10% w / w olive squalane, or about 3% to about 8% w / w, or about 4 to about 7% w / w, or about 5% w / w, or about 0.5% to about 3% w / w, or about 0.5 to about 2% w / w, or about 1% w / w olive squalane; and about 0.1 to about 10% w / w pomegranate seed oil, or about 2 to about 8% w / w, or about 3 to about 7% w / w, or about 4 to about 6% w / w or about 3 to about 5% w / w or about 5% w / w or about 0.5% to about 3% w / w or about 0.5 to about 2% w / w or about 1% w / w of pomegranate seed oil; and about 0.1 to about 3% w / w or about 0.1 to about 2% w / w or about 0.2 to about 1.5% or about 0.2 to about 0.6% or about 0.3% or about 0.5 to about 1.3% or about 0.8 to about 1.2% or about 1% w / w of Vitamin E.

[0140] In various embodiments, the fragrance is selected from the group consisting of rose water, orange blossom, rose gardenia, peony, white jasmine, ylang ylang oil, geranium oil, rose oil, and combinations of any two or more thereof. The fragrance can be added as an oil or a powder.

[0141] In various embodiments, the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalane, vitamin C, beta-carotene, retinol acetate, retinol palmitate, retinol, niacinamide, green tea, green tea extract, caffeine, and any combination of two or more thereof. In various embodiments, the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalane (e.g., olive squalane), vitamin C, beta-carotene, retinol acetate, retinol palmitate, retinol, niacinamide, pomegranate seed oil, flaxseed oil, and any combination of two or more thereof.

[0142] In various embodiments, the skin protectant is selected from the group consisting of ceramides, collagen, elastin, coenzyme Q10, hydrolyzed collagen, hyaluronic acid, sodium hyaluronate, retinol, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-38, acetyl hexapeptide-8, heptapeptide-14, heptapeptide-15-palmitate, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, alpha-hydroxy acids (e.g., lactic acid, glycolic acid), beta-hydroxy acids (e.g., salicylic acid), vitamin B5, seaweed, seaweed extracts, silicone, xylitol, oat extract, oatmeal powder, colloidal oatmeal, aloe vera, and combinations of any two or more thereof. In various embodiments, the skin protectant is selected from the group consisting of ceramides, collagen, elastin, coenzyme Q10, hydrolyzed collagen, hyaluronic acid, sodium hyaluronate, retinol, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-38, acetyl hexapeptide-8, heptapeptide-14, heptapeptide-15-palmitate, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, alpha-hydroxy acids (e.g., lactic acid, glycolic acid), beta-hydroxy acids (e.g., salicylic acid), vitamin B5, seaweed, seaweed extracts, silicone, xylitol, oat extract, oatmeal powder, colloidal oatmeal, aloe vera, sunscreen, and combinations of any two or more thereof.

[0143] In various embodiments, the antimicrobial agent is selected from the group consisting of zinc, salicylic acid, azelaic acid, benzoyl peroxide, and combinations thereof.In various embodiments, the antimicrobial agent is selected from the group consisting of zinc, azelaic acid, benzoyl peroxide, and combinations thereof.

[0144] In various embodiments, the aluminum salt is an aluminum chlorohydrate salt or other salt commonly used in antiperspirants.

[0145] In various embodiments, the mineral pigment is zinc oxide and / or titanium dioxide. Mineral pigments are known to provide protection against, for example, ultraviolet radiation from the sun.

[0146] In various embodiments, the odor absorber or neutralizer is zinc ricinoleate or sodium lauryl sarcosinate.

[0147] In various embodiments, the sunscreen is oxybenzone, avobenzone, octisalate, octocrylene, homosalate, or octinoxate. Mineral sunscreens use zinc oxide and / or titanium dioxide.

[0148] Those skilled in the art will also understand that when the composition is applied in combination with an aqueous phase, the additional additives described can also be provided in the aqueous phase.

[0149] Improving the appearance of skin or signs of aging The compositions described herein are useful for improving the appearance of skin or for improving the signs of aging of skin, which may include, but are not limited to, skin that looks brighter, skin that looks healthier, skin that feels more hydrated, smaller pores, skin that feels softer, skin that looks clearer, increased moisture, reduced sebum production, and reduced appearance of wrinkles, dryness, roughness, dullness, age spots, and impurities.

[0150] Pore ​​size, skin moisture, sebum production, wrinkles, age spots, and impurities can all be measured using a skin analyzer (e.g., Dermo Prime(dp) / viso, CHOWIS, Korea). The AI-powered skin analyzer quantitatively measures changes in various skin parameters such as hydration, sebum, pores, wrinkles, age spots / pigmentation, impurities, keratin, skin tone, dark spots, and skin sensitivity. The device is equipped with a humidity sensor and utilizes advanced optical technology with interchangeable lenses to measure up to 10 different skin measurements and performs precise analysis of high resolution images via the DermoBella app installed on an Android OS or tablet or smartphone. The device is used according to the protocol described in the Examples herein. Skin parameter measurements are taken at the same skin site each time.

[0151] The degree of impurities such as redness can be analyzed by measuring the amount of porphyrins, which appear as scarlet-orange light in response to a specific range of wavelengths of UV light.

[0152] Impurities are seen as scarlet or yellow-green. They can be analyzed separately, but the device combines the two and classifies them as impurities. The index is calculated as a percentage relative to the image size. There is no arbitrary value that classifies the degree of redness based on the amount of porphyrin detected.

[0153] Topical Composition Form In various embodiments, the composition has a viscosity of about 20,000 to about 500,000 cp at 25° C., for example, about 30,000 to about 100,000 cp at 25° C. In various embodiments, the composition has a viscosity of about 20,000 to about 2,000,000 cp at 25° C.

[0154] Viscosity can be measured using a Brookfield LVDVI Prime with a Brookfield Helipath Spindle (S94 or S95 or S96) set at 0.5 RPM at 25° C. One of ordinary skill in the art will appreciate that other methods may be used to measure viscosity, and that viscosity measurements may vary depending on the method used.

[0155] In various embodiments, the composition has a shelf life of at least 6 months at 60% RH (relative humidity) and 25° C. In various embodiments, the composition has a shelf life of at least 12 months at 60% RH (relative humidity) and 25° C.

[0156] two-phase composition Aqueous compositions generally require the addition of preservatives to prevent the growth of pathogens. These preservatives are usually non-selective and cause cell death of the probiotics, and therefore lose their effectiveness quickly. Furthermore, Micrococcus luteus Q24 does not appear to be stable in aqueous formulations. However, it may be advantageous to apply the topical composition of the present invention to an aqueous composition, for example, to improve the spreadability of the composition without affecting the stability or viability of Micrococcus luteus Q24, to improve the sensory features of the composition (e.g., less oily feeling) and / or to act as a vehicle.

[0157] In various embodiments, when applied, the composition is applied in combination with an aqueous phase.

[0158] Suitable aqueous phases will be apparent to those skilled in the art. The aqueous phase may include a suitable combination of solvents, emollients, humectants, emulsifiers, preservatives, viscosity modifiers, prebiotics, and fragrances. Commercially available moisturizers are an example of a suitable aqueous phase.

[0159] Ingredients for the aqueous phase include, but are not limited to, water, sodium CMC and microcrystalline cellulose (Vivapur MCG 811), preservatives (e.g., phenoxyethanol and ethylhexylglycerin), glycerin, sodium surfactin, xanthan gum, fragrance, coco-caprylate, cetostearyl alcohol, glyceryl stearate, cetearyl wheat straw glycosides, phenoxyethanol and sodium stearoyl glutamate.

[0160] The aqueous phase may also contain additional additives as outlined above, such as prebiotics or antioxidants.

[0161] Also described herein is a two-phase composition comprising an oil phase and an aqueous phase, the oil phase comprising Micrococcus luteus Q24, a viscosity modifier, a dispersant, and an oil vehicle. Also described herein is a two-phase composition comprising a separate oil phase and a separate aqueous phase, the oil phase comprising Micrococcus luteus Q24, a viscosity modifier, a dispersant, and an oil vehicle. The composition may comprise any of the aforementioned features. In various embodiments, the oil phase is applied simultaneously with the aqueous phase.

[0162] The two-phase composition can be packaged in a dual chamber bottle that contains separate chambers or containers for the oil and water phases with a nozzle designed to dispense the two phases simultaneously upon application. The phases are stored separately in the chambers and combined upon application. An example of a suitable dispenser is described in U.S. Patent 10,384,224.

[0163] kit Described herein is a kit that includes a topical composition that includes Micrococcus luteus Q24 and an aqueous composition.

[0164] In various embodiments, the kit includes a dispensing system having a first container and a second container, the first container containing an oil phase including Micrococcus luteus Q24 and the second container containing an aqueous phase. Dual chamber bottles such as those described in US 10,384,224 are suitable for inclusion in the kit.

[0165] The kit may also be provided with instructions for use.

[0166] Methods for preparing topical compositions In one aspect, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: a) mixing an oil vehicle and a dispersant; b) adding Micrococcus luteus Q24 and a viscosity modifier to the mixture from step a); c) homogenizing the mixture from step b) to provide a composition; The present invention provides a method for producing a topical composition comprising Micrococcus luteus Q24, comprising:

[0167] The topical compositions described herein can be prepared by first mixing the oil vehicle and the dispersant, adding Micrococcus luteus Q24 to the mixture with constant swirling, adding the viscosity modifier to the mixture, and homogenizing the mixture, for example, for about 1 to 3 minutes, to provide the composition. In various embodiments, the Micrococcus luteus Q24 is added before the viscosity modifier. In other embodiments, the viscosity modifier is added before the Micrococcus luteus Q24. In various embodiments, the mixture is homogenized using a high shear homogeniser or an overhead stirrer.

[0168] Advantageously, the method does not include a heating step. In various embodiments, the method is carried out without heating. Micrococcus luteus Q24 has been found to be heat sensitive. Heating results in loss of cell viability. Therefore, the applicants have developed an alternative composition that allows efficient production of the composition while maintaining optimal viability of the probiotics.

[0169] Micrococcus luteus Q24 can be ground or sieved to a particle size of less than 250 μm before being added to the mixture. In various embodiments, the particle size of Micrococcus luteus Q24 is less than about 250 μm or less than about 100 μm. Suitable grinding and sieving techniques will be apparent to those skilled in the art. For example, Micrococcus luteus Q24 can be ground using a dry powder comill (e.g., Quadro Powder Mills, such as U10 or U21). Smaller particle sizes can be advantageous for dispensing the oil phase from a two-phase dispenser, provide improved spreadability of the composition, improve dispersibility when combined with the aqueous phase, and provide improved sensory properties of the composition, i.e., not large or gritty particles.

[0170] In various embodiments, Micrococcus luteus Q24 has a particle size (Dv90) of less than about 300 μm or less than about 250 μm. Dv90 can be measured by laser diffraction method (Malvern Instruments, USA) for particle size analysis of dry power dispersion. Those skilled in the art will appreciate that particles with Dv90 of less than 300 μm can pass through a 250 μm sieve completely.

[0171] How to use The present invention relates to a method of improving the appearance of skin or at least one sign of aging in a subject in need thereof. The method comprises applying a topical composition of the present invention to the skin of a subject in need thereof. The composition of the present invention can be used as an oil phase serum alone, as a face cream or body moisturizer, in a two-phase moisturizing cream, as a spray or as a spot on cream. The composition can be applied in an appropriate amount to address the subject's skin concerns. Those skilled in the art will appreciate that the composition can be applied to subjects of any age, from the very young to the very elderly. The amount applied can vary according to the subject's age, sex and the concerns being addressed. A typical application regime can include applying the composition of the present invention once a month, once a week, daily or 1-4 times a day.

[0172] The usual dose of the composition when applied to the face or used as a spot-on formulation is typically 0.09 to 0.20 grams, more usually 0.10 to 0.15 grams. The dose is typically about 1×10 5 ~Approx. 1×10 9 cfu. Naturally, larger amounts are used for moisturizing the body. Those skilled in the art will understand that the amount to be dispensed will depend on the concentration of Micrococcus luteus Q24 in the composition to be applied and the form in which it is applied.

[0173] The composition of the present invention can be used as a conventional moisturizer and in any daily skin care regime.A typical application regime can include using the composition of the present invention once or twice a day, usually after cleansing.Long-term or limited-term use of the composition, for example, 1-2 months, is contemplated. EXAMPLES

[0174] material Equipment: The oil phase formulation components were mixed using a homogenizer (IKA Ultra Turrax T25, John Morris group, New Zealand). For clinical trials, the oil phase was filled into dual cylinder single dispensing bottles (Neomix, Salient, France).

[0175] Chemicals: Blis Q24 was fermented and freeze-dried by FoodBowl Auckland. Blis Lyoprotectant Blend was prepared by Callaghan innovation (June 2017). Multiple skin pathogen strains were sourced from a -80°C freezer located at the Blis Technologies Lab. Medium chain triglycerides (caprylic / capric triglycerides), e.g., Miglyol 812 (Ph Eur grade) or Radia 7104, were purchased from Sasol, Hamburg, Germany or Oleon, Malaysia, respectively. Tween 80 (Ph Eur grade) was purchased from Sigma, New Zealand. Hydrophobic silica (Aerosil R972 (pharmaceutical grade)) was purchased from Evonik, Germany (supplied by Chemiplas, Auckland, New Zealand). Aqueous creams were prepared in-house at Blis Technologies and aqueous moisturizers were supplied by Shieling laboratories, New Zealand. Distilled water was used for sample and culture preparation. All other chemicals and solvents were of analytical grade (Sigma, New Zealand). Plastic bottles for storing Q24 oil and water phases for clinical trials and stability studies were purchased from Neomix, France. Skin analyzer (dpViso, Dermobella app) was purchased from CHOWIS (North Korea). Samsung Tablet10.1 was purchased from PB Technologies, New Zealand.

[0176] Example 1: Size optimization of Blis Q24 raw material for inclusion in oil phase The Blis Q24 raw material had a visibly large particle size >500 μm. For the formulation of the oil phase, the intended particle size was to be less than <250 μm to allow for stable dispensing from a double cylinder single dispenser bottle (Neomix, France) and sensory aesthetic when applied to the skin. A filter bag (BagPage+ full-face filter bag with extra-small pore filter <250 μm) (Interscience, New Zealand) was used. Blis Q24 was placed in the inner mesh pocket / sieve (250 μm) of the filter bag and after shaking by hand for 5 minutes, the finely sieved material (<250 μm) was collected in the outer pocket. If necessary, light pressure was applied to the raw material with a rolling pin to further break up large agglomerates. A small slit was then made in the outer pocket of the stomacher bag and the separated sieved material (<250 μm) and unsieved material (>250 μm) were collected and stored separately.

[0177] Example 2: Method for preparing a topical composition Using a 250 ml glass beaker, the required amount of vehicle (see Table 1, MCT) was weighed out followed by the addition of the dispersant Tween 80. The beaker was then gently swirled by hand until a hazy mixture was formed. The Blis Q24 and hydrophobic silica were then added to the MCT / Tween 80 mixture with constant swirling to obtain a homogenous mixture, followed by dispersion using a high shear homogenizer or overhead homogenizer to uniformly disperse all ingredients. [Table 1]

[0178] Example 3: Stability Study The stability of the drug product was tested as per ICH recommended temperature and humidity storage conditions in accordance with the stability testing guideline (ICH Q1AR2), stored in glass or dual chamber bottles at 5°C ± 3°C refrigerated and / or under real-time conditions at 25°C ± 2°C / 60 ± 5% RH.

[0179] For formulation BLT17Q24-2 (aqueous cream), a reduction of less than 1 log reduction was observed within 2 months when stored at 5°C ± 3°C. For formulations BLT17Q24-2 (aqueous cream), BLT17Q24-5 (coconut oil) and BLT17Q24-1 (water), a dramatic reduction in viable cell count was observed within 1 week when the formulations were stored according to 25°C ± 2°C / 60 ± 5% RH. Formulation BLT17Q24-7 (medium chain triglyceride) was stable for at least 6 months (at the end of the study).

[0180] Formulations BLT17Q24-30 and BLT17Q24-31 containing glycerol were not stable regardless of the type of oil. Formulations BLT17Q24-14 (MCT vehicle), BLT17Q24-17 (MCT vehicle), BLT17Q24-19 (olive oil vehicle), and BLT17Q24-21 (olive oil vehicle) were found to be stable for at least 9 months in glass bottles at 25°C ± 2°C / 60 ± 5% RH (Figure 5).

[0181] BLT17Q24-25 (MCT vehicle) was stable in dual chamber bottles at 25°C ± 2°C / 60 ± 5% RH and at 30°C ± 2°C / 65 ± 5% RH for at least 6 months, and in glass vials at 5°C ± 3°C for at least 9 months and at 25°C ± 2°C / 60 ± 5% RH and at least 6 months at 40°C ± 2°C / 75 ± 5% RH. The formulation is expected to be stable for at least 2 years.

[0182] Example 4: Aqueous Phase An example of an aqueous phase is provided. [Table 2]

[0183] Example 5: In vitro testing A cosmetic study using formulation BLT17Q24-28 was conducted in adult volunteers (n=10) from November to December 2020. All participants were female and aged between 22 and 60 years. The study design included measurement of skin parameters using a novel skin analyzer and swab sampling to determine the colonization efficacy of Blis Q24 facial moisturizer.

[0184] Clinical trial sample collection Each participant was assessed in the same room each time and followed the same procedure at each time point. For the measurement of skin parameters using the skin analyzer, the device's sensor was placed on the left cheek in line with the corner of the eye and the tip of the nose. This method was kept consistent at each time point so that measurements were taken from approximately the same area on the cheek or forehead as instructed by the device app. Wrinkles were measured by placing the device's camera on the corner of the eye. Hydration / moisture status was measured by placing the device on the forehead (T-zone) and then on the cheek (U-zone).

[0185] Sebum / oiliness was measured by applying the T-zone side of sebum paper (labeled by CHOWIS) to the forehead and then the U-zone side of the blotting paper to the cheek. The camera of the analyzer was then used to photograph the sebum on the blotting paper. For the measurement of the different parameters, the sensor of the device was not moved when the image was taken and whenever possible, three images were taken for each parameter. The measurements were collected in a step-by-step manner, progressing in the following order based on the instructions of the device's software: moisture, sebum, pore size, blemishes, impurities, and wrinkles.

[0186] Test results FIG. 2 shows the changes in various skin parameters after applying two pumps of the oil phase (BLT17Q24-28) + facial moisturizer combination to the face using a single dispenser bottle for 25 days. Each pump delivers an average of 0.05 g, thereby delivering a total dose that corresponds to a total dose of =0.200 g per day (~0.05 g x 2 pumps twice daily). The total cell count deposited on the face per dose (0.1 g for two pumps) was 3.29E+8 CFU / 0.1 g or 6.6E+8 CFU / day for twice daily administration. These are based on the initial oil phase cell count on day 0 = 3.29E+9 CFU / g.

[0187] Compared to baseline, consistent reductions in pore size, age spots, wrinkles (crow's feet), impurities (porphyrins) and sebum levels, as well as consistent increases in moisture levels (T-zone) were observed in 60%-90% of participants at each time point (days 10, 18, and 25), suggesting the effectiveness of Blis Q24 hydrating serum facial moisturizer.

[0188] FIG. 2 describes the percentage change in skin parameters in all participants from baseline to day 25. Moisture levels / hydration levels had an overall increase of 90%, the largest percentage change among participants in the study. Wrinkles had an overall decrease of 45%. Sebum production decreased by 60% after 10 days. The change in sebum production remained consistent from day 10 to day 25. Further analysis showed that 40% of participants had an overall decrease in pores of 50% or more, 10% of participants had an overall decrease in the appearance of age spots of 50% or more, 66% of participants had an overall decrease in the amount of porphyrins in the skin of 50% or more, and 55% of participants had an overall increase in the amount of moisture present in the T-zone of 50% or more.

[0189] FIG. 3 shows one participant's observations from baseline to day 25, along with the corresponding numerical values ​​assigned by the skin analyzer. This participant had an overall reduction in age spots, imperfections, and wrinkles from day 0 to day 25. Although there was a consistent reduction in pores from day 0 to day 18, their result of 10 on day 25 was higher than the baseline measurement of 7. Visual observation of the skin on day 25 shows improved texture and results such as less shine and tighter looking pores.

[0190] Figure 4 shows an example of the reduction in scores for key parameters of one participant. Application of Blis Q24 moisturizer reduced the size of pores, the number of age spots and wrinkles, and improved skin hydration before and after application.

[0191] Example 6: Comparative Tests - Sedimentation and Caking Three samples of grape seed oil suspension were produced according to the process described in WO2006 / 104403: Example 3 using raw ingredients with three different particle sizes (Dv90: 290 μm, Dv90: 623 μm, Dv90: 1090 μm as measured by laser diffraction particle size analysis using dry powder dispersion (Malvern Instruments, USA)).

[0192] Three versions of serum formulations were manufactured according to the process described in Example 2 above using raw materials with three different particle sizes (Dv90: 290 μm, Dv90: 623 μm, Dv90: 1090 μm).

[0193] Q24, with a Dv90 of 290 μm, was shown to pass through a 250 μm molecular sieve, whereas Q24 with a Dv90 of 623 μm or 1090 μm did not.

[0194] Precipitation Two 10 ml syringes were filled with 6.4 mL of each of the six formulations, a 1 ml headspace gap was left to allow the grape seed oil suspension to mix by shaking, and all syringes were sealed at the tips with blue tac.

[0195] The syringe containing the serum formulation was attached to the bench and the volume of settled Q24 raw material components was measured using syringe measurements at 0, 0.8, 7, 15 and 30 days. The syringe containing the grape seed oil suspension was vibrated and attached to the bench and the volume of settled Q24 raw material was measured at 0, 1, 3, 5, 10, 30 and 60 minutes. All formulation syringes were left untouched and attached to the bench for 30 days.

[0196] The viscosity of the Q24 serum formulation did not result in a change in sedimentation volume for any Q24 raw material particle size, and visually, the raw material with a Dv90 of 290 μm produced the most even distribution. All grape seed oil suspensions had 100% sedimentation of the Q24 raw material, with the largest particle size raw material settling the fastest within minutes.

[0197] Caking Upon completion of the 30 day settling test described above, the grape seed oil suspension was shaken vertically at a constant pace with the Q24 feedstock "cake" on top. Measurements were taken every 10 seconds of the feedstock remaining unsuspended in the "cake" until 100% of the Q24 feedstock had been resuspended.

[0198] None of the serum formulations had any Q24 settling and did not require testing for "caking". All grape seed oil suspension formulations tested had 100% settling of the Q24 raw material. Grape seed oil suspensions made with Q24 raw materials having Dv90 of 290 μm and 623 μm were 100% resuspended within 20 seconds, while the formulation made with raw material with Dv90 of 1090 μm took 40 seconds to resuspend 100%.

[0199] Thus, the grape seed oil suspensions described in WO2006 / 104403 did not exhibit suitable settling and anti-caking properties to be used as topical cosmetic formulations in accordance with the present invention.

[0200] Example 7: Comparative Testing - Viscosity, Spreadability and Stability Four different formulations were prepared. 1. A grape seed oil suspension was prepared according to the process described in WO2006 / 10440: Example 3 using Q24 raw material with a Dv90 of 290 μm. 2. A deodorant stick formulation was manufactured using Q24 raw material with a Dv902 of 290 μm according to the process described in WO2006 / 10440: Example 2. 3. A cetomacrogol cream formulation was manufactured by mixing gamma-irradiated killed Q24 raw material (sieved through a 250 μm sieve) into commercially available cetomacrogol cream (HealthE non-ionic cream, Jaychem, New Zealand) in accordance with the Australian New Zealand Clinical Trials Registry (ANZCTR) study ACTRN12616000022460. 4. Serum formulation BLT17Q24-31 was prepared according to Example 2 using Q24 raw material with Dv90 of 290 μm.

[0201] viscosity Each formulation was filled into a 10 ml syringe, any air bubbles knocked out, and dispensed onto a glass microscope slide by depressing the plunger. The ease or difficulty of dispensing was recorded and the formulations were visually assessed for compatibility of viscosity, distribution of Q24 ingredients, and likely ability to dispense evenly from the two most common forms of packaging for facial moisturizers: a pump bottle or a small round container into which one dipped a finger to sample the formulation before application. [Table 3]

[0202] Formulations 1 and 2 were not considered suitable for dermal application of facial cosmetics as they were too thin and too thick, respectively.

[0203] Spreadability To determine the mechanical spreadability of samples 1 and 4, a circle with a diameter of 2 cm was clearly marked on the underside of the lid of a flat petri dish. Then, 0.5 g of the formulation was placed on the lid evenly covering the 2 cm diameter circle. Another petri dish facing top down with a 200 g mass was placed on top of the formulation and allowed to stand for 20 seconds. The diameter of the circle of spread formulation was then measured. The following equation was used to determine the percentage of spreadability. (Method Saleh A. et al., "Evaluation of Skin Permeation and Analgesic Activity Effects of Carbopol Lornoxicam Topical Gels Containing Penetration Enhancer", The Scientific World Journal, Vol. 2014, Article ID 127495, p. 9, 201) was used to design a slightly modified test method suitable for the format being tested.

[0204] To determine the consistency of microbial growth once spread, 0.1 g of each formulation was placed on top of a human blood agar plate. A cotton swab was then used to spread the formulation evenly down the plate in 14 streaks from side to side. Because the viscosity of the DeoStick did not allow for even spreading down the agar plate, a second method was used in which the formulation was pre-melted and then spread down the plate.

[0205] Formulation 4 showed superior physical and microbial spreadability compared to Formulation 1 and was suitable for body / facial skin application.

[0206] stability Formulation 3 was prepared by weighing 19.8 g of Cetomacrogol 100 BP cream (HealthE, non-ionic cream, B62236, Jaychem, New Zealand) into a Stomacher bag using a sterile spoon. 0.2 g (2% in the final formulation) of Blis Q24 raw material was added to the Cetomacrogol cream, sealed, and mixed by hand until a homogenous cream was formed. The cream was then placed in a Stomacher and mixed for an additional 5 minutes. The Cetomacrogol Q24 cream was then dispensed into 30 ml glass vials and enumerated in triplicate. In a microcentrifuge tube (Eppendorf, USA), 0.1 g of M. luteus Q24 serum or cream was weighed and diluted with 0.9 g of warmed (37°C) phosphate buffered saline and polysorbate 80 (0.1% w / w). The mixture was homogenized by shaking at 2800 rpm for 5 min at ambient conditions (20°C ± 2°C) using a vortex mixer (Autovortex) to obtain a uniform dispersion. 100 μl was then appropriately serially diluted in PBS and spread onto hBaCa agar plates and incubated at 37°C / 5% CO2 for 28-48 h. The number of colonies was counted and the bacterial concentration in CFU / g was calculated using a Q-Count Automatic Colony Counter (Spiral Biotech, New Zealand). The vials were then placed in an incubator set at 25°C / 60% RH for stability testing at 7 and 14 days.

[0207] Formulation 4 was manufactured according to the process described in Example 2. The serum was then dispensed into 30 ml glass vials and enumerated in triplicate. The vials were then placed in an incubator set at 25° C. / 60% RH for stability testing.

[0208] Blis Q24 was found to be very unstable in formulation 3. When stored at 25°C / 60% RH, a 7-log drop was observed after only 7 days, with no viable cell counts observed by day 14. In comparison, Blis Q24 was very stable for at least 24 months at 25°C / 60% RH with varying silica content, percentage of Q24, MCT and olive oil (Figure 5).

[0209] Example 8: Comparative sensory test Formulations 1, 2 and 4 were prepared as described in Example 7. Formulation 5 was prepared by providing a hydrating moisturizer to serum formulation 4 in a dual chamber bottle as described herein.

[0210] Each formulation was set up in a clean beaker and placed on a labeled white background. Each participant was provided with a sterile cotton swab to collect and dose the formulations. Each participant (5 females, 3 males (n=8)) was provided with a scoring form that explained the test method and scoring system for each sensory attribute. Each was asked to follow the instructions provided and individually evaluate and score each formulation for each sensory attribute.

[0211] The properties evaluated were: Consistency Cushioning effect Distribution (diffusion) ·Tackiness / stickiness Absorption rate ·Hydration effect

[0212] Participants were also asked to rate their formulation choices based on appearance, preference, applicability, ease of application, and whether they would recommend to others.

[0213] Formulations 3 and 5 scored significantly higher than formulations 1 and 2 for all parameters tested.

[0214] Example 9: Safety and Efficacy The safety and efficacy of Blis Q24 bacterial cells was evaluated using the EpiDerm 3D Skin Model.

[0215] methodology EpiDerm Human 3D Skin Model Culture (Skin Model) Upon receipt, final development of immature EpiDerm (EPI-201-4D; MatTek Corporation, Ashland, MA, USA) tissues was completed according to the manufacturer's instructions. Briefly, tissues were transferred from the agarose-containing gel in which they were shipped to cell culture plates containing pre-warmed differentiation medium (EPI-201-DM). They were then maintained at 37°C in a 5% CO2 atmosphere for 4 days, during which time fresh differentiation medium was replaced two or three times. Finally, fully developed EpiDerm tissues were transferred to fresh plates containing standard culture medium (EPI-100-NMM) for use in each experiment.

[0216] Dose-response studies on tolerability Maturation of 24 immature EpiDerm tissues was performed as described above. Fully differentiated tissues were cultured with either PBS (negative control) or Blis Q24 at five dose levels (1 × 10 5 cfu / ml ~ 1 × 10 9 cfu / ml) with four replicate tissues per treatment. The apical surface of the tissue was treated with 30 μL of treatment solution, after which the tissue was incubated for 24 hours at 37°C in a 5% CO2 atmosphere. At the end of that period, the tissues were gently washed with PBS and their viability was assessed using the MTT viability assay. (Mosmann, T. (1983). Journal of Immunological Methods, 65(1-2), 55-63; Kubilus, J. (1996). In Vitro and Molecular Toxicology, 9(2), 157-166.)

[0217] Safety and Efficacy Studies Maturation of 36 immature EpiDerm tissues was performed as described above. Fully differentiated tissues were numbered 1-36 and randomly assigned to receive one of the treatments shown in Table 3. This was done so that each of the six combinations of main treatment (PBS, Q24 or Vitamin C) and 0.5% SDS exposure treatment (± 0.5% SDS) were randomly represented in each of five 6-well cell culture plates. The remaining six tissues, which were either for histology or were to receive 5% SDS, were grouped together in the sixth cell culture plate. After maturation, the 36 tissues were cultured in assay medium at 37°C in a 5% CO2 atmosphere for 24 hours, after which baseline (day 0) conditioned medium samples were collected and stored frozen at -80°C for cytokine measurements. After each tissue had been treated with the appropriate main treatment solution each day for 4 days from day 0, the tissues were returned to an incubator maintained at 37°C in a 5% CO2 atmosphere. Tissues treated with Blis Q24 or PBS vehicle were rinsed with PBS to remove residues from the previous day's treatment before containing 30 μL of the appropriate solution applied to the apical surface of the tissue. Alternatively, vitamin C (50 μg / mL) was provided to the cultures. On day 4, conditioned medium samples were collected and stored frozen at -80°C for cytokine measurements. Thirty-three tissues were then exposed to 30 μL of 0.5% or 5% SDS in PBS, or PBS as a negative control. After 1 h, these tissues were washed with PBS and patted dry before being reapplied with the appropriate main treatment (PBS, Blis Q24 or vitamin C) and transferred to a plate containing fresh medium. The remaining three tissues (for histology) were briefly provided with fresh medium and subjected to the appropriate main treatment. All tissues were returned to the incubator for an additional 24 hours at 37°C in a 5% CO2 atmosphere, after which the day 5 conditioned medium was collected and stored frozen at -80°C. All tissues were gently washed with PBS and the three histology tissues were fixed in neutral buffered formalin. The viability of the other 33 tissues was assessed using the MTT viability assay.

[0218] MTT Viability Assay (Safety) EpiDerm tissues on cell culture inserts were added to 6-well cell cultures containing 0.9 mL of 1 mg / mL MTT in DMEM medium (Thermo Fisher Scientific) and incubated at 37 °C for 3 h under 5% CO2. Afterwards, they were washed with PBS and wiped dry on cotton gauze. The remaining water inside the inserts was removed by using a cotton swab, after which each insert and tissue was immersed in 6 mL of 2-propan-2-ol and soaked in a sealed 20 mL scintillation vial in the dark overnight. The resulting purple solution of formazan dye was transferred to a disposable cuvette and absorbance was measured at 570 nm and 650 nm (reference wavelength) on an Ultrospec UV-Visible spectrophotometer (LKB). Data analysis involved subtracting the absorbance at the reference wavelength from the 570 nm reading to give a corrected absorbance value. Each absorbance value was converted to a % viability value by dividing by the mean value of the negative control group (PBS not exposed to 0.5% SDS) and multiplying by 100.

[0219] Histology (Efficacy) Tissues were fixed in neutral buffered formalin (LabServ) and sent to Gribbles Veterinary Lab (Christchurch, NZ) for preparation of HE-stained sections. Light microscopy was performed with a Leica DM6000 B microscope (Leica Microsystems, Switzerland) and images were acquired using Leica Application Suite v4.12 software.

[0220] Quantification of cytokines for anti-inflammatory effects (efficacy) For quantification of IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23 and IL-33, the bead-based multiplexed LEGENDplex™ assay (LEGENDplex™ Human Inflammation Panel 1 (13-plex); BioLegend, San Diego, CA, USA) was used according to the manufacturer's instructions. The relevance of each measured cytokine is described in Table 4. Reactions were performed in duplicate. Analysis was performed on a Cytek™ Aurora flow cytometer (Cytek Biosciences Inc., Fremont, CA, USA). Data were analyzed using Legendplex V8.0 software (BioLegend) and defined as pg / mL.

[0221] statistical analysis Statistical analysis and graphing were performed using Prism 9.2.0 (GraphPad Software). Data were analyzed by one-way or two-way analysis of variance (ANOVA) and corrected for multiple comparisons using Dunnett's or Sidak's method, as appropriate.

[0222] Tolerability The tolerability of Blis Q24 was determined by a dose-response experiment. 5 , 1.31×10 6 , 1.46×10 7 , 1.71×10 8 and 1.63 x 10 9 EpiDerm tissue treated with the 1 × 10 cfu / ml suspension showed >90% viability (Figure 6). 9 Although the viability of CFU / ml was significantly different from the PBS control, the average cell % was still >90% viable, indicating that Blis Q24 was well tolerated by model skin tissue during the 24 hour exposure.

[0223] These in vitro results indicate that 1.5 × 10 8 This compares favourably with in vivo testing of Blis Q24 hydration serum, where no skin irritation was observed even after repeated administration of CFU / dose.

[0224] Conclusions: Blis Q24 at various clinically relevant doses was well tolerated in tissue culture models, demonstrating the safety of strains and products containing Blis Q24.

[0225] safety After dose-response experiments, 1 × 10 8 The dose of CFU / ml was selected for safety and efficacy studies.

[0226] Thirteen cytokines were measured (days 0 and 4) using Blis Q24 (1 × 10 8 CFU / ml), PBS (negative control) and Vit C (positive control). Of the 13 treatment-related effects, only three cytokines were observed: interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-18 (IL-18) are inflammatory cytokines known to be produced by skin cells in response to inflammatory stimuli. IL-6 levels showed a slight but significant increase on day 4 for vitamin C, but remained unchanged over the two days for Blis Q24 and PBS (Figure 7, top left). IL-8 levels decreased only slightly on day 4 for Blis Q24 and PBS (but the change was only significant for the latter) and remained unchanged for vitamin C (Figure 7, top right). For IL-18, there was no significant difference between levels on days 0 and 4, but Blis Q24 and vitamin C showed a trend towards lower values ​​on day 4 (Figure 7, bottom center).

[0227] Conclusion: The results indicate that Blis Q24 is safe as it did not induce an inflammatory immune response in EpiDerm tissues after 4 days of daily administration.

[0228] Efficacy (histology) 1 × 10 in PBS, PBS (negative control) and Vit C (positive control) 8 A CFU / ml dose of biological Blis Q24 suspension was applied to EpiDerm cells (duration 4 days) and treatment-related changes on tissue morphology were recorded (n=1, FIG. 8). Although no obvious treatment-related differences were found, tissues receiving Blis Q24 revealed remnants of nuclei trapped in the stratum corneum, indicating that it may promote stratum corneum production. The stratum corneum was also only slightly thicker in Blis Q24 tissues than in PBS or vitamin C treated tissues. Although more replications are needed to confirm these findings, there appears to be a favorable effect of Blis Q24. It is known that the stratum corneum mainly functions as a barrier between the deeper layers of the skin and the external environment, preventing toxins and bacteria from entering the body. It also helps prevent water from evaporating into the atmosphere, which is important for keeping the skin moist.

[0229] The results indicate that Blis Q24 may help rejuvenate and moisturize the skin by promoting the formation of the stratum corneum.

[0230] Conclusion: Blis Q24 is effective in promoting the growth of the stratum corneum, which is responsible for barrier function and moisture retention. These in vitro results support the results of in vivo studies, where study participants observed increased hydration and reduced pores and wrinkles after applying Blis Q24 Hydration Serum.

[0231] Anti-inflammatory effect Only IL-6, IL-8 and IL-18 had detectable levels to determine the effect of Blis Q24, PBS (negative control and Vit C positive control) treatment (Figure 9-top left).

[0232] Pretreatment with Bris Q24 caused a negligible, non-significant decrease in the amount of IL-6 released in response to 0.5% sodium dodecyl sulfate (SDS); in contrast, vitamin C-treated tissues displayed a negligible increase on day 5 in response to 0.5% SDS exposure. A significant increase in IL-8 levels was evident on day 5 compared to day 4 in response to 0.5% SDS for all three main treatment groups (Figure 14-top right). IL-18 release was also stimulated by 0.5% SDS in all groups, but the change was not significant for the Bris Q24 group (Figure 14-bottom center).

[0233] Conclusion: For the Blis Q24+0.5% SDS group, the negligible decrease in SDS-induced IL-6 release and the blunted increase in IL-18 indicate the anti-inflammatory effect of Blis Q24.

[0234] Example 10: Placebo Study To determine the effect of the formulation of the present invention on cosmetic skin parameters, two studies were conducted. Study 1: Active Q24 serum (oil phase) + water phase vs placebo serum + water phase. Test 2: Live Q24 + cetomacrogol vs Dead Q24 + cetomacrogol.

[0235] Study 1: In this randomized, placebo-controlled, double-blind study, eight participants were divided into two groups. Group 1 (n=4) (active group) received a dual-chamber bottle, with the first chamber containing active Bris Q24 (1×10 9 Group 2 (n=4) (placebo group) also received dual-chamber bottles, with the first chamber consisting of serum base (placebo without Blis Q24) and the second chamber containing the aqueous phase. One participant in group 1 did not complete the study for reasons unrelated to the study.

[0236] Study 2: In this randomized, controlled, double-blind study, eight participants were divided into two groups. Group 1 (n=4) (bio-Q24 group) received a dual-chamber bottle, with the first chamber containing bio-Blis Q24 (1×10 9 Group 2 (n=4) (killed Q24 group) also received dual-chamber bottles, with the first chamber consisting of serum containing gamma-irradiated Blis Q24 (killed bacteria) and the second chamber containing cetomacrogol cream.

[0237] Participants in both studies were then asked to pump the formulations (one pump each of serum and moisturizer or cream), mix them in the palm of their hands, and apply them to their face twice daily (morning and night) for 31 days.

[0238] Whole-face skin quality parameter analysis was performed using an advanced skin analyzer (Chowis, Korea) before the first application (day 0 pre-study), on days 11 and 31 during the study period, and 10 days after the final study (day 41).

[0239] Study 1: Active vs. Placebo The Blis Q24 "active" composition showed improvement in all skin quality parameters versus placebo compared to baseline at various time points (Figure 10). Improvements were seen even after 10 days post-treatment (Tx).

[0240] In this study, an increase in sebum was also observed. The interpretation of the sebum score is difficult, since sebum plays an important multifaceted role: it makes the skin smooth and more impermeable to moisture, protecting it from friction; it reduces water loss from the skin's surface; it protects the skin from bacterial and fungal infections; in addition, sebaceous glands transport antioxidants in and on the skin and provide natural photoprotective activity. Excessive secretion of sebum leads to oily skin.

[0241] It is noted that in this study, none of the participants complained of "oily skin," suggesting that the sebum levels were well within the desired range for the participants, and all showed an increase in sebum and water content. An increase in sebum was also observed in the placebo group. Therefore, an increase or decrease in sebum compared to baseline is not necessarily "desirable" or "undesirable," but rather sebum balance is the key factor.

[0242] Test 2: Live Q24 vs. dead bacteria The Blis Q24 "living" composition showed improvement in all skin quality parameters versus placebo compared to baseline at various time points (Figure 11). Improvements were seen even after 10 days post-treatment (Tx).

[0243] Example 11: Addition of prebiotics Inhibition of common prebiotics CABK12 agar plates were divided into six sections and lawned with a suspension of M. luteus Q24 source P3. Each of the aqueous potential prebiotic substances to be screened was serially diluted in a range of concentrations from 100% to 0.3% with sterile distilled water. Oil-based substances were tested only at 100%.

[0244] 20 μL of each substance at each concentration was pipetted onto a spot on one slice of lawn-grown CABK12 agar plate and incubated at 37°C with 5% CO2 in air for 24 h. [Table 4]

[0245] Conclusions: All substances passed the initial screening test for Q24-free products, except for green tea powder, which was inhibitory at all concentrations tested. The other six potential prebiotics showed some inhibitory effects on Q24, indicating that they may not be suitable for products requiring biological Q24 stability, but may be used in prebiotic-only formulations.

[0246] Effect of prebiotics on the growth of Blis Q24 A batch of M17 broth (Difco #218561) was prepared according to the manufacturer's instructions, excluding the lactose solution. 50 ml of broth was dispensed into sterile 100 ml Schott bottles and 2.5 g (5%) of each potential prebiotic substance was added and mixed well using a magnetic stirrer and stir plate. The mixture was autoclaved at 110°C for 10 minutes and allowed to cool.

[0247] Suspensions of prebiotic candidates were pre-heated to 40°C to allow for improved homogenization of any oily components prior to dispensing into wells. 2 ml of each suspension and M17 only (control) were pipetted into sterile 24-well tissue culture plates. A suspension of Q24 source P3 was made in PBS and adjusted to an optical density of 0.125. 100 μl of suspension was pipetted into each well. Once the suspension was added, each well was mixed by aspirating and dispensing the solution five times with a 1 ml pipette.

[0248] Each sample was enumerated at 0, 3, 6, 15, 26 and 34 hours using the following method: The wells were mixed by dispensing and aspirating five times with a 1 ml pipette at each time point. Then, 100 μl was removed from each well and added to 900 μl of Eppendorf PBS (1 / 10 dilution). These 1 / 10 dilutions were vortexed horizontally at 2600 rpm for 10 minutes. The samples were then serially diluted by transferring 100 μl of the 1 / 10 dilution into fresh 900 μl of PBS, and this was repeated until six serial dilutions had been made. 20 μl of each dilution from each sample was spotted in triplicate onto sheep blood agar plates. The spots were left to dry for 30 minutes and set in a 37°C 5% CO2 incubator for 28-36 hours. The number of colonies on each spot was then counted using an electronic colony counter and averaged to give the final results.

[0249] Results: All tested prebiotic candidates showed a higher increase in Q24 than the control, with most resulting in a 1-2 log increase over 24 hours compared to the control.

[0250] Conclusions: Surprisingly, commonly used prebiotics, typical for topical application, carbohydrates (with the exception of oatmeal), did not support the growth of Q24.

[0251] Traditional substances used as prebiotics for gut bacteria did not work well in terms of increasing the growth of Q24, and some, such as xylitol and manuka honey, were detrimental to the number of live Q24 cells after 24 hours (Figure 12).

[0252] In contrast, oil-based substances commonly used as functional actives and other cosmetic ingredients performed very well, the best of which increased Q24 viable cell counts by 2-log over 24 hours.

[0253] Prebiotic Combinations The same methodology was used as in study 10, except that the time points tested were 0, 6, 18, and 24 h. Olive squalane was selected as one of the preferred prebiotics, and the other three were analyzed in combination with olive squalane and compared to the results of the prebiotic candidates alone (Figure 13).

[0254] Results: The growth of Q24 was increased well above the sum of olive squalane and pomegranate seed oil added separately and showed a synergistic response when both prebiotics were added together, resulting in an increased growth rate of Q24.

[0255] The growth of Q24 was the same as the sum of olive squalane and oatmeal flour (colloidal oatmeal) added separately, indicating no synergistic response with this combination.

[0256] The growth of Q24 was increased well above the sum of olive squalane and vitamin E added separately, and showed a synergistic response when both prebiotics were added together, resulting in an increased growth rate of Q24.

[0257] Conclusion: Both olive squalane + pomegranate seed oil and olive squalane + vitamin E were found to have a synergistic relationship that increased the proliferation rate of Q24 compared to either alone. Olive squalane and oatmeal flour did not show the same response. Formulations made to contain both olive squalane and pomegranate seed oil or vitamin E would be more effective than formulations containing only olive squalane, pomegranate seed oil or vitamin E.

[0258] Prebiotic activity against some skin commensals The same method as above for the Blis Q24 component was used, except that for each of the three selected skin commensals, a suspension with an optical density of 0.125 was prepared.

[0259] The selected skin commensals are: Staphylococcus epidermidis #4 - sensitive to Q24 inhibition Staphylococcus epidermidis E30 (S. epidermidis E30) - resistant to Q24 inhibition S. hominis ATCC27844 - mixed resistance condition dependent It is.

[0260] S. hominis ATCC27844 is available from the American Type Culture Collection (ATCC); S. epidermidis #4 and S. epidermidis E30 are available from BLIS Technologies Ltd upon request.

[0261] Results: No additive growth advantage for skin commensals was observed with or without prebiotics, suggesting that prebiotics selectively promoted the growth of Blis Q24 (Figure 14).

[0262] Conclusion: Olive squalane and pomegranate seed oil, alone or in combination, did not provide any significant effect against skin commensals compared to the control medium, the effect being more pronounced and specific against Blis Q24.

Claims

1. A cosmetic treatment method (excluding a therapeutic method) for improving the appearance of skin or at least one sign of aging, comprising applying a topical composition containing Micrococcus luteus Q24 to the skin, Improving the appearance of the skin or at least one sign of aging includes making the skin appear more radiant, healthier, more moisturized, smaller pores, softer, more translucent, reduced wrinkles, reduced dryness, reduced blemishes, reduced impurities, increased moisture, and reduced sebum production. The particle size (Dv90) of Micrococcus luteus Q24 is less than 300 μm, and Micrococcus luteus Q24 is a living probiotic, by how.

2. The above composition, 1 × 10 ３ ~1 x 10 １２ The method according to claim 1, comprising Micrococcus luteus Q24 in an amount of cfu / g.

3. The method according to claim 1 or 2, wherein the composition comprises a viscosity modifier.

4. The method according to claim 3, wherein the composition comprises a viscosity modifier in an amount of 3 to 15% w / w.

5. The method according to claim 3 or 4, wherein the viscosity modifier is selected from the group consisting of hydrophobic silica, hydrophilic silica, white wax, yellow wax, paraffin wax, jojoba wax, microcrystalline wax, ethylcellulose, stearic acid, xanthan gum, tapioca starch, Carbopol polymer, cocoa butter, shea butter, and any two or more combinations thereof.

6. The method according to any one of claims 1 to 5, wherein the composition comprises a dispersant.

7. The method according to claim 6, wherein the composition comprises a dispersant in an amount of 0.1 to 5% w / w.

8. The method according to claim 6 or 7, wherein the dispersant is selected from the group consisting of polysorbate 80, polysorbate 20, sorbitan oleate, egg lecithin, soy lecithin, polyoxyl 35 castor oil, and any two or more combinations thereof.

9. The method according to any one of claims 1 to 8, wherein the composition comprises an oil vehicle.

10. The method according to claim 9, wherein the oil vehicle is selected from the group consisting of medium-chain triglycerides, vegetable oils, or combinations thereof.

11. The method according to claim 10, wherein the medium-chain triglyceride is tri(caprylic / capric acid)glyceryl.

12. The method according to claim 10, wherein the vegetable oil is selected from the group consisting of sunflower oil, rapeseed oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grape seed oil, and any two or more combinations thereof.

13. The method according to any one of claims 1 to 12, wherein the Micrococcus luteus Q24 does not contain a freeze-drying protective agent.

14. The method according to any one of claims 1 to 13, wherein the composition further comprises one or more additional probiotics.

15. The one or more additional probiotics include: Streptococcus sp., Lactobacillus sp., Limosilactobacillus sp., Lacticaseibacillus sp., Ligilactobacillus sp., Lactiplantibacillus sp., Bifidobacterium sp., Saccharomyces sp. The method according to claim 14, selected from the group consisting of Saccharomyces spp. and any two or more combinations thereof.

16. The method according to claim 15, wherein the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus 24SMB, Streptococcus oralis, Streptococcus salivarius DB-B5, and any two or more combinations thereof.

17. The above composition, 1 × 10 ３ ~1 x 10 １２ The method according to any one of claims 14 to 16, comprising each additional probiotic in an amount of cfu / g.

18. The method according to any one of claims 1 to 17, wherein the composition further comprises an inhibitory activity enhancer, a buffer, an antibacterial agent, a prebiotic, a fragrance, an antioxidant, a colorant, a skin protectant, an antimicrobial agent, an aluminum salt, a mineral pigment, an odor absorber or neutralizer, a sunscreen, and any two or more combinations thereof.

19. The method according to claim 18, wherein the inhibitory activity enhancer is selected from the group consisting of sodium chloride, ethylenediaminetetraacetic acid, arginine, calcium carbonate, and any two or more combinations thereof.

20. The method according to claim 18, wherein the buffer is selected from the group consisting of calcium carbonate, magnesium carbonate, sodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium carbonate, urea, hydrated aluminum oxide, bentonite viscosity, kaolin viscosity, and combinations thereof.

21. The method according to claim 18, wherein the antibacterial agent is selected from the group consisting of xylitol, erythritol, epidermine, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, salivaricin 9, salivaricin MPS, and combinations thereof.

22. The method according to claim 18, wherein the prebiotics are selected from the group consisting of manuka honey powder, olive squalane, pomegranate seed oil, flaxseed oil, coconut oil, colloidal oatmeal, vitamin E, vitamin C, retinol (vitamin A), olive oil, hyaluronic acid, calendula oil, almond oil, tomato oil, allantoin, aloe vera powder, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide solution, fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, niacinamide, sunscreen, and any two or more combinations thereof.

23. The method according to claim 18, wherein the fragrance is selected from the group consisting of rose water, orange blossom, rose gardenia, peony, white jasmine, ylang-ylang oil, geranium oil, rose oil, and any two or more combinations thereof.

24. The method according to claim 18, wherein the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalane, vitamin C, beta-carotene, retinyl acetate, retinyl palmitate, retinol, niacinamide, caffeine, and any two or more combinations thereof.

25. The method according to claim 18, wherein the skin protectant is selected from the group consisting of ceramide, collagen, elastin, coenzyme Q10, hydrolyzed collagen, hyaluronic acid, sodium hyaluronate, retinol, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, palmitoyl tetrapeptide-38, acetyl hexapeptide-8, heptapeptide-14, heptapeptide-15-palmitate, palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, α-hydroxy acid, β-hydroxy acid, vitamin B5, seaweed, seaweed extract, silicone, xylitol, oat extract, oatmeal flour, colloidal oatmeal, aloe vera, sunscreen, and any two or more combinations thereof.

26. The method according to claim 18, wherein the antimicrobial agent is selected from the group consisting of zinc, azelaic acid, benzoyl peroxide, and combinations thereof.

27. The method according to any one of claims 1 to 26, wherein the composition comprises 0.1% to 10% w / w olive squalane and 0.1% to 10% w / w pomegranate seed oil.

28. The method according to any one of claims 1 to 26, wherein the composition comprises 0.1% to 10% w / w olive squalane and 0.1% to 3% w / w vitamin E.

29. The method according to any one of claims 1 to 28, wherein the composition is non-aqueous.

30. The method according to any one of claims 1 to 29, wherein the composition is applied in combination with an aqueous phase, where applicable.

31. The method according to any one of claims 1 to 30, wherein the composition has a viscosity of 20,000 to 2,000,000 cp at 25°C.

32. The method according to any one of claims 1 to 31, wherein the composition has a shelf life of at least 6 months at 25°C and 60% RH.

33. A topical composition comprising Micrococcus luteus Q24, a viscosity modifier, a dispersant, and an oil vehicle, wherein the composition comprises 1 × 10 ４ ~1 x 10 １０ Contains Micrococcus luteus Q24 in cfu / g amounts, The particle size (Dv90) of Micrococcus luteus Q24 is less than 300 μm, and Micrococcus luteus Q24 is a living probiotic, a topical composition.

34. A topical composition comprising Micrococcus luteus Q24, hydrophobic silica, polysorbate 80, and an oil vehicle, The particle size (Dv90) of Micrococcus luteus Q24 is less than 300 μm, and Micrococcus luteus Q24 is a living probiotic, a topical composition.

35. 1×10 ３ ~1 x 10 １２ Micrococcus luteus Q24 at cfu / g, 2-10% w / w hydrophobic silica, 0.5-2% w / w polysorbate 80, and A sufficient amount of oil vehicle, A topical composition comprising, The particle size (Dv90) of Micrococcus luteus Q24 is less than 300 μm, and Micrococcus luteus Q24 is a living probiotic, a topical composition.

36. The topical composition according to claim 35, wherein the oil vehicle is a medium-chain triglyceride.

37. The topical composition according to claim 35 or 36, further comprising 0.1 to 35% w / w of prebiotics(s).

38. The topical composition according to claim 37, wherein the prebiotic(s) are selected from olive squalane, pomegranate seed oil, vitamin E, or a combination thereof.

39. The topical composition according to claim 37 or 38, wherein the prebiotic is selected from olive squalane and pomegranate seed oil; olive squalane and vitamin E; and a combination of olive squalane, pomegranate seed oil, and vitamin E.

40. A topical composition according to any one of claims 33 to 39 for improving the appearance of the skin or at least one sign of aging, A topical composition for improving the appearance of the skin or at least one sign of aging, which includes making the skin appear more radiant, healthier, more moisturized, smaller pores, softer, more translucent, reduced wrinkles, reduced dryness, reduced blemishes, reduced impurities, increased moisture, and reduced sebum production.

41. The use of Micrococcus luteus Q24 in the manufacture of a composition for improving the appearance of the skin or at least one sign of aging, Improving the appearance of the skin or at least one sign of aging includes making the skin appear more radiant, healthier, more moisturized, smaller pores, softer, more translucent, reduced wrinkles, reduced dryness, reduced blemishes, reduced impurities, increased moisture, and reduced sebum production. The particle size (Dv90) of Micrococcus luteus Q24 is less than 300 μm, and Micrococcus luteus Q24 is a living probiotic, for use.

42. A two-phase composition comprising an oil phase and an aqueous phase, wherein the oil phase comprises a topical composition according to any one of claims 33 to 39.

43. A kit comprising a topical composition containing Micrococcus luteus Q24 and an aqueous composition.

44. The kit according to claim 43, comprising a dispensing system having a first container and a second container, wherein the first container comprises an oil phase containing Micrococcus luteus Q24 and the second container comprises an aqueous phase.

45. A method for producing a topical composition containing Micrococcus luteus Q24, comprising the following steps: a) Mix the oil vehicle and the dispersant. b) Add Micrococcus luteus Q24 and a viscosity modifier to the mixture from step a), c) Homogenize the mixture from step b) to provide the composition. A manufacturing method that includes this.

46. The method according to claim 45, wherein the particle size (Dv90) of the Micrococcus luteus Q24 is less than 300 μm.

47. The method according to claim 45 or 46, wherein the particle size of Micrococcus luteus Q24 is less than 250 μm or less than 100 μm.

48. The method according to any one of claims 45 to 47, which is carried out without heating.

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